利用响应面法优化L-苏氨酸发酵条件

采用响应面设计方法对E.coli TRFC苏氨酸发酵培养基及培养条件进行了优化。用部分因子分析法研究了原始发酵培养基及培养条件对响应值的影响程度,发现蔗糖的质量浓度及接种量对苏氧酸产量的影响显著。利用最陡爬坡实验、中心旋转组合设计结合响应面分析确定了蔗糖的质量浓度及接种量(58.739 g/L,3.46%)。在优化条件下进行5L发酵罐实验,L-苏氨酸的产量达到121.20 g/L,比未优化条件下提高了12.43%。     

Development of an industrially stable process for L-threonine fermentation by an L-methionine-auxotrophic mutant of Escherichia coli

Escherichia coli KY10935, an L-methionine-auxotrophic mutant, has the ability to produce 100 g/l of L-threonine under limited conditions of DL-methionine, but occasionally exhibits overgrowth with a concomitant decrease in L-threonine production. We found that the growth of the producer depended not only on the availability of DL-methionine but also on that of iron, and that the dependence on iron caused abnormal fermentations in response to fluctuations in the iron concentration among lots of corn steep liquor, a cheap natural material used as the production medium. To overcome this problem, culture conditions were re-optimized with respect to the amounts of both dl-methionine and iron added in the medium so that the growth was controlled irrespective of the lot of corn steep liquor, to establish a stable industrial process with high productivity.     

pH值对聚唾液酸分批发酵的影响及补料发酵

研究了不同pH值对聚唾液酸发酵过程中菌体生长和产聚唾液酸的影响.发酵初期pH自然下降时有利于菌体生长,菌体生长对数期较长,最大菌体干重可达6.9g/L;发酵中后期pH控制在6.4时有利于聚唾液酸的延续合成,合成对数期比其它pH条件下的合成对数期延长了11h.动力学特性表现为部分相关模式,而在其它pH条件下,动力学特性表现为相关模式.对聚唾液酸的流加补料发酵进行了初步研究,最终使菌体干重达到11.16g/L,聚唾液酸产量达到2.606g/L.     

L-Lysine production by exponential feeding of L-threonine

The effect of L-threonine feeding in the production phase on L-lysine production by Brevibacterium flavum, which requires L-homoserine or L-threonine for cell growth, was investigated considering the concerted inhibition by L-threonine plus L-lysine, and the metabolism related to lysine production. Exponential feeding of L-threonine increased L-lysine production to 70 g/l about three times that without feeding. From the analysis of the metabolic flux, carbon flux of L-lysine synthesis pathway in the production phase after L-threonine feeding was higher than that in the growth phase. The results show that feeding of an inhibitory substance may increase the production, especially when the substance is necessary for the continuation of cell growth and/or production.     

5L发酵罐高密度培养番茄红素工程菌及其发酵条件优化

本研究对产番茄红素的工程菌W-05进行发酵条件优化。首先单因素实验确定培养基种类;培养温度;培养基中的较优碳源、氮源以及无机盐。然后根据单因素实验结果,设置响应面实验确定各因素之间的交互影响,响应面实验结果表明培养基各组分为:蛋白胨10.00 g/L、酵母浸出粉5.00 g/L、甘油7.80 mL/L、硝酸铵3.30 g/L、KH2PO4 1.80 g/L、Na Cl 11.62 g/L时,番茄红素得率达理论值为3.42 mg/L。在5 L发酵罐中,使用优化后的培养基高密度培养工程菌W-05。实验结果显示工程菌W-05高密度培养较优发酵条件为:p H值为7.0,溶氧百分数为20%左右及指数流加补料。此条件对比普通分批培养条件,菌体的生物量和番茄红素产量显著提高(p0.05)。优化发酵29 h后的菌体干重达到16.55 g/L,番茄红素得率为19.93 mg/L。这说明改善培养基成分及发酵条件能大幅提高工程菌的番茄红素得率。     

Continuous plant cell perfusion culture: bioreactor characterization and secreted enzyme production

Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase productivity that reached a plateau fluctuating around 490 units/l/d.     

棘孢木霉液体发酵条件优化

为研究棘孢木霉液体发酵最优条件,以菌丝体干重和孢子数为指标,通过单因素正交实验对棘孢木霉液体发酵培养基进行优化,并测定棘孢木霉的基本成分。实验结果表明最佳发酵条件为:黄豆粉0.25%,KH₂PO₄ 0.3%,MgSO₄ 0.15%,接种量8×106个/mL,装瓶量100 mL/250 mL,蛋白胨1.35%,葡萄糖3.5%,转速160 r/min,温度25 ℃,自然光照,pH7。此发酵条件可使棘孢木霉干重量达到19.453 g/L,孢子数达到4.4812×109个/mL。本研究为棘孢木霉的工业化生产降低成本,缩短发酵周期打下理论基础。     

添加剂对成曲酶活力和酱油质量的影响

以提高酱油成曲酶活力为目标,在发酵培养基的基础上,添加不同碳源、氮源、无机盐进行试验。结果表明,在葡萄糖0.25%、NaNO30.1%、ZnSO40.05%的条件下成曲的蛋白酶活力为2 383.83U/g干基,糖化酶活力为117.82U/g干基,分别较对照组提高了36.75%和46.02%。用该优化组和对照组进行酱油低盐固态发酵比较,优化组得到的酱油,总氮含量和氨基态氮含量等各项质量指标均优于对照组。     

马铃薯淀粉水解条件优化及油脂酵母培养研究

以马铃薯淀粉为底物,葡萄糖含量为评价指标,通过响应面法优化淀粉酶解工艺,将水解液用于发酵生产微生物油脂。最佳酶解条件为马铃薯淀粉质量浓度20 mg/mL,加酶量为干淀粉质量3.2%,α-淀粉酶、葡萄糖淀粉酶的质量比2.2∶1,pH 4.1,酶解时间4.1 h,得到最高葡萄糖含量为15.06 mg/mL。向此水解液中添加一定氮源和无机盐用于油脂酵母菌株Y004-2培养,在250 mL三角瓶中其生物量为14.43 g/L,油脂产量5.16 g/L,在5.0 L发酵罐中生物量为14.27 g/L,油脂产量5.10 g/L,其生物量和油脂产量均略高于以葡萄糖为碳源;且菌株Y004-2在以马铃薯淀粉水解液为碳源的培养基中所得油脂的组成与以葡萄糖为碳源的培养基中所得油脂的脂肪酸组成相同。     

响应面法优化重组大肠杆菌产ADI酶发酵培养基

在单因素优化的基础上,采用响应面分析方法对重组大肠杆菌生产精氨酸脱亚胺酶(ADI)的发酵培养基进行了优化。借助于SAS软件,结合Plackett-Burman和Box-Behnken实验设计对5种培养基组分进行优化。结果表明,最佳培养基组分为胰蛋白胨11.16 g/L,酵母提取物20 g/L,甘油6.3 g/L,磷酸氢二钾16.38 g/L,磷酸二氢钾2.31 g/L。采用优化后的培养基进行摇瓶发酵,ADI酶活达到5.7 U/m L发酵液,比初始培养基(3.72 U/m L发酵液)提高了1.53倍,比LB培养基(2.83 U/m L发酵液)提高了2.01倍。采用优化后的培养基在3 L发酵罐中进行发酵,ADI酶活达到15.17 U/m L发酵液,较LB培养基(4.32 U/m L发酵液)提高了3.51倍。