外添少量电子受体强化丙丁梭菌丙酮-丁醇-乙醇发酵的丁醇合成
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摘要
强化利用丙丁梭菌发酵生产丁醇的主要方法有:添加电子载体强化NADH再生速率、通CO气体抑制氢化酶活性、外添少量丁酸等。但是,上述方法存在着总溶剂产量低、精制成本高、辅料价格昂贵等缺点。本研究通过向丙酮-丁醇-乙醇(ABE)发酵液添加少量电子受体(Na_2SO_4/CaSO_4,2g/L),使得梭菌胞内的电子穿梭传递系统的电子流和质子流发生改变,较多电子e~–和质子H~+走向NADH合成途径,有利于丁醇合成;电子受体添加还可以促进对梭菌生存/丁醇合成的"有益"氨基酸、特别是缬氨酸的胞内积累/分泌,进一步强化了丁醇生产。在7L罐规模的发酵条件下、添加2g/L的电子受体Na_2SO_4,ABE发酵的丁醇浓度达到12.96g/L的最高水平,丁醇/丙酮比也有提高,分别比对照组提高35%和10%。添加Na_2SO_4等廉价电子受体提高了ABE发酵中的丁醇浓度,虽然提高幅度有限,但却可为利用发酵工程技术提高丁醇浓度和丁醇/丙酮比提供一种新的途径。
The common techniques/methods for enhancing butanol synthesis in ABE fermentation by Clostridium acetobutylicum include adding electron carrier to increase NADH regeneration rate,repressing hydrogenase via aerating CO,supplementing small amount of butyrate,etc. However,those methods suffer from the problems of total solvent decrease,purification cost-up to remove pigments,high butyrate cost,etc. In this study,small amount of electron receptors(Na_2SO_4/CaSO_4,2 g/L)were added into the ABE fermentation broth: leading to the electron/proton flow transition/changes in the intracellular electron shuttle-transportation system,directing more electron/proton into the NADH regeneration route which enhanced for butanol synthesis; promoting the intracellular accumulation or secretion of those amino acids beneficial for cells survival/butanol synthesis,particularly valine. In the ABE fermentation in a 7 L fermentor,by adding 2 g/L electron receptor(Na_2SO_4),the butanol concentration reached the highest levels of 12.96 g/L and butanol/acetone ratio also enhanced,the increments were 35% and 10% ,respectively compared with those of control. The addition of the cheap electron receptor,such as Na_2SO_4,could increase the butanol concentration though the increment was limited,but it would supply a new and alternative fermentative method for enhancing butanol synthesis and butanol/acetone ratio.
The common techniques/methods for enhancing butanol synthesis in ABE fermentation by Clostridium acetobutylicum include adding electron carrier to increase NADH regeneration rate,repressing hydrogenase via aerating CO,supplementing small amount of butyrate,etc. However,those methods suffer from the problems of total solvent decrease,purification cost-up to remove pigments,high butyrate cost,etc. In this study,small amount of electron receptors(Na_2SO_4/CaSO_4,2 g/L)were added into the ABE fermentation broth: leading to the electron/proton flow transition/changes in the intracellular electron shuttle-transportation system,directing more electron/proton into the NADH regeneration route which enhanced for butanol synthesis; promoting the intracellular accumulation or secretion of those amino acids beneficial for cells survival/butanol synthesis,particularly valine. In the ABE fermentation in a 7 L fermentor,by adding 2 g/L electron receptor(Na_2SO_4),the butanol concentration reached the highest levels of 12.96 g/L and butanol/acetone ratio also enhanced,the increments were 35% and 10% ,respectively compared with those of control. The addition of the cheap electron receptor,such as Na_2SO_4,could increase the butanol concentration though the increment was limited,but it would supply a new and alternative fermentative method for enhancing butanol synthesis and butanol/acetone ratio.
引文
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[2]WU H,NITHYANANDAN K,ZHANG J,et al.Impacts of acetone-butanol-ethanol(ABE)ratio on spray and combustion characteristics of ABE-diesel blends[J].Applied Energy,2015,149:367-378.
[3]JANG Y S,LEE J Y,LEE J,et al.Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum[J].m Bio.,2012,3(5):e00314.
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[7]PEGUIN S,GOMA G,DELORME P,et al.Metabolic flexibility of Clostridium acetobutylicum in response to methyl viologen addition[J].Applied Microbiology and Biotechnology,1994,42(4):611-616.
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[12]XUE C,ZHAO J,CHEN L,et al.Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum[J].Biotechnology Advances,2017,35(2):310-322.
[13]XUE C,LIU F,XU M,et al.Butanol production in acetone-butanol-ethanol fermentation with in situ product recovery by adsorption[J].Bioresource Technology,2016,219:158-168.
[14]XUE C,LIU F,XU M,et al.A novel in situ gas stripping-pervaporation process integrated with acetone-butanolethanol fermentation for hyper n-butanol production[J].Biotechnology and Bioengineering,2016,113(1):120-129.
