Showing papers in "Bioprocess Engineering in 1996"

Substrate gradients in bioreactors: origin and consequences

Abstract: Gradients of glucose in time and space are shown in a 30 m3 cultivation of Saccharomyces cerevisiae grown in minimal medium to a cell density of 20 gl−1. The fed-batch concept was used with glucose as the limiting component which was fed continuously to the process. As the mean glucose concentration declined throughout the process, the level of glucose was at all times different in three sampling ports (bottom/middle/top) of the reactor. These gradients were furthermore shown to depend on the feed position. This means that if the feed was supplied in the relatively stagnant mixing zone above the top impeller, the gradients were more pronounced than by feed in the well mixed bottom impeller zone. A rapid sampling system was constructed, and continuous glucose samples of every 0.15 s were analysed from a point of the reactor. Fifty samples were collected with this system, but the amount and frequency is possible to change. The results of these series show a variance of the glucose concentration where at one stage, a peak appeared of a relative difference in concentration of 40 mgl−1. The pattern of these rapid glucose fluctuations was shown to depend on the turbulence level at the location of the feed. It was shown, that the fluctuations were more pronounced when the feed was localised in a relatively stagnant area than in the well-mixed impeller area, where the deviation from the mean was negligible. The fluid flow, in the impeller (gassed and ungassed) and bulk area (ungassed) of the reactor, was characterised by turbulence measurements using thermal anemometry. These types of areas resembles well the different areas of sampling as mentioned above. The turbulent frequencies in these areas were in the range of 10−1 to 104 Hz with the highest amplitudes at low frequencies. The spectra depicts a uniform time scale for all zones, especially at the low frequencies. The dominance of low frequency, high amplitude flow variations and the observed short-time oscillations in substrate concentration support the hypothesis of substrate transport over fairly long distances without substantial mixing both in the impeller, but especially, in the bulk zone of the reactor.

Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum

Abstract: Stoichiometric analysis is applied to continuous glycerol fermentation by Clostridium butyricum to calculate theoretical maximum yields and to predict preferred pathways under different conditions. The upper limits of product concentration and productivity as a function of dilution rate in continuous culture is also predicted from product inhibition kinetic. The theoretical maximum propanediol yield (0.72 mol/mol glycerol) which is calculated for a culture without hydrogen and butyric acid formation agrees well with the experimental maximum value (around 0.71 mol/mol). Comparisons of experimental results (product concentration and productivity) with theoretical calculations and those of the glycerol fermentation by Klebsiella pneumoniae reveal that the production of 1,3-propanediol by C. butyricum is far below the optimum performance available with the present strain. One of the reasons is the relatively high formation of butyric acid under the culture conditions so far applied. The distribution of reducing equivalents to propanediol and hydrogen is also suboptimal. The utilization of the reducing power from pyruvate oxidation for propanediol production is about 60–70% of the theoretical maximum under the present experimental conditions.

The use of windows of operation as a bioprocess design tool

Abstract: Bioprocess design problems are frequently multivariate and complex. However, they may be visualised by a graphical representation of the design constraints and correlations governing both the process and system under consideration, namely windows of operation. Windows of operation exist at all stages of process design and find use both in the identification of key constraints from limited information, and also, with more detailed knowledge, the sensitivity of a process to design or operating changes. In this way windows of operation may be used to help understand and optimise a bioprocess design. In this paper the formulation, development and application of windows of operation is discussed for a range of biological processes including fermentation, protein recovery and biotransformation.

Optimization of batch fermentation conditions for dextran production

Abstract: The nutrient medium (containing sucrose, yeast extract and K2HPO4), temperature and initial pH conditions were optimised for batch dextran production in shake flask fermentations using a strain of Leuconostoc mesenteroides NRRL B 512 (F). A 25−1 fractional factorial central composite experimental design was attempted. Multistage Monte Carlo optimization program was used to maximize the multiple regression equation obtained. The optimal values of tested variables for maximal dextran production were found to be: sucrose, 300 g/l; yeast extract, 10 g/l; K2HPO4, 30 g/l; temperature, 23°C and initial pH 8.3 with a predicted dextran yield of 154 g/l.

Continuous production of L-tert-leucine in series of two enzyme membrane reactors

Abstract: The L-tert-leucine synthesis was performed continuously in series of two enzyme-membrane reactors by reductive amination of trimethylpyruvate with leucine dehydrogenase. The necessary “native” cofactor NADH is regenerated with the aid of a second enzyme, formate dehydrogenase.

