Repeated Batch Process Research Articles

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.

Influence of the fermentation parameters and optimisation of isomaltulose production from free Erwinia sp. D12 cells using response surface methodology

The food industry is constantly seeking novel ingredients to improve existing products or to allow for the introduction of new products. Isomaltulose is a reducing sugar with a sweet taste and very similar physical and organoleptic properties to those of sucrose. The strain Erwinia sp. is able to convert sucrose into isomaltulose. A two level rotatory central composite design and response surface methodology were applied to verify the influence and conditions for the production of isomaltulose by Erwinia sp. D12 free-cells in a batch process. The statistical analysis carried out at a confidence level of 90% gave a coefficient of determination of 0.90, and the polynomial model resulted in a response surface and contour curve that indicated the best parameters for the conversion of sucrose into isomaltulose as follows: temperature 35 °C, pH 6.5, wet cell mass 10% and sucrose 35%. The free-cells of Erwinia sp. D12 were recycled during repeated-batch processes to produce isomaltulose from sucrose obtaining high isomaltulose yields.

Evaluation of Zymomonas-based ethanol production from a hydrolysed waste starch stream

Residual starch from flour wet milling, supplemented with crushed wheat grains, was hydrolysed and evaluated for ethanol production by Zymomonas mobilis ZM4 and an industrial ethanol-producing strain of Saccharomyces cerevisiae. The feasibility of replacing yeast extract with ammonium sulphate or corn steep liquor as a nitrogen source in the hydrolysed waste starch (HWS) media was confirmed. With glucose concentrations of 80–110 g L−1, Z. mobilis ZM4 demonstrated superior fermentation kinetics in all three media, with higher specific rates of glucose uptake and ethanol production, greater productivities and ethanol yields close to theoretical. In a repeated batch process (five cycles), rapid cell concentration was achieved between cycles (via cell settling) with the flocculent strain Z. mobilis ZM401. This resulted in very significant productivity increases and illustrated the potential use of this strain for improved technology and cost reductions in larger scale processes.

Repeated production of fatty acid methyl ester with activated bleaching earth in solvent-free system

The enzymatic transesterification of rapeseed oil and methanol with activated bleaching earth (ABE) as a medium was investigated in a solvent-free system. The initial fatty acid methyl ester (FAME) production rate in the solvent-free system was 2.3-fold lower than that in the organic solvent system, but there was not much difference between the overall reaction rates in the solvent and solvent-free systems. When ABE was added to the reaction mixture consisting of rapeseed oil, methanol and lipase, the repeated batch production of FAME was conducted for nine cycles without a significant inactivation of lipase. However, it was only repeated six times in the rapeseed oil-fed batch process, because of the inactivation of lipase. The FAME yield based on used lipase in the repeated batch process was 55.8 g FAME per gram used lipase, which was two-fold higher than that of the oil-fed batch process.

Inulinase biosynthesis using immobilized mycelium of Aspergillus niger

Conidia of Aspergillus niger 20 Osm producing extracellular inulinase were immobilized on pumice stones or polyurethane sponge and used in repeated-batch processes. Some factors affecting inulinase biosynthesis by the mycelium A. niger immobilized on pumice stones were investigated. Maximal inulinase production occurred in 50 ml of medium containing 0.5 g of carrier at 30 °C, pH 6.0 and at an agitation speed of 200 rpm. This procedure enabled repeated-batch enzyme production and as many as six subsequent 24 h batches could be fermented by using the same carrier. This is the first report on inulinase biosynthesis by mycelium of A. niger immobilized on polyurethane sponge using unconventional oxygenation of culture which ensures that the dissolved oxygen concentration remains constant.

Evaluation of a pH-stat feeding strategy on the production and recovery of Fab’ fragments from E. coli

A comparison between two feeding strategies in a gas blending fermentation process to produce Fab’ by Escherichia coli was carried out at 20 L scale. A pH-stat feeding strategy was proposed to address the issue of glycerol oscillations that occur in repeated batch fermentations. This feeding strategy was based on direct coupling of growth and concomitant production of H + and this led to a constant minimum concentration of glycerol (main C-source) in the induction period. This change of environment resulted in a two-fold increase in the production of Fab’ compared to those obtained using a repeated batch strategy. A glycerol yield coefficient ( Y X/glycerol in g cell/ g glycerol) and product yield on biomass ( Y P/ X in mg product/ g cell) of 0.39 and 5.91, respectively, were estimated for the pH-stat feeding fermentation. By contrast, Y X/glycerol and Y P/ X were found to be 13 and 45% lower, respectively, for the repeated batch process. Therefore, considerable improvements in glycerol utilisation and product formation have been obtained using the pH-stat feeding strategy. Product localisation in the cell periplasm of >90% was obtained in both processes and a difference of product loss in the centrifugation step of less than 1% was observed. Overall, a method to improve production of Fab’ fragments without compromising primary recovery of cells by maintaining constant low levels of glycerol has been developed.

A repeated batch process for cultivation of Bifidobacterium longum.

