AQUATIC MICROBIAL ECOLOGY Vol. 20: 299-303,1999 , Published December 30 Aquat Microb Ecol

NOTE

Vertical distribution of photosynthetic sulphur linked to saline gradients in Lake 'El Tobar' (Cuenca, Spain)

' Institute of Aquatic Ecology, University of Girona. Campus de Montilivi. 17071 Girona. Spain * Dept of Microbiology and Ecology. Faculty of Sciences. University of Valencia. Burjassot, 46100 Valencia. Spain

ABSTRACT: The merornictic lake 'El Tobar' was sampled at 3 are subjected to an annual regime of mixing and strat- time points during the annual cycle, coinciding with the ther- ification that provides a high degree of variability in mal stratification period. The photosynthetic microbial com- the physical and chemical parameters, depending on munity was composed of mixoiimnetic oxygenic phototrophs. which were distributed throuqh the mixolimnion, phycoery- the season. However, in some meromictic lakes redox- trine-containing unicellular cyanobacteria and sulphu; photb- clines are placed at such a depth that light is the limit- trophic bacteria placed at the halocline. During the stratification ing factor for bacterial growth during most of the year. period anoxic conditions, formerly confined to the monimo- In Lake 'El Tobar' the vertical structure of both the limnion, moved 1 m upwards, reaching freshwater positions. Consequently, photosynthetic bacteria were re-arranged into water column and the bacterial community is strongly - A-..L,- 1------L ..-- -,..--A :..-L -L -..- --A -- inf!ilenrc+ h.rr tho nrecenrp nf a cham calinitxr nrarliont u uu..t.,.r. ,,u.,,.c-y.LL.r Jnucrulr ,,.ucLu JUJL '."".C U1.U C..*- ' ----1- l i) bedded in the chemocline. In the freshwater layers we found A complete physical and chemical characterization of a population of the purple sulphur bacterium Chromatium the lake as well as a preliminary biological study can minus placed just above a population of the brown-colored be found in Vicente et al. (1993) and Miracle et al. green sulphur bacterium Chlorobium phaeobacteroides. A few centirneters below we found Lamprocystis modesto- (1993). Here we study the seasonal dynamics of the halophilus overlying Chlorobium vibrioforme. This peculiar photosynthetic bacterial populations in this lake, structure was probably due to anoxic conditions reaching which have not been studied to date. This lake has upper positions at the end of the stratification period, thus hypersaline waters in the monimolimnion and low- allowing the growth of freshwater phototrophic bactena. The distribution of the phototrophic bacterial populations reveals mineralised freshwater in the upper 12 m. Thus it was a vertical structure which is strongly influenced by salinity, of interest to identify the dominant physiological whereas light intensity only determines the relative position (freshwater or saline) group of phototrophic sulphur of purple and green bactena. bacteria in the gradients established. The field work was scheduled to cover a whole stratification period. KEYWORDS: Meromictic lake . Phototrophic sulphur bacte- ria . Halocline . Population dynamics . Chromatium . Chloro- Sampling was carried out to cover the onset of thermal bium stratification (spring), complete stagnation (summer) and final steps of the stratification (early fall). Materials and methods. Study area: Lake 'El Tobar' is Accumulation of phototrophlc bacterial biomass in a solution lake located 1250 m above sea level in the the oxic-anoxic boundary of sulphide-containing lakes karstic region of Cuenca (Spain). It is composed of 2 and waterbodies occurs frequently if sufficient light is basins. Field work was done in the smallest and deepest available (Pfennig 1989). An extensive revision of pho- one, which is an elongated circular shaped meromictic tosynthetic bacteria-containing waterbodies can be basin, found on the northern side of the lake, and con- found in van Gemerden & Mas (1995). Meromictic tains a permanently anoxic saline monimolimnion start- lakes are often considered as stable aquatic ecosys- ing at ca 12 m depth (Vicente et al. 1993). tems in which seasonality does not affect the microbial Field measurements: Water temperature, conductiv- community as much as in holornictic lakes. The latter ity, pH, redox potential and dissolved oxygen concen- tration were simultaneously measured in situ with a Hydrolab DS3 multiprobe system.

