Unraveling a novel mechanism for cellulose decomposition in the bacterial phylum Fibrobacteres.

Cellulose is the most abundant organic polysaccharide on Earth and represents a major structural component of plant cell walls. Consequently, lignocellulosic plant biomass is largely recalcitrant to decomposition by microorganisms, and the ability to degrade and utilise cellulosic polysaccharides is limited to only a few bacterial and fungal groups. In nature, two different enzyme mechanisms for cellulose decomposition are utilised by bacteria and fungi; aerobic fungi and bacteria secrete high quantities of extracellular enzymes, whereas anaerobic bacteria and fungi possess cell-surface bound enzyme complexes (cellulosomes). Ruminant herbivores such as domestic cattle rely on symbiotic gut microorganisms for the digestion of plant material. Fibrobacter succinogenes is the type species of the genus Fibrobacter and was first isolated from the bovine rumen where it is established as the most prolific bacterial degrader of plant biomass. This superior efficiency to degrade cellulose in the rumen may be explained by recent evidence that within the genus Fibrobacter, a 'third' mechanism for the degradation of cellulose has evolved. F. succinogenes does not conform to the classical models of cellulose decomposition, and one possible mechanism for cellulose degradation in Fibrobacter involves the removal of individual cellulose fibres and subsequent transport through the outer membrane where they are cleaved by cellulases. Furthermore, molecular approaches have successfully identified members of the genus Fibrobacter in non-gut environments where cellulose is degraded (landfill sites and freshwater lakes), suggesting a greater diversity of fibrobacters than previously thought. Here, our approach is to sequence the genomes of several Fibrobacter strains that represent the breadth of ecological and taxonomic diversity currently detected within the genus. These analyses will include some novel strains of F. succinogenes that we have recently isolated from landfill sites and this is the first isolation of this species from a non-gut environment. Furthermore, these strains can utilise cellulose as the sole source of carbon for growth. Our comparative genomic analyses will enable us to investigate the evolutionary relatedness of the different strains and species within the Fibrobacter genus, with particular emphasis on the mechanism of cellulose degradation that we suspect is conserved across all members of the Fibrobacter genus and is the key physiological attribute that circumscribes the group. We will then focus on phenotypic characterisation of members of the genus, by observing the degradation and utilisation of polysaccharides and their derivatives by each strain, obtaining quantitative data on growth rates and enzyme activities. These data will provide important information on the hydrolytic abilities and substrate specificity of each strain, for which there is a currently a paucity of information. Finally, we will again use high throughput sequencing techniques, but this time focussing on gene expression profiles (mRNA) using transcriptomic approaches that inform on the differential expression of functional genes in response to growth on a range of polysaccharides and their derivatives. We will provide growth substrates that range from simple sugars to complex lignocellulosic plant biomass and compare gene expression profiles to enable us to implicate specific genes in particular degradative processes such as cellulose attachment or the transport of simple sugars. These data will provide a step change in our understanding of the mechanism for cellulose degradation employed by fibrobacters. It is clear that fibrobacters are prolific degraders of cellulose, and their enzymes may therefore find biotechnological application in improving the nutrition of economically important ruminant animals and in the refining of plant biomass for the production of bioethanol.

There are two recognised strategies for the microbial decomposition of cellulose, the most abundant organic polysaccharide in the biosphere and a major component of plant cell walls. The first mechanism, typified by aerobic fungi and bacteria, involves secretion of extracellular cell-free synergistic enzymes; whereas anaerobic bacteria and fungi employ cell surface-bound enzyme complexes (cellulosomes). Arguably, the degradation of cellulosic biomass is best exemplified in the anaerobic microbial communities of ruminant herbivores, of which members of the genus Fibrobacter often represent the most prolific bacterial degraders. The recent molecular detection of novel Fibrobacter species in landfill sites and freshwater lakes suggests a role for fibrobacters in cellulolysis beyond the gut, highlighting the potential importance of fibrobacters in the refining of plant biomass. Genomic data suggest that the type species of the genus, F. succinogenes, employs a novel mechanism for cellulose degradation that appears to have evolved independently of the two classical models for cellulose decomposition. Here, we will employ comparative genomic analyses of ecologically and taxonomically representative Fibrobacter strains to address our hypothesis that despite significant genomic diversity across the genus, the novel mechanism for cellulose decomposition proposed for members of this genus is conserved. We will address the paucity of phenotypic data available for fibrobacters, with the aim of resolving taxonomic uncertainties within the genus. Finally, RNAseq analyses will determine how cellulose metabolism is regulated at the transcriptional level in response to utilisation of lignocellulose and its' derivatives as the sole carbon source. These data will provide novel insights into a 'third' mechanism for the degradation of cellulose, with important implications for the biotechnological application of novel cellulases and our understanding of carbon cycling in the biosphere.

The proposed research will significantly enhance our understanding of one of the most potent enzyme mechanisms for the microbial decomposition of cellulose in the natural world. Tangible outputs would include a greater understanding of ruminant nutrition and the ability to make improvements in the health and production of livestock with regards to global food security, in addition to the identification of novel industrial catalysts that may provide step-change improvements in biotechnological applications of cellulases, such as the generation of sustainable and renewable biofuels, and in waste management processes. This work therefore has the potential to yield significant economical, societal and scientific benefits at local, regional and national scales. A greater understanding of the genomic, physiological and metabolic diversity of fibrobacters, particularly for cellulose metabolism, will be of considerable interest to academic researchers focussed on ruminant nutrition, biotechnology and microbial physiology, in addition to researchers in the general fields of microbial evolution, taxonomy and ecology. There is currently an impetus to promote the UK's expertise and contribution to bacterial systematics and taxonomy on an international scale, and we will therefore address some taxonomic issues regarding the Fibrobacteres phylum in this proposal. A greater understanding of the genetic and physiological diversity of fibrobacters may also enable improvements in the successful detection and isolation of fibrobacters in other terrestrial and freshwater environments where they are known to be present, and may represent important members of saccharolytic communities, such as freshwater lakes. We will produce in silico data and functional annotations of genes/transcripts involved in cellulose metabolism, in addition to the development and application of methodologies for confirmation of these activities in vitro. These data will significantly augment the current sequence repositories of known cellulase activities, which will be of considerable interest to industrial stakeholders in the agri-science, agriculture, and biotechnology/energy sectors. The waste treatment industry would welcome progress on the understanding of microbial biomass decomposition arising from the project, particularly those concerned with landfill site management (where we have implicated fibrobacters as part of the saccharolytic community), anaerobic digestion and for the on-site management and processing of industrial waste. We will interact with staff at Bangor University's Bi-composites centre and the Welsh Institute for Natural Resources (WINR) who provides a forum for engagement with this industrial sector. Improvements in the quality of animal feedstock in terms of ruminant nutrition and livestock production have important societal and economic impacts to the general public, by improving quality of life and addressing issues of sustainability and food security in the face of environmental change, notwithstanding the potential to make improvements in sustainable green energy production. In terms of environmental management, sustainability and stewardship of the planet, these data will also have educational value for members of the public, in addition to students in all levels of education. These issues also have obvious relevance to government policy and third sector organisations promoting a sustainable and secure future.