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Title: Characterising cellulolytic and biofuel generating bacteria using proteomics for future implementation as biofuel producers
Author: Raut, Mahendra
ISNI:       0000 0004 5358 760X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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In natural ecosystems, microbes are mostly found in diverse and complex communities or consortia that can live symbiotically and fulfil most important global biogeochemical cycles. These processes are very difficult or impossible to achieve by a single bacterium. At the global level, scientists have come to know the innate capacity of natural microbial consortia and are starting to understand natural communities and to develop recombinant synthetic consortia for future biotechnology application. In order to overcome key challenges arising due to fossil fuel depletion and contribution to global warming, consolidated bioprocessing (CBP) is thought to be a low cost processing scheme for lignocellulosic biofuel production. Consortia of cellulolytic and biofuel producing microorganisms could be an attractive alternative to single organism approaches. However, proper understanding of the biology of native microbes and their implementation in the development of consortia needs rigorous research study at the system-wide level. Given the immense potential in the conversion of lignocellulosic biomass to biofuels, anaerobic microorganisms are of great interest to researchers. Therefore, this research is focussed on two different anaerobic bacteria: Fibrobacter succinogenes S85, which is an efficient cellulose degrader, but cannot produce biofuels; and Clostridium acetobutylicum ATCC 824, a promising solvents (acetone, ethanol, butanol) producing bacterium that cannot degrade cellulose. The study of these microbes at the systems level will help to understand the biological complexity of these microbes and provide valuable information for future CBP development. Based on capabilities of these microbes, two individual aspects have been proposed and investigated. In this thesis, an investigation of the surface colloidal properties and surface-membrane associated proteins of F. succinogenes involved in cellulose degradation by biotin labelling method using two substrate conditions cellulose and glucose (control) is carried out. Further analysis of the F. succinogenes membrane using high throughput quantitative proteomics using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. This iTRAQ study reveals many novel proteins associated with cellulose degradation, adding valuable information on the mechanism of cellulose degradation in this bacterium. In this thesis, a preliminary technical study comparing two digestion systems (in-gel and in-solution) of soluble proteins from C. acetobutylicum and two peptide separation techniques (SCX and HILIC) is presented. Results reveal that in-gel digestion with HILIC separation is superior to SCX for soluble proteomics from this system. Results are further used in a quantitative proteomics study in the presence of cellobiose and lignin and elucidate the effect of lignin on solvent production and various metabolic processes. This thesis demonstrates that both F. succinogenes and C. acetobutylicum can potentially be used in co-culture to utilise cellulose and pre-treated lignocellulosic waste for bio-augmented bioalcohol production in consolidated bioprocess (CBP) development framework.
Supervisor: Wright, Phillip Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available