Interfaces in Microbial Ecology [Reprint 2014 ed.] 9780674423350, 9780674423336

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Interfaces in Microbial Ecology

Interfaces in Microbial Ecology

K. C. Marshall

Harvard University Press Cambridge, Massachusetts, and London, England

1976

Copyright © 1976 b y the President and Fellows of Harvard College All rights reserved Printed in the United States of America Library of Congress Cataloging in Publication Data Marshall, Kevin C Interfaces in microbial ecology. Bibliography: p. Includes index. 1. Microbial ecology. 2. Surface chemistry. I. Title. QR100.M37 576'. 15 76-7509 ISBN 0-674-45822-2

Preface It is my belief that many microbiologists fail to appreciate the effects of interfaces on microbial populations, despite the widespread occurrence of solid-liquid, gas-liquid, and liquid-liquid interfaces in natural microbial habitats. Conseqently, this monograph was written to emphasize the nature, distribution, and unique physicochemical properties of interfaces, the interaction between microorganisms and interfaces, and the modifying effects of interfaces on the ecology of microorganisms. I have aimed at providing a conceptual basis for more realistic studies on the ecology of microorganisms in natural habitats. This has been achieved by considering the physicochemical and biological properties of bacteria (regarded as living colloids) in relation to the physicochemical properties of different interfaces. Principles derived from this approach provide a reasonable understanding of microbial distribution in natural ecosystems. In addition, I have aimed at creating an awareness of developments in microorganisminterface interactions in seemingly unrelated areas of microbiology. Interest in microbial adhesion to solid surfaces, for example, is evident in medical, dental, aquatic, agricultural, and industrial microbiology; yet it is apparent that some researchers in individual areas of microbiology are blissfully unaware of progress in the other areas. Thus, I have presented a broad coverage of both theoretical and applied aspects of microbial adhesion processes in different ecosystems, and have emphasized similarities existing between the behavior of microorganisms at various types of interfaces. Comprehensive reviews of particular areas have been avoided, although the contents do reflect my particular interest in soil and aquatic ecosystems. I have neglected some important microbial ecosystems, but believe that the principles and applications described herein provide an adequate background for the appreciation of interfacial phenomena in microbial ecology. I am indebted to many colleagues at the University of New South Wales, the University of Tasmania, and Harvard University for

vi

Preface

advice and helpful discussions. In particular, I wish to express my appreciation to Professor Ralph Mitchell, Harvard University, for his encouragement when this monograph was first envisaged. Thanks are due to Mrs. Beverley Humphrey and Professor Geoff Cooper, the University of New South Wales, for comments on the manuscript and for bibliographic assistance. Finally, I thank Mrs. Gay Wylie for her patience and cooperation when typing the drafts and final copy of the manuscript. K. C. Marshall School of Microbiology The University of New South

Wales

Contents 1. Introduction to the Concept of Interfaces in Microbial Ecology Microorganisms in Natural Habitats What Is an Interface? Bacteria as Living Colloidal Systems 2. Liquid-Liquid and Gas-Liquid Interfaces Interfacial Tension Bacteria at Liquid-Liquid Interfaces Factors Influencing Bacterial Behavior at Liquid-Liquid Interfaces Bacteria at Gas-Liquid Interfaces 3. Solid-Liquid and Solid-Gas Interfaces Adsorption of Macromolecules at Solid-Liquid Interfaces Attraction of Ions to Surfaces—The Diffuse Electrical Double Layer Attraction of Bacteria to Solid Surfaces Firm Adhesion of Bacteria to Surfaces Microbe-Microbe Interactions (Aggregate Formation) Adsorption of Colloids and Macromolecules to Bacterial Surfaces 4. Nonspecific Interfacial Interactions in Microbial Ecology: Aquatic Ecosystems Some Definitions Periphytic Microorganisms Microorganisms in the Bulk Liquid Phase (Plankton) Epiphytic Microorganisms Tripton-Associated Microorganisms Benthic Microorganisms Neuston-Associated Microorganisms

1 1 2 5 11 11 12 17 21 27 28 35 36 44 47 49 53 53 54 64 65 67 70 74

viii

Contents

5. Nonspecific Interfacial Interactions in Microbial Ecology: Terrestrial Ecosystems Soil Structure and Microbial Microhabitats Ecology of Soil Microorganisms The Rhizosphere The Phyllosphere Microorganisms and Mineral Dissolution

81 82 88 98 100 102

6. Specific Interfacial Interactions in Microbial Ecology Microbe-Microbe Interactions Microbe-Plant Interactions Microbe-Animal Interactions

107 108 117 119

References Index

129 153

Figures 1.1.

Cells of a kerosene-utilizing bacterium at a kerosenewater interface 1.2. Size range of living organisms and colloidal particles 1.3. pH-electrophoretic mobility curves for Rhizobium 1.4. Effect of pH on surface ionogenic groups of Rhizobium 2.1. Interfacial tensions and bacterial behavior at an oilwater interface 2.2. Orientation of bacteria at an oil-water interface 2.3. Growth of hydrocarbon-utilizing yeast, by substrate concentration and drop radius 3.1. Types of adsorption isotherms 3.2. Adsorption isotherms for bovine serum albumin on hydrophobic and hydrophilic silica 3.3. Zisman plot showing wettability of poly tetrafluoroethylene by the n-alkanes 3.4. Infrared scans of monolayers transferred to germanium prisms 3.5. Gouy-Chapman and Stern models of double layer 3.6. Resolution of a binary mixture of bacteria by ionexchange techniques 3.7. Potential energy of interaction between two colloidal particles 3.8. Energy of interaction between glass and bacterial surfaces 3.9. Reversible sorption of bacteria, by electrolyte valency and concentration 3.10. Electron micrograph showing orientation of Flexi bacter CW7 at a solid-liquid interface 3.11. Polymer fibrils at a solid-liquid interface 3.12. Electron micrographs showing orientation of clay platelets at bacterial surfaces 4.1. Extracellular metabolism of insoluble substrates at solid-liquid interfaces

3 6 9 10 13 15 20 29 30 31 33 35 40 41 42 44 45 46 52 56

χ 4.2. 4.3. 4.4. 4.5. 4.6. 4.7. 5.1. 5.2. 5.3. 5.4. 5.5. 5.6. 6.1. 6.2.

Figures Selective irreversible adsorption of bacteria Effect of chloramphenicol on sorption of bacteria to glass Clay envelope providing Escherichia coli with protection from phage attack Comparison of biologically important parameters with mean diameter of sediment particulates Numbers of bacteria desorbed from sediment at different electrolyte concentrations Bacterial concentration at surface microlayers by different sampling techniques Increase in sorption of bacteria with increasing clay content in soils Scanning-electron micrograph of soil void Water adsorption isotherms for Ca-montmorillonite and for rhizobia Actinomycete growth across drained soil voids Association of microorganisms with root surfaces Erosion of sulfur granule by attached cells of Sulfolobus Attachment of phages to the F-pilus and effect of phage fragments on mating-pair formation Bacterial attack of chlamydospores of Thielaviopsis basicola in soil

58 66 71 73 75 78 84 87 95 97 101 104 111 117

Tables 2.1. Standard surface tension values of pure liquids in air and standard interfacial tension values between water and pure liquids 2.2. Initial spreading coefficients for liquids on water 2.3. Effect of a wetting agent on the partitioning of Mycobacterium phlei from water into oil 3.1. Critical surface tensions of low-energy surfaces 3.2. Critical surface tensions and Zisman plot slopes for germanium prisms immersed in marine bacterial cultures 3.3. Diffusion coefficients and Brownian displacements for uncharged spheres in water 3.4. Electrophoretic mobilities of model particles in natural and artificial seawater 4.1. Survival of Escherichia coli in the presence or absence of phage and/or colloidal montmorillonite 4.2. Adsorption of Serratia marcescens by sediments from salt lakes 4.3. Numbers of protozoa and diatoms in surface microlayer and subsurface seawater samples 5.1. Size distribution and surface area of solid particles found in soils 5.2. Saturation levels of sorbed clays for different Rhizobium species 5.3. Effect of saturating cation on sorption of bacteria to soil and on CO2 evolution from glucose 5.4. Nutritional requirements of soil and rhizosphere microorganisms 5.5. Incidence of silicate-dissolving bacteria, actinomycetes, and fungi in stones 6.1. Effect of selected enzymes and Con A upon the agglutinability of Chlamydomonas gametes 6.2. Distribution of oral bacteria

12 19 21 32

34 38 50 70 72 76 83 89 90 99 103 109 120

Interfaces in Microbial Ecology

1

Introduction to the Concept of Interfaces in Microbial Ecology Ecology is essentially a revolt from the analytic and experimental methods of the laboratory, on the ground that these give no adequate conception of the organism as a whole in relation to its environment, and a return to the methods and aims of the old-fashioned field naturalist, freed however from teleologic bias and with the addition, as far as possible, of the precision Arthur of the T.laboratory. Henrici (1933)

Microorganisms in Natural Habitats Microbial ecosystems are characterized by the presence of diverse microbial species engaged in various activities of functional significance within the ecosystems. Much effort has been devoted to isolating and characterizing microorganisms, and to cataloguing their enormous range of physiological properties. Studies on pure cultures of microorganisms are essential in assessing the potential capabilities of individual species in natural habitats, but the excellent taxonomic, biochemical, and physiological studies undertaken with pure cultures in the laboratory have diverted microbiologists from an original aim of their science; that is, to understand the behavior of microorganisms in various natural habitats. An individual microbial species in a natural habitat is confronted by physical, chemical, and biological interactions rarely encountered under pure culture conditions. They may be subjected to extreme fluctuations in nutrient availability, temperature, pH, or osmotic pressure, as well as varying degrees of stimulation or inhibition by other microorganisms. Relationships of microorganisms to each other and to their chemical and physical environment are the concern of the microbial ecologist. General concepts of ecology as applied to microbial situations have been presented by Brock (1966a) and Alexander (1971) and, consequently, are not considered here. Microbial ecology may be studied at the macrohabitat

2

Interfaces in Microbial

Ecology

level, as in a river estuary, a soil profile, or an animal intestine, or at the microhabitat level, as in the immediate vicinity of one or several microorganisms. Environmental variations within several micrometers are significant in relation to individual microorganisms because of their small size, limited mobility, rapid growth rates, and ability to excrete metabolic by-products that may rapidly alter their microhabitat. A knowledge of the behavior of microorganisms within microhabitats is essential in our attempts to define their activities in different ecosystems. Interfaces are significant physical features encountered by microorganisms in most microhabitats, and these interfaces have profound effects on the activities of microorganisms. It is my intention to indicate the nature, occurrence and physical properties of interfaces, to discuss the interactions between microorganisms and interfaces, and to review present knowledge concerning the effects of interfaces on the ecology of microbial communities. What is an Interface? In physicochemical terms, an interface is the boundary between two phases in a heterogeneous system. Natural microbial habitats are heterogeneous systems consisting of a wide range of liquidliquid, gas-liquid, solid-liquid, and solid-gas interfaces. These phase boundaries possess physical and chemical properties that differ from those of either phase in a two-phase system, and their presence can modify the microhabitats and, in turn, the relationship of microorganisms to each other. The unique physical forces operative at interfaces are weak and are effective only over short distances, but they have a significant effect on the distribution of ions, macromolecules, and larger materials of colloidal size in the vicinity of the interfaces. Microbial activity at or near interfaces is modified by such microenvironmental changes as altered gas availability, pH changes resulting from the attraction or repulsion of H + ions, the concentration or depletion of essential microbial nutrients or of inhibitors (e.g. antibiotics), and altered activity of extracellular enzymes resulting from configurational changes and masking effects in macromolecules adsorbed at an interface.

