Engineered Living Materials 3030929485, 9783030929480

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Wil V. Srubar III   Editor

Engineered Living Materials

Engineered Living Materials

Wil V. Srubar III Editor

Engineered Living Materials

Editor Wil V. Srubar III Department of Civil, Environmental, and Architectural Engineering Materials Science and Engineering Program, University of Colorado Boulder Boulder, CO, USA

ISBN 978-3-030-92948-0 ISBN 978-3-030-92949-7 https://doi.org/10.1007/978-3-030-92949-7

(eBook)

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

Network Formation of Engineered Proteins and Their Bioactive Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seunghyun Sim Living Synthetic Polymerizations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Austin J. Graham and Benjamin K. Keitz Programmable Self-Assembling Protein Nanomaterials: Current Status and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kelly Wallin, Ruijie Zhang, and Claudia Schmidt-Dannert Engineered Living Conductive Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . Lina J. Bird, Fernanda Jiménez Otero, Matthew D. Yates, Brian J. Eddie, Leonard M. Tender, and Sarah M. Glaven

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Photoswitchable Bacterial Adhesions for the Control of Multicellular Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Fei Chen and Seraphine V. Wegner Additive Manufacturing of Engineered Living Materials . . . . . . . . . . . . 149 Lynn M. Sidor and Anne S. Meyer Engineered Living Materials for Construction . . . . . . . . . . . . . . . . . . . . 187 Rollin J. Jones, Elizabeth A. Delesky, Sherri M. Cook, Jeffrey C. Cameron, Mija H. Hubler, and Wil V. Srubar III

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Network Formation of Engineered Proteins and Their Bioactive Properties Seunghyun Sim

Abstract Proteins are one of the main components of the extracellular matrix in natural biological materials. They confer a unique advantage in creating engineered living materials (ELM) because they can be genetically encoded and rationally designed for constructing bioactive network structures. Advances in the design, characterization, and engineering of protein networks have been an important multidisciplinary endeavor and should be considered when designing ELM and understanding their behavior. This chapter describes the network-forming behavior of recombinant proteins, as these proteins, in principle, can be genetically programmed and synthesized directly from living cells residing in ELM. There are three major classes of protein network-forming mechanisms relevant to this topic: (1) phase separation and aggregation-induced recombinant protein networks, (2) self-assembling multi-domain artificial protein networks, and (3) chemically cross-linked recombinant protein networks. We will begin by introducing protein hydrogels and discuss their mechanism of network formation, which is a critical element in designing functionalities and mechanical properties of ELM. After introducing the network-forming mechanisms in protein hydrogels, we will discuss examples of bioactive protein networks equipped with various functionalities before concluding with future directions and remaining challenges in this field. Keywords Protein engineering · Protein network · Protein hydrogel · Bioactive protein network · Artificial extracellular matrix

1 Introduction Natural biological materials have been a key inspiration in the broad scientific field due to their unique ability to self-regulate, self-organize, self-heal, and respond to complex environmental cues. We see this in bone, wood, skin, and biofilm, where

S. Sim (*) Department of Chemistry, University of California, Irvine, Irvine, CA, USA e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 W. V. Srubar III (ed.), Engineered Living Materials, https://doi.org/10.1007/978-3-030-92949-7_1

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living cells produce, regulate, and repair their own surrounding matrix. These emergent properties result from the living cells functioning as an active component in these materials and have yet to be demonstrated in man-made synthetic materials. Inspired and motivated by this striking functionality and complexity of natural biological materials, many efforts have been made in recent years to construct engineered living materials (ELM) where living cells are the producers and modulators of their surroundings. In nature, the main components of these networks made by living cells are self-assembling proteins and exopolysaccharides. Although exopolysaccharides produced by living cells are often the most abundant structural component, engineering and production of designed networks are challenging as they involve a variety of biosynthetic machinery that is not necessarily shared across different species. Proteins, on the other hand, can be genetically encoded, rationally designed for specific purposes, and ported between species. In addition, proteins are an attractive building block for networks interfacing living cells from both functional and structural aspects. They fold into a defined three-dimensional structure, can bind specific partners even in a complex cellular environment, and form protein-protein interactions. As a result, they can assemble into a higher-order network responsible for the mechanical property of the ELM. Moreover, many functional protein motifs are amenable to use in conjunction with structural units that undergo network formation. Self-assembled protein networks in the context of extracellular materials can be classified as hydrogels, as they are extensively hydrated networks housing constituent living cells. Therefore, throughout this chapter, we will use the terms “network” and “hydrogel” interchangeably. Hydrogels are physically or chemically crosslinked polymer networks that swell in water. Their ability to hold large amounts of water stems from a delicate balance of good water solubility and interchain crosslinking. Self-assembling proteins and aggregation-prone proteins constitute physically cross-linked hydrogel as they rely on the physical association of particular structural motifs for the network construction. Proteins that form a covalent bond between two separate domains upon association can serve as a building block for chemically cross-linked hydrogels. Protein hydrogels have a significant advantage over synthetic or bio-derived hydrogels in that one can rationally design protein networks with specific functions and properties in mind, as protein structures can be precisely engineered via genetic modification of DNA. For example, a protein domain known to confer an appealing functionality can be rationally fused to other protein domains that form a network. In addition, protein-based hydrogels tend to be more biocompatible and biodegradable than synthetic polymeric hydrogels. For these reasons, protein hydrogels have been extensively studied for their utility in injectable delivery vehicles, implantable scaffolds for soft-tissue engineering, and matrices for in vitro cell culture. Conformational changes of proteins in response to temperature, pH, light, ligands, and mechanical force further prompted the development of stimuli-responsive protein hydrogels with sensing capability. The main focus of this chapter is understanding the network-forming behavior of proteins as a structural component in engineered living materials, as these proteins,

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in principle, can be genetically programmed to be produced from living cells residing in ELM. We identify three major classes of protein network-forming mechanisms relevant to this topic: (1) phase separation and aggregation-induced recombinant protein networks, (2) self-assembling multi-domain artificial protein networks, and (3) chemically cross-linked recombinant protein networks. We will begin by introducing protein hydrogels and discuss their mechanism of network formation, which is a critical element in designing functionalities and mechanical properties of ELM. After introducing the network-forming mechanisms in protein hydrogels, we will discuss examples of bioactive protein networks equipped with various functionalities before concluding with future directions and remaining challenges in this field.

2 Protein Structure and Self-Assembly Proteins are sequence- and length-controlled linear chains of amino acids joined by peptide bonds. This well-defined linear chain is the most basic structure of proteins, also known as the primary structure. Proteins are synthesized by a sequential biological process. First, the genetic information stored in DNA is transcribed into a messenger RNA (mRNA). The second step is the translation of mRNA by ribosomal catalysis into a linear polypeptide chain, transferring genetic information written in a nucleic acid sequence into a protein sequence. It is possible to produce recombinant proteins with a varying production yield by modifying the DNA of host organisms and harnessing their transcription and translation machinery. Innovation in synthetic and chemical biology gave birth to many tools to manipulate this process, such as engineered ribosomal binding sites, split RNA polymerase, and mutant tRNA synthetase. Notably, advances in co-translational incorporation of noncanonical amino acids have allowed the expansion of native functionalities of proteins, for example, introducing bio-orthogonal reactive handles and photoreactive moieties into recombinant proteins (Link et al. 2003).

2.1

Protein Structures and Their Assembly

Proteins organize themselves into specific three-dimensional structures. The local structural element of proteins with a regular arrangement of peptide backbone is called secondary structures. Their regularity is maintained by the local conformation of the peptide backbone arising from their rotational degree of freedom, hydrogen bonding between amide moieties, and other supramolecular interaction between side chains. The most well-known secondary structures are the α-helix and β-strand. A typical α-helix structure is shown in Fig. 1a: The polypeptide backbone forms a right-handed helix by regular intramolecular hydrogen bonding. Each backbone carbonyl oxygen in an α-helix is hydrogen-bonded to the backbone amide hydrogen

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Fig. 1 (a) A schematic illustration of an α-helix. It is a right-handed helix with 3.6 amino acid residues per helical turn. Each backbone carbonyl oxygen in an α-helix is hydrogen-bonded to the backbone amide hydrogen four residues down from it toward C-terminus. The side chains of the amino acids (designated as R) extend outward from the helical backbone. (b) Schematic illustration of a coiled-coil dimer and its helical wheel representation. Dimer formation is driven by hydrophobic interaction of residues at a and d positions. Residues at e and f positions can stabilize coiledcoil dimers by electrostatic attractions. (c, d) Hydrogen-bonding networks between two β-strands to form (c) parallel and (d) antiparallel β-sheet

four residues down from it toward the C-terminus, resulting in 3.6 amino acids per one complete turn of the helix. While the amide backbone is hydrogen-bonded to itself almost parallel to the helical axis, the side chains of each amino acid extend outward from the backbone. The unique ability of α-helix presenting side chains of its constituent amino acids in a regular fashion has attracted significant interest as a model system for protein folding studies and a design template for creating protein assembly. In particular, coiled-coil structures are formed when two or more α-helices self-assemble (Fig. 1b). Depending on the relative orientation of helical axes, coiledcoil forms either parallel or antiparallel arrangement. The primary structures of a coiled-coil domain comprise heptad repeats—repetition of seven amino acids, often represented as (abcdefg)n. In each helix in coiled-coil, hydrophobic amino acids, including leucine and isoleucine, occupy every three or four residues (a and d ),

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resulting in hydrophobic “face” of the helix. The interaction (burial) of these hydrophobic faces is the major driving force for the self-assembly of coiled-coil domains. The supramolecular interactions of amino acid side chains on each helix affect a helical orientation, oligomerization state, and binding partner specificity of coiled-coil domains. For example, ionic residues at e and g position can stabilize coiled-coil by electrostatic attractions (Fig. 1b). Leucine zippers, a sub-type of coiled-coil and also a common motif in DNA binding proteins, contain a series of leucines spaced seven residues apart. Leucine zippers form coiled-coil structures via paired contacts between hydrophobic faces on the constituent helices. It has been shown that the oligomerization state of coiled-coil configurations in leucine zippers can be affected by the packing characteristics of the hydrophobic residues at positions a and d (Harbury et al. 1993). Examples of leucine zipper coiled-coils with high association numbers have been studied, including a pentamer (n ¼ 5) based on cartilage oligomeric matrix protein (Malashkevich et al. 1996) and a heptamer (n ¼ 7) by engineering GCN4 leucine zipper (Liu et al. 2006). Other minor forms of helix include the 310-helix and polyproline type II (PPII) helix. 310helix is similar to α-helix, but with three residues per turn. PPII helix occurs in proteins with repeating proline residues. It is an extended left-handed helix distinct from other helical secondary structures as it has no internal hydrogen bonding. It adopts a more extended form, with a helical pitch of 9.3 Å per turn, compared to 5.5 Å in the α-helix, and has three residues per turn. The collagen triple helix, as it consists of proline, hydroxyproline, and glycine, adopts a similar conformation to the PPII helix. Examples of self-assembling structures of various helices in naturally occurring protein materials include honey bee silk, collagen, and keratin. The other common secondary structure is the β-strand. β-strands are domains with a fully extended polypeptide chain, and multiple β-strands can laterally selfassemble into β-sheets. Typically, alternating sequences of hydrophobic amino acid and polar amino acid constitute hydrophobic and hydrophilic faces, which drive lateral assembly of β-strands into β-sheets stabilized by hydrogen bonding between carbonyl oxygens and amide hydrogens of adjacent β-strands. Similar to coiled-coil domains, β-sheets are either parallel (same N- to C-terminal direction, Fig. 1c) or antiparallel (opposite N- to C-terminal direction, Fig. 1d) depending on the relative orientation of the constituent β-strands. Sequences containing many amino acids with branched side chains, such as valine, threonine, and isoleucine, are known for their propensity to form β-strands. β-strands can also form β-hairpins, in which two β-strands are connected by a short loop and adopt an antiparallel arrangement, βspirals, or β-turns. Self-assembly and physical association of β-strands can be found in natural structural proteins, including silk, resilin, and elastin. The overall three-dimensional configuration of multiple secondary structures in a single polypeptide chain is called tertiary structure. In other words, it refers to the fully folded state of a protein, with three-dimensional arrangements of peptide backbone and side chains. Therefore, it is closely related to a specific protein function. The quaternary structure of proteins refers to a precise association of more than one polypeptide chain. Non-covalent interactions via hydrogen bonding, hydrophobic interactions, and ionic interactions are commonly found at the interface

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of subunits. One of such examples is GroEL, a barrel-shaped molecular chaperone. It consists of identical 14 subunits arranged into two heptameric rings that are stacked onto each other. Each subunit is held together by elaborate salt bridges between polar amino acids. Alternatively, in some cases, covalent bonding through disulfide bond formation or enzymatic catalysis can be formed between protein subunits. Hemoglobin is a notable example, comprising two pairs of α and β chains, where each subunit is covalently linked to a heme molecule. Recent advances in the de novo computational design of proteins have expanded the toolbox for protein-based materials (Huang et al. 2016). Based on the hypothesis that proteins fold into the lowest energy conformation of the defined sequences, the computational approach using a set of physical principles has now advanced to the point where we can accurately predict the folding of a prescribed sequence. The major driving force for protein folding is the burial of hydrophobic residues away from the solvent, typically water molecules. As a result, we are witnessing striking examples of artificial proteins with sequences unrelated to naturally occurring ones. One notable example is computationally designed multimeric, water-soluble, and channel-forming coiled-coil α-helical barrel (Thomson et al. 2014). Another example is Keating and colleagues’ work in developing a computational framework for designing protein-interaction specificity (Grigoryan et al. 2009). They also reported pairs of synthetic coiled-coils undergoing heterodimeric association called SYNZIPs (Thompson et al. 2012). As discussed in the following section, well-defined interactions between secondary structures have been extensively studied and employed as cross-linking motifs for artificial protein hydrogels. An advanced modeling algorithm, AlphaFold, based on artificial intelligence technology, has been shown to accurately predict protein structures even when no similar structure is known (Jumper et al. 2021).

3 Network Formation of Recombinant Proteins Motivated by the structural role they accomplish in nature, natural proteins have been examined as building blocks for biomaterials in the form of hydrogels, films, and others. In particular, those derived from extracellular matrices, such as collagen and gelatin, were extensively studied in the context of in vitro tissue culture. As our understanding of protein structure, recognition, and self-assembly deepens with the aid of computational frameworks, engineering recombinant proteins offers the unique opportunity to control network structure and functionality. Protein network formation is driven either by non-covalent interactions between domains or by covalent linkages between specific residues. The non-covalent interaction of proteins in a network can be further classified into molecular recognition (self-assembly) and aggregation. Although hydrogels made with short synthetic peptides also constitute an important class of biomaterials, this chapter will only describe recombinant protein networks and the resulting biomaterials. Considering the relevance of protein network in constructing ELM, three important categories of recombinant proteins

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hydrogels, focusing on their network-forming mechanism and some of the physical properties, will be discussed here: (1) phase separation and aggregation-induced recombinant protein hydrogels, (2) self-assembling multi-domain artificial protein hydrogels, and (3) chemically cross-linked recombinant protein hydrogels.

3.1

Phase Separation and Aggregation-Induced Recombinant Protein Hydrogels

Elastin is one of the essential structural components in the extracellular matrix, forming fibers responsible for tensile strength and elasticity of tissues. It is abundant in organs that require constant elastic expansion and contraction as a part of their function, including skin, lungs, and blood vessels. Structurally, elastin combines with other proteins to form elastic fibers, for example, ropelike structures in ligaments. Exported tropoelastins, the elastin precursor protein, assemble with microfibrils comprising fibrillin 1 and are then further cross-linked to each other. Inspired by the elastic mechanical property of organs conferred by elastin, there have been attempts to isolate elastin protein from natural sources. However, the purification of natural elastin poses a multitude of challenges due to its low solubility and tendency both to calcify and aggregate with other structural components (Daamen et al. 2007). Alternatively, the desired mechanical properties of elastin can be recapitulated in recombinant proteins containing multiple elastin-like polypeptides (ELP). ELP contains [VPGXG]n repeats where the X residue can be any amino acid except proline. Notably, ELP proteins exhibit inverse temperature transition behavior: At a lower temperature, ELPs are soluble and adopt random-coil conformation. Upon increasing temperature, ELP proteins become less soluble and eventually aggregate in coacervate phases above a critical temperature. The temperature where this transition from soluble to an aggregated state occurs is called the lower critical solution temperature (LCST), a well-known phenomenon often observed in hydrophilic polymers, including poly(N-isopropylacrylamide). The LCST phenomenon is thermodynamically driven by entropy gained by losing the bound water molecules to the bulk solution. It has been proposed that ELPs adopt a β-spiral structure in higher temperatures above LCST (Urry et al. 1981). The LCST of an ELP is a function of the protein length, concentration, hydrophobicity, and mole fraction of the guest residues (Urry 1997; Meyer and Chilkoti 2004). Therefore, varying the length and the guest residue composition of an ELP alters the protein’s LCST behavior. Due to its LCST behavior, recombinant ELP proteins can be purified via several rounds of temperature cycling and selective centrifugation (McPherson et al. 1996). Temperature-sensitive phase transition leads to aggregation of the more hydrophobic blocks above the LCST and drives the physical selfassembly of ELP networks. Conticello and co-workers have described the physical cross-linking of triblock ELP proteins comprising more hydrophilic mid-block ELP, A, and more hydrophobic end-block ELP, B (Wright and Conticello 2002;

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Wright et al. 2002). These sequences were selected based on their phase transition temperature, with the LCST of B being lower than that of A. At temperatures above its LCST, end-block B undergoes aggregation, while the hydrophilic mid-block A remains solvated, yielding physical and thermoreversible protein hydrogels. The critical temperature and other variables affecting sol-gel transition can be engineered through sequence variation of the triblock proteins. In a study by Chilkoti and co-workers (Betre et al. 2002), recombinant ELP without explicit “more hydrophobic” aggregation domain forms a gel-like coacervate at 37  C, above its LCST (35  C). The ELP coacervate showed three orders of magnitude increase in the complex shear modulus and dynamic viscosity and exhibited similar mechanical properties of the gels that were formed with cartilage extracellular matrix components, suggesting their potential utility for cartilage tissue engineering. As described in Sect. 3.3, ELP hydrogels have also been formed by chemical cross-linking methods after varying the ELP guest residue to incorporate reactive moieties. Silk is another natural protein that features impressive mechanical strength and extensibility. For example, spider dragline silk forms robust and elastic fibers and is three times tougher than synthetic bulletproof material Kevlar (Rising and Johansson 2015). Silk protein contains a repetitive sequence rich in glycine and alanine, which forms β-sheet crystalline domains responsible for high mechanical strength and hydrophilic amorphous regions. Physically cross-linked β-sheet-rich protein hydrogels can be produced from naturally derived silk, such as silkworm fibroin. This process involves chemical and mechanical perturbations, including low pH (Fini et al. 2005), high temperature (Kim et al. 2004), and sonication (Wang et al. 2008). However, harvesting native silk from natural sources faces multitudes of challenges, such as batch-to-batch variation, impurities, and difficulties in farming particular silk proteins, especially spider silk. Successful recombinant productions of silk proteins have been reported using bacterial (Xia et al. 2010), plant (Scheller et al. 2001), and mammalian (Lazaris et al. 2002) hosts. Although a few studies have demonstrated hydrogel formation of recombinant silk proteins (Rammensee et al. 2006; Schacht and Scheibel 2011), most of the efforts to create biomaterials with recombinant silk have focused on processing them into fibers, films, and foams. An alternative approach involves a chimeric recombinant protein containing tandem repeats of the silklike sequence GAGAGS as well as ELP domain (Megeed et al. 2002; Nagarsekar et al. 2003). The addition of ELP reduces the degree of crystallization of the silklike domain and introduces flexibility and solubility. These proteins spontaneously form physically cross-linked hydrogels due to crystalline domains comprising the silklike region and show ELP-like properties, such as temperature responsiveness.

Network Formation of Engineered Proteins and Their Bioactive Properties

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Self-Assembling, Multi-domain Recombinant Protein Hydrogels

Coiled-coil associations have been extensively studied as network-forming motifs for generating recombinant protein networks. In work by Tirrell and colleagues, a telechelic triblock protein debuted as the first example of a rationally designed recombinant protein that forms a hydrogel (Petka et al. 1998). This protein, namely, ACnA, contains two leucine zipper end-blocks (A) separated by a protein spacer mid-block comprising n-repeats of C unit (Cn) that adopts mostly random coil geometry and is highly water-soluble. The leucine zipper forming sequence of A comprises six heptad repeats. It is engineered based on the a/d residue pattern of the Jun oncogene product and a database constructed with naturally occurring coiledcoil proteins for determining residues at b/c/f position. Nine Glu and three Lys residues occupy 12 e/g positions of the A sequence in order to solubilize the coiled-coil structure and control their assembly with pH. The mid-block was based on an alanine and glycine-rich sequence [(AG)3PEG]10. This telechelic multidomain protein, ACnA, forms a hydrogel by the physical association of A blocks, while the mid-block linker [(AG)3PEG]10 remains fully solvated. Temperature and pH affect the association of leucine zipper domains, and as a result, drive the sol-gel transition of this protein. At low pH, the acidic residues at the e and g positions are protonated, and the stability of coiled-coil aggregates increases. With increasing pH, deprotonation of these residues and increased electrostatic repulsion between helices destabilize the association of coiled-coil domains. At high temperatures, ACnA behaves as viscous liquids due to the thermal unfolding of leucine zipper domains. Although ACnA forms a physical protein network by the coiled-coil association of A end-blocks, their low aggregation number (n ¼ 4) and the transient nature of association resulted in a soft hydrogel that erodes rapidly in open solutions near physiological pH. In addition, triblock telechelic proteins tend to form intramolecular loops that do not contribute to network elasticity (Fig. 2a). Intramolecular loops usually form an antiparallel association of the two end-blocks joined by a mid-block. To overcome this limitation, the Tirrell lab showcased several different engineering strategies. Carefully placing cysteine residues on the hydrophobic face of the helix in order to preferentially stabilize intermolecular associations resulted in coiled-coil aggregates that were stabilized via disulfide bond formation (Shen et al. 2005). Extension of a mid-block, Cn, suppresses loop formation, and as a result, ACnA with more extended linker regions were mechanically stiffer (Shen et al. 2007). Similarly, loop formation is reduced in pH or ionic strength conditions that favor the mid-block extension. Kopeček and co-workers designed a series of telechelic triblock proteins, namely, ABA, CBA, ABC, and CBC, with a mid-block spacer B as [(AG)3PEG]10 (Xu et al. 2005; Xu and Kopeček 2008). A and C block sequences are (VSSLESK)6 and (VSSLESK)2-VSKLESK-KSKLESK-VSKLESK-VSSLESK, respectively. Changing one valine and three serine residues from the A block to lysine (underlined) resulted in the C block sequence. These added lysine residues in

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Fig. 2 (a) Schematic illustration of coiled-coil domains. Intermolecular loop formation occurs and competes with network formation in ACnA proteins that form antiparallel coiled-coil aggregates. On the other hand, loop formation is suppressed in PCnP proteins with P domains that aggregates in parallel orientation, as it requires chain stretching of the mid-block. (b) Shear-thinning and elastic recovery of PCnP hydrogels. The shear storage modulus of PCnP hydrogels decreases upon oscillatory strain and recovers to its original modulus within seconds (left). PCnP forms a shearthinning, yet self-supporting, gel (right). Reproduced with permission (Olsen et al. 2010). Copyright 2010, the American Chemical Society. (c) Schematic illustration of the mixing-induced, two-component hydrogel (MITCH) where two domains in component 1 and 2 assemble via molecular recognition. Reproduced with permission (Foo et al. 2009). Copyright 2009, the National Academy of Sciences

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the C block changed the thermal stability, pH responsiveness, and self-association behavior of the protein hydrogel. Controlling network structure by protein engineering effectively modulates network elasticity and erosion rates of the artificial protein network formed by the physical association of the coiled-coil domain. Another type of telechelic triblock protein, PCnP, contains a P zipper domain sequence derived from a rat cartilage oligomeric matrix protein as end-blocks (Shen et al. 2006). PCnP hydrogels are stiffer, exhibit a lower erosion rate than ACnA, and prefer to form homo-oligomeric associations when mixed with A. As A forms interactions with an antiparallel orientation in the aggregate, intramolecular loop formation always competes with network formation. In contrast to A, the P block shows a higher aggregation number (n ¼ 5) and prefers parallel alignment of individual helices. The fact that P exclusively forms parallel orientation for the pentameric coiled-coil aggregate results in PCnP forming a stiffer network (Fig. 2b). The formation of loops in PCnP network is energetically unfavorable because the mid-block needs to stretch. As a result, the P block prefers intermolecular associations that contribute to the network elasticity. In contrast to the trend observed in ACnA, the stiffness decreases with increasing length (n) of the mid-block of the PCnP proteins (Olsen et al. 2010). In the case of ACnP triblock proteins containing both A and P domains, intramolecular loop formation is further suppressed as both A and P helices form homo-oligomeric associations. As a result, ACnP showed the lowest erosion rate and the highest normalized plateau moduli among the three telechelic proteins—ACnA, PCnP, and ACnP—suggesting that ACnP indeed has the fewest number of intramolecular loops (Shen et al. 2006). Another desirable feature of these telechelic triblock PCnP hydrogels is a strong shear-thinning behavior and rapid recovery of elastic strength upon discontinuation of shear stress. In addition to the biocompatibility of protein hydrogel materials, the shear-thinning property is advantageous in the context of injectable biomaterials. Protein hydrogels extruded from a narrow-gauge needle recover to 98% of their initial elastic strength nearly instantaneously and form self-supporting structures (Fig. 2c). A different mid-block, En, comprising ELP repeats, yielded PEnP hydrogel with similar viscoelastic behaviors as PCnP proteins (Dooling et al. 2016; Rapp et al. 2018). Collagen comprises three left-handed helices intertwined to form a right-handed triple helix and further assembles into higher-order fibrous structures. As one of the most abundant proteins found in skin, tendons, and ligaments, collagens are responsible for critical mechanical functions in these tissues. Similar to the case of ELPs, synthetic or recombinant collagen-mimetic peptides containing glycine, proline, and 4-hydroxyproline have been studied to recapitulate collagen’s self-association behavior in protein materials. This motif has also been utilized to construct telechelic triblock protein hydrogels (Skrzeszewska et al. 2009, 2010; Werten et al. 2009). The two end-blocks contain nine Pro-Gly-Pro repeats that form polyproline helices assembling into a collagen-like triple-helical “knot” at the junction points in the protein network and flank a hydrophilic mid-block sequence containing glycine, asparagine, and glutamine. This protein also forms shear-thinning, thermoreversible, and self-healing hydrogels that are capable of releasing a protein cargo via erosion.

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Physical gel formation often requires exposure to high or low pH, temperature change, or high ion concentration, which are not ideal from the cell or biomacromolecule encapsulation perspective as they might lead to loss of function or cell death. To circumvent this issue, Heilshorn and colleagues developed a peptide association pair to trigger gelation upon mixing the two protein components containing separate domains (Fig. 2d) and designated this system as mixing-induced, two-component hydrogel (MITCH) (Foo et al. 2009). Domains suitable for this purpose need to fulfill the three criteria that (1) the association domain sequence must be short enough to be repeated in a single recombinant protein, (2) the domains must not interfere with extracellular signaling machinery, and (3) the domain association should be selective and tunable, and the WW and proline-rich domains were chosen accordingly. The WW domain, named after the conserved tryptophan residue, folds into antiparallel β-sheet structures and associates with the proline-rich domain. A set of two artificial proteins for MITCH comprises several repeats of either one of the domains separated by linker regions that form mostly random coils. Two WW domains, CC43 and Nedd 4.3, were selected because they differ by an order of magnitude in their association constants with a proline-rich domain (PPxY). The sol-gel transition and the viscoelastic properties of the MITCH system can be engineered by varying the stoichiometry and binding strength of the two components (Mulyasasmita et al. 2011). Like other physical protein gels, such as PEnP, MITCH hydrogels are shear-thinning, injectable, and self-healing. Li and co-workers have reported a different two-component protein hydrogel with a pair of complementary leucine zippers, CCE and CCK, that form heterodimeric coiled-coils at neutral pH (Lv et al. 2012). Engineered bio-recognition by splitting a native protein has also shown to be effective in creating physically cross-linked protein hydrogels, as these split protein fragments spontaneously reconstitute the folded conformation of the native protein. The loop elongation variant of GB1 protein, GL5, can be split into two fragments, GN and GC, capable of spontaneous reconstitution. Protein solutions containing repeats of the two fragments produced physical hydrogels (Kong and Li 2015).

3.3

Chemically Cross-Linked Recombinant Protein Hydrogels

Chemical cross-linking—creating covalent connections between constituent protein units—provides a more stable and less dynamic network than the physical protein hydrogels described in the previous section. Although significant progress has been made in the last two decades in polymer-protein conjugates and cross-linking, here we limit our scope to the covalent cross-linking of proteins. Covalent cross-linking of lysine residues is a commonly employed strategy to produce protein hydrogels. An organophosphorus cross-linker, β-[tris(hydroxymethyl) phosphino]-propionic acid (THPP), undergoes a Mannich-type condensation with primary or secondary

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amine residues on proteins. The reaction of THPP with amine is fast and cytocompatible and occurs in physiological conditions. ELP proteins were crosslinked with THPP to yield hydrogels encapsulating various cell types (Lim et al. 2008; Chung et al. 2012a). Resilin is a highly elastomeric protein found in arthropods showing superior resilience and excellent high-frequency responsiveness (Qin et al. 2012). Kiick and colleagues used the resilin-like recombinant protein, RLP-12, which contains 12 repeats of the resilin sequence from the D. melanogaster CG15920 (Li et al. 2011). THPP was employed to cross-link and modulate the mechanical properties of RLP12 hydrogels, exhibiting from 500 Pa to 10 kPa storage moduli. Despite its effectiveness in producing chemically cross-linked protein hydrogel, THPP is no longer commercially available due to its complicated synthetic procedure. Heilshorn and colleagues reported tetrakis(hydroxymethyl)phosphonium chloride (THPC) as an inexpensive and widely applicable cross-linker alternative (Chung et al. 2012b). THPC selectively reacts with amine residues, can modulate the mechanical properties of ELP hydrogels, and is cytocompatible. Glutaraldehyde is another type of cross-linker that reacts to amine residues, and it was employed to cross-link a telechelic protein containing a mid-block with 12 lysine residues sandwiched by end-blocks forming triple helix. This process produced shapememory protein hydrogels dependent on the thermoreversible association of the end-block triple helices (Skrzeszewska et al. 2011). Thiol groups on cysteine residues can form a disulfide bridge under oxidizing conditions and be used for protein hydrogel formation. Chilkoti and colleagues showed that an ELP with periodic cysteine residues undergoes hydrogel formation under mildly oxidative conditions (Asai et al. 2012). The reversibility of the covalent linkage between two sulfur atoms upon exposure to reducing agents such as glutathione confers a significant advantage for producing biodegradable protein hydrogels. Inspired by the biological processes that produce tough networks via enzymatic activities, such as wound healing, extracellular matrix reinforcement, and cell wall synthesis, it has been shown that carefully selected enzymes can construct protein networks. Chilkoti and co-workers chose tissue transglutaminase (tTG), which catalyzes the calcium-dependent acyl transfer reaction between glutamate and various primary amine residues. ELP recombinant proteins containing a 1:6 ratio of lysine (ELP[KV6-112]) or glutamine (ELP[QV6-112]) to valine were incubated with tTG to encapsulate chondrocytes in situ (McHale et al. 2005). On the other hand, the microbial transglutaminase (mTG) does not require proenzymes or calcium ions and achieves a high level of activity over a wide temperature and pH ranges. Using mTG, gelatin hydrogels were cross-linked and studied for their utility in thermal stability, encapsulation of HEK 293 cells, and therapeutic protein transport (Yung et al. 2007, 2010). In addition to transglutaminases, the horseradish peroxidase (HPO) system was harnessed to create di-tyrosine covalent bridges. HPO catalyzes conjugation reactions of phenol and aniline derivatives in the presence of hydrogen peroxide (Teixeira et al. 2012). Cross-linking resilin proteins from Drosophila melanogaster with HPO resulted in both highly elastic and adhesive biomaterials (Qin et al. 2011).

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Photochemical cross-linking is another effective strategy for the generation of protein hydrogels. Visible light irradiation of a protein solution containing a photosensitizer, Ru(bpy)3Cl2, in the presence of electron acceptor, ammonium persulfate, resulted in efficient cross-linking of proteins via tyrosine residues (Fancy and Kodadek 1999). Dixon and co-workers used this technique to cross-link recombinant resilin protein and produce a rubber-like biomaterial (Elvin et al. 2005). Alternatively, reacting ELP recombinant proteins with a heterobifunctional succinimidyl 4,40 -azipentanoate (NHS-diazirine) and subsequent cross-linking through ultraviolet light exposure resulted in the formation of protein films (Raphel et al. 2012). Howarth and colleagues engineered a genetically encodable, highly reactive SpyTag/SpyCatcher covalent bioconjugation system (Zakeri et al. 2012; Veggiani et al. 2014). Fibronectin-binding protein FbaB of S. pyogenes contains a domain called CnaB2 with an intramolecular isopeptide bond between Lys and Asp. By splitting this domain into two and subsequent rational modification, a specific bioconjugation pair, a short peptide (SpyTag) and a protein fragment (SpyCatcher), was created. Upon mixing, SpyTag and SpyCatcher undergo isopeptide bond formation between Asp117 on SpyTag and Lys31 on SpyCatcher (Fig. 3a). The reaction between SpyTag and SpyCatcher is highly specific and works well in diverse pH, temperature, and ionic environments. As the reactive units can be genetically encoded and incorporated into protein building blocks, this technology allows for posttranslational modification of the protein topology (Zhang et al. 2013) and network formation in situ. Tirrell and co-workers employed this strategy to engineer a covalently cross-linked hydrogel comprising trifunctional SpyTag chain, denoted as AAA, and bifunctional SpyCatcher protein, BB (Fig. 3b) (Sun et al. 2014). Instead of using unstructured ELP, Li and colleagues used tandem modular proteins containing folded globular domains such as GB1 and FnIII. Tandem modular proteins that contain either multiple SpyCatcher or SpyTag sequences form cross-linked hydrogels upon mixing with their binding partner (Gao et al. 2016). Stress-relaxation behaviors and viscoelasticity of chemically cross-linked SpyTag/SpyCatcher protein networks can be programmed by incorporating a mechanically interlocked p53dim domain (Yang et al. 2018). Similarly, Zhang and colleagues combined SpyTag/SpyCatcher chemistry with split-GFP and reported a 4-arm recombinant protein that presents four reactive SpyCatcher domains (Yang et al. 2020). Split green fluorescent proteins (GFPs) comprise two fragments of native GFP: a small, 15-amino acid fragment GFP11 and the rest of GFP, GFP1–10. They are highly soluble and reconstitute spontaneously when brought together. Two recombinant proteins, SpyCatcher–ELP–GFP1–10–ELP–SpyCatcher, denoted as B10B, and SpyCatcher–ELP–GFP11–ELP–SpyCatcher, denoted as B11B, were encoded on the same plasmid. Recombinant co-expression of B10B and B11B yielded a 4-arm SpyCatcher with reconstituted GFP (Fig. 3c). After purification, this protein readily forms hydrogels with bi- or trifunctional SpyTag recombinant proteins. The utility of the SpyTag/SpyCatcher system will be discussed again in the next section below.

Fig. 3 (a) Splitting of CnaB2 into the protein SpyCatcher and its peptide reaction partner SpyTag (blue). Reproduced with permission (Veggiani et al. 2014). Copyright 2014, Elsevier. (b) Schematic illustration of covalently cross-linked SpyTag-SpyTag hydrogel formed by mixing the trifunctional protein (AAA) containing SpyTag and the bifunctional protein containing SpyCatcher. Reproduced with permission (Sun et al. 2014). Copyright 2014, the National Academy of Sciences. (c) Schematic illustration describing cellular synthesis of the 4-arm starlike protein, (SpyCatcher)4GFP. Co-expression of the two fusion proteins,

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4 Functional Recombinant Protein Hydrogels Protein engineering offers the unique capability to design bioactive hydrogels, as functional protein modules can be genetically encoded and attached to structural protein domains that serve as junction points. In this regard, the advances in engineering multifunctional protein hydrogels provide a foundation for designing functional ELM. Because recombinant protein materials have been actively investigated in the context of tissue engineering and drug delivery, the most widely investigated functionality to date is cell adhesion and retention. The RGD peptide ligand, in particular, has been utilized as a “minimal cell-binding motif” and encoded into ELP (Lampe et al. 2013; Chung et al. 2012b; Sun et al. 2014; Liu and Tirrell 2008), RLP (Balu et al. 2014; Li et al. 2013, 2016), silk-like proteins (Kambe et al. 2010; Bini et al. 2006; Yanagisawa et al. 2007), and a flexible spacer in MITCH hydrogels (Parisi-Amon et al. 2013) to promote interactions of the protein scaffolds with a variety of cell types. Alternatively, the CS5 cell-binding domain can be conjugated into a structural protein, including ELP, for this purpose (Heilshorn et al. 2005; Panitch et al. 1999). A protein domain derived from extracellular matrix protein, Tenascin-C, binds to integrins. A chimeric protein of this domain and RLP formed ECM-mimetic hydrogels that promote the spreading of living fibroblasts (Lv et al. 2013). In addition to cell-adhesion domains, amino acid sequences that can be cleaved at specific sites by cell-secreted proteases have been installed in protein hydrogels, mimicking the matrix remodeling phenomena in natural biological materials. One commonly employed motif is a substrate sequence of the matrix metalloproteinases (MMPs). Incorporating an MMP substrate sequence into chimeric proteins of RLP (Charati et al. 2009; Li et al. 2013), silk/elastin-like proteins (Price et al. 2015), and silk-like proteins (Gustafson et al. 2013) has been demonstrated. Similarly, in the context of tissue engineering, peptide sequences sensitive to degradation by the proteases, such as tissue plasminogen activator and urokinase plasminogen activator, have been encoded into ELP-based protein hydrogels (Straley and Heilshorn 2009a, b; Madl et al. 2018). Banta and co-workers have engineered functional protein hydrogels based on the coiled-coil association of ACnA proteins. As incorporating fluorescent proteins in the mid-block region of ACnA architecture does not interfere with the coiled-coil association and subsequent gelation, they engineered multicolored protein hydrogels with two different fluorescent proteins and studied network structure by Förster resonance energy transfer (Wheeldon et al. 2007). Based on a similar telechelic architecture, two bifunctional co-assembling recombinant proteins were designed to form a catalytic protein hydrogel (Wheeldon et al. 2008). One protein has a

Fig. 3 (continued) B10B and B11B, and subsequent split GFP reconstitution produced intracellular (SpyCatcher)4GFP. Upon mixing with AA containing two SpyTag peptides, (SpyCatcher)4GFP assembles into a Spy-G network. Reproduced with permission (Yang et al. 2020). Copyright 2020, Elsevier

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telechelic architecture in which a mid-block is sandwiched by self-assembling α-helical leucine zippers and bears a few histidine residues to which exogenously added osmium ion could be attached. The second protein building block is the same α-helix fused to the polyphenol oxidase small laccase (SLAC) that performs catalysis upon dimerization. The combination of the oxidase enzyme and metallopolypeptide yielded an electron-conducting protein hydrogel that reduces molecular oxygen to water. The association of leucine zipper domains in these two protein building blocks allows the incorporation of SLAC enzymes into the protein network, and dimerization of SLAC contributes to additional cross-linking and catalysis. Potential applications of this hydrogel as a cathode in biofuel cells or biosensors have been suggested. Other examples of enzymes incorporated into ACnA design include aldo-keto reductase (Wheeldon et al. 2009) and organophosphate hydrolase (Lu et al. 2010). Chimeric proteins containing a leucine zipper domain fused with other stimuliresponsive self-associating protein domains have been shown to form stimuliresponsive protein hydrogels. One such example utilizes calmodulin, which undergoes a conformational change and binds to calmodulin-binding domains within other proteins in the presence of Ca2+ ions (Topp et al. 2006). When a chimeric calmodulin protein fused to a leucine zipper domain is mixed with a telechelic triblock protein bearing calmodulin-binding domain as end-blocks and Ca2+, a protein network is formed (Fig. 4a). This network undergoes a reversible change in viscosity upon addition and removal of Ca2+. Banta and colleagues engineered various Ca2+-responsive protein building blocks based on the block V repeats-in-toxin (RTX) domain of B. pertussis adenylate cyclase (Dooley et al. 2012, 2014). This domain is intrinsically disordered in the absence of Ca2+ and folds into a β-roll in response to high Ca2+ concentration. Upon addition of Ca2+, the engineered RTX β-roll folds, and the hydrophobic faces which drive oligomerization are exposed. Banta and co-workers engineered chimeric proteins with an engineered β-roll domain that self-assembles upon Ca2+ addition and mixed them with a β-roll mutant that conditionally binds to a specific target, hen egg white lysozyme. This process yielded a protein hydrogel in which gelation and target protein retention is controlled in a Ca2+-dependent manner (Bulutoglu et al. 2017). Conjugating SpyTag and SpyCatcher domains to other functional modules was demonstrated as an effective strategy to produce functional protein hydrogels. Sun and co-workers used the CarH protein, a transcriptional regulator that senses visible light through its C-terminal adenosylcobalamin binding domain (CarHC), for this purpose (Fig. 4b) (Yang et al. 2020; Jiang et al. 2020; Wang et al. 2017). The lightsensing CarHC domains form tetramers when binding to adenosylcobalamin (AdoB12) in the dark and disassemble into monomers upon exposure to a green (522 nm) light. Recombinant proteins SpyTag–ELP–CarHC–ELP–SpyTag, denoted as ACA, and SpyCatcher–ELP–CarHC–ELP–SpyCatcher, denoted as BCB, polymerize upon mixing, and in the presence of AdoB12, form hydrogels that undergo a rapid gel-sol transition with light exposure. Encapsulation and release of fibroblasts and human mesenchymal stem cells, as well as controlled release of proteins, have been demonstrated with this platform, harnessing in situ gel formation upon mixing

Fig. 4 (a) Ca2+-dependent network formation of the triblock artificial proteins. Calmodulin undergoes a conformational change and binds to proteins containing calmodulin-binding domains, including petunia glutamate decarboxylase (PGD) and human endothelial NO synthase (eNOS), in response to Ca2+ ions. Reproduced with permission (Topp et al. 2006). Copyright 2006, the American Chemical Society. (b) Synthesis of photoresponsive CarHC hydrogels. CarHC tetramer undergoes disassembly upon light exposure and releases 40 ,50 -anhydroadenosine. The two telechelic proteins, ACA containing SpyTag and BCB containing SpyCatcher, assemble into polymers and further form a protein network via AdoB12-induced CarHC tetramerization. This network can reversibly be disassembled upon light exposure. Reproduced with permission (Wang et al. 2017). Copyright 2017, the National Academy of Sciences. (c) Reproduced with permission (Huang et al. 2019). Copyright 2019, Springer Nature

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and photoresponsive gel-sol transition. The similar architecture of recombinant proteins bearing metal-binding domains showed the potential of sequestering uranium and chromate from water (Kou et al. 2017). Li and co-workers used SpyCatcher/SpyTag cross-linking to engineer optically responsive protein hydrogel networks, where multimerization of a mutant fluorescent protein, Dronpa145N, serves as an optically controllable physical cross-linker (Lyu et al. 2017). The fluorescence of the Dronpa145N switches off under a strong 500-nm illumination and switches on by 400-nm illumination, which is accompanied by the tetramerization of Dronpa145N. As a result, protein hydrogels produced by SpyCatcher/SpyTag conjugation and physical association of Dronpa145N undergo gel-sol transition upon 500-nm light illumination. Recent pioneering efforts inspired by bacterial biofilms have opened a new chapter of protein network design in the context of ELM. Many bacteria build their own physical habitat called biofilms. As bacterial biofilms are extensively hydrated networks where living cells build and maintain the proteinaceous scaffolds, they are inherently hydrogels. Bacterial fibers are a key building block of bacterial biofilms and provide a significant advantage in constructing protein networks in ELM, as they are genetically encodable, can be secreted from bacterial cells, and in some cases, form cross-β strand amyloids with high tensile strength and mechanical rigidity. Isolated bacterial amyloid fibers have been shown to be effective in enhancing the stiffness of alginate hydrogels (Axpe et al. 2018). Efforts in engineering bacterial fibers, including curli proteins in E. coli and TasA in B. subtilis, have yielded bioinspired ELM with tunable properties. Lu and colleagues demonstrated that conjugation of a short histidine peptide to a major curli substituent, CsgA, yields an extracellular protein fiber displaying reactive handles to load functional modules (Chen et al. 2014). Controlling the production of an engineered CsgA by quorum sensing enabled the autonomous patterning of curli fibers over time. Joshi and co-workers developed Biofilm-Integrated Nanofiber Display (BIND) by appending peptide motifs to the CsgA (Nguyen et al. 2014). Their initial work includes screening the optimal fusion sites on CsgA and a panel of peptide tags that can bind to various exogenous chimeric species. Combination of the covalent binding of a target protein using SpyTag/SpyCatcher chemistry and non-covalent attachment of another target protein via FLAG tag and anti-FLAG antibody allows decoration of the E. coli biofilms with more than one functionality. In addition to the SpyTag/ SpyCatcher conjugation pair, other covalent and non-covalent protein conjugation pairs were examined for this purpose: InaD/EFCA, Tip1/WRESAI, calmodulin/ M13, and SZ21/SZ16 (Nussbaumer et al. 2017). InaD specifically binds to a small peptide EFCA and forms a disulfide linkage, and similarly, Tip1 non-covalently binds to a small hexapeptide WRESAI with a dissociation constant in the nanomolar range (Lu et al. 2014). Similar to the examples discussed in the previous paragraph, calmodulin binds to M13 peptides in a Ca2+-dependent manner (Blumenthal et al. 1985). SZ21 and SZ16 are helical motifs that heterodimerize to form superhelical bundles with each other (Thompson et al. 2012). This technique has expanded our ability to incorporate other functionalities to biofilms, including enzymes (Botyanszki et al. 2015; Nussbaumer et al. 2017), gold nanoparticles (Seker et al.

