固碳微生物菌株的分离鉴定及其固碳能力测定
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摘要
利用微生物回收和固定CO2气体的生物固碳方法,成为解决"温室效应"这一重大环境问题的焦点,本研究目的是分离筛选固碳微生物菌株。利用无碳源无机培养基从活性污泥、沼液和设施土壤中分离筛选出以CO2为碳源的菌株24株,选取其中生长较快的8株菌进行cbbL基因、形态观察、生理生化反应测定和16S rDNA测序分析,将测序结果在BlAST数据库中比对,进行固碳菌株的分子鉴定,并对其菌体含量和RubisCO酶活性进行比较。选取RubisCO酶活性最高的菌株C2-8R,进行土壤施用试验,通过土壤RubisCO酶活性的测定,确定分离筛选的固碳菌的固碳能力。研究表明,可通过无碳源培养基进行固碳微生物菌株的筛选,筛选的固碳菌分别隶属于假单胞菌属和嗜甲基菌属,并可通过RubisCO酶活性来反映微生物的固碳能力。
Currently,as a biological carbon sequestration strategy,microbial recovery and fixing of CO2 gas,has become the focusof interest in dealing with the major environmental problem of greenhouse effect. The purpose of this study is to separate and screen efficientcarbon-fixing microbial strains. By using the carbon-free source inorganic medium,24 kinds of strains with CO_2 as carbon source wereseparated from facilities soil,activated sludge and biogas slurry. Then,the 8 strains of bacteria that grew faster were selected to conduct theirmorphological observations,physiological and biochemical responses as well as to perform cbbL gene and 16 S rDNA sequencing analysis.Further,the sequencing results were compared with the data in BlAST database and the molecular characterization of the 8 carbon-fixing strainswas carried out along with the detection of the strain content and the RubisCO enzyme activity. Finally,the C2-8R with the highest RubisCOactivity among the 8 strains was selected for soil application test,and the carbon sequestration capacity of the C2-8R was determined throughthe detection of the RubisCO enzyme activity in soil. The above results revealed that carbon sequestration strains can be screened throughcarbon-free medium. The screened carbon-fixing bacteria are subordinate to the strains of Pseudomonas sp. and Methanotrophs sp.,and can beused to indicate the microbial carbon-fixing ability through RubisCO enzyme activity.
Currently,as a biological carbon sequestration strategy,microbial recovery and fixing of CO2 gas,has become the focusof interest in dealing with the major environmental problem of greenhouse effect. The purpose of this study is to separate and screen efficientcarbon-fixing microbial strains. By using the carbon-free source inorganic medium,24 kinds of strains with CO_2 as carbon source wereseparated from facilities soil,activated sludge and biogas slurry. Then,the 8 strains of bacteria that grew faster were selected to conduct theirmorphological observations,physiological and biochemical responses as well as to perform cbbL gene and 16 S rDNA sequencing analysis.Further,the sequencing results were compared with the data in BlAST database and the molecular characterization of the 8 carbon-fixing strainswas carried out along with the detection of the strain content and the RubisCO enzyme activity. Finally,the C2-8R with the highest RubisCOactivity among the 8 strains was selected for soil application test,and the carbon sequestration capacity of the C2-8R was determined throughthe detection of the RubisCO enzyme activity in soil. The above results revealed that carbon sequestration strains can be screened throughcarbon-free medium. The screened carbon-fixing bacteria are subordinate to the strains of Pseudomonas sp. and Methanotrophs sp.,and can beused to indicate the microbial carbon-fixing ability through RubisCO enzyme activity.
引文
[1]Adams JM, Piovesan G. Uncertainties in the role of land vegetationin the carbon cycle[J]. Chemosphere, 2002, 49:805-819.
[2]Oexmann J, Hensel C, Kather A. Post-combustion CO-capture fromcoal-fired power plants:Preliminary evaluation of an integratedchemical absorption process with piperazine-promoted potassiumcarbonate[J]. International Journal of Greenhouse Gas Control,2008, 2(4):539-552.
