Repeated Batch Process Research Articles

Investigation of the use of agricultural byproducts for fungal protein production

Cellulose and nutrient salts as well as potato pulp and potato protein liquor (PPL), were used as substrates for the cultivation of Chaetomium cellulolyticum in batch and repeated-batch operations. Using cellulose as the substrate a linear relationship existed between the rates of cell mass formation and acid production. The repeated-batch process was controlled by NaOH consumption using a simple computer model. When the production of cell mass and acid stopped because of a lack of substrate cellulose was fed into the reactor. This occurred within 10 min at which point no NaOH-feed was needed to maintain a constant pH. Repeated-batch operations yielded higher cell concentrations and productivities than batch operations. The relationship between the NaOH and H 2SO 4 consumed, and the fungal mass concentration was complex in cultivation media containing potato pulp and PPL, because various substrates were consumed by the fungus simultaneously and successively. Therefore, for repeated-batch cultivation a constant time interval was used. Repeated-batch cultivation of the fungus on potato pulp and PPL did not yield higher cell concentrations and productivities than did batch cultivation. With the optimal pulp-to-PPL ratio a maximum specific growth rate of 0.61 h 1 was obtained. These investigations indicate, that potato pulp and PPL are well suited to fungal protein production by Chaetomium cellulolyticum for fodder supplement.

Cell size distribution as a parameter for the predetermination of exponential growth during repeated batch cultivation of CHO cells

The routine measurement of the cell size distribution of a Chinese hamster ovary (CHO) cell population during a repeated batch process enables the predetermination of exponential growth even 24 h before the population enters the log phase, due to a short but significantly increased cell size during the lag phase. A prolongation of the stationary phase causes to progressive limitation in asparagine, serine, and ethanolamine. Such extended limitation influences the duration of the following lag phase and obviously induces a synchronization of the cell population that can be monitored easily by a fast cell size analyzing technique. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 793-797, 1997.

N-carbamoyl- d- p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl- d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V max and K m of the immobilized d-hydantoinase at 50°C were 6.28 m m min −1 g −1 biocatalyst and 71.6 m m, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol −1. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.

Immobilization of Pseudomonas sp. BA2 by entrapment in calcium alginate and its application for the production of l-alanine

The conditions for immobilizing the new l-aminoacylase-producing bacterial strain Pseudomonas sp. BA2 by entrapment in calcium alginate gel were investigated. The optimal gel concentration and cell loading were determined. It was demonstrated that the addition of the substrate n-acetyl- l-alanine to the immobilizing matrix enhanced l-aminoacylase activity. The enzymatic properties of immobilized Pseudomonas sp. BA2 were investigated to ascertain which conditions were suitable for the enzymatic reaction. Optimal pH, temperature, and concentration of Tris-maleate buffer were determined. The influence of adding CoCl 2 on the enzymatic reaction rate was studied and the optimal concentration of the activator was determined. Stability studies showed that the immobilized cell preparation is not adequate for use in repeated batch processes. Continuous operation in a stirred tank reactor allowed us to determine the biocatalyst half-life (7 h approximately) but, due to the high l-aminoacylase activity, the reactor productivity (24.5 mmol of l-alanine in 8 h) was noticeably higher than that previously obtained in a packed bed reactor with Aspergillus ochraceus pellets. The results reported in this paper show the potential for using the immobilized Pseudomonas sp. BA2 in calcium alginate to produce l-alanine; however, before large scale production can be undertaken, further biocatalyst stabilization studies have to be made.

Glucose oxidase and catalase activities ofPenicillium variabile P16 immobilized in polyurethane sponge

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L−1; Ca-carbonate concentration, 15 g L−1; temperature, 28°C and aeration rate, 4 VV−1 min−1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.

