高温条件下混菌发酵合成气产乙酸及其群落结构
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摘要
合成气(主要包括CO、H_2和CO_2)通过生物转化生产高附加值的生物燃料和化学品已引起人们广泛关注,微生物菌群作为生物转化的酶催化剂对合成气发酵产物组成和效率十分关键.通过富集得到高温条件下分别稳定转化CO、甲酸钠和合成气的厌氧菌群,探究CO与甲酸钠转化菌混培物和合成气转化菌发酵合成气生成乙酸的能力,并分析其微生物群落结构.结果显示,CO-甲酸钠转化菌混培物与合成气转化菌在合成气发酵前期主要进行CO的产氢反应生成H_2和CO_2以及同型产乙酸反应生成乙酸,CO利用率为100%,CO反应速率分别为6.93和6.34 mmol L~(-1)d~(-1);随后同型产乙酸菌利用H_2和CO_2继续合成乙酸,两者的乙酸最大累积量分别为9.11 mmol/L和8.01 mmol/L.CO-甲酸钠转化菌混培物主要菌群为Thermoanaerobacterium、Romboutsia、Ruminococcus、Clostridium、Eubacterium、Moorella和Desulfotomaculum属,合成气转化混菌则主要含有Romboutsia、Thermoanaerobacterium、Moorella、Eubacterium、Acetonema和Clostridium属,其中同型产乙酸菌广泛分布于Ruminococcus、Clostridium、Eubacterium、Moorella和Acetonema属.本研究表明复配CO和甲酸钠转化菌可用于合成气高温发酵产乙酸,且转化能力优于合成气转化菌,结果可为合成气混菌发酵提供微生物资源和技术参考.
Syngas is a mixture of mainly CO, H_2, and CO_2, which can be produced from the gasification of various organic materials. Syngas fermentation has attracted great attention for its ability to produce valuable biofuels and biochemicals. As biocatalysts for syngas fermentation, microorganisms are important to product profile and conversion efficiency. In this research,the thermophilic CO-, formate-and syngas-converting microorganisms were enriched from cow manure, and the capability for acetate production from syngas by the CO-and formate-converting bacteria mixture and syngas-converting bacteria, as well as their microbial communities, were analyzed. The results showed that the CO-driven hydrogenogenesis and acetogenesis occurred during the initial stage of syngas fermentation and resulted in the formation of H_2 + CO_2 and acetate, respectively. The CO conversion efficiency was 100%, and the CO conversion rates of the CO-and formate-converting bacteria mixture and syngasconverting bacteria were 6.93 and 6.34 mmol L~(-1) d~(-1), respectively. Subsequently, H_2 + CO_2 was mainly converted into acetate via the acetogenic Wood-Ljungdahl pathway, and the final accumulation of acetate for the Co-and formate-converting bacteria mixture and syngas-converting bacteria were 9.11 and 8.01 mmol/L, respectively. The bacterial genera Thermoanaerobacterium,Romboutsia, Ruminococcus, Clostridium, Eubacterium, Moorella, and Desulfotomaculum dominated in the CO-and formate-converting bacteria mixture, and Romboutsia, Thermoanaerobacterium, Moorella, Eubacterium, Acetonema, and Clostridium were the main genera comprising the syngas-converting bacteria; among these genera, Ruminococcus, Clostridium,Eubacterium, Moorella and Acetonema harbor known homoacetogenic species. It is shown that the mixture of CO-and formateconverting bacteria can be used for thermophilic syngas fermentation, and its conversion ability is higher than that of the syngasconverting bacteria. This study identified potential microorganism resources and provides technical references for valuable chemicals and bioenergy production by syngas mixed-culture fermentation.
