ras

Table 4, GENERATION TIMES (HOURS) OF Tetrahymena pyriformis AS A FUNCTION OF
PHOSPHATE MOLARITY AND pH (EXPERIMENT 2).

Initial Phosphate molarity
HH " : — —
p 5 x 10% 1x 107° M 5 x10* M 1x 10% M
8.0 No growth 12.3 19.9 No growth
7.5 No growth 9.3 10.7 No growth



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 X_ (Hours)
Condition Flask 0 24 48
23°C, Aerated A y= 1,250 47,500 51,200

B y = 2,500 42,500 71,800
23°C, Stationary A y= 750 26,000 29,600
B y = 2,000 22,500 39,000
30°C, Aerated A y = 3,000 49,000 48,100
Bo oy = 2,750 49,600 58,700
30°C, Stationary A y = 4,250 28,000 32,500
B y = 5,730 35,000 39,000

33


100

100

+ 80
2
Ey
Ss [34
g &
2 7 09
zg =
3 i=
ez z
& 2
ro) uw
ro)
7%.
a
rs)
&
a

+ 20

: 0

24 48 72 %6 120
TIME (HRS)
Figure 10. Partitioning of organic carbon in a bacterial.
protozoan growth system
Symbols: 4 - total organic carbon

e@ - bacteria
o - protozoa
@ - soluble organic carbon

29


Total Organic Carbon (TOC) was determined either on 5-mt 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-u2 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 mt, depending on the COg 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 TI. pyriformis in a simple
batch culture, with bacteria, Citrobacter, as the carbon source.

Duplicate test systems consisted of 250-mt Erlenmeyer flasks containing
known initial concentrations of washed Citrobacter and T. pyriformis
brought to a volume of 100 mt 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 equation?*

- logioN, - logyoN,
0.301 t

where N, = population at a given time
N, = population after a time lapse, t

t = time lapse between population measurements (in hours).

The data are tabulated as mean generation time (1/k).


WINDOW

10 a
sof
& |
fea)
= ot
=
2
uu
=
ee
40
s
a
a
20
OO cn a
5.4 ws WS 23.5 29.6
CELL VOLUME (yu?) x 1073
Figure Ll.

Size distribution of Tetrahymena pyriformis

16


APPENDIX B

Data from which Generation Times of Tetrahymena pyriformis
as a Function of Phosphate Molarity and pH are Calculated
(Experiment 1).

X = hours;

y = protozoa/m1;

A, B = duplicate flasks

Experimental X_(Hours)
Condition pH Flask 0 24 36 48

5x 10° M 6.0 A&B ys ----No
Phosphate 6.55 A&B y=

7.0 A&B ye

7.5 A&B yeR

8.0 A&B ye
5x 10° M 6.0 A y= 250 2,810 15,900 24,200
Phosphate B y= 250 1,870 53,100 18,400
5x 10° M 6.5 A y= 250 2,340 4,210 18,500
Phosphate B y= 250 1,090 9,530 17,500
5 x 1o° M 7.0 A y= 250 2,180 6,060 14,200
Phosphate B y= 250 1,870 5,310 13,700
5x 10° M 7.5 A y= 250 624 2,960 12,700
Phosphate B y= 250 624 2,340 18,500
5x 10° M 8.0 A y= 250 156 312 1,560
Phosphate B y= 250 468 937. 7,810
5x 10* M 6.0 A y= 250 5,620 20,000 -
Phosphate B y= 250 - - -
5x 10* M 6.5 A y= 250 2,650 17,300 18,200
Phosphate B y= 250 4,840 22,900 31,200
5x 10% M 7.0 A y= 250 781 312 -
Phosphate B y= 250 468 24,800 19,600
5 x 104 M 7.5 A y= 250 7,180 22,100 11,800
Phosphate B y= 250 12,000 26,000 26,500
5x 10* M 8.0 A y= 250 4,530 26,300 23,500
Phosphate B y= 250 2,060 6,560 6,090

34


Abstract
List of Figures

List of Tables

Acknowledgments

Sections

I Conclusions

IL Recommendations

IIr Introduction

Iv Materials and Methods
Vv Results and Discussion
VI References

Vil Appendices

CONTENTS

iii

30

32


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 COg. 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, 93% of the organic carbon was in the form

of bacterial biomass. By 96 hours, about 3% 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 TOC and organism carbon values. Up

to 20% of the organic carbon may be in soluble form, as calculated from
these data.

