Download CARON, DAVID A., KAREN G. PORTER, AND ROBERT W

Notes
433
phate uptake: Are larger cells better competitors
for pulses of phosphate than smaller cells? Oecologia 74: 57 l-576.
TURPIN, D. H., AND P. J. HARRISON. 1980. Cell size
manipulation in natural marine, planktonic, dia318: 55-58.
tom communities. Can. J. Fish. Aquat. Sci. 37:
SAKSHALJG,
E., AND 0. HOLM-HANSEN. 1977. Chem1193-I 195.
ical composition of Skeletonemu cost&urn (Grev.)
WHEELER, P. A., AND D. L. KIRCHMAN. 1986. UtiCleve and Pavlova (Monochrysis) lutheri (Droop)
lization of inorganic and organic nitrogen by bacGreen as a function of nitrate-, phosphate-, and
teria in marine systems. Limnol. Ckeanogr. 31:
iron-deficiency. J. Exp. Mar. Biol. Ecol. 29: l-34.
998-1009.
SUTTLE,C. A., J. G. STOCIUVER,
AND P. J. HARRISON.
1987. Effects of nutrient pulses on community
structure and cell size of a freshwater phytoplankton assemblage in culture. Can. J. Fish. Aquat.
Submitted: 7 June 1989
Sci. 44: 1768-1774.
Accepted: 4 October 1989
-,
-,
K. S. SHORTREED,
AND P. J. HARRISON.
1988. Time-courses of size-fractionated phosRevised: 4 December 1989
et al. [eds.], Tidal mixing and plankton dynamics.
Springer.
PLATT. T.. AND W. G. HARRISON. 1985. Biogenic
fluxes’of carbon and oxygen in the ocean. Nature
Limnd.
Oceanogr..
35(Z), 1990,433-443
Q 1990, by the American Society of Limaolcgy and Ocean@raphy, Inc.
Carbon, nitrogen, and phosphorus budgets for the mixotrophic
phytoflagellate Poterioochromonasmalhamensis(Chrysophyceae)
during bacterial ingestion
Abstract-Growth, grazing, C and nutrient incorporation by the mixotrophic phytoflagellate
Poterioochromonas malhamensis were examined
under various nutrient and light regimes in the
presence of heat-killed bacteria. Poterioochromonas malhamensis readily ingested bacteria in
all culture treatments containing heat-killed bacteria, and growth rates of the protist were much
greater for heterotrophic (bacterivorous) growth
than for phototrophic growth. C incorporation
efficiencies by the phytoflagellate were virtually
identical to P incorporation efficiencies during
heterotrophic growth, but N incorporation efficiencies were somewhat lower in nearly all of the
treatments. Algal growth in cultures with heatkilled bacteria was similar in continuous darkness and in continuous light. Bacterial organic C
was the primary source of protist cellular C for
P. malhamensis during heterotrophic growth.
Based on our observations, we conclude that P.
malhamensis is primarily heterotrophic and
phagotrophic and that it is highly competent in
this mode of nutrition.
Acknowledgments
We are grateful to Kay Ho, Harold King, Douglas
Leeper, and Ee Lin Lim for technical assistance.
The research was supported by NSF grants BSR 8620441 (to KG.P.) and BSR 86-20443 (to D.A.C.).
Contribution 7273 from Woods Hole Oceanographic Institution. Contribution 46 from Lake Oglethorpe
Limnological Association.
Phagotrophic phytoflagellates occur within a number of algal orders. Although algal
phagotrophy has been known for many years
(see Sanders and Porter 1988) only recently
have the ecological implications of this behavior been recognized. There are at least
two important implications of phagotrophy
by photosynthetic
protists. This behavior
may constitute an important source of predation on bacteria and other protists in
aquatic ecosystems. It is now well established that flagellated heterotrophic protists
are important consumers of bacterial and
algal picoplankton in marine and freshwater
communities (e.g. Azam et al. 1983; Sherr
et al. 1983; Davis and Sieburth 1984; Caron
et al. 1985; Sanders et al. 1989). More recently, mixotrophic chrysophytes, exhibiting both photosynthetic and phagotrophic
activity, have been identified as potentially
important bacterivores in some marine and
freshwater ecosystems (e.g. Estep et al. 1984;
Porter et al. 1985; Bird and Kalff 1986; Borass et al. 1988; Porter 1988; Salonen and
Jokinen 1988). Based on the results of these
studies, there is a growing realization that
the magnitude of predation by mixotrophic
algae mav have been overlooked in previous
434
Notes
studies. The potential importance of this
behavior in most natural communities remains largely unknown.
Phagotrophy also may constitute an important nutritional strategy for mixotrophic
algae. The ingestion of particulate organic
material may be a mechanism for the acquisition of organic C, major nutrients (N
and P) or other factors (e.g. vitamins, essential amino acids) required for growth.
The ingestion of particulate organic C contributes significantly to the total C budgets
of some mixotrophs (Bird and Kalff 1987,
1989; Andersson et al. 1989). For autotrophic flagellates, however, supplementing
the uptake of inorganic N or P by grazing
is potentially more important than organic
C nutrition because these organisms are capable of producing organic C photosynthetically. Whether particle ingestion could
be an important source of these elements in
environments with low concentrations of
dissolved nutrients and whether the nutrients contained in food particles could be
utilized more efficiently by these algae than
by heterotrophic protists have not yet been
examined.
