Vaccari D.A., Strom P.F., Alleman J.E. Environmental Biology for Engineers and Scientists
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Lytic infection kills the host cell. With temperate viruses, after entering the host cell
the genome inserts itself into a host chromosome. In that way, it is replicated as the host
cell grows and divides. Eventually, it may direct the format ion of new virions and cause
lysis of the cell, but this may be only after many generations.
Viruses are the cause of many important plant and animal diseases. They may be
spread through contact, food, water, and/or air. Among humans, they are responsible for
the common cold, influenza, polio, herpes, hepatitis, measles, smallpox, and AIDS, among
many others. Viruses also have been implicated as factors in a number of types of cancer.
10.8.2 Viroids and Prions
Viroids consist of a single short, circular strand of RNA, with no protein coat. Fu rther, it
appears that the RNA does not even code for any proteins. Thus, they are not considered
viruses. They are usually found within the nucleus of the infected cell, where the host
enzymes apparently can replicate them. They cause several important plant diseases
and also infect some animals.
Prions are small proteinlike particles that contain no nucleic acid. However, they
are still able to infect host cells, where they are then replicated. They are now known
to be the cause of several fatal brain diseases in mammals, including mad cow disease
(bovine spongiform encephalopathy), scrapie (in sheep and goats), and Creut zfeldt–
Jakob disease in humans (formerly thought to be caused by a ‘‘slow’’ virus).
PROBLEMS
10.1. What are some of the difficulties associated with classification of bacteria as species?
10.2. How do phylogenetic and phenot ypic classification schemes differ?
10.3. What are some of the inorganic chemicals that can serve as energy sources for
prokaryotes?
10.4. A particular prokaryotic microorganism is photosynthetic. Develop a simple
dichotomous key based on Table 10.4 that would allow you to place it in one of
those six major groupings .
10.5. Describe the range of environmental conditions under which microorganisms live.
10.6. Is a virus ‘‘alive’’? Why or why not?
10.7. Recent evidence strongly suggests that Mars at one time had liquid water on its
surface. Since Mar s probably went through a stage at which it was similar to Earth
at the time microbial life evolved here, it has been suggested that microbial life
once existed there also. Speculate on whether microbial life exists on Mars today.
What factors make such life more or less likely?
REFERENCES
American Society for Microbiology. www.asm.org.
Atlas, R. M., 1997. Principles of Microbiology, 2nd ed., Wm. C. Brown, Dubuque, IA.
288
MICROBIAL GROUPS
Barns, S. M., C. F. Delwiche, J. D. Palmer, and N. R. Pace, 1996. Perspectives on archaeal diversity,
thermophily and monophyly from environmental rRNA sequences, Proceedings of the National
Academy Science USA, Vol. 93, pp. 9188–9193.
Bergey’s Manual Trust , www.bergeys.org.
Bitton, G., 1999. Wastewater Microbiology, 2nd ed., Wiley-Liss, New York.
Brock, T. D. (Ed.), 1975. Milestones in Microbiology, American Society for Microbiology,
Washington, DC.
Center for Microbial Ecology, Michigan State University, Ribosomal Database Project, http://
www.cme.msu.edu/RDP/.
Clesceri, L. S., A. E. Greenberg, and A. D. Eaton, 1998. Standard Methods for the Examination of
Water and Wastewater, 20th ed., American Public Health Association, Washington, DC. Updated
regularly, and available at www.wef.org.
Garrity, G. M. (Ed.-in-Chief), 2001–2007 (expected). Bergey’s Manual of Systematic Bacteriology,
2nd ed., Vols. 1 to 5, Springer, New York.
Gordon, R. E., W. C. Haines, and C. H.-N. Pang, 1973. The Genus Bacillus, Agricultural Handbook
No. 427, Agricultural Research Service, U.S. Department of Agriculture, Washington, DC.
Holt, J. G. (Ed.-in-Chief ), 1984–1989. Bergey’s Manual of Systematic Bacteriology, Vols. 1 to 4,
Williams & Wilkins, Baltimore.
Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams (Eds.), 1994. Bergey’s Manual
of Determinative Bacteriology, 9th ed., Williams & Wilkins, Baltimore.
