Effect of cultivation conditions on spore production from Bacillus amyloliquefaciens B128 and its antagonism to Botrytis elliptica

Aims:  To maximize spore production by Bacillus amyloliquefaciens B128, and its antagonism to the fungal pathogen Botrytis elliptica B061. Methods and Results:  In the 5-l stirred-tank bioreactor (STR), with the 0·5 vvm aeration rate, an agitation rate of 200 rev min⁻¹ significantly enhanced the spore yield compared to the same in 300 rev min⁻¹ cultivations. In a 20-l airlift bioreactor (ALR) the maximal spore production was further increased with a controlled aeration rate of 2·5 vvm operated in a 24-mesh net-draft tube mode, and no pH control cultivation. This spore yield in the 20-l ALR was five- and eightfold higher; in addition the cultivation period was 19 h shorter, compared to that obtained from shaker flask and in the 5-l STR cultivations respectively. Conclusions:  Although culture conditions are still to be optimized, by using an ALR with net-draft tube, a scaling up from shaker flasks and STR to ALR of spore production by the strain B128 is technically feasible. Significance and Impact of the Study:  The spore yields obtained using bioreactors were much higher than those previously reported. The freshly produced spore preparations from the B128 strain significantly antagonized the grey mould pathogen B. elliptica.     

High-level production of Fc-fused kringle domain in Pichia pastoris

Recently, as a new non-immunoglobulin-based protein scaffold, a human kringle domain was successfully engineered toward biologically functional agonists and antagonists. In this study, the fed-batch cultivation conditions were optimized for enhanced production of an Fc-fused kringle domain (KD548-Fc) in Pichia pastoris. Fed-batch cultivations were performed in 5-l laboratory-scale bioreactors, and in order to find the optimal conditions for high-level production of KD548-Fc, several parameters including the initial carbon source (glycerol) concentration, temperature, and pH were investigated. When cells were cultivated at pH 4.0 and 25 °C with 9.5 % glycerol in the initial medium, the highest production yield (635 mg/l) was achieved with high productivity (7.2 mg/l/h). Furthermore, functional KD548-Fc was successfully purified from the culture broth using a simple purification procedure with high purity and recovery yield.     

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

Improved laminaribiose phosphorylase production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in a bioreactor: A comparative study of different cultivation methods

Laminaribiose phosphorylase (EC 2.4.1.31) catalyzes a reversible phosphorolysis reaction in which laminaribiose, a very high value sugar is produced. This enzyme is not being produced commercially therefore, to realize the most effective method for producing laminaribiose phosphorylase and obtaining as much activity units as possible per liter of culture, different cultivation methods of Euglena gracilis were compared. Heterotrophic and mixotrophic cultivations of Euglena gracilis in two different pHs, in flask and bioreactor were performed. The reverse phosphorolysis activity of laminaribiose phosphorylase produced under different cultivation methods was measured. The heterotrophic approach showed to be the more effective cultivation method as 47.6 IU/L was obtained compared to 27 IU/L in the mixotrophic one. The heterotrophic cultivation then was further investigated under two different pH values of the culture media. The culture at pH 6.8 resulted in 7.94 IU/L/day whereas only 4.06 was obtained for the culture at pH 4. Cultivation in a bioreactor resulted in a distinctive amount of 191.5 IU/L and an activity yield of 9.7 IU/g glucose compared to 5.4 in flask cultivation. Heterotrophic cultivation of Euglena gracilis in a bioreactor containing a culture media at pH 6.8 and controlled operation conditions showed enhanced laminaribiose phosphorylase activity production per liter and day of cultivation.     

