Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-collumn bioreactor

Summary Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells, Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads. The effect of various aeration rates in a column bioreactor has been investigated. Enzyme production by IC [42 000 units (U)·l^–1] during batch fermentations was comparable to that of a free-cell system. The specific productivity of IC was 8.5 times higher than that of free cells. In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production. Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.0 h^–1 with volumetric productivity (450 U·1^–1) and yield.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Semicontinuous production of red pigment by immobilized cells of<Emphasis Type="Italic">Bacillus</Emphasis> sp. BH-99 using column bioreactor

The semicontinuous production of red pigment by immobilized cells ofBacillus sp. BH-99 was investigated in comparison with free cells. The red pigment produced highest productivity under the conditions of aeration of 0.2 mL/min and 2 mm diameter of gel beads by using 3.0% sodium alginate. Semicontinuous production by immobilized cells showed the highest productivity with replacement of fresh production medium in every 72 h for fourth fermentation cycle following the conditions of red pigment productivity.     

High density cultivation of Anchusa officinalis in a stirred-tank bioreactor with in situ filtration

Previously, Su et al. [Biotechnol Bioeng 42: 884–890 (1993)] reported improved production of rosmarinic acid by Anchusa officinalis in shake-flask cultures using a cultivation strategy that involved intermittent medium exchange. Implementation of this cultivation strategy in 2.5-1 stirred-tank bioreactor cultures is investigated in the present study. Intermittent cell/medium separation in the bioreactor was accomplished by means of automated in situ culture filtration. In the bioreactor culture, rosmarinic acid production was found very sensitive to agitation and aeration conditions as well as dissolved oxygen concentration. A maximum cell density of 35 g dry weight/l and a rosmarinic acid concentration of 3.7 g/l were obtained by maintaining the dissolved oxygen concentration above 30% air saturation, gradually raising the impeller tip speed from 34 cm/s to 72 cm/s, and keeping the aeration rate at 0.44 vvm while increasing the O₂: air ratio in the gas feed stream to 4:1. This result is comparable with the data obtained from shake-flask cultures using the same culture strategy.     

Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor

Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads. Offprint requests to: M. Petruccioli     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Effects of immobilization by entrapment in alginate and scale-up on paclitaxel and baccatin III production in cell suspension cultures of Taxus baccata

Paclitaxel and baccatin III-producing cells of Taxus baccata were immobilized within Ca(2+)-alginate beads. Under established optimum conditions for the biosynthesis of both taxanes, the yields of paclitaxel and baccatin III in shake-flask cultures of free cells increased by factors of up to 3 and 2, respectively, in the corresponding cultures of immobilized cells. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities when both free and immobilized cells were grown in the same optimum conditions in three different bioreactor types (Stirred, Airlift, and Wave) running for 24 days in a batch mode and with the system optimized in each case, there was a considerable increase in the yields of paclitaxel and baccatin III. Among the reactors, the Stirred bioreactor was the most efficient in promoting immobilized cell production of paclitaxel, giving a content of 43.43 mg.L(-1) at 16 days of culture, equivalent to a rate of 2.71 mg.L(-1).day(-1). To our knowledge, the paclitaxel productivity obtained in this study is one of the highest reported so far by academic laboratories for Taxus species cultures in bioreactors.     

Improvement of <Emphasis Type="SmallCaps">d</Emphasis>-lactate productivity in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> by coupling production with growth

Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O₂-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O₂-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli.     

Investigations on neomycin production with immobilized cells of<Emphasis Type="Italic">Streptomyces marinensis</Emphasis> Nuv-5 in calcium alginate matrix

The purpose of this investigation was to study the effect ofStreptomyces marinensis NUV-5 cells immobilized in calcium alginate for the production of neomycin. The effect of various parameters, such as the effect of alginate concentration (1%, 2%, 3%, 4%, and 5% wt/vol), the effect of cation (caCl₂, BaCl₂, and SrCl₂), the concentration of cation (0.01M, 0.125M, 0.25M, 0.375M, and 0.5M), the curing times (1, 6, 11, 16, and 21 hours), and the diameter of the bead (1.48, 2.16, 3.24, 4.46, and 5.44 mm), on neomycin production and bead stability were studied. The effect of maltose (4%, 3%, 2%, and 1% wt/vol) and sodium glutamate (0.6%, 0.3%, 0.15%, and 0.075%) wt/vol) concentration on neomycin production was also studied. Better neomycin production was achieved with optimized parameters, such as alginate at 2% wt/vol, 0.25M CaCl₂, 1-hour curing time, and 3.24 mm bead diameter. Effective neomycin production was achieved with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate concentration. The repeated batch fermentations were conducted (every 96 hours) using the optimized alginate beads, employing the production medium with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate along with minerals salts solution. The increase in antibiotic production was observed up to the 5th cycle, and later gradual decrease in antibiotic production was observed. Comparison of the total antibiotic production with free cells and immobilized cells was also done. An enhanced antibiotic productivity of 32% was achieved with immobilized cells over the conventional free-cell fermentation, while 108% more productivity was achieved over the washed free-cell fermentation. From these results it is concluded that the immobilized cells ofS marinensis NUV-5 in calcium alginate are more efficient for the production of neomycin with repeated batch fermentation.     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Decolorization of Direct Red 28 by mixed bacterial culture in an up-flow immobilized bioreactor

