Download Resource competition and community structure in aquatic micro

Journal of Plankton Research Vol.22 no.8 pp.1591–1610, 2000
Resource competition and community structure in aquatic microorganisms: experimental studies of algae and bacteria along a
gradient of organic carbon to inorganic phosphorus supply
James P.Grover
Department of Biology, University of Texas at Arlington, Box 19498, Arlington,
TX 76019, USA
Abstract. Two microbial communities were grown in chemostats receiving a low supply of inorganic
Phosphorus (P) (10 µM) and different supplies of organic carbon (OC), ranging from 0 to 600 µM,
either as glucose or a mixture of organic substrates. One community was a natural assemblage of lake
plankton and the other was a model community composed of cultured organisms. As the supply ratio
of OC to inorganic P increased, concentrations of dissolved OC increased, concentrations of dissolved
P decreased and abundances of phototrophic algae decreased. Abundances of bacteria and phagotrophic organisms did not consistently change with the OC:P supply ratio. The model community was
first established with a phototroph (Scenedesmus quadricauda) and bacteria; the steady states of this
community were invasible by the mixotroph Ochromonas danica under all OC:P supply ratios used.
When OC supply was high, both microbial communities persisted with higher concentrations of
dissolved OC when mixed substrates, rather than glucose, were supplied. Otherwise, the effects of
organic substrate composition appeared to be secondary to those of the OC:P supply ratio. These
experiments confirm some elements of published theory on resource-based interactions among
heterotrophic bacteria and phototrophic algae.
Introduction
The micro-organisms of the plankton display an impressive diversity of phylogenetic history and ecological interactions. Their nutritional strategies range
widely. The heterotrophic bacterioplankton rely on dissolved organic matter and
mineral nutrients. Some phytoplankton are obligate phototrophs, relying on light
for energy while consuming dissolved mineral nutrients. Other phytoplankton are
facultatively phototrophic (i.e. mixotrophic), capable of photosynthesis but also
of consuming particulate matter, gaining both energy and nutrients through
phagotrophy. Still other planktonic micro-organisms are obligate phagotrophs,
unable to survive without particulate sources of energy and nutrients. Aquatic
ecologists have recently documented the widespread distributions and functional
relationships of these various organisms, which appear to co-exist in virtually
every well lit aquatic ecosystem. Yet, the ecological circumstances permitting
their co-existence remain unclear.
One approach of proven use in untangling the complex relationships of microbial ecology is the chemostat (Jannasch, 1967, 1969), a laboratory model of an
ecosystem with a well developed mathematical representation (Smith and
Waltman, 1995). Using this framework, Thingstad and Pengerud (Thingstad and
Pengerud, 1985) made a pioneering exploration of the co-existence of different
types of planktonic micro-organisms. Their theory explicitly invokes resource
competition and externally mediated resource supplies. For example, considering only heterotrophic bacteria and phototrophic algae competing for an inorganic nutrient, theory predicts co-existence if the supply of dissolved organic
© Oxford University Press 2000
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J.P.Grover
carbon (DOC) is low relative to that of the nutrient. This condition limits heterotrophic bacteria, otherwise assumed to be superior in competition for the inorganic nutrient. If not limited by organic carbon, bacteria are predicted to exclude
algae. When the inorganic nutrient in question is phosphorus (P), experiments
support the assumption of superior bacterial competitive ability (Currie and
Kalff, 1984a,b,c; Pengerud et al., 1987).
Bratbak and Thingstad elaborated this theory to include the commensal supply
of DOC through algal exudation (Bratbak and Thingstad, 1985). Algae nearly
always excrete greater or lesser amounts of DOC (Jørgensen, 1986; Zlotnick and
Dubinsky, 1989), fertilizing their bacterial competitors and making it almost
impossible for algae to exclude bacteria competitively. With or without such
commensalism, both theories predict that as the ratio of DOC supply to inorganic
nutrient increases, bacteria increase, algae decrease and, at a sufficiently high
level, disappear altogether (Grover, 1997).
Thingstad and Pengerud also examined the co-existence of algae, bacteria, and
a phagotrophic predator selectively feeding on the latter (Thingstad and
Pengerud, 1985). They suggested that all three types of organisms would co-exist
over a wide range of resource supply conditions, with phagotrophs restraining
bacterial abundance even at high DOC supply, permitting algae to persist despite
their poor ability to compete for the inorganic nutrient. This theory considered
only obligate phagotrophs, which neither photosynthesize nor consume inorganic
nutrients, but not mixotrophs that photosynthesize and take up both dissolved
and particulate nutrients. Currently, our understanding of how mixotrophy coexists with other nutritional strategies is incomplete. Rothhaupt theorized that
mixotrophs could co-exist with either obligate phototrophs on the one hand, or
with obligate phagotrophs on the other (Rothhaupt, 1996a). Such co-existence
would depend on resource supply conditions, which he demonstrated experimentally. The most elaborate theory to date considers the nutritional strategies
of bacteria, phototrophic and mixotrophic algae, and obligate phagotrophs, when
all compete for a single inorganic nutrient (Thingstad et al., 1996). This theory
does not predict co-existence of all four nutritional types.
This paper applies an experimental approach to studying the co-existence of
nutritionally diverse aquatic micro-organisms. It seeks to test theoretical predictions concerning communities of bacteria, phototrophic algae and phagotrophs
(Bratbak and Thingstad, 1985; Thingstad and Pengerud, 1985). Chemostats were
used to culture both (i) a natural community of micro-organisms and (ii) a model
community assembled from cultured micro-organisms, first by growing algae and
bacteria together, and then inoculating a mixotrophic flagellate. Inorganic P was
chosen as a limiting nutrient for comparability with previous work. Phosphorus
often limits algal growth in the natural community used [Joe Pool Lake, Texas
(Sterner, 1994)], and often limits algal and microbial growth elsewhere
(Schindler, 1977; Morris and Lewis, 1992). Experimental cultures all received the
same P supply but received different supplies of DOC, thus creating a gradient
of OC:P supply ratios.