Syngas is a mixture of mainly CO, H_2, and CO_2, which can be produced from the gasification of various organic materials. Syngas fermentation has attracted great attention for its ability to produce valuable biofuels and biochemicals. As biocatalysts for syngas fermentation, microorganisms are important to product profile and conversion efficiency. In this research,the thermophilic CO-, formate-and syngas-converting microorganisms were enriched from cow manure, and the capability for acetate production from syngas by the CO-and formate-converting bacteria mixture and syngas-converting bacteria, as well as their microbial communities, were analyzed. The results showed that the CO-driven hydrogenogenesis and acetogenesis occurred during the initial stage of syngas fermentation and resulted in the formation of H_2 + CO_2 and acetate, respectively. The CO conversion efficiency was 100%, and the CO conversion rates of the CO-and formate-converting bacteria mixture and syngasconverting bacteria were 6.93 and 6.34 mmol L~(-1) d~(-1), respectively. Subsequently, H_2 + CO_2 was mainly converted into acetate via the acetogenic Wood-Ljungdahl pathway, and the final accumulation of acetate for the Co-and formate-converting bacteria mixture and syngas-converting bacteria were 9.11 and 8.01 mmol/L, respectively. The bacterial genera Thermoanaerobacterium,Romboutsia, Ruminococcus, Clostridium, Eubacterium, Moorella, and Desulfotomaculum dominated in the CO-and formate-converting bacteria mixture, and Romboutsia, Thermoanaerobacterium, Moorella, Eubacterium, Acetonema, and Clostridium were the main genera comprising the syngas-converting bacteria; among these genera, Ruminococcus, Clostridium,Eubacterium, Moorella and Acetonema harbor known homoacetogenic species. It is shown that the mixture of CO-and formateconverting bacteria can be used for thermophilic syngas fermentation, and its conversion ability is higher than that of the syngasconverting bacteria. This study identified potential microorganism resources and provides technical references for valuable chemicals and bioenergy production by syngas mixed-culture fermentation.
引文
1 Munasinghe PC,Khanal SK.Biomass-derived syngas fermentation into biofuels:opportunities and challenges[J].Bioresour Technol,2010,101(13):5013-5022
2郭蔚,刘成,邹少兰,张敏华.同型乙酸茵研究进展及应用前景[J].应用与环境生物学报,2006,12(6):874-877[Guo W,Liu C,Zou SL,Zhang MH.Progress in research and application of homoacetoge[J].Chin J Appl Environ Biol,2006,12(6):874-877]
3 Demirbas A.Progress and recent trends in biofuels[J].Prog Energy Combust Sci,2007,33(1):1-18
4 Kundiyana DK,Huhnke RL,Wilkins MR.Syngas fermentation in a 100-L pilot scale fermentor:design and process considerations[J].J Biosci Bioeng,2010,109(5):492-498
5 Madhukar GR,Elmore BB,Huckabay HK.Microbial conversion of synthesis gas components to useful fuels and chemicals[C]//Humana P.Seventeenth Symposium on Biotechnology for Fuels and Chemicals.Totowa NJ,1996:243-251
6刘超,罗刚,王雯,刘广青.微生物转化合成气制取生物燃料和化学品的研究进展[J].北京化工大学学报(自然科学版),2017,44(5):2-12[Liu C,Luo G,Wang W,Liu GQ.Research progress in biofuel and biochemicals production from syngas fermentation[J].J Beijing Univ Chem Technol(Nat Sci Ed),2017,44(5):2-12]
7 Luo G,Wang W,Angelidaki I.Anaerobic digestion for simultaneous sewage sludge treatment and CO biomethanation:process performance and microbial ecology[J].Environ sci technol,2013,47(18):10685-10693
8 Liu K,Atiyeh HK,Stevenson BS,Tanner RS,Wilkins MR,Huhnke RL.Mixed culture syngas fermentation and conversion of carboxylic acids into alcohols[J].Bioresour Technol,2014,152:337-346
