Mixed carbon source control strategy for enhancing alginate lyase production by marine Vibrio sp. QY102

Medium and culture conditions for alginate lyase production by marine Vibrio sp. QY102 were first optimized using statistical methods including Plackett–Burman design and central composite design. Then, fermentation in 5-L bioreactor showed that alginate acted as easily used carbohydrate for Vibrio sp. QY102, while starch extended its growth phase and stabilized pH variations. Thus, a novel strategy using mixed carbon sources was proposed that starch supported growth while enzyme synthesis was induced by pulse feedings of solid alginate. The optimized process followed that Vibrio sp. QY102 grew on starch until the end of the logarithmic growth phase, and then solid alginate was added as 1 g/L every 3 h. Meanwhile, initial pH 5.0 and natural pH during fermentation was favorable for alginate lyase production. After optimization, the highest alginate lyase production reached 52.8 U/mL, which was 329 % higher than the control. Finally, fermentation scale-up was performed in 30-L bioreactor and the maximum alginate lyase production was obtained as 46.8 U/mL.     

Light regulation on growth, development, and secondary metabolism of marine-derived filamentous fungi

Effects of different light conditions on development, growth, and secondary metabolism of three marine-derived filamentous fungi were investigated. Darkness irritated sexual development of Aspergillus glaucus HB1-19, while white, red, and blue lights improved its asexual behavior. The red and blue lights improved asexual stroma formation of Xylaria sp. (no. 2508), but the darkness and white light inhibited it. Differently, development of Halorosellinia sp. (no. 1403) turned out to be insensitive to any tested light irradiation. Upon the experimental data, no regularity was observed linking development with secondary metabolism. However, fungal growth showed inversely correlation with productions of major bioactive compounds (aspergiolide A, 1403C, and xyloketal B) from various strains. The results indicated that aspergiolide A biosynthesis favored blue light illumination, while 1403C and xyloketal B preferred red light irradiation. With the favorite light sensing conditions, productions of aspergiolide A, 1403C, and xyloketal B were enhanced by 32.9, 21.9, and 30.8 % compared with those in the dark, respectively. The phylogenetic analysis comparing the light-responding proteins of A. glaucus HB 1-19 with those in other systems indicated that A. glaucus HB 1-19 was closely related to Aspergillus spp. especially A. nidulans in spite of its role of marine-derived fungus. It indicated that marine fungi might conserve its light response system when adapting the marine environment. This work also offers useful information for process optimization involving light regulation on growth and metabolism for drug candidate production from light-sensitive marine fungi.     

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.     

Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture

This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.     

Roles of cobalt in biosynthesis stimulation of a cytotoxic compound from marine-derived Aspergillus glaucus

Effects of trace elements on aspergiolide A biosynthesis by marine-derived filamentous fungus Aspergillus glaucus HB 1–19 were investigated. Cobalt (Co²⁺) and nickel (Ni²⁺) stimulated aspergiolide A biosynthesis while zinc (Zn²⁺) inhibited it. Equal cobalt (Co²⁺) addition at 48, 72 and 96 h with total amount of 0.052 mM most effectively enhanced aspergiolide A production. The time courses indicated that mycelia growth, sugar utilization and aspergiolide A biosynthesis were enhanced by Co²⁺. Finally, feeding 0.052 mM Co²⁺ increased mycelia growth, sugar utilization and aspergiolide A production by 12.2%, 13.7% and 46.7%, respectively. The results of organic acids analysis indicated that extracellular pyruvate increased while fumarate decreased in Co²⁺ addition cultures. Moreover, feeding vitamin B₁₂, which combined cobalt to be a cofactor form, increased aspergiolide A production by 59.1%. This indicated Co²⁺ functioned by facilitating pyruvate accumulation and increasing vitamin B₁₂ formation that were beneficial to aspergiolide A biosynthesis that is via polyketide pathway.     

Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum

Investigations on Ganoderma lucidum fermentation suggested that the responses of the cell growth and metabolites biosynthesis to pH and dissolved oxygen tension (DOT) were different. The ganoderic acid (GA) production of 321.6 mg/L was obtained in the pH-shift culture by combining a 4-day culture at pH 3.0 with the following 6-day culture at pH 4.5, which was higher by 45% and 300% compared with the culture at pH 3.0 and 4.5, respectively. The GA production of 487.1 mg/L was achieved in the DOT-shift culture by combining a 6-day culture at 25% of DOT with a following 6-day culture at 10% of DOT, which was higher by 43% and 230% compared with the culture at 25% and 10% of DOT, respectively. A fed-batch fermentation process by combining the above-mentioned pH-shift and DOT-shift strategies resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which is the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactor.     

Statistical optimization of medium composition for aspergiolide A production by marine-derived fungus <Emphasis Type="Italic">Aspergillus glaucus</Emphasis>

Statistical methodologies were employed to optimize submerged culture medium for the production of a novel antineoplastic compound aspergiolide A by a marine-derived fungus Aspergillus glaucus HB1-19 for the first time. Orthogonal design was preformed to determine the initial composition. Then Plackett–Burman design was applied to evaluate the influence of related nutrients, and yeast extract paste, soybean powder and sodium glutamate were confirmed as critical factors in the medium. Response surface methodology (RSM) was finally taken as an effective approach to optimize the combination of the obtained three significant factors. The predicted maximal aspergiolide A production of 62.4 mg/L appeared at the region where the concentrations of sodium glutamate, soybean powder, and yeast extract paste were 2, 1, and 1.07 g/L, respectively. Under the proposed optimized conditions, the experimental aspergiolide A production reached 71.2 mg/L. The correlation between predicted value and measured value of these experiments proved the validity of the response model. After optimization, aspergiolide A production increased 4.22 times compared to that of the original medium. Elemental analysis was finally taken into consideration, and carbon–nitrogen ratio in the medium increased from 20.1:1 to 86.6:1. This great difference was inferred as the most important reason for production enhancement by metabolic pathway analysis.     

Effects of dissolved oxygen and pH levels on weissellin A production by Weissella paramesenteroides DX in fermentation

Experiments carried out with the dissolved oxygen tension (DOT) maintained during fermentation at 0, 10, 50, 70 and 100% showed a direct effect of the dissolved oxygen levels on weissellin A production with no correlative increase on biomass. An estimate of the yield of weissellin A per gram biomass revealed the 50% DOT level as the optimum for increased yields. The effect of pH was studied in experiments carried out without pH control, with pH initially set at 6.0, 5.0 and 4.5 and with pH controlled at 6.0, 5.0 and 4.5. The initial pH value and the pH-drop gradient appear to be the important parameters for weissellin A production. Production was significantly higher with the uncontrolled initial pH compared to that of the controlled initial pH at 6.0, while acidic initial pHs created unfavorable conditions for production. Maintaining a constant pH environment during fermentation led to decreased production levels.     

Enhancing aspergiolide A production from a shear-sensitive and easy-foaming marine-derived filamentous fungus Aspergillus glaucus by oxygen carrier addition and impeller combination in a bioreactor

Production enhancement of a novel antitumor compound aspergiolide A from shear-sensitive and easy-foaming marine-derived fungus Aspergillus glaucus HB1-19 in a 5-l stirred bioreactor was investigated. Two types of impellers, i.e., six-flat-blade disc turbine impeller (DT) and three-sector-blade pitched blade turbine impeller (PB) were used in this work. In cultures with fermentation medium, the combination of upper PB and lower DT led to the maximum dry biomass (13.8 g/l) and aspergiolide A production (19.3 mg/l). However, two PBs brought the highest aspergiolide A yield coefficient (1.9 mg/g dry biomass) despite it produced the lowest dry biomass (5.3 g/l). By contrast, two DTs and the upper DT and lower PB showed insignificant results. Feeding 0.35% (v/v) n-dodecane in cultures with upper PB and lower DT further improved aspergiolide A production by 31.0%, i.e., 25.3 mg/l, which is also 322% higher than that in the ordinary cultures with two DTs.     

