Download The Relationship between Protozoan Populations and Biological

AMER. ZOOL., 13:193-201
(1973).
The Relationship between Protozoan Populations and Biological
Activity in Soils
JOHN D. STOUT
Soil Bureau, Department of Scientific and Industrial Research,
Lower Hutt, New Zealand
SYNOPSIS. The protozoan populations of soil, both free-living and parasitic, are discussed in relation to their size, distribution, biomass, and metabolic activity. Flagellates,
small amoebae, and ciliates are the most widespread and active of free-living forms,
while the distribution of parasites is related to the distribution of their hosts. The
theory of partial sterilization, which first related protozoan populations to soil fertility,
is discussed within the broader context of the soil organic cycle. The role of freeliving protozoa as micropredators and the importance of the soil biomass, as well
as fresh plant debris, as a substrate for microbial activity are emphasized. The nature
and activity of the microbial population are related to plant productivity and the
size of the soil animal biomass. It is suggested that comparatively small changes in
population and biomass may be associated with greatly accelerated metabolic turnover where micropredation takes place. This suggestion is supported by data from
microrespirometry experiments.
PROTOZOAN POPULATIONS OF SOIL
Composition and distribution: the edaphic
fauna
Free-living protozoa are associated with
almost all phases of terrestrial ecosystems.
They are to be found on the leaves of
plants and on the bark of trees; in water
reservoirs in plants and soil; on moss and
lichens; but most commonly and most
characteristically, in the surface horizons
of the soil, particularly in association with
decaying vegetation. The fauna comprises
hundreds of species of free-living protozoa
representing almost all groups except the
Radiolaria, but some are much better represented than others. Flagellates, amoebae,
and ciliates are the most widespread forms.
Heliozoa are represented by only a few
species, and of the shelled amoeboid taxa,
those related to the Foraminifera, such as
Gromia, are uncommon although a large
and varied range of thecate forms are a
conspicuous element in the fauna of organically rich soil. Proteomyxa and Myxomycetes are associated with soils rich in
decaying material. Because of their different ecological requirements the distribution of the taxa varies from soil to soil
and from site to site, but the faunal pattern is closely related to ecological condi193
tions and a number of different species associations have been recognized (Bonnet,
1961).
Protozoan parasites and symbionts are
associated with a number of soil animals.
Symbionts include ciliates in the gut of
oligochaetes and flagellates in the gut of
the termites. These protozoa are host-specific and, consequently, like their hosts,
show a distinctive pattern of geographical
distribution in contrast to most free-living
species. However, some free-living species
may also show restricted geographical distribution, if not at the species level, then,
as with ciliates, at the variety level. Parasites include gregarines which infect earthworms and beetle larvae and which may
be transmitted through the soil, and ciliates, such as Tetrahymena and Colpoda,
which include facultative parasites capable
of infecting a wide range of invertebrate
hosts, such as snails and enchytraeid
worms.
Numbers of protozoa
Numbers of protozoa vary enormously,
not only from site to site and from season
to season but between parallel samples
taken from the same site at the same time.
The flagellates, small limax amoebae, and
ciliates are normally the most numerous,
194
JOHN D. STOUT
although large concentrations of testaceans
may occur under some conditions and the
amoebae of slime molds may multiply to
large numbers prior to fruiting.
Biomass
It follows from the great variation in
numbers that there is similarly a great
variation in biomass, but normally protozoa constitute a relatively insignificant
part of the total microbial biomass or of
the total animal biomass (Stout and Heal,
1967).
conditions are not consistently favorable;
for most of their life the cells will be
either starving, resting, or encysted, and
their rate of respiration will consequently
be greatly reduced.
Reproduction. Under favorable conditions protozoa may reproduce within
hours. It seems probable that in nature
very short periods of favorable conditions
associated with a relatively high rate of
reproduction are balanced by long periods
when the cells do not divide. Unfavorable
conditions are less likely to be due to exhaustion of food than to desiccation, predation, or competition.
