Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

Effects of environmental conditions and methanol feeding strategy on lipase-mediated biodiesel production using soybean oil

Methanol is a commonly used acyl acceptor for lipase-driven biodiesel production, but a high concentration of methanol is detrimental for lipase activity. To overcome this drawback, a simple fed-batch process was developed by optimization of the methanol feeding strategy and reaction conditions. For the feeding strategy, an equal volume of pure methanol was fed twice with specified time intervals into a reactor initially containing a 1:1 molar ratio of soybean oil to methanol in order to adjust the net molar ratio of the oil to methanol to 1:3. In contrast with the batch reaction, a higher agitation speed in the fed-batch process elevated the conversion yield of soybean oil to biodiesel. An agitation speed of 600 rpm and a reaction temperature of 70°C were chosen as the optimal environmental conditions. Residual lipase activities for the fed-batch operation at 40 ∼ 70°C and 600 rpm were 7.1 ± 1.4 times higher than that of the batch method at 40°C with the same agitation speed, indicating that methanol feeding can prevent significant deactivation of lipase. Finally, two times feeding methanol at 2 and 6 hr resulted in a biodiesel productivity of 10.7%/h and 94.9% final conversion yield under the optimal conditions.     

Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high μ for cell growth and high q _p for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l^?1 in 48 h with the yield of 0.051 g g^?1 by applying this strategy, which were 20.5 and 21.4 % over the best results controlled by constant agitation speeds.     

Itaconic acid production using an air-lift bioreactor in repeated batch culture of Aspergillus terreus

Repeated itaconic acid production using an air-lift bioreactor was carried out by three methods—two with cell recycling by means of centrifugation and filtration by a stainless steel filter set inside the bioreactor and one by repeated batch culture without cell recycling. In a flask culture, repeated itaconic acid production was stable for 9 cycles (45 d) and the production rate was 0.47 g/l/h. However, in the air-lift bioreactor, it was difficult to produce itaconic acid in the repeated batch culture with cell recycling for a long period due to a decrease in fluidity resulting from an increase in mycelium concentration. In the method without cell recycling, however, repeated itaconic acid production was stable for 4 cycles (21 d) and the production rate was 0.37 g/l/h.     

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.     

Enhanced acetoin production by Serratia marcescens H32 using statistical optimization and a two-stage agitation speed control strategy

Enhanced acetoin production was carried out by Serratia marcescens H32. First, medium compositions were optimized statistically for shake flask fermentations to produce acetoin. Sucrose and corn steep liquor powder (CSLP) were identified as the most significant factors by Plackett-Burman design. The path of steepest ascent and response surface methodology were then employed to determine the optimal concentrations of the two factors. Acetoin yield was up to 41.5 g/L in flask fermentations using the optimized medium. Furthermore, the optimal medium was used to conduct fermentation experiments in a 3.7-L bioreactor. The influences of different agitation speeds on acetoin production were investigated. Based on a process analysis, a two-stage agitation speed control strategy was proposed, in which the agitation speed was controlled at 700 rpm during the first 8 h and then switched to 600 rpm. A relatively high acetoin concentration (44.9 g/L) and high acetoin productivity (1.73 g/L/h) were achieved by applying this strategy. Fed-batch fermentation based on the two-stage agitation speed control strategy was performed, and a maximum acetoin concentration of 60.5 g/L with productivity of 1.44 g/L/h was achieved.     

Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy

The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g(-1)) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l(-1) h(-1)). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g(-1). Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l(-1)), high pyruvate yield on glucose (0.636 g g(-1)), and high glucose consumption rate (1.95 g l(-1)h(-1)) were achieved by applying this strategy. The productivity (1.24 g l(-1) h(-1)) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.     

Enhanced 2,3-butanediol production by Klebsiella oxytoca using a two-stage agitation speed control strategy

Batch fermentative production of 2,3-butanediol by Klebsiella oxytoca was investigated using various oxygen supply methods though varying agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (q_s) and specific 2,3-butanediol formation rate (q_p), a two-stage agitation speed control strategy, aimed at achieving high concentration, high yield and high productivity of 2,3-butanediol, was proposed. At the first 15 h, agitation speed was controlled at 300 rpm to obtain high μ for cell growth, subsequently agitation speed was controlled at 200 rpm to maintain high q_p for high 2,3-butanediol accumulation. Finally, the maximum concentration of 2,3-butanediol reached 95.5 g l⁻¹ with the yield of 0.478 g g⁻¹ and the productivity of 1.71 g l⁻¹ h⁻¹, which were 6.23%, 6.22% and 22.14% over the best results controlled by constant agitation speeds.     

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.     