Theory predicts several responses to an increased OC:P supply ratio. First,
for communities of phototrophic algae and bacteria only, without phagotrophs:
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(i) bacteria should increase in abundance; (ii) algae should decrease in abundance, and eventually be excluded; (iii) inorganic P should decrease in concentration; and (iv) DOC should increase in concentration. Second, for communities
which also have phagotrophs: (i) bacteria should remain constant in abundance;
(ii) algae should decrease in abundance, though not be excluded; (iii) phagotrophs
should increase in abundance; (iv) inorganic P should decrease in concentration;
and (v) DOC should increase in concentration. In addition to imposing a gradient of OC:P supply ratio, two qualitatively different DOC sources were supplied:
glucose and a substrate mixture. Theory does not currently address effects of
substrate diversity on co-existence of bacteria with other micro-organisms, and
previous experiments have used a single substrate. Therefore, the possible effects
of substrate mixtures require examination.
Method
Continuous cultures
Commercially available bioreactors (Cytolift®; Kontes, Vineland, NJ, USA) were
used as culture vessels. These are jacketed, borosilicate reaction vessels with a
working volume of 600 ml and polypropylene bases and caps. Caps were fitted
with Teflon tubes for sampling, injection and air-sparging. Peristaltic pumps delivered media into vessels, with a balancing overflow to a sterile vented flask. Sparging air was supplied with aquarium pumps, humidified in gas-washing bottles, and
filtered (0.22 µm). Water was circulated through vessel jackets at 25° ± 0.1°C.
Light was supplied with cool-white fluorescent lamps on a 14:10 h light:dark cycle,
at a photon flux of 120–130 µmol quanta m–2 s–1, measured at the position of the
culture vessels. The irradiance used here should be adequate to saturate growth
of Scenedesmus quadricauda (Ahlgren, 1987) and of many other species (Rhee,
1982). It is also within the range of average mixed layer irradiances found in the
habitat where the natural assemblage was obtained [Joe Pool Lake (Sterner,
1994)].
Media reservoirs were vented with 0.22 µm filters to maintain sterility and
contained sufficient medium for an entire experiment (10 l). The entire reactor
system, from reservoir to outflow flask, was sterilized by autoclaving. After each
experiment, sterility of nutrient media in reservoirs was verified by drawing
samples aseptically for examination by epifluorescence microscopy. Bacterial
contamination of media was never found. Each of the experiments reported here
used 10 continuous cultures, arranged in two blocks of five. Each block shared
a set of fluorescent lamps, temperature-controlled circulator and a peristaltic
pump.
Defined nutrient media were based on Guillard’s (Guillard, 1975) WC formulation, as modified by Tilman (Tilman, 1981), with P supplied at 1.0 µM as
Na2HPO4, and various organic carbon supplements. This medium supports
growth of many cultured and natural algae, and has been used previously in
similar experiments on algal–bacterial interactions (Rothhaupt, 1992, 1996a). As
modified, it has N:P [where N is nitrogen (molar)] of 1000. Without additional
supplements, it has only 1.66 µM DOC as vitamins (biotin, cyanocobalamin and
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Table I. Mixture of organic carbon substrates, dispensed to give total carbon concentrations of 100,
300 or 600 µM C, as described in the text
Compound
Molar fraction of C supplied
Weight percent
Alanine
Aspartic acid
Glutamic acid
Glycine
Fructose
Glucose
Sucrose
Dihydroxyacetone
Adenosine
Glucosamine
Sodium acetate
0.09
0.09
0.085
0.085
0.025
0.025
0.025
0.025
0.05
0.25
0.25
4.3
4.9
4.1
5.2
12.2
12.2
11.6
12.2
2.2
14.6
16.6
thiamine) and 20 µM DOC as EDTA, used to chelate trace metals. Two types of
organic carbon supplement were added in the experimental treatments described
below—either glucose alone, or a mixture of 11 substrates (Table I). Natural
waters contain a variety of DOC compounds, mostly uncharacterized and much
of high molecular weight (Stumm and Morgan, 1981). Compounds of low
molecular weight, thought to be more available to bacteria, constitute a variable
but large fraction of the total DOC [16–94% (Jørgensen, 1986)]. The substrate
mixture (Table I) crudely mimics the composition of this material of low
molecular weight, which is roughly 50% proteinaceous and 20% carbohydrate,
with various other structures making up the balance (Stumm and Morgan, 1981).
The mixture contains four amino acids found by Cowie and Hedges (Cowie and
Hedges, 1992) to be common in vascular plant sources for aquatic DOC, and a
variety of other compounds also common in biological materials, and likely to be
present in natural waters.
Natural assemblage experiment
An inoculum was collected from Joe Pool Lake, near Dallas, TX. In this lake,
algal growth at water temperatures above 15°C tends to be limited by P, and is
often co-limited by nitrogen and trace nutrients (Sterner, 1994). Several samples
of surface water and plankton net tows (63 µm mesh) were collected on 23
October 1995, and combined. At the time of sampling, the lake was vertically
isothermal at 21°C, and dissolved nutrient concentrations suggested P limitation.
Soluble reactive phosphorus (SRP) was 0.05 µM, DOC was 284 µM, dissolved
inorganic N was 6.1 µM and dissolved silicate was 92 µM.