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2017), and adhesive proteins (Zhong et al. 2014). Bacillus subtilis, a gram-positive microorganism, produces biofilms containing bacterial filaments of the TasA protein. Engineering TasA by fusing it with various other protein motifs enabled programmable ELM based on B. subtilis biofilm with functional properties, including intrinsic fluorescence, enzymatic activity, and the templated assembly of inorganic nanoparticles (Fig. 4c) (Huang et al. 2019).

5 Future Directions and Challenges This chapter explored achievements in the field of recombinant protein hydrogels in the last few decades. As discussed in previous sections, a rationally designed, programmable, and hydrated protein network offers a unique platform for addressing challenges in biology, medicine, environment, energy, and many other areas. Advances in protein engineering have enabled important discoveries in this field, ranging from fundamental studies on protein network structures to developing functional protein hydrogels. At the same time, the fast-moving frontier of the computational design of artificial proteins will continue to expand the breadth of this field and exert a significant impact. With the frontier of ELM moving toward implementing multifunctional properties, the design capability of recombinant protein networks could drive a multitude of innovations. In this regard, the remaining key challenges include (1) expanding the library of genetically encodable and orthogonal protein-protein interaction/reaction pairs, (2) engineering effective production and secretion systems for recombinant proteins from the constituent cells of ELM, and (3) understanding and designing synergistic effects of protein scaffolds and living cells. The development of SpyTag/SpyCatcher and many other protein-protein recognition motifs has enabled the engineering of protein networks with complex topology and functionalities. They act as a genetically encodable, proteinaceous “glue” that creates junction points in a network or serves as anchor points for attaching functional modules. Expanding the library of employable orthogonal protein-protein interaction/conjugation pairs for multifunctional decoration of protein networks will propel future innovations in the field of recombinant protein hydrogels and ELM. Engineering more efficient or stimuli-responsive bio-recognition, either by rational or de novo computational design, will create protein networks with desired chemical, physical, and functional properties. As seen in artificial telechelic proteins and other protein hydrogels, the concentration of the monomeric protein is a critical factor for the mechanical properties of the resulting materials. Except in several cases, the yields of secreted recombinant proteins from host cells are often low. For instance, the production yield of curli fibers is in the order of milligrams or lower per liter scale (Dorval Courchesne et al. 2017). Although the use of mutant E. coli strains with modified outer membranes has been shown to achieve promising production and secretion yield of some recombinant proteins, the results vary case-by-case and often rely on trial and error (Kleiner-

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Grote et al. 2018). B. subtilis is widely used for the production of industrial enzymes due to well-defined secretion pathways and the absence of an outer membrane. However, yields are often milligram or sub-milligram quantities per liter of culture when it comes to the production and secretion of heterologous proteins (Harwood and Cranenburgh 2008). Engineering bacterial strains and pathways optimized for producing a wide range of network-forming recombinant proteins in large amounts would open possibilities to build ELM with desired mechanical properties. The synergistic effect of living cells and protein networks in ELM beyond their production is often overlooked. Not only do the living cells produce the protein network, but they are also capable of manipulating them. Extensive experimental characterizations complemented by multi-scale modeling will allow us to understand how protein networks in the ELM change over time and respond to environmental perturbations. The ability to understand and engineer the cellular contribution to the mechanical properties over time will allow predictive designs of ELM.

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Living Synthetic Polymerizations Austin J. Graham and Benjamin K. Keitz

Abstract Natural materials, such as biofilms and tissues, sense and respond to environmental signals using genetic, metabolic, and proteomic machinery. This machinery allows natural materials to actuate changes with unmatched spatiotemporal precision. However, natural materials are relatively limited in morphology and functionality compared to synthetic materials. In an effort to enhance synthetic materials with the capabilities of living systems, we describe recent efforts to control synthetic polymerizations using live cells as actuators. Both microbes and eukaryotic cells have been employed in radical and oxidative polymerizations, significantly expanding the synthetic scope available to living systems. In addition, these mechanisms have enabled construction of polymer networks and hydrogels that resemble natural materials like tissues. Future efforts in synthetic biology, combined with new methods for reprogramming metabolism to control abiotic chemistry, will enable more platforms that synergistically enhance synthetic materials with living functions. Keywords Living materials · Synthetic biology · Extracellular electron transfer · Shewanella oneidensis · ATRP · Synthetic chemistry · Organometallic catalysis · Hydrogels

1 Introduction Living organisms detect and respond to a variety of inputs within complex environments. Specifically, living systems exist in a state of dynamic reciprocity, wherein bidirectional flow of chemical, mechanical, and electrical information between cells and their environment dictates micro- and macroscopic properties. This relationship guides the development of cellular “living materials,” including biofilm growth and

A. J. Graham · B. K. Keitz (*) McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA Center for Dynamics and Control of Materials, University of Texas at Austin, Austin, TX, USA e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 W. V. Srubar III (ed.), Engineered Living Materials, https://doi.org/10.1007/978-3-030-92949-7_2

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tissue formation. Inputs such as food, water, light, and oxygen are processed by cellular building blocks, instructing living material properties through growth, morphology changes, and differentiation. At the single cell level, these environmental signals are processed and amplified using genetic, metabolic, and proteomic networks (e.g., biological computation). Biological computation is a central capability of living systems that enables autonomy, self-regulation, and non-equilibrium processing. In a simple example, a physical input such as sunlight serves as a transcriptional activator in plants, kickstarting metabolism through the expression of photosynthesis genes. A more complex example would be tissue homeostasis, where multiple extra- and intracellular signals are simultaneously processed with spatial and temporal precision to maintain proper tissue function across many length scales. This information processing is actuated and amplified using specific biomolecules, including proteins, nucleic acids, and small molecules. Synthetic biology, systems biology, metabolic engineering, and related fields seek to understand and engineer control over biological computation, harnessing the responsiveness of living systems toward outputs ranging from pharmaceutical production to bioremediation (Meng and Ellis 2020; Tang et al. 2020; Voigt 2020). In platforms that capitalize on the self-regulation and sustained growth of living cells, programming both native and new functions through synthetic biology confers added autonomy and intelligence to the engineered application. These cells can then be deployed in dynamic and intervention-less environments. With respect to materials, controlling biological computation toward desired material properties has predominately focused on natural materials already produced by the host organism. Some leading examples in the field are biofilm- and hydrogelbased materials formed from the extracellular amyloid protein CsgA in E. coli (Nguyen et al. 2014). A key advantage of protein-based living materials is the ability to tailor function using amino acid sequence, such that the same general platform can be used to design adhesives, conductors, and bioplastics. These gels also benefit from the flexibility to harbor constituent cells as a method of continued gelation and propagation, or to easily filter out cells for abiotic application. Another common microbially produced material is cellulose, which can be engineered into tissue mimetics or enzymatically functionalized surfaces (Shaffner et al. 2017; Gilbert et al. 2021). Designer control over polysaccharide properties presents an interesting challenge, as genetic sequence does not directly encode properties in the same way as proteins; nonetheless, it has proven an invaluable platform for developing diverse living materials. Organisms have also evolved relationships with inorganic materials, including synthesizing magnetic nanoparticles or forming structural elements like bone and teeth (Furubayashi et al. 2021). In some cases, these relationships can be metabolically tuned to control inorganic material morphology, which informs their function. Coupling enzyme function to inorganic materials presents a novel opportunity to apply bioengineering techniques, such as directed evolution, toward probing and optimizing the functional landscape of these materials. It is possible that features of this space are inaccessible to typical chemical syntheses. Overall, the materials outlined above are notable for their tight evolutionary relationship with the host organism, potentially facilitating genetic control over their synthesis and

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designed function. However, endogenously produced natural materials suffer from a potential lack of design flexibility that primarily stems from their basis in biological building blocks, such as amino acids, carbohydrates, and a few inorganic molecules. In contrast, synthetic materials offer a wider array of chemical, physical, and transport properties that are generally unavailable to natural systems. Given their respective strengths, it would be advantageous to improve the functional versatility of synthetic materials with the dynamic capabilities of biological computation. These “living synthetic materials” could process diverse signals, selfregulate, and coexist in bidirectional communication with their environments by using cells as actuators within the material. However, as nature did not evolve genotype-phenotype relationships between cells and synthetic materials in the same way as with natural materials, generating such relationships de novo is not trivial. This requires reprogramming of metabolic function toward controlling traditionally abiotic processes, such as catalytic nanoparticle assembly or synthetic polymerizations. With respect to synthetic polymerizations, herein we outline some examples that are actuated by living cells (Fig. 1). Specifically, we highlight

Fig. 1 Schematic illustrating an example of a living synthetic polymerization. Biological computation (i.e., sensing environmental signals and computing those signals using genetic, metabolic, and proteomic machinery) can be rewired to control synthetic polymerizations through redoxmediated processes. These mediators can include flavins, peroxides, reduced metals, and glutathione, among others. In addition, extracellular electron transfer pathways, such as the Mtr pathway in Shewanella oneidensis, can also power these reactions. Figure created with BioRender.com

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recent work toward controlling redox-driven polymerization mechanisms using the diverse reducing power of cells, including generation of electron equivalents, secretion of specific enzymes, and direct electron transfer to polymerization catalysts. Synthetic polymerizations are some of the most common platforms for generating scalable, versatile, functional soft materials. Although much of the described work is still in an early stage, rewiring cellular metabolism for controlling these polymerizations is an important step toward sustainable, self-regenerable, and adaptable living materials with the functional diversity of synthetic polymers.

2 Extracellular Electron Transfer Bridges the Biotic/Abiotic Interface In order to program synthetic polymers using living cells, catalytic activity must be controlled by natural biological functions. One evolutionary relationship that couples the biotic and abiotic (i.e., natural and synthetic) worlds is a form of microbial anaerobic respiration known as extracellular electron transfer (EET). In oxygenlimited or redox stratified environments, such as the deep soil and ocean, some microbes have evolved efficient machinery for moving electrons in/out of the cell and across long (cm) distances. This capability allows life to persist in challenging and competitive resource-limited environments. For example, the purple photosynthetic bacteria Rhodopseudomonas palustris TIE-1 is a photoautotrophic microbe that couples photosynthesis and the oxidation of ferrous iron, Fe(II), to carbon dioxide reduction (Guzman et al. 2019). In this case, extracellular metals or poised electrodes serve as electron donors to drive carbon fixation. Alternatively, microbes such as Geobacter spp. and Shewanella oneidensis MR-1 direct metabolic electron flux from carbon oxidation onto inorganic extracellular substrates. In nature, these electron acceptors include metals and metal oxides, such as iron and manganese oxides. However, synthetic materials, including various types of electrodes, nanocrystals, metal-organic frameworks, and soluble metals, can also act as electron acceptors (Shi et al. 2016; Dundas et al. 2018; Springthorpe et al. 2019; Graham et al. 2021). Overall, EET provides an electronic interface between cellular metabolism and redox-driven processes occurring outside of the cell. Geobacter sulfurreducens and S. oneidensis MR-1 are two of the most wellstudied organisms for understanding and applying EET. Despite both species being electroactive, there are several key differences between these bacteria. First, G. sulfurreducens is an obligate anaerobe, which cannot survive under highly oxygenated conditions (Bond and Lovley 2003). In contrast, S. oneidensis is a facultative anaerobe that is famed for its ability to utilize a variety of electron acceptors including oxygen, DMSO, fumarate, nitrate, and various metals. Second, the mechanisms responsible for EET are different between the two bacteria. G. sulfurreducens and related Geobacter species exclusively use protein-based nanowires to connect cellular metabolism to extracellular electron acceptors. The

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exact structure and mechanism of these wires is somewhat controversial, but they are required for Geobacter EET. On the other hand, S. oneidensis MR-1 uses a combination of electron transfer cytochromes bound to the outer membrane and soluble redox shuttles, such as flavins, to facilitate EET (Coursolle et al. 2009; Coursolle and Gralnick 2010; Brutinel and Gralnick 2012; Xu et al. 2016). In one specific example, S. oneidensis uses the Mtr pathway (Fig. 1) to move electrons across the outer membrane and onto extracellular electron acceptors. A final difference between the two bacteria concerns genetic tractability. Genetic transformations in Geobacter species, including the creation of knockouts and the insertion of exogenous DNA, are possible, but time consuming and laborious. Although S. oneidensis is not a model chassis, an increasing number of sophisticated genetic engineering techniques have been demonstrated in this species (Hu et al. 2015; Corts et al. 2019; Dundas et al. 2020; Li et al. 2020). Overall, the oxygen tolerance, relatively high growth rate, well-understood EET machinery, and genetic tractability of S. oneidensis MR-1 make it an ideal organism for applying and manipulating EET. However, it is important to note that Geobacter or other EET active microbes could replace S. oneidensis in many of the EET-driven applications described below. Our understanding of the specific EET pathways in S. oneidensis has facilitated numerous applications using a diversity of metal electron acceptors. The most apparent and well-studied is current generation on electrodes. S. oneidensis colonizes different metal oxide surfaces to respire, which can be used to design a microbial fuel cell (Shi et al. 2016). The diverse and facultative respiratory capabilities of S. oneidensis enable long-distance electron transfer to the oxide surface even in multicellular biofilms, where redox potential is stratified moving away from the electrode. At appropriate potentials, electron transfer can also be redirected into the cells through specific proteins, enabling unfavorable intracellular transformations (termed bioelectrosynthesis). Underpinning these applications is our strong understanding of the Mtr and related pathways and their programmability using synthetic biology. The wide substrate scope of S. oneidensis’ EET machinery enables continued development of new cell-substrate relationships. For instance, EET can be directed toward oxidized soluble metals, such as Pd2+, to generate nanoparticles (Dundas et al. 2018). Manipulating EET through both genetics and metabolism allows control over nanoparticle synthesis rate and localization (e.g., outer membrane-bound or intracellular). In addition, this evolutionary relationship to metals allows cells to tune novel material properties. For example, EET can be used to regulate the optical response of inorganic nanocrystals (Graham et al. 2021). Overall, the tight metabolic relationship between electron transfer via the Mtr pathway and central carbon metabolism in S. oneidensis allows for living control over traditionally abiotic processes like producing current or programming materials. Given even just these few examples, it is apparent that redox-driven chemical transformations are ubiquitous in both living and synthetic systems. However, in practice, synthetic reactions driven by electron flow are usually controlled with chemical reductants or in an electrochemical cell using a potentiostat. Based on early reports that S. oneidensis could affect dehalogenation reactions in the presence

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of soluble metals (Workman et al. 1997), we reasoned that these bacteria may be able to power a wide variety of material-relevant transformations via EET to synthetic catalysts. In this chapter, we describe the efforts of several research groups to validate this and related hypotheses. Specifically, we highlight the ability of S. oneidensis and other electroactive organisms to control polymerization catalysts for the synthesis of polymers and polymer networks. Although EET is an efficient mechanism through which electrogenic bacteria can influence their redox environment, we also provide examples of non-electroactive organisms that are able to influence their extracellular redox environment and redox-driven catalytic reactions through molecule secretion, metabolic activity, and other means. With further development, materials synthesized via the coupling of cellular metabolism to synthetic catalysts will capitalize on the versatility, chemical functionality, and robustness of synthetic materials while still maintaining the autonomy, sensing, and computational abilities of living cells.

3 Synthetic Polymerizations Mediated by Microbes Radical polymerizations are central to synthetic chemistry and yield a diverse array of soft, plastic, and glassy materials. Two well-known classes of “living” radical polymerizations are atom-transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) (Kamigaito et al. 2001; Matyjaszewski 2012). In the context of these polymerizations, “living” refers to the presence of an active/dormant polymer chain end that does not spontaneously decompose or participate in undesirable radical reactions. Both ATRP and RAFT utilize polymer chain ends that continually switch between an active (can add additional vinyl monomers) and a dormant (unreactive) state. The relatively low concentration of radical species allows these polymerizations to achieve high conversions and produce polymers with controlled molecular weights and low polydispersity. The presence of a living chain end also allows for the synthesis of block copolymers, bottlebrush polymers, and other polymer microstructures. There are several different variants of ATRP and RAFT, but critically, there are examples for both where an appropriately poised redox environment promotes reduction of a chemical species, leading to radical initiation. As electrogenic microbes can be potent reducers, we and others reasoned that endogenous cell metabolism could be coopted to kick-start these radical polymerizations. In RAFT, a radical initiator and a chain-transfer agent control the growth of living polymer chains. Many common radical initiators are appropriately poised within a reduction window achieved by microbial culture. Capitalizing on this reducing capability of bacteria, Nothling et al. (2021) employed cultures of E. coli and S. enterica serovar Typhimurium toward activation of a common redox-active aryl diazonium salt. This allowed synthesis of p(oligoethylene glycol methacrylate) via a microbially mediated RAFT mechanism (Fig. 2). The metal-free catalysis employed nontoxic substrates, such that active cell metabolism was maintained within the

Fig. 2 Controlled radical polymerization (BacRAFT) facilitated by the terminal electron flux of E. coli (strain MC4100) and S. Typhimurium (strain TAS2010). The reducing potential generated by growing bacteria activates the redox-active diazonium salt (4-bromobenzenediazonium tetrafluoroborate (4-BT)) to furnish a carbon-centered aryl radical, which subsequently initiates a controlled radical polymerization of methacrylate monomers (OEGMA) in the presence of a chaintransfer agent (TTC-1) via RAFT (Reproduced with permission from Nothling et al. 2021)

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living polymerization. The reaction was well-controlled, producing low dispersity polymers with first-order kinetics and achieving substantial monomer conversion upward of ~70%. Engineered knockout variants of S. enterica demonstrated the importance of redox mediators in the polymerization—glutathione biosynthesis deficiency and accumulation of reduced copper each inhibited and enhanced conversion, respectively. Although redox-controlled RAFT polymerizations are relatively rare (Wang et al. 2017; Lorandi et al. 2019), this example highlights how the careful selection of radical initiators and chain-transfer agents can facilitate biological control over a synthetic polymerization. In ATRP, a radical initiator is activated and deactivated by a transition metal catalyst, and this redox equilibrium controls the polymerization rate of vinyl monomers. By keeping the overall radical concentration low, ATRP yields low dispersity polymers with tight control over molecular weight and minimal chain termination. Unlike most RAFT polymerizations, ATRP is controlled using transition metal catalysts and can be easily actuated using electrochemical control over the catalyst redox equilibrium, which determines the polymerization rate and polymer characteristics. Thus, microbial secretion of reducing equivalents can initiate ATRP in the presence of bacteria. In one example, Magennis et al. (2014) “templated” vinyl monomers on bacterial surfaces for labeling and selection (Fig. 3). The bacteria instructed reduction of a copper catalyst, yielding polymers bound to the cell membrane that were specific to those cells and could be used to identify specific cell populations in co-cultures. Cells could also be fluorescently surface-labeled with tandem copper-catalyzed polymerization and azide-alkyne cycloaddition reactions. In a similar reaction scheme, glycopolymers that were cell-adherent and strainspecific could be polymerized with glycosylated monomers (Luo et al. 2019). The incorporation of biomimetic architectures like sugar molecules provides an interesting handle with which to tune synthetic polymer interactions within biological systems. ATRP initiated by a general reducing environment was also demonstrated using various microbes in the presence of iron, another common organometallic catalyst that is less cytotoxic than copper (Bennett et al. 2020). Overall, the redox environment created by living cells has an appropriate reduction potential for controlling a variety of synthetic polymerizations, and further exploration of reaction conditions, combined with rewiring new metabolic capabilities to catalyst activation, will significantly expand the reaction space available to living organisms.

4 Atom-Transfer Radical Polymerization Powered by Extracellular Electron Transfer While the general reducing capability of microbes is powerful for controlling the above reactions, we reasoned that a more specific mechanism for metal reduction could directly couple synthetic reactions to active cell metabolism. As S. oneidensis is known to reduce a variety of catalytic transition metals, we hypothesized that EET

Fig. 3 A schematic of the bacteria-instructed synthesis process. (a–c) Bacteria induce polymerization in monomer-catalyst suspensions (a) to generate a synthetic extracellular matrix of polymers (b). Recovery of polymers from the suspensions leads to two fractions (c), with polymer obtained from the aqueous phase suspension around the bacteria denoted conceptually as non-templated and a second fraction obtained from a wash of the cell surfaces denoted as

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could serve as a general electron transfer mechanism for organometallic reactions (Fan et al. 2018). As a proof of concept, we chose ATRP for the strong conceptual parallel between EET and a poised electrode (Fig. 4a, b). We first demonstrated that S. oneidensis was capable of polymerizing a vinyl monomer, oligo (ethylene glycol) methyl ether methacrylate, in a microbial broth, Shewanella basal medium (SBM), that was supplemented with mixed metals serving as catalysts (Fig. 4c). Isolating copper within the metal mix improved the reaction kinetics, and E. coli in the same conditions did not polymerize monomer, indicating that specifically EET was required for this process (Fig. 4d). Consistent with previous work, reducing equivalents including glutathione and cell lysate could actuate a small amount of polymerization. Indeed, conversion achieved from cell lysate approached ~40%, which is similar to that achieved by concentrated bacteria cultures that were not performing EET (Magennis et al. 2014), supporting the hypothesis that the reducing environment of microbial culture can nonspecifically drive these reactions. However, living S. oneidensis cells capable of continuous catalyst reduction were required for significant copper reduction and high monomer conversion upward of 90% (Fig. 4e). Overall, p(OEOMA500) polymers formed via EET in the presence of live cells were monodisperse, exhibited first-order kinetics, and achieved high monomer conversion, all indicative of a well-controlled reaction. A primary advantage to using EET as opposed to nonspecific reducing equivalents is the development of a genotype-phenotype linkage to material synthesis. To demonstrate this link, we employed knockouts of specific EET genes in S. oneidensis in ATRP reactions. Although a variety of cytochromes and molecular redox shuttles, such as flavins, are responsible for EET activity in S. oneidensis, we found that the extracellular cytochromes MtrC and OmcA, which anchor on the outer membrane, were the strongest regulators of electron flux to copper, and therefore polymerization (Fig. 5a, b). This was evidenced by a greater decrease in polymerization rate in a ΔmtrCΔomcA knockout compared to a flavin exporter knockout (Δbfe) and a hydrogenase knockout (ΔhydAΔhyaB). Toward further engineering of this genotype-phenotype relationship, plasmid-based complementation of mtrC in the ΔmtrCΔomcA strain almost fully rescued polymerization activity (Fig. 5c). Future efforts in maximizing the role of the Mtr pathway in synthetic polymerizations may investigate downregulation or knockouts of other reduction pathways that may also interact with soluble copper. For example, the flavin exporter

Fig. 3 (continued) templated. (d, e) Incubation of polymers with bacteria results in low binding of cells for which the polymer is non-templated (d), or where a polymer templated with one cell type (shown in red) is incubated with a cell (shown in green) of another type (e). (f) Addition of a polymer, templated by one cell type, with its own “matched” cell population results in the formation of large polymer cell clusters, as the templated polymers sequester the bacteria that “instructed” their formation with high affinity. (g) The same reducing environment at bacterial surfaces that aids the polymer synthesis can also be used to label the cells in situ via pro-fluorescent markers, which react with cell-surface bound polymers containing “clickable” residues (Reproduced with permission from Magennis et al. 2014)

Fig. 4 S. oneidensis-enabled ATRP and initial polymerization kinetics. (a) Electron equivalents generated from S. oneidensis MR-1 reduce a metal catalyst from an inactive state (MOX) to an active state (MRED). The active catalyst reacts with a halogenated initiator or polymer chain to produce a radical (gray arrow) that can polymerize olefins. The radical can also react with the now-deactivated catalyst (MOX) to form a dormant chain (black arrow, right). (b) ATRP initiator (2-hydroxyethyl 2-bromoisobutyrate, HEBIB) and macromonomer (OEOMA500) used in this study. (c) Monomer conversion after 24 h under various conditions with (white) and without (purple) trace metal mix added to bacterial media. (d) Kinetics of monomer conversion in MR-1 or E. coli culture using Cu(II)-EDTA as catalyst (e) Extracellular Cu(II) reduction monitored with the Cu(I)-specific fluorescent dye CF4. Data show mean
SD of three independent experiments. **P < 0.01 (Reproduced with permission from Fan et al. 2018)

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Fig. 5 Electron transfer proteins impact polymerization kinetics. (a) Key proteins involved in extracellular electron transport in MR-1. (b) Effect of gene knockouts on polymerization activity using Cu(II)-TPMA. (c) Rescue of normal polymerization activity via complementation with a plasmid encoding mtrC, using Cu(II)-TPMA as a catalyst. Data show mean
SD of three independent experiments (Reproduced with permission from Fan et al. 2018)

knockout could be augmented with a glutathione knockdown strategy to minimize reducing capacity beyond the specific pathway of interest. Another strategy would be to physically or chemically tether copper to extracellular cytochromes using noncanonical amino acids or metal-binding peptides. This work served as an important foundation toward controlling living synthetic polymerizations using synthetic biology. An outstanding challenge of ATRP, and indeed many organometallic reactions, is oxygen quenching in aerobic environments. As a result, most of these reactions must take place under strictly anaerobic conditions to facilitate control over catalyst redox cycles. For this purpose, it is convenient that EET is an anaerobic respiratory mechanism. However, inspired by enzymatic oxygen consumption in aqueous solutions (Szczepaniak et al. 2021), we further hypothesized that the facultative

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nature of S. oneidensis would enable polymerization in ambient environments by effectively “scrubbing” dissolved oxygen through aerobic respiration, followed by activation of EET pathways (Fan et al. 2020). We first confirmed that dissolved oxygen was consumed in our reaction mixtures after inoculation with bacteria and polymerization reagents (Fig. 6a). The creation of this in situ anaerobic environment enabled synthesis of well-defined polymers in ambient conditions, using only the bacteria to overcome oxygen inhibition (Fig. 6b). Aerobic polymerization also suppressed nonspecific radical production from cell lysate, more tightly coupling catalyst reduction to continuous EET from S. oneidensis (Fig. 6c). Indeed, increasing the concentration of bacteria in the reaction correspondingly increased polymerization rate (Fig. 6d). In contrast to aerobic ATRP powered by enzymatic oxygen depletion, microbial aerobic ATRP is a living reaction that tethers facultative cell metabolism to catalyst reduction. This was further exemplified by the ability to recycle cells after a reaction to complete multiple polymerization cycles. On the other hand, cells could also be lyophilized and treated as a chemical reagent added to an aerobic reaction mixture to initiate a polymerization, highlighting the robust flexibility of this platform. This work also developed the large synthetic material landscape available to S. oneidensis through EET-driven ATRP, as a variety of vinyl monomers were successfully polymerized (Table 1). This included traditionally challenging hydrophobic species such as styrene. In addition, a variety of transition metals, including iron, ruthenium, cobalt, and nickel could all be reduced by S. oneidensis as catalysts

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Fig. 6 S. oneidensis rapidly consumes dissolved oxygen and activates radical polymerization in cultures for which no additional steps were taken to remove oxygen. (a) Dissolved oxygen in S. oneidensis MR-1 cultures growing in SBM and under typical polymerization conditions. Under both conditions, oxygen consumption outcompetes oxygen diffusion. (b) Effect of different biological and polymerization components on monomer (OEOMA500) conversion under aerobic conditions. Monomer, initiator, catalyst, and S. oneidensis MR-1 are all required to achieve significant monomer conversion. (c) Monomer (OEOMA500) conversion using heat-killed (HK) and lysed E. coli MG1655 or S. oneidensis MR-1 cells under anaerobic and aerobic conditions. Lysed cells release intracellular reductants that reduce Cu(II) to Cu(I), which activates polymerization. Under aerobic conditions, adventitious reductants and radicals are quenched by oxygen. Heat-killed cells do not result in polymerization under either condition as they neither consume oxygen nor generate EET flux to reduce the metal catalyst. (d) Polymerization rate constants of OEOMA500 using S. oneidensis MR-1 and Cu(II)-TPMA at varying inoculating OD600 under aerobic conditions. Data show the mean
SD of n ¼ 3 replicates (Reproduced with permission from Fan et al. 2020)

Table 1 Monomer scope under anaerobic and aerobic polymerizations that involve S. oneidensis MR-1. Yields are listed as percentages, followed by experimental Mn and Ð. The target Mn values for the monomer/initiator of 500:1 were polyOEOMA300 (150 kDa), polyOEOMA500 (250 kDa), polyHEMA (65.1 kDa), polyNIPAM (56.6 kDa), polyDMAEMA (78.6 kDa), polyMMA (50.1 kDa), and PS (52.1 kDa). Average and SD values were obtained from n ¼ 3 replicates (Reproduced with permission from Fan et al. 2020)

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for microbial ATRP. Polymerization rate could also be tuned by changing the copper ligand; interestingly, the relationship between catalyst reduction potential and polymerization rate did not follow the expected trend when compared to traditional electrochemical ATRP (Fantin et al. 2015). This suggests that catalyst structure may influence its interaction with S. oneidensis EET machinery, such as MtrC. More work in this area, for example, performing mutagenesis on MtrC to examine the effect of amino acid sequence on catalyst reduction and polymerization, is ongoing. In addition to these biological handles, polymerization could be tuned through chemical means, such as sparging oxygen as a quencher. This did not affect living polymer chain ends, which remained capable of continued monomer addition. Overall, microbial aerobic ATRP powered by facultative S. oneidensis metabolism is highly robust and provides a variety of opportunities for controlling synthetic polymers using a living organism.

5 Extracellular Electron Transfer-Catalyzed Cross-Linking for Living Hydrogel Materials Cells encased within polymeric networks, including synthetic gels, share compositional homology with many natural systems, such as bacteria and extracellular polymeric substance (EPS) in biofilms, and cells and extracellular matrix (ECM) in tissues. In the case of some synthetic cell-polymer composites, the chemistry of the polymer network may not be directly under metabolic or genetic control of the encased organism, but the presence of the organism or its growth can still modulate bulk network properties or enhance cell-material function (Niu et al. 2017; Nagahama et al. 2018). For example, Johnston et al. (2020) demonstrated that encapsulated bacteria and yeast could be cryopreserved in hydrogel networks for up to 1 year without a loss in bioproduction, and that gel encapsulation improved production titer. In addition, encapsulation enabled 3D extrusion printing of the cellgel composites into desired geometries. Similarly, growing Baker’s yeast encased in a polyacrylamide network could predictably actuate macroscopic shape-changing gels (Rivera-Tarazona et al. 2020). By engineering L-histidine auxotrophy in the yeast, researchers could create complex morphologies and tune material responses with spatiotemporal control. Taking this a step further, optogenetic control of L-histidine metabolism in the yeast enabled blue light responsive growth and corresponding deformation. Coordinating work in cellular engineering, polymer chemistry, and network modeling will enable even more cell-polymer composites with enhanced symbiotic function. This compositional homology between tissues and bacteria-loaded synthetic polymers has facilitated a number of therapeutically relevant applications in recent years (Rodrigo-Navarro et al. 2021). Strong control over recombinant protein expression in microbes allows for tailored expression of bioactive compounds, while the hydrogel confers protection and isolation from the tissue environment.

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In one example, a Lactococcus lactis biofilm was engineered to express bone morphogenetic protein 2 on a synthetic substrate (Hay et al. 2018). Constitutive or nisin-driven inducible expression allowed for environmentally and dynamically regulated osteogenic differentiation of human mesenchymal stem cells cultured on top of the biofilm. This 2D platform was expanded into a 3D delivery system using L. lactis loaded into collagen sponges to control differentiation. While collagen is a common naturally derived biomaterial, synthetic substrates could exhibit similar activity while allowing for alternative functionality, such as pore size control to spatially segregate L. lactis and stem cells. In a similar example, vascular endothelial growth factor secretion was coupled to nisin induction in L. lactis to promote angiogenesis and wound healing (Lu et al. 2021a). Highlighting the utility of synthetic platforms, these living materials could be delivered by injection using a heparin-functionalized poloxamer gel loaded with bacteria. Aside from small molecule induction, alternative means of gene regulation can also be applied in therapeutically relevant living materials. In a recent study, optogenetic control over gene expression allowed for blue light-activated secretion of deoxyviolacein, a common antimicrobial and antitumoral drug, using endotoxin-free E. coli (Sankaran et al. 2019). The hydrogels maintained bacteria viability and allowed for continued lightactivated drug release for over a month. These works exemplify the symbiosis of biomaterial engineering and synthetic biology toward dynamic, responsive, and designer living materials. However, in each case, the physicochemical properties of the hydrogel itself are not directly controlled by the resident microbes. The next step toward engineering this dynamic reciprocity in a cell-polymer composite requires connecting cellular metabolism to the chemistry of the surrounding material. In this regard, general microbial reduction (Lu et al. 2021b), as well as EET-driven polymerization, has proven fruitful. As alluded to in the previous examples, hydrogels are an important biomaterial platform that recapture many of the critical components of living systems, including aqueous solubility, electrolyte and small molecule diffusivity, and cell biocompatibility. Therefore, synthetic hydrogels are ideal for developing responsive living materials with genetic links to material properties. Building upon EET-controlled ATRP, we designed polymer precursor solutions functionalized with methacrylate groups that formed crosslinked hydrogels upon catalyst activation by S. oneidensis (Fig. 7) (Graham et al. 2020). Living cells capable of EET were required for cross-linking to occur, and cross-linking activity was directly coupled to S. oneidensis genotype, as knockouts of key EET genes yielded weaker and slower-forming gels. The mechanics of the gels could be tuned using initiator and catalyst concentration, as well as inoculating density of S. oneidensis, implying that these materials are amenable to more traditional methods for tuning network properties. In addition, tracking cell movement via microscopy revealed that gel microstructure, including pore size, was regulated by electron transfer phenotype. Importantly, this work demonstrated that a measurable, macroscopic material property (in this case, storage modulus) was under direct genetic control of the encased bacteria. Using EET as a central conduit, we directly connected biological metabolism to material chemistry, recapturing a process similar to tissue ECM or biofilm EPS formation.

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Fig. 7 Extracellular electron transfer from S. oneidensis controls radical cross-linking of a semisynthetic hydrogel. (a) The Mtr pathway of S. oneidensis directs metabolic electron flux to a metal catalyst, which generates a radical from a halogenated initiator and cross-links acrylate-based functional groups. (b) Chemical structures of the macromer, methacrylated hyaluronic acid (MeHA), and the radical initiator, 2-hydroxyethyl 2-bromoisobutyrate (HEBIB). (c) Cross-linking reaction mixture inoculated with E. coli, which does not possess EET machinery, does not form gels as indicated by liquid flow. The air-liquid interface is highlighted. (d) Cross-linking reaction mixture inoculated with S. oneidensis MR-1 forms a solid gel as confirmed by inversion test. The air-liquid interface is highlighted (Reproduced with permission from Graham et al. 2020)

This genetic basis for controlling hydrogel properties presented an opportunity to couple more sophisticated biological computation to the material. Synthetic biology as a field has taken an engineering approach to controlling genetic and transcriptional machinery in a modular and predictable manner. However, the majority of developed circuitry has been limited to fluorescence as the measurable output. Toward merging progress in synthetic biology with this new living material platform, we demonstrated that a well-studied gene circuit controlling mtrC could function in a quantifiable and predictable manner (Fig. 8a–c). We engineered the bacteria to sense an environmental signal, the sugar molecule isopropyl β-D-1thiogalactopyranoside (IPTG), and respond by activating transcription at the Ptac promoter. An activating Hill function, a common gene expression model, accurately

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Fig. 8 Modeling gene expression allows quantitative prediction of living hydrogel properties. (a) Genetic circuits utilized in this study placing either sfgfp or mtrC under inducible control of IPTG via the LacI repressor and Ptac promoter. (b) In situ rheology of hydrogels cross-linked by S. oneidensis with steady-state induced mtrC levels at various IPTG concentrations. ΔmtrCΔomcAΔmtrF + empty and ΔmtrCΔomcAΔmtrF + mtrC with 0 μM IPTG were also tested but did not form gels on the time scale shown. (c) Hill function analysis of sfGFP fluorescence, denoted as relative expression units by normalization to fluorescence at maximum induction, as a function of IPTG concentration. (d) Hill function analysis of hydrogel storage modulus after 2 h of cross-linking as a function of IPTG concentration. The right y-axis is storage modulus normalized to average modulus at maximum induction. (e) Normalized hydrogel stiffness plotted as a function of relative expression units for corresponding IPTG concentrations. The fit was determined by performing linear regression on the paired data (R2 ¼ 0.80). (ce) Data are shown as mean
SEM, n ¼ 3 biological replicates (Reproduced with permission from Graham et al. 2020)

captured transcriptional output for the fluorescent gene sfgfp in S. oneidensis (Fig. 8c). Further, this simple gene expression model also accurately predicted hydrogel storage modulus when cross-linked using S. oneidensis expressing mtrC on the same circuit (Fig. 8d). The Hill function fit parameters for each response function are in good agreement, implying that the circuit architecture and repressor/ promoter pair are the primary regulators of output activity for either measurement. The approximately linear relationship between normalized transcriptional output and storage modulus further demonstrates that established fluorescent circuits can also predictably capture cross-linking dynamics (Fig. 8e). This analysis suggests that

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different genetic circuit outputs (namely, fluorescence and storage modulus) can be standardized between systems, allowing quantitative modeling and prediction using theory from synthetic biology. Importantly, this will allow parallel development of design rules for living materials that are informed by decades of work in synthetic biology and related fields.

6 Other Living Synthetic Polymerizations Radical polymerizations are powerful material synthesis platforms that are now within the catalytic capabilities of microbes; however, there are other chemical mechanisms that could expand this reaction space further. For example, another redox polymerization is the oxidative polymerization of polypyrrole, a common conducting material. Song et al. (2017) used this approach to enhance electron transfer in S. oneidensis cultures that had been coated with conducting polymer. Fe(III) natively bound to the bacterial membrane via negatively charged phospholipids, and subsequently catalyzed surface-associated pyrrole addition. The coated bacteria remained viable, and the generalizable mechanism enabled polypyrrole coating on non-electroactive bacteria as well, including E. coli, Ochrobactrum anthropi, and Streptococcus thermophilus. Benchmarking new biohybrid mechanisms such as this, emphasizing mild reaction conditions that maintain cell viability, and enhancing live cell function with new nonbiological chemistries will enable design of versatile living materials with unprecedented functionality. Of course, redox-active metabolic transformations are not limited to microbes. Indeed, the redox mechanisms of higher-order organisms are more sophisticated and can similarly be reprogrammed to interface with abiotic catalyses. The Deisseroth and Bao groups recently demonstrated this through an application of oxidative polymerization controlled by rat hippocampal neurons (Liu et al. 2020). Templated synthesis of polyaniline using recombinant ascorbate peroxidase expressed in the neurons enabled increased ion and electron conductivity on polymer-coated cells. Brain tissue slices with polymer-coated neurons exhibited increased capacitance and stability in response to current injection, indicating that the polymer conferred improved electrical conductivity in vivo. The team also demonstrated altered animal mobility in polymer-coated pharyngeal muscle of C. elegans. Interestingly, the overall mobility of C. elegans increased when motor neurons were coated with a conducting polymer, whereas it decreased when coated with an insulating polymer. This work provides an exciting foundation for controlling in vivo biology using synthetic materials that are templated by the organism itself. In the above contexts, living synthetic polymerizations have referred to reactions controlled by the biological activity of live cells, primarily serving as initiation mechanisms. However, orthogonal functionalization of cells using chemically synthesized polymers is another active area of research that is merging these fields. A recent collaboration from the Hawker, Soh, Mitragotri, and O’Malley groups highlighted how pushing interdisciplinary boundaries in polymer engineering and

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cell biology can lead to exciting new directions (Niu et al. 2017). In this study, a grafting-from approach enabled functionalization of cell membranes with modular polymers. A chain-transfer agent initiating a RAFT polymerization could be covalently attached or hydrophobically inserted to membranes of both yeast and Jurkat cells while maintaining high cell viability in all cases. Engineering polymer identity allowed for strong control over surface fluorescence and cell-cell adhesion. The researchers demonstrated greater control over polymer properties and surface density than grating-to or encapsulation studies, which are more common approaches for functionalizing cells with synthetic polymers. Furthermore, polymerization initiation using visible light presents exciting opportunities for in situ, on-demand control over reaction progress. In another approach, Nagahama et al. (2018) similarly grafted synthetically modified carbohydrate polymers from live cell surfaces using strain-promoted azide-alkyne cycloaddition. The modified cells could serve as cross-linking junctions to form tissue-like hydrogels that were enhanced with cellular functions, including growth, repair, and surface-specific adhesion. The mechanical properties of these gels were also impacted by resident cells, with increased cell density leading to decreased gelation time and increased storage modulus. Radical polymerizations have also been demonstrated inside eukaryotic cells, altering properties such as cell migration and cytoskeletal organization (Geng et al. 2019). A variety of new-to-nature polymers could be intracellularly synthesized using visible light to initiate the polymerization, without significantly impacting cell viability. Platforms such as these proof-of-principle studies will evolve in the coming years to generate new cell activities using bioactive synthetic polymers, perhaps as tissue repair substrates, organ models, or microenvironmental diagnostics.

7 Conclusions and Outlook Collectively, the diversity and robustness of microbial and eukaryotic metabolisms present a variety of opportunities for connecting biological computation to synthetic polymerizations. The resultant polymerizations are “living” in that they are dynamic, environmentally responsive, and regulated by the genetic and transcriptional processes of concomitant cells. EET is a particularly advantageous metabolic process for these designs, as it provides a well-studied redox linkage that can theoretically control a broad scope of reactions by either acting directly on the reaction substrates or by biasing the redox equilibrium of a metal catalyst. Specifically, there are a variety of transition metals that can serve as well-established organometallic catalysts, many of which lie within the redox potential window of MtrC. These include iron, copper, nickel, cobalt, and ruthenium. Furthermore, polymerizations are just one class of synthetic reactions that can be powered in this manner; further research in this area could yield living control over small molecule synthesis, nanomaterial function, or drug production, to name a few. In this manner, EET is unique in its ability to serve as a universal transistor for connecting the biotic to the abiotic through electron-powered processes.

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The above examples represent an early sampling of the ways synthetic chemistry can be reprogrammed using biological systems. Discovery of new metabolic capabilities, creative connections between cellular outputs and known chemistries, and development of new living material platforms will continue to advance in the coming years. Additionally, as genetic circuits and transcriptional/translational regulation become more sophisticated and standardized, they can be applied toward new outputs in material synthesis. For example, genetic Boolean logic, toehold switches, and integrase-mediated cellular memory can all be repurposed for functions other than fluorescence, such as controlling EET. Ultimately, merging advancements in synthetic biology, synthetic chemistry, and materials science will allow architectures that combine the advantages of natural materials with the versatility of synthetic materials.