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9 x 10(9) and 3.4 x 10(9) cfu ml(-1) for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l(-1) h(-1), respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l(-1) h(-1), respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.

Modeling and Optimization for High-Throughput-Screening Systems

The problem of cyclic scheduling under the requirement of throughput maximization is considered for a special class of cyclically repeated batch processes. All batches have to follow an identical time scheme. The same resource may be visited more than ouce by the same batch and time window constraints may be stated by the user. It is shown that the cyclic scheduling problem can be transformed into a mixed integer linear optimization problem. The method'sapplication to High-Throughput-Screening processes is demonstrated.

Stability of extracellular proteinase productivity by Bacillus subtilis cells immobilized in PVA-cryogel

Viable Bacillus subtilis cells were entrapped in the PVA-cryogel beads as described earlier [J. Mol. Catal. B Enzym. 11 (2001) 671; Biomol Eng 17 (2001) 55], and applied for synthesis of extracellular proteases (subtilisin and metalloproteinase) in the repeated-batch process. The scope of the present study was to evaluate the dependence of the proteases biosynthesis yield and the immobilized preparation stability on the cells growth phase prior to the entrapment, and their initial concentration in the PVA-beads. Spatial distribution of B. subtilis microcolonies inside the beads, and the cell leakage from the carrier during successive batches were also determined. The depth of the bacterium growth and changes in the biomass content inside the beads were measured using an image processing system. It was observed that the growth rate, spores formation and the proteases biosynthesis by the immobilized B. subtilis cells were strongly affected by the PVA-cryogel microenvironment.

Reuse of microbially treated olive mill wastewater as fertiliser for wheat ( Triticum durum Desf.)

Free cells of Aspergillus niger were grown on olive mill wastewater (OMW) supplemented with rock phosphate (RP) in an air-lift bioreactor in batch and repeated-batch processes. The fungus grew well and reduced the chemical oxygen demand of the waste by 35% and 64% in the batch and repeated-batch (fourth batch) processes, respectively. Total sugar content was consistently reduced (ca. 60%) in both processes while reduction of total phenols was minimal. RP was solubilised and maximum soluble P was 0.63 and 0.75 g l −1 in the batch and repeated-batch (third batch), respectively. Several types of OMW ± RP, microbially-treated or not, were tested in a greenhouse for their fertilising ability on a soil-wheat ( Triticum durum Desf.) model system. Beneficial effects were highest using OMW treated by the repeated-batch process. The treated plants showed an increase in seed biomass, spike number, and kernel weight. Harvest index was highest (0.49 ± 0.04) after treatment with OMW from the repeated-batch process.

Continuous production of l-glutaminase by an immobilized marine Pseudomonas sp BTMS-51 in a packed bed reactor

A marine Pseudomonas sp BTMS-51, immobilized by Ca-alginate gel entrapment was used for the production of extracellular l-glutaminase under repeated batch process and continuous process employing a packed bed reactor (PBR). Immobilized cells could produce an average of 25 U/ml of enzyme over 20 cycles of repeated batch operation and did not show any decline in production upon reuse. The enzyme yield correlated well with the biomass content in the beads. Continuous production of the enzyme in PBR was studied at different substrate concentrations and dilution rates. In general, the volumetric productivity increased with increased dilution rate and substrate concentrations and the substrate conversion efficiency declined. The PBR operated under conditions giving maximal substrate conversion efficiency gave an average yield of 21.07 U/ml and an average productivity of 13.49 U/ml/h. The system could be operated for 120 h without any decline in productivity.

Enhanced separation of filamentous fungi by ultrasonic field: possible usage in repeated batch processes

Usage of ultrasonic field-based filters in retention of filamentous fungal cells was assessed using Rhizopus arrhizus NRRL 1526 as a model organism. Effects of operating conditions, such as power input, harvest pump flow rate, run time and stop time, on the system's separation efficiency (SE) were evaluated by modulating the variables according to a Central Composite Design (CCD). The standard pump with which the ultrasonic filter was equipped was shown to be unsuitable and was, therefore, substituted for with a prime rate reverse pump that made possible separation and recycle of the mycelial biomass. The operating conditions were optimised (run time, 300 s; stop time, 3 s; power input, 6 W; harvest pump flow rate, 4 l per day) and a repeated batch process (three batches for a total of 192 h) was performed during which the SE was maintained always higher than 88%.

Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion.