Upon return to the laboratory, the combined sample was passed through a
50 µm mesh to remove larger zooplankton. Abundant nauplii passed through the
screen, so the sample was rendered anoxic by 2 h of purging with N2, which
appeared to kill the nauplii. The sample was then split into two aliquots. One of
these was enriched with ~10% (by volume) of WC medium with P = 1.0 µM and
no DOC supplement, and the other was enriched with a similar proportion of WC
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Resource competition in algae and bacteria
medium (P = 1.0 µM) with 100 µM C as glucose. These aliquots were incubated
for 4 days at experimental light and temperature conditions, and enriched twice
with increasing proportions of media prepared as above. During pre-conditioning, bacteria increased at rates of 0.39 day–1 (with glucose) and 0.48 day–1 (without
glucose), while algal biomass increased at rates of 0.06 day–1 (with glucose) and
0.42 day–1 (without glucose). To inoculate continuous cultures, the aliquots with
and without glucose were mixed, and 50 ml of the mixture were aseptically transferred to each chemostat culture vessel.
Five replicated nutrient treatments were used in this experiment. All cultures
received WC medium with P = 1.0 µM. One treatment received no organic carbon
supplement; two treatments were supplied with 100 µM C (carbon) either as
glucose or the substrate mixture described above; and two treatments received
600 µM C either as glucose or as a mixture. Experimental treatments thus
consisted of three molar OC:P supply ratios of 0 (no OC supplied), 100 and 600,
and two qualitatively different organic carbon sources. All five treatments were
applied to each of the two blocks of continuous cultures. Dilution rates of all but
two cultures ranged from 0.325 to 0.381 day–1; two cultures (one with no OC
supply, the other with 100 µM C as glucose) had much lower dilution rates of 0.107
and 0.117 day–1. Data from these cultures were included in all analyses because
they were similar to their replicates and did not emerge as outliers in any analyses. All cultures ran for 35 days, during which time several turnovers of culture
volume rendered negligible any residual nutrients from the lake water inoculum,
ensuring that supply was governed by the media provided.
Model system experiment
A unialgal culture of S.quadricauda was used to inoculate continuous cultures.
This strain has been in the personal collection of J.Grover since 1986, and was
used previously in studies of phosphorus-dependent growth and competition
(Grover, 1989, 1991a,b,c). Incidental bacterial contaminants of the Scenedesmus
stock culture served as a source of bacteria for the continuous cultures. In these
experimental cultures, a single rod-shaped morphotype dominated bacteria,
although other morphotypes were present. The dominant rod-shaped bacterium
has since been isolated into pure culture and found to grow well in WC medium
supplemented with glucose, and identified as a Pseudomonas sp. (Codeco, 1998).
After running this model system of algae and bacteria until steady state was
achieved, a mixotrophic flagellate, Ochromonas danica UTEX 1298, was added
to some cultures, to construct a ‘three component’ model community.
As in the natural assemblage experiment, five replicated nutrient treatments
were used with the model system. All cultures again received WC medium with
P = 1.0 µM. One treatment received no organic carbon supplement; two treatments were supplied with 300 µM C as glucose or as a substrate mixture; and two
treatments received 600 µM C as glucose or as a mixture. Experimental treatments thus consisted of three molar OC:P supply ratios, 0, 300 and 600, and two
qualitatively different organic carbon sources.
All five treatments were applied to each of the two blocks of continuous
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cultures. Dilution rates of all cultures ranged from 0.285 to 0.304 day–1. Cultures
ran for 44 days, except for one (receiving 300 µM C as glucose) which ran for
28 days. On day 32, while cultures were in steady state, one culture of each treatment was randomly selected for inoculation with Ochromonas, and the other
culture was maintained as a reference system. Ochromonas stocks were maintained axenically in the medium recommended for its cultivation (Starr and
Zeikus, 1987), but 4 days prior to inoculation, it was pre-conditioned to phagotrophy by transfer to WC medium supplemented with 600 µmol each of glucose
and mixed organic substrates, prepared as for the chemostats. Bacteria from
chemostats were collected by passing 3 ml of chemostat contents through a
1.2 µm Nuclepore filter, and these bacteria were added to the pre-conditioning
culture of Ochromonas. Ochromonas grew well in this culture, and 2 ml of this
suspension were inoculated into selected chemostats. The inoculation of
Ochromonas could have delivered pulses of available P and DOC, in amounts of
up to 0.2 and 4.8 µM, respectively. For the cultures receiving glucose at 300 µM,
the culture which ran for 44 days was inoculated with Ochromonas at day 32, and
the last 12 days of the shortened run for its replicate were used as a steady state
reference condition. Available medium did not allow continuation until new
steady states were achieved, but dynamics of Ochromonas and other species were
documented over 12 days.
Sampling and analysis
Every 2 days, 30 ml samples were taken. Two 10 ml portions were filtered onto
Whatman GF/F filters and frozen for later determinations of chlorophyll a. The
remaining 10 ml were preserved with formalin for bacterial counts. During the
natural assemblage experiment, and from days 36 to 44 in the model system
experiment (after the inoculation of Ochromonas), an additional 10 ml sample
was withdrawn every 4 days and preserved with Lugol’s iodine and neutral formalin for algal counts. Every 4–12 days, samples were withdrawn for SRP and DOC
analyses. SRP samples (50 ml) were filtered through 0.2 µm nitrocellulose filters
and analyzed in duplicate. DOC samples (20 ml) were filtered through precombusted Whatman GF/F filters and frozen in acid-washed polypropylene
bottles for later analysis in triplicate.
