pH-stat控制流加培养红发夫酵母产虾青素

通过酸碱流加培养、混合碳源-氨水流加培养、混合碳源-尿素流加培养等pH-stat控制流加培养红发夫酵母产虾青素。pH-stat控制流加培养与分批培养相比较,生物量及虾青素产量都有不同程度地提高。从提高虾青素产量和降低生产成本综合考虑,混合碳源-氨水流加培养是最理想的方法,培养96 h虾青素产量最高达到19.94 mg/L。

重组海藻糖合成酶工程菌的pH-stat高密度发酵工艺研究

重组海藻糖合成酶工程菌的分批补料发酵中,用氨水调节培养液的pH为6.78～6.80,初糖耗尽后按培养液的pH从6.80起每升高0.01即开始补充葡萄糖至0.7g·L-1。据此补料策略可将发酵过程的乙酸积累控制在6.26g·L-1以下;补料发酵12h后,即获得细胞干重为51.5g·L-1的细胞高密度,细胞密度是分批培养的7.5倍;诱导后重组海藻糖合成酶表达率为15.2%,单位体积内的表达量是分批培养的4.5倍。

Effects of Fed-batch and Nitrogen-deficient Culture on Lipid Yield of Chlorella sp.

[Objective] This study was to realize high-density culture of Chlorella sp. as well as the culture with high lipid yield. [Method] Through analyzing the growth curve of Chlorella sp. cells, dextrose consumption curve, change curves of pH and dissolved oxygen, a fed-batch culture was conducted, followed by a nitrogen-deficient culture aiming at accumulating the lipids in Chlorella sp. cells when a high density of Chlorella sp. cells was obtained. [Result] After four batches of feeding were pro- vided, the biomass of Chlorella sp. reached up to 65.25 g/L, and the lipid content increased from 42.75% to 63.82% in Chlorella sp. cells, with the yield of 43.37 g/L in the following 12 hours of nitrogen-deficient culture. [Conclusion] Reasonable fed- batch can significantly improve the biomass of Chlorella sp., and the nitrogen-defi- cient culture further raises the lipid yield of Chlorella sp.

Process Control for Production of Human-like Collagen in Fed-batch Culture of Escherichia coli BL 21

Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)] and 19.8% (g·g-1 DCW) respectively.

Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB

The production of butyric acid by Clostridium butyricum ZJUCB at various pH values was investigated. In order to study the effect of pH on cell growth, butyric acid biosynthesis and reducing sugar consumption, different cultivation pH values ranging from 6.0 to 7.5 were evaluated in 5-L bioreactor. In controlled pH batch fermentation, the optimum pH for cell growth and butyric acid production was 6.5 with a cell yield of 3.65 g/L and butyric acid yield of 12.25 g/L. Based on these results, this study then compared batch and fed-batch fermentation of butyric acid production at pH 6.5. Maximum value (16.74 g/L) of butyric acid concentration was obtained in fed-batch fermentation compared to 12.25 g/L in batch fermentation. It was concluded that culti- vation under fed-batch fermentation mode could enhance butyric acid production significantly (P<0.01) by C. butyricum ZJUCB.

Run-to-run Optimization for Fed-batch Fermentation Process with Swarm Energy Conservation Particle Swarm Optimization Algorithm

An iterative optimization strategy for fed-batch fermentation process is presented by combining a run-to-run optimization with swarm energy conservation particle swarm optimization (SEC-PSO). SEC-PSO, which is designed with the concept of energy conservation, can solve the problem of premature convergence frequently appeared in standard PSO algorithm by partitioning its population into several sub-swarms according to the energy of the swarm and is used in the optimization strategy for parameter identification and operation condition optimization. The run-to-run optimization exploits the repetitive nature of fed-batch processes in order to deal with the optimal problems of fed-batch fermentation process with inaccurate process model and unsteady process state. The kinetic model parameters, used in the operation condition optimization of the next run, are adjusted by calculating time-series data obtained from real fed-batch process in the run-to-run optimization. The simulation results show that the strategy can adjust its kinetic model dynamically and overcome the instability of fed-batch process effectively. Run-to-run strategy with SEC-PSO provides an effective method for optimization of fed-batch fermentation process.

