Effect of shear stress on anthraquinones production by Rubia tinctorum suspension cultures

Suspension cultures of Rubia tinctorum, an anthraquinones (AQs) producer, were grown both in Erlenmeyer flasks at 100 rpm and in a 1.5 L mechanically stirred tank bioreactor operating at 450 rpm. The effect of hydrodynamic stress on cell viability, biomass, and AQs production was evaluated. Cell viability showed a transient decrease in the bioreactor during the first days, returning to the initial values toward the end of the culture time. The biomass obtained in the bioreactor was 29% lower than that attained in the Erlenmeyer flasks. The H2O2 production in the bioreactor (with peaks at 7 and 10 days) was about 15 times higher than that obtained in the flasks. A clear relationship exists between the maximum concentration of H2O2 generated and AQs produced. The AQs content in the bioreactor was 233% higher than that in the Erlenmeyer flasks. The AQs specific productivity in the stirred tank and in the Erlenmeyer flasks was 70.7 and 28.5 micromol/g FW/day, respectively. This production capability was maintained in the regrowth assays. On the other hand, the negative effects of hydrodynamic stress on viability and biomass concentration observed in the bioreactor culture were reverted in the regrowth cultures. It can be concluded that R. tinctorum suspension cultures are able to grow in stirred tanks at 450 rpm responding to the hydrodynamic stress with higher concentrations of AQs, which suggest the possibility of a technological approach taking advantage of this phenomenon.     

The role of volumetric power input in the growth,morphology, and production of a recombinant glycoprotein by Streptomyces lividans in shake flasks

The impact of flask geometry on Streptomyces lividans growth and morphology, production and O-mannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries. During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51 kW/m³, followed by coiled flasks (CF) with 0.44 kW/m³ and normal Erlenmeyer flasks (NF) with 0.20 kW/m³ (flasks volume of 250 mL, filling with 50 mL and agitated at 150 rpm). During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology. Also, NF cultures were carried out at a filling volume and agitation of 15 mL, 150 rpm (15 mL-NF), and 25 mL, 168 rpm (25 mL-NF), in order to raise P/V closely to the values obtained in CF. However, different growth, morphology and recombinant protein productivity were obtained. These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production. But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V.     

Significance of seed culture methods on mycelial morphology and production of a novel anti-cancer anthraquinone by marine mangrove endophytic fungus Halorosellinia sp. (No. 1403)

The effects of seed culture methods on the mycelial morphology and production of a novel promising anti-cancer anthraquinone 1403C by marine mangrove saprophytic fungus Halorosellinia sp. (No. 1403) was investigated. Inoculums were prepared using different seed culture methods, i.e., mycelia obtained by grinding biomass that was harvested from baffled flask culture (M1); biomass harvested from baffled flask culture (M2); biomass obtained from unbaffled flask culture with glass beads (M3); biomass attained from unbaffled flask culture (Control). The corresponding fermentations using M1, M2 and M3 enhanced 1403C production by 243.5%, 194.8% and 70.2%, respectively, as compared to that using Control (0.33 ± 0.03 g/l). Interestingly, 1403C production increased with the increase of ratio of number of clumps to pellets. Maximum 1403C production from baffled flask cultures was 4.8-fold of that from unbaffled flask culture. Increasing shaking speed from 170 rpm to 260 rpm could highly improve 1403C production by 151.8%.     

Lipolytic enzyme production by Thermus thermophilus HB27 in a stirred tank bioreactor

In this study, lipolytic enzyme production by Thermus thermophilus HB27 at bioreactor scale has been investigated. Cultivation was performed in a 5-L stirred tank bioreactor in discontinuous mode, at an agitation speed of 200 rpm. Different variables affecting intra- and extra-cellular lipolytic enzyme production such as culture temperature and aeration rate have been analysed. The bacterium was able to grow within the temperature range tested (from 60 to 70 °C) with an optimum value of 70 °C for intra- and extra-cellular lipolytic enzyme production.On the other hand, various aeration levels (from 0 to 2.5 L/min) were employed. A continuous supply of air was necessary, but no significant improvement in biomass or enzyme production was detected when air flow rates were increased above 1 L/min. Total lipolytic enzyme production reached a maximum of 167 U/L after 3 days, and a relatively high concentration of extra-cellular activity was detected (40% of the total amount). Enzyme yield was around 158 U/g cells. Moreover, it is noteworthy that the lipolytic activity obtained operating at optimal conditions (70 °C and air flow of 1 L/min) was about five-fold higher than that attained in shake flask cultures     

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

Comparison of the growth ofCinchona ledgeriana Moens suspension cultures in shake flasks and 7 litre air-lift bioreactors

Summary Suspension cultures ofCinchona ledgeriana Moens have been developed which exhibit good growth in shake flasks with dry weight yields of approximately 9.0 g.l^–1. Cultures have been scaled up for growth in a 7 l air-lift bioreactor. A typical growth curve in the fermenter is shown with similar growth rates but a reduced biomass levels when compared to shake flasks. The analysis of both flask and bioreactor grown suspension cultures indicated the presence of quinidine and low levels of quinine.     

