木质素磺酸盐好氧降解菌的筛选及降解特性
|
摘要
木质素是结构复杂的难降解有机物,研究其微生物降解机制对解决制浆造纸黑液处理问题,促进木质纤维素的再生利用具有重要意义.从制浆造纸厂活性污泥样本中分离出5株以木质素磺酸盐为唯一碳源和能源的好氧细菌.选取降解性能优异的菌株,考察温度、pH、碳氮比对该菌株生长和降解效果的影响,并对降解后木质素的官能团、元素组成变化和降解产物进行分析,推测木质素降解机制.结果显示:5株细菌均可有效降解木质素,其中菌株Sphingobacterium sp.HY-H的降解能力最强.通过正交试验确定菌株HY-H的最佳处理条件为温度30℃,pH=7.0,碳氮比5:1.在此条件下,菌株HY-H对木质素磺酸盐(1g L~(-1))的5 d降解率为30.6%.降解后木质素的酚羟基和甲氧基含量减少,羰基和羧基含量增加.分析木质素官能团变化及其降解产物认为,木质素降解过程中主要发生了α羟基氧化、β-O-4键断裂、脱甲基及脱羧作用,最终生成CO_2.本研究表明,菌株HY-H具有较好的木质素磺酸盐降解能力,可用于制浆造纸废水的生物处理.
Lignin is a refractory organic compound with complex structure. For promoting the recycling of lignocellulose, we need to study the microbial degradation mechanism of lignin in mitigating pollution caused by pulp and paper-making black liqour. In this research five aerobic bacterial strains utilizing lignosulfonate as a sole carbon and energy source were isolated from activated sludge in a pulp and paper mill. A strain with better degradation efficiency was then selected to investigate the effect of t emperature, pH and C/N ratio on its growth and degradation performance. Moreover, changes of functional groups and elemental compositions in residual lignosulfonate and the metabolic products were analyzed to speculate the degradation mechanisms. The results showed that all five bacterial strains were capable of degrading lignosulfonate, with Sphingobacterium sp. HY-H showing the best performance. The optimal conditions for strain HY-H were determined by Taguchi's orthogonal matrix method to be initial pH of 7.0, temperature of 30 ℃, and C/N ratio of 5:1. Under these conditions, the lignosulfonate degradation capacity of strain HY-H(ini tial lignosulfonate concentration of 1 g L~(-1)) could reach 30.6% after 5 days. During the process, contents of phenolic hydroxyl and methoxyl groups decreased, but those of carbonyl, carboxyl and sulfonate groups increased. The results indicated that lignosulfonate is degraded to carbon dioxide via oxidation of α-hydroxyl groups, cleavage reaction of β-O-4, demethylation and decarboxylation. The strain HY-H coul d effectively degrade lignosulfonate, thus having a great application prospect for the bio treatment of pulp and paper-making wastewater.
Lignin is a refractory organic compound with complex structure. For promoting the recycling of lignocellulose, we need to study the microbial degradation mechanism of lignin in mitigating pollution caused by pulp and paper-making black liqour. In this research five aerobic bacterial strains utilizing lignosulfonate as a sole carbon and energy source were isolated from activated sludge in a pulp and paper mill. A strain with better degradation efficiency was then selected to investigate the effect of t emperature, pH and C/N ratio on its growth and degradation performance. Moreover, changes of functional groups and elemental compositions in residual lignosulfonate and the metabolic products were analyzed to speculate the degradation mechanisms. The results showed that all five bacterial strains were capable of degrading lignosulfonate, with Sphingobacterium sp. HY-H showing the best performance. The optimal conditions for strain HY-H were determined by Taguchi's orthogonal matrix method to be initial pH of 7.0, temperature of 30 ℃, and C/N ratio of 5:1. Under these conditions, the lignosulfonate degradation capacity of strain HY-H(ini tial lignosulfonate concentration of 1 g L~(-1)) could reach 30.6% after 5 days. During the process, contents of phenolic hydroxyl and methoxyl groups decreased, but those of carbonyl, carboxyl and sulfonate groups increased. The results indicated that lignosulfonate is degraded to carbon dioxide via oxidation of α-hydroxyl groups, cleavage reaction of β-O-4, demethylation and decarboxylation. The strain HY-H coul d effectively degrade lignosulfonate, thus having a great application prospect for the bio treatment of pulp and paper-making wastewater.
