谷氨酸发酵过程葡萄糖自动流加系统

补料（流加葡萄糖）操作是谷氨酸发酵过程最重要的操作之一,工业上有各种各样的补料操作方法。控制葡萄糖浓度于一个较为平稳且适中的水平有利于提高谷氨酸发酵的性能指标。通过在线计量谷氨酸发酵中的氨水耗量并据此在线推定发酵液中的葡萄糖浓度,构建了一个谷氨酸发酵自动在线补料系统。使用该控制系统,谷氨酸发酵过程的葡萄糖浓度可以控制在任意水平,平稳、无波动的谷氨酸发酵可以得到实现。     

在线推定和控制葡萄糖浓度改善谷氨酸发酵性能

谷氨酸发酵过程一般需要定时、人工分批式地添加葡萄糖。该流加操作方式会引起发酵罐内葡萄糖浓度的剧烈波动, 不利于高效、稳定的谷氨酸生产。谷氨酸发酵具有显著的非增殖耦联特征, 产酸期葡萄糖耗量与氨水耗量存在非常明显的关联性。通过在线计量氨水耗量推定糖耗以及葡萄糖浓度, 可比较准确地将谷氨酸发酵产酸期的糖浓度控制在预先设定的水平。当糖浓度控制在5 g/L~10 g/L的低水平时, 最终谷氨酸浓度可以达到80 g/L的较高水平, 高糖浓度下的渗透压效应有望得到缓解, 有利于发酵生产的稳定。     

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。     

On-line optimization of glutamate production based on balanced metabolic control by RQ

In glutamate fermentations by Corynebacterium glutamicum, higher glutamate concentration could be achieved by constantly controlling dissolved oxygen concentration (DO) at a lower level; however, by-product lactate also severely accumulated. The results of analyzing activities changes of the two key enzymes, glutamate and lactate dehydrogenases involved with the fermentation, and the entire metabolic network flux analysis showed that the lactate overproduction was because the metabolic flux in TCA cycle was too low to balance the glucose glycolysis rate. As a result, the respiratory quotient (RQ) adaptive control based “balanced metabolic control” (BMC) strategy was proposed and used to regulate the TCA metabolic flux rate at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux. Compared with the best results of various DO constant controls, the BMC strategy increased the maximal glutamate concentration by about 15% and almost completely repressed the lactate accumulation with competitively high glutamate productivity.     

A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations

A combined predictive and feedback control algorithm based on measurements of the concentration of glucose on-line has been developed to control fed-batch fermentations of Escherichia coli. The predictive control algorithm was based on the on-line calculation of glucose demand by the culture and plotting a linear regression to the next datum point to obtain a predicted glucose demand. This provided a predictive "coarse" control for the glucose-based nutrient feed. A direct feedback control using a proportional controller, based on glucose measurements every 2 min, fine-tuned the feed rate. These combined control schemes were used to maintain glucose concentrations in fed-batch fermentations as tight as 0.49 +/- 0.04 g/liter during growth of E. coli to high cell densities.     

On-line monitoring and controlling system for fermentation processes

A personal computer-based on-line monitoring and controlling system was developed for the fermentation of microorganism. The on-line HPLC system for the analysis of glucose and ethanol in the fermentation broth was connected to the fermenter via an auto-sampling equipment, which could perform the pipetting, filtration and dilution of the sample and final injection onto the HPLC through automation based on a programmed procedure. The A/D and D/A interfaces were equipped in order to process the signals from electrodes and from the detector of HPLC, and to direct the feed pumps, the motor of stirrer and gas flow-rate controller. The software that supervised the control of the stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of glucose and ethanol concentration was programmed by using Microsoft Visual Basic under Microsoft Windows. The signal for chromatographic peaks from on-line HPLC was well captured and processed using an RC filter and a smoothing algorithm. This monitoring and control system was demonstrated to be effective in the ethanol fermentation of Zymomonas mobilis operated in both batch and fed-batch modes. In addition to substrate and product concentrations determined by on-line HPLC, the biomass concentration in Z. mobilis fermentation could also be on-line estimated by using the pH control and an implemented software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.     

