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Nitrogen fixation by diazotrophic cyanobacteria in the Baltic Sea and transfer of the newly fixed nitrogen to picoplankton organisms
Journal of Marine Systems 25 Ž2000. 213–219 www.elsevier.nlrlocaterjmarsys
Nitrogen fixation by diazotrophic cyanobacteria in the Baltic Sea and transfer of the newly fixed nitrogen to picoplankton organisms Ute Ohlendieck a,) , Annegret Stuhr a , Heike Siegmund b a
Institut fur Weg 20, 24105 Kiel, Germany ¨ Meereskunde an der UniÕersitat ¨ Kiel, Dusternbrooker ¨ b Institut fur Germany ¨ Ostseeforschung, Warnemunde, ¨ Received 15 December 1998; accepted 4 June 1999
Abstract Nitrogen fixation Ž15 N2-tracer method. by filamentous cyanobacteria and the flux of the newly fixed nitrogen into picoplanktonic organisms were investigated during three cruises in the central Baltic Sea in July and August 1995 and July 1996. The diazotrophic cyanobacteria consisted mainly of Aphanizomenon and Nodularia and the picoplankton fraction mainly of Synechococcus, a coccoid nondiazotrophic cyanobacterium. The average N2-fixation rate measured during the two July studies Ž3.9 nmol ly1 hy1 in 1995 and 2.4 nmol ly1 hy1 in 1996., was about 13 times higher than in August 1995 Ž0.3 nmol N2 ly1 hy1 .. During the July studies, 5–10% of the total N2-fixation was found in the picoplankton fraction, but none was found in August. The study shows that in the early stage of a cyanobacterial bloom new nitrogen is provided for the pelagic foodweb through release of recently fixed nitrogen by the diazotrophs, whereas in the late stage the input of new nitrogen occurs mainly through lysing of decaying filamentous cyanobacteria cells. Considerable temporal and local variations in nitrogen fixation found in this study indicate that broader studies in time and location are needed to assess the contribution of N2-fixation to the nitrogen budget of the Baltic Sea. q 2000 Elsevier Science B.V. All rights reserved. Keywords: cyanobacteria; Nodularia sp.; Aphanizomenon sp.; Synechococcus sp.; nitrogen fixation; exudation; picoplankton
1. Introduction Filamentous diazotrophic cyanobacteria in the Central Baltic Sea grow intensively in summer when the water column is stratified and the mixed layer is impoverished in dissolved inorganic nitrogen ŽDIN..
) Corresponding author. Tel.: q49-431-597-3983; fax: q49597-3994. E-mail address: [email protected] ŽU. Ohlendieck..
15
N2 -tracer method; Baltic Sea;
Due to their ability to fix N2 , the cyanobacteria can contribute substantially to the input of new nitrogen into the nutrient-poor environment. This nitrogen pool represents a potentially important nitrogen source for other organisms in the pelagic foodweb. There are three major ways in which the fixed nitrogen may enter the marine nitrogen cycle: grazing by zooplankton, lysis of decaying cyanobacteria cells and exudation of dissolved nitrogen products. Grazing of filamentous cyanobacteria has been shown to be of no importance in the Baltic Sea ŽSellner et
0924-7963r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 4 - 7 9 6 3 Ž 0 0 . 0 0 0 1 6 - 6
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U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
al., 1996.. Liberation of fixed nitrogen into the water through lysis of decaying cyanobacteria cells in late summer is thought to be the primary way of making the nitrogen available for other organisms ŽLindahl et al., 1980; Schulz and Kaiser, 1986.. The majority of diazotrophic cyanobacterial biomass occurs in the surface layer because their strong gas vesicles ensure positive buoyancy ŽWalsby et al., 1995.. Due to this, when they decompose, the nitrogen is released in the surface layers. Elevated levels of dissolved nitrogen and dissolved organic carbon ŽDOC. have been found in the mixed layer by several authors during and after decay of cyanobacterial blooms ŽGundersen, 1981; Kuparinen et al., 1984. as well as a well-developed microbial foodweb ŽHoppe, 1981.. Exudation of nitrogenous compounds by actively growing diazotrophic cyanobacteria also seems to be of importance, as some earlier studies indicate. Jones and Stewart Ž1969. showed that more than 50% of the nitrogen recently fixed by Calothrix, a cyanobacterium of the littoral, was released and a part of it was taken up by heterotrophic bacteria, picoplankton and algae. Paerl Ž1984. found a considerable amount of 15 N fixed in cultures of Anabaena that was present in associated bacteria after a short incubation time. Recent studies on Trichodesmium, a diazotrophic cyanobacterium occurring in the tropics, showed that during active growth up to 50% of the newly fixed nitrogen is released into the water as dissolved organic nitrogen ŽDON. ŽGlibert and Bronk, 1994.. In early studies, it was necessary to concentrate samples for nitrogen fixation measurements because the 15 N2-tracer method was not sensitive enough at that time. Nowadays, the availability of highprecision isotope ratio mass spectrometers makes it feasible to carry out 15 N2-tracer incubations on unconcentrated natural water samples, with minimal disturbance of the system ŽMontoya et al., 1995.. This study deals with the utilization of cyanobacterial extracellular products by other organisms. The experiments were conducted on natural plankton communities in the Baltic Sea. Nitrogen fixation by filamentous cyanobacteria was measured in the central Baltic Sea and the flux of the newly fixed nitrogen into picoplanktonic organisms, potential consumers of DON, was investigated during different stages in the development of a cyanobacterial
population. The 15 N2-tracer method was applied, which made it possible to measure the rate of N2fixation directly and to follow the movement of the newly fixed nitrogen into other components of the planktonic foodweb.
