562 ENVIRONMZXTAL AND iNDUSTIUAL MICRORIOLOGY
are concentrating their efforts on the possibility of developing new systems for
nitrogen fixation using recombinant DNA technology.
One area of research is directed toward introducing the 'package" of nitrogen-
fixing genes from bacteria into plant cells. if this were achieved, plants might
be capable of directly fixing nitrogen from the atmosphere. This would be a
tremendous advance not only for agriculture but for the world at large in terms
of producing food more economically and abundantly. Obviously, considerably
more research is necessary before this kind of genetic engineering can be at-
tempted at a practical level. For instance, nitrogenase is easily destroyed by
oxygen, and some means of protection of this enzyme complex from oxygen
would have to be provided in order for a plant cell to be able to fix nitrogen.
Alternatively, it may be possible to modify certain bacteria in a manner so
that they would develop a relationship with the root system of other plants, as
the Rhizobium species grow with legumes. For example, a symbiotic bacterial
nitrogen-fixing system with cereal grains would have a tremendous effect on
grain prodUction both in yield and cost.
BIOCHEMICAL The ultimate source of organic carbon compounds in nature is the carbon
TRANSFORMATIONS OF dioxide present in the atmosphere (or dissolved in water). The process, carbon
CARBON AND CARBON dioxide fixation, was discussed in Chap. 11. Although green plants and algae
COMPOUNDS: THE are the most important agents of carbon dioxide fixation, bacteria are also
CARBON CYCLE capable of synthesizing organic matter from inorganic carbon. The occurrence
of photosynthesis among microorganisms has already been described. Other
Carbon Dioxide Fixation examples of carbon dioxide transformation or incorporation into organic com-
pounds by bacteria are:
I Utilization of carbon dioxide by autotrophic bacteria; the carbon dioxide repre-
sents the sole source of carbon for these organisms and is transformed by a
reduction reaction to carbohydrates. The general reaction is
CO2 + 41-I -* (CH20)X + H20
2 Carbon dioxide fixation by heterotrophic microorganisms is common among bac-
teria. A specific example of this type of reaction is
CH3COCOOH + CO2 -' HOOCC112COCOOH
Pyruvic acid Oxalacetic acid
Organic Carbon The organic carbon compounds that eventually are deposited in the soil are
Compound degraded by microbial activity. The end product, carbon dioxide, is released
Degradation into the air and soil. Fresh air contains approximately 0.03 percent carbon
dioxide by volume. Bacteria and fungi are the principal microorganisms that
degrade organic carbon compounds.
Under most natural systems of vegetation, e.g., forests, the amount of organic
material in the soil remains approximately the same from year to year. This
results from a balance established between the annual litter fall and death of
the plants and the capacity of microorganisms to degrade these tissues.
The most abundant organic material in plants is cellulose. It is readily attacked
by many species of bacteria and fungi. The initial enzymatic attack is by cel-
563 Microbiology of Soil
lulase which splits this long-chain polymer of glucose to cellobiose, which
contains two glucose units. In turn the cellobiose is split to glucose by the
enzyme -glucosidase; glucose is metabolized readily by many microorganisms.
Complete oxidation yields CO2 and H2O. The process can be summarized as
follows:
enzyme
1 Cellulose cellobiose
celtulase
enzyme
2 Cellobiose_ glucose
-glucosidase
enzyme systems
3 Glucose ' carbon dioxide, water, and/or other end products
of many micra-
organiems
An example of the breakdown rate of glucose (and microbial growth) by soil
microorganisms is shown in Fig. 25-13. Similar degradation pathways occur for
the other major plant tissue substances such as hemicellulose, lignin, and pectin.
Carbon dioxide may also originate from the decarboxylation of amino acids as
well as from the dissimilation of fatty acids. All of these transformations may
occur in the soil.
A general summary of the carbon cycle is shown in Fig. 25-14.
Figure 2513. Plate counts rn,
-15.0
of bacteria and fungi and 01
cumulative CO 2 evolution 0 .8
during the incubation of soil —4.0Q
treated with glucose. (Cour- U
1
tesy of B. Behem and G. H.
C
Wagner, Sail Sci Soc Am 20
Proc, 38:591, 1974.) 0
U
U 0'
E E
30
Time, days
Figure 25-14. Carbon cycle
(schematic), showing the
role of microorganisms.
