To gain insight into the microorganisms involved in direct and indirect methane formation from methanol in a laboratory-scale thermophilic (55°C) methanogenic bioreactor, reactor sludge was disrupted and serial dilutions were incubated in specific growth media containing methanol and possible intermediates of methanol degradation as substrates. With methanol, growth was observed up to a dilution of 108. However, when Methanothermobacter thermoautotrophicus strain Z245 was added for H2 removal, growth was observed up to a 1010-fold dilution. With H2/CO2 and acetate, growth was observed up to dilutions of 109 and 104, respectively. Dominant microorganisms in the different dilutions were identified by 16S rRNA-gene diversity and sequence analysis. Furthermore, dilution polymerase chain reaction (PCR) revealed a similar relative abundance of Archaea and Bacteria in all investigated samples, except in enrichment with acetate, which contained 100 times less archaeal DNA than bacterial DNA. The most abundant bacteria in the culture with methanol and strain Z245 were most closely related to Moorella glycerini. Thermodesulfovibrio relatives were found with high sequence similarity in the H2/CO2 enrichment, but also in the original laboratory-scale bioreactor sludge. Methanothermobacter thermoautotrophicus strains were the most abundant hydrogenotrophic archaea in the H2/CO2 enrichment. The dominant methanol-utilizing methanogen, which was present in the 108-dilution, was most closely related to Methanomethylovorans hollandica. Compared to direct methanogenesis, results of this study indicate that syntrophic, interspecies hydrogen transfer-dependent methanol conversion is equally important in the thermophilic bioreactor, confirming previous findings with labeled substrates and specific inhibitors.

A quinone-respiring, enrichment culture derived from methanogenic granular sludge was phylogenetically characterized by using a combined cloning-denaturing gradient gel electrophoresis (DGGE) method, which revealed that the consortium developed was dominated by a single microorganism: 97% related, in a sequence of 1520 base pairs, to Geobacter sulfurreducens. The enrichment culture could grow with acetate, formate or H2 when humic acids, the humic model compound, anthraquinone-2,6-disulfonate (AQDS), or chelated Fe(III) was provided as a terminal electron acceptor. The occurrence of a humic acid- or quinone-respiring microorganism in the microbial community of a wastewater treatment system suggests that this type of microorganisms may play a potential role in anaerobic bioreactors treating humus-containing wastewaters.

In order to study the diversity of ammonia-oxidising bacteria in freshwater habitats, including sediments, a molecular approach focused on the sequencing of 16S rDNA was adopted. 16S rDNA sequences showing affinity with the beta-subgroup of ammonia-oxidising bacteria were recovered by specific PCR of directly isolated DNA from freshwater samples, and samples from brackish water and Glyceria maxima rhizosphere were included in the analysis for comparison. The ammonia oxidiser-like sequences recovered from several locations, which exhibit differences in the composition of their total microbial communities as indicated by denaturing gradient gel electrophoresis, formed a strong monophyletic cluster including Nitrosomonas ureae. This is the first report presenting sequences from an apparently dominant group of Nitrosomonas-like organisms among the beta-subdivision of ammonia-oxidising bacteria in freshwater environments. This group of sequences extends the known diversity within the beta-subgroup of ammonia-oxidisers. The new sequences related to Nitrosomonas ureae do not match with some published primers and probes designed for the detection of Nitrosomonas species, which may explain why these sequences have not previously been detected in freshwater habitats. The sequence diversity detected within this group of sequences was minimal across the environments examined, and no patterns of distribution were indicated with respect to environmental factors such as sediment depth or location.