AQUATIC MICROBIAL ECOLOGY Vol. 9: 127-136, 1995 Published August 31 Aquat rnicrob Ecol Fate of Phaeodactylum tricornutum and nitrogen flow in an experimental microbial food web limited at the top by protozoans France Van Wambeke* Microbiologie Marine, C.N.R.S., U.P.R. 223, Case 907, Campus de Luminy, F-13288 Marseille Cedex 9, France ABSTRACT: The fate of phytoplankton nitrogen through a simplified microbial food web was investi- gated. In a 2-stage culture system, I separated production and degradation processes of a diatom, Phaeodactylum tricornutum. In the first stage, the algae grew axenically and continuously. In the sec- ond stage, maintained in the dark, a 20 pm ciliate grazed on the diatoms and constituted the last trophic step. A microbial loop, based on bacterial utilization of partially degraded P. tricornutum cells, egested with the clliate digestive vacuoles, developed rapidly. Chate production was maintained only because the P. tricornutum grazer was able to feed simultaneously on bacterivorous nanoflagellates, themselves preying upon bacteria using egested material of the chate. Dissolved organic nitrogen rapidly consti- tuted more than 50% of the identified nitrogen forms (&ssolved + particulate; mineral + organic). The nitrogen budget compiled during the succession of the different prey-predator relationships revealed the important role of dissolved organic matter adsorption on particles and aggregate formation includ- ing unstable organic colloids. The temporary importance and turnover of colloidal nitrogen was thus demonstrated. KEY WORDS: Dissolved organic nitrogen . Microbial loop . Phaeodactylum tncornutum . Ciliate INTRODUCTION important role in the nitrogen cycle in the euphotic zone. For example, ammonium is generally described The concept of the microbial loop (Azam et al. 1983) as the main nitrogen form in excretion processes of has brought new interest to the studies of composition microzooplankton (Glibert 1982, Paashe & Kristiansen and utilization of dissolved organic matter (DOM) in 1982). But dissolved free amino acids (Andersson et al. aquatic environments. Of primary production, 10 to 1985) and dissolved combined amino acids (Nagata & 50 % is transformed into bacterial biomass, channelled Kirchman 1991) are also produced by protozoans. to the microbial loop, and subsequently transferred to DON needs now to be included to balance nitrogen higher trophic levels through the grazing food chain budgets (Glibert 1988, Nagata & Kirchman 1991). (Cole et al. 1988). Besides phytoplankton exudates In the euphotic zone, phytoplankton production and (Baines & Paice 1991),digestive by-products of grazing loss occur simultaneously. These processes are so could also constitute a source of DOM for het- tightly coupled that the fate and the turnover of phyto- erotrophic bacteria (Jurnars et al. 1989). In addition to plankton-derived detritus (particulate nitrogen or DOM, interest has recently been focused on dissolved DON) through microbial food webs are difficult to esti- organic nitrogen (DON). The vertical distribution of mate. Thus, I designed an experiment which allowed DON, with high concentrations near the surface where the degradation of phytoplanktonic material to be mineral nitrogen sources are limited (Karl et al. 1993, studied separately from production processes, with a Koike et al. 1993), suggests that DON should play an 2-stage linked culture system (Giide 1991).The objec- tive of this study was to follow the fate of phytoplank- 'E-mail: [email protected] ton nitrogen in a simple microbial food web limited at O Inter-Research 1995 128 Aquat microb Ecol9: 127-136, 1995 the top by a ciliate. Through the investigation of The outflow was partly used for first stage samples. trophic links and the nitrogen budget, 1 examined Another part of the outflow was dispensed drop by importance of colloidal nitrogen. drop (average 1 l d-l) into a plastic enclosure. This sec- ond stage contained 74 1 natural sea water (Gulf of Marseille, France), firstly filtered by gravity through a MATERIALS AND METHODS 150 pm nylon mesh, and then filtered again through a 10 pm Nuclepore filter of 47 mm diameter under low Culture conditions. The 2-stage culture system was vacuum pressure. Aeration and agitation of this second constructed as shown in Fig. 1. Exudates and cellular stage (degrading stage 'D') were provided by blowing content of phytoplankton vary widely with species and air into the microcosm. Water temperature increased phases of growth. Thus, to maintain a constant algal from 18 to 21°C during the experiment which lasted production in the first stage (producing stage 'P'), a about 1 mo. The degrading stage was permanently steady-state growth of Phaeodactylum tricornutum kept in the dark to avoid any new phototrophic pro- algae was controlled in a continuous culture (Van duction. Approximately 1.5 1 was collected every Wambeke unpubl.). P. tricornutum was grown axeni- morning by a siphon tube for all