Excretion of Dissolved Organic Carbon by Phytoplankton of Different Sizes and Subsequent Bacterial Uptake

- - MARINE ECOLOGY PROGRESS SERIES Vol. 132: 249-255, 1996 Published February 29 Mar Ecol Prog Ser - -. - -

Excretion of dissolved organic carbon by phytoplankton of different sizes and subsequent bacterial uptake

Nechama Z. ~alinsky-~ushansky'*~~*,Catherine ~egrand~

'Israel Oceanographic & Limnological Research. Yigal Allon. Kinneret Limnological Laboratory, PO Box 345, Tiberias, Israel 14102 'Department of Life Sciences. Bar-Ilan University, Ramat Gan, Israel ventre de Recherche en Ecologie Marine et Aquaculture de L'Houmeau, BP 5, F-17137 L'Houmeau, France

ABSTRACT: Identical techniques and laboratory equipment were used to compare photosyntheticpar- ticulate assimilationand excretion of dissolved orgdnic compounds (EOC)by different size classesof phytoplankton from marine and freshwater ecosystems.The subsequentassimilation and respiration of these algal exudates by bacteria were nleasured.The percentage of extracellular release of dissolved organlc compounds was lower (4 to 5%) for larger algal cells than for picoeukaryotes (29%).Bacterial ass~milationof the EOC released by th~algae under these conditions seemed to be related more to algal species than to biovolume, although therc may also be an effect on the specific bacterial assemblages. Bacterial uptake and ut~lizat~onof EOC was 19'"n for freshwaterp~coeukaryotes, and 18 to 70";. for marine nanoplankton. Greatvanations were found in the respiration rates of natural bacteria on EOC released by the freshwaterpicoalgae (95";,) and nanoplanktonic saltpond algae (20 to 414'%).

KEY WORDS: Phytoplankton photosynthesis Excretion Subsequentbacterial uptake

INTRODUCTION monly used 14C-technique (Herbland 1974) and the use of different laboratory equipment and procedures The dissolved organic carbon (DOC) pool in aquatic make it hard to compare the photosynthetic and excre- systems is composed of different sources such as runoff tion rates by phytoplankton reported in different stud- from the land, sloppy feeding and excretion from zoo- ies. Most of the data on excretion comes from in situ plankton (Jumars et al. 1989), lysis of plankton cells experiments (Berman & Holm-Hansen 1974, Watanabe (Cole 1985), and excretion by phytoplankton (Brock & 1980). A wide variability of percentage of algal extra- Clyne 1984). Algal excretion of DOC (EOC) is defined cellular release (PER) has been reported in works in as the DOC release during photosynthesis by a healthy situ (0 to 80%), but the average in fast growing algae population. was 13% of total primary production (Baines & Pace Phytoplankton excretion of DOC is considered in 1991). Some studies which gave high PER values were some studies as a continuous process (Larsson & criticized as experimental artifacts (Lancelot 1983). Hagstrom 1982), but others suggest that significant The effect of irradiance intensity cannot be isolated in excretion is a response to extreme environmental in situ experiments from other environmental factors stress, such as changes in the quantity and quality of such as temperature, salinity, oxygen concentration the light (Berman & Holm-Hansen 1974, Verity 1981, and light quality. Some data were obtained with labo- Zlotnik & Dubinsky 1989), 01- limitation of nutrient con- ratory cultures (Verity 1981). Zlotnik & Dubinsky centration (Lancelot 1983) or different physiological (1989) and Feuillade et al. (1990) reported a tight cou- states (Ignatiades & Fogg 1973). Problen~sin the com- pling between the rates of light dependent carbon fixation and of EOC. Another reason for the great variability of EOC values is the difficulty in evaluating bacterial assimilation