[15]WU H,NITHYANANDAN K,ZHOU N,et al.Impacts of acetone on the spray combustion of acetone-butanol-ethanol(ABE)-diesel blends under low ambient temperature[J].Fuel,2015,142:109-116.
[16]Statistical Information on Chemical Industry of Japan.http://www.jpca.or.jp/4stat/02stat/y1seisan.htm.
[17]LUO H,ZHANG J,WANG H,et al.Effectively enhancing acetone concentration and acetone/butanol ratio in ABE fermentation by a glucose/acetate co-substrate system incorporating with glucose limitation and C.acetobutylicum S.cerevisiae co-culturing[J].Biochemical Engineering Journal,2017,118:132-142.
[18]WU Y D,XUE C,CHEN L J,et al.Effect of zinc supplementation on acetone-butanol-ethanol fermentation by Clostridium acetobutylicum[J].Journal of Biotechnology,2013,165(1):18-21.
[19]WU Y D,XUE C,CHEN L J,et al.Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum[J].Scientific Reports,2015,5:16598.
[20]LI X,SHI Z,LI Z.Increasing butanol/acetone ratio and solvent productivity in ABE fermentation by consecutively feeding butyrate to weaken metabolic strength of butyrate loop[J].Bioprocess and Biosystems Engineering,2014,37(8):1609-1616.
[21]李鑫.使用木薯原料和外部代谢调控策略提高丁醇发酵的丁醇/丙酮比[D].无锡:江南大学,2014.LI X.Increasig butanol/acetone ratio in butanol fermentation with the strategies of using cassava substrate and external metabolic regulation modes[D].Wuxi:Jiangnan University,2014.
[22]LI X,LI Z,ZHENG J,et al.Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824using cassava as substrate[J].Bioresource Technology,2012,125:43-51.
[23]钟国清.饲料用硫酸钠含量的快速测定[J].饲料博览,2001(3):26-27.ZHONG G Q.The rapid determination of sodium sulphate in feedstock[J].Feed Expo,2001(3):26-27.
[24]RICHMOND C,HAN B,EZEJI T C.Stimulatory effects of calcium carbonate on butanol production by solventogenic Clostridium species[J].Continental Journal of Microbiology,2011,111(12):123704-123708.
[25]MASION E,AMINE J,MARCZAK R.Influence of amino acid supplements on the metabolism of Clostridium acetobutylicum[J].FEMS Microbiology Letters,1987,43(3):269-274.
[26]HELUANE H,EVANS M R,DAGHER S F,et al.Meta-analysis and functional validation of nutritional requirements of solventogenic Clostridia growing under butanol stress conditions and coutilization of D-glucose and D-xylose[J].Applied and Environmental Microbiology,2011,77(13):4473-4485.
[27]蔡靖,郑平,张蕾.硫酸盐还原菌及其代谢途径[J].科技通报,2009,25(4):427-431.CAI J,ZHENG P,ZHANG L.Sulfate-reducing bacteria and their metabolic pathway[J].Bulletin of Science and Technology,2009,25(4):427-431.
[28]郑礼胜,王士龙.偶氮氯膦Ⅲ做指示剂EDTA滴定法测定水的硬度[J].工业水处理,1993,13(2):30-31.ZHENG L S,WANG S L.EDTA titration method to determine the hardness of water with chlorophosphonazoⅢindicator[J].Industrial Water Treatment,1993,13(2):30-31.
[29]段生兵.基于代谢分析和计算流体力学的头孢菌素C发酵过程优化控制研究[D].无锡:江南大学,2013.DUAN S B.Process control and optimization of Cephalosporin C fermentation based on metabolic analysis and computational fluid dynamics simulation[D].Wuxi:Jiangnan University,2013.
[2]WU H,NITHYANANDAN K,ZHANG J,et al.Impacts of acetone-butanol-ethanol(ABE)ratio on spray and combustion characteristics of ABE-diesel blends[J].Applied Energy,2015,149:367-378.
[3]JANG Y S,LEE J Y,LEE J,et al.Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum[J].m Bio.,2012,3(5):e00314.
[4]JIANG Y,XU C,DONG F,et al.Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicumincreases the butanol ratio[J].Metabolic Engineering,2009,11(4):284-291.
[5]WANG S,ZHU Y,ZHANG Y,et al.Controlling the oxidoreduction potential of the culture of Clostridium acetobutylicumleads to an earlier initiation of solventogenesis,thus increasing solvent productivity[J].Applied Microbiology and Biotechnology,2012,93(3):1021-1030.
[6]GIRBAL L,VASCONCELOS I,SAINT-AMANS S,et al.How neutral red modified carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral p H[J].FEMS Microbiology Reviews,1995,16(2/3):151-162.
[7]PEGUIN S,GOMA G,DELORME P,et al.Metabolic flexibility of Clostridium acetobutylicum in response to methyl viologen addition[J].Applied Microbiology and Biotechnology,1994,42(4):611-616.
[8]KIM B H,BELLOWS P,DATTA R,et al.Control of carbon and electron flow in Clostridium acetobutylicum fermentations:utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields[J].Applied and Environmental Microbiology,1984,48(4):764-770.
