We show the potential of flow cytometry as a fast tool for population identification and enumeration of photosynthetic sulfur bacteria. Purple (PSB) and green sulfur bacteria (GSB) oxidize hydrogen sulfide to elemental sulfur that can act as storage compound to be further oxidized to sulfate generating the reducing power required for growth. Both groups have different elemental sulfur allocation strategies: whereas PSB store elemental sulfur as intracellular inclusions, GSB allocate sulfur globules externally. We used well-characterized laboratory strains and complex natural photosynthetic populations developing in a sharply stratified meromictic lake to show that PSB and GSB could be detected, differentiated and enumerated in unstained samples using a blue laser-based flow cytometer. Variations in cell-specific pigment content and the dynamics of sulfur accumulation, both intra- and extracellularly, were also detected in flow cytometric plots as sulfur accumulation changed the light scatter characteristics of the cells. These data were used to show the potential for studies on the metabolic status and the rate of activity at the single-cell level. Flow cytometric identification and enumeration resulted in faster and more precise analyses than previous approaches, and may open the door to more complex ecophysiological experiments with photosynthetic sulfur bacteria in mixed cultures and natural environments.

A laser in situ scattering and transmissometry (Lisst-100) probe has been used for estimating the particle-size distribution of phytopankton, purple photosynthetic sulphur bacteria (Chromatiaceae), and suspended inorganic sediments in different lakes. Results from Lisst-100 have been compared to laboratory measurements, such as those obtained by using a Galai laser size analyzer (GL), an optical microscope (OM), and a flow cytometer (FC). Although all of these instruments were shown to provide reliable values of the particle number concentration for the given populations, the Lisst-100 was the fastest and most reliable instrument because it did not require manipulation of the samples-which is not the case of GL, OM and FC instruments -and avoided the tedious procedure of microscopic counts. The total particle volume concentration results obtained with Lisst-100 differed from those obtained with GL for populations with large and porous aggregates, such as phytoplankton cells. The difference was attributed to the breakage of fragile algal aggregates resulting from the measuring procedure used by GL. Although for suspended sediment particles both instruments gave the same results for the total particle volume concentration, the particle-size distribution obtained with GL was found always shifted to smaller diameters than with Lisst-100, probably because inorganic sediment particles present compact aggregates. When these aggregates break, they split into a high number of small particles that contribute the same to the total volume concentration as the previous aggregates. Finally, results of the total particle volume concentration with Lisst-100 were in accordance with those obtained with GL for the Chromatiaceae population, because cells remained in a dispersed phase. A good correlation was found between the total particle volume concentration of Chromatiaceae measured with Lisst-100 and the concentration of bacteriochlorophyll a (BChl a), which is the parameter habitually used to estimate the concentration of Chromatiaceae. Therefore, Lisst-100 was found to be a reliable instrument to estimate the Chromatiaceae concentration in aquatic ecosystems.

The relative content of the four main bacteriochlorophyll (BChl) e homologs of several populations of brown‐colored photosynthetic sulfur bacteria grown in different waterbodies have been measured by high performance liquid chromatography and statistically compared by principal component analysis. All the studied populations possessed representative pigment patterns enriched in highly alkylated bacteriochlorophyll e homologs, with average contents of 0.02±0.01%, 24.92±1.01%, 35.2±0.70%, and 39.9±0.71% for bacteriochlorophyll e1, e2, e3, and e4, respectively. These values clearly differ from those obtained for the same species growing under optimal conditions in laboratory batch cultures (4.99±1.11%, 50.34±1.73%, 28.99±0.63%, and 15.6±1.10% for bacteriochlorophyll e1, e2, e3, and e4, respectively). Multivariate statistical analyses grouped samples into two main clusters, both related to the developmental state of the population. Within these clusters, samples were arranged in several groups according to the physiological pigment response of bacterial populations to light limitation. Although bacteriochlorophyll homolog distribution cannot be considered a real taxonomic character, the data presented demonstrate that it can be useful in field studies since it reflects both the physiological status of the cells and the light regime under which the population has been growing.

Two new phototrophic consortia, "Chlorochromatium lunatum" and "Pelochromatium selenoides", were observed and collected in the hypolimnion of several dimictic lakes in Wisconsin and Michigan (USA). The two consortia had the same morphology but different pigment composition. The cells of the photosynthetic components of the consortia were half-moon-shaped. This morphology was used to differentiate them from the previously described motile phototrophic consortia "Chlorochromatium aggregatum" and "Pelochromatium roseum". These phototrophic cells did not resemble any described unicellular green sulfur bacteria. The predominant pigments detected were bacteriochlorophyll d and chlorobactene for the green-colored "Clc. lunatum", and bacteriochlorophyll e and isorenieratene for the brown-colored "Plc. selenoides". Their pigment compositions and the presence of chlorosomes attached to the inner face of the cytoplasmic membrane in both kinds of photosynthetic cells confirmed this new half-moon-shaped morphotype as a green sulfur bacterium. Both consortia were found thriving in lakes with low concentrations of sulfide (< 60 &mgr;M), below the layers of "Clc. aggregatum" and "Plc. roseum". The green consortia were observed in lakes where the oxic-anoxic interface was located at shallow depths (2-7 m), while the brown consortia were found at greater depths (8-16 m). The two newly described consortia were never detected together at the same depth in any lake.

