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.     

Physiological and technological aspects of large-scale heterologous-protein production with yeasts

Commercial production of heterologous proteins by yeasts has gained considerable interest. Expression systems have been developed forSaccharomyces cerevisiae and a number of other yeasts. Generally, much attention is paid to the molecular aspects of heterologous-gene expression. The success of this approach is indicated by the high expression levels that have been obtained in shake-flask cultures. For large-scale production however, possibilities and restrictions related to host-strain physiology and fermentation technology also have to be considered. In this review, these physiological and technological aspects have been evaluated with the aid of numerical simulations. Factors that affect the choice of a carbon substrate for large-scale production involve price, purity and solubility. Since oxygen demand and heat production (which are closely linked) limit the attainable growth rate in large-scale processes, the biomass yield on oxygen is also a key parameter. Large-scale processes impose restrictions on the expression system. Many promoter systems that work well in small-scale systems cannot be implemented in industrial environments. Furthermore, large-scale fed-batch fermentations involve a substantial number of generations. Therefore, even low expression-cassette instability has a profound effect on the overall productivity of the system. Multicopy-integration systems may provide highly stable expression systems for industrial processes. Large-scale fed-batch processes are typically performed at a low growth rate. Therefore, effects of a low growth rate on the physiology and product formation rates of yeasts are of key importance. Due to the low growth rates in the industrial process, a substantial part of the substrate carbon is expended to meet maintenance-energy requirements. Factors that reduce maintenance-energy requirements will therefore have a positive effect on product yield. The relationship between specific growth rate and specific product formation rate (kg product·[kg biomass]^–1·h^–1) is the main factor influencing production levels in large-scale production processes. Expression systems characterized by a high specific rate of product formation at low specific growth rates are highly favourable for large-scale heterologous-protein production.     

A kinetic model for energy spilling-associated product formation in substrate-sufficient continuous culture

It has been demonstrated that excess substrate can cause uncoupling between anabolism and catabolism, which leads to energy spilling. However, the Luedeking-Piret equation for product formation does not account for the energy spilling-associated product formation due to substrate excess. Based on the growth yield and energy uncoupling models proposed earlier, a kinetic model describing energy spilling-associated product formation in relation to residual substrate concentration was developed for substrate-sufficient continuous culture and was further verified with literature data. The parameters in the proposed model are well defined and have their own physical meanings. From this model, the specific productivity of unit energy spilling-associated substrate consumption, and the maximum product yield coefficient, can be determined. Results show that the majority of energy spilling-associated substrate consumption was converted to carbon dioxide and less than 6% was fluxed into the metabolites, while it was found that the maximum product yield coefficients varied markedly under different nutrient limitations. The results from this research can be used to develop the optimized bioprocess for maximizing valuable product formation.     

Controlled gas environments in industrial fermentations

The gas environment is of major importance in controlling aerobic fermentation processes for the manufacture of microbial products. Oxygen and carbon dioxide levels in gas-liquid equilibria affect productivity and energy consumption in such processes and appear to be implicated in the regulation of microbial metabolism. Gas-liquid transfer has been intensively studied by many investigators for Newtonian and non-Newtonian fluids, primarily in terms of oxygen-limitation in biomass and product formation. More recentreports show that microbial growth and product formation are affected by levels of oxygen and carbon dioxide in the gas environment, suggesting that microbial metabolism may be directed towards specific products by the control of such environments. High product concentrations may also be obtained by solid substrate fermentations with mycelial organisms cultured on semi-solid agricultural products at low moisture contents. Such methods are commonly used in the Orient for the manufacture of enzymes and traditional fermented foods and could probably be extended to other microbial products. This review covers fundamental aspects of engineering research in microbial processes that suggest applications for controlled gas environments in submerged culture and solid substrate fermentations of potential industrial interest.     

Fuzzy reasoning system for fault diagnosis of physiological activities in a cultivating process.

Aiming at development of a system which supports cultivating operations, a method to diagnose physiological activities in a cultivating process is presented, and a fuzzy expert system for diagnosing Lactobacillus casei cultivating process is implemented in this paper. This system can calculate specific rates of cell growth, substrate consumption, and product formation with measuring cell mass concentration, substrate concentration, and product concentration by using a turbidity sensor and HPLC. A database is implemented, where standard curves on specific rates representing characteristics of microorganisms are stored according to normalized substrate consumption. Comparing the calculated specific rates with standard values derived from the database, the system diagnoses physiological activities of the microorganisms. As a case study, a knowledge base for diagnosing lactic acid production process is implemented. The use of fault diagnosis on pH malfunctions by the expert system proves its reasonable performance.     