0 Inter-Research 1999 300 Aquat Microb Ecol20: 299-303, 1999

Light measurements were performed around noon et al. (1985). The cell material retained in the filter was with a battery-powered underwater spectroradio- re-suspended in 5 m1 of 100% acetone and stored at meter (LI-1800 UW; Li-Cor, Inc.) operated with a port- -30°C for 24 h to ensure the complete extraction of pig- able LCD terminal. Downwelling irradiance spectra ments. HPLC analyses of pigment extracts were per- between 300 and 850 nm, at 2 nm intervals, were formed as described by Borrego & Garcia-Gil (1994). obtained from the surface to the depth with the mi- Quantitative pigment analyses were performed with nimum light detectable for the spectroradiometer calibration curves prepared according to the extinction (5 X 10-4 pE m-2 S-' nm-l). The irradiance values at coefficients for each bacteriochlorophyll (Bchl) (Taka- each depth were integrated from 400 to 700 nm to hashi & Ichimura 1968, Oelze 1985). Bchl e concentra- obtain the PAR (photosynthetically available radia- tion was calculated according to Borrego et al. (1999). tion). Results and discussion. The vertical distribution of Samples were taken from roughly the center of the physical and chemical variables, and bacterial popu- basin, just over the deepest point, using a special sam- lation~was strongly influenced by the presence of pling device, similar to the one described earlier by an extremely sharp picnocline, which was invariably Jsrgensen et al. (1979), consisting of 2 base-opposed placed at 13.25 m. Conductivity values below this cones which are connected to an impellent pump pow- boundary were higher than 100 mS cm-', indicating ered by a 12 V car battery. This gives a sampling flow high NaCl concentrations (Vicente et al. 1993). Below of 2 l min-' and is specially designed both to avoid dis- this chemocline oxygen was absent. ruption of the multilayered gradients and to minimize Three iayers of distinct optical properiies were ob- turbulence. Water was collected in screw-capped bot- served in the water column (Fig. 1).The first, from the tles of various capacities and preserved from direct surface to about 10 m, was the aerobic layer, whose sunlight in a portable icebox. Samples were processed main absorbing particles were phytoplankton. The for chemical and pigment analysis either at the light extinction coefficient (q) for this layer was fairly moment of sampling or shortly after. constant (0.16 to 0.18 m-') during the sampling period. Cell counb:The numbers of phototrophic sulphur bac- The second layer corresponded to the span of the pho- teria were determined after passage of samples through tosynthetic bacterial community. In this layer, was 0.2 pm pore diameter cellulose acetate filters and sub- more sensitive to the density of these populations. A sequent treatment with erythrosine (Jones 1979). The maximum coefficient of 0.48 m-' was measured in July. number of cells of each was counted on dried fil- Finally, just below the picnocline, where no living bac- ters at 1250x in a Zeiss phase contrast microscope. Pho- teria were detected, a stronger light extinction was totrophic bacteria were identified by using morphologi- measured, with q up to 1.74 m-'. This high optical den- cal and metabolic criteria according to Bergey's manual sity may be due to cell debris and other particulate of systematic bacteriology (Staley et al. 1989). organic matter, which accumulates at this level due to Pigment analysis: Known volumes (0.3 to 1 1) of the sudden increase in water density. PAR reaching water samples were passed through 0.45 pm pore the phototrophic bacterial layer ranged between 2 and diameter Supor@ filters (Gelman Sciences) covered 10 pE m-2 S-'. These values represent a fairly constant with a thin layer of MgC03, as described by Guerrero 0.3 % of surface incident light.

Sanipllng Depthinlen,al 11 date (m) (m-')

March 0-10 0.18 11-13.25 0.35 13.5-14S 171

Fig. 1.Light pen- September 0-9.5 0.16 etration in Lake 10-1075 0.35 'El Tobar' on the 11-1325 0.97 3 sampling dates. 20 1 I I I I I l l Extinction coeffi- -4 - 3 - 2 - 1 0 1 2 cients calculated for the different 2 -1 Layers are indi- log PAR (PE m- S ) cated Garcia-G11 et al.: Vertical distribution of photosynthetic sulphur bacteria 301