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Programmable Self-Assembling Protein Nanomaterials: Current Status and Prospects Kelly Wallin, Ruijie Zhang, and Claudia Schmidt-Dannert

Abstract Protein-based nanomaterials are increasingly engineered as platforms for applications in biomanufacturing, for biomedical purposes, and as structural and organizational components of new types of living materials. Advances in synthetic biology and computational protein design offer tremendous opportunities for the engineering of new types of protein building blocks that self-assemble autonomously into customizable materials with various morphologies and properties. As proteins are genetically encoded, material production is genetically programmable and can be achieved sustainably with microbial cell factories, or in the future, by using cell-free technologies. An overview will be provided of the different types of self-assembling protein materials currently designed, characterized, and functionalized for a range of applications. Finally, opportunities and challenges for the design of genetically programmable materials that self-organize into new types of hierarchical protein materials with emergent functions will be discussed. Keywords Nanomaterial · Protein self-assembly · Protein engineering · Enzyme immobilization · Biomedical · Nanocage · Nanocarrier · Protein scaffold · Protein fiber · Synthetic biology

Abbreviations 1D 2D 3D ACP ADH

one-dimensional two-dimensional three-dimensional atmospheric cold plasma alcohol dehydrogenase

Kelly Wallin and Ruijie Zhang contributed equally with all other contributors. K. Wallin · R. Zhang · C. Schmidt-Dannert (*) Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN, USA e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 W. V. Srubar III (ed.), Engineered Living Materials, https://doi.org/10.1007/978-3-030-92949-7_3

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AF4 AFM AI AmDH AMP AP Bfr BMC CBD CCMV CD CP Cp149 CR Cryo-EM DCS DLS DOX Dps DSC DyP E. coli EDTA ELP ELS ESEM Eut FRET GBS GBS-NN GFP+36 GFP GOX GuHCl HDFM HG12 HRP HuHF IMAC LS Mfp MnP MRI Mx

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asymmetric flow field-flow fractionation atomic force microscopy aggregation index amine dehydrogenase antimicrobial peptide alkaline phosphatase Escherichia coli bacterioferritin bacterial microcompartment chitin binding domain cowpea chlorotic mottle virus circular dichroism bacteriophage P22 coat protein hepatitis B virus Congo red cryo-electron microscopy differential centrifugal sedimentation dynamic light scattering doxorubicin DNA-binding protein from starved cells differential scanning calorimeter dye-decolorizing peroxidase Escherichia coli ethylenediaminetetraacetic acid elastin-like polypeptide electrophoretic light scattering environmental scanning electron microscopy ethanolamine utilization Förster resonance energy transfer group B Streptococcus group B Streptococcus antigen green fluorescence protein with a charge of +36 green fluorescent protein glucose oxidase guanidine hydrochloride hyperspectral dark-field microscopy histidine-rich amphiphile horseradish peroxidase human H ferritin immobilized metal affinity chromatography lumazine synthase mussel foot protein manganese peroxidase magnetic resonance imaging Myxococcus xanthus

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ncAA NTA PAGE PDC PdR PdX PEG PTM Qt RP-HPLC SAXS SbpA SEC SEM S-layer SP SRCD SrtA TCEP TEM TMV TRPS UAA UV-Vis VLP γ-PFD

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noncanonical amino acid nanoparticle tracking analysis polyacrylamide gel electrophoresis pyruvate decarboxylase putidaredoxin reductase putidaredoxin poly(ethylene glycol) posttranslational modification Quasibacillus thermotolerans reverse phase high performance liquid chromatography small angle X-ray scattering Lysinibacillus sphaericus S-layer protein size exclusion chromatography scanning electron microscopy surface layer bacteriophage P22 scaffolding protein synchrotron radiation circular dichroism Staphylococcus aureus sortase tris(2-carboxyethyl) phosphine transmission electron microscopy tobacco mosaic virus tunable resistive pulse sensing unnatural amino acid ultraviolet-visible light viruslike particle gamma-prefoldin

1 Introduction The formation and patterning of materials made by living systems is driven by the self-assembly of simple building blocks into hierarchical, macroscopic structures. Many of these materials combine multiple functions and have properties that are unmatched by synthetic materials. The assembly of these materials is also genetically encoded, which allows them to self-regenerate, remodel, and respond to external cues and events. Due to their infinite chemical (sequence) and structural variability, proteins are key building blocks of these biological materials and are also primary factors that control many of their functions and properties. Learning and then adapting mechanisms that drive the bottom-up assembly of multiscale materials from protein building blocks therefore holds great promise for the design of new types of materials that can be produced sustainably by recombinant production systems such as microbial cell factories or in the future by cell-free transcription and translation from DNA information (Kelwick et al. 2020; Majerle et al. 2019; Hamley 2019; Chiesa et al. 2020).

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Because of the prospect for sustainable manufacturing of new types of potentially transformational materials, we will focus in this chapter on protein-derived nanomaterials. Special emphasis will be placed on self-assembling nanomaterials that rely entirely on the physical and chemical properties of their constituent proteins to drive organized or semiorganized binding into much larger, hierarchical complexes. Consequently, fabrication of protein-based nanomaterials that utilize technologies like 3D printing to impart organization and structure will not be discussed here. Likewise, nucleic acid-based materials will not be covered. Many complex scaffolds and structures have been designed and assembled from nucleic acids (often referred to as “DNA origami”) for a diversity of applications (reviewed in Fu et al. 2020; Bush et al. 2020; Hong et al. 2017). Due to the well-understood mechanisms of DNA assembly and the small number of chemical species available as building blocks, these structures are much easier to design de novo compared to protein structures (Bush et al. 2020). This simplicity is also a major disadvantage of DNA-based structures that can have only a small number of different functional groups, limiting engineering of their surface properties. In contrast, nanomaterials assembled from proteins have access to a diversity of functional groups, including many hydrophilic residues that carry a charge at near-neutral pH. When noncanonical and synthetic amino acids are considered, the range of potential functional groups expands even further. This chapter will therefore outline the state of the art in self-assembling protein materials with a focus on the bioengineering of materials for diverse applications. Computational de novo design of proteins and their assembly (recently reviewed in Pan and Kortemme 2021) will be touched upon as it relates to specific materials but will not be covered in any depth here. As the field of protein nanomaterials is vast and rapidly growing, we will illustrate representative examples of different materials, their properties, design and engineering, and applications with an emphasis on recent reports. Other sections in this book are providing more in-depth discussions of specific systems, examples, and applications that will not be extensively discussed here. In this chapter, we will first introduce the architectures that can be formed by natural self-assembling proteins and provide general remarks on protein selfassembly and genetic programming of such nanomaterials. We will then explore different functionalities that can be engineered into these nanomaterials and discuss various external mechanisms that can be used to control nanomaterial assembly. An overview of the recombinant production and characterization of protein nanomaterials will be followed by a section highlighting a selection of recent examples of such materials in action. Finally, we will conclude with thoughts about the future potential and challenges of the field.

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2 Architectures and Design of Nanomaterials 2.1

Architectures of Protein Nanomaterials

Choosing nanomaterials with appropriate sizes and shapes is crucial for their successful application. A large number of native protein building blocks have been characterized that assemble into structures with different morphologies that include nanowires, nanosheets, nanocages, and nanotubes. This natural diversity provides various possible structures that can serve as platforms for the engineering of different nanomaterials. Here we will provide an overview of protein building block examples that self-assemble into higher-order structures, organized based on the dimensionality of their architectures (i.e., one-, two-, or three-dimensional).

2.1.1

One-Dimensional Strands and Wires

The simplest morphology protein nanomaterials can assume is that of a one-dimensional (1D) strand or wire. These assemblies are generally built from many copies of a single protein species, aligned end-to-end to form a chain or strand. In some materials, multiple strands interact lengthwise to form a thicker, stronger fiber. For example, in the glycine-/proline-/hydroxyproline-rich collagen, three individual chains wind together along their lengthwise axes to form the tropocollagen triple helix. Alignment of multiple tropocollagens generates a strong, flexible fiber (Sheehy et al. 2018; Bielajew et al. 2020; Sorushanova et al. 2019). Many other fiber-forming proteins exist, including silk fibroin which is largely made of antiparallel β-strands, with elasticity provided by α-helices and less-ordered β-regions (Chouhan and Mandal 2020). Amyloid-forming peptides also assemble into large structures, which in vivo play a role in several neurodegenerative disorders. Figure 1 (top) illustrates a general example of an amyloid peptide, which is formed of antiparallel β-strands and assembles with other monomers into a long, structurally stable fiber (Ferguson et al. 2006). Flagella are another example of a more complex self-assembled protein filament. Flagellar structures and compositions vary across domains of life, but in Gram-negative bacteria such as Escherichia coli and Salmonella enterica, flagella are made of the globular protein flagellin. The hydrophobic core region of flagellin is decorated with α-helices, β-strands, and unstructured regions along the surface, which allows interactions with other flagellin monomers to form long protofilaments (Chng and Kitao 2008). An entire flagellum is then assembled from 11 protofilaments, wound together in a helix (O’Brien and Bennett 1972). This specific assembly is necessary for the proper functioning of flagella; changing the directionality of supercoiling during flagellum assembly has effects on bacterial locomotion (Larsen et al. 1974). During biofilm formation, E. coli and S. enterica also produce highly adhesive protein strands known as curli fibers. Assembled from parallel β-sheets stacked perpendicular to the fiber axis, curli are quite robust and tolerant of engineering (Erskine et al. 2018; DeBenedictis et al.

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Fig. 1 Examples of protein-based nanomaterial subunits and morphology. In each case, a small monomeric protein (left column) spontaneously assembles with other copies of itself to form larger structures, with the generalized final morphology shown in the rightmost column. In the top row, a generic β-strand amyloid monomer (Ferguson et al. 2006) assembles into a long fiber. The second row shows a model of the BMC shell protein EutM (Schmidt-Dannert et al. 2018), which selfassembles into hexameric subunits before assembly into 2D sheets. Bacterioferritin (row three) made of predominantly α-helical proteins dimerizes before assembling into the hollow shell of a protein cage (Willies et al. 2009). In the rightmost column, the spherical compartment is shown in cross-section to emphasize the hollow nature of the cage. Finally, row four shows a short β-strand derived from the β-amyloid protein Aβ (Chen et al. 2017). The designed β-strands assemble into dimers and tetramers, which further assemble into hollow nanotubes. In the rightmost column, a nanotube is shown in cross-section to emphasize the hollow nature of the tube. Aβ a β-amyloid protein implicated in Alzheimer’s disease, 2D two-dimensional, BMC bacterial microcompartment, Eut ethanolamine utilization. For structures, PDB accession numbers are provided

2016; Abdali et al. 2020). Many other natural fibers, fibrils, and filaments exist, such as γ-prefoldin (Clark and Glover 2018), microtubules (Kalra et al. 2020), elastins (Wang et al. 2019), and pili [e.g., from Geobacter sp. (Lovley and Walker 2019)], each with their own unique physical and chemical properties.

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Two-Dimensional Sheets and Scaffolds

A number of proteins self-assemble into two-dimensional (2D) structures like sheets and lattices. Such morphologies are useful as layers, scaffolds, and coatings that provide extensive surfaces for functionalization. One such example is the surface layer (S-layer) proteins which form a porous yet highly durable coat on the outside of various bacterial and archaeal organisms. They are composed of highly ordered β-sheets that self-assemble into paracrystalline monolayers, often utilizing divalent cations such as Ca2+ as part of the assembly process. Assembly can be highly robust and occurs rapidly even in vitro (Pum et al. 2013; Raff et al. 2016; Sleytr et al. 2014). Some 2D protein materials are assembled from structural proteins that in their native organism assemble into other morphologies, but upon heterologous expression instead form sheets or lattices. An example of this is the hexameric shell protein EutM from the ethanolamine utilization (Eut) bacterial microcompartment (BMC) (Fig. 1). When expressed as part of the Eut gene cluster, EutM hexamers interact with four other species of structural shell proteins to assemble into a large, hollow spherical structure—the Eut BMC. However, when expressed heterologously without the other Eut shell proteins, the EutM hexamers instead assemble or “tile” into quite robust sheets and rods (Zhang et al. 2018, 2019a). Hexameric shell proteins from other classes of BMC have also been observed to assemble into sheets, crystalline arrays, nanotubes, and other structures when expressed independently (reviewed in Young et al. 2017; Lee et al. 2019a).

2.1.3

Three-Dimensional Cages and Compartments

Compartments, cages, and stacked arrays are examples of three-dimensional (3D) protein structures. Among the many well-studied natural, self-assembling protein compartments are the BMCs (~40–200 nm in diameter) mentioned above (Lee et al. 2019a; Kerfeld et al. 2018), encapsulins (~20–40 nm in diameter) (Jones and Giessen 2021), eukaryotic vault particles (vaults) (~65 nm long and ~40 nm wide) (Frascotti et al. 2021), ferritins (~13 nm in diameter) (Rivera 2017; Arosio et al. 2017), the lumazine synthase cage (LS) (~15 nm in diameter) (Azuma et al. 2018a; Sasaki et al. 2017), and viruslike particles (VLPs) (~25–65 nm in diameter) (Qian et al. 2020; Mateu 2016; Selivanovitch and Douglas 2019). The complexity of the shell-forming processes varies greatly across these groups, with some structures (encapsulins, ferritins, LS, vaults, and some VLPs) assembling from many copies of a single protein, while others (BMCs, some VLPs) contain two or more different proteins in their shells. For example, eukaryotic vault particles are assembled from 78 copies of the major vault protein (Tanaka et al. 2009; Ding et al. 2018) which appear to assemble as each monomer exits the ribosome (Mrazek et al. 2014). Encapsulins are also assembled from a single protein species, which self-assembles into pentameric and/or hexameric subunits before final assembly into the complete

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capsid (Cassidy-Amstutz et al. 2016; Rahmanpour and Bugg 2013), and the iron storage protein bacterioferritin is assembled from 24 copies of a single protein species (Fig. 1) (Willies et al. 2009). In contrast, the outer shell of BMCs contains hundreds or thousands of copies of several different protein species (the numbers vary between BMC types), which form tri-, pent-, and hexameric subunits before assembling into a full compartment. Indeed, because of their large size and complexity, the assembly processes of several classes of BMCs have been studied at length (Faulkner et al. 2019; Greber et al. 2019; Hagen et al. 2018; Uddin et al. 2018; Mahalik et al. 2016; Kerfeld and Melnicki 2016; Pang et al. 2014; Niederhuber et al. 2017; Kerfeld and Erbilgin 2015). However, some complex 3D structures are assembled from short peptides, such as the example shown in Fig. 1, where a seven amino acid subsection of a known amyloid protein (Aβ) was modified to generate a peptide capable of assembling into hollow nanotubes (Chen et al. 2017). Many recent reviews have touched on a range of potential applications for proteinbased compartments in biotechnology and biomedicine as discussed in more detail in Sect. 6 (Demchuk and Patel 2020).

2.2

Design of Self-Assembling Nanomaterials

One quality of proteins that makes them attractive candidates for biomaterial development is the relative ease with which they can be engineered. Changing the exact protein sequence can be accomplished via simple and straightforward molecular biology techniques. Many algorithms for predicting protein structure exist and as our understanding of protein biochemistry expands, so too does the accuracy of protein modeling tools. This has expanded the opportunities for engineering and modifying existing/natural protein structures and provides foundational knowledge for the de novo design of self-assembling proteins. An increasing number of examples of de novo designed proteins, wires, tubes, arrays, and even three-dimensional compartments have been published in recent years (Majerle et al. 2019; Hamley 2019; Suzuki et al. 2016; Chen et al. 2019a; Yeates 2017; Okesola and Mata 2018; Kuan et al. 2018; Matsuura 2018; Cannon et al. 2019; Bale et al. 2016; King et al. 2012; Lai et al. 2012; Padilla et al. 2001; Ben-Sasson et al. 2021). The design of proteins that assemble into structures across multiple length scales that mimic the complexity and heterogeneity of hierarchical materials in nature has yet to be fully realized. However, the inherent programmability of protein-based materials, which allows for controlling self-assembly processes, physicochemical material properties, and functionalization, will eventually enable the fabrication of biorthogonal materials that replicate and augment the properties and functions of biological materials.

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Self-Assembly Process

The assembly of proteins into higher-order structures is governed by many of the same forces that direct the intramolecular folding of individual monomers, simply applied on a larger and intermolecular scale. The sum of these interactions—which are driven by protein primary sequence, subunit secondary/tertiary structure, and subunit orientation within the larger assembly—contributes to the overall chemical and physical properties of the nanomaterials. The hydrophobic effect is the major driver of protein folding due to the favorability of consolidating hydrophobic amino acids away from the aqueous environment. The thermal stability of proteins is due in part to the unfavorability of re-exposing those same amino acids. Protein-protein interactions driven by the hydrophobic effect are similarly robust. However, surface exposure of hydrophobic amino acids can also cause aggregation, misfolding, and nonspecific binding which can become a challenge for nanomaterial design and engineering. When compared to the hydrophobic effect, other noncovalent forces such as hydrogen bonds, chargecharge interactions, and the van der Waals effect are relatively weak. Yet, because the number of hydrophilic noncovalent interactions within a single protein is very large, in total these interactions contribute significantly to protein structure and stability. Protein-protein assemblies driven by charge-charge interactions are often susceptible to disassociation if the solution pH changes sufficiently to alter the protonation state of amino acid side chains at the protein-protein binding site. This property can, however, in some systems, be exploited to control the assembly, disassembly, and reassembly of protein-based structures discussed later (Li et al. 2019). Recent publications have provided detailed discussions of the forces that drive protein assembly into supramolecular structures (Zhang et al. 2020a; Sun and Marelli 2020).

2.2.2

Design and Engineering

In recent years, the cost and tediousness of genetic engineering have both decreased significantly. Short DNA primers of less than 60 nucleotides generally cost less than US$0.25/base pair. Gene synthesis is now commonplace due to its speed and cost efficiency as well as the ability to optimize the precise DNA sequence and codon usage to match the biology of a heterologous host. The production of DNA libraries can also be readily accomplished. Cloning techniques such as Gibson and Golden Gate (Engler et al. 2009) assemblies have eliminated the need for specific restriction enzyme cleavage sites, thus bypassing a somewhat inefficient step in “traditional” cloning while increasing the flexibility of the sequences that can be readily cloned. Advancements in our understanding of genetic elements such as promoters, terminators, and ribosome binding sites enable another level of control over protein assembly. By adjusting these parameters, the timing and rate of protein expression can be optimized and controlled. Libraries of synthetic promoters and other genetic

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elements exist for many of the common heterologous microbial chassis organisms (Schlesinger et al. 2017; Peiroten and Landete 2020; Ito et al. 2020; Kotopka and Smolke 2020; Gilman and Love 2016; Gilman et al. 2021; Wang et al. 2020) that can be harnessed for material fabrication, enabling fine control of protein expression in a range of cell factories. Consequently, in theory large libraries of protein building blocks could be rapidly and cost-effectively tested for the design and fabrication of nanomaterials, provided they can be recombinantly produced and characterized at sufficient throughput and scale. As discussed below and throughout this chapter, implementation of such a rapid design-test-build-learn cycle for the bottom-up engineering of nanomaterials is currently hampered by low throughput bottlenecks in production and, most importantly, characterization of material properties.

3 Functionalization Protein-based materials are genetically encoded and can therefore be readily engineered with additional, emergent functions and properties for a diverse range of applications. For example, the chemical diversity of amino acid side chains opens up possibilities for biorthogonal modifications, labels, and functionalizations. The ability to include unnatural (noncanonical) amino acids (UAA or ncAA) either directly during translation or via posttranslational modification further expands the set of functional groups and chemistries that can be engineered on a protein surface. For example, the in vivo introduction of selenomethionine can provide a unique handle for benzylation for site-specific bioconjugation reactions (Flood et al. 2021). Protein building blocks can be engineered for the attachment or fusion of peptide tags and larger domains, with altered surface charges or interfaces that will facilitate a new function. Such functions may include covalent or noncovalent interactions of the protein materials with other proteins, e.g., as cargo for scaffolding or encapsulation, cellular and subcellular targeting of protein nanomaterials, or interactions with abiotic molecules. In addition, surface functionalization can be used to control material assembly and alter its structural morphology. Considering the large number of peptide sequences and protein domains that are known—or can be readily designed—to facilitate protein-protein interactions and fusion or binding to inorganic molecules, the design space for potential protein building block functionalizations is vast. The only constraint for the genetic introduction of such protein modification is their potential impact on protein assembly. Some nanomaterial building blocks may be less amenable to modification than others and require more extensive design considerations regarding location and/or the use of different functionalizations to eliminate or minimize the detrimental effects on assembly and stability.

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Cargo Protein Localization to Nanomaterials

For many applications, the protein nanomaterial serves as a structure to carry or contain one or more species of cargo protein. As shown in Fig. 2 (left), many wellestablished strategies are available for the covalent attachment of cargo proteins to a protein-based nanomaterial. In the simplest case, the cargo protein is genetically fused to a structural protein of the nanomaterial. Typically, a flexible linker region rich in glycine and serine residues is introduced between the two proteins to minimize detrimental effects on cargo protein function and/or nanomaterial assembly. Linker type and length as well as N- or C-terminal placement often require some degree of optimization. Such a strategy results in a 1:1 ratio of nanomaterial protein and cargo, which may not be optimal for all applications where spatial control over cargo localization would be desired. Alternatively, cargo loading can be accomplished via secondary mechanisms where the attachment of cargo protein can be achieved either during or after nanomaterial formation. This strategy would allow greater control over the loading of cargo proteins, and thus their spatial organization on the material. For example, a very powerful and versatile method is the SpyTag/SpyCatcher system and its variants for covalent ligation of proteins (Reddington and Howarth 2015; Sutherland et al. 2019; Hatlem et al. 2019; Keeble and Howarth 2020). Here, a short SpyTag peptide is fused to one protein partner, and a small SpyCatcher domain is fused to the other protein. When SpyTag and SpyCatcher interact, they spontaneously form an isopeptide bond that covalently links the two proteins. Another protein ligation method uses the sortase (SrtA) from Staphylococcus aureus. This transpeptidase cleaves between the threonine and glycine of a LPXTG motif fused to one protein and joins the carboxyl group of the resulting terminal threonine to the amino group of a pentaglycine motif fused to the other protein partner (Strijbis et al. 2012). Noncovalent binding of cargo to nanomaterials (Fig. 2, right) can be achieved by fusing small peptide or protein domains to structural and cargo proteins that would facilitate charge complementary interactions or high-affinity interactions. Such selective binding can, for example, be achieved with the dockerin/cohesin domains derived from cellulosomes (Mitsuzawa et al. 2009) and other well-known interaction domains like the PDZ and SH3 domains (Behrendorff et al. 2020). In addition, coiled-coil peptide interactions (Lee et al. 2018; Edgell et al. 2020) could be co-opted for cargo attachment. Protein loading into compartments requires specific considerations as the cargo proteins need to be efficiently encapsulated concurrently with compartment assembly. Both native and engineered strategies have been used to accomplish cargo loading in engineered protein compartments. For example, a green fluorescent protein (GFP) variant with significant positive surface charge (GFP+36) was successfully encapsulated inside of a lumazine synthase assembly engineered to possess a significant negative charge along the luminal face (Worsdorfer et al. 2012, 2011). Fusion of GFP+36 to other proteins facilitated their encapsulation as well (Azuma et al. 2016, 2018b). For VLP cargo loading, a statistical encapsulation approach has

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Fig. 2 Methods of co-localizing nonstructural (cargo) proteins to protein-based nanomaterials. These methods include both covalent (green box, left) and noncovalent (blue box, right) mechanisms for protein-protein interactions. Options for forming covalent attachments between the nanomaterial and the cargo protein(s) of interest include genetic fusion (upper left); posttranslational covalent bonding, such as via spontaneous isopeptide bond formation between SpyCatcher and SpyTag (Reddington and Howarth 2015; Sutherland et al. 2019; Hatlem et al. 2019; Keeble and Howarth 2020) (middle left); or enzyme-catalyzed bonding performed by sortase A (Strijbis et al. 2012) (bottom left). Noncovalent co-localization can also be driven by many mechanisms, including charge-charge interactions (upper right); natural encapsulation tags that are known to target proteins to the lumen of, for example, BMCs (Chakraborti et al. 2020) (middle right); and protein interaction pairs found in natural systems, such as modular cohesion-dockerin pairs from the bacterial cellulosome (Mitsuzawa et al. 2009) (bottom right). BMC bacterial microcompartments, SrtA sortase A

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been used, where a very high concentration of the intended cargo is mixed with disassembled shell proteins. Upon modifying conditions (e.g., temperature, pH) to allow assembly, some cargo then ends up inside of each compartment (Schwarz et al. 2017). While this method eliminates the need to modify cargo proteins, it is dependent on the concentration and purity of the cargo and requires that the cargo remains stable and soluble under conditions for shell assembly and disassembly. Native mechanisms for cargo loading in 3D compartments and cages have been characterized for many of the systems described in Sect. 2 (reviewed recently in Chakraborti et al. 2020). Identification of targeting sequences and their corresponding binding sites on the luminal face of compartments has made it possible to load non-native cargo proteins into BMCs, encapsulin, vaults, and LS (recently reviewed in Azuma et al. 2018a; Demchuk and Patel 2020), simply by genetic fusion of the cargo protein of choice to the relevant targeting sequence. As targeting sequences are relatively short (e.g., the targeting peptides for encapsulin systems are ~15–40 amino acids in length (Cassidy-Amstutz et al. 2016; Rahmanpour and Bugg 2013; Tamura et al. 2015; Contreras et al. 2014), their attachment to cargo protein often (but not always) has limited effect on cargo protein function/activity. One feature of these native targeting/encapsulation sequences is that they all function using noncovalent protein-protein interactions. In some systems, such targeting/encapsulation interactions are reversible, and changes in the buffer conditions such as pH and ionic strength may interfere with charge-charge and other noncovalent interactions.

3.2

Material Binding Tags

Numerous binding peptides are known that interact and bind with high affinity to different materials (e.g., metals, metal oxides, minerals, polymers, or carbon). These tags can be used for functionalization to construct nanomaterials for a variety of applications ranging from surface coatings to energy materials (reviewed in Care et al. 2015, 2016). One functionalization that has great potential for the design and fabrication of composite materials is the introduction of biomineralization peptides to protein nanomaterials. In biological systems, many proteins function as matrix formers, facilitators, and modulators of biomineralization via a nonclassical nucleation pathway that gives rise to diverse hierarchical composite materials with extraordinary functions and mechanical properties (Wegst et al. 2015; Frezzo and Montclare 2016). Examples of well-studied systems include the biomineralization of Fe3SO4 and Fe3S4 in magnetotactic bacteria (McCausland and Komeili 2020), the precipitation of hydroxyapatite in tooth enamel (Bai et al. 2020) and bones (Oosterlaken et al. 2021), the biomineralization of calcium carbonate in mollusk shells and fish otoliths (Rozycka et al. 2019; Song et al. 2019; Rivera-Perez et al. 2019), and the silica biomineralization of diatoms and sea sponges (Hildebrand et al. 2018; Kroger et al. 1999; Lechner and Becker 2014; Otzen et al. 2003; Song et al. 2017). Silica biomineralization can also be accomplished utilizing a short peptide

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sequence from the spore coat protein CotB1 from Bacillus bacteria species (Motomura et al. 2016). These and many other biomineralization tags, as well as their potential applications, have recently been reviewed in Qin et al. (2020) and Evans (2019a, b). They have not yet found wide use in protein-based nanomaterial design. Protein engineering can also be used to modify the chemical properties of protein structures to impart or enhance metal binding abilities. For example, mutations introduced into some BMC shell proteins have facilitated the coordination of [4Fe-4S] clusters (Aussignargues et al. 2016) and Cu2+ ions (Plegaria et al. 2019), or form covalent bonds to heme (Huang et al. 2019). The addition of histidine-rich tags has been used to bind iron and/or iron-containing compounds on protein nanowires (Ueki et al. 2019). A single mutation to lumazine synthase (R108C) introduced free sulfhydryl groups on the outer surface of the compartment, and therefore imparted the ability to binding a Gd(III)-based complex used in medical imaging (Song et al. 2015). VLPs have also been engineered to allow binding to Gd-containing medical imaging agents (Eiben et al. 2019).

3.3

Cell Targeting and Artificial Posttranslational Modifications

Biomedical applications of protein-based nanocompartments frequently require surface modifications for cellular and/or subcellular targeting. By displaying a moiety that recognizes or is recognized by cell surface receptors (e.g., antibodies, antigens, and cell targeting peptides), functionalized protein nanoparticles can be targeted to specific cell types. This general strategy has been implemented using protein structures including lumazine synthase (Azuma et al. 2018a), encapsulin (Moon et al. 2014; Choi et al. 2016), vaults (Munoz-Juan et al. 2019), and VLPs (Eiben et al. 2019), and has been a useful strategy for vaccines, drug delivery vehicles, and bioimaging applications. Because of the range of functional groups present in amino acid side chains, it is possible to utilize more traditional synthetic chemistry methods to perform siteselective modifications of nanomaterial surfaces. This can include the installation of artificial posttranslational modifications (PTMs) not accessible through recombinant protein expression. Limitations to in vivo PTM installation can occur if the recombinant host lacks the relevant machinery, or if the host’s modifying enzyme(s) have a substrate specificity incompatible with the recombinant protein to be modified. In vitro chemical or chemoenzymatic approaches also allow the installation of noncanonical PTMs. One recent study sought to mimic a natural PTM (N-terminal myristoylation) by synthetically installing a noncanonical lipid at an N-terminal Ser residue of recombinantly produced elastin-like polypeptides (ELPs) to control their self-assembly and achieve programmable disassembly at low pH (Scheibel et al. 2020). PEGylation is a common strategy used in drug delivery (Yadav and

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Dewangan 2021), and poly(ethylene glycol) (PEG) was recently synthetically attached to the outer surface of purified, fully assembled encapsulins for biomedical applications (Sonotaki et al. 2017). This was accomplished by introducing a lysine residue at the C-terminus of the encapsulin shell protein monomer. Neither the mutation nor the attachment of PEG (~2 kDa) prevented nanocompartment assembly or reassembly following disassembly with guanidine chloride and subsequent salt removal by dialysis (Sonotaki et al. 2017).

4 External Control of Self-Assembly The ability to exert control over the self-assembly of protein building blocks can be important for directing the assembly process toward desired nanomaterial architectures and the creation of responsive materials. As described below, some nanomaterials, such as several types of nanocompartments, can be readily assembled and disassembled. Among them, ferritin is most widely studied for its biomineralization ability and high stability (Jutz et al. 2015). Probably the best described selfassembling protein material with predictable assembly behavior is the ELPs (Varanko et al. 2020) that are widely used in biomedical application because of their stimuli responsiveness. For the majority of other protein nanomaterials, the structure and morphologies of the material can also be influenced by external conditions but to a certain degree and rarely in a predictable manner. The following examples discuss how external factors such as temperature, solvent, ionic strength, pH, and other physical methods impact assembly.

4.1

Temperature and pH

Protein self-assembly is a thermodynamically controlled process, and in some cases, temperature can significantly change self-assembly behavior. As mentioned above, ELPs are well-known for their temperature-responsive behaviors and have inspired the design of other stimuli response peptide materials. For example, short peptides with various VPGVG units were designed to create temperature-sensitive selfassembling peptides that form fibrils with increasing length and diameter when the temperature was raised from 20
C to 80
C (Cao et al. 2019). A toolbox of tailored amphiphilic ELPs was built and fused to fluorescent proteins to demonstrate temperature- and pH-responsive assembly into a diversity of supramolecular complexes (Schreiber et al. 2019). As shown in Fig. 3 with peptide BDP-R40F20 as an example, these modified ELP proteins self-assemble into various morphologies, ranging from spherical coacervates and highly ordered twisted fiber bundles to unilamellar vesicles. Interfacing these peptide modules with other self-assembling nanomaterials could therefore impart similar control over their assembly into new types of hierarchical structures. In addition to these temperature-responsive peptides, proteins are

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Fig. 3 Examples of external factors used to control and drive the assembly of protein nanomaterials. Human H ferritin (HFn; top row) disassembles into subunits in the presence of 8M urea and reassembles after removing urea by dialysis (Ferguson et al. 2006). Second row from the top contains the E. coli bacterioferritin (PDB: 2VXI), which disassembles under high pressure and reassembles under ambient pressure (Le Vay et al. 2020). The exterior surface of the DNA-binding protein (Dps; second from bottom) nanocages was reengineered, such that a highly ordered architecture is formed in the presence of zinc ions. The resulting architecture possesses a body-centered cubic superstructure; the disassembly is reversible and can be regulated by salt concentrations (Chen et al. 2019b). The co-block polypeptide BDP-R40F20, shown in the bottom row, assembles into different structures depending on assembly conditions. Structures were visualized with a green fluorescent dye (BDP) (Schreiber et al. 2019)

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also known to show temperature-induced changes in self-assembly. For example, a recombinant Group B Streptococcus (GBS) fusion antigen (GBS-NN) undergoes an irreversible phase transition at elevated temperature from monomeric to higherorder, self-assembled structures as the result of changes in secondary structure (Rose et al. 2018). The opposite effect was observed for an artificial triblock protein engineered as a viral mimetic protein; here higher temperature led to disassembly that could be reversed by cooling (Dias et al. 2020). Compared to the simple structures of peptides with predictable physicochemical properties, the structural complexity of proteins does not readily offer obvious explanations for changes in self-assembly behavior that can be applied to the design of temperature response protein nanomaterials. Charge-charge interaction-driven assembly can be controlled, at least to a degree, by changes in pH. In some cases, pH changes can therefore be used to drive the assembly, disassembly, and reassembly of protein structures, provided that the proteins can maintain or regain assembly competent structures. For example, protein cages such as ferritin and encapsulin are known to disassemble into subunits under acidic and alkaline conditions. Although ferritin subunits appear to maintain nativelike secondary and tertiary structure at pH 2.0 and pH 11.0, only 60% of the subunits reassembled into 3D compartments after the pH was adjusted back to neutral. The resulting structures also possessed holes and were less structurally sound, suggesting that some subtle changes must have occurred to some or all subunits that interfered with proper assembly (Zhang et al. 2020b). Similarly, the Thermotoga maritima encapsulin completely disassembles into protomers at pH 1 and 14, and reassembles into compartments upon adjustment toward neutral pH. However, cargo protein with a C-terminal targeting peptide failed to be packaged during reassembly, suggesting an alternative reassembly pathway which influences the accessibility of targeting peptide binding site (Cassidy-Amstutz et al. 2016). Acid-solubilized collagen readily self-assembles back into fibrils at room temperature and neutral pH. Self-assembly is fastest at pH 5.0, which is near the isoelectric point of the protein. However, the gel strength and thermal stability of fibrils formed at pH 5.0 is lower compared to fibrils reassembled at higher pH values (Shi et al. 2020). These examples illustrate that like temperature, pH affects the assembly process of every protein differently, and often in ways that are poorly predictable and require experimental characterization for each nanomaterial.

4.2

Salts, Chaotropes, and Solvents

Various organic and inorganic small molecules can be used to control protein assembly as well. The addition of salts and other ionic compounds can promote protein self-assembly via electrostatic attraction. The assembly of archaeal and bacterial S-layer proteins (Sleytr et al. 2014; Rodrigues-Oliveira et al. 2017) into two-dimensional lattices is well-known to be stabilized by divalent cations which interact with acidic surface residues. For example, Mg2+, Ba2+, and Ca2+ act as

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general divalent cations to promote the self-assembly of Lysinibacillus sphaericus S-layer protein (SbpA). A systematic study on divalent ion dependent self-assembly of SbpA identified Ca2+ as a specific ion required to induce self-assembly and determined that S-layer nanosheet growth is affected by Ca2+ and SbpA concentrations (Rad et al. 2015). By taking lessons from S-layer assembly, a self-assembling protein was engineered for the reversible transformation between two different 3D morphologies based on the presence of divalent cations (Chen et al. 2019b). In this example, the DNA-binding protein (Dps), which assembles into a 9 nm protein cages for DNA protection in starved prokaryotic cells, was engineered to display the acidic amino acid Glu and Asp residues on the exterior surface. The addition of zinc ions led to the reorganization of Dps cages into 3D superlattices due to ordered interactions of the divalent metal ions with the Dps monomers. Addition of NaCl and the resulting shielding of the metal ions disassembled the lattice and allowed reassembly back into cages at high salt concentrations (Fig. 3) (Chen et al. 2019b). Unlike S-layer assembly, lattice formation was specific for zinc ions and was not triggered by other divalent ions such as Ca2+, presumably due to the known higher affinity of Glu and Asp residues for Zn2+. Salt-dependent self-assembly is also known for thermostable archaeal ferritins. For example, the assembly of a ferritin from Thermotoga maritima is inducible with NaCl, MgCl2, and CaCl2, while in the presence of the chelator ethylenediaminetetraacetic acid (EDTA), the cages completely disassemble into dimers (Chakraborti et al. 2019). For other protein nanomaterials, ionic strength has been identified as a key factor for controlling assembly. The size of curcumin-loaded bovine serum albumin nanoparticles could, for example, be controlled by simply altering the concentration of salt and/or buffer conditions (Safavi et al. 2017). Chaotropic agents such as urea and guanidine hydrochloride (GuHCl) are also commonly used to regulate the assembly and disassembly of protein materials. Human H ferritin (HuHF), for example, was first disassembled in 8M urea and then reassembled at decreasing urea concentrations in the presence of doxorubicin (DOX) to fabricate HuHF-hybrid nanoparticles for cancer treatment (Fig. 3) (Liang et al. 2014). Reducing agents are widely used in controlling protein nanoparticle and material formation. Disulfide breaking agents such as tris(2-carboxyethyl)phosphine (TCEP) can be used to disrupt a preformed protein or peptide conformation, triggering material formation. Disulfide disruption in a number of proteins can have morphological consequences, such as in the case of lysozyme, which is rich in α-helices, but the addition of reducing agents can lead to a rapid transition to structures with β-sheets that oligomerize to form nanofilms at the air/water interface or assemble into protofibrils that generate microparticles in bulk solution (Zhao and Yang 2020; Li et al. 2018). Compared to salts, the application of chaotropes and reducing agents is less preferred, as they can strongly disrupt the structure of many self-assembling protein building blocks and potential cargo proteins. As described previously, the main driving forces for protein self-assembly are hydrophobic, polar, and electrostatic interactions. Cosolvents, including alcohols, may be used to tune protein interactions in solutions, but the underlying mechanisms are poorly understood. The effect of three different monohydric alcohols on

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α-lactalbumin self-assembly into fibrils was studied. For this protein, the mildly hydrophobic methanol was able to form a solvent layer around the protein compared to ethanol and isopropanol with bulkier hydrophobic chains that largely affected the bulk properties of the solvent. These differences in solvent properties were shown to control α-lactalbumin self-assembly into different structures (Bucciarelli et al. 2020). In another example, ethanol drastically increased the length and diameter of the nanotubes formed by an amphiphilic polypeptide formed from hydrophobic α-helical blocks, indicating that ethanol enhanced the hydrophobic interactions between subunits. In contrast, in the presence of acetonitrile, only twisted ribbons were observed, suggesting that acetonitrile can stabilize the exposed protein edges and promote the segmentation of polypeptide assemblies (Nandakumar et al. 2020). More studies are necessary to understand how different salts, chaotropes, and solvents affect the main forces driving protein self-assembly. A deeper understanding of these effects may benefit our ability to tune the properties of protein nanomaterials.

4.3

Other Control Mechanisms

Most of the strategies described above employ harsh chemical or thermal conditions to promote protein nanomaterial disassembly, which has a significant potential to irreversibly change protein self-assembly behavior and is generally not applicable for loading fragile functional proteins. Therefore, some physical and nonthermal technologies, including hydrostatic pressure (Le Vay et al. 2020), pulsed electric field (Meng et al. 2018), and atmospheric cold plasma (ACP) (Yang et al. 2018), have been expanded to control protein nanomaterial self-assembly. This is especially useful for protein cages, such as ferritin, which is stable even under extreme conditions. For example, E. coli bacterioferritin (Bfr) completely dissociates into dimers under the hydrostatic pressures of 450 MPa. Nearly complete reassembly could be achieved after pressure release, if performed under conditions with appropriate ionic strength and temperature (Fig. 3) (Le Vay et al. 2020). Additionally, ferritin treated with pulsed electric fields (Meng et al. 2018) and ACP (Yang et al. 2018) could disassemble and reassemble under a more moderate pH range. These strategies could modify the native structure of proteins, and generally need incorporating with other factors to achieve revisable dissociation.

5 Production and Characterization 5.1

Heterologous Production and Purification

Both the characterization and commercial application of protein nanomaterials require low-cost and highly efficient production. The heterologous production of

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protein nanomaterials is affected by many factors, such as the codon usage in the coding sequence, vector backbone, genetic elements surrounding the gene (i.e., promoters and terminators), and expression hosts. Therefore, it is often essential to carefully consider protein production strategies on a case-by-case basis, as well as optimize culture conditions to achieve the highest quality protein with the desired properties and functionalization. E. coli is a frequently used host due to its high expression level, fast growth kinetics, inexpensive culturing, scalability, and simple genetic engineering. Hence, a large number of detailed protocols have been established to produce protein nanomaterials with E. coli as an expression host (Zhang et al. 2019b; Huang et al. 2017). The main weakness of E. coli expression system is the presence of endotoxins, which limits the biomedical applications of proteins nanomaterials produced by this organism (Gorbet and Sefton 2005) and requiring the use of alternate heterologous systems. In some situations, eukaryotespecific posttranslational modifications are needed, and other expression hosts must be employed, such as yeast (Werten et al. 2019), insect cell lines (Gopal and Schneemann 2018), plant cells (Dennis et al. 2018), mammalian and avian cells (Fuenmayor et al. 2017), and cell-free systems (Glass et al. 2000; Nooraei et al. 2021). The efficient and thorough purification of protein nanomaterials is another area for system-specific optimization. These processes must take into account not only factors affecting protein solubility and assembly but also any specific conditions that are necessary for the intended industrial and medical applications. The purification protocols of common protein nanomaterials, such as amyloid protein (Jia et al. 2020) and VLPs (Nooraei et al. 2021; Hillebrandt et al. 2020), continue to be optimized to improve protein purity and yield. Some commonly used methods include immobilized metal affinity (IMAC) (Zhang et al. 2019b), size exclusion chromatography (SEC) (Rurup et al. 2015), and reverse phase high performance liquid (RP-HPLC) chromatography (Liao and Chen 2015), as well as centrifugation- and ultracentrifugation-based protocols (Kuadkitkan et al. 2021; Spice et al. 2020). In some case, several purification steps combining different methods are necessary to obtain high-quality products. For example, purification of the heterologously expressed encapsulin from Quasibacillus thermotolerans (Qt) was accomplished through several steps. Cages were first precipitated from cell lysate with NaCl and PEG-8000. After resuspending, the samples were further purified using SEC and ion-exchange chromatography successively (Giessen et al. 2019). These precise techniques may or may not remain effective for other systems, depending on their solubility, morphology, and stability. For some protein nanomaterials, smart design utilizing their specific properties can simplify the purification process. For instance, some encapsulin variants can be purified by heat and ammonium sulfate precipitation because of their thermostability (Lee et al. 2020). Each material therefore requires the development of a system-specific downstream isolation process that considers any unique material properties that could be taken advantage of (e.g., solubilization and reassembly under different conditions), process economics, and the required purify levels for intended applications. Many protocols described in the literature are useful at the laboratory scale but are not necessarily scalable for

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commercial use. Ultracentrifugation, for example, would be an impracticable step for bulk material production.

5.2

Material Characterization

Characterization techniques are critical to assess the physicochemical properties, desired behaviors, and reproducibility of protein-based self-assembling nanomaterials—all of which are of extremely important properties to understand for successful application (Jain and Thareja 2019; Samrot et al. 2020). There is accordingly a diversity of well-established experimental approaches to assess and characterize protein-based nanomaterials (Yildiz et al. 2020). Some of the commonly used techniques in nanoparticle characterization include light scattering (Filipe et al. 2010), electron microscopy (Su et al. 2020), microscopy (ZamoraPerez et al. 2018), separation-based techniques (Bourne et al. 2009), small angle X-ray scattering (SAXS) (Malik et al. 2013), atomic force microscopy (AFM) (An et al. 2020), tunable resistive pulse sensing (TRPS) (Clenet et al. 2018), and spectroscopy (Schreiber et al. 2019, b; Jain and Thareja 2019). In most cases, various parameters including size, ζ-potential, surface morphology, and stability need to be determined. Thus, a combination of techniques is generally necessary to provide precise and comprehensive information about the properties of materials (Pitman and Larsen 2020; Lombardo et al. 2020). Researchers need to select appropriate techniques based on their experimental needs; thus, here we give an overview of frequently used techniques including their advantages and disadvantages (Table 1).

5.2.1

Light Scattering

Light scattering is a widely used technique for measuring the size distribution and hydrodynamic diameter of nanoparticles. It is much more cost-efficient compared with other techniques, such as electron microscopy (Samrot et al. 2020). Light scattering techniques include dynamic light scattering (DLS), electrophoretic light scattering (ELS), differential centrifugal sedimentation (DCS), and nanoparticle tracking analysis (NTA). Among them, DLS is the most common and easiest method for identifying the size and size distribution of protein nanoparticles in solution (Sigmund et al. 2019). ELS is used to measure the electrophoretic mobility of nanoparticles. This mobility can be converted to ζ-potential, which is a key indicator of the stability of colloidal dispersions (Xu 2002). Compared to DLS, DCS allows the size analysis of polydisperse samples (Arosio et al. 2016). NTA is another technique similar to DLS, but which is suitable for both monodisperse and polydisperse protein nanoparticles (Filipe et al. 2010).

