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1.
A starvation-based dissolved oxygen (DO) transient controller was developed to supply growth-limiting substrate to high cell density fed-batch cultures of recombinant Escherichia coli. The algorithm adjusted a preexisting feed rate in proportion to the culture's oxygen demand, which was estimated from transients in the DO concentration after short periods of feed interruption. In this manner, the addition of glucose feed was precisely controlled at a rate that did not exceed the acetate production threshold, thus preventing acetate accumulation. In comparison to exponential feed algorithms commonly used in industry, the implementation of the new feeding strategy increased the final cell density from 32 to 44 g (dry cell weight).L(-1), with less than 16 mM acetate accumulated, producing an ideal culture for subsequent induction. Despite a constant starvation level and relatively low levels of acetate, experimental cultivations still tended to produce acetate towards the end of the process. The use of a simple Monod model provided an explanation as to why this may occur in high cell density cultivations and suggests how it may be overcome.  相似文献   

2.
A probing feeding strategy for Escherichia coli cultures   总被引:1,自引:0,他引:1  
A strain-independent feeding strategy for fed-batch cultures of Escherichia coli is presented. By superimposing short pulses in the glucose feed rate, on-line detection of acetate formation can be made using a standard dissolved oxygen sensor. A simple feedback algorithm is then used to adjust the feed rate to avoid acetate formation. The feasibility of the strategy is demonstrated by both simulation and experiments.  相似文献   

3.
Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.  相似文献   

4.
Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.  相似文献   

5.
A simple pulse-based method for the determination of the maximum uptake capacities for glucose and oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where specific growth rate was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when specific growth rate declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.  相似文献   

6.
Desulfotomaculum nigrificans degraded glucose to acetate, ethyl alcohol, and carbon dioxide. By use of 14C-glucose labeled at different carbon atoms, two pathways of glucose metabolism were detected. They were the Embden-Meyerhof and the Entner-Doudoroff schemes. Because the observed quantities of acetate and carbon dioxide, arising from glucose, were greater than the expected theoretical values, individual fermentations were conducted with 15 uniformly labeled 14C-amino acids. The results indicated that amino acids, supplied by the yeast extract or peptone in the fermentation medium, also contributed to the formation of acetate and carbon dioxide.  相似文献   

7.
This study was conducted by two trials to investigate effects of sequential feeding with low- and high-protein diets on growth traits and plasma metabolites in geese. In Trial I, the effect of sequential feeding under time-restricted feeding system was investigated. Seventy-two White Roman goslings were randomly allotted into either sequential feeding (S1) or control feeding (C1) group. All goslings were fed for 1 h at morning and at evening, respectively, from 2 to 8 weeks of age. S1 group was offered 13% CP diet at morning and 19% CP diet at evening. C1 group was offered the same diet (16% CP; mixed equally with the two diets mentioned above) at both morning and evening. Blood samples were hourly collected for 4 h after feeding at both morning and evening for the determination of the postprandial plasma levels of glucose, triacylglycerol and uric acid at the end of experiment. Results showed that BW, average daily gain (ADG), and daily feed intake (FI) were not different between groups, but the feed efficiency (FE) in S1 group was significantly higher than that in C1 group (P<0.05). The areas under curve (AUC) of plasma postprandial levels of glucose, triacylglycerol and uric acid were not affected by treatment, but the AUC of triacylglycerol and uric acid in morning were lower than those in evening (P<0.05). In Trial II, the effect of sequential feeding under ad libitum feeding system was investigated. Twenty-four goslings were randomly allotted into either sequential feeding (S2) or control feeding (C2) group. Diets were altered at 0600 and 1800 h, respectively, and geese were fed ad libitum from 4 to 8 weeks of age. S2 group was offered 14% CP diet at morning and 20% CP diet at evening. C2 group was supplied the same diet (mixed with the two diets according to the ratio of diets consumed by S2 group on the preceded day) at both morning and evening. Results showed that the ADG in S2 group was higher than those in C2 group (P<0.05). Summarized data from both trials showed that sequential feeding improves daily gain and FE in growing geese.  相似文献   

8.
Three different models: the unstructured mechanistic black-box model, the input–output neural network-based model and the externally recurrent neural network model were used to describe the pyruvate production process from glucose and acetate using the genetically modified Escherichia coli YYC202 ldhA::Kan strain. The experimental data were used from the recently described batch and fed-batch experiments [ Zelić B, Study of the process development for Escherichia coli-based pyruvate production. PhD Thesis, University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia, July 2003. (In English); Zelić et al. Bioproc Biosyst Eng 26:249–258 (2004); Zelić et al. Eng Life Sci 3:299–305 (2003); Zelić et al Biotechnol Bioeng 85:638–646 (2004)]. The neural networks were built out of the experimental data obtained in the fed-batch pyruvate production experiments with the constant glucose feed rate. The model validation was performed using the experimental results obtained from the batch and fed-batch pyruvate production experiments with the constant acetate feed rate. Dynamics of the substrate and product concentration changes was estimated using two neural network-based models for biomass and pyruvate. It was shown that neural networks could be used for the modeling of complex microbial fermentation processes, even in conditions in which mechanistic unstructured models cannot be applied.  相似文献   