Metabolic activity
Nutrition. There are few photosynthetic
protozoa present in most soils, although
Euglena is comparatively widespread. The
great majority of edaphic protozoa are
heterotrophic. Many of the flagellates may
feed osmotrophically. Although most
species of flagellates, amoebae, and ciliates
can be cultured axenically on dissolved
nutrients, in nature it is most probable
that they feed on paniculate food and
particularly bacteria and yeast cells. Indeed, it is known that particulate matter
must be present even in axenic cultures
before the cells will form food vacuoles.
In nature nutrients in solution are likely
to be metabolized more rapidly by the
extracellular enzymes of bacteria or fungi
than ingested by protozoa. Further, the
nutritional requirements of most protozoa
are complex and are more likely to be
satisfied by ingesting bacterial or yeast
cells than by nutrients dissolved in soil
solution.
Excretion. Protozoa tend to be ammoniotelic and do not excrete complex
nitrogenous or phosphatic compounds.
Their carbohydrate metabolism is similar
to other animal cells and may lead to the
excretion of organic acids.
Respiration. As small-celled animals,
protozoa maintain a high rate of respiration under favorable conditions of nutrition and aeration. The metabolic activity
of ciliates is slightly greater than that of
amoebae or flagellates. However, in nature
THE SOIL ORGANIC CYCLE: THE THEORY
OF PARTIAL STERILIZATION
The soil organic cycle has been described as the constant struggle of the vegetation to maintain the fertility of the soil
against the impoverishing effects of leaching and soil weathering. Plant nutrients
drawn from the soil are returned in plant
litter as dead leaves and roots. These contain nitrogen, phosphorus, and, of course,
carbon, but generally in a form no longer
accessible to growing plants. The decomposition, or mineralization, of these residues release them for plant growth and,
consequently, for recycling. Consequently,
the maintenance of soil fertility is dependent upon the maintenance of this cycle:
where it ceases and where plant debris
tends to accumulate, as in peat, the fertility of the soil is depleted and plant
growth tends to be reduced. Thus, in most
soils it is the efficiency of the decomposition of plant residues that is critical in
maintaining soil fertility and also in preserving other soil properties such as soil
structure.
Russell and Hutchinson (1909), in their
classical theory put forward shortly after
the turn of the century, proposed that the
infertility of "sick" soils resulted from protozoa preying on the beneficial soil microflora. Partial sterilization, which appeared
to benefit such "sick" soils, was supposed
to suppress the protozoa and so permit the
growth of the beneficial bacteria. How-
PROTOZOAN POPULATIONS IN SOILS
ever, it is now considered that protozoan
predation is more likely to stimulate than
depress bacterial activity, and further, that
partial sterilization is unlikely to suppress
protozoan activity other than temporarily,
the protozoa becoming active again when
the bacterial population recovers. The effects of partial sterilization are much more
comprehensive than originally considered
by Russell and Hutchinson (1909). Partial
sterilization kills a substantial part of the
soil biomass and so releases appreciable
quantities of readily available nutrients
for further microbial growth; this is reflected not only in enhanced bacterial
growth but also in greatly increased protozoan activity. Repeated partial sterilization reduces the total microbial biomass
so that the response to successive sterilization is gradually diminished. The evidence
of partial sterilization experiments indicates that microbial activity in soil is
largely dependent upon the accumulated
biomass of the soil and is not simply dependent on the accretion of fresh plant
residues of the soil. There are, thus, two
major sources of energy for the maintenance of microbial activity in soil: first, the
energy of the soil biomass, and second,
the accretion of fresh plant nutrients. The
cycling of nutrients in the soil is chiefly
dependent upon heterotrophic organisms
which must derive their energy from one
of these two sources (Fig. 1). Their activity in turn effects the release of plant
nutrients, such as phosphate or nitrogen,
from soil organic matter and, consequently, replenishes the fertility of the soil. The
pattern of decomposition will be determined by the nature of plant residues—
whether or not they are readily comminuted by the soil fauna and whether or
not they consist of organic constituents,
such as lignin, relatively resistant to microbial attack. The rate of decomposition
will be determined by ecological factors,
such as moisture and temperature, and by
the composition of the fauna and microflora.
