Dependence of penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity

The influence of the agitation conditions on the growth, morphology, vacuolation, and productivity of Penicillium chrysogenum has been examined in 6 L fed-batch fermentations. A standard Rushton turbine, a four-bladed paddle, and a six-bladed pitched blade impeller were compared. Power inputs per unit volume of liquid, P/VL, ranged from 0.35 to 7.4 kW/m3. The same fermentation protocol was used in each fermentation, including holding the dissolved oxygen concentration above 40% air saturation by gas blending. The mean projected area (for all dispersed types, including clumps) and the clump roughness were used to characterize the morphology. Consideration of clumps was vital as these were the predominant morphological form. For a given impeller, the batch-phase specific growth rates and the overall biomass concentrations increased with agitation intensity. Higher fragmentation at higher speeds was assumed to have promoted growth through increased formation of new growing tips. The mean projected area increased during the rapid growth phase followed by a sharp decrease to a relatively constant value dependent on the agitation conditions. The higher the speed, the lower the projected area for a given impeller type. The proportion by volume of hyphal vacuoles and empty regions decreased with speed, possibly due to fragmentation in the vacuolated regions. The specific penicillin production rate was generally higher with lower impeller speed for a given impeller type. The highest value of penicillin production as well as its rate was obtained using the Rushton turbine impeller at the lowest speed. At given P/VL, changes in morphology, specific growth rate, and specific penicillin production rate depended on impeller geometry. The morphological data could be correlated with either tip speed or the "energy dissipation/circulation function," but a reasonable correlation of the specific growth rate and specific production rate was only possible with the latter. Copyright 1998 John Wiley & Sons, Inc.     

模拟青霉素分批补料发酵过程的细胞自动机模型

根据青霉素产生菌的生长机理和青霉素分批补料发酵过程的动力学特性,在Paull等建立的形态学结构动力学模型的基础上,建立了模拟青霉素分批补料发酵过程的细胞自动机模型。模型采用三维细胞自动机作为菌体生长空间,采用Moore型邻域作为细胞邻域,其演化规则根据青霉素分批补料发酵过程中菌体生长机理和简化动力学结构模型设计。模型中的每一个细胞既可代表单个产黄青霉菌体细胞,又可代表特定数量的这种菌体细胞,它具有不同的状态。对模型进行的仿真实验结果表明：模型不但能一致地复现形态学结构动力学模型所描述的青霉素分批补料发酵过程的演化特性,而且较形态学结构动力学模型更加直观地刻画了青霉素分批补料发酵过程的演化行为。最后,对所建模型在实际生产过程中的应用问题进行了分析,指出了需要进一步研究的问题。     

A cellular automata model for simulating fed-batch penicillin fermentation process

A cellular automata model to simulate penicillin fed-batch fermentation process(CAPFM)was established in this study,based on a morphologically structured dynamic penicillin production model,that is in turn based on the growth mechanism of penicillin producing microorganisms and the characteristics of penicillin fed-batch fermentation.CAPFM uses the three-dimensional cellular automata as a growth space,and a Moore-type neighborhood as the cellular neighborhood.The transition roles of CAPFM are designed based on mechanical and structural kinetic models of penicillin batch-fed fermentation processes.Every cell of CAPFM represents a single or specific number of penicillin producing microorganisms,and has various state.The simulation experimental results show that CAPFM replicates the evolutionary behavior of penicillin batch-fed fermentation processes described by the structured penicillin production kinetic model accordingly.     

