Method for developing a controlled process. Study of the possible regulation of rifamycin B biosynthesis

A procedure for developing a controlled process for biosynthesis of secondary metabolites is described with reference to rifamycin B as an example. The response of the antibiotic producing culture to changed concentrations of the main nutrients in the initial medium was determined. Mathematical processing of the experimental findings with design of the experiment resulted in defining nutrients such as ammonium sulfate and cornsteep liquor useful for further development of the controlled process.     

Effect of nutritional factors on cellulase enzyme and microbial protein production by Aspergillus terreus and its evaluation

The biomass yield, cellulolytic activity, and protein recovery using Aspergillus terreus GN1 with alkali-treated sugarcane bagasse was studied using different levels (250-600 mg of N/L of broth) of organic and inorganic nitrogen sources. e.g., cattle urine, urea, cornsteep liquor, ammonium sulfate, ammonium nitrate, ammonium iron sulfate, ammonium chloride, and sodium nitrate. Among different levels of alkali-treated bagasse substrate concentrations (0.5-4.0% w/v) tested, 1.0% substrate yielded the highest crude protein content, protein recovery, and cellulolytic activity. The biomass recovery with 1.0% substrate ranged from 290-380 mg/500 mg bagasse substrate in a 50-mL broth with a nitrogen level of 250-600 mg of N/L in all the sources except ammonium iron sulfate, which yielded 402-439 mg/500 mg bagasse substrate. However, crude protein content of biomass obtained with an ammonium iron sulfate nitrogen source was the lowest. Cornsteep liquor nitrogen source at the rate of 600 mg of N/L yielded the maximum crude protein of 32.9%, protein recovery of 22.2 g/100 g of bagasse, and carboxymethyl cellulase and filter paper enzyme activities of 1.1 and 0.09 units/mL, among the organic and inorganic nitrogen sources studied. In general, the organic nitrogen sources and inorganic nonammonium nitrogen sources were utilized preferentially for protein production over the inorganic ammonium nitrogen sources. The fermentation time required under optimum cultural and nutritional conditions for A. terreus GN1 was also evaluated. The crude protein content of the biomass increased gradually up to the seventh day of fermentation, but the protein recovery rate was high up to two or three days. It was observed that the cellulose utilization rate increased after an initial lag of one day up to the third day and gradually increased further, which corresponded positively with protein content, biomass protein recovery, and cellulase enzyme activity. On the seventh day of fermentation, the crude protein content, biomass protein recovery, water-soluble carbohydrate, bagasse cellulose utilization, CMCase, and FPase activities were 32.8%, 20.1 g/100 g of bagasse, 6.2%, 82.7%, 1.0. and 0.08 U/mL, respectively. The final biomass recovered contained 32.8% crude protein content and had an in vitro rumen digestibility (IVRD) coefficient of 68.8%. The biomass contained almost all the essential and nonessential amino acids and was comparable with FAO reference protein. It is concluded that a fermentation time of 72 h gave a faster rate of protein production of 16.9 g/100 g of bagasse with 69.8% bagasse cellulose utilization with 76.0% IVRD. and contained almost all the essential and nonessential amino acids.     

Effect of corn-steep liquor on growth and mosquito larvicidal activity of Bacillus thuringiensis var israelensis de Barjac 1978 and B. sphaericus Neide 1904.

Influence of corn steep liquor on the cell yield and toxicity of three strains of B. thuringiensis var israelensis and two strains of B. sphaericus was studied and compared with peptone-yeast extract using a laboratory fermentor. Large increase in the cell yield of all the three strains of B. thuringiensis var israelensis was observed when cornsteep liquor was used as the sole nitrogen source. Significant increase in toxicity was also observed in B. thuringiensis var israelensis strains B17 and B113. Among the two B. sphaericus strains tested, the strain 1593 showed no significant change in cell yield and toxicity, whereas the strain VCRC B42 showed increased cell yield and toxicity in this medium. The results indicate that cornsteep liquor can effectively replace both peptone and yeast extract in the media presently used for large scale multiplication of the two larvicidal bacilli.     

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.     

