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.

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.

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）.

Fed-Batch Fermentation for Spinosad Production in an Improved Reactor

As a kind of aerobic bacteria, Saccharopolyspora spinosa exhibits a high demand for oxygen. In the fermentation process, the methods of increasing ventilation and improving agitation speed are usually adopted to achieve higher values of dissolved oxygen. These methods decrease the efficiency of spinosad biosynthesis. In this study, an improved reactor was designed to solve these problems. The exhaust gas reflux device, impellers, and baffles were improved. Furthermore, we established the kinetic models for the cell growth, substrate consumption and spinosad generation in batch fermentation process. The simulation results were in good agreement with the experimental data. Spinosad production reached 583.86 mg/L after employing the suitable feeding strategy by fed-batch fermentation in the improved reactor, whereas it was only 157.01 mg/L before optimization. The method described can provide insight to strengthen spinosad production and can be extended to the culturing process of filamentous aerobic bacteria. © 2017 Tianjin University and Springer-Verlag Berlin Heidelberg

Fed-batch培养中补料及接种密度对RABV-G蛋白表达量的影响

目的研究流加(Fed-batch)培养中补料及接种密度对狂犬病毒糖蛋白G(RABV-G)蛋白表达量的影响,初步建立RABV-G蛋白在重组中国仓鼠卵巢(CHO)细胞中Fed-batch培养的较优工艺方案。方法复苏培养1支能够稳定表达狂犬病毒RABV-G蛋白的重组CHO细胞,传代扩增后进行6种方案Fed-batch培养,从Fed-batch培养的第3天开始监测培养上清中目的蛋白表达量和细胞生长参数,第14天结束培养,分析比较不同策略所产生的目的蛋白表达量。结果本研究方案6中5%的Feed1补料及0.5×10~6cells/ml的起始接种密度在Fed-batch培养第14天,糖蛋白表达量达150 mg/L,显著高于其余5种方案,确立为优选方案。结论本研究建立了RABV-G蛋白在重组CHO细胞中流加(Fed-batch)培养的较优工艺方案,为重组狂犬病毒疫苗生产工艺研究提供基础。

Optimal control of a nonlinear state-dependent impulsive system in fed-batch process

Optimal control technique is crucial to improve the yield of microbial fermentation production.In this paper,we propose a nonlinear control system with state-dependent impulses,where the impulsive volume of feeding glycerol and the critical concentration of glycerol for occurring impulse are the control variables,to formulate 1,3-propanediol(1,3-PD)fed-batch production process.We also discuss a quantity of important properties for this control system.Then,we analyze the sensitivity of system state with respect to the kinetic parameters.We further propose a constrained optimal control model governed by the control system with state-dependent impulses.The existence of the optimal impulsive controls is established.For solving this problem,we utilize an exact penalty method to transform the problem into an optimization problem with only box constraints.Moreover,an improved differential evolution method is developed to seek the optimal impulsive strategy.Finally,numerical simulation results demonstrate that,by using the optimal impulsive strategies,final 1,3-PD concentration is considerably increased under the nominal parameter values and disturbances of kinetic parameters have significant effects on the optimal final 1,3-PD yield.

批式流加发酵中的鲁棒脉冲时滞最优控制

本文研究了批式流加发酵中的鲁棒脉冲时滞最优控制问题。首先,提出一个非线性状态依赖的脉冲时滞系统描述批式流加发酵甘油生产1,3-丙二醇(1,3-PD)过程。由于批式流加发酵过程中的动力学参数难以准确估计,本文建立了一个具有连续状态不等式约束的鲁棒脉冲时滞最优控制模型。这里,目标函数为终端时刻1,3-PD浓度及其关于动力学参数的灵敏性的加权和,控制向量为流加发生时甘油的临界浓度及每次流加时甘油的流加体积。然后,通过引入辅助脉冲系统,将该鲁棒最优控制问题转化为等价的标准最优控制问题。进一步,通过约束转换技术,将等价的最优控制问题转化为仅具有盒式约束的罚问题。最后,设计了一种并行差分进化算法求解转化后的罚问题。数值结果表明:当参数受到微小扰动时,尽管牺牲了少量的1,3-PD浓度,但是明显提高了系统的鲁棒性。

高温产2,3-丁二醇甲基芽孢杆菌的筛选及其发酵优化

2,3-丁二醇是一种典型的大宗化学品,广泛应用于化工、食品、医药、燃料等领域。生物法发酵制备2,3-丁二醇是当下的主要方法,然而,大多数报道的菌株为条件致病菌且发酵条件严格,限制了生物法制备2,3-丁二醇的发展。该研究以温泉底土壤为试验材料,经过分离、筛选与鉴定获得了1株高温条件下产2,3-丁二醇的甲基芽孢杆菌。通过摇瓶实验优化发酵工艺,确定最适发酵条件与最优培养基组成,利用优化后的发酵工艺甲基芽孢杆菌发酵24 h产物产量达到32.57 g/L。进一步进行上罐试验,基于不同发酵罐转速条件下甲基芽孢杆菌的产量积累情况,理性设计两阶段发酵策略,并以最优策略分批补料发酵制备2,3-丁二醇,2,3-丁二醇的产量在甲基芽孢杆菌发酵45 h时达到73.19 g/L,转化率达到90.25%。