Growth monitoring and control in complex medium: a case study employing fed-batch penicillin fermentation and computer-aided on-line mass balancing

To broaden the practicality of on-line growth monitoring and control, its application in fedbatch penicillin fermentation using high corn steep liquor (CSL) concentration (53 g/L) is demonstrated. By employing a calculation method that considers the vagaries of CSL consumption, overall and instantaneous carbon-balancing equations are successfully used to calculate, on-line, the cell concentration and instantaneous specific growth rate in the penicillin production phase. As a consequence, these equations, together with a feedback control strategy, enable the computer control of glucose feed and maintenance of the preselected production-phase growth rate with error less than 0.002 h(-1).     

A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations.

A combined predictive and feedback control algorithm based on measurements of the concentration of glucose on-line has been developed to control fed-batch fermentations of Escherichia coli. The predictive control algorithm was based on the on-line calculation of glucose demand by the culture and plotting a linear regression to the next datum point to obtain a predicted glucose demand. This provided a predictive "coarse" control for the glucose-based nutrient feed. A direct feedback control using a proportional controller, based on glucose measurements every 2 min, fine-tuned the feed rate. These combined control schemes were used to maintain glucose concentrations in fed-batch fermentations as tight as 0.49 +/- 0.04 g/liter during growth of E. coli to high cell densities.     

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.     

Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.     

Inline noninvasive Raman monitoring and feedback control of glucose concentration during ethanol fermentation

Raman spectroscopy as a process analytical technology tool was implemented for the monitoring and control of ethanol fermentation carried out with Saccharomyces cerevisiae. The need for the optimization of bioprocesses such as ethanol production, to increase product yield, enhanced the development of control strategies. The control system developed by the authors utilized noninvasive Raman measurements to avoid possible sterilization problems. Real-time data analysis was applied using partial least squares regression (PLS) method. With the aid of spectral pretreatment and multivariate data analysis, the monitoring of glucose and ethanol concentration was successful during yeast fermentation with the prediction error of 4.42 g/L for glucose and 2.40 g/L for ethanol. By Raman spectroscopy-based feedback control, the glucose concentration was maintained at 100 g/L by the automatic feeding of concentrated glucose solution. The control of glucose concentration during fed-batch fermentation resulted in increased ethanol production. Ethanol yield of 86% was achieved compared to the batch fermentation when 75 % yield was obtained. The results show that the use of Raman spectroscopy for the monitoring and control of yeast fermentation is a promising way to enhance process understanding and achieve consistently high production yield.     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

On-line estimation of sugar concentration for control of fed-batch fermentation of lignocellulosic hydrolyzates by Saccharomyces cerevisiae

A feed control strategy, based on estimated sugar concentrations, was developed with the purpose of avoiding severe inhibition of the yeast Saccharomyces cerevisiae during fermentation of spruce hydrolyzate. The sum of the fermentable hexose sugars, glucose and mannose, was estimated from on-line measurements of carbon dioxide evolution rate and biomass concentration by use of a simple stoichiometric model. The feed rate of the hydrolyzate was controlled to maintain constant sugar concentration during fed-batch fermentation, and the effect of different set-point concentrations was investigated using both untreated and detoxified hydrolyzates. The fed-batch cultivations were evaluated with respect to cellular physiology in terms of the specific ethanol productivities, ethanol yields, and viability of the yeast. The simple stoichiometric model used resulted in a good agreement between estimated sugar concentrations and off-line determinations of sugar concentrations. Furthermore, the control strategy used made it possible to maintain a constant sugar concentration without major oscillations in the feed rate or the sugar concentration. For untreated hydrolyzates the average ethanol productivity could be increased by more than 130% compared to batch fermentation. The average ethanol productivity was increased from 0.12 to 0.28 g/g h. The productivity also increased for detoxified hydrolyzates, where an increase of 16% was found (from 0.50 to 0.58 g/g h).     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Pulsed feeding during fed-batch fungal fermentation leads to reduced viscosity without detrimentally affecting protein expression

The goal in this study was to determine if pulsed addition of substrate could be used to alter filamentous fungal morphology during fermentation, to result in reduced broth viscosity. In all experiments, an industrially relevant strain of Aspergillus oryzae was grown in 20-liter fermentors. As a control, cultures were fed limiting substrate (glucose) continuously. Tests were performed by altering the feeding strategy so that the same total amount of glucose was fed in repeated 300-s cycles, with the feed pump on for either 30 or 150 s during each cycle. Variables indicative of cellular metabolic activity (biomass concentration, oxygen uptake rate, base consumed for pH control) showed no significant difference between continuous and pulse-fed fermentations. In addition, there was no significant difference between total extracellular protein expression or the apparent distribution of these proteins. In contrast, fungal mycelia during the second half of pulse-fed fermentations were approximately half the size (average projected area) of fungi during fermentations with continuous addition of glucose. As a result, broth viscosity during the second half of pulse-fed fermentations was approximately half that during the second half of continuous fermentations. If these results prove to be applicable for other fungal strains and processes, then this method will represent a simple and inexpensive means to reduce viscosity during filamentous fungal fermentation.     