2. Materials and methods N2-fixation experiments were carried out during three cruises in the central Baltic Sea in 1995 and 1996: Periods 26r06–11r07r1995 07r08–22r08r1995 15r07–25r07r1996
Areas of investigation Arkona Sea, Middlebank Middlebank, Bornholm Sea Middlebank, Bornholm Sea, Gotland Sea
The experiments in 1995 were carried out during drift experiments conducted in each region. During the cruise in 1996, a larger area was covered Žsee Fig. 1.. 2.1. Nitrogen fixation Water samples were collected using a CTD-rosette Ž12 = 12 l Niskin bottles.. Nitrogen fixation was measured in surface water samples. The samples were incubated in 0.25–, 0.5– or 1–l replicate glass bottles ŽDuran w ., which were filled to overflowing and sealed with a septum cap Žteflon-lined butyl
Fig. 1. The investigation area with drift stations Ž1995. and cruise track Ž1996..
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
rubber.. A gas-tight syringe was used to inject the 15 N2 gas Ž99 at.% 15 N, CAMPRO Scientific. into the bottle and water was allowed to escape through the injection needle to equalize the pressure across the septum. The 15 N2-enrichment in the incubation bottles ranged between 19.7 and 23.4 at.% in 1995 and between 5.4 and 6.1 at.% in 1996 for different incubations. The bottles were gently mixed and then incubated, either attached to an in situ array at the surface or in a deck incubator covered with a neutral density screening to simulate in situ light intensity. The incubation time was 8 to 10 h during daylight. At the end of each experiment, the samples were filtered under a mild vacuum Ž100 cm Hg. through a precombusted Ž6 h at 4508C. 25-mm GFrF filter and stored in a freezer Žy208C.. To investigate the 15 N-enrichment in the picoplankton fraction, one replicate of every sample set was filtered through a 2-mm membrane filter Žon the first cruise. or a 5-mm membrane filter Žon the other cruises., respectively, the filtrate was collected and then also filtered through a GFrF filter. Simultaneously, the picoplankton fraction was incubated with 15 N2 on its own as a control to test if there was any N2 fixed by this fraction. Negative results were obtained in all occasions. 15 N-enrichment by the fractions - 2 and - 5 mm was compared during several experiments. The biomass of the - 5-mm fraction was about 10% higher but there was no difference in the 15 N-enrichment. Back ashore, the samples were dried at 608C, wrapped in tin cups ŽHeraeus CHN cups., formed into pellets and analysed at the Institut fur ¨ Ostseeforschung in Warnemunde as described in Mon¨ toya et al. Ž1995.. The N2-fixation rate was calculated according to Montoya et al. Ž1995.. 2.2. EnÕironmental parameters and phytoplankton composition Hydrographical data were measured by CTD instruments. Analysis of ammonium, nitrate and phosphate concentrations were carried out at sea according to the methods described by Grasshoff Ž1976.. The contribution of filamentous diazotrophic and picoplanktonic cyanobacteria to total chlorophyll-a was calculated using ratios of chlorophyll-a to class specific pigments Žechinenone and zeaxanthin.
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ŽMeyerhofer, in preparation.. The pigment analysis ¨ was carried out by means of the HPLC-technique as described by Mantoura and Llewellyn, 1983, slightly modified by Meyerhofer, 1994. Pigments were iden¨ tified by retention time comparison with pigment standards and by on-line spectra collection using a photo-diode array spectrometric detector. Samples for pigment analysis were filtered through precombusted GFrF filters and stored in a freezer Žy208C. until analysis. The plankton composition was investigated qualitatively on board through light microscopy as well as quantitatively in the laboratory using the Utermohl ¨ method ŽUthermohl, ¨ 1958..
3. Results and discussion 3.1. Nitrogen fixation and the flux of picoplankton fraction
15
N to the
Nitrogen fixation ŽTable 1 and Fig. 2. and the nitrogen fixing capacity of the filamentous cyanobacteria, expressed by the fixation rate per diazotrophic cyanobacteria chlorophyll-a, showed distinct differences between the July studies in 1995 and 1996 on the one hand and the August 1995 study on the other hand. Nitrogen fixation on the cruises in early summer was up to 30 times higher, and on average 13 times higher, than in late summer. Whereas the fixation rate ranged between 2.3 and 5.6 nmol of N2 ly1 hy1 with an average of 3.9 nmol in July 1995 and between 1.4 and 3.1 nmol of N2 ly1 hy1 with an average of 2.4 nmol in July 1996, it amounted only to 0.3 nmol in August 1995. The nitrogen fixing capacity varied between 3.4 and 6.5 nmol of N2 mg diazotrophic-chlorophyll-ay1 hy1 during the July studies Žon average 4.5 nmol in 1995 and 4 nmol in 1996. and between 0.5 and 1.7 nmol of N2 mg diazotrophic-chlorophyll-ay1 hy1 Žon average 1 nmol. in August. There was also a difference between the early summer cruises and the late summer cruise regarding the flux of newly fixed nitrogen into the picoplankton fraction ŽTable 1 and Fig. 2.: 5–10% of the nitrogen fixed by the filamentous cyanobacteria was found in picoplankton biomass during the July studies, but none was found in the picoplankton in August.
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
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Table 1 Nitrogen fixation rates and the flux of the recently fixed nitrogen into the picoplankton fraction Žexpressed as percentages of the total fixation rate. 15
Cruise
Date
Region
Incubation time
h
Incubation condition
N2 fixed wnmol ly1 hy1 x
N2 fixed wnmol mg diazotrophic chl-ay1 hy1 x
N in picoplankton % of total N2 -fixation Ž%.
