Two-stage Fermentation Strategy Research Articles

Assessing the potential of Schizochytrium sp. HX-308 for microbial lipids production from corn stover hydrolysate.

Schizochytrium sp. has received increasing attention as promising commercial resource for the sustainable production of lipids, due to their fast growth rate and high lipid content. However, the price of glucose represents a significant proportion of the total substrate cost. Therefore, in this study, the lignocellulosic hydrolysate of corn stover hydrolysate (CSH) was used as low-cost culture medium to replace glucose in Schizochytrium sp. fermentation. When Schizochytrium sp. HX-308 was fermented with 20% glucose from CSH and 80% of glucose from pure glucose, the lipid production reached 21.2gL-1 , which is lower than that of using 100% of pure glucose. However, the shifts of fatty acid composition indicated that CSH has great potential to enhance the percentage of polyunsaturated fatty acids (PUFAs) in total lipids. However, as the second largest carbon source in CSH, xylose was not utilized by the Schizochytrium sp. HX-308, and further analysis showed that probably because it does not possess a functional xylulose kinase. In addition, the degradation products in lignocellulosic hydrolysate have a strong inhibitory effect on cell growth, so it is necessary to investigate the tolerance of Schizochytrium sp. HX-308 to degradation products. Here, the effects of five typical degradation products on the growth and lipid synthesis were further investigated. Schizochytrium sp. HX-308 showed good tolerance to furan derivatives and organic acids, but low tolerance to phenolic compounds. Furthermore, in order to improve the lipid accumulation using CSH, the two-stage fermentation strategy was developed, resulting in a 54.8% increase compared to that of the one-stage strategy. In summary, this study provides a reference for further fermentation engineering with cheap lignocellulosic biomass as substrate.

Development of in silico strategies to photoautotrophically produce poly-β-hydroxybutyrate (PHB) by cyanobacteria

Poly(3-hydroxybutyrate) (PHB), a biopolymer similar to polypropylene, is currently produced by heterotrophic bacteria. However, the elevated cost of raw material used as carbon and energy sources are promoting the development of new sustainable bioprocess based on photoautotrophic routes costs. In unbalanced growth conditions, some cyanobacteria can accumulate PHB using CO2 as the sole carbon source, becoming an option for PHB production. In this work, we study in silico PHB production with the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). We consider two different strategies for in silico strain design: coupled growth-PHB production and a two-stage fermentation strategy. For both strategies we consider a GEnome-scale Model (GEM) of Synechocystis, which was modified to account for PHB production. The coupled approach relies on a bilevel optimization problem that identifies gene knock-outs needed to obtain a mutant that couples PHB production with biomass formation. For this case, two mutants that successfully achieve the required coupling are obtained, and the best one is analyzed in terms of intervention strategies, flux distributions and productivities. For the two-stage fermentation strategy, we consider two bioreactors in series. The first one is optimized for biomass production under balanced growth conditions, and the second one is optimized for PHB production under nitrogen starvation conditions. Both stages are simulated within dfba, a python tool for solving the Dynamic Flux Balance Analysis (DFBA) problem which provides the solution of a bioreactor model subject to the mass balances of the GEM. Numerical results for the two-stage approach provide a PHB concentration of 4.764 g PHB/L while the mutant provides a lower concentration of 0.391 g PHB/L. Nevertheless, both strategies provide high PHB content per cell dry weight (cdw), which makes results attractive for photosynthetic PHB production.

Sequential Production of ᴅ-xylonate and Ethanol from Non-Detoxified Corncob at Low-pH by Pichia kudriavzevii via a Two-Stage Fermentation Strategy.

Improving the comprehensive utilization of sugars in lignocellulosic biomass is a major challenge for enhancing the economic viability of lignocellulose biorefinement. A robust yeast Pichia kudriavzevii N-X showed excellent performance in ethanol production under high temperature and low pH conditions and was engineered for ᴅ-xylonate production without xylitol generation. The recombinant strain P. kudriavzevii N-X/S1 was employed for sequential production of ᴅ-xylonate and ethanol from ᴅ-xylose, feeding on ᴅ-glucose without pH control in a two-stage strategy of aerobic and shifting micro-aerobic fermentation. Acid-pretreated corncob without detoxification and filtration was used for ᴅ-xylonate production, then simultaneous saccharification and ethanol fermentation was performed with cellulase added at pH 4.0 and at 40 °C. By this strategy, 33.5 g/L ᴅ-xylonate and 20.8 g/L ethanol were produced at yields of 1.10 g/g ᴅ-xylose and 84.3% of theoretical value, respectively. We propose a promising approach for the sequential production of ᴅ-xylonate and ethanol from non-detoxified corncob using a single microorganism.