9 Nam CW,Jung KA,Park JM.Biological carbon monoxide conversion to acetate production by mixed culture[J].Bioresour Technol,2016,211:478-485
10谢丽,杜诗云,卜凡.同型产乙酸菌研究进展及其环境生物技术应用[J].同济大学学报(自然科学版),2018,46(1):67-73[Xie L,Du SY,Bu F.Homoacetogen and its application in environmental biotechnology[J].J Tongji Univ(Nat Sci Ed),2018,46(1):67-73]
11 Drake HL,G??ner AS,Daniel SL.Old acetogens,new light[J].Ann N YAcad Sci,2008,1125(1):100-128
12 Zhang F,Ding J,Zhang Y,Chen M,Ding ZW,Zeng RJ.Fatty acids production from hydrogen and carbon dioxide by mixed culture in the membrane biofilm reactor[J].Water Res,2013,47(16):6122-6129
13张丽娟,符波,罗衎,刘和.同型产乙酸菌富集物的群落解析及转化合成气产乙酸[J].应用与环境生物学报,2014,20(6):1052-1057[Zhang LJ,Fu B,Luo K,Liu H.Microbial community characterization of homoacetogen enrichment culture and its acetate production through syngas bioconversion[J].Chin J Appl Environ Biol,2014,20(6):1052-1057]
14刘和,马琳,刘宏波,符波.一种富集培养同型产乙酸菌的方法.中国:ZL.201110416294.2[P].2014[Liu H,Ma L,Liu HB,Fu B.A method for enriching the homoacetogens.China:ZL.201110416294.2[P].2014]
15 Diender M,Stams AJM,Sousa DZ.Pathways and bioenergetics of anaerobic carbon monoxide fermentation[J].Front Microbiol,2015,6:1275-1293
16 Xu SY,Fu B,Zhang LJ,Liu H.Bioconversion of H2/CO2 by acetogen enriched cultures for acetate and ethanol production:the impact of pH[J].World J Microbiol Biotechnol,2015,31(6):941-950
17 Latif H,Zeidan A A,Nielsen AT,Zengler K.Trash to t reasure:production of biofuels and commodity chemicals via syngas fermenting microorganisms[J].Curr Opin Biotechnol,2014,27:79-87
18 Ryan P,Forbes C,McHugh S,Reilly C,Fleming GTA,Colleran E.Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions[J].Water Res,2010,44(14):4261-4269
19 Alves J I,Stams A J M,Plugge CM,Ma d alena A M,Sou sa DZ.Enrichment of anaerobic syngas-converting bacteria from thermophilic bioreactor sludge[J].FEMS Microbiol Ecol,2013,86(3):590-597
20许科伟.污泥厌氧消化过程中乙酸累积的微生态机理研究[D].无锡:江南大学,2010[Xu KW.A microbial ecological study on the mechanism of acetate accumulation during anaerobic incubation of sewage sludge[D].Wuxi:Jiangnan University,2010]
21田淼,张丽娟,符波,刘宏波,张汝兵,刘和.不同生境微生物转化H2/CO2产乙酸及其在合成气发酵中应用[J].微生物学通报,2017,44(7):1563-1578[Tian M,Zhang LJ,Fu B,Liu HB,Zhang RB,Liu H.Acetate production from H2/CO2 by mixed cultures from diverse ecosystems and their application for syngas fermentation[J].Microbiol China,2017,44(7):1563-1578]
22 Bernardet JF,Segers P,Vancanneyt M,Berthe F,Kersters K,Vandamme P.Cutting a Gordian knot:emended classification and description of the genus Flavobacterium,emended description of the family Flavobacteriaceae,and proposal of Flavobacterium hydatis nom.nov.(basonym,Cytophaga aquatilis Strohl and Tait 1978)[J].Int J Syst Evol Microbiol,1996,46(1):128-148
23 Deng Y,Guo X,Wang Y,He M,Ma K,Wang H,Ruan Z.Terrisporobacter petrolearius sp.nov.,isolated from an oilf ield petroleum reservoir[J].Int J Syst Evol Microbiol,2015,65(10):3522-3526
24 Kovatcheva DP,Nilsson A,Akrami R,Lee YS,De VF,Arora T,B?ckhed F.Dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of Prevotella[J].Cell Metab,2015,22(6):971-982
25 Jadhav UU,Dawkar VV,Ghodake GS,Govindwar SP.Biodegradation of Direct Red 5B,a textile dye by newly isolated Comamonas sp.UVS[J].J Hazard Mater,2008,158(2-3):507-516
26 Song Y,Shin J,Jeong Y,Jin S,Lee JK,Kim DR,Cho BK.Determination of the genome and primary transcriptome of syngas fermenting Eubacterium limosum ATCC 8486[J].Sci Rep,2017,7(1):13694-13705
