Microbial Ecology: Fundamentals and Applications 0201000512

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MICROBIAL ECOLOGY:

FUNDAMENTALS AND APPLICATIONS RONALD

M. ATLAS

University of Louisville

RICHARD BARTHA Rutgers University

A

ADDISON-WESLEY PUBLISHING COMPANY Reading, Massachusetts • Menlo Park, California

London

•

Amsterdam

•

Don

Mills,

Ontario • Sydney

This book

is

in the

Addison- Wesley Series

in the Life

Library of Congress Cataloging

Atlas,

Ronald

M

in

Sciences

Publication Data

1946-

Microbial ecology. Includes bibliographies and index. 1.

author.

Microbial ecology. 11.

biology.

[DNLM:

Title.

QW4

1.

Bartha, Richard, joint 1.

Ecology.

A881m] 576M5

QR100.A87 ISBN 0-201-00051-2

©

2.

Micro-

80-13684

Addison-Wesley Publishing Company, Inc. Philippines copyright 1981 by Addison-Wesley Publishing Company, Inc. Copyright

1981 by

All rights reserved. in a retrieval

No

part of this publication

may

be reproduced, stored

system, or transmitted, in any form or by any means, electronic,

mechanical, photocopying, recording, or otherwise, without the prior written

permission of the publisher. Printed

in the

United States of America.

Published simultaneously in Canada. Library of Congress Catalog Card No. 80-13684.

ISBN 0-201-00051-2

ABCDEFGHIJK-llA-89876543210

PREFACE

A

report published in the

News

Bulletin of the

American Society for Microbiology

in

December,

1975 concluded that “the teaching of microbial

ecology

is

sorely in need of a substantive treatment

well-documented text on the level of The Microbial World (by Stanier, et al.y\ In writing this book we have responded to this need.

in this field in a single

We

have attempted to write a comprehensive text

for the field of microbial ecology. create a thorough text, useful

and

Our aim was

to

flexible microbial ecology

not only to microbiology majors, but

the student will have had introductory courses in

general chemistry, biology and microbiology.

The coverage of the field of microbial ecology in this book is more complete than any other book in this field. The book includes a short historical introduction tracing the emergence of this new field (chapter 1). Microbial diversity and metabolism are reviewed in chapters 2 and 3. Material normally found

in

upper

books, as far as

level general it is

microbiology text-

essential to the

of microbial ecology,

is

understanding

summarized

in these

two

also to students in general ecology, environmental

review chapters. This review considers both pro-

management

karyotic and eukaryotic microorganisms. Ecologi-

science, sanitary engineering, resource

and numerous other related study programs. A thorough treatment of basic principles and approaches is followed by an extensive discussion of various applied aspects of microbial ecology. In addition to serving as a text, the useful as

book should

also be

an introduction and reference source for

scientists in related fields

who wish

to extend their

knowledge and research into microbial ecology, and should help to bridge the communications gap among ecologists and microbiologists. As a teaching tool, this text is designed for a onesemester course on the senior undergraduate or

and intends to familiarize the students with the principles, methodology and practical applications and implications of microbial ecology. As a background, we have assumed that

graduate

level,

parameters (environmental determinants, numbers, biomass and activity) and their measurement are discussed in chapters 4 and 5; chapters 6 and 7 are devoted to habitat and community cal

ecology.

These topics are usually addressed

in

general ecology courses, with consideration given

and animals, but the applicability of ecological principles to microorganisms receives little attention in general microbiology and

to higher plants

ecology texts. Interactions

among microorganisms,

between micro- and macroorganisms, are explored in chapters 8 through 10. These chapters as well as

include discussion of the relationship of micro-

organisms to plant growth and pathology and to animal nutrition and disease. Microbial activities in

biogeochemical cycling and applied aspects of

iii

iv

Preface \

microbial ecology are covered in chapters

The

1

1

microorganisms in biodeterioration control, in soil-, water- and wastemanagement, in various contemporary pollution problems, in resource recovery, in energy production and in biological control receive detailed and up-to-date coverage. Chapters 16 and 17 discuss sampling designs, statistical evaluation of results and modeling approaches that are gaining an increasingly important role in research in microbial ecology. Modeling and the systems approach have received wide use by general ecologists, but hitherto have been given relatively little attention by through

15.

role of

offered at

many

universities.

attention given to algae, fungi,

viruses, or protozoa.

We,

therefore, have included

introductory review chapters as “background equalizers”.

To

students with a sound microbiology

background, these chapters

will

merely serve as

concise refreshers, but to others they will impart

background information needed for the subsequent chapters. Depending on the composition of the class, the instructor may choose to emphasize, skim, or skip these two review chapters. In the chapters of our book that deal with funda-

essential

mental aspects of microbial ecology, along with the presentation of factual knowledge, we took care to discuss the

microbial ecology has gone hand in hand with tech-

developments, and the field will continue to expand as new experimental tools become availnical

able.

Experimental approaches need to be empha-

sized,

because the

common introductory laboratory

courses in microbiology that stress pure culture

procedures do

little

investigation of the

to prepare the student for the

dynamic interactions of organ-

isms and their environment.

pare themselves for making research contributions

grounds and career goals. The study of microbial ecology cuts across traditional academic disciplines and is truly an interdisciplinary science. A challenge in writing this book was to accommodate the rather varied backgrounds of students taking a course in microbial ecology. In our experience, some students have strong backgrounds in biology and microbiology, but not in the relevant environmental sciences, such as soil science or limnology. Others have strong backgrounds in ecology, but not in microbiology. Individuals who have taken an introductory course in microbiology may have been exposed almost exclusively to material concerning little

of

The students who

now

attend these courses have a wide diversity of back-

bacteria with

field

being

microbial ecology are

in

knowledge. The emergence of the

At the option of the instructor, the last two chapters of the book (statistics and modeling) may be treated either as an integral part of the course material, or as an appendix with special relevance for those students and scientists who wish to pre-

microbiologists.

Courses

this

methodology employed

in obtaining

in this field.

An advanced

text should serve as a reference

source to current literature, but the style of citainhibits

most review articles and treatises often efforts to summarize, simplify and inter-

pret. In

order to increase the readability of the text,

tions used in

we chose not

to

list

specific citations for

our state-

ments. Instead, we have provided an extensive

list

of appropriate references, sources, and suggested

readings at the end of each chapter. Inclusion of full titles

with the references should facilitate ready

access to the pertinent literature for terested in pursuing further detail subject.

We

anyone

in-

on a particular

suggest that advanced students should

be required to examine some of these works so that

become familiar with extracting information directly from the scientific literature. Throughout the book we have liberally illustrated general statements with specific figures and tables taken from recent journal articles; the source articles are cited in the legends and are included among the references. They also provide access points to the they

relevant literature

The

field

on

specific topics.

of microbial ecology has experienced

dramatic growth during the past twenty years. While prior to 1960 this area of specialization was

unknown, an impressive body of literature has since accumulated in this field. The rapid devirtually

Preface

velopment discipline

microbial ecology as a

of

was undoubtedly promoted by a

Many

coinci-

including Drs. R. Blakemore, P. Johnson,

J.

McN.

Sieburth, E. Grula, H. Reichenbach, R. Unz,

J.

environmental

Cairns, the American Society for Microbiology,

of today’s environmental problems,

Springer-Verlag, John Wiley and Sons, Academic

dent resurgence quality.

scientific

v

in societal interest in

McGraw

Book

Co., Prentice Hall, W.B.

as well as their potential solutions, are intimately

Press,

interwoven with the rhicrobial component of the

Saunders and Williams and Wilkins. We wish to thank Ms. J. Levy and Mrs. D. Karpoff for typing; Ms. E. Hackett for copy editing; Mr. E. Herzer for artistic assistance; and Mr. P. Owens for photographic contributions. We wish to thank our families for their patience and support during this undertaking, and especially

global ecosystem.

Numerous

practical implications

add relevance and excitement to student interest in the subject that Microbial Ecology:

is

this

new

increasing.

and hope

field,

We

Fundamentals and Appli-

cations helps stimulate interest

in

this

exciting

field.

We

Hill

wish to acknowledge our colleagues, particu-

Michel Atlas, reference librarian, for her profes-

Meeks, W. Mitsch, G. Cobbs, M. Finstein, D. Eveleigh and D. Pramerfor reviewing various sections of this work; their suggestions proved most helpful in improving the

sional contributions to the completion of this book.

larly Drs. J. Staley, J. C.

final product.

We

viduals, societies

are indebted to the

many

indi-

and companies who generously

provided permission to reprint

illustrative material.

Louisville,

New

Kentucky

Brunswick,

June, 1980

New

R.

Jersey

M. A. R. B.

*

,

t

.

I

^

|Sk

>*

Fig. 2.18

A

coenocytic

•

\ ' 1

I

.,*•

—

»V“

35

•-

« ’

mycelium of a phycomycete.

(Courtesy of O. K. Miller.)

Fig. 2.20

Photomicrograph of the basidiomycete

Gomphidius glutinosus showing basidiospores attached to the basidium. (Courtesy of O. K. Miller.)

Fig. 2.19

An

ascus of Schizosaccharomyces octosporus

showing eight ascospores. (Courtesy of Carolina Biological Supply Co.)

Hypochytridiomycota, the Oomycota, the Zygomycota, and the Trichomycota. The Chytridiomycota are differentiated from all other fungi by the production of zoospores which are motile with a single posterior flagellum of the whiplash type.

The Hyphochytridiomycota

also produce zoo-

spores, but the zoospores are motile by tinsel type flagellum.

mycelium of the Phycomycota

means of a

The Hyphochytridiomycota

coenocytic (Fig.

are anteriorly uniflagellate. In both the Chytridio-

Coenocytic mycelia are nonseptate and thus

have multiple nuclei. The Phycomycota produce

mycota and the Hyphochytridiomycota there are haploid zoospores which can generate a vegetative

asexual and sexual spores but the sexual spores are

structure, or thallus. Sexual reproduction occurs in

not borne in or on a specialized structure. The

both groups with formation of a diploid structure.

Ascomycota, Basidiomycota, and Deuteromycota all have septate mycelia. The Ascomycota form

The Oomycota reproduce by flagellated zoospores. The zoospores have one tinsel type flagellum, and one whiplash type flagellum. The Oomycota in-

2.18).

is

sexual spores within a specialized structure called the

ascus (Fig.

2.19).

The Basidiomycota form

clude the water molds, white rusts, and

downy

sexual spores on a specialized structure, the basi-

mildews. Saprolegnia, an opportunistic fish patho-

The Deuteromycota have no known sexual spores and are believed to be imperfect stages of Ascomycota or Basidiomycota. The Phycomycota are classified according to

gen,

dium

types

(Fig.

2.20).

of spores

into

the

Chytridiomycota, the

an example of an Oomycete. Phytophthora infestans is the causative agent of the potato blight is

and was responsible for the great famine. Sexual reproduction in the volves formation

Irish

potato

Oomycota

in-

of oospores which are thick-

36

Microbial Classification and Structure

walled spores that develop from a large nonmotile

female gamete, the oosphere.

The Zygomycota are characterized by formation of a zygospore. The zygospore is a sexual resting spore that usually results from fusion of two gametangia. The Zygomycota lack motile cells. The Mucorales (bread molds) are examples of Zygomycota. The Trichomycota produce zygosporelike structures. Trichomycetes are usually attached by holdfasts to the gut wall of arthropods. The Ascomycota possess an ascus which

Fig. 2.21

may

be considered as specialized sporangia. Asco-

Drawing of

a morel.

(From Lechevalier and

Pramer, 1971; reprinted by permission; copyright

J.

B.

Lippincott Co., Philadelphia.)

Ascomycota, are produced within the ascus. Ascomycetes may be spores, the sexual spores of

filamentous or primarily unicellular (as the asco-

sporogenous yeasts). In the life cycle of a typical filamentous Ascomycete, the ascospore germinates, forming hyphae in which the nucleus divides. Septa then form within the hyphae. The septa of Ascomycetes have a pore through the center and nuclei may pass from one cell into another. The mycelia of filamentous Ascomycetes typically contain a large portion of chitin.

A

variety of asexual spores are

produced by Ascomycetes, including blastospores (spores produced by budding), arthrospores (spores produced by fragmentation of hyphae), and conidia (spores formed at the tip or side of the hyphae). Ascomycetes have specialized hyphal structures, conidiophores, which are fruiting bodies and bear the conidia. The Ascomycetes are divided into two large groups: the Hemiascomycetes, in which the asci are not produced in or on a specific structure, and the Euascomycetes, in which the asci are produced in or on a specific structure called the ascocarp. The ascosporogenous yeasts, e.g., Saccharomyces, are Hemiascomycetes. The Euascomycetes are further divided according to the structure of the

ascocarp into the Plectomycetes,

which the ascocarp has no special opening, the Pyrenomycetes, in which the ascocarp is shaped like a flask, and the Discomycetes, in which the ascocarp is cupped or saucer shaped. The morels and truffles are Discoin

Fig. 2.22

Drawings of

fruiting bodies of various basi-

diomycetes. (From Miller, 1972;

America,

E.

P.

Mushrooms

of North

Dutton Co.; reprinted by permission;

Chanticleer Press.)

the Basidiomycetes are called basidiocarps.

Most some

Basidiomycetes are filamentous but there are yeasts that are Basidiomycetes. Basidiomycetes

and have clamp cell connections. These connections are hook-like structures which serve to bridge two typically have mycelia that are binucleate

The Basidiomycetes are divided into the Heterobasidiomycetes, which have septate basidia, and the Homobasidiomycetes which typically have club-shaped nonseptate basidia. The adjacent

cells.

Heterobasidiomycetes include the rusts and smuts. The Homobasidiomycetes include mushrooms, shell fungi,

puffballs, stinkhorns,

fungi (Fig. 2.22). also

A

and birds nest

variety of asexual spores are

produced by Basidiomycetes. The Deuteromycetes, or imperfect fungi ap-

mycetes (Fig. 2.21).

parently lack a sexual or perfect stage. Deutero-

Basidiomycetes produce sexual spores on the surface of a basidium. The spore bearing bodies of

mycetes

typically- .reproduce

conidia. In

only by means of

most cases the Fungi Imperfecti appear

C lassification of

Aspergillus species

Fungi

37

Penicillium species

H Fig. 2.23

Conidia of various fungi imperfecti: (A-F)

photomicrographs; (G) scanning electron micrograph of Aspergillus', (H,I) schematic drawings of Aspergillus Q.nd

Penicillium. (A-F:

of

W. Rosenzweig;

from H,l:

Ajello, et ai, 1963; G:

from

Courtesy

Ajello, et ai, 1963.

38

Microbial Classification and Structure

Table 2.7

Some

representative genera of Fungi Imperfecti

Genus

Description

A Iternaria

Soil saprophytes

spores

Arthrobotrys

fit

and plant pathogens, “muriform”

together like bricks of a wall.

Soil saprophytes,

some form

organelles for capture of

nematodes. Aspergillus

Common

molds, radially arranged black conidio-

spores.

Aureobasidiurn (Pulullaria)

Short mycelial filaments, lateral blastospores. Often

damage painted

Common

Candida

yeast.

surfaces.

Able

to

decompose concentrated

sugar solutions and hydrocarbons.

Some

cause

mycoses. Coccidioides

C. immitis causes

mycotic infections

in

humans and

animals.

Cryptococcus

Yeasts, saprophytic in soil but

mycoses

Geotrichum

Common

in

soil

some may cause

animals and humans. fungus. Older mycelial filaments break

up into arthrospores.

Helminthosporium

Cylindrical, multiseptate spores.

Many

are eco-

nomically significant plant pathogens.

Histoplasma

H. capsulatum causes

Penicillium

Common in

Trichoderma

human

mycosis.

mold with green conidiospores arranged

brush shape.

Common

soil

saprophyte with characteristic banana-

shaped spores.

Ascomycetes. There are various forms of conidia (Fig. 2.23). Phialospores form on a phialide which is a cell from which a succession of conidia develop without increasing the to be conidial stages of

Meristospores form at the end of a conidiophore that successionally length of the phialide

have been discovered recently, a confusing piece-

meal reclassification has been wisely avoided.

STRUCTURE OF FUNGI

itself.

