Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines

A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads. The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design. Individually, none of the variables tested had a significant affect upon productivity. The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested. This combination of variable levels suggested that better results shold be obtained using a fluidised bed reactor system. However, comparison of the productivities of the two systems showed that the FBR gave the better results. This result can be explained in terms of the relationship of Qs_rAb to .Abbreviations C concentration - D dilution rate - FBR fixed bed bioreactor - FIBR fluidised bed bioreactor - Gln glutamine - Qs cell specific rate - Qv volumetric rate - rAb recombinant antibody - X_v viable cell density - specific growth rate     

A Novel Oscillating Bioreactor BelloCell: Implications for Insect Cell Culture and Recombinant Protein Production

BelloCell is a novel packed bed bioreactor that allows alternating nutrient and gas transfer to a culture. Spodoptera frugiperda Sf-9 grown in the BelloCell (300 ml culture) reached 1.3–1.5×10⁷ cells ml⁻¹ in 7–8 days and the total baculovirus-expressed protein yield was 2.3-times that in a stirred tank bioreactor (600 ml culture). The superior cell and protein yields underline the potential of BelloCell for cell culture and recombinant protein production.     

The influence of variable organic loading on the start-up of an anaerobic fluidised bed reactor

Summary A stepped-loading start-up regime utilising variable organic influent concentrations in the range 1650–11600 mgCOD1^–1 was applied to an anaerobic fluidised bed bioreactor at 37°C. The reactor was sensitive to variable influent COD concentrations, but the stepped-loading aided rapid recovery from transient organic loading shocks. Variable effluent COD levels were produced but a COD removal efficiency of 76% was obtained at a final HRT of 0.5 d and an organic loading rate of 5.3 kg COD m^–3 d^–1.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

A Chinese hamster ovary (CHO) cell line, producing recombinant secreted human placental alkaline phosphatase (SEAP) was investigated under three different culture conditions (suspension cells, cells attached to Cytodex 3 and Cytopore 1 microcarriers) in a biphasic culture mode using a temperature shift to mild hypothermic conditions (33 °C) in a fed-batch bioreactor. The cell viability in both the suspension and the Cytodex 3 cultures was maintained for significantly longer periods under hypothermic conditions than in the single-temperature cultures, leading to higher integrated viable cell densities. For all culture conditions, the specific productivity of SEAP increased after the temperature reduction; the specific productivities of the microcarrier cultures increased approximately threefold while the specific productivity of the suspension culture increased nearly eightfold. The glucose and glutamine consumption rates and lactate and ammonia production rates were significantly lowered after the temperature reduction, as were the yields of lactate from glucose. However, the yield of ammonia from glutamine increased in response to the temperature shift.     

Laboratory-scale evaluation of fluidized bed reactor technology for biotreatment of maleic anhydride process wastewater

Fluidized bed reactor (FBR) technology has emerged in recent years as an attractive approach for the biotreatment of chemical industry wastestreams. A laboratory-scale FBR study was conducted to investigate the feasibility of utilizing FBR technology for the biotreatment of maleic anhydride wastewater generated during manufacturing operations. The maleic anhydride wastestream contains a mixture of maleic acid, fumaric acid, phthalic acid and di-n-butylphthalate (DBP). The FBR removed >98% of chemical oxygen demand (COD) and total organic carbon (TOC) from the wastewater at a chemical loading rate of 4.86 kg of COD m^–3 bed day^–1. Maleic acid, fumaric acid or phthalic acid were not detected in the FBR effluent indicating removal of these diacids. Residues of DBP adsorbed to granular activated carbon (GAC) stabilized at low levels indicating that the >99% removal efficiency for DBP in the FBR resulted from microbial degradation. Solids measurements showed microbial biomass levels on the GAC ranging from 10500 to 32400 mg L^–1 and effluent solids production ranged from 0.027 to 0.041 kg solids kg^–1 COD treated. This laboratory-scale study demonstrated that FBR technology was highly effective for the biotreatment of the maleic anhydride wastestream and may offer several advantages over traditional activated sludge systems.     