Jahn, T. L., and F. F. Jahn, 1949. How to Know the Protozoa, Wm. C. Brown, Dubuque, IA.
Lee, J. J., S. H. Hutner, and E. C. Bovee (Eds.), 1985. Illustrated Guide to the Protozoa, Society of
Protozoologists, Lawrence, KS.
Madigan, M. T., J. M. Martinko, and J. Parker, 2003. Brock Biology of Microorganisms, 10th ed.,
Prentice Hall, Upper Saddle River, NJ.
Pace, N. R., 1997. A molecular view of microbial diversity and the biosphere, Science, Vol. 276,
pp. 734–740.
Patterson, D. J., 2003. Free Living Freshwater Protozoa , ASM Press, Washington, DC.
Prescott, G. W., 1970. How to Know the Freshwater Algae, 2nd ed., Wm. C. Brown, Dubuque, IA.
Prescott, L. M., J. P. Harley, and D. A. Klein, 1999. Microbiology, 4th ed, WCB/McGraw-Hill, New
York.
Ricklefs, Robert E., 1993. The Economy of Nature: A Textbook in Basic Ecology, 3rd ed., W. H.
Freeman, New York
Voyles, B. A., 2002. The Biology of Viruses, 2nd ed., McGraw-Hill, New York.
Woese, C. R., 1987. Bacterial evolution, Microbiological Reviews, Vol. 51, pp. 221–271.
REFERENCES 289
11
QUANTIFYING MICROORGANISMS
AND THEIR ACTIVITY
In Chapter 10 we discussed the great variety of microorganisms and their abilities in a
mainly qual itative way. In many cases in environmental engineering and science, it is
desirable or necessary to deal with microorganisms in a quantitative fashion. What is
their density in a system: How many are there, or what is their mass? How rapidly are
they growing, transforming substrates, or producing products? First, we look at the ele-
mental composition of microorganisms. Next, we consider the microscope, an important
tool of the microbiologist. Then, after some preliminary consideration of sampling and
preparation, we look at a number of ways that the biomass of microorganisms can be
determined, their numbers estimated, and their effect on a system quantified. Finally,
we examine some of the simple models that can be used to help understand or even pre-
dict microbial activities. The focus is on general approaches rather than detailed specific
applications.
11.1 MICROBIAL COMP OSITION AND STOICHIOMETRY
11.1.1 Elemental Makeup
Most microorganisms are 70 to 90% water on a mass basis. The remaining dry weight is
typically about 15% ash (mine rals that remain upon combustion) and 85% volatile
(mainly organic) material. The elemental composition of the dry matter of typical bacteria
such as Escherichia coli is shown in Table 11.1. Of course, these values will vary among
different strains and will also depend on the physiological state of the cell.
Environmental Biology for Engineers and Scientists, by David A. Vaccari, Peter F. Strom, and James E. Alleman
Copyright # 2006 John Wiley & Sons, Inc.
290
It is sometimes useful to write an apparent chemical formula for microorganisms.
Commonly, C
5
H
7
O
2
N has been used for this purpose, and these values have also been
included in Table 11.1. Note that this formula gives reasonably good agreement with
the values from E. coli for C, H, O, and N, which make up 92% of the total dry mass,
but totally ignores the other elements. The elemental composition of some important
cell constituents, including some storage materials, is shown in Table 11.2. Thus, although
C
5
H
7
O
2
N is a useful simplification, it is not a true chemical formula nor an exact stoichio-
metric expression.
The cell’s requirements for C, O, and H are typically supplied by some combination of
organic material, carbon dioxide, elemental oxygen, and water (or occasionally, hydrogen
sulfide or methane). The other requirements can be loosely categorized as macronutri-
ents, micronutrients, and trace elements, although the boundaries between these groups
are not applied uniformly.
TABLE 11.1 Elemental Composition of a Microbial Cell
Atomic Cell Dry Element
Element Symbol Weight Weight (%)
a
Ratio
b
Formula
c
Weight (%)
Carbon C 12.01 50 4.2 5 53.1
Hydrogen H 1.00 8 8.0 7 6.2
Oxygen O 16.00 20 1.3 2 28.3
Nitrogen N 14.01 14 1.0 1 12.4
Phosphorus P 30.97 3 0.097
Sulfur S 32.07 1 0.031
Potassium K 39.10 1 0.026
Calcium Ca 40.08 0.5 0.012
Magnesium Mg 24.30 0.5 0.021
Iron Fe 55.85 0.2 0.0036
Other 1.8
a
Based on E. coli.
b
Apparent stoichiometric formula of E. coli based on cell dry weight.
c
Useful stoichiometric ratio often used to write the components of a cell as a chemical compound formula.