Although there is evidence of some freeliving soil protozoa being able to hydrolyze
cellulose and even humic substances, they
195
are not generally capable of attacking such
complex polymers and are not, therefore,
associated with their degradation. One
notable exception is the symbiotic flagellate population of some termite species
upon whose cellulolytic activity the life
of their hosts depend. Where the protozoa
are primary consumers of plant cells or
plant tissue, therefore, their efficiency is
low since the components which they are
able to metabolize—simple carbohydrates,
amino acids, proteins and fats—constitute
a relatively small proportion of the normal
plant biomass. Further, a low efficiency
in the conversion of plant substrate to new
protozoan cell material means a relatively
low rate of reproduction and, consequently, slow population growth. In general,
therefore, protozoa are unlikely to make
as effective a contribution to primary consumption as either the larger animals,
which are able to comminute plant tissues
and, thereby, extract greater quantities of
available nutrients, or the microflora, with
its wider range of enzymes, particularly
cellulolytic and lignolytic.
When the typical primary consumers,
bacteria and fungi, have effected the initial
decomposition of the plant debris, they
themselves become the substrate for secondary consumers. Of these secondary consumers, protozoa comprise the initial population in most conditions. The activity
of the protozoa reduces the microbial biomass, particularly the bacterial biomass,
and the succession of consumers may then
include other micropredators, such as
nematodes or rotifers, or else the whole
biomass may pass through the gut of larger
animals, such as earthworms, which feed
on the total plant debris.
At each stage of decomposition the initial tissue and its associated biomass may
be considered to consist of three parts: (1)
the fraction that is lost by leaching or by
conversion to carbon dioxide or other volatile residues; (2) the fraction that is resistant to decomposition or that is derived
from such resistant substances, for example, the humic substances derived from
aromatic polymers, which accumulate in
the soil; and (3) the organisms comprising
196
JOHN D. STOUT
SOIL NUTRIENT CYCLE
INPUT
|PLANT AND ANIMAl
RESIDUES
SOLUBLE
(sugars, amino-adds,
phenolics)
INSOLUBLE
(cellulose, lignia
phenolics)
ENDOGENOUS
METABOLISM
BIOMASS
EXOGENOUS METABOLISM
CHEMICAL
OXIDATION
SOIL
PRIMARY
CONSUMPTION
AUTOTROPHIC
GROWTH
SECONDARY
CONSUMPTION
AUTOLYSIS
PREDATKDN
EXCRETION
HETEROTROPHIC
GROWTH
SOLUBLE
INSOLUBLE
IMMOBILIZED
(clay/organic complex)
PLANT UPTAKE
LEACHATES
LOSSES
FIG. 1. The soil nutrient C)cle.
PROTOZOAN POPULATIONS IN SOILS
the biomass. When the biomass of the primary decomposers autolyzes, it also fractionates into two parts: the residues which
are leadily broken down by available soil
enzymes or lost by leaching or as volatile
compounds, and relatively resistant structures such as bacterial cell walls. At this
stage, therefore, the resistant fraction may
not be of plant origin but of microbial
origin. Micropredators, such as protozoa,
which feed either on living or dead bacterial cell walls and which are able to
hydrolyze the entire cell structure, including the cell wall, therefore perform a necessary and valuable role in the pattern of
decomposition. The cell wall material commonly includes lipids, amino acids, and
often polysaccharides. It is a valuable food
for the protozoan predator which is able
to decompose it. Since most of the bacterial nitrogen is excreted as ammonia
some hours after ingestion, protozoan metabolism contributes directly to the mineralization of nitrogen and so to the soil
nitrogen cycle.
The pattern of succession of primary
and secondary decomposers has been followed by Tribe (1961) who emphasized
the role of protozoa as secondary consumers following the initial growth of bacteria and fungi. The pattern of autolysis
of grass leaves has been followed by
autoradiography by Grossbard (1971), and
the overall incorporation of plant residues, particularly rye-grass roots, has been
followed by Jenkinson (1971) using radiocarbon-enriched
material.
Jenkinson
found that about one third of the added
carbon remains in the soil after the first
year and about 15-21% after five years.