Introduction

3

Aspects of the role of interfaces in microbial ecology have been considered briefly by Brock (1966), Codner (1969), and Newman (1974). Before embarking on a detailed consideration of the physical, chemical, and biological significance of interfaces (Chapters 2 and 3), I will present some examples to illustrate the importance of interfaces in certain aspects of microbial ecology. An obvious example of a liquid-liquid interface of biological importance is the oil-water phase boundary. Most oils are immiscible in water, giving a distinct phase boundary to which microorganisms with more hydrophobic surfaces tend to be attracted. Selective colonization of the interface by such organisms does not imply that they utilize the substrates available at the interface. Obviously, the most successful microorganisms colonizing the interfacial region would combine the characteristics of a relatively hydrophobic surface with an ability to utilize all or part of the nonaqueous phase as an energy source. It is these microorganisms that play an important role in the biodegradation of oil pollutants. The growth rate of oil-degrading microorganisms is limited by the available interfacial area. This effect is illustrated in Figure 1.1, Figure 1.1. Cells of a kerosene-utilizing bacterium (Pseudomonas sp.) occupying most of the interfacial area between the aqueous and kerosene phases. Phase-contrast micrograph (x 2,500).

4

Interfaces in Microbial

Ecology

where the complete colonization of kerosene-water interfaces by kerosene-utilizing bacteria (Pseudomonas sp.) obviously limits access to the interface by other bacteria in the bulk aqueous phase. The smaller the droplet size for a given volume of oil, the greater the interfacial area available for microbial colonization. Gas-liquid phase boundaries are found at surfaces of bodies of water, in air bubbles or other gas bubbles in aquatic systems, and in soil voids (pores). Aerobic microorganisms at the interface will have access to oxygen, the availability of which is limited in the aqueous phase by low solubility and microbial utilization. Similarly, gas-liquid interfaces are significant in the ecology of microorganisms capable of utilizing nitrogen, carbon dioxide, carbon monoxide, hydrogen, hydrogen sulfide, methane, ethane, ethylene, and various hydrocarbon vapors. These gases can be produced by certain groups of microorganisms in anaerobic micro-sites in soils and sediments, accumulating as isolated pockets or bubbles of gas and serving as readily available sources of gas for utilization by other groups of microorganisms. Again, these gas-utilizing organisms are dependent upon the aqueous phase for growth, and the interface provides the ideal microhabitat for the most efficient provision of dissolved gas. The most important interfaces affecting microbial behavior in terrestrial, aquatic, and other natural habitats are those of the solid-liquid type. Solid surfaces are very heterogeneous and include colloidal materials (clays, organic matter), sand grains, rock surfaces, larger organisms (algae, aquatic plants, roots in soils, fishes), large man-made surfaces (pipelines, ship hulls, piers), the walls of animal intestines, and the surfaces of human teeth. Opportunities for the selective adhesion of microorganisms are provided by these solid surfaces. Examples of microbial adhesion to surfaces include the primary microbial films found on ship hulls (Corpe, 1970) or hydroelectric pipelines (Tyler and Marshall, 1967). These films are sufficiently extensive to be macroscopically visible, and are economically important because they reduce the efficiency of water flow through pipelines or of ships through water. The adhesion of microorganisms to solid surfaces in microhabitats within complex ecosystems is not generally obvious, but it has a significant effect on the ecology of microorganisms in such systems (Marshall, 1971; Savage, 1972; Gibbons and van Houte, 1971).

Introduction

5

Solid-gas interfaces modify microbial behavior when the relative humidity of the gas phase is high enough to permit growth of some microorganisms. In general, fungi can grow at lower relative humidities than bacteria and are afforded a selective advantage at solid-gas interfaces under these conditions. Microorganisms at solid-liquid interfaces subjected to intermittent drying will be exposed occasionally to solid-gas interfaces. This condition occurs regularly in soils, and in sediments in areas subjected to fluctuating water levels. Survival of desiccated microorganisms at solid-gas interfaces can be influenced by the nature of potentially protective colloids or macromolecules sorbed to the solid surface, the relative humidity of the gas phase, and the nature of the gas phase. The contribution of interfaces to the observed activities of microorganisms in ecosystems has often been ignored. An awareness of the importance of interfacial effects in the ecology of microorganisms adds a further dimension to our understanding of the behavior of these organisms in complex environments. Bacteria as Living Colloidal Systems How are microorganisms attracted to interfaces and why is there a selective adherence by some to solid surfaces? To answer these questions it is necessary to consider the physicochemical properties of both the microorganisms and the interfaces, then relate these properties to the functional characteristics of the organisms involved. Microorganisms range in size from the smallest virus (25nm diam), through an average bacterium (Ιμπι diam), to macroscopically visible filamentous fungi and algae. Viruses and the smallest bacteria (Figure 1.2) overlap the generally accepted size range of colloidal particles (20Ä to 0.2μπι diam). However, most bacteria behave as colloids in aqueous suspension even when the external dimensions of the cells exceed the normal upper limit assigned to colloidal particles. This results in part from the mutual electrostatic repulsion between the negatively-charged bacteria. Much of my discussion on the behavior of microorganisms at interfaces is based on a concept of bacteria as living colloidal systems, and it will become obvious that many principles applicable to interactions between bacteria and interfaces are applicable to other microorganism-interface systems. Let me stress that bacteria are

6

Interfaces in Microbial

Ecology Metres ο

MAN

SMALL SMALL

BIRDS

10 - 2

INSECTS

-• 10

-3

10 ALGAE FUNGI

,

( ι mm ,

10

AND.

PROTOZOA

ι

r

-

10

BACTERIA·

10 °

( 1 pm

VIRUSES COLLOIDS.

10

,, HYDROGEN

ATOM

10"9

( l nm

. 1 io~ 10 ( ι A ;

Figure 1.2. A schematic representation of the size range (logarithmic scale) of living organisms and of colloidal particles.

not inert colloidal particles. They are living organisms and, as such, are capable of growth, metabolism, and, in some instances, of independent motion. These biological characteristics play an important role in the initial attraction of bacteria to interfaces and in the degree of selectivity evident in the adhesion of some bacteria to solid surfaces. The behavior of colloidal suspensions can be appreciated when the electrical properties of the particles are taken into consideration. Colloidal dispersions acquire a surface charge either by ionization of surface groups or by the sorption of ions from solution.

7

Introduction

Some colloidal particles are positively charged (e.g., ferric oxide sols), whereas others are negatively charged (e.g., arsenious sulfide sols). Proteins are amphoteric, acquiring their charge mainly through the ionization of amino (-NH 3 + ) and carboxyl (-COO") groups, and may have a net negative or positive charge depending on the pH of the liquid phase. The isoelectric point of an amphoteric compound is the point at which the net charge on the particle is zero. If the primary charge on a colloidal particle is acquired by the ionization of strongly basic or acidic groups (e.g., sulfonated polystyrene latex particles), then the charge varies little with changes in pH. Air bubbles and oil droplets acquire a negative surface charge by preferential sorption of anions from an aqueous medium, because the anions are smaller (less hydrated) and more strongly polarizing than cations. Charged macromolecular materials, such as proteins, adsorb to oil-water, air-water, and solidwater interfaces, resulting in surface-charge properties that are characteristic of the adsorbed material (Abramson et al., 1942). Obviously, this phenomenon of ion and macromolecular adsorption at interfaces is important as a means of nutrient concentration for microbial utilization in nutrient-deficient natural habitats. The electrokinetic properties of bacteria and other colloidal particles are studied by microelectrophoresis, whereby the charged particles are introduced into a specially constructed cell connected to electrodes, an electric potential is applied, and the velocity of the particles over a known distance is determined (Abramson et al, 1942). The electrophoretic mobility may be calculated from the expression ν =

VKsq/i

where ν is the electrophoretic mobility (μιη sec" l y - 1 cm~-M, ν is the velocity of the particles (μιη sec'^), xs is the specific conductivity (mhos cm"l) of the buffer solution, q is the cross-sectional area (cm^) of the electrophoresis cell, and ί is the current (amps). When a charged particle is suspended in an electrolyte, ions of the opposite charge (counter-ions) are attracted to the particle surface. Most of these ions become loosely associated with the particle to form the so-called diffuse electrical double layer (see Chapter 3 for a more detailed account of this concept). When an electric potential is applied to a colloidal suspension, the charged particles,

