| Tobacco stalk contains nicotine and other toxic components,which have some influence on activity of biological enzyme and growth of microorganism.Because of the special properties of nicotine contained in tobacco stalk,this study adopted discarded tobacco stalk as raw material,aimed at ethanol fermentation,enzymatic hydrolysis and pretreatment of raw materials in the production of cellulosic ethanol to carry out specific research,in order to provide theoretical basis and technical support for bioethanol fermentation of tobacco stalk and other tobacco waste materials.In the study of the effect of nicotine on ethanol fermentation,we investigated the effect of nicotine on ethanol fermentation by Issatchenkia orientalis HN-1,with using industrial strain Saccharomyces cerevisiae 1308 as the control.The results indicated that nicotine could inhibit cell growth of the two strains significantly.Ethanol production of Issatchenkia orientalis HN-1 and Saccharomyces cerevisiae 1308 was 18.17~9.51 g/L and 11.95~5.42 g/L,decreased by 20.81%~58.55% and 40.42%~72.94% compared with the control respectively,when addition amount of nicotine was from 0.1% to 0.5%.Meanwhile,the fermentation period of strain 1308 postponed 12 h compared with the control,and the inhibition of nicotine on glucose consumption was also stronger than strain HN-1.Analysis of byproducts showed that the yield of acetic acid of strain HN-1 was 0.11~0.43 g/L,which was lower than strain 1308 with the yield of 0.20~0.59 g/L.However,glycerol production was 2.20~2.71 g/L,higher than strain 1308 with the production of 1.48~2.33 g/L significantly.Issatchenkia orientalis HN-1 was more suitable for the research and production on bioconversion of tobacco stalk and other waste materials into ethanol compared with Saccharomyces cerevisiae 1308.In the study of the effect of nicotine on enzymatic hydrolysis,we investigated the changes of lignocellulose degrading enzymes activities(including cellulase and xylanase),the relieve degree of nicotine toxicity and the effect of enzymatic hydrolysis of tobacco stalk with adding surfactants PEG6000,Tween80,BSA and Rhamnolipid.The results indicated that CMC cellulase activity and FPA activity of cellulase decreased by 8.97~42.99% and 33.08~69.44% respectively,xylanase activity decreased by 11.11~36.93%,when nicotine concentration was from 0.1% to 0.5%.Total sugar concentration of dilute acid hydrolysate of corn stover gradually decreased with increasing nicotine concentration,the minimum value was 20.40 g/L,decreased by 43.30% compared with the control group,when nicotine concentration was 0.5%.Under nicotine stress,BSA and Tween80 could promote CMC activity and FPA activity increased by 37.83% and 41.89%,31.85% and 29.28%,respectively.Total reducing sugars of tobacco stalk hydrolysate got up to 40.33 g/L and 39.82 g/L,increased by 13.70% and 10.34% compared with the control group,when addition amount of BSA and Tween80 was 0.005 and 0.001 g/g substrate respectively.In the study of pretreatment of raw materials,we investigated the variation trend of main component,nicotine content,effect of enzymatic hydrolysis and ethanol production,through pretreatment of tobacco stalk by using dilute sulfuric acid steam explosion.The results indicated that sulfuric acid impregnation can enhance the effect of steam explosion pretreatment significantly,the content of hemicellulose and acid insoluble lignin were reduced,while the cellulose content was showing higher trend significantly.The nicotine content was reduced significantly after composite pretreatment.It reduced from 0.37% to 0.11%,when dilute sulfuric acid at a concentration of 0.2% to 1.0%.Total sugars of hydrolysate increased by 45.10%,36.16% 39.59% compared with the control group respectively,when concentration of dilute sulfuric acid was 0.6% to 1.0%.Dilute sulfuric acid at a concentration of 0.8% was selected to be the optimum condition with comprehensive consideration involving a higher reducing sugar concentration and lower nicotine content.Under this condition,the initial glucose concentration of hydrolysate was 35.00 g/L,the ethanol production reached to 12.10 g/L,increased by over 1 fold compared with the control group(which was 5.95 g/L). |
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