Continuous production of manganese peroxidase by Phanerochaete chrysosporium immobilized on polyurethane foam in a pulsed packed-bed bioreactor

The bottleneck of the application of manganese peroxidase (MnP) on an industrial scale in pulp biobleaching or in degradation of hazardous compounds is the lack of an efficient production system. Three main problems arise for the continuous production of MnP during secondary metabolism of Phanerochaete chrysosporium: enzyme production occurs only under specific physiological conditions corresponding to C or N limitation, high O(2) tension, and adequate Mn(+2) concentration; the enzyme that is produced is destabilized by extracellular proteases; and excessive growth of the mycelium blocks effective oxygen transfer. To overcome these drawbacks, continuous production of MnP was optimized by selecting a suitable bioreactor configuration and the environmental and operating conditions affecting both enzyme production and stability. The combination between a proper feed rate and the application of a pulsation in a packed-bed bioreactor permitted the maintenance of continuous secretion of MnP while limiting mycelial growth and avoiding bed clogging. Environmental factors as an Mn(+2) concentration of 5000 muM and high oxygen tension enhanced MnP production. The hydraulics of the bioreactor corresponding to a plug flow model with partial mixing and an operating hydraulic rentention time of 24 h were optimal to achieve stable operating conditions. This policy allowed long operation periods, obtaining higher productivities than the best reported in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 130-137, 1997.     

Efficient cellulase production by the filamentous fungus Acremonium cellulolyticus

Cellulase production was investigated in a culture of a strain of Acremonium cellulolyticus. The medium components were optimized for the improvement of cellulase production. The maximum production of cellulolytic enzymes was obtained in a medium containing (grams per liter) 50 Solka Floc, 5 (NH4)2SO4, 24 KH2PO4, 4.7 potassium tartrate hemihydrate, 1.2 MgSO4.7H2O, 1 Tween 80, 4 urea, 0.01 ZnSO4.7H2O, 0.01 MnSO4.6H2O, and 0.01 CuSO4.7H2O, with a pH of 4.0. In the flask culture, 15.5 filter paper units (FPU)/mL of maximum cellulase activity was obtained, 17.42 FPU/mL in a 7-L bioreactor, and 13.08 FPU/mL in a 50-L scale bioreactor for 4-8 d at 30 degrees C. Average production rates were 1.94 FPU/mL.d in flasks, 2.86 FPU/mL.d in the 7-L bioreactor, and 2.56 FPU/mL.d in the 50-L bioreactor. Cellulase production on a small scale was successfully reproduced in the 50-L pilot scale bioreactor. Saccharification activity from A. cellulolyticus was compared with cellulolytic enzymes produced by other strains. The A. cellulolyticus culture broth had a comparable saccharification yield in comparison with those of other Trichoderma enzymes (GC220 or Cellulosin T2) under the same total cellulase activity. Its saccharification yield (percent of released reducing sugar to used dried substrate) was 60%, and its glucose content was 83%.     

Production of manganese-dependent peroxidase in a new solid-state bioreactor by Phanerochaete chrysosporium grown on wood shavings. Application to the decolorization of synthetic dyes

The production of manganese-dependent peroxidase (MnP) byPhanerochœte chrysosporium in a new solid-state bioreactor, the immersion bioreactor, operating with lignocellulosic waste, such as wood shavings, was investigated. Maximum MnP and lignin peroxidase (LiP) activity of 13.4 and 8.48 μkat/L were obtained, respectively. Thein vitro decolorization of several synthetic dyes by the extracellular liquid produced in the above-mentioned bioreactor (containing mainly MnP) was carried out and its degrading ability was assessed. The highest decolorization was reached with Indigo Carmine (98%) followed by Bromophenol Blue (56%) and Methyl Orange (36%), whereas Gentian Violet was hardly decolorized (6%).     

Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.     

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.     

Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture

This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.     

An Integrated Control Strategy for the Fermentation of the Marine-Derived Fungus Aspergillus glaucus for the Production of Anti-cancer Polyketide

An integrated control strategy of pH, shear stress, and dissolved oxygen tension (DOT) for fermentation scale-up of the marine-derived fungus Aspergillus glaucus HB 1–19 for the production of the anti-cancer compound aspergiolide A was studied. Keeping initial pH of 6.5 and shifting pH from 6.0 to 7.0 intermittently during the production phase greatly facilitated biosynthesis of aspergiolide A in shake flask cultures. Thus, a pH-shift strategy was proposed that shifting pH to 7.0 once it went lower than 6.0 by pulsed feeding NaOH solution during the production phase in bioreactor fermentation of A. glaucus HB 1–19. As a result, aspergiolide A production in a 30-L bioreactor was increased to 37.6?mg/L, which was 48.6% higher than that in 5-L bioreactor without pH shift. Fermentation scale-up was then performed in a 500-L bioreactor on the basis of an integrated criterion of near-same impeller tip velocity of early phase, DOT levels, and pH shift. The production of aspergiolide A was successfully obtained as 32.0?mg/L, which was well maintained during the process scale-up. This work offers useful information for process development of large-scale production of marine microbial metabolites.     