Process parameter shifting: Part II. Biphasic cultivation-A tool for enhancing the volumetric productivity of batch processes using Epo-Fc expressing CHO cells

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.     

Monitoring Growth of Microorganisms using the Methods of Mass-Energy Balance and Utilization of Computer on Line with Fermenter in Real Time Processing (in Russian)

The effective means of microbial culture monitoring is the measurement of low-inertial parameters (respiration rate, rates of supply of alkali for pH maintenance and the limiting substrate) and utilization of computer on line with fermenter for recalculation of these rates into the instant values of mass and energy cell yields, specific rates of cell growth and substrate and oxygen consumption, using the method of mass-energy balance. In this paper, the equations of mass-energy balance are presented both in general form and in the form of numerical algorythms for computer programming. The installation for automation of microbial cultivation experiment is described. Experimental data are presented which indicate the effectiveness of the method of indirect measurement of cell biomass yield and specific rates of physiological processes.     

A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding

Mixed substrate feeding strategies are frequently investigated to enhance the productivity of recombinant Pichia pastoris processes. For this purpose, numerous fed batch experiments or time-consuming continuous cultivations are required to optimize control parameters such as the substrate mixing ratio and the applied methanol concentration. In this study, we decoupled the feeding of methanol and glycerol in a mixed substrate fed batch environment to gain process understanding for a recombinant P. pastoris Muts strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates (qs) were controlled separately, and a stepwise increased qGly-control scheme was applied to investigate the effect of various substrate fluxes on the culture. The qs-controlled strategy allowed a parallel characterization of the metabolism and the recombinant protein expression in a fed batch environment. A critical-specific glycerol uptake rate was determined, where a decline of the specific productivity occurred, and a time-dependent acceleration of protein expression was characterized with the dynamic fed batch approach. Based on the observations on recombinant protein expression, propositions for an optimal feeding design to target maximal productivities were stated. Thus, the dynamic fed batch strategy was found to be a valuable tool for both process understanding and optimization of product formation for P. pastoris in a mixed substrate environment.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

Metabolic flux analysis of lactic acid fermentation: effects of pH and lactate ion concentration

A detailed metabolic flux analysis for lactic acid production by Streptococcus lactis has been carried out. A metabolic reaction set was constructed for the metabolism of S. lactis. Fluxes through these reactions were estimated by using accumulation rates of biomass, product and consumption rates of the substrate, which were obtained through experiments. The changes in the flux movement are shown for different pHs and initial lactate concentrations of the medium. The analysis indicated that pH only affected the uptake rates of lactose, whereas lactate ion concentration influenced the movement of flux through the network.     

Products, requirements and efficiency of biosynthesis: a quantitative approach

The question of how many grams of an organism can grow heterotrophically from only 1·0 g of glucose and adequate minerals has been put forward many times. Only a few attempts have been made to answer this question theoretically and these attempts were rather rough. In this paper, it is demonstrated that the yield of a growth process may be accurately computed by considering the relevant biochemistry of conversion reactions and the cytological implications of biosynthesis and growth. Oxygen consumption and carbon dioxide production by these processes are also computed. The weight of the biomass synthesized from 1·0 g of substrate and the quantities of gases exchanged are independent of temperature.These results are obtained by adding the individual equations describing the formation of each compound synthesized by the organism from the substrate supplied. The sum represents an equation which accounts for all substrate molecules required for biosynthesis of the carbon skeletons of an end-product, whose chemical composition is given. It is then calculated how much energy is required for the non-synthetic processes which form a part of biosynthesis, such as intra- and intercellular transport of molecules and maintenance of RNA and enzymes. The additional amount of substrate required to provide this energy by combustion is easily calculated. Adding this substrate to the amount used for skeleton synthesis gives an overall equation which quantifies the substrate and oxygen demand as well as carbon dioxide evolution during biosynthesis of 1·0 g biomass. For example, it requires 1·34 g of glucose with adequate ammonia and minerals to synthesize 1·0 g maize plant biomass in darkness; during this process 0·14 g oxygen are consumed and 0·24 g carbon dioxide are produced. It has been described elsewhere that similar results were obtained experimentally with growing plants.Such results depend considerably upon the chemical composition of the biomass being synthesized and upon the state (oxidized or reduced) of the nitrogen source. Other parameters, such as the number of ATP molecules required for protein synthesis, the possibility for utilization of alternative pathways for synthesis or energy production, the presence or absence of compartmentation of synthetic processes and variations in the P/O ratio between two and three, under many conditions affect results of the computation less than 10%.Since maintenance of cellular structures is not considered, the approach concerns the gross yield of biosynthesis. It predicts therefore the dry matter yield of heterotrophic cells from a given quantity of substrate at high relative growth rates.     