All fungi have eukaryotic cells that are delineated

mem-

elongates and divides. Blastospores are formed by

from

budding of the apical spore. Arthrospores are conidia, formed by fragmentation of a hypha. Im-

brane (Fig. 2.24). The cytoplasmic membrane of eukaryotic cells is a bilipid layer consisting of

perfect fungi are classified according to the type of

hydrophilic and hydrophobic portions.

and the color and shape of the conidia. Some representative genera of Fungi Imperfecti are listed in Table 2.7. While perfect, ascospore-bearing stages of some of the most com-

plasmic

the spore-bearing structure,

mon

representatives {Aspergillus,

Penicillium)

their

environment by a cytoplasmic

membrane

acts as a

As with prokaryotic

cells,

The cyto-

semipermeable barrier. both passive diffusion

and active transport carry materials across the membrane in and out of the cell. There are transport enzymes associated with cytoplasmic membranes of

Structure of Fungi

fungi.

Membranes

of eukaryotic cells usually have

associated sterols which are not typically found

in-

protein.

The nucleolus

ribosomal

is

39

the site of production of

RNA. During division

the nuclear

mem-

The cytoplasmic membrane of most true fungi is surrounded by a cell wall. The cell wall usually is composed of microfibrils of cellu-

system with numerous microtubules. The micro-

combination of cellulose and

tubules disappear following mitosis and the nuclear

prokaryotic

cells.

lose or chitin, or a

an amorphous matrix of the same material. The cell wall is a rigid structure and protects the cell against osmotic shock. The vegetative cells of most slime molds lack a cell wall and are pleomorphic, though some structures in the life cycle of slime molds normally have rigid cell walls. The periplasm is the space between the cell wall and cytoplasmic membrane. Lomasomes, which occur chitin within

within the periplasm of fungi,

may be tubular or or membrane free

membrane structures particles. The function of lomasomes

vesicular

is

not

cells.

The

known. Nuclei are found within

fungal

all

nucleus contains the fungal genome. The

DNA

of

genome is arranged as chromosomes. Chromosomes are linear strands of double-helically

the fungal

coiled

DNA.

Histones, which are basic proteins,

are associated

nucleus nuclear

is

DNA. The

bounded by a nuclear membrane. The

membrane

numerous is

with chromosomal is

a bilipid structure

which has

pores. Within the nucleus the nucleolus

a spherical

body which contains

DNA, RNA, and

Vocuole

Lomosome

brane disappears or contracts and

replaced by a

mitotic apparatus consisting of a spindle-shaped

membrane is resynthesized. Ribosomes occur within

the cytoplasm of

Ribosomes in the cytoplasm of eukaryotic cells are SOS ribosomes and are composed of 60S and 40S subunits. The ribosomes are the sites of protein synthesis. Ribosomes are composed largely of RNA and protein. eukaryotic

cells.

There are several membranous internal structures in eukaryotic cells. The mitochondria, which are the site of oxidative phosphorylation (see Chapter 3), are composed largely of phospholipid membranes. Mitochondria have internal flattened membranes called cristae; they also contain their

own

DNA and ribosomes.

Mitochondrial ribosomes are 70S ribosomes as occur in prokaryotic cells. Eukaryotic cells, thus, have SOS ribosomes in the cyto-

plasm and 70S ribosomes in the mitochondria. The endoplasmic reticulum is a membrane network that runs through the eukaryotic cell. The endoplasmic reticulum of fungi, which is relatively sparse and not closely packed, is continuous with the nuclear envelope but generally not continuous with the cytoplasmic membrane. The SOS ribosomes may attach to the endoplasmic reticulum forming polysomes. Endoplasmic reticulum without ribosomes is called smooth endoplasmic reticulum and with attached ribosomes, rough endoplasmic reticulum. Enzymes

Cell wall

is

may

be bound to the

endoplasmic reticulum.

PI

The Glycogen

Nucleus

golgi apparatus, or dictyosome,

ternal aggregation of

membranes

that

is

is

an

in-

probably

involved in packaging materials for export from the Endoplasmic reticulum

cell.

Fungi

may have

vacuoles which are used for

storage of reserve material. Mitochondrion

Nucleor envelope

The normal storage

products of fungi are glycogen and

may

lipids.

Glycogen

also occur as free rosette-like structures.

Schematic drawing of a typical fungal cell. (From Moore-Landecker, 1972; reprinted by permission;

Fungi produce a large variety of spores. The spores are usually involved in reproduction and dissemination of fungus. Spore production is a key

copyright Prentice-Hall, Englewood

characteristic used in classification of fungi

Ribosomes

Fig. 2.24

Cliffs.)

and has

40

Microbial Classirication and Structure

Table 2.8 Descriptions of

some fungal spores an aecium (an aecium is a structure consisting of binucleate hyphal cells, with or without a peridium, which produce spore chains consisting of aeciospores, alternative with disjunctor cells, by the successive, conjugate division of the nuclei).

produced

Aeciospore:

a binucleate spore

Aplanospore:

a

Arthrospore:

a spore resulting

Ascospore:

a spore, which results

Azygospore:

a zygospore which develops parthenogenetically.

Basidiospore:

a spore borne

Blastospore:

an asexual spore formed by budding.

Chlamydospore:

a hyphal cell, enveloped

in

nonmotile spore.

from the fragmentation of a hypha

from meiosis, borne

(also called oidium).

an ascus.

in

on the outside of a basidium, resulting from karyogamy and meiosis.

by

a thick cell wall,

which eventually becomes separated from the

parent hypha and behaves as a resting spore.

formed asexually, usually

at the tip or side of a

Conidium:

a spore

Dictyospore:

a spore with both vertical

Oospore:

a thick-walled spore which develops

and horizontal

hypha.

septa.

from an oosphere (which

is

a large, naked, nonmotile,

female gamete) through either fertilization or parthenogenesis.

produced from a phialide (which is a small bottle-shaped structure from which spores are produced, and are characteristically formed inside the phialide and extruded).

Phialospore:

a spore

Porospore:

a spore produced

Pseudospore:

a nonmotile

Pycnidiospore:

a

from pores of a conidiophore (which

naked spore; found

conidium borne

in a

in

some

pycnidium (which

is

a specialized

hypha bearing conidia).

Acrasiales.

is

an asexual, hollow fruiting body, lined inside with

conidiophores).

Scolecospore:

an elongated, needle, or worm-like, spore.

Sporangiospore:

a spore borne within a

sporangium (which

is

a sac-like structure, the entire protoplasmic

contents of which become converted into an indefinite

number

Stylospore:

an elongated or cane-shaped pycnidiospore of unknown function.

Teleutospore:

a thick-walled resting spore in

of spores).

some Heterobasidiomycota, notably

the rusts

and smuts,

in

which karyogamy occurs. Uredospore:

a binucleate, repeating spore of the Uredinales.

Zygospore:

a resting spore

been discussed

in the

which

results

from the fusion of two gametangia.

previous section. Table 2.8

shows some of the spores produced by fungi. The vegetative forms of most true fungi are nonmotile, but motile stages are involved in the

many fungi. Some fungal

life

cycle of

spores are motile with flagella.

organisms are different from those of prokaryotes. Eukaryotic flagella have

The

flagella of eukaryotic

two central

fibers

surrounded by nine peripheral

pairs of fibers; the fibers are

The

entire flagellum

is

composed of

protein.

surrounded by a phospho-

membrane. Tinsel flagella bear small filaments which extend from the main axis of the flagella; whiplash flagella lack these small filaments and are lipid

smooth.

Most

true fungi produce filaments

known

as

C lassification of Algae

41

Table 2.9

Pigments of the major groups of algae

substrate by this process. Many lipids can be metabolized to form acetylCoA. Initially complex lipids can be cleaved by lipase enzymes forming fatty acids and, normally, 3.1

1).

glycerol.

process

The

fatty acids are then metabolized in a

known

carbons are

as /^-oxidation. Aliphatic hydro-

initially

metabolized

duced from

yield per six

oxidized to fatty acids and then

in a )3-oxidation.

Acetyl-CoA

is

pro-

fatty acids in )3-oxidation. In /3-oxida-

tion fatty acids that are successively

two carbon

atoms shorter are formed with the liberation each time of acetyl-CoA. The acetyl-CoA so formed can go through a cycle similar to the TCA cycle called the glyoxylate bypass cycle results in the consumpcycle

is

necessary to maintain the intermediates in

NADH

and FADH are generated, which can then go through an electron the cycle. In this process

02

H2O

transport chain generating

showing electron transport chain used during respiration which reoxidizes reduced co-enzymes NADH and FADH and requires an external terminal electron acceptor, e.g., O 2 Three ATP molecules are generated for each NADH molecule; two Fig. 3.10 Oxidative phosphorylation

ATP. Acetate can

larly be utilized to generate energy.

Two

the glycoxylate bypass cycle results in the tion of four acetate molecules

simi-

turns of

consump-

and the formation

.

ATP

molecules are generated for each

FADH

molecule.

of one hexose and two

CO

2

molecules.

Proteins can be metabolized in several ways. Proteins can be hydrolysed to oligopeptides and/or

respiration than by fermentation. This less

substrate need be

energy needs. Hence, substrate

consumed

many

means

that

to satisfy the cell’s

cells will

consume

less

under aerobic conditions than under

anaerobic conditions, a phenomenon

known

as the

The Pasteur effect is significant in microbial ecology when examining the biodegradation of various organic compounds. Pasteur

effect.

Glucose

is

obviously not the only substrate

heterotrophic microorganisms can use for generating

ATP. Other

possible substrates include a great

variety of carbohydrates, lipids and proteins.

Various carbohydrates can be converted to glucose 6-phosphate or fructose 6-phosphate and then go

through the

rest

of the glycolytic pathway (Fig.

amino

acids.

Amino

acids are often deaminated

forming carboxylic acids. These carboxylic acids can either be metabolized to acetyl-CoA or to one of the

TCA

cycle intermediates. In either case, the

molecule enters the place, generating

ATP by amount of ATP tion of

TCA

NADH

cycle at the appropriate

with subsequent produc-

oxidative phosphorylation.

The

dependent on the place of entry into the TCA cycle. There are numerous other substrates that microorganisms can utilize for generating energy, e.g., hydrocargenerated

bons, nucleic acids,

is

etc.

The end products of one organisms’s metabo-

ATP

by other organisms, or even by the same organism under different lism can be used to generate

60

Microbial Metabolism

LACTOSE

GLYCOGEN

SUCROSE

GLUCOSE

FRUCTOSE

1-P

GLUCOSE

1-P

GLUCOSE

6-P

ATP

C. GLUCOSE

1-P

GLUCOSE

6-P

GLUCOSE

6-P

FRUCTOSE

»

GLYCOLYSIS

Fig. 3.11

Pathways

for

ADP

* Pi

6-P

glycolysis

metabolism of

glycolysis

lactose, sucrose

and glycogen.

environmental conditions. Thus, for example, lactic acid, the fermentation end product of lactic acid bacteria, can be converted by another group of bacteria to propionic acid yielding ATP. These propionic acid bacteria gain only one mole of ATP for every three moles of lactic acid they utilize. Fermentation end products, including ethanol and lactate, can also be metabolized under aerobic conditions to yield additional ATP. These processes

A

and then passage through the

TCA

cycle

and

oxidative phosphorylation.

becomes apparent that various microorganisms with distinct metabolic pathway capabilities can generate different amounts of ATP under each defined set of environmental conditions. The efficiency of ATP generation, and the available Thus,

it

substrates in a given ecosystem, determine in part will survive

and which sub-

strates will be utilized in that ecosystem.

-CH2(CH2)xCOOH

-CH2(CH2)x-2COOH

CoA

which microorganisms

LIPID

I

generally involve a reverse step back to form acetyl-

Some microorganisms are incapable of generating their own ATP. Obviously, such organisms

(3-OXIDATION

ACETYL-COA

are totally dependent on other organisms for sup-

plying their energy and hence their survival. Viruses

and

rickettsias, for instance, are

supply

cells to

generate

but not enough; the viruses

rickettsias

(Chlamydiae) totally lack the

ability to generate

membrane

ATP.

ATP

will

pass across the

of rickettsias, but will not enter most

other bacterial

cells.

Viruses disassemble within the

host cells and use the cells for their

virions

rickettsias are able to

ATP

some

and some

ATP; some

dependent on host

ATP

generated by the host

reproduction. Outside the host

do not contain

ATP

cells,

and are metabolically

inert.

CATABOLISM Fig. 3.12

Metabolism of

glyoxylate shunt.

lipids

showing ^-oxidation and

Besides generating energy, the

breakdown of

organic molecules, or catabolism, transforms the

Catabolism

Enzymatic transformations are highly

original substrate into smaller essential biochemical

3.14).

Microorganisms require many types of bio-' chemicals, including carbohydrates, proteins, lipids and nucleic acids, for structural and other purposes. The cataboliir pathways branch off at

specific.

units.

Various microorganisms have enzymes of

distinct specificity

and are thus capable of carrying

out a finite range of transformations. bility in the

The

varia-

kinds of enzymes a microorganism

various points into anabolic or biosynthetic path-

able

ways that produce these macromolecules and their components (Fig. 3.13). Relatively few precursor molecules serve to produce all the required macromolecule components. A continuous supply of

ranges of the individual enzymes, explains

these precursor molecules

necessary.

is

61

to

synthesize,

as

well

as

in

the substrate

some microorganisms can break down strate while others cannot. This

is

is

why

a given sub-

clearly of great

importance in determining whether or not a microorganism will survive in a particular ecosystem.

The transformation processes which produce these units are all enzymatic. Enzymes are protein

The basic aerobic catabolic processes are the same as the heterotrophic transformation pathways

molecules that catalyse biochemical reactions, i.e., reduce the activation energy required to carry out a

already described for generating

particular biochemical transformation.

They

per-

mit chemical reactions to occur rapidly at temperatures that

do not destroy biological systems

(Fig.

intermediates in the glycolytic pathway and the

TCA

cycle are the required precursor molecules for

biosynthesis.

As

these pathways are active in most

aerobic heterotrophic microorganisms, they readily

CARBOHYDRATES

GLUCOSE

r^RlBOSE

I

CO

2

»/>

o VJ < u LU -A

u 3

UJ

cc a.

-PYRIMIDINE

Z

»

PURINE

Fig. 3.13 Interconnections

pathways.

ATP. Various

of catabolic

and anabolic

Microbial Metabolism

62

Many

times a microorganism will be unable to

utilize a particular substrate as sole

This

may

carbon source.

be because the organism lacks the en-

zymatic capability for carrying out the transformation; because the organism cannot generate suffi-

Substrate Cone

Comparison of

Fig. 3.14

(s)

characteristics

of enzymatic

and nonenzymatic reactions. Note that enzymatic reactions exhibit rapid increases in reaction velocity with

increased substrate concentration and temperature and also

show

substrate saturation, optimal and inactivation

temperatures.

ATP

from that carbon source to survive; or because the organism cannot form all the necessary biosynthetic precursor molecules from that source. The latter two explanations do not eliminate the possibility that the microorganism can catabolize

cient

the substrate given an additional supply of energy

or growth factors. In fact,

many organisms can

larly,

cases the glyoxylate bypass replaces the to

because they have an alternate supply of essential

TCA cycle

ATP. When

a

microorganism possesses the enzymatic capability for multiple pathways, it can efficiently generate less

energy versus precursor molecules as

the situation requires, giving such organisms a petitive

advantage

in

survive in a particular

ecosystem and catabolize certain substrates only

Shunts in the basic Embden-Meyerhof glycolysis pathway, such as the hexose monophosphate shunt, provide a more abundant supply of pre-

more or

energy source. Simi-

some

produce the needed precursors.

cursor molecules but a lower yield of

its

and

serve the dual functions of generating energy In

substances which

cannot serve as sole carbon source are catabolized by co-metabolism, a process by which the organism uses a second substrate for

supplying precursors for biosynthesis.

many

growth factors. In a particular ecosystem, intermediate metabolites formed by one group of microorganisms can be translocated and used by another group, a phenomenon ing. It lites

is

not

uncommon

known as cross feed-

for intermediate

metabo-

of aerobic metabolism to reach anaerobic

organisms, allowing them to carry out metabolic activities

of which they would otherwise be in-

capable.

com-

some ecosystems.

