Experimental optimization of fed-batch culture for poly-beta-hydroxybutyric acid production

The optimal feed rate profiles of glucose and ammonium hydroxide were calculated using a proposed model, and implemented for the production of poly-beta-hydroxybutyric acid (PHB) by Alcaligenes eutrophus. By implementing these optimal feed rates with a high glucose feed concentration of 700 g/L and an ammonium hydroxide concentration of 7%(w/w), it was possible to achieve a high final cell concentration of 141 g/L and a high PHB concentration of 105 g/L in 40 h of fed-batch operation. The PHB productivity was as high as 2.63 g/(L hr). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 697-705, 1997.     

Intracellular fluxes in a recombinant xylose-utilizing Saccharomyces cerevisiae cultivated anaerobically at different dilution rates and feed concentrations

A metabolic flux model was constructed for the yeast Saccharomyces cerevisiae comprising the most important reactions during anaerobic metabolism of xylose and glucose. The model was used to calculate the intracellular fluxes in a recombinant, xylose-utilizing strain of S. cerevisiae (TMB 3001) grown anaerobically in a defined medium at dilution rates of 0.03, 0.06, and 0.18 h(-1). The feed concentration was varied from 0 g/L xylose and 20 g/L glucose to a mixture of 15 g/L xylose and 5 g/L glucose, so that the total concentration of carbon source was kept at 20 g/L. The specific uptake of xylose increased with the xylose concentration in the feed and with increasing dilution rate. The excreted xylitol was less than half of the xylose consumed. With increasing xylose concentration in the feed, the fluxes in the pentose phosphate pathway increased, whereas the flux through glycolysis decreased. Under all cultivation conditions, nicotinamide adenine dinucleotide (NADH) was the preferred cofactor for xylose reductase. The model showed that the flux through the reaction from ribulose 5-phosphate to xylulose 5-phosphate was very low under all cultivation conditions.     

Continuous cultivation of the diatom Nitzschia laevis for eicosapentaenoic acid production: physiological study and process optimization

The continuous cultures of the diatom Nitzschia laevis were performed at different dilution rates (D) and feed glucose concentrations (S(0)) to investigate cellular physiological responses and its production potential of eicosapentaenoic acid (EPA). Steady-state cell dry weight, residual glucose concentration, cell growth yield, specific glucose consumption rate, and fatty acid profiles were investigated within the range of D from 0.1 to 1.0 day(-1) (S(0) fixed at 20 g/L) and the range of S(0) from 5 to 35 g/L (D fixed at 0.3 day(-1)), respectively. The highest EPA productivity of 73 mg L(-1) day(-1) was obtained at D = 0.5 day(-1) and S(0) = 20 g/L. However, when the continuous culture achieved high productivities of EPA at certain dilution rates and feed glucose concentrations, glucose in the feed could not be consumed completely. Accordingly, the continuous culture was evaluated in terms of both EPA productivity (P) and glucose assimilation efficiency (E). The parameter eta, defined as the product of P and E, was used as an overall performance index. Since eta is a function of the two independent variables D and S(0), we employed a central composite design to optimize D and S(0) for the highest eta value. Based on the experimental results of the design, a second-order polynomial equation was established to represent the relationship between eta and D and S(0). The optimal values of D and S(0) were subsequently determined as 0.481 day(-1) and 15.56 g/L, respectively by the empirical model. The verification experiment confirmed the validity of the model. Under the optimal conditions, eta value reached 46.5 mg L(-1) day(-1), suggesting a considerably high efficiency of the continuous culture of N. laevis in terms of EPA production and glucose utilization.     

Enhanced production of podophyllotoxin by Podophyllum hexandrum using in situ cell retention bioreactor

The rhizomes of the rare plant Podophyllum hexandrum contain podophyllotoxin, which is a precursor of the anticancer drugs etoposide and teniposide. Batch cultivation of Podophyllum hexandrum was conducted using optimized medium in a 3 L bioreactor, which resulted in biomass and podophyllotoxin concentrations of 21.4 g/L and 13.8 mg/L in 24 and 26 days, respectively. The batch kinetics was used to identify the mathematical model. The model was extrapolated to identify the nutrient feeding rate (150 mL/d) and substrate concentration (105 g/L) in the incoming feed for nonlimiting and noninhibitory glucose concentration in the cell retention bioreactor. An improvement in cell growth to 53 g/L and intracellular podophyllotoxin accumulation of 48.8 mg/L was achieved in 60 days, when the bioreactor was operated in continuous cell retention cultivation mode.     