Growth tolerance of Zygomycetes Mucor indicus in orange peel hydrolysate without detoxification

The capability of two zygomycetes strains, Mucor indicus and an isolate from tempeh (Rhizopus sp.), to grow on orange peel hydrolysate and their tolerance to its antimicrobial activity, was investigated. Both fungi, in particular M. indicus, tolerated up to 2% d-limonene in semi-synthetic media during cultivation in shake flasks, under aerobic as well as anaerobic conditions. The tolerance of M. indicus was also tested in a bioreactor, giving rise to varying results in the presence of 2% limonene. Furthermore, both strains were capable of consuming galacturonic acid, the main monomer of pectin, under aerobic conditions when no other carbon source was present. The orange peel hydrolysate was based on 12% (dry w/v) orange peels, containing d-limonene at a concentration of 0.6% (v/v), which no other microorganism has been reported to be able to ferment. However, the hydrolysate was utilised by M. indicus under aerobic conditions, resulting in production of 410 and 400 mg ethanol/g hexoses and 57 and 75 mg fungal biomass/g sugars from cultivations in shake flasks and a bioreactor, respectively. Rhizopus sp., however, was slow to germinate aerobically, and neither of the zygomycetes was able to consistently germinate in orange peel hydrolysate, under anaerobic conditions. The zygomycetes strains used in the present study demonstrated a relatively high resistance to the antimicrobial compounds present in orange peel hydrolysate, and they were capable of producing ethanol and biomass in the presence of limonene, particularly when cultivated with air supply.     

Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L⁻¹ xylose and 37 g L⁻¹ corn steep liquor, high yields (39.2 g L⁻¹) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g⁻¹ mycelium dry weight, respectively.     

Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L).     

Production of polysaccharide–peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala

The effects of medium components and environmental factors on the production of mycelial biomass and polysaccharide–peptide complexes (exobiopolymers) by Cordyceps sphecocephala J-201 were investigated in submerged cultures. The optimal temperature and initial pH for the production of both mycelial biomass and exobiopolymers in flask cultures were found to be 25 °C and pH 4–5, respectively. The optimal combination of the media constituents was as follows (g l⁻¹): sucrose 40, yeast extract 6, polypepton 2, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄·7H₂O 0.5. The results of bioreactor culture revealed that the maximum concentration of mycelial biomass (28.2 g l⁻¹) was obtained at an agitation speed of 300 rpm and at an aeration rate of 2 vvm, whereas maximum exobiopolymer production (2.5 g l⁻¹) was achieved at a milder agitation speed (150 rpm). There was a significant variance in mycelial morphology between different aeration conditions. Looser mycelial pellets were developed, and their size and hairiness increased as the aeration rate increased from 0.5 to 2.0 vvm, resulting in enhanced exobiopolymer production. The apparent viscosities of fermentation broth increased rapidly towards the end of fermentations at the conditions of high aeration rate and agitation speed, which were mainly due to high amount of mycelial biomass rather than exobiopolymers at the later stages of fermentation. The three different exobiopolymers (FR-I, -II, and -III) were fractionated by a gel filtration chromatography on Sepharose CL-6B. The carbohydrate and protein contents in each fraction were significantly different and the molecular weights of FR-I, FR-II, and FR-III were determined to be 1831, 27, and 2.2 kDa, respectively. The compositional analysis revealed that the three fractions of crude exobiopolymers consisted of acidic and nonpolar amino acids, such as aspartic acid, glutamic acid, glycine, and valine in protein moiety, and of mainly mannose and galactose in sugar moiety.     

Statistical optimization of enzymatic degradation process for oil palm empty fruit bunch (OPEFB) in rotary drum bioreactor using crude cellulase produced from Aspergillus niger EFB1

