orob.

7:199-206 r

MICROBIAL

ECOLOGV

Competition Between Heterotrophic and Autotrophic Microplankton for Dissolved Nutrients Edward J. Brown, Don K. Button, and Douglas S. Lang University of Alaska, Fairbanks, Alaska 99701, USA

Abstract. When a heterotrophic (Rhodotorula rubra) and a phototrophic (Selenastrum capricornutum) plankton were grown together in dilute phosphate (Pi) continuous cultures, coexistence occurred only when the heterotroph was growthrate limited by organic carbon (C). Because of its higher affinity for Pi, and because C starvation does not affect the heterotrophic yeast's ability to transport Pi, the concentration of organic carbon indirectly controlled the biomass of the phototroph. The results support a threshold model of microbial growth. Introduction Phosphorus (P) is the key biomass limiting nutrient in many lakes [13], and kinetic studies beginning with those of Rhee [8] have shown that heterotrophic bacteria are better competitors than are phytoplankton for dissolved inorganic orthophosphate (Pi). If such kinetic studies accurately reflect multi-species competition for Pi, some resource (or inhibitor) other than Pi must restrict heterotrophic production in "phosphatelimited" systems where phytoplankton predominate. Results from at least one whole lake experiment showed that fertilization with organic carbon (sucrose) and nitrogen caused a decline in phytoplankton biomass from the higher levels observed when P was added to the lake. In addition, the carbon/phosphorus ratio of the lake water actually declined after organic carbon was added [ 12]. In this study we have investigated with laboratory experiments how a carbon-limited heterotroph may affect the growth and biomass of a P-limited autotroph. An alga known to have a moderate affinity for Pi [2] was forced to compete for this resource against a heterotrophic yeast known to have a higher affinity for Pi [ 10]. To allow for some growth of the autotroph, the organic carbon (C) concentration was varied from levels that were growth-rate limiting for the heterotroph to levels such that available P limited the biomass of both the yeast and alga. Our experimental design (dual species continuous culture) allowed us to investigate whether or not C starvation affects the Pi affinity of the heterotroph (i.e., does a multiplicative or threshold kinetic model best describe competition for phosphorus and carbon?; see [3]).

Materials and Methods The organisms Selenastrum capricornutum and Rhodotorula rubra, and the general growth conditions and sampling procedures used have been previously described [2, 10]. All experiments were conducted at 25"C 0095-3628/81/0007-0199 $01.60 9 1981 Springer-Verlag New York Inc.

200

E.J. Brown et al.

under a constant illumination of 2.4 x 1016 quanta, s e c - l . c m - 2 (as measured inside the growth chamber containing a typical dilute biomass concentration) provided by cool white fluorescent lighting. For the dual species culture (gnotobiotic) experiments, two 20 liter carboys of sterile media were each connected, through separate variable-speed peristaltic pumps, to a 500 ml round bottom flask growth chamber containing a magnetic stir bar. The modified PAAP medium [2] containing a vitamin mixture, ammonia-nitrogen [ 10], and 870 nMPi were identical in each carboy except that glucose was added to one carboy after autoclaving. The media were buffered to pH 7.2 + 0.2 with Tris (hydroxymethyl)-aminomethanebuffer. Pi in the feed reservoiir was labeled with radiophosphate (32 Pi). By varying the speed of each pump, the glucose concentration [and thus carbon/phosphorus (C0/P0) atomic ratio] of the incoming nutrients was varied for each dilution (growth) rate. The phosphorus concentration was held constant at 870 nM. The experiments were initiated by adding a small inocula of axenic healthy Selenastrum, Rhodotorula, or both species to the growth chamber with a sterile syringe. The data reported are steady state, i.e., at least 3 residence times elapsed between each sampling and samplings were repeated at each dilution rate before changing the influent C0/P 0 by varying pump speeds. Viable counts (plate counts) were used to distinguish yeast from algae. Total culture dry weights (yeast plus algae) were estimated from total cell numbers and mean cell volumes obtained with an electronic particle counter and sizer using a density factor of 1.04 g. ml - 1 and a dry weight/wet weight ratio of 0.33 [2]. Cellular P in the growth chamber was determined as the difference between the incoming Pi supplied (870 nM) and filtrate P (0.45 # m membrane filters) by measuring each radiophosphorus (32p) fraction and multiplying the fraction by 870 nM since the 32pi added to the feed was cartier free. If feed reservoir 32p could not be recovered completely in the growth chamber, the experiment was discontinued due to probable contamination of the glass and/or silicon feed lines. Solution Pi concentrations were determined by measuring 32p in culture filtrate after complexing the filtrate phosphate with a modified [2] molybdate [6] reagent and extracting the complex into isoamyl alcohol. Cell carbon was not measured but estimated as 0.5.(dry weight) for both species.

