恒化培养条件下粪产碱杆菌凝胶多糖的发酵动力学研究
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摘要
通过恒化培养实验研究了不同稀释速率下粪产碱杆菌(Alcaligenes faecalis)的生理代谢特性变化,利用Monod、Luedeking-Piret、Herbert-pirt模型分析了粪产碱杆菌的生长、多糖合成以及底物葡萄糖消耗的动力学变化。结果表明,在铵离子限制条件下的菌体比生长速率与铵离子浓度之间的动力学模型为μ=0.36S/(2.27+S),凝胶多糖比产率与铵离子浓度间的动力学模型为q_p=0.0612S/(2.27+S)+0.14×(1-S/64.94),以及比底物葡萄糖消耗速率与比产率的动力学模型为:q_s=1.5μ+q_p+0.0625。通过对恒化培养模型及生产菌生理代谢参数的分析,统计分析表明该生产菌最佳限制性铵离子浓度为5.75 mg/L,得到控制最适比生长速率为0.21 h~(-1)时,A.faecalis的最高产率可达到0.85 g/L/h,并且最大比产率高达0.171 g/g/h,产率和比产率与批培养过程相比分别提升了118%和200%,能够显著提升凝胶多糖的生产效率。
In this paper,the physiological and metabolic characteristic of A. faecalis at different dilution rates through chemostat cultivation had been studied. The metabolic kinetic parameters on cell growth,curdlan biosynthesis and substrate glucose consumption were obtained with Monod,Luedeking-Piret and Herbert-pirt models. The results showed that the kinetic model for the cell growth rate to ammonium concentration under ammonium ion restriction conditions was μ=0.36 S/(2.27+S),and the kinetic model between the specific biosynthesis rate of curdlan and the ammonium concentration was q_p=0.0612 S/(2.27+S)+0.14×(1-S/64.94),and the kinetic model of glucose consumption rate to the specific yield was q_s=1.5 μ+q_p+0.0625. The statistic physiological metabolic parameters on A. faecalis demonstrated that the optimal ammonium-limited concentration was 5.75 mg/L.And under the specific cell growth rate at 0.21 h~(-1),the highest yield and maximum specific production rate of cudlan could reach 0.85 g/L/h and 0.17 g/g/h,which was 118% and 200% higher than that under batch culture process respectively. It would significantly improve the efficiency of curdlan production.
In this paper,the physiological and metabolic characteristic of A. faecalis at different dilution rates through chemostat cultivation had been studied. The metabolic kinetic parameters on cell growth,curdlan biosynthesis and substrate glucose consumption were obtained with Monod,Luedeking-Piret and Herbert-pirt models. The results showed that the kinetic model for the cell growth rate to ammonium concentration under ammonium ion restriction conditions was μ=0.36 S/(2.27+S),and the kinetic model between the specific biosynthesis rate of curdlan and the ammonium concentration was q_p=0.0612 S/(2.27+S)+0.14×(1-S/64.94),and the kinetic model of glucose consumption rate to the specific yield was q_s=1.5 μ+q_p+0.0625. The statistic physiological metabolic parameters on A. faecalis demonstrated that the optimal ammonium-limited concentration was 5.75 mg/L.And under the specific cell growth rate at 0.21 h~(-1),the highest yield and maximum specific production rate of cudlan could reach 0.85 g/L/h and 0.17 g/g/h,which was 118% and 200% higher than that under batch culture process respectively. It would significantly improve the efficiency of curdlan production.
引文
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[14]梁剑光,朱玲,徐正军.靛酚蓝-分光光度法测定发酵液中氨态氮含量研究[J].食品与发酵工业,2006,32(9):134-137.
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[16]杨勇杰,姜瑞芝,陈英红,等.苯酚-硫酸法测定杂多糖含量的研究[J].中成药,2005,27(6):706-708.
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[18]戚以政,夏杰,王炳武.生物反应工程[M].化学工业出版社,2009:9-23.
[19]Phillips K R,Lawford H G.Theoretical maximum and observed product yields associated with curdlan production by Alcaligenes faecalis[J].Canadian Journal of Microbiology,1983,29:1270-1276.
