Improved performance with multiple fermentors for repeated batch cultivation for non-growth-associated products

Based on batch cultivation data for the production of L-glutamic acid from glucose, a comparative evaluation was made for repeated batch cultivations using one and more fermentors. The problem was formulated as maximizing the productivity of metabolic product with the specified conversion with respect to the cell age and the volume fraction used as seed for the subsequent repeated batch cultivation. Simulations were carried out with the assumption of no lag in product formation for the cases where the total operation time was specified as 200 h with reproducible batch cultivation cycles. The product production was assumed to be solely a function of product concentration. The computation results show the advantage of using more than one fermentor from the viewpoints of productivity and conversion, which will apply in general to non-growth-associated product production with delay time. In particular, three fermentors are recommended for the production of L-glutamic acid chosen as an example in this article.     

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.     

Coiled tube membrane bioreactor for cultivation of hybridoma cells producing monoclonal antibodies.

A coiled tube membrane reactor was developed for the cultivation of mouse-mouse hybridoma cells producing monoclonal antibodies. The cell and antibody concentrations obtained in the membrane reactor were higher than that obtained in a batch spinner flask without a membrane. A mathematical model has been developed to describe the membrane transport coupled growth and product formation, and the physical and kinetic constants of the system were determined.     

Selective synthesis of polysaccharides by Rhizobium trifolii, strain TA-1

Abstract Rhizobium trifolii strain TA-1, produces one of each of the exocellular polysaccharides EPS, CPS and β-1,2-glucans as a major product during cultivation in glutamic acid-mannitol-salts (GMS) medium at 25°. In batch culture, the major exocellular polysaccharide product was acidic exopolysaccharide (EPS) under conditions of air saturation; capsular polysaccharide (CPS) under conditions of N-limitation and moderate oxygen supply; and cyclic β-1,2-glucans at high cell density and severe oxygen limitation.     

Production of cell mass and pertussis toxin by Bordetella pertussis.

The cultivation of Bordetella pertussis affects production of pertussis toxin and biomass. Comparison of batch mode, chemostat operation and pHstat-turbidostatic control showed that productivities for the continuous process were greater than that for the batch operation. Continuous operation in balanced growth at the maximum specific growth rate, provided by the pHstat, resulted in the maximum specific production rate. Because of the strong association of pertussis toxin synthesis and cell growth, the concentration of toxin in the effluent of the continuous processes was greater than the maximum obtained in the batch bioprocess. An expanded Luedeking-Piret model of product formation kinetics fits the observed chemostat data and demonstrates that the production of pertussis toxin from the culture of B. pertussis is predominantly growth associated.     

Linear and cyclic ester Oligomers of succinic acid and 1,4-butanediol: Biocatalytic synthesis and characterization

Abstract

The lipase-catalyzed synthesis of cyclic ester oligomers from non-activated succinic acid (A) and 1,4-butanediol (B) in the presence of immobilized Candida antarctica lipase B was investigated. Batch and pulse fed-batch systems were implemented to increase the formation of cyclic ester products. The substrate conversions after 24 h were 86% and 95% under batch and fed-batch operation, respectively and the product of the reaction was, for both systems, a mixture of cyclic (CEOs) and linear (LEOs) ester oligomers. Fed-batch operation afforded a product containing 71% cyclic ester oligomers (CEOs) as compared with only 52% CEOs in batch operation. Cyclic ester oligomers accumulated as the reaction progressed, with the dimer CEO₁ the most predominant product (i.e. 50% of the total products formed in fed-batch operation). The pulse fed-batch operation system was superior to the batch operation not only because higher substrate conversion and more CEOs were obtained, but also because it resulted in products with a higher degree of polymerization (DP up to 7). Cyclic ester oligomers are produced from the early stage of the reaction simultaneously with the linear ester oligomers by a ring-closure reaction on the active site of the enzyme, and not as a result of ring-chain equilibria.     