We examined these latter questions by
comparing the C, N, and P budgets of the
phagotrophic phytoflagellate Poterioochromonas malhamensis (Pringsheim) Peterfi
growing under various culture conditions.
Poterioochromonas
malhamensis is an 81O-pm spherical, flagellated unicellular
chrysophyte. It was obtained from the University of Texas algal collection (stock number UTEX L 1297). Stock cultures were
maintained in the light at 20°C on f/2-enriched distilled water (Stein 1973) or on organically enriched medium consisting of 1.O
g liter-’ each of glucose, tryptone, yeast extract, and liver extract (Starr 1978).
Heat-killed bacteria were used in all treatments involving bacteria in this study in
order to circumvent the complications of a
live bacterial population releasing or assimilating nutrients during incubations. In this
way, all C and nutrient cycling in the cultures was attributable to the protist. Bacterial activity is a potentially important consideration
for experiments
designed to
examine C and nutrient cycling by photo-
synthetic and phagotrophic protists (Giide
1985; Caron et al. 1988). The uptake of nutrients by live algae also can cause problems
in nutrient cycling experiments where algae
are used as prey, even if the grazing phase
of these experiments is carried out in the
dark (Goldman et al. 1987).
An axenic freshwater bacterial isolate,
Pasteurella sp. (size, -0.647
x 1.5-2.0
pm), was grown at 20°C on a shaker table
in 0.2% yeast extract in distilled water.
Three-day-old,
stationary growth-phase
cultures of the bacterium were heat killed
at 70°C for 30 min. The effectiveness of heat
killing was verified by trial and error at several temperatures and for several incubation
times by testing the viability of the resulting
bacterial suspension in 0.5% yeast extract
broth and on nutrient agar plates. Heatkilled bacteria were centrifuged in sterile,
250-ml polycarbonate centrifuge flasks with
caps in place at 12,000 rpm for 15 min at
5°C. The bacteria were resuspended in sterile distilled water, centrifuged, and resuspended again a total of three times.
Heat-killing
Pasteurella sp. resulted in
some loss of bacterial biomass from the cell
suspension (Table 1). Relative to the live
culture, equivalent volumes of the cell suspension were -20% lower in particulate C,
N, and P immediately after the procedure.
After rinsing and resuspension, however, the
particulate and dissolved constituents in the
cultures remained virtually unchanged for
7 d (length of the experimental incubations
with P. malhamensis). Also, the C: N and
C : P ratios of the heat-killed bacterial cells
were not substantially different than these
parameters for the live culture and were
consistent with the ranges of these values
for live bacteria from natural assemblages
(Bratbak 1985; Nagata 1986). The concentrations of NH,+ and soluble reactive phosphorus (SRP) were high in this test treatment because the bacteria were rinsed only
once. Bacteria used in the experimental
treatments were rinsed three times, and the
residual concentrations of these dissolved
constituents were therefore much lower in
the stock suspension of dead bacteria. Bacterial density and the concentrations of particulate and dissolved constituents were vir-
Notes
435
Table 1. Changes in the particulate and dissolved constituents during heat killing of the freshwater bacterial
isolate Pasteurellu sp. The C and nutrient constituents measured were soluble reactive P (SRP, rg liter-‘), total
dissolved P (TDP, mg liter-‘), particulate P (PP, mg liter-‘), ammonium (NH,+, pg liter-‘), particulate N (PN,
mg liter-‘), and particulate C (PC, mg liter-‘). PP, PN, and PC values were much lower after rinsing because of
dilution and filtration through a LO-Nrn filter.
Sampling
time (d)
Before killing
After killing,
before rinsing
After killing and
1 rinse (T = 0)
1 d after rinse
4 d after rinse
7 d after rinse
Particulate
C:N:Pratio
@Y w
PP
11,100
36.8
7.43
7,500
45.9
182
-
10,900
37.1
6.13
7,300
36.9
143
-
25~6.2: 1
23:6.0: 1
1.44
1.41
1.40
1.86
440
450
500
460
13.0
13.3
12.1
12.9
1.17x10~~0.57
2.42 x 10%0.44
2.30x 10*+0.25
37 : 9.0 : 1
40 : 9.4 : 1
35:8.6:1
29 : 6.9 : 1
38.1
16.8
21.1
21.6
0.14
0.10
0.10
0.11
PN
Bacterial density
(No. ml-l)
TDP
SRP
NH,*
tually unchanged over the length of the
experiment in a control culture of bacteria
not inoculated with the alga.
The heat-killed bacteria were resuspended in a basal salts culture medium (Sanders
et al. 1990) after the final rinse, filtered aseptically through a sterile 5-pm Nuclepore filter with a sterile filter assembly to remove
clumped cells, and diluted to a final concentration in those treatments containing
bacteria. Sterility tests (culture in 0.5% yeast
extract in distilled water) were performed
on the stock suspension of heat-killed bacteria initially and on all cultures during sampling and at the end of the experiment to
ensure that no viable bacteria were present
in any of the cultures.