564 ENVIRONMENTAL MW INDUSTRIAL MICROBIOLOGY
BIOCHEMICAL Sulfur, like nitrogen and carbon, passes through a cycle of transformations
TRANSFORMATIONS Of mediated by microorganisms (see Fig. 25-15). Some species oxidize and others
SULFUR AND SULFUR reduce various sulfur compounds. The microbial transformations of sulfur have
COMPOUNDS: THE counterparts in the microbial transformation of nitrogen. For example, sulfide
SULFUR CYCLE and ammonia are reduction products of the dissimilation of some organic com-
pounds; both ma y be oxidized by various bacterial species. Some of the bio-
chemical changes by microorganisms involved in this cycle maybe summarized
as follows:
1 Sulfur in its elemental form cannot be utilized by plants or animals. Certain
bacteria, however, are capable of oxidizing sulfur to sulfates. The classical example
is Thiobociflus thiooxidons, an autotroph; the reaction involved is
2S + 2l1O + 30 2 -. 21-12SO4
2 Sulfate is assimilated by plants and is incorporated into sulfur-containing amino
acids and then into proteins. Degradation of proteins (proteolysis) liberates amino
acids, some of which contain sulfur. This sulfur is released from the amino acids
by enzymatic activity of many heterotrophic bacteria. The following reaction is
an example:
CH2SH cvateiiit, CH,
desulfurase
HNH2 + H20 + H2S + NH3
cOOH
cyateiric Pyruvic hydrogen
acid sulfide
3 Sulfates may also be reduced to hydrogen sulfide by soil microorganisms. An
example of bacteria involved in this process is the genus Desuifotomoculum, and
the reaction suggested is
4H2 + CaSO4 -. HS + Ca(OH), + 2H20
4 Hydrogen sulfide resulting from sulfate reduction and amino acid decomposition
is oxidized to elemental sulfur. This reaction is characteristic of certain pigmented
(photosynthetic) sulfur bacteria and is expressed as
light
CO2 + 2F12S (CHZO), + H 20 + 25
carboliyd rate
A laboratory technique which facilitates isolation of various sulfur-metabo-
lizing bacteria is the Winogradsky column shown in Fig. 25-16. The column
contains mud, CaSO 4 , plant tissue (a source of carbohydrate-cellulose), and
water. It is exposed to daylight and incubated at room temperature. The mi-
crobiological events can be summarized as follows:
I A variety of heterotrophic microorganisms oxidizes various substrates, depleting
the oxygen supply and creating anaerobic conditions:
Organic matter + 0 2 -* organic acids + CO,
2 Organic acids serve as the electron donors for the reduction of sulfates and sulfites
to hydrogen sulfide by anaerobic sulfate-reducing bacteria, e.g., Desuifotomacu-
I urn;
565 Micineblelogy of Soil
—
-
7 — LigT"-
/
Su
[
/ b°"
c#'
A °
/
-lydr get, /
Bacteria Ireduclion Water
s ide -s-.' ----- -- Sulfates
- .- - I. Monsullur ( Rus t. colared
purple bacteria
0 SO42
Thiobocilli
7.
Q. Purple sulfur
bacteria Red zone)
\O
0
4 Green sulfur - :
:-g ::j.. Sulfate-reducing
0104
bcscterio -Sso4z bacteria
Organic
sulfur
compounds
Gypsum (CuSOi) + plants ports + mud
(Proteins]
Figure 25-16. A Winogradsky column showing
Figure 25-15. Sulfur cycle (schematic), showing the role of
areas of localization of sulfur-metabolizing bac-
microorganisms. (After Bunker.)
Lena. See text for explanation of sequential de-
velopments. (Courtesy of T. Hattori, Microbial
Life in the Soil, Marcel Dekker, Inc.. New York,
1973.)
