Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

Discovery pipelines for marine resources: an ocean of opportunity for biotechnology?

Marine microbial diversity offers enormous potential for discovery of compounds of crucial importance in healthcare, food security and bioindustry. However, access to it has been hampered by the difficulty of accessing and growing the organisms for study. The discovery and exploitation of marine bioproducts for research and commercial development requires state-of-the-art technologies and innovative approaches. Technologies and approaches are advancing rapidly and keeping pace is expensive and time consuming. There is a pressing need for clear guidance that will allow researchers to operate in a way that enables the optimal return on their efforts whilst being fully compliant with the current regulatory framework. One major initiative launched to achieve this, has been the advent of European Research Infrastructures. Research Infrastructures (RI) and associated centres of excellence currently build harmonized multidisciplinary workflows that support academic and private sector users. The European Marine Biological Research Infrastructure Cluster (EMBRIC) has brought together six such RIs in a European project to promote the blue bio-economy. The overarching objective is to develop coherent chains of high-quality services for access to biological, analytical and data resources providing improvements in the throughput and efficiency of workflows for discovery of novel marine products. In order to test the efficiency of this prototype pipeline for discovery, 248 rarely-grown organisms were isolated and analysed, some extracts demonstrated interesting biochemical properties and are currently undergoing further analysis. EMBRIC has established an overarching and operational structure to facilitate the integration of the multidisciplinary value chains of services to access such resources whilst enabling critical mass to focus on problem resolution.

Coupled reductive and oxidative sulfur cycling in the phototrophic plate of a meromictic lake.

Mahoney Lake represents an extreme meromictic model system and is a valuable site for examining the organisms and processes that sustain photic zone euxinia (PZE). A single population of purple sulfur bacteria (PSB) living in a dense phototrophic plate in the chemocline is responsible for most of the primary production in Mahoney Lake. Here, we present metagenomic data from this phototrophic plate--including the genome of the major PSB, as obtained from both a highly enriched culture and from the metagenomic data--as well as evidence for multiple other taxa that contribute to the oxidative sulfur cycle and to sulfate reduction. The planktonic PSB is a member of the Chromatiaceae, here renamed Thiohalocapsa sp. strain ML1. It produces the carotenoid okenone, yet its closest relatives are benthic PSB isolates, a finding that may complicate the use of okenone (okenane) as a biomarker for ancient PZE. Favorable thermodynamics for non-phototrophic sulfide oxidation and sulfate reduction reactions also occur in the plate, and a suite of organisms capable of oxidizing and reducing sulfur is apparent in the metagenome. Fluctuating supplies of both reduced carbon and reduced sulfur to the chemocline may partly account for the diversity of both autotrophic and heterotrophic species. Collectively, the data demonstrate the physiological potential for maintaining complex sulfur and carbon cycles in an anoxic water column, driven by the input of exogenous organic matter. This is consistent with suggestions that high levels of oxygenic primary production maintain episodes of PZE in Earth's history and that such communities should support a diversity of sulfur cycle reactions.

Desulfosporosinus lacus sp. nov., a sulfate-reducing bacterium isolated from pristine freshwater lake sediments.

A novel sulfate-reducing bacterium was isolated from pristine sediments of Lake Stechlin, Germany. This strain, STP12(T), was found to contain predominantly c-type cytochromes and to reduce sulfate, sulfite and thiosulfate using lactate as an electron donor. Although STP12(T) could not utilize elemental sulfur as an electron acceptor, it could support growth by dissimilatory Fe(III) reduction. In a comparison of 16S rRNA gene sequences, STP12(T) was 96.7 % similar to Desulfosporosinus auripigmenti DSM 13351(T), 96.5 % similar to Desulfosporosinus meridiei DSM 13257(T) and 96.4 % similar to Desulfosporosinus orientis DSM 765(T). DNA-DNA hybridization experiments revealed that strain STP12(T) shows only 32 % reassociation with the type strain of the type species of the genus, D. orientis DSM 765(T). These data, considered in conjunction with strain-specific differences in heavy metal tolerance, cell-wall chemotaxonomy and riboprint patterns, support recognition of strain STP12(T) (=DSM 15449(T)=JCM 12239(T)) as the type strain of a distinct and novel species within the genus Desulfosporosinus, Desulfosporosinus lacus sp. nov.

