The theory of fed batch culture with reference to the penicillin fermentation

A batch culture of microbes fed continuously with culture medium is described as a “fed batch culture”. When a portion of a “fed batch culture” is withdrawn at intervals the system is described as a “repeated fed batch culture”. A fed batch culture may reach a “quasi-steady state” in which the specific growth rate (μh^?1) virtually = the dilution rate, that is, the ratio of medium flow rate to culture volume. In a quasi-steady state the specific growth rate gradually decreases. A unique feature of a fed batch culture is that it allows continuous reproduction of the transient conditions between two specific growth rates, which can be chosen at will. Fed batch culture may be used to determine the relation between specific growth rate and the growth-limiting substrate concentration and to determine the maintenance energy. Simple relations are derived to enable the concentrations of products to be predicted. The effects of different cycle times in repeated fed batch culture applied to the pencillin fermentation are discussed. Repeated fed batch culture should find a major application in the production of secondary metabolites which often are synthesised at maximum rates only transiently.     

Enhanced Production of Podophyllotoxin by Fed‐batch Cultivation of Podophyllum hexandrum

Podophyllum hexandrum (Indian May Apple) was successfully cultivated in a 3 L stirred tank bioreactor under low shear conditions in batch and fed‐batch modes of operation. A statistically optimized culture medium was used for the batch cultivation of Podophyllum hexandrum. Under optimum culture conditions of P. hexandrum, the batch culture showed a growth‐associated product formation with a maximum biomass of 21.4 g/L dry cell weight (DCW) basis and a podophyllotoxin production of 13.8 mg/L in 26 d. A mathematical model was developed to design the nutrient feeding strategies for a fed‐batch cultivation to prolong the productive log phase of cultivation. The fed‐batch cultivation was able to enhance the biomass and podophyllotoxin accumulation to 48 g/L (DCW basis) and 43.2 mg/L, respectively, in 60 d. The volumetric productivity of podophyllotoxin in fed‐batch cultivation was found to be 0.72 mg/(L. d) as opposed to 0.53 mg/(L. d) in batch cultivation under optimized culture conditions.     

Computer simulation of the biomass production rate of cyclic fed batch continuous culture

The biomass productivity of cyclically operated variable volume fermenters has been investigated as a function of the initial volume and the feed flow rate. The results of computer simulations show that the productivity-dilution rate curves are similar to those for a constant volume chemostat, which represents a limiting case for the cyclic operation. Both cyclic fed batch and fed batch plus batch operation have been studied for Monod and substrate inhibition kinetics. The results obtained for variable volume operation can be used to estimate the loss in productivity when a chemostat inadvertently undergoes volume fluctuations.     

两阶段温度控制策略提高Lactobacillus kefiranofaciens发酵生产Kefiran

分析了温度对开菲尔基质乳杆菌Lactobacillus kefiranofaciens分批发酵生产胞外多糖Kefiran过程的影响,发现在发酵前期细胞平均比生长速率和Kefiran平均比合成速率达到最大值时的温度较高(33℃),在发酵后期二者达到最大值所需的温度较低(28℃),因此提出两阶段温度控制策略以提高Kefiran分批发酵的产量和发酵强度. 结果表明,两阶段温度控制策略的实施可以进一步提高Kefiran的合成能力,Kefiran产量达到4.65 g/L,比温度控制在28和33℃分别提高了12%和46%,发酵强度分别提高了36%和33%.     