Transmission electron microscopy (TEM) Cryo-transmission electron microscopy (cryo-TEM) Scanning electron microscopy (SEM)

Differential centrifugal sedimentation (DCS) Nanoparticle tracking analysis (NTA)

Dynamic light scattering (DLS) Electrophoretic light scattering (ELS)

Environmental scanning electron microscopy (ESEM) Atomic force microscopy (AFM)

Electron microscopy

Technique Light scattering

Size, surface texture, morphology, and roughness

3D visualization, suitable for both liquid and gas media

Direct visualization of sample, can image liquid samples, does not require sputter-coating

Direct close measurement, can acquire detailed 3D images

Sample is frozen-hydrated state; show 3D structures

Observe each and individual particles, high sample throughput, accurate for both monodisperse and polydisperse samples Direct close measurement

Size (30 nm to 1 μm), ζ-potential, aggregation

Size (>1 nm to 100 μm), aggregation, surface morphology, structure Size (5 nm to 300 nm), aggregation, structure, surface morphology Size (10 nm to 100 μm), aggregation, structure, surface morphology Size (10 nm to 100 μm), aggregation

Short analysis time, samples in natural solution

Ease to use, low sample consumption

Advantages User-friendly

Size (20 nm to 10 μm)

Characterization of: Size (1 nm to 1 μm), size distribution ζ-Potential

Table 1 Common techniques used for the characterization of different protein material properties

Highly influenced by probe damage

Lower resolution than conventional EM, sample thickness limits resolution

Vitrification and sample preparation may still alter properties, lower resolution than conventional TEM Samples are dehydrated, costly, and time consuming

Sample is dehydrated and fixed

Experienced operator required, less reproducible than DLS

Requires known sample properties

Disadvantages Requires monodisperse, homogeneous, clear samples Requires monodisperse, homogeneous, clear samples and known sample properties

An et al. (2020)

Jackson et al. (2015)

Pedersen et al. (2019)

Stewart (2017)

Su et al. (2020)

Filipe et al. (2010)

References Filipe et al. (2010) Varenne et al. (2019), Trampari et al. (2019) Arosio et al. (2016)

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UV-Vis

Spectroscopy

Tunable resistive pulse sensing (TRPS)

Circular dichroism (CD) Fluorescence spectroscopy Small angle X-ray scattering (SAXS)

Size exclusion chromatography (SEC) Asymmetric flow field-flow fractionation (AF4) Native PAGE

Size (75 nm), spectral signature

Hyperspectral dark-field microscopy (HDFM) Fluorescence microscopy

Can measure liquid samples

Simple sample preparation, diverse measurement capabilities

Size (>1 nm to 1 μm)

Size (60 nm to 10 μm), ζ-potential

Easy to use Easy to use

Easy to use

Oligomer status Easy to use

Less adsorption than SEC

Size (1 nm to 100 μm)

Self-assembly status, aggregation Stability, conformational changes Unfolding, aggregation

Requires a short analysis time

Direct visualization of sample, simple sample preparation, realtime analysis Easy to use

Direct close measurement

Size

Oligomerization, cargo status

Size (200 nm)

Light microscopy

Separationbased techniques

Microscopy

Difficult to accurately interpret, cannot be used alone for nonideal or polydisperse samples Potential limit on particle size, requires calibration under sample conditions as measurement

Cannot be applied to large-size nanoparticle Need to include or introduce chromophores Results insufficient for determining structure Needs suitable fluorophore

Low resolution

Low resolution, sample can interact with column

Nanoparticles are below resolution limit Spectral profiles may be difficult to distinguish, especially for non-plasmonic samples Needs suitable fluorophore

Clenet et al. (2018)

Sigmund et al. (2019) Schreiber et al. (2019) Schreiber et al. (2019) Putri et al. (2016) Malik et al. (2013)

Rambaldi et al. (2009)

SchmidtDannert et al. (2018) Bourne et al. (2009)

Zhang et al. (2018) Zamora-Perez et al. (2018)

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Electron Microscopy

Characterization of nanomaterial size and homogeneity can also be performed using various types of electron microscopy, including scanning (SEM) (Pedersen et al. 2019), transmission (TEM) (Su et al. 2020), environmental scanning (ESEM) (Jackson et al. 2015), and cryogenic (cryo-EM) (Stewart 2017) electron microscopies. Compared to SEM, TEM can provide finer details about the size, homogeneity, size distribution, and morphology of protein samples (Su et al. 2020). SEM is generally most useful for acquiring information about the surface topography of a sample (Pedersen et al. 2019). Although cryo-EM is costly and computationally challenging, its major advantage is the preservation of nanoparticles in a native environment, which therefore gives a more accurate image of how the protein looks in solution (Stewart 2017; Montemiglio et al. 2019). ESEM also allows in situ measurement in liquid or gaseous environments and thus provides morphology information under native state, although at lower resolution than cryo-EM (Jackson et al. 2015; Montemiglio et al. 2019).

5.2.3

Spectroscopy

Spectroscopic methods such as circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy are convenient and highly accessible approaches for nanomaterial characterization. In addition, spectroscopy can be used to continuously measure spectroscopic features of a sample to obtain information about, for example, the assembly process of a protein (Pignataro et al. 2020). UV-Vis absorption spectroscopy can be used to detect the light absorption of a protein’s intrinsic chromophores including the side chains of aromatic amino acid residues, as well as any extrinsic chromophores from cofactors or probes which have been co-purified or added to the sample. For example, the Congo red (CR) dye is a widely used probe for amyloidosis identification. When CR binds to exposed hydrophobic regions on amyloids, the absorbance of the dye shifts from 490 to 540 nm, which can readily be observed by using any common spectrophotometer (Yakupova et al. 2019), thereby providing a clear readout of the exposed hydrophobic surfaces in a sample. Turbidity is a commonly used parameter to detect the assembly of particles in solution, and can be quantified using a turbidity meter to measure light absorbance in the range of 350–600 nm. It can be used to compare the influence of different conditions on protein self-assembly (Wiedemann-Bidlack et al. 2007). Another parameter, the aggregation index (AI), is measured between 280 and 350 nm to evaluate the existence of both large aggregates and oligomers. Generally, for a transparent pure protein solution, the AI is lower than 3. An AI between 3 and 30 suggests that some aggregation (including both functional and nonfunctional or denatured aggregates) may have occurred, while a value above 30 demonstrates that the protein is heavily aggregated. Recently, the AI was used to assess the aggregation of the Yersinia pestis outer transmembrane β-barrel Ail under different

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temperatures (Gupta and Mahalakshmi 2020). Thus, these and similar techniques can be straightforward and efficient methods to observe protein assembly. CD is a well-established technique to measure the structural composition of a protein sample. The far-UV (190–250 nm) and near-UV (250–320 nm) spectral regions can be used to observe protein secondary and tertiary structure, respectively. Accordingly, multiple properties of a protein, including conformation changes and thermal stability, can be analyzed by CD (Ranjbar and Gill 2009). For example, CD was applied to determine the relative thermal stabilities and secondary structure of E. coli bacterioferritins (Le Vay et al. 2020). Recent developments in instrumentation have led to new methods for CD spectroscopy, such as synchrotron radiation circular dichroism (SRCD). These new techniques are well-described in many reviews (Wallace 2019; Krupova et al. 2020). In one recent study, both CD and SRCD were used to monitor the thermal denaturation of the Dps nanocage protein from Marinobacter hydrocarbonoclasticus. Compared to CD, SRCD data have a high signal-to-noise level and contain more information, as it is possible to collect data at high energy (e.g., 175 to 195 nm) for SRCD (Pacheco et al. 2020).

5.2.4

Fluorescence Techniques

Fluorescence techniques are among the most widely used methods in the development and characterization of protein nanomaterials and can be detected with both spectroscopy and microscopy. In addition to the huge library of fluorescent proteins that can be genetically attached to proteins, there is also a broad range of fluorescent probes that can be used in the assessment of protein assembly. For example, the dye thioflavin T binds to the surface of amyloid fibrils via hydrophobic interactions with the β-sheet domains and is used in monitoring amyloid fibril assembly (Aliyan et al. 2019) as well as other, similar proteins. The intrinsic fluorescent properties of tryptophan, tyrosine, and cysteine can also be applied to monitor protein assembly. Additional fluorescence spectroscopy techniques that can be employed to characterize protein self-assembly include steady-state anisotropy, time-resolved spectroscopy, and fluorescence correlation spectroscopy. Detailed information for those methods are well-described in specialized book chapters and reviews (Engelborghs and Visser 2014; Limpouchova and Prochazka 2016). A noteworthy strategy for studying protein assembly is Förster resonance energy transfer (FRET). Briefly, two fluorophores (a donor and an acceptor) are introduced in the system. After being excited by light of the appropriate wavelength, the donor fluorophore transfers energy to acceptor fluorophores though dipole-dipole interactions. As energy transfer efficiency is dependent on the distance between the two fluorophores, FRET can be used to indicate distances between fluorophores (Metskas and Rhoades 2020). A recent study employed the FRET to measure the density of the displayed mCerulean3-SpyCatcher and mVenus-SpyCatcher on protein filaments. In this way, the optimal stoichiometry of scaffolds and cargo proteins for self-assembly and loading was identified (Lim et al. 2019).

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Other Techniques

In addition to the common methodologies described above, a range of other techniques are typical for the characterization of different protein nanomaterial. For instance, AFM can be used to measure surface topography and mechanical properties of single nanoparticles and is able to scan protein materials without the addition of metal stains (An et al. 2020). TRPS is also a single-particle technique for size determination, giving measurements in good agreement with TEM micrographs but with simpler sample preparation (Clenet et al. 2018). Another technique, SAXS, allows observation of the self-assembly process over time in solution. However, interpreting results can be challenging, and the method is not suitable for polydisperse samples (Malik et al. 2013). In addition, some approaches and equipment that are accessible to most laboratories have also been expanded to characterize protein nanomaterials. For example, light microscopy is a widely available technique and offers a direct and reliable observation for nanoparticle aggregation. It must be noted, however, that the resolution of light microscopy is too low to visualize single nanoparticles. Hyperspectral dark-field microscopy (HDFM) on the contrary provides high contrast and resolving capability and is therefore suitable for protein nanomaterials, but specific equipment is needed (Zamora-Perez et al. 2018). Separation-based techniques, including SEX (Bourne et al. 2009), asymmetric flow field-flow fractionation (AF4) (Rambaldi et al. 2009), and non-denaturing polyacrylamide gel electrophoresis (PAGE) (Sigmund et al. 2019), are low-cost approaches to determine the oligomerization status or stability of proteins.

6 Applications 6.1

Biomanufacturing and Biocatalysis

Biocatalysis is an increasingly utilized strategy for the production of chemicals by exploiting the specificity and selectivity of enzymes (Sheldon and Woodley 2018). Many potential strategies for optimizing biocatalytic systems draw inspiration from the billions of years evolution has had to arrive at complex but well-ordered metabolic systems. In biological systems, one commonly employed strategy to exert control over enzymatic catalysis is by organizing and isolating subsections of metabolism through the use of membrane-bound organelles, microcompartments, and protein scaffolds. This co-localization serves to isolate pathways forming toxic metabolites, stabilize enzymes, provide microenvironments and conditions required by specific enzymes, sequester reactions to prevent off-target interactions, and control the local concentrations of enzymes or intermediates (Dubey and Tripathi 2021; Zhang and Hess 2017; Schmitt and An 2017). As a result of these factors, the spatial organization of enzymatic systems often enhances its efficiency. Based on these principles, many emerging biocatalytic systems have been designed to include

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Fig. 4 Examples of protein nanomaterials engineered for biocatalysis applications. In the top left, glucose oxidase (GOX) and horseradish peroxidase (HRP) were displayed either separately scaffolded or co-localized on gamma-prefoldin (γ-PFD) filaments to increase enzyme activities (Lim et al. 2019). Bottom left shows NADH-dependent amine dehydrogenase (AmDH) and alcohol dehydrogenase (ADH) were co-immobilized via the SpyTag-SpyCatcher fusion system on scaffolds formed by the EutM bacterial microcompartment (BMC) shell protein to increase chiral amine production (Zhang et al. 2018). The upper right depicts modified PduA from Citrobacter freundii Pdu BMC, which forms hollow filaments when expressed in E. coli. The filaments span the length of the cell and were used to organize pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in vivo to increase ethanol production (Lee et al. 2018). Bottom right shows cowpea chlorotic mottle virus (CCMV), which was used to encapsulate the lipase CalB without decreasing its activity (Schoonen et al. 2016)

a strategy for the immobilization, compartmentalization, and/or organization of the relevant enzymes (Dubey and Tripathi 2021; Vázquez-González et al. 2020; Quin et al. 2017; Schmidt-Dannert and Lopez-Gallego 2019; Xu et al. 2020). Here we focus on the utilization of protein scaffolds, including synthetic protein scaffolds and natural protein cages, which allow finely tuned control of spatial organization, enzyme proximity, and stoichiometry. We will highlight a few recent examples of how these systems are applied to enzyme-mediated synthesis. A more comprehensive recent review can be found here (Seo and Schmidt-Dannert 2021). Until now, a few protein nanomaterials with various morphologies have been employed to build in vivo and in vitro enzyme immobilization system. One of the well-studied platforms is filamentous scaffolds. As shown in Fig. 4, protein

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immobilization on gamma-prefoldin (γ-PFD) ultrastable protein filamentous was accomplished by using SpyCatcher-fused glucose oxidase (GOX) and horseradish peroxidase (HRP). These enzymes were successfully attached to γ-PFD modified to display SpyTag along the filaments, resulting in an increase of about 50% in the in vitro catalytic activity compared to the free enzymes. Systems where GOX and HPR were co-scaffolded or separately scaffolded showed similar catalytic activity, indicating that the activity increase was due to the enhancement of each enzyme through immobilization itself, rather than an effect of physical proximity of the enzymes through co-immobilization (Lim et al. 2019). Another filamentous scaffold is based on Ure2, which is a regulator of nitrogen metabolism from Saccharomyces cerevisiae. Its N-terminal prion domain self-assembles into fibers with an analogous behavior to mammalian prions. Genetic fusion of alkaline phosphatase (AP) and HRP to the C-terminal of the prion-like domain had little effect on fibril assembly. The advantages of this construction include the relative ease of separating enzymes from products, and the potential to recycle enzymes for subsequent reactions. However, for the AP-HRP-Ure2 system, enzymatic activity decreased compared with corresponding wild-type-free enzymes (Zhou et al. 2014). Compared to nanofibers, nanosheets could provide a 2D enzyme displaying platform. As described in Sect. 2, EutM self-assemble into stable nanosheet under broad pH and temperature conditions. The properties of this protein can therefore be used to design scaffolds for biocatalysis. A dual enzyme cascade including an NAD+dependent alcohol dehydrogenase (ADH) and an NADH-dependent amine dehydrogenase (AmDH) could be efficiently co-immobilized onto a EutM-SpyCatcher scaffold when the enzymes were genetically fused with SpyTag. This system improved the conversion efficiency by reducing the time needed to achieve ~90% substrate-product conversion from 48 h to 24 h (Fig. 4) (Zhang et al. 2018). These studies suggest that in some systems both the act of immobilization and the physical proximity of enzymes can play a role in the optimization of biocatalytic systems. Housing enzymes within the cavity of protein cages is a well-studied method to improve biocatalysis efficiency and thermostability in vivo and in vitro. Different from nanofibers and nanosheets, protein cages form a relative closed environment and are often stable even under extreme conditions. Therefore, encapsulation may protect the enzyme from external degradation machineries. It is potentially possible to control the pool of substrates available to encapsulated enzymes by engineering pores on the compartment shell. Commonly used model cages including cowpea chlorotic mottle virus (CCMV), MS2 phage, hepatitis B virus (Cp149), bacteriophage P22, LS, ferritin, and the eukaryotic vault are well reviewed elsewhere (Chakraborti et al. 2020). In one example, the lipase CalB was fused to the CCMV monomer using SrtA-mediated coupling reaction. The ligated shell-lipase protein was still capable of self-assembly into protein cages of the same size. This design protected the lipase from proteases without decreasing its activity (Fig. 4) (Schoonen et al. 2016). Another recent example is based on bacteriophage P22, which assembles from 420 subunits of coat protein (CP) and 100–300 subunits of scaffolding protein (SP) to form a T ¼ 7 icosahedral capsid. An alcohol dehydrogenase cargo enzyme was fused to the CP N- and C-termini without affecting self-assembly

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behavior. Moreover, the wild-type SP and cargo-SP could co-assemble at different ratios, thus allowing fine control over the catalytic activity of the nanoreactors (Sharma and Douglas 2020). Protein nanotubes are also promising structures for building enzyme organization systems. For example, the shell protein PduA from the Citrobacter freundii Pdu BMC was used to achieve in vivo enzyme co-localization and organization. First, a modification to the C-terminus of PduA was made to improve its solubility (PduA*). When overexpressed alone in E. coil, PduA* forms hollow filaments approximately 20 nm in diameter and spanning the length of the cell. Then, as shown in Fig. 4, pyruvate decarboxylase (PDC) and ADH were attached on the filament with heterodimeric coiled-coil interacting peptide pair. As a result, an increase in ethanol production of over 200% was observed (Lee et al. 2018). Each of these examples demonstrates the potential of scaffolds and compartments for optimizing biocatalysis both in vivo and in vitro.

6.2 6.2.1

Biomedical Applications Drug Delivery

One major challenge in drug development is identifying and optimizing a strategy to deliver the active compound to the appropriate tissue(s) in a biologically relevant form. Drug delivery materials can help to improve drug solubility, protect biomolecular drugs from degradation, control release rate, and target delivery to specific cell types. While providing these benefits, drug delivery systems must also be both biocompatible (i.e., nonimmunogenic) and biodegradable. A distinct advantage of protein nanoparticles over other types of drug delivery nanoparticles is that the position of each amino acid is spatially defined, allowing for control over the spatial organization of ligands and targeting (bio)molecules displayed on the nanocage surface. Many nanoparticle-based drug delivery systems utilize protein structures to encapsulate the drug of interest. The ferritin cage is one of the best-studied nanostructures for drug delivery. Ferritin itself is recognized by at least two cellular receptors (transferrin receptor 1 and a class A scavenger receptor) that are overexpressed in tumor cells (Montemiglio et al. 2019; Geninatti Crich et al. 2015), allowing some selective targeting even prior to engineering. Many classes of bioactive molecules including cisplatin (Yang et al. 2007), doxorubicin (DOX) (Liang et al. 2014), and β-carotene (Chen et al. 2014) have been accommodated within ferritin cage using the reversible self-assembly process (Zhang et al. 2020b). In another recent study, three VLPs with different morphologies (MS2 spheres, tobacco mosaic virus (TMV) disks, and nanophage filamentous rods) were evaluated as nanocarriers to deliver the chemotherapeutic DOX. As shown in Fig. 5, the exterior of each VLP was decorated with PEG to improve biodistribution and reduce immune response, while DOX was attached on either the internal (MS2) or external (TMV and nanophage) face of the nanoparticles. Their results showed that

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Fig. 5 Examples of biomedical applications for various protein-based nanostructures. Upper left: The immunogenic epitope E2EP3 from the E2 glycoprotein of the chikungunya was fused to an amyloid core. And the resultant peptides self-assemble to nanofibers and trigger a robust IgG response in mice (Babych et al. 2018). Bottom left: Melittin, a novel antimicrobial peptide, was fused to a beta-sheet-forming synthetic peptide to form antibacterial nanofibers. The result nanomaterial could disrupt the bacterial cell membrane without compromising the mammalian cells (Chen et al. 2019c). Top right: Two encapsulin systems, Myxococcus xanthus (Mx) which encapsulate the ferritin-like proteins MxBCD and Quasibacillus thermotolerans (Qt) which encapsulate native cargo (QtIMEF), were expressed in mammalian cells and could act as gene reporters under TEM (Selivanovitch and Douglas 2019). Bottom right: Three VLPs with different morphologies, including MS2 spheres, TMV disks, and nanophage filamentous rods, were evaluated as nanocarriers to deliver doxorubicin (DOX), and PEG was co-attached to improve biodistribution and minimize the immune response against VLPs (Finbloom et al. 2018). Mel melittin, QtIMEF native cargo from Quasibacillus thermotolerans encapsulin; MxBCD ferritin-like protein from Myxococcus xanthus, PEG poly(ethylene glycol), DOX doxorubicin

glioblastoma-bearing mice showed highest survival rates when treated with the TMV-DOX VLP (Finbloom et al. 2018). The results from these and other studies demonstrate the potential of protein nanoparticles and nanocompartments for increasing drug efficacy. Future studies can focus on the development of other

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protein nanocarriers with various morphologies, sizes, and properties (Jain and Thareja 2019).

6.2.2

Nanoparticle-Based Vaccines

Vaccines are highly effective public health tools to control the spread of severe and fatal diseases. Thus, there will always be interest in the development of vaccines against both known and novel pathogens. Protein-based nanoparticles can also be used to great benefit in vaccines. Attachment of antigens to the surface of nanoparticles has been shown to enhance antigen presentation and increase immunity response (Pan and Cui 2020). A recent review provides a detailed review of nanovaccine design based on self-assembly protein nanostructure (Zottig et al. 2020). Also described in detail are the ways in which the cellular uptake and antigen processing mechanisms of nanoparticles are different from soluble antigens (Zottig et al. 2020). Surface properties, particle size, and antigen display are among the main considerations in nanovaccine design and development. One study fused the immunogenic epitope E2EP3 from the E2 glycoprotein of the chikungunya virus to a 10-mer peptide derived from an endogenous amyloidogenic polypeptide (Fig. 5). The resulting peptides could self-assemble into linear and unbranched fibrils with a diameter ranging from 6 to 8 nm. Mice immunization developed a robust IgG response against the E2EP3 epitope displaying nanofibrils (Babych et al. 2018). These results indicate that amyloid fibers are a suitable platform to anchor antigenic determinants and increase the epitope’s immunogenicity. Another unique feature of nanovaccines is the possibility of functionalizing the nanoparticles to also display adjuvants. For example, one study found that by co-displaying hemagglutinin and flagellin, the exterior surface of the ferritin cage, the humoral immune response of antigen-specific T cells in mice, was greatly enhanced (Lee et al. 2019b).

6.2.3

Antimicrobial Materials

Antimicrobial peptide (AMP)-based nanomaterials are a promising strategy for addressing the rise of antibiotic-resistant microorganisms. Such materials work by displaying peptides known to disrupt bacterial cell membranes on a scaffold or sheet formed from structural proteins. This material could then be used as a coating upon surfaces such as walls and countertops in hospital settings to prevent the growth of bacteria without the use of antibiotics (Mitra et al. 2020). In one example, the natural AMP melittin was fused to a β-sheet-forming synthetic peptide. As shown in Fig. 5, the melittin-bearing monomers self-assembled to form nanofibers with melittin displayed at the nanofiber-solvent interface. This design could disrupt the bacterial cell membrane without compromising the integrity of mammalian cells, creating a novel therapeutic AMP material (Chen et al. 2019c). It is also possible to carefully design AMPs that have both self-assembly and antimicrobial activities. A recent example is a novel AMP named FF8 designed by Shen et al. (2020). This short

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peptide self-assembled into nanofibers with the ability to break the lipid membrane of E. coli and showed enhanced antibacterial activity (Shen et al. 2020). Another possible strategy to develop antimicrobial materials is the encapsulation or display of bacteriolytic enzymes such as lysozymes. For example, T4 lysozyme molecules were encapsulated inside the CCMV VLPs without destroying the enzyme’s catalytically activity (Schoonen et al. 2017). In addition to peptides and enzymes, a wide variety of metal ions are also known to have antibacterial properties. Using protein scaffolds or compartments to precipitate and/or display such metal ions is a potential alternative strategy for the development of antimicrobial materials. For example, when silver nanoparticles were encapsulated within T. maritima encapsulins, the resulting hybrid nanoparticles showed unique antimicrobial properties (Giessen and Silver 2016).

6.2.4

Biosensing and Bioimaging

Biosensors are a promising alternative strategy for detecting the presence of biological and chemical agents including antibiotics, toxic chemicals, pathogenic bacteria, and viruses, as well as various disease biomarkers. This strategy utilizes the substrate selectivity of proteins to design or evolve a sensor that can discern a very specific target molecule, which reduces background or nonspecific reactivity. This recognition is then relayed as an output that can be more easily recognized, such as fluorescence (Li et al. 2020). Biosensors can be cost-effective and have a reduced environmental impact compared to non-biobased sensors while also providing fast results or, potentially, real-time monitoring (Pourakbari et al. 2019). Most currently designed nanoparticle biosensors use nonbiological materials such as carbon- and metal-based structures, magnetic nanoparticles, and quantum dots (Lan et al. 2017). The potential of biosensors built from protein-based nanomaterials is less thoroughly explored, but engineered protein nanotubes have already shown promise. For example, one study used pyrene-labeled peptide amphiphiles including a His-rich HGGGHGHGGGHG (HG12) peptide and linear or branched alkyl chains that self-assemble into nanofibrils. The binding of metal including Cu2+ or Ag2+ to the nanofibrils resulted in changes to the fluorescence profile of the sensor, making it a potential tool for detecting metal ions in solution (Kim et al. 2014). Another promising platform for biosensor development is S-layer proteins (Schuster 2018). Recently, folate was used to functionalize an S-layer lattice, and the resulting nanoparticles were attached to a gold surface. This biosensor was able to detect the presence of folate receptors on the breast cancer cell surface in situ and in real time, which may be used to develop screening tools for early-stage cancers (Damiati et al. 2018). Taken together, these and other studies suggest that protein nanoparticle-based biosensors may have broad applications in medicine and diagnostics. Additionally, protein-based nanomaterials are being developed for bioimaging applications. Assorted metal nanoparticles and dye molecules have been loaded into ferritin and used as contrast agents for magnetic resonance imaging (MRI) and

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optical imaging. One study found that the ferritin-gadolinium nanoparticles generated significantly higher signal than commercially available Gd chelates (Sanchez et al. 2009). Another promising protein cage used for bioimaging is encapsulin, including encapsulins from Myxococcus xanthus (Mx) and Quasibacillus thermotolerans (Qt). The two encapsulin systems were co-expressed in mammalian cells (Fig. 5). As the Mx and Qt encapsulins self-assemble into capsids with different icosahedral symmetries and diameters, when both were co-expressed in mammalian cells, the subunits displayed no cross-assembly and formed only homoligomeric structures. This enabled the use of the encapsulins as in vivo markers for observing gene expression by TEM (Giessen et al. 2019; Sigmund et al. 2019).

6.2.5

Other Applications

Microencapsulation is a technique increasingly studied in food science to increase stability and control the release of aromas, dyes, antioxidants, nutrients, enzymes, preservatives, and microorganisms. Protein nanocarriers are attracting attention for their potential uses in the delivery and release of active food additives, as well as the development of “functional foods.” One possible application of such nanocarriers is to increase the bioavailability of micronutrients such as iron and calcium. Ferrous sulfate and ferrous gluconate are common iron sources, but direct consumption in supplements can have side effects such as constipation, diarrhea, and decreased growth. To avoid these drawbacks, the protein cage ferritin (in this case from soybean) was purified from plant tissue and utilized as a natural dietary supplement that is well-absorbed (Lonnerdal et al. 2006). Inspired by natural ferritin, another group encapsulated calcium ions within phytoferritin. Direct uptake of Ca2+ can be inhibited by compounds such as tannic acid and oxalic acid, which are found in many foods. However, the calcium-containing phytoferritin was absorbed via a different cellular pathway, bypassing the interference and allowing intracellular delivery of Ca2+ (Li et al. 2014). The encapsulation of bioactive nutrients, such as cyanidin-3-O-glucoside, β-carotene, curcumin, rutin, proanthocyanidins, and lutein, is also an efficient strategy to increase in foods and nutrition, as encapsulation significantly improves the stability and solubility of these and other compounds. Water contamination is a global concern, with many causes including agricultural, human, and industrial waste and runoff. Recently, water treatment systems based on nanotechnology have been studied and developed. Possible applications for protein nanomaterials in this area include the absorption of waste and toxic materials onto recoverable materials, and enzyme-catalyzed treatments to remove specific pollutants. Using protein scaffolds to immobilize enzymes and promote biomineralization can be a promising strategy in the design of nanomaterials which can be used in treatment of wastewater and contaminated water (Punia et al. 2021). For example, the enzyme manganese peroxidase (MnP) can be used to degrade the toxic compound phenol. MnP was encapsulated within vaults by genetic fusion to the INT targeting peptide. Packaged MnP-INT was more stable and had a longer lifespan,

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thereby increasing its phenol degradation output (Demchuk and Patel 2020; Wang et al. 2015).

7 Conclusions The different types of protein nanomaterials discussed in this chapter illustrate the large diversity of protein building blocks that can be sourced from biological systems and subsequently engineered into materials with desired structures and functions. These examples also show that we have the genetic engineering tools available to readily customize and fabricate these materials for a broad range of applications. Advances in computational protein design (including de novo protein design, not discussed in-depth in this chapter) provide endless opportunities for the creation of new types of self-assembling protein building blocks. At the same time, these examples also illustrate major bottlenecks and challenges for the fabrication of “next-generation” protein materials. One of the recurring challenges in protein design is our inability to precisely predict the assembly of building blocks into different materials with predefined morphologies and structures. In addition, predictive control of assembly and disassembly is challenging and typically requires the exploration of a variety of conditions for each material. Fundamental and applied research will therefore be required to achieve predictable self-organization of protein building blocks from the nano- to macroscale into hierarchical materials. At the same time, materials need to be designed such that they can incorporate different functions, maintain their structural integrity for their desired applications, and be produced economically in bulk quantities. Together these criteria pose significant challenges for the bottom-up design of genetically programmable materials and will require the development of a robust workflow that integrates computational design, synthetic biology approaches, and material characterization capabilities. While rapid progress has been made in synthetic biology and computational protein design for high-throughput testing, material characterization methods currently lag behind and are not suited for integration into a high-throughput design pipeline based on an iterative design-test-build-learn cycle that could rapidly speed up the fabrication of new types of materials. Efforts have therefore been directed at developing suitable characterization platforms that can analyze material structures and functions with sufficient details to inform design tasks. In conclusion, our current capabilities in genetic engineering and biological design offer tremendous opportunities for the creation of new types of programmable protein materials. Biological systems can provide insight in the design of materials that are extremely robust, have self-healing properties, and incorporate inorganic components (biomineralization) to generate materials with extraordinary mechanical properties with tunable porosities and compressive strengths. For example, incorporation of mineralization processes could not only increase the mechanical strength of materials but also generate new energy materials. Likewise, the development of systems that more closely mimic the heterogeneity of natural

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materials could provide many additional useful nanomaterial properties. For example, increased heterogeneity could possibly lead to fracture-resistant materials, as well as possibly speed up the bulk growth of materials. Learning and then adopting biological material design principles therefore will hold the key for future fabrication of genetically encoded bioorthogonal materials. Acknowledgments Research on the development of self-assembling protein materials in the Schmidt-Dannert Laboratory has been supported by Defense Threat Reduction Agency Grant HDTRA-15-0004, Defense Advanced Research Projects Agency Contract HR0011-17-0038, National Science Foundation CBET-1916030, MnDRIVE, and the University of Minnesota’s Biocatalysis Initiative through the BioTechnology Institute. Kelly Wallin was supported by a predoctoral NIH training grant fellowship (5T32 GM008347) and by an NSF graduate research fellowship (CON-75851, Project 00074041). Author Contributions KKW, RZ, and CSD wrote this paper. Conflicts of Interest The authors declare no conflict of interest.

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Engineered Living Conductive Biofilms Lina J. Bird, Fernanda Jiménez Otero, Matthew D. Yates, Brian J. Eddie, Leonard M. Tender, and Sarah M. Glaven

Abstract Living conductive biofilms are biomaterials comprised of bacterial cells electrically connected to each other and to electrodes across multiple length scales. The properties of living conductive biofilms are due to the ability of some bacteria to form a network of charge carrying proteins across the cell membrane and through the biofilm matrix in a process called extracellular electron transfer. Such bacteria, known as electroactive bacteria, catalyze an array of reactions at electrodes not possible with nonliving conductive films due to the diverse range of their cellular metabolisms. Applications of natural living conductive biofilms include energy harvesting at the bottom of the ocean, bioremediation, and carbon capture for biosynthesis. Recent breakthroughs in bioengineering offer new tools to modify living conductive biofilms in order to improve on existing technologies, such as increasing the electrical current produced by a microbial fuel cell, or creating entirely new ones. For example, fully synthetic living conductive biofilms could be deployed as electrically responsive materials under conditions where natural electroactive bacteria cannot operate. In this chapter, we review our current state of understanding of natural conductive biofilms, tools and methods for studying conductive biofilms, and opportunities for bioengineering of these biomaterials. Keywords Living conductive biofilm · Bioengineering · Bioanode · Biocathode · Geobacter · Shewanella · Microbial electrosynthesis · Interdigitated microelectrode array · Extracellular electron transfer · c-type cytochromes

L. J. Bird · M. D. Yates · B. J. Eddie · L. M. Tender · S. M. Glaven (*) Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC, USA e-mail: [email protected] F. J. Otero College of Science, George Mason University, Fairfax, VA, USA © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 W. V. Srubar III (ed.), Engineered Living Materials, https://doi.org/10.1007/978-3-030-92949-7_4

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1 Introduction Engineered living conductive biofilms are biomaterials based on the ability of some bacteria to form conductive networks among each other, their extracellular matrix, and surfaces. These microorganisms, known as electroactive bacteria, have evolved metabolic strategies that enable them to transport electrons across the cell membrane to insoluble electron acceptors, or take electrons up into the cell from insoluble electron donors, in a process termed extracellular electron transfer. Natural living conductive biofilms have been studied and developed for nearly two decades for energy, bioremediation, and biosynthesis applications. Through genetic modification, living conductive biofilms are envisioned to be further functionalized to behave as sense and respond coatings, as living catalytic polymers with improved reaction rates, and as a bioelectronic interface for wearable or ingestible devices. The advantages that engineered living conductive biofilms provide over conventional, nonliving materials is their ability to adapt to temperature, pH, and salinity, their ability to self-assemble and self-repair, and their programmability. They could take the shape of a natural electroactive bacterium genetically modified with new or improved functionality (e.g., is more conductive). Alternatively, it could mean non-electroactive bacteria engineered with genetic elements required for biofilm conductivity to be used in applications where they may be more robust than natural electroactive species. Use of a bioengineering approach to create leap ahead technologies using living conductive biofilms requires an in-depth understanding of the molecular underpinnings of natural conductivity, tools for manipulating these molecular processes, and appropriate measurement methods to test conferred properties. Due to decades-long research into iron-reducing bacteria, we are now able to take control and manipulate the extracellular electron transfer pathway of sediment-dwelling Shewanella oneidensis. For example, genes for the proteins that facilitate electron transfer across the cell membrane can be functionally expressed in a bacterium that lives in seawater (Bird et al. 2018). Other examples include demonstration of S. oneidensis as a living catalyst for radical polymerization (Fan et al. 2018), and years of work demonstrating functional expression of electron transfer conduits in Escherichia coli (Su et al. 2020; Lienemann et al. 2018; Jensen et al. 2016, 2010; TerAvest et al. 2014; Goldbeck et al. 2013). This foundational work will enable design and engineering of conductive biofilms. In this chapter, we will provide a historic overview of what is known regarding natural conductive biofilms, review approaches and advancements in engineering so-called electroactive bacteria, and provide an outlook for applications using engineered living conductive materials.

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2 Natural Conductive Biofilms Extracellular electron transfer, referred to as EET from this point on, was first discovered in anaerobic bacteria that reduce iron or manganese as part of their respiratory process (Lovley and Phillips 1988; Lovley 1991; Myers and Nealson 1988). Electrons resulting from the oxidation of organic substrates are transported across the cell inner membrane, through the periplasmic space, and across the cell outer membrane (in gram-negative bacteria) to the insoluble extracellular terminal electron acceptor. The EET process was subsequently noted in iron-oxidizing bacteria, where electrons are transported into the cell in a reverse manner to drive reduction reactions, such as CO2 fixation (Bird et al. 2011; Emerson et al. 2010). For both outward and inward EET, the metal or mineral electron donor/acceptor can be replaced with an electrode at a sufficiently reducing or oxidizing potential, providing an infinite sink or source for metabolic electrons (Fig. 1a). Biofilms of electroactive bacteria will spontaneously form on the electrode surface comprised of fixed cells and secreted substances (protein, redox molecules, etc.) enabling EET. When the electrode potential is set to act as an electron acceptor, or anode, electrons released during oxidation of electron donors inside the cell can be captured as current and such biofilms are referred to as bioanodes. When the electrode potential is set to act as an electron donor, or cathode, electrons taken up into the cell via EET provide reducing equivalents for CO2 reduction (Wang et al. 2015), or reduction of other thermodynamically favorable electron acceptors such as O2, sulfate, fumarate, and nitrate (Rabaey and Rozendal 2010; Agostino and Rosenbaum 2018; Gregory et al. 2004; Gregory and Lovley 2005; Strycharz et al. 2008). In this case, the biofilm is referred to as a biocathode and resulting current is measured as a negative value (indicating electrons moving from the electrode into the biofilm). When bioanodes or biocathodes are multiple cell layers thick, electrical conductivity is required to enable access to the electron donor or acceptor by cells not in direct contact with the electrode surface. From this metabolic necessity emerges the concept of a living conductive biofilm. We define a living conductive biofilm as electrode-attached microbial cells conducting electrons over multicell-length distances through fixed mediators for cellular respiration. A key feature of a living conductive biofilm is its electron transfer mediators embedded in the cell membrane and extracellular matrix (described below) that act as electron conduits between the inside and outside of cells, through the extracellular matrix, and across the biofilm/electrode interface.

2.1

Conductive Bioanodes of the Model Electroactive Bacteria Geobacter sulfurreducens and Shewanella oneidensis

To the best of our knowledge, the first modern demonstrations of microorganisms generating electricity without exogenous electron transfer shuttles were by Park et al.

Fig. 1 Schematic and microscopy of conductive bioanodes and biocathodes. (a) Schematic of conductive electrode biofilms of Geobacter sulfurreducens (left), Shewanella oneidensis (middle), and the Biocathode MCL biofilm community (right). (b) Scanning electron micrograph of conductive G. sulfurreducens bioanode grown on a gold interdigitated microelectrode array (Snider et al. 2012). (c) Confocal laser scanning microscopy of S. oneidensis bioanode grown on an

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indium tin oxide (ITO) interdigitated microelectrode array. Cells are stained red with FM4-64FX membrane stain. Scale bar, 10 μm. Adapted from Xu et al. (2018). Copyright 2018 American Chemical Society. (d) Image of Biocathode MCL. Three-dimensional reconstruction of FISH-CLSM image of biocathode MCL on an IDA. The putative electroautotrophic microorganism, ‘Ca. Tenderia electrophaga’ (green to teal), is spatially clustered on the electrode surface. Alphaproteobacteria (orange) and other Gammaproteobacteria (red) are scattered throughout the biofilm. Adapted from Yates et al. (2016a). Work of US Government—no copyright under 17 USC 105

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(2001), Kim et al. (2002), and Bond et al. (2002). Park et al. (2001) and Kim et al. (2002) utilized three-electrode bioelectrochemical reactors to precisely regulate the potential applied to the biofilm electrode (working electrode) vs. a reference electrode with a counter electrode ensuring charge balance. This enabled for the first time application of standard electrochemical methods such as chronoamperometry and cyclic voltammetry using a potentiostat, and created the beginnings of a new line of research called microbial electrochemistry. Chronoamperometry is the measurement of current at a fixed electrode potential for which current typically increases over time as cells grow on the electrode surface by utilizing EET. Cyclic voltammetry characterizes the dependency of EET on the electrode potential while maintaining conditions required by the microorganisms. It is the precise regulation of the driving force of EET, by control of the electrode potential, and precise measurement of resulting rate of EET, by measurement of electrode current, that makes microbial electrochemistry a powerful tool for studying EET. Strains selected for initial studies on bioanodes were based on phylogenetic identity of biofilm species enriched at the anode of sediment microbial fuel cells (Tender et al. 2002), including members of the family Geobacteraceae. The first demonstration of electricity generation by pure culture Geobacter sulfurreducens was by Bond et al. (2002), where the anode compartment of a microbial fuel cell was inoculated with G. sulfurreducens. The resulting bioanode catalyzed electricity generation from oxidation of acetate and conductivity was inferred by microscopy indicating multiple cell layers as well as measurement of increasing biomass correlating to increasing current. This was followed by Fricke et al. (2008) and Marsili et al. (2008b) who recorded the first cyclic voltammetry of G. sulfurreducens bioanodes. These voltammetries revealed now classic dependencies of current on potential applied to the electrode: a sigmoidal shaped current-potential dependency during acetate oxidation by biofilm cells, and matching anodic and cathodic current peaks in the absence of acetate, taken to reflect redox activity of proteins underpinning biofilm EET. The voltammetric current-potential dependencies of G. sulfurreducens bioanodes were remarkable in how closely they compared to those of conductive, wired enzyme electrodes pioneered by Heller and colleagues (Gregg and Heller 1991). Although actual conductivity measurements of G. sulfurreducens biofilms would come later (described below), this comparison led to a simple but enduring model for biofilm conductivity, referred to as the Nernst-Monod model (Torres et al. 2008b; Yi et al. 2009). In this model, microbial cells in a biofilm act as oxidation or reduction catalysts, and conductive redox centers within the biofilm form electron transfer pathways between the catalytic sites inside the cells and the electrode surface. In this model, EET occurs via redox conduction, as occurs in redox polymers (Natan and Wrighton 1989; Dalton et al. 1990; Blauch and Saveant 1992), by discrete electron transfer reactions between redox centers in a manner resembling a bucket brigade (Strycharz-Glaven et al. 2011). This model predicts that EET through living conductive biofilms is inherently a diffusive process, driven by a redox gradient, in which the local concentration of electrons residing within the redox centers of the biofilm changes with distance from the electrode surface (Snider et al. 2012;

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Lebedev et al. 2014). It was Fricke et al. (2008) who first recognized the possible diffusive behavior of biofilm EET. They were confounded by non-turnover voltammetry of their anode grown G. sulfurreducens biofilms, which exhibited peak heights that increased with the square root of scan rate and noted that this was consistent with “diffusion control” of current that could not be explained by knowledge of EET at the time. As microbial electrochemical methods evolved, studies of EET through S. oneidensis bioanodes depicted voltammetry revealing dominant roles not only of bound EET proteins but of soluble (physically diffusing) redox active mediators, a process described in more detail below (Marsili et al. 2008a; Xu et al. 2016). In the case of a G. sulfurreducens bioanode, the existence of a redox gradient has been determined experimentally (Snider et al. 2012) and results from continuous cellular reduction of redox centers throughout the biofilm coupled to continuous oxidation at the biofilm/electrode interface and exhibiting an Arrhenius temperature dependency (Yates et al. 2015; Phan et al. 2016). Redox centers are in a progressively more reduced state with increasing distance from the electrode surface, which in turn results in a less favorable local redox potential for EET. In other words, the driving force for EET experienced by cells in a G. sulfurreducens bioanode drops off with distance from the electrode surface, resulting in reduced metabolic activity (Chadwick et al. 2019).

2.1.1

Conductive Cytochrome Proteins of Geobacter sulfurreducens and Shewanella oneidensis

Once it was determined that G. sulfurreducens biofilms were conductive, the redox centers and other elements enabling electron transfer needed to be established. Years of research on iron reduction by both G. sulfurreducens and S. oneidensis had led to the possibility that membrane-associated, redox active proteins could play a role in transporting electrons out of the cell and through the extracellular matrix (Lovley et al. 2011). Both G. sulfurreducens (Ueki 2021) and S. oneidensis possess a large number of genes encoding c-type cytochromes; proteins with one or more heme groups covalently attached to the protein backbone. c-type cytochromes, or cytochromes c, are a core part of the respiratory pathway, and are thus found in nearly all living organisms. However, the respiratory proteins are typically simpler; the respiratory cytochrome c, a protein found in all respiring organisms, is a small monoheme protein. In contrast, the cytochromes found in EET pathways can contain anywhere from two to ten or more heme groups. The EET pathway in G. sulfurreducens begins at the inner membrane where at least three pathways exist, two of which have been fully characterized (Fig. 2a). The NapC/NirT homolog ImcH is a heptaheme c-type cytochrome with three transmembrane domains anchoring it to the inner membrane (Levar et al. 2014). NapC/NirT homologs like ImcH, and CymA in S. oneidensis, typically transfer electrons from the quinone pool to periplasmic acceptors, but the unique characteristic of ImcH is that it only enables respiration of terminal electron acceptors with a redox potential

Fig. 2 Known and proposed pathways for extracellular electron transfer contributing to living conductive biofilms. (a) Shewanella oneidensis. (b) Geobacter sulfurreducens. Reprinted from Jiménez Otero et al. (2020). Work of US Government—no copyright under 17 USC 105. C. Proposed pathway for extracellular

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electron uptake in “Candidatus Tenderia electrophaga” as part of the Biocathode MCL community. Reprinted from Eddie et al. (2017). Work of US Government—no copyright under 17 USC 105

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positive of
100 mV vs SHE (Levar et al. 2017). In order to respire substrates with a lower redox potential, G. sulfurreducens uses the multiheme cytochrome CbcL. This protein is made up of a HydC/FdnI di-heme b-type cytochrome, usually found in hydrogenases and formate dehydrogenases playing a role as menaquinone oxidoreductase, fused to a periplasmic nonaheme c-type cytochrome (Zacharoff et al. 2016). The ImcH pathway allows for a higher cell yield than CbcL (Levar et al. 2017) suggesting that electron transfer through ImcH creates a larger membrane potential per mol of Fe(III) reduced or mol of electrons transferred to a high potential electrode. In the absence of high redox potential electron acceptors, CbcL is able to reduce low redox potential substrates even if the trade-off is lower cell yield. A third inner membrane electron transfer pathway has been detected electrochemically at extremely low redox potentials, close to the redox potential of the oxidation of acetate, but it is yet to be physiologically identified (Zacharoff et al. 2016). Together, these redox potential-dependent inner membrane electron transfer pathways enable G. sulfurreducens to use a wide variety of extracellular metal oxides spanning 1V in redox potential. For comparison, that range encompasses the redox potential of oxygen, nitrate, and sulfate reduction which require completely separate pathways in organisms such as Escherichia coli. The pathway for electron transfer between the inner and outer membrane in G. sulfurreducens is still not fully characterized, but there are many genes encoding c-type cytochromes predicted to be located in the periplasm, most of them small, triheme c-type cytochromes such as PpcA-E (Morgado et al. 2010), or larger c-type cytochromes consisting of fused triheme homologs of the Ppc family such as the 12-heme GSU1996 or GSU0592, and 27-heme GSU2210 (Pokkuluri et al. 2011). Electron transfer across the outer membrane is enabled by outer membrane conduits in G. sulfurreducens similar in organization to the well-characterized MtrCAB complexes in S. oneidensis (described below). G. sulfurreducens uses at least five different outer membrane conduits (OmbB-OmaB-OmcB, OmbC-OmaCOmcC, ExtABCD, ExtEFG, and ExtHIJKL) in a substrate-dependent manner (Jiménez Otero et al. 2018; Chan et al. 2017). These range from between 23 heme cofactors in the ExtABCD conduit to as little as 5 heme cofactors in ExtHIJKL, although ExtH is a rhodanese-like protein with FeS clusters as electron carriers. Finally, G. sulfurreducens secretes a large number of cytochromes to the extracellular space. Some of these are found as soluble monomers such as PgcA which is involved in Fe(III) oxide reduction (Zacharoff et al. 2017), while some are polymerized into cytochrome nanowires several cell-lengths long such as OmcS and OmcZ. Recent studies have shown that while both OmcS and OmcZ form conductive cytochrome polymers several cell-lengths long, OmcZ wires are 1000-fold more conductive and only polymerized during electrode reduction (Yalcin et al. 2020; Wang et al. 2019). The relationship between G. sulfurreducens outer membrane conduits and secreted extracellular proteins is not resolved and is still an active area of investigation. In S. oneidensis, extracellular electron transport begins in the inner membrane, where electrons resulting from oxidation of some organic electron donor are transferred to a membrane-bound tetraheme cytochrome, CymA (Fig. 2b). From there,

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they are passed to a small periplasmic tetraheme cytochrome, CctA, and then to an outer membrane complex consisting of two decaheme cytochromes and an outer membrane porin that spans the membrane, the MtrCAB complex (Edwards et al. 2018; Coursolle et al. 2010). There are multiple homologs for the outer membrane cytochromes, and the function of all of them is imperfectly understood. However, a variant strain of S. oneidensis MR1 lacking nine of the periplasmic and outer membrane cytochromes shows reduced growth on iron and electrodes (Coursolle and Gralnick 2012; Yates et al. 2021). The phenotype can be rescued by expressing mtrC and mtrA from a plasmid, indicating that the wide variety of multiheme cytochromes found in the genome are, if not fully redundant, at least providing very subtle differences in EET. No other electroactive bacteria have been as closely characterized as G. sulfurreducens and S. oneidensis, but evidence for c-type cytochromes as a biomarker for conductive bioanodes is abundant in both electrochemical and genomic characterization of bioanode microbial communities.