Microbial communities in the chemocline of a hypersaline deep-sea basin (Urania basin, Mediterranean Sea).

The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea. Since this basin is located at a depth of approximately 3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon. In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated. The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface. Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated. Employing 11 cultivation methods, we isolated a total of 70 bacterial strains. The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods. These strains were identified as flavobacteria, Alteromonas macleodii, and Halomonas aquamarina. All 70 strains could grow chemoorganoheterotrophically under oxic conditions. Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically. While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unusually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source. Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.

Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.

Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.

Multitude and temporal variability of ecological niches as indicated by the diversity of cultivated bacterioplankton.

The diversity of cultured planktonic bacteria was analyzed. Bacterial strains were isolated from a eutrophic lake (Zwischenahner Meer, Niedersachsen, Germany) at three different sampling dates (October 1997, April and May 1998). Phylogenetic diversity was assessed by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and sequencing of 16S rRNA gene fragments. Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed a high genomic diversity within the strain collections, which exceeded the diversity of the 16S rRNA gene sequences considerably. The composition of each of the three strain collections was unique since strains isolated at different dates always exhibited different ERIC-PCR fingerprints. Growth tests with 59 different carbon substrates demonstrated that even strains with identical ERIC-PCR fingerprints, isolated on one sampling date, differed in their physiology. The culturable fraction investigated in the present study constituted a relatively small fraction (

A bacteriochlorophyll a antenna complex from purple bacteria absorbing at 963 nm.

A recently isolated species of the photosynthetic purple sulfur bacteria, provisionally called strain 970, was investigated with respect to its antenna function by means of various spectroscopic techniques, including fluorescence and pump-probe absorption difference spectroscopy. The bacterium contains bacteriochlorophyll a and an as yet unidentified carotenoid, perhaps 3,4,3',4'-tetrahydrospirilloxanthin. It has a single antenna complex of the LH1 type, with a Q(y) absorption band situated at the unusually long wavelength of 963 nm at room temperature and 990 nm at 6 K. In contrast to many other species, the reaction center showed two well-separated absorption bands of bacteriopheophytin at 6 K, located at 747 and 762 nm. The primary electron donor showed a bleaching band centered at 925 nm upon photooxidation. Thus, the energy gap between LH1 and the primary electron donor is quite large in this strain: 425 cm(-1). Nevertheless, trapping occurred with a time constant of 65 +/- 5 ps, similar to the rates observed in other purple bacteria. As in other species, no back-transfer from the reaction center to the antenna was observed. Our results show that strain 970 is a unique subject for the study of antenna and reaction center function and organization.

Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes.

The phylogenetic diversity of green nonsulfur bacteria in nine stratified freshwater lakes was investigated. A set of oligonucleotide primers was developed that permitted the selective amplification of 16S rRNA gene sequences of this group. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced, which yielded a total of 19 novel sequence types. Ten of the sequences were related to those of different cultivated members of the C hloroflexus assemblage, whereas nine fell into the T78 group of environmental clones. For the latter subgroup of the green nonsulfur bacteria, no molecular isolate from freshwater plankton has been reported so far. Several of the sequence types occurred in more than one lake, indicating that not only relatives of the C hloroflexus assemblage, but also bacteria of the clone T78 group represent indigenous bacteria of nonthermal stratified freshwater ecosystems. Our results indicate that the natural diversity in the phylum of the green nonsulfur bacteria has been significantly underestimated in the past.

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum.

The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm(-1) higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55+/-5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.

Identification and characterization of ecologically significant prokaryotes in the sediment of freshwater lakes: molecular and cultivation studies.

The aim of this review is to interpret recent studies in which molecular methods were used to identify and characterize prokaryotes in lake sediments and related habitats. In the first part studies based on the phylogenetic diversity of prokaryotes found in lacustrine habitats are summarized. The application of various cultivation-independent methods for the characterization of distinct groups of sediment bacteria is exemplified with morphologically conspicuous, colorless sulfur bacteria in the second part of this review. Finally, traditional and recently developed methods are described which could be used for linking the function of microbial populations with their identification. The potential of these approaches for the study of lake sediments is discussed in order to give a perspective for future studies in this habitat.