Light intensity, inorganic nutrient concentration, and the availability of bacteria
were tested for their effects on the nutritional mode (heterotrophy vs. phototrophy)
of P. malhamensis. Five basic experimental
treatments were used, consisting of various
nutrient
and bacteria additions
to an
N-free and P-free inorganic basal medium
(see Sanders et al. 1990). This basal medium
was enriched with bacteria only (no inorganic N or P added), bacteria and NH,+ (no
inorganic P added), bacteria and POd3- (no
organic N added), bacteria and NH,+ and
POd3-, or NH,+ and POd3- (no bacteria
added). Control cultures consisted of heatkilled bacteria in basal medium (not inoculated with P. malhamensis), an N-free and
P-free algal culture, an algal culture with
NH4+, POd3-, and vitamins but without bac-
PC
53.0
56.0
48.4
53.1
teria, and an algal culture in organically enriched medium (see above) without bacteria.
Concentrations of NH,’ and POd3- were 200
PM and 20 PM after they were added to the
culture medium.
For all treatments, inocula from a phototrophically growing culture of P. malhamensis in bacteria-free f/2-enriched
distilled water were transferred to 900-ml tissue
culture flasks containing 800 ml of culture
medium. Flasks of each of the five experimental treatments were incubated at 20°C
with gentle shaking in continuous low or
high light intensity (4-10 or 400-500 rmol
quanta rnpz ss’) and in continuous darkness
(a total of 15 experimental and 3 control
flasks). Samples incubated in continuous
darkness were wrapped in foil and sampled
in almost total darkness.
Samples for bacterial and algal cell counts
and chemical analyses were removed aseptically at t = 0 and at intervals of several
hours for a period of -7 d in each experimental treatment. Cell densities of the bacterium and the microalga were determined
with epifluorescence microscopy on acridine orange- or DAPI-stained samples preserved with 1.O% glutaraldehyde (Porter and
Feig 1980; Davis and Sieburth
1982).
Growth rates of the alga in the various treatments were determined from regressions of
the linear portion of the increase in the natural Iog of cell density during exponential
growth of the alga in each treatment.
Particulate C (PC) and particulate N (PN)
were analyzed with a Perkin Elmer 240 el-
436
Time (h)
0
50
100
0
150
Time (h)
100
150
Time (h)
Fig. 1. Representative changes in the population densities,
SRP, and DOP in cultures of Poterioochromonas malhamensis
medium for this treatment was the basal salts medium with
were incubated at high light intensity. A. Changes in bacterial
in PN and NH,‘. D. Changes in SRP, PP, and DOP.
emental analyzer on samples retained on
precombusted Gelman GF/F glass-fiber filters. NH,+ concentration was measured in
the filtrate from the PC/PN samples with
standard calorimetric procedures (Parsons
et al. 1984). SRP, total dissolved P (TDP),
and particulate P (PP) were determined with
procedures outlined by Parsons et al. (1984),
with modifications for TDP and PP according to Menzel and Cot-win (1965). SRP was
determined on unfiltered samples. PP was
determined as SRP after persulfate oxidation of samples filtered onto Gelman GA-8
Supor filters (0.2~pm pore size). TDP was
determined as SRP (after persulfate oxidation) in the filtrate of the latter samples. The
50
and in the concentrations of PC, PN, PP, NH,‘,
containing heat-killed Pasteure[la sp. The culture
no inorganic nutrients (N or P) added. Cultures
and algal density. B. Changes in PC. C. Changes
concentration of dissolved organic P (DOP)
was calculated as TDP - SRP. Two complete sets of samples from the 1.5 experimental flasks also were analyzed for SRP
after persulfate oxidation of unfiltered samples. The resulting “total” P concentrations
were highly consistent with the sum of the
SRP, TDP, and PP measurements for these
same samples determined by the procedure
described above.
Phagotrophy was the dominant nutritional mode of P. malhamensis under all experimental conditions when bacteria were
present at densities >, lo6 ml-’ (Fig. 1). Poterioochromonas malhamensis grew rapidly
only in the presence of bacteria or in or-
Notes
437
Table 2. Specific growth rates (d-l) of Poterioochromonas malhamensis cultured under 17 different light
and nutrient conditions and in the presence and absence of heat-killed bacteria. Numbers in parentheses
are averages for all cultures with bacteria at each light
intensity.
Light regime
culture
treatment
Bacteria only
Bacteria + NH,+
Bacteria + POZBacteria + NH,+
+ PO,>NH,+ + PO,‘(bacteria-free)
NH,+ + PO,‘+ vitamins
(bacteria-free)
Organically enriched
medium
(bacteria-free)
Avg of cultures with
bacteria
m
0
100
50
150
Time (hl
Fig. 2. Summary of the changes in algal and bacterial density in 12 cultures ofPoten’oochromonns maihamtwsis with heat-killed Pusteurelfa sp. Data from
the algal cultures with heat-killed bacteria from all four
nutrient regimes and all three light regimes are included.
ganically enriched medium (Table 2). Furthermore, algal growth rates were virtually
identical in all treatments containing bacteria (Fig. 2). Bacterivorous growth of the
alga was more than an order of magnitude
more rapid than growth in inorganic medium without bacteria. Rapid, exponential
growth ceased as the density of bacteria in
the cultures decreased to 0.5-l .O x lo6 ml-’
(Figs. 1A, 2A).