Development of a bioreactor system using an immobilized white rot fungus for decolorization

Abstract: In this paper the fixed film reactor system containing immobilized Phanerochaete chrysosporium cells was employed for decolorization of Red 533 dispersed dye. The inlet dye concentration and the inlet flow rate were shown to affect the decolorization efficiency. Each decolorization process was conducted continuously for 10–20 days or more and the decolorization efficiency remained higher than 80%. The immobilized cultures possessed good biological activities and the biodegrading system also showed capability for a long term operation.

Development of a bioreactor system using an immobilized white rot fungus for decolorization. Part II: Continuous decolorization tests

Abstract: In this paper the fixed film reactor system containing immobilized Phanerochaete chrysosporium cells was employed for decolorization of Red 533 dispersed dye. The inlet dye concentration and the inlet flow rate were shown to affect the decolorization efficiency. Each decolorization process was conducted continuously for 10-20 days or more and the decolorization efficiency remained higher than 80%. The immobilized cultures possessed good biological activities and the biodegrading system also showed capability for a long term operation.

Enhancement of biological treatment performance of saline wastewater by halophilic bacteria

Abstract: Performances of biological treatment processes of saline wastewater are usually low because of adverse effects of salt on microbial flora. High salt concentrations in wastewater cause plasmolysis and loss of activity of cells resulting in low COD removal efficiencies. In order to improve biological treatment performance of saline wastewater, a halophilic organism Halobacter halobium was used along with activated sludge culture. A synthetic wastewater composed of diluted molasses, urea, KH2PO4 and various concentrations of salt (1%–5% NaCl) was treated in an aerobic-biological reactor by fed-batch operation. Activated sludge culture with and without Halobacter were used as seed cultures. Variations of COD removal rate and efficiency with salt concentration were determined for both cultures and results were compared. Inclusion of Halobacter into activated sludge culture resulted in significant improvements in COD removal efficiency. A rate expression including salt inhibition effect was proposed and kinetic constants were determined by using experimental data.

Production of tannase by solid-state fermentation

Abstract: An attempt has been made to optimize the production of enzyme tannase by solid state fermentation (SSF) using the organism Rhizopus oryzae. The best favourable conditions for enzyme production include initial pH 5 with 4 days of incubation period at 40°C and 72% humidity, and 10 g wheat bran soaked in 2.5% tannic acid.

Automatic control of the specific growth rate in fed-batch cultivation processes based on an exhaust gas analysis

Abstract: A new simple strategy for a reliable and robust automatic control of the specific growth rate in fed-batch cultivation processes is presented. Its advantages over model supported control is that the algorithm only needs a minimum of information about the process. Moreover, it is independent of the specific microorganism, the cultivation phase and the biomass level. Also, only a minimum of soft- and hardware is required. Hence, the approach is attractive for industrial production processes that do not have specialized instrumentation. Its accuracy is comparable with model supported control and thus sufficient for most industrial applications. Simulations and experimental tests of the technique performed for the example of a fed-batch cultivation of E. coli demonstrate a good controller performance for various cultivation conditions and process disturbances. Preferred applications will be production systems where the productivity is critically dependent on the growth rate, e.g. in recombinant protein or antibiotic productions.

High density fed-batch cultures for hybridoma cells performed with the aid of a kinetic model

Abstract: Hybridoma fed-batch cultures with either standard medium as feed or concentrated medium as feed and removal of toxic metabolites through dialysis were performed by using model calculations for “a priori” determination of process parameters. In a first step a kinetic model for specific growth and death rate, respectively as well as for substrate uptake and metabolite production rates was formulated. In a bed-batch culture with standard medium as feed the appropriate time for start of the feeding pump and the increase of feed rate were determined “a priori”. The glutamine concentration was controlled at 0.04 mmoll−1. “A priori” calculation and course of the culture coincided rather well. A cell concentration of 3.2×106 cells ml−1, a MAb-concentration of 54 mg MAb l−1 and a MAb-time-space-yield of 0.53 mg MAb l−1h−1 were obtained. For further increase of the efficiency a high density fed-batch process was developed, where concentrated medium is fed to the cells and the accumulating toxic low molecular weight metabolites are removed through a dialysis membrane into a dialyizng fluid. In a membrane dialysis reactor consisting of a culture chamber and a dialyzing chamber, which are separated by a cylindrical dialysis membrane, again model calculations were used to determine feed rate and exchange rate of dialyzing fluid. A viable cell density of 1.2×107 cells ml−1 and a MAb concentration of 425 mg l−1 were reached in a culture with stepwise feeding of 10 x concentrated medium and exchange of dialyzing fluid for removal of low molecular metabolites. The course of the culture could be predicted “a priori” rather well. The MAb-time-space-yield was 2.47 mg MAb l−1h−1, appr. 5 times higher compared to fed-batch cultures with standard medium as feed.

