Ecological Research Series
Effects Of Protozoa On The Fate
Of Participate Carbon
£c
I
55
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LU
03
National Environmental Research Center
Office of Research and Development
U. S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formation,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
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EPA-660/3-73-007
August 1973
EFFECTS OF PROTOZOA ON THE FATE OF PARTICULATE CARBON
by
Harvey W. Holm
Forrest A. Smith
Southeast Environmental Research Laboratory
National Environmental Research Center-Corvallis
Athens, Georgia 30601
Project 16050 GJC
Program Element 1B1023
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 70 cents
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ABSTRACT
Laboratory studies were designed to define the role of protozoa in the
fate of particulate (bacterial) organic carbon. Specific objectives were
(1) to measure the effects of selected environmental parameters on
protozoan growth rates, (2) to measure organic carbon in bacteria and
protozoa, and (3) to quantitate carbon transformations in predator-prey
experimental systems.
A growth system containing 2 X 108 Citrobacter/m-L in 1 X 10~3 M phosphate
of pH 7.5, incubated at 25°C at a shaking rate of 100 rpm, was found to
be an optimal environment for protozoan growth.
The nutrient bacterium, Citrobacter, contained 8.6 X 10"11 mg C/cell,
and Tetrahymena pyriformis contained 1.1 X 10~6 mg C/cell.
T_. pyrif ormis altered the amount and form of carbon in the system while
growing on bacteria. Of the total organic carbon present at the initia-
tion of the predator-prey experiment (93 mg), 93% was in the bacterial
fraction. Within 96 hours, 38% of the carbon was released as C02; 5%
was present as inorganic carbon in the water and the remainder (577o) was
present as organic carbon. The organic carbon in the bacterial fraction
decreased from 86 to 2 mg within 96 hours, while the carbon in the
protozoan biomass increased from 1 to 40 mg. In the bacterial control,
117o of the organic carbon was released as C02 within 96 hours while
negligible amounts of inorganic carbon remained in the water.
This report was prepared in fulfillment of Project Number 310301QPL by
the National Pollutants Fate Research Program, Southeast Environmental
Research Laboratory, National Environmental Research Center-Corvallis,
U. S. Environmental Protection Agency. Work was completed as of
June 30, 1972.
11
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CONTENTS
Abstract ii
List of Figures iv
List of Tables v
Acknowledgments vi
Sections
I Conclusions 1
II Recommendations 2
III Introduction 3
IV Materials and Methods 5
V Results and Discussion 8
VI References 30
VII Appendices 32
111
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FIGURES
No. Page
1 Size distribution of Tetrahymena pyriformis 16
2 Concentration of C02 in effluent air 19
(recorded as ppm)
3 Accumulated carbon emitted as COg 20
(recorded as mg carbon)
4 Total inorganic carbon (TIC) in the medium 22
(recorded as mg carbon/-t)
5 Total organic carbon (TOG) in the medium 23
(recorded as mg carbon/£)
6 Growth of protozoa (Tetrahymena pyriformis) 24
with bacteria as the carbon source
(Experiment 1)
7 Growth of protozoa (Tetrahymena pyriformis) 25
with bacteria as the carbon source
(Experiment 2)
8 Carbon transformation -- bacterial control 26
9 Carbon transformation -- bacterial-protozoan 28
system
10 Partitioning of organic carbon in a bacterial- 29
protozoan growth system
IV
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TABLES
No.
Generation times (hours) of Tetrahymena
pyriformis as a function of temperature
and shaking
pH stability of protozoan growth systems 10
as a function of initial pH and phosphate
molarity
Generation times (hours) of Tetrahymena 11
pyriformis as a function of phosphate
molarity and pH (Experiment 1)
Generation times (hours) of Tetrahymena 12
pyriformis as a function of phosphate
molarity and pH (Experiment 2)
pH stability of protozoan growth systems 13
as a function of initial pH and phosphate
molarity
Generation times (hours) of Tetrahymena 14
pyriformis as a function of bacterial
concentration
Organic carbon content of Tetrahymena 17
pyriformis and Citrobacter
Size distribution of Tetrahymena pyriformis 18
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ACKNOWLEDGMENTS
Technical assistance was received from two individuals of the National
Pollutants Fate Research Program during the carbon balance study.
Mr. John Barnett set up the C02-free air-flow system for the carbon
studies. Mrs. Donna Davis analyzed air samples for carbon dioxide con-
tent during certain phases of the research. The help from these
individuals is gratefully acknowledged.
VI
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SECTION I
CONCLUSIONS
Protozoa have various effects on the fate of particulate carbon (bacteria)
in aqueous environments:
• Total Organic Carbon (TOG) of bacterial-protozoan systems
decreases as protozoa metabolize bacterial cells. Within 120 hours,
49-577» of the organic carbon was transformed to inorganic carbon in
systems containing protozoa, whereas 4-177o of the organic carbon was
similarly transformed in systems containing only bacteria.
• Within 120 hours, the bacterial-protozoan system released over
three times as much C02 as the bacterial control.
• The Total Inorganic Carbon (TIC) concentration in the water of
the bacterial-protozoan system increased from essentially zero to 4 mg
carbon/-^ during the experiment, about five times that of the bacterial
control.
• According to calculations, soluble organic carbon may be produced
in systems containing protozoa.