Manganese peroxidase production by immobilized Phanerochaete chrysosporium BKM-F-1767 in a cell bioreactor

Manganese-dependent peroxidase (MnP) production was performed in an immobilized cell bioreactor in which Phanerochaete chrysosporium BKM-F-1767 was immobilized on polystyrene foam. The immobilized cell culture yielded significantly greater MnP activity than the conventional stationary liquid culture. Cultivation was carried out in batch mode; the effect of glucose concentration was investigated and growth kinetics parameters were found as, micromax=0.59 day(-1), Ks=0.33 g/L and Kss=14.5. Batch operation led to maximum MnP (770.82 U/L) in the culture medium containing 0.05% Tween 80, 10 g/L glucose, and 174 microM Mn2+ at 37 degrees C and pH 4.5. Enzyme productivity was obtained as 110.12 U/day/L.     

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 μg/mL compared with ca. 118 and 990 μg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.     

Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used high purity oxygen supplying strategy to increase viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7 was utilized in this study as a host strain in both 5-L and 30-L scale fermentors. To supply high purity oxygen into a bioreactor, nearly 100 % high purity oxygen from commercial bomb or higher than 93 % oxygen available in-situ from a pressure swing adsorption oxygen generator (PSA) was employed. Under the optimal fermentation of H. polymorpha with high purity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/L and 5.1 g/L in the 5-L fermentor, and 24.8 g/L and 4.5 g/L in the 30-L fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-L and 30-L fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-L fermentor. This study, therefore, proved the positive effect of high purity oxygen to enhance viable cell density as well as target recombinant protein production in microbial fermentations.     

Unstructured models for suspension cultures of Taxus media cells in a bioreactor under substrate-sufficient conditions

For a better understanding of the simulation, optimization, and process control in cell cultures, good kinetic models are necessary for large scale plant cell culture. In this paper, the systematic kinetics of taxol production by Taxus media cell suspension cultures in a stirred 15-L bioreactor under substrate-sufficient conditions and the absence of inducer intervention were studied. A kinetic model of cell growth was established by logistic equation, and kinetic unstructured models of substrate consumption, product synthesis and rheological behavior were constituted, which incorporated energy spilling. These models were verified by comparing the simulation results with those obtained experimentally. These results showed that energy spilling was a key factor that must be considered in constructing unstructured kinetic models of Taxus media cell suspension cultures in a stirred bioreactor under substrate-sufficient conditions. Besides, an optimized operation measure of decreasing energy spilling was proposed. An increase of 17.64% in cell biomass and 14.88% in taxol concentration were obtained when the strategy of limiting added carbon several times was experimentally implemented in a 15-L bioreactor. Results demonstrated that these established models should be helpful in the process prediction and operation optimization to guide the production and amplification of Taxus media cell suspension cultures in a bioreactor.     

Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods

Polysaccharides and ganoderic acids (GAs) are the major bioactive constituents of Ganoderma species. However, the commercialization of their production was limited by low yield in the submerged culture of Ganoderma despite improvement made in recent years. In this work, twelve Ganoderma strains were screened to efficiently produce polysaccharides and GAs, and Ganoderma lucidum 5.26 (GL 5.26) that had been never reported in fermentation process was found to be most efficient among the tested stains. Then, the fermentation medium was optimized for GL 5.26 by statistical method. Firstly, glucose and yeast extract were found to be the optimum carbon source and nitrogen source according to the single-factor tests. Ferric sulfate was found to have significant effect on GL 5.26 biomass production according to the results of Plackett–Burman design. The concentrations of glucose, yeast extract and ferric sulfate were further optimized by response surface methodology. The optimum medium composition was 55 g/L of glucose, 14 g/L of yeast extract, 0.3 g/L of ferric acid, with other medium components unchanged. The optimized medium was testified in the 10-L bioreactor, and the production of biomass, IPS, total GAs and GA-T enhanced by 85, 27, 49 and 93 %, respectively, compared to the initial medium. The fermentation process was scaled up to 300-L bioreactor; it showed good IPS (3.6 g/L) and GAs (670 mg/L) production. The biomass was 23.9 g/L in 300-L bioreactor, which was the highest biomass production in pilot scale. According to this study, the strain GL 5.26 showed good fermentation property by optimizing the medium. It might be a candidate industrial strain by further process optimization and scale-up study.     

