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Aerobic acetone-butanol-isopropanol (ABI) fermentation through a co-culture of Clostridium beijerinckii G117 and recombinant Bacillus subtilis 1A1.

ABSTRACT: An engineered B. subtilis 1A1 strain (BsADH2) expressing a secondary alcohol dehydrogenase (CpSADH) was co-cultured with C. beijerinckii G117 under an aerobic condition. During the fermentation on glucose, B. subtilis BsADH2 depleted oxygen in culture media completely and created an anaerobic environment for C. beijerinckii G117, an obligate anaerobe, to grow. Meanwhile, lactate produced by B. subtilis BsADH2 was re-assimilated by C. beijerinckii G117. In return, acetone produced by C. beijerinckii G117 was reduced into isopropanol by B. subtilis BsADH2 via expressing the CpSADH, which helped maintain the redox balance of the engineered B. subtilis. In the symbiotic system consisting of two strains, 1.7 ?g/L of acetone, 4.8 ?g/L of butanol, and 0.9 ?g/L of isopropanol (with an isopropanol/acetone ratio of 0.53) was produced from 60 ?g/L of glucose. This symbiotic system also worked when oxygen was supplied to the culture, although less isopropanol was produced (0.9 ?g/L of acetone, 4.9 ?g/L of butanol, and 0.2 ?g/L of isopropanol). The isopropanol titer was increased substantially to 2.5 ?g/L when we increased the inoculum size of B. subtilis BsADH2 and optimized other process parameters. With the Bacillus-Clostridium co-culture, switching from the original acetone-butanol (AB) fermentation to an aerobic acetone-butanol-isopropanol (ABI) fermentation can be easily achieved without genetic engineering of Clostridium. This strategy of employing a recombinant Bacillus to co-culture with Clostridium should be potentially useful to modify traditional acetone-butanol-ethanol fermentation for the production of other value-added chemicals.

SUBMITTER: Cui Y

PROVIDER: S-EPMC7322341 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Aerobic acetone-butanol-isopropanol (ABI) fermentation through a co-culture of Clostridium beijerinckii G117 and recombinant Bacillus subtilis 1A1.

Cui Yonghao Y He Jianzhong J Yang Kun-Lin KL Zhou Kang K

Metabolic engineering communications 20200611

An engineered B. subtilis 1A1 strain (BsADH2) expressing a secondary alcohol dehydrogenase (CpSADH) was co-cultured with C. beijerinckii G117 under an aerobic condition. During the fermentation on glucose, B. subtilis BsADH2 depleted oxygen in culture media completely and created an anaerobic environment for C. beijerinckii G117, an obligate anaerobe, to grow. Meanwhile, lactate produced by B. subtilis BsADH2 was re-assimilated by C. beijerinckii G117. I ...[more]

PMID: 32612931

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Project description:BackgroundFurfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulose biomass to monomeric sugars, but the response of acetone butanol ethanol (ABE) producing Clostridium beijerinckii NCIMB 8052 to this compound at the molecular level is unknown. To discern the effect of furfural on C. beijerinckii and to gain insight into molecular mechanisms of action and detoxification, physiological changes of furfural-stressed cultures during acetone butanol ethanol (ABE) fermentation were studied, and differentially expressed genes were profiled by genome-wide transcriptional analysis.ResultsA total of 5,003 C. beijerinckii NCIMB 8052 genes capturing about 99.7% of the genome were examined. About 111 genes were differentially expressed (up- or down-regulated) by C. beijerinckii when it was challenged with furfural at acidogenic growth phase compared with 721 genes that were differentially expressed (up- or down-regulated) when C. beijerinckii was challenged with furfural at solventogenic growth phase. The differentially expressed genes include genes related to redox and cofactors, membrane transporters, carbohydrate, amino sugar and nucleotide sugar metabolisms, heat shock proteins, DNA repair, and two-component signal transduction system. While C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control, ABE production by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase.ConclusionGenome-wide transcriptional response of C. beijerinckii to furfural stress was investigated for the first time using microarray analysis. Stresses emanating from ABE accumulation in the fermentation medium; redox balance perturbations; and repression of genes that code for the phosphotransferase system, cell motility and flagellar proteins (and combinations thereof) may have caused the premature termination of C. beijerinckii 8052 growth and ABE production following furfural challenge at the solventogenic phase.This study provides insights into basis for metabolic engineering of C. beijerinckii NCIMB 8052 for enhanced tolerance of lignocellulose-derived microbial inhibitory compounds, thereby improving bioconversion of lignocellulose biomass hydrolysates to biofuels and chemicals. Indeed, two enzymes encoded by Cbei_3974 and Cbei_3904 belonging to aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) families have been identified to be involved in furfural detoxification and tolerance.

