Optimization of L-phenylalanine production of Corynebacterium glutamicum under product feedback inhibition by elevated oxygen transfer rate.

Production feedback inhibition both on cell growth and on product formation of phenylalanine fermentation might be alleviated by elevated oxygen supply. Batch fermentations by a high phenylalanine producing strain Corynebacterium glutamicum CCRC 18335 at various initial phenylalanine concentrations (P(0)) ranging from 0 to 20 g/L and different oxygen transfer rate coefficients (K(L)a) ranging from 23 to 76 h(-1) were studied. The fermentation parameters with respect to P(0) were strongly dependent on K(L)a. Cell yield favored higher K(L)a and lower P(0). Product yield with respect to varying phenylalanine concentration was evaluated by the relative oxygen availability (ROA). The optimal ROA for phenylalanine formation was strongly dependent on the product concentration. While P(0) was low, the product inhibition was less significant and the maximum product yield occurred while ROA was at 0.5-0.6. While P(0) was high, the product inhibition was significant and the maximum product yield occurred while ROA was at 0.8-0.9. These results suggest that the product feedback inhibition of phenylalanine fermentation processes can be alleviated by a gradual increase in oxygen supply rate while the increasing product concentration is taken into account. The strategy is demonstrated in a fed-batch culture with elevated oxygen supply. The final phenylalanine concentration was 23.2 g/L, which was 45% better than that of the fed-batch fermentation without elevated oxygen supply. Likewise, the maximum productivity was improved by 42% at 0.37 g/(L x h).     

Object-oriented fuzzy expert system for on-line diagnosing and control of bioprocesses

Summary An object-oriented fuzzy expert system to support on-line control of an automated fermentation plant is described. The major elements of the system consist of a fuzzy inference engine, a database, a knowledge base, and an expression evaluater. The expression evaluater calculates specific rates for growth, and substrate and product formation at different physiological states during the cultivation from the measured data. The specific rates are then compared with the standard target rates stored in the database. If differences outside the set tolerances were observed, the inference engine analyses the reasons for the faults on the basis of the knowledge represented in the form of a knowledge network and fuzzy membership functions of the process variables. The fuzzy expert system was developed on the basis of a shell constructed by using the object oriented Smalltalk/V Mac programming environment, with Lactobacillus casei lactic acid fermentation as the example of process application.Visiting scientist from Helsinki University of Technology at RIKEN Correspondence to: P. Linko or I. Endo     

Multivariate statistical monitoring of batch processes: an industrial case study of fermentation supervision

This article describes the development of Multivariate Statistical Process Control (MSPC) procedures for monitoring batch processes and demonstrates its application with respect to industrial tylosin biosynthesis. Currently, the main fermentation phase is monitored using univariate statistical process control principles implemented within the G2 real-time expert system package. This development addresses integrating various process stages into a monitoring system and observing interactions among individual variables through the use of multivariate projection methods. The benefits of this approach will be discussed from an industrial perspective.     

A balanced DO-stat and its application to the control of acetic acid excretion by recombinant Escherichia coli

During fed-batch cultivation of a recombinant Escherichia coli AT2471 harboring plasmid pSY130-14 for phenylalanine production, a large amount of acetic acid was excreted by the cells and accumulated in the culture medium. Acetic acid concentration reached 30-35 g/L at the end of a process conducted without special precautions for the reduction of this excretion. Cell growth stopped when acetic acid concentration was about 15 g/L, resulting in poor growth, 16 g/L cell concentration, and poor production - 8 g/L phenylalanine. A novel control strategy, called a balanced DO-stat. was developed to prevent acetic acid excretion. It represents a model-independent two-loop control structure, which is simple, reliable, and convenient for computer application. Using the balanced DO-stat, implemented in a computer control system, acetic acid concentration was kept at zero during the entire cultivation period. As a result, the cell concentration increased to 36 g/L and phenylalanine concentration reached 24 g/L. Aside from the phenylalanine fermentation, the proposed control approach might be applied to cultivation of other bacterial and yeast strains which have similar mechanism of the excretion of fermentative by-products.     