Phototrophic sulphur bacteria were found form- ing a sharp plate, which overlaid the halocline. This narrow layer contained up to 5 distinct domi- nant species, which were spatially arranged in a vertical span of about 1.5 m (Fig. 2).These species g were Chlorobium phaeobacteroides and Chloro- g 10 b~umvibrioforme plus 3 genera belonging to the purple sulphur bacterial group: (1) low counts of Thiospirillum, which was identified by direct mic- roscopic observations of field samples; (2) Chro- matium minus isolated in pure culture and identi- III: fied according to morphometry and pigment corn- 012345 012345 position (Staley et al. 1989); and (3) a third isolate of 0 purple sulphur bacteria that was obtained in pure culture. According to morphometry and pigment 5 composition we tentatively identified the latter -E bacterium as the moderate halophile Lamprobacter g,o modestohalophilus. This assignment was later cor- nm roborated by molecular analysis. The 16s rRNA sequence of this organism clustered with the y-pro- 15 teobacteria within the halophile purple sulphur bacteria. The EMBL database accession number 20 assigned to this sequence is AJ011498. During the spring phototrophic bacteria were poorly represented, reaching Bchl concentrations between 0.4 and 1.5 pg I-'. The sulphur photo- trophic bacterial assemblages moved upwards dur-

1113 BUIIIIII~~ OS ;:ie - SX~

One feature was especially noticeable about the Pigmentwnc. (pg I") Pigment conc.(pg l.') vertical structure of the studied phototrophic bac- terial assemblage: the Lamprobacter modestohal- Fig. 2. Vertical distribution of photosynthetic pigments. Detailed ophilus population was always found below chlo- distributions at gradient layers (l1 to 13.5 m) are shown in the robium phaeobacteroides. This vertical distribution, right-hand panels with a green sulphur bacterial population overly- ing a one, has not been reported to (Montesinos et al. 1983, Repeta et al. 1989, Ormerod et date. Brown-colored green sulphur bacteria are well al. 1993). This vertical structure was constant through- adapted to low light intensities and therefore are fre- out the period studied. Although it is rather unusual, quently found in the deepest layers of the communities this distribution can be explained by reference to the

Table 1. Mean conductivity values and positions (depth) of maximum cell densities

Mar 25 Jul9 Sep 17 Cond. Depth Cond. Depth Cond. Depth (mS cm-') (m) (mS cm-') (m) (mS cm-') (m)

Chlorobium phaeobacteroides 11.25k3.2 13.0 9.83k3.92 12.5 1.71r0.26 11.0 Chromatium minus 11.25i3.2 13.0 18.95k4.0 12.5 1.81k0.25 11.5 Lamprobacter modestohalophjlus 25.22i10.7 13.0 33.2k11.16 13.0 22.9r4.07 13.0 Thiospirillum sp. 25.22k10.7 13.0 47.0r19.0 13.0 - - Aquat Microb Ecol20: 299-303, 1999