8

Interfaces

in Microbial

Ecology

along with some firmly attached ions and water molecules, migrate towards the appropriate electrode. The remaining counter-ions of the diffuse double layer move in the opposite direction. The dividing line between those materials migrating with the particles and the remainder of the double layer is termed the plane of shear or the slipping plane. The potential at the plane of shear, relative to the bulk medium, is the electrokinetic or zeta (ζ ) potential. Because some counter-ions are present inside the plane of shear, the magnitude of the zeta potential will be smaller than the true surface potential. For relatively large particles such as bacteria, the relationship between electrophoretic mobility and zeta potential (in mV) is given by the Helmholtz-Smoluchowski equation ζ = 4 πην/D where η and D are the viscosity and dielectric constant, respectively, in the diffuse double layer. At the low ionic strengths usually employed, the values of η and D are taken as those for water. In an aqueous medium at 25 °C, this expression reduces to £ = 12.9 ϋ. The charge density (a) in e.s.u. cm"^ at the plane of shear is given by the equation σ = (NDkT/IOOOn)1^ [ l c , < e - z i c £ /kT-l)

+ I c y ( e + Z j £ £ / k T -1)] 1 / 2

where c,· and cy are the concentrations and Z,· and Zy the valences of the cations and anions, respectively, Ν is the Avogadro number, k is the Boltzman constant, Τ is the absolute temperature, and ε is the electronic charge. Calculations based on electrophoretic mobility measurements significantly underestimate the surface charge density at low ionic strengths when no correction is made for bacterial surface conductivity (Gittens and James, 1963; Einolf and Carstensen, 1967). Surface ionogenic characteristics of bacteria are obtained by determining electrophoretic mobilities of the bacteria over a range of different pH values. The mobility vs. pH curves shown in Figure 1.3 for Rhizobium trifolii and R. lupini (Marshall, 1967) are typical of two groups of bacteria with different surface ionogenic characteristics. Cells of R. lupini exhibit a characteristically constant net

Introduction

9

Figure 1.3. pH-electrophoretic mobility curves for: (A) Rhizobium trifolii SU297A; (B) R. lupini UT12 (adapted from Marshall, 1967).

negative charge over the pH range of 4.0 to 10.7, and a decline in the charge to zero at pH values below 4.0. Cells of R. trifolii show a constant net negative charge between pH 4.0 and 9.0, a net increase in negativity between pH 9.0 and 10.7, and a reversal of charge at low pH values. A diagrammatic interpretation of the nature of the surface ionogenic groups of these bacteria at the different pH levels is given in Figure 1.4. The surface ionogenic groups of R. lupini cells are acidic (carboxyl). The carboxyl groups are dissociated at all pH values above 4.0, whereas dissociation is suppressed at pH 2.0, resulting in an uncharged surface. With R. trifolii cells, on the other hand, the net negative charge at pH 7.0 results from an excess of acidic ionogenic groups, but the charge reversal at pH 2.0 indi-

Interfaces in Microbial

10

R . Iupini

R. trifolii COOH

pH 2.0 /

NH

3 +

COOH

COOH

/

Net Charge = COO"

pH 7.0

NH

3 +

COOH

COOH

COOH

COO"

COO

COO"

COO"

COO"

COOH

Net Charge = 0 COO"

COO"

Net Charge = - 4

Net Charge = - 4 NH2

COOH

COOH

+ 1

COO"

COO"

COO"

Ecology

COO-

COO"

COO-

pH 10.7 COO"

Net Charge —

COO"

5

COO"

COO"

Net Charge = - 4

Figure 1.4. Diagrammatic interpretation of the effect of pH on the surface ionogenic groups of two species of Rhizobium (adapted from Marshall, 1967). cates the presence of some dissociated basic (amino) groups at that pH. These amino groups remain dissociated at pH 7.0. The increase in net negative charge at pH 10.7 results from suppressed dissociation of the amino groups. The technique of microelectrophoresis is useful in studying interactions between bacteria and other colloidal or macromolecular materials. These interactions depend on the surface ionogenic characteristics of the bacteria in question. Applications of this technique will be discussed in subsequent chapters.

2

Liquid-Liquid and Gas-Liquid Interfaces Allah is great, no doubt, and Juxtaposition his prophet Arthur Henry

Clough

Most microorganisms require an aqueous environment for normal metabolism and growth. In natural habitats, the continuity of this aqueous phase is interrupted by the presence of a variety of solids, nonaqueous liquids, and gases. In this chapter, I will examine the behavior of bacteria in relation to the distinctive properties of liquid-liquid and gas-liquid interfaces. Interfacial Tension A drop of liquid in air tends to form a spherical shape, illustrating the appreciable magnitude of interfacial forces. The contraction of the liquid surface is a consequence of surface tension (yG). Each molecule in the bulk liquid phase is attracted on all sides to molecules in its vicinity, whereas molecules at a gas-liquid interface experience unbalanced attraction forces and, hence, are in a higher energy state. Since the free energy of any system tends towards a minimum, the area of the interface tends to become as small as possible. A liquid surface is in a turbulent state because of a continual, two-way migration of molecules between the surface and the bulk liquid, and between the surface and the vapor phase. Obviously, surface tension decreases with increasing temperature as a result of increasing thermal motion of the molecules. The surface free energy is the work required to increase the area of the liquid by one cm^. For liquids of low viscosity, surface tension and surface free energy are numerically equal, e.g., the surface tension of water is 72.8 dynes cm~l at 20°C, thus 72.8 ergs of work must be done to increase the area of the water surface by one cm^. Interfacial tension (y,·) is the tension developed at any interface in a two-phase system. For a liquid-liquid interface, the value of yj

Interfaces in Microbial

12

Ecology

TABLE 2.1. Standard surface tension values of pure liquids in air (y 0 ) and standard interfacial tension values between water and pure liquids (yj) at 20° C (adapted from Davies and Rideal, 1963). Liquid

y0

η

dynes cm'·'

dynes cm"^

Water Bromobenzene Benzene Toluene n-Octanol Chloroform Carbon tetrachloride Butanol n-Octane Ethyl ether

72.80 35.75 28.88 28.43 27.53 27.14

38.10 35.00 -

8.50 -

26.90 24.00 21.80 17.01

45.10 1.80 50.80 10.70

may be intermediate between the yQ values of the individual liquids (Table 2.1). When the value of γ( is less than the values of y 0 for both liquids, however, a considerable degree of orientation of polar/nonpolar molecules at the interface is indicated. At a waterbutanol interface, for example, y2· is 1.8 dynes cm"^, whereas y 0 (water) is 72.8 and y 0 (butanol) is 24 dynes cm~l. Interfacial packing and orientation of butanol molecules occurs because the hydrophilic hydroxyl groups are attracted to the water phase, whereas the nonpolar (hydrophobic) hydrocarbon chains remain in the alcohol phase.

Bacteria at Liquid-Liquid Interfaces Mudd and Mudd (1924b) have considered the behavior of bacteria at interfaces in relation to the interfacial tension existing between aqueous and organic phases. The interfacial forces involved when a bacterium is at an oil-water interface are shown in Figure 2.1. If y s o is the tension at the solid (bacterial)-oil interface, y s w that at the solid-water interface, and y o w that at the oil-water interface, then at equilibrium y S o = rsw + Xow *

c o s

0

Liquid-Liquid

and Gas-Liquid Interfaces

13

where Θ is the contact angle between the water phase and the solid bacterial surface. If y s o > y s w + y o w , then the line of contact will move towards the oil side until the particle is completely wetted by and enveloped in the water phase. If y s w > y s o + y o w , then the particle becomes enveloped by the oil phase. Stable equilibrium of the bacteria at the interface occurs under conditions where yso< y s w + y o w a n d ysw < Yso + yow· Although values for the liquidliquid interfacial tensions ( y o w ) are readily available, evaluation of solid-liquid interfacial tensions is difficult. Mudd and Mudd (1924b) found a difference in behavior between normal and acid-fast bacteria at oil-water interfaces. The non-acidfast bacteria (Salmonella typhi, Vibrio percolans, and Bacillus subtilis) were stable at all oil-water interfaces tested, i.e., the situation exists where y s o < y s w + y o w and y s w < y s o + y o w . Stability was greater where y o w was large than where y o w was small. Motile bacteria were able to swim away from the interface towards the

Figure 2.1. Diagrammatic representation of the interfacial tensions acting at the periphery of a bacterium at equilibrium in an oil-water interface. The arrows by their direction and length indicate the direction and intensity of the interfacial tensions on the bacterial surface. A is where y s w > y s o + y o w and the bacterium moves into the oil phase; Β is where y s o > y s w + y o w and the bacterium moves into the water phase.

14

Interfaces in Microbial

Ecology

aqueous phase, but never towards the oil phase. This situation suggests that yso + Tow" ^sw ^ Xsw

t o w ' ^so

or ^ s o ^ Tsw. Acid-fast bacteria, on the other hand, readily passed through the interface into the oil phase with only small numbers remaining at the interface. Little stability exists in this situation, and the condition y s w > y s o must prevail. Reed and Rice (1931) confirmed these observations by determining the numbers of bacteria remaining in the aqueous phase of an oil-water mixture following vigorous shaking to provide maximum opportunity for interface contact. Regardless of Gram-reaction, or the presence or absence of capsular material, non-acid-fast bacteria remained in the aqueous phase. The extent of partitioning of acid-fast bacteria into the oil phase was related directly to the degree of acid-fastness of the various Mycobacterium species tested. The difference in the behavior of the two groups of bacteria was attributed to the nonpolar (hydrophobic) nature of surface components in the acid-fast bacteria, as opposed to their polar (hydrophilic) nature in the non-acid-fast bacteria (Mudd and Mudd, 1924b). Because of the stable equilibrium observed with some non-acidfast bacteria at oil-water interfaces (Mudd and Mudd, 1924b), it is feasible that these bacteria have some hydrophobic groups on their outer surfaces. A perpendicular orientation by certain bacteria at oil-water interfaces was noted by Mudd and Mudd (1924a, p. 639). This phenomenon has been examined in detail by Marshall and Cruickshank (1973), who observed that long filaments of Flexibacter CW7 were tapered (fusiform) and that the pointed end was always directed towards the oil-water interface (Figure 2.2). The same perpendicular orientation to the interface was observed in air-water and solid-water systems. Similar results were obtained with a marine flexibacter (Marshall, 1973) and with Hyphomicrobium vulgare ZV580 (Marshall and Cruickshank, 1973). Marshall and Cruickshank (1973) suggested that the portion of these cells directed towards the interface may be hydrophobic, whereas the majority of the cell surface would be hydrophilic. Because of the

Liquid-Liquid and Gas-Liquid Interfaces

15

Figure 2.2. (A) Flexibacter CW7 showing perpendicular orientation at an oil-water interface, (B) Flexibacter CW7 showing rosette formation with the tapered end of longer cells pointing inwards, and (C) Hyphomicrobium ZV580 showing orientation at an oilwater interface and rosette formation.

