Optimization of a two-stage recombinant fermentation process: the dilution rate effect

The effects of dilution rates on the performance of a two-stage fermentation system for a recombinant Escherichia coli culture were studied. Dilution rate determines the apparent or averaged specific growth rate of a heterogeneous population of cells in the recombinant culture. The specific growht rate affects the genetic parameters involved in product formation in the second stage, such as plasmid stability, plasmid content, and specific gene expression rate. Kinetic models and correlations were developed for these parameters based on experimental data. Simulations of plasmid stability in the first stage showed that for longer fermentation periods, plasmid stability is better at higher dilution rates. However, the plasmid content is lower at these dilution rates. The optimal apparent specific growth rate for maximum productivity in the second stage was determined using two methods: (1) direct search for a constant specific growth rate, and (2) dynamic optimization using the maximum principle for a time-dependent specific growth rate profile. The results of the calculations showed that the optimum constant apparent specific growth rate for maximum over-all productivity is 0.40 h(-1). This coincides with the optimal specific growht rate for maximum plasmid content in the expressed stage. A 3.5% increase in overall productivity can be obtained by using a linear time dependent apparent specific growth rate control, mu(2)(t) = 0.0007t, in the course of the fermentation time.     

General characteristics of optimal feed rate profiles for various fed-batch fermentation processes

General Characteristics of the optimal feed rate profiles have been deduced for various fed-batch fermentation processes by analyzing singular controls and singular arcs. The optimal control sequences depend on the shapes of the specific growth and product formation rates, mu andpi, and the initial conditions. For fed-batch processes described by four mass balance equations, the most general optimal control sequence consists of a period of maximum feed rate, a period of minimum feed rate (a batch period), a period of singular feed rate (variable and intermediate), and a batch period. Degenerate sequences in which one or more periods are missing can result with a particular set of initial conditions. If the fermentation time is not critical, the singular control maximizes the net yield of product and only when the time is also important, it balances a trade off between the yield of product and the specific growth rate which dictates the fermentation time. With the sequence of optimal control known, the optimal feed rate profile determination is reduced to a problem of determining switching times.     

Fermentation of kraft black liquor for the production of citric acid by Candida tropicalis

Summary The production of citric acid by batch fermentation with the yeast strain Candida tropicalis ATCC 20240 was chosen as a potential process for the valorization of kraft black liquor. The effect of nitrogen concentration was studied and direct bioconversion of acetate to citrate was achieved when no nitrogen was supplemented to the medium. The use of kraft black liquor's acetate as a potential substrate for citric acid production was investigated. The acid precipitated liquor was highly inhibitory when its concentration was above 25% of the fermentation broth content. The yields of citric acid at low concentrations of kraft black liquor (5% and 15%) were the same as those recorded in synthetic acetate medium. Other organic acids present in the liquor may affect the yields and rates of citric acid production over acetate. Substrate uptake rates and product formation rates were lower, however, in comparison to synthetic media. The utilization of immobilized biomass improved the process parameters on kraft black liquor and enhanced the fermentation capabilities.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

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.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Model aided dynamic process analysis and optimization for the nourseothricin fermentation

The relation between product formation and growth kinetics could be characterized by two facts: the specific product formation rate depends on the ageing of the population and on the specific growth rate. These relation was formulated and quantified by a mathematical model, which was fitted to experimental data of a representative fermentation run und used to predict an optimal fermentation mode. In the result of this discussion cyclic fed batch fermentation was found to be optimal.     

Kinetics of microbial product formation and its consequences for the optimization of fermentation processes

Different types of product formation kinetics are discussed with respect to their significance for fermentation process economics. Microbial products belonging to various classes are formed in a growth-coupled manner. It is often found that the specific rate of product formation increases with the specific growth rate, approaching a maximum at higher growth rates. It is illustrated that for such types of relationship between the product formation rate and the growth rate process conditions are optimal when the specific rate of product formation is about half-maximal.     