Oil palm empty fruit bunch (OPEFB) was pretreated with 2% (v/v) HNO₃ and degraded by Aspergillus niger EFB1 crude cellulase. Through 2 Level Factorial Design (2LFD), it was found that OPEFB concentration, temperature, incubation time, concentration of Tween 80 and agitation speed have significant effect in reducing sugar production. A standard Response Surface Methodology (RSM) design known as Central Composite Design (CCD) was used to optimize the enzymatic degradation condition of OPEFB in rotary drum bioreactor. Reducing sugar level of 1.183 g/L was obtained with the following optimized degradation conditions: 1.95% (w/v) OPEFB, 0.5% (v/v) Tween 80, 55 °C, 87.5 rpm in the incubation period of 3 days and 16 h. The optimal degradation condition improved reducing sugar production by 1.07 fold compared to that before optimization in shake flasks culture. The optimization strategy of enzymatic degradation of OPEFB inside rotary drum bioreactor led to increase in glucose, xylose, arabinose, galactose and mannose production by 3, 2.5, 1.64, 19.37 and 22.52 fold, respectively. The improvement in reducing sugar and polyoses production were comparable with the reduction in OPEFB cellulose and hemicellulose content by 89.32% and 48.17% respectively after enzymatic degradation in optimized condition.     

Relation between pristinamycins production by Streptomyces pristinaespiralis,power dissipation and volumetric gas–liquid mass transfer coefficient,kLa

During bioreactor cultures, microorganisms are submitted to non-optimal conditions such as nutritional and hydrodynamic stresses which may lead to modifications of the physiological cell response; this is especially true for filamentous microorganisms like Streptomycetes also subjected to significant morphological changes. In the present work, growth and production of pristinamycins by Streptomyces pristinaespiralis in shaking flasks have been related to power dissipation. The filamentous bacteria were grown in different flask conditions with various total and working volumes and at two agitation rates, to test the influence of power dissipation and gas–liquid mass transfer coefficient on growth and antibiotics production. As a first step, computational fluid dynamics–volume of fluid (CFD–VOF) calculations were shown to be able to predict power dissipations for the various operating conditions in Newtonian flow conditions. Then, in non-Newtonian flow conditions (biomass concentration superior to 14 g L⁻¹), the rheological model of Sisko was implemented in CFD simulations for the calculation of the fluid viscosity and then of power dissipation. Whereas microbial growth was correlated to k_La, the antibiotics production onset was linked to the volume mean power dissipation. Once a minimal cell concentration of 15 g L⁻¹ was reached, the concentration of antibiotics was correlated to power dissipation with an optimal range of production, between 5.5 and 8.5 kW m⁻³. Higher power dissipation entailed a drop in production which could be explained by hydrodynamic cell damages.     

Process development of eicosapentaenoic acid production

Eicosapentaenoic acid (EPA), a well-known member of omega-3 fatty acids, is considered to have a significant health promoting role in the human body. It is an essential fatty acid as the human body lacks the ability to produce it in vivo and must be supplemented through diet. Microbial EPA represents a potential commercial source. GC/MS analyses confirmed that bacterial isolate 717, similar to Shewanella pacifica on the basis of 16S rRNA sequencing, is a potential high EPA producer. Two types of bioreactors, a Stirred Tank Reactor (STR) and an Oscillatory Baffled Reactor (OBR), were investigated in order to choose the optimum system for EPA production. The EPA production media was optimised through the selection of media components in a Plackett–Burman (PB) design of experiment followed by a Central Composite Design (CCD) to optimise the concentration of medium components identified as significant in the Plackett–Burman experiment. The growth conditions for the bioreactor, using artificial sea water (ASW) medium, were optimised by applying Response Surface Methodology (RSM). This optimisation strategy resulted in an increase in EPA from 33 mg/l (10 mg/g biomass), representing 8% of the total fatty acids at shake flask level, to 350 mg/l (46 mg/g biomass) representing 25% of the total fatty acids at bioreactor level. During this study the main effects and the interactions between the bioreactor growth conditions were revealed and a polynomial model of EPA production was generated. Chemostat experiments were performed to test the effect of growth rate and temperature on EPA production.     

Fermentation strategies for the production of lipase by an indigenous isolate Burkholderia sp. C20

Burkholderia sp. C20 strain isolated from food wastes produces a lipase with hydrolytic activities towards olive oil. Fermentation strategies for efficient production of this Burkholderia lipase were developed using a 5-L bench top bioreactor. Critical factors affecting the fermentative lipase production were examined, including pH, aeration rate, agitation rate, and incubation time. Adjusting the aeration rate from 0.5 to 2 vvm gave an increase in the overall lipase productivity from 0.057 to 0.076 U/(ml h), which was further improved to 0.09 U/(ml h) by adjusting the agitation speed to 100 rpm. The production of Burkholderia lipase followed mixed growth-associated kinetics with a yield coefficient of 524 U/g-dry-cell-weight. The pH optimum for cell growth and lipase production was different at 7.0 and 6.0, respectively. Furthermore, stepwise addition of carbon substrate (i.e., olive oil) enhanced lipase production in both flask and bioreactor experiments.     