0 lnter-Research 1996 Resale of full article not perrn~tted 250 Mar Ecol Prog Ser 132: 249-255, 1996

and respiration of these algal exudates (Riemann & photosynthetic particulate assimilation (PA) and EOC Ssndergaard 1986). EOC may be important as a source in non-axenic algal cultures. Bacteria from freshwater of primary growth substrates for free-living bacteria non-axenic cultures of picoeukaryotes were obtained (Larsson & Hagstrom 1982). The importance of micro- after filtering dark incubated samples on 0.4 pm bial food chains parallel to the conventional grazing Nuclepore filters that were prerinsed with 50 m1 ones in aquatic food webs is now recognised (Azam et filtered (milllQ) water in order to prevent additlon of al. 1983). The significance of phytoplankton excretion DOC from the filters (Bloem et al. 1989). No chloro- as contributor to the 'microbial loop' has been studied phyll was found in the filtrates containing the bacteria. (Cole et a.1. 1982, Brock & Clyne 1984). EOC contri- Photosynthetic particulate carbon assimilation (PA) buted to about half of the bacterial carbon requirement and excretion (EOC). BOD bottles wlth 100 ml of the in some environments (Larsson & Hagstrom 1982). algal cultures were incubated with I4C-bicarbonate Some studies suggested that a considerable amount of (Steeman-Nielsen 1952) (NaH14C03,CEA, 0.3 11Ci ml-l) the annual primary production passed through the for 3 h under optimal irradiance and temperature con- bactenal. component (Brock & Clyne 1984), and bacte- ditions. At the end of the incubations, the bottles were rial production averaged 20% of planktonic primary immediately f~lteredin the dark. Duplicate subsamples production (Cole et al. 1988). of 15 m1 were filtered under low vacuum (max. 50 mm The goal of this study was to compare (1) the algal Hg), to avoid cell breakage (Herbland 1974).Differential photosynthesis and excretion rates and (2) the propor- filtration was used to measure PA (3and 0.8 pm Nucle- tion of photosynthetic excreted organlc carbon (EOC) pore filters for nano- and picoplankton, respectively), that passes through the bacterial component, in nano- EOC rates (<0.2 pm filtrates), and the subsequent bac- and picoplanktonic eukaryotic algae from salt ponds terial assimilation of EOC (BA) (0.2 pm Nuclepore filters) and lake water, respectively, using identical tech- (Fig. 1).All experiments included dark controls. Unin- niques and laboratory equipment. corporated inorganic radioactive carbon was removed by acidification of the filters in plastic scintillation vials with 0.5 m1 2N HC1, under a fume hood. MATERIALAND METHODS Jn order to measure the amounts of radioactive dissolved organic compounds that were released from the Algal and bacterial cultures. Three algal cultures algae during incubation (EOC), the <0.2 pm filtrates were examined. Naviculafilata (bacillariophyte, 8 to were acidified with 100 p1 0.1 N HCl, bubbled with air 10 pm) and Pavlova lutheri (prymnesiophyte, 4 to for 1 h, and 2 m1 aliquots were measured. Controls were 7 pm) were isolated from salt ponds on the Atlantic run by adding equivalent amounts of '"C-bicarbonate coast of France. The Chlorella-like picoeukaryote (1.5 to 2 pm) was isolated from Lake Kinneret, Israel (Malinsky-Rushansky & Berman 1991). Marine cultures were grown axenically In salt pond water which was prefiltered on 0.2 pm Nuclepore filters and auto- claved after enrichment with di-ammonium-phosphate (DAP) (15 pM (NH,),P04). Two liter cultures were incubated in optimal growth conditions (340 pE m-' S-', photoperiod 15L:9D, 20°C). Algae from late exponential phase cultures were used for the experiments (Legrand 1993). Non-axenic freshwater monoclonal picoeukaryotes were grown till exponential phase, in artificial medium BG-11 (Stanier et al. 1971) under continuous irradiance of 45 pE m-' S-' and temperature of 20°C. The marine natural bactenal population was ob- + acidification tained after filtering salt pond water through 1 pm l Nuclepore filters. DAP was added for 3 d in the dark to the filtrate containing the bacteria. Although micro- scopic observations did not show variations in shape or size of the cultivated bacteria compared to the natural Fig. 1. Methodology of the experiment. PA: Photosynthet~c particulate carbon assimilation, EOC: excretion of organlc populations, we cannot exclude a selection of some photosynthelate compounds, EOt4Ci: initial EOC for rneas- bacterial species. These bacteria were added to the urement of BA, EOI4C: residual EOC, BA: bactenal assimi- marine nanoplanktonic cultures in order to measure lation of EOC, BR:bacterial respiration Malinsky-Rushansky & Legrand: Phytoplankton excret~onof dissolved organic carbon

