Kangaroos ferment forage material in an enlarged forestomach analogous to the rumen, but in contrast to ruminants, they produce little or no methane. The objective of this study was to identify the dominant organisms and pathways involved in hydrogenotrophy in the kangaroo forestomach, with the broader aim of understanding how these processes are able to predominate over methanogenesis. Stable isotope analysis of fermentation end products and RNA stable isotope probing (RNA-SIP) were used to investigate the organisms and biochemical pathways involved in the metabolism of hydrogen and carbon dioxide in the kangaroo forestomach. Our results clearly demonstrate that the activity of bacterial reductive acetogens is a key factor in the reduced methane output of kangaroos. In in vitro fermentations, the microbial community of the kangaroo foregut produced very little methane, but produced a significantly greater proportion of acetate derived from carbon dioxide than the microbial community of the bovine rumen. A bacterial operational taxonomic unit closely related to the known reductive acetogen Blautia coccoides was found to be associated with carbon dioxide and hydrogen metabolism in the kangaroo foregut. Other bacterial taxa including members of the genera Prevotella, Oscillibacter and Streptococcus that have not previously been reported as containing hydrogenotrophic organisms were also significantly associated with metabolism of hydrogen and carbon dioxide in the kangaroo forestomach.

Methanogenic biodegradation involves the cooperative metabolism of syntrophic bacteria that catalyse the initial attack and subsequent degradation of hydrocarbons, and methanogens that convert intermediates such as hydrogen and carbon dioxide, formate, and/or acetate to methane. The identity of syntrophic microbes and the nature of their interactions with other syntrophs and methanogens are not well understood. Furthermore, it is difficult to isolate the organisms responsible for the initial activation and subsequent degradation of hydrocarbon substrates under methanogenic conditions due to the thermodynamic relationships that exist among microbes in methanogenic communities. We used time-resolved RNA stable isotope probing and RT-qPCR to identify the organisms involved in the initial attack on toluene and subsequent degradation reactions in a highly enriched toluene-degrading methanogenic culture. Our results reveal the importance of a Desulfosporosinus sp. in anaerobic toluene activation in the culture. Other organisms that appear to play roles in toluene degradation include Syntrophaceae, Desulfovibrionales and Chloroflexi. The high bacterial diversity observed in this culture and the extensive labelling of different phylogenetic groups over the course of the stable isotope probing experiment highlight the complexity of the relationships that exist in methanogenic ecosystems.

The last decade has witnessed a revolution in the development of methods and technology available to investigate the ecological roles of microorganisms in the environment. As a consequence, microbial ecologists have gained a better understanding of the functional aspects of microorganisms in marine, groundwater and freshwater systems, soils, sediments, hot springs, wastewater treatment plants, landfills, the rhizosphere and the animal gut. This review provides a compilation and critical comparison of the currently available methods linking microbial function with phylogeny, including a description and advantages and limitations of each method. Examples are also provided to illustrate their application. The ongoing improvements of these 'function-identity' methods points to a bright future in our understanding of complex ecological processes and to improved management of microbe dependent ecosystem services.

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