米根霉高密度半连续发酵L-乳酸初步研究
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摘要
微生物的高密度半连续发酵,可以有效提高菌体的发酵密度和发酵液的体积产率,从而缩短生产周期、降低生产成本。本文通过对米根霉菌株的高密度培养与半连续发酵产L-乳酸效果的研究,探索适于高密度半连续发酵的培养基组成和培养条件,确定补料分批法高密度培养米根霉的最佳方式与参数,同时对米根霉高密度半连续发酵的产酸强度及其动力学特性进行研究,建立发酵过程动力学模型。具体结论如下:
(1)通过单因素实验结合正交试验的方法,确定发酵培养基组成主要参数为:葡萄糖160g/L,(NH_4)_2SO_4 6g/L,NaH_2PO_4 0.150g/L,KH_2PO_40.150g/L,MgSO_4·7H_2O 0.25g/L,ZnSO_4·7H_2O 0.22g/L;操作条件为:接种量5%,装液量75ml/250ml,接种时间为24h,发酵温度32℃,摇床转速200r/min时,首批发酵的米根霉生物量可达到12.503g/L。
(2)研究了恒速流加、变速流加和葡萄糖浓度反馈流加等方式对发酵过程中米根霉菌体生物量累积的影响情况,结果表明以葡萄糖浓度反馈流加方式最佳;通过对补料参数进行优化,确定出碳源为葡萄糖160g/L,补料葡萄糖与硫酸铵质量比为20:1,补料时间24h-60h;采用基于葡萄糖浓度的反馈流加方式,在15L发酵罐中初始装液7L,控制温度32℃,转速200r/min,每升发酵液通气量为2L/min时,乳酸产量为117g/L,生物量可达到18.54g/L,比分批培养提高了91.9%。
(3)通过对米根霉高密度半连续产酸效率的考察获得了发酵最佳方式和不同阶段的培养基组成。采用葡萄糖浓度反馈流加进行第一批次发酵,提高菌体生物量;从第2至第5批次补入Ⅱ号培养基以继续累积生物量,培养基组成为:葡萄糖100g/L,(NH_4)_2SO_4 4g/L, KH_2PO_4 0.150g/L,MgSO_4·7H_2O 0.22g/L,ZnSO_4·7H_2O 0.33g/L;从第6批次开始,补加Ⅰ号培养基以提高产酸,培养基组成为葡萄糖100g/L,(NH_4)_2SO_4 2g/L,KH_2PO_40.10g/L,MgSO_4·7H_2O 0.22g/L,ZnSO_4·7H_2O 0.33g/L。发酵15批次,平均产酸强度为3.045g/(L·h),高于分批发酵的1.23g/(L·h)。
(4)建立的米根霉高密度发酵产L-乳酸的发酵过程动力学模型为:
微生物生长模型:(?)
乳酸生成模型:(?)
验证结果表明,该动力学模型能够较好的描述实验过程。
High cell density and semi-continuous fermentation of microorganism can achieve such a result of shortening the fermentation time and reducing the cost by increasing the cell density and volumetric yields efficiency. By the study of high cell density culturing of Rhizopus oryzae and production of L-lactic acid by semi-continuous fermentation, the culture medium ingredient and fermentation condition were optimized, the optimum method and parameter of fed-batch culture were determinated, the fermentation strength and the kinetics were researched as well. The main results were as following:
(1) The culture medium ingredient and fermentation condition were optimized by the combination of single factor experiments and orthogonal experiments. The results were as following: When the key parameters being glucose 160g/L, (NH_4)_2SO_4 6g/L, NaH_2PO_4 0.150g/L, KH_2PO_4 0.150g/L, MgSO_4·7H_2O 0.25g/L , ZnSO_4·7H_2O 0.22g/L; Inoculum size was 5%, inoculums age was 24h, and work volume was 40%;the biomass of Rhizopus oryzae in the first batch of fermentation could achieve 12.503 g/L。
(2) The biomass of Rhizopus oryzae were studied when culture medium were feeding in constant speed, in different speed and feedback by the concentration of glucose which was proved to be the best strategy. The parameters of feeding culture were optimized as follows: the glucose/ammonium sulfate ratio was 20:1(w/w), culture medium feeding speed was feedback by the concentration of glucose from 24h to 60h, the initial work volume was 7L in 15L bioreactor, temperature was 32℃, Rotation rate was 200 r/min, aeration velocity was 2 L/min per liter. The yield of L-lactic acid is 117 g/L; the biomass is 18.54 g/L, which increased by 91.9% over that of batch culture.
(3) The ferment method and the culture medium ingredient in different phase were optimized. Adopt feedback-feeding culture to increase biomass in the first batch of fermentation. Culture mediumⅡwas added to the reactor from 2~(nd) to 5~(th) batch of fermentation to increase biomass further which was composed of glucose100g/L, (NH_4)_2SO_4 4g/L,KH_2PO_4 0.150g/L,MgSO_4·7H_2O 0.22g/L and ZnSO_4·7H_2O 0.33g/L.
Culture mediumⅠwas added to the reactor from 6~(th) batch of fermentation to the production of L-lactic acid which was composed of glucose100g/L, (NH_4)_2SO_4 2g/L, KH_2PO_4 0.10g/L, MgSO_4·7H_2O 0.22g/L, ZnSO_4·7H_2O 0.33g/L. The fermentative strength is 3.045g/(L·h) in average.