Results Table 1 summarizes some P growth kinetic parameters that have been measured under the same growth conditions for several microplartkton species (from [2], [10], and D. S. Lang, M.S. thesis, University of Alaska, 1980). Based on the results shown in Table 1 and their compatibility in dual-species continuous cultures, Selenastrum and Rhodotorula were chosen for the competition studies. Since the apparent maximum growth rate (/s raM) for Selenastrum is I. 2 da - l at 20~ versus 5.8 da- ~for Rhodotorula at 25~ (Table 1), the algae eventually washed out when the steady-state dilution rate (D) exceeded a growth rate (/a) of about 1.5 da- l in dual-species continuous culture (# = D at steady state). However, even when D was well below 1.5 da -l and Pi was the growth-rate limiting nutrient for both species, Selenastrum washed out. This occurred because the steady-state affinity (as) for Pi is lower in Selenastrum than in Rhodotorula (Table 1). Steady-state affinity is calculated as the slope of net steady-state uptake rate (#Q) versus solution growth-limiting nutrient concentration:

= a,

/.tQ (S-St)'

(1)

where Q is the nutrient (P) content per cell dry weight, S is the steady-state solution nutrient (Pi) concentration, and St is the steady-state nutrient (Pi) threshold concentration (the nutrient concentration below which nutrient uptake does not occur). This value is not a half-saturation constant but an initial slope which is an indicator of competitive ability at low substrate concentrations (see [2]). Results (Table 1) have also

5.8

1.6

1.2 2.0 0.6

1.0 1.4

1,397

6,873

2,260 960 2,433

987 950

QMAYa, rdVlP(mgcell) - 1

106

124

40 100 173

14 15

Q 0, nMP(mg cell) - l

30.7

--

2.5 +_.0.5 4.9 -I- 2.2 --

63 + 25 38 c

a s, 1(rag cell-day) - 1

aUniversity of Texas Culture Collection, Austin, Texas. b American Type Culture Collection, Rockville, Maryland. c Tentative. dD. S. Lang, M.S. thesis, University of Alaska, Fairbanks, 1980. e Experiments in reference conducted at 20~ All other experiments with Selenozmon have been conducted at 25~

Rhodotorula rubra

Yeast

Navicula pelliculosa (UTEX 668)

Diatom

Selenastrum capricornutume Scenedesmus quadricauda (UTEX 76) Scenedesmus obliquus (UTEX 1450)

Green algae

Synechococcus N~geli (UTEX 6908) a Synechococcus Niigeli (ATCC 27344) b

Cyanobacter/a

Organism

Apparent /~ MAX, day - 1

Table 1. Phosphorus growth kinetic parameters for several aquatic microorganisms grown under the same conditions

30-80

20-80

50-130 850-1,400 50-130

1-3 1-3

Median cell volume range, #m 3

[10]

Lang d

[2] Lang d I.,ang d

Lang d Lang d

Reference

O

202

E.J . Brown et al.

