Metagenomic analysis of cellulose degrading communities

The carbon cycle in freshwater lake systems comprises two main phases: primary production (photosynthesis) and biodegradation. Sediments and the lower regions of the water column are often anoxic, particularly in summer, and here anaerobic microorganisms degrade the cellulosic phytoplankton biomass, principally to carbon dioxide and methane. In the environment, microorganisms function as communities or consortia from which the isolation and cultivation of individual species is both difficult and a poor representation of the inherent complexity, particularly for anaerobes. In landfill sites, cellulosic waste is the principal source of carbon that is ultimately converted again to carbon dioxide and methane, and in this managed environment it is the activity of anaerobic microorganisms that is entirely responsible for the stabilisation of waste. Cellulose is the principal component of biomass on earth and its degradation and recycling is an important driver of the carbon cycle. Yet the number of species known to be capable of colonising and degrading native crystalline cellulose is limited, and due at least in part to the inadequacies of conventional microbiological methods that rely on the isolation of strains and their cultivation in the laboratory. One alternative is to analyse DNA and RNA extracted directly from environmental samples, and we have applied this molecular ecological approach to the cellulose-degrading community of freshwater lakes and landfill sites. Our strategy has been to suspend cotton (cellulose) baits directly in lakes and in validated landfill leachate microcosms, to enrich and specifically target microorganisms that truly colonise and degrade cellulose in situ. We have indeed been able to publish evidence for the presence of novel species implicated in cellulose degradation in both environments, and more importantly, demonstrate that their occurrence and distribution is quantitatively significant. We now want to exploit the recent advances in high throughput affordable DNA sequencing technology (pyrosequencing) to analyse the metagenomes (total gene content) of cellulose colonised in, and subsequently retrieved from, the environment. We will primarily use RNA as our template, both for identification of the species present and to focus on genes that are actually being expressed in the biofilm and responsible for degradation of the cellulose substrate. This first description of the colonised cellulose expressed gene pool will enable us to design the tools that we will then apply to the identification of larger genetic fragments containing the combinations of ordered genes responsible for adsorption to and degradation of cellulose, and enabling the identification of any entities that are truly novel. In some cases, it may be possible to express these genes and overproduce cellulases for characterisation but in tandem we will also attempt to isolate and cultivate the species responsible for their production in the envrionment. It remains inconceivable that our knowledge of the species responsible for cellulose degradation in the natural aquatic and managed landfill environments is so superficial, but in addition to redressing this imbalance, we hope to identify new sources of cellulases with commercial potential, particularly in the field of second generation biofuel production.