July 1995
03r07r1995 07r07r1995 09r07r1995 18r07r1996 19r07r1996 20r07r1996 22r07r1996 23r07r1996 24r07r1996 12r08r1995 15r08r1995 17r08r1995
Mb AS AS GS, st1 GS, st2 GS, st3 GS, st4 GS, st5 BS, st6 Mb BS BS
09:00–19:00 09:00–19:00 09:00–19:00 10:00–19:00 10:30–18:30 09:30–19:30 11:00–19:00 10:30–19:00 11:00–19:00 09:00–19:00 09:00–19:00 09:00–19:00
10 10 10 9 8 10 8 8.5 8 10 10 10
in situ in situ in situ on deck on deck in situ on deck on deck on deck in situ in situ in situ
2.26 3.74 5.59 2.78 1.36 2.86 1.39 3.09 2.93 0.34 0.19 0.2
5.83 3.47 4.60 6.48 3.39 2.42 3.38 3.46 4.81 1.70 0.54 0.83
7 6 10 7 5 10 5 8 5 0 0 0
July 1996
August 1995
AS s Arkona Sea, BS s Bornholm Sea, Mb s Middlebank, GS s Gotland Sea.
The explanation of these results will draw upon environmental data, data on biomass concentration and results from microscopic investigations. 3.2. EnÕironmental conditions During all of the investigations, the mixed layer was separated from the deeper waters by a seasonal thermocline. All nutrient data Žaverage of three to six samples, depending on the mixed layer depth. refer to the mixed layer ŽFig. 2.. In both July cruises, the mixed layer reached down to 10–15 m with a temperature of about 148C. In August 1995, the hydrographical situation was very different. The mixed layer was only 5–10 m deep and had a temperature of about 208C. The NrP ratio of the nutrients was low during the July cruises Ž1–2 on average. with low concentrations of DIN Žs nitrate and ammonium., 0.1 mmol ly1 on average, and still a considerable amount of phosphate, especially in July 1995 Ž0.14 mmol ly1 in 1995 and 0.06 mmol ly1 in 1996 on average.. As nitrogen fixation is inversely related to available inorganic nitrogen pools and directly correlated with phosphorus availability, as reviewed by Sellner Ž1997., the nutrient conditions found during the July cruises were ideal for growth of diazotrophic cyanobacteria.
In August 1995, the NrP ratio was higher, varying from 3–7 with a slightly higher DIN concentration Ž0.21 mmol ly1 on average. compared to the July cruises. This might have been a critical point for the low N2-fixing capacity at that time. It has been shown that nitrogen fixation ceases when other nitrogen sources are available ŽLindahl et al., 1978; Moisander et al., 1996.. 3.3. Phytoplankton composition 3.3.1. Microscopic inÕestigation The filamentous cyanobacteria consisted mainly of two genera in both years: Aphanizomenon and Nodularia. Anabaena was also found in some regions but was of minor importance. In the July studies, Aphanizomenon dominated with exception of the region around Middlebank in 1996 where Nodularia was more abundant. Nodularia had not yet formed any aggregate but was present as single filaments. From microscopic analysis, both genera were in ‘good condition’, i.e., without any unpigmented cell or disintegrating colony. No attached organisms were visible. In August 1995, Nodularia was the dominant filamentous cyanobacterium. In contrast to early summer, it had formed aggregates in which other organisms such as other bacteria, diatoms, flagellates
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
Fig. 2. Nitrogen fixation rates Žabsolute rates for diazotrophs and the flux into the picoplankton fraction and diazotrophic cyanobacteria chlorophyll-a specific rates., nutrient concentration ŽDINs nitrateqammonium and Phosphate. and NrP-ratio of nutients, and the contribution of diazotrophic cyanobacteria and Synechococcus to total chl-a on the drift stations in 1995 and along the cruise track in 1996. ASs Arkona Sea, BSs Bornholm Sea, Mbs Middlebank, GSsGotland Sea.
217
cus, a picoplanktonic nondiazotrophic cyanobacterium, is shown here as a major contributor to the picoplanctonic size fraction Ž60–85%. besides heterotrophic bacteria and small eukaryotes. It is known that Synechococcus can live on DON ŽCarr and Wyman, 1986.. In July, filamentous cyanobacteria as well as Synechococcus contributed about 30% of total chlorophyll-a each. The remaining portion of about 40% was made up by eukaryotes such as diatoms and flagellates. In August 1995, the contribution of filamentous cyanobacteria was only 10% on average whereas the contribution of Synechococcus has risen to 65%. Thus, the contribution of Synechococcus had more than doubled and the contribution of filamentous cyanobacteria had decreased by two thirds relative to July 1995. The absolute chlorophyll-a values varied for the diazotrophs between 0.4 and 1.1 mg ly1 and for Synechococcus between 0.4 and 0.9 mg ly1 during July. In August, the chlorophyll-a values varied between 0.2 and 0.35 mg ly1 for the diazotrophs and between 1 and 2 mg ly1 for Synechococcus. Besides the low nitrogen-fixing capacity of the diazotrophs, the low active biomass of filamentous cyanobacteria must have been decisive for the low nitrogen fixation rate in August. The active biomass had ceased because the filamentous cyanobacteria were aged and much of the material was decaying whereas both genera were in ‘good condition’ in July 1995 and 1996. Lindahl et al. Ž1980. also found in their study, carried out in the outer Stockholm Archipelago in 1975, significantly higher nitrogen fixation rates Žnine times higher. in July than in August. The physiological state of the diazotrophic cyanobacteria, as well as the nutrient conditions at those times, were similar to the situation found in this study.
and ciliates were abundant. A major part of Nodularia was decaying material, indicated by unpigmented cells.
