康氏木霉的原生质体诱变及玉米秸秆发酵酒精工艺研究
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摘要
本文以玉米秸秆和麦麸为原料，以康氏木霉为出发菌株，对该菌株进行了原生质体诱变，建立了康氏木霉原生质体诱变及选育的方法，测定了选育出的菌株的一些性质，改进了纤维素鉴别培养基和纤维素滤纸酶活的测定方法，建立了一种由玉米秸杆发酵生产酒精的简易方法。
以本实验室保藏的康氏木霉为出发菌株，将该菌株接种至玉米秸杆：麦麸为1：4的固体培养基中28℃培养32h，测得滤纸酶活为13.40±1.58μg/min·mL。将溶壁酶制成酶液，该康氏木霉在酶液的作用下，3h后去壁形成原生质体，原生质体经过紫外线诱变后，选育出40株菌株，经过五代培养后，初筛得到七株菌，最后经过复筛得到纤维素酶活较高的两株菌ZHY1和ZHY2。ZHY1接种至秸杆：麦麸为1：4的固体培养基中28℃培养32h，测得ZHY1菌的滤纸酶活为36.76±1.54μg/min·mL。ZHY2接种至秸杆：麦麸为1：4的固体培养基中28℃培养32h，测得ZHY2菌的滤纸酶活为33.40±2.80μg/min·mL。经过多重比较差异显著性分析，ZHY1和ZHY2的酶活高于出发菌株的酶活并与出菌株的酶活有极显著(p＜0.01)的差异。
初筛培养基为(NH_4)_2SO_4 2g，MgSO_4 0.5g，K_2HPO_4 1g，NaCl 0.5g，纤维素粉2g，刚果红0.4g，琼脂22g，水1000mL，自然pH值(纤维素粉以0.1mol/l的盐酸处理24h，之后水洗至pH值中性，过滤、烘干。琼脂则经流水冲洗24h后烘干)。与两种对照培养基相比，此培养基水解圈效果更明显，根据水解圈沉淀大小，出现早迟便可粗略估计菌株产酶情况。
在测定纤维素滤纸酶活的过程中的酶液浸提的方法为浸提时在摇床上30℃浸提1h，摇床转数为95r/min。这样可较充分的浸提出纤维素酶，与报道方法相比酶活测定结果可以更加准确。
在玉米秸杆生产酒精工艺方面，通过正交实验，建立了一种直接将菌种用于分解纤维素再发酵产生酒精的方法，该法可用于实验室中秸杆发酵的试验。
根据本实验结果每千克玉米秸杆和每4千克麦麸可生产158g酒精。
The material was stalk of maize and wheat bran, the starting bacterium was a kind of Trichoderma koningii in this experiment. The mutagenesis of protoplast of Trichoderma koningii was done by UV. The method of the mutagenesis of protoplast was established. The character of furguses after mutagenesis was determined. In addition, an differential medium which can conveniently segregate cellulose decomposing microorganisms was improved. The method which determine FP cellulose activity was improved. The simple way which produce alcohol from corn straw was established and the economy benefit analysis was done.
The starting bacterium was a kind of Trichoderma koningii which was saved by our laboratory, the Trichoderma koningii was inoculated in the medium made of corn straw / wheat bran = 1 : 4 and foster 32h at 28
℃.The FP cellulose activity of starting bacterium was 13.40+1.58ug/min.mL. The enzyme liquid was made by the enzyme dissolved cell wall. The Trichoderma koningii was operated by the liquid. After 3 hours, the protoplast of Trichoderma koningii was formed. The mutagenesis of protoplast of Trichoderma koningii was done by UV and fourty furguses were gained. After fostering of five era and primary isolation, seven furguses were gained. After further isolation, two furguses were gained and were named ZHYl and ZHY2. ZHY1 was inoculated in the medium made of corn straw / wheat bran = 1:4 and foster 32h at 28
℃. The FP cellulose activity of ZHYl was 36.76+1.54ug/min.mL. ZHY2 was inoculated in the medium made of corn straw / wheat bran =1:4 and foster 32h at 28
℃. The FP cellulose activity of ZHY2 was 33.40+2.80Mg/min.mL. After mulriple compare and analysis of difference prominence.The FP cellulose activity of ZHYl and ZHY2 prominent difference than the starting bacterium.
The component of primary isolation medium included (NH4)2SO42g, MgSO4 0.5g, K2HPO4 1g, NaCl 0.5g, cellulose powder 2g, Congo red 0.4g, agar 22g, water1000mL, nature pH(the cellulose powder was operated by 0. lmol/I hydrochloric acid. After 24 hours, the cellulose powder was washed by water to the pH equal to 7. The agar was washed by water.After 24 hours, the agar was dryed.). The effect of medium is obviouser than the medium which was reported by literature. The cellulose activity of furguses was simply estimated by the size of the red colonies and the time which the red colonies appear.
During determining of cellulose activity, the way distilled enzyme liquid was in shaking bed 1 hours at 30
℃ and the revolution of shaking bed was 95 r/min. The distilling of the cellulase was more sufficient and the result of the cellulose activity was accurater by using this way.