Microbial interactions involving sulfur bacteria: implications for the ecology and evolution of bacterial communities.

A major goal of microbial ecology is the identification and characterization of those microorganisms which govern transformations in natural ecosystems. This review summarizes our present knowledge of microbial interactions in the natural sulfur cycle. Central to the discussion is the recent progress made in understanding the co-occurrence in natural ecosystems of sulfur bacteria with contrasting nutritional requirements and of the spatially very close associations of bacteria, the so-called phototrophic consortia (e.g. 'Chlorochromatium aggregatum' or 'Pelochromatium roseum'). In a similar way, microbial interactions may also be significant during microbial transformations other than the sulfur cycle in natural ecosystems, and could also explain the low culturability of bacteria from natural samples.

Phylogenetic affiliation of the bacteria that constitute phototrophic consortia.

The phylogenetic affiliation of epibionts occurring in three morphologically distinct types of green-colored phototrophic consortia was investigated. Intact consortia of the types "Chlorochromatium aggregatum", "C. glebulum", and a third previously undescribed type, tentatively named "C. magnum" were mechanically separated from accompanying bacteria by either micromanipulation or by chemotactic accumulation in sulfide-containing capillaries. A 540-base-pair-long fragment of the 16S rRNA gene of the epibionts was amplified employing PCR primers specific for green sulfur bacteria. DNA fragments were separated by denaturing gradient gel electrophoresis and subsequently sequenced. The results of this phylogenetic analysis indicated that the symbiotic epibionts, together with only a few free-living strains, form a cluster within the green sulfur bacterial radiation which is only distantly related to the majority of known representatives of this phylum. Consortia with identical morphology but different origin exhibited significant differences in their partial 16S rRNA gene sequences, which could be confirmed by analysis of the 16S rRNA secondary structure. The phylogenetic affiliation of the chemotrophic central rod-shaped bacterium of "C. aggregatum" and "C. magnum" was analyzed by fluorescent in situ hybridization. According to our results and contrary to earlier assumptions, the central bacterium is a member of the beta-subgroup of the Proteobacteria.

Functional exoenzymes as indicators of metabolically active bacteria in 124,000-year-Old sapropel layers of the eastern Mediterranean Sea.

Hydrolytic exoenzymes as indicators of metabolically active bacteria were investigated in four consecutive sapropel layers collected from bathyal sediments of the eastern Mediterranean Sea. For comparison, the organic carbon-poor layers between the sapropels, sediment from the anoxic Urania basin, and sediments of intertidal mud flats of the German Wadden Sea were also analyzed. The sapropel layers contained up to 1.5. 10(8) bacterial cells cm(-3), whereas cell numbers in the intermediate layers were lower by a factor of 10. In sapropels, the determination of exoenzyme activity with fluorescently labeled substrate analogues was impaired by the strong adsorption of up to 97% of the enzymatically liberated fluorophores (4-methylumbelliferone [MUF] and 7-amino-4-methylcoumarin [MCA]) to the sediment particles. Because all established methods for the extraction of adsorbed fluorophores proved to be inadequate for sapropel sediments, we introduce a correction method which is based on the measurement of equilibrium adsorption isotherms for both compounds. Using this new approach, high activities of aminopeptidase and alkaline phosphatase were detected even in a 124,000-year-old sapropel layer, whereas the activity of beta-glucosidase was low in all layers. So far, it had been assumed that the organic matter which constitutes the sapropels is highly refractory. The high potential activities of bacterial exoenzymes indicate that bacteria in Mediterranean sapropels are metabolically active and utilize part of the subfossil kerogen. Since a high adsorption capacity was determined not only for the low-molecular-weight compounds MUF and MCA but also for DNA, the extraordinarily strong adsorption of structurally different substrates to the sapropel matrix appears to be the major reason for the long-term preservation of biodegradable carbon in this environment.

Characterization of abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-feeding termites by molecular and culture-dependent techniques.