[9]TASHIRO Y,TAKEDA K,KOBAYASHI G,et al.High butanol production by Clostridium saccharoperbutylacetonicum N1-4 in fed-batch culture with p H-Stat continuous butyric acid and glucose feeding method[J].Journal of Bioscience and Bioengineering,2004,98(4):263-268.
[10]TASHIRO Y,SHINTO H,HAYASHI M,et al.Novel high-efficient butanol production from butyrate by non-growing Clostridium saccharoperbutylacetonicum N1-4(ATCC 13564)with methyl viologen[J].Journal of Bioscience and Bioengineering,2007,104(3):238-240.
[11]LUO H,GE L,ZHANG J,et al.Enhancing butanol production under the stress environments of co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae integrated with exogenous butyrate addition[J].PLo S One,2015,10(10):e0141160.
[12]XUE C,ZHAO J,CHEN L,et al.Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum[J].Biotechnology Advances,2017,35(2):310-322.
[13]XUE C,LIU F,XU M,et al.Butanol production in acetone-butanol-ethanol fermentation with in situ product recovery by adsorption[J].Bioresource Technology,2016,219:158-168.
[14]XUE C,LIU F,XU M,et al.A novel in situ gas stripping-pervaporation process integrated with acetone-butanolethanol fermentation for hyper n-butanol production[J].Biotechnology and Bioengineering,2016,113(1):120-129.
[15]WU H,NITHYANANDAN K,ZHOU N,et al.Impacts of acetone on the spray combustion of acetone-butanol-ethanol(ABE)-diesel blends under low ambient temperature[J].Fuel,2015,142:109-116.
[16]Statistical Information on Chemical Industry of Japan.http://www.jpca.or.jp/4stat/02stat/y1seisan.htm.
[17]LUO H,ZHANG J,WANG H,et al.Effectively enhancing acetone concentration and acetone/butanol ratio in ABE fermentation by a glucose/acetate co-substrate system incorporating with glucose limitation and C.acetobutylicum S.cerevisiae co-culturing[J].Biochemical Engineering Journal,2017,118:132-142.
[18]WU Y D,XUE C,CHEN L J,et al.Effect of zinc supplementation on acetone-butanol-ethanol fermentation by Clostridium acetobutylicum[J].Journal of Biotechnology,2013,165(1):18-21.
[19]WU Y D,XUE C,CHEN L J,et al.Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum[J].Scientific Reports,2015,5:16598.
[20]LI X,SHI Z,LI Z.Increasing butanol/acetone ratio and solvent productivity in ABE fermentation by consecutively feeding butyrate to weaken metabolic strength of butyrate loop[J].Bioprocess and Biosystems Engineering,2014,37(8):1609-1616.
[21]李鑫.使用木薯原料和外部代谢调控策略提高丁醇发酵的丁醇/丙酮比[D].无锡:江南大学,2014.LI X.Increasig butanol/acetone ratio in butanol fermentation with the strategies of using cassava substrate and external metabolic regulation modes[D].Wuxi:Jiangnan University,2014.
[22]LI X,LI Z,ZHENG J,et al.Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824using cassava as substrate[J].Bioresource Technology,2012,125:43-51.
[23]钟国清.饲料用硫酸钠含量的快速测定[J].饲料博览,2001(3):26-27.ZHONG G Q.The rapid determination of sodium sulphate in feedstock[J].Feed Expo,2001(3):26-27.
[24]RICHMOND C,HAN B,EZEJI T C.Stimulatory effects of calcium carbonate on butanol production by solventogenic Clostridium species[J].Continental Journal of Microbiology,2011,111(12):123704-123708.
[25]MASION E,AMINE J,MARCZAK R.Influence of amino acid supplements on the metabolism of Clostridium acetobutylicum[J].FEMS Microbiology Letters,1987,43(3):269-274.
[26]HELUANE H,EVANS M R,DAGHER S F,et al.Meta-analysis and functional validation of nutritional requirements of solventogenic Clostridia growing under butanol stress conditions and coutilization of D-glucose and D-xylose[J].Applied and Environmental Microbiology,2011,77(13):4473-4485.
[27]蔡靖,郑平,张蕾.硫酸盐还原菌及其代谢途径[J].科技通报,2009,25(4):427-431.CAI J,ZHENG P,ZHANG L.Sulfate-reducing bacteria and their metabolic pathway[J].Bulletin of Science and Technology,2009,25(4):427-431.
[28]郑礼胜,王士龙.偶氮氯膦Ⅲ做指示剂EDTA滴定法测定水的硬度[J].工业水处理,1993,13(2):30-31.ZHENG L S,WANG S L.EDTA titration method to determine the hardness of water with chlorophosphonazoⅢindicator[J].Industrial Water Treatment,1993,13(2):30-31.
[29]段生兵.基于代谢分析和计算流体力学的头孢菌素C发酵过程优化控制研究[D].无锡:江南大学,2013.DUAN S B.Process control and optimization of Cephalosporin C fermentation based on metabolic analysis and computational fluid dynamics simulation[D].Wuxi:Jiangnan University,2013.