The protozoan Tetrahymena pyriforinis requires 3 X 104 bacteria Citrobacter
(2.6 X 10~6 mg carbon) for the production of each protozoal cell con-
taining 1.1 X 10~6 mg carbon, representing a carbon assimilation efficiency
of 42%.
Factors that affected the growth rate of T_. pyriformis included phosphate
concentration, pH, temperature, shaking rate, and bacterial concentration.
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SECTION II
RECOMMENDATIONS
Protozoan populations have a significant role in determining the fate of
pollutants in the environment. Further investigations should be directed
toward clarifying the following points:
• This work shows that protozoa have an impact on the fate of
carbon from one species of bacteria. To better predict the role of
secondary heterotrophs on the fate of particulate carbon, several
protozoa of distinct physiological and ecological types should be examined
for abilities to utilize particulate carbon from several species of
algae and bacteria.
• Influence of environmental factors, such as light and water
flow, on protozoan ecology should be evaluated.
• The fate of other major nutrients (nitrogen and phosphorus)
should be investigated in protozoan growth systems.
• The fate and effects of selected pollutants (e.g., heavy metals,
pesticides, chlorine) should be examined in mixed ecosystems containing
bacteria, algae, and protozoa to determine rate data that are meaningful
for estimating the environmental impact of pollutants.
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SECTION III
INTRODUCTION
'rotozoa are commonly occurring organisms in soils and water. Their
importance in ecosystems is indicated by their ubiquitous nature and by
their large biomass.
Since the majority of protozoa seen in polluted river systems , in ponds ,
in soils , and in sewage treatment facilities4 are non-photosynthetic
secondary heterotrophs, as a group they may be of significance in the
fate of the ubiquitous nutrient, organic carbon. Indeed, some evidence
implicates secondary heterotrophs with several roles in the fate of
carbon.
First, protozoa need vast numbers of bacteria or other microorganisms as
carbon sources . This observation has led some5'6 to believe that
protozoa are harmful because they utilize functional bacteria. Others7j8>9>1°
note that a combination of bacteria and protozoa produces a higher
T T IP
quality sewage treatment than either population alone. Still others '
believe protozoa are the primary agents in the sewage stabilization
process.
Second, in addition to utilizing bacterial cells, certain protozoa can
metabolize soluble organic compounds13. Wilson and Danforth14 reported
a non-photosynthetic strain of Euglena assimilated 58% of a soluble
substrate, acetate, releasing the remainder as carbon dioxide.
Third, protozoa indirectly influence the fate of nutrients in the environ-
ment by stimulating the growth of certain bacteria1 »16.
Only a fraction of the bacterial carbon is thought to be assimilated by
the protozoa; the remainder is released as carbon dioxide and soluble
organics, which may be metabolized by other organisms. Few data are
available that show the rate of carbon assimilation by protozoa with
concomitant release of carbon dioxide. Indirect evidence from BOD tests
presented by Busch17 and confirmed by work of Curds, Cockburn, and
Vandyke as presented by Sykes and Skinner4 suggests that protozoa exhibit
a significant oxygen demand, presumably for metabolizing the primary
heterotrophs (bacteria). Heal18 using data from Warburg manometry, cal-
culated that Acanthamoeba respires 21% of the carbon taken up.
The present study was designed to measure the role of protozoa in the
mineralization of carbon (organic -» inorganic), and to define factors that
might affect the protozoan growth rate and the mineralization process.
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Specific objectives of the study were to determine (1) environmental
conditions producing optimal growth rates of the protozoan Tetrahymena
pyriformis; (2) the carbon content of TT. pyriformis and the bacterium
Citrobacter; and (3) the types and rate of carbon transformations in
bacterial-protozoan ecosystems.
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SECTION IV
MATERIALS AND METHODS
MATERIALS AND ANALYTICAL PROCEDURES
Organisms
A natural aquatic bacterium isolated from Shriner's Pond, Clarke County,
Georgia, was used as the test organism. The bacterium was identified
as a Citrobacter. In all experiments, the bacteria were grown in Payne
and Feisal'sis medium at 25°C for 48 hours, washed twice with dilution
buffer , and resuspended in the appropriate medium for experiments.
An axenic species of protozoa commonly used for laboratory research1 ,
Tetrahymena pyriformis (ATCC 9357) was obtained from the culture collec-
tion of the Department of Microbiology, University of North Dakota,
Grand Forks. The organism was grown in proteose peptone-glucose medium
(1%, 17»)21 for four days, and washed two times in dilution buffer by
centrifugation at 100 X G for 45 seconds before use.
Reagents
Reagent-grade chemicals were used for the preparation of media.
Cylinder air (USP Compressed Air, Breathing Quality) was used for aeration
in the carbon balance studies.
Growth Measurements
Bacteria were counted by two methods. A Coulter Counter equipped with
a 30~n aperture tube was used to determine initial bacterial concentrations
of washed cell suspensions. With an aperture current of 1/2, amplification
of 1/4, and a lower threshold of 12, counting agreed with pour plates.
During the course of the experiments, bacteria were counted by plating
serial dilutions, in duplicate, in Tryptone Glucose Extract Agar (TGE)
pour plates, incubating at 25°C for 48 hours and counting the colonies.
Duplicate samples of protozoa, killed with Lugol's Iodine, were counted
by direct microscopic counts at 100 X using a Levy counting chamber. The
Coulter Counter, with an aperture current of 1 and an amplification of
1, equipped with the Model J plotter and a 200-|j, aperture tube, was used
for biomass determinations.