27 Gerritsen J,Fuentes S,Grievink W,Niftrik L,Tindall BJ,Timmerman HM,Smidt H.Characterization of Romboutsia ilealis gen.nov.,sp.nov.,isolated from the gastro-intestinal tract of a rat,and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Romboutsia gen.nov.,Intestinibacter gen.nov.,Terrisporobacter gen.nov.and Asaccharospora gen.nov[J].Int JSyst Evol Microbiol,2014,64(5):1600-1616
28 Haldrup A,Petersen SG,Okkels F T.The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent[J].Pant Mol Biol,1998,37(2):287-296
29 Xing D,Ren N,Wang A,Wang A,Li Q,Feng Y,Ma F.Continuous hydrogen production of auto-aggregative Ethanoligenens harbinense YUAN-3 under non-sterile condition[J].Int J Hydrogen Energy,2008,33(5):1489-1495
30 Bertsch J,Müller V.Bioenergetic constraints for conversion of syngas to biofuels in acetogenic bacteria[J].Biotechnol Biofuels,2015,8(1):210-222
31 Mohammadi M,Najafpour GD,Younesi H,Lahijani P,Uzir MH,Mohamed AR.Bioconversion of synthesis gas to second generation biofuels:a review[J].Renewable Sustainable Energy Rev,2011,15(9):4255-4273
32 Ukpong M N,Atiyeh HK,Lor me MJ,Liu K,Zhu X,Tanner RS,Stevenson BS.Physiological response of Clostridium carboxidivorans during conversion of synthesis gas to solvents in a gas‐fed bioreactor[J].Biotechnol Bioeng,2012,109(11):2720-2728
2郭蔚,刘成,邹少兰,张敏华.同型乙酸茵研究进展及应用前景[J].应用与环境生物学报,2006,12(6):874-877[Guo W,Liu C,Zou SL,Zhang MH.Progress in research and application of homoacetoge[J].Chin J Appl Environ Biol,2006,12(6):874-877]
3 Demirbas A.Progress and recent trends in biofuels[J].Prog Energy Combust Sci,2007,33(1):1-18
4 Kundiyana DK,Huhnke RL,Wilkins MR.Syngas fermentation in a 100-L pilot scale fermentor:design and process considerations[J].J Biosci Bioeng,2010,109(5):492-498
5 Madhukar GR,Elmore BB,Huckabay HK.Microbial conversion of synthesis gas components to useful fuels and chemicals[C]//Humana P.Seventeenth Symposium on Biotechnology for Fuels and Chemicals.Totowa NJ,1996:243-251
6刘超,罗刚,王雯,刘广青.微生物转化合成气制取生物燃料和化学品的研究进展[J].北京化工大学学报(自然科学版),2017,44(5):2-12[Liu C,Luo G,Wang W,Liu GQ.Research progress in biofuel and biochemicals production from syngas fermentation[J].J Beijing Univ Chem Technol(Nat Sci Ed),2017,44(5):2-12]
7 Luo G,Wang W,Angelidaki I.Anaerobic digestion for simultaneous sewage sludge treatment and CO biomethanation:process performance and microbial ecology[J].Environ sci technol,2013,47(18):10685-10693
8 Liu K,Atiyeh HK,Stevenson BS,Tanner RS,Wilkins MR,Huhnke RL.Mixed culture syngas fermentation and conversion of carboxylic acids into alcohols[J].Bioresour Technol,2014,152:337-346
9 Nam CW,Jung KA,Park JM.Biological carbon monoxide conversion to acetate production by mixed culture[J].Bioresour Technol,2016,211:478-485
10谢丽,杜诗云,卜凡.同型产乙酸菌研究进展及其环境生物技术应用[J].同济大学学报(自然科学版),2018,46(1):67-73[Xie L,Du SY,Bu F.Homoacetogen and its application in environmental biotechnology[J].J Tongji Univ(Nat Sci Ed),2018,46(1):67-73]
11 Drake HL,G??ner AS,Daniel SL.Old acetogens,new light[J].Ann N YAcad Sci,2008,1125(1):100-128
12 Zhang F,Ding J,Zhang Y,Chen M,Ding ZW,Zeng RJ.Fatty acids production from hydrogen and carbon dioxide by mixed culture in the membrane biofilm reactor[J].Water Res,2013,47(16):6122-6129
13张丽娟,符波,罗衎,刘和.同型产乙酸菌富集物的群落解析及转化合成气产乙酸[J].应用与环境生物学报,2014,20(6):1052-1057[Zhang LJ,Fu B,Luo K,Liu H.Microbial community characterization of homoacetogen enrichment culture and its acetate production through syngas bioconversion[J].Chin J Appl Environ Biol,2014,20(6):1052-1057]
14刘和,马琳,刘宏波,符波.一种富集培养同型产乙酸菌的方法.中国:ZL.201110416294.2[P].2014[Liu H,Ma L,Liu HB,Fu B.A method for enriching the homoacetogens.China:ZL.201110416294.2[P].2014]
15 Diender M,Stams AJM,Sousa DZ.Pathways and bioenergetics of anaerobic carbon monoxide fermentation[J].Front Microbiol,2015,6:1275-1293