27


€1

Table 5. pH STABILITY OF PROTOZOAN GROWTH SYSTEMS AS A FUNCTION OF INITIAL
pH AND PHOSPHATE MOLARITY.

pH’ of medium

pH = — — 2
5 x 10° M PO,= 1X 10° M PO,= 5 x 107* M PO,= 1 Xx 10°* M PO,=
hours hours hours hours
o | 24 | 48 o | 24 48 o | 24 48 o | 24 48
7.5 7.4) 7.4] 7.5 7.0] 7.0] 6.9 6.7] 6.8) 6.9 6.4] 6.7 | 6.6
8.0 7.6 7.4] 7.3 7.2) 7.3} 7.1 6.9} 7.0] 6.9

6.4) 6.8 6.5

* pH of medium before addition of organisms

“pH after addition of Citrobacter and Tetrahymena pyriformis



100

80

60

40

20

8

PM) IN EFFLUENT AIR

80

60

CO, AS CARBON (P

40

20

Figure 2.

wi

Experiment 1

il Protozoa and Bacteria

\

X

acteria

oo OO

e.
#9

Concentration of COg in effluent air

Experiment 2

Protozoa and Bacteria

oN

a
4

~

Bacteria »

60

TIME (HRS)

(recorded as ppm)

Symbols:

@ - protozoa and bacteria

o - bacteria

19

120


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 medium'®.
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 xX 10°+7mg carbon, in agreement with a value calculated from literature
data®* The mean carbon content of a protozoan is 1.1 x 10° Sng.

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 u°).

2. The largest biomass is associated with window 8
(831 protozoa with volumes of 9,060 2°, or a total
biomass of 75.2 xX 10°u*).

/ 884.6 x 10°p° \

- acy
7,907 protozoa ” is 1.1 x 10K".

3. The mean protozoan volume

The weight of organic carbon per unit volume of protozoan biomass cal-
culated from the mean organic carbon/protozoan (1.1 X 10°°mg, Table 7)
and the mean protozoan volume (1.1 x 10*u®, Table 8) is 1 x 10°7ug
carbon/u?. 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 C02 released from
the system over that of the control representing endogenous C0z released
by substrate-limited bacteria (Figure 2). In the first 20 hours, the
major portion of the COg evolved was produced by washed bacterial cells;
after the protozoa started growing, however, they produced significant
amounts of COz. The COzg concentration curve is skewed to the right, prob-
ably reflecting a production of COz 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 COg 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 COz were

15


100 7
100
+ 80
Zz
S
_ a
— &
4 So
2 60 =
> =
8 E
ao 2
2
& 6
4 40
a
So
&
a
20 4 20
10F _
a _— ~
—— L 1 1 0
24 48 2 % 120
TIME (HRS)
Figure 9. Carbon transformation -- bacterial-protozoan system
Symbols: A = total carbon
e@ - total organic carbon
o - COzg

@ - total inorganic carbon

28


ot

Table 2.

pH STABILITY OF PROTOZOAN GROWTH SYSTEMS AS A FUNCTION OF

INITIAL pH AND PHOSPHATE MOLARITY.

pH” of Medium

5 x 107? M phosphate 5 x 10°* M phosphate
pir hours hours

0 24 48 0 24 48
6.0 6.2 6.2 6.3 5.9 6.1 6.0
6.5 6.6 6.5 6.6 6.2 6.3 6.5
7.0 6.9 6.9 6.9 6.5 6.5 6.6
7.5 7.4 7.3 7.3 7.0 6.7 7.0
8.0 7.8 7.8 7.8 7.4 6.9 7.0

*pH of medium before addition of organisms

°pH after addition of Citrobacter and Tetrahymena pyriformis



SECTION IT

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:

e 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.

e@ 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.


