Fed-batch culture of yeast Saccharomyces cerevisiae with a DO-stat method by a fuzzy controller

In this research a fuzzy controller was built to perform fed-batch cultures of Saccharomyces cerevisiae with a DO-stat method. The basic principle of fed-batch culture employing the DO-stat method is that a rapid increase of dissolved oxygen concentration due to a lack of substrate (the DO signal) is used as an indicator for substrate feeding. The proposed fuzzy controller can diagnose the state of fermentation and determine a proper feed rate of substrate for the culture of high density and high yield. The results indicate that cell concentration reached to 110?g/l and residual sugar kept below the level of 0.05?g/l.     

Optimum substrate feed rate in fed-batch culture with the DO-stat method

In a fed-batch culture employing the DO-stat method, a rapid increase of dissolved oxygen concentration due to a lack of substrate (the DO signal) is used as an indicator for substrate feeding. The amount of substrate to be fed in response to the appearance of the DO signal is a very important factor for obtaining an optimal fed-batch culture. To select the optimum amount of substrate to be fed at the DO signal, a calculative procedure based on the growth yield, and the relationship between the specific growth rate and substrate concentration is proposed. This procedure is demonstrated in fed-batch cultures of Protaminobacter ruber (a methanol-utilizing bacterium) and Candida brassicae (an ethanol-utilizing yeast). The optimum feed rates calculated with the procedure, 2.5 ml (methanol/l/signal) for P. ruber and 5 ml (ethanol/l/signal) for C. brassicae, both gave good agreement with the cultivation results.     

Fermentation characterization of an L-tryptophan producing Escherichia coli strain with inactivated phosphotransacetylase

Acetate is a primary inhibitory metabolite in Escherichia coli cultivation which is detrimental to bacterial growth and the formation of desired products. It can be derived from acetyl coenzyme A by the phosphotransacetylase (Pta)–acetate kinase (AckA) pathway. In this study, the fermentation characteristics of Pta mutant strain E. coli TRTHΔpta were compared with those of the control strain E. coli TRTH in a 30-L fermentor. The effects of glucose concentration and dissolved oxygen (DO) level were investigated, and the results suggest that DO and glucose concentration are vital influencing parameters for the production of L-tryptophan. Based on our experimental results, we then tested a DO-stat fed-batch fermentation strategy. When DO was controlled at about 20 % during L-tryptophan fermentation in the DO-stat fed-batch system, the pta mutant was able to maintain a higher growth rate at the exponential phase, and the final biomass and L-tryptophan production were increased to 55.3 g/L and 35.2 g/L, respectively. Concomitantly, as the concentration of acetate decreased to 0.7 g/L, the accumulation of pyruvate and lactate increased in the mutant strain as compared with the control strain. This characterization of the recombinant mutant strain provides useful information for the rational modification of metabolic fluxes to improve tryptophan production.     

Extended operation of a pressurized 75-L bioreactor for shLkn-1 production by Pichia pastoris using dissolved oxygen profile control

In this study, a dissolved oxygen (DO)-stat fed-batch process was conducted in a pressurized 75-L bioreactor, resulting in the production of the short version of human leukotactin-1 (shLkn-1) using Pichia pastoris as the host, with control of the DO-stat profile and an extension of the recombinant shLkn-1 production phase. By regulation of the exhaust-gas valve, we were able to maintain the vessel pressure at up to 120 kPa, in order to overcome DO limitations associated with the use of the DO-stat. The lowest DO value was adjusted by varying the feed pump speed, allowing us to control the DO-stat profile. This principle was successfully applied to both glycerol feeding during the growth phase and methanol feeding for the induction of shLkn-1. The extension of the methanol induction phase to a total of 192 h of culture time resulted in a shLkn-1 concentration of 2.5 g/L, and a total of 102 g of cumulative production. During this extended induction period, the C-terminal residue of shLkn-1 was truncated and this was confirmed by both reverse-phase HPLC and mass spectrometry.     

Dissolved-oxygen-stat fed-batch fermentation of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112

This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.     