Analytical Procedures
The Beckman Model 915 Total Organic Carbon Analyzer was used for Total
Organic Carbon (TOG), Total Inorganic Carbon (TIC), and carbon dioxide
(C02) determinations. All analyses were made in duplicate.
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Total Organic Carbon (TOG) was determined either on 5-m-t samples frozen
in Nalgene bottles, or on unstored samples. After appropriate dilutions,
the sample was acidified to pH 4 with sulfuric acid and bubbled for five
minutes with nitrogen gas to remove inorganic carbon.
Total Inorganic Carbon (TIC) was determined on samples collected with a
100-iJL-t syringe and immediately injected into the inorganic channel of the
Beckman Carbon Analyzer.
Carbon dioxide was measured by collecting gas samples from the effluent
line of the flasks (1, 2, or 5 m£, depending on the COs concentration)
with a syringe and needle, and injecting them into the inorganic channel
of the Beckman Carbon Analyzer.
Soluble organic carbon was not measured because the fragility of the
protozoa made physical separation of cells from the medium unreliable.
Measurements of pH were made with a Beckman Zeromatic pH meter equipped
with a Beckman Combination electrode (#39183).
EXPERIMENTAL DESIGN
Phase I - Environmental Factors Affecting Protozoan Growth
Experiments were designed to determine the effects of certain chemical
and physical factors on the growth rate of T. pyriformis in a simple
batch culture, with bacteria, Citrobacter, as the carbon source.
Duplicate test systems consisted of 250-m-L Erlenmeyer flasks containing
known initial concentrations of washed Citrobacter and T. pyriformis
brought to a volume of 100 m-t with phosphate buffer of varying pH and
concentration, depending on the experiment.
The growth rate of T. pyriformis was tested as a function of temperature,
shaking rate, phosphate concentration, pH, and bacterial concentration.
Growth characteristics of T_. pyriformis in various environments were
expressed by growth rate constants, defined as the number of doublings
per unit time and calculated using the following equation22:
- log10N8
0.301 t
where N0 = population at a given time
Nt = population after a time lapse, t
t = time lapse between population measurements (in hours).
The data are tabulated as mean generation time (1/k).
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Phase II - Carbon Content of Bacteria and Protozoa
The organic carbon content of bacterial and protozoal cells was measured
by preparing washed cell suspensions of pure cultures of Citrobacter and
T_. pyriformis, counting each population, and measuring the TOG of each
cell suspension.
Additionally, a size distribution of T_. pyriformis was obtained and the
carbon content of protozoa was calculated as a function of cell size
using the TOG and size distribution data.
Phase III - Effect o_f Protozoa on. Fate of Organic Carbon (Bacteria)
Two comprehensive duplicate experiments were completed to quantitate the
role of growing populations of protozoa in carbon transformations.
Each of the Fernback flasks 3, equipped with ports for air bubbling,
received one-liter volumes of 10 M phosphate, pH 7.5. Two flasks
received washed Citrobacter to an initial concentration of 109/m-t, and
the two remaining flasks received bacteria (10 /m-^) plus washed
T_. pyriformis (initial concentration, 103/m-t).
An incubation system was used in which dry, CDs-free compressed air
was bubbled through the test flasks at a rate of 150 mt/minute. The
flasks were incubated in a water bath at 23°C with gentle reciprocating
shaking.
Carbon dioxide from the effluent line, TOG in the medium, and TIC in
the medium were sampled at selected time periods and analyzed on the
appropriate channel of the Beckman 915 Total Organic Carbon Analyzer.
Samples were collected from the medium for duplicate bacterial counts
(TGE agar pour plates) and protozoan counts (direct microscopic counts
using a Levy Counting chamber).
Samples from Experiment 1 were stored frozen for later analysis of
total organic carbon; samples from Experiment 2 were analyzed at the
time of collection.
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SECTION V
RESULTS AND DISCUSSION
PHASE I - ENVIRONMENTAL FACTORS AFFECTING PROTOZOAL GROWTH
Preliminary results showed that T_. pyriformis grew well on washed
Citrobacter cells resuspended in dilution buffer20. A concentration of
2 X 10B bacteria/m£ was arbitrarily chosen for initial experiments.
Growth rates of T_. pyriformis were measured at 25°C and 30°C in both
stationary and shaking (100 rpm) cultures (Table 1). Although the
protozoan grew under each condition tested, the 25°C shaking system
produced the best growth rate. In the following Phase I experiments,
the system was incubated under these conditions.
The effects of phosphate concentration and pH on the growth of T_.
pyriformis were investigated in buffer solutions containing 2 X 10s
bacteria/m-L. The pH of each test environment (Table 2) was recorded
before and immediately after the addition of the bacteria and protozoa,
at 24 hours and at 48 hours. The pH of the 5 X lO"4^ phosphate system
was significantly changed by addition of the organisms, whereas the pH
of the 5 X 10"3M phosphate systems was not.
The protozoan did not grow in 5 X 10"2M phosphate, but did in 5 x 10~4M
phosphate (Table 3). The initial pH, within the range tested, did not
greatly influence the growth rate.
To further determine optimal growth conditions, growth rates were mea-
sured over a large range of buffer concentrations at two pH values only.