optimal salinity (2%) at which the deepest Chromati- one of Chromatiaceae appears to be the optimal situa- aceae develop. C. phaeobacteroides, in turn, is a fresh- tion for the coexistence of both microbial populations water species and it is therefore more Likely to be without strong negative up-to-down effects. Moreover, found at low conductivity positions. This species, to- these populations can survive below phycoerythrin- gether with Synechococcus sp. and Chromatium minus containing cyanobacteria. This particular arrangement were found in September at conductivity values close of the predominant populations of phototrophic sul- to those of freshwater ecosystems, whereas Thio- phur bacteria is therefore more stable than the in- spirillum sp. and L. modestohalophilus were constantly verted combination, which is usually found in freshwa- found at 13 m, regardless of the season. C. phaeobac- ter communities in which the wide spectral range of teroides and C. minus moved upwards from a depth of intense light absorption by Chromatiaceae populations 13 m in March to 11 m in September. In the latter posi- greatly impairs the growth of the brown-colored tion salinity was similar (1.7 mS cm-') to values found Chlorobiaceae. in other environments where these 2 species are com- In addition to light intensity and quality, salinity was monly found (Garcia-Gil & Abella 1992, Borrego et a1 also a determining factor for the vertical segregation of 1993, Garcia-Gil et al. 1993, Baneras & Garcia-Gil the populations. A freshwater population was found in 1996).Thus, it can be concluded that the phototrophic #c upper positions, whereas unabsorbed light inten- microbial community of Lake 'El Tobar' is vertically sity was still high enough to power the development of arranged according to the salinity gradient. a saline population placed immediately below. In sum- The presence of picopianktonic deep maxima has mary, in Lake 'Ei Tobar' 2 selective factors appear to been described in several meromictic lakes, and can determine the vertical structure of the photosynthetic be explained in terms of adaptation to nutrient com- bacterial community. Firstly, the salinity, which may petition, grazing, sulphide, oxygen and light (Craig segregate 2 distinct purple-plus-green assemblages 1987). Synechococcus sp. have displayed an optimal and, secondly, light (both, intensity and quality), which photon flux density of about 1 % of surface, which is may determine the relative position of green and pur- found in Lake 'El Tobar' at the chemocline level during ple bacteria (Fig. 3). In this double structure, only summer. Phycoerythrin provides Synechococcus sp. saline Chlorobiaceae were hard to find because in cells with a selective advantage in terms of light com- saline environments green bacteria are usually vibrio petition under the green-enriched light conditions at shaped (Imhoff 1995), which makes them difficult to this depth. In addition, phosphate concentrations at distinguish from other heterotrophic bacteria under the chemocline are higher than those of epilimnetic the microscope. However, Bchl d clearly indicates the waters, which are essential for optimal growth of Syne- presence of Chlorobium vibnoforme. Nevertheless, 2 chococcus sp., whose affinity for phosphate has been maxima were observable in some profiles, which shown to be very low (Craig 1987). strongly suggests that 2 complete purple-plus-green Light reaching the upper part of the photosynthetic bacterial structures were sequentially present in Lake bacterial community was practically constant through- 'El Tobar'. out the seasons. All these stratified populations were Another interesting microbiological feature of Lake able to harvest photons under the prevailing light con- 'El Tobar' is the presence of a high salinity picnocline ditions. The light absorption by phycoerythrin-contain- that acts as 'fluid-sediment'. The density of the hyper- ing cyanobacteria was not strong enough to com- pletely eliminate photons at the central spectral range, thus allowing their absorption by the Chlorobiaceae- dominated freshwater community. The Soret band of Bchl e harvests photons in the 470 to 530 nm range, which is complementary to the cyanobacteria (Vila et al. 1996, Cox et al. 1998). These wavelengths are use- less for the green-pigmented Chlorobiaceae (Mon- tesinos et al. 1983, Pfennig 1989, Vila & Abella 1994). Finally, a Chromatiaceae-dominated saline assem- blage was still able to develop with the remaining light. Although this group of phototrophic bacteria is known to have higher light requirements, the wide absorption range of their carotenoids enables them to Fig. 3. Schematic representation of the double dstribution of phototrophic sulphur bacteria found in Lake 'E1 Tobar' The collect ph0ton.s from the whole spectral range (500 to usual vertical structure formed by purple (above) and green 600 nm) reaching the saline layer. The combination of (below) sulphur bacteria is sequentially repeated in freshwa- an upper layer of brown Chlorobiaceae with a deeper ter and saline environments within the same lake Garcia-Gil et al.: Vertical distribution of photosynthetic sulphur bacteria 303