热带假丝酵母β-紫罗兰酮工程菌的构建及其发酵优化

β-紫罗兰酮是一种来源于植物的不规则单萜化合物,具有良好的驱虫、抗菌、抗癌和抗氧化等生物活性,广泛应用于农业、食品、医药和香精香料等行业。热带假丝酵母是潜在的萜类高效合成底盘细胞。为了提高β-紫罗兰酮产量,该研究在课题组前期构建的β-紫罗兰酮工程菌的基础上,通过酶的亚细胞区室组合,构建了一株β-紫罗兰酮合成关键基因PhCCD1,同时在其胞质、过氧化物酶体和脂滴表达的β-紫罗兰酮工程菌PCL-01;然后,对其摇瓶发酵培养基装液量、培养基碳氮比、发酵温度和辅因子Fe^(2+)浓度等条件进行优化;最后进行5 L发酵罐小试。工程菌PCL-01的β-紫罗兰酮摇瓶产量为152.4 mg/L,较出发菌株增加50%;经过摇瓶发酵优化,β-紫罗兰酮产量为185.8 mg/L,提升22%;最后,在5 L发酵罐上可达403.9 mg/L。该研究对于其他萜类物质合成的菌株代谢改造具有借鉴意义,亦可为其他菌种发酵性能的研究提供参考价值。

补料分批发酵优化氧化葡糖杆菌UZ209发酵产酸工艺

醋酸菌作为氧化乙醇生成乙酸的主要菌种,其产酸能力备受关注。氧化葡糖杆菌具有不完全氧化糖和糖醇的特性,可以产生多种类的有机酸,提升食醋风味,但相较于醋酸杆菌属,其产酸能力和乙醇耐受能力较低,限制了其在食醋工业中的应用。本研究通过优化氧化葡糖杆菌UZ209的培养基成分和补料分批发酵方式提高其发酵液总酸含量。结果表明,最佳发酵培养基为:2%葡萄糖、1%酵母浸粉、8%乙醇。补料分批发酵可以显著提高氧化葡糖杆菌UZ209的生长和发酵特性,在乙醇含量为3%的液体发酵培养基中补料分批发酵的最佳方式为按照吸光值补料,最佳补料吸光值为OD6000.8±0.025。在此条件下,5%和6%乙醇补料分批发酵8 d的总酸含量分别为(7.42±0.16)和(6.95±0.45)g/100mL,分别比对照组提高了18.91%和15.04%。

The uncoupled microbial fed-batch fermentation optimization based on state-dependent switched system

In the actual microbial fermentation process,excessive or insufficient substrate can produce inhibitory effects on cells growth.The artificial substrate feeding rules by past experiences have great blindness to keep substrate concentration in a given appropriate range.This paper considers that alkali feed depends on pH value of the solution and glycerol feed depends on glycerol concentration of the solution in the uncoupled microbial fed-hatch fermentation process,and establishes a state-dependent switched system in which the flow rates of glycerol and alkali,the number of mode switches,the mode sequence and the switching times are prior unknown.To maximize the yield of target product 1,3-Propanediol(1,3-PD),we formulate a switching optimal control problem with the flow rates of glycerol and alkali,the number of mode switches,the mode sequence and the switching times as decision variables,which is a mixed-integer dynamic programming problem.For solving the mixed-integer dynamic programming problem,the control parametrization technique,the time scaling transformation and the embedded system technology are used to obtain an approximate parameter optimization problem.By using a parallel optimization algorithm,we obtain the optimal control strategies.Under the obtained optimal control strategies,the 1,3-PD yield at the terminal time is increased significantly compared with the previous results.

HIP1过表达对酿酒酵母工程菌麦角硫因产量的影响

麦角硫因是一种普遍存在于高等动植物体内的天然抗氧化剂,具有减少细胞氧化损伤、抗炎和治疗相关疾病的能力,被广泛应用于食品、化妆品和医药行业。与传统的植物提取法及化学合成法相比,通过微生物合成法制备麦角硫因具有周期短、成本低和产量高等优势。该研究以酿酒酵母Saccharomyces cerevisiae CEN.PK113-5D为出发菌株,利用CRISPR/Cas9系统向其基因组中引入异源酶基因,构建具有完整麦角硫因生物合成途径的酿酒酵母工程菌。为进一步提高其产量,过表达组氨酸特异性转运蛋白Hip1p后,在仅有0.5 mmol/L组氨酸供给条件下,菌株胞内游离组氨酸水平显著提高,且麦角硫因产量提高10%。最终经摇瓶发酵72 h产量达(6.88±0.11)mg/L,而在5 L罐补料分批发酵策略下,培养72 h后麦角硫因产量达237.34 mg/L。该研究为后续代谢工程改造产麦角硫因酿酒酵母工程菌株提供靶基因参考。