Development of physio-chemical pretreatments and mixed microbial cultures for the conversion of lignocellulosic biomass to useful products
Munns, Craig Christopher Robert
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There is increasing interest in producing biofuels; biofuels are preferable to fossil fuels as the biomass from which they are derived is seen as a renewable source, as opposed to fossil fuels which are a finite resource. “First Generation” biofuels are derived from food crops such as grains and sugar cane. The use of food crops is not sustainable in this age of increasing food insecurity. A promising alternative appears to be what is termed “Second Generation” feedstocks, such as energy crops like Miscanthus spp., and agricultural by-products. The problem with the use of second generation feedstocks is firstly that the sugars are locked up in the cell wall polymers (CWP), which need to be released by physio-chemical pre-treatments, that are costly and time consuming. The second problem is that not all the sugars that are released from CWP are able to be utilised by wild type product-forming organisms. However, model chassis organisms can be genetically modified to utilise these sugars and /or produce enzymes to degrade biomass which reduces the time and costs involved in the process. While engineering these organisms to utilise a range of monosaccharides has already been successful, engineering them to produce degradation enzymes is proving to be problematic. A potentially more effective system is to use co-cultures of both cellulose-degrading and product-forming organisms. Since this is a novel approach it is not known whether the two organisms are able to live together without any adverse effects. The aims of this study were firstly to determine whether mixed cultures of both cellulose-degrading and potential product-forming organisms could survive in the presence of one another, secondly whether the cellulose-degrading organisms could degrade potential feedstock down into their monosaccharide building blocks and thirdly whether the potential product-forming organisms could survive and utilise these monosaccharides for growth and potential fermentation. It was discovered that C. hutchinsonii can degrade both paper and Triticum aestivum straw polymers into their monosaccharide components and that B. subtilis can survive on the sugars released by C. hutchinsonii. It was also discovered that C. hutchinsonii and B. subtilis 168 can only tolerate an ethanol concentration of up to 2% (v/v) and that this is below the baseline for a biofuel system to be economically viable. Likewise, C. hutchinsonii and B. subtilis 168 have an even poorer tolerance for butanol; growth is inhibited by < 1% butanol in its growth media. A series of physio-chemical pre-treatments were developed in order to make the monosaccharides present in the cell wall polymers more accessible to microbial saccharification. Sequential pre-treatments, both physical milling and chemical hydrolysis in tandem, had the greatest effect on the bio chemistry of the biomass, but that these physio-chemical pre-treatments produced inhibitory compounds in the medium that retarded microbial growth. Attempts were made to genetically modified Bacillus subtilis 168 to produce lactic acid and ethanol by over expressing the native ldh gene under the highly-expressed promoter of the cspD gene and by integrating the fused pdc:adh gene from Z. mobilis under the same promoter. Transformation of B. subtilis to over express LDH was successful, with PCR confirmation of the correct insertion and enzyme activity for the ldh both in vitro and in vivo, with the latter producing more lactic acid aerobically than the wild type. Transformation of B. subtilis to express pdc:adh and subsequent production of ethanol was not successful.