Manganese Peroxidase production by Bjerkandera sp. BOS55

The use of ligninolytic enzymes in biotechnological applications requires a highly effective production system, with sufficient amounts of the enzymes to be applied in experimental research and herein after at large-scale operations. To reach this final goal, we propose scale-up of ligninolytic production of one of the most well-known enzymes, Manganese Peroxidase (MnP), by Bjerkandera sp. BOS55. Taking into account previous results obtained in shaken flask cultures, MnP production was attempted in stirred fermenters of 2, 10 and 50 l, with levels of activity comparable to those obtained at a lower scale. Additionally, environmental factors as agitation rate, fungus immobilisation and use of buffer were evaluated to maximise MnP production. A fed-batch strategy was proved to reactivate MnP production and to maintain MnP activity for a longer period of time. Operational parameters, such as pH and Redox potential, monitored along the fermentation were found to be useful indicators of MnP production. These variables experimented drastic changes at the MnP peak production, signalling the right moment to collect the enzyme.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

Identifying conditions for inducible protein production in E. coli: combining a fed-batch and multiple induction approach

Background  

In the interest of generating large amounts of recombinant protein, inducible systems have been studied to maximize both the growth of the culture and the production of foreign proteins. Even though thermo-inducible systems were developed in the late 1970's, the number of studies that focus on strategies for the implementation at bioreactor scale is limited. In this work, the bacteriophage lambda P_L promoter is once again investigated as an inducible element but for the production of green fluorescent protein (GFP). Culture temperature, induction point, induction duration and number of inductions were considered as factors to maximize GFP production in a 20-L bioreactor.     

A packed-bed fungal bioreactor for the continuous decolourisation of azo-dyes (Orange II)

The degradation of an azo dye, Orange II, by immobilised Phanerochaete chrysosporium in a continuous packed bed bioreactor for periods longer than 30 days has been carried out. Nearly complete decolourisation (>95%) was achieved when working at a high dye load rate of 0.2 g l⁻¹ d⁻¹, a temperature of 37 °C, a hydraulic retention time (HRT) of 24 h and applying oxygen gas in a pulsed flow. These conditions allowed Manganese peroxidase (MnP) production and the subsequently Orange II decolourisation. A correlation between residual MnP activity in the effluent and decolourisation was established. Apparently, for decolourisation to be effective, a minimum MnP activity was required, no substantial increase in efficiency at MnP activities higher than 10 U 1 ⁻¹ was observed. The treatment caused, the breakdown of the chromophoric group as well as the cleavage of the aromatic ring.     

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

Optimization of manganese peroxidase production by the white rot fungus Bjerkandera sp. strain BOS55

Manganese dependent peroxidase (MnP) is the most ubiquitous peroxidase produced by white rot fungi. MnP is known to be involved in lignin degradation, biobleaching and in the oxidation of hazardous organopollutants. Bjerkandera sp. strain BOS55 is a nitrogen-unregulated white rot fungus which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. Therefore, the strain is a good candidate for use in large scale production of this enzyme. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, incubation temperature and the addition of organic acids. The fungus produced the highest level of MnP (up to 900 U 1⁻¹) when the Mn concentration was 0.2 to 1 mM, the pH value was 5.2, and the incubation temperature was 30°C. A noteworthy finding was that MnP was also produced at lower levels in the complete absence of Mn. The addition of organic acids like glycolate, malonate, glucuronate, gluconate, 2-hydroxybutyrate to the culture medium increased the peak titres of MnP up to 1250 U 1⁻¹. FPLC profiles indicated that the organic acids stimulated the production of all MnP isoenzymes present in the extracellular fluid of the fungus.     

Hyperproduction of cordycepin by two-stage dissolved oxygen control in submerged cultivation of medicinal mushroom Cordyceps militaris in bioreactors

Effect of oxygen supply on cordycepin production was investigated in submerged cultivation of Cordyceps militaris, a famous traditional Chinese medicinal mushroom, in a 5-L turbine-agitated bioreactor (TAB). Initial volumetric oxygen transfer coefficient (kLa) within the range of 11.5-113.8 h(-1) had significant influence on cordycepin production. The highest cordycepin concentration of 167.5 mg/L was obtained at an initial kLa value of 54.5 h(-1), where a moderate dissolved oxygen (DO) pattern was observed throughout cultivation. The possible correlation between cordycepin production and DO level was explored by DO control experiments, and the results showed that DO within the range of 10-80% of air saturation greatly affected the cultivation process. To obtain a high specific cordycepin formation rate (rho) throughout cultivation, a two-stage DO control strategy was developed based on the analysis of the relationship of rho and DO. That is, DO was controlled at 60% from the beginning of cultivation and then shifted to a lower control level of 30% when rho started to decrease. As a result, a high cordycepin production of 201.1 mg/L and a high productivity of 15.5 mg/(L.d) were achieved, which was enhanced by about 15% and 30% compared to the highest titers obtained in conventional DO control experiments, respectively. The proposed DO control strategy was also applied to a recently developed 5-L centrifugal impeller bioreactor (CIB) with cordycepin production and productivity titers of 188.3 mg/L and 14.5 mg/(L.d). Furthermore, the scale-up of the two-stage DO control process from 5-L CIB to 30-L CIB was successfully demonstrated. The work is useful for the efficient large-scale production of bioactive metabolites by mushroom cultures.