棉秸秆纤维素降解菌系构建及固体发酵条件优化
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摘要
【目的】构建棉秸秆降解菌系及其固体发酵条件优化。【方法】根据羧甲基纤维素酶活(CMC)和滤纸酶活(FPA)筛选纤维素降解能力强的菌株,与调节发酵品质的菌株配比,采用响应面法评价初始含水量、接种量、发酵天数及其交互作用对棉秸秆纤维素降解率的影响。【结果】得到24株纤维素降解菌,其中一株透明圈直径与菌落直径的比例最大值6.20,FPA酶活力9.699 U/h,CMC酶活力10.435 U/h,通过鉴定为枯草芽孢杆菌(bacillus subtilis),与产朊假丝酵母(Candida utilis)、植物乳杆菌(lactobacillus plantarum)、地衣芽孢杆菌(Bacillus licheniformis)构建复合菌系按1∶1∶2∶1进行发酵,对模型进行方差分析,方程回归显著(P<0.0001),接种量15.0%,发酵时间35.0 d,水分80.0%时,纤维素降解率达35.91%,接种量对纤维素降解率影响最大。【结论】构建了棉秸秆纤维素降解菌系,确定了固体发酵最佳条件,为棉秸秆饲料化提供了实验及理论依据。
【Objective】To construct cotton straw degrading strains and optimize their solid fermentation conditions.【Method】The cellulose degrading strains were screened based on carboxymethyl cellulase( CMC)and filter paper activity( FPA),and mixed with bacteria strain to adjust the fermentation quality and identify the symbiotic strains mixing ratio,then,the effect of the initial moisture content,inoculation amount,fermentation time and their interactions on cotton stalk cellulose degradation rate were evaluated by using a response surface method.【Result】24 strains of cellulolytic strain bacteria were obtained,among which,the maximum ratio of the diameter of one transparent circle to the diameter of colony was 6. 20,the enzyme activity of FPA was 9. 699 U/h,and the enzyme activity of CMC was 10. 435 U/h. The strain was identified asbacillus subtilis,which was mixed with J1( Candida utilis),J2( Lactobacillus plantarum),J3( Bacillus licheniformis) to construct composite strains at the ratio of 1 ∶ 1 ∶ 2 ∶ 1 for fermentation. The results of variance analysis showed that the regression equation was significant( P < 0. 0001),inoculation amount was 15. 0%,fermentation time was 35 days,water content was 80. 0%,cellulose degradation rate was 35. 91%,the effect of inoculation amount on cellulose degradation was the most significant.【Conclusion】In this experiment,the cellulose-degrading strain of cotton straw was constructed,and the optimum conditions of solid fermentation were determined,which provided the experimental and theoretical basis for the feed conversion of cotton straw.
【Objective】To construct cotton straw degrading strains and optimize their solid fermentation conditions.【Method】The cellulose degrading strains were screened based on carboxymethyl cellulase( CMC)and filter paper activity( FPA),and mixed with bacteria strain to adjust the fermentation quality and identify the symbiotic strains mixing ratio,then,the effect of the initial moisture content,inoculation amount,fermentation time and their interactions on cotton stalk cellulose degradation rate were evaluated by using a response surface method.【Result】24 strains of cellulolytic strain bacteria were obtained,among which,the maximum ratio of the diameter of one transparent circle to the diameter of colony was 6. 20,the enzyme activity of FPA was 9. 699 U/h,and the enzyme activity of CMC was 10. 435 U/h. The strain was identified asbacillus subtilis,which was mixed with J1( Candida utilis),J2( Lactobacillus plantarum),J3( Bacillus licheniformis) to construct composite strains at the ratio of 1 ∶ 1 ∶ 2 ∶ 1 for fermentation. The results of variance analysis showed that the regression equation was significant( P < 0. 0001),inoculation amount was 15. 0%,fermentation time was 35 days,water content was 80. 0%,cellulose degradation rate was 35. 91%,the effect of inoculation amount on cellulose degradation was the most significant.【Conclusion】In this experiment,the cellulose-degrading strain of cotton straw was constructed,and the optimum conditions of solid fermentation were determined,which provided the experimental and theoretical basis for the feed conversion of cotton straw.
