Studies on the maximization of recombinant Helicobacter pylori neutrophil-activating protein production in Escherichia coli: application of Taguchi robust design and response surface methodology for process optimization

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) is a major antigen responsible for the generation of immune response in an infected individual. The cloning and expression of the gene corresponding to neutrophil-activating protein (NAP) were followed by process development for enhanced production and purification. The production process was developed in two parts. In the first part, some of the cultivation medium components (viz. carbon to nitrogen ratio, concentrations of sodium polyphosphate and magnesium sulphate) were optimized using the Taguchi robust experimental design. The intracellular NAP production level after 24 h of cultivation was considered as the target function or the dependent variable. There was a 76.8% increase in the NAP production level. Using this optimal medium composition obtained in the first part, the temperature of cultivation and the pH of cultivation medium were optimized in the second part. The NAP production level at hour 30 of cultivation was considered as the target function or the dependent variable. The optimal values for these two independent variables were 37.2 °C and 6.3 respectively. At this combination of temperature and pH, the theoretical maximum NAP production level was 1280 mg l^–1. This optimal combination was verified experimentally and the NAP production level was found to be 1261 mg l^–1. The optimization of the cultivation conditions resulted in a 61.5% increase in NAP production level. About a 2.91-fold overall increase in NAP production level at hour 24 of cultivation was achieved through process optimization.     

Biotransformation of phosphogypsum in media containing different forms of nitrogen

Studies on the biotransformation of phosphogypsum (a waste product formed in the course of the production of phosphorous fertilizers) with the use of sulfate reducing bacteria (SRB) demonstrated that it is a good source of sulfates and biogenic elements for these bacteria, though the addition of organic carbon and nitrogen is necessary. The aim of this study was to investigate the form of nitrogen and C:N ratio in the medium on the growth of SRB community in cultures containing phosphogypsum. Batch community cultures of sulfate reducing bacteria were maintained in medium with phosphogypsum (5.0 g/l), different concentrations of sodium lactate (1.6 - 9.4 g/l) and different forms (NH4CI, CO(NH2)2, KNO3) and concentrations (0 - 250 mg/l) of nitrogen. The growth of SRB was studied in the C:N ratio of from 2:1 to 300:1. It was found that: 1 - the best source of nitrogen for SRB is urea, followed by ammonium, the worst were nitrates; 2 - the bacteria were also able to grow in medium without nitrogen but their activity was then by approximately 15% lower than in optimal growth conditions; 3 - in medium with KNO3 inhibition of sulfate reduction by approx. 50% was observed; 4 - the highest reduction of nitrates (removal of nitrate) in media with phosphogypsum and nitrates was at limiting concentrations of sodium lactate. This is probably caused by the selection under these conditions (low concentration of hydrogen sulfide) of denitrifying bacteria or sulfate reducing bacteria capable of using nitrates as an electron acceptor.     

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.     

Spirulina platensis growth in open raceway ponds using fresh water supplemented with carbon,nitrogen and metal ions

To investigate the feasibility of using fresh water from Mangueira Lagoon (Rio Grande do Sul, Brazil) for biomass production in open raceway ponds (0.7 m long, 0.18 m wide, 0.075 m deep) we studied the influence of nutrient addition (carbon as sodium bicarbonate, nitrogen as urea, phosphate, sulfate, ferric iron, magnesium and potassium) on the growth rate of the cyanobacteria Spirulina platensis using a 22 factorial design. In unsupplemented lagoon water production of S platensis was 0.78 +/- 0.01 g/l (dry weight basis) while the addition of 2.88 g/l of sodium bicarbonate (without added urea, phosphate, sulfate or metal ions) resulted in 0.82 +/- 0.01 g/l after 400 hours of culture. The further addition of phosphate and metal ions resulted in growth for up to 750 h and a final S. platensis biomass of 1.23 +/- 0.04 to 1.34 +/- 0.03 g/l.     

Medium optimization for antifungal active substances production from a newly isolated Paenibacillus sp. using response surface methodology

Statistics based experimental designs were used to optimize the medium for antifungal active substances production from a newly isolated Paenibacillus polymyxa Cp-S316 in shaker flask cultivation. The medium components having significant effect on the production were first identified using a fractional factorial design. Then steepest ascent method was employed to approach the experimental design space, followed by an application of response surface methodology for further optimization. A quadratic model was found to fit the antifungal active substances production. Response surface analysis revealed that the optimum values of the tested variables for the production of active substances were 12.3 (g/l) lactose, 17.5 (g/l) peptone, 0.4 (g/l) sodium nitrate, 4.5 (g/l) magnesium sulfate and 100 (g/l) potato. A production of 4687.71mug/ml, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of basal medium, 3.05-fold increase had been obtained.     