10° {10°
2 | 3
= —
u
& 10 { 10 S
is) 7 t=)
a 4 &

107 / 10?

boi iiit

106 fl 1 1 1 1 1 102
0 20 40 60 80 100 120

TIME (HRS)

Figure 7. Growth of protozoa (Tetrahymena pyriformis) with
bacteria as the carbon source (Experiment 2)
Symbols: o - bacterial population with no
protozoa present
e - bacterial population with protozoa
4 - protozoa growing on bacteria

25


6 F Experiment 1
4 fF —
a | oN
Ss
= 2
z ws
oO _—_——o-
a O—_ 0 90 0-9
a
S
an 6 F Experiment 2
<x
rs)
He 4 ~
24 L/L
0
0 NZ
0 20 40 60 80 100 =—:120
TIME (HRS)
Figure 4. Total inorganic carbon (TIC) in the medium
(cecorded as mg carbon/2)
Symbols: e - bacteria and protozoa

o - bacteria

22


TABLES

Generation times (hours) of Tetrahymena
pyriformis as a function of temperature
and shaking

pH stability of protozoan growth systems
as a function of initial pH and phosphate
molarity

Generation times (hours) of Tetrahymena
pyriformis as a function of phosphate
molarity and pH (Experiment 1)

Generation times (hours) of Tetrahymena
pyriformis as a function of phosphate
molarity and pH (Experiment 2)

pH stability of protozoan growth systems
as a function of initial pH and phosphate
molarity

Generation times (hours) of Tetrahymena
pyriformis as a function of bacterial
concentration

Organic carbon content of Tetrahymena
pyriformis and Citrobacter

Size distribution of Tetrahymena pyriformis

10

il

12

13

14

17

18


10

FIGURES

Size distribution of Tetrahymena pyriformis

Concentration of COg in effluent air
(recorded as ppm)

Accumulated carbon emitted as COg
(recorded as mg carbon)

Total inorganic carbon (TIC) in the medium
(recorded as mg carbon/t)

Total organic carbon (TOC) in the medium
(recorded as mg carbon/2)

Growth of protozoa (Tetrahymena pyriformis)
with bacteria as the carbon source

(Experiment 1)

Growth of protozoa (Tetrahymena pyriformis)
with bacteria as the carbon source

(Experiment 2)

Carbon transformation -- bacterial control
Carbon transformation -- bacterial-protozoan
system

Partitioning of organic carbon in a bacterial-

protozoan growth system

iv

20

22

23

24

25

26

28

29


Table 3.

GENERATION TIMES (HOURS) OF Tetrahymena pyriformis

AS A FUNCTION OF PHOSPHATE MOLARITY AND pH

(EXPERIMENT 1).

pH Phosphate concentration

5 x 107° M 5x 104M
6.0 No growth 5.6
6.5 No growth 6.9
7.0 No growth 6.6
7.5 No growth 5.4
8.0 No growth 6.6

11


SECTION I

CONCLUSIONS

Protozoa have various effects on the fate of particulate carbon (bacteria)
in aqueous environments:

e Total Organic Carbon (TOC) of bacterial-protozoan systems
decreases as protozoa metabolize bacterial cells. Within 120 hours,
49-57% of the organic carbon was transformed to inorganic carbon in
systems containing protozoa, whereas 4-17% of the organic carbon was
similarly transformed in systems containing only bacteria.

e Within 120 hours, the bacterial-protozoan system released over
three times as much CO, as the bacterial control.

e 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.

e According to calculations, soluble organic carbon may be produced
in systems containing protozoa.

The protozoan Tetrahymena pyriformis requires 3 x 10* bacteria Citrobacter
(2.6 x 10°° mg carbon) for the production of each protozoal cell con-
taining 1.1 x 10-& 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.