Anaerobic or facultative anaerobic hetero-

ANABOLISM OR BIOSYNTHESIS

trophic microorganisms that utilize fermentation

pathways generally do not have an active TCA cycle. Thus, while these organisms can generate ATP and the glycolysis intermediates by pathways already described, they require alternative ways of producing the precursor molecules not supplied by such pathways. To do so they carry out biosynthetic incorporation of CO 2 to produce the precursors that the TCA cycle would otherwise supply (see Fig. 3.16). Producing these components re-

An examination

of microbial anabolic or biosyn-

cycle intermediates. Details of these biosynthetic

pathways is far more complex than the catabolic pathways discussed in the previous section. While many microorganisms can start with and catabolize a single carbon substrate, all microorganisms must produce a tremendous variety of biochemicals for cellular constituents. Not only must a microorganism produce many biochemical molecules but it must assemble these molecules into specific macromolecular arrangements. Every microbial species must have some different biosynthetic pathways to justify its being classified as a distinct species. The variability that must exist in

reactions will be discussed in the next sections.

microbial biosynthetic processes

quires that

some of

the limited energy generated in

glycolysis be used. These

CO

2

fixation reactions

also occur in aerobic systems to replenish

TCA

thetic

is

obvious. In this

Anabolism or Biosynthesis

63

C02

CO by ATP and

Fig. 3.15 Calvin cycle used for assimilation of

CO

autotrophs. This pathway requires

2

,

2

NADPH.

pathways for synthesizing compounds will be discussed when the com-

section only a few

The magnitude of

specific

tential

pounds produced are of general importance or when the pathway for their synthesis is representative of the biosynthesis of similar compounds.

some processes such

this process

biomass of the community. The anabolic

pathway of autotrophic

many

for

determines the po-

CO

is

very similar

of these organisms. Energy

(ATP) and

reducing power

(NADPH),

2

fixation

generated through the

as protein synthesis will

adsorption of light energy or oxidation of inorganic

be discussed in general terms rather than for any

substances as already discussed, are used for reduc-

Also,

specific protein

CO

ing

macromolecule.

which Autotrophic

CO

2

to organic

this

is

compounds. The pathway by

accomplished

is

known

as the Calvin

cycle. 2

Fixation

Autotrophic organisms have the

ability to fix

CO

2

and synthesize organic matter. It is the organic matter formed by autotrophic microorganisms and plants which is utilized by heterotrophic organisms.

While the details of the Calvin cycle are fairly complex, the basic reaction

CO

2

(Fig. is

3.

15)

simple.

reacts with ribulose 1,5-diphosphate to

form

two 3-phosphoglyceric acid molecules. This basic reaction occurs three times in the cycle so that

64

Microbial Metabolism

P-ENOLPYRUVATE

Certain bacteria have

CO2 CH2

still

another pathway for

Some can condense CO2

fixation in addition to the Calvin cycle.

green and purple sulfur bacteria

with acetyl-CoA to form pyruvate. This reaction

is

coupled with the oxidation of reduced ferredoxin; reduced ferredoxin is regenerated by coupling with a light

dependent oxidation of H2S. This

results in

deposition of sulfur granules. In

many

biosynthetic pathways

NADPH

sup-

needed reducing power. It is significant that NADPH, rather than NADH, is used, as it appears to provide a mechanism for separation of biosynthetic activities which involve NADPH from plies the

PYRUVATE

catabolic activities which involve

form oxaloacetate. CO 2 used both by autotrophs and hetero-

Fig. 3.16 Assimilation of

This pathway trophs.

three

is

~P = high

to

energy phosphate.

CO2 molecules

are incorporated, resulting in

a net production of one C3- molecule.

The remain-

ing steps in the cycle are concerned with the reduction of 3-phosphoglyceric acid to glyceraldehyde

3-phosphate, the net product of the cycle, and with regeneration of the ribulose 1,5-diphosphate molecules.

The reduction reactions forming glyceralde-

hyde 3-phosphate require the utilization of six NADPH molecules and six ATP molecules, and the regeneration of ribulose 1,5-diphosphate requires

an additional three ATP molecules. Two glyceraldehyde 3-phosphate molecules formed in the Calvin cycle can be utilized in a reverse glycolytic to

pathway

form a hexose molecule; the net reaction being

6CO2 + 12NADPH + 18ATP 12NADP+ ISADP-b 18P.. In

-

C6H12O6 +

some autotrophic microorganisms

there are

pathways for the fixation of CO2. These alternative pathways can assume greater importance in such organisms than the Calvin cycle. For alternative

example react

in

some

algae, such as Chlorella,

CO2 can

with pyruvate or P-enolpyruvate to form

NADH. NADP

NAD are interconvertable according reaction NAD + ATP ^ NADP + ADP.

and

Heterotrophic

to the

CO2 Fixation

While heterotrophic microorganisms require preformed organic matter, they also must fix CO2 in some anabolic reactions. CO2 fixation by heterotrophs

is

essential for generating

mediates which are used

in

TCA

cycle inter-

anabolic pathways.

Oxaloacetate can be synthesized from the reaction

CO2 and phosphoenolpyruvate, or from the reaction of CO2 and pyruvate, with the expenditure of of

energy in the form of

ATP

(Fig. 3.16).

acetate can be transformed to other

The oxalo-

TCA

cycle

intermediates or can be used as a precursor for synthesis of

amino

acids such as aspartate.

One group of bacteria, the methanogens, convert CO2 to CH4. These bacteria utilize electrons from H2 for CO2 reduction. In this process CO2 acts an electron acceptor. The transfer of electrons from H2 to CO2 does not occur through a conven-

as

tional electron transport chain, but via as yet unidentified electron carriers.

during

ATP

formation occurs

this process.

oxaloacetate. Since oxaloacetate contains four carbons, this is called a C4 pathway, in contrast to the Calvin cycle which

is

a C3 pathway, since the

Fatty Acid Biosynthesis

The

Fatty acids are essential components of lipids and

C4 oxaloacetate formed can be converted via the TCA cycle to C3 forms.

must be synthesized for inclusion into membrane structures. Fatty acids are synthesized from acetyl-

3-P-glyceric acid formed has three carbons.

Anabolism or Biosynthesis

CoA. The acetyl-CoA

CO

form malonyl-CoA, a reaction which requires biotin and uses ATP. The malonyl-CoA reacts with an acyl carrier protein (ACP) to produce malonyl-ACP; acetyl-CoA can also react to form acetyl-ACP. The acetyl-ACP and malonyl-ACP react to form acetoacetyl-ACP which in turn is coupled with the oxidation of NADPH to form butyryl-ACP. This butyryl-ACP can react with acetyl-ACP forming a Ce-ACP. The process of adding acetyl-CoA can be repeated, resulting each time in an increase in chain length of two carbons. Finally, the carrier protein can be removed forming a free fatty acid. These fatty acids can further react with glycerol phosphate to form triglycerides. reacts with

2

NO3*

to

NiN

'

/ \

-

65

/ '

\

(j:ooH

\

c=o

COOH

'

V

H2NtH

/

%

*

I

H-C-H H-C-H

H-i-H H-i-H

COOH

COOH NADPH

a-KETOGLUTARIC ACID

NADP

AMMONIUM

+

L-GLUTAMIC ACID

Formation of the amino acid L-glutamate by reductive amination (or the glutamate dehydrogenase Fig. 3.17

pathway).

organisms must also synthesize additional “unusual” amino acids, e.g., D-amino acids for Storage Product Biosynthesis It is

incorporation into various

often advantageous for microorganisms to pro-

duce storage products for later use as a source of carbon and energy. Such storage products can be used at times of starvation. Glycogen can be synthesized by

many

eukaryotic organisms and by

some prokaryotic organisms. Glycogen

is

cell structures.

Micro-

organisms which are unable to carry out all of the necessary biosynthetic pathways for synthesizing

compounds

these

are restricted to ecosystems

where the required amino acids are available from external sources.

Unlike their precursors from glycolysis and the

a polymer

(uridine

TCA

cycle, all

triphosphate) to form UDP-glucose. UDP-glucose

some

also contain sulfur.

molecules can react to form the glycogen polymer.

organic

Another storage product formed mainly by prokaryotic microorganisms is poly-/3-hydroxy-

thesis.

a particular ecosystem

butryic acid. Synthesis of poly-^-hydroxybutyric

determining the microbial community of that eco-

of glucose. Glucose reacts with

acid

is

UTP

similar to fatty acid biosynthesis already

N

The

system.

or

amino

NH

3

is

acids contain nitrogen

and

The incorporation of essential for amino acid syn-

availability of inorganic nitrogen ions in is

an important factor

in

Some microorganisms can reduce nitrate form the ammonium ions required for direct

described.

ions to

CoA

amination and a limited number of microorganisms can reduce atmospheric nitrogen to the ammonium level. Ammonium ions can be combined directly with some ketocarboxylic acids such as a-ketoglutarate forming L-glutamate (Fig. 3.17). This process is called reductive amination and is coupled

Acetyl-CoA reacts to form acetoacetylwhich can be reduced with NADH, to form

/3-hydroxybutyryl-CoA. As thesis, the

in

fatty acid biosyn-

chain length can be increased by repeated

addition of acetyl-CoA. Subsequent removal of

CoA bial

forms the poly-/3-hydroxbutryic acid. Microcells can accumulate large amounts of these

with the oxidation of

reserve materials.

known

An Amino Acid

There are twenty essential amino acids which tually all

vir-

microorganisms must synthesize for

incorporation into protein molecules.

Some

micro-

to

NADP.

It is

also

as the glutamate dehydrogenase pathway.

important source for the

NADPH

required in

and other biosynthetic reactions is the pentose phosphate pathway. Another pathway for the synthesis of L-glutamate is the glutamine synthetase/glutamate synthase pathway (Fig. 3.18). In this pathway gluthis

Biosynthesis

NADPH

66

Microbial Metabolism

ADP + Pi

ATP

0 -H2N-C-CH2-CH2-CH-COOH+H2O

0 HO-C-CH2-CH2-CH-COOH + NH3

NH,

NHGlutamine

Glutamate

Ketoglutarate

+

2 Glutamate Fig. 3.18

Formation of the amino acid L-glutamate by

the glutamine synthetase/ glutamate synthase pathway.

COOH H 2 NCH

^

HCH HCH

COOH

COOH

Q=0

c^o

HCH H9H

COOH

OXALOACETIC

a-KETOGLUTARIC

ACID

COOH H2NCH I

H^H H^H

L-ASPARTIC

ACID

ACID

COOH

CH20P

CH2OH H2NCH

I

1

HCOH COOH

1

HCH HCH

1

+

COOH

1

COOH

L-GLUTAMIC ACID

COOH

COOH

COOH L-GLUTAMIC ACID

H2NCH HCH

I

nin

COOH

COOH

3 -P-GLYCERIC

a-KETOGLUTARIC

ACID

L-SERINE

ACID Fig. 3.19 Synthesis of

amino

acids by transamination.

tamate reacts with ammonia to form glutamine. This reaction is endothermic and requires ATP. The glutamine then reacts with a-ketoglutarate to form two glutamate molecules. The net result of these reactions can be summarized as: a-ketoglutarate-f

transamination to form other amino acids. By transamination, L-glutamate can react with oxaloace-

NH + ATP ^glutamate ADP + Pi.

The glutamine synthetase/ glutamate synthase pathway operates at low concentrations of ammonia and requires ATP. At high concentrations of ammonia

form L-asparagine. Aspartate also is the precursor for a portion of pyrimidine and purine ring synthesis. Purine and pyrimidine rings are compo-

pathway operates and

formed by transamination. L-glutamate reacts with 3-P-glycerate with a coupled reduction of NAD to form 3-P-hydroxypyruvate, an intermediate which can then react

-I-

3

the glutamate dehydrogenase

does not require

ATP.

Once formed, the amino group of the L-glutamate can be transferred by a process called

form a-ketoglutarate and the amino acid L-aspartate (Fig. 3. 19). Aspartate can go on to react

tate to

with

ammonium

ions, using

ATP as energy source,

to

nents of nucleic acid bases. L-serine

amino

acid which

is

is

another

Anabolism or Biosynthesis 5'

H2S

+

END

THYMINE

S 04 =

CH20H H2NCH

67

»

CH2SH H2NCH

ADENINE

H2O

+

COOH

COOH /

CYTOSINE

HYDROGEN SULFIDE

L-SERINE

Fig. 3.20 Incorporation of sulfur into

amino

GUANINE

WATER

L-CYSTEINE

acids. 5'

with L-glutamate to yield o-ketoglutarate and L-serine (Fig.

amino

OLD 3'

NEW

3. 19).

In addition to the

N

containing amino group,

and methionine also contain sulfur. Cysteine is formed from the reaction of L-serine and H 2 S (Fig. 3.20). The H 2 S the

END

X-A,\

X,

&

*-cA

acids cysteine, cystine

required for this reaction can be assimilated in

X

anaerobic environments or can be produced within the cell by reduction of sulfate ions. The reduction of sulfate to form sulfide requires the expenditure

V

5'

OLD

Fig. 3.21

Structure and replication of

DNA.

ATP

and involves formation of an activated ribose intermediate. L-methionine, of energy as

another S containing amino acid, obtains

sulfur

genetic composition or, in biochemical terms, by

from L-cysteine through a series of reactions. The S containing amino acids are often the active sites of protein molecules and the ability to assimilate inorganic sulfur into those amino acids is quite important. The anabolic pathways for forming the remaining amino acids, especially those containing ring structures, are somewhat more complex and

the arrangement of nucleotide bases in the organ-

will

its

not be discussed here.

Protein Biosynthesis

preceding sections, the presence or absence of specific

enzymes

is

exceedingly important

in

de-

termining the ecosystems in which specific microorganisms will survive. Since all enzymes are pro-

one can not overemphasize the importance of protein biosynthesis and its regutein

molecules,

lation.

microorganism to synthesize a particular enzyme is determined by that organism’s

The

ability of a

ism’s

DNA

(deoxyribonucleic acid). The amino

acid sequence of each protein tion of the

amino

DNA

molecule

is

specified

known

by a

sec-

as a gene. This

acid sequence determines the specificity of

function for the enzyme.

DNA molecule itself

composed of purine and pyrimidine nucleotides. With the exception of a few viruses, the DNA is double stranded. The two strands are complementary and are held together by The

is

hydrogen bonding. Nucleotides

The amino acid molecules formed by these pathways must now be assembled into protein molecules. As has been pointed out several times in the

NEW

the four bases:

in

DNA

contain

adenine, thymine, guanine and

Adenine is complementary to thymine and guanine is complementary to cytosine. In eukaryotic organisms the double stranded DNA molecules are arranged in units called chromosomes. In prokaryotic organisms the double stranded DNA is arranged as a single molecule which may be circular (bacterial chromosome). The DNA molecule can code for production of an identical new DNA molecule by a process called replication (Fig. 3.21). DNA also occurs in extrachromosonal cytosine.

elements called plasmids or episomes.

DNA

repli-

68

Microbial Metabolism

5'

END OH

adenine

HO— P -O— CH,

cation produces a copy of the genetic information

which can then be passed from a parent daughter cell.

The sequence of bases within the

cell to a HO -

DNA

—0 — CH,

1

P

0

mole-

o

acids into a protein molecule

is

A.

OH

0

cule which codes for the incorporation of specific

amino

URACIL

OH

0

//

HO— P — O - CHj

called a

GUANINE

II

'N

o

codon. The sequence of codons which specifies the

complete amino acid sequence for a specific protein is

called a gene.

Each codon

is

composed of

o

HO

three

OH

'nh

f

— P — O — CHj^O.,

CYTOSINE

N^^*^0

J)

nucleotide bases. Using the four nucleotide bases, i

HO

64 different three-base codons are possible. These

r OH 3

64 codons only need to code for the 20 amino acids

found in protein molecules. Some codons do not code for any amino acids and are called nonsense codons. These nonsense codons serve as regulator

some amino by more than one codon and the

RNA.

Fig. 3.22 Structure of

A-G

-

G-T-T - G- G-T-C

END

-

A-A-A-T-T - T-T-T- A-

A

signals to punctuate the message. Also,

acids are coded for

code

is

therefore said to be degenerate.