2.1.2

Conductive Pili and Membrane Extensions

In addition to c-type cytochromes, G. sulfurreducens expresses type IV pili that have been shown to play an essential role in forming conductive biofilms. The reader is directed to recent extensive reviews of the G. sulfurreducens type IV pili by Tabares et al. (2020), and by Lovley and Walker (2019), for an in-depth description of their biology and biotechnological potential. Type IV pili have well-known roles in cell surface attachment, biofilm formation, and secretion; however, groundbreaking work by Reguera et al. (2005), and later Malvankar et al. (2011), also showed that pili themselves have conductive properties with some role in EET in G. sulfurreducens. These “e-pili” have been proposed to conduct charge through stacking of aromatic amino acid residues aligned along the length of the extracellular filament (Lampa-Pastirk et al. 2016). The conductive mechanism itself has been heavily studied using both experimental and theoretical approaches, often with competing interpretations (Lampa-Pastirk et al. 2016; Feliciano et al. 2015; Malvankar et al. 2015; Lebedev et al. 2015). When the gene for the structural monomer for e-pili, pilA, is deleted, electrode biofilms show reduced current, but also do not grow to the same thickness (Richter et al. 2009, 2012). When the arrangement of aromatic residues is modified, biofilm current is also affected (Vargas et al. 2013). Separating the role of e-pili from the role of c-type cytochromes in living, conductive biofilms has not been resolved due to the fact that both are located in the extracellular matrix, where pili likely play a structural role. For example, c-type cytochromes have been shown to interact with pili, and they have also been shown to be involved in cytochrome secretion (Richter et al. 2012). For these reasons, researchers have turned to orthogonal expression of e-pili or purification and assembly of whole filaments or monomers for biotechnological applications as described further below. S. oneidensis solves the problem of accessing the extracellular electron acceptors using two strategies in addition to direct EET through c-type cytochromes: first, while

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direct electron transfer to electrodes does occur (Coursolle et al. 2010), there is good evidence that the outer membrane cytochromes also reduce naturally produced flavins, which act as soluble redox mediators (Kotloski and Gralnick 2013). The second strategy is the use of outer membrane extensions: Shewanella can form chains of interconnected outer membrane vesicles which are redox active due to the inclusion of the outer membrane cytochrome conduits (El-Naggar et al. 2010; Pirbadian et al. 2014; Pirbadian and El-Naggar 2012; Subramanian et al. 2018). These extensions were originally thought to be pili nanowires, but are now believed to extend the reach of individual cells, allowing them to access substrates at a distance.

2.2

Conductive Biocathode Biofilms

Geobacter and Shewanella biofilms can also be configured as biocathodes that use electrodes as electron donors for bioremediation (Gregory and Lovley 2005; Strycharz et al. 2008), for oxygen reduction (Rowe et al. 2018), and potentially for synthesis (Tefft and TerAvest 2019; Ueki et al. 2018). In order to form a robust biocathode with high electron uptake, Geobacter and Shewanella must first be grown as bioanodes and then switched to biocathodes by changing the electrode potential. Cells do not use the electrode as an energy source for growth; however, they can use the reducing power to carry out catalytic reactions while the biofilm remains conductive. This offers opportunities from a bioengineering perspective due to the range of genetic tools available for Geobacter and Shewanella. The ultimate application of conductive biocathodes is for a process termed microbial electrosynthesis. During microbial electrosynthesis, an autotrophic biocathode biofilm uses the electrode as an electron donor for CO2 fixation. If coupled to production of higher value biosynthetic products, such as fuel precursors, microbial electrosynthesis could be used as a means to reduce the carbon footprint of chemical production. The vast majority of autotrophic biocathodes grown for microbial electrosynthesis utilize electrochemically generated H2, and not direct electron transfer mediated by EET proteins, and as a result the biofilms are not conductive. Direct electron transfer to biocathodes for CO2 fixation is inefficient because all demonstrated examples require reverse electron transfer to produce NADH as a reducing equivalent for the Calvin-Benson-Bassham pathway. Increasing the number of microbial electrode catalysts at the biocathode could increase the rate of CO2 fixation but requires a conductive biofilm. The only example of a conductive biocathode is Biocathode MCL (named for the three most abundant community members, Marinobacter, Chromatiaceae, and Labrenzia) (Fig. 1a). Biocathode MCL is an autotrophic biofilm community of approximately 20 bacterial species enriched from seawater at an electrode potential too positive to enable electrochemical generation of H2 (Yates et al. 2016a). Biocathode MCL has been extensively characterized using genomics, transcriptomics, and proteomics (Wang et al. 2015; Malanoski et al. 2018; Eddie et al. 2017; Leary et al. 2015), and has been demonstrated to create a conductive

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biofilm matrix containing c-type cytochromes as the proposed electron mediators (described below (Yates et al. 2016a)). The keystone member of the Biocathode MCL community, “Candidatus Tenderia electrophaga,” fixes CO2 through the Calvin-Benson-Bassham pathway and contains a proposed direct EET pathway similar to that of some neutrophilic, autotrophic iron-oxidizing bacteria, although the strain has not yet been cultivated in isolation (Eddie et al. 2016) (Fig. 2c).

2.3

Biofilm Proton Conduction and Cation Signaling

An understudied aspect of electrode biofilm conductivity is the role of H+ conduction. The oxidation of organic matter by cells in bioanodes not only generates electrons but also protons, and both must be transported across the cell membrane and out of the bulk biofilm to support metabolic activity and charge balance. Current from bioanodes can become limited by proton transport due to an inefficient diffusion of H+ away from the electrode (Torres et al. 2008a). Likewise, CO2 fixation by biocathodes consumes both electrons and H+, and cathodic current may be limited by OH
transport (Popat et al. 2012). As such, as current flows, a H+ and/or OH
gradient occurs between the bulk aqueous medium and the biofilm, which in turn drives the necessary movement of ions. The mechanisms of H+ transport in living biofilms are poorly understood in part due to the reliance of cells on membrane-associated H+ gradients in energy-generating processes, making it difficult to disentangle from bulk movement of ions. For example, the concentration of protons in the periplasm of gram-negative bacteria, and near the cell surface in gram-positive bacteria, can change 1–2 pH units, or 10–100 x [H+]. Protons and to a lesser degree OH
ions are known to have anomalously high mobilities in aqueous solutions (Agmon et al. 2016) due to a phenomenon known as the Grötthuss mechanism (Cukierman 2006). Our modern understanding of this mechanism has become divided between a model where water molecules are either structured to act as a H+ wire or the individual molecules turn to pass H+ from one molecule to the next (Agmon et al. 2016). Biological materials akin to biofilms can have a high H+ conductivity. For example, polysaccharide hydrogels composed of keratan sulfate found in the Ampullae of Lorenzini of sharks and skates having a very high room temperature H+ conductivity of 2 mS cm
1 (Josberger et al. 2016). Proteins can form H+ conductive channels via amino acid side chains that provide a channel to hold water molecules in positions that facilitate transport (Baciou and Michel 1995). Well-studied examples include the channels that transport H+ away from photosynthetic reaction centers in purple photosynthetic bacteria (Okamura et al. 2000) and across membranes in complex IV of the electron transport chain (Karpefors et al. 2000; Brändén et al. 2001). While biofilm H+ conduction has not been explored as a means to genetically engineer a living, conductive material, the opportunity to explore this space is worth mentioning here. In addition to H+ conduction, cation transport plays a role in biofilm cell-cell communication. Bacillus subtilis biofilms have been shown to display neuron-like

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activity with pulses of K+ ions, which cause a signal cascade that is propagated through the biofilm (Prindle et al. 2015). This signal cascade enables metabolic coordination between cells in the interior and exterior of a biofilm, causing a temporary halt to growth of the biofilm and enabling nutrients to penetrate to the interior of the biofilm. K+ signaling can even function as an interspecies communication mechanism, with Pseudomonas cells attracted to the high local K+ concentration (Humphries et al. 2017).

3 Measuring Biofilm Conductivity 3.1

Conductivity Defined

As this chapter is about living conductive biofilms, it behooves us to define just what conductivity is, how it is quantified, and how it is measured in the case of a biofilm. For a redox conductor, the electron transfer mediators are envisioned as occupying fixed sites about which they oscillate, enabling mediators to come into contact with one another at which point an electron transfer can occur (Dalton et al. 1990; Blauch and Saveant 1992). It is the sequential transfer of electrons in this manner, from one to the next mediator in a bucket brigade manner that results in electrical conductivity. Conductivity is the degree to which a material conducts electrical current. It is expressed in units of siemens per meter (S/m) denoted by σ. Conductivity answers the following question: for a 1 m  1 m  1 m cube of a material, if two flat 1 m  1 m electrodes are in pressed into contact with two opposing faces of the cube, and a voltage (V) is applied between the electrodes such that an electrical current (I) flows between the electrodes through the material, and resistance to current flow between each electrode and the material (so-called contact resistance) is negligible, and the voltage is small enough such that current through the material scales linearly with the applied voltage, then what is the slope of the current vs. voltage plot for which I ¼ σV?. As points of reference, σ ¼ 6  106 S/m for copper which is highly conductive, σ < 10
11 S/m for glass which nonconductive, and σ ¼ 5 10
4 S/m for living G. sulfurreducens biofilms (Yates et al. 2016b; Jimenez Otero et al. 2021). Making such measurement for a 1 m3 of any material, yet alone a biofilm, if possible, is not feasible. Actual conductivity measurements involve applying a voltage across two electrodes through the material of interest where the actual scale and geometry used depends on a number of factors not the least of which is the nature of the material (e.g., a liquid or thin film). The result is then extrapolated as if the measurement was performed as described above (Kankare and Kupila 1992). In this way, apples to apples comparisons can be made for conductivity values regardless of the scale and geometry of the measurements.

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Measuring Conductivity of Living Biofilms Interdigitated Microelectrode Arrays for Measuring Biofilm Conductivity

As described above, living conductive biofilms display features analogous to those of redox conducting polymers when classic electrochemical methods are applied (Heller 1990; Pickup et al. 1984; Natan and Wrighton 1989). These features represent electron transfer through multiple interfaces (electrode/biofilm, biofilm/ biofilm, and biofilm/cell) when multicell layer thick (Bond et al. 2012). Measuring conductivity of living conductive biofilms has inherent challenges not associated with their nonliving counterparts. The most obvious being how to control, and therefore measure, the physical parameters associated with the biofilm in order to accurately characterize the conductivity and conditions while ensuring viability of the cells. For biofilms, we (Yates et al. 2015, 2016b, 2018a) have adapted methodology developed by Murray (Chidsey et al. 1986), Wrighton (Natan and Wrighton 1989), and Heller (Rajagopalan et al. 1996) used to study electrical conductivity of thin polymer film coatings. Central to this methodology is utilization of a two-electrode geometry referred to as an interdigitated microelectrode array (IDA) (Fig. 3a). IDAs are very compact, occupying only a few square millimeters of surface area, and when wired up for use easily fit in a 15-mL “Bond style” bioelectrochemical reactor (Marsili et al. 2008b). The geometry and features of IDAs enable very high signal to noise measurements of current conducted through biofilms (Yates et al. 2016b), making them highly sensitive to small differences in conductivity. They are also readily available commercially. An IDA consists of a pair of coplanar electrodes patterned on a smooth insulating surface (e.g., glass or quartz). The electrodes are narrow (e.g., 5 μm wide) and closely spaced (e.g., 5 μm separation), and their interdigitated geometry creates a long and narrow gap between them (e.g., 30 cm long  5 μm wide) (Fig. 3a). By shorting the two electrodes together and applying an appropriate potential to act as an anode or as a cathode, an electroactive biofilm will grow that coats the entire electrode array and gap. Once grown, a bipotentiostat can be used to apply a different potential to each electrode. The difference in these potentials equating to a voltage that drives electrons from the electrode at the more negative potential (referred to as the source) through the biofilm to the electrode at the more positive potential (referred to as the drain), where current conducted through the biofilm (I), is measured directly at either electrode. The long and narrow gap separating the electrodes results in relatively large currents conducted through the biofilm that are readily measurable. At the same time, the small surface area of the electrodes results in relatively small background currents that arise from capacitance charging, which can otherwise confound biofilm conductivity measurements (Yates et al. 2016b).

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Fig. 3 Interdigitated microelectrode array (IDA)-based biofilm electrical conductivity measurements. (a) Schematic of IDA over which a biofilm is grown. Regions not covered by a biofilm including contact pads for electrically connecting to the source and drain electrodes are insulated from exposure to the aqueous medium in which the biofilms are grown and conductivity measurements are made. A bipotentiostat is used to poise both electrodes at the same potential that favors biofilm growth. Then different potentials are applied to the electrodes resulting in electrical current flow from the electrode at the more negative potential (source) through the biofilm to the electrode at the more positive potential (drain), where currents entering (source current) and leaving the biofilm (drain current) are directly measured by the bipotentiostat. A reference and counter electrode complete the electrical circuit. (b) Schematic depiction of an idealized redox gradient that occurs when making gating measurements. Here, the source and drain potentials are changed in unison while maintaining a small (up to 20 mV) difference between them. In the case depicted, as the gate potential (the average of the potential applied to the source and drain) is changed, a redox gradient forms that increases in magnitude and then decreases back to zero. (c) Corresponding idealized generator and collector currents as a function of the gate potential where the current peaks occur at the formal potential of the EET proteins. (d) Schematic depiction of an idealized redox gradient that occurs when making generator-collector measurements. For the case depicted, the source is held at a fixed potential, while the potential of the drain is made progressively more positive. This results in a redox gradient that spans the biofilm between the electrodes that increases in magnitude (becomes more steep) as the drain potential changes. (e) Corresponding idealized generator and collector currents as a function of the drain potential, where the midpoint potential (inflection points in the currents) occurs at the formal potential of the EET proteins forming the EET pathways through which current flows through the biofilm. At any drain potential, the source and drain current should be equal in magnitude but opposite in sign with current conducted through the biofilm taken as the absolute value of either the source or drain current. (f) An example of a real biofilm generator-collector measurement. (g) An example of a real biofilm gating measurement. (a– e) Adapted from Boyd et al. (2015); (e) Adapted from Strycharz-Glaven (2011); (f) Adapted from Snider et al. (2012). Work of US Government—no copyright under 17 USC 105

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Performing Conductivity Measurements Using IDAs

There are two types of IDA-based biofilm conductivity measurements: gating measurements and generator-collector experiments. Gating measurements are performed by slowly sweeping the potentials applied to the source and drain electrodes simultaneously while maintaining a constant offset potential between them (V) (Fig. 3b, c, and f). These measurements are performed over a range of gate potentials (EG), the average potential applied to the source and drain electrodes. In all instances reported for living conducting biofilms, the resulting plot of I vs. EG is peak shaped (Natan and Wrighton 1989; Aoki and Heller 1993; Strycharz-Glaven et al. 2011; Snider et al. 2012; Yates et al. 2015, 2016b, 2018a; Jimenez Otero et al. 2021), indicative of redox conduction (Dalton et al. 1990; Blauch and Saveant 1992; Bond et al. 2012). As such, the value of conductivity (σ) reported for an electroactive biofilm corresponds to that determined at EG for which the maximum current flow occurs through the biofilm for a given V (Yates et al. 2016b). When the source-drain voltage is small (3 orders of magnitude higher solubility. Optimizing physical parameters, such as increasing biofilm thickness, will have trade-offs that must also be considered to achieve the “Goldilocks zone” of biofilm performance where the physical and biological factors are optimized. Biocathode MCL is a conductive biofilm proposed to transport electrons through multistep hopping in a similar manner to G. sulfurreducens, although in this case the electron donor is the electrode and the redox gradient in the biofilm must be considered in reverse where cells close to the electrode surface are more reduced and those at a distance are more oxidized. The rate at which cells at the outer edge of the biofilm reduce O2 and NAD+ may ultimately be limited by the rate of reverse electron transport at the inner cell membrane, a thermodynamically unfavorable reaction requiring energy generated through proton pumping. This again makes inner membrane respiration a target for biofilm conductivity.

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Toward High-Throughput Electrochemical Measurements for Engineered Strains

Developing tools to measure the output from organisms engineered with known conductive proteins is critical to assess their functionality. Typical bioelectrochemical reactors require large volumes of media (10s to 100s of mL), and long setup times making iterative testing of engineered cells cumbersome. This is particularly true during optimization of protein expression (e.g., mix and match EET pathways), or when testing use of genetically encoded sensors and circuits for precise control over EET actuation. Effective characterization of engineered organisms requires tools that enable high-throughput testing. Thus far, the only highthroughput assays demonstrated rely on photometric assays using redox sensitive dyes (Rowe et al. 2021) or nanoparticles (Yuan et al. 2013) that do not mimic the physiological environment of a conductive biofilm. Several platforms have been developed toward high-throughput characterization of electroactive biofilms, each with their own advantages and disadvantages. The research question being asked guides the prioritization of competing factor in the platform design, the number of different engineered organisms that can be measured at a time, separation of the organisms to avoid cross-contamination, fluidic control in the system, and electrochemical control. Interest in high-throughput bioelectrochemical measurements began with a desire to screen organisms for high power production in microbial fuel cells (Hou et al. 2009, 2012) or microbial electrolysis cells (Call and Logan 2011). Soft lithography was used to create a multi-well system with separate chambers with individual fluidic control. Control over the electrochemical potential was not prioritized here or in other systems with a similar goal (Choi et al. 2015; Tahernia et al. 2020), a feature that would be required to adapt them for conductivity measurements. Nano-liter scale measurements using a three-electrode configuration have been demonstrated in a microfluidic reactor with 8-independent and fully separated cultivation chambers (Yates et al. 2021). Here, both wild-type electroactive bacteria and strains modified to express electron transfer proteins under the control of a genetically encoded sensor were successfully tested. More recently, a potentiostat array was developed capable of measuring 128-independent channel simultaneously (Molderez et al. 2021). While individual electrodes were not physically separated in a way that would enable multiplexed testing of different strains, this design represents a step toward high-throughput microbial electrochemistry. Another intriguing possibility for high-throughput microbial electrochemistry is the use of electrochemical surface plasmon resonance imaging (eSPRI). eSPRI was used to show that electron transfer from a cell to the electrode surface could be detected by a change in pixel intensity (Golden et al. 2018). In this case, the resolution of detection is controlled by the resolution of the CCD camera used to capture the light reflected during eSPRI measurements. With high enough resolution, it may be possible to measure single cells which would dramatically increase testing throughput.

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5 Challenges and Outlook In this chapter, we have detailed the current state of knowledge in natural living conductive biofilms, the theory and methods for measuring biofilm conductivity, and research thus far in engineering both native and orthogonal host strains for increased or synthetic biofilm conductivity. The possibility to improve technologies that rely on conductive biofilms, or create entirely new ones, using bioengineering is within reach. However, a number of challenges are apparent. First, the catalog of biological parts, e.g., charge carrying proteins, that can be used to conduct electrons in and out of the cell, as well as through the conductive matrix, is limited. Some of these parts, while known, are derived from organisms that have not been highly studied in the laboratory. This is particularly true for direct electron transfer at biocathodes. In addition, specialized cellular machinery required to orient charge carrying proteins precisely in the cell membrane needs to be controlled in appropriate proportions within the cell. Second, as a biomaterial, living conductive biofilms must be utilized under conditions that suite the host strain. Development of natural or synthetic electroactive bacteria must be done with the intended application in mind, and the potential for successful expression of EET proteins should be considered given lessons learned from G. sulfurreducens, S. oneidensis, and E. coli. Finally, appropriate methods must be used to evaluate engineered biofilm conductivity. A combination of genetic engineering and microbial electrochemistry must be employed to demonstrate a synthetic electrical connection between a bacterial cell and an electrode and possibly between biofilm cells. We envision engineered living conductive biofilms will result in biomaterials that can capture CO2 for biosynthesis using electrons from an electrode as energy to drive the reaction. We also envision that living conductive biofilms, where electron transfer is possible both to and from an electrode, will result in enhanced system performance, e.g., longevity of mission for sensors, and reduction in maintenance because of the ability of conductive biofilms to self-assemble and self-repair. These properties coupled to the endless opportunities to reprogram cell function mean that engineered living biofilms could far surpass their nonliving counterparts.

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Photoswitchable Bacterial Adhesions for the Control of Multicellular Behavior Fei Chen and Seraphine V. Wegner

Abstract Bacteria in nature usually do not only exist as free-floating cells, but predominantly live in social communities, named biofilms. In these consortia, the bacteria function as a collective, perform functions that single bacteria cannot, and show multicellular behavior. Toward engineering biofilms with desired functions, the control of bacterial adhesions is a key step, in particular for regulating the spatial and temporal organization within the biofilm. In this chapter, we review approaches to control bacteria-surface and bacteria-bacteria adhesions and their consequences for bacterial behavior. Among them, the light-responsive methods provide unique advantages including high spatiotemporal resolution as well as noninvasive, biocompatible, and tunable remote control. This is particularly significant for engineering designed multicellular communities with special functions for applications in the fields of biotechnology. Keywords Bacterial adhesion · Multicellular behavior · Photoswitchable · Bacteriasurface adhesion · Bacteria-bacteria adhesion · Light-responsive · Biofilm

1 Introduction Bacteria used to be regarded as isolated unicellular organism that are non-organized and selfish before the concept of multicellularity in bacteria emerged. In fact, bacteria are capable of forming well-organized communities, also known as biofilms (Wood et al. 2011). With benefit from multicellular cooperation, bacteria can

F. Chen Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, China Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany S. V. Wegner (*) Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 W. V. Srubar III (ed.), Engineered Living Materials, https://doi.org/10.1007/978-3-030-92949-7_5

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perform complex tasks by intercellular communication, labor dividing, and collective behaviors. Intercellular communication is one type of the cell-cell interactions and is the basis of coordinated multicellular function. Complex regulatory networks, including quorum sensing circuitries, govern cell differentiation within the biofilm and coordinate matrix component synthesis in a spatiotemporal way. Moreover, forming biofilms is a survival strategy of bacteria to stabilize the local environment of cells and overcome the stresses in adverse conditions. Many bacterial species have been studied in terms of their ability to form biofilms, and these studies have revealed that the extracellular matrix plays a critical role in the biofilm’s proper construction and maintenance. The mesoscale structure of the communities in which various cells specialize is directly linked to the biofilm’s performance. Extracellular signals, in addition to spatial structure, temporally coordinate the expression of particular genes necessary for growth and propagation. Bacteria adopt a variety of methods to enhance their survival and resource utilization since they are multicellular. These processes evolved in response to changing environmental circumstances, guaranteeing colonization and biofilm development at the appropriate time and location. While many more aspects of bacteria’s multicellular existence remain unknown, it’s thrilling to think that some of these processes may be utilized to battle infections and avoid harmful biofilms. Overall, our understanding of biofilms has improved, but the ability to produce bacterial communities that can perform a desired task is still in its infancy although the potential impact for biotechnology and therapy is obvious. This certainly entails controlling multicellularity and regulating it in space and time.

2 Multicellularity in Bacteria: Biofilm Formation In nature, bacteria are predominantly adhering to surfaces, which implicates that bacterial adhesion to surfaces is the first step toward biofilm formation. In 1978 Bill Costerton pioneered the biofilm theory, where he stated that most bacteria grew in a glycocalyx-enclosed biofilm that adhered to surfaces or to other cells and that these adherent bacterial populations dominated in ecosystems including nature, industry, and medical field (Lappin-Scott et al. 2014). Moreover, he noted that the cells in biofilms are physiologically different from the free-floating planktonic cells. The first step of biofilm formation is the initial attachment of free-floating planktonic bacteria to a surface. This process is usually based on physical forces such as electrostatic interactions, hydrophobic interactions, hydrogen bonds, and van der Waals forces (O’Toole et al. 2000). The initial attachment is reversible because of which many adherent bacteria may actually detach from the surface when disrupted by hydrodynamic forces and repulsive forces or in response to nutrient availability (Dunne Jr. 2002; Banin et al. 2005; Wu and Outten 2009). In order to develop a mature biofilm, bacteria have to remain attachment and keep growing after initial attachment.

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In the second stage, bacteria secret exopolysaccharide, form cell groups, and adhere irreversibly (O’Toole and Kolter 1998; Gerke et al. 1998). This brings two factors into focus: the extracellular polymeric substances (EPS) and bacteria adhesions to one another. EPS are a complex consisting of proteins, polysaccharides, lipids, and extracellular DNA secreted by bacteria. Both the periphery of the biofilm and the interior space between the bacteria aggregates are filled by EPS (Sheng et al. 2010). In an analogy if the biofilm is called a “city of microbes” (Watnick and Kolter 2000), EPS would be the “house of biofilm cells” (Flemming et al. 2007), providing structural stability, mechanical strength, cohesion and adhesion, and other biological functions as a highly hydrated gel matrix (Sheng et al. 2010; Flemming et al. 2007; Flemming and Wingender 2010). EPS are considered as the primary matrix material of biofilms and the key determinant for the material properties of biofilms. The interactions among bacteria induce the formation of bacterial aggregates and microcolonies, which has been suggested as one of the mechanisms for the irreversible and permanent attachment at a surface. In the maturation stages, biofilms develop into three-dimensional (3D) structures which are supported by EPS, enabling nutrient transport and waste disposal (Huang et al. 2011). Finally, bacteria within biofilms alter their physiological processes and gene expression according to the environment changes via intercellular communication such as quorum sensing (Stoodley et al. 2002). In biofilms, bacteria interact as a coordinated functional community in order to share nutrients and resist environmental stress such as antibiotics (Hoiby et al. 2010). Biofilms may develop on a variety of surfaces, such as living tissues, medical implants, and industrial water systems. As a result, engineered bacterial communities have a lot of potential for future industrial, medicinal, and environmental applications, such as bacterial factories for the production of complex compounds, biocatalysis, waste treatment, and drug delivery. Controlling bacterial adhesions can be used to design bacterial multicellular communities to accomplish desired activities by forming biofilms with a purposeful spatiotemporal layout.

3 Controlling Bacterial Adhesions with Light Many stimuli-responsive techniques for controlling bacterial adhesion and patterning have been presented. The light-responsive techniques provide the highest spatial and temporal resolution, which is necessary to build stable/viable biofilms with high accuracy. This is particularly important since the microscale organization of bacteria determines the degree of their interaction in a biofilm, as mentioned above. Additionally, light is a noninvasive trigger as it can be applied remotely without disturbing of other processes. In contrast, invasive triggers, such as chemicals, can have unpredictable side effects in the system by interacting with not only the targets but other biomolecules and do not provide the desired high spatiotemporal control. Moreover, it is easy to adjust the light intensity, illumination time, and wavelengths to tune interactions and address different functionalities which respond to different

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colors of light independently. Therefore, the unique advantages of light make it particularly attractive as an external stimulus to regulate diverse biological processes. Unless the organisms are photoresponsive themselves, there are two main approaches to photoregulate systems: either by chemical modification with lightresponsive small molecules or by optogenetic approaches that rely on engineered light-responsive proteins as detailed below.

3.1

Bacteria-Surface Adhesion

Bacteria-surface adhesion initiates the biofilm formation. Therefore, controlling the bacteria-surface adhesion is the most effective method to avoid the formation of biofilm and guide its future development. Moreover, controllable bacterial adhesion allows bacterial cell patterning and engineered biofilm formation with the designed spatial organization, which are important for understanding social cell interactions and chemical exchange within biofilms.

3.1.1

Controlling Bacterial Adhesions Through the Surface Chemistry

The bacteria-surface adhesion is determined by multiple factors such as surface structures of bacteria, the substrate physiochemical properties, and environmental conditions. The most important factors influencing bacteria adhesion include surface charge, hydrophobicity, topography or roughness, and the exposed functional groups (Katsikogianni and Missirlis 2004; Renner and Weibel 2011; Tuson and Weibel 2013). The modification of surface with negative or positive charges is a widely used approach to control the adhesion of bacteria. Since most bacteria have a negatively charged cell surface, adhesion of bacteria is prevented on surfaces with negative charges, while it is promoted on surfaces with positive charges (Yuan et al. 2017). Positively charged polymer surfaces have been reported to be bactericidal because the positive charge can disrupt the bacterial membrane potential or damage the membrane structure (Grace et al. 2016; Murata et al. 2007). Therefore, polycationic surfaces are often suggested to be efficient antibacterial coatings that bind and kill bacteria (Harkes et al. 1991). Surface hydrophobicity is another major parameter affecting bacterial adhesion to surfaces (Halan et al. 2012). Additionally, bacterial adhesion is also influenced by the surface hydrophobicity of bacterial cells. For example, bacteria with a more hydrophobic surface prefer to adhere to hydrophobic areas of the surface, while hydrophilic bacteria attach to the hydrophilic areas (Berne et al. 2018; Absolom et al. 1983). Surface with designed topography has also been reported as a strategy to control bacteria-surface interactions. Perera-Costa et al. investigated the effects of surface

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topography on the adhesion of bacteria by using the polydimethylsiloxane (PDMS) surfaces that contained microtopographic patterns in spatial organization (Chung et al. 2007). Compared to the control, bacterial adhesion was significantly reduced as much as 30–45% on microstructured surfaces. Surface modification with functional groups presents an important approach to control the adhesion of bacteria and biofilm formation. For example, using poly (ethylene glycol) (PEG) as anti-adhesive coatings for surface modification has been widely applied cause it shows a great capacity to resist protein adsorption and cell adhesion (Senaratne et al. 2005). All the methods mentioned above have been widely used for the development of novel antibacterial and antibiofouling strategies. However, adhesive surfaces with bio-specific binding properties and minimized background interferences are required for the fundamental study of bacterial multicellular behaviors such as cell-cell communication, metabolic interactions, and biofilm formation.

3.1.2

Controlling Bacterial Adhesion by Modifying the Surface with Native Adhesion Molecules

To construct an adhesive surface for bacteria with the ability to form a robust, specific, irreversible adhesions, native adhesive molecules have been used as exposed functional groups on surfaces. During the initial attachment step of biofilm formation, bacteria employ specific cell surface receptors, called adhesins, that bind to the substrate through specific receptor ligands for the irreversible attachment (Berne et al. 2015; Busscher and Weerkamp 1987). Bacterial adhesins serve as anchors and act as specific surface recognition molecules, allowing the binding to specific receptor molecules on host cells or target surfaces (Van Houdt and Michiels 2005). Among these adhesins, the best characterized bacterial adhesin is the FimH (Krogfelt et al. 1990). FimH is an α-D-mannoside-specific lectin located at the tips of adhesive organelles, called type 1 fimbriae (Lillington et al. 2014). FimH lectin specifically binds to mannose residues presented in glycoproteins (Bouckaert et al. 2006). This specific adhesin-carbohydrate adhesion of bacteria to mannosylated surfaces has been widely used for medical applications. For example, carbohydrate microarrays have been used for the detection of pathogens and screening of antiadhesion therapeutic agents based on the carbohydrate-binding specificities of bacteria (Pieters 2007; Disney and Seeberger 2004; Hsu and Mahal 2006; Hsu et al. 2006).

3.1.3

Controlling Bacteria-Surface Adhesion by Engineering Bacteria Surface with Adhesion Molecules

An alternative powerful approach to control bacterial adhesion is to engineer the bacterial surface with new adhesion molecules. The ability to modify the surfaces of

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bacteria cells with non-native molecules is vital to engineer bacterial communication, biofilm formation, and cell behavior in synthetic biology. One effective strategy to add molecules to the bacterial cell membrane is called bacterial display, which uses genetic methods to fuse a protein or a peptide with a transmembrane protein. Sankaran et al. reported a novel method to control the specific, dynamic, and reversible bacterial adhesion based on a supramolecular interaction between a peptide displayed on the bacterial surface and cucurbit[8]uril (CB[8]). They genetically modified E. coli by displaying a CB[8]-binding motif at the bacterial surface. The binding of this motif to CB[8] and formation of intercellular complexes induce the bacterial aggregation within the solution in the presence of CB[8] and specific adhesion to CB[8] modified surfaces. Furthermore, these adhesions can be chemically reversed using an excess of CB[8] as a competitor (Sankaran et al. 2015a). Another strategy to modify the bacterial cell surface with adhesion molecules is based on chemical ligation to membrane proteins or carbohydrates. For example, Elahipanah et al. introduced bio-orthogonal groups to engineer the surface of gramnegative bacteria cells by using a liposome fusion-based method. Subsequently, adhesion molecules can be conjugated to these groups for further studies on bacterial adhesion and controlling bacterial behavior (Elahipanah et al. 2016).

3.1.4

Controlling Bacteria-Surface Adhesion by Modifying Surfaces with Light-Responsive Small Molecules

Light-responsive molecules such as azobenzene have been reported as tools to control bacterial adhesion. Azobenzene linkers are responsive to UV light and undergo reversible trans to cis isomerization upon illumination. Weber et al. showed that bacterial adhesion can be reversibly and photochemically controlled by functionalizing the surfaces with azobenzene linked α-D-mannoside (Fig. 1). In this study, a gold surface was functionalized with α-D-mannoside groups through azobenzene linkers (Weber et al. 2014). The orientation of attached mannose can be altered by the photoisomerization of the azobenzene moiety under UV light and mediated the adhesion through the recognition by the receptor FimH on the bacterial surfaces. The bacterial adhesion was blocked upon UV light illumination (365 nm) and could be reestablished upon blue light illumination (Weber et al. 2014; Sankaran et al. 2015b; Voskuhl et al. 2014). Another widely used light-responsive molecules in surface coating are molecules containing nitrobenzyl groups, which are cleavable in response to UV light. To control bacteria-surface interactions, α-D-mannoside was conjugated to a nitrobenzyl linker and then used to modify a non-adhesive surface which was firstly coated with poly(ethylene glycol) (PEG) (Chen et al. 2019). E. coli could adhere to these α-D-mannoside surfaces through their FimH receptors. The α-D-mannoside was selectively eliminated, and the light-exposed regions became non-adhesive when UV light was projected onto these surfaces through a designed photomask. Therefore, only the unexposed areas were attached by bacteria, and a bacterial

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Fig. 1 Photoswitchable adhesion of E. coli cells to the surface immobilized with α-D-mannoside ligands via the azobenzene linkers (Weber et al. 2014). Reproduced with permission from Weber et al. (2014). Copyright 2014 WILEY-VCH Verlag GmbH & Co. KGaA

pattern in a resolution of 10 μm could be easily obtained. This approach can be used for bacterial photopatterning with high spatial resolution without mechanical interference. Furthermore, it provides a method to design biofilms with complex geometries and investigate the influences of bacterial spatial organization on bacterial collective behavior.

3.1.5

Optogenetic Control of Bacteria-Surface Adhesion

Optogenetic methods rely on light-responsive proteins rather than light-responsive small molecules, which can be genetically coded. Photoswitchable proteins have been employed to control bacterial adhesions and pattern bacteria using visible light. In a first example, the Riedel-Kruse group has developed a method with genetically encoded tools for biofilm patterning by photoregulating the expression of the membrane adhesion protein antigen 43 (Ag43) (Jin and Riedel-Kruse 2018). Following the design, these E. coli expressed Ag43 when illuminated with blue light and only formed biofilm in regions of the surface that were illuminated. As a result, biofilm patterns with a high spatial resolution down to 25 μm could be achieved. Huang et al. reported a similar strategy for microprinting living biofilms through optogenetic regulation of the c-di-GMP levels, which regulate biofilm formation in P. aeruginosa (Huang et al. 2018). Remarkably, here two different colors of light were used to increase or decrease c-di-GMP levels. The synthesis of c-di-GMP molecules was activated through the cyclization of the guanosine triphosphate (GTP) molecules upon far-red light illumination, which activated the genes bphO and bphS. On the other hand, c-di-GMP molecules were hydrolyzed upon blue light

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illumination due to the activation of gene blrP1. Overall, the precise control of the cdi-GMP levels in P. aeruginosa through double light illumination can be enabled by combining the far-red and the blue light-responsive modules. Another possibility is to express photoswitchable proteins at the bacterial surface as artificial adhesion receptors. In particular, the protein pMag has been expressed on the surfaces of E. coli such that these could bind to glass surfaces modified with the protein nMag under blue light as pMag and nMag bind to each other under blue light illumination (Chen and Wegner 2017). The binding of pMag and nMag is reversible in the dark, which also allows reversion of the bacterial surface attachment in the dark. The adhesion and detachment could be switched over multiple blue light/dark cycles. Moreover, the blue light intensity as well as different versions of the pMag protein that differ in dark reversion kinetics and binding strength allowed to modulate the bacterial attachments.

3.2

Bacteria-Bacteria Adhesion

Adhesions between bacterial cells to form multicellular clusters are crucial for the development of biofilm structures. Bacteria-bacteria adhesions are also a key factor for regulating spatial organization and heterogeneity within biofilms. This spatial organization has direct consequences for other multicellular behavior such as quorum sensing and metabolic interactions. Quorum sensing is a way of bacteria to communicate through the chemical signals and coordinate their collective behaviors such as growth, movement, and biochemical activities. The unique chemical signals are called autoinducers which are produced by bacteria and secreted out into their environment. Autoinducers increase in concentration according to the cell density, resulting in altering gene expression and inducing multicellular behaviors (Algburi et al. 2017; Nealson et al. 1970). For example, bacteria within biofilms can express gene to resist antibiotics through quorum sensing (Agapakis et al. 2012; Xavier 2011). Quorum sensing is also important for the metabolic interaction regulation in biofilms and influences the structure of the community by encouraging the beneficial species and inhibiting competitors (Berlanga and Guerrero 2016). Manipulation of bacteria-bacteria adhesion has potential applications in the controlling of quorum sensing and engineering multicellular communities. Therefore, multiple strategies have been proposed to control bacteria-bacteria adhesion and associated multicellular behavior.

3.2.1

Controlling Bacteria-Bacteria Adhesion with Native Adhesion Molecules

During the process of biofilm formation, bacteria-bacteria adhesions form under native conditions after the initial attachment to the surface. The antigen 43, a surfacelocated autotransporter protein, is an important determinant for E. coli

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autoaggregation. The interactions between Ag43 α-subunits of adjacent cells in a head-to-tail fashion led to dimer formation and cell aggregation (van der Woude and Henderson 2008). Therefore, bacterial aggregation can be controlled by regulating the Ag43 expression through the gene OxyR or Dam (Ulett et al. 2006). Laganenka et al. showed that autoinducer 2 (AI-2) was an attractant produced by bacteria and induce autoaggregation of E. coli through chemotaxis. Bacteria within autoaggregation showed enhanced resistance to environmental stress and formed biofilm quickly (Laganenka et al. 2016). By altering the gradients of attractant, chemotactic bacterial cells aggregated in various ways and displayed collective phenomena such as moving bands, geometric patterns, and dense multicellular clusters (Woodward et al. 1995; Budrene and Berg 1991, 1995).

3.2.2

Controlling Bacteria-Bacteria Adhesion with External Molecules

Synthetic materials with multivalent interaction sites have been used to induce bacterial aggregation. Examples of these are polysaccharide, polymers, dendrimers, or chemically modified nanoparticles (Bernardi et al. 2013; Wei et al. 2017; Leire et al. 2016; Gupta et al. 2016; Schmidt et al. 2016; Disney et al. 2004). Cationic polymers are regarded as the beginning of the antimicrobial agents that induce bacteria to aggregate. These polymers are positively charged so as to efficiently bind the bacteria which are usually negatively charged and result in the aggregation of these bacteria. Moreover, cationic materials can damage bacterial membranes due to the electrostatic interactions and therefore can be used as antibacterial agents (Leire et al. 2016; Mintzer et al. 2012). Bacteria within aggregates autoinduce quickly and accomplish quorum sensing at bacterial concentrations considerably lower than those necessary for autoinduction without polymers (Xue et al. 2011; Lui et al. 2013; Perez-Soto et al. 2018). For example, Lui et al. reported a cationic polymer poly(N-[3-(dimethylamino)propyl] methacrylamide) induced bacterial aggregation through such electrostatic interactions (Lui et al. 2013). Furthermore, Vibrio harveyi showed enhanced bioluminescence in response to polymermediated clustering, indicating the quorum sensing was activated earlier upon clustering. Glycopolymers carrying carbohydrate functional groups are a second group of polymers that have been widely employed to control bacterial aggregation (Almant et al. 2011). These polymers bind to bacterial lectins on the surface and thereby induce the bacterial aggregation. Pasparakis et al. described a reversible control of bacterial aggregation with thermoresponsive glycopolymers (Pasparakis et al. 2007). In this study, multiple glucose moieties in the polymer were hidden above 40  C and revealed below this temperature. This thermal switchable process enabled the controllable bacterial aggregation based on the interaction of glucose and lectin.

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Controlling Bacteria-Bacteria Adhesion Through Genetic Engineering

Through genetic engineering, bacteria can display designed molecules on the outer membrane and induce bacterial adhesion by the molecules’ interactions. In a remarkable example, Glass et al. reported a synthetic toolbox that enables controlled multicellular self-assembly with high specificity and tunability (Hoiby et al. 2010). For this purpose, nanobodies (Nb) and their corresponding antigens (Ag) were fused to the N terminal of intimin, an autotransporter and surface display system, and displayed on the bacterial surface. Anhydrotetracycline (ATc) or arabinose (Ara) was added as inducers to regulate the expression of the fusion protein. Bacteria expressing Nb and corresponding Ag could aggregate into multicellular clusters with defined patterns due to the specific interaction of Nb-Ag (Fig. 2). Furthermore, this toolbox enabled the rationally design of diverse and complex multicellular patterns.

3.2.4

Controlling Bacteria-Bacteria Adhesion with Photoswitchable Proteins

The optogenetic toolbox provides multiple photoresponsive proteins in a range from UV to red light. Bacterial display is a technique to express a defined protein on the outer surface of bacteria by fusing it to the N terminal of the eCPX, a circular transmembrane protein. To develop light-controllable bacterial cell-cell adhesions with high spatiotemporal resolutions, optogenetic proteins were selected and expressed on the bacterial surface by bacteria display. Blue light switchable proteins, nMag and pMag, were incorporated for the engineering of photoresponsive bacterial strains, providing a noninvasive method for spatiotemporal control of bacterial multicellular behaviors with high tunability (Fig. 3) (Chen and Wegner 2017, 2020). In particular, bacteria displaying nMagHigh and corresponding pMagHigh

Fig. 2 (a) Bacteria-bacteria aggregation based on the interactions of nanobody and antigen. (b) The morphology and patterning of bacterial clusters altered by the ratio of green and red cells (Glass and Riedel-Kruse 2018). Reproduced with permission from Glass and Riedel-Kruse (2018). Copyright 2018 Elsevier

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Fig. 3 Photoswitchable bacterial-bacteria adhesions enable the control of bacteria-bacteria interactions (Chen and Wegner 2020). Reproduced with permission from Chen and Wegner (2020). Copyright 2020 American Chemical Society

would aggregate into multicellular clusters in response to blue light and reversibly disassembled in the dark. Additionally, bacterial collective behaviors, such as quorum sensing, clustering, biofilm formation, and metabolic cross-feeding, could also be regulated through the controlling of bacteria-bacteria adhesion with light. Furthermore, the ability to control and optimize cross-feeding in co-cultures enables the creation of bacterial factories with higher efficiency and better performance of more complicated transformations.