Fed-batch sorbitol to sorbose fermentation by A. suboxydans

Batch kinetics for sorbitol to sorbose bioconversion was studied at 20% sorbitol concentration. The culture featured 90% conversion of sorbitol to sorbose in 20 hours. Increasing the initial substrate concentration in the bioreactor decreased the culture specific growth rate. At 40% initial sorbitol concentration no culture growth was observed. The batch kinetics and substrate inhibition studies were used to develop the Mathematical Model of the system. The model parameters were identified using the original batch kinetic data (S _o =20%). The developed mathematical model was adopted to fed-batch cultivation with the exponential nutrient feeding. The fed-batch model was simulated and implemented experimentally. No substrate inhibition was observed in the fed-batch mode and it provided an overall productivity of 12.6?g/l-h. The fed-batch model suitably described the experimentally observed results. The model is ready for further optimization studies.     

Astaxanthin production by Phaffia rhodozymaenhanced in fed-batch culture with glucose and ethanol feeding

Of various carbon sources, examined for the cultivation of Phaffia rhodozyma, ethanol enhanced the astaxanthin content but severely decreased growth. Therefore, high cell mass was obtained by glucose fed-batch culture with pH-stat, and the ethanol feeding was performed based on DO-stat. As a result of this two-stage fed-batch cultivation, 30 g dry cells per liter were obtained, and the astaxanthin content reached 0.72 mg/g, which was 2.2-fold higher than that without ethanol feeding.     

High cell density cultivation of Pseudomonas putida strain HKT554 and its application for optically active sulfoxide production

Culture conditions with Pseudomonas putida strain HKT554, expressing naphthalene dioxygenase, known as the biocatalyst showing wide substrate specificity, were optimized for high cell density cultivation (HCDC). Culture in a medium TK-B modified from that for HCDC of Escherichia coli with glucose fed-batch and dissolved oxygen stat resulted in a high cell density growth of 114 g dry cell/l at 40 h of cultivation. This system was further applied for S-(+)-methyl phenyl sulfoxide (MPSO) production from methyl phenyl sulfide. Addition of nonpolar organic solvent, such as n-hexadecane, greatly enhanced the MPSO production due to the prevention of substrate evaporation, resulting in a MPSO production up to 39 mM in 30 h with a conversion rate of 95.7 mol%.     

Fed-batch culture automated by uses of continuously measured cell concentration and culture volume

An automated control method of fed-batch culture in which the nutrient feed rate was determined from continuously measured cell concentration and culture broth volume was developed. Theoretical background was elucidated, from which it was found that the method is unique in that it controls specific substrate consumption rate of the microorganism. The method was experimentally applied to the fed-batch cultures of recombinant Escherichia coli HB101. It was observed that the specific substrate feed rate affects not only the specific growth rate but also the growth yield. If some conditions are satisfied, this type of automated fedbatch culture can be applied widely to any microbial systems and seems especially useful when the culture medium is composed of natural complex nutrient(s) because their concentrations are very difficult to detect and control.     

可溶性TRAIL蛋白的高密度培养及补料策略研究

采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略：间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L（WCW）以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。     

Fuzzy neural network for the control of high cell density cultivation of recombinant Escherichia coli

A five-layer fuzzy neural network (FNN) was developed for the control of fed-batch cultivation of recombinant Escherichia coli JM103 harboring plasmid pUR 2921. The FNN was believed to represent the membership functions of the fuzzy subsets and to implement fuzzy inference using previous experimental data. This FNN was then used for compensating the exponential feeding rate determined by the feedforward control element. The control system is therefore a feedforward-feedback type. The change in pH of the culture broth and the specific growth rate were used as the inputs to FNN to calculate the glucose feeding rate. A cell density of 84 g DWC/l in the fed-batch cultivation of the recombinant E. coli was obtained with this control strategy. Two different FNNs were then employed before and after induction to enhance plasmid-encoded β-galactosidase production. Before induction the specific growth rate was set as 0.31 h⁻¹, while it was changed to 0.1 h⁻¹ after induction. Compared to when only one FNN was used, the residual glucose concentration could be tightly controlled at an appropriate level by employing two FNNs, resulting in an increase in relative activity of β-galactosidase which was about four times greater. The present investigation demonstrates that a feedforward-feedback control strategy with FNN is a promising control strategy for the control of high cell density cultivation and high expression of a target gene in fed-batch cultivation of a recombinant strain.     

Bacteriocin Production with Lactobacillus amylovorus DCE 471 Is Improved and Stabilized by Fed-Batch Fermentation

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.     