Any change in the sequence of DNA nucleotide bases is known as a mutation. Mutations change the codons. In most cases this results in a change in the amino acid sequence of the protein. These changes alter the properties of the proteins formed. When these proteins are enzymes, this alters the biochemical activities the organism is capable of performing. Mutations can thereby change the nutritional requirements of a microorganism, changing its ability to compete and survive in an ecosystem. Mutations can also change the role the microorganism plays in the ecosystem, thus affecting other organisms in that ecosystem. It should be noted that the environment can effect mutations. In some ecosystems certain chemicals or

forms of radiation energy will cause mutations. Some of the microorganisms in these ecosystems may have adaptations to protect them against such mutagens. Regardless of whether there is a mutation, the

message of the codon, i.e., the sequence of amino acids coded for by the DNA, must be transferred to form protein molecules. This is accomplished in two stages. First, the base sequence of the DNA is transcribed to messenger ribonucleic acid (Fig. 3.23). The mRNA molecule is transported from the nucleus

in

eukaryotic organisms, or the nuclear area

DNA

T-C-C-A-A-C-C-A-G'T-T-T-A-A-A-A-A-T-T

AA

UU

UC.A

mRNA

A A U U U G G u U G.G CODON IcODON CODON IcODOn'eTC ‘cODON 'codon' A

I

Fig. 3.23 Transcription of

DNA

to

form

mRNA.

prokaryotic microorganisms, to the ribosomes,

in

At the

the anatomical sites of protein synthesis.

ribosome the messenger

RNA

specific protein molecule.

is

translated into the

The process of

transla-

tion (Fig. 3.24) also involves a second type of called transfer

cules carry the

RNA

(tRNA). Transfer

amino

RNA

RNA mole-

acids to the ribosomes.

RNA nucleotide bases pair with their complementary DNA bases (Fig. 3.23). In RNA uracil replaces the thymine of DNA (Fig. 3.22). Except in some viruses, the RNA molecule In transcription,

Only one strand of the DNA, therefore, must be copied to form the mRNA. Once is

single stranded.

the bases are paired in the proper order, the

enzyme

RNA polymerase links the bases to form the mRNA molecule.

The sequence of complementary

bases in the

mRNA

triplet

are also called codons, as in

DNA

which coded them. Once formed, the mRNA moves to and binds to the ribosome where translation occurs (Fig. 3.24). In eukaryotes, the RNA formed in the nucleus the

(hnRNA)

modified before reaching the ribosomes as mRNA. In prokaryotes, the does not appear to be modified before reaching the ribois

mRNA

Carhon

Nutritional Requirements other than

tRNA

moved

then

is

to a

second

site,

69

the peptidyl

This translocation, which requires energy as GTP, allows the next called for charged tRNA to

site.

bring the appropriate site.

A

amino

peptide bond can

the carboxyl group of the

acid to the

now

amino

acyl

be formed between

amino

acid at the

amino

and the amino group of the amino acid at the peptidyl site. Once the peptide bond is formed, acyl site

the peptide can be translocated to the peptidyl site

allowing the next charged

amino

tRNA

to bring the next

amino acyl site. This process is repeated until one of the nonsense codons is reached, this again being a codon that doesn’t call for an amino acid. The nonsense codon thus actually codes for termination. The polypeptide chain then

acid to the

falls

off the ribosome. Generally the formyl

removed and the polypeptide is folded into the finished protein. The folding is determined by the primary amino acid sequence. While it was once thought that the entire mRNA molecule was translated in sequence at the methionine

GTP

GDP

•>]

P, *'1

amino

AMINO— AMINO ACID

— AMINO ACID A

AMINO ACID B ACID C^

acid B

O

6

A

ribosome,

z

o o o

is

is

it

now known

mRNA

ments of the

translation process. TTT

that are skipped during the

It is

important processes.

mRNA mRNA

1

and

2,

and

and

5

Num-

some. The amino acids to be incorporated into the protein molecule are brought to the ribosome by tRNA. There is a specific tRNA molecule for each

The tRNA-amino

complex

called charged

is

acids.

base sequence of the

tRNA. There

tRNA

is

acid

a three

It

also appears that

molecules can be involved

in the

complex than

is

than

The

regula-

certainly

more

originally pictured. This complexity

greater in eukaryotes, where the to

formation

“mRNA”

is

modification following transcription,

in bacteria.

The

synthesis of viral protein

is

further complicated by the fact that the viral genetic

information times

is

is

processed by the host

cell

and some-

incorporated, transcribed and translated

along with normal host

cell

protein synthesis.

is

sequence called for by the mRNA. Initiation of transcription always begins with formyl methionine (f-met)

some

and maturation sections of the same

in differentiation

called the anticodon

complementary to the mRNA codon. This allows for matching the proper amino acid with the which

these

possible that they are

6.

subject

amino

It is

tion of the translation process

is

of the 20

known whether

of different proteins at different times. into polypeptide.

bers indicate sequence of events. Translocation occurs

between steps

not

segments are always skipped or whether their trans-

TTT

lation can be regulated.

Fig. 3.24 Translation of

that there can be seg-

at

tRNA. The one

mRNA

site called

the

first

amino

binds to the riboacyl

site.

The f-met

NUTRITIONAL REQUIREMENTS OTHER

THAN CARBON The previous sections of this chapter concentrated on the metabolism of carbon for both energy pro-

70

Microbial Metabolism

duction and assimilation into organic cellular components. Microorganisms require many nutrients besides carbon sources. All microorganisms require water. Microorganisms may persist in a metabolically inactive state for prolonged periods of time in its absence, but for growth and reproduction water is necessary.

Ecosystems differ greatly in the availability of water for microorganisms. Microorganisms also differ greatly in their water requirements and their abilities to scavenge and store water. Water constitutes a large proportion of the cell cytoplasm. Most

aqueous solution, as does transport of materials in and out of the cell. As was pointed out in the section concerning

enzymatic reactions occur

amino

acids, nitrogen

is

in

a ubiquitous

component of

an essential element found in the nucleic acids of all microorganisms. Nitrogen proteins. Nitrogen

is

components of many

also occurs in the cell wall

microorganisms such as in chitin found in fungi, and murein found in bacteria. Relatively few microorganisms can utilize atmospheric nitrogen. The ability to fix molecular nitrogen is restricted to a few genera of bacteria; eukaryotes do not fix N 2 Those microorganisms that can fix atmospheric nitrogen .

possess the

enzyme nitrogenase. The

fixation of

N

12-15

ATP

NH

3

.

organisms can reduce nitrate as a terminal electron acceptor in respiration pathways, converting nitrate

back to nitrogen simalatory

This process requires

molecules, a very high energy require-

gas. This process

nitrate

reduction.

is

known

as dis-

The reduction of

nitrate to nitrogen gas proceeds via nitrite, nitric

oxide, and nitrous oxide. This process generally results in a substantial loss of nitrogen available

for microbial growth.

Phosphorus

Membranes

is

required by microorganisms.

are largely

composed of phospholipids.

Nucleic acids are linked together by phosphate ester bonds.

ATP,

tains phosphate.

the energy source of the

Some coenzymes,

pyrophosphate (TPP) which

is

e.g.,

cell,

con-

thiamine

involved in carboxy-

Phosphorus are generally supplied by

lation reactions, contain phosphate.

requirements of the

cell

assimilation of inorganic phosphate.

Many

micro-

organisms exhibit an optimal carbon to phosphorus Important reactions that incorporate inorganic phosphate into organic phosphorus containing components include the formation of ATP ration of 30:

1.

and the substrate

level

phosphorylation that occurs

during glycolysis, forming 1,3-diphosphoglycerate.

Oxygen

2

involves the sequential transfer of six electrons to

form two molecules of

chemolitotrophic microorganisms oxidize nitrogen compounds to generate energy. Some micro-

is

an essential component of almost

microbial biochemicals.

all

Oxygen may be supplied

from water, molecular oxygen, inorganic oxidized molecules such as nitrate, sulfate,

CO

2

or a variety

ment. Nitrogen fixation also requires reduced ferredoxin. Most microorganisms can utilize

aerobic microorganisms require molecular oxygen

ammonium

as the terminal electron acceptor in respiration

ions, nitrate ions, or

supply of nitrogen.

amino

Ammonium

acids as a

ions can be in-

corporated directly to form amino acids. Nitrate also can be assimilated into

amino

acids by assimi-

The nitrate is ammonia. ATP

latory nitrate reductase enzymes.

and then to formation does not occur during assimilatory nitrate reduction. The details of this process are not fully understood. Many microorganisms exhibit an optimal carbon to nitrogen ratio of 10:1. Without a sufficient supply of nitrogen, microbial metabolism reduced to

is

nitrite

altered. In addition to requiring nitrogen sources

for assimilation

into cellular

components, some

of oxygen

containing organic molecules.

pathways. Nitrate, sulfate or for

CO may 2

Most

substitute

oxygen as terminal electron acceptor for some

microorganisms.

Many anaerobes cannot survive in

oxygen and require other sources of oxygen to supply their nutritional the presence of molecular

requirements.

Oxygen

incorporated into biochemicals directly from O 2 by oxygenase enzymes and from -OH groups by hydroxylase enzymes. Some prois

metabolism of various hydrocarbons, require the incorporation of molecular oxygen. Steroid biosynthesis also requires O 2 as a cesses,

e.g.,

the

Enzyme

most bacteria lack sterols. Oxygen is produced by cyanobacteria and eukaryotic photosynthetic organisms from the photolysis of water. The evolution of oxygen evolving organisms was ess&htial for the existence of reactant.

It

is

interesting that

higher animals.

amino acids contain sulfur. Sulfur is found in several coenzymes such as S-adeno-

Several also

sylmethionine, biotin, thiamine pyrophosphate, and lipoic acid. Sulfur can be supplied from inorganic or organic sulfides, sulfates, or a variety of other sulfur containing compounds.

Some

bacteria

require reduced forms of sulfur as reducing agents.

For example, the photosynthetic sulfur bacteria utilize hydrogen sulfide to generate NADPH. Sulfate can be reduced by many microorganisms and is incorporated into organic compounds such as amino acids. Most microorganisms are able to carry out assimilatory sulfate reduction. The assimilation of sulfate initially involves an activation step with ATP to form phosphoadenosine phosphosulfate (PAPS). The active sulfate is reduced with NADPH to sulfite and then again with NADPH to hydrogen sulfide. The reduced hydrogen sulfide reacts to form the SH group of an amino acid. Some sulfur reducing bacteria, e.g., Desulfovibrio, utilize SO4" as a terminal electron acceptor carrying out a pro-

cess called dissimalatory sulfate reduction.

H

and

is

the terminal product in this process directly incorporated into

biochemicals.

As

amino

stated earlier,

S

is

not

acids or other cell

some chemolitho-

trophs oxidize elemental sulfur or energy.

2

H

2

S to generate

Some enzymes zinc,

important,

manganese, magnesium,

variety of other metals

may

Magnesium is required for protein synthesis. Without magnesium the ribosomal subunits do not associate tein

is

and translation of nucleic acids

is

Some microorganisms e.g.,

blood

cells to

are dependent on host

not possible.

membrane

transport.

Sodium

chloride concentra-

tions are also important for maintaining cell

envelope structures.

ENZYME CLASSIFICATION The enzymes fall

into six

that catalyze biochemical reactions

major functional

classes (Table 3.2). All

metabolic reactions discussed in catalyzed

by enzymes

in

this

chapter are

one of these enzyme

classes.

Oxidoreductase enzymes catalyze oxidation reduction reactions. Dehydrogenase, oxidase, peroxidase, hydroxylase and oxygenase enzymes

1.

six

major classes of enzymes

Oxidoreductases

2.

Transferases

3.

Hydrolases

4.

Lyases

5.

Isomerases

6.

Ligases (Synthetases)

cells,

supply needed heme proteins.

into pro-

Calcium is an essential component of bacterial endospores as calcium dipicolinate. Calcium also occurs in the cell walls of some algae. Calcium concentrations affect membrane permeability and play a critical role in movement of flagella and cilia. Silica is also required by some microorganisms as a cell wall component. Several ions must be present in critical concentrations for maintenance of membrane transport and permeability properties. Sodium, potassium, and chloride ions are essential for maintenance of

The

an essential component of cytochrome molecules involved in respiration pathways and is also the central element in heme proteins, some of which are important microbial enzymes. organisms. Iron

A

functions.

Table 3.2

Various cations and anions are required by microorganisms. Iron is required by most micro-

nickel, or copper.

be required for other

forming the basis of a

global sulfur cycle.

71

contain other metallic ions such as

The microbial transformations of sulfur are

ecologically

Classification

72

Microbial Metabolism

are

all

oxidoreductase enzymes. Oxidoreductases

are important

enzymes involved

in

fermentation

and respiration pathways. By the nature of the reaction, oxidoreductase enzymes alter the polarity of the substrate molecules. Extracellular oxidoreductase enzymes may increase the solubility of a substrate, cell

making

membrane The

it

available for transfer across the

for intracellular metabolism.

enzymes catalyse the transfer of molecular substituents from one molecule to another. Transferase enzymes include methyltranstransferase

ferases,

acyltransferases,

glycosyltransferases,

alkyltransferases, phosphate transferases, sulfur transferases,

and nitrogenous group transferases.

The methyl donor for many methyltransferases is the coenzyme S-adenosylmethionine. The acyl donor for many acyltransferase enzymes is the coenzyme acetyl-CoA. The transaminase enzymes are involved in the transfer of amino groups from an amino acid to an a-keto carboxylic acid forming a new amino acid. The formation of ATP involves a kinase enzyme which catalyses the transfer of phosphate to

ADP.

The hydrolase enzymes

catalyze the hydrolysis

of various chemical linkages.

The hydrolases

clude the esterases, thioesterases, phosphatases,

and pyrophosphatases. Hydrolase enzymes are very important in the degra-

glycosidases,

peptidases,

Table 3.3

Examples of some microbial enzymes

Type

— name

Substrate

Products

cellulase

cellulose

smaller glucans

amylase

starch, glycogen

invertase

sucrose

+ glucans glucose + fructose

lactase

lactose

glucose

maltase

maltose

glucose

chitinase

chitin

smaller glucosamines

muramidase

peptidoglycans

various

proteinase

protein

proteoses, peptones, peptides,

maltose

+ galactose

amino

acids

+ amino

peptidase

polypeptide

peptides

urease

urea

CO2 + NH3

lipase

neutral lipids

fatty acids

esterase

hydroxyl alcohol esters

fatty

phosphomonoesterase

monophosphate

ester

phosphate

phosphorylase

glycogen, starch

+

glucose -

ATPase

ATP

DNAase RNAase

DNA RNA

phytase

phytic acid

inositol

+

sulfatase

sulfuric acid esters

alcohol

+ SO4”

chlorophyllase

chlorophyll

phytol

acids

+ glycerol acids + alcohol

phosphatase:

P,

in-

+ alcohol

phosphate

ADP + P, nucleotides nucleotides Pi

+ chlorophyllide

a

C ontrol of

dation of large molecules, making them available for incorporation into cellular carbon and for

energy generation.

Some examples

of hydrolase

enzymes are shown in Table 3.3. The lyase enzymes remove groups from substituents

without

hydrolysis,

their

leaving double

bonds. The lyase enzymes include the dehydrogenase and aldolase enzymes. Aldolase enzymes are important in carbohydrate metabolism, catalyzing the

breakdown of hexoses

into trioses in

conserve

its

mental conditions.

The

microorganism to turn on and off the synthesis of particular enzymes in response to environmental conditions is explained by the operon concept (Fig. 3.25). The operon is a region of the DNA which codes for a group of adaptive genes. The operon contains a region called the operator which determines whether protein synthesis of the proteins coded for in that operon is turned on or off; when the operator is in the off position, transcription to mRNA cannot occur. Whether the operator is on or off is determined by whether a specific protein is bound to the operator ability of a

transferase reaction.

inducer,

The

ligase

enzymes, which play an important

role in the repair of

DNA

molecules, bring about

two molecules. The action of ligase enzymes requires energy and therefore ligase enzymes work simultaneously with the hydrolysis of ATP. These enzymes play key roles in biosynthetic reactions. Synthetase enzymes are involved in the linking together of

73

energy and adapt to different environ-

protein synthesis

.

Activity

'

Decarboxylase reactions appear to be forms of dehydrogenase activity with formation of CO 2 Succinate dehydrogenose is an example of a dehydrogenase enzyme that converts succinate to fumarate in the TCA cycle. The isomerase enzymes include intramolecular oxidoreductases and intramolecular transferases. The conversion of 3-phosphoglycerate to 2-phosphoglycerate catalyzed by the enzyme phosphoglyceromutase is an example of an intramolecular glycolysis.