Continuous perfusion culture of encapsulated hybridoma cells

BACKGROUND: The production of Monoclonal antibodies (mAbs) is often performed in batch or fed‐batch operations where low cell densities and low volumetric productivities are achieved. The main bottleneck of both processes is the short operating time with productive cells at maximum cell concentration. RESULTS: The process studied in this work is based on a fluidized‐bed bioreactor culture of encapsulated KB26.5 cells in a liquid core of calcium alginate microcapsules as a culture strategy to produce IgG₃. First, DMEM medium was modified in order to protect the microcapsules from degradation, and later, the optimal operating conditions were set. Under these conditions encapsulated KB26.5 cells reached cell densities of 1.05 × 10⁸cells mL^?1 or 9.8 × 10⁶ cells mL^?1 (referred to the inner capsule volume or total bioreactor volume, respectively), and a mAb volumetric productivity of 2.75 µg mL^?1 h^?1. CONCLUSIONS: The productivity of encapsulated KB26.5 cells in perfusion culture was enhanced significantly in comparison with batch and fed‐batch processes. Continuous operation of the perfusion culture for periods longer than 35 days, represented a volumetric productivity about five‐fold higher than conventional operations. However, the fluidized‐bed also showed limitations such as low cell viability at high cell densities due to the mass transfer limitations of large molecules inside the microcapsules. Copyright © 2011 Society of Chemical Industry     

Lovastatin production by Aspergillus terreus in a two‐staged feeding operation

BACKGROUND: Lovastatin is known to inhibit its own synthesis in the fungus Aspergillus terreus. Therefore, the use of a fermentation strategy that continuously removes some of the lovastatin produced from the bioreactor can enhance its productivity. This paper reports on the effects of dilution rate and the composition of the feed medium on lovastatin production by A. terreus. RESULTS: The feeding strategy consisted of an initial batch/fed‐batch phase and a semi‐continuous culture phase in which the pelleted biomass was retained inside a slurry bubble column. A nitrogen‐free medium was fed at various fixed dilution rates in the semi‐continuous phase. In experiments that were designed to assess the effects of the composition of the medium, the dilution rate was held at 0.42 d^?1, but different feed media were used in separate runs. The best two‐staged production strategy was shown to consist of a 96 h batch/fed‐batch phase that used a nutritionally complete medium. This was followed by a semi‐continuous operation using a medium that was free of both nitrogen and carbon sources. CONCLUSION: Semi‐continuous operation enhanced productivity of lovastatin by 315% compared with a conventional batch operation. The optimal dilution rate in semi‐continuous operation was about 0.42 d^?1. Copyright © 2008 Society of Chemical Industry     

Analysis of fed-batch fermentation processes by graphical methods

Graphical methods are used for many types of engineering calculations. Their appeal is often related to calculational simplicity, but probably the increased understanding that accompanies a graphical calculation method is chiefly responsible for their popularity. In the present work existing graphical concepts which have been used in chemical reaction engineering and fermentation technology have been extended to allow the calculation of biomass and product formation for a variety of fermentation processes. Comparisons in productivity are made among chemostat, batch, fed batch with variable and constant feed rate, and cyclic fed-batch operations.     

Fermentation process development for hyaluronic acid production by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis> ATCC 39920

The development of a fermentation strategy for hyaluronic acid (HA) production by Streptococcus zooepidemicus ATCC 39920 has been explored. The specific HA productivity (Y _P/X ) was affected by the medium carbon-tonitrogen (C/N) ratio rather than the specific growth rate of cells. Accordingly, HA fermentation should be performed in a balanced medium with an optimum C/N ratio of 2: 1 in a batch culture. To improve the performance of the batch culture, the operation conditions for the fill-and-draw culture were investigated. It was found that the timing of medium exchange is critical for successfully performing fill-and-draw operations. Since streptococcal cells at the stationary phase might lose the capacity of HA synthesis, the displacement of the medium in a fill-and-draw culture should be started at the late exponential growth phase.     

Xylitol production by Candida tropicalis from corn cob hemicellulose hydrolysate in a two‐stage fed‐batch fermentation process

BACKGROUND: Xylitol, a sugar alcohol widely used in food and pharmaceutical industries, can be produced through biological reduction of xylose present in hemicellulose hydrolysates by Candida tropicalis. However, the aeration rate and by‐products originating from hemicellulose hydrolysis strongly inhibit the production of xylitol in a fermentation process. A two‐stage fed‐batch fermentation system was developed to reduce these inhibitory effects and to improve xylitol production from corn cob hemicellulose hydrolysates by C. tropicalis. RESULTS: Results of batch fermentations indicated that high xylitol production could be obtained from C. tropicalis at an initial xylose concentration of 80 g L^?1 in corn cob hydrolysate medium at an aeration rate of 0.4 vvm at the micro‐aeration stage. In the two‐stage fed‐batch fermentation process, 96.5 g L^?1 xylitol was obtained after 120 h, giving a yield of 0.83 g g^?1 and a productivity of 1.01 g L^?1 h^?1, which were 12.16% and 65.57% higher than those in a batch fermentation. CONCLUSION: High xylitol production can be achieved in a two‐stage fed‐batch fermentation process, in which the negative effects of aeration rate and inhibitory compounds on xylitol formation can be considerably reduced. Copyright © 2011 Society of Chemical Industry     

