Water Pollution Control Research Series 16050 FGS 07/70
The Interrelation of Carbon and Phosphorous
in Regulating Heterotrophic and Autotrophic
Populations in Aquatic Ecosystems
Department of the Interior*Federal Water Quality Administration
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THI INTERRELATION OF CARBON & PHOSPHORUS EN REGULATING HETEROTROPHIC
AND AUTOTROPHIC POPULATIONS IN AQUATIC ECOSYSTEMS
Pat C. Kerr, Doris F. Paris, and
D. L. Brockway
U. S. Department of the Interior
Federal Water Quality Administration
Southeast Water Laboratory
National Pollutants Fate Research Program
Athens, Georgia 30601
For sale by the Superintendent of Documents, U.S. Government Printing Oifiee
Washington, D C., 20402 - Price 60 cents
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Preface
The research findings presented in tnis report were presented at the
Purdue Industrial Wastes Conference on May 6, 1970. These results,
along with other publicity concerning the research at FWQA's Southeast
Water Laboratory, have been utilized by some individuals and groups
outside FWQA to support a contention that carbon rather than phosphorus
or nitrogen is the limiting nutrient in the control of excessive algal
blooms, a distortion of the conclusions of this research. Interestingly,
most of these arguments were raised well before the research data and
results became available at Purdue.
The research reported here does not alter FWQA's position with regard
to the importance of reducing phosphate discharges to the environment.
The research findings assist in elucidating the highly complex inter-
relationships of the various algal and bacterial nutrients and, if any
single action conclusion were to be drawn, it would be that even more
stringent controls should be placed on carbonaceous (BOD) discharges
than at present. The findings in no way suggest that any less attention
should be given to phosphates and, in fact, additional evidence is
presented that phosphorus plays an important part in stimulating
bacterial growth in water in addition to its role as an algal nutrient.
The FWQA advocates the highest degree of nutrient control necessary
for each receiving water so as to achieve the desired ecological balance.
Research at the Southeast Water Laboratory pertains to the "fate" of
various materials in the aquatic environment and involves, among other
things, the mechanisms of interaction between decomposing organic matter
and the stimulating effect of this decomposition on algae in the
overlying water. The symbiotic relationship between algae and bacteria
is a phenomenon recognized by biologists for many years.
The findings reported are preliminary in that they result from limited
laboratory and field experiments. At this time we cannot say, scienti-
fically, how typical or atypical were the situations studied but it is
unwarranted to extrapolate these results to waters other than those
studied. The research on the role of carbonaceous materials in aquatic
ecosystems is being continued in coordination with the work of FWQA's
National Eutrophication Research Program.
David G. Stephan
Assistant Commissioner
Research and Development
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ABSTRACT
Laooratory and field investigations were conducted on the fate and
cycling of carbon and phosphorus in selected aquatic ecosystems.
Inorganic carbon, as C02, supplied by both bacterial cultures and
cylinder gases, stimulated the growth of the blue-green alga Anacystis
nidulans. The carbon requirement (10"5 ug C02 per cell) for this alga
was determined for a single set of experimental conditions. The
addition of C02 to natural water low in phosphorus ( 5 ug P) and
nitrogen ( 5 ug N) in the laboratory stimulated the growth of indigenous
algal populations. The limiting and luxury cellular concentrations of
phosphorus for starved Anacystis nidulans were found to be 0.3 x 10 ug P
and 3.0 x 10 ug P per cell, respectively.
Diel studies of a stream which received biologically-treated sewage
demonstrated that the dissolved C02 and HC03 continually produced in the
system were essentially depleted by the autotrophic organisms during
daylight hours, while the cpncentration of phosphorus (1.3-2.2 mg/1 P)
remained unchanged.
Addition of organic carbon and inorganic nitrogen and phosphorus alone
and in combination to the waters studied directly stimulated the oxidative
metabolism of the heterotrophic population, which resulted in increased
dissolved C02 and HC03". This increased availability of inorganic carbon,
rather than the direct metabolic removal of dissolved phosphorus by the
algae, appeared to be directly responsible for the growth of the algal
populations in the waters studied.
ii
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TABLES
No.
1 GROWTH OF AnaayeUs nidulans AT DIFFERENT C02 LEVELS
IN THE STANDARD &CDIUM WHICH INCLUDES 2 x 10 '3 M
K2HW4 AND 2 x lO'2 M KN03. ~~ 21
2 SURFACE WATER QUALITY OF LAKE HARWELL NEAR CLEMSCN,
SOUTH CAROLINA. 22
S CO, PRODUCTION BY HETEROTROPHIC POPULATION FOLLOWING
ADDITIONS OF ORGANIC CARBON AND/OR INORGANIC
FERTILIZERS TO A STERILIZED, INFERTILE POND WATER. 31
4 GROWTH OF INDIGENOUS BACTERIAL AND ALGAL POPULATIONS
IN AN INFERTILE POND WATER ( 5 ppb each of N and P)
FOLLOWING ADDITIONS OF ORGANIC CARBON AND/OR INORGANIC
FERTILIZER. 32
iii
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FIGURES
PAGE
I REMOVAL OF u-14 C- GLUCOSE AND u-14C-ACETATE FROM TOE
MEDIA BY ASACJSTIS SIDULANS. GLUCOSE MEDIUM AT pH 6.8.
ACETATE MEDIUM AT pH 4.7. 14
2 GROWTH OF ANACJSTIS SIDULASS IN THE PRESENCE OR ABSENCE
OF A CULTURE OF ESCSERICHIA COLI. 15
3 SCHEMATIC OF APPARATUS USED TO DETERMINE THE INFLUENCE
OF ANY VOLATILE MATERIAL PRODUCED BY A BACTERIAL
CULTURE ON THE GROWTH OF AN ALGAL CULTURE. ALL
CONNECTING TUBING WAS 1/4" ID TYGON. 17
4 C02 REMOVAL BROM THE GAS STREAM DURING ONE CELL CYCLE
OF ASACISTIS NIDULANS. 18
5 GROWTH OF ANACISTIS NIDULANS UTUIZING BACTERIALLY
PRODUCED C02 AS THE ONLY CARBON SOURCE. 19
6 INCORPORATION OF PHOSPHORUS BY ANACISTIS NIDULANS IN
P04-MEDIUM. 25
7 UTILIZATION OF STORED PHOSPHORUS DURING GROWTH OF
ANACISTIS ilDULMS IN TRIS MEDIUM. 26
8 GROWTH OF MACISTIS NIDULANS IN SHRINER'S POND WATER. 28
9 BUFFERING CAPACITY OF 100 ml SHRINER'S POND WATER WITH
OR WIIHOUT ADDED 20-20-5 FERTILIZER. 29
10 DIEL FLUCTUATIONS OF OXYGEN IN WATER OF LAKE LANIER AT
STATION 2 ON JULY 30 AND 31, 1969. 34
11 DIEL FLUCTUATIONS OF NITROGEN IN WATER OF LAKE LANIER
AT THE 1-ft DEPTH OF STATION 1 ON JULY 30 AND 31, 1969. 35
12 DIURNAL FLUCTUATIONS OF C02, HC03~, C03", PO/, AND
ALGAE IN THE WATER OF LAKE LANIER AT THE 1-ft DEPTH OF
STATION 1 ON JULY 2, 1969. 36
13 DIURNAL FLUCTUATIONS OF HCO,", 0)3*, P04~, AND ALGAE
IN THE WATER OF LAKE LANIER AT THE 1-ft DEPTH OF
STATION 4 ON JULY 2, 1969. 37
14 DIEL FLUCTUATIONS OF BACTERIA IN THE WATER AND SEDIMENTS
OF LAKE LANIER AT STATION 1 ON JULY 30 AND 31, 1969. 39
15 DIEL FLUCTUATIONS OF BACTERIA IN THE WATER OF LAKE
LANIER AT STATION 4 ON JULY 30 AND 31, 1969. 40
Iv
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PAGE
16 DIEL FLUCTUATIONS OF DISSOLVED C02 IN THE WATER OF LAKE
LANIER AT THE SAMPLING DEPTHS OF STATION 2 ON JULY 30
AND 31, 1969. 41
17 DIURNAL FLUCTUATIONS OF C02 AND HC03" IN LAKE LANIER
WATER AT THE 1-ft DEPTH OF STATION 1 ON JULY 2 AND 15,
1969. 42
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INTRODUCTION
The increasing demands to control water pollution, to curb eutrophication,
and to develop rational predictive models for water quality management
require a fuller quantitative and qualitative understanding of the funda-
mental biological, chemical, and physical reactions and interactions occurring
in natural waters.
The research described in this paper is a portion of the work being done
by the National Pollutants Fate Research Program at the Southeast Water
Laboratory designed to study the cycling of carbon, phosphorus, and nitrogen
through aquatic ecosystems.
Algal blooms frequently accompany cultural enrichment of waters. Increased
amounts of available nitrogen and phosphorus have been suggested as the
cause of these blooms, Although the importance of carbon in regulating algal
growth has long been known, it received little attention until recently
(Kuentzel, 1969). Increased supplies of carbon (as well as nitrogen and
phosphorus) had to be available to support the increased algal growth asso-
ciated with enriched waters.