Project description:Producing biobutanol from lignocellulosic biomass has shown promise to ultimately reduce greenhouse gases and alleviate the global energy crisis. However, because of the recalcitrance of a lignocellulosic biomass, a pretreatment of the substrate is needed which in many cases releases soluble lignin compounds (SLCs), which inhibit growth of butanol-producing clostridia. In this study, we found that SLCs changed the acetone/butanol ratio (A/B ratio) during butanol fermentation. The typical A/B molar ratio during Clostridium beijerinckii NCIMB 8052 batch fermentation with glucose as the carbon source is about 0.5. In the present study, the A/B molar ratio during batch fermentation with a lignocellulosic hydrolysate as the carbon source was 0.95 at the end of fermentation. Structural and redox potential changes of the SLCs were characterized before and after fermentation by using gas chromatography/mass spectrometry and electrochemical analyses, which indicated that some exogenous SLCs were involved in distributing electron flow to C. beijerinckii, leading to modulation of the redox balance. This was further demonstrated by the NADH/NAD+ ratio and trxB gene expression profile assays at the onset of solventogenic growth. As a result, the A/B ratio of end products changed significantly during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source compared to glucose as the carbon source. These results revealed that SLCs not only inhibited cell growth but also modulated the A/B ratio during C. beijerinckii butanol fermentation.IMPORTANCE Bioconversion of lignocellulosic feedstocks to butanol involves pretreatment, during which hundreds of soluble lignin compounds (SLCs) form. Most of these SLCs inhibit growth of solvent-producing clostridia. However, the mechanism by which these compounds modulate electron flow in clostridia remains elusive. In this study, the results revealed that SLCs changed redox balance by producing oxidative stress and modulating electron flow as electron donors. Production of H2 and acetone was stimulated, while butanol production remained unchanged, which led to a high A/B ratio during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source. These observations provide insight into utilizing C. beijerinckii to produce butanol from a lignocellulosic biomass.

Project description:BackgroundAcetone-butanol-ethanol fermentation has been studied for butanol production. Alternatively, to achieve acetone-free butanol production, use of clostridium strains producing butanol and 1,3-propanediol (1,3-PDO) from glycerol, natural and engineered isopropanol-butanol-ethanol (IBE) producers has been attempted; however, residual 1,3-PDO and acetone, low IBE production by natural IBE producers, and complicated gene modification are limitations.ResultsHere, we report an effective isopropanol and butanol (IB) fermentation using a newly isolated Clostridium sp. A1424 capable of producing IB from various substrates with a small residual acetone. Notably, this strain also utilized glycerol and produced butanol and 1,3-PDO. After 46.35 g/L of glucose consumption at pH 5.5-controlled batch fermentation, Clostridium sp. A1424 produced 9.43 g/L of butanol and 13.92 g/L of IB at the productivity of 0.29 and 0.44 g/L/h, respectively, which are the highest values in glucose-based batch fermentations using natural IB producers. More interestingly, using glucose-glycerol mixtures at ratios ranging from 20:2 to 14:8 led to not only acetone-free and 1,3-PDO-free IB fermentation but also enhanced IB production along with a much higher butanol content (butanol/isopropanol ratio of 1.81 with glucose vs. 2.07-6.14 with glucose-glycerol mixture). Furthermore, when the mixture of glucose and crude glycerol at the ratio of 14:8 (total concentration of 35.68 g/L) was used, high butanol/isopropanol ratio (3.44) and butanol titer (9.86 g/L) were achieved with 1.4-fold enhanced butanol yield (0.28 g/g) and productivity (0.41 g/L/h) compared to those with glucose only at pH 5.5.ConclusionsA newly isolated Clostridium sp. A1424 was able to produce butanol and isopropanol from various carbon sources. The productivity and titer of butanol and total alcohol obtained in this study were higher than the previously reported results obtained using other natural IB producers. Use of the mixture of glucose and glycerol was successful to achieve acetone-free, 1,3-PDO-free, and enhanced IB production with higher yield, productivity, and selectivity of butanol compared to those with glucose only, providing great advantages from the perspective of carbon recovery to alcohols. This notable result could be accomplished by isolating an effective IB producer Clostridium sp. A1424 as well as by utilizing glucose-glycerol mixtures.

Dataset Information

Aerobic acetone-butanol-isopropanol (ABI) fermentation through a co-culture of Clostridium beijerinckii G117 and recombinant Bacillus subtilis 1A1.

Publications

Aerobic acetone-butanol-isopropanol (ABI) fermentation through a co-culture of <i>Clostridium beijerinckii</i> G117 and recombinant <i>Bacillus subtilis</i> 1A1.

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