Organic acids + SO? ^ H2S + CO2
3 Photosynthetic microorganisms such as the purple and green sulfur bacteria (Chro-
matium and Chiorobium) use hydrogen sulfide as the electron donor to reduce
CO,:
sunlight
CO2 + HS (CH20), + S
4 The aerobic sulfur-metabolizing bacteria, Thiobocilius spp., develop in the upper
portion of the column and oxidize reduced sulfur compounds (sulfides, elemental
sulfur, sulfite). Final oxidation product is sulfate; sulfur accumulates:
Reduced sulfur compounds SO + accumulation of S
5 The nonsulfur purple bacteria (Rhodospirillum, Rhodopseudomonas, and Rho-
domicrobium) are facultative phototrophs; they grow aerobically in the dark and
anaerobically in the light and Carl utilize sulfide at low levels. They are capable
of utilizing hydrogen gas as an electron donor in photosynthesis:
560 ENVIRONMENTAL AND INDUSTRIAL MICROBIOLOGY
light
CO2 + H2S (CH 2O) + S
CO2 + 2H2 11911, (CH2 c), + H20
The preceding discussion was concerned with transformations of nitrogen car-
BIOCHEMICAL
bon, and sulfur and their compounds. But this represents only a part of the
TRANSFORMATIONS OF elements and their compounds that are subject to assimilation and dissimilation
OTHER ELEMENTS AND by microorganisms. The metabolic activity of microorganisms (production of
THEIR COMPOUNDS acids) solubilizes phosphate from insoluble calcium, iron, and aluminum phos-
phates. Phosphates are released from organic compounds such as nucleic acids
by microbial degradation. Bacteria change insoluble oxides of iron and man-
ganese to soluble manganous and ferrous salts. The reverse is also possible.
From these examples of biogeochentical changes that take place in the soil, it
should be apparent that microorganisms do, indeed, perform numerous and
essential functions that contribute to the productivity of soil.
Herbicides are chemical substances that kill plants, especially weeds; pesticides,
BIODEGRADATION or as the term denotes, are chemical substances that destroy pests. In the context
HERBICIDES AND
PESTICIDES of soil, we think of those pests which adversely affect economic crops—weeds,
insects, and pathogenic microorganisms. Thus a more specific nomenclature for
substances classified as pesticides would be herbicides, insecticides, fungicides,
and nematocides.
The wide-scale application of herbicides and pesticides, while improving the
crop yield, raises questions as to the short- and long-range effects as they are
deposited in the soil. Are they degraded by soil microorganisms, and if so how
rapidly? Do they have a temporary (or permanent) effect upon the soil micro-
biota? Do they constitute a form of runoff pollution to streams and rivers and
as such affect aquatic plant life? These are some of the questions that concern
the soil microbiologist as well as other soil scientists, biologists, and environ-
mentalists. Naturally, a major research effort is directed toward answering the
questions asked above, as well as others. An ideal pesticide compound would
be one that destroys the pest quickly, and, in turn, the pesticide compound
would be degraded to more elementary nontoxic substances. The soil is the
Figure 25-17. Degradation of Fu
PCP (pentachiorophenol) in
soil by indigenous and inoc- -= 80 1-
ulated bacteria under labo-
ratory conditions at 30°C. J oof-
(Courtesy of R. U. Edgehull
and R. K.-Finn, App] Envi-
U 4o r
0.g
ron Microbial. 45:1122, Botch culture noculum
198S.) 0.2 ml/50 g
£ Control
6 8 10 12 14
Time, cloys
a
I
Ia S
It
4.
p -
p
C-- 14
0
-Pt
-i
h
I
ii Marine plankton:
a Diatoms, copepods, crustacean
'7
/
larvae, protozoa, animal eggs, and
other organisms.
b Diatoms.
1.11 (Figure a courtesy of D. P. Wilson:
Fig. b courtesy ol Dr. Boris Guelt.)
Room air samples:
r
lXl)i a;iire of ;ciuir Ilie(Iiiinl in
N
:1
dish ti, rcu,rn air
I, Odle,tcori if uiui(rci..rgaccJsp,js liv
special air sarccI)lIcic device
Figures a acid I) courtes y if Erivi-
ri iiiiiie,iial Sen-ic-es l3rarich. Natiinial
Cc
Institutes cii health. PtiI,Ije 1-lealili
Service. I
V
.0 4
a
I -c
I
'-: Sr
II
I • at> j';-i
.
4,
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....-.
F.'r
e-
•.-':'•
•-• •,'.