He estimated that about 10% of the added
carbon is in the biomass after one year and
about 4% after four years. This means
that there is a relatively high rate of decomposition in the first year, but although
over 60% is lost from the soil only 10%
is converted to biomass. This indicates
either a very low rate of conversion of
plant residues to soil biomass or, more
likely, the frequent turnover of the biomass. When the remaining plant residues
have either been decomposed or incor-
197
porated into the biomass, the rate of release of radiocarbon from the soil drops
sharply. There is relatively slow reduction
in the biomass, 10% to 4% over three
years, compared with the initial reduction
in plant residue of over 60% in one year
which suggests that the microbial tissue
rather than the plant residues are proving
the least amenable to decomposition.
The rate of turnover of the bacterial
population is very difficult to estimate
since it depends not only on the generation time but also on the mortality. Recent estimates have differed widely—from
a mean value of 16 hours (Gray and Williams, 1971) to a few times each year
(Babuik and Paul, 1970). In both estimates, however, no attempt was made to
determine mortality which in most soils
probably is due largely to predation, either
by micropredators, such as protozoa or
nematodes, or by the larger fauna which
pass large volumes of soil organic material through their gut, such as earthworms. Further, it is well known that micropredators are selective in their diet, prefering some types of bacteria to others and
indeed finding some types inedible or even
toxic.
Bacteria also differ widely in their longevity: Gram-negative bacteria, such as
the Pseudomonadaceae and Enterobacteriacae, survive starvation or desiccation
far less successfully than pleomorphic bacteria, such as Arthrobacter, while bacterial
spores are virtually immortal. Thus, any
estimate of bacterial turnover must take
into consideration both the nature of the
microflora and of the predatory microfauna, as well as the increment of energy
available in fresh plant residues or in the
biomass, and these factors are interrelated.
A continuing increment of fresh plant
residues, rich in readily available nutrients, such as sugars or amino acids, will
differentially stimulate the fast growing
zymogenic flora which consists largely of
Pseudomonadaceae and Enterobacteraceae
(Stout, 1973) and which in turn provides
the most acceptable diet for most soil protozoa. Protozoan populations will increase,
therefore, if other ecological conditions
198
JOHN D. STOUT
are favorable, and there will be much
more rapid rate of turnover of substrate,
bacteria, and protozoa in the soil. An extreme example of nutrient enrichment is
that of whey-irrigated soil when the whey
contains lactose. In such a soil, there is a
marked increase in the zymogenic population, in the number of protozoa, and in
the rate of soil respiration. When there
are only minimal increments of fresh
plant residues, or where they consist of
substances low in immediate nutrient
value, or where the soils are generally unfavorable to biological activity because of
drought, the autochthonous micro flora
will be dominant and the activity of the
protozoa will be reduced, not only because of the smaller increment of food,
but also because of its relatively low nutrient value. For this reason small protozoan populations are correlated less with
the size of the total bacterial population
than with the nature of the soil organic
matter and its rate of decomposition. The
importance of protozoa in the soil organic
cycle is likely to be relatively greater in
soils with a high rate of turnover than in
soils with a low rate of turnover for two
reasons: first, because the nutrient status
of the microflora, particularly the bacterial flora, is more favorable, and second,
because the higher population provides
better opportunities for predation.
In soils with a high energy intake and a
high rate of turnover, a greater proportion of available energy passes to the secondary consumers than in a soil with low
nutrient intake and a low rate of turnover which has longer periods of strictly
endogenous metabolism. It has been estimated that protozoan consumption of bacteria in plots of increasing turnover of
Broadbalk field increases from 15 to 85
times the standing crop of readily available bacteria, and it has also been estimated that the amount of protoplasm
produced and recycled by the small amoebae and flagellates varies from 50 to 300
times the standing crop (Stout and Heal,
1967).
In soils of high productivity, such as
New Zealand pasture soils where there is
a large soil biomass which fluctuates in
size throughout the year, the availability
of nutrients for microbial metabolism from
this source could be as important as that
from fresh plant residues. The earthworm
biomass is directly correlated with plant
productivity, there being 150 kg fresh
weight of earthworms per hectare for every
1000 kg dry herbage produced annually.