to 60 m1 samples, and filtering immediately (blanks),as Subsamples were fixed with formaldehyde (2 ?L final described above. All the samples were analysed for concentration) and counted with an epifluorescent radioactivity after addition of 10 m1 Instagel in a microscope (Zeiss Axioscope). Algal cells were counted Packard Tri Carb 1500 scintillation counter. using their autofluorescence, while bacteria were Extracellular release was expressed as PER of total stained with acridine orange (Hobbie et al. 1977). assimilated I4C: PER = EOC x 100/(PA + EOC) RESULTS AND DISCUSSION where EOC is excreted dissolved organic I4C,and PA is particulate assimilated I4C in the same volume, in- Photosynthetic particulate '"C-carbon assimilation (PA) cluding EOC assimilated by bacteria. Bacterial uptake of EOC (BA) and respiration (BR). Dark values were not subtracted from those meas- In order to measure the bacterial uptake and utilization ured in the light, because different mechanisms of of radioactive EOC, a 10 m1 aliquot from the bacterial carbon fixation were probably functioning in the 2 sample was added to 60 m1 of <0.2 pm filtrate contain- cases (Watanabe 1980). Two experiments were carried ing the radioactive E0I4C (Fig. 1).The pH of the above out, showing the same general trend of results, but in acidified filtrates was first adjusted with sterile NaOH this paper we present in detail the result of the more to in situ values. Final concentrations of bacteria were comprehensive of the 2 experiments. Because experi- slmilar to those observed in natural samples. After 3 h mental samples were always run in dupl~cate,we incubation in the dark, 4 subsamples of 15 ml were show the results in Tables 1 & 2 as ranges. Averages filtered on 0.2 pm Nuclepore filters. The radioactivity were taken for calculations of the photosynthetic para- retained on the filters corresponded to the radioactive meters. In the dark, picoeukaryotes assimilated 40% EOC assimilated by bacteria (BA). of the carbon fixation in the light, while dark carbon In order to obtain residual excreted radioactively fixation was less significant in the nanoplanktonic labelled E014C, 2 of the above filtrates were acidified cultures (average of 0.4 % for Pavlova lutheri and 1.9%) and bubbled with air for 1 h. Both remaining filtrates for Navicula filata) (Table 1). were bubbled without acidification to obtain the re- Photosynthetic rates in picoeukaryotes were similar sidual EOI4C along with the inorganic I4CO2resulting to those found in Lake Kinneret at the period of from bacterial respiration (BR). BR was obtained after picoeukaryote abundance (June), as well as in pico- subtracting the residual E0I4C from the fractions con- eukaryotic cultures from lake isolates (N Z. Malinsky- taining EO14C+ BR. Rushansky unpubl. data). Cell specific rates of carbon Chlorophyll a, algal and bacterial cell counts. assimilation were obtained by dividing PA values by Samples (2 to 10 ml) were filtered on glass fiber initial algal cell numbers, assuming that no significant filters (GF/F Whatman). Chlorophyll a (chl a) was differences occurred during the incubation in the extracted in methanol and measured with a Turner numbers, physiological state and size of the cells. The model 112 fluorimeter (Holm-Hansen & Riemann values of cellular PA for nanoplankton were 2 to 7 1978). The chl a values used in our calculations are times higher than for picoeukaryotes, although no those from the beginning of the experiment (Table 1). relation to the nanoplanktonic cell size was observed. In order to assess the separation between 'algal' and Assimilation numbers showed no relation to algal size 'bacterial' samples after filtration, we checked the chl a and were similar for Navicula filata and picoeukary- content in both fractions. otes, but were almost 2 times higher for Pavlova lutheri.

Table 1. Algal abundance (10"cIIs ml') and chlorophyll a (pg I-')at the bcglnning of the incubation. Photosynthetic particulatr carbon assimilation (PA) (pg C 1 ' h-'), PA per cell (PA/cell, pg C cell ' h 'l,and assimilation numbers (AN) (pg C pg chl-' h ') of axenlc (AX)and non-axenic (+B)algal cultures. Values from dupl~catesamples are presented as ranges Averages were taken for calculations of PA/cell and AN

Navicula Pavlova Picoeukaryotes AX +B AX +B +B

Algal abundance 85.1-87.5 55.4-72.0 Chlorophyll a 67 8-69.0 60.6 PA Light 221 9-236.9 173.5-215.9 Dark 1 6-2.0 5.4-6.0 252 Mar Ecol Prog Ser 132: 249-255, 1996

