混菌发酵对产纤维素酶的影响及菌剂在大豆秸秆降解中的应用
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摘要
为提高秸秆还田过程中的腐解效率,减轻还田产生的病害加重现象,以稻草、麸皮为基本培养基,以黄绿木霉菌(拮抗真菌)、角毛壳菌及绿色木霉菌(纤维素高效降解微生物)为研究材料,测定3种不同菌种及混合菌发酵液处理下滤纸酶、棉花酶与羧甲基纤维素酶的活性变化。通过秸秆翻埋试验测定混菌发酵液和商业菌剂处理大豆秸秆发酵前后纤维素、半纤维素含量变化并探讨最适秸秆翻埋长度和深度。探讨混菌培养对纤维素酶产生能力及对大豆秸秆纤维素与半纤维素降解速率的影响。结果表明:滤纸酶活性、棉花酶活性与羧甲基纤维素酶活性均为黄绿木霉菌、角毛壳菌和绿色木霉菌3株菌混菌发酵的产酶能力最强,混合菌发酵的3种纤维素酶活性分别为385. 12,454. 30和495. 12 U。混菌发酵液处理与商业菌剂处理的大豆秸秆粉纤维素与半纤维素含量与空白对照差异显著,混菌发酵液处理的纤维素与半纤维素降解率分别可达66%和76%。大豆秸秆降解率与秸秆强度的变化说明:在大豆秸秆长度为3 cm,翻埋深度为10 cm时,秸秆降解率最高。添加混合菌剂的处理的秸秆降解率较对照提高50%以上,同时秸秆的强度降低约5倍,穿刺力降低。结果证实了3株不同真菌在秸秆降解中复合应用的可行性,可作为大豆秸秆还田中有效的腐解微生物资源。
In order to improve the decomposition efficiency of straw returning to field and reduce the diseases aggravation,an-tagonistic fungi Trichoderma aureoviride,Chaetomium cupreum and cellulose-decomposing fungus T. viride were used to studythe cellulase production ability of different strains in mixed culture and its effect on the degradation rate of cellulose and hemi-cellulose from soybean straw. The results showed that the cellulase activity,cotton enzyme activity and carboxymethyl cellulaseactivity of three different strains and mixed fermentation at different fermentation time were measured on rice straw and bran asbasic medium. The cellulase activity of these three strains mixed fermentaton was the strongest. The cellulase activity was385. 12,454. 30 and 495. 12 U,respectively. The changes of cellulose and hemicellulose contents before and after fermenta-tion showed that there were significant differences between the two treatments,and the degradation rates of cellulose and hemi-cellulose were 66% and 76% respectively. We also found that the degradation rate of soybean straw was the highest when thelength of soybean straw was 3 cm and the buried depth was 10 cm by the burying experiment. The degradation rate of soybeanstraw was increased by more than 50%,the strength of soybean straw was reduced by about 5 times and the puncture force wasdecreased by adding mixed microbial agent. The results showed that the three strains mixed could be used in the degradation ofsoybean straw,which could provide effective microbial resources for decomposition of soybean straw.
In order to improve the decomposition efficiency of straw returning to field and reduce the diseases aggravation,an-tagonistic fungi Trichoderma aureoviride,Chaetomium cupreum and cellulose-decomposing fungus T. viride were used to studythe cellulase production ability of different strains in mixed culture and its effect on the degradation rate of cellulose and hemi-cellulose from soybean straw. The results showed that the cellulase activity,cotton enzyme activity and carboxymethyl cellulaseactivity of three different strains and mixed fermentation at different fermentation time were measured on rice straw and bran asbasic medium. The cellulase activity of these three strains mixed fermentaton was the strongest. The cellulase activity was385. 12,454. 30 and 495. 12 U,respectively. The changes of cellulose and hemicellulose contents before and after fermenta-tion showed that there were significant differences between the two treatments,and the degradation rates of cellulose and hemi-cellulose were 66% and 76% respectively. We also found that the degradation rate of soybean straw was the highest when thelength of soybean straw was 3 cm and the buried depth was 10 cm by the burying experiment. The degradation rate of soybeanstraw was increased by more than 50%,the strength of soybean straw was reduced by about 5 times and the puncture force wasdecreased by adding mixed microbial agent. The results showed that the three strains mixed could be used in the degradation ofsoybean straw,which could provide effective microbial resources for decomposition of soybean straw.
