Stress Mechanism Of The Inhibitors In Lignocellulose Hydrolysates On The Butanol Production By Clostridium Acetobutylicum

Clostridium acetobutylicum,known as its excellent ethanol-acetone-butanol producing capacity,is investigated at the presence of different inhibitors in the lignocellulose hydrolysates by the method of systems biotechnologies.The systematic analysis of C.acetobutylicum response to the inhibitor stress(furans,weak acids and phenol)provides a theoretical basis for the development of lignocellulose pretreatment techniques,fermentation techniques and strain evolution in bio-butanol industry.Firstly,the study found that formic acid and phenol in lignocellulose hydrolysates showed the highest toxicity for cell growth and butanol production.0.5 g/L formic acid or phenol could seriously inhibit butanol fermentation while furan compounds in low concentration(less than 1 g/L)benefited to butanol production.We therefore selected the three representative inhibitors,i.e.furfural,formic acid and phenol as the main inhibitors,and conducted a metabonomics analysis,respectively,and identified the key intracellular metabolites closely related to stress response.The results showed that intracellular amino acid metabolism,glucose metabolism,C2 metabolism and fatty acid metabolism were changed obviously when C.acetobutylicum was exposed to the three inhibitors respectively.In particular,furfural showed a significant impact on the intracellular reductive metabolism-related compounds;the most significant effect of phenol was referred to fatty acid metabolism and nucleic acid metabolism;formic acid stress led to a significant shrink of intracellular metabolic activities,and the biomass synthesis was blocked.Subsequently,the changes of intracellular proteins under the three inhibitors were investigated by proteomics.The results showed that the proteins involved in the lower glycolysis,Re-TCA cycle,ATP synthesis,acetone-butanol synthesis and redox metabolism,were all inhibited;proteins involved in gluconeogenesis,Ox-TCA cycle,nucleic acid metabolism,fatty acid synthesis,sulfur-containing amino acids metabolism,TPX and molecular chaperones(DnaK,GroEL and HtpG)were all at higher levels.In addition,under the phenol stress,the protein expression pattern of nicotinic acid metabolism,cell wall synthesis,sporulation and flagella synthesis,were distinctly different from the formic acid and furfural groups;Inhibitory effects of formic acid and furfural on the metabolic pathways or modules showed the similar protein expression patterns,revealing the inhibitor-specific stress mechanisms.Finally,we investigated the inhibitory effects of phenolic compounds with different functional groups(syringaldehyde,vanillin,ferulic acid and p-coumaric acid)on the cell growth and butanol production.The RNA-Seq-based transcriptomics analysis showed that these phenolic compounds had strong inhibition on the central carbon and energy metabolism;Key genes of sulfur metabolism,methionine/cysteine synthesis,fatty acid synthesis,riboflavin synthesis,synthesis of glyoxylic acid,degradation of RNA and pentose phosphate pathway,were all up-regulated.In addition,sporulation and purine metabolism were only activated under the stress of syringaldehyde and vanillin;Molecular chaperones were only up-regulated in the presence of vanillin,ferulic acid and coumaric acid;Glycerolipid metabolism was active only in the presence of vanillin,syringaldehyde and ferulic acid.These results showed that the stress response of C.acetobutylicum to the four kinds of phenolic compounds had similarities and differences.