Effect of salt nutrients on mannitol production by Lactobacillus intermedius NRRL B-3693

The effects of four salt nutrients (ammonium citrate, sodium phosphate, magnesium sulfate, and manganese sulfate) on the production of mannitol by Lactobacillus intermedius NRRL B-3693 in a simplified medium containing 300 g fructose, 5 g soy peptone, and 50 g corn steep liquor per liter in pH-controlled fermentation at 5.0 at 37°C were evaluated using a fractional factorial design. Only manganese sulfate was found to be essential for mannitol production. Added manganese sulfate concentration of 0.033 g/l was found to support maximum production. The bacterium produced 200.6±0.2 g mannitol, 61.9±0.1 g lactic acid, and 40.4±0.3 g acetic acid from 300 g fructose per liter in 67 h.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Mycelial waste of tetracycline production as a component of the nutrient medium]

A new composition of the nutrient medium for cultivation of the tetracycline-producing organism was developed with the fermentative hydrolysate of the tetracycline production mycelial waste as a source of nitrogen: 0.02 to 0.04 g/l by amino nitrogen. The use of the medium made it possible to increase the tetracycline yield by 5 to 25 per cent, to exclude cornsteep liquor from the medium composition, to provide a more efficient recovery of the waste and to significantly decrease the environment pollution.     

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%。     

Enhanced PHB production and scale up studies using cheese whey in fed batch culture of Methylobacterium sp. ZP24

Methylobacterium sp. ZP24 produced polyhydroxybutyrate (PHB) from disaccharides like lactose and sucrose. As Methylobacterium sp. ZP24 showed growth associated PHB production, an intermittent feeding strategy having lactose and ammonium sulfate at varying concentration was used towards reaching higher yield of the polymer. About 1.5-fold increase in PHB production was obtained by this intermittent feeding strategy. Further increase in PHB production by 0.8-fold could be achieved by limiting the dissolved oxygen (DO) levels in the fermenter. The decreased DO is thought to increase flux of acetyl CO-A towards PHB accumulation over TCA cycle. Cheese whey, a dairy waste product and being a rich source of utilizable sugar and other nutrients, when used in the bioreactor as a main substrate replacing the lactose, led to further increase in the PHB production by 2.5-fold. A total of 4.58-fold increase in the PHB production was obtained using limiting DO conditions with processed cheese whey supplemented with ammonium sulfate in fed batch culture of Methylobacterium sp. ZP24. The present investigation therefore reflects on the possibility of developing a cheap biological route for production of green thermoplastics.     

Influence of alkali-treated cornsteep liquor containing medium on protein A production byStaphylococcus aureus

Staphylococcus aureus cultivated in liquid media containing untreated cornsteep liquor (CSL) and alkali-treated CSL produced similar biomass yields (6.5–6.9 g/L). However, contents of protein A in the biomass was 0.5% and 1.56% for untreated CSL and treated CSL, respectively. Addition of treated CSL at 20 g/L achieved optimal enhancement of protein A production (0.11 g/L). Probable factors associated in treated CSL for the enhanced protein A production are discussed.     

Identification of required nutrient components of yeast nitrogen base for Candida shehatae ATTCC 22984 fermenting xylose to ethanol

Summary Five components of Yeast Nutrient Base (YNB) of Difco have been identified as required nutrients for Candida shehatae ATCC 22984 in fermenting xylose to ethanol with ammonium sulfate as the nitrogen source. They are potassium phosphate monobasic, magnesium sulfate, zinc sulfate, thiamine hydrochloride, and biotin. The fermentation results in the minimum medium containing only the five required nutrient components plus xylose and ammonium sulfate have been shown to be comparable to those in the full YNB plus xylose.     

氮源对L-苏氨酸发酵的影响

以L-苏氨酸生产菌TRFC为供试菌株,研究了氮源对L-苏氨酸发酵的产量和糖酸转化率的影响。首先通过摇瓶实验确定发酵的最佳无机氮源和有机氮源分别为硫酸铵和酵母粉,进一步利用10L罐补料分批发酵确定硫酸铵和酵母粉的最佳用量,继续优化培养条件,采用发酵中后期流加硫酸铵和糖氨混合补料等措施,L-苏氨酸产量得到进一步的提高。在最优发酵条件下,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。     