重组大肠杆菌分批-补料高密度发酵生产类人胶原蛋白的动力学

The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and growth rates were set at (0.15, 0.2, 0.25h-1) by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase spectively. The model predictions are in good agreement with the experimental data.     

Performance evaluation of batch and unsteady state fed‐batch reactor operations for the production of a marine microbial surfactant

BACKGROUND: Biosurfactants are microbially derived surface‐active and amphipathic molecules produced by various microorganisms. These versatile biomolecules can find potential applications in food, cosmetics, petroleum recovery and biopharmaceutical industries. However, their commercial use is impeded by low yields and productivities in fermentation processes. Thus, an attempt was made to enhance product yield and process productivity by designing a fed‐batch mode reactor strategy. RESULTS: Biosurfactant (BS) production by a marine bacterium was performed in batch and fed‐batch modes of reactor operation in a 3.7 L fermenter. BS concentration of 4.61 ± 0.07 g L^?1 was achieved in batch mode after 22 h with minimum power input of 33.87 × 10³ W, resulting in maximum mixing efficiency. The volumetric oxygen flow rate (K_La) of the marine culture was about 0.08 s^?1. BS production was growth‐associated, as evident from fitting growth kinetics data into the Luedeking‐Piret model. An unsteady state fed batch (USFB) strategy was employed to enhance BS production. Glucose feeding was done at different flow rates ranging from 3.7 mL min^?1 (USFB‐I) to 10 mL min^?1 (USFB‐II). USFB‐I strategy resulted in a maximum biosurfactant yield of 6.2 g l^?1 with an increment of 35% of batch data. The kinetic parameters of USFB‐I were better than those from batch and USFB‐II. CONCLUSION: Comparative performance evaluation of batch and semi‐continuous reactor operations was accomplished. USFB‐I operation improved biosurfactant production by about 35% over batch mode. USFB‐I strategy was more kinetically favorable than batch and USFB‐II. © 2012 Society of Chemical Industry     

Effects of feed water temperature on inorganic fouling of brackish water RO membranes

A bench scale RO process simulator was operated in a batch concentration mode to determine the effects of product water recovery and feed water temperature on flux, rejection, and inorganic fouling by gypsum scale formation for simulated brackish water. As feed water temperature increased, salt rejection and concentration polarization decreased (reducing scale formation potential at a given recovery). However, gypsum crystal nucleation and growth rates increased with temperature. Specifically, at 15 and 25°C gypsum scale formation resulted in slow, steady flux decline at recoveries as low as 10–20%. At these temperatures, many small crystals formed over the entire membrane surface. In contrast, at 35°C flux decline was due to the increasing feed solution osmotic pressure — up to a recovery of about 70%. At this recovery, we observed a sudden, rapid loss of flux and a concomitant spike in feed water turbidity. Relatively few (in number), large crystals formed on the membrane towards the brine outlet of the RO simulator, but the entire membrane surface was covered with “needle-like” crystal fragments. The crystal fragments broke off from growing gypsum rosettes and re-deposited uniformly across the membrane forming a “cake layer” that caused the massive flux decline. These results suggest that high temperature operation of brackish water RO processes could enable higher recovery and lower energy consumption, but operating near the limiting recovery (at elevated temperature) creates an increased risk of a catastrophic fouling event.     