Chlorophyll a samples were extracted in 90% acetone and measured by fluorescence (Welschmeyer, 1994). Bacteria were stained with acridine orange and
counted by epifluorescence microscopy (Daley and Hobbie, 1975). During days
0–32 of the model system experiment, cells of Scenedesmus were counted by
epifluorescence, while counting bacteria. For the entire natural assemblage
experiment, and after day 32 in the model system experiment, algae were counted
by the inverted microscope method (Utermöhl, 1931). Cell volumes for most of
the species observed in the natural assemblage experiment were determined from
measurements of linear dimensions under similar experimental conditions, or
from the literature for less common taxa. These were converted to estimates of
carbon biomass with a conversion factor of 0.2 pg C µm–3 (Rocha and Duncan,
1985). SRP was determined colorimetrically (Strickland and Parsons, 1972), as
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Resource competition in algae and bacteria
were other nutrients in lake samples. To analyze DOC, samples were oxidized
with persulfate in sealed ampoules, and the resulting CO2 was captured in a
stream of N2 gas, and determined by IR spectroscopy (Menzel and Vaccaro, 1964;
McDowell et al., 1987).
All data were plotted against time, and the steady state phase in each culture
was visually identified. We considered cultures to have reached a steady state
when transient exponential growth of cell numbers and chlorophyll a ceased, and
SRP and DOC concentrations stopped consistently decreasing. In the natural
assemblage experiment, algal biomass reached a steady state while slow trends of
increase or decrease of particular species occurred. Such trends are not analyzed
here. After identifying steady states, data from this phase were averaged within
each culture. Nutrient treatment effects were then assessed with an ANOVA for
a randomized complete block design, using the block treatment interaction as
an error term, and assuming block effects to be random (Neter and Wasserman,
1974). To reduce skew and heteroscedasticity, population data (cell density and
biomass, chlorophyll a) were transformed to logarithms prior to analysis.
When the ANOVA indicated that treatment effects were significant, five
planned linear contrasts were calculated to dissect treatment effects. Three of
these tested for effects of increased OC:P supply ratio: (1) cultures with no supply
of organic carbon versus those with an intermediate OC:P supply ratio (100 or
300); (2) cultures receiving no supply of organic carbon versus those with an OC:P
supply ratio of 600; and (3) cultures with an intermediate OC:P supply ratio
versus those with an OC:P supply ratio of 600. A one-tailed test was appropriate
for these contrasts because an increase was hypothesized a priori for some
response variables (DOC, bacteria, phagotrophs), and a decrease for others
(SRP, algae). Contrast (4) compared cultures receiving glucose versus those
receiving mixed organic substrates, and contrast (5) tested for interactions
between OC:P supply ratio and substrate type. For these contrasts, a two-tailed
test was appropriate because there was no hypothesized direction of response.
Variances of contrasts were computed from the contrasts calculated within blocks
(Steel and Torrie, 1980), and a t-test applied, with one degree of freedom (d.f.).
Using a test with low d.f. obviously pushes statistical analysis to its limits, but it
does serve to identify objectively the strongest treatment effects.
In the model community experiment, trends in population densities after
inoculating Ochromonas were analyzed. Natural logarithms of cell density were
regressed against time to estimate the net specific rate of population change (i.e.
the slope coefficient) and its standard error.
Results
Natural assemblage experiment
By day 24, after eight turnovers of culture volume, steady states of chlorophyll a
concentrations and bacterial densities were achieved in all cultures. Earlier, by
day 13, SRP was reduced to <0.2 µM in all cultures, indicating uptake of >80%
of supplied P by resident micro-organisms. SRP remained low throughout the
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J.P.Grover
Fig. 1. Steady state properties in the natural assemblage experiment. Labels on the horizontal axis
indicate experimental treatments: 0 – OC:P supply ratio of 0, no DOC supplied; 100G – OC:P supply
ratio of 100, glucose supplied; 100M – OC:P supply ratio of 100, mixed substrates supplied; 600G –
OC:P ratio of 600, glucose supplied; 600M – OC:P supply ratio of 600, mixed substrates supplied. All
treatments received a P supply of 1 µM. (a) SRP – measurements on days 25 and 35 are averaged;
bars show standard deviation. (b) DOC – measurement on day 31. (c) Chlorophyll a concentration
during steady state; geometric mean and standard deviation. (d) Bacterial density during steady state;
geometric mean and standard deviation. (e) Relative biomasses of phagotrophs (mixotrophs and
heterotrophs) during steady state; mean and standard deviation. (f–g) Abundances of three
phototrophs during steady state; geometric mean and standard deviation.
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Resource competition in algae and bacteria
experiment. Measurements of SRP were made twice during the steady state
phase, on days 25 and 35. Averaging these values within cultures (Figure 1a),
ANOVA did not indicate significant differences between treatments (F = 4.95,
P = 0.075).
Measurements of DOC were made once during the steady state phase, on day
31 (Figure 1b), and ANOVA indicated significant differences between treatments
(Table II). Results were consistent with the hypothesis that increased OC:P
supply ratio would increase DOC. Cultures with an OC:P supply ratio of 600 as
mixed substrates had 3–4 times the steady state DOC of other cultures, and those
with an OC:P supply ratio of 600 as glucose had up to about twice the DOC of
cultures with lower OC:P supply ratios. Contrasts (2) and (3) tested the differences between cultures with an OC:P supply ratio of 600 and cultures with lower
supply ratios, and were significant (Table II). The influence of OC:P supply ratio
on DOC concentration was complex, however. First, there was no difference
between cultures with OC:P supply ratios of 100, compared with those with no
organic C supply [contrast (1) insignificant, Table II]. Second, the type of C
substrate appeared to be important; the significance of contrast (4) suggests that
cultures supplied with mixed C substrates had higher DOC than those with
glucose only (Table II). Finally, this latter difference essentially occurred only in
the cultures with an OC:P supply ratio of 600, leading to significance of contrast
(5) for interaction of OC:P supply ratio and type of C substrate (Table II).
At steady state, chlorophyll a was several-fold lower in cultures with an OC:P
supply ratio of 600 than in other cultures (Figure 1c). Chlorophyll a concentrations were highly variable between replicates of these high OC:P cultures,
however, and treatment effects were not significant (Table II). Bacterial densities
were higher in three of four cultures with an OC:P supply ratio of 600 than in
other cultures (Figure 1d), but variance between replicates in this treatment
group was large, and treatment effects insignificant (Table II).