The relative chemical composition of living organisms demonstrates the
importance of carbon as a nutrient. Carbon accounts for 50-77% of the dry
weight of algae and 45-55% of the dry weight of bacteria.
In aquatic ecosystems, autotrophs fix and transform inorganic forms (002
HCOi") of carbon from the environment into organic compounds, and through
oxidation, heterotrophs return portions of this carbon to the environment
as C02. Essential to the functioning of this cycle are phosphorus, nitrogen,
sulfur, and other nutrients which are required by both the autotrophic and
heterotrophic organisms.
The growth of individual species of organisms depends on the availability
of specific utilizable forms of carbon in the system. Bacteria take up and
utilize most forms of organic carbon. While most algae can take up and
transform organic carbon, they grow (divide) only on C02 and H003~2, Although
the literature concerning algae growth on organic substrates is conflicting,
Allen (1952) , Hoare and Moore (1965) , Pearce and Carr (1967) , and others
report uptake but no increased cell division (when compared to an inorganic
control) in axenic cultures of algae grown on organic media. Also, there is
no evidence that algae can utilize their intracellular carbon compounds to
support growth.
In general, the availability of any ionic species of carbon in an aquatic
ecosystem is dependent upon: 1) direct additions and losses resulting from
flow of the water mass; 2) exchange between sediments and the water and
atmosphere; 3) chemical reactivity in the water; 4) removal or release by
biological activity on soluble compounds, sediments, or other organisms;
5) photolysis of compounds and autolysis of organisms in the water; and
6) secretion, excretion, and exchange phenomena within the biota. The
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availability of carbon or other growth nutrients depends also upon physical
parameters such as temperature, light, and pH.
Foremost, and least understood, among all factors affecting the carbon
cycle are the rates of supply and demand. The literature concerning the
rates of chemical equilibria between the carbonates, water, and atmospheric
002 is devoid of absolute rate coefficients, with most researchers resorting
to adjectives such as fast, relatively fast, or slow. Also, the rates
of replenishment of the required ionic species to the enzymatic sites of
the organisms, by diffusion or mass transport, are poorly understood and
ill defined. However, many of the field studies reported in the literature
demonstrate that the rates of supply of carbon were insufficient to meet
the demands of the autotrophic populations studied.
The quantity of CX>2 exchanged at the air-water interface depends not only
upon the transfer tate but also upon the concentration of 0)3 in air and
water. The saturation concentration in water in equilibrium with normal
air ranges from 0.4 to 1.0 mg (X>2/liter water, depending upon the pH and
buffering capacity.
For terrestrial plants, Quinn and Jones (1936), Brix (1968), Johnson and
Kelley (1969), and others have shown that C02 is the primary factor limiting
their potential growth, even though there is a relatively constant source
of. 002 in the atmosphere (0.6 mg 002/liter air).
In the aquatic system, Birge and Juday (1911) measured the complete removal
of 002 •** severe depletion of HCOs" and COs" from soft, medium, and hard
water lakes of Wisconsin in 1904-1906. They also stressed the importance
of bacterial oxidation of organic material as a source of 002. Pearsall
(19S2) found that in the English Lake District, richer populations of
planktonic diatoms occurred in water containing more CaCOs. Chu (1942)
reported that growth of Pediastmm boryantm increased with increasing
carbonate content up to 22 ppm. The more soluble K2C03 was preferable to
CaC03 as a source of carbon.
Some of the most definitive work is that of Myers (1944) , who studied the
effect of 002 and pH on the growth of Chlorella pyrenoidoea. He found
002 to be fcrowth-limiting even at the tow light intensity of 100 foot
candles and stated:
"The culture obtaining its carbon dioxide by diffusion through
the cotton plug was clearly limited in its rate of growth. A
still greater relative limitation might be expected at higher
light intensities."
Myers further stated:
"The oily carbon dioxide pressure of significance is that existing
within the liquid medium and available to the cells. During
photosynthesis, this carbon dioxide pressure at the surface of
the algal cell can never be as great as that in equilibrium with
the gas phase since a diffusion gradient must always exist....
It is clear that the carbon dioxide pressure within the medium
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must be a complex function of the partial pressure in the gas
phase, the area of the liquid-gas interface, the rate of aerationt
and the rate of uptake of carbon dioxide by the algae. Even in
any one culture in which the first three of the above factors are
held constant, the multiplication of the algae must result in
increasing rates of uptake of carbon dioxide, thus continuously
decreasing the pressure of carbon dioxide within the media. The
effective carbon dioxide concentration must fall off continuously
during the growth curve."
Myers (1947) and Kratz and Myers (1955) reported other data stressing the
importance of C02 in regulating the growth of algae.
Allen (1955) showed the algae in sewage oxidation ponds were utilizing
C02 rather than organic carbon for growth and that this CC^ was produced
by heterotrophic activity.
Wright and Mills (1967) and Wright (1969) reported a downstream reduction
in both primary productivity and macrophyte standing crop on the Madison
River. The downstream decline in productivity was correlated with succes-
sively lower concentrations of free C02. The decline in free (X>2 was due
to photosynthetic removal during the day and to outward diffusion of C02
during both day and night.
In an estuarine environment, Kelley (pers. comm., 1970) has measured changes
in pCC>2, in a dense eel grass (Zoetera) lagoon near Cold Bay, Alaska, during
the summer and fall. At low tide during the summer the water was hyposaturated,
and at high tide the water was nearly saturated with C02. In the fall,
when the eel grass was reaching normal dormancy, the water was super-
saturated with CO?. The pH of the water varied between 6.5 and 11.5 in
summer and 7.8 and 8.3 in the fall.
Evidence in the literature also relates qualitative as well as quantitative
changes in aquatic plant communities to C02» Wright (1969) postulated
that the relative amounts of CC>2 and HC03~ are of importance in control
of species distribution of higher plants as well as the production of biomass.
Bristol's (1969) data indicated that the concentration of 002 is important
not only in regulating the production of biomass but also in controlling
morphogenic response of the heterophyllous amphibious species, Ranunauliia
fldbellofia and Myriophyllim braeilenee. Neither of these species is capable
of utilizing HCO*". Bristow concluded that a concentration of 002 higher
than air saturation may be essential for the normal growth and development
of submerged amphibious plants. Martin et al. (1969) showed that HC03"
content of Pickwick Reservoir was important in regulating the growth of
a rooted aquatic weed, Najas ap.
Data from several studies of artificial fertilization of ponds and lakes
indicate the important role of carbon in the growth of algae. Thomaston
and Zeller (1961) reported that the addition of inorganic nitrogen and
phosphorus fertilizer to fish ponds did not always result in increased
phytoplankton populations. They found plankton growth controlled by the
availability of C02.
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Walker (1949) added lime and cottonseed meal to ponds receiving
swamp drainage (pH 5.2) which contained no carbonates. In the pond
receiving the lime and cottonseed meal, a very thick green bloom was
produced, while in the unlimed ponds, which also received cottonseed meal,
only a very light bloom was produced.
Neess (1946), reviewing the literature from Central Europe, emphasized the
importance of an environment suitable for heterotrophic activity which
would allow rapid cycling of nutrients in fish ponds. He reported that
the water quality of moor ponds (low pH, methane, F^S) inhibited bacterial
activity, resulting in peat accumulations and reduced productivity.
Additions of lime to previously unfertilized and very unproductive moor
ponds with muck bottoms resulted in immediate and phenomenal increases in
yield of phy top lank ton.
Neess also reported literature stressing the importance of the bottom soil
and the additions of organic materials to fish ponds in regulating fish
production, and he stated:
"Plant debris is of particular importance in ponds where
reliance is placed only on inorganic mineral fertilizers."
Neess stated further:
"Cottonseed or soybean meals (C/N=40) have been found to be
unusually effective fertilizers (Embody and Sadler, 1934; Hogan,
1933; unpublished observations of Neess and Hasler at Madison,
Wisconsin) applied either alone or in conjunction with phosphorus."
Waters (1957) found that additions of lime to bog lakes in Northern
Michigan resulted in higher HCC>3~ concentrations, thus increasing G02
utilization by the nanoplankton and producing immediate increases in
phytoplankton yield.
Swingle (1947) reported that C02 is a limiting factor in fish production.
He was able to increase fish yield by the addition of organic material
with and without the addition of inorganic fertilizer. He stated that
addition of stable manure increased fish production 43 Ibs/acre per ton
of manure. Addition of 200 Ibs of superphosphate to the manure increased
production 4.5 Ibs above that for manure alone. He reported the work of
Burr and others demonstrating that algae attain their maximum efficiency
at much higher 002 tensions than normally occur in natural waters.
Swingle emphasized the importance of buffering capacity in regulating
availability of 002. Addition of lime with inorganic fertilizers or with
small amounts of organic material can inhibit fish production. The author
attributed this inhibition to competition for 002 between the lime and the
phytoplankton. Addition of lime with large amounts of organic material
resulted in the growth of extensive phytoplankton populations; in this case,
heterotrophic production of CC$ was sufficient to overcome the reaction
with calcium, allowing more C02 and HC03* to be available to the algae.