模拟青霉素发酵过程中菌体生长动态的细胞自动机模型

在青霉素发酵生产机理及其动力学微分方程模型的基础上,建立了模拟青霉素分批发酵过程中菌体生长动态的细胞自动机模型(CABGM)。CABGM采用三维细胞自动机作为菌体生长空间,采用Moore型邻域作为细胞邻域,其演化规则根据青霉素分批发酵过程中菌体生长机理和动力学微分方程模型设计。CABGM中的每一个细胞既可代表单个的青霉素产生菌,又可代表特定数量的青霉素产生菌,它具有不同的状态。对CABGM进行了统计特性的理论分析和仿真实验,理论分析和仿真实验结果均证明了CABGM能一致地复现动力学微分方程模型所描述的青霉素分批发酵菌体生长过程。最后,对所建模型在实际生产过程中的应用问题进行了分析,指出了需要进一步研究的问题。     

A model for penicillin production with and without temperature shift after the growth phase

Summary A strain of Penicillium chrysogenum producting about 8 g/l of penicillin V, was cultivated in a 10-1 bioreactor. Under carbon (C)-limitation during the production phase a glucose/ammonium sulphate mixture was fed using microprocessor control. When the temperature was shifted from 25° C to 30° C at the end of the active growth phase, the specific penicillin production rate was increased by 30%, while the yield remained constant. Maximal productivity without sporulation was obtained when the net growth rate of the active (respiring and producing) biomass, estimated by measuring the respiration rate under defined conditions, was equal to or higher than 0.004 h^–1. A model was developed for penicillin fermentation during C-limitation possessing the following properties: (1) the model is based on ordinary differential equations; (2) the influence of different nutrients is considered; (3) the model recognizes two cell types (active and inactive); (4) the model describes the influence of a temperature shift at the end of the vigorous growth phase. Offprint requests to: D. Siegmund     

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum

As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q_p) and growth rate (µ) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q_p(µ) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed‐batch experiments, leads to errors in the prediction, because q_p is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate‐limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin‐N synthase (IPNS) was the main rate‐limiting enzyme for penicillin‐G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady‐state as well as the dynamics during chemostat start‐up and fed‐batch cultivation. Biotechnol. Bioeng. 2010;106: 608–618. © 2010 Wiley Periodicals, Inc.     

Evaluation of two unstructured mathematical models for the penicillin G fedbatch fermentation

The mathematical model for the penicillin G fed-batch fermentation proposed by Heijnen et al. (1979) is compared with the model of Bajpai & Reuß (1980). Although the general structure of these models is similar, the difference in metabolic assumptions and specific growth and production kinetics results in a completely different behaviour towards product optimization. A detailed analysis of both models reveals some physical and biochemical shortcomings. It is shown that it is impossible to make a reliable estimation of the model parameters, only using experimental data of simple constant glucose feed rate fermentations with low initial substrate amount. However, it is demonstrated that some model parameters might be key factors in concluding whether or not altering the substrate feeding strategy has an important influence on the final amount of product.It is illustrated that feeding strategy optimization studies can be a tool in designing experiments for parameter estimation purposes.     

Production of penicillin in a fluidized-bed bioreactor using a carrier-supported mycelial growth

A carrier-supported mycelial growth of Penicillium chrysogenum was applied to penicillin fermentation system using celite as a support material. Hyphal growth through the pore matrices of the material showed strong anchorages and provided highly stable biofilm growth. With bioparticles developed in such a manner, both cell growth and penicillin production were observed to increase significantly compared to the conventional dispersed filamentous cultures. Maximum values of specific penicillin production rate were found to be constant regardless of the growth form. A three-phase fluidized-bed fermentor was designed and tested for penicillin production using the bioparticles. Two modes of operation, semicontinuous and repeated fed batch, of the fermentor were tried. It was noted that the overgrowth of free mycelia and the development of fluffy loose bioparticles caused poor mixing and made the fermentor operation quite difficult. Control of the bioparticle size and the extension of production phase were therefore considered important to maintain the reactor productivity at a desired level. From the results of repeated fed-batch operation it was found that the control of bioparticle size could be successfully achieved by phosphate-limiting culture condition. Penicillin production under this condition was also observed to be maintained at a high level (about 80% of the maximum) for at least 1 month.     

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).     

