不同补料控制方式发酵生产头孢菌素C的性能比较

在7 L发酵罐下,对利用顶头孢霉菌(Cephalosporins acremonium)发酵生产头孢菌素C(CPC)过程的最优底物流加工艺进行了研究。提出了一种新式硫铵豆油耦联型的硫铵流加策略。该控制策略可将发酵液中的氨态氮浓度控制在3 6 g/L之间,同时满足了发酵前期细胞生长与CPC合成对氮源和硫源的需求,促进了顶头孢霉菌菌丝分化,为发酵后期的CPC高效生产奠定了前期基础。比较了CPC合成期内间歇、匀速和DO-Stat自动流加3种不同豆油流加方式的发酵性能。研究发现,耦联使用硫铵/后程通富氧空气DO-Stat法进行硫铵和豆油的同时补料和CPC发酵,可将碳源浓度与溶解氧浓度DO同时控制于适中水平,使CPC合成以高浓度和低副产物积累的方式进行,最终CPC浓度和得率分别达到35.77 g/L和13.3%。主代谢副产物脱乙酰氧头孢菌素C(DAOC)的积累量和DAOC/CPC分别仅有0.178 g/L和0.5%。     

混合碳源发酵生产头孢菌素C过程优化

本文对头孢菌素C（Cephalosporin C,CPC）发酵过程中碳源补料控制策略进行了优化研究,提出了一种基于DO—Stat的混合碳源流加策略,提高了发酵整体性能。在7L发酵罐上对使用该策略和传统补油策略的头孢菌素发酵性能进行比较,结果表明,采用补加混合碳源（葡萄糖＋豆油）策略时,CPC终浓度最高,达到36．99g／L,CPC得率也从使用传统单纯补油策略时的11．39％提高到22．19％,代谢副产物去乙酰氧头孢菌素C（DAOC）的积累量少,DAOC／CPC只有0．38％,达到生产要求。     

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.     

Improved industrial fermentation of lincomycin by phosphorus feeding

Phosphorus limitation was found in the fermentation production of lincomycin based on the phosphorus elemental analysis. Phosphorus was thus fed into the fermentation system to improve the lincomycin production, and 16 kg fed-phosphorus increased the lincomycin yield by 14.4% compared to that without the phosphorus feeding. As low concentration of dissolved oxygen limited the growth of mycelia, the phosphorus in the base medium and fed-batch were adjusted to give a more reasonable phosphorus distribution. When the phosphorus in the base medium was decreased to 29.4 kg from 33.4 kg and the fed phosphorus was increased from 16.0 kg to 20.0 kg in a 100 m³ fermenter, the final lincomycin titer increased by 21.6% compared to that the un-fed process. The mycelia growth and lincomycin production rates were also increased at the production stage. The phosphorus feeding and adjustment distribution strategy might be applied to other industrial fermentation processes to improve the process efficiency and productivity.     

Bioprocess strategies for mass multiplication of and metabolite synthesis by plant growth promoting pseudomonads for agronomical applications

The exponential substrate feeding (open-loop) and automated feedback substrate feeding (closed loop) strategies were developed to obtain high cell densities of fluorescent pseudomonad strains R62 and R81 and enhanced production of antifungal compound 2,4-diacetylphloroglucinol (DAPG) from glycerol as a sole carbon source. The exponential feeding strategy resulted in increased glycerol accumulation during the fed-batch cultivation when the predetermined specific growth rate (μ) was set at 0.10 or 0.20 h^?1 (<μ_m = 0.29 h^?1). Automated feeding strategies using dissolved oxygen (DO) or pH as feedback signals resulted in minimal to zero accumulation of glycerol for both the strains. In case of DO-based feeding strategy, biomass productivity of 0.24 g/(L h) and 0.29 g/(L h) was obtained for R62 and R81, respectively. Using pH-based feeding strategy, biomass productivity could be increased to a maximum of 0.51 and 0.54 g/(L h), for the strains R62 and R81, respectively, whereas the DAPG concentration was enhanced to 298 mg/L for R62 and 342 mg/L for R81 strains. These yields of DAPG are thus far the highest reported from GRAS organisms.     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Production enhancement of Rhizopus arrhizus lipase by feeding oleic acid