Hydrodynamic characteristics of two-phase inverse fluidized bed

Abstract: Hydrodynamic characteristics of a new mode of liquid-solid fluidization, termed as “inverse fluidization” in which low density floating particles are fluidized with downward flow of liquid, are experimentally investigated. The experiments are carried out with low density particles (<534 kg/m3) which allow high liquid throughputs in the system. During the operation, three regimes, namely, packed, semi-fluidization and fully fluidization are encountered. Empirical correlations are proposed to predict the pressure drop in each regime. A computational procedure is developed to simulate the variation of pressure drop with liquid velocity.

Optimization of carbon and nitrogen sources in the medium and environmental factors for enhanced production of chitinase by Trichoderma harzianum

Abstract: Statistical design was used to determine the optimal levels of medium components, the optimal initial pH of the enzyme production medium, the temperature of fermentation, age of the organism in the slant growth and the age of the inoculum for the production of chitinase in shake flask fermentations. The use of high concentrations of chitin and ammonium sulphate and exclusion of peptone and urea from the medium resulted in the production of higher level of the enzyme. The optimal concentrations of the medium components were 12.5 kg/m3 and 4.2 kg/m3 for the chitin and ammonium sulphate respectively. The effect of the addition of peptone and urea to the optimized medium was studied. The optimal values of initial pH and temperature were 5.6 and 28 °C respectively. The optimal age of the slant and the inoculum were found to be 105 h and 43 h respectively. The highest level of chitinase before optimization of the above variables was 0.054 U which was maximized to the level of 0.197 U.

Automation of a solid substrate cultivation pilot reactor

Abstract: An automation system for a solid substrate pilot bioreactor is described. The performance of the system in real time experiments is discussed and future improvements are proposed. Good control of temperature and water content of the solid bed was achieved, although the system is not fully automatic and needs human supervision.

Study of charcoal adsorption for improving the production of Xylitol from wood hydrolysates

Abstract: Xylose-containing solutions, obtained from acid prehydrolysis of Eucalyptus wood, were treated with powdered charcoal in order to remove lignin-derived compounds that limit the potential of hydrolysates for making fermentation media. Both the kinetics and equilibrium of adsorption were modelled using equations reported in literature. Charcoal-pretreated hydrolysates were supplemented with nutrients and used for producing xylitol with the yeast Debaryomyces hansenii NRRL Y-7426. The susceptibility to fermentation of culture media made with this procedure was compared with those corresponding to media made from untreated wood hydrolysates or standard xylose solutions. The removal of lignin-derived compounds from hydrolysates was closely related with the efficiency of fermentation.

Citric acid production

Abstract: Selective separation of citric acid and process optimization for its recovery from fermented broth through precipitation and acid hydrolysis were tried. A mathematical model has been proposed for yield. The experimental values are in good agreement with predicted values; the correlation coefficient was found to 0.9198.

Pullulan fermentation in a reciprocating plate bioreactor

Abstract: Reciprocating plate bioreactors are particularly well suited for conducting fermentations which give rise to highly viscous broth. To evaluate their performance for polysaccharide fermentations, a series of pullulan fermentations were performed with a particular emphasis placed on the influence of aeration on both the quantity and quality of the product. Two experiments were conducted at constant aeration rates and two others with constant dissolved oxygen concentrations. For the latter two experiments, the dissolved oxygen concentration was controlled by manipulating either the aeration flow rate or the reciprocating frequency of the perforated plates.

Kinetic behaviour of waste tyre rubber as microorganism support in an anaerobic digester treating cane molasses distillery slops

Abstract: The kinetics of anaerobic digestion of cane molasses distillery slops was investigated using a continuous-flow bioreactor which contained waste tyre rubber as support, to which the microorganisms became immobilized. Hydraulic retention times (HRT) ranging from 1 to 10 days were investigated at an average influent chemical oxygen demand (COD) concentration of 47.7 g/l. The maximum substrate utilization rate, k, and half saturation coefficient, K L, were determined to be 1.82 kg CODremoved/kg VSS day and 0.33 kg COD/kg VSS day. The yield coefficient, Y, and sludge decay rate coefficient, K d, were also determined to be 0.06 kg VSS/kg CODremoved and 0.05 day-1, respectively. Methane production was maximum (6.75 l/l day) at a 2 day HRT corresponding to a biomass loading rate of 2.578 kg COD/kg VSS day. Biogas yield ranged between 0.51 l/g COD (HRT=2 days) and 0.25 l/g COD (HRT=1 day). In addition, the methane percentage in the biogas varied between 70.5% (HRT=10 days) and 47.5% (HRT=1 day). The close relationship between biomass loading rate and specific substrate utilization rate supported the use of Monod equations. Finally, the experimental values of effluent substrate concentration were reproduced with deviations equal to or less than 10% in every case.

