Development of a New <Emphasis Type="Italic">Amycolatopsis orientalis</Emphasis> Strain for High-Yield Eremomycin Production

A multistep random UV mutagenesis of an eremomycin-producing strain Amycolatopsis orientalis VKPM Ac-1125 with rather low productivity (0.5 g/L) and the further selection of the most high-yield mutant strains have resulted in a highly productive E 13-26 strain. After optimization of the fermentation medium by multifactor analysis, the most efficient composition for the fermentation medium was established, %: glycerol, 8; soybean meals, 2.0; MgCl₂, 0.02; KH₂PO₄, 0.02; KNO₃, 0.6; CaCl₂, 0.3; skim milk powder, 0.46; and glucose, 0.8. The maximum productivity of this strain on the optimized medium reached 3.42 ± 0.17 g/L. Therefore, the obtained strain is considered to be very promising for further use in the selection and development of technology for the large-scale eremomycin production.     

Impact of phosphate concentration on docosahexaenoic acid production and related enzyme activities in fermentation of Schizochytrium sp.

Docosahexaenoic acid (DHA) is an important and widely used infant food additive. In this study, the effects of phosphate concentration on lipid and especially DHA synthesis in the oleaginous fungi Schizochytrium sp. HX-308 have been investigated in batch cultures. The maximum DHA yield (8.9 g/L) and DHA productivity (148.3 mg/L h) in 0.1 g/L KH₂PO₄ concentration were higher than the DHA yield (6.2 g/L) and DHA productivity (86.1 mg/L h) in 4 g/L KH₂PO₄ concentration. Furthermore, differences in related enzyme activities (malic enzyme, glucose-6-phosphate dehydrogenase and NAD⁺-isocitrate dehydrogenase) between phosphate-sufficient and phosphate-limitation conditions were assayed. The results showed that the phosphate-limitation condition could maintain higher activities of malic enzyme and glucose-6-phosphate dehydrogenase in addition to lower activity of NAD⁺-isocitrate dehydrogenase. In addition, glucose-6-phosphate dehydrogenase might be the main supplier of NADPH at the early stage of fermentation while malic enzyme might be the provider at the late stage. This information might explain the regulation mechanism of phosphate limitation for lipid production and be useful for further DHA production enhancement.     

Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate

Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH₄)₂SO₄, 0.09 % yeast extract, 0.05 % KH₂PO₄, and 0.05 % MgSO₄·7H₂O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.     

He-Ne laser irradiation of folate-producing Candida utilis and optimization of culture conditions under submerged fermentation

In an attempt to obtain a microbial strain with higher yield of folate for industrial applications, we mutated the wild strain Candida utilis Y1.0 using a novel mutagenic process, i.e., irradiation by a helium–neon (He-Ne) laser with an output power of 20 mW and an exposure time of 20 min. The yield of folate in the mutated cells reached 1,102 ng/mL, which was 20.4-fold that of the wild strain. The mutant strain Y3.636 was relatively stable in terms of folate production through eight successive transfers of cultures and batch fermentation in a 3.7-L stirred-tank fermenter. Optimization further increased the yield of the mutant by 110 %, i.e., to 2,314?±?13 ng/mL. The optimal culture conditions for folate production were: cultivation in fermentation culture medium composed of 62.5 g/L glucose, 15 g/L corn liquor, 3 g/L (NH₄)₂SO₄, 3 g/L MgSO₄, and 1 g/L glutamic acid; inoculum size of 9 %; incubation at 28 °C and 196 rpm for 36 h. A time-course study of cell growth and folate production by mutant strain Y3.636 strongly suggested that folate production in C. utilis is growth-associated.     

Production,Purification, and Antibiofilm Activity of a Novel Exopolysaccharide from Arthrobacter sp. B4

A novel exopolysaccharide (EPS), namely, B4-EPS, is produced by Arthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett–Burman design (PBD), glucose, yeast extract, and KH₂PO₄ were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH₂PO₄. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 hr of batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm formation of P. aeruginosa PAO1 was inhibited in the presence of 50 µg/mL B4-EPS1, which was more effective than the peer published data. Moreover, B4-EPS1 could prevent biofilm formation of other strains. These data suggest B4-EPS may represent a promising strategy to combat bacterial biofilms in the future.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Spore Production Under Solid-State Fermentation of Lignocellulosic Residues

This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47?×?10¹⁰ spores g^?1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82?×?10¹⁰ spores g^?1 biomass) was achieved when 15 g corncobs were moistened with 60 ml of the optimized nutrient medium containing 10 g peptone, 2 g KH₂PO₄, 1 g MgSO₄·7H₂O, and 1 g NaCl per 1 l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105?×?10¹⁰ spores g^?1. Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10?×?10¹¹ spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture.     

