Glucose Feed Rate Research Articles

Process design for recombinant protein production based on the promoter, PmalK.

P(malK) is induced through activation of MalT, by the formation of maltotriose and cyclic adenosine monophosphate (cAMP). The possibility to influence endogenous inducer levels is used to vary the production rates in specifically designed production protocols. Induction based on a batch process protocol on maltose gives low production rates, as the result of a lack of cAMP, which is shown to be of major importance to fully induce this promoter. Two mechanisms are thus used to influence the levels of maltotriose and/or cAMP formation: (1) catabolite derepression achieved from low glucose concentration and (2) catabolite derepression/inducer exclusion from diauxic growth on glucose/maltose. Fed-batch processes based on limited amounts of glucose result in product accumulation of up to 10% of the total protein. Depending on the feed of limiting glucose, different production profiles are developed. The initial increase in the production rate is due to maltotriose formation from endogenous glycogen degradation while, later in the process, production can be further supported by elevated levels of cAMP, provided the feed rate is sufficiently low. The introduction of maltose after a preceding fed-batch process on glucose can be efficiently used to produce maltotriose in combination with cAMP formation in the event of catabolite derepression. This leads to higher production rates and a further increase in product accumulation of up to 30% of the total protein. The diauxic growth phase resulting from the shift in carbon source can be shortened and even avoided by the design of the preceding feed-rate of glucose. It is postulated that proper design of the inoculum and initial phases of production can reduce basal levels of product formation. With this promoter, the production rate can be as high as 65 units mg(-1) h(-1) and the time to reach a maximal production rate can be designed to take up to 8 h. Furthermore, the duration of the production rate can be as long as 7 h.

Ethanol evolution rate: a new parameter to determine the feeding rate for the production of avermectins by Streptomyces avermitilis.

A new parameter ethanol evolution rate (EER) was developed to aid in the determination of glucose feeding rate in avermectin production. The EER characterized the level of primary metabolism and its value was affected mainly by the supply of O2 and glucose. In an abnormal batch, over-feeding of glucose led to 2.5-fold increase of the maximum EER value compared to the normal one, and the production was thus decreased by nearly 80%. Together with other criteria, the EER helped to control utilization of substrate, so it has been successfully used to control glucose feeding in an industrial process.

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

AbstractFed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO2 production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.

Limiting factors in Escherichia coli fed-batch production of recombinant proteins.

Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

Optimization of the glucose feed rate profile for the production of tryptophan from recombinant E coli

AbstractA strain of Escherichia coli was engineered to overproduce L‐tryptophan. A fed‐batch fermentation process was developed, producing 30.8 ± 1.4 g dm−3 with a yield on glucose of 0.132 ± 0.010 g g−1. Specific production rate did not appear to be limited by cloned enzyme activity, but by the carbon flux from central metabolism into the aromatic amino acid pathway. The glucose feed rate profile was modified in an attempt to increase the production rate. Tryptophan production was not affected, but led to glutamic acid excretion at high levels. The high specific glucose consumption rate at the low growth rate led to the high glutamate excretion. A new fermentation process involving modification of the feed profile to limit the formation of by‐products was discovered. The resulting final process increased tryptophan production to 42.3 ± 2.7 g dm−3 with yield on glucose of 0.176 ± 0.006 g g−1. The instantaneous yield realized the theoretical maximum for the majority of the fermentation.© 2002 Society of Chemical Industry

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.

Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone

In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.

Construction of a fuzzy control system for a bioreactor using biomass support particles

A fuzzy control system was applied to a bioreactor in which the flocculent yeast Saccharomyces diastaticus ATCC 60715 was immobilized by entrapment in a reticulated polyurethane foam cube ( 6 mm×6 mm×6 mm, biomass support particle (BSP)). The fuzzy inference algorithm was used to calculate the suitable level of glucose feed rate from the monitored data of the CO 2 evolution rate (CER) and the integrated amount of CO 2 evolution. One of the main concerns is the mass transfer of glucose in the BSP in which S. diastaticus was immobilized. With the BSPs, glucose diffusivity becomes lower and starvation of the cells can occur if the control system is not quick enough to respond to the starvation. The fuzzy control system was successful in controlling the glucose feed pump to maintain a suitable glucose concentration for the smooth continuation of ethanol production.

Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture.

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.

