FEMS Microbiology Ecology 74 (1990) 269-276
Published by Elsevier
269
FEMSEC 50283
Anaerobic free-living protozoa: growth efficiencies
and the structure of anaerobic communities
Tom Fenchel and Bland J. Finlay
' Marine Biological Laboratory (University of Copenhagen), Helsinger, Denmark and
*
Institute of Freshwater Ecology, Windermere
Loboralory, Ambleside, Cumbria, U.K.
Received 28 February 1990
Revision received 15 June 1990
Accepted 27 July 1990
Key words: Anaerobic environments; Phagotrophy; Growth efficiency; Protozoa
1. s1 M A RY
why phagotrophic food chains are short ant I
eukaryote diversity is low in anaerobic habitats.
Most anoxic environments host populations of
phagotrophic, eukaryote microorganisms. Many
physiological properties of these anaerobic
eukaryotes are still incompletely understood and
their role in communities of anaerobic microorganisms has so far drawn little attention. Here
we present theoretical considerations and experimental evidence to show that the net growth efficiency ([assimilated C]/ [assimilated C
dissimilated C ] )and gross growth efficiency (yield
= [assimilated C]/ [consumed C ] ) of anaerobic
protozoa are about 20% and about 25% respectively of those of their aerobic counterparts. This
accords with the observation that the biomass
ratios of predators and their prey is about one
fourth of that found in oxic environments. These
field data also suggest that bacterial numbers are
controlled by protozoan grazing in at least some
anoxic environments. Finally, the results explain
+
Correspondence lo: T . Fenchel, Marine Biological Laboratory,
DK-3000 Helsinger, Denmark.
2. INTRODUCTION
Most anaerobic environments (including the
hypoliminion of stratified lakes and lagoons, sandy
and detrital sediments, sulphureta and anaerobic
sewage) harbour protozoa; these are predominantly ciliates, but heterotrophic flagellates and
rhizopods also occur [l-31. Energy metabolism
and other aspects of cell physiology of anaerobic
protozoa have so far been studied mainly in parasitic and commensal forms, viz.,the protozoan
fauna of the rumen and of the intestinal tract of
mammals and of the termite hind gut [4], but the
biology of free-living forms has recently been the
subject of several studies [S-81.
Protozoa inhabiting anaerobic environments all
seem to have a dissimilatory metabolism which
does not depend on oxidative phosphorylation
and they do not possess cytochromes. They vary
in their sensitivity to oxygen. Some forms (e.g.
Plagiopyla nasuta) can grow at low oxygen ten-
0168-6496/90/$03.50 6 1990 Federation ofEuropean Microbiological Societies
270
sions ( < 2% atm sat) while others (e.g. Parablepharisma collare) require strictly anaerobic and
reducing environments and are sensitive to even
trace levels of 0, [6,7,Fenchel and Finlay, unpublished observations]. Some flagellates (the diplomonads) and the rhizopods (e.g. Entamoeba
spp.) do not have any organelles resembling
mitochondria; for energy metabolism they depend
on fermentation and substrate level phosphorylation, with ethanol, acetate and lactate as the principal end products [4]. All anaerobic ciliates that
have been studied, and some flagellates, have
mitochondria-like structures which are involved in
energy metabolism. In some cases (e.g. the ciliates
Plagiopyla and Metopus and the trichomonad
flagellates) these organelles are capable of oxidising pyruvate to acetate while producing CO, and
H, and the organelles are then called ‘hydrogenosomes’ [4,8]. Some of the species that excrete
H, (such as the two above mentioned ciliates)
harbour symbiont methanogenic bacteria [5,71. Attempts to make carbon and redox balances for the
metabolic processes have so far not been attempted; this is mainly due to the difficulty of
obtaining axenic or even monoxenic cultures of
the free-living forms.
Here we describe attempts to determine the
growth efficiencies of anaerobic protozoa from
theoretical considerations, from direct estimates,
and by comparing maximum growth rates of
anaerobes with those of aerobic protozoa. Finally
we discuss the implications for the structure of
phagotrophic food chains in anoxic environments.
3. MATERIALS AND METHODS
3.1. Materials
The following species were studied in cultures:
Metopus contortus Quennerstedt, Metopus palaeformis Kahl, Plagiopyla frontata Kahl and Parablepharisma collare Kahl (all ciliates) and the
flagellate Hexamita sp. Metopus palaeformis is a
freshwater species which was isolated from a
landfill site in Cumbria, U.K. The other species
are all marine and were isolated from anaerobic
and sulphide-containing sands collected in shallow
water in Nivi Bay situated 15 km south of Helsingerr, Denmark.