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Swingle discussed the importance of bacteria in nutrient cycling and stated
that the bacterial populations were capable of completely utilizing the
nitrogen and phosphorus available in the water under certain conditions.
The phosphorus content of ponds ana lakes has long been of interest because
phosphorus has been considered a limiting factor in the production of algae
and, hence, of fish in many natural waters. One of the early studies of
the phosphorus and nitrogen content of natural waters was made by Juday
and Birge (1931) who, in the 1920's, determined the nitrogen and phosphorus
content of 479 lakes in Wisconsin. The arithmetic mean of the entire set
of phosphorus determinations was 0.023 mg of phosphorus/liter; the average
soluble phosphate content was found to be 0.003 mg of phosphorus/liter;
and the average organic phosphorus content was 0.020 mg of phosphorus/liter.
In another study of the phosphorus content of a pond, Hutchinson (1941)
reported the overall mean content of Linsley Pond as 0.021 mg phosphorus/liter,,
Mackereth (1953) stated that measurements of dissolved pnosphate in Lake
Windermere have shown the concentration of phosphate phosphorus to vary
from below 1.0 mg phosphorus/liter to a maximum of 2 mg phosphorus/liter
However, Mackereth said it bore no relationship to the size of the popu-
lation of Aaterianella formoaa, the dominant algal species in this lake.
Rigler (1956), from his observations of the phosphorus cycle in the waters
of Toussaint Lake, estimated that 62-651 of the phosphorus is found as
paniculate organic bound and only 15-301 as soluble organic. He also
found that fractions previously described as soluble may actually be asso-
ciated with particles between 0.1 u and 0.45 u.
\j
Rigler used ^P tracer to demonstrate that both attached and suspended
bacteria added to autoclaved lake water utilized inorganic phosphorus for
growth. Rigler concluded that the growth of large bacterial populations
must be an important factor in the removal of phosphate from solution.
Hayes and Phillips (1958) showed that uptake of phospnorus by bacteria and
higher aquatic plants prevented the elements from being incorporated into
lake sediments. They concluded that bacteria are more efficient than higher
plants in removing phosphorus from water and actually keep the phosphorus
available in the water column under both aaarobbcc and aerobic conditions
When the bacteria were killed by antibiotic treatment, only 101 of the 3Z'
was found in the water after one week; while in the control, 661 of the
*2p remained in the water. Earlier work reported by Hutchinson and Bowen
(1950) on Linsley Pond showed the same general phenomenon.
Rigler (1956) stated that bacteria and algae take up significant amounts
of phosphate from solution. Using 32p as a tracer, he found that in 20
minutes an equilibrium had been attained, with approximately 931 of the 32p
taken up by plankton. The plankton were separated into bacterial and algal
fractions by filtering the culture through Nos. 10 and 20 bolting cloth,
No. 2 Whatman paper and a Millipore filter. He found that 981 of the algae
had been removed by the Whatman paper, and they contained 21.3% of the 3zp.
The Millipore filter removed the bacteria and the other 21 of the algae and
contained 68.4% of the 32p. Since the total phosphate concentration in solu-
tion remained 0.8 ug/liter throughout the experiment, he concluded that the
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incorporation of 32P represented turnover rather than uptake. He reported
turnover times of 3.6 and 5.4 minutes for two similar experiments. From
these experiments, Rigler concluded that the bacteria are largely responsible
for thfi turnover of inorganic phosphate and thus, "might be competing with
the algae for the inorganic phosphate".
Bacteria are reported to contain a larger percentage of phosphorus on a
dry weight basis than algae. McKinney (1962) stated that bacteria contain
2.5* phosphorus and Taylor (1946) reported 2.721 phosphorus for B strain
E. ooli. Sanders (pers. comm.) found the phosphorus content of suspended
stream bacteria ranged from 1.07 to 2.02%; that of attached bacteria ranged
from 1.5 to 3.11 phosphorus.
Bacteria cells not only contain small amounts of phosphorus, but also
apparently do not increase their uptake of this element when the phosphorus
concentration in the medium is increased.
Levin (1963), using 30% raw sewage, 1.5mM sodium succinate/liter, 0.5mM
glucose/liter, and varying concentrations of KfyPCty, found that regardless
of initial concentration, the absolute amount of dissolved orthophosphate
taken up by the bacteria was remarkably similar. Within the concentration
range of approximately 5 - 33 mg of dissolved orthophosphate/liter the rate
and amount of uptake were independent of concentration.
Porcella (1969) reported approximately 10'1° mg of phosphorus utilized
per cell of Selenastmm capricornutwn. Al Kholy (1956) reported a maximum
phosphorus content of 1.50 x 10"6 ug and a minimum content of 0.10 jc 10~6 ug
phosphorus per cell of Chlorella. He found no growth below this minimum
concentration. Mackereth (1953) found that 1 ug of phosphorus supported
the growth of 16 x 106 cells of Asterionella formoea and that the limiting
amount was 0.06 ug phosphorus per 106cells.
Both Al Kholy (1956) and Mackereth (1953) demonstrated phospnorus uptake
and storage by algae from external concentrations of 1 ug/liter or less.
This accumulated phosphorus was used to support growth and cell division
in the absence of an external supply of the element.
Batterton and van Baalen (1968) reported the normal level of pnosphorus
in Anaoystis nidulana was approximately 3.7 ug Pi/mm3 cells. This value
fell to 0.5 ug Pi/mm3 cells under prolonged phosphorus starvation. Even
at low cellular phosphate levels, cells were viable and continued to divide
slowly.
Goldberg, Walker, and Whisenand (1951) demonstrated that the size of the
population of Asterionella japoniea (a marine diatom) was the same, when the
cells were grown in 2.7, 4.2, 8.4, or 12.8 ug-atoms phosphorus/liter. This
alga also exhibited luxury consumption of phosphorus and was capable of
dividing in the absence of an external supply. These data indicated
that the final phosphate content of the diatoms and the maximum cell popu-
lation were essentially independent of the initial inoculum and the initial
phosphate level.
Ketchum (1939), Chu (1942); Lund (1950), Hunenik and Hanna (1969); and
others provide data indicating comparably small amounts of phosphorus
required for the growth of
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Plants require large amounts of carbon and are incapable of growing on
their cellular carbon compounds. Conversely, plants require small amounts
of nitrogen and phosphorus and possess the capability of growing on cellular
nitrogen and phosphorus compounds.
Our literature review indicates:
(1) Removal_of all the C02 and severe depletion of the HOOs"
and C03~ in soft and hard water lakes has been attributed
to green plants,
(2) Additions of nitrogen and phosphorus to eome waters do not
stimulate algal growth. These waters have been shown to
contain little available C02 and "
(3) Bacterial oxidation of organic carbon is a principal
source of C02 in aquatic ecosystems.
Since many industrial and domestic wastes include organic material, increased
organic carbon as well as nitrogen and phosphorus has been added to the
receiving water. Addition of organic carbon should stimulate the hetero-
trophic population, thus resulting in the production of more CO? which could
be utilized by the aquatic plants. Because of their generally faster metabolic
rates, growing populations of heterotrophic microorganisms may need more
nitrogen and phosphorus than autotrophic organisms. If this assumption is
correct, addition of nitrogen and/or phosphorus to some waters should stimulate
the heterotrophic oxidation of organic carbon.
This report includes some of our research findings to date on the relation-
ships of carbon, nitrogen, and phosphorus in regulating the growth of
heterotrophic and autotrophic populations in aquatic ecosystems.
Emphasis has been on the roie of carbon and/or nitrogen plus phosphorus in
regulating the growth of an axenic culture of the Blue -Green alga, Anaeyatia
nidulone, of isolated heterotrophic populations in sterile media, and of
mixed natural populations of heterotrophs and autotrophs in non-sterile media.
For clarity and ease of presentation in this report, the work has been
divided into eight phases:
Phase I: Stimulation of Algal Growth by Bacteria.
Phase II: C02 Requirement of Anocyatia nidulona.
Phase III: Stimulation of Algal Growth by 002.
J^hase IV: Phospnorus Requirements of Eacherichia ooH and Anaayatis
Phase V: Regulation of the Growth of Anooyatia nidulona by addition
of Calfoon and/or Nitrogen plus Phosphorus to Sterilized, Infertile Pond Water.
Phase VI: Regulation of Growth of Natural Heterotrophic Organisms by
Addition of Carbon and/or Nitrogen plus Phosphorus to Sterilized, Infertile
Pond Water.
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Phase VII: Regulation of the Growth of Natural Populations of
Heterotrophs and Autotrophs by the Addition of Carbon and/or Nitrogei
plus Phosphorus to Nonsterile, Infertile Pond Water.
Phase VIII: Growth of Heterotrophic and Autotrophic Peculations
and Nutrient Cycling in an Enriched Lake.