Effect of dissolved carbon dioxide on penicillin fermentations: Mycelial growth and penicillin production

The effect of dissolved carbon dioxide on the specific growth rate and the penicillin production rate of Penicillium chrysogenum was examined experimentally. The dissolved carbon dioxide was found to inhibit the specific growth rate and the penicillin production rate when the aerated submerged penicillin fermentation was exposed to influent gases of 12.6 and 20% carbon dioxide, respectively. Upon exposure to influent gases of 3 and 5% carbon dioxide, no pronounced metabolic inhibition was noted.     

Macrokinetic model for methylotrophic Pichia pastoris based on stoichiometric balance

A macrokinetic model for Pichia pastoris expressing recombinant human serum albumin is proposed. The model describes the balances of some key metabolites, ATP and NADH, during glycerol and methanol metabolism. In the glycerol growth phase, the metabolic pathways mainly include phosphorylation, glycolysis, tricarboxylic acid cycle, and respiratory chain. In the methanol growth phase, methanol is oxidized to formaldehyde at first. Then, while a part of formaldehyde is oxidized to formate, the rest is condensed with xylulose-5-monophosphate to form glyceraldehyde-3-phosphate, and further assimilated to form cell constituents. The metabolic pathways following glyceraldehyde-3-phosphate were assumed to be similar to those in the glycerol growth phase. Based on the model, the macrokinetic bioreaction rates such as the specific substrate consumption rate, the specific growth rate, the specific acetyl-CoA formation rate as well as the specific oxygen uptake rate are obtained. The specific substrate consumption rate and the specific growth rate are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, i.e., the medium volume, the concentrations of the biomass, the substrate, and the product, is set up. Experimental results demonstrate that the model can describe the cell growth and the protein production with reasonable accuracy.     

A cellular automata model for simulating fed-batch penicillin fermentation process

A cellular automata model to simulate penicillin fed-batch fermentation process (CAPFM) was established in this study, based on a morphologically structured dynamic penicillin production model, that is in turn based on the growth mechanism of penicillin producing microorganisms and the characteristics of penicillin fed-batch fermentation. CAPFM uses the three-dimensional cellular automata as a growth space, and a Moore-type neighborhood as the cellular neighborhood. The transition rules of CAPFM are designed based on mechanical and structural kinetic models of penicillin batch-fed fermentation processes. Every cell of CAPFM represents a single or specific number of penicillin producing microorganisms, and has various state. The simulation experimental results show that CAPFM replicates the evolutionary behavior of penicillin batch-fed fermentation processes described by the structured penicillin production kinetic model accordingly. __________ Translated from ACTA BIOPHYSICA, 2005, 21(2) [译自: 生物物理学报, 2005,21(2)]     

Study of the effect of microconcentrations of glucose on the biosynthesis of penicillin]

A method of "acute" experiments with significant dilution of the fermentation broth is proposed for studying the effect of easily assimilable substrates on growth of the culture and production of the antibiotic by it. The effect of microconcentrations of glucose on the parameters of the process kinetics was studied with respect to penicillin. The specific effect of microconcentrations of glucose on the specific rate of the culture growth and productivity was noted.     

Metabolic characterization of a L-lysine-producing strain by continuous culture

Continuous culture experiments with the L-producer, Corynebacterium glutamicum, were carried out to characterize the effect of specific growth rate on fermentation yields, specific rates, productivities, and fluxes through the primary metabolism. The specific productivity of L-lysine exhibited a maximum with respect to specific growth rate, with an initial growth-associated behavior up to specific growth rates of about 0.1 h(-1), and a constant specific productivity for specific growth rates in the range of about 0.1 to 0.2 h(-1). The productivity dropped at specific growth rates larger than about 0.2 h(-1). The yield of L-lysine on glucose increased approximately linearly with decreasing specific growth rate over the entire range studied, as did the respiratory quotient. A direct relationship was established between the culture respiratory quotient and the L-lysine yield. By explicitly accounting for glucose used for biomass synthesis, it was shown that the strain synthesizes L-lysine with an intrinsic yield, or efficiency, of about 0.41 mol L-lysine/mol glucose, compared with the theoretical yield of 0.75 mol/mol. Metabolic flux modeling based on the continuous culture data suggests that the production of ATP is not likely to be a limiting factor in L-lysine production, and that a high TCA cycle activity, coupled with a tightly controlled split of metabolite flow at the PEP node, is likely the cause of the large discrepancy between theoretical and actual yields in L-lysine fermentations.     