引文
[1]宋大利,侯胜鹏,王秀斌,等.中国秸秆养分资源数量及替代化肥潜力[J].植物营养与肥料学报,2018,24(1):1-21.(Song D L,Hou S P,Wang X B,et al. Nutrient resource quanti-ty of crop straw and its potential of substituting[J]. Journal ofPlant Nutrition and Fertilizers,2018,24(1):1-21.)
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[3]唐萍.秸秆综合利用方案评价-以肥东县为例[D].合肥:合肥工业大学,2010.(Tang P. Evaluation of mroposals of strawscomprehensive utilization:For example of county of Feidong[D].Hefei:Hefei University of Technology,2010.)
[4]花莉,张成,马宏瑞,等.秸秆生物质炭土地利用的环境效益研究[J].生态环境学报,2010,19(10):2489-2492.(HuaL,Zhang C,Ma H R,et al. Environmental benefits of biocharmade by agricultural straw when applied to soil[J]. Ecology andEnvironmental Sciences,2010,19(10):2489-2492.)
[5]曹志宏,黄艳丽,郝晋珉.中国作物秸秆资源利用潜力的多适宜性综合评价[J].环境科学研究,2018,31(1):179-186.(Cao Z H,Huang Y L,Hao J M. Multi-suitability compre-hensive evaluation of crop straw resource utilization in China[J].Research of Environmental Sciences,2018,31(1):179-186.)
[6] Madari B,Machado P L O A,Torres E,et al. No tillage and croprotation effects on soil aggregation and organic carbon in a RhodicFerralsol from southern Brazil[J]. Soil and Tillage Research,2005,80(1-2):185-200.
[7] Zhang Y H P,Himmel M E,Mielenz J R. Outlook for cellulase im-provement:Screening and selection strategies[J]. BiotechnologyAdvances,2006,24(5):452.
[8] Abdulla H M,EI-Shatoury S A. Actinomycetes in rice straw decom-position[J]. Waste Management,2007,27(6):850-853.
[9] Ma H,Liu W W,Liu P,et al. Enhanced enzymatic saccharificationof rice straw by microwave pretreatment[J]. Bioresource Technol-ogy,2009,100(3):1279-1284.
[10] Lundell T K,Mkel M R,Hildén K. Lignin-modifying enzymes infilamentous basidiomycetes:Ecological,functional and phyloge-netic review[J]. Journal of Basic Microbiology,2010,50:5-20.
[11] Fang X,Qin Y Q,Li X Z,et al. Progress on cellulase and enzy-matic hydrolysis of lignocellulosic biomass[J]. Biotechnology,2010,26(7):864-869.
[12]张国秀.里氏木霉纤维酶高产菌株遗传改造及新型糖苷水解酶的挖掘[D].上海:华东理工大学,2017.(Zhang G X. Ge-netic modification of trichoderma reesei Hyper-cellulolytic mutantand mining for new glycoside hydrolases[D]. Shanghai:East Chi-na University of Science and Technology,2017.)
[13]李洪林,刘凤艳,龚振平,等.稻杆还田对水稻主要病害发生的影响[J].作物研究,2012,26(1):7-10.(Li H L,Liu F Y,GongZ P,et al. Effect of straw return back to paddy field on occurence ofrice major disease[J]. Crop Research,2012,26(1):7-10.)
[14]张雪松,曹永胜,曹克强.保护性耕作与小麦主要土传病害问题和治理对策[J].西北农林科技大学学报,2005,33(S1):47-48.(Zhang X S,Cao Y S,Cao K Q. Management ofwheat soil-borne diseases under the conservative farming system[J]. Journal of Northwest Science and Technology University ofAgricultural and Forestry(Natural Science Edition),2005,33(S1):47-48.)
[15]孙秀娟.稻秆集中掩埋还田对赤霉病菌(Fusarium graminea-rum Sehw)和二化頓(Chilo suppressalis Walker)幼虫存活的影响[D].南京:南京农业大学,2012.(Sun X J. Effects of strawcentralize-buried in soil on suevtval dynamics of phytoalexin(Fusari-um graminearum Sehw)and stem-borer(Chilo suppressalis Walker)larvae[D]. Nanjing:Nanjing Agricultural University,2012.)
[16]方诩,秦玉琪,李雪芝,等.纤维素酶与木质纤维素生物降解转化的研究进展[J].生物工程学报,2010,26(7):864-869.(Fang X,Qin Y Q,Li X Z,et al. Progress on cellulase and enzy-matic hydrolysis of lignocellulosic biomass[J]. Chinese Journal ofBiotechnology,2010,26(7):864-869.)
