Study On Inhibitory Mechanisms In Butanol Fermentation With Lignocellulose Hydrolysate

As a new type of bio-energy, butanol has some advantages such as high energy density, low corrosion and so on. Fossil energy crisis led to a stirring of interest in biomass energy. Compared with chemical methods, the procedure of butanol produced by microbial fermentation is more environmentally friendly and easier to operate. Lignocellulose is the most abundant and cheapest biomass in the earth, but it can not be directly used for butanol fermentation. Lignocellulose hydrolysis by acid or alkali treatment can produce simple saccharides that can be utilized by microorganisms, but at the same time the toxic substrance may inhibit cell growth. As a result, lignocellulose hydrolysate cannot be used as substrate directly for ABE fermentation. In this study, clostridium acetobutylicum TSH06 was chosen as the start strain. The tolerance of TSH06 to these inhibitors was evaluated. Also gene transcription and protein expression level in TSH06 were measured by RT-PCR and proteomics technology respectively to understand the mechanism. The results provide theoretical basis for the ABE fermentation using hydrolysate without detoxification.Firstly, cell growth of TSH06 was investigated in hydrolysate without detoxification. Hemicellulose hydrolysate produced from corn cob by acid hydrolysis was used as start substrate. The hydrolysate with different dilution was used as medium for butanol fermentation, with P2 medium(with the same concentration of xylose and glucose) as control. Comparing pH adjustment with NaOH and ammonia, ammonia is suitable for TSH06 cell growth. Using diluted hemicellulose hydrolysate as medium, Na+ concentration(0.34 mM) had an adverse effect on TSH06. There was no significant difference in growth with P2 medium or hydrolysate adjusted with NH4 Cl into P2 medium. While at the stable phase of TSH06, the biomass of TSH06 in P2 medium with NH4 Cl was greater than that in P2 medium, indicating that NH4+ can promote cell growth of TSH06.Furthermore, different dilution of hydrolysate was used to explore the tolerance of TSH06 to the toxic inhibitors. TSH06 could grow and ferment in 50% or lower degrees of diluted hydrolysate and produce butanol(4.16-5.16 g·L-1), which was lower than that in P2 medium(butanol concentration of 8.83 g·L-1). The acetic acid was about 3.18-4.16 g·L-1, which is higher than that fermentation with P2 medium(1.61 g·L-1). RT-PCR was carried out to determine the transcription change of related key genes. Compared with fermentation with P2 medium, TSH06 cultured in the 50% hydrolysates had a higher transcription leve of key genes in the organic acid synthesis pathway(ak,pta,bk,ptb). As for the organic acid reflux pathway(ctf) and butanol production pathway(adhE, bdhA, bdhB), a lower transcription level was observed.Proteomics analysis through iTRAQ was conducted to compare the protein expression of TSH06 in culture with hydrolysate and P2 medium. At the beginning of fermentation, low expression level was observed for enzymes from glycolysis and PTS system in TSH06 cultured in hydrolysate, leading to a low sugar comsuption rate. At the same time, acetic acid produciton in the fermentation with hydrolysate increased sharply from 1.5 g·L-1 to 3.5 g·L-1, as compared to 1.0 g·L-1 in the culture with P2 medium. This phenomenon could be the result of changes of the entire carbon metabolism coused by inhibitors in hydrolysate. In the fermentation with hydrolysate, presence of inhibitor in hydrolysate can cause DNA damage and mismatch. As a result, the expression level of proteins in nucleic acid metabolism increased, including recombination protein RecR(RecR), single DNA binding protein(SSB) and DNA mismatch repair protein(MutS). The change of these proteins might be due to stress reaction from toxic inhibitors. The above physiological variation in bacteria is the melocular bassis which account for the limited utillizaiton of hemi-cellulose hydrolysate.