Abstract
BackgroundAn industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes.Methodology/Principal FindingsIn this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v).Conclusion/SignificanceThe findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.
Highlights
Organic fuels, chemicals, and materials developed from plant biomass are among the leading options to meet sustainability requirements of the future [1]
Conclusion/Significance: The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism
The Populus hydrolysate used in these experiments was analyzed by HPLC and gas chromatograph-mass spectrometer (GCMS)
Summary
Chemicals, and materials developed from plant biomass are among the leading options to meet sustainability requirements of the future [1]. The most economic process combines the cellulase production, biomass hydrolysis, and sugar fermentation into a single step by a cellulose-fermenting microorganism in a consolidated bioprocessing (CBP) scheme [1,2]. Various pretreatment methods are designed to make the sugars more available for subsequent hydrolysis and fermentation steps through the breakdown of the cell wall and the degradation of cellulose, hemicelluloses, and lignin matrix [4,5]. Most pretreatment processes produce compounds that are inhibitory to the organism used for fermentation. These inhibitory compounds come from the partial degradation of biomass components and include carboxylic acids (primarily acetic acid), furfural, 5-hydroxymethylfurfural (HMF), phenolic compounds, and inorganic salts [4]. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes
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