Enzyme Synthesis and

region. If the protein

is

there, the

mRNA

for that

operon region is unable to line up with the codon bases and the mRNA cannot be formed. If an enzyme is inducible, the blocking or repressor protein normally binds to the operator, and

when

turned

is

off.

An

appropriate

present, can bind with the repressor

from binding with the operator and thus turning on protein synthesis. An appropriate inducer can be a substrate which the protein preventing

it

OPERATOR REPRESSOR GENE

CODON

INDUCIBLE GENE

CODON

CODON

REPRESSOR PROTEIN

the formation of storage materials, e.g., glycogen

synthetase catalyzes the formation of glycogen

from glucose

1-P. Ligase

tant role in repair of

enzymes play an impor-

DNA

OPERATOR REPRESSOR GENE

CODON

INDUCIBLE GENE

CODON

CODON

molecules. INDUCER REPRESSOR PROTEIN

CONTROL OF ENZYME SYNTHESIS AND ACTIVITY While the

DNA

OPERATOR

—m

REPRESSOR GENE

CODON

mill contains the necessary coding for

1

1

all

REPRESSIBLE GENE

CODON

CODON

Ml

III

nw

REPRESSOR PROTEIN

the protein molecules that the particular micro-

organism can synthesize, not

all

of these protein OPERATOR

molecules are being synthesized at

all

times.

Some

enzymes, called constitutive enzymes, are synthesized all the time but other enzymes, called adaptive enzymes, are inducible or repressible and are synthesized only at certain times.

protein synthesis

means

that a

The regulation of microorganism can

REPRESSOR GENE III

III

CODON 1

1

1

REPRESSIBLE GENE

CODON

CODON

m

m

OFF

REPRESSOR

REPRESSOR PROTEIN

Fig. 3.25 Regulation of protein synthesis at transcrip-

tion level according to the operon

mechanism.

74

Microbial Metabolism

lac

structural genes (a)

Repressor gene

Promoter

i

Gene

Gene

Operator

Gene a

v

I

I Transcription proceeds

mRNA

DNA

RNA^ ^ polymerase

RNA

polymerase

\

^ ^ RNA /

f

moves to promoter site

*

f

precursor

Repressor

units

protein

Translation

\

'

in

progress

on ribosomes Lactose

Repressor inactivated

(b)

Repressor gene

Promoter

/

Operator

V

z

J-.L-T

a '

'

Z1

CRPcAMP. Complex

mRNA

RNA

polymerase

/ Repressor

Moves to operator when lactose is absent;

protein

blocks transcription

/

III ^RNA precursors

Fig. 3.26 Catabolite repression in

which

needed the promoter site of

to activate a molecule that binds at

the

DNA

AMP

is

to initiate transcription: (a) system operative;

system blocked. (From Avers, 1976; reprinted by permission of D. van Nostrand Co.; copyright Litton Educa-

(b)

tion Pub., Inc.)

enzymes

to

be synthesized catabolize. Thus, in

some microorganisms, glucose can induce formation of the

enzymes involved

The regulation of

of

in glycolysis.

a repressible

enzyme system

works in the opposite way. The repressor protein normally cannot bind to the operator and so the proteins of the operon can normally be synthesized. Only when a specific inhibitor substance is present will the repressor protein bind to the operator and turn off protein synthesis. Often the specific inhibitor

is

product repression allows a microorganism to stop making a substance once it has made enough

a metabolic end product. This type of

end

it.

Another form of repression lite

is

known as catabo-

repression (Fig. 3.26). Transcription requires

binding of

RNA

polymerase

the operon. This binding of

at a site adjacent to

RNA

polymerase

in

enzymes requires a binding protein (CAP) which is activated by 3',5' cyclic AMP.’The normal function of the bindthe case of catabolite repressible

ing protein

is

to activate the transcription process.

C ontrol of

CAP,

catabolite

region of the

activation

DNA

known

This type of control

is

protein,

as the

binds at a

promoter region.

considered a positive control

mechanism

since transcription

binding of

CAP. Addition

promoted by the

'

F^nzyme Synthesis and Activity

75

chromosomal DNA. The receptor sites may be on the same or different chromosomes. The structural genes are nonrepetitive

DNA

sequences. This

such as glucose, however, can decrease the cyclic

model permits simultaneous activation of genes and synthesis of enzymes involved in a particular biochemical pathway by a single stimulus molecule

AMP available for activating CAP, thus effectively

even when the structural genes are not clustered

decreasing or preventing transcription and syn-

together on the chromosome.

of certain substances,

enzymes controlled by these regions of

thesis of

the

is

DNA.

Operons occur in bacteriophages as well as in bacteria. Such viral operons control the enzymes involved in DNA or RNA synthesis. The existence of operons in eukaryotic organisms identical to

those of bacteria has not definitely been established.

Some

fungi appear to possess operon controlled

gene

is

If

a given structural

subject to transcriptional control by

more

than one receptor sequence, then overlapping genes could be activated by distinct effector molecules.

By proposing that a single structural gene can be activated by more than one activator molecule, and that one activator molecule can activate more than one structural gene, very complex biochemical responses in eukaryotes can be explained. The Britten-Davidson model is theoretical but presents some interesting concepts for explaining control of

However, the enzymes involved in most biochemical pathways in eukaryotic organisms are coded for by genes located at different loci of the

gene expression

DNA

re-

poses an optimal integrated control network that

Some

could regulate simultaneously different regions of

functions.

rather than being clustered together as

quired for the existence of operon control.

enzymatic

activities

clearly inducible

is

of eukaryotes though are

or repressible.

It

is

likely that

mechanisms exist in eukaryotic organisms for turning on and off enzyme synthesis. Some of these probably resemble operon control, others are probably under positive control analogous to that

the

mechanisms probably exist. One model proposed by Britten and Davidson genetic control of enzyme synthesis in eukary-

plored,

for

otes this

is

similar to the

model the

CAP activation in

bacteria. In

DNA has regions called sensor genes.

eukaryotes.

effectively pro-

It

DNA and in which identical DNA regions could

serve multiple functions.

Another control mechanism

several

controlling catabolic repression. Other, yet unex-

in

in

eukaryotes can

RNA

occur after transcription. Since the nucleus of eukaryotes

(hnRNA)

is

subject to modi-

messenger RNA, it is possible that control occurs during this modification process. Different RNA molecules can be linked together or destroyed during this process. fication prior to acting as

Thus, even the

if

RNA need

regions of the

DNA

are transcribed,

not leave the nucleus as

tRNA

in the

form following transcription and thus prosynthesis can be controlled. The specificity of

original

The attachment of an effector molecule to the sensor gene promotes the transcription of an adjacent region of DNA termed the integrator gene. The integrator gene codes for an activator molecule which may be RNA or a protein. The activator molecules diffuse and bind to specific receptor sites of the DNA. The function of the activator molecule is analogous to the AMP-CAP complex in bacteria and promotes the transcription of adjacent structural genes. The receptor sites for the

tein

binding of activator molecules are repeated as sequences at different regions of the identical

sequences of structural genes. In the

DNA

in the

the it

RNA

modification process obviously exists, as

would be required

Still

for control of gene expression.

another control process possible

in

eukaryotes

based on controlling the rate of synthesis of a molecule. There are identical particular sequences in eukaryotic chromosomes. These is

mRNA

DNA

repetitive

trol

DNA

sequences

may

function as a con-

mechanism

Davidson

as proposed in the Brittenmodel, or may represent repetitive latter case,

various numbers of repetitive regions, coding for

76

Microbial Metabolism

the

same

on

structural gene could be turned

at

any

point in time, allowing for genetic control of the rate of synthesis of specific

The above discussion has concerned the genetic control of enzyme synthesis. Both prokaryotic and eukaryotic microorganisms have control mechanisms that permit enzyme regulation even after synthesis of the enzyme has been completed. An enzyme that already has been synthesized can be inactivated in a variety of ways. Allosteric inhibition occurs

by the

allosteric interaction

acts at an allosteric

enzymes.

specific action of a small

mole-

cule acting at a site other than the active site that

an enzyme. Both constitutive and adaptive enzymes can be regulated in this manner.

that enzyme.

ADP,

for example,

is

an

allosteric

The fact that the same reaction

activator of phosphofructokinase.

ADP that

is

an

ATP

allosteric activator for is

an

allosteric inhibitor, allows the cell a

redundant responsive capability in regulating the production of energy as ATP in the cell. The activation of CAP by cyclic that promotes transcription of portions of the DNA is an analogous example of such activation.

AMP

In addition to allosteric inhibition,

alters the properties of

Enzymes

The reverse type of occurs when a small molecule site of an enzyme to activate

also a substrate in this reaction.

are subject to inhibition

chemicals.

The

activity

enzymes

by a large number of of an enzyme may be

that are susceptible to allosteric in-

hibition possess a site which can interact with low

molecular weight molecules, resulting

in

an altered

enzymatic activity. This allosteric site of the enzyme is in addition to the enzyme’s active site. The interaction of the small molecule at the allosteric site alters the conformation of the enzymes. Interaction at the enzyme’s allosteric site generally

shows the same high degree of specificity shown by the active site for a substrate. tion

is

End product

inhibi-

often an expression of allosteric inhibition.

The end product of a metabolic pathway is often an allosteric inhibitor of an enzyme near the beginning of the pathway.

When

sufficient concentrations of

the end product are formed, the at the beginning.

As compared

pathway

is

shut off

to feedback repres-

sion of protein synthesis, feedback inhibition of

enzyme action occurs more rapidly and thus allows an organism to respond more quickly to changes in its

environment.

An example

of allosteric

occurs

inhibition

with the enzyme aspartate transcarbamoylase,

which

initiates the

tion of

CTP

allosteric lase.

pathway leading

to the

(cytidine triphosphate).

forma-

CTP

an inhibitor of aspartate transcarbamoy-

Another example of

is

allosteric inhibition

occurs in glycolytic pathways where ATP is an allosteric inhibitor of phosphofructokinase, which catalyses the reaction of fructose 6-phosphate to fructose 1,6-diphosphate. Interestingly,

ATP

is

IS] Fig. 3.27 Competitive

and noncompetitive enzyme

in-

hibition. (v) reaction rates; (s) substrate concentration; right

graph linear

plot; left

graph double reciprocal

plot.

References and Suggested Readings

altered

if

the conformation of the

changed. Not only can simple chemicals, e.g., NaCl, H 2 S, Pb, etc., cause such changes, but other

environmental factors, such as temperature, pH, and pressure may similarly cause such conformational changes that alter enzyme action. The effects of such enzyme inhibitors can be seen as a change in the rate

of the reaction.

Some enzyme

inhibitors

cause competitive inhibition. The differences that these

two types of inhibition have on enzymatic

activities are illustrated in

Figure 3.27.

petitive inhibitor reduces the

maximal

A noncom-

rate ( Vmax) of

an enzyme reaction. A competitive inhibitor competes with the normal substrate for the active site of the enzyme. Normally, a competitive inhibitor is structurally quite similar to the normal substrate. Competitive inhibition does not decrease the Vmax, but rather alters the apparent affinity (Km) of the enzyme for the normal substrate. In some cases rather than inhibit, interactions of enzymes with various substances enhance the normal enzymatic activity. Cooperative effects occur when an enzyme has more than one active site and when binding of a substrate at one of the active sites increases the affinity of the enzyme for a substrate at one of the other active

'

Anderson, R. L., and W. A. Wood. 1969. Carbohydrate Metabolism in Microorganisms. Ann. Rev. Microbiol., 33:539-575.

and E. H. Davidson. 1969. Gene Regulation for Higher Cells: a Theory. Science,

Britten, R. J.,

165:349-357. Avers, C.

New

1976. Cell Biology. D. van

J.

Nostrand Co.,

York.

Conn,

and

E. E.,

Stumpf. 1972. Outlines of John Wiley and Sons, New

P. K.

Biochemistry. 3rd ed.

York. Dagley, tion to

New

and D. E. Nicholson. 1970. An IntroducMetabolic Pathways. John Wiley and Sons, S.,

York.

Davis, B. D., R. Dulbecco, H. N. Eisen, H. S. Ginsberg, and W. B. Wood. 1973. Microbiology. 2nd ed.

New

Hoeber,

Dixon, M., and

York.

E. C.

Academic

Press,

Gunsalus,

I.

New

Webb.

Y. Stanier (eds.). 1961.

Metabolism; Vol. Academic Press, New York. Bacteria, Vol.

Enzymes.

York.

and R.

C.,

1964.

1:

II:

The

Biosynthesis.

Lehninger, A. L. 1973. Bioenergetics. 2nd ed. Benjamin, Menlo Park, California.

Mandelkern,

An

Introduction to Macromolecules. Springer- Verlag, New York. L. 1972.

sites.

The regulation of enzyme

activity

plays an

forms a major basis by which abiotic environmental factors, e.g., temperature, ammonia ions, etc., can directly affect the metabolic activities of microorganisms. It provides feedback mechanisms whereby microimportant role

in

microbial ecology.

can be maintained. It also provides a mechanism through which the metabolic activities of one organism can directly influence the metaactivities

activities

of another organism, creating a

basis for positive

and negative

species relationships.

The

results

such metabolic interrelationships in later chapters.

and interand importance of intra-

will be

Mandelstam, J., and K. McQuillen. 1973. Biochemistry of Bacterial Growth. 2nd ed. Halsted Press, New York.

It

organisms can regulate their activities and an ecological balance between synthetic and degradative

bolic

REFERENCES AND SUGGESTED READINGS

enzyme molecule

is

77

considered

Stanier, R. Y., 1970.

M. Doudoroff, and

E. A. Adelberg.

The Microbial World. Prentice-Hall, Englewood

Cliffs, N.J.

West, E.

S.,

R.

W. Todd, H.

S.

Mason, and

J.

T.

VanBrugen. 1966. Textbook of Biochemistry. Macmillan,

New

York.

Handler, and E. L. Smith. 1973. Principles of Biochemistry. 5th ed. McGraw-Hill, York. White, A.,

P.

New

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PART

III

ECOLOGICAL PARAMETERS

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CHAPTER FOUR

DETERMINATION OF MICROBIAL NUMBERS, BIOMASS AND ACTIVITIES

A

quantitative approach,

more than anything

else,

distinguishes ecologists from naturalists. Naturalists

came

many

to perceive

interrelationships of

organisms and their surroundings. Ecologists strive to go beyond the mere recognition of such

complexities of enumerating microorganisms and

some of

the approaches that are used for this

purpose.

interrelationships; they attempt to quantify inter-

Microorganisms are extremely diverse, and the methods used to enumerate one group of microorganisms may be inappropriate for enumeration

relationships in terms of numbers, biomass, ac-

of another group.

tivity,

The

growth, death, cycling, and transfer

rates.

physical and chemical characteristics of en-

vironments that influence these processes also need quantitative description. Only such quantitative

knowledge leads to a deeper understanding of the structure and functioning of ecosystems. The following chapters discuss the meaning and the measurement of these parameters. The microbial ecologist often needs to determine the numbers of microorganisms that are present in a sample or specimen. In the case of a pure culture, the question

organisms are there?”

is

“How many

micro-

relatively simple to answer.

Dealing with the mixed communities of environmental samples, however, the problem becomes exceedingly complex and,

in

most

cases, defies a

simple and absolute answer. Quantitation is possible, but the numbers obtained need to be qualified

by the technique that is used. Furthermore, the enumeration technique needs to be chosen with care to assure

its

relevance to the question that

asked. In this chapter

we

will

is

being

consider some of the

It

is

first

necessary to define

which microorganisms are to be enumerated. Are all microorganisms to be enumerated, or are only certain groups to be counted? Are numbers of microorganisms to be converted to estimates of biomass? The methods used for enumerating viruses, bacteria, fungi, algae and protozoa are quite different. Special techniques must be used for enumerating specific physiological groups, such as psychrophilic bacteria, or obligately anaerobic bac-

Enumeration of such defined groups requires special sampling and processing procedures. Precise differential criteria must be used for enumerating an individual microbial species, such as Escherichia coli, which is often used as an indicator of sewage contamination. There are three basic approaches used for enumerating microorganisms: the direct observateria.

tion, or direct count, procedures; the indirect, or

viable count, procedures; and procedures that

measure

specific biochemical constituents of micro-

organisms. Each approach has

its

advantages and

limitations.