Growth characteristics in fed-batch culture of hybridoma cells with control of glucose and glutamine concentrations

An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one-half and the lactate concentration by one-third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 x 10(6) cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation. (c) 1994 John Wiley & Sons, Inc.     

基于差分进化算法的酿酒酵母分批补料培养在线自适应控制

酿酒酵母分批补料培养中,葡萄糖添加过量会导致乙醇大量积累,破坏细胞结构及功能,降低葡萄糖利用效率;葡萄糖添加不足会限制细胞生长。为解决这一矛盾,提出了一种基于差分进化算法的在线自适应控制策略,并利用计算机仿真方法对该策略、传统的间歇流加、分段恒速流加及PID控制策略的控制性能进行了研究和比较。结果表明,在该控制策略下,发酵液中的乙醇浓度能够被稳定地控制在1g/L的低水平,而细胞浓度却达到34.45g/L的高水平,比采用间歇流加、分段恒速流加及PID控制策略的批次分别提高了243%、18%和29%。由此可知,该自适应控制策略能够将葡萄糖流加速率控制在适宜水平,避免乙醇过量积累的同时保证细胞的快速增殖。     

A cell retention internal filter reactor for ethanol production using tapioca hydrolysates

Summary Ethanol fermentation with Saccharomyces cerevisiae was carried out in a bioreactor equipped with a sintered stainless steel filter to obtain a high cell density as well as a high productivity. With a 100 g glucose/l feed, the maximum yeast concentration was 208 g/L similar to that obtained in external membrane cell recycle cultures. However, with tapioca hydrolysates, treated with activated carbon column, the yeast concentration attained was 42g/L.     

Characterization of an immobilized cell, trickle bed reactor during long term butanol (ABE) fermentation

Acetone-butanol-ethanol (ABE) fermentation was performed continuously in an immobilized cell, trickle bed reactor for 54 days without, degeneration by maintaining the pH above 4.3. Column clogging was minimized by structured packing of immobilization matrix. The reactor contained two serial glass columns packed with Clostridium acetobutylicum adsorbed on 12- and 20-in.-long polyester sponge strips at total flow rates between 38 and 98.7 mL/h. Cells were initially grown at 20 g/L glucose resulting in low butanol (1.15 g/L) production encouraging cell growth. After the initial cell growth phase a higher glucose concentration (38.7 g/L) improved solvent yield from 13.2 to 24.1 wt%, and butanol production rate was the best. Further improvement in solvent yield and butanol production rate was not observed with 60 g/L of glucose. However, when the fresh nutrient supply was limited to only the first column, solvent yield increased to 27.3 wt% and butanol selectivity was improved to 0.592 as compared to 0.541 when fresh feed was fed to both columns. The highest butanol concentration of 5.2 g/L occurred at 55% conversion of the feed with 60 g/L glucose. Liquid product yield of immobilized cells approached the theoretical value reported in the literature. Glucose and product concentration profiles along the column showed that the columns can be divided into production and inhibition regions. The length of each zone was dependent upon the feed glucose concentration and feed pattern. Unlike batch fermentation, there was no clear distinction between acid and solvent production regions. The pH dropped, from 6.18-6.43 to 4.50-4.90 in the first inch of the reactor. The pH dropped further to 4.36-4.65 by the exit of the column. The results indicate that the strategy for long term stable operation with high solvent yield requires a structured packing of biologically stable porous matrix such as polyester sponge, a pH maintenance above 4.3, glucose concentrations up to 60 g/L and nutrient supply only to the inlet of the reactor.     

Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation.

