Whey Fermentation by Anaerobiospirillum succiniciproducens for Production of a Succinate-Based Animal Feed Additive

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with Anaerobiospirillum succiniciproducens. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO₂, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO₂, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO₂, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.     

Broth recycling to reduce process noise resulting from concentrated substrate addition in fed-batch cultivation of Escherichia coli

In this work feed hardware for fed-batch cultivation is presented (broth recycle feed injection system or BRFIS). BRFIS proved superior to conventional submerged or dripped feed systems in reducing dissolved oxygen (DO) oscillations during Escherichia coli fed-batch cultivation (5 min coefficient of variation of 0.7% for BRFIS as compared to 26% or greater for conventional feeding hardware in a 2 L test reactor). Hence, BRFIS is useful for fed-batch cultivation systems where the DO signal is used in measurement or control.     

Controlled fed-batch by tracking the maximal culture capacity

Fed-batch processes are well established in the biotech industry. The major reason to apply this technique is to avoid overflow metabolism and/or accumulation of toxic substrates. The basic idea of this approach is to control the physiological state of the culture, rather than just the typically exponential feed rate profile, by challenging a fed-batch cultivation repetitive in useful time intervals. The feed rate is reduced for a short period and culture responses are analysed in real time and on-line. Thus it is possible to get a positive response at the earliest detectable point of potential overfeeding. During the disturbance minute amounts of overflow metabolites will deplete simultaneously. This highly dynamic approach was applied successfully to industrially relevant production systems such as yeasts (Saccharomyces cerevisiae, Pichia pastoris) and bacteria (Escherichia coli).     

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.     

Constant-volume fed-batch operation for high density cultivation of hyperthermophilic aerobes

When hyperthermophilic archaebacteria are cultivated under aerobic conditions, the loss of the culture volume becomes significant due to vaporization of water with aeration. To eliminate the problems caused by evaporation of the medium, we have devised a constant-volume fed-batch system, where the loss of water due to evaporation is compensated by feeding additional water into the fermentor. The feeding strategy for maintaining a constant volume during fed-batch cultivation was developed from the water balance and the cell growth and substrate consumption kinetics, and the developed method was applied to high density cultivation of Sulfolobus solfataricus DSM 1617. The maximum cell density obtained was 22.6 g/L, which is the highest value reported for hyperthermophiles in fed-batch operations.     

Dynamic metabolic flux analysis (DMFA): a framework for determining fluxes at metabolic non-steady state

Metabolic flux analysis (MFA) is a key tool for measuring in vivo metabolic fluxes in systems at metabolic steady state. Here, we present a new method for dynamic metabolic flux analysis (DMFA) of systems that are not at metabolic steady state. The advantages of our DMFA method are: (1) time-series of metabolite concentration data can be applied directly for estimating dynamic fluxes, making data smoothing and estimation of average extracellular rates unnecessary; (2) flux estimation is achieved without integration of ODEs, or iterations; (3) characteristic metabolic phases in the fermentation data are identified automatically by the algorithm, rather than selected manually/arbitrarily. We demonstrate the application of the new DMFA framework in three example systems. First, we evaluated the performance of DMFA in a simple three-reaction model in terms of accuracy, precision and flux observability. Next, we analyzed a commercial glucose-limited fed-batch process for 1,3-propanediol production. The DMFA method accurately captured the dynamic behavior of the fed-batch fermentation and identified characteristic metabolic phases. Lastly, we demonstrate that DMFA can be used without any assumed metabolic network model for data reconciliation and detection of gross measurement errors using carbon and electron balances as constraints.     

Towards dynamic metabolic flux analysis in CHO cell cultures

Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.     

Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain

By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.     

Fed-batch culture for L-lysine production via on-line state estimation and control

Computer application for fed-batch culture of Brevibacterium flavum for L-lysine production has been developed. The organisms are auxotrophic mutants for L-homoserine and are resistant to S-(2-aminoethyl)-L-cysteine. Adaptive control is applied for substrate addition. The sugar concentration is estimated using online respiratory measurement. During the period of fed-batch culture, the total sugar concentration is maintained at a given value. The cultivation strategy results in high productivity and high conversion yield.     

The way to adequate control of microbial processes passes via real-time knowledge-based supervision.

Knowledge-based supervision is viewed as a major tool for achieving high-performance control of microbial processes. By providing an adequate insight into the integral state of the cell culture, knowledge-based supervisory systems allow for monitoring and handling various important phenomena which usually remain outside the scope of the conventional control approach. The present paper focuses on the development of a computer system for knowledge-based supervision of bioprocesses. Its application to the control of fed-batch cultivation of recombinant Escherichia coli for phenylalanine production is also discussed.     

Adaptive,model-based control by the Open-Loop-Feedback-Optimal (OLFO) controller for the effective fed-batch cultivation of hybridoma cells

Although fed-batch suspension culture of animal cells continues to be of industrial importance for the large scale production of pharmaceutical products, existing control concepts are still insufficient. Changes in cell metabolism during cultivation and between similar cultivations, the complexity of the cell metabolism, and the lack of on-line state variables restrict the transfer of available control strategies established in bioprocess engineering. A process control strategy designed to achieve optimized process control must account for all these difficulties and fit sophisticated requirements toward adaptability and flexibility. The combination of a fed-batch process and an Open-Loop-Feedback-Optimal (OLFO) control provides a new approach for cell culture process control that couples an efficient cultivation concept to a capable process control strategy. The application of an adaptive, model-based OLFO controller to a hybridoma cultivation and experimental results are presented.     