Control of carbon source supply and dissolved oxygen by use of carbon dioxide concentration of exhaust gas in fed-batch culture

Accurate and automatic control strategies for a feedback-control system of volatile carbon source feeding and dissolved oxygen (DO) level were investigated. To maintain the optimal ethanol concentration for microbial growth, carbon dioxide concentration in exhaust gas was used as a stepwise control parameter of ethanol feeding. A proportional-differential (PD) control program was used to correct the errors. The coefficient of stepwise control was calculated stoichiometrically, and parameters of PD were experimentally preset and were not changed during cultivation. DO was also controlled by the PD control and the stepwise program based on carbon dioxide concentration of the exhaust gas. Agitation speed and partial pressure of oxygen of the inlet gas were changed stepwise in accordance with the oxygen consumption rate. The stepwise coefficients were estimated from stoichiometry and material balance of molecular oxygen. The PD control program was only used for the agitation speed control to correct the fluctuations of DO level. The parameters did not need to be changed during cultivation. By use of these sophisticated control programs for fed-batch culture of Candida brassicae, ethanol concentration and DO level were accurately controlled at 3.4–3.7 g/l and 2.2–2.8 ppm, respectively, while cell mass concentration reached about 80 g/l. No manual operation was needed.     

Growth and carotenoid production of Phaffia Rhodozyma in fed-batch cultures with different feeding methods

The growth and carotenoid production of Phaffia rhodozyma in fed-batch cultures with different feeding methods and grown at specific growth rates similar to the batch culture was compared. With constant feeding, exponential feeding, DO-stat and pH-stat fed-batch cultures of Phaffia rhodozyma, the highest biomass (17.4 g/l) and lowest carotenoid content (307 g/g cell) of Phaffia rhodozyma was from the DO-stat fed-batch culture. The lowest biomass (14.7 g/l) and highest carotenoid content (412 g/g cell) was from the exponential, fed-batch culture.     

Development and optimization of two-stage cyclic fed-batch culture for hG-CSF production using L-arabinose promoter of Escherichia coli

The long-term process for producing human granulocyte-colony stimulating factor (hG-CSF) was developed using two-stage cyclic fed-batch culture, in which hG-CSF expressing-recombinant Escherichia coli was directed by an L-arabinose promoter system. For the optimization, the preinduction growth rate during the growth stage and the feeding strategy during the production stage were investigated. The maximum harvest volume during the production stage was predicted before long-term cyclic operation. Based on those optimized strategies, the two-stage cyclic fed-batch culture was performed for 12 cycles (86 h). The cell growths in both stages were maintained at 45-50 g/L and 71-77 g/L, respectively. hG-CSF was stably produced at a level of 8-9 g/L and the plasmid stability was maintained at more than 90%. Volumetric productivity by the two-stage cyclic fed-batch culture was 0.643 g/L/h, which was about 280% higher than that of conventional DO-stat fed-batch culture.     

Broth recycling to reduce process noise resulting from concentrated substrate addition in fed-batch cultivation of Escherichia coli

In this work feed hardware for fed-batch cultivation is presented (broth recycle feed injection system or BRFIS). BRFIS proved superior to conventional submerged or dripped feed systems in reducing dissolved oxygen (DO) oscillations during Escherichia coli fed-batch cultivation (5 min coefficient of variation of 0.7% for BRFIS as compared to 26% or greater for conventional feeding hardware in a 2 L test reactor). Hence, BRFIS is useful for fed-batch cultivation systems where the DO signal is used in measurement or control.     

High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development

A fed-batch process was developed for high cell density culture of the diatom Nitzschia laevis for enhanced production of eicosapentaenoic acid (EPA). Firstly, among the various medium components, glucose (Glu) was identified as the limiting substrate while nitrate (NO₃⁻), tryptone (Tr) and yeast extract (Ye) were found to promote cell growth by enhancing specific growth rate. Therefore, these components were considered essential and were included in the feed medium for subsequent fed-batch cultivation. With the optimized ratio of NO₃⁻:Tr:Ye being 1:2.6:1.3 (by weight), the relative proportions of glucose to the nitrogen sources in the feed were investigated. The optimal ratios of Glu:NO₃⁻ for specific growth rate and EPA productivity were both determined to be 32:1 (by weight). Finally, based on the residual glucose concentration in the culture, a continuous medium feeding strategy for fed-batch fermenter cultivation was developed, with which, the maximal cell dry weight and EPA yield obtained were 22.1 g l⁻¹ and 695 mg l⁻¹, respectively, which were great improvements over those of batch cultures.     

Cell engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> allows high cell density accumulation without fed-batch process control

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h⁻¹, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.     

Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast

A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of Mut^S recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.