Initial pH values were high because, in the previous experiment, addition
of bacteria caused the pH (pH 7.5 buffer 5 X lO'^M) to drop to pH 7.0
(Table 2), an ideal pH for many microbiological systems. Maximal growth
rates (Table 4) occurred in 1 X 10~3M phosphate, pH 7.5. Under these
conditions, the pH of the system remained near 7.0 for the duration of
the study (Table 5). T_. pyriformis did not grow in either 5 X 10~3M
or 1 X 10~4M phosphate.
The effect of bacterial concentration on the protozoan population was
tested in a shaking system at 25°C containing 1 X 10~3M phosphate at
pH 7.5, 5 x 102 protozoa/m-L, and varying concentrations of bacteria.
Data (Table 6) show that the concentration used in all of the initial
experiments (2 X 108/m-L) does not limit the growth rate, and was therefore
a suitable choice for this work. Concentrations as high as 10 /m-L did
not adversely affect the growth kinetics of the protozoa.
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Table 1. GENERATION TIMES OF Tetrahymena pyriformis AS A
FUNCTION OF TEMPERATURE AND SHAKING.
Temperature
25°C
30°C
Generation time (hours)
Shaking (100 rpm)
9.4
11.4
Stationary
10.1
16.8
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Table 2. pH STABILITY OF PROTOZOAN GROWTH SYSTEMS AS A FUNCTION OF
INITIAL pH AND PHOSPHATE MOLARITY.
o
PH*
6.0
6.5
7.0
7.5
8.0
pH of Medium
5 X 10~2 M phosphate
hours
0
6.2
6.6
6.9
7.4
7.8
24
6.2
6.5
6.9
7.3
7.8
48
6.3
6.6
6.9
7.3
7.8
5 X 10 4 M phosphate
hours
0
5.9
6.2
6.5
7.0
7.4
24
6.1
6.3
6.5
6.7
6.9
48
6.0
6.5
6.6
7.0
7.0
apH of medium before addition of organisms
bpH after addition of Citrobacter and Tetrahymena pyriformis
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Table 3. GENERATION TIMES (HOURS) OF Tetrahymena pyriformis
AS A FUNCTION OF PHOSPHATE MOLARITY AND pH
(EXPERIMENT 1).
pH
6.0
6.5
7.0
7.5
8.0
Phosphate concentration
5 X 10~B M
No growth
No growth
No growth
No growth
No growth
5 X 10~4 M
5.6
6.9
6.6
5.4
6.6
11
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Table 4. GENERATION TIMES (HOURS) OF Tetrahymena pyriformis AS A FUNCTION OF
PHOSPHATE MOLARITY AND pH (EXPERIMENT 2).
Initial
PH
8.0
7.5
Phosphate molarity
5 X 10'3 M
No growth
No growth
1 X 10~3 M
12.3
9.3
5 X 10~4 M
19.9
10.7
1 X 10~4 M
No growth
No growth
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Table 5. pH STABILITY OF PROTOZOAN GROWTH SYSTEMS AS A FUNCTION OF INITIAL
pH AND PHOSPHATE MOLARITY.
„
7.5
8.0
pH of medium
5 X 10"u M P04 =
hours
0
7.4
7.6
24
7.4
7.4
48
7.5
7.3
1 X 10 a M P04 =
hours
0
7.0
7.2
24
7.0
7.3
48
6.9
7.1
5 X 10~4 M P04 =
hours
0
6.7
6.9
24
6.8
7.0
48
6.9
6.9
1 X 10~4 M P04 =
hours
0
6.4
6.4
24
6.7
6.8
48
6.6
6.5
pH of medium before addition of organisms
pH after addition of Citrobacter and Tetrahymena pyriformis
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Table 6. GENERATION TIMES (HOURS) OF Tetrahymena pyriformis
AS A FUNCTION OF BACTERIAL CONCENTRATION.
Bacteria/in^
5 X 107
1 X 10s
2 X 108
5 X 108
1 X 109
Generation time
8.8
9.0
7.9
8.1
7.7
14
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PHASE II - CARBON CONTENT OF BACTERIA AND PROTOZOA
Because of their fragility, the protozoa could not be separated from the
mixed population culture for carbon content determination. Measurements
were therefore made on axenic cultures of T_. pyriformis grown in proteose
peptone-glucose medium and Citrobacter grown in Payne and Feisal's medium19.
Size distribution plots of protozoa after washing showed little evidence
of cell breakage from the washing procedure (Figure 1). Windows 1, 2,
and 3 represent debris in the system.
According to the measured data (Table 7), each Citrobacter contains
8.6 X 10~i:Lmg carbon, in agreement with a value calculated from literature
data24. The mean carbon content of a protozoan is 1.1 X !CT6mg.
Table 8 is a tabulation of the size distribution data obtained from
Figure 1. Three points should be noted:
1. More protozoa are found in window 7 than any other
(volume = 7,852 p,3 ) .
2. The largest biomass is associated with window 8
(831 protozoa with volumes of 9,060 n3, or a total
biomass of 75.2 X 10 p, ).
/ 884.6 X Kfu,3 N^, , 3
3. The mean protozoan volume ' — / is 1.1 X 10 M. .
7,907 protozoa '
The weight of organic carbon per unit volume of protozoan biomass cal-
culated from the mean organic carbon/protozoan (1.1 X 10~6mg, Table 7)
and the mean protozoan volume (1.1 x 104|i3, Table 8) is 1 X 10~7p,g
carbon/(i3 . From this value, we can assign carbon content to protozoa
of varying sizes.
