Two-stage oxygen supply strategy for enhanced lipase production by Bacillus subtilis based on metabolic flux analysis

Lipase production by Bacillus subtilis CICC20034 was assessed by metabolic flux distribution analysis. Lipase production was tested under various oxygen supply conditions in a synthetic medium to obtain the optimal oxygen supply profile. Based on the metabolic flux analysis, a two-stage oxygen supply strategy (TOS) that maintained high oxygen supply conditions during early fermentation phase, and then step-wisely reduced aeration to keep a stable, smooth, and adequate changing dissolved oxygen (DO) level profile throughout the production phases was carried out. With the proposed control strategy, the final lipase activity in batch fermentation significantly increased and reached a high level at 0.56 U/ml, corresponding to a 51% increase. The relevant metabolic flux analysis verified the effectiveness of the proposed control strategy. By applying TOS in composite medium, the final lipase activity reached 5.0 U/ml.     

Production of gluconic acid from glucose by Aspergillus niger: growth and non-growth conditions

Batch fermentation of glucose to gluconic acid was conducted using Aspergillus niger under growth and non-growth conditions using pure oxygen and air as a source of oxygen for the fermentation in 2 and 5 l stirred tank reactors (batch reactor). Production of gluconic acid under growth conditions was conducted in a 5 l batch reactor. Production and growth rates were higher during the period of supplying pure oxygen than that during supplying air, and the substrate consumption rate was almost constant. For the production of gluconic acid under non-growth conditions, conducted in the 2 l batch reactor, the effect of the pure oxygen flow rate and the biomass concentration on the gluconic acid production was investigated and an empirical equation suggested to show the dependence of the production rate r_p on the biomass concentration C_x and oxygen flow rate Q, at constant operating conditions (30 °C, 300 rpm and pH 5.5). Biomass concentration had a positive effect on the production rate r_p, and the effect of Q on r_p was positive at high biomass concentrations.     

Improvement of the intracellular environment for enhancing l-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply

l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H₂O₂-forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H₂O₂ synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3 g/L and 0.326 g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H₂O₂ synthesis and increasing the ATP supply.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

A pH control strategy for increased β-carotene production during batch fermentation by recombinant industrial wine yeast

The effects of different pH values, ranging from 4.0 to 7.0, on cell growth and β-carotene production by recombinant industrial wine yeast Saccharomyces cerevisiae T73-63 in a synthetic grape juice medium was investigated. Based on the kinetic analysis of the batch fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 7.0 for the first 24 h and then shifted to 5.0 after 24 h. Using this strategy, the highest β-carotene production (50.39 mg/l) and the formation rate (1.40 mg/l/h) were increased by 19.1% and 18.6%, respectively, compared to the maximum values of constant pH fermentation. The oxidative stress during β-carotene production was also determined in terms of the catalase (CAT) and superoxide dismutase (SOD) activities. Oxidative stress appears to be induced by the lowering of pH as indicated by the increase in activities of CAT and SOD due to pH shift from pH 7.0 to pH 5.0. Pre-treating cells with ascorbic acid (an antioxidative agent) reversed the improvement of β-carotene production while addition of H₂O₂ enhanced it. Considering that induction of oxidative stress is associated with increased β-carotene production, it was concluded that the enhancement of β-carotene production by the low-pH strategy involved the induction of oxidative stress.     

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL⁻¹ in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

Integrated sophorolipid production and gravity separation

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fermentation broth, while cells and other media components are recirculated and returned to the bioreactor. The capability of the separation system to recover an enriched sophorolipid product phase was demonstrated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recoveries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume. These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high volume applications such as enhanced oil recovery economically feasible.     

Achieving high strength vinegar fermentation via regulating cellular growth status and aeration strategy

Implementing of high strength vinegar fermentation is still the mission of vinegar producers. The aim of this study was to carry out high acidity vinegar fermentation efficiently based on comprehensive analysis on bacterial fermentation kinetics characteristics of Acetobacter pasteurianus CICIM B7003-02. In practice, semi-continuous vinegar fermentation was optimized with an optimal discharge/charge ratio of 34% of working volume (v/v), which resulted in a proper growth status of Acetobacter and beneficial to acetification. Then, a two-stage aeration protocol was adopted in the vinegar fermentation in line with the Acetobacter theoretical oxygen demand, by which both vinegar stoichiometric yield and acetification rate were improved effectively. As the final result, a titer of 93.09 ± 0.24 g/L acetic acid was achieved, the average acetification rate was enhanced to a level of 1.83 ± 0.01 g/L/h, and the vinegar stoichiometric yield was promoted to 93.97 ± 0.16%. The strategy and practice worked out from this study provided a valuable reference for performing large scale vinegar fermentation with higher strength.     