3.4. The significance of the newly fixed nitrogen for the pelagic foodweb
3.3.2. Pigment analysis The contribution of filamentous cyanobacteria and Synechococcus to total chlorophyll-a, as derived from pigment analyses, is shown in Fig. 2. Synechococ-
Due to the low nitrogen fixation rate in August, there could have been just very little transfer of newly fixed nitrogen to other organisms. If there had been some, this nitrogen would most probably have
218
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
been consumed by organisms directly associated with the aggregates, so that this flux would not have been measurable by the size fractionation method. The most important means of nitrogen input at that stage of a bloom would be lysing of decaying cyanobacteria cells, which has been shown in other studies ŽGundersen, 1981; Kuparinen et al., 1984.. However, in the early phase of the cyanobacterial development, newly fixed nitrogen was transferred from filamentous cyanobacteria to picoplanktonic organisms, accounting for between 5% and 10% of total nitrogen fixation. As grazing of filamentous cyanobacteria in the Baltic is of no importance ŽSellner et al., 1996., the release of nitrogen by actively growing nitrogen-fixing cyanobacteria would be the only flux of new nitrogen from diazotrophic cyanobacteria to the nutrient impoverished upper euphotic zone, available for the pelagic foodweb at that time of the year. This nitrogen flux supports the microbial loop and leads to an increase of biomass of organisms that depend on bound nitrogen compounds. In this study, there was a pronounced increase of the Synechococcus biomass from July to August ŽFig. 2.. This biomass increase can be attributed partly to the utilisation of nitrogen released by diazotrophic cyanobacteria in the early stage of the bloom, as shown in this study. But it can also be attributed to uptake of nitrogen released by decaying cells and uptake of mineralisation products in the late stage of a bloom. Unfortunately, neither the temporal development of the Synechococcus biomass increase nor the development of the nitrogen fixation rate Žnor the amount of exudation products. could be followed continuously during this study. Therefore, one cannot weigh the relative importance of either of these processes. Besides the pronounced difference in N2-fixation between the July and the August studies, there was also a significant variability in nitrogen fixation between the different regions investigated and even within the drift studies. This could have been caused either by patchiness of the diazotrophic biomass or by variability in their nitrogen fixing capacity. There was indeed patchiness in biomass locally as well as temporally, as can be seen in the chlorophyll-a data ŽFig. 2.. But there was also considerable variability in the nitrogen fixing capacity. The reason for this variability is not easy to judge; there are various
possibilities, such as patchiness of dissolved nitrogen, patchiness of diazotrophs in ‘good condition’ and the aging of colonies or light inhibition of nitrogen fixation.
4. Summary and conclusions Summing up, one can say that nitrogen fixation was high in the early stage of the cyanobacterial development. Nitrogen was released from actively growing cells, providing new nitrogen for the microbial loop in the oligotrophic mixed layer. At the late stage, nitrogen fixation rates were low and nitrogen supply into the water seemed to occur mainly through lysing of decaying cyanobacteria cells. Though the N2-fixation rate is low at that stage, the released nitrogen further supports the microbial foodweb. The high variability in nitrogen fixation at different times and locations during the vegetation period of diazotrophic cyanobacteria found in this study indicates that it is necessary to conduct studies on a broader and more systematic basis Žsee also Stal and Walsby, 1998. in order to record data on N2-fixation, which make it possible to work out a reliable budget for nitrogen input through nitrogen fixation into the Baltic Sea.
Acknowledgements We are greatly obliged to Maren Voss and the Institut fur mass ¨ Ostseeforschung, Warnemunde ¨ spectrometry facility for advice and for the analysis of all 15 N-samples and to Rolf Boje for support, discussions and for including us in the project. Further, we thank the captains and crews of the RrV Alkor, the RrV Littorina and the RrV Alexander Õon Humboldt for their support at sea, Joseph P. Montoya for advice, Frank F. Jochem for inspiring discussions, Michael Meyerhofer for providing ¨ HPLC-data and Uschi Junghans and Petra Krischker for their technical assistance at sea. We also thank Anthony E. Walsby and two anonymous reviewers for their careful reading of the manuscript and for their helpful comments. This research was supported by the European Commission Environment RTD Programme DG XII, Contract No EV5V-CT94-0404
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
to the Institut fur ¨ Meereskunde an der Universitat ¨ Kiel.
References Carr, N.G., Wyman, M., 1986. Cyanobacteria: their biology in relation to the oceanic picoplankton. In: Platt, T., Li, W.K.W. ŽEds.., Photosynthetic Picoplankton. Can. Bull. Fish. Aquat. Sci. pp. 159–204. Glibert, P.M., Bronk, D.A., 1994. Release of dissolved organic nitrogen by marine diazotrophic cyanobacteria, Trichodesmium spp. Appl. Environ. Microbiol. 60, 3996–4000. Gundersen, K., 1981. The distribution and biological transformations of nitrogen in the Baltic Sea. Mar. Pollut. Bull. 12, 199–205. Hoppe, H.-G., 1981. Blue–green algae agglomeration in surface water: a microbiotope of high bacterial activity. Kiel. Meeresforsch. Sonderh. 5, 291–303. Jones, K., Stewart, W.D.P., 1969. Nitrogen turnover in marine and brackish habitats. The production of extracellular nitrogen by Calothrix scopulorum. J. Mar. Biol. Assoc. U. K. 49, 475–488. Kuparinen, J., Leppanen, J.-M., Sarvala, J., Sundberg, A., Virta¨ nen, A., 1984. Production and utilization of organic matter in a Baltic ecosystem off Tvarminne, southwest coast of Finland. ¨ Rapp. P.-V. Reun.-Cons. Int. Explor. Mer 183, 180–192. Lindahl, G., Wallstrom, ¨ K., Brattberg, G., 1978. On nitrogen fixation in a coastal area of the northern Baltic. Kiel. Meeresforsch. Sonderh. 4, 171–177. Lindahl, G., Wallstrom, ¨ K., Brattberg, G., 1980. Short-term variations in nitrogen fixation in a coastal area of the northern Baltic. Arch. Hydrobiol. 98, 88–100.