9.
The aim of this study is to determine the medium feeding strategy to maximize the invertase productivity of recombinant Saccharomyces Cerevisiae using a fed-batch mode of operation. The yeast contains the plasmid, pRB58, which contains the yeast SUC2 gene, coding for the enzyme invertase. The expression of this gene is repressed at high glucose levels. A Goal-oriented model is development to describe the kinetics of fed-batch fermentations. This simple model could quantitatively describe previous experimental results. A conjugate gradient algorithm is then used, in conjunction gradient algorithm is then used, in conjunction with this mathematical model, to compute the optimum feed rate for maximization of invertase productivity. The optimal feeding procedure results in an initial high cell growth phase followed by a high invertase production phase. (c) 1993 Wiley & Sons, Inc.  相似文献   

10.
A nonlinear model of a recombinant Escherichia coli producing porcine growth hormone (pGH) fermentation was developed. The model was used to calculate a glucose feeding and temperature strategy to optimize the production of pGH. Simulations showed that the implementation of optimal feed and temperature profiles was sensitive to the maximum specific growth rate, and a mismatch could result in excessive acetate production and a significant reduction in pGH yield. An optimization algorithm was thus developed, using feedback control, to counter the effects of uncertainty in the specific growth rate and thus determine an optimal operating strategy for pGH production. This policy was experimentally implemented in a 10 L fermenter and resulted in a 125% increase in productivity over the previous best experimental result with this system—in spite of significant plant-model mismatch.  相似文献   

11.
This study comprises two trials that investigated the effects of feeding time and time-restricted feeding on the fattening traits and plasma metabolite levels of White Roman geese. In Trial I, 24 geese aged 8 weeks of each sex were allowed free access to a fattening diet for 1 h either in the morning (morning-feeding group) or afternoon (afternoon-feeding group). At 12 weeks of age, blood samples were collected hourly for 4 h, beginning 1 h after feeding to determine the plasma levels of glucose, triacylglycerols and uric acid. The results showed a lower (P<0.05) daily feed intake (DFI) and daily gain (DG) and higher (P<0.05) feed efficiency (FE) for the morning-feeding group compared with those of the afternoon-feeding group. In addition, the postprandial plasma levels of glucose, triacylglycerols and uric acid did not differ (P>0.05) between groups. In Trial II, 12 geese aged 8 weeks of each sex were randomly assigned to either the ad libitum feeding group (control group) or time-restricted feeding group (restricted group). The geese in the control group were fed a fattening diet ad libitum, whereas those in the restricted group were allowed access to the diet for 2 h every morning. All geese were killed at 13 weeks of age and their carcass traits were evaluated. The results showed a lower DFI and DG and higher FE for the restricted group compared with those of the control group (P<0.05). In addition, the restricted group exhibited lower visceral and abdominal fat and higher empty digestive tract and liver weights than those of the control group (P<0.05). The results showed that time-restricted feeding in the morning resulted in superior DG and FE compared with feeding in the afternoon. Moreover, time-restricted feeding implemented in the morning during the fattening period reduced DFI and increased FE in geese compared with ad libitum feeding.  相似文献   

12.
In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process. Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000  相似文献   

13.
A method was developed to predict spoilage of minced meat at chill temperatures, based on the difference in proton efflux from and influx into bacterial cells. This difference depends on the number of organisms present, the available glucose in the meat sample and the ability of the organisms to metabolize amino acids. The proton efflux/influx of a meat filtrate containing bacteria was measured at 25°C with a pH/ion meter in the presence of peptone with or without glucose. There was a noticeable rate of change of mV h-1 of the meat filtrate prior to the organoleptic detection of spoilage which may be used semi-predictively to determine the remaining shelf-life of meat at different storage temperatures. The method could be investigated further, encompassing type and relative numbers of organisms, incubation temperature, meat type and composition (i.e. available glucose) to produce a spoilage prediction model. The method does not require sophisticated equipment, only a standard pH/ion meter, is cheap, needing only peptone and glucose, is relatively simple, and takes less than 2 h to perform.  相似文献   

14.
In glutamate fermentation, intermittent feeding is the most widely used glucose feed strategy. This feeding strategy causes severe fluctuations of glucose concentration and osmotic pressure in fermentation broth, which deteriorates the viability of the cell and reduces glutamate production in turn. In order to maintain glucose concentration at stable and constant levels, an on-line prediction and feedback control system based an empiric mass balance model was developed. However, the control system did not work properly and sometimes glucose concentration could even decline to 0 level (glucose exhaustion), as the model parameter varies in different runs. As a result, a novel model-based adaptive feedback control system incoporating with an artificial neural network (ANN) based pattern reconition unit for on-line diagnosizing the fault of glucose exhaustion was proposed and applied for glutamate fermentation. This adaptive control system could accurately detect glucose exhaustion when it occurs, and then immediately updates the control parameter based on some pre-defined rule. With the proposed control system, glucose was automatically fed, and its concentration could be maintained at desired levels constantly. As a result, glutamate concentration was 17 ~ 30% higher than that of the traditional fermentations using the intermittent glucose feed strategy.  相似文献   

