汽爆秸秆中副产物对产乙醇过程影响及生物解毒效能研究
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摘要
利用废弃的纤维素类物质生产燃料酒精是解决能源危机的有效途径之一。纤维素乙醇生产中,原料必须经过预处理,但是预处理后产生的某些副产物会抑制酶解或发酵。本文以目前应用最广泛的蒸汽爆破预处理法为研究对象,考查了玉米秸秆蒸汽爆破预处理后产生的几种有机物和无机物对发酵的影响。并且根据这一结果,研究了本实验室筛选的解毒菌株FLZ10对这些物质的去除作用,并以此对该菌株的生物解毒作用做进一步的研究。
首先,利用AFM技术观察预处理前后玉米秸秆表面结构的变化,证明汽爆预处理和酸爆预处理技术可以起到打破纤维素原料紧密结构的作用。根据对汽爆秸秆原料的洗液及醪液分析结果,研究了预处理和发酵副产物对乙醇产量的影响。结果表明,无机离子Mg~（2+）和Fe~（2+）对乙醇产量有明显的抑制作用,乙醇产量分别降低34.43%和11.48%;乙醇产量会随着全盐量的增加而降低;有机物甲酸≤2g/L浓度范围内不表现明显的抑制作用而是有轻微的促进作用;乙酸在低浓度时表现对发酵的促进作用,浓度达15g/L时,抑制明显,乙醇产量降低91.69%,并随着乙酸浓度增加抑制增强;糠醛浓度≤1g/L时对乙醇产量有明显的促进作用;当5-HMF得浓度≥2.0g/L时,对乙醇产量产生抑制,乙醇产量降低54.03%。
其次,以副产物的抑制作用为依据,研究FLZ10菌剂对这些物质的去除效果。结果表明,无机离子方面,FLZ10对Ca~（2+）、Mg~（2+）、Al~（3+）、Fe~（2+）均有明显的去除效果,培养48h去除率分别为70%、50%、90%、80%。有机物方面,35℃时以葡萄糖为碳源的培养基中,FLZ10对乙酸、丙酸、丁酸于96h的去除率可分别达24.20%、30.92%、27.73%。35℃时以葡萄糖为碳源的培养基中,FLZ10对糠醛和5-HMF的去除率分别为56.52%、69.21。在实际发酵中,FLZ10的生物解毒效果明显,可取得与水洗解毒相同的效果,并且可辅助水洗解毒,乙醇终产量可达50g/L。
再次,为了降低生物解毒成本,对FLZ10菌剂培养基进行优化。根据测得几种菌剂的CB酶活和FPA酶活及其对秸秆洗液中抑制物的去除,表明50℃比40℃更利于酶的作用,而40℃比50℃更利于FLZ10对发酵抑制物的代谢。以原培养基做参照,菌剂优化试验培养基中全麸皮培养基的CB酶活和FPA酶活均最高;SBR和EGSB出水培养基对有机酸的去除效果最好;全麸皮培养基对甲酸、糠醛和5-HMF的去除效果均最佳。但是几种培养基在40℃时,对抑制物的去除效果差别并不是很大,经综合评价,确定可以用价格低廉的玉米秸秆粉代替结晶纤维素制作菌剂培养基。
最后,讨论了几种微量元素营养物的添加对发酵的贡献。经过单因素分析和正交分析,得到实验结果表明,微量元素营养物可以促进发酵,营养物质中对乙醇产量的影响大小依次为泛酸>钴>VB1>锌>锰>镍>肌醇。七种物质应选的加入量分别为六水氯化镍50μm/L、四水氯化锰25mg/L、七水硫酸锌6mg/L、八水氯化钴30μm/L、肌醇150mg/L、泛酸10ml/L、VB1150mg/L。
Fuel ethanol production from lignocellulosic materials is an effective way to relief fossil energy crisis. The lignocellulosic materials must be pretreated before hydrolysis in cellulosic ethanol conversion. However, some byproducts released from pretreatment process may inhibit the enzymic hydrolysis or fermentation. The influences of the inhibitors liberated from steam explosion, which is the widely used pretreated method, was detected in this study. Based on the inhibition result, fungi FLZ10 was used for removing the inhibitors and biodetoxification was further studied.
