Efficient acetic acid production by repeated fed-batch fermentation using two fermentors

Summary In acetic acid fermentation, the number of viable cells decrease as the acetic acid concentration increases to more than about 40 g/l, which means that the productivity attainable by conventional fed-batch and repeated fed-batch operations using one fermentor is limited. In this paper, based on a fed-batch experiment using Acetobacter aceti 2096, a mathematical model was developed. The optimization carried out showed the superiority of repeated fed-batch operation using two fermentors. The performance evaluation was made with respect to productivity and product concentration. It was shown to be attractive in practice to use multiple fermentors, in particular for high product concentrations. Experiments were then conducted to ascertain the simulation results. Offprint requests to: T. Kobayashi     

Fed-batch production of citric acid by Candida lipolytica grown on n-paraffins

This study reports on the effects of fermentor agitation and fed-batch mode of operation on citric acid production from Candida lipolytica using n-paraffin as the carbon source. An optimum range of agitation speeds in the 800-1000 rpm range corresponding to Reynolds numbers of 50000-63000 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Application of multiple fed-batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. The three-cycle fed-batch system increased overall citric acid yields to 0.8-1.0 g citricacid/g n-paraffin, approximately a 100% improvement in product yield from those observed in the single cycle fed-batch system and a 200% improvement over normal batch operation. The three-cycle fed-batch mode of operation also increased the final citric acid concentration to 42 g/l from about 12 and 6g/l for single fed-batch cycle and normal batch modes of operation, respectively. Increased citric acid concentrations in three-cycle fed-batch mode was achieved at longer fermentation times.     

Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate

The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains performed better in fed-batch mode than when all hydrolysate was added at once. However, the difference between strain performances varied significantly in the two fermentation modes. Large differences were observed between strains during the fed-batch experiments in the in vitro ability to reduce the furan compounds furfural and 5-hydroxymethyl furfural (HMF). A common feature among the strains was the induction of NADPH-coupled reduction of furfural and HMF, with the exception of strain CBS 8066. This strain also performed relatively poorly in both batch and fed-batch fermentations. Strain TMB3000--previously isolated from spent sulphite liquor fermentation--was by far the most efficient strain with respect to specific fermentation rate in both pulse addition and fed-batch mode. This strain was the only strain showing a significant constitutive NADH-coupled in vitro reduction of HMF. The ability to induce NADPH-coupled reduction together with the level of the apparently constitutive NADH-coupled reduction appeared to be key factors for selecting a suitable strain for fed-batch conversion of lignocellulose hydrolysate.     

Improvement in the bioreactor specific productivity by coupling continuous reactor with repeated fed-batch reactor for acetone-butanol-ethanol production

In comparison to the different fermentation modes for the production of acetone, butanol and ethanol (ABE) researched to date, the continuous fermentation is the most economically favored. Continuous fermentation with two or more reactor cascade is reported to be the most efficient as it results in a more stable solvent production process. In this work, it is shown that a continuous (first-stage) reactor coupled to a repeated fed-batch (second stage) is superior to batch and fed-batch fermentations, including two-stage continuous fermentation. This is due to the efficient catalyst use, reported through the specific product rate and rapid glucose consumption rate. High solvents are produced at 19.4 g(ABE) l?1, with volumetric productivities of 0.92 g(butanol) l?1 h?1 and 1.47 g(ABE) l ?1 h?1. The bioreactor specific productivities of 0.62 and 0.39 g g?1(cdw) h?1 obtained show a high catalyst activity. This new process mode has not been reported before in the development of ABE fermentation and it shows great potential and superiority to the existing fermentation methods.     

Efficient production of gene products by repeated fed-batch fermentation using multiple fermentors

In the cultivation of genetically engineered microorganisms, cell growth decreases after induction, and it becomes eminent when a product such as β-galactosidase is harvested, indicating that conventional repeated fed-batch operation cannot be used to increase productivity. In this study, the use of multiple fermentors to overcome such difficulties was attempted. A mathematical model was developed based on fed-batch experiments using Escherichia coli JM103 harboring the pUR2921 plasmid encoding the β-galactosidase structural gene. Computer simulation demonstrated that improved performance can be attained by repeated fed-batch operation using multiple fermentors. Experiments were then conducted to ascertain the operating conditions.     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Multi-objective optimization in Aspergillus niger fermentation for selective product enhancement