[3]Rodríguez N, Alonso M, Grasa G. Process for capturing CO2 arisingfrom the calcination of the CaCO3 used in cement manufacture[J].Environ Sci Technol, 2008, 42(18):69-80.
[4]张炜,李义连.二氧化碳储存技术的研究现状和展望[J].环境污染与防治, 2006, 28(12):950-953.
[5]Grntzis T. Susurface sequestration of carbon dioxide-an overviewfrom an Alberta(Canada)perspective[J]. International Journalof Coal Geology, 2000, 43(1/4):287-305.
[6]Freund P, Ormerod WG. Progress toward storage of carbondioxide[J]. Energy Conversion and Management, 1997, 38(S1):199-204.
[7]Lal R. Carbon sequestration[J]. Philosophical Transactions of theRoyal Society B, 2008, 363(4):815-830.
[8]Dixon RK, Brown S, Houghton RA, et al. Carbon pools and flux ofglobal forest ecosystems[J]. Science, 1994, 263(14):185-190.
[9]Masakazu M, Masahiro I. The biological CO2 fixation and utilizationproject by rite(2)-screening and breeding of microalgae with highcapability in fixing CO2[J]. Energy Conversion and Management,1997, 38(5):493-497.
[10]Grimston MC, Karakoussis V, Fouquet R, et al. The European andglobal potential of carbon dioxide sequestration in tackling climatechange[J]. Climate Policy, 2001, 1(2):155-171.
[11]Miltner A, Kopinke FD, Kindler R. Non-phototrophic CO2 fixationby soil microorganisms[J]. Plant and Soil, 2005, 269(1-2):193-203.
[12]Shively JM, English RS, Baker SH, et al. Carbon cycling:theprokaryotic contribution[J]. Current Opinion in Microbiology,2001, 4:301-306.
[13]Richarda KR. A brief overview of carbon sequestration economicsand policy[J]. Environmental Management, 2004, 33(4):545-558.
[14]袁红朝,秦红灵,刘守龙,等.长期施肥对稻田土壤固碳功能菌群落结构和数量的影响[J].生态学报, 2012, 32(1):0183-0189.
[15]胡佳俊,王磊,李艳丽,等.非光合CO2同化微生物菌群的选育/优化及其群落结构分析[J].环境科学, 2009, 30(8):2438-2444.
[16]Xu HH, Abita FR. Ribulose-1, 5-bisphosphatecarboxylase/oxygenasegene expression and diversity of Lake Eric planktonicmicroorganisms[J]. Appl Environ Microbiol, 1996, 62:1913-1921.
[17]Nanba K, King GM, Dunfield K. Analysis of facultative lithotrophicdistribution and diversity on volcanic deposits by use of the largesubunit of ribulose-1, 5-bisphosphatecarboxylase/oxygenase[J].Appl Environ Microbiol, 2004, 70:2245-2253.
[18]Elsaied H, Naganuma T. Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes fromdeep-sea microorganisms[J]. Appl Environ Microbiol, 2001,67:1751-1765.
[19]周盛,韦彬勤,张琼,等.一种能同时固定CO2和N2的微生物——兼性固CO2、N2菌的分离鉴定及其验证实验[J].环境科学学报, 2013, 33(4):1043-1050.
[20]吴小红,简燕,陈晓娟,等.自养微生物同化CO2的分子生态研究及同化碳在土壤中的转化[J].生态学报, 2014, 34(3):701-709.
[21]王竞,周集体,张晶晶,等.固定CO2氢细菌的筛选及其培养条件优化[J].应用与环境生物学报, 2000, 6(3):271-275.
[22]范秀容,李广武,沈萍.微生物学实验[M].第2版.北京:高等教育出版社, 1995:107-112.
