Larry J. Forney

CH4-consuming microorganisms and the formation of carbonate crusts at cold-seeps

To understand the role played by microorganisms in the formation of cold seep carbonates, we conducted an integrated microbial, mineralogical and organic geochemical study of methane-related authigenic carbonate crusts formed on eastern Mediterranean mud volcanoes. We show that supersaturation with respect to carbonate minerals is induced by microbial anaerobic oxidation of methane. Combined lipid biomarker analysis and 16S rRNA gene surveys identified a highly diversified methane-consuming archaeal community possibly comprising novel species, implying that the anaerobic oxidation of methane is phylogenetically widespread and directly implicating these organisms in the process of crust precipitation. Moreover, pore-water sulphate gradients produced by co-occurring methane-based sulphate reduction exert the main control on aragonite versus magnesian calcite precipitation. We propose that this may be the dominant mode of carbonate crust formation at cold seeps world-wide, in agreement with aquatic chemistry predictions and explaining carbonate mineralogy.

Comparison of deep‐sea sediment microbial communities in the Eastern Mediterranean

Bacterial and archaeal communities in sediments obtained from three geographically-distant mud volcanoes, a control site and a microbial mat in the Eastern Mediterranean deep-sea were characterized using direct 16S rRNA gene analyses. The data were thus in relation to the chemical characteristics of the (stratified) habitats to infer community structure-habitat relationships. The bacterial sequences in the different habitats were related to those of Actinobacteria, Bacilli, Chloroflexi, Alpha-, Beta-, Gamma-, Delta- and Epsilonproteobacteria and unclassified bacteria, including the JS1 group. The archaeal sequences found were affiliated with those of the Methanosarcinales, Thermoplasmales, Halobacteriales and Crenarchaea belonging to marine benthic group I and B, as well as MCG group archaea. In each sample, the communities were diverse and unique at the phylotype level. However, at higher taxonomic levels, similar groups were found in different sediments, and similar depth layers tended to contain similar communities. The sequences that dominated in all top layers (as well as in the mat) probably represented organisms involved in aerobic heterotrophy, sulfide-based chemoautotrophy and methanotrophy and/or methylotrophy. Sequences of organisms most likely involved in anaerobic methane oxidation, sulfate reduction and anaerobic heterotrophy were predominantly found in deeper layers. The data supported the notion of (1) uniqueness of each habitat at fine taxonomic levels, (2) stratification in depth and (3) conservation of function in the sediments.

Microbial community changes in biological phosphate-removal systems on altering sludge phosphorus content

Biomarkers (respiratory quinones and cellular fatty acids) and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes were used to characterize the microbial community structure of lab-scale enhanced biological phosphate-removal (EBPR) systems in response to altering sludge phosphorus (P) content. All the data suggest that the microbial community structures of sludge samples with a P content between 8 and 12.3% (sludge dry weight) (i.e. good EBPR activity) were very similar, but differed from those with 2% P content (i.e. no EBPR activity). For all samples analysed, ubiquinones Q-8 and Q-10, menaquinone MK-8(H4), and fatty acids C16:0, C16:1 omega9c and C18:1, omega11c were the major components. The dominance of Q-8, Q-10 and MK-8(H4) suggested that large numbers of organisms belonging to the beta and alpha subclasses of the Proteobacteria and the Actinobacteria from the high G+C Gram-positive bacteria, respectively, were present. DGGE analysis revealed at least 7-9 predominant DNA bands and numerous other fragments in each sample. Five major DGGE fragments from each of the 2% and 12% P-containing sludge samples, respectively, were successfully isolated and sequenced. Phylogenetic analysis of the sequences indicated that both 2% and 12% P-containing sludge samples shared three common phylotypes that were separately affiliated with a novel bacterial group from the gamma subclass of the Proteobacteria, two MK-8(H4)-containing actinobacteria previously isolated from the 2% P-containing sludge, and a Caulobacter spp. in the alpha subclass of the Proteobacteria. The phylogenetic analysis also revealed phylotypes unique to both sludge samples. Changes in sludge P content therefore had an effect on the composition and abundance of the predominant microbial populations, though specific phylotypes could not be unequivocally associated with EBPR.

Microbial Community Structure in Three Deep-Sea Carbonate Crusts

Carbonate crusts in marine environments can act as sinks for carbon dioxide. Therefore, understanding carbonate crust formation could be important for understanding global warming. In the present study, the microbial communities of three carbonate crust samples from deep-sea mud volcanoes in the eastern Mediterranean were characterized by sequencing 16S ribosomal RNA (rRNA) genes amplified from DNA directly retrieved from the samples. In combination with the mineralogical composition of the crusts and lipid analyses, sequence data were used to assess the possible role of prokaryotes in crust formation. Collectively, the obtained data showed the presence of highly diverse communities, which were distinct in each of the carbonate crusts studied. Bacterial 16S rRNA gene sequences were found in all crusts and the majority was classified as α-, γ-, and δ-Proteobacteria. Interestingly, sequences of Proteobacteria related to Halomonas and Halovibrio sp., which can play an active role in carbonate mineral formation, were present in all crusts. Archaeal 16S rRNA gene sequences were retrieved from two of the crusts studied. Several of those were closely related to archaeal sequences of organisms that have previously been linked to the anaerobic oxidation of methane (AOM). However, the majority of archaeal sequences were not related to sequences of organisms known to be involved in AOM. In combination with the strongly negative δ13C values of archaeal lipids, these results open the possibility that organisms with a role in AOM may be more diverse within the Archaea than previously suggested. Different communities found in the crusts could carry out similar processes that might play a role in carbonate crust formation.

Characterization and distribution of esterase activity in activated sludge

The location and activity of esterase enzymes in activated sludge from three municipal wastewater treatment plants were characterized using model substrates and denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) of particulate, freeze-thaw (primarily periplasmic enzymes and those associated with outer cell surfaces) and extracellular fractions of activated sludge bacteria. Particulate and freeze-thaw fractions had a similar spectrum of substrate specificity and contained significant levels of protein and esterase activity against model substrates, C2-C18 monoesters of p-nitrophenol and C2-C8 diesters of fluorescein. Esterase activity was highest with substrates that had short alkyl chains (C4) and decreased as the chain lengths increased beyond C8. Extracellular fractions contained very low levels of protein (<0.1 mg/l) and showed no esterase activity against any of the model substrates tested. Multiple bands were observed upon analysis of particulate and freeze-thaw fractions by non-denaturing PAGE in combination with activity staining using various alpha-naphthol ester substrates (C2-C8). Our results indicate that esterase enzymes in activated sludge are fairly diverse from a structural standpoint but exhibit a high level of functional redundancy, with different enzymes catalyzing the same reactions in different sludges. Extracellular esterase activity was totally absent for the substrates we tested and the esterase activity that we observed was closely linked to a particulate floc or cellular material.