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Mantoura, R.F.C., Llewellyn, C.A., 1983. The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase high-performance liquid chromatography. Anal. Chim. Acta 151, 297– 314. Meyerhofer, ¨ M., 1994. Plankton pigments and their derivatives as biomarkers for the description and assessment of phytoplankton succession and sedimentation in the North Atlantic. Ber. Inst. Meereskd., Kiel 251, 188. Moisander, P., Lehtimaki, ¨ J., Sivonen, K., Kononen, K., 1996. Comparison of 15 N2 and acetylene reduction methods for the measurement of nitrogen fixation by Baltic Sea cyanobacteria. Phycologia 35, 140–146. Montoya, J.P., Voss, M., Kahler, P., Capone, D.G., 1995. A ¨ simple, high-precision, high-sensitivity tracer assay for N2 fixation. Appl. Environ. Microbiol. 62 Ž3., 986–993. Paerl, H.W., 1984. Transfer of N2 and CO 2 fixation products from Anabaena oscillarioides to associated bacteria during inorganic carbon sufficiency and deficiency. J. Phycol. 20, 600–608. Schulz, S., Kaiser, W., 1986. Increasing trends in plankton variables in the Baltic Sea — a further sign of eutrophication? Ophelia, Suppl. 4, 249–257. Sellner, K.G., 1997. Physiology, ecology, and toxic properties of marine cyanobacteria blooms. Limnol. Oceanogr. 42 Ž5., 1089–1104, Žpart 2.. Sellner, K.G., Olson, M.M., Olli, K., 1996. Copepod interactions with toxic and non-toxic cyanobacteria from the Gulf of Finnland. Phycologia 35, 177–182. Stal, L.J., Walsby, A.E., 1998. The daily integral of nitrogen fixation by planktonic cyanobacteria in the Baltic Sea. New Phytol. 139, 665–671. Walsby, A.E., Hayes, P.K., Boje, R., 1995. The gas vesicles, buoyancy and vertical distribution of cyanobacteria in the Baltic Sea. Eur. J. Phycol. 30, 87–94.
Nitrogen fixation by diazotrophic cyanobacteria in the Baltic Sea and transfer of the newly fixed nitrogen to picoplankton organisms Ute Ohlendieck a,) , Annegret Stuhr a , Heike Siegmund b a
Institut fur Weg 20, 24105 Kiel, Germany ¨ Meereskunde an der UniÕersitat ¨ Kiel, Dusternbrooker ¨ b Institut fur Germany ¨ Ostseeforschung, Warnemunde, ¨ Received 15 December 1998; accepted 4 June 1999
Abstract Nitrogen fixation Ž15 N2-tracer method. by filamentous cyanobacteria and the flux of the newly fixed nitrogen into picoplanktonic organisms were investigated during three cruises in the central Baltic Sea in July and August 1995 and July 1996. The diazotrophic cyanobacteria consisted mainly of Aphanizomenon and Nodularia and the picoplankton fraction mainly of Synechococcus, a coccoid nondiazotrophic cyanobacterium. The average N2-fixation rate measured during the two July studies Ž3.9 nmol ly1 hy1 in 1995 and 2.4 nmol ly1 hy1 in 1996., was about 13 times higher than in August 1995 Ž0.3 nmol N2 ly1 hy1 .. During the July studies, 5–10% of the total N2-fixation was found in the picoplankton fraction, but none was found in August. The study shows that in the early stage of a cyanobacterial bloom new nitrogen is provided for the pelagic foodweb through release of recently fixed nitrogen by the diazotrophs, whereas in the late stage the input of new nitrogen occurs mainly through lysing of decaying filamentous cyanobacteria cells. Considerable temporal and local variations in nitrogen fixation found in this study indicate that broader studies in time and location are needed to assess the contribution of N2-fixation to the nitrogen budget of the Baltic Sea. q 2000 Elsevier Science B.V. All rights reserved. Keywords: cyanobacteria; Nodularia sp.; Aphanizomenon sp.; Synechococcus sp.; nitrogen fixation; exudation; picoplankton
1. Introduction Filamentous diazotrophic cyanobacteria in the Central Baltic Sea grow intensively in summer when the water column is stratified and the mixed layer is impoverished in dissolved inorganic nitrogen ŽDIN..
) Corresponding author. Tel.: q49-431-597-3983; fax: q49597-3994. E-mail address: [email protected] ŽU. Ohlendieck..