Bacterivorous growth in P. malhamensis
was rapid, and chloroplast autofluorescence
was just barely visible by epifluorescence
light microscopy. When bacteria were reduced below - lo6 ml-‘, however, rapid algal growth ceased, giving the appearance of
a stationary growth phase (Fig. 2A). Algae
cultured in the light became light green dur-
Hi%
Low
Dark
1.8
1.5
1.8
1.6
1.6
1.6
1.5
1.5
1.5
1.7
1.5
1.5
CO.1
KO.1
(1.6)
(1.5)
co.1
CO.1
1.5
(1.7)
ing this growth phase, chloroplast autofluorescence became readily apparent by epifluorescence microscopy, and clumping of
the cells was visible macroscopically. Algal
cells in the inorganic medium without bacteria remained autofluorescent and viable
in the light, but changes in population density were negligible throughout the experiment.
Bacterivory by P. malhamensis was the
primary mode of nutrition in all experimental treatments containing bacteria. Light
had virtually no effect on the growth rate of
P. malhamensis with heat-killed bacteria
present in the medium (Table 2). The average algal growth rates in the bacterized
cultures were 1.7, 1.6, and 1.5 d-’ for cultures incubated at high light intensity, low
light intensity, and in continuous darkness
(four treatments in each light regime). These
small differences in average growth rate are
within the variability associated with the
method used to calculate growth rates. The
growth rate of the alga in culture vessels
with bacteria also was unaffected by the concentrations of inorganic N (NH,‘), inorganic P (POd3-), or the addition of vitamins.
The similarity of the growth curves of the
alga in the bacterized cultures, and of the
losses of bacteria from these cultures, in-
Notes
438
Table 3. Maximal ingestion (bacteria alga’ h-l) and
clearance (nl alga-’ h-l) rates for P. malhamensis on
heat-killed bacteria (Pasteurella sp.) in the presence of
various nutrient enrichments. (Nutrient concentrations given in text.)
T~~t~Wlt
High light
Bacteria alone
Bacteria + NH.,+
Bacteria + PO,+
Bacteria + NH,+ + PO,)Low light
Bacteria alone
Bacteria + NH.,+
Bacteria + POa3Bacteria + NH,+ + PO,‘Continuous darkness
Bacteria alone
Bacteria + NH,+
Bacteria + POa3Bacteria + NH.,+ + POd3-
Ingestion
Clearance
103
77
77
49
0.8
0.9
0.8
0.9
64
94
47
70
0.6
0.9
0.9
0.5
72
52
57
83
0.9
0.6
0.8
0.6
dicated the ineffectiveness of inorganic nutrient concentrations or light in altering the
bacterivorous nature of P. malhamensis (Fig.
2). Growth curves for the alga were virtually
identical in all treatments containing heatkilled bacteria. The patterns of disappearance of the bacteria in these cultures also
were identical, indicating that grazing rates
were similar in all treatments.
“Maximal”
ingestion (bacteria consumed
alga-’ h-l) and clearance rates (nl of water
filtered alga-’ h-l) were calculated according
to the equations of Heinbokel (1978) for
each experimental treatment. These rates
were based on changes in the cell densities
of the bacteria and algae between consecutive samples. The highest rates calculated
for each parameter were taken as maximal.
In all cases, maximal ingestion rates occurred during the first sampling interval
during which the bacterial density decreased significantly.
Maximal
clearance
rates occurred over the last sampling interval during which the bacterial density decreased significantly. The maximal rates of
bacterivory by P. malharnensis were high
in all cultures containing bacteria (Table 3).
The presence or absence of light, inorganic
N, inorganic P, or vitamins had no consistent effect on bacterivory by P. malhamensix The overall average for maximal inges-
tion rate was 70 bacteria alga-i h-i for
cultures with bacteria.
No significant changes in PC, PN, PP,
NH4+, SRP, or DOP occurred during the
experimental period in the vessels with heatkilled bacteria but not inoculated with P.
malhamensis. In contrast, the concentrations of PC, PN, and PP in the cultures of
P. malhamensis with bacteria decreased
rapidly during exponential growth of the alga
(e.g. Fig. lB,C,D). Concentrations of NH4+,
SRP, and DOP increased concomitantly.
Most of the bacterial N and P released by
the alga during bacterivory was eventually
released as NH,+ and SRP (Fig. lC,D). In
addition, however, some of the C, N, and
P in the consumed bacterial biomass was
released by the alga as dissolved organic material. Dissolved organic C and N were not
determined, but small amounts of DOP (=
TDP - SRP) amounting to lo-20% of the
PP concentrations were measured in all algal cultures with heat-killed bacteria (Fig.
1D). We assume that a similar proportion
of DON was released because, on average,
the amount of NH,’ appearing in the cultures was lO-25% less than the amount of
PN disappearing from the cultures (e.g. in
Fig. lC, the loss of PN x 1.40 - 0.35 =
1.05 mM, the increase ofNH,+ z 0.70 mM,
1.05-0.70=0.35,and0.35+1.40=25%).
It is possible that some dissolved organic
material released by the alga was reassimilated and remineralized by the alga during
the experimental period. It is also possible,
however, that some of the observed dissolved organic material in these cultures may
have been due to cell breakage during filtration rather than to algal excretion. It is
difficult, therefore, to estimate the exact importance of dissolved organic material in
these cultures. Nevertheless, the overall
outcome is that most of ,the bacterial biomass released as dissolved material is eventually released in remineralized form.