Development of a bioreactor system using an immobilized white rot fungus for decolorization. Part I : Cell immobilization and repeated-batch decolorization tests

Abstract: In this research the wood-rotting fungus Phanerochaete chrysosporium culture was shown to be immobilized very well on the porous foam material. The biomass concentration increased to about 2-3 g/l in 4-5 days. Repeated-batch decolorization tests using immobilized Phanerochaete chrysosporium cells were conducted for 16 days with initial concentrations of 50-500 ppm of Red 533 dispersed dye, a decolorization efficiency of 80% or higher was achieved within a period of one or two days. The ability of the immobilized culture to perform a long-term decolorization operation was confirmed.

Extraction of organic acids from kiwifruit juice using a supported liquid membrane process

Abstract: In order to extract or remove organic acids from kiwifruit juice, we evaluated their separation and transport rates through supported liquid membranes (SLMs). The liquid membrane consisted of an organic solution composed of a carrier (Aliquat 336/Alamine 336) and a linear alcohol (oleyl alcohol) and was loaded on a microporous polypropylene support (commercial grade Celgard 2500/2400). These SLMs were evaluated (i) in a batch cell to determine the permeability and (ii) in a continuous spiral membrane module to study the effects of various process parameters – flow of feed and strip solutions, membrane composition, recycling mode of operation and kiwifruit juice at natural pH. It was observed that there exists an optimum for each system: pH 2.5– 3.0 for Alamine 336/oleyl alcohol and pH 4.5 for Aliquat 336/oleyl alcohol. At this pH the flux rates of citric acid and malic acid was greater (6–8 times) than that of quinic acid. The flux rates decreased (greatly for citric acid) with the flow rate of feed and strip solutions and increased (considerably for citric acid) with the SLM composition . The recycling of feed and strip solutions significantly improved the removal efficiency. The SLM system retained its performance over a period of a few days. The SLM process allowed extraction of the above three organic acids (ascorbic acid was removed in trace amounts) from kiwifruit juice at a rate of a few percent (5%) in a single-pass processing.

Pilot-scale verification of a computer-based simulation for the centrifugal recovery of biological particles

Abstract: The development of a model for simulating the recoveries by pilot-scale disc-stack centrifugation of whole yeast cells, yeast cell debris and protein precipitates prepared by ammonium sulphate salting-out is presented. The model is based on the grade efficiency concept and incorporates the effects of hindered settling at high biomass concentrations and the breakage of shear-sensitive material within the centrifuge feed-zone to give an accurate prediction of solid/liquid separation. The simulations have been proven by comparison with data from pilot-scale verification trials. The trials have highlighted where improvements to the models were required to increase their accuracy. The value of verification trials in proving the validity of models is commented upon.

Influence of nutrients on cell growth and xanthan production by Xanthomonas campestris

Abstract: Xanthan production by Xanthomonas campestris was performed in a batch fermentation study with a view to achieving maximum yields. The factors influencing the production — mainly the nutrients, were investigated. Trace elements such as phosphate and magnesium at 6.0 g/l and 0.2 g/l enhanced the yields.

Effects of downcomer-to-riser cross sectional area ratio on operation behaviour of external-loop airlift bioreactors

Abstract: Experiments performed in two external-loop airlift bioreactors of laboratory and pilot scale, (1.880–1.189) · 10−3 m3 and (0.170-0.157)m3, respectively, are reported. The A D /A R ratio was varied between 0.111–1.000 and 0.040–0.1225 in the laboratory and pilot contractor respectively.

Citric acid production in submerged and solid state culture of Aspergillus niger

Abstract: In this work, the citric acid production in solid state culture was performed, evaluating the isolated effect and interactions of particle size and liquid phase employed, by means of the factorial design of first order. The results indicate that the particle size is the most determinant variable. An analysis comparing submerged and solid state in optimal conditions was performed. When solid state culture was used, the productivity of citric acid was doubled, reducing the fermentation time from 14 to 6 days, compared to the submerged culture, obtaining a maximum citric acid concentration of 21.24 g/l.