CFD analysis of the turbulent flow in baffled shake flasks

In this work, computational fluid dynamics (CFD) technique is used to simulate the complicated unsteady-state turbulent flow field formed in baffled flask. The baffled flask shows advantages both in mass transfer capacity and in shear formation in comparison with unbaffled flasks. Detailed investigations of power consumption, mass transfer and shear rate are carried out in baffled flasks under shaking frequencies ranging from 100 rpm to 250 rpm, and filling volumes from 50 mL to 150 mL. The results show that the specific power input and specific interface area are both greatly influenced by shaking frequency and filling volume. For the positive effect of shaking frequency on both mass transfer coefficient (k_L) and specific interface area (a), the volumetric mass transfer coefficient (k_La) increases greatly with shaking frequency. Results also show that filling volume has no significant effect on k_L but negative effect on specific interface area. Shear force formed in baffled flask shows great dependent on shaking frequency, but it is insensitive to the filling volume. Based on these investigations, correlations linking these parameters are proposed. Finally, cultivations of filamentous fungus conducted in unbaffled and baffled flasks validated the simulating results.     

Studies on phosphate uptake by Acinetobacter calcoaceticus under aerobic conditions

Acinetobacter calcoaceticus was cultivated in a well-aerated stirred tank reactor and its phosphate uptake capacity was investigated. Statistical media optimization was done to figure out favourable growth conditions of Acinetobacter calcoaceticus NRRLB-552. Plackett–Burman design was used to figure out the key nutrients (sodium acetate, ammonium chloride and calcium chloride) featuring high growth and/or uptake of phosphate. The optimal concentrations for these nutrients were (sodium acetate 5.0 g/l, ammonium chloride 0.67 g/l, calcium chloride 0.05 g/l) obtained by central composite design (CCD) protocols and verified in shake flask cultivations. Predicted and experimental dry cell weights obtained using the optimized media were 2.046 and 2.54 g/l indicating 97% agreement. The optimal values of pH and temperature for growth and phosphate uptake were found to be 7.69 and 31.86 °C, respectively, using CCD. Batch kinetics was also established in shake flask and fermenter using optimized medium and environmental conditions. Phosphate uptakes of 21 mg/g biomass and 36 mg/g biomass were obtained in shake flask and fermenter, respectively. The possible inhibition of nutrients (carbon, nitrogen and phosphate) was also established under shake flask cultivation conditions. Growth of the bacteria was inhibited at a concentration higher than 0.4% carbon and 0.6% nitrogen. However increasing concentration of phosphate did not show any inhibitory effect on growth. The above kinetics and inhibition data will serve as suitable database for the development of a mathematical model for growth and its use will be able to facilitate appropriate reactor design for the removal of phosphates from industrial effluents.     

Scopolamine,anisodamine and hyoscyamine production by Brugmansia candida hairy root cultures in bioreactors

Hyoscyamine, anisodamine (6β-hydroxyhyoscyamine) and scopolamine are tropane alkaloids produced by plants belonging to the Solanaceae family such as Brugmansia candida. These alkaloids were traditionally used in medicine because of their anticholinergic activity. Further therapeutical properties for anisodamine were recently described renewing the interest in these alkaloids. The scaling-up of hairy root cultures is an interesting strategy for the pharmaceutical production of these compounds instead of the isolation from plants. In this work, B. candida hairy roots were cultured in a modified 1.5 L stirred tank in order to analyze an alternative production system of scopolamine and anisodamine. It was found that these cultures produced an increased biomass and alkaloids concentration compared to the processes carried out in Erlenmeyer flasks. Anisodamine was the predominant alkaloid reaching a maximum concentration of 10.05 ± 0.76 mg/g DW in modified bioreactor culture system. The results obtained in this work are potentially applicable for the rational scale-up of the process.     

Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed. (c) 1993 John Wiley & Sons, Inc.     

Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture

Successful scale-up of Azadirachta indica suspension culture for azadirachtin production was done in stirred tank bioreactor with two different impellers. The kinetics of biomass accumulation, nutrient consumption and azadirachtin production of A. indica cell suspension culture were studied in a stirred tank bioreactor equipped with centrifugal impeller and compared with similar bioreactor with a setric impeller to investigate the role of O₂ transfer efficiency of centrifugal impeller bioreactor on overall culture metabolism. The maximum cell mass for centrifugal impeller bioreactor and stirred tank bioreactor (with setric impeller) were 18.7 and 15.5 g/L (by dry cell weight) and corresponding azadirachtin concentrations were 0.071 and 0.05 g/L, respectively. Glucose and phosphate were identified as the major growth-limiting nutrients during the bioreactor cultivation. The centrifugal impeller bioreactor demonstrated less shearing and improved O₂ transfer than the stirred tank bioreactor equipped with setric impeller with respect to biomass and azadirachtin production.