引文
[1]华彬彬.分解木质纤维素复合菌系的形成机理及应用[D].北京:中国农业大学博士论文,2017.HUA Bin-bin.(2017).Formation mechanism and application of lignocellulose degradation microbial community[D].Ph D Dissertation.China Agricultural University,Beijing.(in Chinese)
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[3]庞秀芬,王浩菊,段若依,等.混合菌群发酵秸秆合成菌体蛋白及其动力学分析[J].生物资源,2017,39(3):217-222.PANG Xiu-fen,WANG Hao-ju,DUAN Ruo-yi,et al.(2017).Fermentation of straw protein by mixed bacteria and its kinetic analysis[J].Biological Resources,39(3):217-222.(in Chinese)
[4]解恒参,赵晓倩.农作物秸秆综合利用的研究进展综述[J].环境科学与管理,2015,40(1):86-90.XIE Heng-can,ZHAO Xiao-qian.(2015).Review of research progress on comprehensive utilization of crop straw[J].Environmental Science and Management,40(1):86-90.(in Chinese)
[5]魏如腾.高效降解玉米秸秆复合菌系的构建[D].太谷:山西农业大学硕士论文,2016.WEI Ru-teng.(2016).Construction of efficient degradation of corn straw composite strains[D].Master Dissertation.Shanxi Agricultural University,Taigu.(in Chinese)
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[7]崔宗均,李美丹,朴哲,等.一组高效稳定纤维素分解菌复合系MC1的筛选及功能[J].环境科学,2002,(3):36-39.CUI Zong-jun,LI Mei-dan,PIAO Zhe,et al.(2002).Screening and function of a group of efficient stable cellulose decomposing bacteria MC1[J].Environmental Science,(3):36-39.(in Chinese)
[8]Kato,S.,Haruta,S.,Cui,Z.J.,Ishii,M.,&Igarashi,Y.(2005).Stable coexistence of five bacterial strains as a cellulose-degrading community.Appl Environ Microbiol,71(11):7,099-7,106.
[9]侯敏,包慧芳,王宁,等.高效降解棉酚菌株的选育及脱毒条件的研究[J].新疆农业科学,2016,53(6):1 114-1 121.HOU Min,BAO Hui-fang,WANG Ning,et al.(2016).Screening and breeding of highly-effected degrading gossypol strains and study on defoxication technology and conditions[J].Xinjiang Agricultural Sciences,53(6):1,114-1,121.(in Chinese)
[10]侯敏,包慧芳,王宁,等.菌酶共降解棉秸秆的工艺研究[J].新疆农业科学,2016,53(8):1 467-1 473.HOU Min,BAO Hui-fang,WANG Ning,et al.(2016).Technology for codegradation of cotton stalk by microorganism and enzyme[J].Xinjiang Agricultural Sciences,53(8):1,467-1,473.(in Chinese)
[11]叶明.微生物学实验技术[M].合肥:合肥工业大学出版社,2009:371-373.YE Ming.(2009).Experimental technique in microbiology[M].Hefei:Hefei University of Technology Press:371-373.(in Chinese)
[12]张彦庭,马一.纤维素酶高产菌株的筛选及其降解秸秆的研究[J].现代农业科技,2013,(20):203-204.ZHANG Yan-ting,MA Yi.(2013).Screening of cellulase producing strain and its degradation of straw[J].Modern Agricultural Science and Technology,(20):203-204.(in Chinese)
[13]Miller,G.L.(1959).Use of dinitrosalicylic acid reagent for determination of reducing sugar.Analytical Biochemistry,31(3):426-428.
[14]Mandels,M.,&Andreotti,R.E.(1978).Problems and challenges in the cellulose to cellulase fermentation.Process Biochemistry,13(5):265-275.
[15]冯继华,曾静芬,陈茂椿.应用Van Soest法和常规法测定纤维素及木质素的比较[J].西南民族学沈学报(自然科学版),1994,20(1):55-56.FEN Ji-hua,ZEN Jin-fen,CHEN Mao-chun.(1994).Application Van Soest method and conventional method the comparison of cellulose and lignin[J].Journal of Southwest University(Natural Science Edition),20(1):55-56.(in Chinese)
[16]Liu,Y.P.,Zheng,P.,Sun,Z.H.,Ni,Y.,Dong,J.J.,&Zhu,L.L.(2008).Economical succinic acid production from cane molasses by actinobacillus succinogenes.Bioresource Technology,99(6):1,736-1,742.
[17]Sharma,S.,Malik,A.,&Satya,S.(2009).Application of response surface methodology(rsm)for optimization of nutrient supplementation for cr(vi)removal by aspergillus lentulus aml05.Journal of Hazardous Materials,164(2-3):1,198-1,204.
