红壤中秸秆降解的微生物演替及应用研究
摘要
微生物在秸秆降解中发挥了巨大作用，而多项研究表明，秸秆中的木质素成分是秸秆降解速度的限制因素。鉴于此，本论文即从筛选高产木质素酶的菌株入手，结果从重阳木茎内皮中筛选到一株有较高漆酶(木质素酶之一)活性的菌株B3，并将其制成秸秆降解剂，模拟大田试验研究了它对秸秆的降解效果及对后季作物生长的影响。
本论文包括三个部分：1．调查了中科院红壤生态站内不同土壤中的微生物数量及区系组成，跟踪检测秸秆自然降解状况。结果表明，水田以细菌为主，旱地中真菌和放线菌数量最多，林地中有大量的高等担子菌；秸秆自然降解较慢，土壤微生物也随着降解的进行而呈现一定的变化规律；当调节C/N比为25：1时，秸秆的腐解残留率比对照组下降10.67％。2．通过形态学观察及测定其rDNA ITS序列对菌株B3进行鉴定，该菌株属于子囊菌亚门的腐皮壳属(Diaporthe)。纤维素类物质CMC-Na是其最佳碳源诱导物，正交优化后的发酵条件是马铃薯200g/L,CMC-Na 20g/L，NH_4Cl 1g/L,KH_2PO_43g/L,MgSO_4·7H_2O 1.5g/L,VB_10.01g/L,pH4.0，培养温度25℃；同时发现菌株B3分泌的漆酶有较好的耐热性，在酸性条件下能很好的生长，可以适应红壤酸性条件，因此根据其最佳发酵条件在7L发酵罐内发酵制成秸秆降解剂。3．通过盆栽试验和扫描电镜观察检测了秸秆降解剂对秸秆的降解效果，结果发现，该降解剂在15d内就能破坏秸秆的结构，使秸秆不再漂浮，不影响作物的生根；进一步检测其对后季作物生长的影响发现，该降解剂还能促进后季作物的生长，薄板层析和免疫学检测均显示菌株B3分泌了IAA和ABA。
Many studies have shown that lignin was the limited factor of straw degradation. In this study we screened a strain named B3 from the stem's inner bark of Bischofia polycarpam Airy Shaw which yielded high level of ligninase. The strain B3's ability in straw degradation and its effect on the next season crop's growth were then studied by making it into microbial agent in the potted experiment. Brief results are followed:
1. The changes of microorganisms' number in different kind of Red Soil from CAS Ecological Experiment Station were surveyed. The results showed in the paddy field the bacteria were predominated, in the dry land the proportion of fungi and actinomyces was distinctly higher than the other two kinds of soil, in the woodland abundant species of basidiomycetes were found; the natural degradation speed was low, with the degradation the number and composition of microorganisms changed regularly; when the C / N ratio was adjusted to
25 '?1, the degradation remnant ratio reduced 10.67% than the control.
2. By morphological and molecular methods the strain B3 was identified as Diaporthe and could exist under acidic condition, whose laccase had good thermostability. CMC-Na could induce laccase maximally. The composition of fermentation medium was potato 200g / L, CMC-Na 20g / L, NH4Cllg / L, KH2PO4 3g / L, MgSO4 7H2O 1.5g / L, VB10.01g / L, mixed solution of trace metals 7ml / L, and the optimal conditions were pH of medium was 4.0, and incubation with 150r / min at 25 C for 4d. According to that it was made into microbial agent in 7 liter bioreactor.
3. The microbial agent's ability in straw degradation was detected by potted experiment and scanning electron microscope. The result indicated that after 15d the structure of straw was destroyed and did not float longer, which did not affect crop's rootage; furthermore the effect on the next crop's growth was studied, and the results shown that the microbial agent could accelerate the next crop's growth. The strain B3 secreted incretion IAA and ABA by TLC and immunological analysis.