PHASE III - EFFECT OF PROTOZOA ON FATE OF ORGANIC CARBON (BACTERIA)
Populations of T. pyriformis increased the amount of COg released from
the system over that of the control representing endogenous COs released
by substrate-limited bacteria (Figure 2). In the first 20 hours, the
major portion of the COs evolved was produced by washed bacterial cells;
after the protozoa started growing, however, they produced significant
amounts of COs. The COs concentration curve is skewed to the right, prob-
ably reflecting a production of C02 by endogenous metabolism of the
protozoan population. Under microscopic examination, protozoan cells
were seen to be decreasing in size during this time.
Figure 3 shows the cumulative release of COs from the test systems. Note
the similarity between the COg production in the bacterial control and the
bacterial-protozoan test systems during the first 20 hours of the experiment
Overall, the protozoan-bacterial system released three to four times as
much as the bacteria alone. Between hours 24 and 96, 28.6 mg C as C02 were
15
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100
U-l
CO
60
40
o:
20
WINDOW
li J I 1^1 I I L l*°l I I I 1^1 I I 1 II I I I 1
I I I I I I I I I I I I I I I I I I I I T
5.4 11.5 17.5 23.5 29.6
CELL VOLUME
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Table 7. ORGANIC CARBON CONTENT OF Tetrahymen.a pyriformis
AND Citrobacter.
Organism
Citrobacter
T. pyriformis
Organisms/nvC,
1 X 109
3.25 X 10b
TOG, mg/m£
0.086
0.36
mg organic
carbon /organism
8.6 X 10"11
1.1 X 10~B
17
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Table 8. SIZE DISTRIBUTION OF Tetrahymena pyriformis.
Window
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Average cell volume
(V?)
604
1,812
3,020
4,228
5,436
6,644
7,852
9,060
10,268
11,476
12,684
13,892
15,100
16,308
17,516
18,724
19,932
21,140
22,348
23,556
24,764
25,972
27,180
28,388
29,596
Protozoa /window
(0.73 rot)
_
-
-
419
797
887
936
831
719
556
485
435
342
298
244
209
143
137
89
121
66
81
35
56
21
7,907
protozoa/
0.73 ml
Biomass /window
(M-3)
_
-
-
17.7 X 105
43.3 X 10b
58.9 X 10B
73.5 X 10b
75.2 X 106
73.8 X 105
63.8 X 105
61.5 X 105
60.4 X 10E
51.6 X 105
48.5 X 105
42.7 X 10b
39.1 X 105
28.5 X 10B
28.9 X 105
19.8 X 10b
28.5 X 105
16.3 X 10B
21.0 X 105
9.5 X 105
15.9 X 10B
6.2 X 10b
884.6 X 105
I//0.73 mt
Mean protozoal volume =
884.6 X 10b
7.9 X 103 protozoa
= 1.1 X 104
18
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tt 30
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LLJ
5
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50
40
30
20
10
0
Experiment 1
Experiment 2
20 40 60 80 100 120 140
TIME (MRS)
Figure 3. Accumulated carbon emitted as C02
(recorded as mg carbon)
Symbols: • - bacteria and protozoa
o - bacteria
20
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produced by the growth of 39 X 106 protozoa, a production of 0.7 X 10~6
mg C as C02 per protozoa.
Protozoa can also contribute inorganic carbon to water. The TIC concen-
tration in the medium of the test system is illustrated as a function of
time in Figure 4. When the protozoan population began growing (about 20
hours), inorganic carbon accumulated in the water. When the water became
saturated, the excess was released into the atmosphere and measured as
C02.
The amount of organic carbon in the system, as shown in Figure 5, decreased
markedly as a function of time in systems containing protozoa. In experi-
ment 1 (carbon values obtained from frozen samples), 837o of the initial
organic carbon remained in the bacterial control flask, compared to 517,
in systems containing protozoa. In experiment 2 (unstored samples), 96%
and 437o of the initial organic carbon remained in the bacterial control
and in the bacterial-protozoan system, respectively, at the termination
of the experiment.
The protozoa utilized vast numbers of bacteria during their period of
growth (Figures 6 and 7). According to calculations from Figures 6 and 7,
each protozoan required 3 x 10* bacteria for growth. From the carbon
values reported in Table 7, 2.6 X 10~6mg bacterial carbon was required
to produce protozoan biomass of 1.1 X 10~6mg, representing a 427,
efficiency of carbon assimilation.
As mentioned previously, the soluble organic carbon in the test system
was impossible to measure. By use of formulation similar to that of
Heal18, the amount of carbon solubilized by protozoa was estimated;
carbon solubilized/protozoa = bacterial carbon ingested/protozoa
- (C03-C produced/protozoa + organic carbon/protozoa)
= 2.6 X 10~6mg - (0.7 X 10~6mg + 1.1 x 10~8mg)
= 0.8 X 10~6mg/protozoan
Autolysis of bacteria in this system was considered to be an insignificant
factor in altering carbon form because the bacterial standing crop remained
fairly constant in the bacterial control system (Figures 6 and 7). Also,
after the period of maximal protozoan growth, the bacterial population
increased, presumably using soluble carbon released by protozoa.
