Bioreactor operation for transgenic Nicotiana tabacum cell cultures and continuous production of recombinant human granulocyte-macrophage colony-stimulating factor by perfusion culture

The production of hGM-CSF was investigated in both a flask and a 5-l bioreactor, using transgenic Nicotiana tabacum suspension cells. While the maximum cell density and secreted hGM-CSF in the flask were 15.4 g l⁻¹ and 6.5 μg l⁻¹, respectively, those in the bioreactor were 15.6 g l⁻¹ and 7.6 μg l⁻¹. No detectable growth inhibition, shorter production of hGM-CSF and reduced cell viability in the batch bioreactor were observed under the specific conditions used compared with the flask culture. To improve the productivity, a perfusion culture was carried out in the bioreactor, with three different perfusion rates (0.5, 1.0 and 2.0 day⁻¹). In all cases, the hGM-CSF in the medium was significantly increased during the overall culture period (16 days), with maximum values 3.0-, 9.4- and 6.0-fold higher than those obtained in the batch cultures, respectively, even though the intracellular hGM-CSF content was not significantly varied by the perfusion rate. In terms of the total amount of hGM-CSF secreted, 205.5, 1073.2 and 1246.3 μg accumulated in the perfusate within 16 days at the perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. It was concluded that the beneficial effect of perfusion on the production of hGM-CSF originated from the reduced proteolytic degradation due to the lower protease activity caused by the perfusion. Additionally, the cell growth and physiology in the perfusion culture were somewhat negatively affected by the increased perfusion rate, although the dry cell density steadily increased, and as a result, 19.4, 22.4 and 22.9 g l⁻¹ of maximum cells were obtained with perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. This work highlighted the importance of proteolytic degradation in plant cell cultures for the production of secretory proteins and the feasibility of perfusion strategies for the continuous production of foreign proteins by the prevention of protein loss due to proteolytic enzymes.     

High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size

High-density cultivation of Perilla frutescens cells for anthocyanin production was carried out in both batch and fed-batch modes in a 500-ml shake flask. In fed-batch cultures, a high cell density of 27.7 g dry cells l⁻¹ and a total anthocyanin production of 3.87 g l⁻¹ by intermittent feeding of all medium components except hormones were obtained. In batch cultures, both initial sucrose concentration and inoculum size showed a conspicuous effect on the kinetics of cell growth, sugar consumption, and secondary metabolite (anthocyanins) production by suspended P. frutescens cells. At an inoculum size of 50 g wet cells l⁻¹, the maximum cell density of 38.3 g dry cells l⁻¹ was obtained after 11 days of cultivation at an initial sucrose concentration of 60 g l⁻¹, the highest pigment production of>5.8 g l⁻¹ was attained after 10 days of cultivation at an initial sucrose concentration of 45 g l⁻¹. These amounts of cell mass and anthocyanin pigments were 3.3 and 24 times higher than those at an initial sucrose concentration of 15 g l⁻¹ and inoculum size of 15 g wet cells l⁻¹, respectively.     

The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture

The growth of the Spodoptera frugiperda cell line Sf9 was studied in batch and continuous culture. The results of batch cultivations showed that glucose was the preferred energy and carbon source limiting the cell density in both TNM-FH and IPL-41 media. Continuous culture using IPL-41-based feeding medium with different glucose (2.5, 5 and 10 g l⁻¹) and yeast extract concentrations (4, 8 and 16 g l⁻¹) showed that in serum-supplemented medium the maximum cell density was limited by glucose and yeast extract concentration. The transition to glucose limitation caused a decrease in growth rate and viability. A high cell density culture (18 × 10⁶ ml⁻¹) was obtained using a glucose concentration of 10 g l⁻¹ and a yeast extract concentration of 8 g l⁻¹ in the feeding medium. A yeast extract concentration of 16 g l⁻¹ inhibited growth. Unlike mammalian cell cultures, lactate, alanine and ammonia were not involved in growth inhibition. Lactate did not accumulate under aerobic conditions. Ammonia accumulation, if observed, was insignificant. The level of alanine synthesized and excreted into the culture medium never reached an inhibitory level. During glucose limitation alanine did not accumulate and ammonia was released. However, even in the presence of glucose significant amounts of Asp, Glu, Gln, Asn, Ser, Arg and Met were utilized for energy production. The amino groups of these amino acids were transferred to pyruvate or used for nucleic acid synthesis and excreted in the form of alanine into the culture medium. The consumption of His, Lys, Thr, Gly, Val, Leu, Phe, Tyr, Trp and Ile by growing Sf-9 cells was almost equal to their concentration in the biomass.     