16

Interfaces in Microbial

Ecology

similar orientation at any type of interface, it was suggested that the more hydrophobic portion of the cell is rejected from the aqueous phase towards the nonaqueous phase. Thus, over most of the bacterial surface the condition y s o > y s w exists, but at the pole attracted to the interface the condition y s w > y s o exists. The formation of rosettes by Flexibacter and Hyphomicrobium (Figure 2.2) may resemble the phenomenon of micelle formation in ionic surfactant molecules (Marshall and Cruickshank, 1973). At low concentrations, these polar/nonpolar molecules behave as normal electrolytes, but at a well-defined concentration they exhibit abrupt changes in surface tension, osmotic pressure, and electrical conductivity. This results from micelle formation, whereby the surfactant molecules aggregate with the nonpolar hydrocarbon chains oriented inwards, leaving the polar groups facing outwards in contact with the water phase. Rosette formation by certain bacteria may result from a similar inward orientation of hydrophobic portions of cells, giving an aggregate with a hydrophilic outer surface. It is significant that the portion of the cell attracted to an interface always forms the inner region of the rosette, i.e., the tapered ends of the larger Flexibacter filaments and the nonhyphal end of the mother cell of Hyphomicrobium (Figure 2.2). Bacteria may reach a liquid-liquid interface by Brownian motion, motility (in motile organisms), buoyancy, currents within the aqueous phase, or by movements of the interface itself. Microscopic study of thin organic liquid-water films reveals that encroachment of the organic phase on the aqueous phase is a major cause of bacterial accumulation at the interface. Mudd and Mudd (1924a) found no evidence for direct attraction of bacteria to the interface through microscopically visible distances. A chemotactic response by bacteria (Adler, 1969) to nutrients, gases, or inhibitory substances accumulating at interfaces might have considerable significance in microbial ecology and will be discussed in detail in Chapters 3 and 4. Similarly, those bacteria (Walsby, 1974) and higher protists capable of regulating their buoyancy could take advantage of conditions existing at certain gas-liquid and liquidliquid interfaces. The movement of microorganisms towards interfaces by currents within the aqueous phase should be of considerable ecological significance. Once a bacterium enters an oil-water interface, it is trapped and cannot escape unless work is done to remove the bacterium (Mudd and Mudd, 1924a).

Liquid-Liquid

and Gas-Liquid Interfaces

17

Factors Influencing Bacterial Behavior at Liquid-Liquid Interfaces Different interfacial

tensions

Accumulation of bacteria at organic liquid-water interfaces occurs more rapidly and to a greater extent in systems of high interfacial tension than in those of low interfacial tension (Mudd and Mudd, 1924a). Within an hour of preparation of cyclohexanewater films (y,· = 60.60 dynes cm~l), for example, many portions of the interface were immobilized by masses of bacteria accumulated at the interface. On the other hand, cyclohexanol-water films (y^ = 3.92 dynes cm"^) remained mobile and bacteria were observed streaming along the interface several days after preparation of the films. Mudd and Mudd observed that movements of bacteria in the interface were jerky and spasmodic when the interfacial tension was high, whereas motion in the interface was vigorous but smooth when the interfacial tension was low. No significant differences were observed between Gram-positive and Gram-negative bacteria as regards these phenomena. Streaming of bacteria along the interface was a characteristic in films of every composition, whether of high or low interfacial tension and whether the organic fluid and water were miscible or immiscible. Marshall and Cruickshank (1973) reported streaming of flexibacters where the perpendicular orientation of the cells at oil-water interfaces gave the appearance of a mobile "picket fence." Streaming of bacteria in the interface is related to the composition of the film, to local inequalities in interfacial tension, often resulting from surface active substances produced by certain bacteria, or to minute currents arising from a mixing of the two phases across and along their boundary line (Mudd and Mudd, 1924a). When the interface in organic liquid-water films moves it sweeps bacterial masses ahead in the aqueous phase. With films of low interfacial tension, the phase boundary may be drawn out into pointed or peninsular projections. If these peninsulas become too drawn out they either retract or break off, often leaving a droplet of water containing bacteria in the organic phase. With films of high interfacial tension, the interface tends to be drawn out into more rounded projections. Interface

contact

For those microorganisms capable of utilizing an organic liquid

18

Interfaces

in Microbial

Ecology

as an energy source, the degree of spreading of an immiscible (or poorly miscible) liquid will determine the interfacial area available to the microorganisms. This is particularly important in the biodegradation of large-scale oil spillages in aquatic environments. When an immiscible liquid is placed on a water surface it may remain as a lens on the surface, it may spread as a monolayer, or it may spread as a uniform film thick enough for the oil-water and oil-air interfaces to be independent. The initial spreading coefficient (5) is a measure of the likelihood of an immiscible liquid spreading on another liquid and, for oil on water, is given by the equation 5

—

Yaw ~ (/oa

^ow)

where y is the surface tension, and o, a, and w are oil, air, and water, respectively. For an oil to spread on water, S should be zero or a positive value. Examples of initial spreading coefficients for several liquids on water are presented in Table 2.2. The work of adhesion between two immiscible liquids, such as oil and water, is equal to the work required to separate one cm^ of the liquid-liquid interface to form two separate liquid-air interfaces. This is expressed by the Dupr£ equation ^A

=

yoa

Yaw" Xow

where is the work of adhesion at the oil-water interface. A typical value for the work of adhesion between water and a paraffinic oil is 43 ergs cm"^. The work of cohesion for a single liquid corresponds to the work required to pull apart a column of liquid of one cm^ cross-section, and is equal to twice the value of the surface tension of the liquid, Wc

= 2 /aw

where W c is the work of cohesion. Thus, the value of W c for water is 145.6 ergs cm"^. The initial spreading coefficient can be related to work of adhesion and cohesion by substitution in the Dupre equation S = WA - 2 y o a = WA

-Wc

From this relationship, it can be seen that spreading will occur when the oil coheres to itself less firmly than it adheres to the

Liquid-Liquid

and Gas-Liquid Interfaces

19

TABLE 2.2. Initial spreading coefficients for several liquids on water at 20°C (adapted from Davies and Rideal, 1963). Liquid

yaw

-

^oa

n-Hexadecane 72.8 n-Octane 72.8 n-OctanoI 72.8

-

(30.0 + (21.8 +

+

(27.5 +

w ) 52.1) = 50.8) = 8.5) =

S - 9.3 + 0.2 +36.8

Conclusion Will not spread Will just spread Will spread freely

water. The value of 5 will be reduced if impurities are present in the aqueous phase, because y a w is lowered more than y o w by the impurity. On the other hand, impurities in the oil phase can reduce y o w to a point where S becomes positive. Complete spreading of oil over a water surface still provides limited opportunities for interfacial contact. As mentioned previously, Reed and Rice (1931) obtained significant partitioning of the acidfast Mycobacterium tuberculosis from water into an oil phase only after vigorous shaking to increase the degree of interfacial contact. Another example of the importance of interfacial contact is the growth of the hydrocarbon-utilizing yeast Candida guilliermondi Y-8. Borzani et αϊ. (1971) have provided evidence for the attachment of yeast to hydrocarbon droplets during the exponential phase of growth and for a negligible growth rate of unattached cells. They have developed a model based on the assumption that the exponential phase of growth ends when the droplets become saturated with cells. This model predicts that the duration of the exponential phase increases as the droplet radius decreases (interfacial area increases) over a range of initial hydrocarbon substrate concentrations (Figure 2.3). pH Reed and Rice (1931) reported no change in the efficiency of partitioning of acid-fast bacteria into the oil phase at pH values of 1.6 to 6.0 in the aqueous phase. In more alkaline conditions, the aqueous phase became cloudy as a result of soap formation. Nevertheless, the acid-fast bacteria still passed into the oil phase, whereas non-acid-fast bacteria remained in the aqueous phase. Wetting

agents

Surface-active agents (or surfactants) function by lowering the

20

Interfaces in Microbial

2 SUBSTRATE

4

Ecology

6 CONC.

8

10

(gl"')

Figure 2.3. Computed curves relating the exponential phase duration with the initial substrate concentration for different values of the initial hydrocarbon drop radius for Candida guilliermondii Y-8 (redrawn from Borzani et ah, 1971). surface tension of a liquid. The hydrophilic group of these molecules has a strong attraction to water and is capable of "solubilizing" long hydrocarbon chains. Examples of surfactants are (i) Anionic—sodium dodecyl sulfate; (ii) Cationic—cetyl trimethyl ammonium bromide; and (iii) Nonionic—polyethylene oxides. At low concentrations, ionic surfactants behave as normal electrolytes, but at a well defined concentration they exhibit an abrupt change in properties as a result of micelle formation (see page 16). The concentration of the surfactant where this aggregation occurs is termed critical micelle concentration. With increasing concentration, a surfactant progressively lowers the surface tension of water until micelle formation occurs. Beyond this point, the surface tension remains constant because the micelles are not surface active. When non-acid-fast bacteria were suspended in a 0.06% sodium

Liquid-Liquid

and Gas-Liquid

Interfaces

21

oleate solution, their behavior at a mineral oil-water interface resembled that reported for low interfacial tension systems (Mudd and Mudd, 1924a). A quantitative study by Reed and Rice (1931) revealed that sodium oleate reduced the partitioning of the acid-fast Mycobacterium tuberculosis from the water to the oil phase, the effect being inversely proportional to the concentration of the wetting agent (Table 2.3). Dispersed growth of hydrophobic tubercle bacilli has been achieved by the use of certain nonionic surface active agents in the liquid growth medium (Dubos, 1945). These agents include the polyoxyethylene esters of oleic acid (Atlas G-2144), lauric acid (Atlas G-2124), sorbitan monooleate (Tween 80), sorbitan monostearate (Tween 60), sorbitan monopalmitate (Tween 40), and sorbitan monolaurate (Tween 20). The suspension of Flexibacter CW7 in 0.05% Tween 80 prevented both the perpendicular orientation of these cells at oil-water interfaces and firm adhesion of the bacteria to solid surfaces (Marshall and Cruickshank, 1973). Immune

serum

Mudd and Mudd (1926) found that acid-fast bacteria sensitized with homologous immune serum behaved like non-acid-fast bacteria at oil-water interfaces. This result was confirmed by the quantitative studies of Reed and Rice (1931). The hydrophobic surface of these bacteria reacts with serum antibody to give rise to a more hydrophilic surface. Bacteria at Gas-Liquid Interfaces Air-water interfaces are the most important of gas-liquid systems TABLE 2.3. The effect of a wetting agent on the partitioning of Mycobacterium phlei from water into oil (from Reed and Rice, 1931). % sodium oleate used