Carbon and nutrient incorporation
efficiencies of P. malhamensis (expressed as
percent) feeding phagotrophically
on bacteria were calculated from the concentration
of PC, PN, and PP in the algal cultures at
the end of exponential growth of the algae
(when algal biomass highly dominated the
particulate fraction) divided by these con-
Notes
centrations at the beginning of the experiment (when bacterial biomass constituted
all of the particulate fraction). It was assumed in these calculations that the contribution of bacterial biomass and defecated
particulate material in the cultures at the
end of the exponential growth phase of the
alga was negligible. Although the former was
certainly true (bacterial biomass remaining
in the cultures at the end of the exponential
growth phase was calculated to be less than
a few percent of the particulate material),
significant amounts of defecated particulate
material may have been present. Therefore,
incorporation
efficiencies cannot be truly
equated with growth efficiencies in these
cultures. We estimate that our incorporation efficiencies may have overestimated the
true growth efficiencies by not more than
10%.
Relatively large percentages of the prey
C, N, and P were incorporated into algal
biomass during bacterivorous growth of P.
malhamensis. Overall, the incorporation efficiencies of the alga for bacterial N, C, and
P were 33, 43, and 45% at the end of bacterivorous growth (Fig. 3). The average incorporation efficiencies for C and P were not
significantly different from one another, but
these latter efficiencies were significantly different from the average N incorporation efficiency. The N incorporation efficiency was
less than the C incorporation
efficiency in
all treatments with bacteria and less than
the P incorporation efficiency in all of these
treatments except one.
There were no apparent correlations between the incorporation
efficiencies of P.
malhamensis in bacterized cultures and the
type of experimental treatment (i.e. with
light regime or nutrient additions). Average
N, C, or P incorporation
efficiencies were
comparable for the four experimental treatments with bacteria in each of the three light
regimes (Table 4). Likewise, incorporation
efficiencies averaged for all bacterized treatments in each of the three light regimes were
similar for the four types of bacterized treatments (Table 4). Thus, neither light nor the
presence of dissolved inorganic nutrients
significantly affected the growth efficiency
of the alga when it was consuming bacteria.
Poterioochromonas malhamensis contin-
439
60
q
Nitrogen
P 50
B0
40
El
ig
30
3
t
2o
24
10
n
Exponential
Algal Growth
stationary
Phase
Fig. 3. Average incorporation efficiencies of Poterioochromonas malhamensis fed heat-killed Pasteurella
sp. in 12 cultures at the time of maximal algal density
(“exponential”
phase) and 2 d after the depletion of
bacterial prey (“stationary” phase). Error bars are f 1
SD.
ued to remineralize significant amounts of
N, P, and C even after rapid population
growth stopped (Fig. 3). The C, N, and P
incorporation
efficiencies of the alga decreased an average of 13% for all bacterized
cultures between the time of maximal algal
density (the end of bacterivorous growth)
and 2 d after the onset of the stationary
growth phase. The losses of C or nutrients
during this phase were not related to the
light regime and presumably were due to
cannibalism or the metabolism of cellular
reserves by the alga. Strictly speaking, these
losses during this growth phase no longer
represented incorporation efficiencies of the
alga because the bacterial prey already had
been exhausted.
We hypothesized that P. maIhamensis
might incorporate a larger percentage of the
N or P from the bacterial biomass when
these nutrients were not added to the culture
medium. Alternatively,
we hypothesized
that the presence of high concentrations of
inorganic nutrients might depress the rate
of bacterial ingestion by the alga because
these nutrients would be readily available
as dissolved constituents. Neither of these
possibilities occurred. These results for P.
malhamensis contradict a report of enhanced predation in the absence of growth-
Notes
440
Table 4. Summary of C, N, and P incorporation efficiencies (mean % + 1 SD) of Poterioochromonas malhamensis during bacterivorous growth in three different light regimes and four nutrient conditions (total of 12
treatments). Four treatments were performed in each light regime (the four nutrient conditions) and three light
regimes were used for each nutrient condition (high light, 400-500 pmol quanta me2 s-l; low light, 4-10 rmol
quanta rndz s-l; and continuous darkness).
Light regime
Nutrient
High
Low
Carbon
Nitrogen
Phosphorus
44+7
38+6
41k8
42-t3
31k4
44+4
limiting,
dissolved inorganic nutrients
closely related species of Ochromonas
Nutrient
continuous
darkness
43k2
3Ok2
49*7
by a
(Keller et al. 1988), but they agree with the observations of Andersson
et al. (1989) for
another Ochromonas species in which heterotrophy was the primary mode of nutrition and in which bacterivory was not important
in nutrient
acquisition
for
photosynthetic growth. Based on the large
percentage of the bacterial C that was incorporated into the alga, it is improbable
that P. malhamensis consumed bacteria
solely as a source of major nutrients or solely as a source of some specific growth factor.
Instead,
P. malhamensis
apparently
switched nearly completely from phototrophic growth to heterotrophic growth when
sufficient bacteria were present in the culture medium. The processes involved in
switching between phototrophy and heterotrophy in this species are examined elsewhere (Sanders et al. 1990).
Phototrophy contributed negligibly to the
growth of P. malhamensis in this study. Algal densities in cultures containing inorganic nutrients and vitamins did not change
significantly during the experiment. Previous studies and our own work have demonstrated that P. malhamensis is capable of
limited phototrophic growth (Handa et al.