shown that the maximum transient P storage capacity (QMAxt) is much higher for the alga [2.26 (#MP)(mg cell)-l] than for the yeast [1.40 (/zMP)(mg cell) -l] and that the alga can survive on less P per cell dry weight [Qo = 40 (nMP)(mg cell)- l ] than can the yeast [Q0 = 106 (nMP)(mg cell)-l]. This minimum P per cell dry weight is the reciprocal maximum yield of cells on P. Figure la shows the relationship between internal P storage capacity (in terms of cell carbon/phosphorus ratio; C/P) and dilution rate for Pi-limited continuous cultures of Rhodotorula. The maximum yield of the yeast is 9.4 (mg cell)(nMP) -z and it occurs at the lowest dilution rate and then decreases proportionately to a minimum yield of 1.6 (mg cell)(nMP) -Z at/ZMAX- However, unlike that of P and other mineral nutrients [6], the yield of yeast (12 mg-cell per mM organic C) remains constant with dilution rate even when the heterotroph is C starved. Figure lb shows that when the C0/P0 ratio is below 128, the yeast cell yield on P is always at its lowest, corresponding to the lowest measured cell C/P ratio of 64. The cell C/P value remains 64 and is independent of growth rate as long as C0/P0 is 128 or less (Fig. lb). When the C0/P0 ratio of the incoming nutrient supply exceeds 740, the yeast becomes P limited rather than C limited at all growth rates (Fig. lb, arrow), and thus the cellular C/P level is only a function of D as illustrated in Fig. la. When C0/P0 is between 128 and 740, the cellular C/P ratio is a multiple function of D and C0/P0 (Fig. lb and Fig. lc, shaded area). If C limitation has no significant effect on the Pi affinity of the yeast (threshold model) and allelopathic interactions are negligible, then using the results shown in Fig. 1 and our phosphate-limited growth kinetic descriptions of Selenastrum [2] and Rhodotorula [ 10] we should be able to predict not only the final survivor in a competition experiment between these two species but also the exact biomass of each organism that would result for each dilution rate and incoming C0/P0. Batch culture experiments in which algae were grown in "nutrient-amended yeast filtrate" and vice versa showed no indications of allelopathic interactions. Figure 2 shows the results of a steady-state competition experiment. In this experiment the incoming Pi concentration (870 nMP) and dilution rate (0.80 da-l) were held constant, while the incoming organic C concentration was varied from background levels of 1.10 mg.1-1 to 19.3 mg-I -l (as glucose). The results (Fig. 2) show that C limitation has no apparent effect on the ability of the yeast to sequester Pi or store P. The concentration of Pi remaining in solution for all steady states with C0/P0 values through 117 was 40 -t- 10 nM (Fig. 2), which approximates the concentration remaining in single-species phosphate-limited Selenastrum continuous cultures at D = 0.80 da- 1 [ 1]. When the C0/P0 reached 272, phosphate in solution was below 40 nM, and Selenastrum was no longer visible in the culture. The biomass of Rhodotorula continued to increase with increasing C0/P0 to its maximum predicted biomass of 7.0 (mg cell)l- 1 at C0/P0 = 740, indicating that Rhodotorula biomass was then Pi limited at D = 0.80 da-l (see Fig. lb, c). Note that the combined biomass of yeast and algae declined to a minimum as C0/P0 approached 100. This occurred because the yeast biomass was C limited, yet the yeast had sequestered most of the available Pi, leaving little in excess of 40 nM for the P-starved algae. The theoretical combined biomass minimum for these two species based on yield data and available Pi should occur when Co/P0 is 117 at D = 0.80 da- 1. When the total concentration of incoming Pi was increased, the combined species biomass increased proportionately but with the same C0/P0 relationships shown in Fig. 2 for D = 0.80 da -1 . For other dilution rates below 1.5 da -I , a family of relationships correlating combined biomass to C0/P0 occurs since both stored P and Pi in solution vary with D when each organism is P starved.

Microplankton Competition for Phosphorus

203

Fig. 1. Cellular yields (as C/P ratios) of Rhodotorula rubra when growth is P limited plotted as function of dilution rate (a), when growth is P limited or C limited plotted as a function of C0/P 0 for several dilution rates (b), and when growth is P limited or C limited plotted as a function of both dilution rate and C0/P 0 (c). The same data are plotted in b and c. The solid heavy line in Fig. 2b shows cellular C/P when carbon is limiting and is thus independent of D but dependent on available C 0 (written as C 0/P0). The dashed lines in the shaded area of Fig. 2b indicate cellular C/P levels that will result when P becomes growth limiting and those levels are dependent on D as in Fig. 2a, and a function of C0/P 0 between 128 and 740. When C0/P 0 exceeds 740, Rhodotorula is P limited for all dilution rates from 0.80 to DMAX. Each point represents mean values from several steady states, except the C/P value for D = 5.8 da - 1, which was obtained in batch culture.