[18]毋锐琴,杜双奎,李志西,等.细菌纤维素发酵培养基的优化及超微观结构分析[J].生物工程学报,2008,24(6):1 068-1 074.WU Rui-qin,DU Shuang-kui,LI Zhi-xi,et al.(2008).Optimization of bacterial cellulose fermentation medium and analysis of ultra micro structure[J].Journal of Biological Engineering,24(6):1,068-1,074.(in Chinese)
[19]孙展英,陈雨璇,张晓晗,等.不同组合菌种固态发酵马铃薯渣对其营养价值影响研究[J].资源开发与利用,2015,(16):68-74.SUN Zhan-ying,CHEN Yu-xuan,ZHANG Xiao-han,et al.(2015).Effects of different combinations of solid state fermentation of potato residue on its nutritional value[J].Resource Development and Utilization,(16):68-74.(in Chinese)
[20]魏敏,雒秋江,潘榕,等.对棉花秸秆饲用价值的基本评价[J].新疆农业大学学报,2003,26(1):1-4.WEI Min,LUO Qiu-jiang,PAN Rong,et al.(2003).Initial Evaluation on Nutritional Value of Cotton Stalk[J].Xinjiang Agricultural Sciences,26(1):1-4.(in Chinese)
[21]Sánchez,C.(2009).Lignocellulosic residues:biodegradation and bioconversion by fungi.Biotechnology Advances,27(2):185-149.
[22]李立波,任晓冬,窦森.固态发酵中2种微生物降解玉米秸秆效果的对比研究[J].农业环境科学学报,2017,36(10):2136-2 142.LI Li-bo,REN Xiao-dong,DOU Sen.(2017).Comparative study of the degradation efficiency of 2 types of microorganisms on the degradation of corn stalks in solid-state fermentation[J].Journal of Agro-Environment Science,36(10):2,136-2,142.(in Chinese)
[23]刘云鹏,苏瑛杰,孙旸,等.裂褶菌对玉米秸秆木质纤维素的降解作用研究[J].吉林农业大学学报,2017,(9):38-44.LIU Yun-peng,SU Ying-jie,SUN Yang,et al.(2017).Research of degradation of lignocellulose in corn stalk by Schizophyllum commune[J].Journal of Jilin Agricultural University,(9):38-44.(in Chinese)
[24]王毅.农林废物堆肥化中细菌对木质素的降解及产酶研究[D].长沙:湖南大学硕士论文,2008.WANG Yi.(2008).Research of lignin degrading ability and the enzyme production by bacteria in agriculture and forestry waste composting[D].Master Dissertation.Hunan University,Changsha.(in Chinese)
[25]张斯童,兰雪,李哲,等.微生物降解玉米秸秆的研究进展[J].吉林农业大学学报,2016,38(5):517-522.ZHANG Si-tong,LAN Xue,LI Zhe,et al.(2016).Research progress of microbial degradation of corn straw[J].Journal of Jilin Agricultural University,38(5):517-522.(in Chinese)
[26]王尧悦,曹平华,陈玉林,等.一株纤维素降解菌的鉴定及其对饲料粗纤维的降解效果[J].西北农林科技大学学报(自然科学版),2016,44(7):16-24.WANG Yao-yue,CAO Ping-hua,CHEN Yu-lin,et al.(2016).Identification of a cellulose decomposing strain and its application for degrading different cellulosic materials[J].Journal of Northwest A&F University(Nat.Sci.Ed.),44(7):16-24.(in Chinese)
[27]汤小朋,赵华,汤加勇,等.黑曲霉固态发酵改善木薯渣品质的研究[J].动物营养学报,2014,26(7):2 026-2 034.TANG Xiao-peng,ZHAO Hua,TANG Jia-yong,et al.(2014).Study on improving the quality of cassava residue by solid-state fermentation of Aspergillus niger[J].Chinese Journal of Animal Nutrition,26(7):2,026-2,034.(in Chinese)
[28]美合热阿依·木台力甫,乌斯满·依米提.乳酸菌与纤维素分解菌混合菌剂对玉米青贮饲料发酵品质的影响[J].饲料工业,2016,37(23):51-54.Mihray Mutallip,Wusiman Yimit.(2016).Effects of mixed additive of lactobacillus and cellulose decomposition bacteria on the quality of corn stover silage[J].Feed Industry,37(23):51-54.(in Chinese)