^j,;n.i
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567 Microbiology of Soil
"sink" which receives the pesticide, and it is the soil microbiota that we depend
upon to degrade the compound. As an example to illustrate the results of
research on this topic, Fig. 25-17 shows the rate of disappearance (degradation)
of a pesticide deposited in the soil. This aspect of soil microbiology, namely,
the impact of and the fate of, pesticides deposited in the soil, is a subject of
growing concern.
QUESTIONS 1 Describe how the physical composition of soil influences the magnitude and
diversity of the microbial flora.
2 Describe one contribution made by Winogradsky and one by Beijerinck to
our knowledge of soil microbiology.
3 Assume that you made a microscopic count on a soil sample and a standard
nutrient agar plate count from the same sample. What generalizations are
likely with respect to the comparability of the counts?
4 How could one proceed to enumerate by cultural techniques, the various
physiological groups of microorganisms present in soil?
5 Compare the microbial flora of soil in the region of the rhizosphere to that
in an area at a distance from the rhizosphere.
6 What is meant by the term mineralization? Give an example.
7 Assume that some protein material is buried in the soil. Trace the changes
it may undergo as a result of microbial attack. Identify bacteria capable of
bringing about each of the changes.
8 Distinguish between symbiotic and nonsymbiotic nitrogen fixation. Name
several genera of bacteria that are nonsymbiotic nitrogen fixers.
9 What are the components of the bacterial nitrogen-fixing system?
10 How may the process of nitrogen fixation be determined experimentally?
11 Describe the process by which Rhizobium spp. invade the root system of a
leguminous plant.
12 How is recombinant DNA technology being explored to develop new means
of nitrogen fixation?
13 Tremendous amounts of plant material, largely cellulose, are deposited an-
nually on the earth's surface. Insofar as microbiological events are con-
cerned, what happens to this cellulose?
14 Illustrate, with reactions, the manner in which organically bound sulfur is
released by microbial dissimilation.
15 Describe the mineralization process as related to the dissimilation of organic
phosphorous compounds.
REFERENCES Alexander, M.: Introduction to Soil Microbiology, Wiley, New York, 1977. A general
book useful for more detailed information on the topics presented in this chapter
as well as other subjects related to soil microbiology.
Gray, T. R. C., and U. Parkinson (eds.): The Ecology of Soil Bacteria: An International
Symposium, University of Toronto Press, Toronto, 1968. The papers include
identification of soil bacteria, factors influencing their development, effects on
soil and metabolic interrelations between bacteria and between bacteria and
other microorganisms, as well as the microbial flora in the plant-root environ-
ment. -
ENVIRONMENTAL AND INDUSTRIAL MICROBIOLOGY
Griffen, D. M.: Ecology of Soil Fungi, Syracuse University Press, Syracuse, New
York, 1972. A view of general problems of soil fungal ecology is presented in
the first section. The second part provides a critical review on the physical
ecology of soil fungi.
Hattori, Tsutomu: Microbial Life in the Soil, an Introduction. Marcel Dekker, New
York, 1973. The subject matter is presented by interweaving relevant material
from other disciplines. The coverage of microbial distribution, physiology, and
interactions is good, and interrelationships between colloidal systems of the soil
and microbes are examined in depth.
Payne, W. J.: Denitrification, Wiley, New York, 1981. A small book (214 pages) which
provides an in-depth discussion of microbiological denitrificotion.
Richards, B. N.: Introduction to the Soil Ecosystem, Longman, Inc., 1974. A good
overview of the characteristics and activities of organisms found in soil is given.
The ecosystem concept and soil as an ecosystem are presented in detail.
Starr, M. R. at al. (ads.): The Prokaryotes: A Handbook on Habitats, Isolation and
Identification of Bacteria, vols. 1 and 2, Springer-Verlag, New York, 1961. These
two volumes provide extremely comprehensive coverage of all groups of pro-
caryotes. One can obtain good coverage of groups of soil bacteria in these
volumes.
Stewart, W. D. P. (ad.): Nitrogen Fixation of Free-Living Microorganisms, Cambridge,
New York, 1976. A series of papers discussing the distribution of the nitrogenase
enzyme in certain bacteria and blue-green algae and their role in the soil and
aquatic ecosystems and methods of measuring nitrogen fixation.