[17]孙美娜,张凡凡,王永强,等.棉花秸秆纤维素降解菌的筛选鉴定与降解棉秆效果研究[J].新疆农业科学,2018,55(1):16-23.(Sun M N,Zhang F F,Wang Y Q,et al. Screening andidentification of cellulolytic srains from cotton straw and its effecton degradation of cotton stalk[J]. Xinjiang Agricultural Sciences,2018,55(1):16-23.)
[18] Baldrian P,Valof P. Degradation of cellulose by basidiomycetousfungi[J]. FEMS Microbiology Reviews,2008,32(3):501-502.
[19] Adsul M,Bastawde K,Varma A,et al. Strain improvement ofPenicillium janthinellum NCIM 1171 for increased cellulose pro-duction[J]. Bioresource Technol,2007,98(7):1467-1473.
[20]方星星.里氏木霉与黑曲霉混合发酵产纤维素酶的研究[D].合肥:合肥工业大学,2012.(Fang X X. Study on cellulase pro-duction by mixed fermentation of Trichoderma ressei and Aspergillusniger[D]. He Fei:Hefei University of Technology,2012.)
[21]孙冬梅,杨谦,宋金柱,等.黄绿木霉固定化生产纤维素酶及酶学特性的研究[J].林产化学与工业,2006,26(2):79-82.(Sun D M,Yang Q,Song J Z,et al. Studies on cellulase produc-tion by immobilized cells and characteristics of cellulase[J].Chemistry and Industry of Forest Products, 2006, 26(2):79-82.)
[22]孙冬梅,杨谦,张军政.黄绿木霉诱变菌株对大豆根腐病镰刀菌的拮抗[J].大豆科学,2005,24(3):171-175.(Sun DM,Yang Q,Zhang J Z. Antagonism of Trichoderma aureoviridemutant strain aganist Fusarium spp. the pathogen of soybean rootrot[J]. Soybean Science,2005,24(3):171-175.)
[23]孙冬梅,杨谦,宋金柱.黄绿木霉菌对大豆根腐病镰刀菌的拮抗作用[J].中国油料作物学报,2005,27(3):59-63.(Sun D M,Yang Q,Song J Z. Antagonism of Trichoderma aureo-viride against Fusarium spp which causes soybean root rot[J].Chinese Journal of Oil Crop Sciences,2005,27(3):59-63.)
[24]张海燕,杨谦.角毛壳菌产纤维素酶条件的研究[J].河南工业大学学报(自然科学版),2006,27(4):15-18.(Zhang HY,Yang Q. Study on cellulase ferment production of Chaetomiumcupreum[J]. Journal of Henan University of Technology(NaturalScience Edition),2006,27(4):15-18.)
[25]张海燕,杨谦.角毛壳菌生物防治相关基因的筛选[J].中国生物防治,2007,23(2):191-194.(Zhang H Y,Yang Q. Selec-tion of biocontrol-related genes from Chaetomium cupreum[J].Chinese Journal of Biological Control,2007,23(2):191-194.)
[26] Lan T Q,Wei D,Yang S T,et al. Enhanced cellulase produc-tion by Trichoderma viride in a rotating fibrous bed bioreactor[J].Bioresource Technology,2013,133:175-182.
[27]唐国涛,邢沙沙,黄榕彬,等.脂麻秆中纤维素与半纤维素的含量测定[J].作物研究,2012,26(1):53-55.(Tang G T,Xing S S,Huang R B,et al. Determination of content of celluloseand hemicellulose in stem of Sesamum indicum[J]. Crop Re-search,2012,26(1):53-55.)
[28]杨腾腾,周宏,王霞,等.微生物降解纤维素的新机制[J].微生物学通报,2015,42(5):928-935.(Yang T T,Zhou H,Wang X,et al. A new microbial strategy for cellulose degradation[J]. Microbiology China,2015,42(5):928-935.)
[29] Beguin P,Aubert J P. The biological degradation of cellulose[J].FEMS Microbiology Reviews,1994,13(1):25-58.
[30] Dashtban M,Schraft H,Qin W. Fungal bioconversion of lignocel-lulosic residues,opportunities&perspectives[J]. InternationalJournal of Biological Sciences,2009,5(6):578-595.
[31]石娜娜,顾颖慧,郑颖,等.大豆秸秆纤维素降解菌的筛选及鉴定[J].农技服务,2016,33(1):95-81.(Shi N N,Gu YH,Zheng Y,et al. Screening and identification of cellulose de-grading strains of soybean straw[J]. Agricultural Service,2016,33(1):95-81.)