Data from experiment 2, used to construct carbon balances, are summarized
in the following graphs. Figure 8 shows the partitioning of carbon in
the bacterial control. By 120 hours, 1270 of the total carbon was present
as atmospheric C02, whereas less than 17, was in the form of TIC in the
medium.
21
-------
o
CO
C£
-------
80
60
40
£3 20
o
o
QO
a:
o 80
00
<
o
o 60
40
20
0
Experiment 1
\_ Experiment 2
0 20 40 60 80 100 120 140
TIME (MRS)
Figure 5. Total organic carbon (TOG) in the medium
(recorded as mg carbon/^)
Symbols: • - bacteria and protozoa
o - bacteria
23
-------
10
- in?
o
0£
Q_
40 60 80
TIME (HRS)
120
Figure 6. Growth of protozoa (Tetrahymena pyriformis) with
bacteria as the carbon source (Experiment 1)
Symbols: o - bacterial population with no
protozoa present
• - bacterial population with protozoa
A - protozoa growing on bacteria
24
-------
10'
£10"
i—
o
<:
CO
107
106
105
104
o
OL
Q_
103
10*
0 20 40 60 80 100 120
TIME (MRS)
Figure 7. Growth of protozoa (Tetrahymena pyriformis) with
bacteria as the carbon source (Experiment 2)
Symbols: o - bacterial population with no
protozoa present
• - bacterial population with protozoa
A - protozoa growing on bacteria
25
-------
O
en
120
100
80
60
40
20
0
24 48 72 96
TIME (HRS)
120
120
100 ^
80 g
60
40
20
0
O
Q_
Figure 8. Carbon transformation -- bacterial control
Symbols: A - total carbon
• - total organic carbon
o - C02
• - total inorganic carbon
26
-------
Figure 9 summarizes the carbon conversions of the bacterial-protozoan
system. By 120 hours, 42% of the organic carbon remained and 40% of the
total carbon was converted to C02• Less than 5% of the organic carbon
initially present was found in the medium as TIC.
Figure 10 shows the partitioning of organic carbon in the bacterial-
protozoan system. Initially, 937» of the organic carbon was in the form
of bacterial biomass. By 96 hours, about 37» of the initial carbon remained
in viable bacterial cells. The protozoa accounted for 44%, of the organic
carbon at 96 hours. The remaining organic carbon, represented as soluble
organic carbon, was calculated from TOG and organism carbon values. Up
to 207o of the organic carbon may be in soluble form, as calculated from
these data.
27
-------
o
CO
100
90
80
70
i 60
\
50
40
30
20
10
0
100
80
o
CD
60 ^
-------
24 48 72 96 120
TIME (MRS)
Figure 10.
Partitioning of organic carbon in a bacterial'
protozoan growth system
Symbols: A - total organic carbon
• - bacteria
o - protozoa
• - soluble organic carbon
29
-------
SECTION VI
REFERENCES
1. Brinley, F. J. Biological Studies, Ohio River Pollution Survey:
I. Biological Zones in a Polluted Stream. Sewage Works Journal
14:147-52, 1942.
2. Bamforth, S. S. Ecological Studies on the Planktonic Protozoa
of a Small Artificial Pond. J. of Limnol. and Oceanog. 3_:39
1958.
3. Alexander, M. Introduction to Soil Microbiology. New York, John
Wiley & Sons, Inc., 1961. 472 p.
4. Sykes, G., and F. A. Skinner. Microbial Aspects of Pollution.
New York, Academic Press, 1971. 289 p.
5. Russell, E. J., and H. B. Hutchinson. The Effect of the Partial
Sterilization of Soil on the Production of Plant Food. Agric. Sci.
3_: 111-44, 1909.
6. Fairbrother, T. H., and A. Renshaw. The Relation between Chemical
Constitution and Antiseptic Action in the Coal Tar Dyestuffs.
J. Soc. Chem. Ind. Lond. (London). 4^:134-44, 1922.
7. Heukelekian, H., and M. Gurbaxani. Effect of Certain Physical and
Chemical Agents on the Bacteria and Protozoa of Activated Sludge.
Sewage Works Journal. 2^:811-17, 1949.
8. Butterfield, C. T. Studies of Sewage Purification. II. A Zoogloea-
forming Bacterium Isolated from Activated Sludge. Public Health
Reports. 5jO:671-84, 1935.
9. Curds, C. R., and G. J. Fey. The Effect of Ciliated Protozoa on
the Fate of Escherichia coli in the Activated Sludge Process.
Water Research. _3:853-67, 1969.
10. McKinney, R. E., and A. Gram. Protozoa and Activated Sludge.
Sewage and Industrial Wastes. 2_8:1219-31, 1956.
11. Pillai, S. C., T. K. Wadhwani, M. J. Gurbaxani, and V. Subrahmanyan.
Relative Efficiency of Bacteria and Protozoa in the Flocculation
and Oxidation of Organic Matter Suspended in Water. Current Science.
_16:340-41, 1947-
12. Pillai, S. C., and V. Subrahmanyan. Role of Protozoa in the Activated
Sludge Process. Nature. 150:525, 1942.
30
-------
13. Kidder, G. W., and V. C. Dewey. The Biochemistry of Ciliates on Pure
Culture. In: Biochemistry and Physiology of Protozoa, Vol. 1,
Lwoff, A. (ed.). New York, Academic Press, 1951. p. 323-400.