Flagellates capable of phagotrophy were common in these cultures. Most of
them were small (<10 µm greatest linear dimension) and appeared to belong to
chrysophyte genera such as Ochromonas, Poteriochromonas, Spumella and Paraphysomonas, which are principally or exclusively phagotrophic (Sandgren, 1988).
Definitive identification could not be made during routine counts, however.
Cryptophyte flagellates, also potential phagotrophs (Tranvik et al., 1989), were
present in many cultures at much lower densities than chrysophytes. Ciliates were
seen only rarely and were not counted. Phagotrophic flagellates sometimes
composed a large fraction of algal biomass (Figure 1e). Phagotrophs dominated the
two cultures receiving mixed organic substrates at an OC:P supply ratio of 600, and
were also relatively abundant (though highly variable) in one culture receiving
glucose at an OC:P supply ratio of 600. In this latter culture, eukaryotic organisms
were sparse and their counts highly variable. Phagotrophs had low relative abundance in the other culture receiving glucose at an OC:P supply ratio of 600, and
they ranged up to ~50% of algal biomass in cultures with lower OC:P supply ratios.
Overall, there were no significant treatment effects on the relative biomass of
phagotrophs analyzed after an arcsin–square root transformation (Table II).
The relative biomass of phototrophic algae is the complement of Figure 1e. In
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Table II. Summary of statistical tests for the natural assemblage experiment. The ANOVA F-tests had 4,4 d.f.; the contrast t-tests had 1 d.f. Contrast tests were
not carried out unless the ANOVA F-test was significant (P < 0.05). One-tailed tests are reported for contrasts (1)–(3), based on hypothesized responses to
DOC:P supply ratio. Hypothesized decreases of SRP, algae and phagotrophs are supported if contrasts (1)–(3) are positive. Hypothesized increases of DOC
and bacteria are supported if contrasts (1)–(3) are negative. Two-tailed tests are reported for contrasts (4) and (5)
ANOVA
Response
SRP
DOC
Chlorophyll a
Bacteria
Proportion phagotrophs
Oocystis pusilla
Chlorella vulgaris
Chlorella ellipsoidea
F
4.95
103.30
2.51
1.03
2.56
12.54
7.83
8.49
P
0.075
0.0003
0.20
0.49
0.2
0.016
0.036
0.031
Contrast (1)
Intermediate versus
zero DOC:P
Contrast (2)
High versus
zero DOC:P
Contrast (3)
Contrast (4)
High versus
Glucose versus
intermediate DOC:P mixed substrates
Contrast (5)
DOC:P substrate type
t
t
t
t
–
–2.17
–
–
–
4.30
2.34
2.93
P
–
0.12
–
–
–
0.075
0.13
0.11
–
–103
–
–
–
12.3
10.2
10.5
P
–
0.003
–
–
–
0.026
0.031
0.03
–
–40.9
–
–
–
14.9
284
41.8
P
–
0.008
–
–
–
0.022
0.001
0.008
t
–
16.6
–
–
–
13.5
12.7
7.60
P
–
0.038
–
–
–
0.047
0.050
0.083
–
–49.9
–
–
–
–2.62
–2.30
–2.46
P
–
0.013
–
–
–
0.23
0.25
0.25
Resource competition in algae and bacteria
all but one culture, chlorophytes composed at least 60% of algal biomass (excluding mixotrophs). Despite the insignificance of treatment effects for the biomass
of phototrophs in aggregate, at species level, the three most common phototrophs
were strongly affected by treatments. Oocystis pusilla was present in all cultures
and was the most abundant phototroph (by biomass) in six of 10 cultures. In the
four exceptions, either Mougeotia thylespora or Synedra acus was the most abundant phototroph, but as these species were found in only a few cultures their
responses to experimental treatments could not be examined. In addition to
O.pusilla, Chlorella vulgaris and Chlorella ellipsoidea occurred in all cultures,
though as relatively minor components of biomass.
These three phototrophic species all showed similar responses to treatments
(Figure 1f–h). Biomasses at steady state were lower in cultures with an OC:P
supply ratio of 600 than in other cultures, and were often lower when glucose,
rather than mixed organic substrates, was supplied. For O.pusilla, treatment
effects were significant (Table II). Its biomass was significantly lower in cultures
with OC:P supply ratios of 600 than in other cultures [contrasts (2) and (3),
Table II], and was also significantly lower in cultures supplied with glucose rather
than mixed organic substrates [contrast (4), Table II]. There was no interaction
between OC:P supply ratio and substrate type [contrast (5) insignificant,
Table II]. For C.vulgaris, treatment effects were also significant (Table II). The
pattern of contrasts was similar to that for O.pusilla; steady state biomass of
C.vulgaris was significantly lower in cultures with an OC:P supply ratio of 600
than in other cultures, and was also significantly lower with glucose rather than
mixed substrates [contrasts (2)–(4), Table II]. Treatment effects were significant
for C.ellipsoidea, and the pattern of contrasts was similar to that for the other
phototrophs (Table II), although insignificance of contrast (4) suggests only a
weak influence of glucose versus mixed organic substrates.
Model system experiment
By day 22, after six turnovers of culture volume, steady states of chlorophyll a
concentrations and bacterial densities were achieved in all cultures. Earlier, by
day 7 for all cultures, SRP was reduced to <0.2 µM and SRP remained at these
levels subsequently, indicating that resident micro-organisms took up >80% of
supplied P. For analyzing steady state responses to nutrient treatments, the period
from day 22 to 32 was used (on day 32, half the cultures were inoculated with
Ochromonas, disturbing the steady state). The one culture that ran only 28 days
was in steady state by day 18, so the last 10 days of its run were used in analysis.