本论文包括三个部分：1．调查了中科院红壤生态站内不同土壤中的微生物数量及区系组成，跟踪检测秸秆自然降解状况。结果表明，水田以细菌为主，旱地中真菌和放线菌数量最多，林地中有大量的高等担子菌；秸秆自然降解较慢，土壤微生物也随着降解的进行而呈现一定的变化规律；当调节C/N比为25：1时，秸秆的腐解残留率比对照组下降10.67％。2．通过形态学观察及测定其rDNA ITS序列对菌株B3进行鉴定，该菌株属于子囊菌亚门的腐皮壳属(Diaporthe)。纤维素类物质CMC-Na是其最佳碳源诱导物，正交优化后的发酵条件是马铃薯200g/L,CMC-Na 20g/L，NH_4Cl 1g/L,KH_2PO_43g/L,MgSO_4·7H_2O 1.5g/L,VB_10.01g/L,pH4.0，培养温度25℃；同时发现菌株B3分泌的漆酶有较好的耐热性，在酸性条件下能很好的生长，可以适应红壤酸性条件，因此根据其最佳发酵条件在7L发酵罐内发酵制成秸秆降解剂。3．通过盆栽试验和扫描电镜观察检测了秸秆降解剂对秸秆的降解效果，结果发现，该降解剂在15d内就能破坏秸秆的结构，使秸秆不再漂浮，不影响作物的生根；进一步检测其对后季作物生长的影响发现，该降解剂还能促进后季作物的生长，薄板层析和免疫学检测均显示菌株B3分泌了IAA和ABA。
Many studies have shown that lignin was the limited factor of straw degradation. In this study we screened a strain named B3 from the stem's inner bark of Bischofia polycarpam Airy Shaw which yielded high level of ligninase. The strain B3's ability in straw degradation and its effect on the next season crop's growth were then studied by making it into microbial agent in the potted experiment. Brief results are followed:
1. The changes of microorganisms' number in different kind of Red Soil from CAS Ecological Experiment Station were surveyed. The results showed in the paddy field the bacteria were predominated, in the dry land the proportion of fungi and actinomyces was distinctly higher than the other two kinds of soil, in the woodland abundant species of basidiomycetes were found; the natural degradation speed was low, with the degradation the number and composition of microorganisms changed regularly; when the C / N ratio was adjusted to
25 '?1, the degradation remnant ratio reduced 10.67% than the control.
2. By morphological and molecular methods the strain B3 was identified as Diaporthe and could exist under acidic condition, whose laccase had good thermostability. CMC-Na could induce laccase maximally. The composition of fermentation medium was potato 200g / L, CMC-Na 20g / L, NH4Cllg / L, KH2PO4 3g / L, MgSO4 7H2O 1.5g / L, VB10.01g / L, mixed solution of trace metals 7ml / L, and the optimal conditions were pH of medium was 4.0, and incubation with 150r / min at 25 C for 4d. According to that it was made into microbial agent in 7 liter bioreactor.
3. The microbial agent's ability in straw degradation was detected by potted experiment and scanning electron microscope. The result indicated that after 15d the structure of straw was destroyed and did not float longer, which did not affect crop's rootage; furthermore the effect on the next crop's growth was studied, and the results shown that the microbial agent could accelerate the next crop's growth. The strain B3 secreted incretion IAA and ABA by TLC and immunological analysis.
引文
[1] 李忠佩，程励励，林心雄．红壤有机质的分解、积累与平衡．红壤生态系统研究(第六集)[M]．江西：江西科学技术出版社，2001．36～51
[2] 曹玉凤、李建国．生物技术在处理农作物秸杆饲料中的应用．饲料研究，1999，(1)：25～26．
[3] 段佐亮．我国作物秸秆燃烧甲烷、氧化亚氮排放量变化趋势预测(1900—2020)．农业环境保护，1995，14(3)：111～116．
[4] 范秀莲，曲英杰．秸秆还田与可持续化发展[J]．农机化研究，2002，(2)：172．
[5] 王玉松，杨育文．秸秆还田 提高生物质资源利用率[J]．农村能源，1999，88(6)：20～21．
[6] 曾木祥，王蓉芳，彭世琪等．我国主要农区秸秆还田试验总结[J]．土壤通报，2002，33(5)：336～339．
[7] Jiang Y, Tian S Z, Zhao L P, Effect of the 14~(th) International Congress of Soil Science.Vol..Ⅱ, 1990,420～421,Kyoto,Japan.
[8] 赵兰坡、姜岩．施用有机物料对土壤酶活性的影响Ⅰ有机物料对酶活性的效应．吉林农业大学学
报，1987，9(4)：43～50．
[9] 窦森、陈恩凤，须湘成．有机物料对土壤胡敏酸的光学性质．土壤学报，1995，32(1)：41～49．
[10] 吴景贵、席时权、姜岩．玉米秸秆腐解过程的红外光谱研究．土壤学报，1999，36(1)．
[11] 陈文新．土壤和环境微生物学．北京农业大学出版社，91～94，1990．
[12] 史玉英、沈其荣．纤维素分解菌群的分离和筛选．南京农业大学学报，1996，19(3)：59～62．
[13] 赵小蓉、林启美、孙炎鑫等．纤维素分解菌对不同纤维素类物质的分解作用．微生物学杂志，2000，20(3)：12～14．
[14] 陈叶福，郭雪娜，王正祥等．木质素生物降解与纸浆工业废水脱色[J]．工业微生物，2001，31(4)：49～53．
[15] Eaton D.C. et al. Decolorization of kraft bleach plant effluents. Tappi J., 1980, 63: 103～106.
[16] Bharrti S.G. et al. Role of lignin from paper mill waste waters. Bull. Environ. Contamin. Toxicol., 1992,49: 738～742.