At first, the surface structures of corn-stovor were observed by AFM technology, to prove the results of breaking the compact lignocellulosic structures after pretreatment. According to the analysis of hydralysates, the effects of the byproducts to the fermentation were studied. It was showed that the ethanol yield were strongly inhibited by Mg~（2+）and Fe~（2+）, the decrease of ethanol yield is 34% and 11%respectively. As the total salts decreased, the ethanol yield has reduced. It has no obvious influence on the ethanol yield when the concentration of formic acid≤2g/L and furfural≤1g/L.The ethanol yield has been inhibited when the concentration of acetic acid≥15g/L and 5-HMF≥2.0g/L, the decrease of ethanol yield is 92% and 54%respectively, but the low concentration have promotion to the fermentation.
Secondly, according to the studies of inhibition, the removals of inhibitors with FLZ10 have been studied. The removal rate of inorganic ions, Ca~（2+）、Mg~（2+）、Al~（3+）、Fe~（2+）are 70%、50%、90%、80%.When T=35℃,t=96h,the removal rate of organics, acetic acid、propionic acid、butyric acid are 24%、31%、28% in the culture medium with glucose as carbon source.. In the actual fermentation, the effect of bio-detoxification and washing detoxification were equivalent. The ethanol yield using bio-detoxification based washing detoxification is 50g/L.
Once more, the optimizations of culture medium of FLZ10 agent have been carried out. As standards for monitoring cellulase activity, CB and FPA activities were detected, and the concentrations of inhibitors in corn-stover washing water, including formic acid、acetic acid、furfural、5-HMF,were detected for the bio- detoxification ability. The results were performed that the CB and FP activities in 50℃were higher than they were in 40℃, and the removal rates of inhibitors in 40℃were higher than they were in 50℃. The culture medium which using SBR or EGSB effluent as nutrients abtained the best rate for removing acetic acids; The culture medium of entire wheat bran was turned out the most replacement of formic acid,furfural and 5-HMF. However, there were little differences in inhibitors removing rate among different culture medium. Based on the comprehensive analysis, cheap crystalline cellulose (Avicel ) can be replaced by cheap corn stover powder in thedetoxified bioagent medium.
At last, contribution of growth factors was performed. According to orthogonal analysis ,it showed that the effects of growth factors rang from strong to weak were pantothenic acid > Co > VB1 > Zn > Mn > Ni > inositol, the optimized actual concentration were NiCl2·6H2O 50μm/L、MnCl2·4H2O 25mg/L、ZnSO4·7H2O 6mg/L、CoCl2·8H2O 30μm/L、inositol 150mg/L、pantothenic acid10ml/L、VB1150mg/L.
首先,利用AFM技术观察预处理前后玉米秸秆表面结构的变化,证明汽爆预处理和酸爆预处理技术可以起到打破纤维素原料紧密结构的作用。根据对汽爆秸秆原料的洗液及醪液分析结果,研究了预处理和发酵副产物对乙醇产量的影响。结果表明,无机离子Mg~（2+）和Fe~（2+）对乙醇产量有明显的抑制作用,乙醇产量分别降低34.43%和11.48%;乙醇产量会随着全盐量的增加而降低;有机物甲酸≤2g/L浓度范围内不表现明显的抑制作用而是有轻微的促进作用;乙酸在低浓度时表现对发酵的促进作用,浓度达15g/L时,抑制明显,乙醇产量降低91.69%,并随着乙酸浓度增加抑制增强;糠醛浓度≤1g/L时对乙醇产量有明显的促进作用;当5-HMF得浓度≥2.0g/L时,对乙醇产量产生抑制,乙醇产量降低54.03%。
其次,以副产物的抑制作用为依据,研究FLZ10菌剂对这些物质的去除效果。结果表明,无机离子方面,FLZ10对Ca~（2+）、Mg~（2+）、Al~（3+）、Fe~（2+）均有明显的去除效果,培养48h去除率分别为70%、50%、90%、80%。有机物方面,35℃时以葡萄糖为碳源的培养基中,FLZ10对乙酸、丙酸、丁酸于96h的去除率可分别达24.20%、30.92%、27.73%。35℃时以葡萄糖为碳源的培养基中,FLZ10对糠醛和5-HMF的去除率分别为56.52%、69.21。在实际发酵中,FLZ10的生物解毒效果明显,可取得与水洗解毒相同的效果,并且可辅助水洗解毒,乙醇终产量可达50g/L。
再次,为了降低生物解毒成本,对FLZ10菌剂培养基进行优化。根据测得几种菌剂的CB酶活和FPA酶活及其对秸秆洗液中抑制物的去除,表明50℃比40℃更利于酶的作用,而40℃比50℃更利于FLZ10对发酵抑制物的代谢。以原培养基做参照,菌剂优化试验培养基中全麸皮培养基的CB酶活和FPA酶活均最高;SBR和EGSB出水培养基对有机酸的去除效果最好;全麸皮培养基对甲酸、糠醛和5-HMF的去除效果均最佳。但是几种培养基在40℃时,对抑制物的去除效果差别并不是很大,经综合评价,确定可以用价格低廉的玉米秸秆粉代替结晶纤维素制作菌剂培养基。