Table 7. ORGANIC CARBON CONTENT OF Tetrahymena pyriformis
AND Citrobacter.

mg organic
Organism Organisms/m2 | TOC, mg/m? | carbon/organism
Citrobacter 1 x 10? 0.086 8.6 x 10°32
T. pyriformis 3.25 x 10° 0.36 1.1 x 107°

17


Ecological Research Series



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 T. pyriformis and the bacterium
Citrobacter; and (3) the types and rate of carbon transformations in

bacterial-protozoan ecosystems.


SECTION IIT

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 facilities* 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 some®*® to believe that
protozoa are harmful because they utilize functional bacteria. Others?9%1°
note that a combination of bacteria and protozoa produces a higher
quality sewage treatment than either population alone. Still others
believe protozoa are the primary agents in the sewage stabilization
process.

11,12

Second, in addition to utilizing bacterial cells, certain protozoa can
metabolize soluble organic compounds’*, Wilson and Danforth'* 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 bacteria’® »1®

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 Busch?” and confirmed by work of Curds, Cockburn, and
Vandyke as presented by Sykes and Skinner* suggests that protozoa exhibit
a significant oxygen demand, presumably for metabolizing the primary
heterotrophs (bacteria). Heal’® 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.


Data from which Generation Times of Tetrahymena pyriformis

APPENDIX D

as a Function of Bacterial Concentration are Calculated.

X = hours;

Experimental
Condition

5 X 10” bacteria/ml

10°

bacteria/ml

10° bacteria/ml

10° bacteria/ml

bacteria/ml

10” bacteria/ml
bacteria/ml

10° bacteria/ml
10° bacteria/ml

bacteria/ml

Experimental
Condition

5 x 10’ bacteria/ml
10° bacteria/ml

2X 10° bacteria/ml
5 X 10° bacteria/ml

10° bacteria/ml

y = protozoa/m1;

Experiment 1

A, B = duplicate flasks

X_ (Hours)

Flask i) 24 48

A y= 500 10,750 -

B y= 500 8,750 750

A y= 500 9,750 2,750

B y= 500 9,500 3,000

A y= 500 10,750 -

B y= 500 9,250 1,250

A y= 500 14,250 40,750

B y= 500 13,750 35,500

A y= 500 15,250 67,000

B y= 500 14,500 67,750

Experiment 2
A&B y= 500 2,187 9,687
A&B y= 500 4,062 8,437
A&B ye 500 6,406 14,531
A&B y= 500 7,031 16,563
A&B ye 500 8,400 19,062

Experiment 3

X_(Hours)
0 24 36 48

A&B y= 500 3,359 8,281 14,062
A&B y= 500 3,437 10,156 14,453
A&B y= 500 4,297 18,593 22,734
A&B y= 500 3,281 29,687 38,906
A&B y= 500 3,828 33,125 35,781



faaewe

10°

_

TT TT Wa
eo
?
PROTOZOA / ML

0,
a No

2.

104

nD:

BACTERIA/ML
3
EAA

po

107 b

RJ N="

E 4
L 4

10° _L r Ll L 1 102
0 20 40 60 80 100 = 120

TIME (HRS)

Figure 6. Growth of protozoa (Tetrahymena pyriformis) with
bacteria as the carbon source (Experiment 1)
Symbols: o - bacterial population with no
protozoa present
e@ - bacterial population with protozoa
A 7 protozoa growing on bacteria

24


80
[ Experiment 1
60 +
40
aN a a ee
a2 a
eo L
=
210 Py 3.
2 to.
= ——a Oy
oO 80 F
vn
2 l \
S 60 | Experiment 2 \
ra
40 - \
20 +
0 f 1 L a 1 mn
0 20 40 60 80 100 = 120 140
TIME (HRS)
Figure 5. Total organic carbon (TOC) in the medium
(recorded as mg carbon/2)
Symbols: e - bacteria and protozoa

o - bacteria


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 CO,-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


Table 6. GENERATION TIMES (HOURS) OF Tetrahymena pyriformis
AS A FUNCTION OF BACTERIAL CONCENTRATION.