Lactic acid bacteria have been identified as typical and numerically significant members of the gut microbiota of Reticulitermes flavipes and other wood-feeding lower termites. We found that also in the guts of the higher termites Nasutitermes arborum (wood-feeding), Thoracotermes macrothorax, and Anoplotermes pacificus (both soil-feeding), lactic acid bacteria represent the largest group of culturable carbohydrate-utilizing bacteria (3.6-5.2x10(4) bacteria per gut; 43%-54% of all colonies). All isolates were coccoid and phenotypically difficult to distinguish, but their enterobacterial repetitive intergenic consensus sequence (ERIC) fingerprint patterns showed a significant genetic diversity. Six different genotypes each were identified among the isolates from R. flavipes and T. macrothorax, and representative strains were selected for further characterization. By 16S rRNA gene sequence analysis, strain RfL6 from R. flavipes was classified as a close relative of Enterococcus faecalis, whereas strain RfLs4 from R. flavipes and strain TmLO5 from T. macrothorax were closely related to Lactococcus lactis. All strains consumed oxygen during growth on glucose and cellobiose; oxygen consumption of these and other isolates from both termite species was due to NADH and pyruvate oxidase activities, but did not result in H2O2 formation. In order to assess the significance of the isolates in the hindgut, denaturing gradient gel electrophoresis was used to compare the fingerprints of 16S rRNA genes in the bacterial community of R. flavipes with those of representative isolates. The major DNA band from the hindgut bacterial community was further separated by bisbenzimide-polyethylene glycol electrophoresis, and the two resulting bands were sequenced. Whereas one sequence belonged to a spirochete, the second sequence was closely related to the sequences of the Lactococcus strains RfLs4 and TmLO5. Apparently, those isolates represent strains of a new Lactococcus species which forms a significant fraction of the complex hindgut community of the lower termite R. flavipes and possibly also of other termites.

Detection of abundant sulphate-reducing bacteria in marine oxic sediment layers by a combined cultivation and molecular approach.

The depth distribution and diversity of sulphate-reducing bacteria (SRB) was analysed in the upper intertidal zone of a sandy marine sediment of the Dutch island Schiermonnikoog. The upper centimetre of the sediment included the oxic-anoxic interface and was cut into five slices. With each slice, most probable number (MPN) dilution series were set up in microtitre plates using five different substrates. In the deeper sediment layers, up to 1 x 10(8) cm(-3) lactate-utilizing SRB were counted, corresponding to 23% of the total bacterial count. From the highest positive dilutions of the MPN series, 27 strains of SRB were isolated in pure culture. Sequencing of a 580 bp fragment of the 16S rDNA revealed that 21 isolates had identical sequences, also identical with that of the previously described species Desulfomicrobium apsheronum. However, the diversity of the isolates was higher with respect to their physiological properties: a total of 11 different phenotypes could be distinguished. Genomic fingerprinting by enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) revealed an even higher diversity of 22 different genotypes. A culture-independent analysis by PCR and denaturing-gradient gel electrophoresis (DGGE) revealed that the partial 16S rDNA sequence of the isolated D. apsheronum strains constituted a significant fraction of the Desulfovibrionaceae. The high subspecies diversity suggests that this abundant aggregate-forming species may have evolved adaptations to different ecological niches in the oxic sediment layers.

Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments.

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and beta-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, beta-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species.

Selective enrichment and characterization of Roseospirillum parvum, gen. nov. and sp. nov., a new purple nonsulfur bacterium with unusual light absorption properties.