Improvement of welan gum production and redistribution of metabolic flux under pH control process in Alcaligenes sp. CGMCC2428

Batch fermentations of welan gum from Alcaligenes sp. CGMCC2428 at pH values of 5.5 ～ 7.0 were studied. Based on the kinetic analysis, a pH control process for improving welan production was developed. By maintaining the culture pH at 7.0, the process significantly improved the maximal welan concentration and productivity to reach 25.1 ± 0.65 g/L and 0.42 ± 0.003 g/L/h, respectively, compared with those in native pH processes where pH value would decrease from 7.0 to 5.1 (18.5 ± 0.72 g/L and 0.28 ± 0.002 g/L/h). This improvement may be due to the increased metabolic flux of glucose-1-phosphate to welan induced by pH control process. Furthermore, scale-up fermentation under controlled pH was implemented using 300-L fermentor. The highest welan concentration of 27.5 ± 0.97 g/L was obtained. This simplified process proved effective in industry fermentation for high welan production.     

Selection method of pH conditions to establish Pseudomonas taetrolens physiological states and lactobionic acid production

Microbial physiological responses resulting from inappropriate bioprocessing conditions may have a marked impact on process performance within any fermentation system. The influence of different pH-control strategies on physiological status, microbial growth and lactobionic acid production from whey by Pseudomonas taetrolens during bioreactor cultivations has been investigated for the first time in this work. Both cellular behaviour and bioconversion efficiency from P. taetrolens were found to be negatively influenced by pH-control modes carried out at values lower than 6.0 and higher than 7.0. Production schemes were also influenced by the operational pH employed, with asynchronous production from damaged and metabolically active subpopulations at pH values lower than 6.0. Moreover, P. taetrolens showed reduced cellular proliferation and a subsequent delay in the onset of the production phase under acidic conditions (pH?<?6.0). Unlike cultivations performed at 6.5, both pH-shift and pH-stat cultivation strategies performed at pH values lower than 6.0 resulted in decreased lactobionic acid production. Whereas the cellular response showed a stress-induced physiological response under acidic conditions, healthy functional cells were predominant at medium operational pH values (6.5–7.0). P. taetrolens thus displayed a robust physiological status at initial pH value of 6.5, resulting in an enhanced bioconversion yield and lactobionic acid productivity (7- and 4-fold higher compared to those attained at initial pH values of 4.5 and 5.0, respectively). These results have shown that pH-control modes strongly affected both the physiological response of cells and the biological performance of P. taetrolens, providing key information for bio-production of lactobionic acid on an industrial scale.     

Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods

Polysaccharides and ganoderic acids (GAs) are the major bioactive constituents of Ganoderma species. However, the commercialization of their production was limited by low yield in the submerged culture of Ganoderma despite improvement made in recent years. In this work, twelve Ganoderma strains were screened to efficiently produce polysaccharides and GAs, and Ganoderma lucidum 5.26 (GL 5.26) that had been never reported in fermentation process was found to be most efficient among the tested stains. Then, the fermentation medium was optimized for GL 5.26 by statistical method. Firstly, glucose and yeast extract were found to be the optimum carbon source and nitrogen source according to the single-factor tests. Ferric sulfate was found to have significant effect on GL 5.26 biomass production according to the results of Plackett–Burman design. The concentrations of glucose, yeast extract and ferric sulfate were further optimized by response surface methodology. The optimum medium composition was 55 g/L of glucose, 14 g/L of yeast extract, 0.3 g/L of ferric acid, with other medium components unchanged. The optimized medium was testified in the 10-L bioreactor, and the production of biomass, IPS, total GAs and GA-T enhanced by 85, 27, 49 and 93 %, respectively, compared to the initial medium. The fermentation process was scaled up to 300-L bioreactor; it showed good IPS (3.6 g/L) and GAs (670 mg/L) production. The biomass was 23.9 g/L in 300-L bioreactor, which was the highest biomass production in pilot scale. According to this study, the strain GL 5.26 showed good fermentation property by optimizing the medium. It might be a candidate industrial strain by further process optimization and scale-up study.     