[29]孙展英,陈雨璇,张晓晗,等.不同组合菌种固态发酵马铃薯渣对其营养价值影响研究[J].资源开发与利用,2015,(16):68-74.SUN Zhan-ying,CHEN Yu-xuan,ZHANG Xiao-han,et al.(2015).Effects of different combinations of solid state fermentation of potato residue on its nutritional value[J].Resource Development and Utilization,(16):68-74.(in Chinese)
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[2]郑梦莉,王凯军,张佩华,等.农作物秸秆饲料化技术研究进展[J].中国饲料,2017,(11):5-14.ZHENG Meng-li,WANG Kai-jun,ZHANG Pei-hua,et al.(2017).Research progress on feed technology of crop straw[J].Feed in China,(11):5-14.(in Chinese)
[3]庞秀芬,王浩菊,段若依,等.混合菌群发酵秸秆合成菌体蛋白及其动力学分析[J].生物资源,2017,39(3):217-222.PANG Xiu-fen,WANG Hao-ju,DUAN Ruo-yi,et al.(2017).Fermentation of straw protein by mixed bacteria and its kinetic analysis[J].Biological Resources,39(3):217-222.(in Chinese)
[4]解恒参,赵晓倩.农作物秸秆综合利用的研究进展综述[J].环境科学与管理,2015,40(1):86-90.XIE Heng-can,ZHAO Xiao-qian.(2015).Review of research progress on comprehensive utilization of crop straw[J].Environmental Science and Management,40(1):86-90.(in Chinese)
[5]魏如腾.高效降解玉米秸秆复合菌系的构建[D].太谷:山西农业大学硕士论文,2016.WEI Ru-teng.(2016).Construction of efficient degradation of corn straw composite strains[D].Master Dissertation.Shanxi Agricultural University,Taigu.(in Chinese)
[6]薛红蕾.高效棉秆降解菌系的构建及其特性研究[D].武汉:华中科技大学硕士论文,2007.XUE Hong-lei.(2007).Construction and characterization of high efficient cotton stalk degradation strains[D].Master Dissertation.Huazhong University of Science and Technology,Wuhan.(in Chinese)
[7]崔宗均,李美丹,朴哲,等.一组高效稳定纤维素分解菌复合系MC1的筛选及功能[J].环境科学,2002,(3):36-39.CUI Zong-jun,LI Mei-dan,PIAO Zhe,et al.(2002).Screening and function of a group of efficient stable cellulose decomposing bacteria MC1[J].Environmental Science,(3):36-39.(in Chinese)
[8]Kato,S.,Haruta,S.,Cui,Z.J.,Ishii,M.,&Igarashi,Y.(2005).Stable coexistence of five bacterial strains as a cellulose-degrading community.Appl Environ Microbiol,71(11):7,099-7,106.
[9]侯敏,包慧芳,王宁,等.高效降解棉酚菌株的选育及脱毒条件的研究[J].新疆农业科学,2016,53(6):1 114-1 121.HOU Min,BAO Hui-fang,WANG Ning,et al.(2016).Screening and breeding of highly-effected degrading gossypol strains and study on defoxication technology and conditions[J].Xinjiang Agricultural Sciences,53(6):1,114-1,121.(in Chinese)
[10]侯敏,包慧芳,王宁,等.菌酶共降解棉秸秆的工艺研究[J].新疆农业科学,2016,53(8):1 467-1 473.HOU Min,BAO Hui-fang,WANG Ning,et al.(2016).Technology for codegradation of cotton stalk by microorganism and enzyme[J].Xinjiang Agricultural Sciences,53(8):1,467-1,473.(in Chinese)
[11]叶明.微生物学实验技术[M].合肥:合肥工业大学出版社,2009:371-373.YE Ming.(2009).Experimental technique in microbiology[M].Hefei:Hefei University of Technology Press:371-373.(in Chinese)
[12]张彦庭,马一.纤维素酶高产菌株的筛选及其降解秸秆的研究[J].现代农业科技,2013,(20):203-204.ZHANG Yan-ting,MA Yi.(2013).Screening of cellulase producing strain and its degradation of straw[J].Modern Agricultural Science and Technology,(20):203-204.(in Chinese)
[13]Miller,G.L.(1959).Use of dinitrosalicylic acid reagent for determination of reducing sugar.Analytical Biochemistry,31(3):426-428.
[14]Mandels,M.,&Andreotti,R.E.(1978).Problems and challenges in the cellulose to cellulase fermentation.Process Biochemistry,13(5):265-275.