High-throughput screening of Hansenula polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale

Most large-scale production processes in biotechnology are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode, which results in the microorganisms being subjected to different physiological conditions. This significantly affects strain selection. To demonstrate differences in ranking during strain selection depending on the operational mode, screenings were performed in batch and fed-batch modes. Two model populations of the methylotrophic yeast Hansenula polymorpha RB11 with vector pC10-FMD (P_FMD-GFP) (220 clones) and vector pC10-MOX (P_MOX-GFP) (224 clones) were applied. For fed-batch cultivations in deep-well microtiter plates, a controlled-release system made of silicone elastomer discs containing glucose was used. Three experimental set-ups were investigated: batch cultivation with (1) glucose as a substrate, which catabolite represses product formation, and (2) glycerol as a carbon source, which is partially repressing, respectively, and (3) fed-batch cultivation with glucose as a limiting substrate using the controlled-release system. These three experimental set-ups showed significant variations in green fluorescent protein (GFP) yield. Interestingly, screenings in fed-batch mode with glucose as a substrate resulted in the selection of yeast strains different from those cultivated in batch mode with glycerol or glucose. Ultimately, fed-batch screening is considerably better than screening in batch mode for fed-batch production processes with glucose as a carbon source.     

Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes.

The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose.     

Modeling and simulation of Streptomyces peucetius var. caesius N47 cultivation and epsilon-rhodomycinone production with kinetic equations and neural networks

This study focuses on comparing different kinetic growth models and the use of neural networks in the batch cultivation of Streptomyces peucetius var. caesius producing epsilon-rhodomycinone. Contois, Monod and Teissier microbial growth models were used as well as the logistic growth modeling approach, which was found best in the simulations of growth and glucose consumption in the batch growth phase. The lag phase was included in the kinetic model with a CO2 trigger and a delay factor. Substrate consumption and product formation were included as Luedeking-Piret and logistic type equations, respectively. Biomass formation was modeled successfully with a 6-8-2 network, and the network was capable of biomass prediction with an R2-value of 0.983. Epsilon-rhodomycinone production was successfully modeled with a recursive 8-3-1 network capable of epsilon-rhodomycinone prediction with an R2-value of 0.903. The predictive power of the neural networks was superior to the kinetic models, which could not be used in predictive modeling of arbitrary batch cultivations.     

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli

Abstract: The production of the fusion protein staphylococcal protein A/E. coli β-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g 1^-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g 1^-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g 1^-1.     

Prediction of the course of continuous fermentation on the basis of analysis of the batch process

A way of predicting the conditions of product formation in a two-state continuous cultivation on the basis of analyzing the batch process is described. The prediction applies to processes where the product formation is proportional to the growth rate and where the relationship between culture activity and the external conditions is linear. Using the example of glucose oxidase production byAspergillus niger the prediction is borne out. The difference between the predicted and found values does not exceed 15%. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Gene‐expression‐based quality scores indicate optimal harvest point in Bordetella pertussis cultivation for vaccine production

The evolution of vaccine product quality during batch cultivation of Bordetella pertussis, the causative agent of whooping cough, was investigated with the goal to determine the optimal harvest point. The process was explored by measuring mRNA expression at frequent intervals during cultivation. The genes that are involved in virulence are already known for this product and changes in their expression levels are proposed to be indicative for product quality. A quantitative product quality score is calculated based on the expression levels of these virulence genes, which allows comparison of expected product quality between culture samples. Product quality scores were maximal throughout the logarithmic growth phase, but dropped significantly at the start of the stationary phase. This showed that the decreasing lactate and glutamate concentrations towards the end of the batch are critical for product quality. On‐line measurement of these nutrients allows the cultivation process to be harvested at the optimal harvest point, increasing process robustness and consistency. Biotechnol. Bioeng. 2009;103: 900–908. © 2009 Wiley Periodicals, Inc.     

Experimental and modelling study of different process modes for retroviral production in a fixed bed reactor

Pseudotype vectors are promising for gene transfer in many gene therapy approaches, however, low-vector concentration in batch cultures and high temperature-dependent decay do limit sufficiently large-scale production. To overcome these obstacles, the kinetic relations of cell growth and vector formation in different culture modes need to be understood. Effective optimisation of process modes is needed to achieve sufficient yields. Experimental and modelling studies were carried out in order to analyse the impact of different process modes such as perfusion, perfused fed-batch or repeated-batch on vector titer and productivity. Retroviral pseudotype vector, derived from the murine leukaemia virus carrying the HIV-1 envelop protein MLV (HIV-1) were produced using a 200 ml fixed bed reactor for high cell density cultivation on macroporous carriers. After starting the cultivation in batch mode, the reactor was either run in perfusion, perfused fed-batch or repeated-batch. A mathematical model of the bioreaction was developed on the basis of experimental data measured in culture dishes. The ability of the model to describe all different process modes of fixed-bed cultivation without additional fitting of the parameters was proven by three long-term cultivations for more than 400 h. The results of optimisation with the aid of the model, leads to the conclusion that perfusion with optimised harvest cycles and fed flows, result in a higher yield in comparison to batch or fed batch culture.     