Some approaches to adaptive control of fed-batch culture of the cellobiase producer aspergillus japonicus

Biosynthesis of cellobiase by an Aspergillus japonicus culture under various modes of cultivation was studied using a fermenter-computer system. Two modes of fed-batch cultivation were developed: temperate and intensive. Both modes used the double algorithm of glucose supply based on the process control by the preset profile of CO₂ concentration in exhaust gas and consumption of the pH stabilizing titrant. Intensive fed-batch proved the most efficient mode: it maintained conditions for derepressed cellobiase Liosynthesis and decreased the carbon and energy limit for enzyme production. Intensive fed-batch is, thus, the most adapted to cellobiase biosynthesis conditions and nutrient requirements of the producing culture.     

A simple kinetic model for myeloma cell culture with consideration of lysine limitation

A simple kinetic model is developed to describe the dynamic behavior of myeloma cell growth and cell metabolism. Glucose, glutamine as well as lysine are considered as growth limiting substrates. The cell growth was restricted as soon as the extracellular lysine is exhausted and then intracellular lysine becomes a growth limiting substrate. In addition, a metabolic regulator model together with the Monod model is used to deal with the growth lag phase after inoculation or feeding. By using these models, concentrations of substrates and metabolites, as well as densities of viable and dead cells are quantitatively described. One batch cultivation and two fed-batch cultivations with pulse feeding of nutrients are used to validate the model.     

Improving bioreactor cultivation conditions for sensitive cell lines by dynamic membrane aeration

Although the importance of animal cell culture for the industrial (large scale) production of pharmaceutical products is continuously increasing, the sensibility of the cells towards their cultivation environment is still a challenging issue. In comparison to microbial cultures, cell cultures which are not protected by a cell wall are much more sensitive to shear stress and foam formation. Reactor design as well as the selection of ‘robust’ cell lines is particularly important for these circumstances. Nevertheless, even ‘sensitive’ cell lines are selected for certain pharmaceutical processes due to various reasons. These sensitive cell lines have even higher requirements regarding their cultivation environment. Important characteristics for the corresponding reactor design are a high (volumetric) gas mass transfer coefficient, low volumetric power input, low shear stress, low susceptibility to bio-fouling, the ability to cultivate sticky cells and sufficient mixing properties. Membrane aeration has been a long-known possibility to meet some of these requirements, but has not often been applied in recent years. The reasons lie mainly in low gas mass transfer rates, a limited installable volume-specific membrane surface area, restrictions in scalability and problems with membrane fouling. The dynamic membrane aeration bioreactor aeration is a simple concept for bubble-free oxygen supply of such sensitive cultures. It overcomes limitations and draw-backs of previous systems. Consisting of an oscillating, centrally arranged rotor (stirrer) that is wrapped with silicone membrane tubing, it enables doubling the gas mass transfer at the same shear stress in the investigated cultivation scales of 12, 20, 100, and 200 L. Continuous cultivation at these scales allows the same product output as fed-batch cultivation does at tremendously larger reactor volumes. Apart from introducing this novel technology, the presentation comprises selected cultivation results obtained for blood coagulation factor VIII in continuous mode and a therapeutic monoclonal antibody in fed-batch mode in comparison to reference trials.     

Microbioreactor-assisted cultivation workflows for time-efficient phenotyping of protein producing Aspergillus niger in batch and fed-batch mode

In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor.     

A serum substitute for fed-batch culturing of hybridoma cells

We developed a substitute for serum to produce fed-batch cultures of hybridoma cells in serum-free medium and confirmed that the cells could be successfully cultivated this way. Our substitute consisted of 12 components. The specific production rates of lactate and ammonia, which are harmful byproducts from the cells, were significantly reduced compared with a conventional serum-containing batch culture. This reduction led to a higher cell concentration and a longer production lifetime. As a result, the final concentration of monoclonal antibody was 400 mg/L, or five times greater than that in the conventional serum-containing batch culture. The developed substitute is expected to enable fed-batch cultivation in a serum-free condition.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

Enhanced process monitoring of fed-batch penicillin cultivation using time-varying and multivariate statistical analysis

On-line monitoring of penicillin cultivation processes is crucial to the safe production of high-quality products. In the past, multiway principal component analysis (MPCA), a multivariate projection method, has been widely used to monitor batch and fed-batch processes. However, when MPCA is used for on-line batch monitoring, the future behavior of each new batch must be inferred up to the end of the batch operation at each time and the batch lengths must be equalized. This represents a major shortcoming because predicting the future observations without considering the dynamic relationships may distort the data information, leading to false alarms. In this paper, a new statistical batch monitoring approach based on variable-wise unfolding and time-varying score covariance structures is proposed in order to overcome the drawbacks of conventional MPCA and obtain better monitoring performance. The proposed method does not require prediction of the future values while the dynamic relations of data are preserved by using time-varying score covariance structures, and can be used to monitor batch processes in which the batch length varies. The proposed method was used to detect and identify faults in the fed-batch penicillin cultivation process, for four different fault scenarios. The simulation results clearly demonstrate the power and advantages of the proposed method in comparison to MPCA.     

Experimental optimization of a real time fed-batch fermentation process using Markov decision process

This article describes a methodology that implements a Markov decision process (MDP) optimization technique in a real time fed-batch experiment. Biological systems can be better modeled under the stochastic framework and MDP is shown to be a suitable technique for their optimization. A nonlinear input/output model is used to calculate the probability transitions. All elements of the MDP are identified according to physical parameters. Finally, this study compares the results obtained when optimizing ethanol production using the infinite horizon problem, with total expected discount policy, to previous experimental results aimed at optimizing ethanol production using a recombinant Escherichia coli fed-batch cultivation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 317-327, 1997.