Citric acid production by a novel Aspergillus niger isolate: I. Mutagenesis and cost reduction studies

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid production from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the mutation doubled the specific substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and specific productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substitutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

甲醇抑制时重组毕赤酵母发酵的特性研究

本文对毕赤酵母进行了恒化培养研究。以甲醇为唯一碳源时,在稀释率较低时(D<0.048 h^-1),连续培养系统操作很稳定。但在稀释率高时(D>0.048h^-1),连续培养系统的定态点不止一个,实验不能维持,故采用比生长速率恒定的分批流加培养进行研究。结果表明,毕赤酵母的生长符合Andrew普遍化底物抑制模型。综合考虑水蛭素的生成、底物的消耗,在生产中维持甲醇浓度为限制性浓度(0.5 g/L),且维持比生长速率为0.02 h^-1时,水蛭素Hir65的比生成速率达到最大值0.2 mg/(g·h)且甲醇的比消耗速率为0.04 g/(g·h)。     

Product formation kinetics in genetically modified <Emphasis Type="Italic">E. coli </Emphasis>bacteria: inclusion body formation

A data-driven model is presented that can serve two important purposes. First, the specific growth rate and the specific product formation rate are determined as a function of time and thus the dependency of the specific product formation rate from the specific biomass growth rate. The results appear in form of trained artificial neural networks from which concrete values can easily be computed. The second purpose is using these results for online estimation of current values for the most important state variables of the fermentation process. One only needs online data of the total carbon dioxide production rate (tCPR) produced and an initial value x of the biomass, i.e., the size of the inoculum, for model evaluation. Hence, given the inoculum size and online values of tCPR, the model can directly be employed as a softsensor for the actual value of the biomass, the product mass as well as the specific biomass growth rate and the specific product formation rate. In this paper the method is applied to fermentation experiments on the laboratory scale with an E. coli strain producing a recombinant protein that appears in form of inclusion bodies within the cells’ cytoplasm.     

Production of ethanol by Clostridium thermosaccharolyticum: II. A quantitative model describing product distributions

The study of the continuous culture of Clostridium thermosaccharolyticum on xylose showed multiple steady states and hysteresis. A quantitative model based on the biochemistry and physiology of xylose fermentation by C. thermosaccharolyticum was developed. The objective in developing this model was to bring together the observations both of this study and of other researchers on the fermentation of xylose. The model equations were written based on the metabolic pathway for xylose utilization by C. thermosaccharolyticum and the requirement that the carbon, ATP, and NADH within the cell be balanced. Given the specific growth rate mu and the specific xylose utilization rate q(s), a set of product distributions (ethanol, acetate, and lactate) satisfying these balances was obtained. This set was plotted on a triangular plot and named the permitted region. The product distributions within this permitted region were shown to be affected by the environmental parameters such as iron concentration and hydrogen partial pressure. The model predicted trends in product distribution which correlate with experimentally observed phenomena. The model was also used to analyze the continuous-culture data from our experimental work.     

Kinetic modeling of <Emphasis Type="Italic">Moorella thermoacetica</Emphasis> growth on single and dual-substrate systems

Acetic acid is an important chemical raw material that can be produced directly from sugars in lignocellulosic biomass. Development of kinetic models that capture the bioconversion dynamics of multiple sugar systems will be critical to optimization and process control in future lignocellulosic biorefinery processes. In this work, a kinetic model was developed for the single- and dual-substrate conversion of xylose and glucose to acetic acid using the acetogen Moorella thermoacetica. Batch fermentations were performed experimentally at 20 g L^?1 total sugar concentration using synthetic glucose, xylose, and a mixture of glucose and xylose at a 1:1 ratio. The product yield, calculated as total product formed divided by total sugars consumed, was 79.2, 69.9, and 69.7 % for conversion of glucose, xylose, and a mixture of glucose and xylose (1:1 ratio), respectively. During dual-substrate fermentation, M. thermoacetica demonstrated diauxic growth where xylose (the preferred substrate) was almost entirely consumed before consumption of glucose began. Kinetic parameters were similar for the single-substrate fermentations, and a strong linear correlation was determined between the maximum specific growth rate μ _max and substrate inhibition constant, K _s . Parameters estimated for the dual-substrate system demonstrated changes in the specific growth rate of both xylose and glucose consumption. In particular, the maximum growth rate related to glucose tripled compared to the single-substrate system. Kinetic growth is affected when multiple substrates are present in a fermentation system, and models should be developed to reflect these features.     

Modeling of an integrated fermentation/membrane extraction process for the production of 2-phenylethanol and 2-phenylethylacetate

An unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-phenylethanol and 2-phenylethylacetate by Kluyveromyces marxianus CBS 600 was developed. The extent to which this model, based only on data from the conventional fermentation and separation processes, provided an estimation of the integrated process was evaluated. The effect of product inhibition on specific growth rate and on biomass yield by both aroma compounds was approximated by multivariate regression. Simulations of the respective submodels for fermentation and the separation process matched well with experimental results. With respect to the in situ product removal (ISPR) process, the effect of reduced product inhibition due to product removal on specific growth rate and biomass yield was predicted adequately by the model simulations. Overall product yields were increased considerably in this process (4.0 g/L 2-PE+2-PEA vs. 1.4 g/L in conventional fermentation) and were even higher than predicted by the model. To describe the effect of product concentration on product formation itself, the model was extended using results from the conventional and the ISPR process, thus agreement between model and experimental data improved notably. Therefore, this model can be a useful tool for the development and optimization of an efficient integrated bioprocess.     