The way which made the Trichoderma koningii decompose the cellulose directly to produce alcohol was determined. This way is vary simple, and the way can produce alcohol from corn straw in laboratory.
158g alcohol can be transformed by 1 kg corn straw and 4 kg wheat bran.
以本实验室保藏的康氏木霉为出发菌株，将该菌株接种至玉米秸杆：麦麸为1：4的固体培养基中28℃培养32h，测得滤纸酶活为13.40±1.58μg/min·mL。将溶壁酶制成酶液，该康氏木霉在酶液的作用下，3h后去壁形成原生质体，原生质体经过紫外线诱变后，选育出40株菌株，经过五代培养后，初筛得到七株菌，最后经过复筛得到纤维素酶活较高的两株菌ZHY1和ZHY2。ZHY1接种至秸杆：麦麸为1：4的固体培养基中28℃培养32h，测得ZHY1菌的滤纸酶活为36.76±1.54μg/min·mL。ZHY2接种至秸杆：麦麸为1：4的固体培养基中28℃培养32h，测得ZHY2菌的滤纸酶活为33.40±2.80μg/min·mL。经过多重比较差异显著性分析，ZHY1和ZHY2的酶活高于出发菌株的酶活并与出菌株的酶活有极显著(p＜0.01)的差异。
初筛培养基为(NH_4)_2SO_4 2g，MgSO_4 0.5g，K_2HPO_4 1g，NaCl 0.5g，纤维素粉2g，刚果红0.4g，琼脂22g，水1000mL，自然pH值(纤维素粉以0.1mol/l的盐酸处理24h，之后水洗至pH值中性，过滤、烘干。琼脂则经流水冲洗24h后烘干)。与两种对照培养基相比，此培养基水解圈效果更明显，根据水解圈沉淀大小，出现早迟便可粗略估计菌株产酶情况。
在测定纤维素滤纸酶活的过程中的酶液浸提的方法为浸提时在摇床上30℃浸提1h，摇床转数为95r/min。这样可较充分的浸提出纤维素酶，与报道方法相比酶活测定结果可以更加准确。
在玉米秸杆生产酒精工艺方面，通过正交实验，建立了一种直接将菌种用于分解纤维素再发酵产生酒精的方法，该法可用于实验室中秸杆发酵的试验。
根据本实验结果每千克玉米秸杆和每4千克麦麸可生产158g酒精。
The material was stalk of maize and wheat bran, the starting bacterium was a kind of Trichoderma koningii in this experiment. The mutagenesis of protoplast of Trichoderma koningii was done by UV. The method of the mutagenesis of protoplast was established. The character of furguses after mutagenesis was determined. In addition, an differential medium which can conveniently segregate cellulose decomposing microorganisms was improved. The method which determine FP cellulose activity was improved. The simple way which produce alcohol from corn straw was established and the economy benefit analysis was done.
The starting bacterium was a kind of Trichoderma koningii which was saved by our laboratory, the Trichoderma koningii was inoculated in the medium made of corn straw / wheat bran = 1 : 4 and foster 32h at 28
℃.The FP cellulose activity of starting bacterium was 13.40+1.58ug/min.mL. The enzyme liquid was made by the enzyme dissolved cell wall. The Trichoderma koningii was operated by the liquid. After 3 hours, the protoplast of Trichoderma koningii was formed. The mutagenesis of protoplast of Trichoderma koningii was done by UV and fourty furguses were gained. After fostering of five era and primary isolation, seven furguses were gained. After further isolation, two furguses were gained and were named ZHYl and ZHY2. ZHY1 was inoculated in the medium made of corn straw / wheat bran = 1:4 and foster 32h at 28
℃. The FP cellulose activity of ZHYl was 36.76+1.54ug/min.mL. ZHY2 was inoculated in the medium made of corn straw / wheat bran =1:4 and foster 32h at 28
℃. The FP cellulose activity of ZHY2 was 33.40+2.80Mg/min.mL. After mulriple compare and analysis of difference prominence.The FP cellulose activity of ZHYl and ZHY2 prominent difference than the starting bacterium.
The component of primary isolation medium included (NH4)2SO42g, MgSO4 0.5g, K2HPO4 1g, NaCl 0.5g, cellulose powder 2g, Congo red 0.4g, agar 22g, water1000mL, nature pH(the cellulose powder was operated by 0. lmol/I hydrochloric acid. After 24 hours, the cellulose powder was washed by water to the pH equal to 7. The agar was washed by water.After 24 hours, the agar was dryed.). The effect of medium is obviouser than the medium which was reported by literature. The cellulose activity of furguses was simply estimated by the size of the red colonies and the time which the red colonies appear.
During determining of cellulose activity, the way distilled enzyme liquid was in shaking bed 1 hours at 30
℃ and the revolution of shaking bed was 95 r/min. The distilling of the cellulase was more sufficient and the result of the cellulose activity was accurater by using this way.
The way which made the Trichoderma koningii decompose the cellulose directly to produce alcohol was determined. This way is vary simple, and the way can produce alcohol from corn straw in laboratory.
158g alcohol can be transformed by 1 kg corn straw and 4 kg wheat bran.
引文
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