15
N2 -tracer method; Baltic Sea;
Due to their ability to fix N2 , the cyanobacteria can contribute substantially to the input of new nitrogen into the nutrient-poor environment. This nitrogen pool represents a potentially important nitrogen source for other organisms in the pelagic foodweb. There are three major ways in which the fixed nitrogen may enter the marine nitrogen cycle: grazing by zooplankton, lysis of decaying cyanobacteria cells and exudation of dissolved nitrogen products. Grazing of filamentous cyanobacteria has been shown to be of no importance in the Baltic Sea ŽSellner et
0924-7963r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 4 - 7 9 6 3 Ž 0 0 . 0 0 0 1 6 - 6
214
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
al., 1996.. Liberation of fixed nitrogen into the water through lysis of decaying cyanobacteria cells in late summer is thought to be the primary way of making the nitrogen available for other organisms ŽLindahl et al., 1980; Schulz and Kaiser, 1986.. The majority of diazotrophic cyanobacterial biomass occurs in the surface layer because their strong gas vesicles ensure positive buoyancy ŽWalsby et al., 1995.. Due to this, when they decompose, the nitrogen is released in the surface layers. Elevated levels of dissolved nitrogen and dissolved organic carbon ŽDOC. have been found in the mixed layer by several authors during and after decay of cyanobacterial blooms ŽGundersen, 1981; Kuparinen et al., 1984. as well as a well-developed microbial foodweb ŽHoppe, 1981.. Exudation of nitrogenous compounds by actively growing diazotrophic cyanobacteria also seems to be of importance, as some earlier studies indicate. Jones and Stewart Ž1969. showed that more than 50% of the nitrogen recently fixed by Calothrix, a cyanobacterium of the littoral, was released and a part of it was taken up by heterotrophic bacteria, picoplankton and algae. Paerl Ž1984. found a considerable amount of 15 N fixed in cultures of Anabaena that was present in associated bacteria after a short incubation time. Recent studies on Trichodesmium, a diazotrophic cyanobacterium occurring in the tropics, showed that during active growth up to 50% of the newly fixed nitrogen is released into the water as dissolved organic nitrogen ŽDON. ŽGlibert and Bronk, 1994.. In early studies, it was necessary to concentrate samples for nitrogen fixation measurements because the 15 N2-tracer method was not sensitive enough at that time. Nowadays, the availability of highprecision isotope ratio mass spectrometers makes it feasible to carry out 15 N2-tracer incubations on unconcentrated natural water samples, with minimal disturbance of the system ŽMontoya et al., 1995.. This study deals with the utilization of cyanobacterial extracellular products by other organisms. The experiments were conducted on natural plankton communities in the Baltic Sea. Nitrogen fixation by filamentous cyanobacteria was measured in the central Baltic Sea and the flux of the newly fixed nitrogen into picoplanktonic organisms, potential consumers of DON, was investigated during different stages in the development of a cyanobacterial
population. The 15 N2-tracer method was applied, which made it possible to measure the rate of N2fixation directly and to follow the movement of the newly fixed nitrogen into other components of the planktonic foodweb.
2. Materials and methods N2-fixation experiments were carried out during three cruises in the central Baltic Sea in 1995 and 1996: Periods 26r06–11r07r1995 07r08–22r08r1995 15r07–25r07r1996
Areas of investigation Arkona Sea, Middlebank Middlebank, Bornholm Sea Middlebank, Bornholm Sea, Gotland Sea
The experiments in 1995 were carried out during drift experiments conducted in each region. During the cruise in 1996, a larger area was covered Žsee Fig. 1.. 2.1. Nitrogen fixation Water samples were collected using a CTD-rosette Ž12 = 12 l Niskin bottles.. Nitrogen fixation was measured in surface water samples. The samples were incubated in 0.25–, 0.5– or 1–l replicate glass bottles ŽDuran w ., which were filled to overflowing and sealed with a septum cap Žteflon-lined butyl
Fig. 1. The investigation area with drift stations Ž1995. and cruise track Ž1996..
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
rubber.. A gas-tight syringe was used to inject the 15 N2 gas Ž99 at.% 15 N, CAMPRO Scientific. into the bottle and water was allowed to escape through the injection needle to equalize the pressure across the septum. The 15 N2-enrichment in the incubation bottles ranged between 19.7 and 23.4 at.% in 1995 and between 5.4 and 6.1 at.% in 1996 for different incubations. The bottles were gently mixed and then incubated, either attached to an in situ array at the surface or in a deck incubator covered with a neutral density screening to simulate in situ light intensity. The incubation time was 8 to 10 h during daylight. At the end of each experiment, the samples were filtered under a mild vacuum Ž100 cm Hg. through a precombusted Ž6 h at 4508C. 25-mm GFrF filter and stored in a freezer Žy208C.. To investigate the 15 N-enrichment in the picoplankton fraction, one replicate of every sample set was filtered through a 2-mm membrane filter Žon the first cruise. or a 5-mm membrane filter Žon the other cruises., respectively, the filtrate was collected and then also filtered through a GFrF filter. Simultaneously, the picoplankton fraction was incubated with 15 N2 on its own as a control to test if there was any N2 fixed by this fraction. Negative results were obtained in all occasions. 15 N-enrichment by the fractions - 2 and - 5 mm was compared during several experiments. The biomass of the - 5-mm fraction was about 10% higher but there was no difference in the 15 N-enrichment. Back ashore, the samples were dried at 608C, wrapped in tin cups ŽHeraeus CHN cups., formed into pellets and analysed at the Institut fur ¨ Ostseeforschung in Warnemunde as described in Mon¨ toya et al. Ž1995.. The N2-fixation rate was calculated according to Montoya et al. Ž1995.. 2.2. EnÕironmental parameters and phytoplankton composition Hydrographical data were measured by CTD instruments. Analysis of ammonium, nitrate and phosphate concentrations were carried out at sea according to the methods described by Grasshoff Ž1976.. The contribution of filamentous diazotrophic and picoplanktonic cyanobacteria to total chlorophyll-a was calculated using ratios of chlorophyll-a to class specific pigments Žechinenone and zeaxanthin.
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ŽMeyerhofer, in preparation.. The pigment analysis ¨ was carried out by means of the HPLC-technique as described by Mantoura and Llewellyn, 1983, slightly modified by Meyerhofer, 1994. Pigments were iden¨ tified by retention time comparison with pigment standards and by on-line spectra collection using a photo-diode array spectrometric detector. Samples for pigment analysis were filtered through precombusted GFrF filters and stored in a freezer Žy208C. until analysis. The plankton composition was investigated qualitatively on board through light microscopy as well as quantitatively in the laboratory using the Utermohl ¨ method ŽUthermohl, ¨ 1958..
