Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

Enhancement of enzymatic production of cyclodextrins by adding polyethylene glycol or polypropylene glycol

Enzymatic production of cyclodextrins (CDs) from soluble starch was studied using either Bacillus macerans or Bacillus ohbensis cyclomaltodextrin glucanotransferase (CGTase). The production yield of CDs was found to be increased up to 1.5–2 times by the addition of low molecular weight polyethylene glycol (PEG 400) or polypropylene glycol (PPG 425) to the reaction medium. Such results were interpreted as being due to a conformational change of the substrate as well as reduction of hydrolytic activity of the enzyme in the presence of these additives.     

不同培养方式对细菌纤维素产量和结构性质的影响

考察了自行筛选的Acetobacter xylinum NUST4.2在静置培养和发酵罐培养获得的细菌纤维素(BC)的产量、基本结构和性能的差异。结果表明:静置培养时产纤维素7.5g/L,产率为0.052g/L/h,在机械搅拌发酵罐中培养3d产量达3.13g/L,产率达0.043g/L/h;SEM分析显示静置培养和发酵罐培养得到的纤维素均具有网状结构,但静置获得的纤维素丝带相互缠绕且层状重叠,更加致密,丝带更细;FT-IR分析知搅拌不改变纤维素的化学结构,但能减弱分子间氢键,和XRD结合分析可知静置培养的纤维素具有更高结晶指数,更高Iα含量和更大晶粒尺寸,但不改变晶型,仍为纤维素I型,说明搅拌会干扰纤维素初始纤丝的结晶,有利于形成更小的晶粒和较Iα稳定的Iβ。与棉纤维素相比,静置培养获得的纤维素的热稳定性更好,而发酵罐培养获得的纤维素则阻燃性更好。     

Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1

A newly isolated strain of Kluyveromyces marxianus YS-1 was used for the production of extra cellular inulinase in a medium containing inulin, meat extract, CaCl2 and sodium dodecyl sulphate (SDS). Fermentation medium pH 6.5, cultivation temperature 30 degrees C and 5% (v/v) inoculum of 12 h-old culture were optimal for enzyme production (30.8 IU/ml) with a fermentation time of 72 h at shake flask level. Raw inulin (2%, w/v) extracted from dahlia tubers by processing at 15 kg/cm2 for 10 min was optimum for bioreactor studies. Maximum enzyme production (55.4 IU/ml) was obtained at an agitation rate of 200 rpm and aeration of 0.75 vvm in a stirred tank reactor with a fermentation time of 60 h.     

Schizophyllan production by newly isolated fungus <Emphasis Type="Italic">Schizophyllum commune</Emphasis> IBRC-M 30213: optimization of culture medium using response surface methodology

Schizophyllan (SPG) is a commercially attractive biopolymer produced by Schizophyllum commune. An investigation on the potential for SPG production by Iranian native S. commune was conducted based on culture medium, fermentation conditions and bioreactor type, . Nine native fungal strains were isolated from the northern forest of Iran at different times. Based on growth rate and SPG production, one strain was selected for further study. Optimal medium composition and inoculum size for maximizing SPG production and minimizing biomass were determined using central composite design by setting sucrose, yeast extract, inoculum size, carboxymethyl cellulose and oleic acid in the ranges of 50–200 g/L, 1–4 g/L, 2–10%, 2–12 g/L and 0.032–0.222%, respectively. The results showed that optimal results were obtained at 93.47 g/L sucrose, 1.87 g/L yeast extract, 7.68% inoculum size, 9.07 g/L carboxymethyl cellulose and 0.13% oleic acid, with maximum SPG production of 9.97 g/L and minimum biomass of 35.18 g/L. Under these optimal conditions, the production of SPG was studied in stirred tank and bubble column bioreactors. The results revealed greater production in the stirred tank because of better mixing of the culture medium. The SPG produced was characterized using rheometery, Fourier transform infrared spectroscopy, nuclear magnetic resonance), scanning electron microscopy and gel permeation chromatography. The results of these characterizations demonstrated the similarity of the SPG produced by S. commune IBRC-M 30213 to commercial SPG. Thus, the SPG produced shows good potential as a polysaccharide for use in various industries.     

Repeated hybridoma batch culture with cell recycle

At the end of a hybridoma batch culture, the cells are usually discarded after separation from the culture broth. If, however, they are aseptically recycled into the reactor, the production process can be resumed simply by the addition of fresh medium. This cycle can then be repeated several times consecutively. In a test case, with a mouse hybridoma, we found antibody yields for each cycle in the same range as for a standard batch. In a 15 1 stirred tank reactor we could, within 6 days, produce 2.8 g of monoclonal antibody (MAb). This type of reactor operation allowed a doubling in the reactor volumetric productivity (mg/l/day).     

Comparison of manufacturing techniques for adenovirus production

We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers. The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and 7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell. Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced lignin peroxidase synthesis by Phanerochaete Chrysosporium in solid state bioprocessing of a lignocellulosic substrate

Summary A solid state fermentation (SSF) process for the production of lignin peroxidase was optimized to enhance enzyme production by Phanerochaete chrysosporium. Optimization of the corncob SSF medium caused a significant reduction in fermentation time to give maximum lignin peroxidase yield. Supplementation of the SSF medium by low concentrations of peptone, yeast extract and Tween-80 enhanced lignin peroxidase production. Maximum yield of lignin peroxidase was 13.7 U/gds (units per gram dry substrate) noted after 5 days of SSF with 70% moisture and 20% (v/w) inoculum.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

Effect of pH on exocellular riboflavin production byEremothecium ashbyii

Summary The production of riboflavin byEremothecium ashbyii grown in a chemically defined medium in batch culture was affected by pH of the medium. Highest yields were obtained at constant pH of 4.5 and 5.5, while little or no riboflavin was detected at either pH 3.5 or 8.5. The medium pH also affected cell morphology. When the organism was grown in a stirred tank reactor and an airlift vessel at pH 4.5 very similar levels of riboflavin were obtained.     