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MATERIALS $ METHODS
Organisms
Axenic cultures of the Blue-Green alga, Anacystis nidulans (Starr Culture
t625) were obtained from the University of Indiana Algae Collection.
Natural populations of algae were obtained from Lake Hartwell, South
Carolina, and Shriner's Pond near Athens, Georgia.
Eaeherichia coli was obtained from W. J. Payne, University of Georgia
Natural populations of heterotrophs were isolated from the water of Shriner's
Pond, Oconee River, and Sycamore Valley Lake, Athens, Georgia.
Growth Media
rhe standard algae medium, pH 6.8, used in the laboratory was that of Allen
and Arnon (1955) containing Arnon AS micronutrients. Unless otherwise
indicated E. ooli were cultured in the basal salts medium of Payne and Feisal
(1963), with either 0.25% or II glucose as the carbon source.
Isotopes and Reagents
Carrier-free 2pQ4 Was obtained from Mallinckrodt/Nuclear.* Specific
activity was calculated by dividing the disintegrations/minute/liter by the
ug phosphorus/liter.
All chemicals were reagent grade except the commercial fertilizer. Commercial
fertilizer components were obtained from the Royster Fertilizer Company,
Athens, Georgia, and mixed according to the formulation for 20-20-5 fish pond
fertilizer. This mixture was used as a source of nitrogen (N) and phosphorus
(P) at the same concentrations (0.8 mg N/liter, 0.6 mg P/liter, and 0.33 mg
KCl/liter) recommended by the Georgia Extension Service for fertilizing fish
ponds.
Gases
C02 and air mixtures (51 C02, 1% C02, and compressed air) were obtained in
commercial cylinders. Zero grade helium was used as the carrier gas for the
gas chromatograph and for purging air from the gas collection and reaction
flasks.
Glassware
All glassware used in the phosphorus experiments was acid washed and rinsed
in double distilled water just prior to use.
Mention of commercial products does not constitute endorsement by the
Federal Water Quality Administration.
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Growth Measurements
The growth of algae in the laboratory and in the field was measured by
direct cell counts. Algae were fixed in 5% phenol containing 0.51 crystal
violet and enumerated by direct microscopic counts at 1500X with a Petroff-
Hausser Bacteria Counter.
Bacteria in all field studies and in all laboratory experiments, unless
otherwise indicated, were enumerated by the Millipore filter technique
(Millipore Corp. 1969), using 47 mm, 0.45 u filters and Trypticase Glucose
Yeast Broth Medium, at pH 7.0, and incubated at 27°C for 48 hours.
Contamination
Extreme caution was exercised in maintaining the algae cultures free from
bacterial contamination. Microscopic examination at 1500X and growth in
various liquid media in shake cultures at 23°C were enployed before, during,
and after individual experiments. Cultures found to be contaminated were
discarded.
Sampling
Aliquants taken in the laboratory for biological and chemical analyses were
withdrawn from the culture flasks with sterile 6" stainless steel needles
and syringes.
Field samples were taken with a 2-liter Kemmerer bottle at desired locations
and depths. All biological and chemical analyses were performed on aliquants
from the same water sample.
Sediments for bacterial analysis were also removed with the Kemmerer bottle
and immediately placed in sterile bottles. All bacterial samples were stored
in ice and returned immediately to the laboratory (travel time approximately
two hours) where they were cultured.
Water samples from Shriner's Pond used in the laboratory experiments were
collected and returned to the laboratory (1 hr) where they were filtered
through 47mm, 0.45 u Millipore filters, autoclaved, and stored until used.
Chemical analyses were performed on the autoclaved water by the Analytical
Chemistry Section, Technical Services Program, Southeast Water Laboratory.
Analytical Procedures
Quantitative chemical determinations were made in dilution triplicate.
Analytical tests were checked for chemical interferences. Substances have
been found in samples which interfered with all three phosphate methods used.
N and P determinations were made on samples filtered through 47mm, 0.45 u
Millipore filters. Inorganic carbon determinations were made on samples
filtered through 13mm, 0.22 u Millipore filters.
10
-------
Total inorganic carbon (C02, HC03", and CDj ) was determined on aliquants
stored in 2 -ml gas-tight bottles. For analysis, 1 ml was removed with a
syringe and injected into a 130-ml he liun- filled, tared, reaction flask
with side arms, stop cocks, and rubber septum. The flask was re-weighed
and sufficient H2$04 injected into the sample to reduce the pH to 4. At
this pH all inorganic carbon should be released as CO? with little or no
oxidation of the soluble organic fraction. After shaking for 5 minutes,
200-ul samples of the head gas in the flask were injected into a Model
1532 Varian Aerograph Gas Chromatograph. From the total CX>2 in the ali-
quant and the pH of the original samples, calculations were made of the
amounts of C02, HC03~, and COj* that could have existed in the original
water sample.
002 deterroinations were performed on gas samples by flushing the gas
stream directly through the helium-filled, 130-ml reaction flasks (2 minutes) ,
and injecting 50 ul of this sample into the gas chromatograph.
Total organic carbon was determined with the Beckman Total Carbon Analyzer
Model 915 by the Analytical Chemistry Section, Technical Services Program,
Southeast Water Laboratory.
Phosphate-P concentrations were determined by the Fiske-Subbarow procedure
(Fiske and Subbarow, 1925) , the Lowry modification of the Fiske-Subbarow
procedure (Lowry et al.t 1954) and the Lowry-Lopez method (Lowry and Lopez,
1946) .
Since the absolute amounts of inorganic and total P in all initial samples
were identical when the first two methods were used, it was suspected that
organic P was being hydrolyzed to P04 by the acid in the reagents. Exami-
nation of the procedures showed that hydrolysis was apparently occurring.
Rigler (1968) suspected this hydrolysis to be a source of error. Since the
Lowry-Lopez procedure is done in a buffered solution, hydrolysis is reduced,
if not eliminated.
Nitrate-N concentrations were determined by the Brucine method (Standard
Methods, 1965).
Ammonia and total Kjeldahl-N were determined by the Kjeldahl procedure
(Standard Methods, 1965) by the Analytical Chemistry Section, Technical
Services Program, Southeast Water Laboratory.
Dissolved oxygen was determined by the Azide modification of the Winkler
method (Standard Methods, 1965).
Radiophosphorus disintegrations were counted in a low background Nuclear-
Chicago planchet counter.
The pH determinations were made with either a battery-operated Beckon
Model N jJH meter (field) or a Zeromatic model pH meter (lab). In all cases
the meters were standardized with two buffer solutions just prior to use.
Light measurements in the field were made with a submersible Weston photo-
meter at all depths each daylight sampling time. Light intensities in
11
-------
the laboratory were measured with a Weston Model 756 Illumination Meter.
Light quality measurements were also made in the laboratory with an ISCO
spectroradiometer at the surface of the water bath.
Three white light sources are available in the laboratory:
(1) 200 ft-c fluorescent source
(2) 2000 ft-c fluorescent - incandescent source
(3) 4000 ft-c fluorescent - incandescent source
12
-------
EXPERIMENTAL RESULTS
Phase I: Stimulation of Algae Growth by Bacteria.
Initial growth experiments were performed to verify the reports of Kratz
and Myers (1955), Smith ft al. (1967), Hoare et aZ. (1967), and Pearce
and Carr (1967) concerning the obligate photoautotrophic nature of the
Blue-Green alga, A. nidulcms* The studies were performed with axenic cultures
of A. nidulane in shake flasks at 22*C under the 200 ft-c light source.
Specific uptake studies using 14C-labeled acetate, glycine, serine, and
glucose, respectively, according to standard isotope techniques, were
conducted at various pH's for each compound. Results indicate that acetate
and serine were removed from the media, but glycine and glucose were not
removed from the media. The results also indicate that growth in the algae
medium containing these substrates was no greater than growth in the standard
algae medium alone.
Figure 1 shows the comparative uptake of glucose and acetate for a 12-
hour period at pH 6.8 and 4.7, respectively. The acetate curve at pH 4.7
was chosen because that is the pK and represents maximum uptake.
A second series of experiments was performed to demonstrate the growth
stimulatory effect of the presence of a bacterial culture on the growth
of
Axenic cultures of A. nidulana*eTe grown in 500 ml of the standard culture
medium, modified by adding 0.5 g NbCl. 0.5 g (NH^SOj, and 0.25 g glucose
per 100 ml, in 1-liter Erlenmeyer flasks on a rotary shaker and were main-
tained at 22*C under the 200 ft-c light source. A 3" long dialysis bag
was suspended in each flask. Twenty milliliters of the modified medium
were added to the bag in each control flask while 15 ml of medium plus
5 ml of a heavy inoculum (331 transntittance with a 420 mm filter) of
B, eoli were added to the bag in each test flask. Appropriate dialysis
bags were added to all flasks every eight hours. SincesU nidutona does
not take up glucose, this sugar was used as the carbon source for the bacteria.
All cultures were stoppered with cotton plugs to allow gas exchange with
the atmosphere.