Production of Ammonium Lactate by Fed-batch Fermentation of Rhizopus oryzaefrom Corncob Hydrolysate

L-(+)-Lactic acid production from corncob hydrolysate as a cheap carbohydrate source by fed-batch fermentation of Rhizopus oryzaeHZS6 was studied. After 96 h of fermentation in a 5 L fermentor, the final concentration of ammonium L-(+)-lactate, average productivity(based on initial xylose concentration) and maximum dry cell weight were 132.4 g/L, 1.38 g/(L·h), and 8.9 g/L respectively. The optical purity of L-(+)-lactate was 98.8%.

Improved 5-Aminolevulinic Acid Production with Recombinant Escherichia coli by a Short-term Dissolved Oxygen Shock in Fed-batch Fermentation

5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen （DO） shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombi-nant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase （ALAS） activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 rain in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 rain. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 （72 mmol.L-1）, which is the highest yield in the fermentation broth reported up to now.

Strategies for Optimizing Feed Rate of Fed-Batch Yeast Fermentation by Fuzzy-Neural Network

In this paper,a novel fuzzy neural network model,in which an adjustable fuzzy sub-space was designed by uniform design,has been established and used in fed-batch yeast fermentationas an example.A brand-new optimization sub-network with special structure has been built andgenetic algorithm,guaranteeing the optimization in overall space,is introduced for the feed rateoptimization.On the basis of the model network,the optimal substrate concentration and theoptimal amount of fed-batch at different periods have been studied,aided with the optimizationnetwork and the genetic algorithm separately.The above results can be used as a basis for theestablishment of a fuzzy neural network controller.

NonliNonlinear GPC with In-place Trained RLS-SVM Model for DOC Control in a Fed-batch Bioreactor

In this study, Saccharomyces cerevisiae (baker's yeast) was produced in a fed-batch bioreactor at the optimal dissolved oxygen concentration (DOC) and growth medium temperature. However, it is very difficult to control the DOC using conventional controllers because of the poorly understood and constantly changing dynamics of the bioprocess. A generalized predictive controller (GPC) based on a nonlinear autoregressive integrated moving average exogenous (NARIMAX) model is presented to stabilize the DOC by manipulation of air flow rate. The NARIMAX model is built by an improved recursive least-squares support vector machine, which is trained by an in-place computation scheme and avoids the computation of the inverse of a large matrix and memory reallocation. The proposed nonlinear GPC algorithm requires little preliminary knowledge of the fermentation process, and directly obtains the nonlinear model in matrix form by using iterative multiple modeling instead of linearization at each sampling period. By application of an on-line bioreactor control, experimental results demonstrate the robustness, effectiveness and advantages of the new controller.

On-line Scheduling Algorithm for Penicillin Fed-batch Fermentation

An on-line scheduling algorithm to maximize gross profit of penicillin fed-batch fermentation is proposed. According to the on-line classification method, fed-batch fermentation batches are classified into three categories. Using the scheduling strategy, the optimal termination sequence of batches is obtained. Pseudo on-line simulations for testing the proposed algorithm with the data from industrial scale penicillin fermentation are carried out.

Remote Control of Fed-Batch Fermentation Systems

Bioreactor operation requires continuous monitoring of fermentation parameters and real-time control over bioreactor devices. Remote monitoring and control of the bioreactor＇s computer via the Internet avoids the necessity of personnel being continually onsite during operation. A two liter Sartorius-stedim Biostat~ A Plus fermentation system was networked and interfaced with the commercial software from GoToMyPC to allow remote control of the fermentation system utilizing the internet. The fermentation vessel was equipped with hardware calibrated for monitoring and controlling culture parameters during experimentations. The uniform resource locator controlled night-vision web camera allowed continuous monitoring of the glass fermentation vessel during the day and at night. The main window screen of the laboratory computer can be securely accessed from any portable device （i.e. laptop） capable of establishing an Internet connection and executing the commercial software from GoToMyPC. The secured internet protocol address, provided by GoToMyPC, assures that the system can only be controlled by authorized users who have been given access to the account. This interface permits the remote control of the Biostat A Plus fermentation system and possibly other automated or potentially automated culturing systems at the convenience of the user（s）.