14. Wilson, B. W. , and W. F. Danforth. The Extent of Acetate and
Ethanol Oxidation by Euglena gracilis. J. Gen. Microbiol.
L3:535-42, 1958.
15. Prokesova, V., and M. Legner. Interrelations between Bacteria and
Protozoa during Glucose Oxidation in Water. Int. Revue ges
Hydrobiol. _5_l:279-93, 1966.
16. Nikoljuk, V. F. Some Aspects of the Study of Soil Protozoa. Acta
Protozoologica. _7:99-109, 1969.
17. Busch, A. W. BOD Progression in Soluble Substrates. Sewage and
Industrial Wastes. _30:1336-49, 1958.
18. Heal, 0. W. Quantitative Feeding Studies on Soil Amoebae.
In: Progress in Soil Biology, Graff, 0., and J. E. Satchell (ed.).
Amsterdam, North Holland Publishing Co., 1967. p. 120-125.
19. Payne, W. J., and V. E. Feisal. Bacterial Utilization of Dodecyl
Sulfate and Dodecyl Benzene Sulfonate. Appl. Microbiol. 11:339-44,
1963.
20. Standard Methods for the Examination of Water and Wastewater. 12th
Ed. Am. Public Health Assoc., Inc. New York. 1965.
21. Schuster, G. J., and J. W. Vennes. Synchronous Division of
Tetrahymena pyriformis in a Biphasic Medium. North Dakota Academy
of Science. Vol. 14. 1960.
22. Stanier, R. Y., M. Doudoroff, and E. A. Adelberg. The Microbial
World. Englewood Cliffs, Prentice-Hall, Inc., 1970. 873 p.
23. Kerr, P. C., D. F. Paris, and D. L. Brockway. The Interrelation
of Carbon and Phosphorus in Regulating Heterotrophic and Auto-
trophic Populations in Aquatic Ecosystems. U. S. Department of
the Interior, Federal Water Pollution Control Administration.
Washington, D. C. Water Pollution Control Research Series 16050 FGS.
July 1970. 53 p.
24. Lamanna, C., and M. F. Mullette. Basic Bacteriology. Baltimore,
Williams and Wilkins Co., 1959. 853 p.
31
-------
SECTION VII
APPENDICES
A. Data from which General-ion Times of Tetrahymena
pyriformis as a Function of Temperature and
Aeration are Calculated 33
B. Data from which Generation Times of Tetrahymena
pyriformis as a Function of Phosphate Molarity
and pH are Calculated (Experiment 1) 34
C. Data from which Generation Times of Tetrahymena
pyriformis as a Function of Phosphate Molarity
and pH are Calculated (Experiment 2) 35
D. Data from which Generation Times of Tetrahymena
pyriformis as a Function of Bacterial Concentration
are Calculated 36
32
-------
APPENDIX A
Data from which Generation Times of Tetrahymena pyriformis
as a Function of Temperature and Aeration are Calculated.
X = hours; y = protozoa/ml; A,B = duplicate flasks
Experimental
Condition Flask
23°C, Aerated A
B
23°C, Stationary A
B
30°C, Aerated A
B
30°C, Stationary A
B
0
y = 1,250
y = 2,500
y = 750
y = 2,000
y = 3,000
y = 2,750
y = 4,250
y = 5,730
X (Hours)
24
47,500
42,500
26,000
22,500
49,000
49,600
28,000
35,000
48
51,200
71,800
29,600
39,000
48,100
58,700
32,500
39,000
33
-------
APPENDIX B
Data from which Generation Times of Tetrahymena pyriformis
- _t 1 _._ >«-_-! • .t J -_TT /-!« 1 1 « J_« J
Experimental
Condition
5 X 10~2 M
Phosphate
5 X 10~3 M
Phosphate
5 X 10 M
Phosphate
5 X 10~3 M
Phosphate
5 X 10~3 M
Phosphate
5 X 10~3 M
Phosphate
5 X 10~4 M
Phosphate
5 X 10~4 M
Phosphate
5 X 10~4 M
Phosphate
5 X 10~4 M
Phosphate
5 X 10~4 M
Phosphate
of Phosphate Molarity and
(Experiment 1) .
y = protozoa/ml; A, B =
pH Flask
6.0
6.5
7.0
7.5
8.0
6.0
6.5
7.0
7.5
8.0
6.0
6.5
7.0
7.5
8.0
A & B
A & B
A & B
A & B
A & B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
y =
y -
y -
y =
y -
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
y =
0
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
pH are Calculated
duplicate flasks
X (Hours)
24
•NT ,,
M/-v
"KTr\
•NT _
1ST,-.
2,810
1,870
2,340
1,090
2,180
1,870
624
624
156
468
5,620
2,650
4,840
781
468
7,180
12,000
4,530
2,060
36
15,900
53,100
4,210
9,530
6,060
5,310
2,960
2,340
312
937
20,000
17,300
22,900
312
24,800
22,100
26,000
26,300
6,560
48
24,200
18,400
18,500
17,500
14,200
13,700
12,700
18,500
1,560
7,810
-
18,200
31,200
19,600
11,800
26,500
23,500
6,090
34
-------
APPENDIX C
Data from which Generation Times of Tetrahymena pyriformis
as a Function of Phosphate Molarity and pH are Calculated
(Experiment 2).