Production of protease using wastewater from the manufacture of shochu

The production of protease using wastewater from a shochu distillery was investigated in order to devise a process for the treatment of shochu distillery wastewater. Aspergillus usami mut. shirousami IFO 6082 was selected from among eight strains for production of protease. Production of 240 U/ml of proteolytic activity was achieved after 72 h in a jar-fermentor culture under the following conditions: initial pH, 5; 30°C; aeration, 1 vvm; and agitation, 600 rpm. The protease was purified by column chromatography on Sephadex G-150 and isoelectrofocusing. The molecular weight of the purified enzyme determined by SDS-PAGE was 56 kDa, and the isoelectric point was pH 4.0. The optimum pH for the reaction was about 4.0, so the enzyme is therefore an acid protease. The optimum temperature for proteolysis ranged between 55 and 60°C, while the enzyme was unstable at temperatures above 60°C.     

Establishment of cultivating strategy for highly aggregated mycelia of Morchella esculenta in a stirred-tank bioreactor

Mycelia of Morchella esculenta were found to aggregate rapidly in a submerged culture, which caused the decrease in dispersed mycelia and the problem of diffusion limitation. The effect of different agitation schemes on the growth of mycelia was investigated in a stirred-tank bioreactor. At the constant speed of 100 or 300?rpm, rapid aggregation caused the biomass concentration to drop to zero in 30?h, which was even worse than achieved under static culture. Intermittent agitation maintained a higher mycelium fragment concentration for 48?h and enhanced the biomass concentration to 4.73?g/L at 120?h. The operation with a polytron connection disrupted effectively mycelium aggregation, thus increasing the specific growth rate, biomass concentration and maximum productivity to 0.0613 1/h, 7.73?g/L and 0.0878?g/L?h at 88?h, respectively. Moreover, logistic equations and genetic algorithm (GA) were used for the simulation of biomass growth and estimation of all kinetic coefficients. The operating strategy developed in this study could be used for the production of highly aggregated mycelia, which could also achieve a high cell-density culture in a stirred tank reactor.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.     

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.     

Effects of methocel A15LV, polyethylene glycol, and polyvinyl alcohol on CD13 and CD33 receptor surface content and metabolism of HL60 cells cultured in stirred tank bioreactors

Flow cytometry was used to examine the effect of hydrodynamic forces in a stirred tank bioreactor on the CD13 and CD33 receptor surface content of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 400 rpm reduced the HL60 cell apparent growth rate and increased the CD13 receptor surface content per cell, on average, by 95%. In contrast, this step increase in agitation rate to 400 rpm decreased the CD33 receptor surface content per cell, on average, by 10%. The protective effects of 0.1% Methocel A15LV, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) on CD13 and CD33 receptor surface content were examined under agitation at 300 rpm in parallel 2 L bioreactor runs. The average CD33 receptor surface content was unaffected by the presence of Methocel A15LV or PEG, while PVA had a slight protective effect. In contrast, in terms of CD13 receptor content, HL60 cells agitated at 300 rpm with Methocel A15LV, PEG, or PVA behaved like cells agitated at 80 rpm with no media additives (McDowell and Papoutsakis, 1998). That is, Methocel A15LV, PEG, and PVA prevented the transduction of mechanical forces which affect CD13 cell content. HL60 cells cultured with 0.1% A15LV, PEG or PVA under conditions of mild agitation (60 rpm) in spinner flasks exhibited glucose consumption and lactate production rates that were approximately 20% lower than values of cultures containing no additive. Under conditions of agitation at 300 rpm in the 2 L bioreactor, the presence of A15LV, PEG, and PVA reduced the HL60 glucose consumption and lactate production rates by approximately 50%. Thus, media additives can dramatically reduce lactate accumulation in agitated bioreactors due to cell growth, in addition to providing protection from cellular injury.     

Effects of Minerals on Neomycin Production by Streptomyces fradiae

A study was made on the mineral requirements of Streptomyces fradiae strain 3535 for neomycin production. It was observed that optimal levels of the elements Ca, Fe, and Zn per milliliter of a synthetic medium for neomycin production were 10.8, 1.0, and 0.115 μg, respectively. K₂HPO₄ was required at a concentration of 0.1% for maximal yield of neomycin, whereas NaCl and the metals Mn and Cu were without any effect. High doses of Zn (0.23 μg/ml or above) caused destruction of neomycin after the fifth day of fermentation.     