Enhancing ectoine production by recombinant Escherichia coli through step-wise fermentation optimization strategy based on kinetic analysis.

In this study, the recombinant ectoine-producing Escherichia coli ET01 was constructed by introducing the ectABC operon from Halomonas venusta ZH. To further improve ectoine production, the regulation of the fermentation process was systematically investigated. First, the effects of the initial glucose concentrations and glucose feeding mode on ectoine production were analyzed. Using a combination of pH-feedback feeding and glucose-controlled feeding, the ectoine titer reached 25.5g/L, representing an 8.8-fold increase over standard batch culture. Then, the effects of dissolved oxygen (DO) levels (50, 40, 30, or 20%) on ectoine production were studied, and a DO control strategy was developed based on the fermentation kinetics. When the final optimized two-stage fermentation strategy was used, the ectoine titer reached 47.8g/L, which was the highest level of ectoine produced by E. coli fermentation. The fermentation regulation strategy developed in this study might be useful for scaling up the commercial production of ectoine.

Production of bioethanol and xylitol from non-detoxified corn cob via a two-stage fermentation strategy

A novel two-stage fermentation strategy was applied to produce xylitol and ethanol from the whole acid-pretreated corn cob slurry. The acid-pretreated corn cob was used without filtration and detoxification by the two-stage fermentation with the robust Kluyveromyces marxianus CICC 1727-5. In the first stage, xylose in the slurry after dilute acid pretreatment of lignocellulosic biomass was used to produce xylitol under micro-aeration conditions. In the second stage, simultaneous saccharification fermentation was carried out, and the ethanol was produced from glucose releasing from the solid. Important parameters, such as aeration rate, cellulase loading during xylose utilization and SSF fermentation were studied for best performance. The two-stage fermentation strategy removed the inhibition of glucose on xylose, and little xylose was left in the fermentation broth. Under the optimized condition, the maximum ethanol and xylitol concentration were 52 g/L and 24.2 g/L corresponding to the yield of 0.41 g/g and 0.82 g/g, respectively.

Improving RNA content of salt-tolerant Zygosaccharomyces rouxii by atmospheric and room temperature plasma (ARTP) mutagenesis and its application in soy sauce brewing.

Derived from RNA, 5'-ribonucleotides, especially Inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP), can enhance the umami taste of soy sauce. In this study, the RNA content of three different salt-tolerant yeasts was examined. The most valuable strain was subjected to atmospheric and room-temperature plasma (ARTP) mutagenesis, which improved its RNA content by 160.54%. Regular fermentation with RNA-enhanced strain failed to increase the amount of 5'-ribonucleotides in the soy sauce due to hydrolysis by phosphatase. A two-stage fermentation strategy was then carried out. Aroma compounds were mainly synthesized in the first stage, and RNA-enriched biomass was massively produced in the second stage followed by heat treatment to inactivate phosphatase. After the proposed strategy was applied, IMP and GMP in the soy sauce reached 68.54 and 89.37 mg/L, respectively. Moreover, the amounts of key aroma compounds and organic acids significantly increased. Results may provide new insights for improving the quality of soy sauce through microorganism breeding and fermentation control.

Enhanced acarbose production by Streptomyces M37 using a two-stage fermentation strategy.

In this work, we investigated the effect of pH on Streptomyces M37 growth and its acarbose biosynthesis ability. We observed that low pH was beneficial for cell growth, whereas high pH favored acarbose synthesis. Moreover, addition of glucose and maltose to the fermentation medium after 72 h of cultivation promoted acarbose production. Based on these results, a two-stage fermentation strategy was developed to improve acarbose production. Accordingly, pH was kept at 7.0 during the first 72 h and switched to 8.0 after that. At the same time, glucose and maltose were fed to increase acarbose accumulation. With this strategy, we achieved an acarbose titer of 6210 mg/L, representing an 85.7% increase over traditional batch fermentation without pH control. Finally, we determined that the increased acarbose production was related to the high activity of glutamate dehydrogenase and glucose 6-phosphate dehydrogenase.

A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield.

5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host,Corynebacterium glutamicum The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future.

Enhanced production of β-glucuronidase from Penicillium purpurogenum Li-3 by optimizing fermentation and downstream processes

β-Glucuronidase from Penicillium purpurogenum Li-3 (PGUS) can efficiently hydrolyze glycyrrhizin into the more valuable glycyrrhetic acid monoglucuronide. However, a low productivity of PGUS and the lack of an effective separation strategy have significantly limited its industrial applications. Therefore, the production of PGUS has been improved by optimizing both the fermentation and purification strategies. A two-stage fermentation strategy was developed where PGUS was first grown with glucose and then PGUS was produced in the presence of glycyrrhizin as an inducer. By using this strategy, the biomass was increased 1.5 times and the PGUS activity increased 5.4 times compared to that when glycyrrhizin was used as the sole carbon source. The amount of PGUS produced was increased another 16.6% when the fermentation was expanded to a 15-L fermenter. An effective protocol was also established to purify the PGUS using a sequential combination of hydrophobic, strong anionexchange and gel filtration chromatography. This protocol had a recovery yield of 6% and gave PGUS that was 39 times purer than the crude PGUS. The purified PGUS had a specific activity of 350 U·mg–1.