3. Results and discussion 3.1. Nitrogen fixation and the flux of picoplankton fraction
15
N to the
Nitrogen fixation ŽTable 1 and Fig. 2. and the nitrogen fixing capacity of the filamentous cyanobacteria, expressed by the fixation rate per diazotrophic cyanobacteria chlorophyll-a, showed distinct differences between the July studies in 1995 and 1996 on the one hand and the August 1995 study on the other hand. Nitrogen fixation on the cruises in early summer was up to 30 times higher, and on average 13 times higher, than in late summer. Whereas the fixation rate ranged between 2.3 and 5.6 nmol of N2 ly1 hy1 with an average of 3.9 nmol in July 1995 and between 1.4 and 3.1 nmol of N2 ly1 hy1 with an average of 2.4 nmol in July 1996, it amounted only to 0.3 nmol in August 1995. The nitrogen fixing capacity varied between 3.4 and 6.5 nmol of N2 mg diazotrophic-chlorophyll-ay1 hy1 during the July studies Žon average 4.5 nmol in 1995 and 4 nmol in 1996. and between 0.5 and 1.7 nmol of N2 mg diazotrophic-chlorophyll-ay1 hy1 Žon average 1 nmol. in August. There was also a difference between the early summer cruises and the late summer cruise regarding the flux of newly fixed nitrogen into the picoplankton fraction ŽTable 1 and Fig. 2.: 5–10% of the nitrogen fixed by the filamentous cyanobacteria was found in picoplankton biomass during the July studies, but none was found in the picoplankton in August.
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Table 1 Nitrogen fixation rates and the flux of the recently fixed nitrogen into the picoplankton fraction Žexpressed as percentages of the total fixation rate. 15
Cruise
Date
Region
Incubation time
h
Incubation condition
N2 fixed wnmol ly1 hy1 x
N2 fixed wnmol mg diazotrophic chl-ay1 hy1 x
N in picoplankton % of total N2 -fixation Ž%.
July 1995
03r07r1995 07r07r1995 09r07r1995 18r07r1996 19r07r1996 20r07r1996 22r07r1996 23r07r1996 24r07r1996 12r08r1995 15r08r1995 17r08r1995
Mb AS AS GS, st1 GS, st2 GS, st3 GS, st4 GS, st5 BS, st6 Mb BS BS
09:00–19:00 09:00–19:00 09:00–19:00 10:00–19:00 10:30–18:30 09:30–19:30 11:00–19:00 10:30–19:00 11:00–19:00 09:00–19:00 09:00–19:00 09:00–19:00
10 10 10 9 8 10 8 8.5 8 10 10 10
in situ in situ in situ on deck on deck in situ on deck on deck on deck in situ in situ in situ
2.26 3.74 5.59 2.78 1.36 2.86 1.39 3.09 2.93 0.34 0.19 0.2
5.83 3.47 4.60 6.48 3.39 2.42 3.38 3.46 4.81 1.70 0.54 0.83
7 6 10 7 5 10 5 8 5 0 0 0
July 1996
August 1995
AS s Arkona Sea, BS s Bornholm Sea, Mb s Middlebank, GS s Gotland Sea.
The explanation of these results will draw upon environmental data, data on biomass concentration and results from microscopic investigations. 3.2. EnÕironmental conditions During all of the investigations, the mixed layer was separated from the deeper waters by a seasonal thermocline. All nutrient data Žaverage of three to six samples, depending on the mixed layer depth. refer to the mixed layer ŽFig. 2.. In both July cruises, the mixed layer reached down to 10–15 m with a temperature of about 148C. In August 1995, the hydrographical situation was very different. The mixed layer was only 5–10 m deep and had a temperature of about 208C. The NrP ratio of the nutrients was low during the July cruises Ž1–2 on average. with low concentrations of DIN Žs nitrate and ammonium., 0.1 mmol ly1 on average, and still a considerable amount of phosphate, especially in July 1995 Ž0.14 mmol ly1 in 1995 and 0.06 mmol ly1 in 1996 on average.. As nitrogen fixation is inversely related to available inorganic nitrogen pools and directly correlated with phosphorus availability, as reviewed by Sellner Ž1997., the nutrient conditions found during the July cruises were ideal for growth of diazotrophic cyanobacteria.
In August 1995, the NrP ratio was higher, varying from 3–7 with a slightly higher DIN concentration Ž0.21 mmol ly1 on average. compared to the July cruises. This might have been a critical point for the low N2-fixing capacity at that time. It has been shown that nitrogen fixation ceases when other nitrogen sources are available ŽLindahl et al., 1978; Moisander et al., 1996.. 3.3. Phytoplankton composition 3.3.1. Microscopic inÕestigation The filamentous cyanobacteria consisted mainly of two genera in both years: Aphanizomenon and Nodularia. Anabaena was also found in some regions but was of minor importance. In the July studies, Aphanizomenon dominated with exception of the region around Middlebank in 1996 where Nodularia was more abundant. Nodularia had not yet formed any aggregate but was present as single filaments. From microscopic analysis, both genera were in ‘good condition’, i.e., without any unpigmented cell or disintegrating colony. No attached organisms were visible. In August 1995, Nodularia was the dominant filamentous cyanobacterium. In contrast to early summer, it had formed aggregates in which other organisms such as other bacteria, diatoms, flagellates
U. Ohlendieck et al.r Journal of Marine Systems 25 (2000) 213–219
Fig. 2. Nitrogen fixation rates Žabsolute rates for diazotrophs and the flux into the picoplankton fraction and diazotrophic cyanobacteria chlorophyll-a specific rates., nutrient concentration ŽDINs nitrateqammonium and Phosphate. and NrP-ratio of nutients, and the contribution of diazotrophic cyanobacteria and Synechococcus to total chl-a on the drift stations in 1995 and along the cruise track in 1996. ASs Arkona Sea, BSs Bornholm Sea, Mbs Middlebank, GSsGotland Sea.