A hybrid bioreactor for high density cultivation of plant cell suspensions

Summary A hybrid bioreactor was developed for the production of secondary metabolites from high density cultivation of plant cell suspensions. Some of the advantages of both air-lift and cell-lift by agitation were combined. The addition of a decanting column also made it possble to run a perfusion system for high density culture or to run a two-stage culture efficiently. Cell growth and the production of berberine from Thalictrum rugosum in the hybrid bioreactor are reported in this paper. A cell density up to 31 g/l was obtained by perfusion without any problems in mixing or loss of cell viability and the specific berberine productivity was comparable to that in shake flasks. The maximum berberine concentration was 88 mg/l at 3 weeks operation and declined thereafter.Offprint requests to: D.-I. Kim     

The improvement of cephalosporin C production by fed-batch culture ofCephalosporium acremonium M25 using rice oil

The objective of this study is to improve cephalosporin C (CPC) production by optimization of medium and culture conditions. A statistical method was introduced to optimize the main culture medium. The main medium for CPC production was optimized using a statistical method. Glucose and corn steep liquor (CSL) were found to be the most effective factors for CPC production. Glucose and CSL were optimized to 2.84 and 6.68%, respectively. CPC production was improved 50% by feeding of 5% rice oil at day 3rd and 5th day during the shake flask culture ofC. acremonium M25. The effect of agitation speeds on CPC production in a 2.5-L bioreactor was also investigated with fed-batch mode. The maximum cell mass (54.5 g/L) was obtained at 600 rpm. However, the maximum CPC production (0.98 g/L) was obtained at 500 rpm. At this condition, the maximum CPC production was improved about 132% compared to the result with batch flask culture.     

Itaconic acid production from xylose in repeated-batch and continuous bioreactors

Summary Itaconic acid production from xylose by immobilized Aspergillus terreus TKK 200-5-2 mycelia was optimized both in repeated shake-flask fermentations and in continuous column bioreactors using statistical experimental design and empirical modelling. Using continuous 9-1 scale air-lift bioreactors, a pH of 2.5, aeration rate of 0.6 v/v per minute and residence time of 160 h gave the highest itaconic acid concentration. In air-lift bioreactors a cubic carrier size of 0.5 cm gave a 3.3-fold higher product concentration than 1-cm cubes. Packed-bed column reactors had a higher production rate than air-lift reactors. Offprint requests to: H. Kautola     

Lipase catalyzed transesterification of soybean oil using ethyl acetate,an alternative acyl acceptor

Methanol, the acyl acceptor usually used in the commercial process of biodiesel production, is associated with some problems such as immiscibility with oils and lipase deactivation. To overcome these barriers, ethyl acetate was proposed as an alternative acyl acceptor for the production of biodiesel from soybean oil using an immobilized lipase, Novozym 435, Ethyl acetate mixed well with soybean oil, and only slightly inhibited the lipase activity by 5%. The effects of various environmental parameters, such as the composition of soybean oil and ethyl acetate, lipase content, and reaction temperature, were investigated to determine the optimal conditions for biodiesel production. As a result, the highest biodiesel production yield, 63.3 (±0.6)%, was obtained by using an ethyl acetate and soybean oil mixture with a 6∶1 molar ratio, with 8% of the immobilized lipase based on the weight of oil added at 70°C and 600 rpm.     

The Effects of Nutrient Concentration,Addition of Thickeners,and Agitation Speed on Liquid Fermentation of Steinernema feltiae

Entomopathogenic nematode production in liquid fermentation still requires improvements to maximize efficiency, yield, and nematode quality. Therefore, this study was aimed at developing a more suitable liquid medium for mass production of Steinernema feltiae, by assessing the effects of nutrient concentration, thickeners (primarily agar), and agitation speed on infective juvenile (IJ) yield. Base medium (BM) contained yeast extract (2.3%), egg yolk (1.25%), NaCl (0.5%), and corn oil (4%). All media were inoculated with Xenorhabdus bovienii, and 2 d later, with 2-d-old S. feltiae juveniles. For the nutrient concentration experiment, we evaluated the base medium versus a modified base medium containing all the components, but with 3× concentrations of yeast extract (6.9%), egg yolk (3.75%), and corn oil (12%). The nematodes and bacteria were cultured in 150-ml Erlenmeyer flasks containing 50 ml of liquid medium at (25°C) and 180 rpm on a rotary shaker incubator. To assess the effect of thickeners, IJs were inoculated in BM with agar (0.2%), carrageen (0.2%), and carboxymethyl cellulose (0.2% and 0.5%). The addition of 3× more nutrients relative to the BM resulted in a significantly lower yield of nematodes. For agar and agitation speed experiments, five levels of agar in the BM (0%, 0.2%, 0.4%, 0.6%, and 0.8% agar) and two agitation speeds (180 and 280 rpm) were evaluated for production. Increasing agitation speed from 180 to 280 rpm and higher levels of agar in the medium (> 0.2%) significantly increased the yield of bacteria. At the lower agitation speed, media amended with 0.4% and 0.6% agar produced higher nematode yields compared to media without agar. Media with 0.2% and 0.8% agar resulted in intermediate levels of nematode production. At the higher agitation speed, media supplemented with 0.8% agar resulted in the lowest yield of nematodes when compared to the other media tested. Results indicated that increasing nutrient concentration levels was detrimental to nematode production. Also, media containing agar (0.4% and 0.6%) increased nematode yields when cultures were grown at low agitation speed. When IJs were used as the inoculum, 0.2% agar also enhanced recovery and nematode yield at the higher agitation speed.