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{ 5
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
\
X
4
\
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