analyses. cally in N-replete conditions in a 5 1 culture vessel Microbial counts. Algae, heterotrophic nanoflagel- under continuous light. The medium reservoir was lates (HNAN) and bacteria were fixed with formalin filled with Whatman GF/F pre-filtered sea water sup- (final concentration 2%), and stained with DAPI. Bac- plemented with 50 pM N-NO3, 5 pM P-PO, and 30 pM teria were filtered through 0.2 pm Nuclepore filters Si-silicate, then autoclaved. This medium was also and counted (Porter & Feig 1980). Algae and hetero- supplemented with vitamins and metals (Antia and trophic nanoflagellates were filtered through 0.8 pm Cheng's medium diluted at 1/10, without Tris). The Nuclepore filters and counted (Sherr & Sherr 1983). axenic culture was maintained at a dilution rate of Sea water subsamples were fixed with Lugol's solution 0.5 d-' with the medium inflow of 2.5 1 d-' and a con- and ciliates were counted using Utermohl's sedirnenta- tinuous outflow of the biomass and excretion products tion technique. of the first stage culture vessel of 2.5 l d-'. This system Nitrogen conversion factors. Nitrogen conversion maintained constant growth of the prey and avoided was estimated as follows: influence of its nutritional state on nitrogen regenera- Phaeodactylum tricornutum: The first stage outflow tion (Goldman et al. 1985). was regularly sampled for counts and particulate nitro- gen analysis. The average nitrogen content of algae in the first stage outflow was 1.09 pg N cell-'. - first stage Ciliates and flagellates: Nitrogen equivalent was cal- samplinq culated from abundance and biovolume (306 pm3 for HNAN and 8615 pm3 for a ciliate, see 'Results') using 110 fg C pm-3 (Turley et al. 1986, Weisse et al. 1990) and a C/N (by atom) ratio of 7 (Goldman et al. 1987a). Bacteria: As this experiment provided no data on Degrading batch bacterial biovolume, I used Lee and Fuhrman (1987) nutrient conversion factors of 20 fg C bacterium-', with a C/N reservoir ratio (by weight) of 4. Chemical analysis. After filtration through 0.2 pm cellulose acetate filters (Millipore), sea water samples were kept frozen until nutrients were measured using a Technicon Analyzer, except for ammonium, which was immediately determined. All the parameters were measured according to Strickland & Parsons (1972). Dissolved free amino acids (DFAA) were analyzed by high performance liquid chromatography. The pre- column derivatization method with o-phthaldialde- hyde (Lindroth & Mopper 1979) was applied as previ- ously described (Hermin et al. 1986).Dissolved organic First stage nitrogen (DON) was determined in the 0.2 pm Milli- second stage (in the dark) pore filtrate by the UV-H202oxidation method (Arm- strong et al. 1966). DON was calculated from the dif- Fig 1. The 2-stage culture system ference between nitrate concentration in the UV Van Warnbeke: Fate of Phaeodactylum tricornutum nitrogen through a microbial food web treated sample and dissolved inorganic nitrogen con- mensions of 27 X 2.5 pm, is considered as 2 cones joined centration (NO3+ NO2 + NH,) in the unaltered sample. at their bases. The calculated corresponding volume Ammonium concentration was measured only after (v01 P) was 45 pm3, in the range of those classically used Day 10. Therefore, before this date, the method pro- for this alga: 38.5 to 59.9 pm3 (Bonin et al. 1986). vided the sum DON + NH,, and not only DON. Non- identified organic nitrogen corresponded to DON minus DFAA nitrogen after Day 10, and (DON + NH,) RESULTS minus DFAA nitrogen before. Particulate organic nitrogen and carbon were measured with a Perkin Microbial populations Elmer 240 Elemental Analyzer on samples retained on pre-combusted Whatman GF/F glass fiber filters. A ciliate population preying on Phaeodactylurn tri- Data analysis. In the degrading stage, the continuous cornutum developed rapidly in the degrading stage inflow of 1 1 d-' from the first stage did not correspond and constituted the higher trophic level. Ciliates to the daily outflow (1.5l sample). The turnover rate of increased from nondetectable levels to 250 ciliates the water in such a system varied from 1.4 to 2 % d-l. ml-' on Day 18 (Fig. 2b). This ciliate population ap- Daily concentration variations of any component C peared homogenous: the typical form of the cell was an (nutrient, amino acid, cell), as calculated by simple dif- ellipsoid of average linear dimensions 22.5 X 32.5 pm ference between observed data (units I-'), simultane- (biovolume was 8615 pm3), morphologically similar to ously reflected the sum of inflow fluxes, outflow flux the oligotrich ciliate Strombidium sp. The first increase and a term K, which corresponds to the sum of biolog- in algae numbers, from Days 2 to 5, corresponded to ical and physical fluxes inside the culture, i.e. the sum the continuous inflow from the producing batch of of production sources minus the sum of losses, and about 8000 algae ml-' d-l. Then algae numbers consequently represents the true net daily variation.