High-yield spore production from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> by solid state fermentation

Bacillus licheniformis was grown for 48 h at 37°C in solid state fermentation; a maximum of 1.7 × 10¹¹ spores/g dry substrate were obtained using rice straw powder (300 g/kg) and wheat bran (700 g/kg) supplemented with glucose (40 g/kg), peptone (20 g/kg), yeast extract (20 g/kg), KH₂PO₄ (10 g/kg) and CaO (5 g/kg) with an initial moisture content of 65%.     

Optimization of submerged fermentation conditions for immunosuppressant mycophenolic acid production by Penicillium roqueforti isolated from blue-molded cheeses: enhanced production by ultraviolet and gamma irradiation

Mycophenolic acid (MPA) is a promising drug owing to its immunosuppressive and biological activities. In this study, two strains of Penicillium roqueforti designated as AG101 and LG109 were selected among several strains isolated from Roquefort cheese samples on the basis of their activity for MPA-producing ability. The appropriate fermentation conditions necessary for MPA biosynthesis by the two respective fungal strains were investigated. These conditions included selection of the cultivation medium, agitation rate, incubation temperature, fermentation time, pH value, inoculum size, and fermentation medium volume. Maximum MPA productivities were maintained when the fermentation process was carried out using a medium composed of (g l^?1): Sucrose, 30; peptone, 5.0; KH₂PO₄, 1.0; MgSO₄·7H₂O, 0.5 and KCl, 0.5; pH 6.0, inoculated with an inoculum size of 6.0 % (v/v), and incubated at 25 °C for 10 days at 120 rpm. The potentiality of both P. roqueforti strains for further improvement of MPA production was applied by mutagenesis through exposure to irradiation by ultraviolet rays (UV, 254 nm) for different periods of time and gamma rays at various doses (KGy). The dry cell weight of both irradiated fungal strains showed a greater reduction when irradiated either with UV or gamma rays. However, the MPA yield of both strains was increased by 1.27–1.39 fold when irradiated with UV rays and by 2.11–2.33 fold when irradiated with gamma rays, as compared with the respective controls (non-irradiated cultures). These findings indicate the future possibility to reduce the cost of producing fermentation-based drugs.     

Optimization of the fermentation process of <Emphasis Type="Italic">Cordyceps sobolifera</Emphasis> Se-CEPS and its anti-tumor activity in vivo

Background

Cordyceps sobolifera (C. sobolifera) isolated from cicadae was used as the starting fungus to produce selenium-enriched C. sobolifera extracellular polysaccharide (Se-CEPS). An orthogonal experimental design based on a single-factor experiment was used to optimize the C. sobolifera fermentation conditions, including the potato juice, peptone, and KH₂PO₄ concentrations. Ultraviolet (UV) and infrared (IR) analyses of CEPS and Se-CEPS were conducted, as well as an in vivo anti-tumor analysis.

Results

Under optimal conditions (i.e., 40 potato juice, 0.4 KH₂PO₄, and 0.5 % peptone), the fermentation yield of Se-CEPS was 5.64 g/L. UV and IR spectra showed that Se-CEPS contained a characteristic absorption peak of a selenite Se?=?O double bond, demonstrating the successful preparation of Se-CEPS. Activity tests showed that Se-CEPS improved the immune organ index, serum cytokine content, and CD8⁺ and CD4⁺ T lymphocyte ratio in colon cancer CT26 tumor-bearing mice, thereby inhibiting tumor growth. When combined with 5-FU, Se-CEPS reduced the toxicity and enhanced the function of 5-FU.

Conclusion

The result of these experiments indicated that orthogonal experimental design is a promising method for the optimization of Se-CEPS production, and the Se-CEPS from C. sobolifera can improve the anti-tumor capacity of mice.

     

一株维氏气单胞菌发酵培养基优化及其制备疫苗免疫效力比较

采用单因素试验、响应面试验法对维氏气单胞菌（Aeromonas veronii）发酵培养基的氮源、碳源、无机盐和磷酸盐成分及用量进行优化组合,确定优化培养基组成：胰蛋白胨10.8 g/L,葡萄糖5.0 g/L,牛肉膏3.0 g/L,磷酸二氢钾2.0 g/L,硫酸镁0.4 g/L,NaCl 5.0 g/L。并与基础培养基的发酵活菌数、制备的灭活疫苗免疫效力进行比较,经过验证试验绘制维氏气单胞菌在优化培养基条件下的7 L发酵罐生长曲线。在优化发酵培养基条件下,维氏气单胞菌活菌数为5.94×10⁹ cfu/mL,比基础培养基增幅43.13%;制备的灭活疫苗相对保护率为77.78%,比基础培养基提高了14.81%。7 L发酵罐发酵培养10 h,活菌数达到最大8.85×10⁹ cfu/mL。通过对发酵培养基的优化,可以获得低成本、优质高效的维氏气单胞菌发酵菌液,为今后维氏气单胞菌灭活疫苗规模化发酵培养提供参考。     

Optimization of Fermentation Medium and Conditions for Mycelial Growth and Water-soluble Exo-polysaccharides Production by Isaria farinosa B05

Abstract

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K₂HPO₄ 0.1%, and MgSO₄ 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25°C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.     

