The major fraction of microbial communities cannot be cultivated by artificial means in the laboratory. In order to access the full diversity of microbial life in the open environment it is necessary to employ culture independent methods. Molecular biology and now metagenomics have enabled the phylogenetic and functional investigation of microbial communities without isolation and cultivation of organisms and has led to a new appreciation of the breadth of diversity of microbes on Earth and to the discovery and characterisation of new enzymes. Here, molecular biological techniques have been applied to the study of microbial communities specifically in anaerobic environments and with an emphasis on those involved in the primary degradation of plant cellulosic biomass. Quantitative PCR was used to assess the presence of cellulolytic bacteria both in landfill leachate and specifically in association with cotton cellulose “baits” maintained in leachate microcosms. Lineages of clostridia previously associated with cellulose degrading strains were detected in all five of the landfill leachate samples, and Fibrobacter spp. were detected at low abundance (2.3% of total bacteria) in one sample. Clostridia Group III and Fibrobacter spp. were enriched on the surface of a bait (17% and 29% of total bacteria, respectively) that was rapidly degraded by the colonising community and were present in low abundance (< 1%) and absent, respectively, on another colonised by a community which did not exhibit any degradation of the cellulose. The observed correlation between high levels of cellulose degradation and presence Fibrobacter spp. demonstrates a cellulolytic role outside of the gut environment for these organisms the first time. A metatranscriptome was prepared from a set of cotton cellulose baits maintained in a lake sediment for 2-8 weeks, and Illumina sequencing was used to generated ca. 7 million paired-end reads. Just under one million putative protein coding sequences were identified and of these, MEGAN analysis determined that 40% had no blast hit to the NCBI NR database suggesting that a large number of unknown sequences were present. Analysis of this metatranscriptome and a metagenome produced from the same site revealed that bacteria accounted for 75% of the protein coding sequences and 97% of the metagenome. Genes with matches to cellulolytic lineages of clostridia were found to be present and Fibrobacter sequences were also detected in both of these datasets further demonstrating their presence in the wider environment as probable cellulose degraders ORF prediction and HMM searching were used to search for expressed cellulases in the metatranscriptome and identified 503 sequences with high similarity to glycoside hydrolase protein families, representing carbohydrate active enzymes with possible cellulolytic activity. Of these 112 were also found to have representatives in the metagenome with 100% sequence similarity. All of these sequences had a low level of identity to entries in the NCBI NR database indicating the discovery of previously unknown genes. A fosmid library was produced from the same DNA used to generate the metatranscriptome and it is possible that full-length copies of the expressed genes identified in silico will have been captured. This fosmid library can be interrogated accordingly using probe and PCR primer sequences designed using the curated metatranscriptome dataset. In this way, potentially novel cellulases can be discovered for biochemical characterisation, genetic manipulation and biotechnological exploitation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:617412 |
| Date | January 2013 |
| Creators | Houghton, James |
| Contributors | McCarthy, Alan; Allison, Heather |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/14933/ |
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