[23]TolliJ,KingGM.Diversityandstructureofbacterialchemoautotrophic communities in pine forest and agroecosystemsoils[J]. Applied&Environmental Microbiology, 2005, 71(12):8411-8418.
[24]布坎南RE,等.伯杰氏细菌鉴定手册[M].第8版.北京:科学出版社, 1984:729-797.
[25]东秀珠,蔡妙英.一般细菌分类手册[M].北京:科学出版社,2001:145-155.
[26]Follett RF. Soil management concepts and carbon sequestration incropland soils[J]. Soils and Tillage Research, 2001, 61(1/2):77-92.
[27]Smith P. Carbon sequestration in croplands:the potentialin Europe and the global context[J]. European Jourmal ofAgronomy, 2004, 20(3):229-236.
[28]Yang HQ, Xu ZH, Fan MH. Progress in carbon dioxide separationand capture:A review[J]. Journal of Environmental Sciences,2008, 20:14-27.
[29]李娟,赵秉强,李秀英,等.长期不同施肥制度下几种土壤微生物学特征变化[J].植物生态学报, 2008, 32(4):891-899.
[30]Boyle NR, Morgan JA. Computation of metabolic fluxes andefficiencies for biological carbon dioxide fixation[J]. MetabolicEngineering, 2011, 13(2):150-158.
[31]袁红朝,秦红灵,刘守龙,等.固碳微生物分子生态学研究[J].中国农业科学, 2011, 44(14):2951-2958.
[32]徐敏,刘国祥,胡征宇.耐受极高浓度CO2藻类的研究及其在固碳领域的应用[J].中国科学院研究生院学报, 2005, 22(5):529-535.
[33]Ragsdales SW, Wood HG. Enzymology of the acetyl-CoA pathwayof CO2fixation[J].CriticalReviewsinBiochemistryandMolecular Biology, 1991, 26(3/4):261-300.
[2]Oexmann J, Hensel C, Kather A. Post-combustion CO-capture fromcoal-fired power plants:Preliminary evaluation of an integratedchemical absorption process with piperazine-promoted potassiumcarbonate[J]. International Journal of Greenhouse Gas Control,2008, 2(4):539-552.
[3]Rodríguez N, Alonso M, Grasa G. Process for capturing CO2 arisingfrom the calcination of the CaCO3 used in cement manufacture[J].Environ Sci Technol, 2008, 42(18):69-80.
[4]张炜,李义连.二氧化碳储存技术的研究现状和展望[J].环境污染与防治, 2006, 28(12):950-953.
[5]Grntzis T. Susurface sequestration of carbon dioxide-an overviewfrom an Alberta(Canada)perspective[J]. International Journalof Coal Geology, 2000, 43(1/4):287-305.
[6]Freund P, Ormerod WG. Progress toward storage of carbondioxide[J]. Energy Conversion and Management, 1997, 38(S1):199-204.
[7]Lal R. Carbon sequestration[J]. Philosophical Transactions of theRoyal Society B, 2008, 363(4):815-830.
[8]Dixon RK, Brown S, Houghton RA, et al. Carbon pools and flux ofglobal forest ecosystems[J]. Science, 1994, 263(14):185-190.
[9]Masakazu M, Masahiro I. The biological CO2 fixation and utilizationproject by rite(2)-screening and breeding of microalgae with highcapability in fixing CO2[J]. Energy Conversion and Management,1997, 38(5):493-497.
[10]Grimston MC, Karakoussis V, Fouquet R, et al. The European andglobal potential of carbon dioxide sequestration in tackling climatechange[J]. Climate Policy, 2001, 1(2):155-171.
[11]Miltner A, Kopinke FD, Kindler R. Non-phototrophic CO2 fixationby soil microorganisms[J]. Plant and Soil, 2005, 269(1-2):193-203.
[12]Shively JM, English RS, Baker SH, et al. Carbon cycling:theprokaryotic contribution[J]. Current Opinion in Microbiology,2001, 4:301-306.