SRP was measured twice during steady state. Averaging these two values, SRP
declined as the OC:P supply ratio increased (Figure 2a). Treatment effects were
significant, and contrasts (1)–(3) indicate that cultures with an OC:P supply ratio
of 600 had lower SRP than all other cultures, and that cultures with an OC:P
supply ratio of 300 had significantly lower SRP than cultures with no organic C
supply (Table III). Contrasts (4) and (5) for the effect of organic substrate type
and the interaction of substrate type and OC:P supply ratio were both insignificant (Table III).
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Fig. 2. Steady state properties in the model system experiment. Labels on the horizontal indicate
experimental treatments: 0 – OC:P supply ratio of 0, no DOC supplied; 300G – OC:P supply ratio of
300, glucose supplied; 300M – OC:P supply ratio of 300, mixed substrates supplied; 600G – OC:P ratio
of 600, glucose supplied; 600M – OC:P supply ratio of 600, mixed substrates supplied. All treatments
received a P supply of 1 µM. (a) SRP – measurements on days 24 and 32 are averaged; bars show
standard deviation. (b) DOC – measurements on days 24 and 32 are averaged; bars show standard
deviation. (c) Chlorophyll a concentration during steady state; geometric mean and standard
deviation. (d) Bacterial density during steady state; geometric mean and standard deviation.
DOC was also measured twice during steady state. Averaging these two values,
DOC increased as the OC:P supply ratio increased, and was also higher when
mixed organic substrates were supplied rather than glucose alone, and especially
so at an OC:P supply ratio of 600 (Figure 2b). This pattern led to significant treatment effects (Table III), and to significant effects of OC:P supply ratio [contrasts
(1)–(3)], organic substrate type [contrast (4)] and interaction between substrate
type and OC:P supply ratio [contrast (5)].
Steady state concentrations of chlorophyll a were at least 10-fold lower in
cultures with a supply of organic C than in cultures without (Figure 2c). In
cultures with an OC:P supply ratio of 600, Scenedesmus was essentially excluded;
chlorophyll a concentrations were near experimental blank values, and algal cells
were virtually absent (a few cells at most seen during microscopic counts). In the
cultures with an OC:P supply ratio of 300, chlorophyll a was lower in cultures
receiving mixed organic substrates than in those receiving glucose alone. Similar
patterns were observed in cell densities of Scenedesmus (data not shown).
Steady state densities of bacteria were ~5-fold higher in cultures with an OC:P
supply ratio of 600 than in cultures with no organic C supply, and intermediate in
the cultures with an OC:P supply ratio of 300 (Figure 2d). Treatment effects,
however, were insignificant (Table III).
1602
Table III. Summary of statistical tests for the model system experiment. The ANOVA F-tests had 4,4 d.f.; the contrast t-tests had 1 d.f. One-tailed tests are
reported for contrasts (1)–(3), based on hypothesized responses to DOC:P supply ratio. Hypothesized decreases of SRP and chlorophyll a are supported if
contrasts (1)–(3) are positive. Hypothesized increases of DOC and bacteria are supported if contrasts (1)–(3) are negative. Contrast tests were not carried out
unless the ANOVA F-test was significant (P < 0.05). Two-tailed tests are reported for contrasts (4) and (5)
Contrast (1)
Intermediate versus
zero DOC:P
Contrast (2)
High versus
zero DOC:P
Contrast (3)
Contrast (4)
High versus
Glucose versus
intermediate DOC:P mixed substrates
Contrast (5)
DOC:P substrate type
F
23.5
36.6
36.0
3.13
t
57.5
–6.54
38.1
–
t
t
t
P
0.005
0.002
0.002
0.15
P
0.006
0.048
0.008
–
22.2
–29.7
21.4
–
P
0.014
0.011
0.015
–
P
12.7 0.025
–12.9
0.025
8.56 0.037
–
–
t
P
0.40 0.76
25.6
0.025
–28.7
0.022
–
–
P
5.19 0.12
–19.2
0.033
–74.5
0.009
–
–
1603
Resource competition in algae and bacteria
Response
SRP
DOC
Chlorophyll a
Bacteria
ANOVA
J.P.Grover
Fig. 3. Trend analysis after inoculation of Ochromonas in the model system experiment. Experimental treatments labeled as in Figure 2. The net specific rate of change is shown for each population,
with open bars showing rates in cultures into which Ochromonas was inoculated, and shaded bars
showing rates in reference cultures with the same nutrient treatment; bars show standard errors. (a)
Ochromonas. (b) Bacteria. (c) Chlorophyll a. (d) Scenedesmus.
On day 32, an inoculum of Ochromonas was delivered to a randomly selected
replicate of each treatment type. Data from days 34–44 were then used to estimate trends in population variables as specific rates of change. In all cultures, the
estimated trend for Ochromonas was positive (Figure 3a), suggesting successful
invasion by this organism. Averaged over all cultures, the rate of increase of
Ochromonas was 0.31 day–1. Given a dilution rate of ~0.3 day–1, Ochromonas thus
appeared capable of sustaining a reproductive rate of ~0.6 day–1 under the range
of culture conditions used. There was large variation between cultures, and the
trend estimates are based on only three samples. Nevertheless, these trends
suggest that the mixotrophic strategy of Ochromonas was at least viable, and
perhaps highly beneficial in these cultures.
Results also suggest reliance by Ochromonas on phagotrophic nutrition in at
least some cultures. After inoculation with Ochromonas, rapid bacterial declines
(at net rates <–0.17 day–1) were observed in the culture with an OC:P supply ratio
of 600 and that with an OC:P supply ratio of 300 as mixed organic substrates,
while bacteria remained unchanged or increased slightly in corresponding reference cultures without Ochromonas (Figure 3b). After inoculation with Ochromonas, the bacterial decline was relatively modest (–0.07 day–1) in the culture
with an OC:P supply ratio of 300 as glucose, and bacteria increased slightly in the
culture with no supply of organic C. Either Ochromonas relied less on phagotrophic nutrition than on phototrophy in these cultures, or the bacteria reproduced at rates sufficient to compensate losses to phagotrophy, or both.