Table 2. Excretion of organic compounds (EOC) (pg C I-' h-') by 3 algal marine nanoplanktonic cultures had simi- axenic (AX) and non-axenic (+B)cultures, subsequent bacterial assimilation lar PER values (Table 21. (BA) (pg C 1.' h-'), and percentage of extracellular release (PER] (%). Values =he PER values (3 to 12 %) in our from duplicate samples are presented as ranges. Averages were taken for calculations of EOC+BA and PER experiments agreed with those found in other studies (Table 3). Smaller algae pm) showed higher (>l0%) PER values Na vicula Pa v10va Picoeukaryotes (4 AX +B AX +B +B (Table 3), perhaps due to their generally higher metabolic rate (Reynolds 1984). EOC 7.77-7.83 6.6-7.8 2.4-2.8 1.2-1.6 3.98-4.02 In in situ studies, PER varied from 0 to BA 0 0 0 0.9-1.5 0.4 70% (Sundh 1989, Gomes et al. 1991). EOC+BA 7.8 7.2 2.6 2.6 4.4 PERd 3.3 3.6 3.8 3.8 12.4 Higher PER values were found at low and very high irradiances, when photosynthe- "EOC + BA/PA +EOC + BA sis was reduced, near the surface or at the bottom of the euphotic zone (Berman & Holm-Hansen 1974, Watanabe 1980, Table 3. Excretion of organic compounds by different phytoplankton~ccul- tures. Modified from Learand (19931 Larsson & Hagstrom 1982). In our experiments, we used optimal light intensities Algal species Si~e(pm) PER (X) Source and temperature for growth, probably resulting in lower PER values compared Synechococcus sp. 10-15 Zlotnik & Dubinsky (1989) to those observed in nature with different ChloreUa -like 12.4 This study environmental factors. lsochrysis galbana 10 Zlotnik & Dubinsky (1989) Chlorella vulgaris 4 Zlotnik & Dubinsky (1989) Pavlova lutheri 3.6-4.5 Ignatiades & Fogg (1973) 3-4 This study Bacterial uptake of algal excreted Navicula filata 3-5 This study organic compounds (EOC) Leptocylindrus danicus 1.3-9 Verity (1981) ~keletonemacostatuni 3-20 4.6-9 ~~natiades& Fogg (1973) Oscillatoria 3-30 <3 We tried to estimate the bacterial 1 Nalewajko (1966) uptake and utilization of algal EOI4C and Chlamydomonas provasolu 5-30 5.6 Saks (1982) to differentiate between the 'assim~lation' Chaetoceros affinis 9-30 9 Myklestad et al.. (1989) (BA) an.d the 'respiration' (BR) (Fig. 1). Thalassiosjra fluviatilis 18-50 5 Sharp (1977) Bacterial numbers in the filtrate contain- l ing algal EO1'C corresponded approxi- mately to those found in the original algal Our results (Table 1) suggest that the photosynthetic cultures during the light experiments. Here we give parameters measured (PA and EOC) are not related to values as percentages to minimize the effect of algal size an.d physiological state, but to the species. changes in specific activity of I4CO2 and EOI4C in our In order to measure if all the chlorophyll was retained experiments (Tab1.e 4). in the algal fraction, the bacterial fractions were filtered In the light experiments (Fig. I),PER was calculatea on GF/F filters. All picoeukaryotic chlorophyll was re- relative to total carbon fixed, including BA in the non- tained on 0.8 pm filters, and the loss for nanoplanktonic axenic algal cultures, and residual EOC, as in the chlorophyll was less than 7 'h.This suggests that assimilation numbers found for picoeukaryotes are correctly Table 4. Bacterial assmilation (BA) and respiration (BR) 01 evaluated, while those found for nanoplankton could be EOC. All values are in % slightly overestimated. Navicula Pavlova Picoeukaryotes