烟曲霉TMS-26产紫杉醇补料分批发酵条件初步探索

内生真菌发酵法是解决紫杉醇药源短缺问题的有效途径之一。本研究以摇瓶分批发酵为基础,进行摇瓶补料分批发酵研究,探究了苯丙氨酸、甘氨酸、苯甲酸钠乙酸钠混合液、3,5-二硝基水杨酸、H₂O₂、CuSO₄在发酵周期（13d）中,不同添加时间点对TMS-26菌体量及紫杉醇产量的影响,发现在第8天添加苯丙氨酸、甘氨酸、3,5-二硝基水杨酸时,其产量分别达到了(664.80±40.34)µg/L、(628.72±30.44)µg/L、(641.36±19.62)µg/L;在第9天添加CuSO₄时,其产量达到了(697.46±15.76)µg/L;在第10天添加H₂O₂、苯甲酸钠乙酸钠混合液,其产量分别达到了(615.78±36.28)µg/L、(792.54±10.04)µg/L。在摇瓶补料分批发酵研究结果的基础上,进行了5L罐发酵工艺放大研究,探究了前体和诱导子通过进行一次补加和恒速补加的方式对Aspergillus fumigatus TMS-26菌体量及紫杉醇产量的影响,结果表明恒速补加苯丙氨酸乙酸钠混合液,紫杉醇产量达到了746.17µg/L。通过本次研究,优化了TMS-26产紫杉醇摇瓶补料分批发酵和5L罐发酵工艺,为后续实现紫杉醇工业化生产奠定基础。     

Pattern analysis techniques to process fermentation curves: application to discrimination of enological alcoholic fermentations

In fermentation processes, kinetic curves are generally aimed at control purposes. However, these curves could also contain information about inherent features of the product (such as origin, quality, etc.). This article presents several pattern analysis techniques used to classify fermentation curves. An application to alcoholic fermentation is presented as an illustration: it aims at retrieving the origin of a must from its fermentation curve. The fermentation kinetics of five vineyard musts, harvested over 9 years on the same parcels, were recorded. From these curves two sets of variables were generated: The first (p(1)) gathers all the kinetic curve points. The second (p(2)) contains a restrained number of variables, generated by the expert knowledge of the enologist. The set p(2) was processed by two very different techniques: a linear one (factorial discriminant analysis) and a nonlinear one (artificial neural networks). The set p(1) was processed by a new chemometric technique, the discriminant partial least-squares regression. For all the sets and the techniques used the selection of variables was studied. The interest in the latter is largely demonstrated both by theoretical and practical discussions. The discrimination results (up to 94% of good predictions) enhance the interest of the on-line measurements and their use in such pattern analysis tools.     

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.     

前体及诱导子和发酵条件对烟曲霉TMS-26产紫杉醇发酵体系优化

紫杉醇是一种广谱的抗癌药物,因其具有独特的抗癌机制、良好的抗癌效果和供不应求的市场等特征而备受关注。紫杉醇具有重大经济效益,但产量受到制约,价格极为昂贵,通过内生真菌发酵法生产紫杉醇能在一定程度上缓解其来源困难的问题。在产紫杉醇内生真菌TMS-26发酵液中添加前体物质和诱导子,并通过对接种量、装液量、初始pH和发酵时间等条件进行优化研究。单因素及正交试验表明在PDB培养基中加入苯丙氨酸20mg/L、苯甲酸钠30mg/L、乙酸钠8g/L、甘氨酸15mg/L、CuSO₄ 0.05mg/L、H₂O₂ 6mmol/L、3,5-二硝基水杨酸15mg/L时能有效提高紫杉醇产量,比优化前增产46.64%,达到446.28µg/L,并且发现最适菌株TMS-26的发酵条件为pH7.5、接种量5%、装液量120mL/250mL、发酵时间为10d。     

An expert system applied to the physiological analysis of early stage of beer fermentation

A fuzzy expert system was applied to the knowledge analysis of yeast physiology in the early stage of beer fermentation, when the wort was aerated. We used ergosterol and glycogen concentration in the wort as a suitable marker of physiological state of the cell population. The amount of both compounds influences the rate of fermentation, cell growth and the final taste of beer. The concentrations of ergosterol and glycogen including the number of cells can not be measured immediately during the relatively short aeration period, and incomplete experimental data are therefore found in laboratory logbooks. We therefore suggested that the fuzzy relation between the directly measurable dissolved oxygen concentration and the rate of ergosterol or glycogen formation should be identified and a fuzzy expert system should be used to analyze the behavior of the yeast.     

Studies on the phenylalanine hydroxylase system in vivo. An in vivo assay based on the liberation of deuterium or tritium into the body water from ring-labeled L-phenylalanine.