A new type of phototrophic purple bacterium, strain 930I, was isolated from a microbial mat covering intertidal sandy sediments of Great Sippewissett Salt Marsh (Woods Hole, Mass., USA). The bacterium could only be enriched at a wavelength of 932 (+/- 10) nm. Cells were vibrioid- to spirilloid-shaped and motile by means of bipolar monotrichous flagellation. The intracytoplasmic membranes were of the lamellar type. Photosynthetic pigments comprised bacteriochlorophyll a and the carotenoids spirilloxanthin and lycopenal. The isolated strain exhibited an unusual, long-wavelength absorption maximum at 911 nm. Sulfide or thiosulfate served as electron donor for anoxygenic phototrophic growth. During growth on sulfide, elemental sulfur globules formed outside the cells. Elemental sulfur could not be further oxidized to sulfate. In the presence of sulfide plus bicarbonate, fructose, acetate, propionate, butyrate, valerate, 2-oxoglutarate, pyruvate, lactate, malate, succinate, fumarate, malonate, casamino acids, yeast extract, L(+)-alanine, and L(+)-glutamate were assimilated. Sulfide, thiosulfate, or elemental sulfur served as a reduced sulfur source for photosynthetic growth. Maximum growth rates were obtained at pH 7.9, 30 degrees C, 50 micromol quanta m-2 s-1 of daylight fluorescent tubes, and a salinity of 1-2% NaCl. The strain grew microaerophilically in the dark at a partial pressure of 1 kPa O2. The DNA base composition was 71.2 mol% G + C. Sequence comparison of 16S rRNA genes indicated that the isolate is a member of the alpha-Proteobacteria and is most closely related to Rhodobium orientis at a similarity level of 93.5%. Because of the large phylogenetic distance to known phototrophic species of the alpha-Proteobacteria and of its unique absorption spectrum, strain 930I is described as a new genus and species, Roseospirillum parvum gen. nov. and sp. nov.

Specific detection of green sulfur bacteria by in situ hybridization with a fluorescently labeled oligonucleotide probe.

An oligodeoxynucleotide probe (GSB-532) specific for green sulfur bacteria was developed. Highly stringent hybridization conditions were established using whole cells of Chlorobium limicola DSM249 immobilized on glass slides. At a formamide concentration of 10%, the optimum specificity was reached at 47 degrees C. When a conventional fixation procedure was used, a conspicuous autofluorescence developed within the cells. This autofluorescence was due to the liberation of bacteriochlorophyll by the detergent Triton X-100 and a subsequent conversion to bacteriophenophytin and related compounds. The signal-to-noise ratio could be increased by a final dehydration of the samples with methanol. Finally, the method was adapted to the hybridization of natural samples collected on polycarbonate membrane filters. In situ hybridization of pure cultures, various enrichments, and natural samples from the chemocline of a freshwater lake confirmed that probe GSB-532 hybridized exclusively to cells of green sulfur bacteria. Our protocol allows the highly specific detection of green sulfur bacteria in water samples and a rapid screening of natural bacterial communities. Employing probe GSB-532, the phylogenetic affiliation of the epibionts in "Chlorochromatium aggregatum" and "Pelochromatium roseum" could be demonstrated for the first time.

Grazing of the copepod Diaptomus connexus on purple sulphur bacteria in a meromictic salt lake.

A meromictic lake ecosystem (Mahoney Lake, BC, Canada) was investigated to elucidate the significance of chemocline bacteria in the total carbon cycle under natural conditions. In this lake, primary production by oxygenic phototrophs was insufficient to support the observed net secondary production of the calanoid copepod Diaptomus connexus and the rotifer Brachionus plicatilis, indicating the presence of additional food sources for consumers. Mahoney Lake harbours the densest population of phototrophic sulphur bacteria ever reported in a natural body of water. This layer is located at the interface between oxic and anoxic water layers and is dominated by the purple sulphur bacterium Amoebobacter purpureus. The transfer rates of A. purpureus carbon to D. connexus determined in stratified mesocosms were very low (0.71 ngC copepod(-1) day(-1)) and accounted for only 0.6% of the observed net biomass increase in the zooplankter. Stable stratification within the mesocosms prevented an upwelling of A. purpureus into the oxic part. However, measurements of carbon fluxes, infrared fluorescence microscopy and stable carbon analysis provided cumulative evidence that, under in situ conditions, the cell carbon of purple sulphur bacteria indeed enters the aerobic food chain via the grazing activity of D. connexus. Based on a two-source isotopic mixing model, A. purpureus represents at least 75-85% of the diet of D. connexus. Autumnal upwelling into oxic water layers and aggregation of A. purpureus cells appear to be the main factors determining the high carbon flux from purple sulphur bacteria to zooplankton under natural conditions, and most probably also play a key role in other aquatic ecosystems. Through this pathway, over 53% of the reduced organic matter of purple sulphur bacteria trapped in anoxic bottom waters is returned to the oxic realm.