Bacteria/mt _ Generation time
5 x 107 8.8
1 x 10° 9.0
2 x 10° 7.9
5 x 10° 8.1
1 x 10° 7.7

14


13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24,

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.

Wilson, B. W., and W. F. Danforth. The Extent of Acetate and
Ethanol Oxidation by Euglena gracilis. J. Gen. Microbiol.
18:535-42, 1958.

Prokesova, V., and M. Legner. Interrelations between Bacteria and
Protozoa during Glucose Oxidation in Water. Int. Revue ges
Hydrobiol. 51:279-93, 1966.

Nikoljuk, V. F. Some Aspects of the Study of Soil Protozoa. Acta
Protozoologica. 7:99-109, 1969.

Busch, A. W. BOD Progression in Soluble Substrates. Sewage and
Industrial Wastes. 30:1336-49, 1958.

Heal, O. 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.

Payne, W. J., and V. E. Feisal. Bacterial Utilization of Dodecyl
Sulfate and Dodecyl Benzene Sulfonate. Appl. Microbiol. 11:339-44,
1963.

Standard Methods for the Examination of Water and Wastewater. 12th
Ed. Am. Public Health Assoc., Inc. New York. 1965.

Schuster, G. J., and J. W. Vennes. Synchronous Division of
Tetrahymena pyriformis in a Biphasic Medium. North Dakota Academy
of Science. Vol. 14. 1960.

Stanier, R. Y., M. Doudoroff, and E. A. Adelberg. The Microbial
World. Englewood Cliffs, Prentice-Hall, Inc., 1970. 873 p.

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.

Lamanna, C., and M. F. Mullette. Basic Bacteriology. Baltimore,
Williams and Wilkins Co., 1959. 853 p.

31


SECTION VII

APPENDICES

Data from which Generation Times of Tetrahymena
pyriformis as a Function of Temperature and
Aeration are Calculated

Data from which Generation Times of Tetrahymena
pyriformis as a Function of Phosphate Molarity
and pH are Calculated (Experiment 1)

Data from which Generation Timesof Tetrahymena
pyriformis as a Function of Phosphate Molarity
and pH are Calculated (Experiment 2)

Data from which Generation Times of Tetrahymena

Ppyriformis as a Function of Bacterial Concentration
are Calculated

32

33

34

35

36


40 +

T

30

ACCUMULATED CO, AS CARBON (MG)

30

10¢

Experiment 1

Figure 3.

20 40 60 80 100 120 8140
TIME (HRS)

Accumulated carbon emitted as C02

(recorded as mg carbon)

Symbols: e - bacteria and protozoa
o - bacteria

20


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 10° Citrobacter/mt in 1 x 107° 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 107*? mg C/cell,
and Tetrahymena pyriformis contained 1.1 x 10°° mg C/cell.

T. pyriformis 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 COg; 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,
11% of the organic carbon was released as COg 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.

ii


SELECTED WATER Report No.| 2. Accession No
RESOURCES ABSTRACTS

INPUT TRANSACTION FORM w
4. Title 5. Report Date
6
EFFECTS OF PROTOZOA ON THE FATE OF PARTICULATE CARBON 8. Performing Orgastization
Report No.
7. Author(s) a
70. Project M1
Holm, Harvey W., and Smith, Forrest A. Project No.

9, Organization 310301QPL
Southeast Environmental Research Laboratory 1h Coutract) Grant Wa,
National Environmental Research Center-Corvallis
U. S. Environmental Protection Agency 12. Type -f Report and
Athens, Georgia 30601 Period Cevered

12, Sponsoring Organixation == sy, gg Environmental Protection Agency Final Report

15. Supplementary Notes

Environmental Protection Agency report number,
EPA-660/3-73-007, August 1973.

15, 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 10° Citrobacter/mt in 1 x 107°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 101? mg C/cell, and Tetrahymena
pyriformis contained 1.1 x 10° mg C/cell.