Production of tetramethylpyrazine by batch culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> with optimal pH control strategy

The effects of initial culture pH ranging from 5.0 to 7.5 on biomass content, precursor 3-hydroxy-2-butanone (HB) accumulation, and 2,3,5,6-tetramethylpyrazine (TTMP) formation by Bacillus subtilis CCTCC M 208157 were investigated in shake flask fermentation. Weak acidic conditions were found to favor cell growth and precursor HB accumulation, while TTMP could be synthesized more efficiently in conditions with initial pH towards neutrality. Batch bioprocess of TTMP fermentation by Bacillus subtilis CCTCC M 208157 at various controlled pH values ranging from 5.5 to 7.0 was then examined in 7.5-l fermentor. The results suggested that optimum pH for cell growth and precursor HB accumulation was 5.5 with maximum cell growth rate (Q _x) and precursor HB accumulation rate (Q _HB) of 0.833 g l⁻¹ h⁻¹ and 1.118 g l⁻¹ h⁻¹, respectively, while optimum pH for TTMP formation was 7.0 with maximum TTMP formation rate (Q _TTMP) of 0.095 g l⁻¹ h⁻¹. A pH-shifted strategy was accordingly developed to improve TTMP production in bioreactor fermentation by shifting the culture pH from 5.5 to 7.0 after 48 h of cultivation. By applying the strategy, final TTMP concentration of 7.43 g l⁻¹ was obtained, being 22.2% greater than that of constant-pH fermentation.     

Improvement for the production of clavulanic acid by mutant Streptomyces clavuligerus

AIMS: To improve the production of clavulanic acid through the development of strains, the selection of a production medium and a pH shift strategy in a bioreactor. METHODS AND RESULTS: Streptomyces clavuligerus mutant 15 was selected by antibacterial activities. As a result of pH control in a 2.5 l bioreactor, the highest productivity (3.37 microg x ml(-1) x h(-1)) was obtained at a controlled pH of 7.0. CONCLUSIONS: The highest level of production obtained was an increase of about 36% compared with a non-controlled pH. When the production of clavulanic acid reached the maximum level, the pH was shifted from 7.0 to 6.0 for reduction in decomposition rate. The maximum concentration of clavulanic acid was maintained for 24 h as a result of the pH shift control, and a significant reduction in the decomposition of clavulanic acid was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: Clavulanic acid decomposition was considerably reduced as a result of the pH shift control. The results of this study can be applied for the efficient production of beta-lactamase inhibitory antibiotics.     

Staphylococcus aureus Enterotoxin B Release (Excretion) Under Controlled Conditions of Fermentation

Release of Staphylococcus aureus enterotoxin B (SEB) into the culture medium was initiated during the mid-log phase of growth. A medium consisting of 4% N-Z Amine A (Sheffield), 0.2% dextrose, and 1% yeast extract supported maximum production of SEB. Although pH of the medium during cultivation did not significantly affect the growth curve of the organism, the time required for detectable excretion was affected, as was the final yield. Optimal conditions for SEB production were achieved with pH control at 7.0; alkaline control (pH 8.0) produced only minimal amounts of toxin, whereas acid control (pH 6.0) resulted in 50% reduction in yield. Slightly less SEB was produced when there was no extrinsic pH control, and cultures were buffered only by media constituents and by-products of growth. With pH control at 7.0, deletion of 0.2% dextrose from the medium resulted in 40% reduction in the 8-h yield. There was also a delay in production during early stages of fermentation.     