% bacteria in oil phase in olive oil-water (2:1) mixture

5.0 2.5 1.0 0.3 0.1

28

0.01

52

62 63 76 84

22

Interfaces in Microbial

Ecology

with respect to growth and metabolism of microorganisms, but gases other than air are significant in certain restricted natural habitats. Microbial activity in anaerobic sites results in the production of gases, such as hydrogen, carbon dioxide, carbon monoxide, or methane, which may serve as energy or carbon sources for other groups of microorganisms. Those microorganisms with ready access to the gas-liquid interface should benefit, because of the limited solubility and rapid microbial utilization of some of the gases. Few direct microscopic observations have been made of bacterial behavior at air-water or other gas-liquid interfaces. Mudd and Mudd (1924a) noted that bacteria streamed along air-water interfaces in the same manner as at oil-water interfaces. Similarly, Marshall and Cruickshank (1973) reported that Flexibacter and Hyphomicrobium cells exhibited the same perpendicular orientation at air-water interfaces as at oil-water interfaces, and suggested that this results from the hydrophobic pole of the cells being attracted to any nonaqueous phase. Microlayers

at gas-liquid

interfaces

Natural aquatic systems do not possess completely uncontaminated air-water interfaces. A variety of materials accumulate at these interfaces and markedly alter their physicochemical and biological properties. The surface microlayer has been defined by Parker and Barsom (1970) as a reservoir of hydrophobic substances with low evaporative potentials and debris with specific gravities below that of the aqueous medium. The maximum depth of surface microlayers in natural waters varies considerably, from molecular monolayers to several millimeters in thickness. Materials accumulating at air-water interfaces include natural oils, living organisms, dust, and synthetic compounds that may originate from atmospheric fallout, rain, runoff, or the sub-surface water. Some substances (e.g., long-chain alcohols and fatty acids, proteins) form monolayers on the surface of water as a result of hydrophilic groups orienting towards, and hydrophobic groups orienting away from, the water phase. Monomolecular films can exist in different physical states, depending on the magnitude of the lateral adhesive forces between the molecules. Condensed films occur where the molecules are closely packed and oriented almost vertically, as is found, for example, with the strong lateral cohesion

Liquid-Liquid and Gas-Liquid

Interfaces

23

between the hydrocarbon chains of palmitic or stearic acids. Expanded films are coherent, but occupy a much larger area than condensed films. For example, some chains of oleic acid orient horizontally with they hydrophilic double bonds in contact with the water surface, thereby resulting in less cohesion between the hydrocarbon chains. Protein and other macromolecular layers are of significance in microbial ecology because they represent a potential concentration of nutrients and because they alter the interfacial tension and other properties, such as the wettability of solid surfaces (see Chapter 3). In dilute protein solutions, as found in natural aquatic systems, proteins rapidly sorb at high energy interfaces and unfold to form monolayers. The extent of this unfolding is illustrated by globular proteins of diameter exceeding 100A, which spread at interfaces to form monomolecular films of 8 to lOA. Proteins tend to unfold to a greater extent at oil-water than at air-water interfaces. Adam (1937) developed a rapid method for determining surface tension of natural water surfaces based on the behavior at the surface of drops of a hydrocarbon oil, the spreading coefficient of which was modified by dissolving increasing amounts of a more hydrophilic liquid in the oil. He reported significant lowering of surface tension near sewage outfalls, in small harbors, and in other confined areas of water. Using this technique, Sturdy and Fischer (1966) showed that slicks accumulating at the sea surface above kelp ( M a c r o c y s t i s pyrifera)

beds modified the surface

tension. They recorded y a w values of 70 to 72 dynes cm~l in open, rippled water, 60 to 70 dynes cm~l at the edge of slick areas, and 50 to 60 dynes cm~l directly over the kelp beds. Natural ocean slicks contain organic oils, derived mainly from diatoms, with lowerorder interference colors on water than found with petroleum slicks (Dietz and Lafond, 1950). Jarvis et al. (1967) reported that the properties of surface-active organic materials collected from the upper 0.15 mm of different waters were similar, and suggested that competition for sites at the air-water interface resulted in a selective accumulation of high molecular weight, polar/nonpolar groups. Surface slicks result in wave dampening, as well as influencing sea surface temperatures, diffusion of gases across the air-water interface, and the stability of surface foams. The effects of surface microlayers on microbial activity in natural aquatic habitats will be discussed in Chapter 4.

24

Interfaces in Microbial

Bubbles as gas-liquid interfaces in natural

Ecology

habitats

Bubbles may be formed by mechanical incorporation of air into the liquid phase (e.g., by wave motion), by decreased solubility of dissolved gases due to temperature or pressure changes, or by the biological production of gases in soil, sediment, or aqueous environments. Where the predominant gas within the bubble can be oxidized by specific microorganisms (e.g., hydrogen, methane, ethylene, and carbon monoxide), then the surface of stabilized bubbles affords an excellent habitat for the selective growth of such microorganisms. The accumulation rate of surface-active materials in microlayers was shown by Jarvis (1967) and Garrett (1967) to be related to the rate of bubble appearance at the water surface; in the absence of bubble transport or thermal currents, it was a function of the molecular diffusion rate. Lemlich (1972) has proposed the term adsubble (or adsorptive bubble separation) processes to encompass those natural or man-made phenomena and techniques in which dissolved or suspended material is segregated within or removed from a liquid by adsorption or attachment at the surfaces of rising bubbles. Both bubble fractionation and foam fractionation are effective in removing substances present in low concentrations in the bulk aqueous phase and depositing surface-active substances at the air-water interface. An otherwise surface-inactive substance may be concentrated at bubble surfaces after complexing with a surface-active substance. The accumulation of organic materials at bubble surfaces has been implicated in the formation of organic aggregates in natural waters (Baylor and Sutcliffe, 1963), although Barber (1966) has presented evidence suggesting an interaction between bubbles and bacteria in the formation of organic aggregates. Certainly, Carlucci and Williams (1965) have found that adsubble processes were responsible for the concentration of bacteria at water surfaces. Organic marine particles have been classified by Gordon (1970a) into aggregates, flakes, fragments, and unclassifiable particles. The aggregates appear to be chiefly carbohydrate, the flakes chiefly protein, and the fragments completely carbohydrate. Aggregates were most abundant in surface layers, whereas the abundance of flakes and fragments was constant with depth.

Liquid-Liquid and Gas-Liquid

Interfaces

25

jet drops and water-to-air transport of bacteria When a bubble breaks at a surface it rapidly collapses and a vertical jet of water rises at high speed from the bubble cavity. This jet becomes unstable, breaking up into four or five drops that continue to heights that are reproducible from one bubble to the next under experimental conditions (Blanchard, 1964). These jet drops can be collected in large numbers and analyzed for their chemical and microbiological content. Blanchard demonstrated that surfaceactive organic films at air-water interfaces are ejected into the air by this jet drop mechanism. More recently, Blanchard and Syzdek (1970, 1972) and Bezdek and Carlucci (1972) have shown a significant ejection of bacteria into the air on jet drops. Concentration of bacteria in jet drops was determined from the following relationship (Blanchard and Syzdek, 1972): C =

Cd/Cb

where C is the concentration factor, C^ is the concentration of bacteria in the jet drops, and Cy the concentration of bacteria in the bulk water phase. Several thousand jet drops were sampled in order to obtain statistically meaningful bacterial counts. Only the top jet drop from each bubble burst was collected. Using a special bubble aging tube, Blanchard and Syzdek demonstrated that C for Serratia marcescens increased with bubble age, indicating that bacteria from the bulk water phase became attached to the bubble as it passed through the water and were ejected into the air on the jet drops. Surface-active organic materials also adsorbed to the bubble surface, resulting in a lowered ejection height and a decrease in jet drop size following bubble burst. These effects were observed during a bubble-aging time of 10 to 20 seconds, the same time period over which C increased with bubble age. This relationship suggests that the bubble interfacial tension is altered by the adsorbed organic materials, favoring the adsorption of bacteria at the the bubble surface. Blanchard and Syzdek (1972) have reported values of C from 1 to 100 for Serratia marcescens, the value increasing with drop size up to a drop diameter of 80μτη. They suggested that this puzzling relationship might result from the time required for the bacteria on the bulk air-water interface to move into the bubble cavity and onto

26

Interfaces in Microbial

Ecology

the rising jet. This time must be much faster for small bubbles than for larger bubbles. It was assumed that adsorption of bacteria on the bubble surface was negligible, for the age of the bubbles was less than 0.2 seconds on bursting. Values of C were found to decrease with an increase in Cy within the range of 10^ to 10^ per ml. for drops of about 40μπι diameter. Blanchard and Syzdek stated that since the surface film is essentially two-dimensional and probably accommodates only a certain number of bacteria per unit area ( = C s ), an increase in Cy only decreases the time required for bacteria in the bulk suspension to migrate to the surface to give the concentration C s . Since C = Cj/Cy and Crf = kCs (where k is a constant of proportionality), then C = kCs/Cy. If kCs is constant, however, then C must be proportional to 1 /Cy. Bezdek and Carlucci (1972) reported that the value of C depended on the bulk density of the bacteria. The role of jet drops in the water-to-air transfer and concentration of bacteria is important in the dispersal of microorganisms. This aspect, along with the effects of liquid-liquid and gas-liquid interfaces on microbial ecology, will be examined in detail in Chapters 4 and 5.