EOC uptake (total) Algal excretion of dissolved EOI4Ci - EOI4Ca 72.0 18.2 19.3 14C-organic compounds (EOC) BA+BR" 69.8 18.2 18.6 EOC respirationC 19.6 40.6 95.1 The excretion of photosynthetic carbon was percep- aAssimilated EOC, calculated as the difference between tible in all 3 algal cultures. The PER relative to total Initial andresldual EOC carbon fixed was calculated, including BA, from the '~ssimilated EOC, calculated as the sum of bactenal assimdation and respiration non-axenic cultures (Table 2) PER values were about 'Respired EOC from total assimilated EOC, calculated as 3-fold higher for freshwater picoeukaryotes (120h] BR X 100/(EO1'Ci - EOi4C) than for marine nanoplanktonic algae (3 to 4 %). The 2 ivIalinsky-Rushansky& Legrand: Phytoplankton excretion of (Jizsolved organic carbon 253

method of Riemann & Ssndergaard (1986). The differ- culture, bacteria more adapted to using the specific ences in values of residual EOC found in axenic and composition of EOC released inay have been selected. non-axenic cultures of Pavlova lutheri were probably The composition of EOC could also be affected by due to bacterial assimilation of EOC, which was mostly volatile and acid unstable compounds that may escape recovered in the bacterial fraction BA (Table 2). In after acidification and bubbling, and this may explain terms of carbon, BA was 1.4 % of particulate assimi- the slightly higher values (0.7 % for picoeukaryotes lated carbon for picoeukaryotes and 1.9% for l? lutheri, and 2.2% for N. Eilata) found for bacterial utilization and accounted for 107i, and 46% of the EOC excreted (EOC uptake), when calculated from the difference by picoeukaryotes and P lutheri, respectively, assum- between initial and residual E014C, compared to those ing that the specific activity was the same in all corn- calculated froin the sum of bacterial assimilation and partments. BA of EOC was not observed in the non- respiration (Table 4). axenic cultures of Navicula Eilata in the light, probably Large variations were found in the respiration rates because of a filtration error. It is unlikely, however, that of EOC by natural bacterial population from the salt there was no BA, because significant BA of labelled pond (20 to 41 %) and from freshwater (95%).Respira- E014C was found during the dark incubation. tion rates were calculated as the percentage of BR In the dark experiments, we attempted to quantify of the total EOC uptake (Table 4). Respiration rates bacterial utilization of total algal EOC, including BA obtained for bacterial salt pond population were simi- and BR, by adding radioactive EOL4C (obtained as lar to those found for bacteria in complex nutritive ~0.2pm filtrate from the algal cultures) to natural medium (Payne & Wiebe 1978). The high percentage marine or freshwater bacterial populations. We assumed for BR (95%) of picoeukaryotic EOC can explain the that no algae passed the 0.2 pm filters and in conse- low percentage (0.3 %) found for BA of EOC. High BR quence, all radioactivity subsequently found in par- rates on EOC (80 to 95 %) were also reported by Billen ticles was considered to result from bacterial uptake of et al. (1980) and Itturiaga & Zsolnay (1981). algal EOI4C. Most of the picoeukaryotic EOC (95 %) was respired The percentage of bacterial utilization of excreted by bacteria, while only a small part (5%) of the EOC organic compounds EO14C, calculated as the differ- used was assimilated and used to increase the biomass. ence between the initial and the residual E0I4C, was However, no increase in bacterial cell numbers was about 3.5-fold higher for EOC from Navicula Eilata observed. More than half of the EOC released by (72%) than from Pavlova lutheri (18%) and pico- Pavlova lutheri and Navicula filata (59 and 80%, eukaryotes (19%) (Table 4) and could result from dif- respectively), was utilized to increase the bacterial bio- ferences in the efficacy of bacteria in assimilating EOC mass, but increase in bacterial cell numbers was noted from different sources and of different composition only with EOC released from N. filata (from 86.3 X (Bell 1984). Bacterial uptake depends on the kinds of 104 + 1.2 X 104 to 96.7 X 104 + 1.2 X 104cells ml-l). This algal EOC released and probably also on the bacterial finding could be related to the high percentage of species and their origin. Bacterial assemblages were EOC uptake (72%) which was then relatively effi- almost certainly different in freshwater and in salt ciently utilized (80%) to increase cell numbers. We ponds. We used the same bacterial population with the have no information on slight changes in bacterial cell E014C from the 2 marine nanoplanktonic cultures, and sizes that could result from the increase in biomass the difference in response suggests a different compo- without increase in cell numbers. sition of E0I4C. For the non-axenic picoeukaryote