Improvement of ganoderic acid production by fermentation of Ganoderma lucidum with cellulase as an elicitor

Two-stage cultivation of Ganoderma lucidum was performed for the enhanced production of ganoderic acid (GA). Cellulase was identified to be an effective elicitor for the improvement of GA production, and GA titer reached 1334.5 mg/l compared to the control (779.6 mg/l) using lactose as the substrate without cellulase addition. Loading of 5 mg/l cellulase on day 3 resulted in the maximal GA titer of 1608 mg/l. To our knowledge, this is the first time that cellulase was used as the elicitor to enhance GA production. Submerged fermentation in a 2.0-l bioreactor was also conducted with cellulase as the elicitor, and as a result the maximal GA titer of 1252.7 mg/l was obtained on day 12. This is so far the best GA production obtained in submerged fermentation of G. lucidum.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

A cultivation technique for <Emphasis Type="Italic">E. coli</Emphasis> fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O₂ transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.Revisions requested 13 April 2005; Revisions received 6 May 2005     

Nutrition and bioprocess development for efficient biosynthesis of an antitumor compound from marine-derived fungus

An integrated nutrition and bioprocess strategy was developed for improving the biosynthesis of an antitumor compound, 1403C, by a marine-derived fungus, Halorosellinia sp. (no. 1403). First, statistical design strategies were synthetically applied to optimize the nutritional composition. The resulting 1403C production reached 2.07 g/l, which was 143.5 % higher than the original production. However, it only produced 0.44 g/l of 1403C in 5-l bioreactor fermentation. Thus, the operating parameters including culture pH, dissolved oxygen, agitation speed, impeller type and inoculum level were considered to improve the fermentation process, and an effective control strategy for 1403C production by Halorosellinia sp. submerged in a 5-l bioreactor was established. When inoculating 0.22 g/l dry biomass, controlling dissolved oxygen not lower than 30 % during the growth phase but ranging between 30 and 40 % during the stationary phase, using a double-layer six-flat-blade Rushton disc turbine agitated at 400 rpm, keeping short-term low pH and rapid-rising pH with glucose starvation, the highest 1403C production was finally obtained at 1.32 g/l, which was promoted by 200 % compared to before optimization. Fermentation scale-up was finally performed in a 500-l bioreactor, and 1403C production of 1.09 g/l was obtained.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

Strategy for the Improvement of Prodigiosin Production by a Serratia marcescens Mutant through Fed-Batch Fermentation

A Serratia marcescens mutant for prodigiosin production was obtained by u.v. mutation with rational screening methods and a two-step feeding strategy was used to increase its productivity. In flasks, the mutant strain B6 gave a 2.8-fold higher prodigiosin production than that of the parent strain with glycerol as a carbon source. In a 5-l bioreactor, with a two-step feeding strategy in which glucose was selected as the initial carbon source in the fermentation media and glycerol was fed as a ‘prodigiosin inducer’, it gave a 7.8 times higher prodigiosin production (583 mg/l) than the parent stain with the original cultivation mode.     

Metarhizium anisopliae conidia production in packed-bed bioreactor using rice as substrate in successive cultivations

This paper presents a study on scale-up and cost reduction of the production of spores of Metarhizium anisopliae IBCB 425, entomopathogenic fungus used in sugarcane crops. Rice was mixed with sugarcane bagasse (9:1 w/w) for substrate composition, assuring adequate physical structure for cultivation in packed-beds. Spores yield from only rice in bench-scale packed-bed bioreactor was 56 % of the one obtained from the mixture 9:1 w/w. In comparison to plastic packages used in bioindustries, equivalent spores yields per gram of substrate have been achieved in bench and pilot-scale bioreactors built by cylindrical jacketed modules, that provide better control of operational and environmental variables, attested by little variability among replicates. Although non-negligible temperature rise (5 °C above the ideal) occurred within the pilot-scale bioreactor, spores production was not harmed in comparison to bench-scale. By reusing rice up to three successive cultivations, a 2.5-fold increase of spores yields was achieved in comparison to single use. Temperatures and CO₂ profiles corroborates the fungus adapted differently to substrate at each usage. Such results are valuable for industrial producers of commercial formulations of the fungus spores, allowing process modernization by using packed-bed bioreactors and production costs and rice demand reduction by recycling the substrate.     