[2]李逢雨,孙锡发,冯文强,等.麦秆、油菜秆还田腐解速率及养分释放规律研究[J].植物营养与肥料学报,2009,15(2):374-380.(Li F Y,Sun X F,Feng W Q,et al. Nutrient releasepatterns and decomposing rates of wheat and rapeseed straw[J].Plant Nutrition and Fertilizer Science,2009,15(2):374-380.)
[3]唐萍.秸秆综合利用方案评价-以肥东县为例[D].合肥:合肥工业大学,2010.(Tang P. Evaluation of mroposals of strawscomprehensive utilization:For example of county of Feidong[D].Hefei:Hefei University of Technology,2010.)
[4]花莉,张成,马宏瑞,等.秸秆生物质炭土地利用的环境效益研究[J].生态环境学报,2010,19(10):2489-2492.(HuaL,Zhang C,Ma H R,et al. Environmental benefits of biocharmade by agricultural straw when applied to soil[J]. Ecology andEnvironmental Sciences,2010,19(10):2489-2492.)
[5]曹志宏,黄艳丽,郝晋珉.中国作物秸秆资源利用潜力的多适宜性综合评价[J].环境科学研究,2018,31(1):179-186.(Cao Z H,Huang Y L,Hao J M. Multi-suitability compre-hensive evaluation of crop straw resource utilization in China[J].Research of Environmental Sciences,2018,31(1):179-186.)
[6] Madari B,Machado P L O A,Torres E,et al. No tillage and croprotation effects on soil aggregation and organic carbon in a RhodicFerralsol from southern Brazil[J]. Soil and Tillage Research,2005,80(1-2):185-200.
[7] Zhang Y H P,Himmel M E,Mielenz J R. Outlook for cellulase im-provement:Screening and selection strategies[J]. BiotechnologyAdvances,2006,24(5):452.
[8] Abdulla H M,EI-Shatoury S A. Actinomycetes in rice straw decom-position[J]. Waste Management,2007,27(6):850-853.
[9] Ma H,Liu W W,Liu P,et al. Enhanced enzymatic saccharificationof rice straw by microwave pretreatment[J]. Bioresource Technol-ogy,2009,100(3):1279-1284.
[10] Lundell T K,Mkel M R,Hildén K. Lignin-modifying enzymes infilamentous basidiomycetes:Ecological,functional and phyloge-netic review[J]. Journal of Basic Microbiology,2010,50:5-20.
[11] Fang X,Qin Y Q,Li X Z,et al. Progress on cellulase and enzy-matic hydrolysis of lignocellulosic biomass[J]. Biotechnology,2010,26(7):864-869.
[12]张国秀.里氏木霉纤维酶高产菌株遗传改造及新型糖苷水解酶的挖掘[D].上海:华东理工大学,2017.(Zhang G X. Ge-netic modification of trichoderma reesei Hyper-cellulolytic mutantand mining for new glycoside hydrolases[D]. Shanghai:East Chi-na University of Science and Technology,2017.)
[13]李洪林,刘凤艳,龚振平,等.稻杆还田对水稻主要病害发生的影响[J].作物研究,2012,26(1):7-10.(Li H L,Liu F Y,GongZ P,et al. Effect of straw return back to paddy field on occurence ofrice major disease[J]. Crop Research,2012,26(1):7-10.)
[14]张雪松,曹永胜,曹克强.保护性耕作与小麦主要土传病害问题和治理对策[J].西北农林科技大学学报,2005,33(S1):47-48.(Zhang X S,Cao Y S,Cao K Q. Management ofwheat soil-borne diseases under the conservative farming system[J]. Journal of Northwest Science and Technology University ofAgricultural and Forestry(Natural Science Edition),2005,33(S1):47-48.)
[15]孙秀娟.稻秆集中掩埋还田对赤霉病菌(Fusarium graminea-rum Sehw)和二化頓(Chilo suppressalis Walker)幼虫存活的影响[D].南京:南京农业大学,2012.(Sun X J. Effects of strawcentralize-buried in soil on suevtval dynamics of phytoalexin(Fusari-um graminearum Sehw)and stem-borer(Chilo suppressalis Walker)larvae[D]. Nanjing:Nanjing Agricultural University,2012.)
[16]方诩,秦玉琪,李雪芝,等.纤维素酶与木质纤维素生物降解转化的研究进展[J].生物工程学报,2010,26(7):864-869.(Fang X,Qin Y Q,Li X Z,et al. Progress on cellulase and enzy-matic hydrolysis of lignocellulosic biomass[J]. Chinese Journal ofBiotechnology,2010,26(7):864-869.)