Critical limitations in biological production of chemicals: process or genetic solutions?

Abstract: Production of bulk chemicals by biological processes is presently limited by failure of contemporary biological and bioreactor technology to deliver high product concentrations in high space-time yields in fluids of sufficiently low water content for subsequent down-stream processing operations. Limitations in the bioreactor portion of the process can arise due to failure to process sufficient substrate, substrate inhibition, inadequate rates or yields, and product inhibition. Various process approaches for addressing many of these limitations have been demonstrated or conceptualized. Less developed but potentially effective are genetic strategies addressing these process limitations. Ideally, the most effective combination of genetic and process approaches should be integrated in a synergistic fashion to maximize the economic potential of biological production of chemicals.     

A simple model-based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess

A predictive control algorithm coupled with a PI feedback controller has been satisfactorily implemented in the heterologous Rhizopus oryzae lipase production by Pichia pastoris methanol utilization slow (Mut(s)) phenotype. This control algorithm has allowed the study of the effect of methanol concentration, ranging from 0.5 to 1.75 g/L, on heterologous protein production. The maximal lipolytic activity (490 UA/mL), specific yield (11,236 UA/g(biomass)), productivity (4,901 UA/L . h), and specific productivity (112 UA/g(biomass)h were reached for a methanol concentration of 1 g/L. These parameters are almost double than those obtained with a manual control at a similar methanol set-point. The study of the specific growth, consumption, and production rates showed different patterns for these rates depending on the methanol concentration set-point. Results obtained have shown the need of implementing a robust control scheme when reproducible quality and productivity are sought. It has been demonstrated that the model-based control proposed here is a very efficient, robust, and easy-to-implement strategy from an industrial application point of view.     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

Enzymatic resolution of (S)-(+)-naproxen in a continuous reactor

An enzymatic method for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen ethoxyethyl ester catalyzed by Candida cylindracea lipase. The reaction has been carried out in a continuous-flow closed-loop column bioreactor packed with Amberlite XAD-7, a slightly polor resin on which the lipase has been immobilized by adsorption. Various immobilization conditions as well as the properties of the immobilized lipase have been studied. The performance and the productivity of the bioreactor were evaluated as a function of the critical reaction parameters such as temperature, substrate concentration, and product inhibition. By using a 500-mL column bioreactor, 1.8 kg of optically pure (S)-(+)-Naproxen were produced after 1200 h of continuous operation with a slight loss of the enzymatic activity.     

Upstream processes in antibody production: evaluation of critical parameters

The demand for monoclonal antibody for therapeutic and diagnostic applications is rising constantly which puts up a need to bring down the cost of its production. In this context it becomes a prerequisite to improve the efficiency of the existing processes used for monoclonal antibody production. This review describes various upstream processes used for monoclonal antibody production and evaluates critical parameters and efforts which are being made to enhance the efficiency of the process. The upstream technology has tremendously been upgraded from host cells used for manufacturing to bioreactors type and capacity. The host cells used range from microbial, mammalian to plant cells with mammalian cells dominating the scenario. Disposable bioreactors are being promoted for small scale production due to easy adaptation to process validation and flexibility, though they are limited by the scale of production. In this respect Wave bioreactors for suspension culture have been introduced recently. A novel bioreactor for immobilized cells is described which permits an economical and easy alternative to hollow fiber bioreactor at lab scale production. Modification of the cellular machinery to alter their metabolic characteristics has further added to robustness of cells and perks up cell specific productivity. The process parameters including feeding strategies and environmental parameters are being improved and efforts to validate them to get reproducible results are becoming a trend. Online monitoring of the process and product characterization is increasingly gaining importance. In total the advancement of upstream processes have led to the increase in volumetric productivity by 100-fold over last decade and make the monoclonal antibody production more economical and realistic option for therapeutic applications.     