TABLE 11.2 Elemental Composition (Mass %) of Some Important Microbial
Cell Components
Stoichiometric Formula C H O N P S
Glucose C
6
H
12
O
6
40 6.7 53.3
Cellulose, starch, glycogen (C
6
H
10
O
5
)
n
44.4 6.2 49.3
Chitin (C
8
H
13
O
5
N)
n
47.3 6.5 39.4 6.9
Protein
a
(C
5.35
H
7.85
O
1.45
N
1.45
S
0.1
)
n
54.0 6.7 19.5 17.1 2.7
DNA
b
(C
9.75
H
12
O
6
N
3.5
P
1
)
n
38.3 4.1 31.4 16.1 10.1
PHB (C
4
H
6
O
2
)
n
55.8 7 37.2
Palmitic acid
c
C
16
H
32
O
2
74.9 12.6 12.5
a
Assuming about equal prevalence of all the amino acids.
b
Assuming 50% G þ C content (equal prevalence of all four bases).
c
A common fatty acid.
MICROBIAL COMPOSITION AND STOICHIOMETRY
291
For microorganisms, N and P are typically considered macronutrients (for plants, K
would be added). These are needed in a mass ratio of about 5 : 1. The required C/N/P
ratio, as a rule of thumb, is commonly said to be 100 : 5 : 1. However, in this case,
much of the carbon is used as an energy source rather than to make cell constituents.
As can be seen in Table 11.1, the C/N ratio of a typical cell itself is around 3.6
(or 4.3 in the formula C
5
H
7
O
2
N) rather than 20.
The term micronutrients usually includes S and Fe, and probably K, Ca, and Mg. Trace
nutrients would include the many other elements, such as cobalt (Co), nickel (Ni), copper
(Cu), and zinc (Zn), needed in only very small amounts, usually for specific enzymes.
Roles that a number of elements play in cell metabolism are summarized in Table 11.3.
Some organisms may need fairly high concentrations of another element for a special
purpose. Diatoms, for example, need substantial amounts of silicon (Si) to construct their
silica shells, and many testate amoeba need calcium for theirs. Other organisms may
TABLE 11.3 Roles of Various Elements within Microorganisms
Element Symbol Important Cellular Roles
Carbon, hydrogen, oxygen C, H, O Major constituents of organic matter
Nitrogen N Proteins; nucleic acids; peptidoglycan
Phosphorus P Nucleic acids; membrane phospholipids; coenzymes;
energy utilization (phosphorylation and ATP); present
as phosphate (PO
4
3
)
Sulfur S Amino acids cysteine and methionine, which give
proteins much of their three-dimensional structure;
coenzymes (including CoA)
Iron Fe Cytochromes and other heme and nonheme proteins;
enzyme cofactor
Potassium K Major inorganic ion (K
þ
) in all cells; enzyme cofactor
Calcium Ca Major divalent ion (Ca
2þ
); enzyme cofactor; endospores;
some amoeba tests (shells)
Magnesium Mg Major divalent ion (Mg
2þ
); enzyme cofactor; active in
substrate binding; chlorophyll
Cobalt Co Coenzyme (vitamin) B
12
Copper Cu Specialized enzymes, including cytochrome oxidase and
oxygenases
Manganese Mn Specialized enzymes, including superoxide dismutase;
enzyme cofactor
Molybdenum Mo Specialized nitrogen enzymes (nitrate reductase,
nitrogenase, nitrite oxidase) and some dehydrogenases
Nickel Ni Urease; required for autotrophic growth of hydrogen
oxidizers
Selenium Se Specialized enzymes, including glycine reductase and
formate dehydrogenase
Tungsten W Some formate dehydrogenases
Vanadium V Some nitrogenase enzymes
Zinc Zn Specialized enzymes, including RNA and DNA
polymerases; enzyme cofactor
Silica Si Cell walls of diatoms (algae)
Sodium, chlorine Na, Cl Transport processes; osmoregulation; required by
halophilic bacteria
292
QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY
require small amounts of other elements. Molybdenum (Mo), for example, is needed in
trace amounts by the nitrifying bacterium Nitrobacter to oxidize nitrite.