198 1). The phototrophic growth rate of P.
malhamensis was slightly greater in an acidic medium (Sanders et al. 1990) but bacterivory was the main source of C for the
alga regardless of the pH of the medium or
the light regime. In this study, phototrophic
nutrition of the alga was important only for
survival in the absence of bacterial biomass
that was sufficient for heterotrophic growth.
Bacteria only
41k2
31*5
48k4
Bacteria
+ N
45*4
33*9
49k6
condition
Bacteria
+ P
42k5
32k4
43*11
Bacteria
+N+P
45*4
35+7
40+5
Overall, the behavior of P. malhamensis
in bacterized cultures was highly analogous
to results that have been obtained with
purely heterotrophic nanoflagellates. Maximal ingestion rates and clearance rates of
P. malhamensis observed in this study are
within the range of rates published for some
heterotrophic flagellates of similar size, although they are certainly not the highest
rates ever observed (e.g. Fenchel 1986; Davis and Sieburth 1984; Geider and Leadbeater 1988). The C, N, and P incorporation
efficiencies of the alga during bacterivory
also were comparable to the incorporation
efficiencies of some obligately heterotrophic
nanoflagellates (e.g. Kopylov et al. 1980;
Fenchel 1982a; Sherr et al. 1983; Geider
and Leadbeater 1988).
In conceiving this experiment, we hypothesized that the concentration
of dissolved inorganic nutrients in the bacterized
cultures without NH,+ or POd3- might remain low if P. malhamensis was an efficient
mixotroph:
that is, if the nutrients contained in the ingested bacterial biomass were
efficiently retained by the alga and converted into algal biomass by using photosynthesis to supplement the C budget of the
cell. This result was not observed. The retention of N in the particulate fraction during grazing by P. malhamensis on average
was less than the retention of C (or P) and
was not affected by NH,+ availability (Fig.3;
Table 4). This result is consistent with expectations for a purely heterotrophic nanoflagellate
based on the relatively
low C : N
ratio of bacterial biomass (Table 1; see Nagata 1986; Lee and Fuhrman 1987) the percentage of C incorporated by P. malhamen-
Notes
sis, and the expected C : N ratio of protist
biomass (Goldman et al. 1987). It is also
consistent with the observation that heterotrophic nanoflagellates apparently catabolize protein-rich compounds more readily
than other cellular components (Fenchel
1982b; Caron et al. 1985; Goldman et al.
1985; Andersen et al. 1986).
In general, the cycling of C and nutrients
by the chrysophyte alga P. malhamensis was
very similar to previous studies on nutrient
and C cycling by the omnivorous (but obligately heterotrophic)
marine nanoflagellate, Paraphysomonas imperjbrata (Goldman et al. 1985, 1987; Caron et al. 1985;
Andersen et al. 1986). The relative proportions of N, P, and C incorporated and remineralized by the alga were very similar
to patterns observed in C and nutrient cycling by P. imperforata (Fig. 3). The proportions of particulate C, N, and P retained
in the particulate
fraction during algal
phagotrophy indicate an inability by the alga
to retain “excess” nutrients contained in its
prey for subsequent use during phototrophic growth.
The release of remineralized N and P from
the particulate fraction of the P. malhamensis cultures in the light even after the
bacterial population was depleted and autofluorescence
increased is particularly
striking (Fig. 3). This reduction may indicate the “autophagy”
of cellular components during the stationary growth phase of
the species, similar to results obtained for
Ochromonas (Fenchel 1982a). Alternatively, this result may indicate a shift to a cannibalistic mode of nutrition once bacterial
prey were depleted. This latter alternative
is highly probable because we frequently observed cannibalism by this alga, and the high
density of algal cells in these cultures at the
end of the experimental period increases the
probability of encounters between the cells.
The tendency of the alga to cannibalize other algal cells rather than rely on photosynthesis for growth further indicates the importance of phagotrophy for the nutrition
of this species.
The information
that we have obtained
on the mixotrophic
chrysophyte P. malhamensis indicates that this alga strongly
favors phagotrophy as its primary mode of
441
nutrition. Under all conditions tested, P.
malhamensis rapidly ingested bacteria when
they were present in the culture medium at
densities > 1O6 ml-*. The alga is a highly
competent bacterivore and should be able
to compete successfully with other bacterivorous protists in planktonic communities.
Under conditions that will support phagotrophy of P. malhamensis, it supplies organic C and major nutrients for growth. In
these situations, C and nutrient incorporation and remineralization
are analogous to
C and nutrient cycling by truly heterotrophic protists.
Poterioochromonas
malhamensis developed visible autofluorescence (i.e. increased
its chlorophyll content) only when bacteria
were not present in sufficient densities, and
population growth of the alga under our experimental conditions occurred only during
baterivory. On the basis of the present study,
we conclude that phototrophy is of minor
importance for the growth of this species.
Presumably the ecological significance of
mixotrophy for P. malhamensis involves a
long-term survival strategy (phototrophy)
for times when bacterial density is insufficient to support its primary mode of nutrition (phagotrophy). Moreover, major nutrients (N and P) obtained during bacterivory
are not retained efficiently by the alga for
subsequent use during phototrophic growth.
For the mixotrophic
P. malhamensis,
phagotrophy and phototrophy seem to be
nearly mutually exclusive but in series promote the growth and survival of the organism.