The Cost of Maintenance Processes in Plant Cells

The most important maintenance processes in plants are proteinturnover and active transport processes to maintain certainion concentrations in the cells. In this paper an attempt ismade to calculate the total energy cost of these processes fromwhat is known about their specific costs and what has been observedabout their rates. Because of insufficient reliable data aboutrates of individual maintenance processes, only approximatevalues can be obtained. The average turnover rate of leaf proteins may be about 100mg protein per g proteins per day at normal temperature in leavesassimilating at moderate light intensities. This process consumes28–53 mg glucose per g protein per day, which equals 7–13mg glucose per g dry weight per day in leaves. It is likelythat the rates of protein turnover and of CO₂-assimilation arerelated. The cost of maintaining ion concentrations is estimatedto be about 6–10 mg glucose per g dry weight per day inleaves. The sum of these figures is lower than is indicatedby measurements of maintenance respiration. One reason for theunderestimation may be that the protein turnover rates usedin the calculations apply to plants with lower photosyntheticrates than the plants in which the maintenance respiration wasmeasured. Effects of water stress and salinity, temperatureand other environmental factors on the rate of maintenance processesare discussed. The consumption of assimilates for maintenance of plant cellsis a significant, negative factor in plant productivity. A betterunderstanding of the maintenance processes may give a clue howto manipulate plant characteristics or the environment to reducethe amount of assimilates consumed in these processes. It issuggested that reduction in protein turnover rates may be onesuch manipulation.     

Recent advances in production of succinic acid from lignocellulosic biomass

Production of succinic acid via separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are alternatives and are environmentally friendly processes. These processes have attained considerable positions in the industry with their own share of challenges and problems. The high-value succinic acid is extensively used in chemical, food, pharmaceutical, leather and textile industries and can be efficiently produced via several methods. Previously, succinic acid production via chemical synthesis from petrochemical or refined sugar has been the focus of interest of most reviewers. However, these expensive substrates have been recently replaced by alternative sustainable raw materials such as lignocellulosic biomass, which is cheap and abundantly available. Thus, this review focuses on succinic acid production utilizing lignocellulosic material as a potential substrate for SSF and SHF. SSF is an economical single-step process which can be a substitute for SHF — a two-step process where biomass is hydrolyzed in the first step and fermented in the second step. SSF of lignocellulosic biomass under optimum temperature and pH conditions results in the controlled release of sugar and simultaneous conversion into succinic acid by specific microorganisms, reducing reaction time and costs and increasing productivity. In addition, main process parameters which influence SHF and SSF processes such as batch and fed-batch fermentation conditions using different microbial strains are discussed in detail.     

Kinetics of multiproduct acidogenic and solventogenic batch fermentations

Summary Large amounts of data indicated that most of the metabolic processes of the acidogenic (acid producing) and the solventogenic (solvent producing) fermentations were regulated by product accumulation. A simple unstructured model simulated microbial growth, product formation and substrate utilization in six different fermentations, where five different microorganisms produced various combinations of ten different products. Specific growth rates of these microorganisms decreased proportionally with overall product accumulation. The products were excreted in non-growth associated pattern. Excretion of some of these products were inhibited by the overall product accumulation similarly as the microbial growth. A substrate consumption model which considered the biomass and individually all the products as separate substrate sinks simulated the data satisfactorily.     

Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation

Polyhydroxyalkanoates (PHA) have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. In this paper, several factors affecting the production cost of PHA, such as PHA productivity, content and yield, the cost of the carbon substrate, and the recovery method were reviewed. A sensitivity analysis was also carried out with respect to these factors and with a view to scale-up. Several production processes were designed on the basis of the reported fermentation and recovery results, and were economically evaluated. PHA productivity only affects equipment-related costs, but PHA content has multiple effects on the process economics. Development of an economical and efficient recovery method is also important to the overall economics of PHA production. Received: 10 August 1998 / Accepted: 26 September 1998