The rate of release of deuterons into the body water from 2,3,4,5,6-pentadeutero-L-phenylalanine has been shown to be a valid measure of the activity of the phenylalanine hydroxylase system in vivo. At a dose of 0.5 g/kg, the rate of release of deuterons is linear for 60 to 90 min. Male rats, which had previously been shown to have 22 to 25% more phenylalanine hydroxylase activity in liver extracts than female rats, produced deuterons from deuterated phenylalanine at a rate 20 to 30% greater than female rats. p-Chlorophenylalanine, which irreversibly inhibits phenylalanine hydroxylase in vivo, caused a similar degree of inhibition of the rate of deuteron formation as was found when phenylalanine hydroxylase was measured in extracts from the same group of animals. Methotrexate, which inhibits the phenylalanine hydroxylase system by preventing regeneration of the tetrahydropteridine cofactor, caused parallel inhibition of the in vivo assay as well as when the conversion of phenylalanine to tyrosine was measured in liver slices. Randomly ring-tritiated phenylalanine can be used interchangeably with ring-deuterated phenylalanine if greater sensitivity is needed in the in vivo assay for phenylalanine hydroxylase. However, a dose of 20 to 30 muCi/kg is required. The in vivo deuterium release assay described in this paper should be useful in studying the physiological control of the phenylalanine hydroxylating system. It also may be of value in differentiating between individuals who are heterozygotes for phenylketonuria and those who are homozygotes for hyperphenylalaninemia.     

L-苯丙氨酸生产的代谢工程研究

L-苯丙氨酸是一种重要的食品和医药中间体。工业上一般采用酶法和发酵法来生产L-苯丙氨酸。代谢工程的兴起,使得更加理性的改造菌株成为可能,这更加促进了发酵法的广泛应用。主要介绍了代谢工程在L-苯丙氨酸生产菌的改造中的应用情况,其中涉及苯丙氨酸生物合成途径中相关基因及其酶的调控、中央代谢途径的改造和芳香族氨基酸生物合成支路的修饰。并探讨了将来的发展前景。     

An on-line advisory control system for the lactic acid fermentation process

A fuzzy expert system was developed for online diagnosing and controlling of bioprocesses. The system was constructed in object-oriented Smalltalk/V for diagnosing and controlling of bioprocesses. Lactic acid fermentation with an industrial strain ofLactobacillus casei was chosen as the model system. The performance of the fuzzy expert system and the knowledge base utilizing experts' knowledge and several facts obtained from the experiments were successfully validated with on-line fermentations. The fuzzy expert system could diagnose a fault on-line and give reasonable advice to the process operator. In order to achieve the diagnosing faculty, a database, a knowledge base, and both backward and forward chaining procedures were implemented employing the object-oriented programming environment. A defuzzifier was implemented in the system to achieve on-line control. In order to realize a decision-making system with a human operator and a fuzzy expert system, a new control strategy namedAdvice was also introduced. Several cultivations were carried out in order to collect knowledge on the effects concerned with inoculum properties to the process and to construct a database including standard time-course profiles. The performance of the fuzzy expert control system was successfully tested with on-line experiments.Visiting scientist from HUT at RIKEN     

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.     

Monitoring and control of the physiological state of cell cultures

Advances in bioprocess engineering depends ultimately on the level of understanding and control of the physiological state of the cell population. Process efficiency is strongly influenced by changes in the cellular state which should be monitored, interpreted, and, if possible, properly manipulated. In most control systems this function is not explicitly considered, which hampers process development and optimization. Conventional control logic is based on direct mapping of the growth environment into process efficiency, thereby bypassing the cell state as an intermediate control objective. Today, this limitation is well realized, and explicit monitoring and control of cellular physiology are considered to be among the most challenging tasks of modern bioprocess engineering. We present here a generic methodology for the design of systems capable of performing these advanced monitoring and control functions.The term "physiological state" is quantified by a vector composed of several process variables that convey significant information about cellular state. These variables can be selected among different classes, including specific metabolic rates, metabolic rate ratios, degees of limitation, and others. The real-time monitoring of many of these is possible using commercial sensors. The definition and calculation of representative sets of physiological state variables is demonstrated with examples from several fermentor cultures: recombinant Escherichia coli for phenylalanine production, bioluminescent E. coli (harboring lux genes driven by a heat shock protein promoter) for detection of environmental pollutants, plant cell culture of Perilla frutescensfor anthocyanin production, and perfusion cultures of recombinant mammalian cells (NS0 and CHO) for therapeutic protein production.If the physiological state vector is on-line calculated, the fermentation process can be described by its trajectory in a space defined by the vector components. Then, the goal of the control system is to maintain the physiological state of the cell as close as possible to the trajectory, providing maximum efficiency. A control structure meant to perform this function is proposed, along with the mechanism for its design. In contrast to conventional systems which work in a closed loop in respect to the cell environment, this scheme operates in a closed loop in respect to the cell state. The discussed control concept has been successfully applied to the recombinant phenylalanine production, resulting in physiologically consistent operation, total computer control, and high process efficiency. Initial results from the application of the method to perfusion mammalian cell cultures are also presented. (c) 1996 John Wiley & Sons, Inc.     