On inflamed endothelium the cell surface protein E-selectin isexpressed which supports the initial process of attachment -capturing and rolling of leukocytes. A recombinant CHO cell linesecreting a soluble E-selectin-IgG chimera was cultivated competitively under serum free conditions in three different bioreactor systems: a 1 l Super-Spinner, a 2 l stirred tank bioreactor equipped with a spinfilter, and a 100 l stirred tankbioreactor. In the smallest system 25.4 mg E-selectin-IgG wereproduced in 62 days using a repeated batch process whileachieving a maximal viable cell density of 3.7 x 10(6) cells ml(-1). Using continuous perfusion mode a total amount of35.2 mg were produced with a maximal viable cell density of1.65 x 10(7) cells ml(-1) in the 2 l bioreactor within 29 days. Large scale cultivation in a 100 l stirred tankbioreactor yielded 105.6 mg in three batches with a maximal viable cell density of 9.7 x 10(5) cells ml(-1) within 15 days. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatants were concentrated via ultra filtration. Purificationwas performed by affinity chromatography with rProtein A. Integrity of the E-selectin-IgG protein was checked with SDS PAGE. Its activity was verified in a cellular adhesion assay performed with HL-60 cells and a recombinant CHO cell line expressing membrane-anchored E-selectin constitutively, and E-selectin expressing HUVECs, respectively. Soluble E-selectin-IgG was used to block adhesion to these cell layerscompetitively. A concentation of 18.8 and 37.5 mug ml(-1)was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively.

Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H2O2, decomposed by catalase to O2 and water. The highest gluconic acid productivity of 8.2 g l−1 h−1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O2 at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.

Repeated-batch production of pigments by immobilised Monascus purpureus

Extracellular pigment production by immobilised Monascus purpureus C322 has been studied in repeated-batch processes using different immobilising carriers such as Ca-alginate, polyurethane sponge, active carbon and pearlite. With Ca-alginate, pigment production was maximum (30.5 UA 470 as process mean production, three batches) while the cell leakage was negligible (0.4 g l −1 free biomass) and the bead mechanical stability good; with this carrier, an extended repeated-batch fermentation (nine batches, 55 days) was carried out: the process pigment productivity was 3.87 UA 470 day −1.

Onion alcohol production by repeated batch process using a flocculating yeast

Alcohol production from worthless onions by a repeated batch process using a flocculating yeast was investigated. The juice from the cultivar red onion R-3, which contained 67.3 g/l of total sugar, was smoothly fermented by a repeated batch operation using the flocculating yeast IR-2. The productivity, the final ethanol concentration and the ethanol yield were about 6.5 g/l/h, 30.6 g/l and 91.9%, respectively. The onion alcohol produced could be used as a favorable source of onion vinegar or alcoholic beverage.

Effective onion vinegar production by a two-step fermentation system

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/ l total sugar, was smoothly converted to onion alcohol containing 30.6 g/ l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/ l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/ l/h and 37.9 g/ l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.

Immobilization of Paenibacillusmacerans NRRL B-3186 cyclodextrin glucosyltransferase and properties of the immobilized enzyme

Cyclodextrin glucosyltransferase from Paenibacillus macerans NRRL B-3186 was immobilized on aminated polyvinylchloride (PVC) by covalent binding with a bifunctional agent (glutaraldehyde). The immobilized activity was affected by the length of the hydrocarbon chain attached to the PVC matrix, the amount of the protein loaded on the PVC carrier, and glutaraldehyde concentration. The activity of the immobilized enzyme was 121 units/gram carrier, the specific activity calculated on bound protein basis was 48% of the soluble enzyme. Compared to the free enzyme, the immobilized form exhibited: a higher optimal reaction temperature and energy of activation, a higher K m (Michaelis constant) and lower V max (maximal reaction rate), improved thermal stability and resistance to chemical denaturation. The operational stability was evaluated in repeated batch process and the immobilized enzyme retained about 85% of the initial catalytic activity after being used for 14 cycles.

Transient growth- and product-formation kinetics of acetic acid bacteria

Rates of growth and product formation under non-stationary conditions were measured in fermentations of industrial acetic acid bacteria. A repeated-batch process, where conditions change rapidly, and a slower shift experiment in CSTR culture were examined. Significant deviations from the steady-state kinetics determined in continuous fermentations were found for cell growth as well as for the formation of acetic acid.

Repeated-batch and continuous production of chitinolytic enzymes by Penicillium janthinellum immobilised on chemically-modified macroporous cellulose

Conidiospores of the fungus Penicillium janthinellum P9 were immobilised on polyurethane sponge and chemically-modified macroporous cellulose and cultivated for the production of chitinolytic enzymes in shaken culture, repeated-batch processes. The highest enzyme production (342 U l −1) was obtained using the macroporous cellulose BR-L01T(70) (Biomaterial Co. Ltd., Japan); with this carrier cell leakage was negligible. The immobilised mycelium of P. janthinellum P9 was then used for repeated-batch and continuous production of chitinolytic enzymes and N-acetyl- β- d-glucosamine in a fluidised-bed reactor (1.0 l working volume). While the repeated-batch cultivation resulted in almost the same enzyme activity (338 U l −1) obtained in shaken culture, with the continuous operation a steady enzyme production of about 450 U l −1 was reached and maintained for 168 h at a feed rate of 0.036 l h −1. At this feed rate, enzyme volumetric productivity and total N-acetyl- β- d-glucosamine production were 14.76 U l −1 h −1 and 15.9 g, respectively. Carrier colonisation and hyphal growth were followed by observations under the scanning electron microscope.