Stimulation of murine granulocyte macrophage-colony stimulating factor production by Pluronic F-68 and polyethylene glycol in transgenic Nicotiana tabacum cell culture

Pluronic F-68, PEG 8000, or PEG 20 000 added to cell suspension cultures of transgenic Nicotiana tabacum promoted cell growth and the production of the recombinant murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in a 5-l stirred tank bioreactor. The specific growth rates were enhanced from 0.27 d^–1 to 0.47 d^–1, 0.37 d^–1 and 0.4 d^–1 when Pluronic F-68, PEG 8000, or PEG 20 000 was added, respectively. The maximum cell density was also increased most to 13.6 g l^–1 when Pluronic F-68 was added (11.3 g l^–1 in the control culture). In terms of mGM-CSF production, PEG 8000 gave the greatest stimulation and with 2 g PEG 8000 l^–1, mGM-CSF increased from 1.6 to 6.6 ng ml^–1.     

Immobilization of the slime mould Dictyostelium discoideum for the continuous production of recombinant human antithrombin III

Recombinant vegetative Dictyostelium discoideum cells were immobilized inside a porous matirx by an inorganic membrane that was permeable to nutrients but not to cells, in order to produce recombinant human antithrombin III. Cells so entrapped could reach up to 15 times higher biomass densities compared with organisms growing freely in suspension. The high cell concentration maintained in the immobilized cell bioreactor caused an increase in specific and volumetric productivity. In continuous operation a maximum volumetric antithrombin productivity of 56 ng h ^–1 ml ^–1 catalyst bulk volume was attained at a dilution rate of 0.016 h ^–1. In addition, the good retention of metabolic activity for several weeks as well as the convenient form of storage and regeneration of the catalytic system were shown. Correspondence to: H. Tiltscher     

Continuous production ofl-lactic acid from whey permeate by immobilizedLactobacillus casei subsp.casei

Summary The production of l-lactic acid from whey permeate, a waste product of the dairy industry, by fermentation with the lactic acid bacterium Lactobacillus casei subsp. casei was investigated. A fermentation medium consisting of permeate and supplements, which enables exponential growth of the organisms, was developed. A fast method for determination of free and immobilized biomass in solid-rich media, based on measurement of cellular ATP, was evolved. Continuous fermentations in a stirred tank reactor (STR) and in a fluidized bed reactor (FBR) with immobilized biomass were compared. In the STR a volumetric productivity of 5.5 g/l per hour at 100% substrate conversion [dilution rate (D) = 0.22 h^–1] was determined. In the FBR porous sintered glass beads were used for immobilization and a maximum biomass concentration of 105 g/kg support was measured. A productivity of 10 g/l per hour was obtained at D = 0.4 h^–1 (substrate conversion 93%) and of 13.5 g/l per hour at D = 1.0 h^–1 (substrate conversion 50%). Offprint requests to: W. Krischke     

Bioreactor culture of heterotrophic sandalwood (Santalum album L.) cell suspensions utilizing a cell-lift impeller

Summary Growth and phenolic production by two heterotrophic suspension cultures (SW-1 and SW-2) of sandalwood cultivated in a 2.5 L bioreactor were investigated. Cultures of SW-1 cell suspensions resulted in a maximum phenolic content of 32.5 mg L^–1 compared to 12.5 mg L^–1 produced by SW-2 cell suspensions. Fresh weight doubling time (Td) was 5.8 days and the specific growth rate () was 0.12 d^–1 during exponential growth for both cell lines. The pH of the culture medium decreased from 5.5 to 3.5 during the exponential growth phase of SW-1 and SW-2 cell suspensions. The dissolved oxygen content also dropped steadily during culture and remained at 40% throughout exponential growth phase. These results should provide a basis for developing sandalwood cell cultures for bioproduction of useful compounds.Abbreviations 2,4-D: 2–4 dichlorophenoxyacetic acid - BA N⁶-benzyladenine - Eh Medium oxidation-reduction potential - K_La Oxygen transfer coefficient - MS Murashige and Skoog (1962) basal medium - SW-1 and SW-2 Sandalwood suspension lines     

