Reduction of contamination in an industrial fermentation plant

Information is presented concerning revisions of the fermentation air system equipment which improved the sterility situation in the production of monosodium glutamate. Basically, the revision were a relocation and elevation of the intake to the air compressors and installation of a retention chamber after the compressors to take advantage of the heat of compression. The extended high temperature retention ensures sterile air to the final air filter before the fermentor. Contamination losses—mainly from phage—have been materially reduced relative to the level prevailing before the change.     

On-line ultrasonic velocity monitoring of alcoholic fermentation kinetics

In this work, fundamental aspects on the ultrasonic velocity monitoring of alcoholic fermentations in synthetic broths (glucose, fructose and sucrose) and natural media (must and wort) are reported. Results are explained in terms of monosaccharide catabolism, polysaccharide hydrolysis, gas production and microorganism growth. The effect of each one of these subprocesses upon ultrasonic velocity has been independently studied. It is shown that, regarding the sound propagation, the simplest systems behave as ternary dissolutions of sugar and ethanol in water, where, in the course of time, substrates are transformed into metabolites according to the fermentation reaction. A semi-empirical approach, based on the excess volume concept and the density and velocity measurements of binary mixtures, has been used to calculate these magnitudes in the ternary mixtures and to obtain the concentrations of the main solutes throughout the fermentations, reaching a good correlation (especially for the media of simplest composition). In all the processes analyzed, the data obtained from the ultrasonic measurements followed the changes caused by the yeast metabolism, asserting the potential of mechanical waves to monitor fermentations and, in general, biotechnological processes.     

The acetone-butanol fermentation in pilot plant and pre-industrial scale

A summary of literature data concerning pilot or preindustrial scale trials of the acetone-butanol fermentation throughout its history is given. The recent pilot plant trials in Austria are also described for the first time. Some aspects of the current development of the acetone-butanol fermentation in general, especially from a technical point of view are also discussed.     

Optimization of an industrial microalgae fermentation

Optimization of cellular productivity of an industrial microalgae fermentation was investigated. The fermentation was carried out at Coors Biotech Products Company, Fort Collins, Colorado. A mathematical model was developed based on the data collected from pilot plant test runs at different operating conditions. Pontryagin's maximum principle was used for determining the optimal feed policy. A feedback control algorithm was also studied for maximizing the cellular productivity. During continuous operation, the optimum dilution rate was determined by an adaptive optimization scheme based on the steepest descent technique and a recursive least squares estimation of model parameters. A direct search algorithm was also applied to determine the optimum feed rate. Comparison of the theoretical results of the different optimization schemes revealed that the direct search algorithm was preferable because of its simplicity. The experimental results of real time application of the feedback algorithm agreed fairly well with those of the theoretical analyses. (c) 1994 John Wiley & Sons, Inc.     

On-line study of growth kinetics of single hyphae of Aspergillus oryzae in a flow-through cell

Using image analysis the growth kinetics of the single hyphae of the filamentous fungus Aspergillus oryzae has been determined on-line in a flow-through cell at different glucose concentrations in the range from 26 mg L-1 to 20 g L-1. The tip extension rate of the individual hyphae can be described with saturation type kinetics with respect to the length of the hyphae. The maximum tip extension rate is constant for all hyphae measured at the same glucose concentration, whereas the saturation constant for the hyphae varies significantly between the hyphae even within the same hyphal element. When apical branching occurs, it is observed that the tip extension rate decreases temporarily. The number of branches formed on a hypha is proportional to the length of the hypha that exceeds a certain minimum length required to support the growth of a new branch. The observed kinetics has been used to simulate the outgrowth of a hyphal element from a single spore using a Monte Carlo simulation technique. The simulations shows that the observed kinetics for the individual hyphae result in an experimentally verified growth pattern with exponential growth in both total hyphal length and number of tips.     