(4) Kinetic models of L-lactic acid production by high cell density culture of Rhizopus oryzae were established.
The model of Rhizopus oryzae growth : (?)
The model of L-lactic acid yield : (?)
The results showed that the models could describe the process exactly.
(1)通过单因素实验结合正交试验的方法,确定发酵培养基组成主要参数为:葡萄糖160g/L,(NH_4)_2SO_4 6g/L,NaH_2PO_4 0.150g/L,KH_2PO_40.150g/L,MgSO_4·7H_2O 0.25g/L,ZnSO_4·7H_2O 0.22g/L;操作条件为:接种量5%,装液量75ml/250ml,接种时间为24h,发酵温度32℃,摇床转速200r/min时,首批发酵的米根霉生物量可达到12.503g/L。
(2)研究了恒速流加、变速流加和葡萄糖浓度反馈流加等方式对发酵过程中米根霉菌体生物量累积的影响情况,结果表明以葡萄糖浓度反馈流加方式最佳;通过对补料参数进行优化,确定出碳源为葡萄糖160g/L,补料葡萄糖与硫酸铵质量比为20:1,补料时间24h-60h;采用基于葡萄糖浓度的反馈流加方式,在15L发酵罐中初始装液7L,控制温度32℃,转速200r/min,每升发酵液通气量为2L/min时,乳酸产量为117g/L,生物量可达到18.54g/L,比分批培养提高了91.9%。
(3)通过对米根霉高密度半连续产酸效率的考察获得了发酵最佳方式和不同阶段的培养基组成。采用葡萄糖浓度反馈流加进行第一批次发酵,提高菌体生物量;从第2至第5批次补入Ⅱ号培养基以继续累积生物量,培养基组成为:葡萄糖100g/L,(NH_4)_2SO_4 4g/L, KH_2PO_4 0.150g/L,MgSO_4·7H_2O 0.22g/L,ZnSO_4·7H_2O 0.33g/L;从第6批次开始,补加Ⅰ号培养基以提高产酸,培养基组成为葡萄糖100g/L,(NH_4)_2SO_4 2g/L,KH_2PO_40.10g/L,MgSO_4·7H_2O 0.22g/L,ZnSO_4·7H_2O 0.33g/L。发酵15批次,平均产酸强度为3.045g/(L·h),高于分批发酵的1.23g/(L·h)。
(4)建立的米根霉高密度发酵产L-乳酸的发酵过程动力学模型为:
微生物生长模型:(?)
乳酸生成模型:(?)
验证结果表明,该动力学模型能够较好的描述实验过程。
High cell density and semi-continuous fermentation of microorganism can achieve such a result of shortening the fermentation time and reducing the cost by increasing the cell density and volumetric yields efficiency. By the study of high cell density culturing of Rhizopus oryzae and production of L-lactic acid by semi-continuous fermentation, the culture medium ingredient and fermentation condition were optimized, the optimum method and parameter of fed-batch culture were determinated, the fermentation strength and the kinetics were researched as well. The main results were as following:
(1) The culture medium ingredient and fermentation condition were optimized by the combination of single factor experiments and orthogonal experiments. The results were as following: When the key parameters being glucose 160g/L, (NH_4)_2SO_4 6g/L, NaH_2PO_4 0.150g/L, KH_2PO_4 0.150g/L, MgSO_4·7H_2O 0.25g/L , ZnSO_4·7H_2O 0.22g/L; Inoculum size was 5%, inoculums age was 24h, and work volume was 40%;the biomass of Rhizopus oryzae in the first batch of fermentation could achieve 12.503 g/L。
(2) The biomass of Rhizopus oryzae were studied when culture medium were feeding in constant speed, in different speed and feedback by the concentration of glucose which was proved to be the best strategy. The parameters of feeding culture were optimized as follows: the glucose/ammonium sulfate ratio was 20:1(w/w), culture medium feeding speed was feedback by the concentration of glucose from 24h to 60h, the initial work volume was 7L in 15L bioreactor, temperature was 32℃, Rotation rate was 200 r/min, aeration velocity was 2 L/min per liter. The yield of L-lactic acid is 117 g/L; the biomass is 18.54 g/L, which increased by 91.9% over that of batch culture.
(3) The ferment method and the culture medium ingredient in different phase were optimized. Adopt feedback-feeding culture to increase biomass in the first batch of fermentation. Culture mediumⅡwas added to the reactor from 2~(nd) to 5~(th) batch of fermentation to increase biomass further which was composed of glucose100g/L, (NH_4)_2SO_4 4g/L,KH_2PO_4 0.150g/L,MgSO_4·7H_2O 0.22g/L and ZnSO_4·7H_2O 0.33g/L.
Culture mediumⅠwas added to the reactor from 6~(th) batch of fermentation to the production of L-lactic acid which was composed of glucose100g/L, (NH_4)_2SO_4 2g/L, KH_2PO_4 0.10g/L, MgSO_4·7H_2O 0.22g/L, ZnSO_4·7H_2O 0.33g/L. The fermentative strength is 3.045g/(L·h) in average.
(4) Kinetic models of L-lactic acid production by high cell density culture of Rhizopus oryzae were established.
The model of Rhizopus oryzae growth : (?)
The model of L-lactic acid yield : (?)
The results showed that the models could describe the process exactly.
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