Discussion T h e c o n c e n t r a t i o n o f an essential nutrient is considered to be the factor that m o s t often c o n t r o l s m i c r o p l a n k t o n g r o w t h rate, b i o m a s s , and c o m m u n i t y structure in aquatic s y s t e m s [ 14]. T h u s kinetic m o d e l s relating microbial growth to either external or internal nutrient c o n c e n t r a t i o n s are w i d e l y used (see [2]). F r o m the parameters obtained by fitting steady-state and transient data to kinetic models, investigators have been able to learn m o r e about the m i c r o p l a n k t o n c o m m u n i t y - l i m i t i n g nutrient relationship. F o r e x a m p l e , f r o m the P - d e p e n d e n t g r o w t h kinetic constants that w e h a v e m e a s u r e d (Table

E.J. Brown et al.

204 tC

~

~1~

~

C~176

i

6-

i2~ Algae E 4

zoo

400

600

800

~redorninate ~ , 4

~

o,-- - ' ~ )

(

}

t~"~~ 50

100

Yeast predominate

~ , 150

Incoming nutrients BackgroundC L.. . . . . I

0

I

I 200 (Co/Po) I

t.9 4.9 Added glucose (rng liter-')

l 250

, 300

I

7.9

Fig. 2. Continuous culture dry weight of Selenastrum capricornutum ~ o), Rhodotorula rubra (: :), and both species (_~ ~ as a function of available C and Pi (C0/P0) ratio and Pi in solution versus C0/P 0 (inset). Each point with error bars represents the mean value of two steady-state measurements. Those without error bars represent one measurement. Total dry weights are the sum of each species' mean dry weights.

1), it seems apparent that the two species of unicellular cyanobacteria should predominate in P-limited "steady-state" environments. The presence of significant numbers of small unicellular cyanobacteria in marine oligotrophic systems [4] may therefore be attributed to their high affinities for mineral nutrients, possibly Pi. On the other hand, all but one of the eukaryotic algae studied can store more P than can the cyanobacteria or the yeast (Q~,,va, Table 1). Tlius in environments with fluctuating levels of mineral nutrients (possibly Pi), the eukaryotic species should predominate. In environments with fluctuating levels of essential nutrients, it is often not possible to identify a single nutrient which limits the growth rate and biomass of the microplankton community. In such nonsteady-state systems, regulation of microplankton growth may be controlled by multiple nutrients more or less simultaneously. Many models have been proposed to describe the effects of multiple nutrient limitation on the growth of microorganisms [ 1]. The various models assume gradations ranging from no multiple effects on growth among nutrients (threshold models) to very strong effects on growth among nutrients (multiplicative models). For most combinations of nutrients, no

MicroplanktonCompetitionfor Phosphorus

205

particular model has been fully verified or rejected [1]. Our data (Fig. 1) show that there is no apparent multiplicative effect of C limitation on the yeast's ability to transport Pi, even in the presence of a competing P-starved phytoplankton (Fig. 2). These data support a threshold model of nutrient-limited growth as do the data that Rhee [9] obtained for a species of Scenedesmus. The results from the type of competition experiment reported here are also useful for experimentally validating predictions based on kinetic constants derived from studies of single species. We have suggested from the growth kinetic descriptions of Selenastrum and Rhodotorula [2] that the alga and yeast could never coexist when P is the only nutrient limiting their growth. The results shown in Fig. 2 validate that conclusion. The major ecological implication of such results is that although the total biomass of oligotrophic microplankton communities may be limited by mineral nutrients such as P, microplankton community structure may be controlled by the type and amount of organics available to heterotrophs. As total nutrient concentrations increase toward eutrophy, and in systems with periodic nutrient perturbations, factors such as zooplankton grazing, diel periodicities, and allelopathy [5] may be more important in determining microplankton species composition. Finally, these results suggest that productivity of some eukaryotic phytoplankton (Table 1) may be maximized only when organic C is low and the environment is periodically perturbed by mineral nutrients. In oligotrophic Lake 227, Schindler [12] reported as much as a 35-fold increase in total lake organic C from spring to fall with total lake carbon/phosphorus ratios ranging from approximately 200 to 6,500. The C and P actually available for growth of the microbial population were, at any given time, unknown in Lake 227, hut clearly the ratios vary enough to favor heterotrophs with high P affinities at certain times and eukaryotic autotrophs with lower P affinities at other times. Further, the decline of the carbon/phosphorus ratio and phytoplankton biomass upon addition of sucrose to Lake 227 [12] could have resulted from the removal of available P to algae by heterotrophs responding to the additional available C in a manner similar to that shown in Fig. 2. The laboratory results that we report here and field studies such as those of Schindler [ 12] illustrate the value that laboratory-derived growth kinetic descriptions coupled with field measurements of solution nutrient ratios (not just concentrations) have as predictors of species composition and biomass in aquatic systems.