A multi-objective optimization formulation that reflects the multi-substrate optimization in a multi-product fermentation is proposed in this work. This formulation includes the application of ε-constraint to generate the trade-off solution for the enhancement of one selective product in a multi-product fermentation, with simultaneous minimization of the other product within a threshold limit. The formulation has been applied to the fed-batch fermentation of Aspergillus niger that produces a number of enzymes during the course of fermentation, and of these, catalase and protease enzyme expression have been chosen as the enzymes of interest. Also, this proposed formulation has been applied in the environment of three control variables, i.e. the feed rates of sucrose, nitrogen source and oxygen and a set of trade-off solutions have been generated to develop the pareto-optimal curve. We have developed and experimentally evaluated the optimal control profiles for multiple substrate feed additions in the fed-batch fermentation of A. niger to maximize catalase expression along with protease expression within a threshold limit and vice versa. An increase of about 70% final catalase and 31% final protease compared to conventional fed-batch cultivation were obtained. Novel methods of oxygen supply through liquid-phase H₂O₂ addition have been used with a view to overcome limitations of aeration due to high gas–liquid transport resistance. The multi-objective optimization problem involved linearly appearing control variables and the decision space is constrained by state and end point constraints. The proposed multi-objective optimization is solved by differential evolution algorithm, a relatively superior population-based stochastic optimization strategy.     

A substrate feeding strategy--using an oxystat for L-phenylalanine fermentation by Corynebacterium glutamicum

Summary A substrate feeding strategy using an oxystat was first successfully applied to a fed-batch phenylalanine fermentation. The control method allowed the fermentation to be under low dissolved oxygen tension, which was favourable phenylalanine formation, and to be from substrate inhibition during the course of fed-batch operation. The final product concentration was 3 times higher than in a batch culture.     

Extractive fermentation by Zymomonas mobilis and the use of solvent mixtures

Summary A systematic and comprehensive screening of 1330 solvents, individually and as mixtures, was undertaken with the aim of improving on present extractive fermentation technology. The screening resulted in the selection of a solvent mixture of 5% by volume 4-heptanone and 95% Adol 85 NF (primarily oleyl alcohol). The performance of this solvent mixture in an extractive fed-batch fermentation configuration was compared with that of pure Adol 85 NF. The solvent mixture had a distribution coefficient 12% higher than that of pure Adol 85 NF and had no significant inhibitory effect on the fermentation under typical operating conditions.     

Fed-batch sorbose fermentation using pulse and multiple feeding strategies for productivity improvement

Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600 g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal. The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of 320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better strategy for fed-batch sorbose fermentation.     

微生物法生产二羟基丙酮的研究进展

以下综述了微生物发酵法制备二羟基丙酮的研究进展。利用微生物发酵法生产二羟基丙酮比化学合成法具有更大的优势,氧化葡萄糖酸杆菌是二羟基丙酮工业发酵生产中最有应用价值的菌株。发酵过程中底物、产物、氧气、菌体量等各种因素都会对二羟基丙酮产量产生影响,在各种发酵方式中反复流加工艺和固定化发酵工艺最有前途。重组菌株的构建和发酵工艺的优化是将来微生物发酵生产二羟基丙酮的发展方向。     

Investigation of the performance of fermentation processes using a mathematical model including effects of metabolic bottleneck and toxic product on cells

A number of recent research studies have focused on theoretical and experimental investigation of a bottleneck in a metabolic reaction network. However, there is no study on how the bottleneck affects the performance of a fermentation process when a product is highly toxic and remarkably influences the growth and death of cells. The present work therefore studies the effect of bottleneck on product concentrations under different product toxicity conditions. A generalized bottleneck model in a fed-batch fermentation is constructed including both the bottleneck and the product influences on cell growth and death. The simulation result reveals that when the toxic product strongly influences the cell growth and death, the final product concentration is hardly changed even if the bottleneck is removed, whereas it is markedly changed by the degree of product toxicity. The performance of an ethanol fermentation process is also discussed as a case example to validate this result. In conclusion, when the product is highly toxic, one cannot expect a significant increase in the final product concentration even if removing the bottleneck; rather, it may be more effective to somehow protect the cells so that they can continuously produce the product.     

利用甜菜糖蜜补料发酵生产丁醇

从土壤中分离出1株适合利用甜菜糖蜜发酵生产丁醇的丙酮丁醇梭菌(Clostridium acetobutylicum)2N,通过优化发酵条件,得到最适发酵温度为33℃,玉米浆最适添加量为15g/L,发现甜菜糖蜜中还原糖质量浓度高于50g/L时影响菌株的生长和溶剂生产。以补料分批发酵方式降低底物抑制,33℃发酵48h后,丁醇和总溶剂的质量浓度分别达到14.15g/L和19.65g/L,丁醇质量分数超过70%。     

A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza A virus Hemagglutinin protein

The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.     

