Development of balanced medium composition for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3

Aim:  To develop optimum fermentation environment for enhanced rifamycin B production by isolated Amycolatopsis sp. RSP-3.
Methods and Results:  The impact of different fermentation parameters on rifamycin B production by isolated Amycolatopsis sp. RSP-3 was investigated using Taguchi methodology. Controlling fermentation factors were selected based on one variable at a time methodology. The isolated strain revealed more than 25% higher production compared to literature reports. Five different nutritional components (soyabean meal, glucose, potassium nitrate, calcium carbonate and barbital) and inoculum concentration showed impact on rifamycin B production at individual and interactive level. At optimized environment, 65% contribution was observed from selected fermentation parameters.
Conclusions:  Soyabean meal and calcium carbonate were the most significant factors among the selected factors followed by barbital and potassium nitrate. Glucose, however, showed the least significance on rifamycin B production with this strain. A maximum of 5·12 g l⁻¹ rifamycin B production was achieved with optimized medium containing (g l⁻¹) soyabean meal, 27; glucose, 100; potassium nitrate, 4; calcium carbonate, 3 and barbital, 1·2.
Significance and Impact of the Study:  The present study signifies identification of balanced medium component concentrations for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3. This strain requires organic and inorganic nitrogen sources for effective product yield. Yet at individual level, organic nitrogen source has c. nine-fold higher influence compared to inorganic one.     

根霉液态发酵产生纤溶酶的培养条件优化

目的:目前临床使用的溶栓药物疗效肯定,但还存在许多缺陷,而且价格昂贵,因此研制新型溶栓药物的需求迫切。方法:研究了根霉Rhizopus chinensisYY-15液体摇瓶发酵产生纤溶酶的工艺条件。采用单因素试验对液体发酵培养基的碳源、氮源、碳氮比、初始pH进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果:实验范围内菌株液体发酵产纤溶酶的适宜培养基组成为:麸皮水浓度3%(w/v),豆粕浓度5%(w/v),初始培养基pH5.0。适宜培养条件为接种量6%,培养时间72h。优化条件下的摇瓶液体发酵纤溶酶产量平均达98.31 U/ml。     

重组Bacillus subtilis产B.stearothermophilus环糊精葡萄糖基转移酶的发酵优化

利用本研究室已构建的重组菌Bacillus subtilis/pBSMuL3-α/β-CGTase对产B.stearothermophilus环糊精葡萄糖基转移酶的发酵产酶进行了优化,考察了培养基中重要成分:碳源、有机氮源、无机氮源、有机与无机氮源质量比、碳源与氮质量比、金属离子种类等单因素对该重组菌产α/β-CGTase的影响,并采用正交实验对发酵培养基进行优化,对优化结果分析可知,重组菌B.subtilis/pBSMuL3-α/β-CGTase发酵产α/β-CGTase的最优培养基成本为:葡萄糖5 g/L,氮源(鱼骨蛋白胨∶NH4Cl=3∶1)25 g/L,1 mmol/L Mg^2+。在最优条件下发酵培养,α/β-CGTase的酶活由原来TB发酵培养基的9.20 U/mL提高至20.32 U/mL,是优化前酶活的2.2倍,为α/β-环糊精葡萄糖基转移酶的工业应用提供了理论支持。     

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Tannase production by Paecilomyces variotii

Surface response methodology was applied to the optimization of the laboratory scale production of tannase using a lineage of Paecilomyces variotii. A preliminary study was conducted to evaluate the effects of variables, including temperature ( degrees C), residue (%) (coffee husk:wheat bran), tannic acid (%) and salt solutions (%) on the production of tannase during 3, 5 and 7 days of fermentation. Among these variables, temperature, residues and tannic acid had significant effects on tannase production. The variables were optimized using surface response methodology. The best conditions for tannase production were: temperature (29-34 degrees C); tannic acid (8.5-14%); % residue (coffee husk:wheat bran 50:50) and incubation time of 5 days. The supplementation of external nitrogen and carbon sources at 0.4%, 0.8% and 1.2% concentration on tannase production were studied in the optimized medium. Three different nitrogen sources included yeast extract, ammonia nitrate and sodium nitrate along with carbon source (starch) were studied. Only ammonia nitrate showed a significant effect on tannase production. After the optimization process, the tannase activity increased 8.6-fold.     