Minimal nutritional requirements for immobilized yeast

The effect of reduced nutritional levels (particularly nitrogen source) for immobilized K. fragilis type yeast were studied using a trickle flow, "differential" plug flow type reactor with cells immobilized by adsorption onto an absorbant packing matrix. Minimizing nutrient levels in a feed stream to an immobilized cell reactor (ICR) might have the benefits of reducing cell growth and clogging problems in the ICR, reducing feed preparation costs, as well as reducing effluent disposal costs. In this study step changes in test feed medium nutrient compositions were introduced to the ICR, followed by a return to a basal medium. Gas evolution rates were monitored and logged on a continuous basis, and effluent cell density was used as an indicator of cell growth rate of the immobilized cell mass. Startup of the reactor using a YEP medium showed a rapid buildup of cells in the reactor during the initial 110 h operation. The population density then stabilized at 1.6 x 10(11) cells/g sponge. A defined medium containing a complex mix of essential nutrients with an inorganic nitrogen source (ammonium sulfate) was able to maintain 90% of the productivity in the ICR as compared to the YEP medium, but proved unable to promote growth of the immobilized cell mass during startup. Experiments on reduced ammonium sulfate in the defined medium, and reduced yeast extract and peptone in YEP medium indicated that stable productivity could be maintained for extended periods (80 h) in the complete absence of any nutrients besides a few salts (potassium phosphate and magnesium sulfate). It was found that productivity rates dropped by 35-65% from maximal values as nitrogenous nutrients were eliminated from the test mediums, while growth rates (as determined by shed cell density from the reactor) dropped by 75-95%. Thus, nutritional deficiencies largely decoupled growth and productivity of the immobilized yeast which suggests productivity is both growth- and non-growth-associated for the immobilized cells. A yeast extract concentration of 0.375 g/L with or without 1 g/L ammonium sulfate was determined to be the minimum level which gave a sustained increase in productivity rates as compared to the nutritionally deficient salt medium. This represents a 94% reduction in complex nitrogenous nutrient levels compared to standard YEP batch medium (3 g/L YE and 3.5 g/L peptone).     

Acid Protease Produced by Trametes sanguinea,a Wood-destroying Fungus

A wood-destroying fungus, Trametes sanguinea, produced a potent acid protease in a submerged culture. Maximum proteolytic activity of the culture was attained after 140-hours cultivation in a medium containing dextrin and corn steep liquor. The acid protease was obtained in crystalline form from the mycelium-free culture filtrate by the following successive treatments: acetone precipitation, ionexchange column chromatography, ammonium sulfate fractionation, dialysis, and crystallization by acetone. Throughout the over-all process, the acid protease was purified approximately 30-fold with about 8% recovery of the original activity.     

Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of nutrients levels using response surface methodology

The optimization of nutrient levels for the production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation (SSF) was carried out using response surface methodology based on the central composite rotatable design. The design contains a total of 54 experimental trials with the first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. The design was employed by selecting potato starch, magnesium chloride, ferrous sulfate, corn steep liquor and pearl millet flour as model factors. Among the five independent variables studied, except magnesium chloride, all the nutrients were found significant. 16.5% potato starch, 2.5% corn steep, 0.015% ferrous sulfate and 14% pearl millet flour have been found optimal for the production of thermostable pullulanase. The strain SV2 produced 10% more pullulanase in the nutritionally optimized solid-state fermentation medium containing only four nutrients.     

An optical-density-based feedback feeding method for ammonium concentration control in Spirulina platensis cultivation

Cultivation of Spirulina platensis using ammonium salts or wastewater containing ammonium as alternative nitrogen sources is considered as a commercial way to reduce the production cost. In this research, by analyzing the relationship between biomass production and ammonium- N consumption in the fed-batch culture of Spirulina platensis using ammonium bicarbonate as a nitrogen nutrient source, an online adaptive control strategy based on optical density (OD) measurements for controlling ammonium feeding was presented. The ammonium concentration was successfully controlled between the cell growth inhibitory and limiting concentrations using this OD-based feedback feeding method. As a result, the maximum biomass concentration (2.98 g/l), productivity (0.237 g/l·d), nitrogen-to-cell conversion factor (7.32 gX/gN), and contents of protein (64.1%) and chlorophyll (13.4 mg/g) obtained by using the OD-based feedback feeding method were higher than those using the constant and variable feeding methods. The OD-based feedback feeding method could be recognized as an applicable way to control ammonium feeding and a benefit for Spirulina platensis cultivations.     