T. 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 CO,; 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, 11% of the organic carbon
was released as CO, within 96 hours while negligible amounts of inorganic carbon
remained in the water. (Holm - Southeast Environmental Research Laboratory)

17a. Descriptors *protozoa, *Aquatic microbiology, *Aquatic bacteria, *Carbon cycle,
*Cycling nutrients, Carbon dioxide, Aquatic microorganisms, Food chain, Secondary
productivity, Growth rates, Predation.

17b, Identifiers

*Carbon transformation, Carbon utilization, Population dynamics, Organic carbon,
Citrobacter, Tetrahymena pyriformis.

I7c. COWRR Field & Group Q5R

18. Availability 19. Security Class. 21, No.of Send To:
(Repor:) Pages
. WATER RESOURCES SCIENTIFIC INFORMATION CENT!
2. Sexusty C! ss. 2 Prive US) DEPARTMENT OF THe INTERIOR eR
(Page) WASHINGTON, D.C. 20240

=o. Harvey W. Holm I

Southeast Environmental Research Laborator:



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
I. pyriformis, counting each population, and measuring the TOC 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 TOC and size distribution data.

Phase III - Effect of 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®*, 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 10°/mt, and
the two remaining flasks received bacteria (10°/mt) plus washed

T. pyriformis (initial concentration, 10° /mt) .

An incubation system’ was used in which dry, COg-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, TOC 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
(IGE 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.


Table 1.

GENERATION TIMES OF Tetrahymena pyriformis AS A
FUNCTION OF TEMPERATURE AND SHAKING.

Generation time (hours)

Temperature Shaking (100 rpm) Stationary
25°C 9.4 10.1
30°C 11.4 16.8



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


10.

ll.

12.

SECTION VI

REFERENCES

Brinley, F. J. Biological Studies, Ohio River Pollution Survey:
I. Biological Zones in a Polluted Stream, Sewage Works Journal.
14:147-52, 1942.

Bamforth, S. S. Ecological Studies on the Planktonic Protozoa
of a Small Artificial Pond. J. of Limnol. and Oceanog. 3:398-412,
1958.

Alexander, M. Introduction to Soil Microbiology. New York, John
Wiley & Sons, Inc., 1961. 472 p.

Sykes, G., and F. A. Skinner. Microbial Aspects of Pollution.
New York, Academic Press, 1971. 289 p.

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.

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). 41:134-44, 1922.

Heukelekian, H., and M. Gurbaxani. Effect of Certain Physical and
Chemical Agents on the Bacteria and Protozoa of Activated Sludge.
Sewage Works Journal. 21:811-17, 1949.

Butterfield, C. T. Studies of Sewage Purification. II. A Zoogloea-
forming Bacterium Isolated from Activated Sludge. Public Health
Reports. 50:671-84, 1935.

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.

McKinney, R. E., and A. Gram. Protozoa and Activated Sludge.
Sewage and Industrial Wastes. 28:1219-31, 1956.

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.

Pillai, S. C., and V. Subrahmanyan. Role of Protozoa in the Activated
Sludge Process. Nature. 150:525, 1942.

30


Table 8. SIZE DISTRIBUTION OF Tetrahymena pyriformis.

Average cell volume Protozoa/window Biomass /window

Window (uw?) (0.73 mt) (Tp)

1 604 - -

2 1,812 - -

3 3,020 - -
4 4,228 419 17.7 x 10°
5 5,436 797 43.3 x 10°
6 6,644 887 58.9 X 10°
7 7,852 936 73.5 x 10°
8 9,060 831 75.2 x 10°
9 10,268 719 73.8 x 10°
10 11,476 556 63.8 x 10°
ll 12, 684 485 61.5 x 10°
12 13,892 435 60.4 x 10°
13 15,100 342 51.6 x 10°
14 16,308 298 48.5 x 10°
15 17,516 244 42.7 x 10°
16 18,724 209 39.1 x 10°
17 19,932 143 28.5 x 10°
18 21,140 137 28.9 x 10°
19 22,348 89 19.8 x 10°
20 23,556 121 28.5 x 10°
21 24,764 66 16.3 x 10°
22 25,972 81 21.0 x 10°
23 27,180 35 9.5 xX 10°
24 28,388 56 15.9 x 10°
25 29,596 21 6.2 x 10°
7,907 884.6 x 10°
protozoa/ u2/0.73 mt

0.73 mt

884.6 x 10° uy 3
Mean protozoal volume = —ooooooeoooC—iaS XX 10* pf
7.9 X 10° protozoa

18


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 formaticn,
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.