The growth rate of A. niouLona was doubled when grow*, in the presence of
the cultures of E. eoli (Fig. 2). After 43 hours the algal cultures with
bacteria contained an average of 6.5 x 10' algae/ml and the control cultures
contained an average of 3.7 x 10' algae/ml.
Since there is much evidence that A. nidulons is an obligate photoautotroph,
it was suspected that CX>2 produced by the bacteria stimulated the growth
of this algae.
Phase II: Carbon Dioxide Requirement of A. nidulana»
To test the hypothesis stated in Phase I, experiments were designed to
sparge the 002 produced in a bacterial culture through an axenic culture
of algae such that it would serve as the sole carbon source for sustained
algal growth.
13
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Flasks were set up with two in series and one control in parallel (Fig. 3)
in the 23°C shaking water bath under the 2000 ft-c light source. The
bacterial population (Fig. 3, Flask 1) was not counted; however, the 1400 ml
of Nutrient Broth was inoculated with 125 ml of a culture of approximately
401 transmittance using a 420 mm filter. The bacterial culture flask
was replaced at 8-12 hour intervals to insure continued bacterial production
of C02. The test (Fig. 3, Flask 2) and control (Fig. 3, Flask 3) algal
cultures contained 1.3 x 107 and 1.4 x 10' algae/ml, respectively, at time
zero.
The bacteria-algae train was gassed (300 ml/min) with CO?-free air obtained
by flowing cylinder air through an Ascarite (a quantitative 002 adsorber)
column. This C02~free air passed through the bacterial culture and into
the algae flask where it was released at the bottom of the algal culture.
The control algae were gassed with 300 ml/min of the same C02-free air as
the bacteria (Fig. 3).
Analysis of the gas leaving the bacterial culture showed that the bacteria
were producing up to 115 mg C02/hr (Fig. 4, top curve). The difference
between the influent and effluent gas concentrations was the quantity of
C02 sufficient to support the growth of the algae during one cell cycle
(Fig. 4, bottom curve). Repeated experiments showed that this biphasic
curve is reproducible and characteristic of A. nidulane under the specified
conditions.
By determining the area under the C02 uptake curve in Fig. 4, it was
calculated that 1.4 x 10s ug C02 supported the growth of 1.3 x 10*° algae.
Therefore, A, nidulana under the specified conditions, removed from the gas
stream a calculated amount of 10~5 ug C02/cell during one cell cycle.
The data indicate that C02 was utilized continually throughout the cell
cycle except for a short period prior to cell division. Since algae cannot
use stored carbon for growth, they require a constant adequate supply during
this uptake period for maximum growth.
In a 48-hour repetition of the same experiment, A. nidulana grew through
four divisions (Fig. 5) when gassed with air flushed through the bacterial
culture. During the same period, the control algae did not divide.
To make certain the growth stimulant was CC>2, a similar experiment was
conducted with an Ascarite column inserted into the line between the two
flasks to remove the C02 before it reached the algae. No cell division
occurred in either the test or control cultures of A. n-idulana. It was
therefore presumed that the growth stimulant was bacterially produced CC^.
Phase III. Stimulation of Algae Growth By C02
Having indirectly determined that C02 was a growth stimulating material,
experiments were performed in the laboratory to determine the effect of CC>2
concentration on the growth of A. nidulans where no other nutrient could be
limiting.
16
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The algae were grown in the standard culture medium in Fernbach flasks
in the shaking water bath under the 4000 ft-c light source. Flasks 1-4
were bubbled with either Ob-free air, air (0.03% C(>2), 1% C02 in air
or 5% C02 in air. Flask 5 had air blown over the surface of the medium.
The experiment was continued until the last culture stopped dividing at
168 hours. The C02-free air produced no growth (Table 1), while the "»ost
growth occurred in the 51 C02 where the algae underwent 5 doublings.
It should be emphasized that the pH was constant at 6.8 in all cultures
throughout the experiment and that increased growth associated with
increased concentration of C02 was a carbon effect that was not related
to pH.
If the availability of C02 regulates the growth of an algae in laboratory
cultures, the addition of CO? to natural waters should stimulate the
indigenous population. Samples of water obtained from Lake Hartwell, Souti
Carolina, and Shriner's Pond were brought into the laboratory and gassed
with air or 51 C02 in air.
A 700-ml untreated sample of the Lake Hartwell water in a 2-liter flask
was put on a rotating shaker under the 200 ft-c light source at 23°C. The
water initially was clear and had a mixed diatom population of 10$ cells/ml.
Water quality data are shown in Table 2. After being bubbled with 51 CX>2 in
air for four days, the water was brownish and contained 1010 algae/ml. The
pH of this sample dropped from 7.1 to 3.5 in the first 30 minutes and remained
there throughout the experiment.
Water obtained from Shriner's Pond had an initial population of 1.6 x 10$
algae/ml and contained less than 5 ug each of N and P/liter. One-liter
flasks containing 500-ml samples were gassed with either air or 51 CC«2 in
air. From an initial population of 1.6 x 10s algae/ml the culture gassed
with air alone increased to 2 x 105 algae/ml, while the culture gassed with
5t C02 increased to 5*6 x 10s algae/ml in 48 hours.
When these relatively unbuffered, low nutrient waters were gassed with 51
C02, the pH dropped precipitously so that the algae growing under these
conditions had to possess the genetic potential to utilize free (X>2. If
such an inoculum were not present, there would have been no increase in the
algae population.
Phase IV: Phosphorus Requirements of Eacherickia ooli and Anaayatis nidulana.
This phase was concerned with determining the P requirements of A. nidulana
and E. coli and the competition between these two organisms for inorganic P.
The limiting and luxury cellular concentrations of P can be ascertained
only under conditions where P is the sole nutrient regulating the growth of
the organisms, and where physical parameters are not limiting. Because algal
cultures must be sparged with C02 to insure an adequate supply of carbon,
and because of the difficulty in maintaining a stable pH, it was impossible
to obtain reliable uptake and growth data for A. nidulana in solutions
containing small amounts of P. The same pH problem occurred in bacterial
20
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Table 2
SURFACE WATER QUALITY OF LAKE HARTWELL
NEAR CLEMSON, SOUTH CAROLINA
September 4, 1969
pH 7.1
Alkalinity as calciun carbonate 14 mg/1
Temperature 27°C
Dissolved oxygen 8.5 mg/1
Total dissolved carbon as C 5 mg/1
Total dissolved phosphorus as P 0.04 mg/1
Hardness as calciun carbonate 10 mg/1
22
-------
cultures, presumably caused by secretions of C02 and acids by the organisms.
It is difficult to measure, by the usual chemical methods, the small amounts
of P (1.0 ug/liter) that are removed by growing populations of algae and
bacteria.
For the above reasons, radioactive 32P was used with the standard Allen and
Arnon medium and the basal salts meditm of Payne and Feisal to ascertain
the limiting and luxury cellular concentrations of P for A. nidulans and
E. ooli» Since there was sufficient buffering capacity in these media to
maintain pH control, more reliable data relative to the uptake of P could
be obtained.
When phosphorus-starved cells are suspended in phosphate-rich medium con-
taining 32p tracer, uptake is maximal with less dilution of the 32P in the
medium by exchange with cellular 3lP than in the P-replete cells. The amount
of P in P-starved cells calculated from the specific activity (specific
activity * dpm/ug P) should be a more reliable measure of the P metabolized
by the cells since there would be less exchange. The maximum amount of P
stored by the cells would be the luxury amount of P. If the same cells
containing stored "p + 31p are placed in phosphate-free medium until cell
division ceases (because of phosphate limitations), then the amount of P
in the cells would be the limiting cellular concentration.
A. nidulans grown in Fernbach flasks in the 23°C shaker water bath was
continuously bubbled with 5% C02 in air under the 4000 ft-c light source.
Phosphorus-deficient cells were obtained by growing them in Allen and Arnon
medium with Tris buffer substituted for the potassitm dibasic phosphate.
When cell division ceased, they were harvested by centrifugation and washed
three times with phosphate-free medium. Phosphorus-replete cells were grown
in the standard Allen and Arnon medium, harvested, and washed with phosphate
medium.
After harvesting, identical flasks of Allen and Arnon medium containing
32p as a tracer were inoculated with deficient and non-deficient cells.
Samples for cell counts and 32p activity in cells and in cell-free medium
were taken aseptically at 0 time, 4 hours, 8 hours, 16 hours, and 24 hours.
After the first 24 hours, samples were taken daily until cell division
ceased.
To determine the amount of 32p in the cells and media, 1-ml aliquants of the
cultures were filtered through 0.45 u Millipore filters, which were washed
three times with 5 ml of 0.1 M K^PCty and three times with 5 ml of distilled
water. The activity in the cells was counted by placing the Millipore filter
in the planchet counter.
The initial pH was 6.8 in both cultures; when cell division ceased, the pH
was 7.5 for the deficient cells and 7.8 for the non-deficient cells.
