pH control for fed-batch culture of Bacillus thuringiensis

Summary Fed-batch culture of Bacillus thuringiensis for thuringiensin (-exotoxin) production was carried out in a modified airlift reactor. The feeding policy was based on the pH value in the broth. The feed rate of the substrate was regulated by an appropriate pH value. The yield of thuringiensin based on the fed-batch culture increased 30% in comparison with that of batch culture.     

Production of cyclic adenosine monophosphate by Arthrobacter sp. A302 using fed-batch fermentation with pH-shift control

The production of cyclic adenosine monophosphate (cAMP) by Arthrobacter sp. A302 was studied in a 5 L stirred tank fermentor under a range of pH values (6.5–8.0) and glucose feeding rates. In batch fermentation under a controlled pH, the optimum pH for cell growth was 7.5 with dry cell density (X) of 11.43 g L, and the optimum pH for cAMP accumulation was 7.0 with cAMP concentration of 7.41 g L. In order to achieve the high X and cAMP yield simultaneously, a pH-shift control strategy was proposed based on kinetic analysis of specific cell growth rate (μ) and specific cAMP formation rate (q _s ). In this method, pH was controlled to 7.0 for the first 30 h of fermentation, and then subsequently shifted to 7.5 and maintained until the end of the process. Application of this approach significantly enhanced the cAMP concentration. Thereafter, cAMP production was further improved by combining the above-mentioned pH-control system and fed-batch process with glucose at a constant feeding rate of 1.0 g L⁻¹ h⁻¹. Under optimum conditions, the final cAMP production was 10.87 g L, which is 110.0, 46.7, and 27.7% higher than that of the pH-uncontrolled, pH-controlled, and pH-shift controlled methods, respectively.     

Fed-batch culture of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> based on motile intensity

The operation of a fed-batch culture is more complicated than that of batch or continuous culture. Thus, an appropriate feeding strategy for fed-batch cultures should be carefully designed. In this study, a simple feeding strategy for fed-batch culture of Bacillus thuringiensis based on motile intensity is described. The feeding strategy consisted of two steps: (1) initiating feeding at the peak of motile intensity; (2) terminating feeding at low motile intensity (or non-motility) of the cells. In addition, the motile intensity of B. thuringiensis was used to determine the optimum environmental conditions (pH, temperature, and dissolved oxygen) and optimum medium composition. Using this fed-batch strategy, the production of thuringiensin increased 34% compared with batch culture using the same environmental conditions and medium composition. The proposed strategy for fed-batch culture helps to avoid overfeeding of substrate and facilitates on-line control. A comparison of several alternative strategies for fed-batch culture demonstrated that strategies such as glucose-stat and DO-stat result in a lower productivity than that obtained using the motility intensity method.     

3-Phenyllactic acid production by substrate feeding and pH-control in fed-batch fermentation of Lactobacillus sp. SK007

3-Phenyllactic acid (PLA), which is produced by some strains of lactic acid bacteria (LAB), is a known antimicrobial agent with a broad spectrum. Batch and fed-batch fermentation by the strain Lactobacillus sp. SK007 for PLA production have been reported. With batch fermentation without pH-control, PLA production yield was 2.42 g L⁻¹. When fed-batch fermentation by Lactobacillus sp. SK007 was conducted in 3 L initial volume with pH-control at 6.0 and intermittent feeding, which was developed after fermentation for 12 h and every 2 h with 120 mL 100 g L⁻¹ PPA phenylpyruvic acid (PPA) and 50 mL 500 g L⁻¹ glucose each time, PLA production yield reached 17.38 g L⁻¹. The final conversion ratio of PPA to PLA was 51.1%, and the PLA production rate was 0.241 g L⁻¹ h⁻¹. This indicated that PPA was the ideal substrate for PLA fermentation production, and fed-batch fermentation with intermittent PPA feeding and pH-control was an effective approach to improve PLA production yield.     

Effect and control of glucose feeding on bacitracin production by fed-batch culture of Bacillus licheniformis

The effect of glucose feeding on bacitracin production was investigated by fed-batch culture of Bacillus licheniformis. In batch culture, bacitracin secretion was induced after the glucose initially contained in the medium was completely consumed. The concentration of bacitracin, however, increased to no more than 340 units·ml⁻¹ in the batch cultivations. Therefore, additional glucose was supplied after exhaustion of the initial glucose. The effect of glucose feeding on bacitracin biosynthesss was investigated in two ways, the pH-stat modal feeding method and the CO₂-dependent feeding method. A kinetic study of bacitracin production found that some glucose was necessary, even during the bacitracin production phase. Excessive feeding of glucose, however, caused a reduction in bacitracin biosynthetic activity. When 50 g·l⁻¹ of defatted soy bean meal (SBM) was used, the bacitracin concentration reached 670 units·ml⁻¹ with the pH-stat modal feeding method and 610 units·ml⁻¹ with the CO₂-dependent feeding method, respectively. The yield of bacitracin from consumed glucose was better for the pH-stat method. Using this control strategy, the highest concentration of bacitracin (940 units·ml⁻¹) was obtained with 150 g·l⁻¹ of SBM.     

