A survey of the relative abundance of specific groups of cellulose degrading bacteria in anaerobic environments using fluorescence in situ hybridization

Aims: The utility of fluorescence in situ hybridization (FISH) for detecting uncultured micro-organisms in environmental samples has been shown in numerous habitats. In this study a suite of three FISH probes for cellulolytic bacteria is described and their efficacy is demonstrated by quantifying the relative abundance of the target micro-organisms in a range of industrial biomass samples. Methods and Results: The probes were designed from data derived from an artificial landfill leachate reactor study and 16S rRNA gene databases. The original biomass sample proved to be well described by the three probes targeting a total of 51% of the bacterial (EUBMIX targeted) cells in quantitative FISH experiments. Conclusions: Three probes were developed and applied to samples from a range of industrial digesters. The CSTG1244 probe, specific for organisms closely related to Clostridium stercorarium, were observed in the widest range of samples (7 of the 19 samples tested). The CTH216a FISH probe, specific for organisms closely related to Clostridium thermocellum, described the highest proportion of the bacterial population within any one sample (46% in an anaerobically digested sludge sample). Finally, the BCE216a probe, specific for organisms closely related to Bacteroides cellulosolvens, achieved the lowest level of hybridisation of the three probes tested. Significance and Impact of the Study: This study demonstrates that the three groups of anaerobic cellulolytic micro-organisms were present in different bioreactors but at variable abundances ranging from low (where other organisms would have been responsible for cellulolysis) to high. We showed the potential of using group specific FISH probes and quantitative FISH in environmental studies. The utility of using newly designed FISH probes was demonstrated by their ability to detect and quantify the target bacterial groups in samples from a range of industrial wastewater digesters.