[17] Falcon M.A. et al. Isolation of microorganisms with lignin transformation potential from soil of Tenerife island. Soil Biol. Biochem., 1995, 27: 121～126.
[18] Buswell J.A. and Odier E. Lignin biodegradation. CRC Crit. Rev. Biotechnol., 1987, 6: 1～60.
[19] Slávīková Elena et al. Modification of lignin by Geotrichum klebahnīī., World J. of Microbiol.Biotechnol., 2001, 17:1～3.
[20] 王宜磊，孙迅，邓振旭．木素生物降解研究进展[J]．微生物学杂志，1998，18(1)：48～51．
[21] Thurston C.F. The structure and function of function of fungal laccases. Microbiology, 1994, 140:19～21.
[22] Palaez F. et al. Screening of 68 species of basidiomycetes for enzymes involved in lignin degradation. Mycol. Res., 1995, 99: 37～42.
[23] In-Young Lee et al. Enhanced production of laccase in Trametes vesicolor by the addition of ethanol. Biotechnol. Lett., 1999, 21: 965～968.
[24] Annia I. Ruiz et al. Improved activity and stability of an immobilized recombinant laccase in organic solvents. Biotechnol. Lett., 2000, 22: 229～233.
[25] 钞亚鹏，钱世钧．真菌漆酶及其应用[J]．生物工程进展，2001，21(5)：23～27．
[26] M Smith, Thurston C.F. and Wood D.A., Fungal laccase: Role in delignification and possible industrial application. In: Multi-copper Oxidases.
[27] Pratima Bajpai. Biotechnol. Prog, 1999, 15: 147～157.
[28] 孙波，赵其国．红壤退化中的土壤质量评价指标及评价方法[J]．地理科学进展，1999，18(2)：
118～128．
[29] 路季梅，刘洪顺．江南红壤丘陵区农业气候特点与作物生产的气候相宜性[J]．南京农业大学学报，1999，22(2)：15～20．
[30] 陈国潮，何振立．红壤不同利用方式下微生物量的研究[J]．土壤通报，1998，29(6)：276-278．
[31] 陶水龙，林启美，赵小蓉．土壤微生物量方法研究进展[J]．土壤肥料，1998，(5)：15-18．
[32] 任天志，Stefano Grego．持续农业中的土壤生物指标研究[J]．中国农业科学，2000，33(1)：68～75．
[33] Doran J.W. et al., Defining soil quality for sustainable environment[A]. Soil Society of America Special Publication[M]. No.35, Madison, Wisconsin, 1994, 3～234.
[34] Humpheg AE. The hydlolysis of cellulosic matelialo to uletrl products, Adv. Chem sel, 1979(181) :25～53.
[35] Wood TM. & Maccpae SI. In bioconversion of cellulose substance into energy, chemicals and microbial protein, 1978, Edited by T. K. Ghose Gall, India, 111～141.
[36] 须湘成、张继宏．有机物料在不同土壤中腐解残留率的研究．土壤通报，1985，16(1)：21～26．
[37] 刑来君、李明春．普通真菌学．高等教育出版社，208～214，1999．
[38] 杨林书、阎成．秸秆资源化考略．农业环境保护，1993，12(6)：271～273，283．
[39] 方靖、高培基．纤维二糖脱氢酶在纤维素降解中的作用研究．微生物学通报，2000，27(1)：15～18．
[40] 李泓泉．微生物处理秸秆的进展．饲料博览，1992，46(2)：46．
[41] 赵守贤．用生物技术开发秸秆资源．饲料工业，1997(2)：34～36．
[42] 陈坚．环境生物技术．中国轻工业出版社，256～257，1999．
[2] 曹玉凤、李建国．生物技术在处理农作物秸杆饲料中的应用．饲料研究，1999，(1)：25～26．
[3] 段佐亮．我国作物秸秆燃烧甲烷、氧化亚氮排放量变化趋势预测(1900—2020)．农业环境保护，1995，14(3)：111～116．
[4] 范秀莲，曲英杰．秸秆还田与可持续化发展[J]．农机化研究，2002，(2)：172．
[5] 王玉松，杨育文．秸秆还田 提高生物质资源利用率[J]．农村能源，1999，88(6)：20～21．
[6] 曾木祥，王蓉芳，彭世琪等．我国主要农区秸秆还田试验总结[J]．土壤通报，2002，33(5)：336～339．
[7] Jiang Y, Tian S Z, Zhao L P, Effect of the 14~(th) International Congress of Soil Science.Vol..Ⅱ, 1990,420～421,Kyoto,Japan.