Extraction of microalgae derived lipids with supercritical carbon dioxide in an industrial relevant pilot plant

Microalgae are capable of producing up to 70% w/w triglycerides with respect to their dry cell weight. Since microalgae utilize the greenhouse gas CO₂, they can be cultivated on marginal lands and grow up to ten times faster than terrestrial plants, the generation of algae oils is a promising option for the development of sustainable bioprocesses, that are of interest for the chemical lubricant, cosmetic and food industry. For the first time we have carried out the optimization of supercritical carbon dioxide (SCCO₂) mediated lipid extraction from biomass of the microalgae Scenedesmus obliquus and Scenedesmus obtusiusculus under industrrially relevant conditions. All experiments were carried out in an industrial pilot plant setting, according to current ATEX directives, with batch sizes up to 1.3 kg. Different combinations of pressure (7–80 MPa), temperature (20–200 °C) and CO₂ to biomass ratio (20–200) have been tested on the dried biomass. The most efficient conditions were found to be 12 MPa pressure, a temperature of 20 °C and a CO₂ to biomass ratio of 100, resulting in a high extraction efficiency of up to 92%. Since the optimized CO₂ extraction still yields a crude triglyceride product that contains various algae derived contaminants, such as chlorophyll and carotenoids, a very effective and scalable purification procedure, based on cost efficient bentonite based adsorbers, was devised. In addition to the sequential extraction and purification procedure, we present a consolidated online-bleaching procedure for algae derived oils that is realized within the supercritical CO₂ extraction plant.

     

Modeling of an industrial alcohol fermentation and simuiation of the plant by a process simulator

The aim of the present study was the development of a general simulation module for fermentation within the framework of existing chemical process simulators. This module has been applied to an industrial plant which produces ethanol from beet molasses and fresh beet juice by Saccharomyces cerevisiae. An unstructured mechanistic model has been developed with kinetic laws that are based on a chemically defined reaction scheme which satisfies stoichiometric constraints. This model can be applied to different culture conditions and takes into account secondary byproducts such as higher alcohols. These byproducts are of prime importance and need to be correctly estimated because a sequence of distillation columns follow the fermentor in the plant. Important measurement campaigns have been performed on the plant to validate the model. Plant operation has been successfully simulated using the same kinetic model for both continuous and fed-batch modes of production. (c) 1995 John Wiley & Sons, Inc.     

Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation

We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations—such as glucose overbatching and insufficient aeration—on growth, metabolism, and titer.     

Solid state fermentation reactors: from lab scale to pilot plant

Relatively many workers in the world are studying different aspects in SSF processes but few are working on reactor design and scale-up. From about 10 years, we are developing reactors from lab scale to pilot plant, based on the same technology, reactor design and flowsheet to allow fermentation with a deep layer (up to 1 m in the pilot plant). These reactors have all a forced aeration and the possibility or not to agitate. Regulations of temperature and water content of the culture are monitored by a special device.     

Growth kinetics of Apple plant cell cultures

Summary Aerobic batch cultures of Apple fruit cells were carried out using sucrose as the sole carbon source. The determination of the growth parameters was used in order to estimate the maintenance coefficient m and the maximum growth yield Y_G; their values at 25°C are respectively 0.32. 10^-2 g sugar/g cell dry weight.h and 0.57 g cell dry weight/g sugar. Then, a mathematical expression of the cell growth is discussed, with respect to the specific growth rate evolution during cultivation.     

Observed quasi-steady kinetics of yeast cell growth and ethanol formation under very high gravity fermentation condition

Using a generalSaccharomyces cerevisiae as a model strain, continuous ethanol fermentation was carried out in a stirred tank bioreactor with a working volume of 1,500 mL. Three different gravity media containing glucose of 120, 200 and 280 g/L, respectively, supplemented with 5 g/L yeast extract and 3 g/L peptone, were fed into the fermentor at different dilution rates. Although complete steady states developed for low gravity medium containing 120 g/L glucose, quasi-steady states and oscillations of the fermented parameters, including residual glucose, ethanol and biomass were observed when high gravity medium containing 200 g/L glucose and very high gravity medium containing 280 g/L glucose were fed at the designated dilution rate of 0.027 h⁻¹. The observed quasi-steady states that incorporated these steady states, quasi-steady states and oscillations were proposed as these oscillations were of relatively short periods of time and their averages fluctuated up and down almost symmetrically. The continuous kinetic models that combined both the substrate and product inhibitions were developed and correlated for these observed quasi-steady states.     