L-asparaginase production by isolated Staphylococcus sp. - 6A: design of experiment considering interaction effect for process parameter optimization

AIMS: Evaluation of fermentation process parameter interactions for the production of l-asparaginase by isolated Staphylococcus sp. - 6A. METHODS AND RESULTS: Fractional factorial design of experimentation (L18 orthogonal array of Taguchi methodology) was adopted to optimize nutritional (carbon and nitrogen sources), physiological (incubation temperature, medium pH, aeration and agitation) and microbial (inoculum level) fermentation factors. The experimental results and software predicted enzyme production values were comparable. CONCLUSION: Incubation temperature, inoculum level and medium pH, among all fermentation factors, were major influential parameters at their individual level, and contributed to more than 60% of total l-asparaginase production. Interaction data of selected fermentation parameters could be classified as least and most significant at individual and interactive levels. Aeration and agitation were most significant at interactive level, but least significant at individual level, and showed maximum severity index and vice versa at enzyme production. SIGNIFICANCE AND IMPACT OF THE STUDY: All selected factors showed impact on l-asparaginase enzyme production by this isolated microbial strain either at the individual or interactive level. Incubation temperature, inoculum concentration, pH of the medium and nutritional source (glucose and ammonium chloride) had impact at individual level, while aeration, agitation and incubation time showed influence at interactive level. Significant improvement (ninefold increase) in enzyme production by this microbial isolate was noted under optimized environment.     

响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

紫色红曲霉Mp-24高产Monacolin K发酵工艺响应面优化

将单因素实验结果与响应面法相结合,对高产Monacolin K的紫色红曲霉Mp-24菌株进行发酵工艺条件优化。通过摇瓶发酵对碳源、氮源、碳源含量、氮源含量、培养时间等进行单因素优化,确定Mp-24菌株摇瓶发酵适宜条件：乳糖为碳源、酵母膏为氮源、碳源含量7%、氮源含量2%、培养时间12 d,Monacolin K产量为167 mg/L。应用Box-Behnken中心组合试验设计建立数学模型,进行响应面分析优化发酵条件,结果显示最佳发酵工艺条件为：碳源(乳糖)8%,氮源(酵母膏)3%,培养时间11 d,在此条件下Monacolin K的含量达到247.8 mg/L,比优化前提高1.5倍。     

Studies on fermentative production of rifamycin using Amycolatopsis mediterranei

The fermentative production of rifamycin by Amycolatopsis mediterranei (MTCC17) has been studied. Both qualitative and quantitative aspects of the fermentation were determined by optimizing cultural conditions and medium design to improve the production of rifamycin. A pH value of 7.0, a temperature of 26°C, an aeration rate of 250rev/min for a 50ml volume, a level of inoculum of 10% grown aeration for 48h and a fermentation period of 11days were found to be optimum. Among the nitrogen sources, and culture conditions, peanut meal and aeration were found to be critical for rifamycin production respectively. The above mentioned exercise increased the yields of rifamycin from 350mg/l to 2000mg/l.     