The marine species were grown in 125
Hypo-Vials capped with butyl stoppers and
aluminum seals. The medium consisted of 100
diluted seawater (S: 18%). It was boiled With
dried grass (400 mg 1 - I , filtered and a few drops
of a resazurin solution (Sufficient to colour the
medium) were added as a redox indicater. Two
boiled wheat grains were added to each vial. T h e
medium was then bubbled with purified N-pgas
( < 5 ppm 0,according to the supplier). In Some
case; we reduced the medium with drops of a
neutral sulphide solution (10 mM) Or with a few
crystals of dithionite before the vials were capped.
Dithioite sometimes produced Poor growth a n d
its use was discontinued. In some cases we sim
PlY
left the vials until bacterial growth had reduced
the medium as evident from the decolouration of
the resazurin (24-48 h). The vials were finally
inoculated with protozoa (and the mixed assemblage of bacteria which serve as food) through the
butyl stoppers with a hypodermic syringe. During
growth of the protozoa, the medium typically contained about 1 (0.7-4) mM sulphide and maintained a pH around 7. The freshwater species
Metopus palaeformis was grown in SES medium
(autoclave 33 g sieved soil in 1 1 distilled water (1
h), decant clear supernatant, add 20 mg K,HPo,,
20 mg MgSO, * 7 H 2 0 and 200 mg KNO,, buffer
with 100 mg NaHC03 per 100 ml, outgas with N,
and CO, to pH 7 and add 1 mg powdered cereal
leaves per ml as substrate for the bacterial flora
which serve as food) in Hypo-Vials. Eventual yield
ranged from 100 to 200 cells ml-’ for the large
Metopus contortus and Plagiopyla frontata to almost lo5cells ml for the small Hexamita. Cultures
were kept at 20°C and t h s temperature also applies to all experimental results.
3.2. Experimental methoris
Cell volumes of ciliates were determined by
squeezing glutaraldehyde-fixed cells between a
slide and a cover slip supported by feet of vaseline
until the cells had plane parallel sides. We then
measured the surface area by connecting the microscope to a video camera and a semiautomatic
image analyser (MOP-Videoplan, Kontron). W e
271
also estimated the thickness of the preparation by
focusing on the upper and lower surface of the
preparation while reading the micrometer scale of
the fine adjustment of the microscope. In this way
we calculated cell volumes for about 10 individuals of each species of ciliate. The cell volume of
Hexamit3 was estimated as a prolate ellipsoid on
the basis of measurements of the length and diameter.
For the estimation of growth rate constants we
used Hypo-Vial cultures. Samples of 1 or 2 ml
were withdrawn every 24 h with a hypodermic
syringe and counted directly in dishes or in a
Sedgewick-Rafter chamber under the dissection
microscope or (in the case of Hexamitu) in a
haemcytometer cell. Growth rate constants were
estimated by linear regression of the logarithm of
the counts during the exponential growth phase
(Fig. 1).
Direct estimates of growth yield (gross growth
efficiency) were made by growing Metopus contortus cells in capillaries with plane sides ('Microslide-tubes', Camlab, Cambridge); the internal di-
,
O
O
t
101
j
f
50
i
100
150
200
250
hours
Fig. 2. Growth of 3 cultures of Metopus contorrus fed with
suspensions of baker's yeast in capillaries.
mensions were 0.4 x 4.0 mm and the length of the
capillaries was about 3 cm. We filled the capillaries
with a suspension of bakers yeast (about lo6 cells
ml-') in oxygen-free seawater and then injected a
few pl of concentrated Metopus cells. The ends of
the capillary were then sealed with Vaseline. The
whole procedure took place in a N,-atmosphere.
Numbers of Metopus were counted daily for about
three generations (Fig. 2). A! intervals the number
of yeast cells eaten per ciliate per unit time was
determined by direct observation under the microscope. On each occasion 10-15 cells were observed for 2-5 min. On the basis of the rate of
consumption (U),the growth rate constant ( p )
and the average volumes of the yeast cells ( V , )
and ciliates (Vc), respectively, the growth efficiency (in terms of volumes) can be calculated as
y = [ P x Kl"
V,l.