81

82

Determination of Microbial Numbers, Biomass and Activities

considering which microorganisms are to be enumerated, the properties of the ecosystem to be examined must be taken into account. Obviously, different approaches must be In

addition

to

used for enumerating microorganisms from such diverse ecosystems as the guts of animals, the sur-

and the sediment of the deep sea. How samples are obtained and processed is determined in part by the physical and chemical properties of the ecosystem being examined, and in part by which microorganisms are to be enuface layer of soils,

merated.

The enumeration process can be broken down into three distinct phases: sampling; sample pro-

and the actual counting procedure. These three phases are integral parts of the enumeration procedure and results must be interpreted considercessing;

ing the entirety of

all

Fig. 4.1

Various collection vessels: (A) screw cap

jar;

(B) whirl-pak bag; (C) glass stoppered bottle.

phases of the process.

SAMPLING The enumeration process begins with sampling. Sampling procedures must insure that samples are representative and are not contaminated with foreign microorganisms,

i.e.,

sampling procedures

must insure that the microorganisms that are eventually enumerated came from the ecosystem being examined. The term “representative” also implies that the sample reflects the diversity and density of organisms in the entirety of the sampled environment. In

many environments,

tion of microorganisms

patchy. as

Any

single

compared

is

sample

to the

the distribu-

not homogeneous but is,

of necessity, minute

whole environment being

sampled and therefore may easily over- or underestimate the true abundance. This source of error can be minimized by processing composite samples, prepared from individual ones collected by the use of a suitable sampling grid. Confidence

Fig. 4.2 Soil corer used for collecting

uncompacted

soil

cores.

limits of extrapolations

from samples to the

entire

environment should be statistically determined. Sampling designs and statistical procedures are described in greater detail in Chapter 16. Sampling procedures must also insure that numbers of microorganisms are not altered, either positively or

nonquantifiable manner during collection of the sample. In some enumeration negatively,

in

a

procedures, sample processing and even the counting procedure are coupled with sampling.

Sampling

83

B

/,

/

C

D

A E

Fig. 4.3 Sipre corer for collection of ice

and frozen

soil

Fig. 4.4 J -Z

sampler for collection of water samples

at

shallow depths: (A) bracket; (B) messenger; (C) glass tube

cores.

to be tially

The simplest sampling

situation occurs

such as at the surface of some terrestrial and aquatic

when

a pooled sample

is

inlet tube; (E)

par-

when

the samples to be collected are readily accessible,

ecosystems; and

broken by messenger; (D) rubber evacuated sample bottle.

accept-

on a shovel and pail. When concern collecting soil from a particular depth, is

generally used (Fig. 4.2).

The

soil

is

given to

a soil corer

corer should be

from the top 10 cm. In these situations, the sample can simply aseptically be placed in a

designed to prevent compaction during sample

suitable container such as a sterile bottle or bag.

ing deep soil samples.

The sampling vessel should not react with the sample. For most field collections polyethylene

collecting frozen soil

able, e.g.,

bottles,

or whirl-pak bags, are used to prevent

recovery.

The corer may be motorized

A

for obtain-

Sipre corer can be used for

and ice samples (Fig. 4.3). There are various sampling devices available

The samplers genof an evacuated chamber which can

for collecting water samples.

breakage during transport (Fig. 4.1). Many soil microbiologists, recognizing the

erally consist

abundance of microorganisms in the soil relative to any contaminant from air or nonsterile containers, do not use aseptic technique, but pragmatically rely

the chamber.

be opened at a given depth, allowing water to

The J-Z sampler

consists of

fill

an evacu-

ated glass bottle or a compressed rubber bulb with a sealed glass tube inlet (Fig. 4.4).

The device

is

84

Determination of Microbial Numbers, Biomass and Activities

A

lowered to a desired depth and a weighted messenger sent

down

the line to break the glass inlet tube

allowing the water to enter the flask or bulb. The

bag water sampler consists of an evacuated polyethylene bag with a sealed rubber tube inlet (Fig. 4.5). The sampling bag is mounted on a spring-loaded holder and lowered to the desired depth. A messenger is then lowered which releases a knife blade that cuts the rubber inlet hose. This releases the springs which spread the sampling bag drawing in a water sample. Since the sampling devices are lowered through a water column to the desired depth the possibility exists that some of the collected sample is contaminating water from a different depth. Contamination may also originate from the sampling wire used to lower the device. Some more elaborate sampling devices have an arm that swings away from the sampling wire into the current while the sample is withdrawn (Fig. 4.6). Niskin

Fig. 4.5 Sterile Niskin

butterfly

(A) spring loaded holder; (B)

water collecting bag:

sterile plastic bag;

(C) rub-

ber inlet tube; (D, E) knife blade for opening inlet

when

sterile

triggered by messenger.

Fig. 4.6 Microbial

water sampling device designed to

avoid contamination from the hydrographic wire: (A) vane maintains direction into current; (B) when triggered by a messenger the sampling arm swings out and sterile

cover of sample tube pulls away; (C) syringe

plunger retracts to draw

in

sample; (D) sampling

inlet

sampling apparatus. (From Jannasch and Maddux, 1967; reprinted by permission; J. Mar.

closes; (E) detail of

Res.)

Sampling

decompression during sample recovery a specialized sampling device is required for collecting water samples from great depths. Devices have been designed that will open at depth allowing a sample to enter and will close again maintaining the appropriate pressure during recovery. Some deep sea samplers have a built-in In order to avoid

,

85

which allows concentration of bacteria in the water sample (Fig. 4.7). Water samples collected for enumeration of algae and/or protozoa are often collected with a Nansen or VanDorn sampling bottle (Fig. 4.8). These devices are not suitable for bacteriological sampling since they cannot be sterilized. For colfilter

Sampling device for collection of undecompressed water samples from great depths: (a) transfer unit; Fig. 4.7

(b) filter sampler,

1)

nuclepore

filter,

B) intake

ton for maintaining pressure and regulating

through

filter.

For other

details of

lid,

K)

How

pis-

rate

apparatus and

its

Fig. 4.8

Nansen water

collection device, used largely tor

operation see original reference. (From Jannasch and Wirsen, 1977; reprinted by permission; Amer. Soc.

water chemistry studies: (A) hydrographic wire; (B) messenger weight; (C,E) trigger mechanism; (D,G) rubber

Microbiol.)

stoppers; (F) collection cylinder; (H,l) outlet ports.

86

Determination of Microbial Numbers, Biomass and Activities

Arms

Citch holdint bucket arms

slip

Tension now bucket

past catch

Ring

Fig. 4.9

front

Grab sampler

and

side views during operation.

strikes the sediment the

showing

Fig. 4.10 Gravity coring device for collection of sediment

the sampler

(From Ross, 1970; reprinted by permission; copyright Prentice-Hall, Englewood Cliffs, N.J.)

for retrieving sediment

When

jaws of the device are triggered

to close, collecting a disturbed sample.

(From Barnes,

1959; reprinted by permission; copyright

George Allen

and Unwin, Publishers, London)

and protozoa nets are generally towed from a boat. The planktonic organisms may be funneled into a collection bottle where they are concentrated. Some plankton samplers directly measure the amount of water lection of planktonic algae

being filtered; this

is

core samples.

necessary for quantification.

For collecting sediment samples from marine or freshwater ecosystems there are a variety of grab

samplers or corers. Grab samplers have two jaws

which are spring loaded and triggered when the sampler reaches bottom or when a messenger is lowered. When triggered, the jaws of a grab sampler snap shut, digging into the sediment, and collect a sample (Fig. 4.9). Most grab samplers do not prevent contamination of the sample with overlying water.

Core samplers generally drive into the sediment

upon impact.

In*

order to core into the sediment,

most gravity corers are quite heavy.

A corer used for

Sampling

Fig. 4.11

Andersen microbiological

sampler with

air

87

six

petri plates for collection of different size propagules.

collecting sediment for bacteriological

procedures

may

enumeration

be sterilized and lined with poly-

ethylene (Fig. 4.10).

Upon

be removed and sectioned.

recovery, the cores can

As with

soil corers, sedi-

a

moving

son

air

vehicle such as an aircraft. In the Ander-

sampler, air

is

drawn through

series of 6 grids of decreasing

An

agar plate

is

a graded

pore size (Fig.

4.1

1).

placed under each grid to collect

ment corers should not compress the sample. Box corers, which are frequently used by benthic biolo-

microorganisms and other airborne particles that, by their inertia, impact on the agar surface. Air

but only rarely by marine microbiologists,

velocity increases with decreasing grid openings,

recover sediment in a relatively undisturbed state.

thus impacting increasingly smaller particles on

Box

successive agar plates.

gists

corers are extremely heavy and require heavy

duty winches for operation. Sampling microorganisms from

Enumerating microorganisms from plants and air

requires

that processing be coupled with sample collection.

Microbial numbers in air are generally quite low

and

it

is

impractical to collect large volumes of air

air

different

filter

types

sampling device

may

ucts,

e.g.,

faecal material, can also be collected

The filter mounted on

be scraped or washed from the tissue and thus collected. For example, in the oral cavity it is generally

filter.

The pore

of microorganisms.

may

such as sap or blood, or dissection, to recover a particular tissue such as root or gut. Excreted prod-

be varied for collection of

by passing a measured volume of

through a membrane

membrane

vital fluid

determine the numbers of microorganisms associated with macroorganisms. In some cases only the surface of a particular tissue is to be examined. In such instances microorganisms may

for later processing. Air samples are, therefore, generally collected

animals generally requires collection of a

be stationary or

size of the

and used

to

88

Determination of Microbial Numbers, Biomass and Activities

wash microorganisms from a tooth surface for enumeration. Likewise, it is possible to wash microorganisms from a leaf or stem surface. Such nondestructive sampling repossible to scrape and

quires that the microorganisms not be tightly bond-

ed to the plant or animal. Concern must be given to the recovery efficiency of such methods. In other cases the tissue or cells

may

be pre-

served together with the associated microorganisms. This

is

especially useful

when

the interest

is

in

examining the anatomical relationship as well as enumerating microbial populations. Such ap-

Densities of microorganisms on surfaces are often high.

For enumeration of microorganisms within

or on the surfaces of other microorganisms, tech-

niques suitable for the collection of the larger

organism are generally employed. Thus, to enumerate viruses within protozoa, sampling techniques for the collection of protozoa would be utilized. A simple sampler for collecting microorganisms on the surface of an animal is a balloon (Stotzky, 1978). After the balloon has been pressed on the surface it can be inflated, spreading out the microorganisms so that they can be plated and con-

proaches are useful when considering associations between microbial populations, e.g., between phage

veniently counted.

and bacteria, as well as when examining associations bewteen plants or animals and microbiota. In still other cases, the plant or animal tissue may be macerated. This is acceptable for enumeration of microorganisms associated with the entire tissue

which are present within an ecosystem in relatively low numbers may require the use of attractants or

rather than with a particular layer.

It

is

also the

approach used for the enumeration of microorganisms from many food products. Microorganisms may be recovered from an airwater interface of aquatic habitats by adsorption onto a suitable material. Sterile nuclepore filters, teflon or cellophane

may

be used for this purpose.

The enumeration of some microorganisms

“baits.” In

some

cases

isms.

is

sufficient to provide a

surface that will, by absorption, concentrate the naturally occurring dissolved nutrients.

The buried

technique of Cholodny-Rossi has seen extensive use in the observation and enumeration of slide

microorganisms nique, a glass soil

and sediments. In this techmicroscope slide is implanted into the in soils

or sediment (Fig. 4.12).

The

glass slide

is

later

recovered with microorganisms that had become attached to the slide surface, and they are subjected

Schematic representation of buried slide method for collection and enumeration of micro*organFig. 4.12

it

Sample Processing

to microscopic observation

and counting. Assum-

ing that the clean glass slide surface

and

is

nonselective

acts like the surface of mineral particles in soil,

the types

and proportions of the observed organ-

isms can be considered representative of the

community

in general.

buried slide technique

microscope grids

in

A modern is

soil

variation on the

the exposure of electron

aquatic or other natural en-

vironments. The retrieved grids are electron microscopically observed to reveal detail

than

light

more morphological

them

is

the use of flattened

glass capillaries instead of glass slides.

According

to their use in soil or in sediment, these devices are

designated “pedoscopes” or “peloscopes” respec-

The

capillaries resemble

sediment

The

freely.

may

enter

flattened optical surface of the

capillaries facilitates microscopic observation

and

may

also

counting after be

filled

retrieval.

The

capillaries

with nutrient solutions to attract micro-

organisms.

It

should be recognized that,

case, the technique bears

in this

some resemblance

to an

enrichment culture and will, therefore, alter the original composition of the natural microbial community of the environment.

microscopy. Another variation of

the buried slide technique

tively (Fig. 4. 13).

or soil pore spaces, and microorganisms

89

Major achievements attributed capillary techniques in soils include:

tion of microbial landscapes of

silt

to the use of 1)

the observa-

deposits, e.g.,

abundance of Caulobacter, algae and flagellate protozoa in wet soils, the development of Metallogenium indicative of iron-manganese deposition and the extensive growth of Gallionella when ferrous iron is present; 2) the enumeration of new genera which had remained unknown due to their unusual characteristics; and 3) the observation of the

life

cycle stages in natural habitats.

SAMPLE PROCESSING Only rarely are naturally occurring microbial populations found in concentrations that are convenient for counting. Normally the microorganisms in the sample must be concentrated or diluted. Samples with too many microorganisms may be brought to appropriate concentrations by serial dilutions (the successive diluting of the sample with an appropriate diluent). Samples with too few microorganisms are normally concentrated by centrifugation or by passage through membrane filters. The conditions employed for processing have a great effect on the eventual numbers of microorganisms that are enumerated.

count procedures are to be used the samples may be preserved. Formalin or glutaraldehyde are often used as preservatives. Preserved If direct

Fig. 4.13 Peloscope for in situ observation of micro-

organisms

in

observation

sediment (Pedoscope

in soil).

is

a similar device for

(From Aristovskaya,

specimens may be stored for varying periods of time with no change in numbers of enumerable microorganisms.

1973; reprinted

by permission; Swedish National Research Council.)

is

The preparation of samples more complex. If the counts

for viable counts

are accurately to

90

Determination of Microbial Numbers, Biomass and Activities

reflect the

numbers of

the microorganisms present

sample at the time of sample collection, then processing must be accomplished rapidly. Microorganisms may rapidly reproduce in the collection in the

vessel yielding erroneously elevated counts.

ditions used in processing samples

many microorganisms

originally

may

Con-

also

present

in

Table

4.1

mixing time on viable counts of

Effect of

soil bacteria

Viable count on soil extract agar

Mixing time in waring blender

incubated 12 days at 25 C. Soil

1

Soil 2

7.4X10'

2.9X10"

kill

the

5 sec

1

1

min

8.1X10^

4.2X10"

counts.

2

min

8.7X10"

3.9X10"

samples are to be diluted for viable count procedures, microorganisms must be evenly dis-

4 min

8.4X10"

7.0X10"

min

7.1X10"

7.1X10"

original sample, thus yielding artificially depressed

If

8

persed in a diluent. This

is

a difficult task, espe-

and sediment samples. Microorganisms are more often than not bound to particles. In order to maximize the numbers of individual microorganisms in suspension, the sample may be shaken, either by hand or mechanically. They may be vibrated, mechanically or by some oscillation; ground in a mortar or ball mill; or mixed in a blender. Dispersants such as sodium pyrophosphate may be added to enhance dispersion. There is an optimal mixing time for each dispersion technique that will result in maximal numbers of enumerated microorganisms (Table 4. 1). One should be also aware of the fact that agitation will break up filamentous organisms, such as fungi and actinomycetes, and will release spores that would otherwise cially for soil

remain attached. This leads to a predictable overestimation of spore forming and filamentous organisms in agitated samples.

Numbers

regularly in-

•indicates optimal mixing time.

After Jensen, 1968.

Table 4.2 Effect of chemical composition of diluent

counts of

on viable

soil bacteria

Viable count on

agar 25

C

soil extract

— 8 day incubation at with

1

min delay

between preparation of dilutions and plating.

Diluent

1.9X10’

Tap water Pyrophosphate

0.