4 Engineering Living Material with Bacteria Biofilms, shells, and skeletal tissue are examples of natural multicellular structure with unique properties that would be useful for material generation and pattern formation (Dunlop and Fratzl 2010; Eder et al. 2018). They can respond to external signals by remodeling, growing, and self-healing when damaged, incorporating inorganic elements to form hybrid biobiotic materials, and archiving patterns at various scales. These systems inspire the design of composite systems with environmental responsiveness, and the inclusion of living materials could offer solutions

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(Brenner et al. 2007). However, the size and shape of living materials cannot be easily controlled and made into useful stuffs for further construction. Therefore, Engineered Living Materials (ELMs) emerge and become a relatively new research field (Nguyen et al. 2018; Huang et al. 2019). ELMs are those materials embedded with cells that contribute to the functionalization. Advanced functionalities such as chemical synthesis, sense-and-respond, growth, and self-repair can be imparted to materials by living cells. This is a new type of material that takes advantage of the characteristics of living creatures. In the subject of ELMs, scientists are attempting to create living biomaterials that combine the structural qualities of standard construction materials with the characteristics of living systems, such as the capacity to quickly grow in situ, self-repair, and adapt to their surroundings. For example, An Xin et al. have reported a method to combine living bacteria and 3D-printed materials for the creation of mineralized composites with ordered microstructures (Xin et al. 2021). They have successfully manufactured bionic composites by using bacteria-assisted mineralization within polymer scaffolds of 3D printing. Synthetic-living hybrid bionic materials can be developed by controlling the interactions of living organisms and printed materials. Its bionic method can produce mineralized compounds with high mineral content and well-ordered mineral orientations that are superior to other existing approaches. It demonstrates how to use living bacteria to create self-growing materials and opens the door to a new class of engineering materials that can self-grow in the same way as living animals can. This technique may easily be developed by regulating the activity of live organisms selectively in order to generate unique living materials with specified microstructures. The development of synthetic biology has contributed to the creation of gene circuits with sophisticated topology and environmental sensitivity, which make it possible for the engineering of novel cellular functions (Gilbert and Ellis 2019). The combination of toolboxes from synthetic biology and materials science has lately resulted in a broad range of living materials that recapitulate some of the distinguishing biology properties. Allen Y. Chen et al. reported a method to engineer E. coli biofilms with synthetic biology tools for the dynamical organization of bioticabiotic composite materials (Chen et al. 2014). To regulate the production of curli amyloid, genetic circuits with inducible expression and circuits for cellular communication were incorporated into E. coli. On this basis, E. coli cells can produce amyloid-based materials that are externally controllable or, through the organization of amyloid fibrils on various length scales, undergo autonomous pattern formation. Nanomaterials such as gold nanoparticles (AuNP) and quantum dots (QD) have been connected with Curli fibrils to produce biofilm-based materials, including gold nanowires, environmentally friendly electrical switches, and new QD materials with lifetime modulated fluorescence. Their research sets the groundwork for using modified cells to synthesize, pattern, and regulate useful composite materials. Based on the light controllable bacterial adhesion, biomimetic mineralization with diverse hierarchically organized structures that are similar to or better than their natural counterparts has been produced. Developing cell-controlled methods to create living materials with dynamic patterns or even composite with graded

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functions represents a promising research direction. Drawing inspiration from functionally classified compounds found in nature, Yanyi Wang et al. developed lightinducible E. coli biofilms in conjunction with biomimetic mineralization to create living composites with fine spatial structure and gradual gradient control of mineral density (Wang et al. 2021). To construct a light-inducible biofilm-forming strain, the plasmid of lightreceiver-CsgA-Mfp3S-pep was designed. The fusion sequences encoding the CsgA protein and Mfp3S-pep were placed downstream of the lightsensitive transcription control element pDawn so that the expression of the fusion protein was strictly regulated by illumination with blue light. Previous studies suggested that CsgA domain was a major component of the E. coli biofilm and Mfp3S peptide could initiate HA mineralization. Upon light illumination, CsgA– Mfp3S-pep fusion protein was expressed and thereby promoted the HA mineralization with faster mineralization rate and denser mineral formation. They demonstrate that structural biofilm proteins in E. coli fused with mussel foot protein (Mfp) analogs allow HA mineralization and interfacial binding of engineered biofilms. When compared to traditional non-living composites, the resulting composites essentially harness the environmental reactivity of living materials. Even after mineralization, the cells within the composites remained alive and could respond to environmental cues. Furthermore, they found that the biomass density of the biofilms determines the mineral density, as well as the ultimate mechanical properties of the compounds. This research provides information on the likely methods of production of functionally graded compounds in nature and paves the way for the development of living functional organic-inorganic compounds with adaptive, selfhealing, and other previously unattainable material properties. By including genetic circuits with multiple inputs that respond to multiple forms of light or other inputs (chemical inducers, fluctuations in pH, temperature, etc.), the ability of the system to provide a more complex environmental response could be harnessed. Christopher A. Voigt et al. presented an approach to pattern E. coli with light on different materials by controlling the expression of curli fibers, which anchor the formation of a biofilm (Moser et al. 2019). Light of various colors is utilized to produce variations of the structural protein CsgA coupled to various peptide tags. This method can be used to shape the formation of composite materials, such as protein layers and gold nanoparticles, by projecting color images onto the substance containing bacteria. It’s used to design cells onto 3D printing materials, polymers (polystyrene), and textiles (cotton). Furthermore, the living materials respond to sensory information such as tiny chemicals (IPTG and DAPG) and light from lightemitting diodes. This work improves the ability to design attractive life materials in which cells perform a variety of functions.

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5 Living Therapeutic Systems with Bacteria Engineered live therapeutic systems are often considered the next generation of drugs for a variety of reasons including the prevention and treatment of infections, the suppression of tumors, and the treatment of metabolic diseases (Charbonneau et al. 2020; Ozdemir et al. 2018). Unlike standard systemic therapy, living therapies function as factories within the body, self-replicating, manufacturing, and pumping forth medicines (Heavey et al. 2021; Pedrolli et al. 2019). Programmable attenuated bacterial strains express foreign genes or produce in vitro and in vivo products such as enzymes, proteins, and immunotoxins that have been identified and used as cancer therapies in vivo (Riglar and Silver 2018; Zhou et al. 2018). Despite the fact that bacteria have long been used as therapeutic agents, problems such as pathogenicity and insufficient release of therapeutic drugs from intracellular bacteria have not yet been solved (O’Toole et al. 2017). Microorganisms are taught to execute useful and intelligent activities using synthetic biology approaches. These designed biological entities are subsequently integrated into properly created polymeric matrices, yielding composite materials with extremely diverse functions, a broad range of tunability, and in situ controllability. Light has proven to be an advantageous stimulus because it allows noninvasive, spatiotemporal resolution, adjustable control over the variation of exposure dose, and multiplexing at different wavelengths (Rapp and DeForest 2021). To replicate the molecular interactions that occur inside live tissues, an ideal biomaterial for use in medical applications would be able to transmit and respond to signals from attached or encapsulated cells. Recent developments in this field include the timed release of sequestered growth factors, as well as in situ changes in mechanical properties in response to an external stimulus, which may then influence cellular activity in real-time. Bin Liu et al. developed a living therapeutic system based on engineered Salmonella bacteria labeled with a photosensitizer MA and transduced with VEGFR2 plasmid (Liu et al. 2021). The metabolically incorporated MA can generate ROS under light irradiation. The strain of Salmonella bacteria used in this work is VNP20009, which is genetically modified as an auxotrophic mutant for adenine. This strain can obtain adenine from the tumor sites and exhibit proliferation when located there, resulting in tumor-specific targeting. Thus, engineered bacteria can colonize and express exogenous genes in tumor tissues. Singlet oxygen generated by MA upon light illumination damage the bacterial membrane and thereby release VEGFR2 plasmid into the host cells. The expression of VEGFR2 protein could break immunological tolerance and activate autoimmune antiangiogenic responses, resulting in the inhibition of tumor growth. Living therapeutic systems encapsulated with bacteria for drug-producing or drug delivery have been widely reported. The potential for continuous delivery of bioactive chemicals while decreasing design complexity is highlighted by “living” hydrogels with embedded microorganisms. Sankaran Shrikrishnan et al. created a proof-of-concept design for a light-regulated “living biomaterial” (Sankaran et al. 2018). Bacterial behavior can be precisely regulated by light in their design,

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allowing for real-time modulation of cell-material-bacteria interactions. E. coli was genetically engineered to express a cellular adhesion protein (RGD) at its surface in response to pharmacological stimulation (IPTG), allowing the bacteria to connect with mammalian cells. They next created a photo-activatable variant of IPTG (PA-IPTG), which would only stimulate protein synthesis in response to light stimulation. They next planted mouse embryonic fibroblasts (MEFs) onto the light-regulated living biomaterial and investigated how light exposure affected MEF behavior. They discovered that in the absence of light, the MEFs did not interact with the bacteria and did not stretch out much across the material. The surface adhesion protein RGD sequence was produced and identified by MEF receptors during light exposure, allowing the development of focal adhesions. In their following studies, they built on this technique by integrating optogenetic technologies (Sankaran and del Campo 2019; Sankaran et al. 2019). When bacteria are engineered to generate and release a red fluorescent protein in response to blue light irradiation and then embedded in agarose hydrogels, living materials capable of releasing proteins into the surrounding media when exposed to light are created (Sankaran and del Campo 2019). They also made a living hydrogel using an endotoxin-free active strain of E. coli that was metabolically and optogenetically engineered to produce deoxyviolacein, an antibacterial and antitumor drug, in a light-regulated manner (Sankaran et al. 2019). The porous hydrogel matrix maintains a live and functional bacterial population while also allowing for the diffusion and dispersion of the manufactured medicine in levels regulated by light dose into the surrounding medium.

6 Outlook Controlling the adhesion of bacteria in high spatiotemporal resolution is critical for controlling biofilm development, organization, and microstructure. Various ways for controlling bacterial adhesions, particularly utilizing light, and their effects on bacterial multicellular behavior have been discussed above. These new techniques enable the engineering of multicellular communities, the understanding of fundamental bacterial behavior in biofilms, and the creation of biofilms with new functionalities for biotechnological applications. The development of single-celled life forms to multicellular life forms has inspired the design of composite systems with living properties and resulted in an explosion of studies in the fields of engineering living materials and living therapeutic systems. Understanding and regulating multicellularity in the realm of synthetic biology allows for more than simply bacterial community sophistication. Acknowledgments The authors thank the Deutsche Forschungsgemeinschaft (DFG WE 5745/21) for funding.

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Xin A, Su Y, Feng S, Yan M, Yu K, Feng Z, Hoon Lee K, Sun L, Wang Q (2021) Growing living composites with ordered microstructures and exceptional mechanical properties. Adv Mater 33(13):2006946 Xue X, Pasparakis G, Halliday N, Winzer K, Howdle SM, Cramphorn CJ, Cameron NR, Gardner PM, Davis BG, Fernandez-Trillo F, Alexander C (2011) Synthetic polymers for simultaneous bacterial sequestration and quorum sense interference. Angew Chem Int Edit 50(42):9852 Yuan Y, Hays MP, Hardwidge PR, Kim J (2017) Surface characteristics influencing bacterial adhesion to polymeric substrates. RSC Adv 7(23):14254 Zhou S, Gravekamp C, Bermudes D, Liu K (2018) Tumour-targeting bacteria engineered to fight cancer. Nat Rev Cancer 18(12):727

Additive Manufacturing of Engineered Living Materials Lynn M. Sidor and Anne S. Meyer

Abstract Recently, traditional additive manufacturing techniques have been modified and enhanced in order to facilitate the printing of engineered living materials. This chapter discusses these recent advances in bioprinting, including the developing techniques of thermal inkjet bioprinting, direct-write bioprinting, laser direct-write bioprinting, fused deposition modeling, and multiphoton lithography. For every technique described in this chapter, we will survey how an additive manufacturing method has been modified to work with living materials, the types of cells each method was redesigned for, their applications, future directions, and the remaining problems to solve for this expanding field. The creation of spatially patterned, highresolution, free-standing, and faithfully reproducible bioprinting methods will further advancements in tissue engineering, biomedical engineering, material sciences, and more. The applications for these three-dimensionally printed engineered living materials are innumerous. Keywords Additive manufacturing · 3D printing · Bioprinting · Bio-ink · Tissue engineering · Biomaterials · Computer-aided design (CAD) · Computer-aided manufacturing (CAM) · Direct-write · Laser printing · Fused deposition modeling · Multiphoton lithography

1 Overview Traditionally, additive manufacturing has been defined as a process that utilizes information from a computer-aided design file to create a three-dimensional object, printing the materials in a layer-by-layer approach to build a three-dimensional product. The field of additive manufacturing is composed of numerous manufacturing techniques, most of which are non-biological and are typically what comes to mind when discussing additive manufacturing. The emergence of biological additive

L. M. Sidor · A. S. Meyer (*) Department of Biology, University of Rochester, Rochester, NY, USA e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 W. V. Srubar III (ed.), Engineered Living Materials, https://doi.org/10.1007/978-3-030-92949-7_6

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manufacturing techniques, or bioprinting, is the topic of this chapter. We will cover why it is interesting to create additively manufactured living materials, what techniques are used to make them, what we can make now, and what we might be able to make in the future.

2 What Is Additive Manufacturing? Additive manufacturing, or AM, can be broken down into three main types, powder, solid, and liquid, and then into subcategories under each of these. Non-biological AM methods customarily use plastics or metals to build their three-dimensional (3D) parts. First, the information in a computer-aided design (CAD) file is conventionally translated into a stereolithography (STL) file, which essentially converts the geometric CAD data into triangular approximations and slices the file into the layers needed to render the object (Wong and Hernandez 2012). How the process continues after this depends on the AM technique that is being used to 3D print the object. Table 1 outlines some of the various techniques used for the non-biological production of a 3D printed object. Overall, these methods for additive manufacturing, as a rule, use conditions that are not conducive to biological manufacturing: the use of ultraviolet lasers and lamps, the high temperatures and pressures of narrow printheads, harsh chemicals, working within a vacuum, and more. How can these techniques be modified in order to bioprint living materials?

3 Why Create Spatially Patterned Engineered Living Materials? The need to develop methods for biological manufacturing, or bioprinting, living materials in an environmentally friendly and sustainable way is only increasing, and the market to produce spatially patterned materials is expanding with it. Traditional additive manufacturing processes, like the examples described in Table 1, employ the use of massive quantities of harsh and polluting chemicals. In order to print living materials, bioprinting needs to overcome a number of challenges, specifically, transporting the liquid biological ink, or bio-ink, through the printhead at moderate temperatures and pressure; the ability to layer the living material without collapsing; avoiding nutrient depletion of the living materials throughout the layers; and production of the living material in a sustainable way without the use of harsh chemicals. Researchers have been working to develop engineered living materials that are able to combine the structural components and innate ability of cells with genetic engineering to add new functionalities. In order to imbue these living materials with three-dimensional spatial patterning, they will require some kind of structural scaffold on which the cells can organize and grow. The ability for

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Table 1 Non-biological additive manufacturing and 3D-printing Technique Stereolithography (SL or SLA) (Wong and Hernandez 2012)

PolyJet (Wong and Hernandez 2012)

Fused deposition modeling (FDM) (Wong and Hernandez 2012)

Selective laser sintering (SLS) (Wong and Hernandez 2012)

Components and process CAD files are first translated into STL files and are reduced into slices of information, which will be used to build up the structure. The process requires the curing/solidification of a photo-sensitive polymer (resin) to a platform using an ultraviolet laser An inkjet head moves in the x and y directions to deposit a photopolymer which is cured by ultraviolet lamps upon the completion of each layer. A gel-type polymer is used to support overhanging features and is removed after the process is finished via water jetting Thin plastic materials are fed into and melted by a printhead to a thickness of ~0.25 mm Plastics used: polycarbonate (PC), acrylonitrile butadiene styrene (ABS), polyphenylsulfone (PPSF), PC-ABS blends, medical grade PC (PC-ISO), and polylactic acid (PLA) Powder is sintered (fused) utilizing a carbon dioxide laser beam to specific locations in each layer of the structure. A powder bed is sequentially lowered via pistons with each finishing layer Possible materials: plastics, metals, metal combinations, metals and polymers, metals and ceramics

Advantages • Reusable materials: when layers are finished, the excess is drained and can be reused • Can use multiple materials to build a structure; however each layer requires draining and refilling with new materials

Disadvantages and common errors • Overcuring: any section of the structure overhanging from the base is not fused with the platform • Layer thickness is variable because of resin’s innate highviscosity • Surface finishing must be done by hand

• High resolution since layers are 16 μm thick • Multicolored parts can be built

• Weaker parts compared to SL or SLS

• No post-process chemicals required • No resins to cure • Less expensive machinery • More cost-effective overall

• Lower resolution across z-axis • If the final surface needs to be completely smooth, a lengthy finishing process will be required

• Wide range of materials can be used to create structures • Unused powder can be recycled

• Accuracy is limited to powder particle size • Oxidation needs to be avoided by using an inert gas atmosphere • Entire process occurs near the melting point of the material

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Table 1 (continued) Technique Electron beam melting (EBM) (Wong and Hernandez 2012)

Laminated/laser engineered net shaping (LENS) (Wong and Hernandez 2012)

Prometal (Wong and Hernandez 2012)

Laminated object manufacturing (LOM) (Wong and Hernandez 2012)

Electron-beam freeform fabrication (EBF) (Taminger and Hafley 2014)

Components and process Similar to SLS, but in a vacuum chamber. An electron laser beam melts metal powder via high voltages (30–60 KV) Within a closed chamber with an argon atmosphere, a highpowered laser beam melts metal powder. The molten metal is injected into a specific location, where it solidifies once cooled For making stainless steel injection tools and dies. In a steel powder bed, jets spurt out liquid binder. Pistons sequentially lower the bed once a layer is completed and feed in materials for each layer. At the end, residual powder must be removed The materials begin as sheets, which are bonded together using pressure, heat, and a thermal adhesive coating. Then, a carbon dioxide laser cuts the material layer to the shape corresponding to the CAD and STL files. This process continues layer by layer For building near-netshape metal parts. A focused electron beam is used in a vacuum to create a molten metallic substrate pool. The electron beam moves across the surface of the substrate to solidify it, while a metal wire is fed into the molten metal pool. This is repeated in a layer-by-layer manner

Advantages • Variety of metals can be used • Potential for use in outer space, since it is all done within a vacuum • Variety of metals can be used in combinations (i.e., with stainless steel, nickel alloys, alumina, etc.) • Can be used to repair parts

Disadvantages and common errors • Oxidation needs to be avoided—uses a vacuum chamber

• Residual stress from uneven heating and cooling, which can be problematic for highprecision processes (i.e., turbine blade repair)

• No post-processing is required for moldbased fabrication of injection tools

• If a functional part is made, it will need to undergo sintering, infiltration, and a finishing process

• Low cost • No post-processing • No supporting structures required • No phase change or deformation of materials • Can be used to build large parts • Can be used for models with paper, composites, or metals • Easily scalable • Can be used to add details to components already fabricated

• The cut material is subtracted and therefore wasted • Low surface definition • Materials determine machinability and mechanical properties • Very difficult to build complex internal cavities • Vacuum chamber size limits size of part • Wire feedstock availability also limits part size

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scientists to print a scaffold at the submillimeter scale will revolutionize the capabilities of bioprinting living materials. The applications for standardized, reproducible, and nontoxic bioprinting strategies are virtually endless.

4 Types Numerous bioprinting methods are currently being developed, examples of which we will cover in this chapter. Bioprinting is typically considered to fall under the “liquid-based” category of additive manufacturing, but it can also be thought of as combining both liquid and solid approaches since many of the methods use liquid bio-inks in combination with solidification processes to hold the living material in place. We have constructed a figure illustrating how certain additive manufacturing techniques have been transformed to be suited for living materials, showing the bioprinting applications in purple (Fig. 1). The remainder of this chapter will explore each of these bioprinting processes in greater detail, and we will take a closer look at specific examples of each type of technique.

5 Thermal Inkjet: Tissue Engineering and Regeneration In today’s medical research climate, issues surrounding tissue and organ failure are a major concern. Tissue and organ failures can be due to a variety of factors: aging, diseases, accidents, birth defects, and more. Currently, the standard approach for organ replacements is organ transplantation, but critical shortages of available organs around the world leave many patients in severe need. It is not surprising then that tissue engineering and regenerative medicine to help overcome this shortage are major focuses for biomedical researchers. Tissue engineering, a branch of biomedical engineering, combines traditional engineering and material science methods with cells in order to create man-made biological tissues that are suitable replacements for the native tissue(s) they hope to replicate. Engineered tissues can also assist medical researchers to better understand the fundamental science of physiological conditions. However, there are limitations to conventional tissue engineering approaches when tissues become too thick and complex. Such limitations include the following: pre-formed scaffolds are not effective for cell seeding and cell penetration on the scaffold; precise control and placement of multiple cell types within engineered tissues have not been solved; and the use of pre-designed 3D patterns does not allow for the fabrication of vascular and microvascular systems simultaneously with the scaffold (Cui et al. 2012). The Lotz laboratory has proposed and shown that thermal inkjet bioprinting can help scientists overcome these limiting factors for more accurate engineered tissues (Cui et al. 2012). This thermal inkjet printing approach combines solid freeform fabrication with precise cell placements in both 2D and 3D.

Fig. 1 Map of the non-biological and biological additive manufacturing and 3D printing processes. This schematic groups the different additive manufacturing methods into three material-based groups: Liquid (blue), Solid (yellow), and Powder (green). Each group is split into the seven main branches of additive manufacturing: Inkjet, Material Extrusion, Light Polymerized, Laminated, Wire, Power Bed, and Power Fed. Branching from these seven categories are the individual methods described in Table 1 and in our chapter. The blue-to-purple gradient boxes represent methods used for both non-biological and biological printing. The purple boxes represent the bioprinting applications

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Fig. 2 Thermal inkjet vs. direct-write bioprinting, (a) Inkjet printing deposits bio-ink as a dot-todot representation of a previously designed pattern. Layers are built from the bottom-up to create the final 3D structure. Often, the layers will be printed within a scaffold, which is to be rinsed away after fabrication. (b) Direct-write bioprinting uses similar printers to inkjet printing, but dispenses bio-ink in a continuous line as opposed to single dots. These lines are printed layer-by-layer to create the 3D-printed final product. Image (a) and (b) adapted with permission from Springer Nature Customer Service Centre GmbH: Springer, Cell Biochemistry and Biophysics. A Review of 3D Printing Techniques and the Future in Biofabrication of Bioprinted Tissue. Satyajit Patra, et al., © (2016)

There are multiple types of inkjet printing: thermal, piezoelectric, and electromagnetic. Inkjet printing utilizes a printing technique in which a digital pattern is printed onto a substrate via tiny ink drops in a non-contacting manner (Mohebi and Evans 2002). Typically, inkjet printers use heat or mechanical compression to extrude ink droplets; thermal inkjet printing uses small air bubbles that, once heated, collapse within the printhead to provide the necessary force to push out ink droplets (Fig. 2a). The size of droplets can vary tremendously for this technique, ranging from 10 to 150 pL, because of temperature gradients, frequency of the current pulse, and the ink’s viscosity (Hock et al. 1996; Canfield et al. 1997; Hudson et al. 2000). While inkjet printing has successfully been used by the biomedical field to print DNA molecules directly onto glass slides (Okamoto et al. 2000; Goldmann and Gonzalez 2000), there are still challenges to using inkjet printers to print mammalian cells. Mammalian cells, if printed, can undergo various types of damage, for example, cell membrane damage and cell lysis have been documented after sonication at 15–25 kHz during piezoelectric inkjet printing (Seetharam and Sharma 1991). Thermal inkjet printing has been shown to be a superior approach for mammalian cell printing because, despite the fact that the nozzles can reach 300
C, the mammalian cells are only exposed to heat for 2 μs and thereby reach a temperature 4–10
C above ambient temperatures, resulting in 90% cell viability on average (Cui et al. 2010). Additionally, thermal inkjet printers are easier to access, maintain, and modify than piezoelectric inkjet printers, making them a prime candidate for allowing wide adoption of tissue engineering and regenerative medicine applications (Cui et al. 2012). Current approaches to bioprinting tissues face challenges to accomplish singlecell manipulation, which is important for attaining higher degrees of cell

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organization, i.e., for specific anatomic structures like neurons (Mironov et al. 2009). Additionally, some printing methods require the use of complex equipment that requires long periods of time for printing but still results in a low throughput of cells deposited (Odde and Renn 1999, 2000). These approaches also do not necessarily use biocompatible methods. For example, laser printing vaporizes biological sample solutions and then extrudes the remaining substances, thus drying out the samples, not to mention being a high-cost system. Thermal inkjet instead uses a water-based bio-ink that will minimally clog the printhead, allowing for the control of the cell concentration by manipulation of the bio-ink while also keeping the cells in an aqueous environment. Thermal inkjet printing, despite being a suitable way to print living systems, also faces some of the same concerns of cell death and/or damage from printing. The printing nozzles on thermal inkjet printers can have small diameters in order to lead to higher printing resolution, but the small diameter of the nozzle puts mechanical stress on the cells, and when coupled with the extrusion heat, it is possible that the printing process can damage the cell membranes and alter the phenotype of the cells (Tirella et al. 2011). More thorough research still needs to be conducted to evaluate whether the thermal inkjet printing process affects the physiology of the printed cells, including cell viability, apoptotic factors and apoptosis, heat shock protein expression, cell membrane pore sizes, and cellular repair pathway processes.

5.1

Applications

Thermal inkjet printing has many potential applications within the fields of tissue engineering and regeneration. Currently, this method has been shown to be applicable in direct human cartilage repair, neocartilage formation, human microvasculature fabrication, and more. Cartilage repair is a critical area of research, as most common approaches for cartilage repair are highly invasive and complex, do not result in long-lasting or healthy cartilage (Rasanen et al. 2007), involve removing healthy cartilage and placing it at the defect sites (Kalson et al. 2010), could result in even more cartilage and tissue necrosis and degeneration (Shapiro et al. 1993), and do not produce cartilage with the organization, extracellular matrix composition, and mechanical properties of native cartilage (Hunziker 2002). Direct cartilage repair instead offers scientists the ability to print cartilage that mimics native cartilage more closely without causing further cartilage degeneration. This method is also modular in that it is adaptable to the size and thickness of repair needed (Cui et al. 2012). The Lotz laboratory described one successful approach for printing cartilage within a poly(ethylene glycol) hydrogel. This process uses a thermal inkjet printer that was modified to deposit human articular chondrocytes and poly(ethylene) glycol dimethacrylate in a layer-by-layer manner into a cartilage defect within an osteochondral plug, referred to as 3D biopaper, thus simulating cartilage repair. This method was shown to be quicker than manual zonal cartilage fabrication, which

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requires the use of UV exposure for a minimum of 11 min, and resulted in 80% less UV light exposure for the chondrocytes. This hydrogel was also shown to be intact and bound to native tissue after sectioning, and the chondrocytes displayed an even distribution throughout the hydrogel. Additionally, this printed cartilage was reported to have bonded securely to surrounding tissues. The addition of growth factors, like FGF-2 and TGF-β1, to the growth media was shown to induce cell proliferation and increase chondrogenic extracellular matrix accumulation, thus reducing the delay in neocartilage formation. This work demonstrated that direct cartilage repair via inkjet printing was able to maintain precise control over cell placement upon deposition, increased cell viability, and showed proper integration with host tissues. Thermal inkjet printing has also been applied to printing engineered vasculature systems. The human cardiovascular system is lined internally with endothelial cells, which are able to adapt to their environments via alterations in number and relative positioning. Tissues require blood supply for proper function, and this supply is dependent on the endothelial cells, which are some of the only cells that are capable of forming the capillaries that are spread all throughout the body. Endothelial cells, in conjunction with fibrin, make tissue growth and repair possible. Fibrin also participates in wound healing, and fibrin gels have been used in surgery for sealants and adhesives as well as in tissue engineering. A thermal inkjet printer was modified to print a combination of human microvascular endothelial cells and fibrin in order to synthesize microvasculature. Following a 3-week culturing period, the printed cells had aligned with the fibrin and proliferated to form a lining. Imaging showed that this lining was tubular and maintained its angiogenesis functions, allowing the researchers to conclude that thermal inkjet printing could also be used to form human microvasculature, opening the door to even more tissue engineering applications.

6 Direct-Write: Tissue Engineering and Organ Printing Direct-write additive manufacturing is also commonly called direct ink writing or robocasting. Direct-write AM is an extrusion-based method in which liquid ink is dispensed through small nozzles. The ink extrusion rate is controlled digitally to build a 3D structure layer-by-layer. Direct-write is related to both inkjet technologies and material extrusion technology, like fused-deposition modeling, since the processes can use very similar methodologies and principles (Fig. 2b).

6.1

The BioAssembly Tool

The Williams laboratory has developed a direct-write tool, which is called the BioAssembly Tool, or BAT (Smith et al. 2004). BAT utilizes CAD and computer-

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aided manufacturing (CAM). This tool has been used to construct 3D heterogeneous tissue models and biomaterials in a spatially organized manner. This deposition machine uses a multihead through-nozzle in order to print these biomaterials and their components (i.e., cells and cofactors). This tool is composed of many sub-components: an XY coordinate system-controlled stage with a water jacket temperature control, numerous Z-direction deposition heads all of which have individual controlling cameras and individual ferroelectric temperature controls, a fiber-optic light source used to both light the field on which deposition will occur and to cure photopolymers, and a piezoelectric humidifier. To lay down materials, the BAT is controlled by a software program that allows the users to manipulate parameters such as the 3D geometry of the deposition pathway, speed of deposition, air pressure in the heads, and more. The resolution of the BAT has been shown to exceed 5 μm in the XYZ directions. These researchers have developed versions of the BAT that are also able to meet biohazard level II requirements, where the BAT is surrounded within a positively pressurized and ventilated case. The “ink” used by the BAT consists of a solution of Pluronic F-127 (PF-127) and cells. Pluronics are a class of biomedical polymers called poloxamers. These polymers are amphipathic molecules that have the innate ability to form micelles in aqueous environments, to form hydrogels when they are in concentrated water-based solutions, and to act as surfactants—lowering the surface tension between two liquids. These properties make Pluronics useful in bioprinting applications such as the BAT. The PF-127 solution can be mixed with other components to print a variety of cells and tissues. Human fibroblast cells were successfully mixed with PF-127, printed onto a polystyrene slide, and the cell viability was assessed via trypan blue penetration test, indicating that the cell viability was approximately 60% following extrusion. Additionally, the BAT’s ability to print a variety of additional cell types was also evaluated. Bovine aortic endothelial cells (BAECs) were mixed with collagen I (Col I), which served as the printing matrix instead of PF-127, and printed onto flat sheets of polyethylene terephthalate (PET). The printed lines of the BAECs and Col I appeared to have consistent widths and an even cell distribution. If the bio-ink of BAECs and Col I were added too forcefully or before collagen polymerization occurred fully, then the middle of the printed line failed to jellify and washed away, leaving higher concentrations of cells at the edges. This issue was solved by waiting 30 s before printing a new layer on top of another and by incubating for 2 min before adding the medium. The viability of the printed cells was demonstrated by cell elongation after 24 h of incubation and by counting the number of both elongated and round cells. The cell viability was calculated to be 86% for a 25-gauge printing pen tip. Most dead cells were found around the edges of the printed lines, and the authors concluded that the delay between printing layers gave the constructs time to dry out. Additionally, this BAEC and Col I bio-ink was used to showcase the CAD/CAM abilities of the BAT. They printed a vascular tree structure derived from an angiogram of a pig heart. The vascular-based structure was printed in a layer-bylayer manner using a computer script modeling the structure seen on the angiogram.

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These printed cells were shown to have the same proliferation and pattern persistence of their previous printed cells.

7 Pluronic-Alginate Bio-ink Systems Pluronic bio-inks have been used to print patterned 3D structures, but their cellsupporting properties are limited by a dependence on both temperature and concentration of their sol-gel transitions. These systems undergo rapid degradation upon cooling or immersions. Conversely, alginate systems offer the advantage of excellent structural support in solutions, but the rapid cross-linking of this system prevents complex interlayer adhesion. Instead, a combination of both of these systems has allowed researchers to integrate the desired properties from each in order to create a new bio-ink. The Perriman laboratory has developed such a Pluronic-alginate bio-ink (Armstrong et al. 2016), which has complex phase behaviors and was used in a 3D printing process to engineer bone and cartilage structures by depositing human mesenchymal stem cells (hMSCs). This printing method involved extruding the shear-thinning cell-laden gel onto a heated stage, which causes the rapid solidification of the Pluronic gel, and this structure’s gelation was stabilized through alginate cross-linking using calcium chloride. The Pluronic component also served as a sacrificial template, which was dissolved after alginate cross-linking, and led to the creation of complex micropatterning of the gel. Shear-thinning hydrogels, like those created by this group, have properties such as Herschel-Buckley flow and stress relaxation which make them easier to eject through needles and therefore advantageous to use for 3D bioprinting over other biomaterials (Zandi et al. 2021). Since the bio-ink was a mixture of sodium alginate and Pluronic F127, and then required calcium chloride washes, experiments were needed to test the proper concentrations and develop a method that would work for the conflicting conditions needed for each component. It was found that 13 wt% of PF-127 and 6 wt% alginate produced smooth prints with reliable shape retention, and a wash of 5 mM calcium chloride for 10 min was the most optimal to sustain the 3D structure during longterm culturing (Fig. 3b). This bio-ink was printed using a MendelMax 2.0 3D printer, which was modified such that a syringe pump was added to extrude the bio-ink onto a stage heated to 37
C (Fig. 3a). This elevated temperature drives the gelation of the PF-127, therefore enabling the printing of self-supporting 3D structures. In order to cross-link the alginate and further stabilize the printed structures, calcium chloride washes were performed at room temperature following printing. This temperature facilitated the dissolving of PF-127 and its diffusion out of the printed gel structure. The PF-127 was seen to diffuse completely away from the gel, leaving alginate only. The end result was a macroscopically templated alginate print. Examination of the micro- and nanostructures of the gel via scanning electron microscopy revealed porous architectures within the hydrogel, and larger pores were observed with increasing amounts of PF-127. This trend led them to conclude

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Fig. 3 Pluronic-alginate bio-ink systems. (a) The modified MendelMax 2.0 3D printer, fitted with an extruder head for printing with gel-loaded syringes. (b) A series of hollow square-based prisms, 3D printed using the 13% F127, 6 wt% alginate hybrid gel (scale bar ¼ 5 mm). (c) Post-crosslinking photographs of a full-sized ear and nose, with a height of 0.64 and 1.72 cm, respectively (scale bar ¼ 1 cm). Image a, b, and c adapted from Armstrong et al. (2016) © (2016) Wiley

that the PF-127 enabled the macrostructure formation of the hydrogel. The embedded pores and the overall porosity of the gels are important for tissue engineers; the larger pores are able to improve nutrient transport and provide enough space for extracellular matrices deposition by the printed cells. Additionally, the diffusion of calcium ions into the hydrogel during the washes created channels within the gel that were perpendicular to the gel’s surface, creating an even more highly patterned architecture. After determining the mechanical properties and porosity of the hydrogels, they printed various anatomical structures, including an ear and a nose, that were able to be printed faithfully from their design files. They also demonstrated that the heat from the print bed was able to induce gelation of the bio-ink even at the tip of the nose, which was 17.2 mm at its final height (Fig. 3c). Additionally, the hMSCs were added into the bio-ink to determine whether the addition of the cells had any effect on the printing process or stability of the printed structures. No difference was observed in the extrusion efficiency of the cell-laden bio-ink, nor were any structural defects observed over a period of 5 days, demonstrating that this method results in strong structure fidelity. The viability of the printed hMSC cells was determined using confocal microscopy, showing 83%  6% viable cells after 7 days of culturing. Therefore, they concluded that the addition of the hMSCs did not alter the structural integrity of the gel. Since the cells were embedded in a high density throughout the gel, treatment of the printed bio-structures with growth factors led to successful induction of both chondrogenesis and osteogenesis.

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This Pluronic-alginate direct-write bioprinting has created the capability of fabricating cross-linked biomaterial structures at high resolutions which have long-term structural integrity. The Pluronic component of this gel makes it possible to pattern the hydrogel at macro- and microscopic levels, making the gel more porous. These structured, porous gels provide the possibility for the printing of more complex physiological tissue constructs.

8 Spheroid Organ Printing One of the main goals for tissue engineering is to be able to manufacture functional tissues and organs that mimic the natural tissues and organs found in humans. Tissue engineering focuses on the regeneration, repair, and replacement of damaged organs and tissues. Biocompatibility of these man-made structures is a vitally important concern for tissue engineers. Most current techniques rely on the use of a biodegradable solid scaffold, meaning that engineers design a porous structure out of biodegradable polymers that is used to support and template cell attachment and new tissue growth. Solid scaffold-based approaches, where a hollow mold is printed and then filled with the bio-ink components, must take multiple considerations into account for tissue engineering. First, growth of the cells is directly dependent on their ability to attach to the substrate onto which they are printed. Second, the organ and tissue shape retention are directly linked and dependent on the scaffold design. Third, the substrate on which the cells are printed also dictates cell differentiation, migration, proliferation, and orientation. Fourth, the porosity of the scaffold corresponds to cell seeding, construct viability, and possible vascularization. Lastly, the mechanical properties of the scaffold must be maintained by newly synthesized tissues within the tissue construct after the biodegradation of the solid scaffold (Hutmacher 2001; Ma and Elisseeff 2006). While these solid scaffolds have been used successfully to produce functional tissues, they still have limitations to overcome. Future goals will need to include promoting the vascularization of thicker tissues, the precise placement of multiple tissue types inside the porous 3D scaffold, successful recreation of the organo-specific cell densities of tissues, reducing the rigidity of the solid scaffolds that can limit stem cell differentiation, and improving the biodegradability of the scaffold itself (Khademhosseini et al. 2006; Langer 2007). Organ printing, a layer-by-layer biomanufacturing technique, is a scaffold-free biotechnology. Organ printing offers several advantages over solid scaffold printing: its ability to be automated can help scientists manipulate scale and reproducibility and accomplish mass production; several cell types can be printed in 3D space simultaneously; structures can be created with high cell densities; it can overcome the limitations to creating thick tissues that are present for scaffold-based printing; and it can be performed in situ. A method developed by the Markwald laboratory began by looking at how tissues and organs develop naturally during embryonic development, without the need for scaffolds, albeit embryonic development happens

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at a slower rate than would be required for tissue engineering (Mironov et al. 2009). Organ printing is based on the developmental biology principle that tissues and organs are self-assembling systems, meaning that they can self-organize without the need for external influence and factors. Additionally, organs should not require the need for a solid scaffold or template in order to assemble correctly. Without a solid scaffold, cultured precursor cells form aggregates and selfassemble into multi-cellular spheroids. Cell-cell adhesion is one of the driving forces that shape these spheroids. Spheroids are classified as microtissues, meaning that they share higher cell density comparable to normal tissues and organs (Archilli et al. 2012). In order to create 3D tissues without need of a scaffold, this group utilized three specific varieties of the many different types of tissue spheroids: (1) solid (non-lumenized) vascular tissue spheroids; (2) mono-lumenized vascular spheroids, which are cyst-like spheroids encapsulating one large central lumen; and (3) histotypical microvascularized tissue spheroids. The layer-by-layer printing of these vascular tissue spheroids was performed simultaneously with the deposition of layers of organo-specific tissue spheroids, in order to direct the proper bioassembly of a vascular tree. This combination allows for post-printing tissue fusion and accelerated tissue maturation. Therefore, this method is well-suited to the creation of bioengineered vascular trees capable of perfusion that can be integrated into other 3D tissue and organ constructs, seen in Fig. 4. This approach is a tremendous advancement toward the goal of eventually being able to print viable human organs, which has been limited by researchers’ lack of ability to create built-in and naturallike hierarchically branched vascular and microvascularized structures. These methods and the production of tissue constructs still need to be researched and tested further.

9 Laser Direct-Write: Indirect Cell Patterning Laser forward-transfer techniques offer an alternative to lithographic processes while maintaining the high-resolution patterns (Arnold et al. 2007). Such techniques fall into the direct-write category. These methods utilize a pulsed laser to transfer material from a source film onto a substrate either in close proximity or in direct contact with the film. The sources used are usually laser-transparent substrates which are coated with the material of interest, often called the target. The pulses of the laser propagate through the source substrate and are absorbed by the film, while simultaneously the material is ejected from the source and thrust onto the nearby substrate. Complex patterning in 3D is facilitated by scanning and modulation of the laser beam (Arnold et al. 2007). Laser printing, which traditionally is used to add, remove, or modify a target material without the need for physical contact, has been adapted for bioprinting through multiple methods (Hopp et al. 2004). One such method is laser-induced forward transfer, or LIFT. LIFT uses a single laser pulse to detach and transfer an absorbing thin film from the donor substrate to the nearby target or acceptor

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Fig. 4 Spheroid organ printing of a vascular tree. Bioprinting of segments of an intraorgan branched vascular tree using solid vascular tissue spheroids: (a) kidney intraorgan vascular tree; (b) bioprinted segment of vascular tree; (c) physical model of the bioassembly of tube-like vascular tissue construct using solid tissue spheroids; (d) bioassembled ring-like vascular tissue constructs of tissue spheroids fabricated from human smooth muscle cells. Tissue spheroids are labeled with green and red fluorescent stains in order to demonstrate the absence of cell mixing during the process of tissue fusion; (e–g) sequential steps of the morphological evolution of ring-like vascular tissue construct during the tissue fusion process. Reprinted with permission from Mironov et al. (2009) © (2009), with permission from Elsevier

substrate. LIFT provides the basis for matrix-assisted pulsed laser evaporation direct-write, or MAPLE DW, which has been used to produce mesoscopic electronic devices. In MAPLE DW, the material that is to be transferred is combined with a matrix material that has a high absorption coefficient for the laser that will be used. Following modification for use in bioprinting, MAPLE DW has been applied to successfully deposit multiple biomaterials and biomolecules in order to create microarrays and microfluidic sensors which were able to maintain their native protein structures and integrity and also to bioprint bacterial cells and Chinese hamster ovaries (Wu et al. 2001; Chrisey et al. 2003). Another laser printing method that has been developed is called absorbing filmassisted laser-induced forward transfer, or AFA-LIFT. AFA-LIFT uses similar methods to both LIFT and MAPLE DW, but its approach uses biomaterials which are placed onto an absorbing film-coated holder and are then transferred in a gentle manner such that the biomaterial maintains its structure and viability, in the case that cells are used. AFA-LIFT does not require the transferred material to be embedded into a matrix material, like MAPLE DW does. However, if living cells are used, then some kind of nutrient and supplement medium will be required to sustain the cells. The Chrisey laboratory has demonstrated that AFA-LIFT can be used to transfer

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viable Trichoderma conidia fungal cells, which were deposited onto a thin silver absorbing layer (Fig. 5a) (Hopp et al. 2004). Trichoderma fungal species are able to produce extracellular degrading enzymes, which is directly linked to their role in decomposition of plant litter (Klein and Eveleigh 1998). Trichoderma fungi have also been linked to causing opportunistic infections in immunocompromised humans, though they are primarily soil organisms (Kredics et al. 2003). The Chrisey laboratory used AFA-LIFT to print films of conidia, asexually produced fungal spores, at different fluences (Hopp et al. 2004), which were then incubated for 20 hours. Using optical microscopy, they observed islands of germinated Trichoderma conidia at the same spatial positions of the donor spots. The density of the germ islands was dependent on the fluence, and the highest density of cells within the islands was located at the centers of the irradiated spots on the donor layer, even though some fungi spread out and grew away from the center. This experiment led to the conclusion that AFA-LIFT is a viable bioprinting technique that can be used to transfer organisms and maintain their viability. AFA-LIFT, while not patterning the cells in three dimensions, can also be used to transfer larger groups of freshly isolated cells, including rat Schwann, astroglial, and pig lens epithelial cells; these cells were able to proliferate, differentiate, grow to a mature phenotype, and could be maintained in culture (Hopp et al. 2005). Another LIFT technique, used by the Chichkov laboratory, uses gold as a light absorbing layer, to determine how laser printing affects skin cells and human mesenchymal stem cells (hMSCs) when they are printed into distinct patterns (Koch et al. 2010). Their LIFT method, which is similar to the previously described laser printing methods, starts with two parallel glass slides. The upper slide is the donor slide and is coated with the light absorbing gold layer and a layer of biomaterial to be transferred, and the bottom slide is the collector slide. Next, laser light is pulsed through the upper slide through the gold layer, which becomes locally evaporated. The absorption in the gold layer results in the generation of a high gas pressure which will propel the biomaterial toward the bottom slide. The collector slide is also coated with a thick hydrogel layer to protect the laser-propelled cells from desiccation and to cushion their impact onto the slide. This hydrogel is intended to extend the printed cells’ viability but is not necessary for them to survive the printing process. The cell deposition is controlled by a computerized scanning setup using XY and Z translational stages. These stages are synchronized with the laser pulses using a computer-based real-time system. This setup allows for the printing of patterns with a speed of 1200 printed cell droplets per minute. The cell types that were tested, both skin and hMSC cells, were successfully printed using the LIFT technique. The transfer efficiency was greater than 90%. The survival of the skin cells was shown to be 98%  1% following printing and 90%  10% for the hMSCs. No differences were detected in the proliferation ability, apoptosis rate, or amount of DNA damage of the skin cells and the hMSCs following LIFT. The immunophenotype of the hMSCs was also unaltered by the LIFT printing. This technique is a safe and reliable, computer-controlled system to laser print different cell types, with the potential to become a promising tool for tissue engineers who are working to create viable tissue replacements ex vivo.