根霉12号固体发酵产生纤溶酶工艺条件的研究*

研究了根霉12号固体发酵产生纤溶酶的工艺条件。采用单因素试验、均匀设计方法对固体发酵培养基的碳源、氮源、碳氮比、初始pH、加水量、无机盐加量进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果表明,实验范围内根霉12固体发酵产纤溶酶的适宜培养基组成为:麸皮∶豆粕=1∶2,初始pH5.0,加水量0.75ml/g物料, MnSO4H2O和 (NH4)2SO4加量分别为0.25%和 1.42%(对物料)。适宜培养条件为接种量107个孢子/g物料,培养时间72h。优化条件下的纤溶酶产量平均达791.81u/g物料。     

培养基的组成及培养条件对茶树菇多糖产量的影响

本文研究了不同碳源,氮源以及发酵条件对茶树菇多糖产量的影响,并通过正交试验优化了深层发酵培养基。结果表明,茶树菇深层发酵最适的碳源为玉米粉,最适的氮源为酵母粉,最适的发酵条件为温度25℃,摇瓶装液量100mL/250mL,转速150r/min,接种量15%,起始pH自然。     

An economic approach for L-(+) lactic acid fermentation by Lactobacillus amylophilus GV6 using inexpensive carbon and nitrogen sources

AIMS: Development of cost-effective production medium by applying statistical designs for single-step fermentation of starch (corn flour - CF) to L-(+) lactic acid, using inexpensive nitrogen sources as substitutes for peptone and yeast extract in MRS medium by amylolytic Lactobacillus amylophilus GV6. METHODS AND RESULTS: A two-level Plackett-Burman design was employed for screening various available crude starches (flours) for L-(+) lactic acid production by Lact. amylophilus GV6 using red lentil flour (RL) and bakers yeast cells (YC) as substitutes for commercial peptone and yeast extract in MRS medium in anaerobic submerged fermentation. Of all the tested flours, CF was found to be the most significant. Central composite rotatable design was employed to determine maximum production of L-(+) lactic acid at optimum values of process variables, CF, RL, YC, CaCO(3) and incubation period (IP). minitab analyses showed that lactic acid production was significantly affected by the linear terms CF, RL, CaCO(3) and IP. The interactions of CF-RL, CF-YC, CF-CaCO(3), RL-YC and RL-CaCO(3) and the square terms CF and IP were significant. The maximum lactic acid production of 29 g/37 g of starch present in 50 g of CF was obtained at optimized concentrations of CF 5%, RL 0.7%, YC 0.8%, CaCO(3) 0.8% and IP 2.9 days. CONCLUSIONS: Successful application of Plackett-Burman design helped in identifying CF as the best carbon source among the tested flours for L-(+) lactic acid production using inexpensive nitrogen sources. Further optimization of the process variables by response surface methods (RSMs) led to maximum production of lactic acid (29 g lactic acid from 37 g of starch present in 50 g of flour). SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus amylophilus GV6 showed 78.4% lactic acid production efficiency (g lactic acid produced/g starch taken) and 96% lactic acid yield efficiency (g lactic acid produced/g starch utilized). Information from the present studies provides a better understanding on production of L-(+) lactic acid on fermentation of CF using inexpensive nitrogen sources and on changes in the production as a response from interaction of factors. Use of inexpensive nitrogen sources and starch as substrate in MRS medium for single-step fermentation of lactic acid can become an efficient, economic and viable process. This report is on optimization of inexpensive nitrogen sources completely replacing peptone and yeast extract in single-step submerged fermentation of starch (present in CF) to lactic acid with high production efficiency.     

樟绒枝霉(Malbranchea cinnamomea)固体发酵产木聚糖酶的发酵条件优化

[目的] 研究樟绒枝霉(Malbranchea cinnamomea) CAU521利用农业废弃物固体发酵产木聚糖酶的发酵条件.[方法]采用单因素试验法优化影响菌株产酶的各个条件,包括碳源种类、氮源种类、初始pH、初始水分含量、培养温度及发酵时间共6个因素.[结果]获得的最佳产酶条件为:稻草为发酵碳源、2％(W/W)的酵母提取物为氮源、初始pH 7.0、初始水分含量80％和发酵温度45℃.在此条件下发酵6d后木聚糖酶的酶活力达到13 120 U/g干基碳源.[结论]樟绒枝霉固体发酵产木聚糖酶的产酶水平高,生产成本低,具有潜在的工业化应用前景.     