Co-fermentation of cassava waste pulp hydrolysate with molasses to ethanol for economic optimization

Cassava waste pulp (CWP)–enzymatic hydrolysate was co-fermented with molasses (CWP-EH/molasses mixture) with the aim to optimize ethanol production by Saccharomyces cerevisiae TISTR 5606 (SC 90). The optimal fermentation conditions for ethanol production using this mixture were 245 g/L initial total sugar supplemented with KH₂PO₄ (8 g/L), at 30 °C for 48 h of fermentation under an oxygen-limited condition with agitation at 100 rpm, producing an ethanol concentration of 70.60 g/L (0.31 g ethanol/g total sugar). The addition of cassava tuber fiber (solid residue of CWP after enzymatic hydrolysis) at 30 g/L dry weight to the CWP-EH/molasses mixture increased ethanol production to 74.36 g/L (0.32 g ethanol/g total sugar). Co-fermentation of CWP-EH with molasses had the advantage of not requiring any supplementation of the fermentation mixture with reduced nitrogen.     

Biosynthesis of protein by microscopic fungi in solid state fermentation. II. Optimization of aspergillus oryzae A. or. 11 cultivation for protein enrichment of starchy raw materials

Study was made of the effect of medium humidity, source and dose of nitrogen, dose of phosphorus, dose of inoculum and aeration on protein biosynthesis by strain Aspergillus oryzae A. or. 11 in solid state fermentation. It was found that for a maximal protein yield the medium ought to contain about: 60% of water, 36% of starchy raw materials d. m. (e.g. 28.8% of coarse rye meal and 7.2% of beet pulp), 0.85% of nitrogen sources (1.6% of (NH₄)₂SO₄ and 1.1% of urea), and 0.35% of phosphorus source (1.3% of KH₂PO₄). pH of medium should be near 6.5. The dose of inoculum should not be lower than 10⁸ spores/100 g medium. The duration of culture ought to be 20–22 h at 30–35°C, with aeration at least 40 dm³ of air/h × 1000 g medium. Under these conditions the protein yield is about 6.0–6.3 g/100 g of starting medium d.m. at the cost of utilization of about 25 g total carbohydrates.     

Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material

Synthesis of amylase by Aspergillus niger strain UO-01 under solid-state fermentation with sugarcane bagasse was optimized by using response surface methodology and empirical modelling. The process parameters tested were particle size of sugarcane bagasse, incubation temperature and pH, moisture level of solid support material and the concentrations of inoculum, total sugars, nitrogen and phosphorous. The optimum conditions for high amylase production (457.82 EU/g of dry support) were particle size of bagasse in the range of 6–8 mm, incubation temperature and pH: 30.2°C and 6.0, moisture content of bagasse: 75.3%, inoculum concentration: 1 × 10⁷ spores/g of dry support and concentrations of starch, yeast extract and KH₂PO₄: 70.5, 11.59 and 9.83 mg/g of dry support, respectively. After optimization, enzyme production was assayed at the optimized conditions. The results obtained corroborate the effectiveness and reliability of the empirical models obtained.     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

Improved gellan gum production by a newly-isolated Sphingomonas azotifigens GL-1 in a cheese whey and molasses based medium

Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na₂HPO₄ 14.58 g/L and KH₂PO₄ 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 ± 1.55 g/L. 14.75 ± 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na₂HPO₄ 9.71 g/L and KH₂PO₄ 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 × 10⁶ and 0.89 × 10⁶ Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer.     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi

An efficient hairy root induction system for an important endangered medicinal plant, Dracocephalum kotschyi, was developed through Agrobacterium rhizogenes-mediated transformation by modifying the co-cultivation medium using five bacterial strains, A4, ATCC15834, LBA9402, MSU440, and A13 (MAFF-02-10266). A drastic increase in transformation frequency was observed when a Murashige and Skoog medium lacking NH₄NO₃ KH₂PO_4, KNO₃ and CaCl₂ was used, resulting in hairy root induction frequencies of 52.3 %, 69.6 %, 48.6 %, 89.0 %, and 80.0 % by A4, A13, LBA9402, MSU440, and ATCC15834 strains, respectively. For shoot induction, hairy roots and unorganized tumors induced by strain ATCC15834 were placed on an MS media supplemented with 0.1, 0.25, 0.5, and 1 mg/l BA plus 0.1 mg/l NAA. The high frequency of shoot regeneration and number of shoot were obtained in the medium containing 0.25 mg/l BA and 0.1 mg/l NAA. Root induction occurred from the base of regenerated shoots on the MS medium supplemented with 0.5 mg/l IBA after 10 days.     

Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.