2.3X10’

1%

Ringer’s solution

1.6X10’

Winogradsky’s solution

3.0X10’

After Jensen, 1968.

crease with duration and severity of the agitation.

reached where further fragmentation of the mycelia affects their viability. Eventually, a point

Beyond

is

counts start to decline again. The optimal treatment time varies from sample to sample, precluding an absolute standardization this point,

samples must be concentrated by filtration, appropriate filters must be selected. Filters with If

pores that are too large

organisms;

filters

fail to collect

the micro-

with pore sizes that are too small

of methodology.

clog easily and cannot

The chemical composition of the diluent also affects the numbers of enumerable microorganisms.

particular chemical composition of the filter

filter sufficient

volumes. The

may

also affect the viable counts of microorganisms. Ex-

Different samples require different chemically defined diluents for obtaining maximal counts.

cessive use of

Varying concentrations of sodium chloride, magnesium phosphate, and other ions are required to maintain viability of certain groups of microorgan-

Special precautions must be taken during processing for enumeration of certain physiological

isms (Table

4.2).

may

disrupt

vacuum or pressure during filtration and kill some fragile microorganisms.

groups of microorganisms. For example, enumeration of obligate anaerobes requires that diluents be

3

91 step

A.

VIRUS CONCENTRATION

WATER SOURCE

^

sparged free of oxygen and

all

processing be car-

under an oxygen-free atmosphere. Enu-

ried out PUMP

meration of obligate psychrophiles requires that the

and

diluent

all

glassw'are such as pipettes be pre-

chilled to avoid killing these

There are a number of ways of recovering viruses from environmental samples. Viruses can be concentrated from water by repetitive adsorption and elution processes. In one such procedure, following acidification, viruses in large volumes of water can be adsorbed onto epoxy, fiberglass, or

BY PASS VALVE

U2mm

STEP

B.

DIA.

COX FILTER

STEP C. VIRUS RECONCENTRATION

VIRUS ELUTION

n

POSITIVE

POSITIVE

ADJUSTED K27-COX

)lf*l

RESERVOIRjJ 11

LITERI

U?7

ing in a

J

]

ELUATE

^ 1

DEPTH FILTER DIA.

47nim DIA.

COX

COX

such as occur at

r

FILTER

EFFLUENT

ELUATE RESERVOIR

many thousandfold

viruses. Viruses -

FILTER

^

Adsorbed viruses can then be eluted with an alkaline solution. The sorption and elution process can be repeated resultnitrocellulose filters (Fig. 4.14a).

PRESSURE

PRESSURE ELUENT

microorganisms.

INJECTOR

10

pH

1

1.

in

concentration of the

can also be eluted from particles sewage sludge, using glycine buffer

Eluted viruses can be concentrated onto

an aluminum hydroxide floe with lowering of the pH to 3.5. Viruses remaining in the solution at pH 3.5 can be adsorbed to and later eluted from mem-

A Oyster ineil

brane ADSORPTION:

Homogenize In dIstllM water

(1:7

wM

:

dures.

< 2.(n) ppm NaCI

conductivity

Somewhat

during concentration proce-

different procedures are required

from animal or plant tissues. As an example. Figure 4.14b shows a scheme for viral recovery from oysters. for recovery of viruses

Centrifuge

»

Use of appropriate buffers maintains

infectivity of viruses

HCI pH S.O

filters.

Discard supernatant

I Resuspend sediment

ELl/TION:

(conductivity

pH 7.3 glycine-sallne

In

6.000 ppm NaCII

DIRECT COUNT PROCEDURES

Centrifuge

Direct count procedures yield the highest estimates

Discard sediment

of FILTRATION:

Filter

through Mllllpore AP25

and Cox 0.45 /jm porosity

Filter

through Mllllpore

AP23 fIbergUss

fitter

fiberglass filter series

HCI

CONCENTRATION:

I Concentrate

Concentrate

filtrate

by uftraflttration lUFI

filtrate by

precipitation at pH

numbers of microorganisms.

counts allow for observation of microorganisms that are bound to particles, or are contained within other organisms, as well as for the observation of

4.

individual free microorganisms. Centrifuge

Supernatartt

Resuspend sediment In 0. 1 N NapHPO^adjust to pH'i7.4

Treat concentrate

with antibiotics;

methods do not require separation of microorganisms from their surrounding particles or cells. Thus,

with direct

observational

methods,

possible to enumerate phages on bacterial viruses in plant cells, bacteria

fungal mycelia in

on sediment

it

is

cells,

particles,

soil, etc.

assay lor viruses

Scheme

and concentration of viruses; (A) from water samples; (B) from shellfish. (A: from Sobsey, et ai, 1973; B: from Sobsey, et al., 1978; reprinted by permission; Amer. Soc. Microbiol.) Fig. 4.14

Direct observa-

tional Discard

B

Ideally, direct

for

recovery

however, several major drawbacks to direct observational methods. First, it is impossible to distinguish living from dead microorgan-

There

isms.

are,

Secondly,

in

some samples with

excessive

92

Determination of Microbial Numbers, Biomass and Activities

amounts of background debris distinguish

it is

The

often difficult to

do not allow

Both chambers are designed to fill with a known volume. The chambers have grid systems which can be used for counting. Phase contrast microscopy is useful for counting with such chambers as it eliminates the necessity of staining prior to enumeration. The thin section technique can be used for observation of various microorganisms from dif-

for

further study of the observed microorganisms, in-

cluding determination of species or physiological

groups that are present in the sample. For large microorganisms such as protozoa, algae,

and fungi,

direct observation in a

haemo-

cytometer can be used. Smaller organisms, such as bacteria, are enumerated in the Petroff-Hauser

chamber. The

ferent ecosystems.

has a thin cover glass allowing

latter

the use of short focal length, high

power

chamber prevents

distortion of the cover glass by capillary forces.

microbial forms. Thirdly, direct ob-

servational methods generally

special construction of the

the sample

which

objectives.

is

may

In the thin section technique

be impregnated with a plastic resin

allowed to harden.

Some samples do

Table 4.3

Length of fungal mycelium

in

tundra

soils

Mean Mycelium Country and location

Sites

Canada

Mesic

(Devon

Meadow

Raised Beach

Is.)

Kingdom

United

(m /g oven dry

(Moor House)

1005 199

Blanket Bog

4968

Juncus Moor

1667

Limestone Grass

826

Antarctica

Hut Bank

6328

(Signy

Mountain Moss

2783

Is.)

Grassland

288

Old Moraine

84

New Moraine

4

Marble Knolls Marble Schist

144 Soils

44

Norway

Wet Meadow

5000

(Hardangervidda)

Dry Meadow

1000

Wood

7000

Ireland

Blanket Bog

995

Sweden

Mire

Birch

United

Wood

Kingdom

Canada

1900

(Mt. Allen)

2500 2800

After

Dowding and Widden,

3260

1974.

m m m

341

580 801

50

soil)

not

Count Procedures

Direct

The sample is microtome and observed

require impregnation with plastic.

the soil particles can be seen

then sectioned with a

are broken.

when

93

the soil particles

Samples that can be sectioned include soil and sediment particles and plant and animal cells. The sectioned samples may be stained prior to microscopic observation. Prepared sections may be viewed by electron microscopy or light microscopy, e.g., light* microscopy is suitable for the observation of fungal mycelia on soil particles. Electron microscopy is required for observation of viruses within animal or plant tissues. The agar film technique of Jones and Mollison (1948) has been modified and used for direct observation of bacteria and fungi. In this technique the sample is mixed with agar and pipetted onto glass slides, forming a thin film. The dried agar film

mixed with a fluorescent stain such as acridine orange. The technique may be applied for

can be stained, e.g., with phenolic aniline blue. The slides can then be microscopically examined and the

counting bacteria as well as larger microorganisms, such as algae. Samples may be directly pipetted

number of microorganisms

onto a

microscopically.

film technique

is

quantitated.

The agar

extensively used for enumeration

of fungi. Since most fungi form mycelia, enumeration of fungi generally involves

measurement of

hyphal lengths rather than individual fungal Lengths of mycelia can be measured with a distance measuring device.

An example

in

Table

4.3. If

map

of typical

measurements of fungal mycelia for tundra

shown

cells.

cence microscopy to estimate the biomass of living fungi.

In

diacetate,

method a vital stain, fluorescein used. The procedure appears only to

this is

hyphae; fluorescence enzymatic cleavage of the stain

stain metabolically active

occurs only after

molecule. The addition of vital stain permits

mation of

total

versus

or

living

active

esti-

fungal

hyphae. Fluorescence microscopy can be used for direct observation of microorganisms. In this technique the

sample

is

slide or

passed through a

filter

viewed. Polycarbonate nuclepore

and the

filters

filter

have been

found to be superior to cellulose filters for direct counting of bacteria because they have uniform pore size and a flat surface that retains all the bacteria on top of the filter (Table 4.5). Nitrocellulose filters

also retain

all

is

trapped inside the

average hyphal diameter

is

counted.

measured and the cell volumes are calculated, the total fungal biomass can be estimated (Table 4.4). A problem that has been found with this technique is that total fungal biomass increases with increased dispersion since hyphae hidden among

film

technique for enumeration of fungi with fluores-

soils

also

combine the agar

also possible to

is

It

It is

many

of the bacteria, but

where they cannot be

filter

necessary to stain the nuclepore

filters

with irgalan black or other suitable dye to give a black background against which fluorescing micro-

organisms can be counted.

When

acridine orange

Comparison of Nuclepore (polycarbonate) and

Measurement of fungal mycelia and biomass

in soil

Length of Hyphae

Fungal Biomass

m/g

g/m^

Sartorius (cellulose nitrate)

filters for direct

COUNT Sartorius

counts

(#/ml)

Nuclepore

174

0.31

Site

(0.5 fjim)

Soil 2

619

2.69

Estuary

8.6X10^

1.7X10^

Soil 3

457

1.39

Reservoir

4.0X10'

7.2X10'

Soil 4

1602

7.66

Pond

1.7X10"

4.0X10"

Soil

1

Miller, unpublished data.

is

used as a stain, bacteria and other microorganisms

Table 4.5

Table 4.4

are

After Hobbie, et

al.,

1977.

(0.2

/luti)

94

Determination of Microbial Numbers, Biomass and Activities

Table 4.6

Comparison of

direct epifluorescent counts

and viable

plate counts

Marine water

Soil

Sample

Direct count

Viable count

Direct count

Viable count

A

5.0X10*

3.1

XIO’

2.2X10^

1.3X10'

B

i.ixio’

6.2X10’

8.2X10"

7.6X10’

C

2.0X10’

1.7X10*

1.3X10^

2.1X10"

Atlas, unpublished data.

X 10®

scopy, though these forms

it is

usually not possible to cultivate

on laboratory media.

It

is

not clear

whether these are “degenerate” nonviable representatives of known bacteria, or forms with yet un-

known In

physiological requirements.

some

cases there

is

a high positive correla-

tion between direct fluorescent microscopic counts

and viable plate counts (Fig. 4.15); in other cases the correlation is low (Table 4.7). The value of the direct count epifluorescent microscopy approach to enumeration is that it is applicable to a variety of habitats without the bias that

is

inherent in viable plate count procedures.

It

allows the estimation of numbers of microorgan-

isms in marine, freshwater and

soil habitats despite

the great differences in population sizes

and physio-

logical types that occur in these various habitats.

Direct counts are often directly proportional to bio-

plate count

mass, and thus can be used to estimate microbial between direct microbial counts by fluorescence microscopy and viable plate counts for soil Fig. 4.15 Correlation

samples.

(From

Trolldenier, 1973; reprinted by permis-

sion of Swedish National Research Council.)

fluoresce orange or green.

The green or orange

color correlates with the physiological state of the microorganism, but attempts to separate living

from dead microorganisms by the color of fluorescence have been misleading and are unsatisfactory. Counts by direct epifluorescent microscopy are typically two orders of magnitude higher than counts obtained by cultural techniques (Table 4.6). Many small and unusually shaped bacteria are observable and countable with epifluorescence micro-

biomass. However, tedious size measurements must be made to accurately convert direct microscopic

counts to biomass estimates.

A

recently developed technique permits simul-

taneous determination of total numbers of microorganisms by epifluorescent microscopy and the

numbers of microorganisms carrying out respiration. The method is based on the fact that electron transport systems in respiring microorganisms will

reduce 2-(/?-iodophenyl)-3-(/7-nitrophenyl)-5phenyl tetrazolium chloride (INT) to INT-formaRespiring bacteria deposit INT-formazan intracellularly as dark red spots. A relatively short incubation period with INT, ca. 20 minutes, is adezan.

Direct

Count Procedures

95

Table 4.7

Counts of bacteria (numbers/ml) Season/ Year

Area

Viable count

Direct count

/

4°C

20° C

9.9X10'

6.6X10'

4.5X10'

8.2X10^

9.6X10'

7.3X10'

1.8X10'

6.1X10^

i.ixio'

Beaufort Sea

Winter ’76

Ice

Summer

Water

’75

Winter ’76

Summer

’76

5.2X10'

5.0X10'

2.7X10'

Summer

’75

3.0X10'

1.0X10^

5.2X10^

1.4X10'

1.3X10^

2.4X10^

N.W. Gulf of Alaska

Water N.E. Gulf of

Alaska Winter ’76

Water After Kaneko,

et al.,

1978.

quate for sufficient INT-formazan to accumulate in a quantity that can be detected by microscopy.

the

Acridine orange staining can be carried out after this incubation period. In combination with

Table

fluorescent staining, the proportion of the total

microorganisms that accumulate INT-formazan can be readily observed. This allows for separation of actively metabolizing and nonactive microorganisms. When applied to Baltic Sea water samples, 6%-12% of the bacteria were found to be active. In fresh-water samples, teria

were active.

An example

5%-36%

of the bac-

of estimates of total

biomass and active bacteria made using this method is shown in Table 4.8. Another technique for determining bacterial numbers and the spectrum of actively metabolizing cells is to use autoradiography combined with epifluorescent microscopy. Natural bacterial populations incubated with tritiated glucose are filtered

onto 0.2 fim Nuclepore filters. The filters are placed on glass slides coated with a film emulsion. The bacteria are stained with acridine orange following incubation. The bacteria stain orange or green. Actively metabolizing bacteria are associated

with dark silver grains deposited in the vicinity of

cell.

method

An example

of an application of this

to nearshore water

samples

is

shown

in

Between 2.3% and 56.2% of the total number of bacteria in these samples were actively metabolizing cells. Autoradiography also is useful in estimating natural growth rates in situ. Organisms can be exposed to tritiated thymidine in their natural habitat. Following recovery of micro4.9.

organisms for enumeration, the proportion of the cells that have incorporated the thymidine into their DNA can be determined by combined autoradiography-direct epifluorescent counting. In this manner cells that have actively divided within the

habitat can be quantitated. Addition of trace amounts of tritiated thymidine does not greatly disturb the habitat so that the results are representative of natural growth rates.

Fluorescent antibody techniques may also be used for direct observation of microorganisms (Fig. 4.16). The fluorescent antibody technique involves staining the microorganism with a fluorescent dye that is coupled to an antibody. The anti-

an appropriate microbial antigen. Visualization of the staining reaction is by means of fluorescent microscopy. The fluorescent antibody

body

reacts with

Determination of Microbial Numbers, Biomass and Activities

96

Table 4.8

Number and biomass

of optically differentiated populations from aquatic environments

Water

Sample

No. of

Biomass of

Total

respiring

respiring

Share (%)

Share (%) of biomass

bacteria

of respiring

of respiring

bacteria

bacteria

temp.