Fermentation fault management and on-line state estimation using methods of cluster analysis

The results of the cluster analysis of fermentation data are used for the supervision and on-line state estimation. The results of the classification are presented as the average over all fermentation runs belonging to the class as well as the standard deviation. With the help of the class information the on-line fermentation is associated with the best suiting class. Faults in the data such as spikes or total failure of the sensors are detected as the class information automatically supplies tolerance regions for the measurements. In case of a fault a reliable extrapolation for the time of the fault can be calculated. The approach is implemented in the real-time expert system tool G2 and is applied to data of the carbon dioxide evolution rate (CER) of an industrial antibiotic fermentation process.     

氧载体、表面活性剂及H2O2对L-phe发酵影响的研究

用添加氧载体(油酸、豆油)、表面活性剂(Triton-X100)及H2O2的方法,改善L-苯丙氨酸发酵体系中的氧传递速率,以提高苯丙氨酸的产量。实验结果表明,在发酵0h添加1%的豆油、3%的油酸均可使产酸提高,分别可以使L-phe产量提高21.1%和39.5%;发酵0h同时加入3%油酸和0.05%Triton-X100时,提高产量78.95%;发酵12h添加0.075%H2O2,可以提高产苯丙氨酸产量18.42%。     

A substrate feeding strategy--using an oxystat for L-phenylalanine fermentation by Corynebacterium glutamicum

Summary A substrate feeding strategy using an oxystat was first successfully applied to a fed-batch phenylalanine fermentation. The control method allowed the fermentation to be under low dissolved oxygen tension, which was favourable phenylalanine formation, and to be from substrate inhibition during the course of fed-batch operation. The final product concentration was 3 times higher than in a batch culture.     

Co-extraction during reactive extraction of phenylalanine using Aliquat 336: interfacial mass transfer

Reactive liquid-liquid extraction can be used to separate hydrophilic fermentation products that would not otherwise partition into nonpolar solvents. However, during extraction of the target solute other compounds present in the extraction medium will also react with the ion exchange reagent and are thus co-extracted. In this study the effect of co-extraction on the interfacial flux of the target solute phenylalanine has been investigated for reactive extraction using Aliquat 336. The effect of co-extracting compounds has been included in a new interfacial flux balance, and experimental results reveal that the interfacial concentrations are equal to the final equilibrium conditions of the system. Using this information a simple mass transfer model has been developed from which film mass transfer coefficients may be determined. Co-extraction of other compounds present in the feed was found to reduce the interfacial flux of the target solute by reducing the driving force. Co-extraction did not affect the value of the film mass transfer coefficient, and therefore, co-extraction does not effect the transport properties of the solute to the interface. Extraction from a multicomponent fermentation broth resulted in a reduced flux, which arises from a reduction in the driving force caused by high levels of co-extraction. Furthermore, the flux was also reduced as the result of a mass transfer resistance caused by soluble surface-active compounds present in the fermentation broth adsorbing to the interface. The biomass associated with the fermentation broth was also found to reduce the solute flux, and it is believed that this is due to blockage of the interfacial area.     

克雷伯氏肺炎杆菌LDH526产1,3-丙二醇的甘油自动流加策略

1,3-丙二醇是一种重要的化工原料,主要作为平台化合物用于合成聚酯,如聚对苯二甲酸丙二醇酯。经基因工程改造的克雷伯氏肺炎杆菌LDH526能以甘油作为唯一碳源合成1,3-丙二醇,最终发酵浓度超过90 g/L。甘油浓度是影响1,3-丙二醇合成的关键因素。为了实现对甘油浓度的精确控制,设计并优化了基于发酵动力学的甘油自动流加策略。通过将底物流加速率与易观察变量p H和发酵时间偶联,实现了发酵过程中甘油流加的自启动和甘油浓度的动态控制。发酵72 h,1,3-丙二醇的浓度可稳定超过95 g/L。自动控制甘油流加的发酵过程具有可重复性、连续性以及人工工作量少的特点,有望从实验室规模扩大到生产规模。     

Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.