Enhancement of erythromycin A production with feeding available nitrogen sources in erythromycin biosynthesis phase

Effects of feeding different available nitrogen sources from 80 h in erythromycin biosynthesis phase on the erythromycin A (Er-A) production were investigated in 50 l fermenter. Feeding corn steep liquor and yeast extract, the Er-A production was enhanced, while the biotransformation from erythromycin C (Er-C) to Er-A had no increase. When ammonium sulphate was fed at high feeding rate, the maximal Er-A production and ratio of Er-A to Er-C were 7953 U/ml and 98.18:1 at 184 h, respectively, which were higher than that of the control (6742 U/ml and 5.47:1). The feeding ammonium sulphate process was successfully scaled up from 50 l to 25 m³ fermenter. The maximal Er-A production reached 7938 U/ml at 203 h, which was enhanced by 22.1% compared with the control (6501 U/ml at 192 h). The ratio of Er-A to Er-C was 24.05:1, which was higher than that of the control (4.77:1).     

Bacterial fermentation of cheese whey for production of a ruminant feed supplement rich in curde protein.

A simple and efficient process for the production of a ruminant feed supplement, rich in crude protein (defined as total N X 6.25), by bacterial fermentation of cheese whey has been developed. The lactose in unpasteurized whey is fermented to lactate acid by Lactobacillus bulgaricus at a temperature of 43 degrees C and pH 5.5. The lactic acid produced is continually neutralized with ammonia to form ammonium lactate. The fermented product is concentrated by evaporation to a solids content of about 70% and adjusted to pH 6.8 with additional ammonia. The concentrated product contains about 55% crude protein. Approximately 6 to 8% of the crude protein is derived from bacterial cells. 17% from whey proteins, and 75 to 77% from ammonium lactate. The efficiency of conversion of lactose to lactic acid usually exceeds 95%. The fermentation time is greatly reduced upon the addition of 0.2% yeast extract or 0.1% corn steep liquor as a source of growth factors. Whey containing lactose at concentrations up to 7% can be fermented efficiently, but at higher concentrations lactose is fermented incompletely. The process has been scaled up to a pilot plant level, and 40 tons of concentrated product were produced fro animal feeding trials, without ever encountering putrefactive spoilage.     

Bacterial fermentation of cheese whey for production of a ruminant feed supplement rich in curde protein.

A simple and efficient process for the production of a ruminant feed supplement, rich in crude protein (defined as total N X 6.25), by bacterial fermentation of cheese whey has been developed. The lactose in unpasteurized whey is fermented to lactate acid by Lactobacillus bulgaricus at a temperature of 43 degrees C and pH 5.5. The lactic acid produced is continually neutralized with ammonia to form ammonium lactate. The fermented product is concentrated by evaporation to a solids content of about 70% and adjusted to pH 6.8 with additional ammonia. The concentrated product contains about 55% crude protein. Approximately 6 to 8% of the crude protein is derived from bacterial cells. 17% from whey proteins, and 75 to 77% from ammonium lactate. The efficiency of conversion of lactose to lactic acid usually exceeds 95%. The fermentation time is greatly reduced upon the addition of 0.2% yeast extract or 0.1% corn steep liquor as a source of growth factors. Whey containing lactose at concentrations up to 7% can be fermented efficiently, but at higher concentrations lactose is fermented incompletely. The process has been scaled up to a pilot plant level, and 40 tons of concentrated product were produced fro animal feeding trials, without ever encountering putrefactive spoilage.     

Medium optimization for the production of penicillin V acylase fromBacillus sphaericus

Summary Bacillus sphaericus (NCIM 2478) produced high levels of penicillin V acylase (100 U / g dry cells) when grown on cornsteep liquor — minerals medium at 25°C for 20 h. Supplementation of the medium either with 1 % (w/v) whole wheat bran or its aqueous extract brought about more than 70 % increase in the total enzyme activity. Moreover, deletion of Na₂HPO₄, MgSO₄, CaCl₂, FeSO₄, CuSO₄ and KCl from the medium affected neither growth nor enzyme production.NCL Communication No; 4964.     

氮源流加对Alcaligenes eutrophus 积累聚β-羟基丁酸的影响

以真养产碱杆菌（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ）为聚β－羟基丁酸９ＰＨＢ）的生产菌株,在分析了ＰＨＢ发酵过程参数变化的基础上,进一步探讨了ＰＨＢ合成期不同的硫酸铵流加速度对ＰＨＢ合成的影响。研究结果表明,在ＰＨＢ合成阶段,培养基中氮源的完全缺乏。导致细胞合成ＰＨＢ能力的下隆;在ＰＨＢ合成期,不同的氮源流加速率对ＰＨＢ合成过程存在着显著的影响,当流加速率较小时,尽管最终胞内ＰＨＢ含量很高