Production of carrot somatic embryos in a bioreactor

Somatic carrot embryos were grown as batch cultures in a stirred 10-l bioreactor. Embryo production in the bioreactor was comparable to that obtained in shake-flasks. A production of about 50·10³ embryos/l per day was commonly achieved with an inoculum density of 0.1% volume of tissues/volume of medium. Regularly changing of the medium increased embryo viability. A filtering unit coupled to the bioreactor was developed in order to calibrate embryos. The characteristics of the population of harvested embryos are described. Correspondence to: J.-P. Ducos     

High cell density fermentation of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> DSM 2003 for glycolic acid production

Gluconobacter oxydans has a lower biomass yield. Uniform design (UD) was applied to determine the optimum composition of the critical media and their mutual interactions for increased biomass yield of Gluconobacter oxydans DSM 2003 in shake flasks. Fed-batch fermentation process for biomass was optimized in a 3.7-l fermentor. By undertaking a preliminary and improved fed-batch fermentation-process strategy, a cell density of 6.0 g/l (DCW) was achieved in 22 h and 14.1 g/l (DCW) in 35 h, which is the highest cell density of G. oxydans produced thus far in a 3.7-l bioreactor. The biomass production was increased by 135% compared with that using the original cultivation strategy. Bioconversion of ethylene glycol to glycolic acid was catalyzed by the resting cells of G. oxydans DSM 2003, and conversion rate reached 86.7% in 48 h. In summary, the approach including high-density fermentation of G. oxydans DSM 2003 and bioconversion process was established and proved to be an effective method for glycolic acid production.     

Continuous semi-solid cultivation for the production of cellulase by Trichoderma reesei mutants using a polyurethane foam carrier and a liquid medium

The production of cellulase was investigated in semi-solid state culture using the immobilized mycelium of Trichoderma reesei mutants on polyurethane foam impregnated with lactose medium. An extremely high value of about 2.6 FPU/ml was reached after the cultivation of T. reesei D-78085 on a 0.5% lactose medium in continuous culture at a pH medium of 4.0 when a bioreactor with vertical polyurethane foam plates was used. The enzyme yield on lactose was 520 FPU/g of lactose metabolized in comparison with 160 FPU/g using a stirred tank bioreactor.     

Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 μL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96‐well microtiter plate, to preserve compatibility with the current high‐throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO₂ production rate was obtained from the online measurement of cumulative CO₂ production during the cultivation. The online data obtained by the sensor array and the CO₂ production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench‐scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro‐scale fermentations, e.g. using the 96‐well microtiterplate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Optimization of culture conditions for production of the anti-tubercular alkaloid hirsutellone A by Trichoderma gelatinosum BCC 7579

AIMS: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. METHODS AND RESULTS: The fungus was initially cultured in shake flasks at 25 degrees C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l(-1), obtained from basal condition, to 610.55 mg l(-1) and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min(-1) at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l(-1) of hirsutellone A within 6 days. CONCLUSIONS: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with an initial pH of 7. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future.     

Accumulation of organic acids in cultivations of Neisseria meningitidis C

Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 l min⁻¹. A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O₂) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O₂ control, varying glucose concentration from 1.0 to 3.0 g l⁻¹, nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O₂ control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O₂ control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.     

12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors.

A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.     

Scale-up and optimization of culture conditions of a human heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa

Summary Three different stirred bioreactors of 0.5 to 12 l volume were used to scale up the production of a human monoclonal antibody. Inoculation density and stirrer speed were evaluated in batch cultures, whereas dilution rate and pH were optimized in chemostat cultures with respect to high specific antibody production rate and high antibody yield per time and reactor volume. The cell line used for the experiments was a heterohybridoma, producing immunoglobulin M (IgM) against lipopolysaccharide of Pseudomonas aeruginosa. Cells were cultured in spinner flasks of 500 ml liquid volume for adaptation to stirred culture conditions. Subsequently cells were transferred to the 1.5-1 KLF 2000 bioreactor and to the 12-1 NLF 22 bioreactor for pilot-scale cultures. Chemostat experiments were done in the 1.5-1 KLF bioreactor. Cell density, viability, glucose and lactate and antibody concentration were measured during culture experiments. In batch cultures in all three stirred bioreactors, comparable maximal cell densities and specific growth rates were achieved. Chemostat experiments showed that at a pH of 6.9 and a dilution rate of 0.57 per day the specific antibody production rate was threefold higher than similar experiments done at pH 7.2 with a dilution rate of 0.36 per day. By optimizing pH and dilution rate in chemostat cultures the daily yield of human IgM increased nearly threefold from 6 to 16 mg/day and per litre of reactor volume. The yield per litre of medium increased twofold. Correspondence to: U. Schürch