A strategy for Rhizopus arrhizus lipase production enhancement by feeding oleic acid was developed. The oleic acid was proved to have strong inducing effect on lipase production, but high concentration oleic acid could repress lipase production. The decrease rate of oleic acid concentration using peanut oil as initial carbon source was figured out according to the change of oleic acid concentration in the fermentation broth. Our feeding strategy designed based on the decrease rate of oleic acid could avoid the repression of lipase production that is caused by high concentration of oleic acid in the fermenting liquor, and this strategy worked as a new feeding method showing excellent performance. The maximum lipase activity was gained by feeding dilute oleic acid every 12 h starting at 60 h, which maintained the oleic acid concentration around 18 mg/L, and the lipase activity was 31% higher than that of no feeding.     

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.     

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor

Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.     

A two-stage oxygen supply strategy for enhanced l-arginine production by Corynebacterium crenatum based on metabolic fluxes analysis

In this study, the metabolic flux distribution analysis of a new l-arginine (Arg) overproducing strain, Corynebacterium crenatum, was carried out under various oxygen supply conditions in order to explore the optimized oxygen supply profile. The metabolic flux analysis indicated that a relatively higher l-arginine production could be obtained under high oxygen supply (HOS) condition overall. However, during the late fermentation phases, a much more stable l-arginine production could be rather achieved under medium oxygen supply (MOS) condition. As a result, a two-stage oxygen supply strategy, which maintained HOS condition during early fermentation phase, and then step-wisely reduced agitation to keep a stable, smooth and moderate dissolve oxygen levels (DO) changing profile throughout the production phases, was proposed. With the proposed control strategy, the final l-arginine concentration of the batch fermentation was largely increased and reached to a high level of 36.6 g L^?1, which was 16% and 51% higher than those obtained under the HOS and MOS conditions. The two-stage oxygen supply strategy could also accelerate glucose consumption rate and thus shorten fermentation time under the same batch initial fermentation condition. The relevant metabolic flux analysis verified the effectiveness of the proposed control strategy.     

Xylitol production from non-detoxified corncob hemicellulose acid hydrolysate by Candida tropicalis

The interest on use of lignocellulose for producing chemicals is increasing as these feedstocks are low cost, renewable and widespread sources of sugars. Corncob is an attractive raw material for xylitol production due to its high content of xylan. In this study, hemicellulose hydrolysate from corncobs without detoxification was used for xylitol production by Candida tropicalis CCTCC M2012462. Compared with prepared xylose medium, xylitol production with dilute acid hydrolysate medium does not seem to influence specific xylose reductase activity. The decrease in xylitol productivity with dilute acid hydrolysate medium is a result of a lower biomass concentration and lag-phase time. It appears that biomass growth rate is essential for xylitol production. In xylitol fermentation with a low initial inhibitors concentration and substrate feeding strategy, a maximal xylitol concentration of 38.8 g l⁻¹ was obtained after 84 h of fermentation, giving a yield of 0.7 g g⁻¹ xylose and a productivity of 0.46 g l⁻¹ h⁻¹.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

Enhancement of pilot scale production of l(+)-lactic acid by fermentation coupled with separation using membrane bioreactor

Traditional batch fermentation leads to a higher energy consumption and lower production capability because of longer culture time. In this work, a pilot scale bioreactor composed of a 3000 L fermentor and external ceramic microfiltration equipment was used to perform cell-recycle fermentation. Repeat feeding medium was also used to relieve the substrate inhibition. In such pilot system, the maximum yield and productivity of l(+)-lactic acid production reached 157.22 ± 3.42 g/L and 8.77 ± 0.15 g/L/h which were 4.23% and 315.64% higher than those of batch fermentation, respectively, when equal amount of sugar was consumed. The cost of l(+)-lactic acid production was successfully reduced about two-thirds by the increase of yield and productivity. 12 rounds of cell-recycle fermentations were successfully achieved in the pilot system. The membrane filtration productivity reached to 61.27 ± 2.74 L/m²/h which increased 172.80%, while the cell damaging rate dropped to 3.88 ± 0.18% which decreased 85.77%, compared with those of the ultrafiltration. Furthermore, the ceramic microfiltration membrane showed advantages in tolerance for the temperature, pressure and acid, compared with the organic ultrafiltration membrane. The experimental results indicated that the method could give a reference for low cost production of l(+)-lactic acid in an industrial scale.     