1604
Resource competition in algae and bacteria
In all cultures inoculated with Ochromonas, there was an increase in chlorophyll a, and there were no corresponding increases in reference cultures without
Ochromonas (Figure 3c). Some of this increase is due to the growth of
Ochromonas and indicates a potential for increased phototrophy associated with
the invasion of the mixotroph. Scenedesmus might also increase in cultures inoculated with a mixotroph, as bacterial competitors would be selectively ingested
and perhaps some of their P content recycled (Rothhaupt, 1996b). There was
some evidence for such an increase. In four cultures, the net growth rate of
Scenedesmus was higher in the presence of Ochromonas than in its absence, and
in three cultures with Ochromonas, Scenedesmus had positive net growth, while
net growth was zero or negative in all cultures without Ochromonas (Figure 3d).
Discussion
These experiments tested theoretical predictions of how nutrient concentrations
and microbial abundances would vary in response to defined supplies of DOC
relative to inorganic P (Bratbak and Thingstad, 1985; Thingstad and Pengerud,
1985). The experiments supported the predictions that as OC:P supply ratio
increases, (i) DOC increases, (ii) dissolved inorganic P decreases and (iii) algal
biomass decreases. Taken together, these results support hypotheses that bacteria
and phototrophic algae compete for inorganic P, and that bacteria are superior
competitors for inorganic P. Given these assumptions, a high OC:P supply ratio
relieves C-limitation of bacterial growth, allowing them to deplete inorganic P to
levels at which algae cannot replace themselves. With bacterial growth P-limited,
unconsumed DOC accumulates.
This interpretation is strengthened by further work with purified cultures of
Scenedesmus and Pseudomonas obtained from the model system (Codeco, 1998).
At steady state, chemostats of pure Pseudomonas supplied with OC:P from 126
to 629 had an approximately linear decline of SRP with OC:P, from 0.98 µM to
0.02 µM. After inoculation at low density into these cultures, Scenedesmus invaded
those with OC:P ≤ 422, but not the culture with OC:P = 629. In those cultures
where Scenedesmus invaded, it reached steady state biomasses that declined with
OC:P supply ratio, and the new steady state SRP ranged from 0.05 to 0.2 µM.
These dynamics strongly suggest that C-sufficient Pseudomonas reduces P below
the level required by Scenedesmus at steady state, while C-limited Pseudomonas
do not. Other experiments with bacteria and algae grown in chemostats also
support this scenario (Currie and Kalff, 1984a; Pengerud et al., 1987).
Broadly, the results obtained in this study agree sufficiently with earlier similar
studies to suggest a general response of planktonic algae and bacteria to gradients
of OC:P supply, mediated through a competitive mechanism. Nevertheless, the
results of such laboratory studies depend partially on conditions chosen by the
experimenter (e.g. temperature, irradiance). The conditions adopted in this study
are representative of the habitat where the natural assemblage was obtained (Joe
Pool Lake, Texas) at the time of sampling (autumn) (Sterner, 1994), but may be
less representative of other times of year or other habitats. In particular, dominance by chlorophytes of natural assemblages cultured continuously with low P
1605
J.P.Grover
supply has not often been observed [cf. (Sommer, 1983; Tilman and Kiesling, 1984;
Tilman et al., 1986)], and could be a particular outcome of these experimental
conditions.
The experiments reported here provide evidence that commensalism is also an
important mechanism in algal–bacterial interactions. In chemostats with no external supply of DOC, bacteria persisted at relatively high abundance. Many others
have observed this [e.g. (Currie and Kalff, 1984a; Bratbak and Thingstad, 1985;
Rothhaupt, 1992)] and indeed, the difficulty of maintaining bacteria-free cultures
of algae is well known. In the study presented here, cultures without a supply of
organic C had DOC concentrations of ~50–100 µM at steady state, most of which
can only have originated with algae. Dilution rates pose a lower bound to
turnover on this organic matter and on algal biomass, and given this, the
minimum DOC production by algae can be roughly estimated. On an absolute
basis, this was 15–30 µmol C l–1 day–1 in both the natural community and model
system experiment. Because the model system had lower algal biomass, its exudation rates relative to estimated net production are ~65%, as opposed to ~12% in
the natural community. These are comparable with the range of relative rates
reported from similar chemostat experiments [6–50% (Bratbak and Thingstad,
1985)], and in physiological studies [1–55% (Zlotnick and Dubinsky, 1989)].
Algal communities that excrete large fractions of fixed carbon as DOC may be
especially susceptible to competition from bacteria for inorganic nutrients.
Some theoretical predictions were not supported by the results reported here.
Bacteria did not consistently increase with the OC:P supply ratio. Such an
increase is predicted by theory that does not include phagotrophic predators of
bacteria (Bratbak and Thingstad, 1985; Thingstad and Pengerud, 1985). Possibly,
the failure of bacteria to show the predicted response in the natural assemblage
experiment is due to top-down control of bacteria by phagotrophs, which is
predicted by theory including the latter (Thingstad and Pengerud, 1985).
However, the predicted increase of phagotrophs with OC:P supply ratio was not
seen in the natural assemblage experiment. In the model system experiment,
during the time when phagotrophs were absent, there was a statistically insignificant tendency for bacteria to increase with OC:P supply ratio. Possibly, the
predicted increase in bacterial abundance was simply too weak to detect with an
experiment having few replicates.