saline waters lying at the bottom causes the sedimen- Guerrero R, Montesinos E, Pedros-Ali6 C, Esteve I,Mas J, van tation rate to drop drastically. This forces the down- Gemerden H, Hofman PAG, Bakker JF (1985) Phototro- ward-moving organic matter to accumulate in a thin phic sulphur bacteria in two Spanish lakes. Vertical distri- bution and limiting factors. Limnol Oceanogr 30:919-931 layer. A dense plate of heterotrophic bacteria occurs Imhoff JF (1995) and physiology of purple bacteria due to both decomposition and sulfide production. Vib- and green sulphur bacteria. In: Blanckenshp RE, Madi- rio-shaped bacteria were abundant in samples.starting gan MT. Bauer CE (eds) Anoxygenic phototrophic bacte- at 13 m. Below this layer no signs of bacterial activity ria. Kluwer Academic Publishers, Dordrecht, p 1-15 Jones JG (1979) Direct counts on membrane filters In: Fresh- were observed due to the extremely high salinity level. water Biological Association (eds) A guide to methods for However, it is necessary to further study the hetero- estimating microbial numbers and biomass in freshwaters, trophic populations and their role in the anaerobic Vol39. Titus Wilson & Son Ltd, Ambleside, p 19-39 nutrient cycle. Jsrgensen BB, Kuenen JG, Cohen Y (1979) Microbial trans- formation of sulphur compounds in a stratified lake (Solar Lake, Sinai). Lirnnol Oceanogr 24:799-822 Acknowledgements. This work was supported by the R+D Miracle MR. Armengol-Diaz J, Dasi MJ (1993) Extreme mer- project of the CICYT, CLI96-1103-C02-01 and AMB 95-0323 omixis determines strong differential planktonic vertical to L.J.G.-G. Thanks are due to M. D. Sendra and V Oliveras distributions. Verh Int Ver Limnol 25:?05-710 for their assistance in sampling and analytical procedures and Monteslnos E, Guerrero R,Abella CA, Esteve I (1983) Ecology to J. Figueras for helping with molecular biology work. and physiology of the competition for Light between Flnally we are also indebted to Prof. Dr M. R. Miracle for her Chlorobium limicola and Chlorobium phaeobacteroldes in valuable help with field work and for &scussion. natural habitats. Appl Environ Microbiol 46(5):100?-1016 Oelze J (1985) Analysis of bacteriochlorophylls. Meth Micro- biol 18:25?-284 Ormerod JG, Aukrust TW, Johnsen IJ (1993) Frugal Chloro- LITERATURE CITED bium. the ultimate phototroph. In: Guerrero R, Pedros- Alio C (eds) Trends in microbial ecology. Spanish Society Baneras L, Garcia-Gil W (1996) Role of photosynthetic nucro- for Microbiology, Barcelona, p 59-62 bial populations in the phosphorus exchange through the Pfennig N (1989) Ecology of phototrophic purple and green oxic anoxic boundary in a meromictic eutrophic lake. Arch sulphur bacteria. In: Schlegel HG, Bowien B (eds) Autotro- Hydrobiol Spec Issues Adv Lirnnol48:171-181 phic bacteria. Science Tech Publ, Springer-Verlag, Berlin, Borrego CM, Garcia-Gil U (1994) Separation of bacteri- p 97-116 ochlorophyll homologues from green photosynthetic sul- Repeta DJ, Simpson DJ, Jsrgensen BB, Jannasch HN (1989) v. r - . - ..-.--:..-!--L A,.--:- c-,.- &L,. A:...;,... pnur bdclelld uy ~ave~reci-pi~drr iiFiC. Xluiuaylli;l ;\=a LVLU~IICCL", "II"*JyS.II.C ,,*I"L"J,l.l1*r~1., IIV1.l C2.C -.l...-- 41~157-163 tion of bacteriochlorophyll in the Black Sea. Nature 342: Borrego CM, Garcia-Gil W, Baneras L, Brunet RC (1993) 68-7 2 Changes in the composition of phototrophic sulphur bac- Staley JT, Bryant MP, Pfennig N, Holt JG (eds) (1989) terial communities in three basins of Lake Banyoles Bergey's manual of systematic bacteriology, Vol 3. Wil- (Spain). Verh Int Ver Limnol25:?20-725 liams & Wilkins, Baltimore Borrego CM, Arellano JB, Abella CA, Gillbro T, Garcia-Gil W Takahashi M, Ichimura S (1968) Vertical distribution and (1999) The molar extinction coefficient of bacteriochloro- organic matter production of phototrophic sulphur bacte- phyll e and the pigment stoichiometry in Chlorobium ria in Japanese lakes. Limnol Oceanogr 13:644-653 phaeobacteroides. Photosynth Res 60:25?-264 van Gemerden H, Mas J (1995) Ecology of phototrophic sul- Cox R, Miller M, Aschenbriicker J, Ma YZ, Gillbro T (1998) phur bacteria. In: Blankenship RE, Madigan MT, Bauer The role of bacteriochlorophyll e and carotenoids in light CE (eds) Anoxygenic photosynthetic bactena. Advances harvesting in brown-colored green sulphur bacteria. In: in Photosynthesis, Vol 11. Kluwer Academic Publishers, Garab G (ed) Photosynthesis: mechanisms and effects. Dordrecht, p 49-85 Kluwer Academic Publishers, Dordrecht, p 149-152 Vicente E, Camacho A, Rodrigo MA (1993) Morphometry and Craig SR (1987) The distribution and contnbution of pico- physico-chemistry of the crenogenic merornictic Lake 'El plankton to deep photosynthetic layers in some mero- Tobar' (Spain). Ver Int Ver Limnol 25:698-304 mictic lakes. Acta Acad Aboensis 47:55-81 Vila X, Abella CA (1994) Effects of light quality on the physi- Garcia-Gil U, Abella CA (1992) Population dynamics of pho- ology and the ecology of planktonic green sulphur bacte- totrophic bacteria in three Mferent basins of Lake Bany- ria in lakes. Photosynth Res 41:53-65 oles (Spain). 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Editorial responsibility: Tom Fenchel, Submitted: November 1 1,1998; Accepted: November 26,1999 HeIsing0r, Denmark Proofs received from author(s): December 24, 1999