Other elements, such as sodium and chloride, may be present in fairly high concentra-
tions within a cell. However, even if they are required for survival (e.g., for osmoregula-
tion), they usually are not referred to as nutrie nts.
Figure 11.1 provides a comparison of a typical microbial cell’s major and minor ele-
ments to their presence within the water, land, and air of our planet. In general, there are
large reservoirs in water or soil for most of the cell’s requirements. One exception is nitro-
gen, most of which is present in the atmosphere as N
2
, a form that is available only to
nitrogen-fixing organisms (Section 13.2.1).
11.1.2 Growth Factors
Many microorganisms are able to grow in a system with a single organic carbon and
energy source, such as a sugar, and inorganic forms of all othe r nutrients (e.g., N as
ammonium). This means that they are able to synthesize all of the other organic molecules
0.001
0.01
0.1
1
10
100
CONHPS KCaMgFeSiAlNaCl
0.001
0.01
0.1
1
10
100
CONHPS KCaM
g
Fe Si Al Na Cl
0.001
0.01
0.1
1
10
100
CONHPS KCaMgFeSiAlNaCl
0.001
0.01
0.1
1
10
100
CONHPS KCaMgFeSiAlNaCl
Figure 11.1 Elemental distribution of microbial biomass (top) relative to (bottom) the
hydrosphere, lithosphere, and atmosphere (consecutively).
MICROBIAL COMPOSITION AND STOICHIOMETRY 293
that they require. Photo- and chemoautotrophs, in fact, may be able to grow without the
need for any organic substances at all, since they get their carbon from CO
2
.
However, other microbes may require a few or many specific essential organic mole-
cules that they are unable to synthesize. Referred to as growth factors, they usually fall
into one of three categories:
1. Vitamins, which are typically components of certain coenzymes
2. Amino acids, the building blocks of proteins
3. Purines and pyrimidines, the nitrogen-containing bases of nucleic acids
In some cases the growth factor required may depend on the other compounds present.
The filamentous bacterium Sphaerotilus natans, for example, can grow with ammonium
as the only nitrogen source if vitamin B
12
(cyanocobalamin) is present, but otherwise
requires methionine (a sulfur-containing amino acid).
In growing microorganisms in the laboratory, the growth medium (plural, media)is
considered defined if its exact chemical composition is known. Growth factors can be
added individually as part of a defined medium when required, but often a preparation
such as yeast extract, made from natural products and containing many compounds of
unknown composition, will be used instead. Such ‘‘undefined’’ media are referred to as
complex.
11.1.3 Molecular Makeup
Most of the dry mass of cells is composed of macromolecules. As indicated in Table 11.4,
proteins typically account for over half of the total . Nucleic acids, polysaccharides, and
lipids are also major components.
11.2 MICROSCOPY
The fact that microbes are extremely small and relatively transparent imposes subst antial
constraints on ‘‘seeing’’ them. Thus, a variety of micro scopes and microscopic techniques
have been developed to magnify them.
TABLE 11.4 Typical Molecular Composition of Bacteria
Typical (Approx.)
Cell Fraction Molecular Weight
(% dry weight) (g/mol) Cellular Role
Proteins 52 10
5
Structure and enzymes
RNA 16 10
5
–10
6
Genetic
DNA 3 10
9
Genetic
Polysaccharides 17 10
3
–10
6
Structure, genetic, storage
Lipids 9 10
3
Structure, storage
Inorganics and small 3 10
2
Enzyme cofactors,
organics osmoregulation
294
QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY
11.2.1 Light Microscopes
Rather than the simple single lens instruments of van Leeuwenhoek (Section 10.2.1),
today the compound light microscope is the conventional tool used (Figure 11.2). It
routinely gives magnifications of up to 1000 power () by combining a 10 ocular
(eyepiece) lens with an objecti ve of up to 100. The total magnification is determined
by multiplying the two toge ther. Objectives of 4, 10, 20, and 40 are also common , and
oculars of 15 are available.