[17]孙美娜,张凡凡,王永强,等.棉花秸秆纤维素降解菌的筛选鉴定与降解棉秆效果研究[J].新疆农业科学,2018,55(1):16-23.(Sun M N,Zhang F F,Wang Y Q,et al. Screening andidentification of cellulolytic srains from cotton straw and its effecton degradation of cotton stalk[J]. Xinjiang Agricultural Sciences,2018,55(1):16-23.)
[18] Baldrian P,Valof P. Degradation of cellulose by basidiomycetousfungi[J]. FEMS Microbiology Reviews,2008,32(3):501-502.
[19] Adsul M,Bastawde K,Varma A,et al. Strain improvement ofPenicillium janthinellum NCIM 1171 for increased cellulose pro-duction[J]. Bioresource Technol,2007,98(7):1467-1473.
[20]方星星.里氏木霉与黑曲霉混合发酵产纤维素酶的研究[D].合肥:合肥工业大学,2012.(Fang X X. Study on cellulase pro-duction by mixed fermentation of Trichoderma ressei and Aspergillusniger[D]. He Fei:Hefei University of Technology,2012.)
[21]孙冬梅,杨谦,宋金柱,等.黄绿木霉固定化生产纤维素酶及酶学特性的研究[J].林产化学与工业,2006,26(2):79-82.(Sun D M,Yang Q,Song J Z,et al. Studies on cellulase produc-tion by immobilized cells and characteristics of cellulase[J].Chemistry and Industry of Forest Products, 2006, 26(2):79-82.)
[22]孙冬梅,杨谦,张军政.黄绿木霉诱变菌株对大豆根腐病镰刀菌的拮抗[J].大豆科学,2005,24(3):171-175.(Sun DM,Yang Q,Zhang J Z. Antagonism of Trichoderma aureoviridemutant strain aganist Fusarium spp. the pathogen of soybean rootrot[J]. Soybean Science,2005,24(3):171-175.)
[23]孙冬梅,杨谦,宋金柱.黄绿木霉菌对大豆根腐病镰刀菌的拮抗作用[J].中国油料作物学报,2005,27(3):59-63.(Sun D M,Yang Q,Song J Z. Antagonism of Trichoderma aureo-viride against Fusarium spp which causes soybean root rot[J].Chinese Journal of Oil Crop Sciences,2005,27(3):59-63.)
[24]张海燕,杨谦.角毛壳菌产纤维素酶条件的研究[J].河南工业大学学报(自然科学版),2006,27(4):15-18.(Zhang HY,Yang Q. Study on cellulase ferment production of Chaetomiumcupreum[J]. Journal of Henan University of Technology(NaturalScience Edition),2006,27(4):15-18.)
[25]张海燕,杨谦.角毛壳菌生物防治相关基因的筛选[J].中国生物防治,2007,23(2):191-194.(Zhang H Y,Yang Q. Selec-tion of biocontrol-related genes from Chaetomium cupreum[J].Chinese Journal of Biological Control,2007,23(2):191-194.)
[26] Lan T Q,Wei D,Yang S T,et al. Enhanced cellulase produc-tion by Trichoderma viride in a rotating fibrous bed bioreactor[J].Bioresource Technology,2013,133:175-182.
[27]唐国涛,邢沙沙,黄榕彬,等.脂麻秆中纤维素与半纤维素的含量测定[J].作物研究,2012,26(1):53-55.(Tang G T,Xing S S,Huang R B,et al. Determination of content of celluloseand hemicellulose in stem of Sesamum indicum[J]. Crop Re-search,2012,26(1):53-55.)
[28]杨腾腾,周宏,王霞,等.微生物降解纤维素的新机制[J].微生物学通报,2015,42(5):928-935.(Yang T T,Zhou H,Wang X,et al. A new microbial strategy for cellulose degradation[J]. Microbiology China,2015,42(5):928-935.)
[29] Beguin P,Aubert J P. The biological degradation of cellulose[J].FEMS Microbiology Reviews,1994,13(1):25-58.
[30] Dashtban M,Schraft H,Qin W. Fungal bioconversion of lignocel-lulosic residues,opportunities&perspectives[J]. InternationalJournal of Biological Sciences,2009,5(6):578-595.
[31]石娜娜,顾颖慧,郑颖,等.大豆秸秆纤维素降解菌的筛选及鉴定[J].农技服务,2016,33(1):95-81.(Shi N N,Gu YH,Zheng Y,et al. Screening and identification of cellulose de-grading strains of soybean straw[J]. Agricultural Service,2016,33(1):95-81.)