Total no.

biomass

bacteria

(°C)

(XlO'ml)

(mgC m^)

(X10^/ml)

(mgC

m^)

Westensee (lake near Kiel) 14

March 1977

18

July 1977

5.0

3.1

0.7

23

17.6

4.2

1.5

36

2.0

1.1

5.8

0.1

0.8

9

14

1.9

3.7

16.7

0.2

3.4

5

20

4.0

2.4

0.3

12

0.5

1.4

0.1

7

1.8

1.5

Pond (Botanical Garden) 4 January 1978

Pond

(Kiel City)

January 1978

14

Baltic

Sea

(Kiel Firth)

March 1977

28

Baltic

Sea

(Kiel Bight) 2

March 1977

10

After

January 1978 Zimmermann,

et al.,

6

0.8

0.1

6.1

13

1978.

technique

is

extremely specific for an individual

microbial species and

may

for autecological studies,

permit a i.e.,

new approach

the studies of indi-

vidual microorganisms in their natural environ-

ments. The fluorescent antibody technique requires preparation of a suitable antibody. This is

accomplished by exposing an animal to the desired antigen and collecting the serum. The antibody containing blood serum is fractionated and the gamma globulin fraction collected. A fluorochrome, usually fluorescein isothiocyanate

(FITC)

is

con-

jugated with the antibody. The conjugated fluorescent antibody

Photomicrograph of fluorescent antibody of Rhizohium on clover roots. Arrows point to

Fig. 4.16

staining

is

purified by

column chroma-

tography. Problems with the fluorescent antibody

technique include nonspecific fluorescence of the

some samples, and

root hair tips which had a bright yellow-green fluores-

background

(From Dazzo and Brill, 1977; mission; Amer. Soc. Microbiol.)

of specificity of thie antigen antibody reaction which

cence.

reprinted by per-

may

in

the high degree

preclude staining of even different strains of

Direct

Count Procedures

97

Table 4.9 Microbiological variables measured in water samples taken from above sandy sediments at beaches of the Kiel Fjord

and the

Kiel Bight (4 to 13 July 1977)“

Colonyforming units/ml

Station

X

Total no. of bacteria /ml

No. of active

Total

X

biomass

bacteria/ ml

X

Active bacteria

{%

of

10^ (plate

10^ (direct

(mg/ml X

10^ (autora-

total no. of

counts)

counts)

10'')

diography)

bacteria)

Actual uptake rate of glucose

(g/ml per

X

10’^)

A

261

41.7

50.2

19.8

47.5

18.9

A'

188

57.8

65.4

32.5

56.2

23.5

26.5

32.1

1

1.0

41.5

11.9

B

81.4

B'

187

64.2

62.4

29.9

46.6

10.3

C

433

68.8

74.2

3.1

4.5

2.6

C'

98.2

67.2

70.9

23.8

35.4

6.6

D

3.4

52.2

58.0

1.2

2.3

1.2

D'

3.1

51.4

40.9

6.1

11.9

7.8

E

5.5

39.4

43.6

16.6

42.1

6.9

E'

2.9

65.8

60.1

19.2

29.2

4.8

F

7.4

56.1

60.8

28.3

50.5

3.8

F'

1.7

50.0

44.7

3.9

7.8

5.2

to

F mark

stations at the west side; A' to F' are corresponding stations at the east side of the Kiel Fjord

spectively. Stations

D

A/

A'

are located at the inner part, B/B' at the center part, and

E/E'and F/F'are located

D',

C /C' at

and the

h,

Kiel Bight, re-

the outer part of the Kiel Fjord. Stations

at the Kiel Bight.

After Meyer-Reil, 1978.

same

which the antibody was prepared. Conversely, cross reactions between unrelated strains occasionally occur, and it is extremely the

species for

laborious to conclusively exclude this potential

Table 4.10 Quantification of R. japonicum^

IISDA

110, in the

rhizosphere of soybean plants grown in the field

source of error. Fluorescent

antibody techniques have been

applied to studies on selected microbial species in their natural habitat.

A number of bacteria, includ-

ing ecologically important organisms involved in

Sample (Weeks After

No. R. japonicum 110/gsoil*

Planting)

Uninoculated

1

10-inoculated

2

4.1X10'

6.2X10^*

key geobiochemical cycling reactions, have been examined in their natural habitats. It has been pos-

3

4.9X10'

1.9X10'’

example, to follow Rhizobium japonicum

4

4.9X10'

3.6X10'

association with specific plant roots,

5

N.T.

6.4X10'

presence of other Rhizobium species. As an example. Table 4. 10 shows the quantification of R. japonicum in the rhizosphere of soybean plants.

6

N.T.

1.7X10"

sible, for

in soil

even

and

its

in the

Growth or death

of specific microbial species can be

(N.T.= Not Tested) *= Based on counts of 20 microscopic After Bohlool and Schmidt, 1973.

fields per

sample

98

Determination of Microbial Numbers, Biomass and Activities

by successive counting using this method. Fluorescent antibody techniques are also

followed

in situ

has been used to quantitate Legionella

pneumo-

the quantification of pathogenic microorganisms in

which causes Legionnaire’s disease, not only in blood samples from patients, but also from lake waters which appear to be natural habitat reservoirs

plant and animal tissues, e.g,, the detection of Tre-

of this pathogen.

often used for the detection and sometimes also for

ponema

pallidum, which causes syphilis,

samples. Recently the fluorescent antibody

in

philia,

blood

method

The scanning

electron microscope provides a

useful tool for enumerating microorganisms visualizing their natural distribution surfaces.

on

and

particle

The scanning electron microscope pro-

duces a three-dimensional image (Fig. 4.17). Results

of enumerations with scanning electron

microscopy have been found to be comparable to results obtained by epifluorescent microscopy (Table 4.11). Caution must be used in electron microscopic techniques because of the possibility of producing artifacts when specimens are metal coated, to increase contrast for observation, and when specimens are placed under high vacuum in the electron microscope.

numerous viewing

As magnification

may have

fields

before a microorganism

is

to be

increases

scanned

observed. Therefore,

electron microscopic observation has been applied

mainly to samples with naturally dense or artificially enriched microbial communities. Both the buried slide technique and capillary

methods were included pling methods. In

some

in the discussion of

chamber that can be viewed microscopically. The capillary tubes used in these techniques are extremely thin and flattened, and can be viewed directly under a microscope. With recovered to a slide or

Scanning electron micrograph showing detrital bacteria on a decomposing submerged leaf. (Courtesy of C. Versfeld.)

Table

4.1

Comparison of enumerations by epifluorescent and scanning electron microscopy for estuary water samples

Count (#/ml) Scanning 0.2 fjim

Estuary

1

Estuary 2 After Bowden, 1977.

EM

Nucleopore

methods may microorganisms

cases, these

use artificial substrates to attract

Fig. 4.17

sam-

Epifluorescence 0.2 ^im

Nuclepore

4.34X10^

4.81X10'

3.30X10'

2.92X10'

0.2 fjun Sartorius

2.00X10' *•

0.78X10'

Viable Count Procedures

99

may be These methods may

time the plates are prepared (pour plate method).

also be coupled with the fluorescent microscopy

tions, for a period of time, to allow the bacteria to

techniques described above.

multiply forming macroscopic colonies.

capillaries

and buried

slides, the

stained prior to examination.

specimens

A

The

plates are incubated,

Coulter particle counter can be used to estimate directly numbers of microorganisms. The

colony originates

Coulter counter measures electronically the number

this

of particles within set

can be

set to

‘size

ranges.

count particles

bacteria to protozoa.

The

in the size

size

ranges

ranges from

The problem with Coulter

under specified condi-

The

colo-

The assumption is that each from a single bacterial cell. For

nies are then counted.

assumption to be valid

is

it

necessary that the

bacteria be widely dispersed in or Plates with too

many

on the agar.

colonies cannot be accurately

may

counted because one colony

more

represent

that nonliving small particles

than one original bacterium. Plates with too few

are counted together with microorganisms. Its ad-

colonies also must be discarded from the counting

counter analysis

is

it

can be used to estimate numbers of

procedure for

microorganisms

in a particular size range, e.g., to

sible to

vantage

is

that

give separate estimates of bacteria

and protozoa.

filters

statistical considerations. It

concentrate microorganisms on

and place the membrane

is

pos-

membrane

filters directly

agar surface. The colonies develop on the

on an

mem-

brane and can then be counted. Staining of the colonies, or rendering the filter transparent, facili-

VIABLE COUNT PROCEDURES There are two basic approaches to viable count procedures: 1) the plate count technique; and 2) the

tates the

Most Probable Number (MPN) technique.

enumeration may contain organic contaminants. For this reason, when specific nutritional groups of microorganisms are to be enumerated, the agar is replaced with an alternative solidifying agent, normally silica gel. Silica gel plates are more difficult to prepare than agar plates, and therefore are

All

viable count procedures require separation of

microorganisms into individual reproductive units. The microorganisms must be in suspension for accurate viable counts.

Plate

counting of small colorless colonies.

Agar used

used only

Count

for

when

making media for

bacteriological

necessary.

The main considerations

for plate count pro-

a misnomer.

cedures are the composition of the medium; the in-

count procedures are selective for certain microorganisms; the degree of selectivity varying

cubation conditions, and the length of incubation.

with the particular viable count procedure. This

hensively

one is trying to estimate the total viable microbial biomass within an ecosystem. Selectivity, however, allows for differentiation and estimation of numbers of particular

for viable counts. Rather, selected conditions will

The concept of a

total viable

count

is

All viable

selectivity

is

a disadvantage

if

types of microorganisms.

The agar

plate count

method

is

widely used for

enumeration of viable microorganisms, especially bacteria. Most bacteria lack the enzymes necessary for utilization of agar. The agar typically is mixed with a nutrient solution and added to petri plates. Dilutions of samples are then spread on the top of the agar (surface spread method). Alternatively the sample suspension can be mixed with the agar at the

No

attempt all

made

examine comprethe conditions that may be employed will

be

to

be discussed as examples of the types of considera-

must be taken into account in designing plate count procedures and interpreting viable enumeration results. Ideally, the plate count procedure should create optimal conditions for all of the microorgantions that

isms to be enumerated.

When

designing plate count

procedures, maximal counts of the desired group of microorganisms are used as an indicator of optimal conditions. Generally, media with relatively high nutrient concentrations are used for enumerations

of total viable heterotrophs.

100

Determination of Microbial Numbers, Biomass and Activities

Media

enumeration of nonnitrogen fixing heterotrophs must contain a useable source of carbon, nitrogen, and phosphorus. The media must also contain required cations and anions, such as iron, magnesium, sodium, calcium, chloride, and sulfate. The carbon source often used for enumerafor

tion of total heterotrophs

is

a protein digest such as

tion of

microorganisms from

Blood, with

its

heme

colony unless spent

same organism medium.

ing the carbon source or

yields the highest

number

of microorganisms enumerated

4.12 and 4.13).

Ammonium

(Tables

or nitrate ions are

often used as the nitrogen source. Phosphate

normal form of phosphorus added

Growth

factors

are also

microorganisms. Yeast extract

the

to the media.

required by is

is

many

incorporated into

many media as a source of vitamins. Additionally, extracts may be prepared from the particular environment being studied and incorporated into the media. Many marine media are prepared with filtered seawater to supply unknown growth factors. Soil extract, a sterile aqueous solution obtained by autoclaving equal amounts of water and soil, is incorporated into many media used for the enumera-

Table 4.12

Comparison of various carbon sources

for total viable

of the

It is

a

filter-sterilized is

particular

culture fluid

incorporated into the cul-

not usually clear

why

a particular

medium

numbers of microorganisms from

ecosystem.

Clearly,

nutritional

re-

quirements, including growth factors, must be met

by the media. Most successful media have nutrient concentrations that are much higher than those

Concentrations of nutrients in many of these media used for total enumerations are high enough to be toxic to at least some microorganisms. The real advantage of viable enumeration profound

in the

cedures

only

is

natural ecosystem being studied.

that conditions can be adjusted so that

members of

Plate count

a defined group are enumerated.

media may be designed

to be selective

Selective plate count procedures

or differential.

growth of the desired group of microorganisms. Growth of other groups of microorganisms is precluded by media composition and/or incubation conditions. Differential media do not preclude the growth of other microare designed to favor the

organisms,

counts of marine bacteria

often incorporated

is

media for enumeration of microorganisms from animal tissues. Single cells of extremely fastidious microorganisms that require a variety of unknown growth factors will often fail to form a

ture

concentration alters the

ecosystems.

into

peptone or tryptone. Beef extract is often added. For some total enumerations, a carbohydrate, such as glucose, is incorporated into the media. Changits

proteins,

soil

but permit detection of the desired

group by some distinguishing characteristic.

C

Count

source

Bacto peptone

1.3X10^

Phytone

9.9X10^

Comparison of various media

Proteose peptone 3

i.ixio'

soil bacteria

Glucose

9.9X10^

Maltose

8.7X10^

Glycerol

i.oxio'

Acetate

1.0X10^

Table 4.13

Plating

medium

Soil

Soil extract agar Soil extract agar

9.7X10^

1% glucose 0. 1% peptone +

Succinate

1.1X10^

0.1%

After Simidu, 1972.

•

yeast extract

After Jensen, 1968.

1

Soil 2

1.4X10’

7.7X10^

1.1X10’

8.9X10'

+

0.

Citrate

for total viable counts of

Viable

Bacteriophage can be detected plate counts.

A

spread as a lawn

by selective

susceptible strain of bacteria in a thin

is

agar layer resting on top

of a thicker cell-free support agar layer (double layer plate).

The agar medium

is

designed to sup-

growth of the susceptible bacterium. The sample to be examined for phage is incorporated into the top agar layef. The development of each phage particle results in bacterial cell lysis. Areas of port

bacterial lysis can

be seen as clearing zones or

plaques in the opaque bacterial lawn (Fig. 4.18).

The number of plaques that develop indicates the number of infectious phage in the sample. Use of different bacterial strains allows for selection and enumeration of different infectious phage. The phage infectection, which limits infection to a narrow host range, precludes “total” phage counts. An analogous assay procedure can be used for enumerating plant and animal viruses. Suitable plant or animal cells can be grown as monolayers

selectivity of

in tissue culture. Eagle’s

growth of

medium

cells in tissue culture.

is

often used for

Samples that have

been treated with antibiotics to prevent bacterial

growth are added to the surface of the cells.

Plaques,

i.e.,

tissue culture

holes in the continuous tissue

culture cell network,

form where the

cells are in-

fected

by viruses.

Plant

(

ount Procedures

101

pathogenic viruses are

sometimes enumerated by counting infection centers on specially prepared leaf surfaces. An example of the application of selective plating for enumeration of not only phage, but also for several bacterial

species

is

shown

in

Table

4. 14.

The

bacterial species

were enumerated by using different selective-differential media; the

phage by using specific susceptible

host bacteria.

Media can be designed for the selective enumeration of fungi. The viable plate count technique, though, is generally not the method of choice for enumerating fungi, since this technique favors enumeration of non-filamentous fungi and spores.

Some

filamentous fungi,

the Basidiomycetes

e.g.,

by plate count techniques. Nevertheless, plate counts are suitable for enumeration of some fungi, such as yeasts. Many fungi utilize carbohydrates and therefore

are

notoriously

underestimated

carbohydrates are generally incorporated into media designed for enumeration of viable fungi. To prevent overgrowth of fungal colonies by bacteria, which may be more numerous than fungi in the sample, bacterial inhibitors are generally added to the media. The stain, rosebengal, and the antibiotics, streptomycin and neomycin, are often added to fungal enumeration media as bacterial inhibitors. A simple technique for suppression of bacteria volves the lowering of the 4.

5-5. 5.

pH

of the

medium

in-

to

Most fungi are unaffected by this pH, while

most bacteria are suppressed. For enumeration of photosynthetic microorganisms it is necessary to incubate in the light. Photosynthetic bacteria and some algae can be enumerated by plate count procedures. To prevent growth of heterotrophic bacteria, carbon sources are generally omitted from media used in enumeration of photosynthetic microorganisms. For enumeration of some photoorganotrophic bacteria

(Rhodospirillaceae)

to provide Fig. 4.18

Enumeration of phage by plaque assay on agar

(From Brock, 1979; reprinted by permission of Prentice-Hall, Englewood Cliffs, N.J.) plate seeded with bacteria.

it

is

necessary, however,

some simple organic

acids. All

photo-

synthetic microorganisms require mineral nutrients

and some require,

in

addition, vitamins such as

biotin, thiamin or vitamin

B 12 Reducing conditions .

102

Determination of Microbial Numbers, Biomass and Activities

Table 4.14

Comparison of

total mesophilic vibrios, V. alginolyticus

vibrio bacteriophages isolated

from Pacific oysters

and

V.

parahaemolyticus counts with the numbers of specific

(C. gigas)

and No. of Bacteriophage g of Sample

Specific Host Strain

Source Total Vibrios/g of Sample

Mesophilic alginolyticus

lyticus

K-4^

TCnE2A‘*

800

10