An optical-density-based feedback feeding method for ammonium concentration control in Spirulina platensis cultivation

Cultivation of Spirulina platensis using ammonium salts or wastewater containing ammonium as alternative nitrogen sources is considered as a commercial way to reduce the production cost. In this research, by analyzing the relationship between biomass production and ammonium- N consumption in the fed-batch culture of Spirulina platensis using ammonium bicarbonate as a nitrogen nutrient source, an online adaptive control strategy based on optical density (OD) measurements for controlling ammonium feeding was presented. The ammonium concentration was successfully controlled between the cell growth inhibitory and limiting concentrations using this OD-based feedback feeding method. As a result, the maximum biomass concentration (2.98 g/l), productivity (0.237 g/l·d), nitrogen-to-cell conversion factor (7.32 gX/gN), and contents of protein (64.1%) and chlorophyll (13.4 mg/g) obtained by using the OD-based feedback feeding method were higher than those using the constant and variable feeding methods. The OD-based feedback feeding method could be recognized as an applicable way to control ammonium feeding and a benefit for Spirulina platensis cultivations.     

Productivity improvement of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation via robust optimization

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.     

Application of computer to monitoring and control of fermentation process: Microbial conversion of ML-236B Na to pravastatin

An automatic feeding process for microbial hydroxylation of ML236B sodium salt (ML-236B Na; compactin) by Streptomyces carbophilus SANK 62585 was developed. The hydroxylated product, pravastatin sodium salt (pravastatin; trade name Mevalotin), is an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase (HMG-CoA reductase) used as cholesterol-lowering drug. The hydroxylation activity of S. carbophilus was induced by the addition of ML236B Na to culture broth but inhibited by high concentration of ML236B Na. In order to obtain high conversion yield, it was necessary to maintain optimum ML236B Na concentration throughout the fermentation by continuous feeding. For this purpose, we developed an on-line monitoring method, which mainly consisted of a cross-flow filtration module, high-performance liquid chromatography (HPLC) analyzer, feed pump, and microcomputer for regulation of ML236B Na concentration. An algorithm for control of ML236B Na feed rate based on feedback and feed-forward control where conversion rate after Deltat was estimated by using regression analysis of the five latest values of conversion rate. In a fed-batch culture employing this system, the concentration of ML236B Na was maintained at optimum level during the fermentation and the productivity of pravastatin was increased threefold over that obtained in manual control culture. (c) 1993 John Wiley & Sons, Inc.     

不同补料控制方式发酵生产头孢菌素C的性能比较

在7 L发酵罐下,对利用顶头孢霉菌(Cephalosporins acremonium)发酵生产头孢菌素C(CPC)过程的最优底物流加工艺进行了研究。提出了一种新式硫铵豆油耦联型的硫铵流加策略。该控制策略可将发酵液中的氨态氮浓度控制在3 6 g/L之间,同时满足了发酵前期细胞生长与CPC合成对氮源和硫源的需求,促进了顶头孢霉菌菌丝分化,为发酵后期的CPC高效生产奠定了前期基础。比较了CPC合成期内间歇、匀速和DO-Stat自动流加3种不同豆油流加方式的发酵性能。研究发现,耦联使用硫铵/后程通富氧空气DO-Stat法进行硫铵和豆油的同时补料和CPC发酵,可将碳源浓度与溶解氧浓度DO同时控制于适中水平,使CPC合成以高浓度和低副产物积累的方式进行,最终CPC浓度和得率分别达到35.77 g/L和13.3%。主代谢副产物脱乙酰氧头孢菌素C(DAOC)的积累量和DAOC/CPC分别仅有0.178 g/L和0.5%。     

A probing feeding strategy for Escherichia coli cultures

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.