Effects of medium glucose concentration and pH on docosahexaenoic acid content of heterotrophic Crypthecodinium cohnii

The effects of medium glucose concentration and pH on growth and docosahexaenoic acid (DHA, C22:6 ω-3) content of Crypthecodinium cohnii were investigated. Over a range of glucose concentrations (5–40 g l⁻¹) investigated, the highest specific growth rate (0.12 h⁻¹), highest cell dry weight concentration (3.13 g l⁻¹) and highest growth yield on glucose (0.6 g g⁻¹) were obtained at 20 g l⁻¹ glucose. However, the highest degree of fatty acid unsaturation (3.2) and highest DHA proportion (53.4% of total fatty acids) were achieved at 5 g l⁻¹ glucose. Low glucose concentrations enhanced the degree of fatty acid unsaturation and DHA formation. Medium pH also affected cell growth, fatty acid unsaturation and DHA proportion. When medium pH was 7.2, the highest specific growth rate (0.089 h⁻¹), highest cell dry weight concentration (2.73 g l⁻¹), highest growth yield on glucose (0.564 g g⁻¹), highest degree of fatty acid unsaturation (3.4) and highest DHA proportion (56.8% of total fatty acids) were obtained. Results suggest that glucose concentration and pH value could be effectively manipulated to achieve maximum DHA production by C. cohnii.     

Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes

The advantages of nanofiltration membranes coupled with a CSTR were demonstrated for the semicontinuous production of lactic acid from whey permeate. Lactic acid was removed from the growth medium while lactose was kept in the bioreactor with the bacterial cells; moreover, Mg²⁺ ions were also recycled in the bioreactor at 96% and the nanofiltrate color was greatly reduced. The highest volumetric productivity achieved with this device was 7.1 g l⁻¹ h⁻¹ and the lactate concentration was 55 g l⁻¹. The specific productivity was 3.54 h⁻¹. More than 99% of the membrane fouling after 44 h of fermentation was reversible. The initial permeate flux was restored easily by a water rinse. The performance of this type of membrane bioreactor was discussed.     

Optimisation of docosahexaenoic acid production in batch cultivations by Crypthecodinium cohnii

The heterotrophic micro alga Crypthecodinium cohnii was cultivated in media containing glucose, yeast extract and sea salt. Increasing amounts of yeast extract stimulated growth but influenced lipid accumulation negatively. Sea salt concentrations above half the average seawater salinity were required for good growth and lipid accumulation. C. cohnii was able to grow on a glucose concentration as high as 84.3 g l⁻¹, although concentrations above 25 g l⁻¹ decreased the growth rate. Comparison of growth at 27 and 30°C showed that the higher incubation temperature was more favourable for growth. However, lipid accumulation was higher at the lower incubation temperature. In a bioreactor the biomass concentration increased from 1.5 to 27.7 g l⁻¹ in 74 h. In the final 41 h of the process the lipid content of the biomass increased from 7.5 to 13.5%. In this period the percentage of docosahexaenoic acid of the lipid increased from 36.5 to 43.6%. The total amounts of lipid and docosahexaenoic acid after 91 h were 3.7 and 1.6 g l⁻¹, respectively.     

