Abstract
In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb_3974 relative to Cb_3904 and Cb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb_3904 and Cb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb_3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.
Highlights
Renewable feedstocks such as lignocellulosic biomass (LB) and organic municipal wastes are sources of cheap sugars with potential to lower the overall cost of fuels and chemicals production
The lignocellulose-derived microbial inhibitory compounds (LDMICs) generated during LB pretreatment and hydrolysis include furfural, 5-hydroxymethyl furfural (HMF) and a collection of lignin-derived phenolic compounds[2]
Both enzymes were found to be active on HMF and the phenolic compound, benzaldehyde, which is co-generated during LB pretreatment
Summary
Renewable feedstocks such as lignocellulosic biomass (LB) and organic municipal wastes are sources of cheap sugars with potential to lower the overall cost of fuels and chemicals production. These techniques include the use of chemical additives such as dithionite, dithiothreitol, sulfite and calcium hydroxide (over-liming), enzymatic treatments with laccases and peroxidases, liquid-liquid extraction with ethyl acetate or trialkyl amine, liquid-solid extraction with activated carbon or ion exchange resins for inhibitor removal[6,7,8,9,10,11,12,13,14,15] Effective, these techniques introduce additional detoxification steps, with the attendant increase in overall cost, which diminishes the economic competitiveness of ABE fermentation for bio-butanol production. Both genes were chromosomally integrated into Cb genome via double-cross homologous recombination to generate Cb_3974 (AKR) and Cb_3904 (SDR) Both strains were tested for the capacity to detoxify furfural and select lignin-derived microbial inhibitory compounds during ABE fermentation. Development of Cb_3974 and Cb_3904 represents a significant step towards fermentation of LB-derived sugars to biobutanol
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