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l^?1 and 138.8 mg l^?1 h^?1, respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca²⁺ and Mg²⁺ for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl₂ and MgCl₂ concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca²⁺/Mg²⁺ feeding to 117 h with Ca²⁺/Mg²⁺ feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 × 10⁶ and 9.12 × 10⁶ cells ml⁻¹, respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.     

Distribution of substrates carbon in sophorose lipid production by Torulopsis bombicola

Summary Torulopsis bombicola (ATCC 22214) produced sophorose lipid to 80 g/l in batch culture containing 11% glucose and 10% soybean oil as carbon and energy sources. According to the carbon mass balance analysis, 13% and 37% of input carbon were channeled to cells and to products, respectively, and 50% of the total input carbon was channeled to CO₂ gas in batch culture. In fed-batch culture with intermittent oil feeding, however, the carbon fractions incorporated into sophorose lipid and cells were 60% and 12%, respectively, and the carbon fraction evolved as CO₂ gas was 30%. In conclusion, yield of sophorose lipid based on total input carbon substrates was increased from 0.37 g/g-substrate in batch culture to 0.6 g/ g-substrate by employing a fed-batch culture.     

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.     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

Mass production of thiostrepton by fed-batch culture of Streptomyces laurentii with pH-stat modal feeding of multi-substrate

Summary High concentration production of an antibiotic, thiostrepton, was achieved by the fed-batch culture of Streptomyces laurentii. To produce thiostrepton efficiently, the pH of the medium had to be maintained in a very narrow range between 6.0 and 6.2. As the substrates, not only glucose but also natural nutrients such as defatted soy bean meal and corn steep liquor were demanded. All of these substrates (multi-substrate) had to be supplied during the cultivation. pH was used as the indicator to detect the deficiency of substrates. When the glucose in the medium had been exhaustively consumed, the pH increased immediately; the multi-substrate solution (MS-solution) was then supplied. The composition of the feeding solution was determined from data obtained in batch cultures. During the fed-batch culture, glucose concentration was kept lower than 5 g/l by regulating the feed amount of MS-solution with this pH-stat modal control. By this control strategy a high concentration of thiostrepton, 10.5 g/l, was obtained, while total cell mass concentration reached 157 g/l. The productivity of thiostrepton was greatly increased compared with the conventional batch culture.     

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。     

Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures

The effects of cultural parameters such as carbon and nitrogen source and environmental factors including temperature and pH were investigated on spore and mycelial yield of Trichoderma viride, which has potential as a biocontrol agent against species of Fusarium in batch culture and fed-batch culture where there was limiting nutrient. The results obtained indicated that growth and sporulation of T. viride were greatly influenced by various carbon and nitrogen sources, and by environmental factors such as pH and temperature. Mannitol, wheat bran and rice bran as sole carbon sources appear to stimulate high mycelial growth and spore yield in fed-batch culture. Growth and sporulation were also favoured by NaNO₃, peptone and NH₄SO₄ as the nitrogen sources in fed-batch and batch cultures_. Maximum growth and sporulation was between pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good for mycelium growth of T. viride while temperatures between 30 to 45 °C were good for sporulation. The amount of spore and mycelium produced and the time required for attainment of maximum spore yield increased with increasing carbon and nitrogen source in batch culture. The final spore yield obtained in fed-batch culture was two times higher than the apparent spore-carrying capacity of batch culture. These results show that T. viride is capable of growing and sporulating with varied nutritional and environmental conditions, and, therefore, this strain of T. viride may be useful as a biocontrol agent under diverse physiological and environmental conditions.     

Fed-batch propionic acid production by Propionibacterium acidipropionici

The batch fermentations were conducted using lactose as the substrate at pH 6.5 and temperature 30°C. Average batch kinetic data was eventually used to develop an unstructured mathematical model. The kinetic parameters of the model were determined by non-linear regression technique using the batch experimental results. Parametric sensitivity analysis showed the maximum specific substrate consumption rate (r_S^max) and the maintenance energy constant (m_S) to be the most sensitive parameters. The experimental observations in batch fermentation were close to the model predictions. The batch model was extrapolated to identify nutrient feeding strategies, which were tested successfully for two different fed-batch fermentations. It demonstrated enhanced propionic acid productivity. The developed model was found suitable for the design of feeding strategies to increase propionic acid production in fed-batch mode of reactor operation.     

Enhanced production of the nonribosomal peptide antibiotic valinomycin in Escherichia coli through small-scale high cell density fed-batch cultivation

Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L^?1 by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L^?1. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD₆₀₀ >50 and a valinomycin titer of appr. 10 mg L^?1. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli.     

Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor

In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed. Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b). In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model. Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies. Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model. The formation of toxic byproducts, such as ammonia and lactate (Hassellet al., 1991), was greatly reduced. The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model. Ammonia formation was also decreased compared with both the batch and fed-batch cultures. Most importantly, the monoclonal antibody concentration reached 900 mg l^?1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively.     

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.     

A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 10⁴ U·mg^?1, corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.