On the other hand, genuine complexities in the biology of bacteria and
phagotrophs could frustrate the theoretical predictions. Bacteria make a number
of physiological adjustments to resource conditions (Vadstein and Olsen, 1989;
Chrzanowski and Kyle, 1996). Moreover, strong interactions based on allelopathy
or commensalism often modify the action of resource competition in bacteria
(Grover, 1997). The internal dynamics of bacterial populations and communities
could complicate the response of total cell numbers to resource conditions in
ways not anticipated by current theory.
Our understanding of the ecology of phagotrophs, including mixotrophs and
obligate heterotrophs, is currently limited. The model system of algae and
bacteria was open to invasion by mixotrophs, but it is not clear whether
mixotroph abundance at steady state would have shown any pattern in response
1606
Resource competition in algae and bacteria
to OC:P supply ratio. Pengerud et al. found co-existence of bacteria, phototrophic
algae and an obligate phagotroph over a range of OC:P supply ratios in
chemostats, with little influence of the supply ratio on their abundances
(Pengerud et al., 1987). Because DOC was high in their experiments, and
dissolved P undetectable, it appears that the OC:P supply ratios were not low
enough to induce C-limitation of bacteria. However, co-existence of bacteria,
phototrophic algae and an obligate phagotroph in chemostats with no organic C
supply has also been observed (Rothhaupt, 1992). Co-existence of mixotrophs,
phototrophs and bacteria, as well as co-existence of bacteria, mixotrophs and
obligate phagotrophs, have both also been observed under varied conditions of
mixed culture (Rothhaupt, 1996a). However, chemostat experiments with mixotrophs present across a range of OC:P supply ratios appear not to have been
carried out. Were such experiments to be carried out, they could help answer a
lingering question: can obligate phototrophs persist in systems where C-limitation
of bacteria does not occur, or is the only viable strategy to eat one’s bacterial
competitors for inorganic nutrients as suggested by Nygaard and Tobiesen
(Nygaard and Tobiesen, 1993)? Theory does predict that obligate phototrophs
could persist if phagotrophic organisms sufficiently restrain bacteria (Thingstad
et al., 1996), but the conditions for such co-existence are complex and as yet insufficiently tested.
The experiments reported here and those of Pengerud et al. (Pengerud et al.,
1987) show that in the absence of phagotrophic consumers of bacteria, rather high
OC:P supply ratios (>250–300 by moles) are required for the competitive exclusion of obligate phototrophs by bacteria. At high OC:P supply ratios, bacteria
switch from C- to P-limitation, and become formidable competitors with algae.
The switch point occurs at an OC:P supply ratio higher than the cellular OC:P
ratio of P-limited bacterial cells [up to 150–160 (Vadstein and Olsen, 1989;
Chrzanowski and Kyle, 1996)] because slow-growing, P-limited bacteria have
high respiratory demands for C (Thingstad, 1987).
The switch from C- to P-limitation of bacteria may be influenced by the qualitative nature of DOC substrates. For most responses measured here, the effect
of substrate type (glucose versus mixed substrates) was small relative to that of
OC:P supply ratio. However, DOC concentrations at steady state were much
higher when mixed substrates rather than glucose were supplied at the highest
OC:P ratio. The mixed substrates may include material more refractory than
glucose. Alternatively, bacteria receiving mixed substrates might have had a
lower C demand, relative to P demand, than those metabolizing glucose. The
substrate mixture contained substantial reduced N, while the bacteria supplied
with glucose were forced to rely on nitrate. The energetic costs of reducing nitrate
could account for the higher C demand of bacteria supplied only with glucose. If
DOC substrates have an N composition suited to bacterial needs, the switch to
P-limitation might occur at an OC:P supply ratio lower than ~300.
The results found here and in similar studies suggest that high supply ratios of
labile DOC to inorganic P will characterize any natural habitats in which
phototrophs and bacteria are simultaneously P-limited and compete. Such habitats are predicted to have bacterial dominance over algae and an accumulation
1607
J.P.Grover
of DOC, despite such heterotrophic dominance. Crude calculations suggest that
some fresh waters may have sufficient organic C supply for this to occur. Along
a gradient from oligotrophic to eutrophic lakes, total P concentration ranges from
0.1 to 30 µM (Rast et al., 1991), while DOC concentrations range from 300 to
2000 µM C (Jørgensen, 1986). Conservatively assuming that 15% of DOC is
readily available to bacteria (Jørgensen, 1986), the molar ratio of labile
DOC:total P ranges up to ~500 in oligotrophic lakes and tends to be lower in
eutrophic lakes. If supply ratios are similarly high, bacterial dominance over algae
due to competition for P could result. Chrysophytes, an algal group in which mixotrophy is common, are most abundant in oligotrophic lakes with low P supplies
(Kalff and Watson, 1986; Sandgren, 1988). This pattern may be consistent with
the hypothesis that obligate phototrophy is a less viable strategy than mixotrophy
or heterotrophy when the OC:P supply ratio is high. Marine coastal sites with high
riverine inputs also seem to have P-limited bacteria and accumulation of DOC
(Zweifel et al., 1995; Thingstad et al., 1998), and this process also appears seasonally in oceanic sites (Cotner et al., 1997). The gradient of resource conditions
produced in laboratory studies such as those presented here may help illuminate
the mechanisms structuring microbial communities in such natural habitats.
Acknowledgements
I am grateful to A.J.Downey III, K.Massey, C.Napier, and especially D.Hinshaw
and J.L.Robinson, for technical assistance. T.H.Chrzanowski provided a subculture of Ochromonas, and much helpful advice on many aspects of this study.
J.Cole and three anonymous reviewers provided helpful comments on an earlier
draft of this paper. Funding was provided by NSF grant number DEB-9418096.
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Received on August 30, 1999; accepted on March 23, 2000
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