Bioreactor seaweed cell culture for production of bioactive oxylipins

Liquid cell suspension cultures derived from marine plants have the potential to biosynthesize novel biomedicinal compounds in a controlled environment. Of particular interest are the eicosanoids and related oxylipins emanating from the 15-lipoxygenase manifold of the arachidonic acid cascade, which is active in the brown algaLaminaria saccharina. Filamentous cell clumps ofL. saccharina isolated from female gametophytes were cultured in an illuminated bubble-column bioreactor in GP2 artificial seawater nutrient medium at 13 °C and air flow rate of 0.35 L air min^–1 L^–1 culture (vvm). Growth kinetics and biomass productivity data were obtained as a function of incident light intensity (2.4 to 98mol photon m^–2 s^–1) and initial cell density (27 to 149 mg DCW L^–1). Maximum cell densities exceeded 1200 mg DCW L^–1 after a 20 day cultivation time at optimal conditions of 98mol photon m^–2 s^–1 and 118 mg DCW L^–1 initial cell density. Qualitative analysis of chloroform/methanol extracts of the cell culture biomass by GC-MS confirmed the presence of the hydroxy fatty acids 13-HODTA and 13-HOTE, the likely products of 15-lipoxygenase catalyzed oxidation of linoleic or linolenic acids.     

Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations

Addition of 40 g NaCl l^–1 to a chemically defined medium containing 140 g glucose l^–1 in shake-flask culture improved glycerol production by Candida krusei from 16.5 g l^–1 to 47.7 g l^–1. With 40 g NaCl l^–1 at a dilution rate of 0.065 h^–1, glycerol concentration, glycerol yield (based on glucose consumed), and productivity in a four-stage cascade bioreactor were higher by 240%, 27% and 28%, respectively, than in a single-stage continuous culture system.     

Evaluation of plastic composite-supports for enhanced ethanol production in biofilm reactors

Biofilms are a natural form of cell immobilization that result from microbial attachment to solid supports. Biofilm reactors with polypropylene composite-supports containing up to 25% (w/w) of various agricultural materials (corn hulls, cellulose, oat hulls, soybean hulls or starch) and nutrients (soybean flour or zein) were used for ethanol production. Pure cultures ofZymomonas mobilis, ATCC 31821 orSaccharomyces cerevisiae ATCC 24859 and mixed cultures with either of these ethanol-producing microorganisms and the biofilm-formingStreptomyces viridosporus T7A ATCC 39115 were evaluated. An ethanol productivity of 374g L^–1 h^–1 (44% yield) was obtained on polypropylene composite-supports of soybean hull-zein-polypropylene by usingZ. mobilis, whereas mixed-culture fermentations withS. viridosporus resulted in ethanol productivity of 147.5 g L^–1 h^–1 when polypropylene composite-supports of corn starch-soybean flour were used. WithS. cerevisiae, maximum productivity of 40 g L^–1 h^–1 (47% yield) was obtained on polypropylene composite-supports of soybean hull-soybean flour, whereas mixed-culture fermentation withS. viridosporus resulted in ethanol productivity of 190g L^–1 h^–1 (35% yield) when polypropylene composite-supports of oat hull-polypropylene were used. The maximum productivities obtained without supports (suspension culture) were 124 g L^–1 h^–1 and 5 g L^–1 h^–1 withZ. mobilis andS. cerevisiae, respectively. Therefore, forZ. mobilis andS. cerevisiae, ethanol productivities in biofilm fermentations were three- and eight-fold higher than suspension culture fermentations, respectively. Biofilm formation on the chips was detected by weight change and Gram staining of the support material at the end of the fermentation. The ethanol production rate and concentrations were consistently greater in biofilm reactors than in suspension cultures.This is Journal Paper No. J-16356 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253     