[13]Richarda KR. A brief overview of carbon sequestration economicsand policy[J]. Environmental Management, 2004, 33(4):545-558.
[14]袁红朝,秦红灵,刘守龙,等.长期施肥对稻田土壤固碳功能菌群落结构和数量的影响[J].生态学报, 2012, 32(1):0183-0189.
[15]胡佳俊,王磊,李艳丽,等.非光合CO2同化微生物菌群的选育/优化及其群落结构分析[J].环境科学, 2009, 30(8):2438-2444.
[16]Xu HH, Abita FR. Ribulose-1, 5-bisphosphatecarboxylase/oxygenasegene expression and diversity of Lake Eric planktonicmicroorganisms[J]. Appl Environ Microbiol, 1996, 62:1913-1921.
[17]Nanba K, King GM, Dunfield K. Analysis of facultative lithotrophicdistribution and diversity on volcanic deposits by use of the largesubunit of ribulose-1, 5-bisphosphatecarboxylase/oxygenase[J].Appl Environ Microbiol, 2004, 70:2245-2253.
[18]Elsaied H, Naganuma T. Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes fromdeep-sea microorganisms[J]. Appl Environ Microbiol, 2001,67:1751-1765.
[19]周盛,韦彬勤,张琼,等.一种能同时固定CO2和N2的微生物——兼性固CO2、N2菌的分离鉴定及其验证实验[J].环境科学学报, 2013, 33(4):1043-1050.
[20]吴小红,简燕,陈晓娟,等.自养微生物同化CO2的分子生态研究及同化碳在土壤中的转化[J].生态学报, 2014, 34(3):701-709.
[21]王竞,周集体,张晶晶,等.固定CO2氢细菌的筛选及其培养条件优化[J].应用与环境生物学报, 2000, 6(3):271-275.
[22]范秀容,李广武,沈萍.微生物学实验[M].第2版.北京:高等教育出版社, 1995:107-112.
[23]TolliJ,KingGM.Diversityandstructureofbacterialchemoautotrophic communities in pine forest and agroecosystemsoils[J]. Applied&Environmental Microbiology, 2005, 71(12):8411-8418.
[24]布坎南RE,等.伯杰氏细菌鉴定手册[M].第8版.北京:科学出版社, 1984:729-797.
[25]东秀珠,蔡妙英.一般细菌分类手册[M].北京:科学出版社,2001:145-155.
[26]Follett RF. Soil management concepts and carbon sequestration incropland soils[J]. Soils and Tillage Research, 2001, 61(1/2):77-92.
[27]Smith P. Carbon sequestration in croplands:the potentialin Europe and the global context[J]. European Jourmal ofAgronomy, 2004, 20(3):229-236.
[28]Yang HQ, Xu ZH, Fan MH. Progress in carbon dioxide separationand capture:A review[J]. Journal of Environmental Sciences,2008, 20:14-27.
[29]李娟,赵秉强,李秀英,等.长期不同施肥制度下几种土壤微生物学特征变化[J].植物生态学报, 2008, 32(4):891-899.
[30]Boyle NR, Morgan JA. Computation of metabolic fluxes andefficiencies for biological carbon dioxide fixation[J]. MetabolicEngineering, 2011, 13(2):150-158.
[31]袁红朝,秦红灵,刘守龙,等.固碳微生物分子生态学研究[J].中国农业科学, 2011, 44(14):2951-2958.
[32]徐敏,刘国祥,胡征宇.耐受极高浓度CO2藻类的研究及其在固碳领域的应用[J].中国科学院研究生院学报, 2005, 22(5):529-535.
[33]Ragsdales SW, Wood HG. Enzymology of the acetyl-CoA pathwayof CO2fixation[J].CriticalReviewsinBiochemistryandMolecular Biology, 1991, 26(3/4):261-300.