Effect of iron concentration on hydrogen fermentation

The effect of the iron concentration in the external environment on hydrogen production was studied using sucrose solution and the mixed microorganisms from a soybean-meal silo. The iron concentration ranged from 0 to 4000 mgFeCl₂ l⁻¹. The temperature was maintained at 37°C. The maximum specific hydrogen production rate was found to be 24.0 mlg⁻¹ VSSh⁻¹ at 4000 mgFeCl₂ l⁻¹. The specific production rate of butyrate increased with increasing iron concentration from 0 to 20 mgFeCl₂ l⁻¹, and decreased with increasing iron concentration from 20 to 4000 mgFeCl₂ l⁻¹. The maximum specific production rates of ethanol (682 mgg⁻¹ VSSh⁻¹) and butanol (47.0 mgg⁻¹ VSSh⁻¹) were obtained at iron concentrations of 5 and 3 mgFeCl₂ l⁻¹, respectively. The maximum hydrogen production yield of 131.9 mlg⁻¹ sucrose was obtained at the iron concentration of 800 mgFeCl₂ l⁻¹. The maximum yields of acetate (389.3 mgg⁻¹ sucrose), propionate (37.8 mgg⁻¹ sucrose), and butyrate (196.5 mg g⁻¹ sucros) were obtained at iron concentrations of 3, 200 and 200 mgFeCl₂ l⁻¹, respectively. The sucrose degradation efficiencies were close to 1.0 when iron concentrations were between 200 and 800 mgFeCl₂ l⁻¹. The maximum biomass production yield was 0.283 gVSSg⁻¹ sucrose at an iron concentration of 3000 mgFeCl₂ l⁻¹.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.     

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l⁻¹ glucose. An average ethanol concentration of 124.6 g l⁻¹ or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h⁻¹. The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.     

Immobilization of Acidithiobacillus ferrooxidans by a PVA–boric acid method for ferrous sulphate oxidation

Acidithiobacillus ferrooxidans was immobilized in poly(vinyl alcohol) (PVA) by a PVA–boric acid method, and spherical beads of uniform size were produced. Biooxidation of ferrous iron by immobilized cells was investigated in repeated batch culture and continuous operation in a laboratory scale packed-bed bioreactor. During repeated batch culture, the cell-immobilized gels were stable and showed high constant iron-oxidizing activity. In continuous operation in a packed-bed bioreactor, biooxidation of ferrous iron fits a plug-flow reaction model well. A maximum Fe²⁺ oxidation rate of 1.89 g l⁻¹ h⁻¹ was achieved at the dilution rate of 0.38 h⁻¹ or higher, while no obvious precipitate was detected in the bioreactor.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Production of microbial rennin from Mucor miehei in a continuously fed fermenter

In this study, microbial production of rennin, a milk-clotting enzyme, from a commercial strain of Mucor miehei NRRL 3420 has been investigated in a continuously fed fermenter for prolonged times. The spherical film-type growth of the culture has been accomplished in the fermenter and the effects of medium pH, mixing and dilution rates, and feed -glucose concentration on milk-clotting activity have been elaborated. In the fermenter, optimum operational parameters have been determined as 400 rpm, 0.125 day⁻¹, and 7.5 g l⁻¹ for mixing rate, dilution rate, and feed -glucose concentration, respectively. Under these conditions, the fermenter operated 575 h continuously producing 1.24 IU ml⁻¹ maximum milk-clotting activity without concentration. In the fermenter sample at maximum milk clotting activity, the R factor and specific milk-clotting activity were determined as 1.55 × 10⁻³ IU PU⁻¹ and 5.28 IU mg⁻¹ medium protein, respectively, denoting competitive characteristics of a commercial rennet after concentration.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).     

Oxygen limitation improves glycerol production by Candida krusei in a bioreactor

An oxygen limitation strategy based on dynamic enzyme activity was applied to improve glycerol accumulation and decrease the residual sugar level in a fermentation of Candida krusei in a bioreactor. By applying oxygen limitation at 88 h when the activities of two glycerol synthetic enzymes cytosolic glycerol-3-phosphate dehydrogenase (ctGPD) and glycerol-3-phosphatase (GPP) were low and the activity of mitochondrial glycerol-3-phosphate dehydrogenase (mtGPD) which catalyzes the glycerol dissimilation was high, the glycerol dissimilation was efficiently reduced. The final glycerol concentration reached 51.8 g l⁻¹ at 96 h and 54.9 g l⁻¹ at 116 h, which was 18 and 60% higher than the control (without oxygen limitation), respectively. The residual sugar was consumed completely while it was 11.2 g l⁻¹ at the end of fermentation in the control. Under oxygen limitation, ethanol production was detected at a final concentration of 3.6 g l⁻¹. This work suggests a metabolic flux shift by oxygen limitation in the bioreactor.     

