AQUATIC MICROBIAL ECOLOGY Vol. 14: 271-280,1998 Published April 6 Aquat Microb Ecol Influence of bacteria on phytoplankton cell mortality with phosphorus or nitrogen as the algal-growth-limiting nutrient Corina P. D. ~russaard'~',Roe1 Riegman2 'Department of Microbiology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway 'Department of Biological Oceanography, Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg. Texel, The Netherlands ABSTRACT: The effects of bacteria on phytoplankton mortality were studied with phosphorus or nitro- gen as the algal-growth-limiting nutrients. Experiments were performed with the &atom Ditylum brightwellii using batch cultures, steady state continuous cultures and batch-mode cultures which were starved for the limiting nutrient after being preconditioned in a chemostat. With phosphorus lim- iting algal growth, the specific death rates of D. bnghtwellii generally increased upon bacterial inocu- lation. Bacteria enhanced algal mortality very likely due to competition with D. brightwellii for the lim- iting phosphate. With nitrogen as the algal-growth-limiting nutlient, however, the presence of hactena had either no pronounced effect on or led to a reduction of the specific death rates of D. brightwellu. Remineralized ammonium was probably partly utilized by N-starved cells of D. bnghtwellii, leading to reduced death rates of the algal cells. Bacteria thus indirectly prolonged survival of D. bnghtwellii pop- ulation~under N starvation. This study shows that bacteria can affect phytoplankton survival, which in turn may influence algal species succession. The degree of bacterial influence on algal death kinetics depends largely on the type of nutrient limiting algal growth and on the culture conditions. KEY WORDS: Algal cell mortality . Ditylum brightwellii . Bacteria. N and P deficiency . Starvation INTRODUCTION affinity for the limiting dissolved orthophosphate (Thingstad et al. 1993).Accordingly, bacteria would be In the field, phytoplankton populations experience also more efficient competitors for ammonium, but loss of cells due to grazing, sinking and cell lysis. whether bacteria also promote extra algal mortality Under axenic culture conditions, algal cell lysis was when ammonium is the algal-growth-limiting sub- found to be directly affected by the degree of nutrient strate is still unknown. deficiency (Brussaard et al. 1997). However, phyto- Under ammonium or phosphate limitation, bacteria plankton under natural growth conditions will have to will only be superior to phytoplankton when organic compete for the growth-limiting nutrient with bacteria. carbon is present in sufficiently high amounts to pre- Laboratory work by Currie & Kalff (1984) and Bratbak vent C limitation (Bratbak & Thingstad 1985, Roth- (1987) suggested that bacteria are better competitors haupt 1992, Kirchman 1994, Ietswaart & Flynn 1995). for inorganic phosphorus than algae. Bratbak (1987) Bacterial growth rates in marine ecosystems are often found that the diatom Skeletonema costatum is nega- considered to be carbon rather than mineral nutrient tively affected by bacterial activity under phosphorus limited (Bratbak & Thingstad 1985, Kirchman et al. depletion. Small bacteria, with a higher surface:vol- 1990, Brussaard et al. 1995). Besides exudation of ume ratio, have been reported to have the highest organic components, interpreted as overflow of photo- synthetically fixed carbon (photosynthetic extracellu- lar release, PER; Mague et al. 1980, Bjarnsen 1988, Myklestad et al. 1989, Obernosterer & Herndl 1995), 0 Inter-Research 1998 272 Aquat Microb Ecol algal cellular components released through cell lysis (GF/C Whatman, approximately 50% of total bacterial may be an important C source for bacteria. Several cells passed through the filter). The filtrate (5 ml) was field studies suggested that phytoplankton cell lysis used as bacterial inoculum for the axenic culture of D. products are potential C sources for bacteria, allowing bnghtwellii. The mixed bacterial community (domi- balancing of their C budgets (Lancelot & Billen 1985, nated by rods) thus consisted of a consortium of bacte- Riemann & S~ndergaard1986, Christoffersen et al. rial species presumably adapted to the specific culture 1990, Brussaard et al. 1996). Laboratory studies on conditions. The concentrations of bacteria at the start microbial decomposition of phytoplankton cells of the experiments were between 0.25 and 0.4 X 106 showed rapid mineralization rates (Fukami et al. 1981, ml-' (= 20 to 30 ng C ml-l, using a conversion factor of Newel1 et al. 1981, Garber 1984, Hansen et al. 1986, 0.22 X 10-l2 g C pm-3 biovolume). The axenic cultures Biddanda 1988). Bacterial mineralization of the rela- received 5 m1 of 0.2 pm filtrate (sterile cellulose acetate tively N- and P-rich lysed algal cellular components dsposable filters, Schleicher & Schuell) containing no (Garber 1984, Caron et al. 1988, Jiirgens & Gude 1990, bacteria. No heterotrophic nanoflagellates grew in the Goldman & Dennett 1991) might also be an important cultures. process to supply algae with ammonium or phosphate, Because pH values varied between 8.0 and 8.7 in potentially influencing algal survival and species suc- these batches, the experiments were repeated includ- cession under nutrient deficiency. ing cultures buffered with 20 mM HEPES at pH 8.0 + This report presents data on the bacterial influence 0.1. This pH range had no significant effect on algal on phytoplankton cell mortality with either phosphorus growth and mortality under either phosphate, ammo- (P) or nitrogen (N) as the algal-growth-limiting sub- nium or nitrate depletion (for all limitations p > 0.1; strate. The experiments were performed with algal data not shown). Time interval between the duplicate populations in batch cultures, in steady state continu- unbuffered experiments was 1 mo. A List of culture ous cultures, and with algal populations precultured in conditions is presented in Table 1. continuous cultures before being starved by switching Media were based on aged, nutrient-poor, 0.2 pm fil- off the medium supply. Batch cultures start with high tered, autoclaved seawater from the Central North concentrations of inorganic nutrients, which may Sea, acidified with HCl (pH < 3.5).The algal-growth- resemble early spring bloom situations typical for tem- limiting factor was either NH,' (concentration at the perate waters as argued by Bratbak (1987). In field sit- start of the experiments was 15 FM), NO3- (starting uations, algae may also encounter prolonged periods concentration 15 pM) or Pod3-(starting concentrations of nutrient limitation and exhaustion (e.g. summer 10 and 2.5 PM). Concentrations of the other macronu- periods in coastal areas or oligotrophic waters). Such trients in the sterile medium were 2.7 mM NaHC03, conditions are not well represented by ordinary batch 150 pM Na2Si03.9H20,and either 300 pM N (150 pM cultures in which the physiological state of the algal NH4C1 and 150 pM NaN03) or 25 pM NaH2POd3-, cells constantly changes, but are better mimicked by depending on the type of limiting nutrient. The basic nutrient-limited continuous cultures and by nutrient- medium was enriched with micronutrients and vita- starvation experiments with algal cultures precondi- mins according to Brussaard et al. (1997). The pH of tioned under nutrient Limitation in continuous cultures. the medium was aseptically adjusted to 8.0 + 0.1. Light was provided in a 16/8 h light/dark cycle at a density of 100 to 125 pmolphotons m-' S-'. Temperature was kept MATERIAL AND METHODS at 13°C. Batch cultures were gently mixed twice a day by hand, which gave the most reliable results com- Batch culture systems. Batch culture expenments pared to the use of several other mixing devices. were performed with the axenic diatom Ditylum Axenic algal cultures were checked for bacterial cont- bnghtweLlii (West) Grunow (#358 Bigelow culture col- amination at least once a week by epffluorescence lection). During earlier experiments with this axenic D. microscopy after staining with acridine orange. brightwellii strain, no virus-like particles were ob- Continuous culture systems. Continuous culture ex- served extra- and intracellularly using transmission periments were conducted with Ditylum bnghtwellii electron microscopy. To create xenic cultures of D. (Table 1).The 900 m1 cultures were held at 13.0 2 0.2OC brightwellii (unialgal cultures containing bacteria), under a 16/8 h light/dark regime, receiving 100 to axenic cultures (75 m1 each) were inoculated directly 125 pm01 photons m-' S-' during the light period. Auto- at the start of the experiments with a mixed community claved, aged, nutrient-poor seawater was enriched of marine bactena. The latter was obtained from xenic with sterile NaHC03 (2.7 mM final concentration), cultures of D. brightwellii (originally isolated from NaSi03 (300 pM), trace metals and vitamins (Brussaard Dutch coastal waters) at the end of the exponential et al. 1997).Phosphorus-limited media contained 3 pM phase by filtration through a sterile glass fiber filter NaH'P0, and 300 pM nitrogen (NaN03 and NH4Cl Brussaard & Rlegman: Influence of bactel-ia on phytoplankton mortality 273 at 150 pM each). Nitrogen-limited media contained supply was stopped (by turning off the pump) to study 50 pM N (NaNO, and NH,Cl, 25 pM each) and 50 pM the influence of bacteria on algal cell mortality under NaH2P04.The pH of the medium was adjusted to 8.0 * nutrient starvation (batch-mode cultures; Table 1).The