When cell division had ceased, the cells in both flasks were harvested,
washed with phosphate-free medium, inoculated into separate flasks of
phosphate-free medium, and grown until cell division again ceased. Samples
for cell counts and for radioactivity measurements were taken on the same
23
-------
schedule as in the first part of the experiment. The initial pH of the
phosphate-free cultures was 7.1, and the final pH was 7.0 for the deficient
cells and 6.8 for the non-deficient cells. The cultures were shown to be
neither light- nor nutrient-limited by subculturing techniques.
The maximum concentration of inorganic P in phosphorus-deficient cells
during the experiment was 3.0 x 10"8 ug/cell, observed at the 16-hour
sampling time (Fig. 6). At 24 hours, cell number had doubled and cell
concentration had dropped to 1.5 x 10"8 ug P.
By the 16-hour sampling time, the non-deficient population had doubled
and the P concentration had increased from 1.5 x 10"8 to 1.8 x 10"8 ug/cell.
The concentration of P in both groups of cells was always highest just
before cell division and lowest following cell division.
In the phosphate-free medium, both phosphorus-deficient and non-phosphorus-
deficient cells divided twice (second and fourth days) (Fig. 7). Sampling
continued through the seventh day and showed no further cell division. The
cell concentration of P at.the time of the last division was 0.25 x 10"8 ug
P for replete and 0.3 x 10»8 ug for deficient cells. When the concentration
dropped below this figure, there was no cell division. On the seventh day,
the concentration per cell had dropped to 0.03 and 0.06 x 10~8 ug P for
replete and deficient cells, respectively. When transferred to medium
containing phosphate, these cells again started to divide.
From the amounts of P in the cells under outlined experimental conditions,
it would seem that A. nidulcois required small amounts of P for cell division
and that when cellular P concentration was 0.9 x 10~8 ug initially, cell
division could take place in the absence of an external P source.
Similar experiments were performed using E. coli as the test organism.
The experimental procedures were the same as described above, except the
organisms were grown in different media and were gassed with air. The
standard basal salts medium of Payne and Feisal plus* II glucose was used
for the P medium and the phosphate-free medium was obtained by again
substituting Tris buffer for the phosphate buffer.
The initial pH of the phosphorus-containing medium was 7.0, and the final
pH was 6.1 for the deficient cells and 6.0 for the non-deficient cells.
The initial pH of the cells and phosphate-free medium was 7.4, and the
final pH was 6.0. Samples for cell counts and for determination of
radioactivity in cells and in cell-free medium were taken every 30 minutes
for two hours and hourly thereafter. Cell counts for the bacteria were
determined by the same direct microscopic technique used to enumerate the
algae. Cells were viable at the end of the experiment.
The greatest incorporation of P into starved cells was observed at the
30-minute sampling time. The cellular concentration of P-starved cells
was 350 x 10~° ug/cell at that time, and the cell count had dropped from
1.05 x 107 to 0.7 x 107.
After 18 hours in medium containing P, cells containing 1.6 x 10~8 ug P/cell
were inoculated into medium containing no P. In the absence of an external
24
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source of P, about one-half of the cells of E. ooli underwent division by
the three-hour sampling time. Sampling continued for 10 hours and there
was no further cell division.
Phase V: Regulation of the Growth of Anacyatis nidulans by Additions
of Carbon and/or Nitrogen plus Phosphorus to Sterilized, Infertile Pond Water.
A. nidulane was inoculated into two sterile, 1-liter flasks containing
Shriner's Pond water ( 5 ug each of P and N/liter). To one flask,
commercial 20-20-5 fertilizer was added. Both cultures were grown in
shake culture at 23°C under the 200 ft-c light source. During the first
96 hours both cultures were in shake equilibrium with the air which entered
the flasks through cotton plugs. After cell division ceased, air was
bubbled through the flasks for another 102 hours. After the cells stopped
dividing again, 51 C02 in air was bubbled through the cultures for an
additional 66 hours.
Figure 8 shows no difference between the final sizes of the population of
A. nidulane in the fertilized and unfertilized water. However, increasing
the amount of available C02 stimulated growth in both cultures. In both
cultures, when air was the source of CO?, cessation of growth was accom-
panied by a pH in the range of 9.8 to 10.1 indicating that even though there
was a constant source of C02 (0.03%) it was not sufficient to support larger
populations of algae. The growth rate of A, nidulane was faster when 51
CC>2 in air was bubbled through the culture containing the fertilizer, but
at the tine growth ceased at 66 hours, (assumed to be due to light limita-
tion) the maximum size of the two populations was identical irrespective of
the concentration of P and N.
The difference in growth rate between the two systems is probably a reflection
of the buffering capacity. Addition of fertilizer to the water does increase
the buffering capacity (Fig. 9), which would tend to prevent precipitous
changes in pH.
Phase VI; Regulation of the Growth of Natural Heterotrophic Organisms by
Addition of Carbon and/or Nitrogen plus Phosphorus to Sterilized, Infertile
Pond Water,
To elucidate further the relative roles of carbon and N plus P in regulating
growth of heterotrophic organisms, a mixed natural population of heterotrophs
was inoculated into flasks containing sterile Shriner's Pond water (SPW).
Additions to the water were glucose (0.25%) and 20-20-5 commercial fertilizer.
Effects of the following combinations were observed:
Shriner's fond water (SPW) + fertilizer
SPW + glucose
SPW + fertilizer * glucose
SPW (control)
27
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Carbon dioxide evolution was used as a measure of heterotrophic metabolic
activity (Levin, 1963, used a similar method for determining metabolic
activity in activated sludge). The heterotrophs were cultured in the
dark. C02-free air was bubbled through the cultures and finally into
saturated solutions of barium hydroxide. The cultures were grown for
72 hours, and the C02 produced was trapped continuously as barium carbonate.
Samples of the medium were analyzed for dissolved inorganic carbon at 0
time and 72 hours. Barium carbonate was determined gravimetrically, and
the amounts of C02 produced were calculated.
The production of CC>2 resulting from the metabolic activity of the micro-
organism was stimulated by the addition of inorganic N and P, glucose, and
glucose plus N and P (Table 3). Duplicate experiments using reagent grade
chemicals in the same proportions produced similar results.
Phase VII: Regulation of the Growth of Natural Populations of Heterotrophs
and Autotrophs by the Addition of Carbon and/or Nitrogen plus Phosphorus
to Non-sterile, Infertile Pond Water.
Growth of natural bacterial and algal populations in Shriner's Pond water
was compared to the growth obtained when 0.4 mg of the 20-20-5 fertilizer,
0.25 g glucose, or combined fertilizer and glucose was added to 100 ml of
water in 250-ml Erlenmeyer flasks. Shake cultures at 23°C were bubbled
with air under the 200 ft-c light source. The initial pH of the cultures
was 7.1. The number of bacteria, number of algae, and amount of dissolved
CC>2 were determined at 0 time and at the end of 30 hours. Table 4 shows
the increased number of bacteria and algae and the increase in dissolved
C02 in those flasks containing fertilizer, glucose, or fertilizer plus
glucose.
Additions of fertilizer and of fertilizer plus glucose resulted in the
growth of such extensive bacterial populations that it was impossible to
obtain reliable counts from the dilutions used for plate counts. During
the same 30-hour period, the number of bacteria in the control decreased.
In the culture containing the glucose, the pH was 5 at the end of 30 hours,
while the pH of the other cultures varied from 6.5-7.0. The smaller number
of viable cells in the glucose culture may have been due to the low pH
causing either a direct inhibition of growth or lysis of the cells.
Addition of fertilizer or glucose alone resulted in a 2.5 to threefold
increase of algae (largely mixed diatoms, Chlamydomonae sptf Chlorella sp.)
while the addition of glucose plus fertilizer resulted in a sixfold increase
of algae. The control algal population did not grow during the 30-hour
period.
Dissolved inorganic carbon decreased in the control while it increased
in the fertilizer, glucose, and the fertilizer plus glucose treated flasks.
It should be stressed that these amounts of inorganic carbon represent only
the excess C(>2 produced by the bacteria that was not removed by the algae
or lost to the atmosphere.
30
-------
Table 3
C02 PRODUCTION BY HETEROTROPHIC POPULATION
FOLLOWING ADDITIONS OF ORGANIC AND/OR
INORGANIC FERTILIZERS TO A STERILIZED INFERTILE POND WATER
(<5 ppb each of N and P)
C02 + HC03"
(difference between Milligrams C02
Treatment . 0 time and 72 hours) trapped as
Shriner's Pond Water (SPW) 3.6 0
SPW + 20-20-5 fertilizer 10.0 0.003
SPW + glucose 9.2 0.005
SPW + fertilizer + glucose . 12.0 0.03
31
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Phase VIII; Growth of Heterotrophic and Autotrophic Populations and
Nutrient Cycling in an Enriched Lake.
It stimulation of heterotrophic populations by the addition of carbon,
N,, and P is responsible for the blooms of algae occurring in enriched
waters, large numbers of bacteria should be found associated with high
pre-dawn concentrations of dissolved inorganic carbon, and large numbers
of algae.
Since algal cultures are largely synchronous, nutrient uptake is cyclic.