16 Xu SY,Fu B,Zhang LJ,Liu H.Bioconversion of H2/CO2 by acetogen enriched cultures for acetate and ethanol production:the impact of pH[J].World J Microbiol Biotechnol,2015,31(6):941-950
17 Latif H,Zeidan A A,Nielsen AT,Zengler K.Trash to t reasure:production of biofuels and commodity chemicals via syngas fermenting microorganisms[J].Curr Opin Biotechnol,2014,27:79-87
18 Ryan P,Forbes C,McHugh S,Reilly C,Fleming GTA,Colleran E.Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions[J].Water Res,2010,44(14):4261-4269
19 Alves J I,Stams A J M,Plugge CM,Ma d alena A M,Sou sa DZ.Enrichment of anaerobic syngas-converting bacteria from thermophilic bioreactor sludge[J].FEMS Microbiol Ecol,2013,86(3):590-597
20许科伟.污泥厌氧消化过程中乙酸累积的微生态机理研究[D].无锡:江南大学,2010[Xu KW.A microbial ecological study on the mechanism of acetate accumulation during anaerobic incubation of sewage sludge[D].Wuxi:Jiangnan University,2010]
21田淼,张丽娟,符波,刘宏波,张汝兵,刘和.不同生境微生物转化H2/CO2产乙酸及其在合成气发酵中应用[J].微生物学通报,2017,44(7):1563-1578[Tian M,Zhang LJ,Fu B,Liu HB,Zhang RB,Liu H.Acetate production from H2/CO2 by mixed cultures from diverse ecosystems and their application for syngas fermentation[J].Microbiol China,2017,44(7):1563-1578]
22 Bernardet JF,Segers P,Vancanneyt M,Berthe F,Kersters K,Vandamme P.Cutting a Gordian knot:emended classification and description of the genus Flavobacterium,emended description of the family Flavobacteriaceae,and proposal of Flavobacterium hydatis nom.nov.(basonym,Cytophaga aquatilis Strohl and Tait 1978)[J].Int J Syst Evol Microbiol,1996,46(1):128-148
23 Deng Y,Guo X,Wang Y,He M,Ma K,Wang H,Ruan Z.Terrisporobacter petrolearius sp.nov.,isolated from an oilf ield petroleum reservoir[J].Int J Syst Evol Microbiol,2015,65(10):3522-3526
24 Kovatcheva DP,Nilsson A,Akrami R,Lee YS,De VF,Arora T,B?ckhed F.Dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of Prevotella[J].Cell Metab,2015,22(6):971-982
25 Jadhav UU,Dawkar VV,Ghodake GS,Govindwar SP.Biodegradation of Direct Red 5B,a textile dye by newly isolated Comamonas sp.UVS[J].J Hazard Mater,2008,158(2-3):507-516
26 Song Y,Shin J,Jeong Y,Jin S,Lee JK,Kim DR,Cho BK.Determination of the genome and primary transcriptome of syngas fermenting Eubacterium limosum ATCC 8486[J].Sci Rep,2017,7(1):13694-13705
27 Gerritsen J,Fuentes S,Grievink W,Niftrik L,Tindall BJ,Timmerman HM,Smidt H.Characterization of Romboutsia ilealis gen.nov.,sp.nov.,isolated from the gastro-intestinal tract of a rat,and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Romboutsia gen.nov.,Intestinibacter gen.nov.,Terrisporobacter gen.nov.and Asaccharospora gen.nov[J].Int JSyst Evol Microbiol,2014,64(5):1600-1616
28 Haldrup A,Petersen SG,Okkels F T.The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent[J].Pant Mol Biol,1998,37(2):287-296
29 Xing D,Ren N,Wang A,Wang A,Li Q,Feng Y,Ma F.Continuous hydrogen production of auto-aggregative Ethanoligenens harbinense YUAN-3 under non-sterile condition[J].Int J Hydrogen Energy,2008,33(5):1489-1495
30 Bertsch J,Müller V.Bioenergetic constraints for conversion of syngas to biofuels in acetogenic bacteria[J].Biotechnol Biofuels,2015,8(1):210-222
31 Mohammadi M,Najafpour GD,Younesi H,Lahijani P,Uzir MH,Mohamed AR.Bioconversion of synthesis gas to second generation biofuels:a review[J].Renewable Sustainable Energy Rev,2011,15(9):4255-4273
32 Ukpong M N,Atiyeh HK,Lor me MJ,Liu K,Zhu X,Tanner RS,Stevenson BS.Physiological response of Clostridium carboxidivorans during conversion of synthesis gas to solvents in a gas‐fed bioreactor[J].Biotechnol Bioeng,2012,109(11):2720-2728