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Monitoring of low concentrations of glucose in fermentation broth

A highly sensitive glucose sensor, operating in flow-injection analysis (FIA) mode, was developed for the detection of glucose in fermentation broth. The assay system is based upon the post-column reaction of the peroxide formed in the glucose-oxidase-catalysed reaction and subsequent spectrophotometric detection of the coloured product formed. The sensor system was characterised and calibrated using standard solutions, and later used for quantification of glucose in fermentation media. Two types of enzyme column were used: one operated in packed-bed mode and the other in expanded-bed mode. Both columns were integrated into a FIA system and were found to give good analytical results. Glucose concentrations as low as 0.1 mg/l and 5 mg/l could be detected in packed- and expanded-bed modes respectively. Glucose concentrations were measured during typical fed-batch fermentation conditions in this system, and the results are presented.     

Noninferior periodic operation of the metabolite producing biological reactor

The optimal periodic operation of the biological reactor in which the metabolites belonging to Type I or II in Gaden's classification are produced was investigated from the viewpoint of the multiobjective programming problem. In addition to the productivity of the desired product, its concentration and the conversion of the substrate which have a large influence on the performance of the separation process following the fermentation process were adopted as the objective functions. The growth of cells was assumed to be inhibited both by the substrate and the product, and the Luedeking-Piret model was employed for the specific production rate. The optimal periodic operation was determined by use of the optimization method due to Miele. It was clarified that the noninferior set for the periodic operation was generally composed of the repeated batch portion and the repeated fed-batch one.     

裂殖壶菌发酵产DHA过程呼吸参数的在线检测分析

利用尾气分析仪对发酵过程的尾气中的O2、CO2含量进行实时检测,建立了裂殖弧菌发酵生产DHA过程中的呼吸参数在线检测方法,实现了裂殖壶菌补料分批发酵过程及双阶段供氧控制发酵过程中的呼吸参数在线检测分析。通过呼吸参数在线检测分析,从氧消耗机制方面解释了双阶段氧传递控制工艺能获得较高生物量、油脂和DHA含量的原因,从而为该工艺过程提供了理论指导。根据发酵过程中菌体生长不同时期的呼吸参数的变化情况,建立了基于呼吸商变化的在线补料控制方法,设计了一种基于RQ-Stat的补料工艺。RQ-Stat补料方式最终获得的油脂含量、DHA产量和产率比间歇式补料工艺分别提高了11.58%、12.19%和11.40%。     

重组质粒pVAX1-PENK大规模制备研究

目的：建立质粒pVAX1-PENK的大规模制备2--艺。方法：对大肠杆菌工程菌DH5α-pVAX1-PENK进行补料发酵,利用自行发明的连续碱裂解过程对菌体进行裂解,经超滤浓缩后,用Sepharnse 6 Fast Flow层析柱分离DNA与RNA,再经Plasmidselect Xtra层析柱分离超螺旋质粒DNA与开环或线性质粒DNA,最后经Source 15Q层析柱精制质粒DNA。结果：发酵获得质粒pVAX1-PENK的产率为182mg／L,经碱裂解及层析分离后,最终制备的质粒DNA超螺旋比例大于98％,总回收率为60.5％,纯度（D260nm／D280nm）为1.8～2.0。结论：建立的质粒DNA生产工艺可以制备大量高纯度的质粒DNA,并避免了使用动物源性的酶及有毒试剂。     

Development of Suitable Solvent System for Downstream Processing of Biopolymer Pullulan Using Response Surface Methodology

Downstream processing is an important aspect of all biotechnological processes and has significant implications on quality and yield of the final product. Several solvents were examined for their efficacy on pullulan precipitation from fermentation broth. Interactions among four selected solvents and their effect on pullulan yield were studied using response surface methodology. A polynomial model was developed using D-optimal design and three contour plots were generated by performing 20 different experiments and the model was validated by performing optimization experiments. The results indicated that lower concentration of ethanol in combination with the other three solvents has resulted in higher yield of polymer from fermentation broth and the optimized solvent system was able to recover 1.44 times more pullulan as compared to the conventional ethanolic precipitation method. These observations may help in enhancing efficiency of pullulan recovery from fermentation broth and also result in reduced cost of production for the final product.