X = hours; y = protozoa/ml; A, B = duplicate flasks
Experimental X (Hours)
Condition
5 X 10~3 M
Phosphate
5 X 10~3 M
Phosphate
10~3 M
Phosphate
10~3 M
Phosphate
5 X 10~4 M
Phosphate
5 X 10~4 M
Phosphate
10~4 M
Phosphate
10~4 M
Phosphate
£H
7.5
8.0
7.5
8.0
7.5
8.0
7.5
8.0
Flask
A & B
A & B
A & B
A & B
A & B
A & B
A & B
A & B
y =
y =
y =
y =
y =
y =
y =
y =
0
500
500
500
500
500
500
500
500
24 48
-
937 156
310 2,260
1,400 7,500
312 5,070
1,560 2,650
-
2,030
35
-------
APPENDIX D
Data from which Generation Times of Tetrahymena pyriformis
as a Function of Bacterial Concen
X = hours; y = protozoa/ml;
Experiment
Experimental
Condition Flask
5 X 107 bacteria/ml
108 bacteria/ml
2 X 10s bacteria/ml
5 X 10s bacteria/ml
109 bacteria/ml
5 X 107 bacteria/ml
108 bacteria/ml
2 X 10s bacteria/ml
5 X 108 bacteria/ml
109 bacteria/ml
Experimental
Condition
5 X 107 bacteria/ml
108 bacteria/ml
2 X 108 bacteria/ml
5 X 108 bacteria/ml
109 bacteria/ml
A y =
B y =
A y =
B y =
A y =
B y =
A y =
B y =
A y =
B y =
Experiment
A & B y =
A & B y =
A & B y =
A & B y =
A & B y =
Experiment
A & B y =
A & B y =
A & B y =
A & B y =
A & B y =
.tration are Calculated.
A, B = duplicate flasks
X (Hours)
0
500
500
500
500
500
500
500
500
500
500
2
500
500
500
500
500
_3
0
500
500
500
500
500
24 48
10,750
8,750
9,750 2
9,500 3
10,750
9,250 1
14,250 40
13,750 35
15,250 67
14,500 67
2,187 9
4,062 8
6,406 14
7,031 16
8,400 19
X (Hours)
24 36
3,359 8,281
3,437 10,156
4,297 18,593
3,281 29,687
3,828 33,125
750
,750
,000
,250
,750
,500
,000
,750
,687
,437
,531
,563
,062
48
14,062
14,453
22,734
38,906
35,781
36
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Re.n6r.iMfi.
Acc-i-ssian
w
Title
EFFECTS <0F PROTOZOA ON THE FATE OF PARTICULATE CARBON
5. Report Date
,6,, .
7. Authoi(s)
Holm, Harvey W., and Smith, Forrest A.
9. Organization
Southeast Environmental Research Laboratory
National Environmental Research Center-Corvallis
U. S. Environmental Protection Agency
Athens, Georgia 30601
iiig Organization .
ReporiNo.
10, Project No.
310301QPL
!. Canti'fict'!Ginnt "ffo.
'13 Type ,•/ Repofi a
Period Covered
U,
S. Supplemental y Notes
Environmental Protection Agency report number,
n< s> Environmental Protection Agency Final Report
ction Agenc
EPA-660/3-73-007 , August 1973.
16. Abstract
Laboratory studies were designed to define the role of protozoa in the fate of par-
ticulate (bacterial) organic carbon. Specific objectives were (1) to measure the
effects of selected environmental parameters on protozoan growth rates, (2) to measure
organic carbon in bacteria and protozoa, and (3) to quantitate carbon transformations in
predator-prey experimental systems.
A growth system containing 2 X 10s Citrobacter/m-L in 1 x 10"3M phosphate of pH 7.5,
Incubated at 25°C at a shaking rate of 100 rpm, was found to be an optimal environment
for protozoan growth.
The nutrient bacterium, Citrobacter, contained 8.6 X 10"11 mg C/cell, and Tetrahymena
pyriformis contained 1.1 x 10~B mg C/cell.
£. pyriformis altered the amount and form of carbon in the system while growing on
bacteria. Of the total organic carbon present at the initiation of the predator-prey
experiment (93 mg), 93% was in the bacterial fraction. Within 96 hours, 38% of the
carbon was released as COS; 5% was present as inorganic carbon in the water and the
remainder (57%) was present as organic carbon. The organic carbon in the bacterial
fraction decreased from 86 to 2 mg within 96 hours, while the carbon in the protozoan
biomass increased from 1 to 40 mg. In the bacterial control, 117o of the organic carbon
was released as COS within 96 hours while negligible amounts of inorganic carbon
remained in the water. (Holm - Southeast Environmental Research Laboratory)
I7a. Descriptors *protozoa, *Aquatic microbiology, *Aquatic bacteria, *Carbon cycle,
*Cycling nutrients, Carbon dioxide, Aquatic microorganisms, Food chain, Secondary
productivity, Growth rates, Predation.
l~b. Identifiers
*Carbon transformation, Carbon utilization, Population dynamics, Organic carbon,
Citrobacter, Tetrahymena pyriformis.
17c. COWRR. Ficlrl & Group Q5B
18. Availability
19, Security Class.
(Report)
W. Se. '.Tity Ci ',$.
21. No. of
Pages
2. Pi '".M -
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 2O24O
Ab:n';»*•'-,•. Harvey W. Holm
Southeast Environmental Research Laboratory
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