[15]冯继华,曾静芬,陈茂椿.应用Van Soest法和常规法测定纤维素及木质素的比较[J].西南民族学沈学报(自然科学版),1994,20(1):55-56.FEN Ji-hua,ZEN Jin-fen,CHEN Mao-chun.(1994).Application Van Soest method and conventional method the comparison of cellulose and lignin[J].Journal of Southwest University(Natural Science Edition),20(1):55-56.(in Chinese)
[16]Liu,Y.P.,Zheng,P.,Sun,Z.H.,Ni,Y.,Dong,J.J.,&Zhu,L.L.(2008).Economical succinic acid production from cane molasses by actinobacillus succinogenes.Bioresource Technology,99(6):1,736-1,742.
[17]Sharma,S.,Malik,A.,&Satya,S.(2009).Application of response surface methodology(rsm)for optimization of nutrient supplementation for cr(vi)removal by aspergillus lentulus aml05.Journal of Hazardous Materials,164(2-3):1,198-1,204.
[18]毋锐琴,杜双奎,李志西,等.细菌纤维素发酵培养基的优化及超微观结构分析[J].生物工程学报,2008,24(6):1 068-1 074.WU Rui-qin,DU Shuang-kui,LI Zhi-xi,et al.(2008).Optimization of bacterial cellulose fermentation medium and analysis of ultra micro structure[J].Journal of Biological Engineering,24(6):1,068-1,074.(in Chinese)
[19]孙展英,陈雨璇,张晓晗,等.不同组合菌种固态发酵马铃薯渣对其营养价值影响研究[J].资源开发与利用,2015,(16):68-74.SUN Zhan-ying,CHEN Yu-xuan,ZHANG Xiao-han,et al.(2015).Effects of different combinations of solid state fermentation of potato residue on its nutritional value[J].Resource Development and Utilization,(16):68-74.(in Chinese)
[20]魏敏,雒秋江,潘榕,等.对棉花秸秆饲用价值的基本评价[J].新疆农业大学学报,2003,26(1):1-4.WEI Min,LUO Qiu-jiang,PAN Rong,et al.(2003).Initial Evaluation on Nutritional Value of Cotton Stalk[J].Xinjiang Agricultural Sciences,26(1):1-4.(in Chinese)
[21]Sánchez,C.(2009).Lignocellulosic residues:biodegradation and bioconversion by fungi.Biotechnology Advances,27(2):185-149.
[22]李立波,任晓冬,窦森.固态发酵中2种微生物降解玉米秸秆效果的对比研究[J].农业环境科学学报,2017,36(10):2136-2 142.LI Li-bo,REN Xiao-dong,DOU Sen.(2017).Comparative study of the degradation efficiency of 2 types of microorganisms on the degradation of corn stalks in solid-state fermentation[J].Journal of Agro-Environment Science,36(10):2,136-2,142.(in Chinese)
[23]刘云鹏,苏瑛杰,孙旸,等.裂褶菌对玉米秸秆木质纤维素的降解作用研究[J].吉林农业大学学报,2017,(9):38-44.LIU Yun-peng,SU Ying-jie,SUN Yang,et al.(2017).Research of degradation of lignocellulose in corn stalk by Schizophyllum commune[J].Journal of Jilin Agricultural University,(9):38-44.(in Chinese)
[24]王毅.农林废物堆肥化中细菌对木质素的降解及产酶研究[D].长沙:湖南大学硕士论文,2008.WANG Yi.(2008).Research of lignin degrading ability and the enzyme production by bacteria in agriculture and forestry waste composting[D].Master Dissertation.Hunan University,Changsha.(in Chinese)
[25]张斯童,兰雪,李哲,等.微生物降解玉米秸秆的研究进展[J].吉林农业大学学报,2016,38(5):517-522.ZHANG Si-tong,LAN Xue,LI Zhe,et al.(2016).Research progress of microbial degradation of corn straw[J].Journal of Jilin Agricultural University,38(5):517-522.(in Chinese)
[26]王尧悦,曹平华,陈玉林,等.一株纤维素降解菌的鉴定及其对饲料粗纤维的降解效果[J].西北农林科技大学学报(自然科学版),2016,44(7):16-24.WANG Yao-yue,CAO Ping-hua,CHEN Yu-lin,et al.(2016).Identification of a cellulose decomposing strain and its application for degrading different cellulosic materials[J].Journal of Northwest A&F University(Nat.Sci.Ed.),44(7):16-24.(in Chinese)
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