Redox potential-driven repeated batch ethanol fermentation under very-high-gravity conditions

When Saccharomyces cerevisiae was cultivated under ～200 g glucose/l condition, the time point at which glucose was completely utilized coincided with the moment at which the slope of a redox potential profile changed from negative or zero to positive. Based on this feature, a redox potential-driven glucose-feeding fermentation operation was developed, and resulted in a self-cycling period of 14.25 ± 0.4 h. The corresponding ethanol concentration was maintained at 88.4 ± 1.0 g/l with complete glucose conversion, and the cell viabilities increased from 80% in the transition period to 97.2 ± 1.1%, implying the occurrence of yeast acclimatization. In contrast, a pre-determined 36-h manually adjusted period was chosen to oscillate yeast cells under ～250 g glucose/l conditions, which resulted in 106.76 ± 0.7 g ethanol/l and 15.19 ± 1.3 g glucose/l remaining at the end of each cycle. Compared to the equivalent batch and continuous ethanol fermentation processes, the annual ethanol productivity of the reported fermentation operation is 2.4% and 13.2% greater, respectively in ～200 g feeding glucose/l conditions.     

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r = 0.8–0.9) and dilution (up to 0.48 h^?1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160 g/l at r = 0.9 and dilution rate of 0.2 h^?1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5 g/l h^?1 could be achieved with feeding concentrations of 80–200 g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48 g/l h^?1.     

Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production

To improve the growth of recombinant Pichia pastoris with a phenotype of Mut^S and expression of angiostatin, the effects of glycerol, sorbitol, acetate and lactic acid which were, respectively, added together with methanol in the expression phase, were studied in a 5-l fermentor. Methanol concentration was automatically controlled at 5 g/l by a methanol monitor and control system, while the feeding of the other carbon source was manually adjusted. The angiostatin production level was 108 mg/l when glycerol was added at an initial rate of 2.3 g/h and gradually increased to 9.9 g/h within an induction period of 96 h. The angiostatin concentration was 141 mg/l as sorbitol was used, while only 52 mg/l were obtained on acetate. The highest angiostatin production of 191 mg/l was achieved as lactic acid was used; whose feeding rate was gradually increased from 2.6 to 11.3 g/h. Lactic acid accumulated during the induction phase and reached 6.3 g/l at the end of fermentation. However, the accumulation of lactic acid did not interfere with angiostatin production, indicating that lactic acid to be a non-repressive carbon source. The average productivity and specific productivity of angiostatin obtained on lactic acid and methanol were, respectively, 2.96 and 0.044 mg/(g h), 1.7- and 2.5-fold of those obtained in the fermentation fed with glycerol and methanol.     

Multi-fed batch culture integrated with three-stage light irradiation and multiple additions of copper ions for the hyperproduction of ganoderic acid and Ganoderma polysaccharides by the medicinal mushroom Ganoderma lucidum

To further enhance the accumulation of the bioactive metabolite ganoderic acid (GA) by fermentation of the medicinal mushroom Ganoderma lucidum, a novel integrated strategy was developed by simultaneously adopting a strategy of multiple Cu²⁺ additions, three-stage light irradiation and multi-pulse feeding of carbon and nitrogen sources. Maximal GA content (i.e., 4.1 mg/100 mg DW) and production (i.e., 720.8 mg/L) were obtained using the novel integrated strategy. Not only the biomass but also the total GA production obtained in this work is the highest reported for a shaker flask culture of G. lucidum. This work is useful for the large-scale production of GA by G. lucidum fermentation.     

2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.