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cus, a picoplanktonic nondiazotrophic cyanobacterium, is shown here as a major contributor to the picoplanctonic size fraction Ž60–85%. besides heterotrophic bacteria and small eukaryotes. It is known that Synechococcus can live on DON ŽCarr and Wyman, 1986.. In July, filamentous cyanobacteria as well as Synechococcus contributed about 30% of total chlorophyll-a each. The remaining portion of about 40% was made up by eukaryotes such as diatoms and flagellates. In August 1995, the contribution of filamentous cyanobacteria was only 10% on average whereas the contribution of Synechococcus has risen to 65%. Thus, the contribution of Synechococcus had more than doubled and the contribution of filamentous cyanobacteria had decreased by two thirds relative to July 1995. The absolute chlorophyll-a values varied for the diazotrophs between 0.4 and 1.1 mg ly1 and for Synechococcus between 0.4 and 0.9 mg ly1 during July. In August, the chlorophyll-a values varied between 0.2 and 0.35 mg ly1 for the diazotrophs and between 1 and 2 mg ly1 for Synechococcus. Besides the low nitrogen-fixing capacity of the diazotrophs, the low active biomass of filamentous cyanobacteria must have been decisive for the low nitrogen fixation rate in August. The active biomass had ceased because the filamentous cyanobacteria were aged and much of the material was decaying whereas both genera were in ‘good condition’ in July 1995 and 1996. Lindahl et al. Ž1980. also found in their study, carried out in the outer Stockholm Archipelago in 1975, significantly higher nitrogen fixation rates Žnine times higher. in July than in August. The physiological state of the diazotrophic cyanobacteria, as well as the nutrient conditions at those times, were similar to the situation found in this study.
and ciliates were abundant. A major part of Nodularia was decaying material, indicated by unpigmented cells.
3.4. The significance of the newly fixed nitrogen for the pelagic foodweb
3.3.2. Pigment analysis The contribution of filamentous cyanobacteria and Synechococcus to total chlorophyll-a, as derived from pigment analyses, is shown in Fig. 2. Synechococ-
Due to the low nitrogen fixation rate in August, there could have been just very little transfer of newly fixed nitrogen to other organisms. If there had been some, this nitrogen would most probably have
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been consumed by organisms directly associated with the aggregates, so that this flux would not have been measurable by the size fractionation method. The most important means of nitrogen input at that stage of a bloom would be lysing of decaying cyanobacteria cells, which has been shown in other studies ŽGundersen, 1981; Kuparinen et al., 1984.. However, in the early phase of the cyanobacterial development, newly fixed nitrogen was transferred from filamentous cyanobacteria to picoplanktonic organisms, accounting for between 5% and 10% of total nitrogen fixation. As grazing of filamentous cyanobacteria in the Baltic is of no importance ŽSellner et al., 1996., the release of nitrogen by actively growing nitrogen-fixing cyanobacteria would be the only flux of new nitrogen from diazotrophic cyanobacteria to the nutrient impoverished upper euphotic zone, available for the pelagic foodweb at that time of the year. This nitrogen flux supports the microbial loop and leads to an increase of biomass of organisms that depend on bound nitrogen compounds. In this study, there was a pronounced increase of the Synechococcus biomass from July to August ŽFig. 2.. This biomass increase can be attributed partly to the utilisation of nitrogen released by diazotrophic cyanobacteria in the early stage of the bloom, as shown in this study. But it can also be attributed to uptake of nitrogen released by decaying cells and uptake of mineralisation products in the late stage of a bloom. Unfortunately, neither the temporal development of the Synechococcus biomass increase nor the development of the nitrogen fixation rate Žnor the amount of exudation products. could be followed continuously during this study. Therefore, one cannot weigh the relative importance of either of these processes. Besides the pronounced difference in N2-fixation between the July and the August studies, there was also a significant variability in nitrogen fixation between the different regions investigated and even within the drift studies. This could have been caused either by patchiness of the diazotrophic biomass or by variability in their nitrogen fixing capacity. There was indeed patchiness in biomass locally as well as temporally, as can be seen in the chlorophyll-a data ŽFig. 2.. But there was also considerable variability in the nitrogen fixing capacity. The reason for this variability is not easy to judge; there are various
possibilities, such as patchiness of dissolved nitrogen, patchiness of diazotrophs in ‘good condition’ and the aging of colonies or light inhibition of nitrogen fixation.
4. Summary and conclusions Summing up, one can say that nitrogen fixation was high in the early stage of the cyanobacterial development. Nitrogen was released from actively growing cells, providing new nitrogen for the microbial loop in the oligotrophic mixed layer. At the late stage, nitrogen fixation rates were low and nitrogen supply into the water seemed to occur mainly through lysing of decaying cyanobacteria cells. Though the N2-fixation rate is low at that stage, the released nitrogen further supports the microbial foodweb. The high variability in nitrogen fixation at different times and locations during the vegetation period of diazotrophic cyanobacteria found in this study indicates that it is necessary to conduct studies on a broader and more systematic basis Žsee also Stal and Walsby, 1998. in order to record data on N2-fixation, which make it possible to work out a reliable budget for nitrogen input through nitrogen fixation into the Baltic Sea.
Acknowledgements We are greatly obliged to Maren Voss and the Institut fur mass ¨ Ostseeforschung, Warnemunde ¨ spectrometry facility for advice and for the analysis of all 15 N-samples and to Rolf Boje for support, discussions and for including us in the project. Further, we thank the captains and crews of the RrV Alkor, the RrV Littorina and the RrV Alexander Õon Humboldt for their support at sea, Joseph P. Montoya for advice, Frank F. Jochem for inspiring discussions, Michael Meyerhofer for providing ¨ HPLC-data and Uschi Junghans and Petra Krischker for their technical assistance at sea. We also thank Anthony E. Walsby and two anonymous reviewers for their careful reading of the manuscript and for their helpful comments. This research was supported by the European Commission Environment RTD Programme DG XII, Contract No EV5V-CT94-0404
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to the Institut fur ¨ Meereskunde an der Universitat ¨ Kiel.
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