Galacto-oligosaccharide production with immobilized β-galactosidase in a packed-bed reactor vs. free β-galactosidase in a batch reactor

We report here that the usage of immobilized enzyme in a continuous packed bed reactor (PBR) can be a good alternative for GOS production instead of the traditional use of free enzyme in a batch reactor. The carbohydrate composition of the product of the PBR with immobilized enzyme was comparable to that of the batch reactor with free enzyme. The stability of the immobilized enzyme at a lactose concentration of 38% (w/v) and at 50 °C was very high: the half-life time of the immobilized enzyme was approximately 90 days. The enzymatic productivity of GOS production using immobilized enzyme in a PBR can be more than six times higher than that of GOS production with free enzyme in a batch reactor. Besides, when aiming for an equal volumetric productivity to the batch process in designing a PBR, the volume of the PBR can be much smaller than that of the batch reactor, depending on the enzyme dosage and the run time of a single batch.     

利用甘蔗糖蜜半连续发酵生产琥珀酸

为获得较高的琥珀酸发酵产量和生产强度,对Actinobacillus succinogenes CGMCC1593两级双流半连续发酵甘蔗糖蜜生产琥珀酸的工艺过程进行了研究。通过对一级罐初始总糖浓度、补加培养基体积分数和批次发酵时间等发酵条件的优化,琥珀酸产量较分批发酵36 h提高12.9％,与补料分批发酵结果接近;生产强度较分批发酵和补料分批发酵分别提高111％和114%。     

Osmolality Effects on CHO Cell Growth,Cell Volume,Antibody Productivity and Glycosylation

The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in late stage culture. Herein, we explore the effect of osmolality on CHO cell growth, specific monoclonal antibody (mAb) productivity and glycosylation achieved with the addition of NaCl or the supplementation of a commercial feed. Although both methods lead to an increase in specific antibody productivity, they have different effects on cell growth and antibody production. Osmolality modulation using NaCl up to 470 mOsm kg⁻¹ had a consistently positive effect on specific antibody productivity and titre. The addition of the commercial feed achieved variable results: specific mAb productivity was increased, yet cell growth rate was significantly compromised at high osmolality values. As a result, Feed C addition to 410 mOsm kg⁻¹ was the only condition that achieved a significantly higher mAb titre compared to the control. Additionally, Feed C supplementation resulted in a significant reduction in galactosylated antibody structures. Cell volume was found to be positively correlated to osmolality; however, osmolality alone could not account for observed changes in average cell diameter without considering cell cycle variations. These results help delineate the overall effect of osmolality on titre and highlight the potentially negative effect of overfeeding on cell growth.     

Candida krusei 的分批培养与补料分批培养生产甘油

采用Candida krusei分批发酵生产甘油,因菌体生长形成副产物,甘油总得率仅为0.377g/g。生长阶段改用流加培养,限制葡萄糖的供应,然后补入剩余葡萄糖。这样,菌体对葡萄糖的得率系数和甘油总得率比分批培养分别提高了0.258g/g和0.058g/g,最终甘油浓度提高了10g/L 左右。     

Lipase production in continuous culture of Candida rugosa

The yeast Candida rugosa produces multiple extracellular lipases. The production of extra‐ and intracellular lipases was investigated in continuous cultures using a sole or different mixtures of carbon sources. Also, the effect of different C:N ratios was tested. Lipase productivity in continuous cultures increased by 50% compared with data obtained from batch fermentations and depended on the dilution rate applied. Maximum yields relative to consumed substrate were obtained with oleic acid at low dilution rate. It was found that during nitrogen limitation, lipase activity was suppressed. All carbon source mixtures tested allowed both cell growth and lipase production, but extra‐ and intracellular lipase activities were affected by the combination of substrates used. Maximum extracellular lipolytic productivity was attained with lactic and oleic acid mixtures, probably due to the non‐repressor effect of these carbon sources. The chemical composition of the biomass also depended on the type of substrate used and was related to the accumulation of lipidic compounds as intracellular inclusions, which were observed when oleic acid was used as the carbon source. The results obtained were compared with previous data from batch and fed‐batch cultures in order to select the best process strategies for the lipase production with C rugosa. The best lipase yields were obtained in fed‐batch fermentations using oleic acid. Copyright © 2003 Society of Chemical Industry     

Yeast‐mediated stereo‐selective reduction of estrone by continuous cell culture with dual stirred tanks for product yield improvement