Fuzzy control of ethanol concentration its application to maximum glutathione production in yeast fed-batch culture

A fuzzy logic controller (FLC) for the control of ethanol concentration was developed and utilized to realize the maximum production of glutathione (GSH) in yeast fedbatch culture. A conventional fuzzy controller, which uses the control error and its rate of change in the premise part of the linguistic rules, worked well when the initial error of ethanol concentration was small. However, when the initial error was large, controller overreaction resulted in an overshoot.An improved fuzzy controller was obtained to avoid controller overreaction by diagnostic determination of "glucose emergency states" (i.e., glucose accumulation or deficiency), and then appropriate emergency control action was obtained by the use of weight coefficients and modification of linguistic rules to decrease the overreaction of the controller when the fermentation was in the emergency state. The improved fuzzy controller was able to control a constant ethanol concentration under conditions of large initial error.The improved fuzzy control system was used in the GSH production phase of the optimal operation to indirectly control the specific growth rate mu to its critical value mu(c). In the GSH production phase of the fed-batch culture, the optimal solution was to control mu to mu(c) in order to maintain a maximum specific GSH production rate. The value of mu(c) also coincided with the critical specific growth rate at which no ethanol formation occurs. Therefore, the control of mu to mu(c) could be done indirectly by maintaining a constant ethanol concentration, that is, zero net ethanol formation, through proper manipulation of the glucose feed rate. Maximum production of GSH was realized using the developed FLC; maximum production was a consequence of the substrate feeding strategy and cysteine addition, and the FLC was a simple way to realize the strategy. (c) 1993 John Wiley & Sons, Inc.     

浓香型白酒发酵过程微生物合成正丙醇途径解析

【目的】揭示浓香型白酒窖内发酵过程与正丙醇合成相关的微生物和代谢途径。【方法】通过对浓香型白酒窖内发酵过程酒醅中微生物的宏转录组进行分析,解析与正丙醇合成相关的微生物和代谢途径,并验证相关微生物合成正丙醇的能力。【结果】浓香型白酒窖内发酵过程中有3条可能的酒醅微生物合成正丙醇的途径。真菌主要通过2-甲基苹果酸代谢途径和苏氨酸代谢途径合成正丙醇,细菌则主要通过丙酸代谢途径合成并参与苏氨酸代谢途径。宏转录组测序分析表明,这3条途径对白酒窖内发酵过程正丙醇的合成与积累均有贡献,并且微生物通过这3条途径合成正丙醇的时期和能力存在较大差异。此外,对分离自酒醅的酵母和乳酸菌合成正丙醇能力分析发现,它们均与浓香型白酒窖内发酵过程正丙醇的合成有关。【结论】本研究揭示了浓香型白酒窖内发酵过程中正丙醇合成相关的微生物和代谢途径,为阐明白酒发酵过程中正丙醇的形成机制奠定了理论基础。     

Maximization of production of secreted recombinant proteins in Pichia pastoris fed-batch fermentation

Pontryagin's Maximum Principle has been applied for optimization of secreted proteins from Pichia pastoris fed-batch fermentation. The objective of this work is to maximize the total accumulated product per unit operation time under different given conditions and system constraints. To obtain optimal solutions, an automated curve-fitting software, Table Curve 2D, was employed to construct the necessary mathematical models and solve the complicated functions. In the solution processes, the end of the glycerol batch phase was defined as the initial state of the system, the end of the methanol fed-batch phase as the final state, the cell mass produced along with product accumulated as state variables, and the specific growth rate (mu) as the control variable. Initially, a relationship between the specific production rate (rho) and mu was established. Then, according to Pontryagin's Maximum Principle, the admissible range of mu and its trajectories for the optimal operations were determined. Four representative cases with different combinations of the operation time along with the initial and final states were evaluated. A close correlation was obtained between the predicted values of the model equation with the experimental results from the Pichia pastoris fed-batch fermentations producing secreted alpha-galactosidase. The approaches proposed here greatly simplify the computational processes and validate the optimization strategy as a generalized approach to maximize the yield from fed-batch fermentations.