A pilot plant for mammalian cell culture

Studies of the possible viral etiology of human leukemia have required large quantities of cultured cells derived from human hematopoietic tissues. Since cultures sufficiently large and free from contamination could not readily be produced according to existing methods, a pilot, cell culture plant has been constructed for the production of mammalian cells in mass quantity. 500-ml to 20-liter trophocell units have already proved to be scientifically and economically practical, as they provide good reliability, excellent growth rates, and sustained yield of human cells. 200-liter stainless steel culture units have now been added to the trophocell system. Five complete 200 liter units are now in operation. The design of the original stainless steel unit was based on that of a stainless steel, jacketed soup kettle. There are no openings in the vessel other than those in the lid, which provide convenient access points for sampling, sensor probes, etc. Environmental parameters, e.g., liquid level, temperature, and pH, are monitored and controlled with commercially available apparatus. Many initial problems connected with the new 200 liter units have been resolved, but operational and design problems remain in the areas of stable instrumentation, cell harvesting, salvaging and reuse of unspent media components, establishment of physiologic steady stale, recovery of virus-containing cells with reculture of the remaining unaffected cells, and the recovery and separation of cell components and special products such as immunoglobulins, interferons, and hormones. A definitive cell plant with culture units of 20, 50, 250, and 1250 liters is now being constructed.     

Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation

Proteolytic degradation of recombinant proteins is an industry-wide challenge in host organisms such as Escherichia coli. These proteases have been linked to stresses, such as the stringent and heat-shock responses. This study reports the dramatic up-regulation of protease activity in an industrial recombinant E. coli fermentation upon induction. The objective of this project was to detect and characterize up-regulated proteases due to recombinant AXOKINE overexpression upon IPTG induction. AXOKINE is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE was expressed in both the soluble and inclusion body fractions in E. coli. Sodium dodecyl sulfate gelatin-polyacrylamide gel electrophoresis (SDS-GPAGE) was used to analyze the up-regulated protease activity. Western blot analysis showed degraded AXOKINE in both the soluble and insoluble fractions. Protease inhibitors were used to characterize the proteases. The proteases were ethylenediaminetetraacetic acid (EDTA) sensitive. The protease activity increased in the presence of phenyl-methyl sulfonyl-fluoride (PMSF), a serine protease inhibitor. The incubation buffer composition was varied with respect to Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent. A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0.     

Study of process variables in industrial acetic fermentation by a continuous pilot fermentor and response surfaces

The modeling and optimization of industrial processes requires an intensive study of the factors involved. In this work, a continuous pilot system for studying the industrial process of acetic fermentation is developed. A Doehlert design is applied to the five variables involved in the pilot process. This experimental design allows reduction of the experimental burden and the maximum amount of information to be obtained, studying the factors at different levels depending on their significance. The experimental system provides a robust measure of the specific growth rate and the rates of substrates consumption and acetic acid production, related to the flow of effluent stream evaluated in the steady state. The results demonstrate the growth-associated kinetics of substrates and product, and the yield factors are calculated with low values of variances for the coefficients, i.e., within the range 1-11%. The specific growth rate suits the quadratic model proposed. The response surfaces generated by the model are applied to explain the behavior of the bacterial growth and, therefore, the effects of the process variables studied over the acetic acid production. Very low levels of ethanol or oxygen make the acetification rate decrease, and a saturation effect with high levels of ethanol or oxygen is also deduced. The effects of the aeration rate, agitation, and overpressure suggest a kind of inhibition of the acetic acid production caused by the oxygen that has not been practically studied before. The temperature strengthens the inhibitory effect of the ethanol and the oxygen. The conclusions of this work consolidate the structure of a hybrid model for the acetic fermentation.