SUMMARY The spectral distribution of light reaching the populations of phototrophic bacteria in the metalimnion of stratified lakes is a selective factor determining the community composition. At deep metalimnia, light spectra are enriched in photons of the central part of the spectrum (500–600 nm) and benefit Chromatiaceae, brown‐coloured Chlorobiaceae and phyco‐erythrine‐containing cyanobacteria. Their carotenoids (okenone, spiriloxanthine, isorenieratene) and phycoerythrines allow these phototrophic bacteria to use light from the narrow central spectral wavebands. Otherwise, shallow metalimnetic communities receive light from a wide range (400–800 nm) and their composition is more diverse and usually enriched in green‐coloured Chlorobiaceae, which are unable to take advantage of the central part of the spectrum. Gilvin compounds (humic substances dissolved in water), have strong effects on light absorption, especially at shorter wavelengths. Therefore, light spectra in lakes with high gilvin contents are enriched in photons of long wavelengths (> 600 nm). Several Wisconsin lakes with different gilvin contents were studied during the period of summer stratification in 1994. Spectral distribution of light reaching their metalimnia changed with increasing gilvin contents (measured as g440). In the latter, phototrophic metalimnetic bacterial communities were absolutely dominated by green‐coloured Chlorobiaceae. Intermediate lakes could experiment changes on their community composition depending on variations in gilvin content, as happened in Little Long lake. The dynamics of this lake was studied during summer 1995. The ratio of green‐coloured species in respect to brown‐coloured species increased after a sudden increase of gilvin due to strong rainfall. These results agree with the photosynthetic advantage of green‐coloured Chlorobiaceae under red‐light illumination, inferred from laboratory experiments, and suggest a bacteriochlorophyll‐dependent, light‐harvesting strategy of these phototrophic sulphur bacteria.

The merornictic lake 'El Tobar' was sampled at 3 time points during the annual cycle, coinciding with the thermal stratification period. The photosynthetic microbial community was composed of mixoiimnetic oxygenic phototrophs. which were distributed throuqh the mixolimnion, phycoerytrine-containing unicellular cyanobacteria and sulphu; photbtrophic bacteria placed at the halocline. During the stratification period anoxic conditions, formerly confined to the monimolimnion, moved 1 m upwards, reaching freshwater positions. Consequently, photosynthetic bacteria were re-arranged into -A -. . L , - -

The presence of unusual bacteriochlorophyll (BChl) d forms in natural populations of green photosynthetic sulfur bacteria is reported. These new pigment forms share spectral properties with the standard BChl d previously described in green sulfur bacteria, i.e. absorption maxima at 429 nm and 656 nm at the Soret and Q y bands, respectively, but greatly differ in the HPLC analysis. Two types of natural BChl d were found: type I, observed in populations of Pelodictyon clathratiforme and the green consortium`Chlorochromatium aggregatum', was composed of three main homologs unevenly distributed along the chromatographic run. BChl d type II was only observed in photosynthetic communities dominated by Chloronema spp., and eluted as a cluster of seven homologs characterized by their high apolarity. The distinct chromatographic behavior suggests differences in the chemical structures of these pigments, which are suspected to be located at the C-17 position of the macrocycle by esterification of alcohols longer than farnesyl. The possible origin of these new pigment forms as well as their potential role in light adaptation in natural habitats is discussed. z

The 'Salada de Chiprana' (Chiprana Lake) is a hypersaline (30-73 per thousand), permanent and shallow lake of endorheic origin in a semi-arid region of the Ebro depression (Aragon, Spain). Magnesium sulfate and sodium chloride represent the main salts of this athalassohaline environment. Anoxic conditions occurred periodically in the bottom layers of the lake during the study period. When stratified, high sulfide concentrations (up to 7 mM) were measured in the hypolimnion. Physical and chemical conditions gave rise to the development of very dense green sulfur bacteria blooms (10.7 mg l(-1) of BChl c and 16.7 mg l(-1) of BChl d) at 0.5-1 m from the bottom. Microscopic observations revealed that cells morphologically similar to Chlorobium vibrioforme were dominant in the phototrophic bacterial community, but Prosthecochloris aestuarii was also found sometimes at lower concentrations, as revealed by both microscopic observation and flow cytometric analyses. Deep agar dilution series allowed to obtain several axenic cultures of phototrophic bacteria. They were identified according to their morphology, pigment composition and phylogenetic relationships (16S rDNA sequence analysis). Two of the sequenced strains (CHP3401 and CHP3402) belonged to the green sulfur bacteria and were related to Prosthecochloris aestuarii SK413(T) and Chlorobium vibrioforme DSM260(T), respectively. HPLC analyses of both natural samples and Chlorobium vibrioforme isolates indicated that these strains contained both BChl c and BChl d. Phylogenetic results suggested that Chlorobium vibrioforme strains DSM260(T) and CHP3402, all sequenced strains of Prosthecochloris aestuarii and strain CIB2401 constitute a separate cluster of green sulfur bacteria, all of them isolated from marine to hypersaline habitats.