Methanogenic food web in the gut contents of methane-emitting earthworm Eudrilus eugeniae from Brazil.

The anoxic saccharide-rich conditions of the earthworm gut provide an ideal transient habitat for ingested microbes capable of anaerobiosis. It was recently discovered that the earthworm Eudrilus eugeniae from Brazil can emit methane (CH4) and that ingested methanogens might be associated with this emission. The objective of this study was to resolve trophic interactions of bacteria and methanogens in the methanogenic food web in the gut contents of E. eugeniae. RNA-based stable isotope probing of bacterial 16S rRNA as well as mcrA and mrtA (the alpha subunit of methyl-CoM reductase and its isoenzyme, respectively) of methanogens was performed with [(13)C]-glucose as a model saccharide in the gut contents. Concomitant fermentations were augmented by the rapid consumption of glucose, yielding numerous products, including molecular hydrogen (H2), carbon dioxide (CO2), formate, acetate, ethanol, lactate, succinate and propionate. Aeromonadaceae-affiliated facultative aerobes, and obligate anaerobes affiliated to Lachnospiraceae, Veillonellaceae and Ruminococcaceae were associated with the diverse fermentations. Methanogenesis was ongoing during incubations, and (13)C-labeling of CH4 verified that supplemental [(13)C]-glucose derived carbon was dissimilated to CH4. Hydrogenotrophic methanogens affiliated with Methanobacteriaceae and Methanoregulaceae were linked to methanogenesis, and acetogens related to Peptostreptoccocaceae were likewise found to be participants in the methanogenic food web. H2 rather than acetate stimulated methanogenesis in the methanogenic gut content enrichments, and acetogens appeared to dissimilate supplemental H2 to acetate in methanogenic enrichments. These findings provide insight on the processes and associated taxa potentially linked to methanogenesis and the turnover of organic carbon in the alimentary canal of methane-emitting E. eugeniae.

Emission of methane by Eudrilus eugeniae and other earthworms from Brazil.

Earthworms emit denitrification-derived nitrous oxide and fermentation-derived molecular hydrogen. The present study demonstrated that the earthworm Eudrilus eugeniae, obtained in Brazil, emitted methane. Other worms displayed a lesser or no capacity to emit methane. Gene and transcript analyses of mcrA (encoding the alpha subunit of methyl-CoM reductase) in gut contents of E. eugeniae suggested that Methanosarcinaceae, Methanobacteriaceae, and Methanomicrobiaceae might be associated with this emission.

Occurrence of methanogenic Archaea in highly polluted sediments of tropical Santos-São Vicente Estuary (São Paulo, Brazil).

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos-São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30 degrees C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH(4)/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.

Microbial community evaluation of anaerobic granular sludge from a hybrid reactor treating pentachlorophenol by using fluorescence in situ hybridization.

We used in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes concurrently with microscopic examinations and methane measurements to characterize the microbial community of an anaerobic hybrid reactor treating pentachlorophenol (PCP) with a mixture of fatty acids (propionic, butyric, acetic and lactic) and methanol. Archaeal cells detected with probe ARC915 prevailed in anaerobic granular sludge without and with the addition of PCP in a range of 2.0 to 21.0 mg/L to the reactor. This group accounted for 81 and 90% of the DAPI-stained cells before and after the addition of 21 mg/L of PCP, respectively. In these conditions, cells detected with the Methanosarcinales specific probe (MSMX860) were the only methanogenic Archaea found and accounted for 59 to 87.6% of the DAPI-stained cells. No cells were detected by the Methanomicrobiales (MG1200), Methanobacteriaceae (MB1174) and Methanococcaceae (MC1109) specific probes. Bacterial cells detected with probe EUB338 were found in very low numbers, which ranged from 5.7 to 1.0% of the DAPI-stained cells. This finding agrees with the scanning electron microscope examinations, in which cells morphologically resembling Methanosaeta and Methanosarcina were predominantly observed in the granular sludge. Results contributed to the investigation of the importance of the methanogens during PCP degradation.