Algal photosynthetic assimilation (PA) and excretion Table 5. Uncorrected (UN) and corrected (COP.) values of total photosynthetic particulate carbonassimilation (PA)and (EOC) corrected for total bacterial uptake excretion (EOC)in non-axenic algal cultures.Values of PA and EOC are in pg C 1 ' h-', and of PER in O/u Values of gross photosynthetic carbon fixation may be underestimated if EOC and bacterial uptake are not Na vicula Pavlova Picoeukaryotes taken into account. Total bacterial uptake was re- UN COR UN COR UN COR evaluated as the sum of EOC assimilated but not respired, which was found in the bacterial fraction during EOCa 7.2 7.2 1.4 3.4 4.0 12.8 PA~ 194.7 202.0 63.4 66.8 31.2 44.0 the light experiment with phytoplankton (Table l), plus PER 3.6 3.6 3.8 5.0 12.4 29.1 the amount of EOC respired (derived from Table 4), assuming that specific activity was the same during aTotalEOC, corrected for bacterial assimilation and respiration (EOC + BA + BR) the incubation period. With this correction, total PA was b~otalPA, corrected for total excretion (PA +EOC tot) 591') higher for Pavlova lutheri and 29'% higher for picoeukaryotes (Table 5). Photosynthetic parameters 254 Mar Ecol Prog Ser 132 249- 255, 1996

for Navicula filata may be slightly (.c4'%)underesti- environment. BR of EOC (as pg C 1-Ih') from freshwa- mated, because there was no correction due to bacterial ter picoeukaryotes was 2 and 7 times higher than for uptake and utilization of EOC. The percentage of cxtra- marine hr. filata and P luthen,respectively. Assuming cellular release (PER) was 22% and 57'!:, higher for F? that the average biovolumc of bacteria from salt ponds luthen and pi.coeukaryotes, respectively. Our results and Lake Kinneret in our experiments are 0.071 and show underestimates (1.0 to 26":,) s~milarLo those noted 0.025 I.lmi, respectively, with a carbon content of by Baines & Pace (1991) in measurements of primary 0.22pg C pm-' (Bratbak & Dundas 1984).the residence production of natural communities when dissolved or- time for EOC in bacteria was lower in those growing ganic production is ignored. with N. filata and F? luthen(0.4 to 4.6 d) than in those growing with picoeukaryotes (22 d). We conclude that bactenal uptake of algal EOC can Fluxes of carbon through algal excretion be an important component of the carbon flux and should be determined as part of total photosynthetic Carbon fluxes (given as pg C 1" h-') from algal carbon fixation. Because we used identical techniques, photosynthetic carbon assimilation to the bacterial we could compare and conclude that excretion was biomass via algal excretion are summarized and com- higher in picoeukaryotes than in nanoplanktonic cul- pared in Fig. 2. Values were higher with freshwater tures. BA of EOC depended more on the algal spec~es pic.n::ukaryotes compared to the 2 marine algal cultures and the bacterial specific assemblage than on the algal (12.8 and 3.4 to 7.2 yg C I-' h-', respectively). Although biovolume. BR rates on EOC were much h~gherin absol.ute carbon excretion was 2-fold higher for Navic- freshwater picoeukaryotes in cultures. ula filata,the PER was similar in the 2 marine algal cultures (4 to 5 %), and was 29% for picoeukaryotes. The Acknowledgements. This work was supported by a 'Bourse efficiency of EOC uptake by bacteria was highest with Chateau-briand' from The Ministry of Foreiqn Affairs. France, IFREhIER Poitou-Charentes grant and Grant 1932-1-93 from EOC from N. filata (70@4))and sim~larwith EOC from the Xiinistry of Scicncr and the Arts, Jerusalem, Israel. The Pavlova lutheri and picoeukaryotes (18 and 19'%, experiments were made in the Laboratoire CNRS-IFREXIER, respectively), suggcstlng no relation to algal size and Crema-L'Hourncau, France. We thank T Berman, A. Herb- land and D. Delmas for their useful comrncnts on thc nldnu- -- script, as well as L. Joassard for technical help with the fPavlova 1uther-i ~icoeukar~otes chlorophyll samples. This manuscript is a part of the PhD m thesis of N.Z.M , Bar Ilan Univers~ty.Israel.

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This article was subn~rttedto the ed~tor Manuscr~ptfirst received. December 1, 1994 Revlsed version accepted: Allgust 24, 1995