4. RESULTS A N D DISCUSSION
4.1. Theoretical considerations
The growth of cells is closely related to their
power generation and it has been established that
1 mol of ATP is required for producing 4 g cell C
[9]. In the case of an aerobic organism the comwill
plete oxidation of 1 mol of C6H,,0, (72 g
yield 32 mol ATP. To synthesise the 4 X 32 g cell
carbon the cells must assimilate this amount of
organic C in addition to the 72 g C necessary for
the dissimilatory metabolism so that the net growth
c)
2
0
200
400
hours
Fig. 1. Growth of 2 anaerobic batch cultures of Plagiopyla
frontata.
272
efficiency becomes En= [32 x 4]/[(32 x 4) + 721
= 0.64. A net growth efficiency of around 60% is
consistent with empirical values for a wide range
of aerobically growing unicellular organisms and
growing tissue of metazoa [lo]. Phagotrophic
organisms cannot usually digest all their food with
an efficiency of 100% and they may also lose
dissolved organic material by leakage through the
cell membrane. The amount of the ingested
material which thus becomes unavailable to the
cells is undoubtedly variable according to the
organism and the nature of the food particles;
however, 30%is a typical figure for many studied
protozoa [ll].If we accept this value, about 30%
of the ingested food will be used for dissimilatory
metabolism, 40 for synthesis of new cell material
and 30% will be lost so the gross growth efficiency
(or cell yield) will be 40%.
In the case of the anaerobic protozoa the energy conserved as ATP per mol glucose fermented
has not been determined with certainty. The glyce
lytic pathway will yield 2 mol of ATP and the
subsequent complete oxidation of pyruvate to
acetate will yield another 2 mol ATP [12]. However, not all anaerobic protozoa possess hydro-
genosomes and all studied forms (mainly parasitic
ones) seem to excrete some metabolites which are
less oxidised than acetate. On the other hand, the
presence of symbiont methanogens may yield Some
extra energy on the basis of H 2 or acetate. We will
here assume that 3 mol ATP per mol glucose is a
probable average. The net growth efficiency of the
anaerobic protozoa (using the same consideration
as for the aerobes) then becomes 0.14 (or about
one fifth of that of aerobes). Since the Substrates
of the fermenting organisms are not completely
oxidised to c02,it is possible that some of the
metabolites are assimilated. However, even if d l
cell carbon is based on this (which is unlikely) the
calculation shows that net growth efficiency Would
only increase from 14 to 16%.There is no reason
to assume that anaerobes differ from aerobes with
respect to the efficiency of digestion or loss of
dissolved organic material. Therefore, again assuming a loss of 30% we find that the gross
growth efficiency of anaerobic protozoa is 10% o~
about one fourth of that of aerobes.
4.2. Direct measurements of gross growth efficiency
The generation time of Metopus contortw fed
Or------
-
-1 ’
r
-c
-_,
x
m
-0
-2
4 - _
o aerobic
flagellates
I anaerobic
-
o aerobic ciliates
b anaerobic
4
-3.
-
-
,deprived of endosymbiont
methanogens
L
213
baker's yeast in capillaries (Fig. 2) was 75 h (p =
0.0092 h-') or slightly longer than in the HypoVial cultures (60.2 h). The rate of consumption of
yeast cells (based on the observation of 44 cells)
was 98 h-'. The average cell volume of the yeast
cells was 135 pm3 and that of the Metoptis cells
1.3 X lo5 pm3. With these figures, the yield (gross
growth efficiency) can be calculated as 9.0% in
terms of cell volumes.
When this result is evaluated, it must be taken
into consideration that the carbon content per
unit volume of the ciliate and of the yeast cells
may not be exactly the same. Also, the yeast cells
do not seem to constitute optimal food particles
insofar as they did not support maximum growth
rate. This may be because the yeast cells were in
the upper size range of what Metopus can ingest.
It was often observed that the ciliates were incapable of ingesting yeast cells which had been captured by the oral membranelles. However, the
result does support the prediction of a gross growth
efficiency of about 10% and thus about 25% of
that characteristic of aerobic protozoa.
4.3. Growth rate constants
Growth rate constants in batch cultures of protozoa vary according to temperature, but also
according to the nature of the food particles and
possibly other external factors such as salinity,
pH, etc. Nevertheless, an apparent maximum
growth rate constant can often be approximated
in cultures and be reproduced under a fairly wide
range of conditions [ll]and we are confident that
our estimates are close to the maximum rates
attainable for the different species.