Correlation of pigment production with mycelium morphology in extractive fermentation of Monascus anka GIM 3.592

Extractive fermentation with nonionic surfactants is a potential method for producing Monascus pigments. In this study, the correlation between mycelium morphology and pigment production was investigated in extractive fermentation of Monascus anka GIM 3.592. The results demonstrated that pigment biosynthesis was associated with mycelial morphology and the accumulation of granular inclusions in cells. The physiological status in terms of hyphal and pellet diameters exhibited an excellent correlation with pigment accumulation, especially the yield of extracellular pigment in extractive fermentation (r > 0.85, p < 0.05). Nonionic surfactants could reduce pigment yield by influencing the morphology of hyphae and mycelium pellets. High yields of both intracellular and extracellular pigments could be achieved by controlling variations in hyphal diameters in two-stage extractive fermentation. Moreover, continuous extractive fermentation led to stable pigment production, with a relatively high productivity of total pigments reaching 72.3 AU/day. This study proposed a simple method for monitoring pigment biosynthesis in extractive fermentation using mycelium morphology as an indicating factor.     

Impact of pH and butyric acid on butanol production during batch fermentation using a new local isolate of Clostridium acetobutylicum YM1

The effect of pH and butyric acid supplementation on the production of butanol by a new local isolate of Clostridium acetobutylicum YM1 during batch culture fermentation was investigated. The results showed that pH had a significant effect on bacterial growth and butanol yield and productivity. The optimal initial pH that maximized butanol production was pH 6.0 ± 0.2. Controlled pH was found to be unsuitable for butanol production in strain YM1, while the uncontrolled pH condition with an initial pH of 6.0 ± 0.2 was suitable for bacterial growth, butanol yield and productivity. The maximum butanol concentration of 13.5 ± 1.42 g/L was obtained from cultures grown under the uncontrolled pH condition, resulting in a butanol yield (Y_P/S) and productivity of 0.27 g/g and 0.188 g/L h, respectively. Supplementation of the pH-controlled cultures with 4.0 g/L butyric acid did not improve butanol production; however, supplementation of the uncontrolled pH cultures resulted in high butanol concentrations, yield and productivity (16.50 ± 0.8 g/L, 0.345 g/g and 0.163 g/L h, respectively). pH influenced the activity of NADH-dependent butanol dehydrogenase, with the highest activity obtained under the uncontrolled pH condition. This study revealed that pH is a very important factor in butanol fermentation by C. acetobutylicum YM1.     

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.     

Production of polysaccharides from Ganoderma lucidum (CCRC 36041) under limitations of nutrients

This research studied the production of polysaccharides from Ganoderma lucidum under the various limitations of nutrients, including carbon-source, nitrogen-source, phosphate-source, magnesium-source, and dissolved oxygen. The different responses of polysaccharide production were observed under different limitations of nutrients. The concentration of polysaccharides was from 1.79 g/L decreasing to 0.91 g/L when the concentration of glucose was from 60 g/L decreasing to 20 g/L. The highest specific polysaccharide production was found at 60 g/L glucose media with 0.299 g/g-cell, and the lowest molecular weight was found in carbon-source limitation. Under nitrogen-source limitation the concentration of cells was low, but both polysaccharide production (1.61 g/L) and specific polysaccharide production (0.492 g/g-cell) were the highest. The lowest molecular weight of polysaccharides was found under nitrogen-source limitation. Both the phosphate-source and magnesium-source limitations showed low cell growth. With the phosphate-source limitation both low polysaccharide production and a lower molecular weight of polysaccharides was found. In the magnesium-source limitation low polysaccharide production, but a higher molecular weight of polysaccharides, was found. For the factor of oxygen supply the best polysaccharide production was found with sufficient oxygen for the first 5 days’ cultivation and then, after changing to oxygen limitation for another 5 days’ cultivation. On the other hand, the highest molecular weight of polysaccharides was found from the beginning with oxygen limitation throughout the process of fermentation.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Effects of oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) and the metabolic characterization of Bacillus subtilis NX-2

The effects of different oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) (PGA) were investigated in the batch fermentation of Bacillus subtilis NX-2. n-Hexane, n-heptane, and n-hexadecane enhanced the PGA concentration and molecular weight. The PGA concentration reached a maximum of 39.4 ± 0.19 g L^?1, and the highest molecular weight obtained was (19.0 ± 0.02) × 10⁵ Da with the addition of 0.3% n-heptane. However, n-dodecane decreased the PGA concentration and molecular weight to final values of 20.1 ± 0.10 g L^?1 and (8.4 ± 0.02) × 10⁵ Da, respectively. Analysis of the intracellular nucleotide levels of B. subtilis NX-2 with n-heptane and n-dodecane additives showed that the lowest NADH/NAD⁺ ratio and ATP levels were obtained with the n-dodecane additives, which can explain the decreased PGA yield and molecular weight. The metabolic flux distribution of B. subtilis NX-2 with n-heptane and n-dodecane additives was also investigated. Flux distribution was primarily directed to the EMP and TCA cycles with n-heptane additives. The flux of 2-oxoglutarate to intracellular glutamate and the flux distribution from extracellular to intracellular glutamate both increased to improve PGA production.     

The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7 g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639 g/(l h).