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.     

Computer control of the penicillin fermentation using the filtration probe in conjunction with a structured process model

A structured model for the penicillin fermentation is presented. This model includes three different cell types: (1) hyphae tips, (2) penicillin-producing cells, and (3) degenerated, metabolically inactive cells. Cell degeneration has been described previously as a gradual loss of cytoplasmic material by endogenous metabolism. The rate at which such loss of cytoplasm (and activity) proceeds can be expressed as a linear function of the specific growth rate. At growth rates above some minimum value (0.0115 h(-1)) cell degeneration can be prevented. This model served as the control basis during open-loop as well as closed-loop computer control of the fermentation. Closed-loop control was achieved through feedback information of biomass concentration using a filtration probe and was required when complex nutrients contributed significantly to the overall biomass production.     

A maximum production strategy of lysine based on a simplified model derived from a metabolic reaction network

The aim of this study is to develop a strategy for maximum production of a target product with a simplified model derived from a metabolic reaction network through an example of lysine production. Based on the model, a search for the optimal specific growth rate profile was conducted among the available conditions of batch fermentation based on the derived model, when the total fermentation time was fixed. The optimal specific growth rate was obtained as a boundary control: initially, the specific growth rate was maintained at a maximum value and was subsequently switched to a critical value giving the maximum specific production rate. To make the specific growth rate follow this optimal profile as accurately as possible in batch mode, first, an appropriate initial concentration of leucine was employed in the experiment. Second, the feeding strategy of leucine was further studied. The specific growth rate profile with feeding was closer to the optimal one and the amount of lysine produced at the final stage of fermentation was increased about twofold, compared to that in the batch fermentation. Finally, the strategy was summarized as an algorithm for general use of this method.     

Penicillin hydrolysis rate: Its effect on the kinetics of penicillin fermentation

The influence of the penicillin hydrolysis rate and dilution rate or specific growth rate on the productivity of a penicillin fermentation is quantified. Comparison of the theoretical with the experimental data suggests that hydrolysis largely accounts for the apparent critical minimum growth rate necessary to maintain maximum productivity.     

Mathematical model for aerobic culture of a recombinant yeast

A mathematical model was formulated to simulate cell growth, plasmid loss and recombinant protein production during the aerobic culture of a recombinant yeast S. cerevisiae. Model development was based on three simplified metabolic events in the yeast: glucose fermentation, glucose oxidation and ethanol oxidation. Cell growth was expressed as a composite of these metabolic events. Their contributions to the total specific growth rate depended on the activities of the pacemaker enzyme pools of the individual pathways. The pacemaker enzyme pools were regulated by the specific glucose uptake rate. The effect of substrate concentrations on the specific growth rate was described by a modified Monod equation. It was assumed that recombinant protein formation is only associated with oxidative pathways. Plasmid loss kinetics was formulated based on segregational instability during cell division by assuming constant probability of plasmid loss. Experiments on batch fermentation of recombinant S. cerevisiae C468/pGAC9 (ATCC 20690), which expresses Aspergillus awamori glucoamylase gene and secretes glucoamylase into the extracellular medium, were carried out in an airlift bioreactor in order to evaluate the proposed model. The model successfully predicted the dynamics of cell growth, glucose consumption, ethanol metabolism, glucoamylase production and plasmid instability. Excellent agreement between model simulations and our experimental data was achieved. Using published experimental data, model agreement was also found for other recombinant yeast strains. In general, the proposed model appears to be useful for the design, scale-up, control and optimization of recombinant yeast bioprocesses.