When protozoa, spanning a wide range in cell
sizes, are compared the growth rate constant of
protozoa is known to scale with cell volume
according to the power function p = a Wb, where
W is cell volume and a and b are constants, the
latter taking the value about - 0.25 [ll].The lines
in Fig. 3 have a slope of -0.25 rather than being
regression lines for the data points shown. The
figure shows published data for aerobic ciliates
and heterotrophic flagellates [11,13 and references
therein]. Also shown are the present data for the
anaerobic protozoa and a previously published [6]
value for the anaerobic ciliate Trimyema compres-
sum (corrected to 20°C assuming a Q,, of 2.5). In
the case of Plagiopyla and Metopus contortus
growth rate constants for cells deprived of
methanogen symbionts [Fenchel and Finlay, unpublished data] are shown in addition to values
for normal symbiont-containing ciliates.
The studied anaerobic protozoa have minimum
generation times from 60 h for the large Metopus
contorrus (100 h for methanogen-free cells) down
to about 20 h for the flagellate Hexamita. In
contrast, aerobic, heterotrophic flagellates and
small ciliates have minimum generation times
around 4 h and large, aerobic ciliates typically
have minimum generation times in the range 10-20
h. It is noteworthy, that the constant a in the
equation describing the relation between cell size
and growth rate constant seems to take a value
some 1.5 times higher in ciliates than in flagellates; this (with the reservation that we only have
one data point) seems also to apply to the
anaerobes.
Provided that the digestion efficiencies and the
efficiencies with regard to the concentration of
food particles are similar in aerobic and anaerobic
protozoa (and there is no a priori reason assuming
otherwise) then growth rate constants should be
proportional to cell yield. The fact that the growth
rate constants of anaerobic protozoa seem systematically to be about 25% of the values of
similarly sized aerobic protozoa indicates that t h s
also applies to gross growth efficiencies.
4.4. Prey /predator ratios in aerobic and anaerobic
environments
Theoretical models of steady-state phagotrophic
food chains suggest that there is a characteristic
ratio between the biomasses of predators and their
prey [14,15]. The value of this ratio is derived from
the ratios of individual predator and prey sues
and the size-dependent rates of metabolism and
growth, and is thus a function of growth efficiencies. Specifically, the ratio between predator and
prey biomass is proportional to gross growth efficiency of the predator. In (aerobic) plankton
food chains with a fairly simple community size
structure, theory predicts a predator/prey biomass ratio of slightly less than unity [14,15].
In Tables 1 and 2 we have accumulated data on
274
the biomass ratios between protozoa and their
food organisms in plankton in water bodies with
anoxic bottom waters so that the predator/prey
biomass ratios in the aerobic and anaerobic parts
of the systems can be compared. It is evident that
in all cases, this ratio is 3-7 times higher for
aerobic systems. Pooling all the data the ratio:
[predator/prey],,,,/[predator/prey],,
is on the
average 0.24 (S.D.: 0.07, range: 0.14-0.36).
Natural ecosystems and especially eutrophic
systems (as listed in Tables 1 and 2) are never in a
steady state and so these figures represent only
approximations (but this has been somewhat compensated for in that some estimates are seasonal
averages based on several samples). Our knowledge of the details of the food chain (that is, who
eats who) may also be incomplete. Yet the results
Table 2
Gypsum lagoon in Spain
Biovolume (lo6am3 d-')
Bacteria
Aerobic
Anaerobic
Heterotrophic
nanoflagellates
3.6
3.0
7.9
Spanish meromictic lake
1.6
Aerobic
Anaerobic
4.3
1.2
0.2
11.1
-
0.4
0.8
0.07
Ciliates
16.1
28.1
15.7
5.1
-
Ratio
predator/pre+,
1.7
Food
organisms '
-
Ratio
predator/prey
'
Biovolume(10' pm3 m1-I)
Bacteria
Heterotrophic
nanoflagellates
Aerobic
Anaerobic
Table 1
-
Predator/prey ratios of aerobic and anaerobic environments
-
1
Ratio
Predator/prey
0.98
0.2
Unpublished data collected by B.J. Finlay from the p
.
ARCAS I1 in 1987.
Unpublished data collected by B.J.Finlay from Laguna de la
Cruz in 1987.