引文
1李科,靳艳玲,甘明哲,刘晓风.赵海.木质纤维素生产燃料乙醇的关键技术研究现状[J].应用与环境生物学报,2008,14(6):877-884[Li K,Jin YL,Gan MZ,Liu XF,Zhao H.Progress in research of key techniques for ethanol production from lignocellulose[J].Chin J Appl Environ Biol,2008,14(6):877-884]
2 Boerjan W,Ralph J,Baucher M.Lignin biosynthesis[J].Ann Rev Plant Physiol Mol Biol,2003,54:519-546
3苏维丰,柴立元,王云燕.造纸黑液综合治理的研究进展[J].工业水处理,2004,24(11):4-8[Su WF,Chai LY,Wang YY Research progress on comprehensive treatment of black licuor[J].Ind Water Treatment,2004,24(11):4-8]
4孙宪昀,曲音波,刘自勇.青霉木质纤维素降解酶系研究进展[J].应用与环境生物学报,2007,13(5):736-740[Sun XY,Qu YB,Liu ZY.Progress in research of lignocellulose-degrading enzymes from Penicillium[J].Chin J Appl Environ Biol,2007,13(5):736-740]
5 Bruce A,Palfreyman JW.Forest Products Biotechnology[M].London:Taylor&Francis,1998
6 Bugg TDH,Ahmad M,Hardiman EM,Rahmanpour R.Pathways for degradation of lignin in bacteria and fungi[J].Nat Prod Rep,2011,28(12):1883-1896
7蒋荣清,袁兴中,曾光明,黄红丽,喻曼,梁运姗,张嘉超.一组高效木质素降解复合菌的筛选[J].应用与环境生物学报,2010,16(2):247-251[Jiang RQ,Yuan XZ,Zeng GM,Huang HL,Yu M,Liang YS,Zhang JC.Screening of an efficient lignin-degrading composite microorganisms[J].Chin J Appl Environ Biol,2010,16(2):247-251]
8 Chandra R,Raj A,Purohit HJ,Kapley A.Characterisation and optimisation of three potential aerobic bacterial strains for kraft lignin degradation from pulp paper waste[J].Chemosphere,2007,67(4):839-846
9 Chen Y,Chai L,Tang C,Yang Z,Zheng Y,Shi Y,Zhang H.Kraft lignin biodegradation by Novosphingobium sp.B-7 and analysis of the degradation process[J].Bioresour Technol,2012,123:682-685
10 Odier E,Janin G,Monties B.Poplar lignin decomposition by gramnegative aerobic-bacteria[J].Appl Environ Microbiol,1981,41(2):337-341
11 Ahmad M,Taylor CR,Pink D,Burton K,Eastwood D,Bending GD,Bugg TDH.Development of novel assays for lignin degradation:comparative analysis of bacterial and fungal lignin degraders[J].Mol Biosyst,2010,6(5):815-821
12张晓琰,彭学,政井英司.木质素芳香族化合物降解菌Sphingobium sp.SYK-6的研究进展[J].微生物学报,2014,54(8):854-867[Zhang XY,Peng X,Masai E.Recent advances in Sphingobium sp.SYK-6 for lignin aromatic compounds degradation-a review[J].Acta Microbiol Sin,2014,54(8):854-867]
13 Liu Z,Liang J,Du W,Wang D,He Y.Study on the aerobic biodegradation of lignin by activated sludge reactor[C]//International Symposium on Water Resource and Environmental Protection,Xi'an,2011:1999-2002
14 Lin SY,Dence CW.Methods in Lignin Chemistry[M].Berlin:Springer,1992
15 Song YJ.Characterization of aromatic hydrocarbon degrading bacteria isolated from pine litter[J].Korean J Microbiol Biotechnol,2009,37(4):333-339
16 Zhang C,Zeng G,Yuan L,Yu J,Li J,Huang G,Xi B,Liu H.Aerobic degradation of bisphenol A by Achromobacter xylosoxidans strain B-16 isolated from compost leachate of municipal solid waste[J].Chemosphere,2007,68(1):181-190