Paecilomyces sp. ZB is a cell factory for the production of gibberellic acid using a cheap substrate in solid state fermentation

Gibberellic acid from the fungi has been widely used in agriculture. In this study, more than 20 fungal isolates were screened and Paecilomyces sp. ZB shown to produce more gibberellic acid than other fungal isolates. Cow dung was used as low cost substrate for gibberellic acid production in solid state fermentation (SSF). Carbon, nitrogen and ionic sources stimulated gibberellic acid production in SSF. Lactose emerged as the significant carbon source supporting more gibberellic acid production (731 µg/g). Among the nitrogen sources, glycine appeared to influence the production of more gibberellic acid (803 µg/g). The process parameters were optimized to enhance gibberellic acid production using a two-level full factorial design and response surface methodology. The amount of gibberellic acid production was influenced mainly by moisture and pH of the substrate. Gibberellic acid production was 1312 µg/g under the optimized conditions and the predicted response was 1339 µg/g. The gibberellic acid yield increased twofolds after medium optimization. The extracted gibberellic acid was sprayed on the growing Mung bean plant and it stimulated the growth of the plant effectively. To conclude, cow dung is a new alternative to produce gibberellic acid in SSF.     

A complete industrial system for economical succinic acid production by Actinobacillus succinogenes

An industrial fermentation system using lignocellulosic hydrolysate, waste yeast hydrolysate, and mixed alkali to achieve high-yield, economical succinic acid production by Actinobacillus succinogenes was developed. Lignocellulosic hydrolysate and waste yeast hydrolysate were used efficiently as carbon sources and nitrogen source instead of the expensive glucose and yeast extract. Moreover, as a novel method for regulating pH mixed alkalis (Mg(OH)₂ and NaOH) were first used to replace the expensive MgCO₃ for succinic acid production. Using the three aforementioned substitutions, the total fermentation cost decreased by 55.9%, and 56.4 g/L succinic acid with yield of 0.73 g/g was obtained, which are almost the same production level as fermentation with glucose, yeast extract and MgCO₃. Therefore, the cheap carbon and nitrogen sources, as well as the mixed alkaline neutralize could be efficiently used instead of expensive composition for industrial succinic acid production.     

Biosynthesis of short-chain-length-polyhydroxyalkanoates during the dual-nutrient-limited zone by Ralstonia eutropha

Biosynthesis of PHAs by Raltonia eutropha during the dual nutrient-limitation-zone was investigated with mixed organic acids as carbon sources and (NH₄)₂SO₄ as nitrogen source. Two different methods of maintaining the dual-nutrient-limitation zone were adopted by feeding mixed acids and (NH₄)₂SO₄ at determined rates into the fermentation cultures which were initially free of carbon sources (method A) or nitrogen sources (method B). The results indicate that, firstly, with the increase of the width of the dual-nutrient-limitation zone, the yield of short-chain-length-polyhydroxyalkanoates also increases and it suggests that most of the short-chain-length-polyhydroxyalkanoates were biosynthesized during the dual-nutrient-limitation zone. Secondly, in contrast with the dual-nutrient-limitation method of limiting the nitrogen source first (method B), the dual-nutrient-limitation method of limiting the carbon source first (method A) was more favourable for the production of short-chain-length-polyhydroxyalkanoates, and the maximum production of short-chain-length-polyhydroxyalkanoates of these two methods are 3.72 and 2.55 g/l, respectively.     