Biomass segregation in sage cell suspension culture

The biomass of sage (Salvia officinalis L.) cell suspension culture was composed of single cells and cell aggregates. The development of aggregated cell culture from a single-cell suspension was monitored by particle size distribution for four particle size classes. Particle size distribution was compared between the biomass grown in bioreactor and shake flasks. The size of the particles had a strong influence on content of secondary metabolite, ursolic acid (UA). The single cell biomass fraction accumulated up to 7.7 mg UA g^–1 DW which was up to 50 times higher compared to aggregated biomass fractions.     

Co-metabolic degradation of trichloroethylene by Pseudomonas putida in a fibrous bed bioreactor

Co-metabolic degradation of trichloroethylene (TCE) by Pseudomonas putida F1 was investigated in a novel bioreactor with a fibrous bed. A pseudo-first-order rate constant for TCE degradation was 1.4 h^–1 for 2.4 to 100 mg TCE l^–1. Competitive inhibition of toluene on TCE removal could be prevented in this bioreactor. 90% TCE was removed over 4 h when 95 mg toluene l^–1 was presented simultaneously.     

Citric acid production by immobilized Aspergillus niger in a fluidized bed reactor

Summary Citric acid production by immobilized of Aspergillus niger in a fluidized bed reactor was performed, evaluating the productivity and the stability of the process when pulsing device was used. The application of a pulsing flow to fluidized bed reactor and the feed nitrogen limited allow to control of bioparticles morphology avoiding bed compactation. When operated at optimum pulsation frequency (0.3 s^–1) the stability of the bioreactor was maintained for more than 30 days, increasing the citric acid production in more than 52.2%.     

Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis

Lactose of sweet whey permeate was converted into sodium lactate byLactobacillus helveticus. To increase the, productivity of the lactic acid fermentation and to reduce the amounts of effluents, the bioreactor was coupled with an ultrafiltration module and an electrodialysis unit. Without the electrodialyzer, with total cell recycling and at a dilution rate of 0.88 h^–1, a cellular concentration of 64 gl^–1 and a productivity of 22 gl^–1 h^–1 were obtained. When the electrodialysis unit is coupled, the outlet concentration of lactate was stabilized at 85±5 gl^–1.     

Application of a membrane recycle bioreactor for continuous ethanol production

Summary A series of continuous fermentations were carried out with a production strain of the yeast Saccharomyces cerevisiae in a membrane bioreactor. A membrane separation module composed of ultrafiltration tubular membranes retained all biomass in a fermentation zone of the bioreactor and allowed continuous removal of fermentation products into a cell-free permeate. In a system with total (100%) cell recycle the impact of fermentation conditions [dilution rate (0.03–0.3 h^–1); substrate concentration in the feed (50–300 g·1^–1); biomass concentration (depending on the experimental conditions)] was studied on the behaviour of the immobilized cell population and on ethanol formation. Maximum ethanol productivity (15 g·1^–1·h^–1) was attained at an ethanol concentration of 81 g·1^–1. The highest demands of cells for maintenance energy were found at the maximum feed substrate concentration (300 g·1^–1) and at very low concentrations of cells in the broth.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics

The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (Qs _rAb) increased in parallel with increases in the specific growth rate (). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qs _product increased as decreased. This report shows that the relationship between cell growth and Qs _product for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregate, Qs _product increased with increases in . In such systems, the cells have a rounded morphology. When cells were grown on microcarriers, Qs _product decreased as increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qs _product to are correlated with changes in cell morphology. The relationship between Qs _product and is also affected by the choice of cell line. Correspondence to: A. J. Racher