Isolation of enriched-yielders and fed-batch production of alkaline protease from the newly isolated <i>Bacillus</i>sp. BHA

An alkalophilc and thermophilic Bacillus sp. BHA that produced a thermostable alkaline protease was isolated from decaying protein substrates. The isolate was found to grow in pH range 7 - 11 with an optimum pH 9.0 and temperature up to 55℃. The activity of alkaline protease of Bacillus sp. BHA (68.98 APU/ml) was found higher than the standard strains of Bacillus amyloliquefaciens MTCC 610 (8.98 APU/ml) and Bacillus subtilis MTCC 8349 (12.14 APU/ml, used in this study, and was comparable (68.98 APU/ml, equivalent to 30.38 APU/mg) to the activity of the commercially produced standard protease procured from Novo Nordisk, Denmark (30.35 APU/mg). Hence, the proteolytic activity produced by this isolate was further investigated in batch and fed-batch process. Sucrose was the best carbon source for the production of protease activity by that isolate. Different organic nitrogen sources (casein, peptone and beef extract) at 1% (w/v) with varying levels of sucrose (1% - 4% w/v) initially repress enzyme synthesis. The duration and extent of repression decreased with increased concentration of sucrose. Maximum protease activity was found in basal medium with 4% (w/v) sucrose and 1% (w/v) yeast extract. Yeast-extract was thought to be an inducer of enzyme synthesis. Further, the basal medium was unique with respect to the enzyme production, as protease production was growth associated with the peak enzyme production being detected at the time of maximum growth. Interestingly, a rise in 34.2% (104.86 APU/ml) of protease activity was detected at incubation temperature of 50℃ and when culture filtrate was assayed at 60℃, signifying a high temperature stability of the produced protease by this isolate. Additional studies on the enzyme characterization were resulted in recognition of highly significant properties of the activity towards casein at pH 9.0 and stability at high temperature with retention of 96% the enzyme activity at 60℃. The parametric study under feed intervals had enabled improvement in the maximum protease activities attainable from batch cultures in excess of 21.78% and 26.32% via two feeding strategies. A small continual increase in enzyme activity (132.46 APU/ml during 24 h - 120 h) and enhancement in protease production in excess of 36.84% was observed by fed-batch process than the batch experiment.

恒pH补料分批培养技术培养谷胱甘肽合成酶系

采用一种新的补料分批培养技术 ,培养重组大肠杆菌生产谷胱甘肽合成酶系 .在分批培养和补料分批培养期间 ,采用不同的pH控制模式 :在发酵前期采用分批培养 ,加入碱以补偿pH值的降低 ;而在发酵后期 ,采用一种新的恒 pH补料分批培养方式 ,加入葡萄糖和碱调节发酵液的 pH .在这种模式中 ,根据培养过程中的pH变化确定pH参数 .实验结果表明 :同时设置发酵液的 pH上限和下限可避免恒 pH补料分批培养过程中葡萄糖的周期性缺乏问题 ;对于缓冲能力不同的发酵液 ,应设置不同的pH参数来进行

恒pH葡萄糖流加方法培养重组大肠杆菌生产人肿瘤坏死因子-α及其动力学研究

研究了一种新型的流加方法──恒pH流加葡萄糖法，用于培养重组大肠杆菌生产人肿瘤坏死因子-α。流加后培养液中菌体OD(600)达到9．0，是在LB培养基培养的15倍，而α-肿瘤坏死因子的比活保持（1．05±0．11）×105u/mg，并建立了菌体生长的动力学方程。