Comparative evaluation of various control schemes for fed-batch fermentation

The crucial problem associated with control of fed-batch fermentation process is its time-varying characteristics. A successful controller should be able to deal with this feature in addition to the inherent nonlinear characteristics of the process. In this work, various schemes for controlling the glucose feed rate of fed-batch baker's yeast fermentation were evaluated. The controllers evaluated are fixed-gain proportional-integral (PI), scheduled-gain PI, adaptive neural network and hybrid neural network PI. The difference between the specific carbon dioxide evolution rate and oxygen uptake rate (Qc–Qo) was used as the controller variable. The evaluation was carried out by observing the performance of the controllers in dealing with setpoint tracking and disturbance rejection. The results confirm the unsatisfactory performance of the conventional controller where significant oscillation and offsets exist. Among the controllers considered, the hybrid neural network PI controller shows good performance.

An Improved Probing Controller for Substrate Feeding in Fed-Batch Cultures of E. Coli: Simulations and Experiments

A strategy for feedback control of the glucose feed rate in cultivations of E. coli is discussed. By making probing pulses in the feed rate it is possible to detect and avoid undesirable by-product formation using a standard dissolved oxygen sensor. The feasibility of an improved and simplified probing algorithm is demonstrated by simulations as well as laboratory-scale experiments.

Characteristics of a DO-controlled fed-batch culture of Escherichia coli

The DO-controlled glucose limited fed-batch technique was investigated in an E. coli process for production of a recombinant protein. The k L ac * value (oxygen transfer rate at zero oxygen concentration) was calculated from on-line gas analysis data during the process. In the investigated processes with induced production of recombinant protein, the k L ac * value decreased drastically several hours after induction. The reason for the decrease was found in increasing concentrations of DNA in the medium and increased viscosity due to cell lysis. The consequences of such a dramatic decrease in the volumetric oxygen transfer coefficient on the glucose feed and specific rates are described in computer simulations and experimental data.

On-line control of feeding of medium components to attain high cell density

On line control coupled with an expert system was constructed for the control of a fed-batch culture with the aim of achieving a high cell density. During the cultivation, the expert system could monitor the extents of sufficiencies in the amounts of chemical elements in the medium every 10 min, and suggest a modification to the feeding control policy if the amount of a particular component was inadequate for cell growth. However, we often encounter such a kind of cultivations in which some particular carbon source such as glucose and ethanol should be controlled at low concentration in many culture processes, because the excess feeding might cause the growth inhibition by its own accumulation in the culture broth, or the biproduct accumulation like as lactate or acetate, unsuitable substances for smoothed growth. In this study, we developed an online control system based on production rules which managed the glucose feed rate from DO signal. The online control was carried out by the control computer connected with the other computer for expert system, because a relatively long time (several minutes) was needed for the inference of the expert system and the influence of the starvation of carbon source on the cell growth is not negligible even in several minutes. The on-line control system with expert system was applied to the production of cell mass of E. coli W3110 and the final cell concentration reached 137 g-dry cell weight/l.

Acetic acid accumulation in aerobic growth of recombinant Escherichia coli

A correlation between μ HAc (specific acetic acid accumulation rate) and μ (specific growth rate) for a recombinant Escherichia coli BL21 strain was defined under typical conditions to achieve high cell densities (fed-batch process, dissolved oxygen concentration higher than 30% saturation, semi-synthetic medium). The feeding rate of glucose was continuously adjusted in order to support constant values of μ (0.4, 0.3, 0.2, 0.1 and 0.05 h −1) during each of the five runs. An exponential correlation between μ and μ HAc showed significant increments in μ HAc for μ values higher than 0.30 h −1. The metabolic activity of acetic acid synthesis was not suppressed, even at μ as low as 0.05 h −1, a result which is not in agreement with previous published data.

A structured model for hyphal differentiation and penicillin production using Penicillium chrysogenum

A structured kinetic model describing growth, differentiation, and penicillin production in submerged Penicillium chrysogenum fermentations is reported. The filamentous hyphae are divided into four distinct regions on the basis of the activities and structure of hyphal compartments, viz., actively growing (mainly apical) regions, nongrowing or penicillin producing regions, vacuoles, and degenerated or metabolically inactive regions. A mechanistic approach is taken to give quantitative descriptions of differentiation and degeneration as a consequence of vacuolation. The growth and degeneration of vacuoles are expressed in the form of a population balance. The model assumes that newly generated vacuoles appear by differentiation of healthy regions, grow in size with limitation of available substrate, and eventually give rise to empty hyphal compartments. In the model the penicillin production is related to the amounts of the nongrowing regions of the hyphae. The model is used for successful predictions of the amounts of the four hyphal regions and the penicillin G production rate throughout the fed-batch fermentations of an industrial P. chrysogenum strain under different glucose feeding regimes. Quantitative information on proportions of the hyphal regions was obtained from image analysis measurements and the parameters of the kinetic model were identified. When the glucose feed rate to the production culture is switched between a high and a low value, the model can successfully predict the dynamic changes of differentiation and the resulting penicillin production caused by the variations in the nutrient conditions. The use of image analysis to characterize differentiation as a basis for structured modeling of the penicillin fermentation appears to be very powerful, and the method has great potential for use in process simulation and control of antibiotic fermentations.