Effect of nitrogen source and nitrogen concentration on the production of pyruvate by Torulopsis glabrata

The effect of nitrogen sources including yeast extract, peptone, soybean hydrolyzate and some inorganic nitrogen sources, as well as the nitrogen concentration on the fermentative production of pyruvate by Torulopsis glabrata WSH-IP12 was investigated. The addition of yeast extract greatly inhibited pyruvate accumulation, while peptone was shown to be the most favorable nitrogen source. In flask culture, 15 g l(-1) peptone was needed to consume 80 g l(-1) glucose with 23.4 g l(-1)of pyruvate accumulated. Pyruvate production was markedly dependent on the ratio of carbon to nitrogen (C:N), its production was improved by increasing the concentration of glucose and peptone proportionally and reduced by exclusively increasing the glucose concentration. In a glucose fed-batch culture, cell growth and pyruvate production slowed after 28 h. However, cell growth and pyruvate production recovered after further nitrogen, in the form of peptone and ammonium sulfate, was added to the culture. A final concentration of pyruvate of 54.5 g l(-1) was achieved at 64 h (yield to glucose consumed of 0.471 g g(-l)). By using aqueous ammonia instead of potassium hydroxide for pH control, 57.3 g l(-1) pyruvate with a yield of 0.498 g g(-1) was produced by 55 h. This result further indicates that nitrogen level plays an important role in the production of pyruvate.     

Use of response surface methodology for selection of nutrient levels for culturing Paecilomyces variotii in eucalyptus hemicellulosic hydrolyzate

Eucalyptus hemicellulose was hydrolyzed by treating eucalyptus wood chips with sulfuric acid. The hydrolyzate was used as the substrate to produce single-cell protein by growing Paecilomyces variotii IOC-3764 for 72 or 96 h. The influences of rice bran, ammonium sulfate and fermentation time were verified by a 23 full-factorial central composite design. At the optimum process conditions, the cell concentration was 12.06 g/l, which was obtained when the microorganisms were cultivated for 89 h in a medium composed of 10 g/l rice bran, 2.0 g/l nitrogen and 1.1 g/l sodium phosphate. The mathematical model Y = 10.65 + 2.40X2 + 2.36X3 + 1.16X2X3 - 2.10X2(2) - 1.06X3(2) describes biomass production by P. variotii in eucalyptus hemicellulosic hydrolyzate with a determination coefficient of R2 = 0.9561, where X2 and X3 are ammonium sulfate and fermentation time, respectively.     

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。     

北柴胡不定根系生物转化合成皂苷研究

通过在北柴胡不定根系培养体系中添加目的产物柴胡皂苷的前体物质和生物合成促进物质以及进行高糖渗透胁迫试验,探讨了利用不定根系生产柴胡皂苷的条件和方法。结果表明,在1/2 MS培养基中添加硫酸镁、硝酸钾、丙酮酸钠以及高糖渗透胁迫均有利于柴胡皂苷的合成。1/2 MS培养基+硫酸镁0.37 g/L+硝酸钾5.7 g/L+丙酮酸钠2.0 mmol/L+蔗糖50 g/L是最适合柴胡皂苷转化合成的培养基,此条件下使柴胡皂苷的含量提高了9.74倍。     

Statistical optimization of the medium composition by response surface methodology to enhance schizophyllan production by Schizophyllum commune

The response surface methodology (RSM) involving central composite design (CCD) was employed to optimize the fermentation medium for the cell growth and schizophllan production by Schizophyllum commune CGMCC 5.113 in submerged culture at pH 6.5 and 26 degrees C. The four variables involved in this study were glucose, yeast extract, ammonium nitrate, and magnesium sulfate. The statistical analysis of the results showed that, in the range studied, glucose and yeast extract had a highly significant effect on schizophyllan production. The optimal medium for schizophyllan production calculated from the regression model of RSM was as follows: glucose, 18 g/l; yeast extract, 0.5 g/l; NH4NO3, 0.48 g/l; and MgSO4, 0.05 g/l, with a predicted maximum schizophyllan production of 11.74 g/l. These predicted values were experimentally validated. The excellent correlation between predicted and measured values justifies the validity of the response model. The results of bioreactor fermentation also show that the optimized medium enhanced schizophyllan production (12.80 g/l) by S. commune in a 5-1 fermenter.     

Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts.

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacter sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources.     

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.     

Factors affecting microbial sulfate reduction by Desulfovibrio desulfuricans in continuous culture: limiting nutrients and sulfide concentration

The effects of sulfate and nitrogen concentrations of the rate and stoichiometry of microbial sulfate reduction were investigated for Desulfovibrio desulfuricans grown on lactate and sulfate in a chemostat at pH 7.0. Maximum specific growth rates (mu(max)), half-saturation coefficients (K(sul)), and cell yield (Y(c/Lac)) of 0.344 +/- 0.007 and 0.352 +/- 0.003 h (-1), 1.8 +/- 0.3 and 1.0 +/- 0.2 mg/L, and 0.020 +/- 0.003 and 0.017 +/- 0.003 g cell/g lactate, respectively, were obtained under sulfate-limiting conditions at 35 degrees C and 43 degrees C. Maintenance energy requirements for D. desulfuricans were significant under sulfate-limiting conditions. The extent of extracellular polymeric substance (EPS) produced was related to the carbon: nitrogen ratio in the medium. EPS production rate increased with decreased nitrogen loading rate. Nitrogen starvation also resulted in decreased cell size of D. desulfuricans. The limiting C : N ratio (w/w) for D. desulfuricans was in the range of 45 : 1 to 120 : 1. Effects of sulfide on microbial sulfate reduction, cell size, and biomass production were also ivestigated at pH 7.0. Fifty percent inhibition of lactate utilization occurred at a total sulfide concentration of approximately 500 mg/L. The cell size of D. desulfuricans decreased with increasing total sulfide concentration. Sulfide inhibition of D. desulfuricans was observed to be a reversible process. (c) 1992 John Wiley & Sons, Inc.     