Semi-continuous fumaric acid production by Rhizopus arrhizus immobilized in polyurethane sponge

Fumaric acid was produced in repeated batch processes using Rhizopus arrhizus NRRL 1526 immobilized in polyurethane sponge particles and cultivated in a fluidized-bed reactor. Fermentation conditions were established for concentration of carrier-particles and optimal concentrations of glucose nolasses (as glucose equivalent) and ammonium sulphate. Acid production was optimized (12·3 g/litre, 35·56% and 0·256 g/litre h of fumaric acid accumulation, yield and volumetric productivity of the whole process, respectively) under the following conditions: carrier-particle concentration, 20% v/v) of the working volume; glucose molasses, 50 g/litre; ammonium sulphate, alternation of cycles with 0·0 and 0·5 g/litre. The time of fermentation was 48 h per batch for a total of eight batches. Scanning electron microscopy revealed that the fungus began to grow within the particle cells but soon after the end of the first batch growth was essentially confined to the outer part of the polyurethane particle, where a peripheral layer of mycelium began to form.

Repeated Batch Acid Proteinases Biosyntesis by Immobilized Humicola Lutea Cells Cultivated in Bioreactor

Fungal strain Humicola lutea 120–5 was immobilized in poly[hydroxyethylmethacrylate (HEMA)-ethyleneglycoldimethacrylate (EDMA)] gel. Immobilized cells were cultivated in column and airlift bioreactors with different height to diameter (H/D) ratio for acid proteinases production during batch and repeated-batch processes. Influence of aeration rate in the bioreactors on enzyme production was studied. Optimal conditions for maximum proteinases production were obtained during repeated batch cultivation of immobilized cells (IC) in an airlift bioreactor with H/D=4 and aeration rate of 6 wm.

Immobilization–Stabilization of Kerase, a Serine Protease fromStreptomyces fradiae, by Covalent Attachment to Porous Glass

Kerase, a serine protease from Streptomyces fradiae, was immobilized on porous glass by covalent attachment of the enzyme through its amino groups. Chemical modification studies have shown that the amino groups can be used to establish covalent attachment of this protease to porous glass, modification of four lysine residues (44.4% of the accessible amino groups) resulting in a 6.5% loss of enzymatic activity. After immobilization, the optimal reaction pH range changed from 7.5–8.5 to 9–10, the protease being stable over a broad pH range of 6–12, while the soluble protease was irreversibly denatured in the alkaline pH range (pH > 8). Moreover, the optimal reaction temperature was changed from 55 to 65°C, showing higher thermal stability. Kerase immobilized onto porous glass was stable for at least 28 days when working in a repeated-batch process of three cycles per day, with an activity loss of 22.1 ± 3.1 %.

Kerase immobilization by covalent attachment to porous glass

Kerase, a serine protease from Streptomyces fradiae, was immobilized on porous glass (SIKUG®) by covalent attachment, through amino groups on the enzyme. Modifications of four lysine residues (44·4% of the accessible or superficial amino groups) results in a loss of 6·5% of the enzymic activity. After immobilization, the optimal reaction pH changed from a range of 7·5–8·5 to 9–10. The immobilized protease was stable in a broad pH range, 6–12, while the soluble protease was irreversibly denaturated at alkaline pHs (pH > 8). The optimal reaction temperature was displaced from 55 to 65°C, showing a higher thermal stability of the immobilized enzyme. Kerase immobilized onto porous glass was stable for at least 28 days, working in a repeated-batch process of three cycles per day, with an activity loss of 22·1 ± 3·1%.

Glucose oxidase, catalase and gluconic acid production by immobilized mycelium of Penicillum variabile P16

Conidia of Penicillium variabile P16 producing glucose oxidase were immobilized in different carriers and used in repeated-batch processes. Limited free-cell growth and good mechanical stability of the carriers were obtained with Ca-alginale, agar and polyurethane sponge. During prolonged experiments, the polyurethane sponge appeared to be the best carrier in relation to glucose oxidase and catalase activities. The maximum mean volumetric productivity of gluconate was obtained using agar as immobilizing agent.

Production of Ethanol from Reclaimed Paper Using a Combination of a Reversibly Soluble-autoprecipitating Cellulase and Flocculating Yeast Cells.