Effect of growth conditions on the production of manganese peroxidase by three strains of Bjerkandera adusta

We were looking for a strain of Bjerkandera adusta that produces high titres of manganese peroxidase under optimal conditions for large-scale enzyme purification. We have chosen two strains from the University of Alberta Microfungus Collection and Herbarium, UAMH 7308 and 8258, and compared the effects of growth conditions and medium composition on enzyme production with the well-characterized strain BOS55 (ATCC 90940). Of four types of cereal bran examined, rice bran at 3% (w/v) in 60 mM phosphate buffer pH 6 supported the highest levels of enzyme production. Using 100 mL medium in 500-mL Erlenmeyer flasks, maximum enzyme levels in the culture supernatant occurred after about 10 days of growth; 5.5 U x mL(-1) for UAMH 7308, 4.4 U x mL(-1) for UAMH 8258, and 1.7 U x mL(-1) for BOS55, where units are expressed as micromoles of Mn-malonate formed per minute. Growth as submerged cultures in 10-L stirred tank reactors produced 3.5 U x mL(-1) of manganese peroxidase (MnP) by UAMH 8258 and 2.5 U x mL(-1) of MnP by 7308, while enzyme production by BOS55 was not successful in stirred tank reactors but could be scaled up in 2-L shake flasks containing 400 mL rice bran or glucose-malt-yeast extract (GMY)-Mn-glycolate medium to produce MnP levels of 1.7 U x mL(-1). These results show that the two strains of B. adusta, UAMH 7308 and 8258, can produce between two and three times the manganese peroxidase level of B. adusta BOS55, that they are good candidates for scale up of enzyme production, and that the rice bran medium supports higher levels of enzyme production than most previously described media.     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

Production of laccases in submerged process by <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis> PS-2001 in relation to carbon and organic nitrogen sources,antifoams and Tween 80

Some conditions in media composition for laccases production, such as different sources of carbon and organic nitrogen, antifoams and a surfactant, were studied in liquid cultures of Pleurotus sajor-caju strain PS-2001. Cultivation with fructose or glucose as carbon sources produced maximum enzyme activities of 37 and 36 U mL⁻¹, respectively. When sucrose was present in the medium, the best results were obtained using 5 g L⁻¹ of this carbohydrate, on the 11th day of the process, attaining laccase titres of 13 U mL⁻¹. In a medium without casein, practically no enzyme was produced during the experiments; among the sources of nitrogen studied, pure casein led to the highest titres of laccase activity. Different concentrations of pure casein and sucrose were also tested. As to the different concentrations of casein, the addition of 1.5 g L⁻¹ resulted in the highest titres of laccase activity. Negligible levels of manganese peroxidase activity were also detected in the culture medium. In low concentrations, polypropylene glycol or silicon-based antifoams and the surfactant Tween 80 have no significant influence on the formation of laccases by P. sajor-caju. However, enhanced concentration of polypropylene glycol negatively affected the production of laccases but favored the titres in total peroxidases, lignin peroxidase and veratryl alcohol oxidase.     

Use of diluted medium in repeated-batch fermentation for production of lignin peroxidase by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Lignin peroxidase has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by the white rot fungus, Phanerochaete chrysosporium, include the improvement of yield results and cell maintenance. In the present work, Phanerochaete chrysosporium was immobilized in polyurethane foam and used for repeated-batch fermentations with various dilution of the initial medium (D), and lignin peroxidase production was investigated. The peak of 283 ± 17.5 U lignin peroxidase/l production rate was obtained at a D of 1/5, with significantly lower production rates seen at higher and lower dilution ratios. When six cycles of repeated-batch fermentation were conducted using a D of 1/5, the results revealed that at least four cycles of repeated-batch fermentation were possible with a high lignin peroxidase production rate under a cut-off value of 178 ± 3.87 U/l. Furthermore, the cell-free culture broth could be successfully concentrated to 2,800 U/l by ultrafiltration. Thus, the present study shows that optimizing the dilution of the utilized nutritional medium can improve repeated batch production of lignin peroxidase from immobilized P. chrysosporium, in terms of both cycle number and output.     

Separation and retention of human blood cells by surface-affinity chromatography.

This review describes the surface-affinity chromatography of human peripheral blood cells on polyethylene glycol (PEG) and polypropylene glycol (PPG) chemically bonded columns. The affinity of surface blood cells to bonded PEG and PPG stationary phases is apparently selective, and granulocytes and lymphocytes are more strongly retained on the column than erythrocytes and platelets. The retention factors of granulocytes and lymphocytes increased with increase in the hydrophobicities (Delta log K values) of PPG-agarose column packing beads. Hydrophobic interactions contributed to the retention of the blood cells on the PPG-bonded agarose columns.     

Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.     

Two-stage batch process for the production of ajmalicine by Catharanthus roseus: The link between growth and production stage

The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. (c) 1995 John Wiley & Sons, Inc.     

Inhibition of fungal exopolysaccharide production by chemical antifoams

Attempts to control foam production in exopolysaccharide synthesising cultures of Acremonium persicinum in aerobic fermentation with polypropylene glycol (PPG) 2025 led to a marked reduction in yield. Similar effects were seen with polysaccharide producing cultures of Epicoccum purpurascens , but not with Aureobasidium pullulans. The effects of other chemical antifoams on polysaccharide production in A. persicinum were examined, and no inhibition was noticed with any silicone-based compound.     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.