In a high-producing New Zealand pasture
the earthworm biomass alone may reach
a peak figure of 2700 kg fresh weight per
hectare in the autumn and may fall to
1300 kg fresh weight per hectare during
the summer months. Part of the earthworm mortality will be due to predation
by birds but the greater part of the 1400
kg fresh weight of biomass lost during the
year will remain in the soil and will become available as a substrate for other
soil organisms, of which microorganisms
are likely to be the most active. This recycling of energy in the biomass is facilitated by the nature of the biomass tissue
which consists of readily accessible carbon
compounds and other nutrients, such as
nitrogen, phosphorus, and sulphur and
trace minerals. There is not likely to be
any limiting nutrient, such as nitrogen,
as with plant tissues. The larger the biomass becomes the more important is the
recycling within the biomass, and the
more important is the respiration dependent upon the release of energy from the
biomass (by autolysis or secretion). Conversely, the endogenous respiration of the
biomass is less important. Some indication
of the relative importance of endogenous
and exogenous respiration is given by
respiratory measurements. When soil plots
under very different productivity regimes
are compared, the respiratory rate (release
of carbon dioxide per unit of soil carbon)
is very similar. However, when fresh substrate such as glucose is added, the highproducing plots metabolize the added substrate much more rapidly than the lowproducing plots, and this is correlated
with a larger bacterial population and
particularly a larger population of Enterobacteriaceae (Stout and Dutch, 1968); that
is, high-producing soils not only have a
199
PROTOZOAN POPULATIONS IN SOILS
larger biomass but also a more marked
response to added nutrients and, therefore, a more rapid rate of cycling. This
cycling involves not only fresh plant residues but also the recycling of energy within the biomass. For this reason micropredators, such as protozoa, which accelerate the rate of recycling play an important role in soil ecology.
INTERACTIONS OF PROTOZOA WITH OTHER
SOIL ORGANISMS: INTERACTION WITH
BACTERIAL AND YEAST POPULATIONS
While protozoa are normally thought
of as predators of the bacterial and yeast
microflora, under normal conditions the
death of protozoan cells will also release
nutrients for further bacterial and yeast
growth. Thus, the growth of protozoa with
bacteria or yeasts provides three possibilities: (1) The protozoa may feed on the
bacterial or yeast population, resulting in
stimulated growth and metabolic activity
of the protozoa and reduction of the microfloral population; (2) the protozoan
cells may die and release nutrients which
stimulate metabolic activity of the microfloral population; (3) there may be no
marked interaction where the microflora
is unacceptable to the protozoa which may
encyst.
The ecology of soil protozoa is characterized by two important features: first,
the activity of populations is confined to
small spaces and consequently small populations; and second, activity is restricted
to brief periods when moisture conditions
are favorable. Experiments designed to
study interactions of soil populations must
simulate these two features, that is, they
must study the interactions of restricted
populations over relatively short periods.
For this reason elaborate model systems,
such as chemostats, are not applicable to
problems of soil ecology. One technique
which does permit the study of interactions of relatively small microbial populations confined to small spaces is the ampulla diver technique used in microrespirometry. A simplified form of this technique using an open gradient has recent-
ly been developed (Nex0 et al., 1973) and
preliminary experiments give some indication of how small population changes can
effect marked changes in respiratory activity. A typical experiment is shown in
Figure 2 where the results are plotted as
the movement of experimental and control divers in an open gradient since this
shows most dramatically the contrast between the control divers, the divers with
pure cultures, and those in which both
protozoa and bacteria are present. These
experiments indicate that: (1) the endogenous respiration of small populations of
bacterial and yeast cells is almost negligible, but respiration is significantly stimulated by the depth of protozoan cells; (2)
the endogenous respiration of ciliate cells
is very much lower than the respiration
of feeding and dividing cells; and (3) even
where there are minimal population
changes of predator and prey the respiration of the two populations together is
much greater than either separately. In
one set of experiments, the respiratory activity of the ciliate Colpoda steinii and the
yeast Rhodotorula together was about
twice that of the two organisms measured
separately, although there was no division
of Colpoda during the experiment and the
population of Rhodotorula was only reduced by about 5%.