Acknowledgments. The investigators thank Alan Braley for computerprogrammingand Lewis Molot and Betsy Robertsonfor reviewingthe manuscript.This researchwas supportedby NSF GrantDEB-7708427A01 and Office of Water Researchand TechnologyGrant 14-34-0001-9002(A-066-ALAS).

References 1. Ahlgren, G.: Effectson algal growth rates by multiple nutrient limitation. Arch. Hydrobiol.89, 43-53 ( ! 980) 2. Brown, E. J., and D. K. Button: Phosphate-limited growth kinetics of Selenastrum capricornutum (Chlorophyceae).J. Phycol. 15, 305-311 (1979) 3. Fredrickson, A. G.: Behaviorof mixed cultures of microorganisms.Annu. Rev. Microbiol. 31, 63-87 (1977)

206

E.J. Brown et al.

4. Johnson, P. W., and J. McN. Sieburth: Chroococcoid cyanobacteria in the sea: A ubiquitous and diverse phototrophic biomass. Limnol. Oceanogr. 24, 929-935 (1979) 5. Keating, K. I.: Allelopathic influence on blue-green bloom sequence in a eutrophic lake. Science 196, 885--887 (1977) 6. Murphy, J., and J. P. Riley: A modified single solution method for the determination of phosphate in natural waters. Anal. Chem. Acta 27, 31-36 (1962) 7. Nyholm, N.: A mathematical model for microbial growth under limitation by conservative substrates. Biotechnol. Bioeng. 18, 1043--1056 (1976) 8. Rhee, G.-Y.: Competition between an alga and an aquatic bacterium for phosphate. Limnol. Oceanogr. 17,505-514 (1972) 9. Rhee, G.-Y.: Effects of N:P atomic ratios and nitrate limitation on algal growth, cell composition, and nitrate uptake. Limnol. Oceanogr. 23, 10-25 (1978) 10. Robertson, B. R., and D. K. Button: The phosphate-limited continuous culture ofRhodotorula rubra: kinetics of transport, leakage and growth. J. Bacteriol. I38, 884-895 (1979) 11. Saks, N. M., and E. G. Kahn: Substrate competition between a salt marsh diatom and a bacterial population. J. Phycol. 15, 17-21 (1979) 12. Schindler, D. W.: Whole lake eutrophication experiments with phosphorus, nitrogen and carbon. Verh. Int. Verein. Limnol. 19, 3221-3231 (1975) 13. Schlinder, D. W.: Evolution of phosphorus limitation in lakes. Science 195,260-262 (1977) 14. Tempest, D. W., and O. M. Neijssel: F_go-physiologicalaspects of microbial growth in aerobic nutrientlimited environments. In M. Alexander, (ed.): Advances in Microbial Ecology, Vol. 2, pp. 105-153. Plenum Press, New York (1978)

Competition between heterotrophic and autotrophic microplankton for dissolved nutrients.

When a heterotrophic (Rhodotorula rubra) and a phototrophic (Selenastrum capricornutum) plankton were grown together in dilute phosphate (Pi) continuo...
562KB Sizes 0 Downloads 0 Views