Aerobic mixed bacterial culture comprised of five isolates (Bacillus vallismortis, B. pumilus, B. cereus, B. subtilis and B. megaterium) identified by 16srDNA analysis was developed from wastewater samples from the aeration tank of an effluent treatment plant of a textile and dyeing industry and evaluated for its ability to decolorize azo dye Direct Red 28 in an up-flow immobilized packed bed bioreactor using marble chips as support matrix. The bioreactor was operated under two parameters: an aeration rate of 0.4 and 0.6 mmol/min at a flow rate of 60, 90 and 120 ml/h, respectively. At a constant aeration rate of 0.4 mmol/min and with flow rates of 60, 90 and 120 ml/h, optimum decolorization of 91, 75 and 72% was observed, while at an aeration rate of 0.6 mmol/min and flow rates of 60, 90 and 120 ml/h, optimum decolorization of 93, 78 and 72% was observed over 10 days. The study concluded that across the two aeration rates and the respective flow rates, the higher aeration rate of 0.6 mmol/min along with a flow rate of 60 ml/h was best suited to decolorize Direct Red 28 in the packed bed bioreactor. Spectral changes of the input and output of the bioreactor by UV–visible spectroscopy indicated decolorization of the dye solution by degradation in addition to the visual observation of the biosorption process.     

Continuous production of bacitracin by immobilized living whole cells of Bacillus sp.

Whole cells of Bacillus sp., a bacitracin-producing bacteria, were immobilized in polyacrylamide gel. The continuous production of bacitracin by an immobilized whole-cell-containing air-bubbled reactor was examined with 0.5% peptone solution. The bacitracin productivity (28 units/ml/hr) obtained with this system was higher than that with a batch system. The effluent bacitracin concentration increased with increasing aeration rate and reached a steady-state maximum above the aeration rate of 3.0 liter/min. A high bacitracin productivity was retained for at least eight days when the gel was washed with sterilized saline at a flow rate of 250 ml/hr for 2 hr once a day. The half-life of the immobilized whole-cell system was about 10 days. Bacitracin productivity by the immobilized whole-cell reactor was higher than that by a conventional continuous fermentation process at high dilution rates.     

A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - SSPM Selby Sitka proliferation medium - SSMM Selby Sitka maturation medium The following was presented at the NERC TBLG '95 Meeting as the Bioreactor Workshop     

Beneficial effects of enhanced aeration using perfluorodecalin in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cultures for lipase production

The inadequate supply of oxygen to biomass is a critical factor to the productivity of most aerobic submerged fermentations. This happens because oxygen is sparingly soluble in the aqueous media. The use of a second liquid phase of perfluorocarbon (PFC), an oxygen-carrying compound, in the culture medium can increase the availability of oxygen to the microorganisms. The effect of perfluorodecalin on Yarrowia lipolytica cultures was investigated in shake-flask cultures. It was found that the specific growth rate of Y. lipolytica, a strictly aerobic yeast, increases with increasing PFC concentration. Extracellular lipase production was increased with 20% (v/v) of PFC and agitation of 250 rev/min. It was shown that the PFC presence benefitted lipase production and not just its secretion to the extracellular medium.     

High bioreactor production and emulsifying activity of an unusual exopolymer by Chromohalobacter canadensis 28

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2‐L bioreactor at agitation speeds at interval 600‐1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale‐up of bioreactors mass transfer parameters were estimated and lowest values of K_La obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ‐polyglutamic acid (PGA) showed higher viability than control group.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

Effects of oxygen volumetric mass transfer coefficient on transglutaminase production by Bacillus circulans BL32

The effects of oxygenation in cultures of Bacillus circulans BL32 on transglutaminase (TGase) production and cell sporulation were studied by varying the agitation speed and the volume of aeration. Kinetics of cultivations has been studied in batch systems using a 2 L bioreactor, and the efficiency of agitation and aeration was evaluated through the oxygen volumetric mass transfer coefficient (k_La). It was adopted a two-stage aeration rate control strategy: first stage to induce biomass formation, followed by a second stage, in which cell sporulation was stimulated. A correlation of TGase production, spores formation, and oxygen concentration was established. Under the best conditions (500 rpm; 2 vvm air flow, followed by no air supply during stationary phase; k_La of 33.7 h⁻¹), TGase production reached a volumetric production of 589 U/L after 50 h of cultivation and the enzyme yield was 906 U/g cells. These values are 61% higher than that obtained in shaker cultures and TGase productivity increased 82%, when k_La varied from 4.4 to 33.7 h⁻¹. The maximal cell concentration increased four times in relation to shaker cultures and the cultivation time for the highest TGase activity was reduced from 192 h to just 50 h. These results show the importance of bioprocess design for the production of microbial TGase, especially concerning the oxygen supply of cultures and the induction of cell sporulation.     

Immobilization of 293 cells using porous support particles for adenovirus vector production

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10⁷ cells cm⁻³-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.