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Fig. 5 Laser direct-write. (a) Experimental arrangement for the absorbing layer-assisted laser-induced forward transfer of Trichoderma conidia. (b) Schematic of the BioLP™ apparatus. The BioLP™ apparatus uses a microscopic objective to focus a laser beam onto the interface between a quartz substrate and the laserabsorptive metal or metal oxide layer. The focused beam causes the thermal and/or photomechanical volatilization of a small amount of solution near the interface and results in ejection of a small number of cells. Movement of the support and substrate is achieved with automated X, Y translational stages. Imaging of the transfer is possible via a CCD camera arranged confocally. Image (a) reprinted from Hopp et al. (2004), with the permission of AIP Publishing. Image (b) adapted with permission from Springer Nature Customer Service Centre GmbH: Springer, Applied Physics A. Biological laser printing of three-dimensional cellular structures. J.A. Barron, et al., © (2004)

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Tissue engineering techniques are constantly evolving to be able to rapidly and accurately print viable cells into complex 3D structures, using both cell-by-cell and layer-by-layer approaches. The Ringeisen laboratory has developed a Biological Laser Printing, or BioLP™, technique, which is able to print patterned, viable prokaryotic cells as well as 3D-patterned, viable eukaryotic cells (Fig. 5b) (Barron et al. 2004). The BioLP™ uses laser printing methods very similar to LIFT and MAPLE DW, but differs from them in that it uses a laser-absorbing interlayer that absorbs incident laser energy. This layer, which is usually titanium or titanium oxide approximately 75–85 nm thick, causes all of the interactions between the laser and biomaterials to be indirect. This indirect interaction results in the reduction and possible elimination of damages to the biomaterial which can be caused by ultraviolet radiation. The remainder of the BioLP™ process is similar to the previous laser printing techniques; the transfer of the biomaterial occurs when a focused laser is pointed at the interface of an optically transparent material and the laser-absorptive layer. The biomaterial which is to be transferred is coated opposite the absorptive layer. The interaction between the laser and the material at these interfaces results in photoabsorption of the laser and then heat transfer through the laser-absorption layer. This heating through the laser-absorbing layer then triggers rapid heating of the biomaterial that is in contact with the laser-absorbing layer. This rapid heating results in the vaporization of a small portion of the biomaterial layer, and then the biomaterial is propelled toward the second substrate. Through this process, the laserabsorbing coating should remain attached to the transport support, again resulting in the reduction of contamination. Overall, characteristics of the applied laser energy influence the area of the absorbent layer; the amount of transferred biomaterial is influenced by the laser energy, laser spot size, and the composition and thickness of the biomaterial itself. The BioLP™ has been applied to build cellular constructs in 3D using layer-bylayer methods. Human osteosarcoma cells were deposited in two layers on top of a basement membrane which was spread on a glass substrate. The cells’ viability was tested using live/dead stain and imaging of the cells. The laser printed cells were thereby demonstrated to be 95% viable. The first layer of cells was shown to grow in a 2D manner and adhere to the basement membrane and even through it to the glass substrate, while the second later of printed cells grew in a ball-like manner, similarly to how osteosarcoma cells grow when they are grown in matrices without accessible adherence surfaces. Therefore, 3D cellular structures able to be printed in just two layers using the BioLP™. Laser printing techniques have immense potential in addressing two primary focuses of tissue engineering: creating skin substitutes for burn victims and exploiting the ability of mesenchymal stem cells (MSC) to repair and regenerate, which has many applications for cellular repair and in the overall regeneration of tissues. The complex composition and organization of highly specific cells within tissues is a major obstacle for tissue engineers. In order to be able to fabricate more complex structures, integration of vasculature, glands, and the precise deposition of cells and biomaterials all need to be addressed and implemented (Koch et al. 2010). While inkjet printing is commonly used to accomplish printing biomaterials, the

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drawback of inkjet techniques is the high shear force of the inkjet nozzle which can lead to cell damage. Inkjet is therefore more suitable to printing material fluids with low viscosity and low cell density. In contrast, laser printing does not require the use of a nozzle, and it can be used with higher viscosity liquids and higher cell densities, making it a more promising platform for future technique development.

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Advanced Manufacturing of Microbial Communities: FDM, DW, and MPL

Bacteria are an excellent source for engineered living materials. Numerous species of bacteria, both synthetically modified and unmodified, are amenable to culturing and manipulation. Bacteria have evolved enzymes that can perform advanced chemical reactions that produce materials. The combination of AM and 3D printing techniques with bacterial materials has allowed for the development of complex, patterned biomaterials.

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Modifications of an FDM 3D Printer

The Meyer laboratory developed the first microbial 3D printers, which have the ability to deposit bacteria in specific 3D patterns (Lehner et al. 2017; Schmieden et al. 2018). The method deployed by this laboratory is centered around the modification of a commercially available 3D printer which has been altered to print a mixture of bacteria and alginate. This mixture will solidify into a gel once it comes into contact with a printing surface that is treated with calcium chloride (Lehner et al. 2017). This laboratory has also created a low-cost bioprinter utilizing easily obtainable K’NEX and electronic parts, called the Biolinker (Schmieden et al. 2018). The modifications performed on an inexpensive fused deposition modeling 3D printer to create a bioprinter can be seen in Fig. 6. The extruder, the component of the printer that ejects liquid material for deposition in successive layers, was replaced with a pipette tip nozzle attached to a system of tubing. This external transportation allows the bio-ink to move under ambient temperatures appropriate for microbes. This practice differs from traditional AM techniques which commonly use plastics, where the extruder must be hot enough to melt the material for deposition. Additionally, a second pipette tip was attached to the printhead, making the changeover between different bio-inks easier and faster (Fig. 6b). In order to produce a continuous and adjustable flow of bio-ink, a syringe pump was added to extrude the bio-ink through the external tubing to the pipette nozzle. An external computer controls the trajectory of the movable printhead in a programmable manner, allowing for the creation of printed shapes. Printed shapes and designs can be created in silico via CAD software programs, which can be translated into slices using printer-specific

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Fig. 6 Modification of a 3D printer. (a) Overview of all bioprinter components. 1, syringe pump; 2, syringe filled with bio-ink; 3, extruder holder; 4, one of three step-motors for positioning; 5, breadboard and hardware of the printer; 6, frame of the printer. (b) Detailed view of the modified extruder. 3a, active pipette tip; 3b, secondary pipette tip for layering materials; 3c, tubing system. Image reprinted from Lehner et al. (2017) © 2017, ACS. https://pubs.acs.org/doi/10.1021/ acssynbio.6b00395

software. All of these modifications to this 3D printer can be implemented on other 3D printers that feature accessible and removable extruders.

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FDM: Bio-ink Properties and Bacterial Viability

The bio-ink that the Meyer laboratory developed for use with this modified 3D printer is a custom mixture of live bacteria, liquid growth medium, and dissolved alginate (Lehner et al. 2017). This mixture flows through the printhead(s) in liquid state and then rapidly solidifies into the desired patterned shape upon contacting the printing surface. The printing surface contains calcium ions, which triggers alginate molecule cross-linking when it comes into contact with the alginate-bacteria mixture. This cross-linking results in the formation of a stable, biocompatible aerogel scaffold within seconds (Kuo and Ma 2001; Almqvist et al. 2001). The optimal calcium chloride and alginate concentrations for successful printing were identified to be 1M and 2.5% w/v, respectively. If the concentrations were too low, poor gelation and poor printing resolution were observed, whereas too much led to premature gelation which would block the pipette tip and prevent printing. The reproducibility and consistency of this specific bioprinter was tested by measuring the width of printed monolayers at multiple positions. No statistical differences in width were measured between different positions within individual monolayers or between positions in different printed monolayers, determining that the printer, with the optimized bio-ink, was able to print structures in a consistent and uniform manner. The bioprinter’s printing resolution was optimized by adjusting

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printhead movement speed and syringe pump rates both simultaneously and in parallel. The printing of multilayered structures with this printer required that the printhead increases its z-position by 0.15 mm/layer. As layers are deposited on top of previous layers, the new layers are still able to solidify because the calcium ions on the printing surface have diffused upward through the previously printed layers. Notably, each newly added layer resulted in a fractional increase in width of the printed structure, which was due to the small delay in solidification of the alginate after printing. When depositing layers one through six, the width increased an average of 0.14  0.01 mm per layer, but after the sixth layer the width plateaued and remained constant. This bioprinter was therefore able to print 3D structures with resolution capabilities at the submillimeter scale. This resolution could potentially be improved if a commercial 3D printer containing a more accurate printhead were modified similarly. Applications for printed layered bacterial structures could require the spatial separation of bacterial strains between the layers. To determine the amount of mixing between the layers of bacteria, bilayered structures of bacteria were printed, with one strain of bacteria expressing fluorescent yellow protein and the other fluorescent blue. Separate tubing and pipette tips were used for each layer to limit the mixing of bacteria outside of the layers. Upon analysis of the layer mixing using confocal microscopy, good separation of bacteria between the layers was observed even after 24 h of incubation, as seen in Fig. 7. Many applications of printed bacteria will require the bacteria to be able to survive in the alginate gel. To test the viability of the printed bacterial cells at various time points, the bacteria were deposited in alginate hydrogels and were incubated from 0 to 48 h. The gels were then dissolved in a solution of sodium citrate, which chelates the calcium ions thus dissolving the gel. The dissolved samples were then grown on LB plates to determine the amount of colony forming units remaining in the samples at the different timepoints. The bacteria were seen to grow within the alginate gels between 0 and 24 h, after which the cell viability remained steady through the 48 h timepoint. Compared to nonprinted bio-ink

Fig. 7 Internal structures of printed biofilm layers. Modified strains of E. coli expressing two different fluorescent proteins were printed in two layers using the Biolinker, one on top of the other. A layer of bio-ink containing blue fluorescent bacteria was printed onto a glass coverslip, followed by a layer of bio-ink containing yellow fluorescing bacteria. After each printing cycle, calcium chloride was added to solidify the alginate in the bio-ink. 30 min after solidification of the second layer, the internal structure of the printed bacterial layers was inspected along the z-axis by confocal microcopy. Scale bar is 100 μm. Image adapted with permission from Schmieden et al. (2018). © 2018, American Chemical Society

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bacteria, the printed bacteria had a 50% reduction in viability immediately upon printing. After incubation within the alginate gel, the number of viable bacteria in the printed structure demonstrated an overall increase in viability of approximately 200% compared to the nonprinted bio-ink bacteria. The differences between the nonprinted bio-ink bacteria and the printed bacteria can be explained by the nutrient availability within each substance; the nonprinted bacteria had a limited media supply compared to the added nutrients that could be found within the printed substrate gel, combined with the decrease in the density of the bacteria following printing. These experiments concluded that the printed bacteria are able to survive for as long as 2 weeks, such that there is sufficient time for microbial-mediated material production and patterning to occur, and showed robust fluorescence within the layers for the first week. The long-term viability of the cells could potentially be even further extended by adding additional nutrients during the incubation period and providing a way to remove waste products from the gel.

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FDM: Modeling Biofilms

Biofilms are complex aggregates of bacterial cells suspended in a self-produced matrix (Hall-Stoodley et al. 2012) and can grow on almost any available surface. Biofilms can contain pathogenic bacteria, which can be antibiotic resistant, and can cause serious health complications for patients. Research surrounding biofilms is therefore centered around how to combat these types of biofilms (Li and Lee 2017; Sharma et al. 2016), how to control their formation (Nagar and Schwarz 2015), modeling biofilm growth and structure (Pu et al. 2018; Chen and Wegner 2017), and characterizing corrosion caused by biofilms (Li et al. 2013). On the other hand, biofilms can also have less nefarious and more beneficial applications, including wastewater treatment (Lewandowski and Boltz 2011), bioleaching (Vera et al. 2013; Olivera-Nappa et al. 2010), or producing living materials with various potential applications (Schaffner et al. 2017). Biofilms have the ability to express a diverse set of biopolymers that allows them to adapt their mechanical properties when put under stress, letting them acclimate to their surrounding environmental conditions (Schaffner et al. 2017). Bacteria can perform an enormous range of chemical reactions (i.e., forming calcium carbonate (Douglas and Beveridge 1998), magnetite (Bazylinski and Frankel 2004), biopolymers (Rehm 2010), etc.), and the combination of bacterially produced biomaterials with the physical resilience of biofilms creates the potential for the development of numerous applications. Despite the vast potential applications for biofilms, the 3D shape, structure, and metabolic dynamics of biofilms have still not been thoroughly explored and standardized (Schaffner et al. 2017). The complexity of biofilms also often limits the applicability of the research being done. For example, biofilms are highly heterogeneous, with imbalanced distributions of biomass, nutrients, metabolic products, and more. The microbial populations within the biofilms can also differ genetically to bacteria in solitary, or planktonic, lifestyles, with biofilm bacteria frequently

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displaying resistance to cleaning products and disinfectants (Stewart and Franklin 2008) and expressing a suite of biofilm-forming genes (DePas et al. 2013). In nature, bacterial cells switch from planktonic growth to biofilm growth when exposed to stress conditions, which can include temperature fluctuations, changes in salt concentration, or other environmental variations. The extracellular matrices of biofilm hydrogels are primarily composed of proteinaceous amyloid fibers, and some species of bacteria produce a specific fiber called curli fibers (McCrate et al. 2013; Barnhart and Chapman 2006). Extracellular matrices of biofilms also contain cellulose and other polysaccharides, of which the exact composition depends on the bacterial strains within the biofilms and the growth conditions. Curli fibers are composed of CsgA, a secreted protein that self-assembles on CsgB, a membrane nucleator protein (Wang et al. 2007; Hammar et al. 1996). These curli fibers interconnect the biofilm and help it adhere to surfaces. Two operons control the curli fiber gene expression: csgBAC and csgDEFG (Hammar et al. 1995). CsgD positively regulates curli fiber formation and other biofilm components; CsgE, F, and G participate in the export and folding of CsgA and B (Evans and Chapman 2014). To make biofilm-inspired living materials that are useful tools for both engineering and research, control over the spatial arrangement of the bacterial cells and the formation of extracellular matrix components is required. A number of methods have been developed to immobilize bacteria while maintaining their metabolic activities, including adsorption onto surfaces, cell cross-linking, encapsulation, and entrapment (Cassidy et al. 1996). Immobilizing the bacteria within a hydrogel is an approach that offers an optimal living environment for the bacteria, allowing for high water content that permits the flow of nutrients into the gels and waste out of the gels (Schaffner et al. 2017). Combinatorial control over the extracellular matrices of a biofilm and the spatial distribution and concentration of cells can help researchers make more tunable and patternable biofilms. The Meyer laboratory created a method that combines 3D printing with genetic control over biofilm formation, thus making initial strides in this direction (Schmieden et al. 2018). Their 3D printing method, described in the previous section, prints the bacterial cells within a 3D polymeric matrix. However, the stability of the printed bacterial structure is constrained by the chemical composition of the matrix polymers (Lehner et al. 2017), and the biofilm-forming ability of the bacterial cells is also not controlled in this method. To overcome these challenges, the Meyer laboratory has developed a technique that utilizes bacterial cells that can be induced to form biofilms, through inducing the expression of CsgA after printing the bacteria, thus advancing the 3D printing of bacteria to include patterned biofilminspired material production (Schmieden et al. 2018). In order to produce a 3D model for biofilms, the Meyer laboratory first engineered bacteria to express biofilm-forming proteins following induction. Curli fiber proteins are composed of CsgA, monomers of which assemble to create the fibers outside of the cell, and a CsgB outer membrane protein, which attaches the fiber to the outer membrane (Wang et al. 2007; Hammar et al. 1996). Mathematical modeling indicated that the rate-limiting step for curli fiber formation is CsgA production, so that

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curli fiber formation and growth rate can be controlled by the genetic control and induction of the csgA gene alone, without requiring inducible expression of the other curli-related genes. To achieve this, a csgA knockout bacterial strain was created that carried an inducible vector containing the csgA gene. The biofilm formation was tested via microtiter plate assays (Schmieden et al. 2018; Zhou et al. 2013). The cells were grown in 96-well plates for 5 days in the presence or absence of the inducer molecule. Planktonic bacterial cells were washed away; the remaining biofilm cells were stained with crystal violet, which was then dissolved by ethanol; and its optical density was measured at 595 nm. The data showed that the bacteria containing the csgA gene, once induced, have a 131% increase in biofilm formation. Therefore, the bacterial containing the inducible plasmid with CsgA alone was sufficient to control the formation of engineered biofilms in a solution. Next, the biofilm formation by the engineered bacteria printed within an alginate gel was assessed. The gelation of the alginate is reversible; a strong calciumcomplexing agent like citrate can wash out the calcium, thereby dissolving the gel. Printed bacteria that did not contain the cgaA gene were mostly washed away once the alginate gel was treated with citrate, whereas the localization of the printed CsgA-expressing bacteria was not significantly affected by the citrate treatment. These experiments determined that the CsgA-expressing cells were stably and homogeneously anchored within the bio-ink gel. Overall, it was demonstrated that the curli fibers were able to hold the bio-ink together, mimicking the extracellular matrix of natural biofilms. The Meyer laboratory even demonstrated that they could print the CsgA-expressing bacteria in spatially patterned shapes, which remained patterned on a macroscopic level even when treated with citrate.

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FDM: Regenerative Photosynthetic Living Materials

The printing of microalgae, a photosynthetic unicellular organism, has unique suitability for applications as algae are able to adapt to harsh conditions and are a sustainable option for biotechnology, bioremediation, biofuel, wastewater treatment, and more. Bioprinting of algae has been demonstrated by multiple groups, primarily using scaffolds made of alginate (Anja Lode et al. 2015), carrageenan (Malik et al. 2020), silk (Zhao et al. 2019), and starch (An et al. 2019). The Meyer laboratory has expanded the possible applications for printed microalgae by printing it onto a substrate of bacterial cellulose, which has excellent tensile strength and toughness while also being a natural, robust biopolymer (Balasubramanian et al. 2021). Bacterial cellulose is composed of a nanofibrous architecture which has absorptive capabilities, which is expanded on later in this chapter in the section on Direct Ink Writing using Flink. This photosynthetic living material has applications for air purification systems, wastewater treatment, artificial leaves, and more. The Meyer laboratory utilizes a regenerative bioprinting approach, using a modified fused deposition bioprinter similar to the previously described printer described in the “Modifications of a FDM 3D Printer” section, to print their

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photosynthetic living material. Their bio-ink, composed of alginate and the microalgae Chlamydomonas reinhardtii, is printed onto a substrate composed of bacterial cellulose and calcium chloride that sits on a bed of microalgae growth nutrients, resulting in the formation of an alginate hydrogel. In order to use the microalgae as a living material, they first optimized the growth conditions for the C. reinhardtii. This algae species can grow under various growth conditions: (1) photoautotrophically, with CO2 as a carbon source, in the presence of light; (2) chemo-/heterotrophically, in the absence of light, using alternate carbon sources; or (3) photomixotrophically, a combination of (1) and (2) with both CO2 and light provided. This alga can also use cellulose in photomixotrophic conditions as an alternative carbon source (Blifernez-Klassen et al. 2012). To test the microalgae growth in various growth conditions, microalgae were printed over top of a bacterial cellulose layer which was sitting on a bed of carbonsupplemented agar media. The nutrients are able to diffuse upward through the cellulose to support the microalgal growth. The photomixotropically grown bioprinted structures had higher green coloration (due to increased chlorophyll content) and higher cell densities compared to photoautotrophic and chemotrophic conditions. Overall, the bioprinted microalgae were able to grow following printing and could remain viable within their matrices for at least 4 weeks. The algae’s ability to survive on the bacterial cellulose alone, with no carbon supplemental agar, was also tested. The algae could survive at least 3 days without the supplemental agar, and their growth could be revived upon the addition of the carbon supplemented agar. However, if the algae were without contact to the carbon supplement for longer than 5 days, the density of the cells was irreversibly decreased. The printing process was shown not to alter the morphology of the microalgal cells. The photosynthetic algae used by this group was able to live longer and divide more after printing compared to printed bacteria, likely since they were able to feed themselves through the light source they were exposed to. When the microalgae were printed on top of the cellulose, the printed structure was able to be peeled off from the agar layer, thus indicating that the material was self-standing. It was also possible to remove the microalgae from the cellulose and even to reattach it to a new cellulose surface, which could make this process applicable to even more applications (i.e., brand labels). Stability to physical distortions was also examined. Following folding, twisting, or crushing, the bioprinted structures on cellulose were able to resume their shapes. Microalgae structures not printed onto cellulose were unable to resume their shapes following manual distortions, thereby demonstrating the enhanced mechanical properties given by the bacterial cellulose. Additionally, the stability of the printed structures following repeated submersions in water was evaluated. The bioprinted structures did not exhibit any dissolution or change in shape, even following an extended 1 week submersion. No microalgae could be detected in the water following this incubation, indicating that there was very minimal escape of the microalgae into the water. The complete sequestration of the microalgae in the hydrogel makes this approach advantageous because it does not raise any environmental concerns of microorganism escape. Lastly, citrate treatment was used to dissolve this hydrogel, after which

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the microalgal cells could be recovered, recultured with fresh carbon supplements, and reused to produce future bioprinted structures. This finding demonstrates that microalgal living materials are capable of regeneration and reusability and therefore have the potential to become versatile materials which could be printed and shipped across the world, or even into outer space, where they could be used on-site to produce more of themselves.

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FDM: Printing Bacterial Spores

Some bacteria are able to survive under immensely adverse conditions by forming endospores. Spores are small, tough spherical structures which can lie dormant. The cell membrane of spores is multilayered, consisting of a virtually impermeable inner membrane, a germ cell membrane, a thick peptidoglycan cortex, an outer membrane, a basement layer, the inner coat, the outer coat, and the crust (McKenney et al. 2012). The DNA in these spores is highly protected and is very tightly packed by specialized proteins. These spores are able to survive extreme conditions: high temperatures, freezing, oxidizing agents, acid and alkaline solutions, genotoxic agents, solvents, high pressure, X-rays, gamma radiation, UV light, and desiccation (Setlow 2006; Moeller et al. 2008). Spores are able survive in this state nearly indefinitely. The Voigt laboratory has altered a fused deposition modeling 3D printer—the MakerBot Replicator—in order to print materials containing Bacillus subtilis spores (González et al. 2020). Similar to how the Meyer laboratory modified a commercial 3D printer, the Voigt laboratory also reconfigured theirs. The nozzle was redesigned in order to mix two liquids, one polymer maintained at a higher temperature and one cell-and-media mixture maintained at a lower temperature, to form the bio-ink just prior to printing. The printhead temperature was kept at 75
C in order to print the polymer at a high enough temperature to keep it liquid, an elevated temperature that did not damage the printed spores but was too high for successful printing of living meso- or thermophilic bacteria species that were non-spore-forming. The cells were exposed to the higher temperature for less than 20 min, and the mixture was rapidly cooled to 16
C following printing using thermoelectric cooling. This immediate cooling following extrusion was seen to improve the overall resolution of the structural details in their printed structures. Upon testing the biocompatibility of various polymers and their bacterial cells, agarose and B. subtilis spores were found to produce the most consistent structures while also having the highest percentage of viable cells post-printing. The ideal concentration of agarose was determined to be 4% w/v; less than this resulted in lack of proper structure formation, and more than this resulted in clogging of the nozzle, similar to the Meyer laboratory’s results. Various complex geometries were able to be printed using these materials and cells. To gain the ability to print cells at specific locations, a second printhead was added which allowed them to create structures with sharp boundaries between sections that did and did not contain cells. Additionally, the spores were

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demonstrated to be evenly distributed through the hydrogel following printing. The printed spores located near the surface of the printed structures were preferentially able to germinate, due to the increased amount of oxygen and nutrients that were available at the surface. If the printed structure was torn or damaged, thus exposing more internalized spores to a surface, they too would germinate following incubation in media. Next, the survival of the spores within the 3D printed structures was tested. The structures were dehydrated and stored. Following rehydration, the structures were able to return to their original shape, and the spores were shown to survive for up to 1 month of storage, the maximum duration tested. The authors predicted that the spores would be able to survive indefinitely in storage following this desiccation and rehydration procedure. Additionally, the ability of the printed spores to survive extreme stresses was tested. Following exposure to 100% ethanol for 1 week, the spores survived but demonstrated delayed outgrowth; spores survived after 1 week of treatment with high osmolarity; spores survived acidic conditions (pH 1) but were killed by alkaline conditions (pH 13); spores survived treatment with 365 nm UV light but were killed by 254 nm UV sterilization light; spores survived high (80
C) and lower (50
C) temperatures; spores survived 10 min of X-ray treatment; and spores survived 1 h of gamma radiation but were killed by 6 or 12 h. Overall, the authors demonstrated that these spores could survive and function in drastically fluctuating and stressful environmental conditions, which opens the door to applications which harness these abilities. Once germinated, these printed spores have the capacity to work as biosensors. The Voigt laboratory illustrated the versatility of this approach by using four genetically encoded sensors that were incorporated into the genome of the B. subtilis. These sensors respond to small molecules, such that the presence of the correct small molecule induced an engineered sensor to drive the expression of GFP, which was then quantified. Induction of these responses was observed to begin within 6 h, and full induction occurred by 12 h. The printed B. subtilis spores were also programmed to respond to the presence of Staphylococcus aureus. S. aureus is a well-known bacterium that has evolved an antibiotic-resistant strain known as MRSA, methicillin-resistant S. aureus. This ability to detect S. aureus is an important application for this printing method, as S. aureus poses multiple health concerns for humans, livestock, and the environment. The 3D printer was used to print patches of material that could fit into a modeled human wound. The B. subtilis were engineered to sense a quorum signal from the S. aureus, autoinducer peptide, or AIP and respond by producing antibiotics which would kill the S. aureus but not the B. subtilis itself. This approach has the potential to be applied in a variety of settings, such as creating printed objects with the embedded ability to kill bacterial pathogens on their surface.

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Direct Ink Writing: Bioremediation and Biomedical Applications of Flink

Following development of the bacterial printing method presented by the Meyer laboratory, the Studart laboratory also developed a technique that 3D prints bacteria such that they have control over the spatial distribution and concentration of cells in complex 3D hydrogel structures (Rasanen et al. 2007). This technique uses a newly designed bio-ink, called “Flink” for “functional living ink.” Flink was shown to be compatible with the immobilization medium they used to 3D print cells via multimaterial direct ink writing (DIW), a method described above in the directwrite section of this chapter (Schaffner et al. 2017). Flink was designed to have good viscoelastic and shear-thinning properties while using biocompatible ingredients. Their hydrogel bio-ink was devised to have suitable rheological properties, in order to be able to properly print distortion-free and accurate 3D structures while maintaining a high survival rate for the bacteria. Their new bio-ink was formulated through the use of nontoxic, water-soluble biocomponents hyaluronic acid (HA), κ-carrageenan (κ-CA), and fumed silica (FS) combined with bacterial growth media. The HA and κ-CA increased the viscosity of the solution, making the hydrogel able to retain enough water to promote bacterial growth. The use of these biocomponents in a 1:1:1 ratio created suitable rheological behavior for the bio-ink (i.e., a 3 wt% Flink contains 1 wt% of each of the biocomponents). Using a DIW approach, bacteria could be incorporated into specific regions of the printed structures with high accuracy, since the bio-ink cartridges could be loaded with different bacterial strains at various concentrations to be extruded at any point within the printed structure. Multiple types of bacteria, each with their own nutrient requirements, metabolic activities, and functionalities, were able to be combined together within the same 3D printed structure. Additionally, the viability and proliferative ability of the bacteria were not hindered by the printing process. The bio-ink was able to be further modified so that, following printing, resilient selfsupporting structures could be created. This was accomplished by exchanging HA for GMHA, a chemically modified glycidyl methacrylate HA. The replacement of HA with GMHA did not affect the viscosity of the hydrogel, and the GMHA allowed the hydrogel to be cross-linked via low-dose UV exposure. The resulting hydrogels were strong and had high mechanical stabilities. Using these modified hydrogels, bacteria could be immobilized within complexly shaped hydrogels, which were strong enough to be handled and swollen in various media conditions, demonstrating that the hydrogels had the strong structural and mechanical stability that is needed for scaffolds. The DIW-printed hydrogels were able to mimic healthy environments of bacteria, which enabled control over the metabolic activity and growth of embedded bacteria, demonstrated using two examples: (1) the phenol degradation capabilities of Pseudomonas putida and (2) in situ formation of bacterial cellulose by Acetobacter xylinum. To test the ability of P. putida to degrade phenol following printing, the

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Fig. 8 Bioremediation and biomedical applications of 3D-printed bacterial strains. (a) Staining of Pseudomonas putida DNA within a printed grid by ethidium bromide before (top) and after (bottom) incubation in phenol-containing medium (343 nm). (b) Bacterial cellulose nanofibril network created by Acetobacter xylinum printed within 3 wt% Flink, imaged under SEM. (c) A doll face was scanned, and 4.5 wt% Flink containing A. xylinum was deposited onto the face using a custom-built 3D printer. In situ cellulose growth led to the formation of a cellulose-reinforced hydrogel that, after removal of all biological residues, could serve as a skin transplant. Image reprinted with permission of AAAS from Schaffner et al. (2017). © Schaffner, M, et al., some rights reserved; exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 License

bacteria strain was immobilized within a 3D-printed Flink GMHA grid. This gridded structure had a high surface area which maximized the contact between the printed bacteria and the liquid medium. The printed grid was first photo-cross-linked and then incubated in minimal media containing phenol as the only possible carbon source for the bacteria. The phenol concentration was monitored over time using UV-visible adsorption spectroscopy. The phenol concentration was seen to decrease, as a result of degradation by the printed bacteria (Fig. 8a). Additionally, with a second phenol incubation period, the bacteria showed an even higher phenol degradation efficiency. These experiments demonstrate a possible bioremediation technique utilizing bacteria printed within these unique hydrogels. The ability of A. xylinum bacteria to produce cellulose when exposed to oxygen within a 3D-printed structure was also demonstrated. These bacteria were embedded within a hydrogel containing κ-CA, HA, and FS and then incubated for up to 7 days. This bio-ink was purposefully not cross-linked, therefore promoting biofilm formation and the production of bacterial cellulose. Following incubation, the ink components were washed away, leaving behind a network of nanofibrillated bacterial cellulose (Fig. 8b) that was confirmed by scanning electron microscopy and by staining the cellulose. The formation of the cellulose was shown to be directly dependent on the oxygen availability within the printed structures and the viscosity of the hydrogel ink. The cellulose was formed along the outer surfaces of the printed structures, where the oxygen levels were the highest, allowing the authors to conclude that the oxygen availability was directly correlated to the growth of the bacteria at different depths within the 3D structure. These results illustrate how this 3D bacteria printing process can be useful for the production of thin films and coatings. Patterned cellulose coatings may even have application in medical

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transplants, since they have been linked to a decreased organ rejection (Bottan et al. 2015). To further demonstrate how this technique can be used to create intricate functional materials, a 3D printer was custom built to deposit the A. xylinum onto a substrate shaped like a human face (Fig. 8c). The resulting cellulose mask illustrates the possibility of forming personalized skin replacements and the potential for many biomedical applications for 3D-patterned bacterial cellulose.

17

MPL: 3D Bacterial Communities

The organization of cellular and chemical landscapes over small, micrometer distances can profoundly impact the phenotypic states of biofilms. These bacterial communities are spatially dependent on intercellular interactions, meaning that organisms within biofilms release distance-dependent signaling that can be sensed because of the close proximity of the cells. The microbes can then help protect each other from environmental risks by aggregating together. Chemical sensing can help bacteria adapt to dynamic environmental conditions. Research into the organization of microscopic bacterial aggregates is essential to understanding such interactions. Various methods using microfluidic devices, microcavities, and more have been developed to discern the size, shape, and physical attributes of these environments within bacterial communities, but they have not been successful in allowing researchers to define the 3D geometry of bacterial aggregates and orientation of multiple populations of bacteria within these structures. To tackle these challenges, the Shear laboratory has developed a multiphoton lithography (MPL)-based method which they have demonstrated can create “designer” ecosystems that can help researchers determine the interactions of bacterial populations in 3D (Connell et al. 2013). In this method, bacteria are first mixed at 37
C with a solution containing gelatin and a photosensitizing molecule that will promote chemical cross-linking of polypeptides following photoexcitation via the direct cross-linking of unmodified gelatin. Once this mixture is cooled to ambient temperatures, bacteria are suspended in the gel at various 3D positions. The geometrical microstructures are printed around bacteria at specified 3D coordinates in the gel using MPL. These gelatin-based barriers are permeable to chemical species, including metabolic waste generated during cell growth, which is unique compared to other photolithographic techniques. These sealed microcontainers create microenvironments that can support bacterial proliferation, and the MPL-based encapsulation process does not affect cellular viability. This method can create basically endless 3D geometrical arrangements of small groups of bacteria. MPL bacteria printing was applied to demonstrate the spatially localized interactions between Staphylococcus aureus and Pseudomonas aeruginosa bacteria. Communities of these bacteria were able to be spatially defined using the MPL method, meaning that they could be co-printed within the same structure as a mixed culture or printed in spatially distinct regions within the same structure. When the two bacterial

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species were co-printed, the mixed populations were able to proliferate and remain viable within their microcontainers. Additionally, different complex geometries were constructed by sequentially printing these bacteria: first a core of S. aureus and then a surrounding shell of P. aeruginosa. These precisely defined microbial microenvironments have made it possible for researchers to investigate cell-cell interactions more closely. The Shear laboratory investigated whether the antibiotic resistance of P. aeruginosa may influence the antibiotic susceptibility of S. aureus. Since S. aureus is susceptible to β-lactam-based antibiotics like ampicillin, the researchers hypothesized that it could be sheltered by the ability of P. aeruginosa to produce β-lactamases that would break down the ampicillin. Within the concentric spheres containing the two bacterial species, the P. aeruginosa proved to be substantially protective to the encapsulated S. aureus upon exposure to ampicillin. The more β-lactamases that were produced, the greater the protection that the P. aeruginosa provided. This protection was not seen to be affected by the densities of either bacterial population, showing that even a low density of P. aeruginosa surrounding the S. aureus was able to dramatically reduce the ampicillin toxicity. This MPL method from the Shear laboratory gives scientists the opportunity to study microbial interactions at the micrometer resolution in just about any 3D geometry. The largest hindrance to this method, however, is the high cost for this type of fabrication equipment.

18 18.1

What Can Each Method Be Used for in the Future? Thermal Inkjet: Tissue Engineering and Regeneration

Since thermal inkjet printing has been shown capable of printing living materials with minimal harmful side effects, the possible variety of attractive applications is seemingly limitless. Scientists are hopeful to be able to use this system for gene transfections and drug delivery, as well as for further tissue engineering and regeneration applications. The development of a hand-held thermal inkjet printer or a digitally controlled printhead would allow for more precise control over tissue repairs. Scientists could use such devices in conjunction with 3D reconstructions of lesions and defects in tissues, potentially increasing their capability of bioprinting more complex tissues and vasculatures.

18.2

Direct-Write: Tissue Engineering and Organ Printing

Tissue restoration is a major topic of research for tissue engineers, for which tissue engineers need precise spatial control over the organization of 3D scaffolds. The ability to print cells within scaffolds in specific patterns, without the need for cellular invasion, will help scientists improve the fabrication of tissues. The BAT direct-

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write system can offer a critical step in this direction, permitting tissue engineers to place cells in distinct locations within a printed scaffold. The BAT prints in continuous linear rod-like structures, comparable to liquid droplets, and has the ability to move along the XYZ axes simultaneously, making the BAT a highly flexible system. The BAT has been shown to be able to print multilayered anatomically based structures. Similar to an inkjet printer, the BAT’s bio-ink components are combined before it is loaded into the system, adding the ability to use a multi-head pen system to deposit multiple cellular solutions in the same printing sessions. Perhaps by utilizing these features of the BAT, direct-write can be used by tissue engineers to print constructs into specialized architectures using a variety of cell types, each cell type having the correct spatial organization needed. Additionally, the Pluronicalginate direct-write bioprinting gels have enhanced mechanical and rheological properties and have made significant advances toward being able to regenerate and fabricate physiological tissues in vitro.

18.3

Fused Deposition Modeling: Spheroid Organ

The use of spheroid organs takes scientists a step closer to creating useable 3D printed organs with built-in vasculature. This microtissue approach is appealing because it is adaptable for industrial-scale robotic and automated biofabrication. However, technical challenges arise when creating microtissues such as these: largescale production, reproducing built-in vascular tree networks in 3D-printed tissues and organs, development of a continuous digital bioprinter, and more. However, this developmental biology-based approach to organ printing is expected to enhance the tissue engineering field as it is further researched, tested, and implemented.

18.4

Laser Direct-Write: Indirect Cell Patterning

Work with the BioLP™ technique illustrated that in the future this method can be used to build more complex 3D structures using a layer-by-layer approach. This method could be enhanced via the incorporation of growth factors, cytokines, and other components that could result in more sophisticated and advanced tissue growth, which could overall become superior to the traditional scaffold-based techniques. The LIFT method also offers highly versatile applications for the printing of viable, unharmed cells that can be used to create tissue replacements. The AFA-LIFT method to print viable fungi also illustrates how laser printing has been adapted to promote the survival of different cell types. The ability to precisely print undamaged biomaterials makes laser printing an exciting and cutting-edge application for researchers and tissue engineers.

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181

Microbial Communities: FDM, DIW, and MPL

The FDM 3D printing method for bacteria has allowed for the development of stable, patterned biofilm-inspired bacterial materials. While these engineered biofilms allow for control over the spatial arrangement, species of bacteria, genetic characteristics of the bacteria, and bacterial density, they still do not precisely mimic natural biofilms. The technique is able to deposit engineered biofilms in a standardized, reproducible manner, enabling researchers the opportunity to use them to evaluate nefarious biofilms that can coat medical devices and pathogenic biofilms that contain curli fibers. More research needs to be done to properly establish which of the features of the artificial curli-fiber-producing biofilm are representative of the natural biofilms. Additionally, the FDM-based fabrication of microalgae living materials illustrates another bioprinting approach that is simple, cost-effective, eco-friendly, and scalable. The use of biodegradable components and naturally occurring materials lays the groundwork for the possibility of developing more advanced bioprinted materials that can take advantage of the regenerative and reusable properties found in nature. The DIW-based bioremediation and biomedical applications for printed Pseudomonas putida and Acetobacter xylinum, respectively, illustrate how advanced manufacturing can revolutionize the environmentally friendly production of biologically generated functional materials while allowing researchers to have precise and accurate control over the printed structures, cells, and biocomponents. The ability to print viable bacteria spores can give scientists the added benefit of simpler preparation and longer storage times. This printing method results in spores that are able to survive a variety of extreme conditions, and this method can be used to make useful and robust biosensors. Genetic engineering of bacterial strains capable of this same spore formation and spore survival will offer scientists the chance at creating application-ready living materials that are able to survive in fluctuating and stressful environmental conditions. The MPL-based bacteria printing strategy creates a platform which can be used to study resource competition, symbiosis, population-dependent antibiotic resistance, and more. This method provides researchers a strategy to study how bacteria adapt to thrive in the heterogeneous environments found in nature. Taking this method a step further, it could be possible to use this approach to nest bacterial microcolonies within animal hosts to study how infections develop in vivo.

19

Summary

Additive manufacturing of living materials is a rapidly progressing field, in which new techniques and applications are constantly emerging. Spatially patterned living materials are important to researchers across many disciplines: tissue engineering, biomedical engineering, and material science, just to name a few. The applications

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for the methods described in this chapter are virtually endless. However, some key challenges need to be considered for the use of living materials in 3D-printed constructs: long-term survival of the living material will be dependent on the nutrient supply; proper boundaries need to be constructed in order to prevent engineered living cells from being able to escape their structures; waste products will need to be properly removed from the printed structures; and new bio-inks must be developed that would allow researchers to print their living material in hundreds of layers without the structure collapsing (Gona and Meyer 2020).

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Engineered Living Materials for Construction Rollin J. Jones, Elizabeth A. Delesky, Sherri M. Cook, Jeffrey C. Cameron, Mija H. Hubler, and Wil V. Srubar III

Abstract The design and construction of sustainable and durable civil infrastructure provides modern societies higher qualities of life. Continued population growth and urbanization, however, is increasing global demand for building materials, like concrete and steel, whose production are not without environmental consequences. Reducing the environmental impacts of construction materials through the development of innovative, sustainable, and durable material technologies is critical if urban environments are ever to thrive in harmony with the natural world. Civil engineers can aspire to achieve equilibrium with the natural world by drawing inspiration from nature and implementing many of its design principles. This chapter provides a stateof-the-art review of the field of engineered living materials (ELMs) that are specifically designed for construction applications. ELM technologies based on microorganisms, fungal mycelium, and plants are reviewed in light of their biological functions and end-use applications. In addition, challenges that new ELM technologies designed for the built environment must overcome, including economic

R. J. Jones · E. A. Delesky Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, Boulder, CO, USA S. M. Cook Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, Boulder, CO, USA Environmental Engineering Program, University of Colorado Boulder, Boulder, CO, USA J. C. Cameron Department of Biochemistry, University of Colorado Boulder, Boulder, CO, USA Renewable and Sustainable Energy Institute, University of Colorado Boulder, Boulder, CO, USA M. H. Hubler · W. V. Srubar III (*) Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, Boulder, CO, USA Materials Science and Engineering Program, University of Colorado Boulder, Boulder, CO, USA e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 W. V. Srubar III (ed.), Engineered Living Materials, https://doi.org/10.1007/978-3-030-92949-7_7

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feasibility, uncertainty, scale-up, long-term organism viability, and biocontainment, are also reviewed and discussed herein. Keywords Engineered living materials (ELMs) · Self-healing materials · Living building materials (LBMs) · Biocementation · Living façades · Mycotecture · Soil stabilization · Algae building technology

1 Rethinking Infrastructure The design and construction of civil infrastructure affords modern societies higher qualities of life by providing water and shelter—two of humanities most basic needs. As the global population continues to rise and urbanize, concerns regarding the impact of the built environment on climate change are becoming ever more predominant. It is estimated that buildings alone are responsible for one-third of global energy consumption and 40% of total carbon dioxide (CO2) emissions (IEA 2020). Building material scientists and structural engineers are now being called to action to improve the resilience and sustainability of civil infrastructure by producing and using durable materials with lower carbon footprints. A grand opportunity for reducing the global climate impact of the built environment includes reducing the so-called embodied carbon and other environmental impacts associated with building materials. Embodied carbon refers to the carbon dioxide (CO2) emissions attributed to the manufacturing of building material manufacture. Concrete, for example, is one of the most widely consumed materials on Earth, second only to water. Its manufacturing, however, is responsible for 8–9% of the world’s anthropogenic CO2 emissions (Monteiro et al. 2017). In 2012, concrete also accounted for 9% of global industrial water withdrawals (Miller et al. 2018). Urbanization and growing populations are also leading to unprecedented rates of construction and demolition waste and municipal solid waste generation (Kaza et al. 2018). Understanding, quantifying, and reducing these impacts through the development of new, durable, low-carbon construction are a critical need if urban development is ever to be in harmony with the natural world.

1.1

Emergence of Engineered Living Materials

Nature has developed a multitude of sustainable ecosystems over billions of years, yielding balanced, self-sufficient communities of organisms that rely solely on natural resources. Civil infrastructure can aspire to achieve the same equilibrium by drawing inspiration from nature and implementing many of its design principles. Civil engineers have long incorporated natural materials into the design and construction of buildings. Biogenic materials, such as timber, straw, and hemp, have been used for centuries to build durable, long-lasting buildings. However, because many of these materials take time, land, water, fertilizer, and human resources to grow, researchers are now looking into the potential of industrial

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biotechnology to accelerate the production of high-performance biologically derived—and biologically active—materials for construction applications. Over the past decade, the field of engineered living materials (ELMs) has emerged and evolved into a new, standalone discipline. ELM researchers have since fused bioengineering design-build-test-learn approaches with classical materials science to yield breakthrough innovations in the synthesis of complex, biologically active materials for construction and other fields (Srubar 2020).

1.2

Summary of ELM Taxonomy

A taxonomy of ELMs was recently illustrated by Srubar (2020) to facilitate crossdisciplinary discussions of ELM advancements and to provide a framework to highlight current trends and future directions within the field (Fig. 1). This chapter will adhere to these taxonomic classifications in our review of structural and non-structural ELMs derived from bacteria, fungi, and non-woody plants for construction applications.