最后,讨论了几种微量元素营养物的添加对发酵的贡献。经过单因素分析和正交分析,得到实验结果表明,微量元素营养物可以促进发酵,营养物质中对乙醇产量的影响大小依次为泛酸>钴>VB1>锌>锰>镍>肌醇。七种物质应选的加入量分别为六水氯化镍50μm/L、四水氯化锰25mg/L、七水硫酸锌6mg/L、八水氯化钴30μm/L、肌醇150mg/L、泛酸10ml/L、VB1150mg/L。
Fuel ethanol production from lignocellulosic materials is an effective way to relief fossil energy crisis. The lignocellulosic materials must be pretreated before hydrolysis in cellulosic ethanol conversion. However, some byproducts released from pretreatment process may inhibit the enzymic hydrolysis or fermentation. The influences of the inhibitors liberated from steam explosion, which is the widely used pretreated method, was detected in this study. Based on the inhibition result, fungi FLZ10 was used for removing the inhibitors and biodetoxification was further studied.
At first, the surface structures of corn-stovor were observed by AFM technology, to prove the results of breaking the compact lignocellulosic structures after pretreatment. According to the analysis of hydralysates, the effects of the byproducts to the fermentation were studied. It was showed that the ethanol yield were strongly inhibited by Mg~（2+）and Fe~（2+）, the decrease of ethanol yield is 34% and 11%respectively. As the total salts decreased, the ethanol yield has reduced. It has no obvious influence on the ethanol yield when the concentration of formic acid≤2g/L and furfural≤1g/L.The ethanol yield has been inhibited when the concentration of acetic acid≥15g/L and 5-HMF≥2.0g/L, the decrease of ethanol yield is 92% and 54%respectively, but the low concentration have promotion to the fermentation.
Secondly, according to the studies of inhibition, the removals of inhibitors with FLZ10 have been studied. The removal rate of inorganic ions, Ca~（2+）、Mg~（2+）、Al~（3+）、Fe~（2+）are 70%、50%、90%、80%.When T=35℃,t=96h,the removal rate of organics, acetic acid、propionic acid、butyric acid are 24%、31%、28% in the culture medium with glucose as carbon source.. In the actual fermentation, the effect of bio-detoxification and washing detoxification were equivalent. The ethanol yield using bio-detoxification based washing detoxification is 50g/L.
Once more, the optimizations of culture medium of FLZ10 agent have been carried out. As standards for monitoring cellulase activity, CB and FPA activities were detected, and the concentrations of inhibitors in corn-stover washing water, including formic acid、acetic acid、furfural、5-HMF,were detected for the bio- detoxification ability. The results were performed that the CB and FP activities in 50℃were higher than they were in 40℃, and the removal rates of inhibitors in 40℃were higher than they were in 50℃. The culture medium which using SBR or EGSB effluent as nutrients abtained the best rate for removing acetic acids; The culture medium of entire wheat bran was turned out the most replacement of formic acid,furfural and 5-HMF. However, there were little differences in inhibitors removing rate among different culture medium. Based on the comprehensive analysis, cheap crystalline cellulose (Avicel ) can be replaced by cheap corn stover powder in thedetoxified bioagent medium.
At last, contribution of growth factors was performed. According to orthogonal analysis ,it showed that the effects of growth factors rang from strong to weak were pantothenic acid > Co > VB1 > Zn > Mn > Ni > inositol, the optimized actual concentration were NiCl2·6H2O 50μm/L、MnCl2·4H2O 25mg/L、ZnSO4·7H2O 6mg/L、CoCl2·8H2O 30μm/L、inositol 150mg/L、pantothenic acid10ml/L、VB1150mg/L.
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