It is resolution, however, rather than magnification itself, that is the primary limitation
of observation with a light microscope. For the average human eye, as two points move
closer together they would become one point—could no longer be resolved as separate—
at a distance of about 75 mm. (Most prokaryotes, remember, are about 1 to 2 mm in size.)
At 1000 this would occur at a distance of 0.075 mm. However, visible light has wave-
lengths of about 450 to 720 nm (0.45 to 0.72 mm), which effectively limits resolution to
about 0.2 mm. Any additional magnification does not lead to further resolution if visible
light is used; the object simply looks grainy or fuzzy.
When the compound microscope is used for standard bright-field microscopy
(Figure 11.3), the specimen, typically in water, is placed on a glass slide and a small
glass coverslip is placed over it. The sample is then seen against a bright background.
This is particularly useful in determining the color of cells, such as cyanobacteria
and algae, and observing the yellow of sulfur inclusions. However, for most colorless
Figure 11.2 Modern compound light microscope. This model includes a fluorescence attachment
and a camera.
MICROSCOPY 295
microorganisms, there is little visible contrast between them and the water, and thus little
detail is observable. This is a primary reason that so much effort was put into development
of staining procedures (Section 10.4.4 and Figure 10.15) by early microscopists.
A disadvantage of traditional staining techniques is that they normally involve killing
the cells. Thus, observations of motility and any other activity of the microorganisms is
lost. One variation that is sometimes used is dark-field microscopy (Figure 11.4), in
which objects are illuminated from the side. This makes them appear bright against a
dark background and can improve visibility of some transparent or fine structures, such
as the flagella of eukaryotes.
A major advance for observing live cultures was the development of phase-contrast
microscopy. This specialized modification of the compound microscope makes small
changes in refractive index visible, allowing improved observation of both the cell surface
and intern al structures (Figure 11.5). Frederik Zernike of the Netherlands was awarded a
Nobel Prize in Physics in 1953 for this invention.
Another modification is the fluorescence microscope (Figure 11.6). Fluorescent com-
pounds absorb light at one wavelength to become ‘‘excited’’ or ‘‘activated’’ and then emit
the energy at another wavelength. A variety of fluorescent molecules have been developed
that can penetrate or be taken up by cells. The specimen is then exposed to the specific
activation wavelength of light, and through filters, the specific emission wavelength is
observed.
The size of microorganisms can be measured using an ocular micrometer. This is a
small ‘‘ruler’’ placed in one of the eyepieces, so that it appears superimposed on the mag-
nified image. This scale is then calibrated at each magnification using a stage micro-
meter, a special glass slide with a precisely etched 1-mm scale broken down into
tenths and hundredths of a millimeter (Figure 11.7). Usually, the ocular micrometer is
designed to have markings at 10-mm intervals at 100 magnification, and at 1 mm for
1000, but this may depend on the specific microscope.
Ocular lens
Objective lens
Condenser
White light source
Cover slip
Sample
Glass slide
Platform
Stage
Figure 11.3 Bright-field microscopy: an original image viewed against a light or bright background.
296
QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY
Example 11.1 At 100 (10 ocular and 10 objective) a ciliated protozoan is observed
to have a length of 9.3 units on the ocular micrometer scale. If from a previous calibration
it is known that at 100 each micrometer unit is 10 mm, what is the length of the organ-
ism? What is the field diameter if it is observed to correspond to 161 units?
Ocular lens
Objective lens
White light source
Cover slip
Sample
Glass slide
Condenser
Platform
Stage
Platform
Stage
Platform
Stage
Figure 11.4 Dark-field microscopy: a bright reflected image is viewed against a darkened or black
background.
Figure 11.5 Phase-contrast microscopy. Object (a), with high refractive index ð nÞ, bends and
retards the light more; after further retardation at the edges of the phase plate, the recombined light
(retarded and unretarded) creates destructive interference, leading to a darker appearance. Object
(b), with a low n, does not bend or retard the light, creating no interference, and thus appears bright.
Thus, contrast was created between the two nearly transparent objects that was not otherwise visible.
A special condenser (not shown) with a phase ring is also required.
MICROSCOPY 297