| As an advanced biofuel,butanol has attracted great attention by the researchers due to the high energy value and low corrosiveness.The overall economics of butanol production could be enhanced by using the abundant sourced,and low-price of lignocellulose.However,it must be pretreated before butanol fermentation,in which various compounds are formed during the process.Among them,phenolic compounds derived from lignin degradation was the most toxicity inhibitor for butanol fermentation.In addition,the solubility of phenolics is very poor,and it is hard to measure the kinds and concentration in the biomass hydrolysate.The inhibitory mechanism of solventogenic clostridia response to phenolic stress was unclear.Based on the above problems,the typical Clostridium acetobutylicum ATCC 824 was selected as the mode strain,and the comprehensive effects of several typical phenolics(vanillin,vanillic acid,p-hydroxybenzoic acid,and p-hydroxybenzaldehyde)on butanol fermentation were investigated to elucidate the physiological mechanism of C.acetobutylicum to phenolic stress.The major results were summarized as follows:(1)The effects of different concentrations of phenolics on butanol fermentation were studied in 100 mL anaerobic bottles.Results showed that low concentration of vanillin(less than 0.1 g/L)was beneficial for butanol fermentation,and the inhibitory effect was increased with higher vanillin concentration.When adding vanillin and other phenolic compounds in fermentation medium,it was found that the effect of vanillin/ferulic acid on butanol fermentation was similar to the control without phenolics addition.Additionally,when combining vanillin with vanillic acid,p-hydroxybenzoic acid and p-hydroxybenzaldehyde as the inhibitors,the butanol fermentation performances were inhibited severely.Thus,a synergistic inhibitory effect presents in anaerobic bottles under without pH control strategy.(2)An optimized pH-control strategy was developed in a 5 L anaerobic fermenter for butanol production: the pH of was controlled at 5.0 during acidogenesis,and it was not controlled during solventogenesis.The results indicated when using the pH-control strategy,the final butanol titer was increased from 1.16 g/L to 9.42 g/L with a 712% increment,and the “acid crash” phenomenon was alleviated.(3)Under the optimized pH-control strategy,the effects of phenolics on butanol fermentation were studied in a 5 L fermenter.The results showed that the lower concentration of vanillic acid/vanillin(less than 0.2 g/L)can increase the cell growth of C.acetobutylicum.Also,the inhibitory effects were different under various lignin-derived phenolics stress(4)Typical phenolic acids and aldehydes were selected as the stress factors for C.acetobutylicum.The transcriptome profiles of the cells under different stresses were analyzed by the RNA-seq technology.The results showed that the inhibitory effect of phenolic aldehydes was stronger than the phenolic acids on butanol fermentation.Meanwhile,phenolics changed the expression of membrane transporter related genes,which led to the decrease of carbon consumption efficiency.In addition,some genes related to heat shock proteins were up-regulated under phenolic stress.Furthermore,the two-component system and sporulation related genes were also affected by phenolics.Based on the above results obtained by fermentation kinetics and transcriptome analysis,the physiological mechanism of C.acetobutylicum response to the lignin-derived phenolics was revealed.In summary,our study will not only provide theoretical basis for metabolic engineering modification in C.acetobutylicum for enhanced tolerance to phenolics,but also provide shoring of foundation for high-efficient bioconversion of lignocellulosic biomass to biobutanol. |
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