BACKGROUND: β‐Estradiol is an important hormone for the treatment of breast cancer and osteoporosis. β‐Estradiol can be produced via Saccharomyces cerevisiae mediated reduction of estrone. However, substrate inhibition and low production yield have been observed in the batch cell culture. RESULTS: An innovative continuous cell culture with dual stirred tanks in series was designed to solve the above problems. The growth medium was fed continuously to the incubation tank where the cells were incubated aerobically; the viable cells were then supplied continuously to the reaction tank in which the yeast‐mediated anaerobic reduction of estrone was performed with continuous feed of the substrate medium and continuous withdawal of the reaction cell culture. Thus, an increase in cell productivity from about 3‐fold to 7‐fold was obtained when compared with the batch cell culture. The β‐estradiol yield was improved to 64.8% on the second reaction day, accompanied by an accumulation of 12.9 mg β‐estradiol on the third reaction day. The yield was about 10% more and the accumulated recovery of β‐estradiol was 4.3‐fold better than with the batch cell culture. The diastereomeric excess value (%de) of β‐estradiol was more than 99%. CONCLUSION: A high product yield with excellent stereo‐selectivity was achieved in a short reaction period with the developed continuous cell culture and the dual stirred tank. Copyright © 2010 Society of Chemical Industry     

Enhanced recombinant protein synthesis in batch and fed‐batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis

BACKGROUND: The formation of acetate as a metabolic by‐product in Escherichia coli fermentation is well known to have detrimental effects on cell growth and productivity. Various bioprocess and genetic approaches have previously been made to limit acetate accumulation, however, they tend to be conservative, limiting overall process productivity, or lead to other problems such as a decrease in maximum specific growth rate and decreased product yield on carbon. RESULTS: In this work, the utility of electrodialysis is examined as a potentially generic approach for in situ acetate removal and its impact on recombinant protein production. Using the induced synthesis of recombinant green fluorescent protein (GFP) in E. coli Tg1 (pGLO) as an example, it is shown that in situ removal of acetate to below inhibitory levels (～1 g L^?1) provides significant improvements in cell growth rate as well as specific biomass and recombinant protein yields. Experiments were performed in a 7.5 L stirred‐tank bioreactor using an external single cell‐pair electrodialysis module with an effective ion exchange membrane area of 0.01 m 2 . For this system increases in specific recombinant protein yield of up to 4‐fold have been observed dependent upon the time of induction, the mode of operation and the level to which acetate concentration is reduced in the fermentation broth. CONCLUSIONS: The implementation of ED can significantly increase the level of recombinant protein synthesis in batch and fed‐batch fermentation. The approach is considered to be generic, readily implemented and has wide application for the production of recombinant enzymes and proteins. Copyright © 2009 Society of Chemical Industry     

Recycle optimization in non-linear productive chromatography—I Mixing recycle with fresh feed

This paper examines the chromatographic separation of two solutes interacting non-linearly with the sorbent, when only partial separation is achieved at the column outlet, and an intermediate cut of the effluent is recycled. It is shown that, for given feed composition and column size, there is an optimum of the amount of fresh feed injected per cycle; alternately, for given feed composition and amount treated per cycle, there is an optimal column length. This optimum is such that interferences of concentration fronts are minimized in the column. The process is analysed using the “equilibrium theory”, which neglects hydrodynamic dispersion and mass-transfer resistances. Complete analytical solutions are given in the optimal situation, for the case of mass-action law equilibria with unity exponents (Langmuir type equilibrium).The theoretical predictions are compared to experimental results for the separation of Na⁺ from K⁺ by H⁺ eluent in chloride solutions, on a commercial cation-exchanger. The results are also compared to “classical” chromatography (without recycle), and to “two-way” chromatography, presented in a previous paper[1]. With respect to “classical chromatography”, for a given product purity, the optimal recycle process is shown to improve product richness (i.e. concentration of product with respect to solvent) and all performance criteria (eluent consumption, sorbent immobilization, productivity). With respect to “two-way” chromatography, it improves sorbent immobilization and productivity, but yields a poorer Na⁺ product, thus uses more eluent.