Studies on biodegradation of a mixture of toxic and nontoxic pollutant using Arthrobacter species

Abstract: The effect of a nontoxic easily degradable substrate, glucose, on the biodegradation of toxic pollutant, phenol, was studied in batch reactors using a phenol degrading culture (Arthrobacter species). The effect of glucose on phenol degradation was determined at different glucose concentrations. The effect of different inoculum on substrate removal in a phenol and glucose mixture was also studied. Results indicated that when a mixed substrate (phenol and glucose) was used, phenol acclimated population showed an initial preference for phenol and utilised glucose after phenol removal. However phenol degradation rate was reduced in the presence of glucose. It was also observed that phenol degradation was completely inhibited when the glucose concentration exceeds 2 g/l. The substrate removal pattern changed completely when inoculum was drawn from mixed substrate acclimatised culture. The glucose utilisation started immediately and the rate of glucose utilisation was not affected by the presence of phenol. The phenol degradation also started simultaneously. In presence of phenol only, the rate of phenol degradation for the culture acclimatised to mixed substrates was lower than that of phenol acclimatised culture. These results indicate that nontoxic substrate can affect the biodegradation of toxic pollutants is suitable and acclimatisation may be necessary for biodegradation of mixed substrate.

Kinetic study of Bacillus thuringiensis var. israelensis in lab-scale batch process

Abstract: Fermentations of Bacillus thuringiensis have long traditions for the production of δ-endotoxin as bioinsecticide in agriculture and forestry. A process analysis study of endoprotein production by batch fermentation is presented in this paper. General fermentation characteristics are given and the influence of dissolved oxygen to sporulation and toxicity is discussed. Kinetics of growth and product formation is examined and a model based on Monod with an inhibition constant is used to describe the process. The model should provide a useful framework for further process verification and optimisation in practice.

Simulation of glucose isomerase reactor: optimum operating temperature

Abstract: A design equation for immobilized glucose isomerase (IGI) packed bed reactor is developed assuming enzyme deactivation and substrate protection. The developed equation is used to simulate the performance of the reactor at various temperatures (50–80 °C). Enzyme deactivation is significant at high temperature. Substrate protection showed to have significant effect in reducing enzyme deactivation and increasing the enzyme half-life. Factors affecting the optimum operating temperature are discussed. The optimum operating temperature is greatly influenced by the operating period and to a lesser extent with both initial glucose concentration and glucose conversion.

Production of ethanol from dilute glucose solutions - A technical-economic evaluation of various refining alternatives

Abstract: In order for ethanol to be competitive with gasoline, the production cost of ethanol must be lower than it is today. One economically crucial step in the production of ethanol from lignocellulosics is refining, since hydrolysis yields dilute glucose solutions which, after fermentation, result in a dilute ethanol solution, 2–3 wt-% ethanol. A technical-economic investigation of various energy-saving alternatives to conventional distillation has been performed. The energy-saving techniques investigated were: multi-column distillation, distillation with mechanical vapour recompression, distillation with an absorption heat transformer, phase separation with potassium carbonate and extraction of ethanol from the fermentation broth with Aldol 85. The most economical refining step for the production of ethanol from a dilute glucose solution, around 5 wt-%, was found to be multicolumn distillation.

Citric acid production: Part 1: Strategies for reduction in cycle time for targeted yields

Abstract: Techniques for possible higher and rapid production of citric acid from the well known industrial medium i.e. molasses has been reported using Aspergillus niger. This includes optimization of the total reducing sugar (TRS) and nutrients like nitrogen and phosphorous. The long and unproductive lag periods normally associated with this type of fermentation has been reduced. These strategies are discussed in detail.

Incomplete mixing in large bioreactors — a study of its role in the fermentative production of streptokinase

Abstract: The production of streptokinase in a batch fermentation has been analysed for the role of incomplete macromixing of the broth. The analysis is based on a kinetic model exhibiting inhibition by the substrate and a primary metabolite (lactic acid), and a mixing model comprising two continuous flow reactors (CFRs) with closed-loop recycle. The inoculum is introduced into one region (one CFR) and the mixing process determines its distribution, growth and reactivity. By varying the dilution rates of the CFRs, any degree of macromixing can be simulated. For dilution rates larger than 1.0 h−1 almost complete macromixing is achieved, for which an analogy has been drawn with micromixing. Increasing the volume of the inoculated region relative to the noninoculated region improves the maximum attainable activity of streptokinase and shortens the time for this. In such a situation an imperfectly mixed bioreactor is superior to a perfectly mixed one, implying that good productivity requires a large inoculated region and incomplete macromixing. These inferences are supported by earlier studies of fluid mixing and relaxation times in bioreactors.