3

Solid-Liquid and Solid-Gas Interfaces To doubt everything or to believe everything are two equally convenient solutions; both dispense with the necessity of reflection. Poincare

A n interface involving a solid phase has a degree of permanence not normally associated with liquid-liquid or gas-liquid interfaces. Consequently, the fate of ions, macromolecules, and even microorganisms attracted to a solid surface is of concern in microbial ecology because of the permanent changes that may be induced in microbial populations b y the presence of the solid surface. The altered balance in microbial populations results from the selective sorption to surfaces of both molecular and microbial species. Such effects increase as the available interfacial area increases. The fundamental aspects of adsorption phenomena have been deduced primarily for solid-gas interfaces, but the principles are applicable in other systems. Physical adsorption is characterized b y low heats of adsorption (< 10 kcals mole Ί ) , b y reversibility, and b y the rapid establishment of the adsorption equilibrium. The attractive forces involved are of the nonspecific London-van der Waals type. The adsorbed layer is often more than one molecule thick. Chemical adsorption (chemisorption) is a specific process, it is accompanied b y much higher heat changes (20 to 100 kcals mole "1) and is characterized by the formation of monomolecular layers.

Chemisorption probably

involves a reaction

between

adsorbed gas molecules and solid surfaces, leading to a firmer attachment than found in physical adsorption. Physical adsorption probably occurs in all instances of adsorption, but is masked when the stronger chemisorption occurs simultaneously.

Adsorption

increases with increasing partial pressure of gas until the solid is saturated, the total amount of gas adsorbed depending on the surface area, chemical nature, and previous history of the solid. Sorp-

28

Interfaces

in Microbial

Ecology

tion is also temperature dependent; the higher the temperature the less gas is adsorbed. Adsorption isotherms are graphical representations of the relationship between the amount of adsorbed material and the equilibrium pressure (or other parameter) at a constant temperature. Five characteristic types of adsorption isotherms have been observed in the adsorption of gases to solids (Figure 3.1). Type I isotherms (Langmuir-type) exhibit a rapid rise in the amount of gas adsorbed with increasing pressure, up to a limiting value. This limiting value indicates that adsorption is restricted to a monolayer. The sigmoid Type II isotherm represents multilayer adsorption on nonporous solids, with the point X representing the formation of an adsorbed monolayer. Isotherm Types III and V show no initial phase of rapid gas adsorption, possibly because adsorption forces in the first monolayer are weak. Type IV isotherm resembles Type II but levels off on approaching the saturation vapor pressure. It is believed that this reflects condensation occurring in narrow capillaries in porous solids at pressures lower than the normal saturation vapor pressure.

Adsorption of Macromolecules at Solid-Liquid Interfaces The behavior of macromolecules at solid-liquid interfaces has not been studied as extensively as at liquid-liquid or gas-liquid interfaces, due to a lack of suitable methodology. Macromolecular adsorption at the solid-liquid interface is important biologically because it results in altered wetting properties at the surface, an improved nutritional status near the surface, and an altered activity of some of the macromolecules, particularly enzyme proteins. That surface properties influence the adsorption of macromolecules is illustrated by MacRitchie's (1972) finding of greater adsorption of bovine serum albumin at the surface of Aerosil R-972, a colloidal silica with chemically bonded methyl groups giving a hydrophobic surface, than at the surface of the hydrophilic silica Aerosil 200 (Figure 3.2). This is consistent with the view that protein adsorption, at relatively uncharged interfaces, is governed by the lowered free energy resulting from the reduction of interactions between nonpolar groups and water molecules. The sharp rise in the isotherm for the hydrophobic silica at higher concentrations of protein resulted from interfacial coagulation. The extent of

Solid-Liquid

and Solid-Gas

Interfaces

29

Ιο Λ α < ζ Ο


Figure 3.1. Types of adsorption isotherms (see text for detailed description). adsorption on the hydrophilic silica decreased dramatically as the net charge on the serum albumin increased on either side of the isoelectric point (pH 4.9), whereas little change was observed with the hydrophobic silica (Figure 3.2). This indicates that the free energy of adsorption is greater at the hydrophobic than at the hydrophilic surface. Alterations in the wettability of a solid surface following the adsorption of macromolecules may be determined by contact angle measurements. A drop of liquid either spreads over a surface or remains as a discrete drop with its edge having a definite angle of contact (Θ) with the solid surface. The surface tension (y) at the solid-air interface is given by the equation 7sa

=

rsl + y a l c o s 0

where s, a, and 1 refer to the solid, air, and liquid phases, respec-

30

Interfaces in Microbial Ecology/

- 2 0

- 1 0

LOG (gm

BULK

0

CONCENTRATION

100 gm

soln. )

Figure 3.2. Adsorption isotherms for bovine serum albumin on hydrophobic Aerosil R-972 (solid lines) and hydrophilic Aerosil 200 (dotted lines) at different pH values. (A) pH 5.5; (B) pH 3.2 and 9.0; (C) pH 5.5; (D) pH 3.2; (E) pH 2.1; (F) pH 7.5 (adapted from MacRitchie, 1972). tively. Since y s a and y s l cannot be measured accurately, the above equation can be combined with a form of the D u p ^ equation (see Chapter 2) to give ^sl = raid +

cos

Θ)·

This is known as Young's equation, and shows that the contact angle is determined by the relative magnitudes of the adhesion of the liquid to the solid (lV s l) and the self-cohesion of the liquid (2 y a l). A finite contact angle results when the liquid adheres to the solid less than it coheres to itself. The solid is completely wetted when Θ is zero. When Θ is less than 90 ° the liquid wets the solid, and when© is larger than 90° it does not wet the solid. Surface rough-

Solid-Liquid and Solid-Gas Interfaces

31

ness increases the contact angle when the normal value for Θ exceeds 90°, but decreases the angle if Θ is less than 90°. Although clean glass has a contact angle of zero for water, sorbed materials alter the wetting properties by masking the hydrophilic-OH groups on the glass surface with groups of variable affinity for water. A useful measurement in studies of biological adhesion is the critical surface tension (yc). This is a measure of the highest surface tension a liquid can have and still spread over a given solid surface by molecular attraction (Baier et al, 1968). The critical surface tension for wetting by a homologous series of liquids is defined on a "Zisman plot" (Figure 3.3) as the intercept of the horizontal axis at cos 0 = 1 with the extrapolated straight line plot of cos Θ against yQ of the liquids. The surface depicted in Figure 3.3, polytetrafluoroethylene (Teflon), shows a critical surface tension of 18.5 dynes cm~l. The yc intercept reflects the solid-gas interfacial tension,

0

10

15

0-9 30

ΙΛ Ο

0-8

Ο Ζ < υ < t— ζ Ο υ

2 uptake b y phototrophs and - ^ S (as sulfate) uptake by both phototrophs and heterotrophs, Monheimer found that axenic algal cultures gave a ratio of ^^C to - ^ S approximating the theoretical ratio of 500 to 1. Much lower ratios were recorded consistently in natural aquatic habitats, as a result of significant uptake of unlabeled organic carbon by heterotrophs. While indicating a close association in aquatic habitats between heterotrophs and primary producer phototrophs, these results do not give any measure of the relative contribution of epiphytic and other heterotrophic bacteria to total productivity. It is likely, however, that many of the heterotrophs would exist as epiphytes if the dissolved organic matter levels in the waters were low.

Tripton-Associated Microorganisms Particulate matter in fresh and marine aquatic systems may be inorganic, organic, or both. The largest fraction of particulate matter in the oceanic euphotic zone is organic debris or detritus (Parsons and Strickland, 1962). Manheim et al. (1972) found a predominance of combustible organic particulates at sites beyond 100 km from shore, but reported mainly inorganic particles encompassed by organic debris in transitional zones, and mineral particulates closer inshore. Mineral materials ranged from a low magnesian

68

Interfaces in Microbial

Ecology

calcite-aragonite off Florida to montmorillonitic-kaolinitic

clay

combinations from Alabama to Texas. Appreciable amounts of organic material are present in suspended matter in freshwater systems (Paerl, 1973). The amount and composition of mineral particulates present obviously depends on soil characteristics in the lake or river catchment areas. Because of their small size, suspended particulates provide a very large interfacial area for potential concentration of nutrients from the aqueous phase. In addition, some of the organic detritus should be available as a substrate for microbial growth. Oceanic detritus contains significant amounts of protein and carbohydrate (Parsons and Strickland, 1962), and at least 2 0 % of deep oceanic particulate matter is readily hydrolyzed by a mixture of α-amylase, trypsin, and chymotrypsin (Gordon, 1970b). The highest concentrations of hydrolyzable organic carbon are found in shallow layers, reflecting the greater abundance of living organisms in these waters. One might expect that microorganisms adhering to particulates in oligotrophic waters would account for most of the respiratory activity in such ecosystems. Floodgate (1972) contends that the continued sorption of organisms from water onto particulate surfaces constitutes an energy input that, in turn, leads to a succession of microorganisms with an energy turnover. However, experimental evidence for this activity of tripton-associated microorganisms is conflicting. Pomeroy and Johannes (1968) found that bacteria, protozoa, and phytoplankton were associated with marine detritus, and reported a good correlation between primary productivity and respiration rates in a range of oceanic conditions. They suggested that the major site of metabolic activity in oceans may be on organic aggregates, and demonstrated that organic carbon availability was more important than temperature in controlling respiration rates. Pomeroy and Johannes also noted that significant respiration was associated with organic particulate matter sinking below the euphotic zone. A good correlation between microbial biomass and particulate organic matter in seawater was reported by Seki (1970a), who also estimated an average concentration of bacterial-containing aggregates of 3 χ 1 0 3 m l ' 1 in subarctic waters (Seki, 1970b). The majority of these particulates were in excess of 5μητι diameter and were composed of bacteria attached to plankton debris. Seki calculated, on the basis of l^C-labeled glucose uptake measurements,