A filter fermenter-apparatus and control equipment

Laboratory equipment for cultivation of microorganisms with the removal of cell-free medium is described. Separation is accomplished by filtration through a membrane which is built into a top-driven fermenter. A system is presented which controls the flow rate by automatic cleaning of the filter. Flow schemes for batch and continuous fermentations are shown. The filter fermenter is useful when studying continuous culture with feedback of biomass or processes where growth or product formation rates are influenced by inhibitory metabolites. Results are presented from experiments with Saccharomyces cerevisiae, Pedicoccus pentosaceus, and Zymomonas mobilis.     

Effects of Temperature Variation on the Fatty Acid Composition of a Psychrophilic Candida Species

During the batch cultivation of a psychrophilic Candida species, the proportions of oleic and linolenic acid esters in the cells underwent a cyclic variation regardless of the incubation temperature.     

Unstructured model for L-lysine fermentation under controlled dissolved oxygen

An unstructured model was developed for batch cultivation of Corynebacterium lactofermentum (ATCC 21799) under controlled dissolved oxygen. The model is capable of predicting batch experiments performed at various initial substrate concentrations. By extending the batch culture model to a fed-batch model and using a heuristic approach to optimize the fed-batch cultivation, it is shown that fed-batch cultivation is superior to batch operation due to increased productivity at high substrate concentrations.     

Investigation of the use of agricultural byproducts for fungal protein production

Cellulose and nutrient salts as well as potato pulp and potato protein liquor (PPL), were used as substrates for the cultivation of Chaetomium cellulolyticum in batch and repeated-batch operations. Using cellulose as the substrate a linear relationship existed between the rates of cell mass formation and acid production. The repeated-batch process was controlled by NaOH consumption using a simple computer model. When the production of cell mass and acid stopped because of a lack of substrate cellulose was fed into the reactor. This occurred within 10 min at which point no NaOH-feed was needed to maintain a constant pH. Repeated-batch operations yielded higher cell concentrations and productivities than batch operations. The relationship between the NaOH and H₂SO₄ consumed, and the fungal mass concentration was complex in cultivation media containing potato pulp and PPL, because various substrates were consumed by the fungus simultaneously and successively. Therefore, for repeated-batch cultivation a constant time interval was used. Repeated-batch cultivation of the fungus on potato pulp and PPL did not yield higher cell concentrations and productivities than did batch cultivation. With the optimal pulp-to-PPL ratio a maximum specific growth rate of 0.61 h¹ was obtained. These investigations indicate, that potato pulp and PPL are well suited to fungal protein production by Chaetomium cellulolyticum for fodder supplement.     

Continuous production of acetone and butanol with shear-activated Clostridium acetobutylicum

Summary When using shear activation of Clostridium acetobutylicum by pumping the cells through capillaries, the cell growth, glucose consumption and product formation rates are considerably increased. Shear-activated continuous cell culture can be used as an inoculum with a welldefined fermentation activity for batch cultures. Different runs of such batch cultivation yield well-reproducible results which could not be obtained from inocula of other cultures or even of heat-shocked spores. The cells can attain a growth rate higher than 1.6 h^-1.The shear-activated continous culture growth is affected already at a butanol concentration lower than 1.6 g/l^-1.     

Cell engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> allows high cell density accumulation without fed-batch process control

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h⁻¹, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.     

应加强对单细胞生物分批培养生长曲线中减速期的认识和教学

根据单细胞生物分批培养过程中比生长速率(μ)的变化,其生长曲线分为延滞期、加速期、对数期、减速期、稳定期和衰亡期6个时期.与其他生长时期相比,在减速期生物的生长、基质的利用、产物的合成和基因表达谱等方面有显著的不同,并对发酵生产有着重要作用.然而,长期以来,对减速期的认识和教学相当薄弱,亟需加强对减速期的认识和教学.