1.3

Summary of Biofunctions Applicable to Construction

To create ELMs for construction applications, a living, biological component must be integrated into the manufacturing process of a building material for the express purpose of achieving a target material performance (e.g., structural, thermal) and maintaining biological activity. Beyond merely joining two dissimilar entities, considerations must be made for the involvement of the organism in material manufacturing, survivability, and biological activity of the organism within the bulk and the impact of the organism and its biofunctions on initial and long-term material properties.

Fig. 1 Taxonomic classifications of engineered living materials (ELMs) with regard to scale, design, living organism, material properties, and application domain. Figure reproduced with permission (Srubar 2020)

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Biofunctions of interest to construction include (1) biocementation and bioadhesion, (2) self-healing, (3) biomass growth, (4) biofiltration and bioremediation (including air detoxification), (5) thermal regulation, and (6) environmental sensing and response. These biofunctions are described further in Sect. 2.1 and Sect. 3.1.

1.4

Summary of Reviewed ELM Technologies

The incorporation of living organisms into structural or non-structural building components can result in a variety of ELM building material technologies, some of which are illustrated in Fig. 2. Structural technologies reviewed herein include biocemented soils, biological concrete masonry units (bio-CMUs), self-healing concrete, and living building materials (LBMs). The non-structural technologies reviewed herein include mycotecture, vegetative façades, algae-building technologies, and living surfaces.

2 Engineered Living Structural Materials Structural materials are load-bearing materials, which require not only mechanical strength and stiffness but also environmental durability. The biofunctions utilized to produce structural ELMs include those that can impart or improve mechanical strength and/or prevent or heal damage from environmental degradation. The most

Fig. 2 An overview of select ELM construction technologies. Targeted applications of the technologies are shown (e.g., soil, building structure, building envelope, façade) to highlight where their implementation would be most effective in the context of a living city. Images for the figure are reproduced with permissions (Van Paassen 2011; Ednie-Brown 2013; Susorova et al. 2014; Jones et al. 2018; Biloria and Thakkar 2020; Strain 2020; Van Mullem et al. 2020)

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common biofunctions used to produce structural ELMs are detailed in Sect. 2.1, and resultant structural ELM technologies are discussed in Sect. 2.2.

2.1

Biofunctions for Producing Structural ELMs

Multiple biofunctions have been utilized to produce structural, load-bearing ELMs. Relevant biofunctions include biocementation and bioadhesion, self-healing, and biomass growth (see Fig. 3).

2.1.1

Mechanism of Biocementation and Bioadhesion

Biocementation involves the application of a naturally occurring microbial process, namely, microbially induced calcium carbonate (CaCO3) precipitation or, more commonly, microbially induced calcite precipitation (MICP). Biologically precipitated calcium carbonate (CaCO3) minerals can bridge aggregates, like sand grains, to impart mechanical strength (Whiffin 2004; Mujah et al. 2017). In most applications, biomineralizing organisms such as Sporosarcina pasteurii (S. pasteurii) are used to precipitate CaCO3 through urea hydrolysis. In urea hydrolysis, urea (CO(NH2)2) is hydrolyzed by urease catalysis to produce carbonate (CO32
) and ammonium (NH4+). The ammonium ion will spontaneously hydrolyze to ammonia and carbonic acid, thereby increasing the local pH around the microbe (Phillips et al. 2013). In the presence of calcium ions at elevated pH, the carbonate precipitates with the calcium into calcium carbonate (CaCO3), which can take on several polymorphs—calcite, vaterite, and aragonite. Calcite is the desired polymorph of CaCO3 for biocementation purposes due to its high stiffness and strength (Heveran et al. 2019). The following chemical reactions describe urea hydrolysis and CaCO3 precipitation: urease

þ COðNH2 Þ2 þ H2 O ! CO2
3 þ 2NH4

ð1Þ

Ca2þ þ CO2
3 ! CaCO3

ð2Þ

As depicted in Fig. 3, MICP can be used to build effective bridges between soil, sand, and other aggregate particles. Resulting products include MICP-stabilized soil (i.e., biocemented soil) or concrete masonry units (i.e., bio-CMUs). Biocemented soils or bio-CMUs are produced by combining ureolytic biomineralizing organisms and their media with soil or aggregate, which enables the microorganisms to undergo MICP and effectively biocement individual soil or aggregate particles together. While the biocementation literature generally refers to binding particles with biologically precipitated mineral bridges, this biofunction can be expanded to include bioadhesion using biopolymers, such as polysaccharides or proteins, or any biogenic molecule that facilitates binding. Examples include collagen, chitin,

Fig. 3 An overview of biofunctions that have been leveraged to produce structural ELMs. Biocementation and Bioadhesion: An example illustration of precipitated calcium carbonate (CaCO3) minerals that can bridge sand grains to provide structural strength. An SEM image showing precipitated CaCO3 bridges between sand grains. Self-healing: An example illustration of the mechanism of self-healing concrete, in which spore-forming biomineralizing bacteria

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embedded within the concrete matrix can proliferate in the presence of a crack, mineralize, and seal the crack. A photograph substantiating the capacity bacteria to heal a 0.4 mm crack over 28 days. Biomass Growth: An example representation of “growing” (i.e., producing) subsequent generations of ELMs from one parent materials by utilizing the exponential rate of biomass production. A photograph of structural ELMs grown using the biomass and biominerals produced by photosynthetic organisms. Figures and images for the figure have been reproduced with permissions (Jonkers 2007; Mujah et al. 2017; De Belie et al. 2018; Heveran et al. 2020)

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cellulose, starch, polyhydroxybutyrate (PHB), or extracellular polymeric substances (EPS). A prime example of a combination of biocementation and bioadhesion in nature is the formation of stromatolites. Stromatolites are produced by photosynthetic marine cyanobacteria that produce EPS biofilm mats that adhere sand particles together. The cyanobacteria also precipitate CaCO3, which mineralizes, strengthens, and toughens the EPS-bound sand particles (Awramik et al. 1976). The continuous biocementation and bioadhesion result in the large, macroscale stromatolite structures observable in many parts of the world. In another example of bioadhesion, fungal mycelium has been used to bind lignocellulosic materials into functional materials using fungal biopolymers (e.g., chitin, chitosan, and other polysaccharides and proteins) (Jones et al. 2020).

2.1.2

Mechanism of Self-Healing

In principle, self-healing is the ability of a material to autonomously repair damage. Biologically induced self-healing of concrete using MICP was first demonstrated at Delft University of Technology in the Netherlands (2007). Since then, three common microbial metabolic pathways have been explored for MICP-based healing in concrete: (1) the ureolytic pathway (as described in Sect. 2.1.1), (2) aerobic conversion of organic compounds, and (3) denitrification (De Belie et al. 2018). In aerobic conversion of organic compounds, inorganic carbon, typically CO2, is produced through the decomposition of organic carbon, such as sugars. The production of CO2 increases the dissolved inorganic carbon in the local medium, which in turn increases CO32
concentrations in alkaline conditions. As in Eq. (2), CaCO3 will precipitate if both Ca2+ and CO32
are present. In denitrification, nitrate acts as an electron acceptor for the decomposition of organic carbon into inorganic carbon, which is advantageous in anaerobic conditions. As microcracks form in self-healing concrete materials, embedded bacteria effectively heal the microcracks through MICP, as shown in Fig. 3. The crack exposes the bacteria to water and/or air, and, in the presence of proper nutrients, calcium ions, and favorable environmental conditions, MICP facilitates crack sealing. The selfhealing mechanism has important implications for increasing the service life of concrete, where a major degradation mechanism is cracking and subsequent exposure and corrosion of steel rebar. Crack filling can protect the rebar from exposure and corrosion, potentially increasing material service life. Self-healing is not limited to MICP-induced crack sealing in concrete. For example, self-healing mechanisms in plants and animals are well documented (Speck and Speck 2019) and can likely be integrated into other ELMs for construction. No matter the biopolymer or biomineral system, most living organisms possess the innate ability to sense damage and respond with a mechanism for repair.

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Mechanism of Biomass Growth

Fundamentally speaking, biomass growth has been utilized for thousands of years in the production of cellulosic building materials, such as timber, straw, or hemp. While typical materials derived from these biomass resources take long timescales to grow (on the order of months to years) and are no longer living, the feasibility of constructing load-bearing structures using living plants (e.g., bamboo), in a process known as Baubotanik, has been demonstrated (Ludwig et al. 2010). Utilization of biomass growth has recently expanded to include several subcategories of ELMs that have demonstrated the successive regeneration, exponential manufacturing, and selfassembly of construction materials. While these ELM subcategories implement the broad mechanism of biomass growth, each targets a specific niche application. Utilizing rapid biomass growth to successively regenerate and exponentially manufacture LBMs, a class of structural ELMs was recently demonstrated by Heveran et al. (2020). Successive regeneration and exponential manufacturing utilize a living parent ELM to propagate subsequent generations of materials, in much the same way a starter culture of microorganisms can be used to seed multiple other cultures. The process capitalizes on the exponential growth rates of microorganisms to perpetuate an increase in the number of ELMs without the exogenous addition of new living components. The work by Heveran et al. utilized temperature and humidity switches that regulated the metabolic activity of cyanobacteria to demonstrate that three successive regenerations of LBMs—eight LBMs in total— could be exponentially “grown” from one parent generation. The successive regeneration and exponential manufacturing concept is visualized in Fig. 3. Although humans have been growing building materials for millennia, in situ bottom-up, self-assembly, and autonomous growth of living structures is an emerging topic in recent ELM literature (Nguyen et al. 2018; Heveran et al. 2020; Gilbert et al. 2021). Currently, most ELM technologies for construction applications involve incorporating abiotic (i.e., non-living) materials (e.g., sand, aggregate) with living organisms to produce hybrid living materials. Future research will certainly probe whether it is possible for organisms to autonomously form ordered, functional ELMs for large-scale construction applications from genetically encoded single seeds or cells. The emergence of this new structural ELM classification is further discussed in Sect. 2.2.5.

2.2

Structural ELM Technologies

Structural ELM technologies leverage these biofunctions to ultimately produce loadbearing material with structural and biological properties. Exemplar structural ELM technologies include biocemented soil, bio-CMUs, self-healing concrete, and LBMs (Fig. 4). The details of these technologies are further discussed in the following sections.

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Fig. 4 Exemplar structural ELM technologies. The background color of each image denotes the predominant biofunction of the technology previously shown in Fig. 3, namely, brown, biocementation and bioadhesion; gray, self-healing; green, biomass growth. Biocemented Soil: a sample of biocemented soil with a volume of 43 m3. Bio-CMUs: a commercially available bio-CMU. Self-healing Concrete: a large-scale implementation of self-healing concrete. LBMs: a bench-top scale LBM truss. Images for the figure have been reproduced with permissions (Van Paassen 2011; Ednie-Brown 2013; Strain 2020; Van Mullem et al. 2020)

2.2.1

Biocemented Soil

Biocemented soils were the seminal application of microbial biocementation to civil engineering (Whiffin 2004). In these early works, sand was inoculated with S. pasteurii, and the resulting materials demonstrated an eightfold increase in shear strength and threefold increase in stiffness (Whiffin 2004). Since then, S. pasteurii has been the organism of choice for biocemented soil. Some other investigators have used indigenous ureolytic bacteria isolated from the soil itself (Burbank et al. 2011; Al-Thawadi et al. 2012). To produce biocemented soil, the primary target biofunctions include biocementation and, because the microorganisms can survive in soil, self-healing, which provide strong, resilient soils that continuously strengthen through MICP. Most applications of biocemented soils still lie within geotechnical engineering, a subdiscipline of civil engineering. These applications include slope stabilization, settlement reduction, erosion control, and liquefaction prevention (Mujah et al. 2017). Biocemented soil is produced using three primary top-down approaches: (1) injection of MICP solutions by pumping, (2) mixing, or (3) surface percolation (Mujah et al. 2017). Pumping MICP solutions into confined columns in the soil is the dominant method, likely due to ease of control. Pumping into the soil has been used in scaled-up applications (Van Paassen 2011; Gomez et al. 2015).

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Despite the popularity of pumping, one notable disadvantage is that pumping can result in inhomogeneity of the biocemented soil (Mujah et al. 2017). By contrast, mixing results in higher degrees of homogeneity at the expense of a significant disturbance of the soil and its natural stability. Surface percolation involves spraying MICP solutions over soils and relying on the natural percolation of microorganisms and the media. While this approach eliminates the need for heavy machinery, the depth of solution penetration can be limited (Mujah et al. 2017). Typically, the target material properties in biocemented soils include mechanical stiffness, shear strength, compressive strength, permeability, and self-healing capacity. Biocementation has been shown to improve mechanical properties, such as stiffness, shear strength, and compressive strength, and lead to reduced permeability. A few studies have also investigated the self-healing capacity of biocemented soils by demonstrating that damaged soils can heal when exposed to additional growth medium (Montoya and Dejong 2013; Harbottle et al. 2014). Most recently, the microorganism Sporosarcina ureae (S. ureae) was investigated for its ability to form spores, survive harsh conditions for extended periods, and ultimately heal damaged soil structures (Botusharova et al. 2020). In that work, the researchers report that S. ureae spores retained viability for at least 6 months and were capable of MICP when the biocemented soil was damaged and exposed to fresh media. Large-scale field experiments of up to 100 m3 and 1000 m3 soil volumes have been performed using the injection-pumping method (Van Paassen 2011). A 100 m3 experiment resulted in a biocemented soil volume of 43 m3 and is highlighted in Fig. 4. In the 1000 m3 experiment, geoelectrical resistivity measurements and CaCO3 measurements performed on excavated biocemented soil indicated successful biocementation. The main challenges to biocementation of soils in this scaled-up application included the inhomogeneity of treated soils and the cost of the treatment. The company bioMASON’s Project Medusa focused on biocementing soil for military-related vertical takeoff and landing operations (bioMASON 2021). For Project Medusa, a surface percolation approach was taken for soil cementation. The biocementation technique developed on the project involves inoculating soil by spraying it with ureolytic organisms and then subsequently “feeding” the organisms by spraying the soil with cementation solutions. Utilizing MICP in vertical takeoff and landing operations offers benefits, such as significant reductions in material and equipment transport and manpower when compared to building traditional concrete runways, which is desired for military operations.

2.2.2

Bio-CMUs

Biomineralizing organisms can be mixed with fine aggregate (i.e., sand) and larger aggregates to produce bio-CMUs for building applications. Bio-CMUs have been commercialized by at least two American and Swedish companies, bioMASON and BioZEment, respectively. While self-healing is theoretically possible for bio-CMUs, commercial products focus exclusively on utilizing the mechanisms of biocementation to produce concrete- and masonry-like alternatives.

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Like most biocemented soil applications, the production of bio-CMUs utilizes the biomineralization capabilities of ureolytic microorganisms, with S. pasteurii being the microorganism of choice (Dosier 2011). In this top-down approach to ELM fabrication, aggregate is first placed in a formwork or mold before being saturated with biocementation solutions and incubated until minerals precipitate and a solid material is formed. In many cases, the biocementation solutions are refreshed until desired mechanical properties are achieved. bioMASON reports that their bio-CMUs can exhibit compressive strengths of 4000–6000 psi and flexural strengths of 550–750 psi—properties that are comparable to masonry units fabricated using portland cement.

2.2.3

Self-Healing Concrete

Self-healing concrete utilizes the same fundamental biomineralization processes utilized to produce biocemented soils and bio-CMUs. However, the primary objective in self-healing concrete, as previously discussed, is crack sealing in response to microstructural damage as opposed to increasing the strength of loose aggregates during material fabrication. Self-healing is often measured in terms of crack closure and mechanical strength recovery. Ultimately, steel rebar in reinforced concrete is what provides concrete with tensile capacity, and the ability to cover and protect the rebar from corrosion is of high interest. When microcracks form in concrete, it is usually on the tensile surface, thereby damaging the protective cover that the concrete matrix provides for the embedded steel. In studies using Bacillus species and lightweight aggregate, 100% crack closure was achieved in cracks varying from 0.1 to 0.8 mm (Wiktor and Jonkers 2011; Zhang et al. 2017). Self-healing concrete has been researched extensively, and more details can be found in previously published comprehensive reviews (Souradeep and Kua 2016; De Belie et al. 2018; Jakubovskis et al. 2020). In self-healing concrete applications, the most effective production of CaCO3 was determined to be metabolic conversion of organic compounds (e.g., urea) under aerobic conditions (Jonkers et al. 2010; Jakubovskis et al. 2020). While ureolytic organisms typically exhibit substantial CaCO3 production, the ammonium by-product can be detrimental to steel reinforcement. Denitrifying organisms provide a chemical environment favorable for steel reinforcement, but express insufficient long-term microbial viability and crack sealing (De Belie et al. 2018). The most robust organisms for self-healing concrete are from the Bacillus genus, such as B. pseudofirmus DSM 8715 and B. cohnii DSM 6307, which remain viable and form spores in the harsh concrete environment (pH > 13) (Jonkers et al. 2010; Jakubovskis et al. 2020). Bacillus species convert calcium lactate to CaCO3, and CO2 from the metabolism of calcium lactate can further react with available portlandite (Ca(OH)2) in the concrete matrix to produce additional CaCO3. This process is represented by the following chemical reactions:

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CaC6 H10 O6 þ 6O6 ! CaCO3 þ 5CO2 þ 5H2 O

ð3Þ

5CO2 þ CaðOHÞ2 ! 5CaCO3 þ 5H2 O

ð4Þ

Most recent self-healing concrete applications utilize microbial encapsulation approaches to improve microbial viability. Hydrogel-based nutrient capsules or lightweight aggregates, such as perlite or clay, are first impregnated with bacteria spores before being added directly to fresh concrete. The spores exhibit higher degrees of germination due to encapsulation. The impregnated hydrogels or lightweight aggregates surround the cells with essential nutrients and result in favorable growth conditions. As concrete damage occurs, the bacteria are exposed to air, water, nutrients, and carbon—all the elements necessary for germination and MICP. Use of lightweight aggregate, for example, helped researchers obtain a relatively constant microbial viability of ~107 CFU per gram of material over 72 days (Jakubovskis et al. 2020). The primary goal of engineering self-healing concrete is to ensure the long-term integrity of the concrete’s strength, durability, self-healing capacity, and microbial viability. However, some strategies employed to produce self-healing concrete can affect other properties of the concrete. Use of lightweight aggregate for self-healing, for example, can reduce the compressive strength of the concrete by 30–40%, but can improve freeze-thaw resistance and decrease the weight of the concrete by 20–50% (Jakubovskis et al. 2020). Long-term microorganism viability remains a major challenge for self-healing concrete. Early tests of viability demonstrated that only 1% of B. cohnii spores remained viable after 9 days in concrete. The company Basilisk, which is commercializing self-healing concrete, utilizes bacteria that can supposedly survive up to 200 years in concrete. It will take decades before conclusive data are obtained to substantiate the claim. The only large-scale implementations of self-healing concrete to-date include the work done by Davies et al. (2018), Van Mullem et al. (2020), and the Basilisk projects. The roof slab cast with bacterial concrete from the Van Mullem et al. (2020) work is shown in Fig. 4.

2.2.4

LBMs

As discussed in the seminal work by Heveran et al. (2020), LBMs are a novel subclass of structural, load-bearing ELMs that leverage the biomineralization capability of microorganisms to produce a strong, tough binder for aggregates without the need for portland cement (Fig. 4). The ~500 psi compressive strength of LBMs is comparable to that of cementitious mortar and greater than that of adobe brick. It was demonstrated that the inclusion of bacteria significantly improved the mechanical properties of LBMs compared to abiotic specimens (Heveran et al. 2020). Notably, microbes in LBMs have been shown to exhibit a survival rate of 9% for 30 days, nearly 10 times higher than that for self-healing concrete. Increased cell viability within LBMs is attributable to the hydrogel-sand matrix, which creates a favorable

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chemical environment compared to portland cement concrete. While LBMs exhibit lower strengths seen in traditional concrete structures, they demonstrate improved cell viability and the capacity for self-regeneration, as described in Sect. 2.1.3. Both ureolytic and photosynthetic biomineralization pathways have been utilized to produce LBMs. The photosynthetic cyanobacterium, Synechococcus sp. PCC 7002, was incorporated in LBMs by Heveran et al. (2020). Cyanobacteria produce CaCO3 minerals through a carbon concentrating mechanism. CO2 is converted into sugars using the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), where HCO3
is concentrated within the cell and OH
is exported outside of the cell, increasing localized pH, and promoting CaCO3 precipitation when in a calcium-rich environment (Kamennaya et al. 2012). In contrast, the ureolytic pathway for MICP was utilized by Qiu et al. (2021) by employing S. pasteurii and an engineered E. coli strain that expresses the urease operon from S. pasteurii (HB101:pBU11) to produce LBMs (Liang et al. 2018). With a focus on improving microbial viability and structural properties, the work by Qiu et al. investigated the effect of various design factors on the properties and performance of LBMs. The results illustrated that the inclusion of trehalose, a natural microbial desiccation protectant, helped maintain bacterial viability at ambient conditions and low relative humidity without affecting mechanical properties of the LBMs produced using Synechococcus sp. PCC 7002. LBMs produced using heterotrophic ureolytic E. coli, which was previously engineered by the authors to exhibit MICP (Liang et al. 2018), demonstrated the most mechanical enhancement compared to the abiotic controls, but these LBMs did not exhibit as high of viabilities as the LBMs produce using Synechococcus sp. PCC 7002. In terms of scale, the size of LBMs is currently limited, as LBMs have only been fabricated at bench-top scale with 200  200  200 cubes for compression testing and 100  100  400 prisms for fracture and flexure testing. While the preliminary results are promising, there is a need for scale-up of these materials to demonstrate viability for structural applications. LBMs offer the exciting potential to be applied as biodegradable temporary relief structures or as temporary military bases in locations where the delivery of traditional construction materials is impeded. Additionally, the full recyclability of LBMs is a notable advantage over traditional portland cement concrete (Heveran et al. 2020).

2.2.5

Emergence of Autonomously “Grown” ELM Structures

Materials that grow and self-assemble into large structures in situ represent an emergent class of ELMs. While there has been limited success with fully grown ELM structures, promising early examples exist. Grown structural materials have been attempted with plants in multiple studies, such as guided growth using trees and bamboo to form living cellulosic structures (Ludwig et al. 2010; Vallas and Courard 2017). In other studies, grown structural materials have been fabricated by encouraging fungal growth into formwork to create structural fungi masses with higherorder structure (Dessi-Olive 2019).

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Other bottom-up fabrications of hierarchical composites of organic and inorganic components produced by microorganisms are a promising route for autonomously grown, self-assembled structural materials, given that there are numerous examples of such hybrid living structural materials found in nature (e.g., bones, stromatolites). Biologically produced synthetic nacres, for example, are an emerging area of research (Spiesz et al. 2019). Further developments could lead to biotic selfassembly of other biogenic polymers and minerals to produce load-bearing materials with emergent mechanical properties. Bacterial cellulose is now being utilized as a matrix for bottom-up living organic-inorganic composites, due to its relatively high mechanical properties and biocompatibility compared to other polymeric matrices (Nguyen et al. 2018; Gilbert and Ellis 2019). Diatoms and coccolithophores, siliceous and calcareous microalgae that form complex microstructures, have been proposed as cellular factories for hierarchical structuring (Nguyen et al. 2018; Srubar 2020). For bottom-up ELMs, a significant grand challenge lies in the programmability of organisms to assemble structural ELMs beyond the single cell (Srubar 2020), lending credence to other fabrication approaches, like 3D printing (Balasubramanian et al. 2019), to help these emerging ELM technologies and their applications scale beyond the benchtop.

3 Engineered Living Non-structural Materials Non-structural building materials, like insulation, flooring, or roofing, are not required to bear the mechanical loads of a building; they serve other functions to improve building performance. The biofunctions that can be utilized to produce engineered living non-structural materials for buildings can facilitate improved energy efficiency, acoustics, or indoor environmental quality for building occupants. Common biofunctions imparted to non-structural ELMs are detailed in Sect. 3.1. Non-structural ELM technologies for buildings are detailed in Sect. 3.2.

3.1

Biofunctions for Producing Non-structural ELMs

Biofunctions utilized to produce non-structural ELMs can improve a building’s performance. Categories of such biofunctions include biofiltration and bioremediation including air purification, thermal regulation, and sensing and response. Representations of the biofunctions are shown in Fig. 5.

3.1.1

Biofiltration and Bioremediation

Biofiltration and bioremediation are biological processing techniques commonly utilized in environmental engineering and industrial biotechnology. Biofiltration

Fig. 5 Common biofunctions for non-structural ELMs. Biofiltration and Bioremediation: a graphical representation of the capacity for plants to remove concentrations of volatile organic compounds, or VOCs, from indoor air. An image of photobioreactors with annotations demonstrating the capacity for photosynthetic organisms to provide oxygen and clean water from CO2, wastewater, and sunlight. Thermal Regulation: a schematic demonstrating how a

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vegetative façade can absorb solar radiation and reduce heat transfer into a building. A rendering of the Process Zero Concept Building that utilizes an algae photobioreactor façade. Sense and Response: a flow chart demonstrating different possibilities for microbial biosensors, including the bioreceptor, signal transducers, and measurable outputs. A photograph of bioluminescent bacteria in flasks, demonstrating a potential signal transducer of a microbial biosensor. Figures and images for the figure have been reproduced with permissions (Dela Cruz et al. 2014; Susorova 2015; Rajkumar et al. 2017; Brodl et al. 2018; Elrayies 2018; Biloria and Thakkar 2020)

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and bioremediation are commonly utilized in water and wastewater treatment, air pollution mitigation, and soil remediation. In addition to these applications, biofiltration and bioremediation functions can be imparted to new classes of ELMs for construction. Biofiltration and bioremediation of water and air can be accomplished using bacteria, vegetation, and microalgae. Ureolytic organisms have been used to purify groundwater. In one study, co-precipitation of strontium and calcite was observed when the microorganisms were added to artificial groundwater (Mitchell and Ferris 2005). It is well-known that plants have natural mechanisms for removal and degradation of volatile organic compounds (VOCs) and excess CO2 from the air (Dela Cruz et al. 2014). Due to human respiration, CO2 levels can be twice as high indoors as outdoors (Lee and Chang 1999). VOCs are also a common indoor pollutant (EPA 2021). Microalgae cultivation presents compelling opportunities for tertiary wastewater removal of inorganic nitrogen and phosphorous (AbdelRaouf et al. 2012), as well as CO2 removal, since microalgae excel at carbon sequestration through photosynthesis. A high potential exists for researchers to incorporate beneficiated waste streams in the production of non-structural ELMs. Treatment of waste gas and water streams, such as flue gas, indoor air, outdoor air, and reclaimed wastewater, by vegetative façades and algae building technologies has been proposed or mentioned in numerous studies (Elrayies 2018; Kisser et al. 2020; Megahed and Ghoneim 2021). In one study from South Africa, researchers fabricated bio-CMUs with CaCO3 precipitated by S. pasteurii using the urea present in human urine (Lambert and Randall 2019).

3.1.2

Thermal Regulation

In developed countries, buildings account for 20–40% of total energy consumption (Pérez-Lombard et al. 2008). The operation of heating, ventilation, and air conditioning systems consume approximately half of the energy used in buildings. The performance and optimization of building envelopes, which regulate thermal transport and contribute to a building’s overall energy efficiency, has been a major focus of the building design and construction community over the past few decades. ELMs with improved thermal properties could enhance the performance of building envelopes, thereby reducing building energy consumption. Energy production and energy savings have been demonstrated to some extent by plants and microalgae in vegetative façades and other algae building technologies. Algae photobioreactor façades can reduce heat transfer into a building all the while generating biofuels (Biloria and Thakkar 2020). In addition to biofuels, algae facades could produce other bioproducts, such as food and pharmaceutical supplements. Vegetative façades can help regulate the local microclimate and contribute to cooling effects by evapotranspiration (Susorova 2015). Regulation of moisture and heat transfer, a process known as hygrothermal regulation, can lead to significant energy savings in buildings (Kreiger 2019). Walls covered by plants, for example, have been shown to reduce temperature extremes and temperature variations and

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regulate the relative humidity near wall surfaces (Sternberg et al. 2011; Vox et al. 2018; Thomsit-Ireland et al. 2020).

3.1.3

Sense and Response

Environmental responsiveness describes the ability for a material to sense and respond to external stimuli. These “smart” materials include materials that are shape-changing, self-actuating, self-sensing, self-diagnostic, and self-healing (Bogue 2014). Common examples of non-ELM “smart” materials include temperature-sensitive shape-memory alloy actuators that can apply stress or displacement in response to external stimuli or smart glass that changes opacity in response to light intensity or temperature. A multitude of “smart” sense and response mechanisms can be found in nature. The ability of ELMs to harness and utilize these capabilities in smart living materials that exhibit environmental responsiveness has been explored extensively by ELM researchers over the past few years to produce a wide variety of non-load-bearing materials and biological sensors that sense and respond to their environment (Nguyen et al. 2018; Gilbert and Ellis 2019; Huang et al. 2019). From a construction perspective, self-healing concrete could be considered an early example of imparting a sense-and-response mechanism to a load-bearing structural ELM. The environmental stimulus in self-healing concrete is crack formation and exposure to environmental triggers (e.g., CO2, H2O) that enable the embedded microorganisms to undergo self-healing through MICP. Future generations of self-healing concrete could have engineered bacteria that directly sense stress and strain in the material and respond with MICP. More recent examples of non-structural ELMs that rely on sense and response mechanisms include living surfaces and microbial biosensors. Living surfaces are essentially living cells embedded in polymeric matrices that respond to external stimuli, such as heat, moisture, light, or even food spills (Nguyen et al. 2018). Microbial biosensor technologies have employed genetically engineered bacteria to build biologic “circuitry” that can read and analyze external stimuli (e.g., heat, light) and respond intelligently (Inda and Lu 2020). Microbial biosensor technologies can be employed in a variety of ELMs that could enable “coding” of organisms by genetic engineering to recognize environmental inputs, convert biological responses into physiochemical signals, and respond accordingly. For building materials, environmental stimuli that could be utilized include heat, moisture, concentrations of volatile molecules and particles in the air (e.g., toxins, viruses, VOCs, CO2), air flow, light intensity, pressure, and deformation. As a response, building materials could perform a multitude of biofunctions. Upon stimulation, for example, materials could adjust building envelope characteristics (e.g., geometry, opacity), adjust hygrothermal properties, or neutralize air contaminants. Building health could be monitored with microbial biosensors. Utilizing bioluminescence as a response, light could be transmitted at different frequencies to electronics (alarms or sprinklers) or even human eyes. Structures that glow red

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could indicate to pedestrians or building occupants that air quality is unhealthy. In short, opportunities abound for imparting environmental responses to non-structural ELMs.

3.2

Non-structural ELM Technologies

Non-structural ELM technologies produced to-date include mycotecture, vegetative façades, algae building technology, and living surfaces. These technologies are detailed in the following sections. Representations of these ELM technologies are shown in Fig. 6.

3.2.1

Mycotecture

With origins as a biodegradable, environmentally friendly alternative to packaging and insulation materials, the field of mycotecture has emerged as a commercially viable ELM building technology. The company Ecovative Design envisioned utilizing MycoComposite, a mix of fungal mycelium and agricultural waste products from cotton or hemp, to generate biodegradable packaging (Holt et al. 2012). The biobinding and material growth capabilities of the mycelium composites quickly evolved into research thrusts toward the development of similar mycelium-based composite materials for construction applications. Selecting appropriate fungal species are a critical component of mycotecture. Fungal mycelium must be non-hazardous to humans and compatible with the selected substrate to ensure sufficient growth. Species and substrate compatibility dictate the properties of the composite. Since the growth substrate is often a plantbased waste by-product with high cellulose and lignin content, fungal species capable of digesting cellulose and lignin, such as Trametes, Ganoderma, and Pleurotus genera, or the phylum Basidiomycota, are typically implemented to ensure good mycelium growth (Holt et al. 2012; Vallas and Courard 2017; Jones et al. 2020). The top-down fabrication process for mycotecture materials is as follows. First, low-cost, fibrous plant waste is hydrated and homogenized. Then, the substrate is sterilized before being inoculated with fungal mycelium. The composite is then cast into a mold and allowed to incubate for 4–7 days at ambient conditions to facilitate mycelium material growth. Finally, the materials are demolded and dehydrated. Mycotecture materials have exhibited low densities (60–300 kg/m3) and good thermal conductivity (0.04–0.08 W/mK), yielding a competitive material to traditional insulation materials (Jones et al. 2020). Further bolstering its viability as an insulation material, mycelium composites have demonstrated 70% acoustic absorption at 1000 Hz, as well as some fire-retardant properties due to the mycelium substrate (Jones et al. 2020). Based on its material properties, Jones et al. (2020)

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Fig. 6 Representative examples of non-structural ELM technologies. The background color of each image denotes the predominant biofunction of the technology previously shown in Figs. 3 and 5, namely, brown, biocementation and bioadhesion; green, biomass growth; blue, biofiltration and bioremediation; orange, thermal regulation; purple, sense and response. Mycotecture: an image of a mycelium composite. Vegetative Façades: an example of a vegetative façade found at the University of Chicago. Algae Building Technology: a photograph of the BIQ algae building in Hamburg, Germany, demonstrating implementation of algae photobioreactors. Living Surfaces: a photograph of fungi proposed to be used as living surfaces. Images for the figure have been reproduced with permissions (Gerber et al. 2012; Susorova et al. 2014; Jones et al. 2018; Biloria and Thakkar 2020)

also hypothesized that mycotecture composites can replace “foam, timber and plastic insulation, door cores, panels, flooring, [and] furnishings.” Large-scale mycotecture applications have included a vault with dimensions of 2.5  2.5  2.5 meters and a volume of 2.75 m3 (~800 kg of mycelium material) that was produced by growing the materials within a prefabricated form. One challenge for larger mycotecture applications is that formwork strategies must be as rigorous as those of traditional concrete forms to fabricate a consistent final product (DessiOlive 2019). Mycelium composites have also been used as an insulation component of a living tree house (Vallas and Courard 2017). Taken together, these large-scale installations demonstrate the promise and scalability of mycotecture composites as commercially viable ELMs for construction applications.

3.2.2

Vegetative Façades

Green façades and living walls are two designations of vegetative façades that have been gaining significant attention in the building design and construction community over the last two decades (Susorova 2015; Radić et al. 2019). Vegetative façades can

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provide biofiltration and bioremediation and thermal regulation functions by improving air quality and providing moisture and heat buffering capacity to building envelopes. Plant species used in vegetative façades are influenced by the region of implementation, since the façades depend on the local climate to ensure flora prosperity. Aesthetics and targeted functions are other aspects that are considered in species selection. Vegetative façades are fabricated through various top-down approaches. In many applications, plant vines are structured so that they proliferate up a wall. Plants have also been built into façade frames that hold growth medium and help transfer weight into the structure. Vegetative façades offer a variety of benefits to the local environment, including improved thermal regulation of building envelopes, biofiltration, bioremediation, and acoustic attenuation. A recent study reported that building envelopes can be improved using vegetative façades, which can lead to 30% savings in energy used to heat buildings (Radić et al. 2019). Vegetative façades sequester CO2 and increase urban O2 concentrations, as well as reduce urban NO2 concentrations by up to 40% (Radić et al. 2019). Vegetative façades can also lead to noise reductions of up to 40 dB (Radić et al. 2019). Vegetative façades have been implemented at the building scale (Fig. 6). Dozens of vegetative façades have been installed across the world, with a multitude of companies offering design and installation services of vegetative façades to improve sustainability and energy efficiency of the built environment.

3.2.3

Algae Building Technology

Algae are photosynthetic organisms that require light, nutrients, and CO2 to grow. Algae can proliferate in either fresh- or saltwater systems. The Bio Intelligent Quotient (BIQ) algae house in Hamburg, Germany, showcased the potential use of photobioreactor façades. The BIQ algae house has 120 SolarLeaf flat panel photobioreactors mounted on the façade, which covers 200 m2 of the low-energy residential building (Wilkinson et al. 2016). Algae biofiltration is beneficial for two main reasons. Algae sequester CO2 and algal biomass can be used to produce energy. Through photosynthesis, algae consume CO2 and produce biomass. Subhadra and Grinson-George (2011) substantiated that each kg of algae growth equates to approximately 2 kg of sequestered CO2. The biomass can be collected and transformed into biogas in a hydrothermal process (Talaei et al. 2020). Algae façades can be used to improve the carbon neutrality of a building by using the building’s indoor air ventilation system, which replenishes high-CO2 indoor air with fresh air, as a source of CO2 for the photobioreactors. Building envelope enhancement using algae provides enhanced thermal performance via three main functions: adaptive shading, thermal insulators, and solar thermal collectors (Wilkinson et al. 2016; Talaei et al. 2020). An increase in biomass allows adaptive shading in accordance with culture density. The absorption of sunlight for photosynthesis provides solar thermal collection (Talaei et al. 2020). Wilkinson et al. (2016) reported that algae photobioreactors could provide noise

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abatement, but the capability of algae to provide acoustic benefits has not yet been directly investigated. Algae building technologies are produced using a top-down approach. Algae media is introduced into a pre-built, flat-plate, hollow glass panel within a façade frame. Species such as Arthrospira platensis, Chlorella vulgaris, and Haematococcus pluvialis have been used in such algae photobioreactors (Talaei et al. 2020). However, species selection is heavily dependent on the local climate for implementation of the façade. For a healthy and productive photobioreactor, there are a few necessary requirements: medium light (1 k–10 k lux), growth temperature 16–27  C, nutrients (salinity, CO2, ammonia, phosphate, oxygen), pH between 7 and 9, circulation, and a sufficient photoperiod (i.e., the amount of light that is received per day) (Biloria and Thakkar 2020). Due to their nutritional requirements, algae photobioreactors could be integrated with on-site wastewater processing and air purification for large commercial buildings. Algae can remove nitrogen and phosphorous from wastewater, a crucial step in water remediation (Lage et al. 2018). Currently, however, few buildings possess on-site wastewater processing capabilities. Those that do only process greywater, which does not have sufficient nutrients to support algal systems. Greywater algal systems could be supplemented with nutrients from other nearby sources. On-site blackwater processing, which has yet to become mainstream, could also provide higher concentrations of nutrients for algal systems. The ability of algae to sequester CO2 directly from indoor air and from commercial CO2 sources, such as flue gases that output large quantities of CO2, has also been investigated (Lage et al. 2018). On-site algae photobioreactors would consume the CO2 and further convert it into bioenergy through the generation of biomass. While utilizing algae façades for wastewater, air purification, or flue gas remediation has been proposed in literature (Elrayies 2018), they have yet not been thoroughly investigated for these specific purposes. Biloria and Thakkar (2020) performed a life cycle analysis to compare a solar building façade to an algae photobioreactor façade that was not connected to on-site wastewater, air, or flue-gas sources. The researchers determined it would take 24 years for current state-of-the-art photobioreactors to turn a profit and 36 years for that profit to exceed the solar façade. While these results suggest that algae facades are currently cost-prohibitive, it is important to consider the efficiencies that will result from market maturation and future technological developments, such as deep integration with on-site wastewater, air purification, and point-source CO2 sequestration. When the initial costs of energy derived from solar was compared to energy derived from fossil fuels, it was found to be prohibitively expensive. However, solar is now an economical alternative to conventional fossil-fuel energy sources (Mathiesen 2016).

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Living Surfaces

Living surfaces are an emerging ELM technology (Nguyen et al. 2018; Gilbert and Ellis 2019; Huang et al. 2019; Inda and Lu 2020). The earliest examples of living surfaces were introduced in 2012 (Gerber et al. 2012). Living surface materials consist of living cells housed in polymeric matrices that are subsequently applied to interior walls. Ultimately, living surfaces embody the true nature of ELMs, which are capable of sense and response in the presence of environmental stimuli. Organisms that have been—or could be—employed in living surface research include bacteria, fungi, microalgae, or consortiums of organisms. Early living surface examples from 2012 employed fungi as a self-cleaning surface. The fungi surfaces demonstrated an ability to metabolize and effectively clean food spills (Gerber et al. 2012). Lichen, which are symbiotic consortia of fungi and algae or cyanobacteria, have recently been investigated as interior building materials for hygrothermal regulation (Kreiger 2019). In the future, microbial biosensor technology could also be integrated with living wall surfaces (Nguyen et al. 2018; Inda and Lu 2020).

4 Grand Challenges and Final Remarks The challenges that structural and non-structural ELMs must overcome include economic feasibility, uncertainty, scale-up, long-term organism viability, and biocontainment. Despite these obstacles, the recent commercial success of some ELM technologies has provided a roadmap for the development and commercialization of other structural and non-structural ELMs for construction applications.

4.1

Economic Feasibility in a Commoditized Market

As industrial biotechnologies, many ELMs have high capital costs associated with their production. ELMs are competing with commodity materials like concrete, which is considered cheap, accessible, and high-performing due to its proven strength and long-term durability. While current ELM technologies may be more expensive, capitalizing on waste streams and industrial bioprocesses that have already scaled can improve economic competitiveness. Additionally, cost feasibility would be aided by new environmental regulations, such as a carbon tax, that would economically penalize the excessive carbon emissions associated with the production of traditional building materials, enabling ELMs to be more economically competitive.

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Uncertainty

ELM systems are not well defined and, like many biological systems, can be highly variable and unpredictable. Biological systems tend to exhibit a lot of inherent heterogeneity. In general, new technologies also carry uncertainty due to the lack of broad implementation and empirical evidence of their performance. There is a need for consistent and repeatable manufacturing that provides reliable results in terms of the final product and its properties. When property owners invest in the economic venture of erecting buildings, they expect consistent, quality products that will serve their long-term needs. Defaulting to dependable, proven technologies is a common method of risk management. Establishing strong manufacturing and operating methods, predictive modeling tools, and cost-benefit analyses will be necessary to inform decision-making for the future implementation of ELMs in construction.

4.3

Scale-Up

Scale-up is a difficult task for any new biological technology. Large-scale implementation takes time to perfect and additional troubleshooting. Increasing the scale of ELM production introduces obstacles when considering large quantities of biological organisms and timescales required for their growth. Depending on the species, organisms can take a long time to proliferate to generate necessary population sizes for a single building application. For example, Armillaria ostoyae is a species of fungus that has possibly produced the largest living organism. In Oregon, an A. ostoyae mycelium network covers over 3.5 square miles and could weigh up to 35,000 tons (Schmitt and Tatum 2008). While this scale is comparable to the urban and building scale, this organism is up to 9000 years old (Schmitt and Tatum 2008). In contrast, algae can double in biomass on the order of hours (Dauta et al. 1990). While native organisms can take a long time to grow, speeding up natural processes through metabolic engineering can enable new biotechnologies to compete with commoditized construction material production.

4.4

Organism Viability

Since organisms have nutritional and environmental requirements to remain viable, they often cannot withstand harsh environments. Enzyme-based stabilization, synthetic coatings, microfluidic techniques, and/or natural protective mechanisms are technological pathways researchers can take to increase viability (Srubar 2020). Optimally, mass transfer through microfluidic techniques similar to a circulatory system or cell specialization, in which “root” cells could be attached to a nutrient

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source and transfer products to the rest of the ELM, could be implemented into ELMs for improved longevity of organisms. Competing organisms can be a threat to nutrition-rich ELMs. Typical competing organisms include fungi and bacteria, which thrive in the typical wet, nutritional environments seen in ELMs to sustain the original incorporated organisms. It is necessary to investigate strategies like natural protective mechanisms, antibiotics, and fungicides, where the organisms within the ELM have an engineered resistance to allow the outperformance of competitors. Using extremophiles that are among the select few that can function in certain environmental conditions is another approach to limit competition. For example, the nutrient and energy-poor conditions in which photosynthetic cyanobacteria thrive make it challenging for other non-photosynthetic organisms to compete.

4.5

Biocontainment

The proliferation of genetic material from ELMs in the environment can be a concern, as invasive species have been shown to be incredibly disruptive to local ecosystems. ELMs have rightfully earned a descriptor as biohazardous materials, which insinuates problematic issues regarding disposal at the end of life if ELMs cannot be recycled. Fortunately, many ELMs incorporate recycling and regeneration in their material design to extend service lives and mitigate potential biohazard disposal considerations. Selection of indigenous and wild-type organisms should remain a major consideration in ELM design to avoid spreading of genetically modified organisms. Eventually, strategies will need to be developed for ELMs to contain them within normal supply chains.

4.6

Final Remarks

Structural and non-structural ELMs are nascent building material technologies, and, while they offer an incredible opportunity for technological advancement, they also introduce a multitude of challenges that must be considered prior to large-scale implementation. The convergence of multi-disciplinary thought is generating revolutionary solutions to complex infrastructure challenges. Ample opportunities exist for creating new, living urban ecosystems that are self-reliant and self-sustaining through the integration of the built environment and the natural world. Acknowledgments This research was made possible by the Department of Civil, Environmental, and Architectural Engineering, the College of Engineering and Applied Sciences, and the Living Materials Lab at the University of Colorado Boulder with financial support from the United States (US) Defense Advanced Research Projects Agency (Award No. HR0011-17-2-0039). This work represents the views of the authors and not necessarily those of the sponsors.

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