Aquatic

Ecosystems

69

that each aggregate contained an average of 30 bacterial cells, and that bacterial biomass constituted 1% of the oceanic particulate organic matter. An analysis by Hargrave (1972) of his own and other work revealed that the number of bacteria on detritus, and their metabolic activity, is proportional to the available particle surface area, despite the fact that bacteria colonize only a small area of the particle surfaces. A correlation between particulate protein and bacterial distribution in water columns near Hawaii was reported by Gundersen et al. (1972). Contrary to these findings, Williams (1970) has suggested that the most active heterotrophic microorganisms in marine waters are planktonic and not associated with particles (see pages 64-65). In support of this proposal, Hobbie et al. (1972) reported that the numbers of bacteria associated with particulates from the open ocean were so low that their contribution to the respiratory levels measured in water samples must be insignificant. In an extensive survey of oceanic organic particulates, Wiebe and Pomeroy (1972) emphasized that, of the predominant flake and floe particulates, flakes rarely had associated bacteria and floes had adherent bacteria but always in low numbers. Paerl (1973) overcame some of the problems of observing small bacteria on particulates by using scanning electron microscopy, and published photographs of bacteria and fungi on detrital material from Lake Tahoe, California. Sizes of attached bacteria ranged from 0.2 to 3.0μπι, indicating that many would be difficult to observe by light microscopy. Paerl claimed that detrital particles from depths beyond 75m were larger and smoother with little obvious bacterial attachment, and suggested that most bacterial decomposition by detritus occurs only in near-surface waters. An important aspect of microbial ecology in aquatic habitats is the effect of sorption of colloidal materials to microbial surfaces. ZoBell (1943) suggested that sorption to bacterial surfaces of colloidal inorganic materials, such as clays, might inhibit microbial activity. Evidence for the sorption of colloidal clay and organic matter to bacteria was presented in Chapter 3, and further implications of such interactions in soils will be considered in Chapter 5. Roper and Marshall (1974) demonstrated in aqueous suspensions that Escherichia coli is protected from bacteriophage attack by an envelope of sorbed colloidal material around the cell (Table 4.1 and

70

Interfaces

in Microbial

Ecology

Figure 4.4). More recent studies (Roper and Marshall, unpublished data) indicate that a number of microbial predator-prey interactions are interrupted to varying degrees by adsorption of colloidal materials to the microorganisms. Such microbe-particulate interactions must influence the survival of certain species in aquatic habitats and, hence, could alter the balance between populations in aquatic microbial communities. Benthic Microorganisms Materials found in marine and freshwater sediments include pebbles, sand, clays, and detritus, all of which provide surfaces for the potential sorption of microorganisms. According to Rubentschik et al. (1936), the type of sediment material determines the degree of sorption of an individual bacterial species (Table 4.2). This degree TABLE 4.1. Survival of Escherichia coli in the presence and absence of phage and/or colloidal montmorillonite at two different electrolyte concentrations (from Roper and Marshall, 1974).

Treatment

I/Ls*

No. of E. coli

Output/

(χ ΙΟ" 7 )

Input***

72

125.3

Ε. coli + phage Ε. coli

72 719

4.0 108.2

Ε. coli + phage Ε. coli + clay

719 72

0.015 139.9

E. coli

%ο Survival**

8.89 3.19

0.28 7.67

0.014 0.001 9.92 66.16 Ε. coli + phage + clay Ε. coli + clay

72 719

6.57 8.78

92.6 123.8 61.81

Ε. coli + phage + clay

719

76.5

5.43

*1/L S = reciprocal of the specific conductivity of the suspension. " P e r c e n t survival = (No. of E. coli recovered in phage treatment/No. recovered in control treatment) χ 100. ***Output/input = total No. o f f . coli recovered following dilution to achieve desorption/No. in initial inoculum (14.1 χ 1 0 7 ) .

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of sorption will vary between different bacterial species. Tsernoglou and Anthony (1971) presented data indicating an inverse correlation between the logarithm of the heterotrophic bacterial population and the median size of untreated sediment samples. Direct counts gave values ranging from 3,000 to 15,000 bacteria mm'^ of sediment particle surface, corresponding to only about 0.2% of the available surface being colonized. An examination of marine pebbles (Batoosingh and Anthony, 1971) revealed low levels of surface colonization by bacteria, ranging from 0.3% for indirect counts to 2% for direct counts. The metabolism of microorganisms attached to sand grains was

Figure 4.4. An electron micrograph showing Escherichia coli coated with colloidal montmorillonite and showing phage particles excluded from the bacterial surface, as indicated by the white arrows. Freeze-dried, gold-palladium shadowed specimen (from Roper and Marshall, 1974).

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TABLE 4.2. Adsorption of Serratia marcescens by sediments from salt lakes (adapted from Rubentschik et al., 1936). Sediment sample Nonplastic black-grey mud Gray plastic mud Black mud Black sand

Percentage of added bacteria sorbed to sediment 68.0 - 79.1

Gray sand

45.8 - 84.8 71.2 3 9 . 5 - 61.9 19.3 - 20.5

Grayish-black sand

2 1 . 0 - 22.4

examined by Munro and Brock (1968) using radioautographic techniques, and they concluded that bacteria are the dominant heterotrophs on these surfaces. A detailed analysis by Hargrave (1972) of original and published data revealed a negative linear correlation between the logarithm of the particle diameter and the logarithms of the numbers of bacteria, the percent organic matter and nitrogen, and the oxygen consumption of different sediment particulates (Figure 4.5). Detritus consumed up to three times more O2 per unit dry weight than did sand. The narrow range of oxygen uptake per cm^ by different particles led Hargrave to suggest that all the microbial surface communities have similar metabolic rates, which, in turn, may be limited by the O2 availability in natural sediments. Strzelczyk and Mielczarek (1971) reported that benthic bacterial isolates showed a much lower metabolic activity on a range of substrates than either epiphytic or planktonic bacteria. They made the point that different sites within aquatic ecosystems must contain organic matter of different quality, which must be colonized by bacterial communities adapted to different specific substrates. The presence of mineral particulates in sediments will modify the metabolic activities of microorganisms. Rubentschik et al. (1936) demonstrated that adsorption of Nitrosomonas on oxidized mud inhibited ammonium oxidation, whereas the activity of sulfatereducing bacteria was stimulated following adsorption. McCabe and Frea (1971) studied the sorptive interactions between clay (kaolin) particles, an aquatic streptomycete, its extracellular collagenase, and the enzyme substrate (collagen). In a cell-free system, they found that kaolin-enzyme and kaolin-substrate interactions result

Aquatic

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Ecosystems

PARTICLE

DIAMETER

73

(jam)

Figure 4 . 5 . Comparison on logarithmic axes of mean particle diameter and: (A) "internal surface" of sand (cm^ lOcrrf^); (B) viable counts of bacteria in different sediments (x 10"^ g~l); (C) organic matter O 2 consumption (mgC>2 hr~l); (D) detritus O 2 consumption (mgC>2 g"-*· h r ' l ) ; (E) dotted line indicating a slope of - 1 . 0 ; (F) percent organic carbon in beach sediments; (G) percent organic nitrogen in beach sediments; (H) sand O 2 consumption (mgÜ2 hr"l). Solid lines are calculated regression lines (data compiled from various sources by Hargrave, 1972).

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in a multiple enhancement of enzyme activity. The enzyme is concentrated on kaolin surfaces, and the adherence of the kaolinenzyme complex to collagen places the enzyme in direct contact with the substrate. In view of these results it is interesting to note the consistent isolation of actinomycetes from ocean sediments and their general absence from the bulk aqueous phase (Weyland, 1969), which suggests that these microorganisms are adapted to a benthic rather than a planktonic existence. The numbers of Serratia marcescens

desorbed from saline sedi-

ments on shaking were found by Rubentschik et al. (1936) to increase with time up to 60 seconds, and then to remain unchanged on further shaking. Only 6 % of the original inoculum added to the sediment was recovered. Desorption of microorganisms from sediment or clay suspensions is dependent on the electrolyte concentration (Roper and Marshall, 1974). Few bacteria are desorbed from sediment at high salinities, but, following successive washings in distilled water, large numbers are desorbed on the attainment of a critical electrolyte concentration (Figure 4.6). This point coincides with the dispersion of the colloidal material in the sediment. The release of bacteria and dispersion of colloidal materials result from interparticle repulsion effects at low electrolyte concentrations (see Chapter 3, pages 39-43). Roper and Marshall (1974) demonstrated that bacteria sorbed to sediment materials are protected from phage attack. Montmorillonite and extracted sediment organic matter provided a similar degree of protection, suggesting that the protective mechanism is purely physical and not associated with any chemical properties of the colloidal materials. The phages were prevented from direct contact with the host bacteria because both the phages and the bacteria were adsorbed to the sediment or other colloidal materials. Interruption of predator-prey interactions by such sorption phenomena might account for increased recovery rates of enteric bacteria in certain sediments (Hendricks, 1971; VanDonsel and Geldreich, 1971).

Neuston-Associated Microorganisms The physical and biological factors involved in the concentration of microorganisms at or near the air-water interface have been con-

Aquatic

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75

υ Ζ < co o£ Ο ιη

C 1 0 4 . "High count" surface layers (>1(P ml 1) overlying "low count" waters were found only in Pacific Ocean samples collected in an area near Central America known for upwelling. "High count" surface and subsurface samples were encountered sporadically in Caribbean and Atlantic waters. As a measure of biochemiTABLE 4.3. Numbers of protozoa and diatoms per liter of surface microlayer and subsurface seawater samples (adapted from Harvey, 1966). Sampling depth Microorganisms

60 μ m

10cm

13m

Unidentified flagellates ( < 1 5 μηι)