Accumulation of γ-aminobutyric acid by Enterococcus avium 9184 in scallop solution in a two-stage fermentation strategy.

SummaryIn this study, a new bacterial strain having a high ability to produce γ‐aminobutyric acid (GABA) was isolated from naturally fermented scallop solution and was identified as E nterococcus avium. To the best of our knowledge, this is the first study to prove that E . avium possesses glutamate decarboxylase activity. The strain was then mutagenized with UV radiation and was designated as E . avium 9184. Scallop solution was used as the culture medium to produce GABA. A two‐stage fermentation strategy was applied to accumulate GABA. In the first stage, cell growth was regulated. Optimum conditions for cell growth were pH, 6.5; temperature, 37°C; and glucose concentration, 10 g·L−1. This produced a maximum dry cell mass of 2.10 g·L−1. In the second stage, GABA formation was regulated. GABA concentration reached 3.71 g·L−1 at 96 h pH 6.0, 37°C and initial l‐monosodium glutamate concentration of 10 g·L−1. Thus, compared with traditional one‐stage fermentation, the two‐stage fermentation significantly increased GABA accumulation. These results provide preliminary data to produce GABA using E . avium and also provide a new approach to process and utilize shellfish.

Two-Stage Fermentation for 2-Ketogluconic Acid Production by Klebsiella pneumoniae

2-Ketogluconic acid production by Klebsiella pneumoniae is a pH-dependent process, strictly proceeding under acidic conditions. Unfortunately, cell growth is inhibited by acidic conditions, resulting in low productivity of 2-ketogluconic acid. To overcome this deficiency, a two-stage fermentation strategy was exploited in the current study. During the first stage, the culture was maintained at neutral pH, favoring cell growth. During the second stage, the culture pH was switched to acidic conditions favoring 2-ketogluconic acid accumulation. Culture parameters, including switching time, dissolved oxygen levels, pH, and temperature were optimized for the fed-batch fermentation. Characteristics of glucose dehydrogenase and gluconate dehydrogenase were revealed in vitro, and the optimal pHs of the two enzymes coincided with the optimum culture pH. Under optimum conditions, a total of 186 g/l 2- ketogluconic acid was produced at 26 h, and the conversion ratio was 0.98 mol/mol. This fermentation strategy has successfully overcome the mismatch between optimum parameters required for cell growth and 2-ketogluconic acid accumulation, and this result has the highest productivity and conversion ratio of 2-ketogluconic and produced by microorganism.

Exploring two-stage fermentation strategy of polyhydroxyalkanoate production using Aeromonas hydrophila

With consideration of sustainable development, this study explored the fermentation strategy of cost-effective production of biodegradable polymer- polyhydroxyalanoates (PHAs) for feasibility of eco-friendly materials recycling during wastewater treatment. As prior studies showed that Aeromonas hydrophila NIU01 was a promising PHA-producing bacterium, this follow-up study tended to seek for optimal nutrient-supplementation strategy to stimulate maximal PHA accumulation of A. hydrophila NIU01 for cellular production. As maximal PHA production took place at growth-limiting conditions, two-stage fermentation was much more appropriate for practical applications compared to batch mode of operation. Moreover, this optimal two-stage operation strategy maximized cellular PHA production under nitrogen-limiting conditions at C/N molar ratio of 60/1. For materials recycling, this operation strategy could be applicable to simultaneous PHB production and wastewater decolorization using A. hydrophila.

Screening and Evaluation of Polyhydroxybutyrate-Producing Strains from Indigenous Isolate Cupriavidus taiwanensis Strains