核桃蛋白水解物水解度测定方法比较

为比较测定核桃分离蛋白(WPI)水解物水解度最佳方法,采用Alcalase 2.4L、Protamex水解WPI,对不同时间段核桃蛋白水解液水解度测定方法进行比较。结果表明,采用内切蛋白酶Alcalase 2.4L水解WPI时,pH-Stat法和TNBS法测定结果基本一致,而甲醛滴定法测定结果偏低;而用复合蛋白酶Protamex水解WPI时,pH-Stat法和甲醛滴定法测定结果接近,但且均低于TNBS法,TNBS法测定结果与另外两法差异显著(ρ<0.05)。由于制备核桃多肽主要采用内切蛋白酶,所以可认为pH-Stat法测定WPI水解液水解度较为准确。

Study on the Substrate Inhibition and the Method of Disarming Inhibition in the Fermentation Process of Hyaluronic Acid

[Objective] The research aimed to search and obtain the fermentation method of hyaluronic acid with the high yield.[Method] In the production process of hyaluronic acid by the microbial fermentation method,the influence of substrate concentration on the fermentation was explored.[Result] The glucose had the biggest influence on the hyaluronic acid fermentation.The low concentration of glucose only could synthetize a little bacteria and hyaluronic acid,and the high concentration of glucose could inhibit the growth of bacteria and the formation of hyaluronic acid.Via the exploratory experiment,the fed-batch fermentation could disarm the substrate inhibition.The concrete method was adding 2% glucose before the start of fermentation,and adding 2% glucose at the 14th hour of fermentation,and adding 2% glucose again at the 22nd hour of fermentation.[Conclusion] In the microbial fermentation process,the fed-batch fermentation could disarm the substrate inhibition and obtain the hyaluronic acid with the larger relative molecular mass and the higher yield.

酪蛋白水解物对轮枝链霉菌SK-1产谷氨酰胺转胺酶的影响

酪蛋白经胰酶水解后 ,可以被轮枝链霉菌SK - 1所利用 .采用pH -STAT法得到了DH=3、DH =6、DH =9、DH =1 2等 4个水解度的酪蛋白水解物 .当酪蛋白水解物作氮源时 ,其水解度 (即肽相对分子质量的大小 )对轮枝链霉菌SK - 1产谷氨酰胺转胺酶酶影响很大 ,其中以DH =3的酪蛋白水解物效果最好 ,而且肽的浓度对轮枝链霉菌SK - 1产谷胺酰胺转胺酶也有一定的影响 ,其中以

不同水解度的豆饼粉对轮枝链霉菌发酵生产谷氨酰胺转胺酶的影响

谷氨酰胺转胺酶在食品行业应用广泛,其可用轮枝链霉菌发酵生产。豆饼粉是一种廉价的富含蛋白质的工业原料,直接用作氮源不能被轮枝链霉菌利用。本文使用pH-STAT法,将豆饼粉水解成DH为3%、5%、7%、9%和12%等一系列的酶解液,并将其用作轮链霉菌的氮源。研究发现,在培养基中添加质量浓度为20g/L、DH=5%的豆饼粉酶解液可显著提高谷氨酰胺转胺酶的产量,其酶活为4.2U/mL,具有一定的应用价值。

碱性蛋白酶对螺旋藻活性蛋白的酶解研究

螺旋藻中的藻蓝蛋白作为一种具有生物活性功能的天然藻蓝素蛋白,其理论研究和应用近年来得到广泛的重视。通过测定螺旋藻破碎后上清液中蛋白质含量及其活性藻蓝蛋白含量,建立了一种提取螺旋藻蛋白质较适的方法和条件,最高提取率达到94.6%,活性藻蓝蛋白的得率为9.87%。利用正交实验,对提取的胞内蛋白质采用pH—stat方法进行碱性蛋白酶的水解,确定了其最佳酶促水解条件,即pH值为7.0,酶底物比为2.2%,反应时间为160min,反应温度为50℃,此时水解度与htot之比为120.20%,N溶解指数为27.73%。该研究对螺旋藻蛋白中生物活性物质的开发奠定了基础。