David A. Caron
Biology Department
Woods Hole Oceanographic Institution
Woods Hole, Massachusetts 02543
Karen G. Porter
Zoology Department
University of Georgia
Athens 30602
Robert W. Sanders
Division of Environmental
Research
Philadelphia Academy of Natural Sciences
19th and the Parkway
Philadelphia, Pennsylvania 19 103
References
ANDE~SEN, 0. K., J. C. GOLDMAN, D. A. CARON, AND
M. R. DENNIZTT. 1986. Nutrient cycling in a microflagellate food chain: 3. Phosphorus dynamics.
Mar. Ecol. Prog. Ser. 31: 47-55.
ANDERSSON,A., S. FALK, AND G. SAMUEISSON. 1989.
Nutritional characteristics of a mixotrophic nanoflagellate, Ochromonas sp. Microb. Ecol. 17: 252262.
ADAM, F., AND OTHERS. 1983. The ecological role of
water-column microbes in the sea. Mar. Ecol. Prog.
Ser. 10: 257-263.
BIRD, D. F., AND J. KALFF.
1986. Bacterial grazing
by planktonic algae. Science 231: 493-495.
-,
AND -.
1987. Algal phagotrophy: Regulating factors and importance relative to photosynthesis in Dinobryon (Chrysophyceae). Limnol.
Oceanogr. 32: 277-284.
-,
AND -.
1989. Phagotrophic sustenance
of a metalimnetic phytoplankton
peak. Limnol.
Oceanogr. 34: 155-162.
BORASS.M. E.. K. W. ESTEP.P. W. JOHNSON.AND J.
MdN. SIEXVJRTH. 1988. Phagotrophic’ phototrophs: The ecological significance of mixotrophy.
J. Protozool. 35: 249-252.
BRATBAK, G. 1985. Bacterial biovolume and biomass
estimations. Appl. Environ. Microbial. 49: 148%
1493.
CARON, D. A., J. C. GOLDMAN, AND M. R. DENNETT.
1985. Nutrient cycling in a microllagellate food
chain. 2. Population dynamics and carbon cycling.
Mar. Ecol. Prog. Ser. 24: 243-254.
-,
-,
AND -.
1988. Experimental
demonstration of the roles of bacteria and bacterivorous Protozoa in plankton nutrient cycles. Hydrobiologia 159: 2740.
DAVIS, P. G., AND J. McN. SIEBURTH. 1982. Differentiation of the phototrophic and heterotrophic
nanoplankton populations in marine waters by
epifluorescence microscopy. Ann. Inst. oceanogr.
Paris SS(supp1.): 249-260.
-,
AND -.
1984. Predation of actively
growing bacterial populations by estuarine and
oceanic microflagellates. Mar. Ecol. Prog. Ser. 19:
237-246.
ESTEP,K. W., P. G. DAVIS, P. E. HARGRAVES, AND J.
McN. SIEBURTH. 1984. Chloroplast-containing
microflagellates in natural populations of North
Atlantic nanoplankton,
their identification
and
distribution; including a description of five new
species of Chrysochromulina (Prymnesiophyceae).
Protistoloaica 20: 613634.
FENCHEL, T. i982a.b. Ecology of heterotrophic microflagellates. 2. Bioenergetics and growth. 3. Adaptations to heterogeneous environments. Mar.
Ecol. Prog. Ser. 8: 225-231; 9: 25-33.
-.
1986. The ecology of heterotrophic microflagellates. Adv. Microb. Ecol. 9: 57-97.
GEID& R. J., AND B. S. C. LEADBEATER. 1988. Kinetics and energetics of growth of the marine
choanoflagellate Stephanoeca diplocostata. Mar.
Ecol. Prog. Ser. 47: 169-177.
GOLDMAN, J. C., D. A. CARON, 0. K. ANDERSEN,AND
M. R. DENNETT. 1985, 1987. Nutrient cycling in
a microflagellate food chain: 1. Nitrogen dynamics. 4. Phytoplankton-microflagellate
interactions.
Mar. Ecol. Prog. Ser. 24: 231-242; 38: 75-87.
GUDE, H. 1985. Influence of phagotrophic processes
on the regeneration of nutrients in two-stage continuous culture systems. Microb. Ecol. 11: 193204.
HANDA, A. K., R. A. BRESSAN,H. QUADER, AND P.
FILNER. 198 1. Association of formation and release of cyclic AMP with glucose depletion and
onset of chlorophyll synthesis in Poterioochromonas malhamensis. Plant Physiol. 68: 460-463.
HEINBOKEL, J. F. 1978. Studies on the functional role
of tintinnids in the Southern California Bight. 1.
Grazing and growth rates in laboratory cultures.
Mar. Biol. 47: 177-189.
KELLER, M. D., AND OTHERS. 1988. Phagotrophy by
a photosynthetic marine phytoplankter
demonstrated by fluorescence-labelled bacteria and flow
cytometry. Eos 69: 1116.
KOPYLOV, A. I., T. I. MAMAYEVA, AND S. F. BATUNIN.
1980. Energy balance of the colorless flagellate
Purubodo attenwtus (Zoomastigophora,
Protozoa). Oceanology 20: 705-708.
LEE, S., AND J. A. FUHRMAN. 1987. Relationships
between biovolume and biomass of naturally derived marine bacterioplankton.
Appl. Environ.