15.
Summary Maintaining high metabolic activities for extended periods by feeding small amounts of the growth limiting nutrient was examined for the production of cycloheximide by Streptomyces griseus. Batch studies indicated that increased initial phosphate levels led to increased cell concentrations, stimulated glucose utilization, and over a limited range (<0.6 g/l KH2PO4) did not adversely affect cycloheximide production rates. Semi-continuous phosphate feeding was observed to permit limited cell growth, and to enhance metabolic activities (i. e. glucose utilization). The effect of semi-continuous phosphate feeding on antibiotic production depended on the feed rate, with high feed rates suppressing production.  相似文献   

16.
《Process Biochemistry》2004,39(9):1057-1062
Optimization of the fermentation medium for maximization of actinorhodin production by Streptomyces coelicolor A3(2) was carried out. Response surface methodology (RSM) was applied to optimize the medium constituents. A 24 full-factorial central composite design (CCD) was chosen to explain the combined effects of the four medium constituents, viz. sucrose, glucose, yeast extract (YE) and peptone, and to design a minimum number of experiments. The P-values of the coefficients for linear, quadratic and cross-product effect of sucrose and glucose concentration were <0.0001, suggesting that these were critical variables having the greatest effect on the production of actinorhodin in the complex medium. The optimized medium consisting of 339 g/l sucrose, 1 g/l glucose, 1.95 g/l YE and 2.72 g/l peptone predicted 195 mg/l of actinorhodin which was 32% higher than that of the unoptimized medium. The amounts of glucose, YE and peptone required were also reduced with RSM.  相似文献   

17.
Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.  相似文献   

18.
A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.  相似文献   

19.
A standardised methodology is required for classification of starchy diets. One important question is what feeding status is optimal to describe glycaemic and insulinaemic responses to the respective starchy diet. Four horses were fed, in a randomised order, four different feeding protocols relative to offering hay before or after cracked corn (CC): (i) ad libitum hay for 12 h before feeding CC and ad libitum hay after CC intake for 9 h (+CC+), (ii) ad libitum hay for 12 h before feeding CC and no hay after CC intake for 9 h (+CC-), (iii) feed restriction for 12 h before feeding CC and ad libitum hay after CC intake for 9 h (-CC+) and (iv) 1.2 kg hay/100 kg body weight (BW) per day, divided into two equal portions and offered at 0900 h and 1900 h, feed restriction for 12 h before feeding CC and no hay after CC intake for 9 h (-CC-). CC intake was adjusted to a starch intake of 2 g/kg BW. The different hay offerings did not affect basal plasma glucose and insulin levels. A significant rise in plasma glucose and insulin was found after CC intake for all diets. The highest peak glucose levels were analysed for -CC+, and the lowest glucose peaks were found for +CC- (diet P < 0.05). The highest insulin peaks were monitored for -CC+ (31.27 ± 18.19 μU/ml) and lower peaks for +CC- (13.36 ± 2.93 μU/ml) (diet P < 0.05). Insulin for -CC- and +CC- returned to resting values about 300 min after CC feeding. For +CC+ and -CC+, insulin levels were still above resting levels 510 min after CC intake (diet P < 0.05). The present data suggest that feed restriction for 12 h before feeding the starchy diet and no further roughage intake during blood sampling period provide the best-defined conditions.  相似文献   

20.
Studies were conducted on the viability of Micrococcus varians strain M95 and Lactobacillus plantarum strain L4 upon freezing and freeze-drying using five cryoprotectants (sucrose, lactose, sodium glutamate, peptone, dry nonfat milk) singly or in combinations with gelatin, glutamic acid, and sodium acetate. The number of survivals was determined immediately after treatment and after storage at room temperature or refrigeration temperatures, under vacuum or in air. Dry nonfat milk and peptone introduced at the levels of 8 and 5%, respectively, to broth culture, were found to be the best cryoprotectants providing a 100% viability determined immediately after the treatment of the strains under investigation.Immediately after freezing and freeze-drying, the numbers of viable micrococci remain high, the percentage viability in the presence of almost all the protectants used being 100%. During storage, those numbers decrease rapidly, reaching zero in 3 months upon storage at room temperature in air. The storage ability of lactobacilli is considerably better and, regardless of the fact that the percentage viability decreases, sufficient numbers of viable cells remain after 6 months of storage at both test temperatures.The best results are obtained on storing the microoganisms under vacuum in ampoules under reduced temperatures (+5 °C).  相似文献   

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