真氧产碱杆菌在双营养（碳、氮）限制区内合成聚羟基烷酸酯

研究了真氧产碱杆菌以混合有机酸为碳源,硫酸铵为氮源,在双营养（碳、氮）限制区内聚羟基烷酸酯的生物合成。结果表明：双营养限制区的长度与聚羟基烷酸酯的产量呈正相关。同时,在对两种不同的双营养限制区实现方式进行比较后发现,首先限制碳源的双营养限制方式比首先限制氮源的双营养限制方式更有利于聚羟基烷酸酯的合成;在这两种不同营养限制方式下,PHAs的最高产量分别为3.72 g/L和2.55 g/L。     

Structured kinetic model to represent the utilization of multiple substrates in complex media during rifamycin B fermentation

Industrial fermentations typically use media that are balanced with multiple substitutable substrates including complex carbon and nitrogen source. Yet, much of the modeling effort to date has mainly focused on defined media. Here, we present a structured model that accounts for growth and product formation kinetics of rifamycin B fermentation in a multi-substrate complex medium. The phenomenological model considers the organism to be an optimal strategist with an in-built mechanism that regulates the sequential and simultaneous uptake of the substrate combinations. This regulatory process is modeled by assuming that the uptake of a substrate depends on the level of a key enzyme or a set of enzymes, which may be inducible. Further, the fraction of flux through a given metabolic branch is estimated using a simple multi-variable constrained optimization. The model has the typical form of Monod equation with terms incorporating multiple limiting substrates and substrate inhibition. Several batch runs were set up with varying initial substrate concentrations to estimate the kinetic parameters for the rifamycin overproducer strain Amycolatopsis mediterranei S699. Glucose and ammonium sulfate (AMS) demonstrated significant substrate inhibition toward growth as well as product formation. The model correctly predicts the experimentally observed regulated simultaneous uptake of the substitutable substrate combinations under different fermentation conditions. The modeling results may have applications in the optimization and control of rifamycin B fermentation while the modeling strategy presented here would be applicable to other industrially important fermentations.     

Iso-migrastatin titer improvement in the engineered <Emphasis Type="Italic">Streptomyces lividans</Emphasis> SB11002 strain by optimization of fermentation conditions

The heterologous production of iso-migrastatin (iso-MGS) was successfully demonstrated in an engineered S. lividans SB11002 strain, which was derived from S. lividans K4-114, following introduction of pBS11001, which harbored the entire mgs biosynthetic gene cluster. However, under similar fermentation conditions, the iso-MGS titer in the engineered strain was significantly lower than that in the native producer — Streptomyces platensis NRRL 18993. To circumvent the problem of low iso-MGS titers and to expand the utility of this heterologous system for iso-MGS biosynthesis and engineering, systematic optimization of the fermentation medium was carried out. The effects of major components in the cultivation medium, including carbon, organic and inorganic nitrogen sources, were investigated using a single factor optimization method. As a result, sucrose and yeast extract were determined to be the best carbon and organic nitrogen sources, resulting in optimized iso-MGS production. Conversely, all other inorganic nitrogen sources evaluated produced various levels of inhibition of iso-MGS production. The final optimized R2YE production medium produced iso-MGS with a titer of 86.5 mg/L, about 3.6-fold higher than that in the original R2YE medium, and 1.5 fold higher than that found within the native S. platensis NRRL 18993 producer.     

枯草芽孢杆菌发酵条件优化及其破乳效能

本文对枯草芽孢杆菌在不同碳源、氮源培养基的生长及破乳效能进行了研究, 并通过正交试验对枯草芽孢杆菌的发酵条件进行优化结果表明, 单一碳源葡萄糖和混合碳源葡萄糖 + 液体石蜡的培养基可提高枯草芽孢杆菌的发酵产量; 单一碳源葡萄糖、混合碳源葡萄糖 + 汽油和以硝酸铵 + 酵母膏为氮源的菌液有较高的破乳效能; 在正交试验中, 培养温度对枯草芽孢杆菌的发酵产量影响最大, 其最优组合为: 培养温度25oC, 摇床转数140 r/min, 培养pH值7.0, 接菌量6 mL, 培养时间24 h。摇床转数对枯草芽孢杆菌的发酵产物的破乳效能影响最大, 优化结果为: 培养温度25oC, 摇床转数140 r/min, pH值7.0, 培养时间20 h。