Overexpression of an alkaline lipase gene from Proteus vulgaris K80 in Escherichia coli BL21/pKLE

Over-expression of Proteus vulgaris K80 lipase gene in Escherichia coli BL21 (DE3)/pKLE was achieved, with the enzyme being produced in an as active soluble form, using the T7 RNA polymerase system in a modified M9 salt or M9ZB medium. d-Lactose (55 mM) was used to induce gene expression and gave twice the lipase activity achieved with 0.4 mM IPTG. The expression of the lipase gene depended on the feeding rate of glucose being optimal at 12 g l−1 h−1.

Effect of controlling lactate concentration and periodic change in DO concentration on fermentation characteristics of a mixed culture of Lactobacillus delbrueckii and Ralstonia eutropha for PHB production

A mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly β-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermentor. Based on the experimental studies on the effect of lactate concentration on the cell growth of both microorganisms, the lactate concentration was controlled at less than 5 g/ l using an on-line enzymatic lactate and glucose sensors with the FIA (flow injection analysis) system, and by manipulating the glucose feeding rate. Since L. delbrueckii prefers anaerobic conditions while R. eutropha prefers aerobic conditions, we studied the effect of DO concentration on fermentation characteristics of each microorganism. For the mixed culture, we considered the control scheme of a two inputs and three outputs multivariable system. It was experimentally shown that the periodic fermentation resulted in superior PHB yield with relatively high productivity as compared with the cases where DO concentration was controlled to be constant either at less than 1 ppm or 3 ppm.

Continuous production of gluconic acid by Aspergillus niger immobilized on a cellulosic support: study of low pH fermentative behaviour of Aspergillus niger

Bioconversion of glucose to gluconic acid at low pH using Aspergillus niger immobilized on cellulose fabric was investigated. Glucose solution (100 g l −1) was made to flow through capillaries of a vertical fabric support, used for immobilization and is oxidized to gluconic acid at the interface. Conditions of temperature, humidity, airflow, and glucose feed rate have been optimized. The system could be run continuously for a period of 61 days utilizing the entire available glucose. The emerging broth contained a product concentration of 120–140 g l −1 of gluconic acid which is higher than the expected [maximum of 109 g gluconic acid 100 g glucose −1] as a result of evaporative concentration during the downward flow.

Control of a mixed culture of Lactobacillusdelbrueckii and ralstonia eutropha for the production of PHB from glucose via lactate

The mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly β-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermentor. Noting that the high lactate concentration inhibits the cell growth for both cells, we attempted to control it at low level. The lactate concentration was measured on-line using an on-line enzymatic lactate sensor with FIA system. The control system was 2 × 2 multivariable system where glucose and lactate concentrations were controlled at their set points. Since the lactate was produced by L. delbrueckii and consumed by R. eutropha, it was quite difficult to control the lactate concentration with the conventional control policy due to interaction of both control loops. In order to compensate for the interaction between the two control loops, we considered to manipulate the glucose feeding rate depending on the difference between the on-line measured or estimated lactate concentration and its set point. The manipulation was made by adjusting the on period of on-off control so that the proportinal control property was retained. The usefulness of the proposed control policy was verified by both computer simulation and by the fed-batch experiments. Since the lactate concentration could be estimated on-line by the amount of NaOH solution supplied to keep the culture pH at constant level to some extent, we also studied the effect of its accuracy on the control quality and thus PHB production.

THE FUZZY NEURAL NETWORK CONTROLLER IN YEAST FED-BATCH FERMENTATION

On the basis of the optimized substrate feeding rate of glucose acquired from an artificial neural network model of yeast fed-batch fermentation, an adaptive fuzzy neural network controller (FNNC) was developed, and on-line experiments were performed to examine the feasibility of the controller for actual use. The objective is to obtain a high concentration of cell mass in the shortest fed-batch culture time. Experimental results reveal that FNNC-controlled on-line feeding of substrate performs well.