17高永超,王加宁,孔学,陈贯虹,邱维忠,迟建国.石油降解菌多食鞘氨醇杆菌的发酵条件优化[J].生物技术,2009,19(1):74-77[Gao YC,Wang JN,Kong X,Chen GH,Qiu WZ,Chi JG.Optimization of the liquid fermentation condition of the petroleum degradation bacteria Sphingobacterium multivorum[J].Biotechnology,2009,19(1):74-77]
18刘和,沈超峰,王侃,陈英旭.多食鞘氨醇杆菌共代谢降解五氯酚[J].中国环境科学,2004,24(3):294-298[Liu H,Shen CF,Wang K,Chen YX.Cometabolic degradation of pentachlorophenol by Sphingobacterium multivorum[J].China Environ Sci,2004,24(3):294-298]
19 Kratzl K,Claus P,Lonsky W,Gratzl JS.Model studies on reactions occurring in oxidations of lignin with molecular oxygen in alkaline media[J].Wood Sci Technol,1974,8(1):35-49
20 Jokela J,Pellinen J,Salkinojasalonen M.Initial steps in the pathway for bacterial degradation of two tetrameric lignin model compounds[J].Appl Environ Microbiol,1987,53(11):2642-2649
21 Prim N,Pastor FIJ,Diaz P.Biochemical studies on cloned Bacillus sp.BP-7 phenolic acid decarboxylase PadA[J].Appl Microbiol Biotechnol,2003,63(1):51-56
22 Bugg TDH,Winfield CJ.Enzymatic cleavage of aromatic rings:mechanistic aspects of the catechol dioxygenases and later enzymes of bacterial oxidative cleavage pathways[J].Nat Prod Rep,1998,15(5):513-530
23 Masai E,Katayama Y,Fukuda M.Genetic and biochemical investigations on bacterial catabolic pathways for lignin-derived aromatic compounds[J].Biosci Biotechnol Biochem,2007,71(1):1-15
24 Camarero S,Ibarra D,Martinez MJ,Martinez AT.Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes[J].Appl Environ Microbiol,2005,71(4):1775-1784
2 Boerjan W,Ralph J,Baucher M.Lignin biosynthesis[J].Ann Rev Plant Physiol Mol Biol,2003,54:519-546
3苏维丰,柴立元,王云燕.造纸黑液综合治理的研究进展[J].工业水处理,2004,24(11):4-8[Su WF,Chai LY,Wang YY Research progress on comprehensive treatment of black licuor[J].Ind Water Treatment,2004,24(11):4-8]
4孙宪昀,曲音波,刘自勇.青霉木质纤维素降解酶系研究进展[J].应用与环境生物学报,2007,13(5):736-740[Sun XY,Qu YB,Liu ZY.Progress in research of lignocellulose-degrading enzymes from Penicillium[J].Chin J Appl Environ Biol,2007,13(5):736-740]
5 Bruce A,Palfreyman JW.Forest Products Biotechnology[M].London:Taylor&Francis,1998
6 Bugg TDH,Ahmad M,Hardiman EM,Rahmanpour R.Pathways for degradation of lignin in bacteria and fungi[J].Nat Prod Rep,2011,28(12):1883-1896
7蒋荣清,袁兴中,曾光明,黄红丽,喻曼,梁运姗,张嘉超.一组高效木质素降解复合菌的筛选[J].应用与环境生物学报,2010,16(2):247-251[Jiang RQ,Yuan XZ,Zeng GM,Huang HL,Yu M,Liang YS,Zhang JC.Screening of an efficient lignin-degrading composite microorganisms[J].Chin J Appl Environ Biol,2010,16(2):247-251]
8 Chandra R,Raj A,Purohit HJ,Kapley A.Characterisation and optimisation of three potential aerobic bacterial strains for kraft lignin degradation from pulp paper waste[J].Chemosphere,2007,67(4):839-846
9 Chen Y,Chai L,Tang C,Yang Z,Zheng Y,Shi Y,Zhang H.Kraft lignin biodegradation by Novosphingobium sp.B-7 and analysis of the degradation process[J].Bioresour Technol,2012,123:682-685