' In the aerobic layer ciliates fed on cryptomonads, Other
photosynthetic flagellates. and on heterotrophic flagellat,,
In the anaerobic zone the most important food items were
Chrornarrum and pigmented and non-pigmented flagellate,.
In the aerobic zone the ciliate community was dominated by
species of Prorodon and of pleuronema. In the anaerobic
zone the ciliates belonged to the genera Ludo and
Caenomorpho .
a
Ratios of biovolumes of predators and prey in aerobic and
anaerobic environments
Stratified waters of a marine Danish fjord a
Bacterivorous/
Ratio
Bacteria
preda tor/prey
(ng c mI I ) protozoa
(ng c m1-l)
Surface
Oxycline
Anoxic
25.0 (12.3)
40.6 (10.1)
76.3 (18.0)
7.3 (4.6)
19.6 (19.0)
5.7 (2.6)
Stratified pond, English Lake Distnct
0.29 (0.15)
0.59 (0.64)
0.08 (0.03)
'
Bacteria
Heterotrophic
(ng C d-')nanoflagellates
(ng C m1-I)
Aerobic
Anaerobic
80
57
172
326
Ratio
predator/prey
0.47
0.17
~~
Stratified lake, English Lake District
Ratio between biovolume of heterotrophic nanoflagellates and
bacteria
-
~~~
July
September
Aerobic layer
Anaerobic layer
0.1 7
0.21
0.046
0.046
Data from 13). Means (S.D.) based on 7 sampling occasions
from April to October, 1987.
Data from [16]. Average values for 3 sample depths in the
aerobic and anaerobic zone, respectively.
' Unpublished data collected by U.-G. Berninger. Samples
taken at 4 m and 15 m depth in Esthwaite Water, corresponding co aerobic and anaerobic conditions. respectively.
a
'
d o demonstrate a significant difference in the
structure of phagotrophic communities of aerobic
and anaerobic plankton, respectively and this is
related to the difference in growth efficiency be.
tween anaerobic and aerobic eukaryotes.
Our findings probably represent the best explanation for the low diversity and short food
chains of anaerobic communities. The phagotrophs
of anaerobic environments are predominantly
bacterivorous protozoa. In addition, a few species
of predatory ciliates which prey on flagellates,
algal cells or small ciliates occur in small numbers
[3] in some habitats, but this seems to be the limit
to the length of phagotrophic food chains in
anaerobic habitats. In contrast, aerobic aquatic
habitats support longer food chains (up to at least
5 levels) including not only protozoa of various
275
sizes, but also metazoan predators. If we assume
10% as a reasonable figure for the gross growth
efficiency of anaerobic phagotrophs and the production of bacteria is unity, then the production
of the second food chain level of phagotrophs
(protozoa preying on bacterivorous protozoa) will
be 0.01. This seems to be too low to support the
production of a further anaerobic trophic level. If,
on the other hand, we assume that gross growth
efficiency in aerobic phagotrophs is 40%, then the
5th level of a phagotroph food chain will still
represent 1%of the production of the first level.
From an evolutionary point of view it may
seem strange that among the eukaryotes only some
mainly bacterivorous protozoa (belonging, to many
different and unrelated taxa) have evolved to live
in anoxic environments. Among multicellular
organisms there are many examples of species
which can endure anaerobiosis for long periods or
thrive at very low oxygen tensions, but multicellular anaerobes which complete their entire life cycle
in the absence of oxygen seem to be non-existent.
There does not seem to be any obvious physiologjcal reason for this. We therefore offer the following explanation: the low growth efficiency of
anaerobic eukaryote phagotrophs Limits the length
of food chains in anaerobic habitats; there is
simply not enough food for larger phagotrophs,
which depend on relatively large food particles,
and so must occupy higher trophic levels.
ACKNOWLEDGEMENTS
We wish to acknowledge the laboratory assistance of Ms Jeanne Johansen. The study was
supported by the Danish Natural Science Research Council (11-8391) and the Natural Environment Research Council (U.K.). Metopus
palaeformis was isolated by B.J.F. while working
on contract No. E/5A/CON/1256/2099 funded
by the Department of Energy (U.K.) (through
ETSU, Harwell). Collections in Spain were obtained with the help of Drs. M.R. Miracle and E.
Vincente (Valencia) and financial support from
the British Council. Dr. U.-G. Berninger kindly
supplied some unpublished data from English
lakes.
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