[20]Beard,Daniel A,Liang,et al.Energy balance for analysis of complex metabolic networks[J].Biophysical Journal,2002,83(1):79-86.
[21]吴剑荣,詹晓北,刘惠,等.氨水流加用于粪产碱杆菌热凝胶发酵[J].生物工程学报,2008,24(6):1035-1039.
[2]Wu D,Li A,Ma F,et al.Genetic control and regulatory mechanisms of succinoglycan and curdlan biosynthesis in genus Agrobacterium[J].Applied Microbiology and Biotechnology,2016,100(14):6183-6192.
[3]Kai A,Ishino T,Arashida T,et al.Biosynthesis of curdlan from culture media containing 13C-labeled glucose as the carbon source[J].Carbohydrate Research,1993,240(2):153-159.
[4]Maeda I,Saito H,Masada M,et al.Properties of gels formed by heat treatment of curdlan,a bacterial β-1,3-glucan[J].Journal of the Agricultural Chemical Society of Japan,2008,31(10):1184-1188.
[5]蒋樟丽,韩福霞,金泽霖,等.粪产碱杆菌AF01产可溶性胞外多糖发酵条件的优化[J].食品工业科技,2016,37(23):165-169.
[6]刘惠,詹晓北,吴剑荣.不同的底物流加方式对Alcaligenes faecalis发酵生产热凝胶的影响[J].生物技术,2008,18(4):78-81.
[7]孙永生,王磊,詹晓北,等.氮源NH4Cl浓度对粪产碱杆菌发酵生产热凝胶的影响[J].生物工程学报,2005,21(2):328-331.
[8]Ogawa K,Wanatabe T,Tsurugi J,et al.Conformational behavior of a gel-forming(1-3)-β-d-glucan in alkaline solution[J].Carbohydrate Research,1972,23(3):399-405.
[9]Yu L J,Wu J R,Zheng Z Z,et al.Changes in gene transcription and protein expression involved in the response of Agrobacterium sp.ATCC 31749 to nitrogen availability during curdlan production[J].Applied Biochemistry and Microbiology,2011,47(5):487-493.
[10]Zhang H T,Zhan X B,Zheng Z Y,et al.Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp.ATCC 31749 under simulated dissolved oxygen gradients conditions[J].Applied Microbiology and Biotechnology,2011,91(1):163-175.
[11]Varma A,Boesch B W,Palsson B O.Biochemical production capabilities of Escherichia coli[J].Biotechnology and Bioengineering,2010,42(1):59-73.
[12]Zhang Q,Sun J Y,Wang Z J,et al.Kinetic analysis of curdlan production by Alcaligenes faecalis with maltose,sucrose,glucose and fructose as carbon sources[J].Bioresource Technology,2018,259:319-324.
[13]史其峰,詹晓北,郑志永.粪产碱杆菌在不同底物限制性条件下连续培养[J].食品与生物技术学报,2008,27(5):67-72.
[14]梁剑光,朱玲,徐正军.靛酚蓝-分光光度法测定发酵液中氨态氮含量研究[J].食品与发酵工业,2006,32(9):134-137.
[15]Miller G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Chemistry,1959,31(3):426-428.
[16]杨勇杰,姜瑞芝,陈英红,等.苯酚-硫酸法测定杂多糖含量的研究[J].中成药,2005,27(6):706-708.
[17]Edwards J S,Palsson B O.The Escherichia coli MG1655 in silico metabolic genotype:Its definition,characteristics,and capabilities[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(10):5528-5533.
[18]戚以政,夏杰,王炳武.生物反应工程[M].化学工业出版社,2009:9-23.
[19]Phillips K R,Lawford H G.Theoretical maximum and observed product yields associated with curdlan production by Alcaligenes faecalis[J].Canadian Journal of Microbiology,1983,29:1270-1276.
[20]Beard,Daniel A,Liang,et al.Energy balance for analysis of complex metabolic networks[J].Biophysical Journal,2002,83(1):79-86.
[21]吴剑荣,詹晓北,刘惠,等.氨水流加用于粪产碱杆菌热凝胶发酵[J].生物工程学报,2008,24(6):1035-1039.