4,470

0

0

Unidentified flagellates ( > 1 5 μηι) Dinoflagellates

30

0

0

31,270

3,900

1,100

Ciliates

330

370

100

Diatoms

930

3,770

16,100

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cal activity, Sieburth compared the ratios of lipolytic: proteolytic: amylolytic bacteria isolated from different sites, and found ratios for the "high count" Pacific flora of 93:92:24 and the "high count" Caribbean and Atlantic flora of 46:53:54. Atypical pseudomonads dominated the Pacific samples, whereas types 2 and 4 pseudomonads were dominant in the Caribbean and Atlantic samples. Isolates from "low count" waters were shown to have less proteolytic and more amylolytic activity than isolates from "high count" waters. The proteolytic:amylolytic ratios were similar for "low count" isolates regardless of latitude, depth or oceanic site. These results indicate a predominance of protein substrate in "high count" areas and of carbohydrate substrate in "low count" areas. DiSalvo (1973) reported that bacterial adhesion to glass surfaces in contact with the neuston was a thousand-fold greater than to surfaces exposed to subsurface water, which may be evidence for a higher bacterial metabolic activity, including the production of extracellular bridging polymer, in surface microlayers. Microbial enrichment in surface microlayers in freshwater systems was reported by Hatcher and Parker (1974), but the results varied with the sampling method employed (Figure 4.7). The drum sampler (Harvey, 1966) gave the most consistent indication of bacterial concentration in surface microlayers. Hatcher and Parker also detected significant increases in ammonium, nitrate, nitrite, orthophosphate, sulfate, iron, zinc, and manganese in surface microlayers, and suggested that this chemical and biological enrichment results in higher metabolic and recycling rates in these layers. An accumulation of inorganic ions and heavy metals in sea-surface microlayers has been reported by Duce et al. (1972) and Barker and Zeitlin (1972). As pointed out by Parker and Barsom (1970) and Hatcher and Parker (1974), an important consequence of bacterial concentration in surface microlayers is the generation of bacterial aerosols by the jet-drop mechanism (Blanchard and Syzdek, 1970 and 1972), resulting in widespread dispersal of the bacteria. For instance, Adams and Spendlove (1970) have detected aerosolized Escherichia coli and other coliform bacteria up to 1.2km downwind of a sewage treatment plant. The survival and transport of these aerosolized bacteria depended on temperature, humidity, wind velocity, and the presence or absence of solar radiation. An interesting suggestion (Singer and Ames, 1970) concerning the survival of bacteria at

78

Interfaces

Drum

Screen

in Microbial

Tray

Ecology

Dip

Sampler

Figure 4.7. Percentages of days when significant differences in viable counts of bacteria between surface microlayer and subsurface samples were obtained with different sampling techniques (from Hatcher and Parker, 1974). air-water interfaces is the possible selection of bacteria possessing DNA with high mole % GC values. This DNA could resist damage by ultraviolet irradiation as it would be too low in thymine for thymine dimerization to occur. Microbial concentration at air-water interfaces could influence the biodegradation rate of oils spilt on natural waters, because some neuston microorganisms must decompose natural organic oils responsible for surface slick formation (Dietz and Lafond, 1950) and, hence, may metabolize certain fractions of crude oil. The accumulation of inorganic ions in surface microlayers may help stimulate biodegradation in an otherwise nutrient-deficient situation. A potentially useful technique for following the biodegradation of

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79

interfacial oil films, even of monomolecular dimensions, is the internal reflection spectroscopy method described by Baier (1970). Although some bacteria grow in the oil phase (LaRock, 1975), bacteria and fungi capable of hydrocarbon oxidation generally grow at the oil-water interface, their rate of growth being limited by the available interfacial area. Dispersal of the oil into small droplets greatly increases the interfacial area and, hence, the rate of microbial decomposition. The growth of the aerobic kerosene decomposing fungus Cladosporium resinae increases as the depth of kerosene above the kerosene-water interface increases (D. Parberry, personal communication). This observation is explained by the finding of Mimura et αϊ. (1969) that the transfer of oxygen from oil to water is faster than that from air to oil. They demonstrated that with an increase in the proportion of oil to water, more'oxygen becomes available to an aerobic microorganism (Aerobacter aerogenes), resulting in a progressive increase in its growth rate.

Nonspecific Interfacial Interactions in Microbial Ecology: Terrestrial Ecosystems The simplest and most lumpish fungus has a peculiar interest to us, compared to a mere mass of earth, because it is so obviously organic and related to ourselves, however remote. It is the expression of an idea; growth according to a law; matter not dormant, not raw, but inspired, appropriated by spirit. If I take up a handful of earth, however separately interesting the particles may be, their relation to one another appears to be that of mere juxtaposition generally. I might have thrown them together thus. But the humblest fungus betrays a life akin to our own. It is a successful poem in its kind. There is suggested something superior to any particle of matter, in the idea or mind which uses and arranges the particles. Henry David Thoreau Journal, Oct. 10,1858 Microorganisms in terrestrial habitats normally are found in an aqueous phase, but are surrounded by a variable array of solid components in the form of soil particle, rock, plant, and animal surfaces. Opportunities arise in these systems for gas-liquid and even gas-solid interfaces to be of some significance in the ecology of terrestrial microorganisms. The nonaqueous components are so variable in type and extent that the nature and area of interfaces are difficult to define with any degree of precision. An awareness of their importance, however, does help in any interpretation of microbial activities within such complex ecosystems. In this chapter, I intend to discuss several typical terrestrial ecosystems where nonspecific interfacial interactions are of importance. These include soils, the rhizosphere and phyllosphere of plants, and microbial dissolution of minerals. Many interfacial effects involving microorganisms in animal systems appear to be of a specific nature and some will be considered in Chapter 6.

82

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Soil Structure and Microbial Microhabitats

Soils are the most complex of microbial habitats. The characteristics of solid soil components are variable in three dimensions, changing with depth as well as with area, this variability affecting the physical and chemical properties of the soils, which, in turn, affect both plant and microbial growth. Soil groups Soils consist of mineral grains and organic materials occurring in a remarkable variety of arrangements. The traditional way of classifying soils into groups is based on the morphological features of soil profiles, where the soil profile is defined as the complete vertical succession of genetic horizons down to the parent material or parent rock (Brewer, 1964). The development of different structured layers or horizons in a soil profile depends on the nature of the parent material, the climate, and the topography, all of which influence the degree of weathering, leaching of soluble materials, and translocation of smaller particulate fractions. Classification of soil groups is subject to limitations resulting from subjective elements in the framing of central concepts and in the choice of keying properties (Stace et ah, 1968). Examples of modern soil classifications are those of the U.S. Department of Agriculture (1960) and Northcote (1971). Soils may be classified into broad textural classes on the basis of particle size analysis (Table 5.1). With decreasing particle size there is an increase in both particle number and surface area per g of soil. The interfacial area increases with increasing proportions of the finer fractions, and, consequently, the opportunities for interactions between soil particles and microorganisms must increase. For example, Minenkow (1929) reported that sorption of Bacillus cereus var. mycoides and Serratia marcescens increased with increasing clay content in a wide range of soils tested (Figure 5.1). The actual relationship between microorganisms and soil particles is more complex than indicated by the results in Figure 5.1, because particle size analysis of soils fails to account for the spatial distribution of various solid components within the soil mass. Because of their small size (large surface area) and their cation exchange properties, clay minerals have profound effects on the

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83

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γη rq 4, with the resultant crumbling of the outer surfaces. The reported sources of reduced sulfur compounds for oxidation by the thiobacilli were atmospheric pollutants in Paris limestone samples, and seepage from soil water in Angkor Wat (Cambodia) sandstone samples.

6

Specific Interfacial Interactions in Microbial Ecology Dust as we are, the immortal spirit grows Like harmony in music; there is a dark Inscrutable workmanship that reconciles Discordant elements, makes them cling together In one society. William Wordsworth The Prelude

Specific interfacial interactions involve an interlocking of complementary molecular structures on the surfaces of the interacting bodies. It is obvious that organisms capable of adhering specifically to a particular animate or inanimate surface must gain some selective advantage in ecosystems where numerous microbial species exist. Elucidation of the exact mechanism of such complementary molecular interactions has been achieved in few systems. Investigations of microbial systems have been stimulated to some extent by studies of cellular recognition between different species of sponge cells (Moscona, 1963 and 1968; Humphreys, 1965). Attachment and aggregation of dissociated sponge cells is mediated by specific glycoprotein particles of about 200A diameter localized at cell surfaces. These "specific cell-ligands" were separated from cells by washing in seawater free of divalent cations. The washed cells were unable to aggregate, but regained this ability on addition of the specific ligands to the cell suspension. This aggregation effect is species specific, since, when dissociated cells of two sponge species, Microciona and Haliclona, were mixed, aggregates formed only between like cells. In addition, specific cell-ligands from Microciona enhanced aggregation of Microciona cells but not of Haliclona cells, and vice versa. The activity of the ligands was not destroyed by treatment with the enzymes DNase, RNase, trypsin, collagenase, hyaluronidase, and Iysozyme, but was destroyed by α-amylase and pronase, as expected with glycoproteins. Antiserum prepared against the purified ligand preparation from Microciona

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Ecology

cells agglutinated these cells but not Haliclona cells. Somewhat similar results have been reported by Lilien (1968) and Daday (1972) for embryonic chick neural retinal cell aggregation. Microbe-Microbe Interactions Mating

contact

Sexual agglutination in the yeast Hansenula wingei results from interactions between two complementary glycoproteins on the surfaces of the respective mating types (Crandall and Brock, 1968). These glycoproteins were termed 5-factor, from strain 5, and 21factor, from strain 21. The interaction of these two factors, or of each factor with cells of the opposite type, is rapid, specific, and readily reversible. The 5-factor is an agglutinin and, according to Crandall and Brock, is multivalent in the immunological sense. Biologicallyactive particles are heterogeneous, varying in size from 3.5S to 100S. The activity of the 5-factor is destroyed by mercaptoethanol and proteolytic enzymes, but not by heat or alkali. The 21-factor is not an agglutinin, but reacts with the 5-factor and inhibits its biological activity. This factor is homogeneous in size (2.9S) and is thought to be univalent. It is resistant to mercaptoethanol and proteolytic enzymes, but is inactivated by heat and alkali. Crandall and Brock have suggested that the specific binding site on each factor is in the protein moiety. Neither the 5-factor nor the 21-factor is synthesized in diploid hybrids, and Crandall and Brock suggest that this results from a repression of each sex factor by some genomic element of the opposite mating type. Two types of nonconjugating mutants were isolated from strain 5, a nonagglutinative mutant lacking the ability to synthesize the 5-factor, and an agglutinative but nonconjugative mutant. The existence of this latter mutant type indicates that the glycoprotein sex factors are not solely responsible for conjugation, other factors being necessary for the process of cell fusion. The role of specific binding sites in conjugation of isogamous dioecious Chlamydomonas species has been studied by Weise (1965) and Weise and Hayward (1972). Differences were established between different species and between gamete types ( + or strains) on the basis of their enzyme and concanavalin A (Con A)

Specific Interfacial

109

Interactions

ο ε α

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