[8] 赵兰坡、姜岩．施用有机物料对土壤酶活性的影响Ⅰ有机物料对酶活性的效应．吉林农业大学学
报，1987，9(4)：43～50．
[9] 窦森、陈恩凤，须湘成．有机物料对土壤胡敏酸的光学性质．土壤学报，1995，32(1)：41～49．
[10] 吴景贵、席时权、姜岩．玉米秸秆腐解过程的红外光谱研究．土壤学报，1999，36(1)．
[11] 陈文新．土壤和环境微生物学．北京农业大学出版社，91～94，1990．
[12] 史玉英、沈其荣．纤维素分解菌群的分离和筛选．南京农业大学学报，1996，19(3)：59～62．
[13] 赵小蓉、林启美、孙炎鑫等．纤维素分解菌对不同纤维素类物质的分解作用．微生物学杂志，2000，20(3)：12～14．
[14] 陈叶福，郭雪娜，王正祥等．木质素生物降解与纸浆工业废水脱色[J]．工业微生物，2001，31(4)：49～53．
[15] Eaton D.C. et al. Decolorization of kraft bleach plant effluents. Tappi J., 1980, 63: 103～106.
[16] Bharrti S.G. et al. Role of lignin from paper mill waste waters. Bull. Environ. Contamin. Toxicol., 1992,49: 738～742.
[17] Falcon M.A. et al. Isolation of microorganisms with lignin transformation potential from soil of Tenerife island. Soil Biol. Biochem., 1995, 27: 121～126.
[18] Buswell J.A. and Odier E. Lignin biodegradation. CRC Crit. Rev. Biotechnol., 1987, 6: 1～60.
[19] Slávīková Elena et al. Modification of lignin by Geotrichum klebahnīī., World J. of Microbiol.Biotechnol., 2001, 17:1～3.
[20] 王宜磊，孙迅，邓振旭．木素生物降解研究进展[J]．微生物学杂志，1998，18(1)：48～51．
[21] Thurston C.F. The structure and function of function of fungal laccases. Microbiology, 1994, 140:19～21.
[22] Palaez F. et al. Screening of 68 species of basidiomycetes for enzymes involved in lignin degradation. Mycol. Res., 1995, 99: 37～42.
[23] In-Young Lee et al. Enhanced production of laccase in Trametes vesicolor by the addition of ethanol. Biotechnol. Lett., 1999, 21: 965～968.
[24] Annia I. Ruiz et al. Improved activity and stability of an immobilized recombinant laccase in organic solvents. Biotechnol. Lett., 2000, 22: 229～233.
[25] 钞亚鹏，钱世钧．真菌漆酶及其应用[J]．生物工程进展，2001，21(5)：23～27．
[26] M Smith, Thurston C.F. and Wood D.A., Fungal laccase: Role in delignification and possible industrial application. In: Multi-copper Oxidases.
[27] Pratima Bajpai. Biotechnol. Prog, 1999, 15: 147～157.
[28] 孙波，赵其国．红壤退化中的土壤质量评价指标及评价方法[J]．地理科学进展，1999，18(2)：
118～128．
[29] 路季梅，刘洪顺．江南红壤丘陵区农业气候特点与作物生产的气候相宜性[J]．南京农业大学学报，1999，22(2)：15～20．
[30] 陈国潮，何振立．红壤不同利用方式下微生物量的研究[J]．土壤通报，1998，29(6)：276-278．
[31] 陶水龙，林启美，赵小蓉．土壤微生物量方法研究进展[J]．土壤肥料，1998，(5)：15-18．
[32] 任天志，Stefano Grego．持续农业中的土壤生物指标研究[J]．中国农业科学，2000，33(1)：68～75．
[33] Doran J.W. et al., Defining soil quality for sustainable environment[A]. Soil Society of America Special Publication[M]. No.35, Madison, Wisconsin, 1994, 3～234.
[34] Humpheg AE. The hydlolysis of cellulosic matelialo to uletrl products, Adv. Chem sel, 1979(181) :25～53.
[35] Wood TM. & Maccpae SI. In bioconversion of cellulose substance into energy, chemicals and microbial protein, 1978, Edited by T. K. Ghose Gall, India, 111～141.
[36] 须湘成、张继宏．有机物料在不同土壤中腐解残留率的研究．土壤通报，1985，16(1)：21～26．
[37] 刑来君、李明春．普通真菌学．高等教育出版社，208～214，1999．
[38] 杨林书、阎成．秸秆资源化考略．农业环境保护，1993，12(6)：271～273，283．
[39] 方靖、高培基．纤维二糖脱氢酶在纤维素降解中的作用研究．微生物学通报，2000，27(1)：15～18．
[40] 李泓泉．微生物处理秸秆的进展．饲料博览，1992，46(2)：46．
[41] 赵守贤．用生物技术开发秸秆资源．饲料工业，1997(2)：34～36．
[42] 陈坚．环境生物技术．中国轻工业出版社，256～257，1999．