Algal metabolism is dependent upon light as an energy source, so diurnal
and diel measurements of the amounts of carbon, N, and P in water reflect
the cycling and relative importance of the nutrients in regulating the
heterotrophic and autotrophic populations. Those substances necessary
for the growth (increased number) of any population should be decreasing
in the water during periods of active increase in size of the population.
Field studies were conducted on July 2, 15, and 30-31, 1969, in the
Flat Creek area of Lake Sidney Lanier, Georgia, to follow the cycling of
inorganic carbon (C02, HC03~, C()T=), N (inorganic and total dissolved),
P (inorganic and total dissolved), oxygen, algae, and bacteria.
Flat Creek received the effluent from a secondary sewage treatment plant
at Gainesville, Georgia, approximately seven miles upstream from the test
area. Sampling stations 1,2, and 3 were located approximately 200 yards
apart in the Flat Creek area of the lake. Station 4 was located one-fourth
mile farther out in open water. A well-established algal bloom was present
in Flat Creek from station 1 to station 3.
Water samples were taken with a Kemmerer bottle at one foot below the
surface, one foot above the bottom, and at various intermediate depths,
except at station 4 where no samples were taken below 20 feet (the top of
the thermocline). Sediment samples were collected for bacterial analysis.
Emphasis has been placed on the cycling of carbon and P and the biological
activities of the heterotrophic and autotrophic populations. Oxygen, N,
and pH data are now presented. Diel cycling of oxygen (Fig. 10) occurred
with the maximum in the late afternoon. Highest concentrations (12-13 mg/liter)
were found at the 1-, 3-, and 5-ft levels. Minimal values occurred at all
depths at 7:00 AM. The greatest fluctuation in the amount of oxygen occurred
at the 8-ft depth where it ranged from 2 to 11 mg/liter.
Since both nitrate and ammonia-N were present at all times, N was not
limiting the system (Fig. 11). The pH increased during the day (7:00 AM
to 7:00 PM) from 6.5 to 10 at the three stations in Flat Creek, and from
8.9 to 10.6 at station 4.
Comparison of the data from highly enriched station 1 (Fig. 12) with that
from enriched station 4 (Fig. 13) revealed that approximately five times
more algae (240 x 104 cells/ml) were at station 1 than at station 4
(50 x 104 cells/ml). The high, pre-dawn concentration of C02 (7 mg/liter)
at station 1 was removed from the water by 1:00 PM. Bicarbonate decreased
from 27 mg/liter at 7:00 AM to 4 mg/liter at 4:00 PM. This depletion of
inorganic carbon at station 1 is especially significant because these waters
33
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were constantly being gassed with bubbles containing 8-9$ CC^. Even with
a constant source of 0)2, there was no accumulation of CC>2 or HCOj" in the
water until sunset, when the uptake by the algae was at a minimum.
There was no detectable C02 in the 1-ft samples at station 4. Bicarbonate
was removed from the water by 1:00 PM, then increased, and leveled off at
4 ing/liter by 4:00 PM. The concentration of CQ$~ in this water was much
higher than at station 1.
The amount of C02 at station 1 in the early morning is very large (7mg/liter)
in comparison with the 0.4 to 1 mg/liter that can be obtained from air
equilibrium. Since this is a soft-water (hardness, 30 mg/liter as CaC03)
lake (Little, pers. comm. 1970), CC>2 contribution from carbonates would be
minimal.
The amount of P remained constant or increased slightly at both stations
during the same period that the inorganic carbon was being depleted.
Phosphorus was high in these waters (1.3-2.2 mg/liter).
Figures 14 and 15 show the diel fluctuation in the size of the bacterial
population at stations 1 and 4. The population density of the bacteria
from the three depths at station 1 was approximately 100 times larger than
the density from the five depths at station 4. At each station, the number
of bacteria increased with depth. Since station 4 was 60 ft. deep, and no
samples were taken below the thermocline, the reported values probably do
not reflect the total biological activity. Bubbles rising through the water
column indicated additional biological activity near the bottom of the lake.
The largest number of heterotrophs and presumably the majority of the
heterotrophic activity were associated with the bottom, especially with the
upper layers of the sediments. Metabolic activity of the benthos was indi-
cated by the concentrations of C02 at the various depths of station 2 over
a 24-hour period (Fig. 16). There can be no doubt that the bottom in this
area was a source of large amounts of CC^.
Accumulation of C02 in the bottom waters from 4:00 to 7:00 PM, and the
build-up of 0)2 through the water column during the night was evident.
Since the largest population of bacteria was found on the bottom and this
is a soft-water system presumably this C02 originated from biological
oxidation of organic substrates.
Data plotted in Fig. 17 demonstrate the importance of the physical environ-
ment (in this case, light) in regulating both biological activity and
nutrient cycling. July 15, 1969, was cloudy and overcast with periods of
intermittent sunshine before noon, and clear skies in the afternoon. July 2
was clear and sunny. Though the algal populations were essentially identical
(240 x 104 cells/ml) on July 2 and July 15, 1969, both the rates and quantity
of CO? and HC03" removed from the water were lower on the cloudy day. On
July 2, the total amount of CC>2 in solution (7mg/liter) was removed from
the water by 1:00 PM; on the cloudy day, July 15, only 2.5 mg of C02/liter
were removed by 1:00 PM. When the skies cleared on July 15, another 2.5 mg
were removed in only 3 hours (1:00-4:00 PM). On July 2, 23 mg HC03"/liter
were removed from the waters from 7:00 AM to 4:00 PM; on July 15, only
13 mg HC03"/liter were removed from solution (7:00 AM to 1:00 PM). The
38
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FIG. 17
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dissolved HCO-r was increasing during the remaining hours of the experiment
(1:00 PM to 10:00 PM).
The observed differences in pH paralleled those occurring in the carbon
cycling. On July 2, the pH ranged from 6.5 to 10; on July 15, the pH
increased from 7.1 to 8.
43
-------
DISCUSSION
Data collected in this laboratory indicate the importance of CO? in regulating
the growth of axenic cultures of A. nidulcns and natural populations of
algae. This effect is observed when either commercial air mixtures containing
C02, or CC>2 produced by heterotrophic oxidation of organic carbon is used
as a carbon source. Lange (1967) demonstrated similar results with
Osoillatoria Piibesoens and Miarocystis aeruginoaa Kutz. Allen (1955) demon-
strated that Chlorella and Scenedesmus were growing in sewage oxidation ponds
on the C02 produced by the heterotrophic oxidation of organic carbon. Cyclic
uptake of carbon during a cell cycle of A. nidulans resulted in the calculated
removal of 10"5 ug C02/cell. Birge and Juday (1911) , in classic ecological
studies of the_lakes of Wisconsin, described the seasonal cycling of CC>2,
HC03~, and C03= in soft, medium, and hard-water lakes. The authors attributed
removal of carbon available to green plants both directly by biological fixa-
tion and indirectly by chemical precipitation of carbonates as the pH rises.
Birge and Juday emphasized the importance of bacterial oxidation or organic
material as a principal source of CC>2 in aquatic systems. They reported
that there was no diurnal, only seasonal, cycling of carbon in the lakes of
Wisconsin during 1904-1906.
Carbon, N, and P compounds function both as nutrients and as buffers.
Buffering capacity is important in controlling the chemical availability of
carbon, N, P, and trace elements. As the data in Phase V indicate, addition
of 20-20-5 fertilizer increased the buffering capacity of Shriner's Pond
water. In enriched waters, the buffering capacity could be increased directly
by the addition of charged compounds and indirectly by the amino acids,
organic acids, and C02 resulting from increased biological activity. Such
increases in buffering capacity could be very important in regulating algal
blooms since more carbon could be held in solution in the forms (C02 and
HC03~) most available to the plants. Increased buffering capacity would also
tend to "protect" the heterotrophic and autotrophic populations. Since
enzymatic reactions are pH dependent, biological activity would be favored
in situations where precipitous changes in pH are prevented. Thus, any
increase in buffering capacity would enhance the biological generation of
CC>2, its availability in solution, and the biological utilization of C02.
Data obtained in 1969 leave no doubt that the Flat Creek-Lanier ecosystem
was in a dynamic state and exhibited diel fluctuation in nutrient cycling
and biological activity. Thus, it is difficult, if not impossible, to
correlate actual biological response with instantaneous measurements of
dissolved nutrients. C02 and HCOj" are of extreme importance in regulating
the size of the algal population in Flat Creek, but measurements of these
ionic forms in this water in the afternoon revealed no CC>2 and a minimal
~ value of 4 mg/liter.
Conversely, afternoon measurements of the P concentration would yield high
values of 1.3-2.2 mg/liter. Observation of the diurnal fluctuation of total
P alone would reveal that there was no appreciable biological activity
occurring in this ecosystem, while in fact, it is an area of intense metabolic
activity.
44
-------
Both Lund (1950) and Purdy (1937) cited evidence that instantaneous
measurements of nutrients in water are not reliable indications of
potential activity. Lund (1950) stated:
"The amount in solution in the water at any one moment
is not a reliable indication of the potential algal productivity,
though it might be were other substances in excess of requirements."