Polyhydroxyalkanoate (PHA) is a biodegradable material with many potential biomedical applications, including medical implants and drug delivery. This study developed a system for screening production strains in order to optimize PHA production in Cupriavidus taiwanensis 184, 185, 186, 187, 204, 208, 209 and Pseudomona oleovorans ATCC 29347. In this study, Sudan black B staining, Infrared (IR) and Gas Chromatography (GC) analysis indicated that the best strain for PHA synthesis is C. taiwanensis 184, which obtains polyhydroxybutyrate (PHB). Cultivation of C. taiwanensis 184 under a pH of 7.0, at 30 °C, and at an agitation rate of 200 rpm, obtained a PHB content of 10% and PHB production of 0.14 g/L. The carbon and nitrogen types selected for analysis of PHB production by C. taiwanensis 184 were gluconic acid and NH4Cl, respectively. Optimal carbon/nitrogen ratio for PHB production was also determined. This study demonstrated a PHB content of 58.81% and a PHB production of 2.44 g/L when the carbon/nitrogen ratio of 8/1 was selected for C. taiwanensis 184. A two-stage fermentation strategy significantly enhanced PHB content and PHB production. Under a two-stage fermentation strategy with nutrient-limited conditions, C. taiwanensis 184 obtained a PHB content of 72% and a PHB concentration of 7 g/L. Finally, experimental results confirmed that optimizing the growth medium and fermentation conditions for cultivating the indigenous C. taiwanensis 184 strain substantially elevated PHB content from 10% to 72% and PHB production from 0.14 g/L to 7 g/L, respectively.

Two-stage fermentation process for alginate production by Azotobacter vinelandii mutant altered in poly-β-hydroxybutyrate (PHB) synthesis

A two-stage fermentation strategy, based on batch cultures conducted first under non-oxygen-limited conditions, and later under oxygen-limited conditions, was used to improve alginate production by Azotobacter vinelandii (AT6), a strain impaired in poly-beta-hydroxybutyrate (PHB) production. The use of sucrose as carbon source, as well as a high oxygen concentration (10%), allowed to obtain a maximum biomass concentration of 7.5 g l(-1) in the first stage of cultivation. In the second stage, the cultures were limited by oxygen (oxygen close to 0%) and fed with a sucrose solution at high concentration. Under those conditions, the growth rate decreased considerably and the cells used the carbon source mainly for alginate biosynthesis, obtaining a maximum concentration of 9.5 g l(-1), after 50 h of cultivation. Alginate concentration obtained from the AT6 strain was two times higher than that obtained using the wild-type strain (ATCC 9046) and was the highest reported in the literature. However, the mean molecular mass of the alginate produced in the second stage of the process by the mutant AT6 was lower (400 kDa) than the polymer molecular mass obtained from the cultures developed with the parental strain (950 kDa). The use of a mutant of A. vinelandii impaired in the PHB production in combination with a two-stage fermentation process could be a feasible strategy for the production of alginate at industrial level.

A fermentation strategy for production of recombinant protein subjected to plasmid instability

The appearance of plasmid-losing cells in a recombinant Escherichia coli culture was observed when the cell mass became doubled after induction, which corresponded to the timing of cell fission. Accordingly, a two-stage fermentation strategy capable of maintaining plasmid stability without selective pressure in a recombinant E. coli culture was proposed. In the first stage (cell growth stage), a high cell density culture was obtained by incubating the cells in the R medium. In the second stage (producing stage), the cells were devoted to producing the recombinant protein by introducing the fresh LB medium supplemented with isopropyl-β-d-thiogalactopyranoside (IPTG). It was necessary to prevent the doubling in the cell mass after induction; otherwise cell fission would occur and generate plasmid-losing cells. The present strategy is expected to be extensively applicable in recombinant E. coli cultures.

Scale-up fermentation of recombinant Candida rugosa lipase expressed in Pichia pastoris using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml(-1) and a cell wet weight of ca. 500 g l(-1) at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.

A phenomenological model to represent the kinetics of growth by Corynebacterium glutamicum for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.

Production of a desulfurization biocatalyst by two-stage fermentation and its application for the treatment of model and diesel oils.

For the production of oil-desulfurizing biocatalyst, a two-stage fermentation strategy was adopted, in which the cell growth stage and desulfurization activity induction stage were separated. Sucrose was found to be the optimal carbon source for the growth of Gordonia nitida CYKS1. Magnesium sulfate was selected to be the sulfur source in the cell growth stage. The optimal ranges of sucrose and magnesium sulfate were 10-50 and 1-2.5 g x L(-1), respectively. Such a broad optimal concentration of sucrose made the fed-batch culture easy, while the sucrose concentration was maintained between 10-20 g x L(-1) in the actual operation. As a result, 92.6 g x L(-1) of cell mass was acquired by 120 h of fed-batch culture. This cell mass was over three times higher than a previously reported result, though the strain used was different. The desulfurization activity of the harvested cells from the first stage culture was induced by batch cultivation with dibenzothiophene as the sole sulfur source. The optimal induction time was found to be about 4 h. The resting-cell biocatalyst made from the induced cells was applied for the deep desulfurization of a diesel oil. It was observed that the sulfur content of the diesel oil decreased from 250 mg-sulfur x L-oil(-1) to as low as 61 mg-sulfur x L-oil(-1) in 20 h. It implied that the biocatalyst developed in this study had a good potential to be applied to a deep desulfurization process to produce ultra-low-sulfur fuel oils.