Microbial. 53: 1298-I 303.
MENZEL, D. W., AND N. CORWIN. 1965. The measurement of total phosphorus in seawater based
on the liberation of organically bound fractions by
persulfate oxidation. Limnol. Oceanogr. 10: 280282.
NAGATA, T. 1986. Carbon and nitrogen content of
natural planktonic bacteria. Appl. Environ. Microbiol. 52: 28-32.
PA~ZSONS,
T. R., Y. MAITA, AND C. M. LALLI. 1984.
A manual of chemical and biological methods for
seawater analysis. Pergamon.
PORTER, K. G. 1988. Phagotrophic phytoflagellates
in microbial food webs. Hydrobiologia
159: 8997.
-,
AND Y. S. FEIG. 1980. The use of DAPI for
identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948.
-,
E. B. SHERR, B. F. SHERR, M. PACE, AND R.
W. SANDERS. 1985. Protozoa in planktonic food
webs. J. Protozool. 32: 409-415.
SALONEN, K., AND S. JOK~NEN. 1988. Flagellate grazing on bacteria in a small dystrophic lake. Hydrobiologia 161: 203-209.
SANDERS,R. W., AND K. G. PORTER. 1988. Phagotrophic phytoflagellates. Adv. Microb. Ecol. 10:
167-192.
-,
-,
S. J. BENNETT, AND A. E. DEBIASE.
1989. Seasonal patterns of bacterivory by flagellates, ciliates, rotifers and cladocerans in a freshwater planktonic community. Limnol. Oceanogr.
34: 673-687.
-,
-,
AND D. A. CARON. 1990. Relationship between phototrophy and phagotrophy in the
mixotrophic chysophyte Poterioochromonas malhamensis. Microb. Ecol. 19: 97-109.
Notes
B. F., E. B. SHERR, AND T. BERMAN. 1983.
Grazing, growth, and ammonium excretion rates
of a heterotrophic microflagellate fed with four
species of bacteria. Appl. Environ. Microbial. 45:
1196-1201.
STARR,
R. C. 1978. The culture collection of algae at
the University of Texas at Austin. J. Phycol. 14:
47-100.
SHERR,
Limnol. Oceanogr.. 3X2),
Q 1990, by the American
1990,443-447
Society of Limnology
and Oceanography,
STEIN,
443
J. R. [ED.].
1973. Handbook
methods. Cambridge.
of phycological
Submitted: 12 June 1989
Accepted: 16 August 1989
Revised: 18 December 1989
Inc.
Distribution of labile dissolved organic carbon in
Lake Michigan
Abstract -Bioassay-measured,
labile dissolved
organic carbon (LDOC) concentrations
were
compared in near-bottom and near-surface Lake
Michigan water between April and October 1986.
In five of seven experiments, the LDOC concentration was higher in near-bottom
water.
LDOC reached 40.2% of the total DOC pool in
the near-bottom water in late May and 13.8% in
the near-surface water in early July. Concentration in near-bottom water was highest during early stratification; concentration in surface water
varied less but was highest in early July. The data
suggest that an allochthonous source of labile organic C may be important.
Identifying primary sources of labile dissolved organic carbon (LDOC) in pelagic
systems has taken on new interest in light
of recent evidence that a major portion of
the C fixed via autotrophic production passes
through heterotrophic bacteria (Love11 and
Konopka 1985; Scavia et al. 1986; Scavia
and Laird 1987; Nagata 1987) and that much
of the bacterial production is grazed (Scavia
and Laird 1987; Nagata 1987). This transfer
of LDOC to primary consumers captures C
that might otherwise be lost.
Recently, we reported that in Lake Michigan a disequilibrium
between phytoplankton organic C production (supply) and bacterial C use (demand) on seasonal and spatial
scales may exist (Scavia and Laird 1987).
Acknowledgments
We thank M. T. Babbitt for technical assistance and
W. S. Gardner and G. L. Fahnenstiel for their suggestions and improvements to the manuscript.
GLERL Contribution 66 1.
Although annual bacterial C demand (236
g C m-2 yr-I) appeared to be balanced by
annual phytoplankton net C production (225
g C me2 yr-I), summer bacterial C demand
(1,087 mg C mm2 d-‘) could not be satisfied
by net summer phytoplankton C production
(627 mg C mm2 d-l), indicating that a large
discrepancy exists between DOC demand
and supply. This discrepancy was even more
dramatic when restricted to the summer
epilimnion. We suggested that seasonal and
spatial disequilibrium
between organic C
supply and demand could explain the apparent deficit. The hypothesis suggested that
temporal disequilibrium
is driven when
bacterial production is low (winter or early
spring) and LDOC accumulates, exceeding
bacterial demand. Also, spatial disequilibrium is driven by accumulation of organic
matter in cold, deep regions through sedimentation of phytoplankton,
detritus, and
other decaying material. These supplies of
LDOC
would then become available
throughout the water column during winter
mixing.
Strayer (1988) generalized the issue of
secondary production in ecosystems and
questioned the need for our disequilibrium
hypothesis. He based his analysis on model
calculations demonstrating that because organic C is recycled, total consumer C demand may exceed organic C inputs to ecosystems. Extension of that model analysis
(Scavia 1988) corroborated that consumer
demand can be met by autotrophic production on an annual water-column
basis in
Lake Michigan; it also suggested, however,