120 -
r oo 4120 Z
100 F a a
rN > s
a ea 7100S
a g0 F =<
2 | } a0 5
Z 60 + %
: /°:
S ag b a)
40 440 &
| a
20 F + 20
L ee ee
0 peop * :— 0
24 48 72 % 120
TIME (HRS)
Figure 8. Carbon transformation -- bacterial control

Symbols: 4 - total carbon
e - total organic carbon
o = C02
@ - total inorganic carbon

26


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 pH Flask 0 24. 48

5x 10° M

Phosphate 7.5 A&B y= _ 500 - -

5x 10° M

Phosphate 8.0 A&B y= 500 937 156

10° M

Phosphate 75 A&B yes 500 310 2,260

10° M

Phosphate 8.0 A&B y= 500 1,400 7,500

5x 104 M

Phosphate 7.55 A&B y= 500 312 5,070

5 x 10* M

Phosphate 8.0 A&B y= 500 1,560 2,650

10“ M

Phosphate 7.5 A&B yes= 500 - -

10 * M

Phosphate 8.0 A&B yes= 500 2,030 -

35


produced by the growth of 39 x 10° protozoa, a production of 0.7 x 10°
mg C as COg 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), 83% of the initial
organic carbon remained in the bacterial control flask, compared to 51%

in systems containing protozoa. In experiment 2 (unstored samples), 96%
and 43% 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 107° mg bacterial carbon was required

to produce protozoan biomass of 1.1 x 10 °mg, representing a 42%
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
Heal**, the amount of carbon solubilized by protozoa was estimated:

carbon solubilized/protozoa = bacterial carbon ingested/protozoa
- (COg-C produced/protozoa + organic carbon/protozoa)
= 2.6 x 10°Smg - (0.7 x 107Smg + 1.1 x 107mg)
= 0.8 x 107-°mg/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, 12% of the total carbon was present
as atmospheric COz, whereas less than 1% was in the form of TIC in the
medium.

21


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's’TM medium at 25°C for 48 hours, washed twice with dilution

2 r ; . .
buffer®°, and resuspended in the appropriate medium for experiments.

An axenic species of protozoa commonly used for laboratory research’,
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%, 1%)? 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-,, 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-uy aperture tube, was used
for biomass determinations.

Analytical Procedures

The Beckman Model 915 Total Organic Carbon Analyzer was used for Total
Organic Carbon (TOC), Total Inorganic Carbon (TIC), and carbon dioxide
(COz) determinations. All analyses were made in duplicate.

5


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 buffer®°. A concentration of
2 x 10° bacteria/mt 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 “10°
bacteria/mt. 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 10°*M phosphate system
was significantly changed by addition of the organisms, whereas the pH
of the 5 x 10°°M phosphate systems was not.

The protozoan did not grow in 5 x 10°°M phosphate, but did in 5 x 107*M
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 10°*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°°M phosphate, pH 7.5. Under these
conditions, the pH of rhe system remained near 7.0 for the duration of
the study (Table 5). « pyriformis did not grow in either 5 x 107 Su

or 1 x 10°*M phosphates

The effect of bacterial concentration on the protozoan population was
tested in a shaking system at 25 °c containing 1 x 10° bavt phosphate at

pH 7.5, 5 x 10° protozoa/mt, and varying concentrations of bacteria.

Data (Table 6) show that the concentration used in all of the initial
experiments (2 x 10°/mt) does not limit the growth rate, and was therefore
a suitable choice for this work. Concentrations as high as 10°/mt did
not adversely affect the growth kinetics of the protozoa.