Development of physio-chemical pretreatments and mixed microbial cultures for the conversion of lignocellulosic biomass to useful products
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Date
07/07/2017Author
Munns, Craig Christopher Robert
Metadata
Abstract
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.