In this present work we report on a repeated batch process for ethanol production from a cellulosic material, reclaimed paper, by simultaneous saccharification and fermentation using soluble-autoprecipitating (S-AP) cellulase and flocculating yeast cells in a reactor with a separator. A continuous production process with a modified reactor for improving productivity is also reported

Production of polygalacturonases from Kluyveromyces marxianus fermentation: preliminary process design and economics

A simple one-stage purification scheme for the recovery of polygalacturonase from K. marxianus fermentation is described. The optimum pH for ion exchange is 4·5, which, being compatible with the fermentation conditions, means that the broth can be centrifuged and directly applied to an ion-exchange column; the process gives more than 90% recovery of a highly purified enzyme. Economic analysis of a repeated batch process shows that recovery of other by-products (cells and ethanol) is only of marginal importance, whilst pectinase production is highly profitable. There are strong economies of scale, up to a fermentation batch scale of around 20 m 3, with 100 batches per year. Under typical conditions our analysis predicts a payback time of under a year.

Utilization of unhydrolyzed cheese whey for the production of extracellular polysaccharide by Xanthomonas cucurbitae PCSIR B-52

A strain of Xanthomonas cucurbitae PCSIR B-52 produced extracellular polysaccharide using partially deproteinized cheese whey without hydrolysis. A synthetic lactose-salt medium was also utilized to determine the optiomum level of lactose desirable for successfull fermentation. The amount of extracellular polysaccharide was maximised at 7.8 g l −1 in the presence of 40 g l −1 lactose. The bacterium efficiently consumed cheese whey, particularly in the presence of corn steep liquor and penicillin waste mycelium in shaken flasks. The polysaccharide, bacterial cell mass and viscosity gradients were improved as a result of efficient oxygen transfer in a mechanically agitated fermentor. A depletion in dissolved oxygen tension resulted during the exponential growth phase. The fermentation pattern of extracellular polysaccharide was also studied by repeated batch process.

CHITOSANASE PRODUCTION BY <i>ALCALIGENES FAECALIS</i> IMMOBILIZED IN CALCIUM ALGINATE GEL BEADS

Alcaligenes faecalis cells were immobilized in calcium alginate gel beads and employed for production of chitosanase by repeated batch processes. The activity of the immobilized cells increased with extending the culture period, and reached a maximum, 3.6U/ml in 6 days. This value corresponded to about 1/3 of that of free cells. In repeated batch experiments the activity of the immobilized cells increased gradually in the early cycles, and it decreased slightly after the four cycle. Chitosanase was produced by the immobilized cells in an enzyme production medium containing 0.1% glucose, 0.5% peptone, 0.1% yeast extract, 0.1% KH2PO4, 0.02%MgSO4•7H2O, 0.1%NaCl, 0.5%CaCl2•H2O and 0.5% colloidal chitosan.

Light-dependent porphyrin production by suspended and immobilized cells of Rhodobacter sphaeroides

This study was undertaken to determine optimum conditions for porphyrin production by suspended and immobilized cells of Rhodobacter sphaeroides. Cell suspensions of R. sphaeroides strain NR3 previously grown photosynthetically excreted large amounts of porphyrins with coproporphyrin III as the major component under anaerobic-light conditions. The most pronounced excretion was found in the presence of 10 mM glycine, 10 mM succinate, and 0.1 mM Mg 2+ (or Ca 2+) and at pH between 7.5 and 8.0 and a high light intensity ( ≧ 15,000 lx ). The amount of porphyrins produced after 6 d of incubation by cell suspensions with an initial cell dry weight of 0.54 g was 170 μmol· l −1. Extracellular porphyrin formation was severely inhibited by levulinate, 2, 2′-dipyridyl, or chloramphenicol. Immobilization of cells with agar or carrageenan resulted in a marked decrease in the formation rate. However, intermittent treatment with a growth medium restored the ability of the immobilized cells to produce porphyrins effectively. Agar-immobilized cells in a repeated batch process including the intermittent treatment stage for re-activation produced higher levels of porphyrins than did cell suspension.

Glucoamylase and ?-amylase production by immobilized Aspergillus niger

Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and α-amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and α-amylase produced was altered by immobilization.

Optimal control of an enzyme reaction subject to enzyme deactivation. I. Batch process

AbstractThis paper is concerned with the study of an enzymatic system in a repeated batch process where the enzyme is subject to deactivation. The particular system studied was the enzymatic hydrolysis of Penicillin G to 6‐aminopenicillanic acid. Utilizing standard optimization techniques, pH and temperature control policies were determined that would maximize the product yield.