10 Odier E,Janin G,Monties B.Poplar lignin decomposition by gramnegative aerobic-bacteria[J].Appl Environ Microbiol,1981,41(2):337-341
11 Ahmad M,Taylor CR,Pink D,Burton K,Eastwood D,Bending GD,Bugg TDH.Development of novel assays for lignin degradation:comparative analysis of bacterial and fungal lignin degraders[J].Mol Biosyst,2010,6(5):815-821
12张晓琰,彭学,政井英司.木质素芳香族化合物降解菌Sphingobium sp.SYK-6的研究进展[J].微生物学报,2014,54(8):854-867[Zhang XY,Peng X,Masai E.Recent advances in Sphingobium sp.SYK-6 for lignin aromatic compounds degradation-a review[J].Acta Microbiol Sin,2014,54(8):854-867]
13 Liu Z,Liang J,Du W,Wang D,He Y.Study on the aerobic biodegradation of lignin by activated sludge reactor[C]//International Symposium on Water Resource and Environmental Protection,Xi'an,2011:1999-2002
14 Lin SY,Dence CW.Methods in Lignin Chemistry[M].Berlin:Springer,1992
15 Song YJ.Characterization of aromatic hydrocarbon degrading bacteria isolated from pine litter[J].Korean J Microbiol Biotechnol,2009,37(4):333-339
16 Zhang C,Zeng G,Yuan L,Yu J,Li J,Huang G,Xi B,Liu H.Aerobic degradation of bisphenol A by Achromobacter xylosoxidans strain B-16 isolated from compost leachate of municipal solid waste[J].Chemosphere,2007,68(1):181-190
17高永超,王加宁,孔学,陈贯虹,邱维忠,迟建国.石油降解菌多食鞘氨醇杆菌的发酵条件优化[J].生物技术,2009,19(1):74-77[Gao YC,Wang JN,Kong X,Chen GH,Qiu WZ,Chi JG.Optimization of the liquid fermentation condition of the petroleum degradation bacteria Sphingobacterium multivorum[J].Biotechnology,2009,19(1):74-77]
18刘和,沈超峰,王侃,陈英旭.多食鞘氨醇杆菌共代谢降解五氯酚[J].中国环境科学,2004,24(3):294-298[Liu H,Shen CF,Wang K,Chen YX.Cometabolic degradation of pentachlorophenol by Sphingobacterium multivorum[J].China Environ Sci,2004,24(3):294-298]
19 Kratzl K,Claus P,Lonsky W,Gratzl JS.Model studies on reactions occurring in oxidations of lignin with molecular oxygen in alkaline media[J].Wood Sci Technol,1974,8(1):35-49
20 Jokela J,Pellinen J,Salkinojasalonen M.Initial steps in the pathway for bacterial degradation of two tetrameric lignin model compounds[J].Appl Environ Microbiol,1987,53(11):2642-2649
21 Prim N,Pastor FIJ,Diaz P.Biochemical studies on cloned Bacillus sp.BP-7 phenolic acid decarboxylase PadA[J].Appl Microbiol Biotechnol,2003,63(1):51-56
22 Bugg TDH,Winfield CJ.Enzymatic cleavage of aromatic rings:mechanistic aspects of the catechol dioxygenases and later enzymes of bacterial oxidative cleavage pathways[J].Nat Prod Rep,1998,15(5):513-530
23 Masai E,Katayama Y,Fukuda M.Genetic and biochemical investigations on bacterial catabolic pathways for lignin-derived aromatic compounds[J].Biosci Biotechnol Biochem,2007,71(1):1-15
24 Camarero S,Ibarra D,Martinez MJ,Martinez AT.Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes[J].Appl Environ Microbiol,2005,71(4):1775-1784