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.     

Control of the production of individual fusicoccins at different dissolved oxygen concentrations

The regulatory effect of different concentrations of dissolved oxygen on the production of fusicoccins by the fungus Fusicoccum amygdali Del. was studied. The maximum output of total fusicoccins was obtained by using a profiled dissolved oxygen tension (DOT) regime, in which the DOT was maintained at 15–20% during the biomass growth phase and at 5–8% during the fusicoccins production phase. In comparison with the profiled regime, the maintenance of DOT at 15–20% during the whole fermentation shortened the fusicoccins production phase. The fermentation performance at a low DOT (5–8%) inhibited both the accumulation of biomass and the production of fusicoccins. At high DOT (40–50%), an accelerated accumulation of the biomass with an expressed autolysis of mycelia took place, and the production of fusicoccins was lowered. The qualitative composition of individual fusicoccins varied substantially at different DOTs. Fusicoccins, A, C, D, J, H, 16-O-demethyl-J, detretpentenylfusicoccin and some minor fusicoccin metabolites were found in the fermentation broth using the method of liquid secondary ion mass spectrometry. It was established that the profiled DOT regime (15–20% to 5–8%) provided both the maximum concentration of fusicoccins and an enhanced accumulation of the main metabolite – fusicoccin A (FC A). The performance of the fermentation at a DOT of 15–20% decreased the content of FC A by 2–6% in comparison with the profiled DOT regime, and increased the content of fusicoccin C to 14–20% of the total fusicoccins. Fermentation at DOT of 5–8% was characterized by the highest content of the precursors of FC A, the less oxidized fusicoccins H and J, the contents of which were in range 7–12% and 16–17% of total fusicoccins, respectively.     

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

Enhanced production of valienamine by Stenotrophomonas maltrophilia with fed-batch culture in a stirred tank bioreactor

Valienamine is an important medicinal intermediate with broad use in the synthesis of some stronger α-glucosidase inhibitors. In order to improve valienamine concentration in the fermentation broth and make the downstream treatment easy, a fed-batch process for the enhanced production of valienamine by Stenotrophomonas maltrophilia in a stirred tank bioreactor was developed. Results showed that supplementation of validamycin A in the process of cultivation could increase the valienamine concentration. One-pulse feeding was observed to be the best strategy. The maximum valienamine concentration of 2.35 g L⁻¹ was obtained at 156 h when 86.4 g of validamycin A was added to a 15-L bioreactor containing 8 L fermentation medium with one-pulse feeding. The maximum valienamine concentration had a great improvement and was increased above 100% compared to batch fermentation in the stirred tank bioreactor. The pH-controlled experiments showed that controlling the pH in the process of one-pulse feeding fermentation had not obvious effect on the production of valienamine.     

Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations

Single-use stirred tank bioreactors on a 10-mL scale operated in a magnetic-inductive bioreaction block for 48 bioreactors were equipped with individual stirrer-speed tracing, as well as individual DO- and pH-monitoring and control. A Hall-effect sensor system was integrated into the bioreaction block to measure individually the changes in magnetic field density caused by the rotating permanent magnets. A restart of the magnetic inductive drive was initiated automatically each time a Hall-effect sensor indicates one non-rotating gas-inducing stirrer. Individual DO and pH were monitored online by measuring the fluorescence decay time of two chemical sensors immobilized at the bottom of each single-use bioreactor. Parallel DO measurements were shown to be very reliable and independently from the fermentation media applied in this study for the cultivation of Escherichia coli and Saccharomyces cerevisiae. The standard deviation of parallel pH measurements was pH 0.1 at pH 7.0 at the minimum and increased to a standard deviation of pH 0.2 at pH 6.0 or at pH 8.5 with the complex medium applied for fermentations with S. cerevisiae. Parallel pH-control was thus shown to be meaningful with a tolerance band around the pH set-point of ± pH 0.2 if the set-point is pH 6.0 or lower.