Such experiments give a better insight
into how very small changes in the activity
of microbial populations in soil can affect
significant changes in soil metabolism.
Such changes in the biomass will take
place continuously with the fluctuating
conditions which obtain in soil, whereas
major changes in biomass and in the accretion of fresh plant residues occur over
longer periods.
CONCLUSIONS
The role of protozoa in soil ecology can
be considered under the following headings:
1) The role of symbionts such as the
termite flagellates or oligochaete ciliates
whose presence is essential to the life of
their hosts.
200
JOHN D. STOUT
RESPIRATION OF TETRAHYMENA A N D PSEUDOMONAS
66
(
2
3
4
TIME (HOURS)
HG. 2. Relative oxygen uptake of bacteria and domonas, I — Tetrahymena pyriformis)
proto/oa separately and together. (P = Pseu-
PROTOZOAN POPULATIONS IN SOILS
2) The role of parasites which affect
populations of soil animals, such as earthworms, enchytraeids, snails, or beetle larvae.
3) The role of protozoa as food ensuring the healthy development of other
animals, e.g., Eisenia foetida (Miles, 1963).
4) The role of protozoa in modifying
the microbial population of soils, e.g., by
removing the enteric bacteria or by influencing the incidence of phytopathogenic fungi (Nikoljuk, 1965).
5) The role of protozoa in attacking
plant residues or humic substances (Schonborn, 1965).
6) The role of protozoa in accelerating
the turnover of the soil biomass and consequently of the turnover of soil organic
matter.
REFERENCES
Kabuik, L. A., and E. A. I'aul. 1970. The use of
Hiioresceii) isothiocyanate in the determination
o£ the bacterial biomass of grassland soil. Can.
J. Microbiol. 16:57-62.
Bonnet, L. 1961. Caracteres generaux des populations ihccamoebiennes endogees. Pedobioloeia
1:6-24.
Cray, T. R. C, and S. T. Williams. 1971. Micro
bial productivity in soil, p. 255-286. In D. E
Hughes and A. H. Rose [ed.], Microbes and
biological productivity, Twenty-first Symposium
201
of the Society of General Microbiology, University Press, Cambridge.
Grossbard, E. 1971. The utilization and translocation by microorganisms of carbon-14 derived
from the decomposition of plant residues in soil.
J. Gen. Microbiol. 66:339-348.
Jenkinson, D. S. 1971. Studies on the decomposition
of C" labeled organic matter in soil. Soil Sci.
111:64-70.
Miles, H. B. 1963. Soil Protozoa and earthworm
nutrition. Soil Sci. 95:407-409.
Xex0, B. A., K. Hamburger, and E. Zeuthen. 1973.
Simplified microgasometry with gradient divers.
C. R. Trav. Lab. Carlsberg. (In press)
Xikoljuk, V. E. 1965. Antagonistic interrelationship
between some soil protista and certain phytopathogenous fungi affecting cotton plants, p.
118-119. In Excerpta Med., Int. Cong. Ser. No. 91,
2nd Int. Conf. Protozool., London, July-August
1965, Progress in Protozoology.
Russell, E. J., and H. B. Hutchinson. 1909. On the
effect of partial sterilization of soil on the production of plant food. J. Agric. Sci. 3:111-114.
Schonborn, W. 1965. Untersuchungen fiber die
Erna'hrung bodenbewohnender Testaceen. Pedobiologia 5:205-210.
Stout, J. D. 1973. Response of some soil bacteria
and yeasts to glucose. Soil Biol. Biochem.
(In press)
Stout, J. D., and M. E. Dutch. 1968. Rates of
organic matter decomposition measured by Warburg respiratory experiments. Trans. 9th Int.
Congr. Soil Sci. Adelaide 3:203-209.
Stout, J. D., and O. W. Heal. 1967. Protozoa, p.
149-195. In A. Binges and F. Raw [ed.], Soil
biology. Academic Press, London.
Tribe, H. T. 1961. Microbiology of cellulose decomposition in soil. Soil Sci. 92:61-77.