A coupled two-stage continuous fermentation for solvent production by Clostridium acetobutylicum

The effects of nitrogen and phosphate in batch and continuous AEB fermentations were tested. Both nitrogen- and phosphate-limited fermentations favored acid formation but not solvent production. A coupled two-stage continuous fermentation was performed for 30 days with a nitrogen-limited first stage fermentation for enhanced acid production. The bacteria from the acidogenic phase (first stage) fermentation were continuously pumped into a 14-l second stage fermentor with supplemental glucose and nitrogen for solvent production. The second stage fermentor had a maximum butanol productivity of 0.4 g l⁻¹ h⁻¹ (total solvent production was 0.6 g l⁻¹ h⁻¹) at a dilution rate of 0.06 h⁻¹.     

Concentration of L-phenylalanine with a reverse osmosis membrane

A high flux, thin film composite reverse osmosis (RO) membrane was used to concentrate L-phenylalanine (L-Phe) from clarified bioreactor harvest media. At pH 10±0.5 and 50°C, concentrations of 100 g l⁻¹ were easily achieved and at fluxes from 17 to 119 1 m⁻² h⁻¹. Rejection coefficient for L-Phe was inversely proportional (as the log) to retentate concentration. A preliminary system study showed that stages in a cascade could be used to recover essentially all of the product from clarified harvests. The study shows the importance of empirical evaluation as the basis of design and suggests that bioprocess applications of RO are likely to be case specific.     

Continuous production of lactic acid from whey permeate by Lactobacillus helveticus in two chemostats in series

The effect of dilution rate on the production of lactic acid from whey permeate by Lactobacillus helveticus has been investigated. In the first chemostat of a two-stage system, total conversion (98.1%) and maximum lactic acid concentration (43.7 g l⁻¹) were obtained at a dilution rate (D_Itot) of 0.06 h⁻¹. Maximum volumetric productivities of lactic acid (8.27 g l⁻¹ h⁻¹) and biomass (1.90 g l⁻¹ h⁻¹) occurred at D_Itot of 0.40 h⁻¹. The fraction of -lactate in the product was found to increase with dilution rate and reached a maximum of 66% at the same dilution rate. The maximum specific growth rate (μ_max) on this medium was 0.7 h⁻¹. A Y_ATP (max) value of 22.4 g dry weight (mol ATP)⁻¹ and a maintenance coefficient of 8.0 mmol ATP (g dry weight h)⁻¹ were determined. The second stage, in series with the first, confirmed these results and further showed that the total residence time could be reduced by 50%, compared with a single chemostat for the same nearly complete level of substrate conversion.     

Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene

Fermentation characteristics of recombinant Saccharomyces cerevisiae containing a xylose reductase gene from Pichia stipitis were investigated in an attempt to convert xylose to xylitol, a natural five-carbon sugar alcohol used as a sweetener. Xylitol was produced with a maximum yield of 0.95 g g⁻¹ xylitol xylose consumed in the presence of glucose used as a co-substrate for co-factor regeneration. Addition of glucose caused inhibition of xylose transport and accumulation of ethanol. Such problems were solved by adopting glucose-limited fed-batch fermentations where a high ratio of xylose to glucose was maintained during the bioconversion phase. The optimized two-substrate fed-batch fermentation carried out with S. cerevisiae EH13.15:pY2XR at 30°C resulted in 105.2 g l⁻¹ xylitol concentration with 1.69 g l⁻¹ h⁻¹ productivity.     

High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development

A fed-batch process was developed for high cell density culture of the diatom Nitzschia laevis for enhanced production of eicosapentaenoic acid (EPA). Firstly, among the various medium components, glucose (Glu) was identified as the limiting substrate while nitrate (NO₃⁻), tryptone (Tr) and yeast extract (Ye) were found to promote cell growth by enhancing specific growth rate. Therefore, these components were considered essential and were included in the feed medium for subsequent fed-batch cultivation. With the optimized ratio of NO₃⁻:Tr:Ye being 1:2.6:1.3 (by weight), the relative proportions of glucose to the nitrogen sources in the feed were investigated. The optimal ratios of Glu:NO₃⁻ for specific growth rate and EPA productivity were both determined to be 32:1 (by weight). Finally, based on the residual glucose concentration in the culture, a continuous medium feeding strategy for fed-batch fermenter cultivation was developed, with which, the maximal cell dry weight and EPA yield obtained were 22.1 g l⁻¹ and 695 mg l⁻¹, respectively, which were great improvements over those of batch cultures.