Purdy (1937) reported that Mohlman et al in 1931, "pointed out the necessity
of caution in accepting a single daily sample at a given station as repre-
senting the average conditions."
Both field and laboratory data indicated that growing populations of algae
and bacteria require small amounts of P. The luxury concentration of P
in A. nidulans is 3.0 x 10~8 ug/cell; the limiting cellular concentration
is 0.25 to 0.3 x 10-8 ug/cell.
The cellular P concentration appeared to regulate the uptake of P by
A. nidulane. Phosphorus-starved and phosphorus-replete cells were innoculated
into media containing 2 x 10-3 M I^HPC^. Preceding the first division,
the starved cells incorporated F times more phosphorus than the replete
cells. Division of the starved cells had occurred by the 24th hour; division
had occurred by the 16th hour in the non-starved cells.
Algae and bacteria possess the genetic potential to store N and P beyond
their immediate metabolic needs. A. nidulans can undergo two doublings in
the absence of an external supply of P. Both of these findings have been
demonstrated earlier for the marine diatom, Nitzachia alosterium (Ketchum,
1939).
The data from these studies support the thesis that during periods of
active growth, the nutrient most critical in regulating the size of biological
populations is the one that is most nearly removed relative to need from the
environment. (Note the earlier discussion of removal of C02 and HCOj" from
the water in the Flat Creek area while there was no measurable removal of
P.)
Lund (1950) states:
"On this view, the large phosphate concentrations in waters
such as those described in Mackenzie (1940) and Gardiner (1941)
may be considered to be due to there being an excess supply over
that which the algal cells can take up. This may also be the
explanation of the customary rise of phosphate-phosphorus in the
lakes of the English Lake District in mid-winter when algal numbers
are at a minimum due to lack of light."
If P and/or N are not present in the water in available forms, the growth
of the population may or may not be regulated by these nutrients. If no
P and/or N were added, the population could reach a size that would be
determined by the amount of P and N stored in their cells and turned over
by other organisms. The extent of growth due to P storage occurring under
natural conditions is not well established, but laboratory values indicate
population increases for more than a month with some algae (Levin, 1963).
45
-------
Lund's (1950) data indicate that Asterlonella containing luxury amounts
of P sinks to the deeper waters where the growth of the organism is regu-
lated by decreased light. During the spring turn-over, these cells serve
as the inoculum for that season's population. Thus, the cells contain
luxury amounts of P at the beginning of the growing season.
Nutrients not removed from the water are still capable of supporting the
growth of other organisms, bat only -if there is an added adequate supply
of the nutrient that is available in the smallest amount relative to need.
These data indicate the importance of the biological production of C02
in regulating the size of autotrophic populations in the three waters
studied containing high (1.3-2.2 mg/liter, Flat Creek-Lanier), intermediate
(0.04 mg/liter, Lake Hartwell), and low (<0.005 mg/liter, Shriner's Pond)
concentrations of P. Increased production of C02 by mixed natural hetero-
trophic populations has been shown to accompany the addition of glucose,
soluble N and P, and glucose plus soluble N and P to infertile water from
a Georgia fishpond.
Additions of N and P to this same sterile, infertile water (<0.005 mg each
N and P/liter) had no effect on the growth of an axenic culture of
A. nidulans. Additions of increasing amounts of (X>2 increased the growth
of the algae. Since the final size of the algal population was the same
irrespective of whether N and P had been added, growth of the algae in this
water is regulated by the amount of carbon present more than by the amount
of N and P.
These findings offer a deeper understanding of the changes that can accompany
the cultural (or natural) enrichment of the waters studied. Additions of
N and P alone can result in transient increases in the size of the hetero-
trophic and autotrophic populations. Additions of organic carbon alone can
stimulate the growth of both populations until either the allochthonous
carbon or the N and/or P are depleted. Additions or organic carbon, N, and
P can result in sustained growth of large populations of both heterotrophs
and autotrophs.
Additions of organic carbon and/or N plus P to these waters directly stimu-
lated the heterotrophic population. This stimulation increased the amount
of 002 an^ HOOj" available for the aquatic plants. This increase in inorganic
carbon is apparently directly responsible for the increased size of the algal
populations in the waters studied.
46
-------
SUMMARY
In our studies of the Blue-Green alga, A. nidulans, we observed that:
1. COo produced by bacteria stimulated the growth of axenic
cultures of A. nidulans.
2. CC>2 is important in regulating the size of the population
of axenic cultures of A. nidulans and natural algal
populations.
3. 10"5 ug C02 was sufficient for the division of a cell
of A. nidulans.
4. The limiting and luxury cellular concentrations of P
for P-starved A. nidulans are 0.3 x 10"8 and 3.0 x 10'8
ug/cell respectively.
5. The removal of P from solution by A. nidulane is
dependent upon the cellular concentration of P.
6. Carbon uptake is cyclic during a cell cycle of A. nidulans.
7. Additions of carbon to a sterilized pond water are more
important than additions of N and P in regulating the
population size of axenic cultures of A. nidulans.
Laboratory studies of heterotrophic populations demonstrated that:
1. Additions of glucose, soluble N and P, and glucose plus
soluble N and P to a sterilized pond water stimulated the
production of C02 by isolated natural heterotrophic
populations.
2. Additions of N and P, glucose, and glucose plus fertilizer
to non-sterile water from an infertile farm pond resulted
in increased numbers of bacteria, increased amounts of
dissolved C02 and increased numbers of algae.
The biological and nutritional interactions studied in an enriched area,
Flat Creek, receiving the effluent from a secondary treatment plant showed:
1. The diel cycling of CO?, HC03", C03=, 02, and pH in these
waters is indicative of an area of high biological activity.
It is similar to the seasonal cycling described in the works
of Birge and Juday (1911).
2. Large amounts of CC>2 were being produced by the organisms
growing on the bottom of this creek. Bubbles containing
8-9% CC>2 were constantly rising through the water column.
3. In the afternoon, the water was supersaturated with oxygen
which was not depleted at night.
47
-------
4. Light affected the removal of nutrients from
the water.
5. Growing populations of bacteria were associated
with high pre-dawn concentrations of inorganic
carbon and growing populations of algae.
6. Removal of certain nutrients from the waters accompanied
the growth of biological populations. Inorganic carbon
reached very high levels at night, but was almost
depleted during the day. Phosphorus levels stayed
almost constant. The nutrient, in this case, carbon,
most nearly depleted, relative to need, should be the
one regulating the size of the population.
7. Repetitive diel fluctuation in numbers of both
bacteria and algae was observed during these studies.
The most rapid increase in algal number in the first
foot of water usually occurred in the early morning
and was correlated with the most rapid rate of removal
of 002 from the water. The maximum increase and
decline in the bacterial population occurred from
7:00 PM to 7:00 AM and was correlated with the peak
concentration and removal of oxygen from the water.
Concomitant with the increased number of bacteria and
removal of oxygen from the water, the dissolved CC>2
and HC03~ content of the water increased.
8. Instantaneous measurements of the amounts of nutrients
in the water cannot be used to accurately predict
biological activity of dynamic ecosystems.
Studies on these natural waters containing low, intermediate, and high
concentrations of N and P stress the importance of carbon in regulating
the growth of heterotrophic and autotrophic populations.
48
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ACKNOWLEDGMENT
We wish to thank the staff of the National Pollutants Fate Research
Program (Dr. Walter M. Sanders III, Chief), and the staff of the
Impoundment Studies Activity and the Analytical Chemistry Section
of the Technical Services Program, Southeast Water Laboratory, for
their invaluable assistance.
49
-------
REFERENCES
1. Al Kholy, A. A., "On the Assimilation of Phosphorus in Chlorella
pyrenoidosa," Physiol. Plantarwnt 9, 137-143 (1956).
2. Allen M. B.f "The Cultivation of Myxophyceae," Arch. Mikrobiol.,
17, 34-53 (1952).
3. Allen, M. B., "General Features of Algal Growth in Sewage Oxidation
Ponds," State Water Poll. Cont. Bd., Sacramento, Calif., Publication
No. 13 (1955)
4. Allen, M. B., and Arnon, D. I., "Studies on Nitrogen-Fixing Blue-
Green Algae. I. Growth and Nitrogen Fixation by Andbaena cylindrical
Lemm," Plant Physiol.t 30, 366-372 (1955).
5. Batterton, J. C., and Van Baalen, C., "Phosphorus and Phosphate Uptake
in the Blue-Green Alga, Anacystis nidulans," Canad. J. Mcrobiol.,
14, 341-348 (1968).
6. Birge, E. A., and Juday, C., The Inland lakes of Wisconsin. The
Dissolved Gases of the Water and Their Biological Significance^
Wisconsin Geological and Natural History Survey, Bull. No. XXII (1911).
7. Bristow, J. Michael, "The Effects of Carbon Dioxide on the Growth
and Development of Amphibious Plants," Canad. J. Bot.t 47, 1803-1807
(1969).
8. Brix, Holger, "Influence of Light Intensity at Different Temperatures
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53
*IJ. S. GOVERNMENT PRINTING OFFICE : 1970 O - 398-776
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