A silicone membrane was used to study butanol separation from model butanol solutions and fermentation broth. Depending upon the butanol feed concentration in the model solution and pervaporation conditions, butanol selectivities of 20.88-68.32 and flux values of 158.7-215.4 g m(-)(2) h(-)(1) were achieved. Higher flux values (400 g m(-)(2) h(-)(1)) were obtained at higher butanol concentrations using air as sweep gas. In an integrated process of butanol fermentation-recovery, solvent productivities were improved to 200% of the control batch fermentation productivities. In a batch reactor the hyper-butanol-producing mutant strain C. beijerinckii BA101 utilized 57.3 g/L glucose and produced 24.2 g/L total solvents, while in the integrated process it produced 51.5 g/L (culture volume) total solvents. Concentrated glucose medium was also fermented. The C. beijerinckii BA101 mutant strain was not negatively affected by the pervaporative conditions. In the integrated experiment, acids were not produced. With the active fermentation broth, butanol selectivity was reduced by a factor of 2-3. However, the membrane flux was not affected by the active fermentation broth. The butanol permeate concentration ranged from 26.4 to 95.4 g/L, depending upon butanol concentration in the fermentation broth. Since the permeate of most membranes contains acetone, butanol, and ethanol (and small concentrations of acids), it is suggested that distillation be used for further purification.     

Preparation and performance of immobilized yeast cells in columns containing no inert carrier

Schizosaccharomyces pombe was cultivated in a medium of glucose (10 g/L) malt extract (3 g/L), yeast extract (3 g/L), and bactopeptone (5 g/L) to form flocs. More than 95% of the cell population were flocculated. Variation in glucose concentration (from 10 to 100 g/L) did not affect flocculation. Yeast extract helped induce flocculation. Application of the immobilized yeast for the continuous production of ethanol was tested in a column reactor. Soft yeast flocs (50-200 mesh) underwent morphological changes to heavy particles (0.1-0.3 cm diameter) after continuously being fed with fresh substrates in the column. Productivity as high as 87 g EtOH L(-1) h(-1) was obtained when a 150 g/L glucose medium was fed. The performance of this yeast reactor was stable over a two-month period. The ethanol yield was 97% of the theoretical maximum based upon glucose consumed.     

Software sensing for glucose concentration in industrial antibiotic fedbatch culture using fuzzy neural network

In order to control glucose concentration during fed-batch culture for antibiotic production, we applied so called “software sensor” which estimates unmeasured variable of interest from measured process variables using software. All data for analysis were collected from industrial scale cultures in a pharmaceutical company. First, we constructed an estimation model for glucose feed rate to keep glucose concentration at target value. In actual fed-batch culture, glucose concentration, was kept at relatively high and measured once a day, and the glucose feed rate until the next measurement time was determined by an expert worker based on the actual consumption rate. Fuzzy neural network (FNN) was applied to construct the estimation model. From the simulation results using this model, the average error for glucose concentration was 0.88 g/L. The FNN model was also applied for a special culture to keep glucose concentration at low level. Selecting the optimal input variables, it was possible to simulate the culture with a low glucose concentration from the data sets of relatively high glucose concentration. Next, a simulation model to estimate time course of glucose concentration during one day was constructed using the on-line measurable process variables, since glucose concentration was only measured off-line once a day. Here, the recursive fuzzy neural network (RFNN) was applied for the simulation model. As the result of the simulation, average error of RFNN model was 0.91 g/L and this model was found to be useful to supervise the fed-batch culture.     

Kinetics of ethanol fermentations in membrane cell recycle fermentors

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow-fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.     

The effect of amino acid supplementation in an industrial Chinese Hamster Ovary process

The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Abundance of serine and glucose increased lactate production resulting in low viability and low productivity. In the next step, we developed an amino acid feed without serine to avoid the metabolic boost. Supplemented amino acids improved cell viability by 9%; however, mAb production did not increase significantly. In the final step, we limited glucose concentration (<5.55 mmol/L) in the cell culture to avoid the metabolic boost while supplementing an amino acid feed including serine. Data analysis showed that we were able to (a) replace depleted amino acids and avoid metabolic boost, (b) increase viability by 12%, (c) enhance mAb production by 0.5 g/L (total by approximately 10 g), and (d) extend the overall process time of an already developed bioprocess.     

Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control

Alcaligenes eutrophus NCIMB 11599 was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from glucose by the automatic fed-batch culture technique. The glucose concentration of the culture broth was controlled at 10 to 20 g/L by two methods: using exit gas data obtained from a mass spectrometer and using an on-line glucose analyzer. The effect of ammonium limitation on PHB synthesis at different culture phases was studied. The final cell concentration, PHB concentration, and PHB productivity increased as ammonia feeding was stopped at a higher cell concentration. High concentrations of PHB (121 g/L) and total cells (164 g/L) were obtained in 50 h when ammonia feeding was stopped at the cell concentration of 70 g/L. The maximum PHB content reached 76% of dry cell weight and the productivity was 2.42 g/L h with the yield of 0.3 g PHB/g glucose.     

A Mathematical model for ethanol production by extractive fermentation in a continuous stirred tank fermentor

Extractive fermentation is a technique that can be used to reduce the effect of end product inhibition through the use of a water-immiscible phase that removes fermentation products in situ. This has the beneficial effect of not only removing inhibitory products as they are formed (thus keeping reaction rates high) but also has the potential for reducing product recovery costs. We have chosen to examine the ethanol fermentation as a model system for end product inhibition and extractive fermentation and have developed a computer model predicting the productivity enhancement possible with this technique together with other key parameters such as extraction efficiency and residual glucose concentration. The model accommodates variable liquid flowrates entering and leaving the system, since it was found that the aqueous outlet flowrate could be up to 35% lower than the inlet flowrate during extractive fermentation of concentrated glucose feeds due to the continuous removal of ethanol from the fermentation broth by solvent extraction. The model predicts a total ethanol productivity of 82.6 g/L h if a glucose feed of 750 g/L is fermented with a solvent having a distribution coefficient of 0.5 at a solvent dilution rate of 5.0 h(-1). This is more than 10 times higher than for a conventional chemostat fermentation of a 250 g/L glucose feed. The model has furthermore illustrated the possible trade-offs that exist between obtaining a high extraction efficiency and a low residual glucose concentration.     

非离子表面活性剂对生物丁醇发酵的影响

传统的丙酮-丁醇发酵的产物浓度过低(丁醇终浓度约为1.3 wt%),导致后期分离成本过高,从而影响了该过程的经济性,限制了其工业化进程。本文研究了高添加量的小分子非离子表面活性剂对生物丁醇发酵的影响。以吐温80为例,实验表明,当表面活性剂添加量超过其临界胶束浓度后,丁醇发酵的终浓度会随着表面活性剂添加量的增加而增加。当添加量达到5 wt%时,丁醇终浓度可以达到1.6 wt%,远高于该菌种的抑制浓度(0.8 wt%)。为阐明表面活性剂的作用机理,实验考察了吐温80对丁醇的增溶效应以及对发酵菌体表面亲疏水性的影响。结果表明,吐温80对丁醇的增溶效果很小,而对菌体表面的亲疏水性有较明显的影响。     

Demonstration of the Crabtree effect in Phaffia rhodozyma during continuous and fed-batch cultivation

By monitoring cell yield and fermentation products during fed-batch and continuous growth, Pfaffia rhodozyma was shown to exhibit the Crabtree effect. In fed-batch culture at feed concentrations of 27 and 55 g glucose/l there was good agreement between the observed biomass formation and that predicted by a mass balance model. At 125 g glucose/l in the feed, biomass formation was less than predicted and fermentation products such as ethanol and acetic acid accumulated in the culture medium. In continuous culture with a feed concentration of 10 g glucose/l, the Crabtree effect became apparent at a dilution rate of 0.1 h -1 . Aerobic fermentation did not occur provided the sugar substrate was maintained at a concentration of less than 0.5 g/l. Although the cell yield coefficient was reduced from 0.5 g/g to 0.16 g/g during aerobic fermentation, the carotenoid content of the cells was unaffected.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

无载体固定化酵母细胞木薯淀粉质原料酒精连续发酵研究

以木薯粉糖化液为发酵底物,在总发酵体积(有效)为15L的悬浮床生物反应器中,对一株粟酒裂殖酵母变异株进行一级和二级连续发酵研究。结果表明,二级连续发酵系统可明显改善一级系统的不足,并取得了平均流加糖液浓度150g/L,发酵强度为97g/L.h,流出液酒精浓度727g/L,残糖浓度374g./L,总糖利用率达90%的较好结果;整个系统在连续一个月的运行中从未发现染菌现象,发酵操作稳定。     

Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH(4)OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L . h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.