Optimization of carbon and nitrogen sources and growth factors for the production of an aquaculture probiotic (Pseudomonas MCCB 103) using response surface methodology

AIM: To develop a new medium for enhanced production of biomass of an aquaculture probiotic Pseudomonas MCCB 103 and its antagonistic phenazine compound, pyocyanin. METHODS AND RESULTS: Carbon and nitrogen sources and growth factors, such as amino acids and vitamins, were screened initially in a mineral medium for the biomass and antagonistic compound of Pseudomonas MCCB 103. The selected ingredients were further optimized using a full-factorial central composite design of the response surface methodology. The medium optimized as per the model for biomass contained mannitol (20 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5 g l(-1)), urea (3.3 g l(-1)) and mineral salts solution (20 ml l(-1)), and the one optimized for the antagonistic compound contained mannitol (2 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5.1 g l(-1)), urea (3.6 g l(-1)) and mineral salts solution (20 ml l(-1)). Subsequently, the model was validated experimentally with a biomass increase by 19% and fivefold increase of the antagonistic compound. CONCLUSION: Significant increase in the biomass and antagonistic compound production could be obtained in the new media. SIGNIFICANCE AND IMPACT OF THE STUDY: Media formulation and optimization are the primary steps involved in bioprocess technology, an attempt not made so far in the production of aquaculture probiotics.     

Production of biomass and nutraceutical compounds by Spirulina platensis under different temperature and nitrogen regimes

The cyanobacterium Spirulina platensis has been used by humans because of its nutritional and possibly medicinal effects. Our study evaluated the influence of temperature and nitrogen concentration in the medium on the production of biomass by this cyanobacterium and the biomass composition in protein, lipid and phenolic compounds. We found that at 35 degrees C there was a negative effect on biomass production but a positive effect on the production of protein, lipids and phenolics, the highest levels of these compounds being obtained in Zarrouk's medium containing 1.875 or 2.500 g l(-1) sodium nitrate. Higher biomass densities and productivity were obtained at 30 degrees C than at 35 degrees C, but nitrogen concentration appeared to have no effect on the amount of protein, lipid or phenolics, indicating that at 30 degrees C the concentration of sodium nitrate in Zarrouk's medium (2.50 g l(-1)) can be reduced without loss of productivity, an important cost-saving factor in large-scale cultivation.     

Improvement in raw sago starch degrading enzyme production from Acremonium sp. endophytic fungus using carbon and nitrogen sources

Attempts were made to improve the growth of endophytic fungus Acremonium sp. and its raw sago starch degrading enzyme (RSSDE) production using different nitrogen and carbon sources at varying pH values and temperatures. It was observed that growth and enzyme activity levels were highest with peptone and sodium nitrate as the nitrogen sources and raw sago starch as the carbon source of which the optimum concentrations were 0.5 g/l, 3 g/l, and 20 g/l, respectively. Cell growth and RSSDE production reached their optimum at pH 5.0 and incubation temperature of 30 degrees C. Under these conditions, the enzyme production was significantly increased by 19- to 22-folds compared to the activity obtained in the original basal medium.     

Culture media optimization for growth and phycoerythrin production from Porphyridium purpureum

Porphyridium spp. is a red micro alga and is gaining importance as a source of valuable products viz., phycobiliproteins (PB), sulfated exopolysaccharides, and polyunsaturated fatty acids with potential applications in the food and pharmaceutical industries. In the present study, the effects of the major media constituents of Porphyridium species were studied using response surface methodology (RSM) on biomass yield, total PB and the production of phycoerythrin (PE). A second order polynomial can be used to predict the PB and PE production in terms of the independent variables. The independent variables such as the concentrations of sodium chloride, magnesium sulfate, sodium nitrate, and dipotassium hydrogen phosphate influenced the total PB and PE production. The optimum conditions showed that total PB was 4.8% at the concentration of sodium chloride 26.1 g/L, magnesium sulfate 5.23 g/L, sodium nitrate 1.56 g/L, and dipotassium hydrogen phosphate 0.034 g/L. In case of optimum PE production (3.3%), the corresponding values are 29.62, 6.11, 1.59, and 0.076 g/L, respectively. PE production depends greatly on the concentrations of chloride, nitrate, and sulfate as well as phosphate of which the former possess the maximum effect.     

植物乳杆菌ZJ316生产细菌素

[目的]研究植物乳杆菌ZJ316生长和产细菌素的最佳培养基成分和发酵条件,以提高该菌产plantaricin ZJ316的能力.[方法]改变培养基成份和发酵条件,考察不同氮源、碳源等培养基成分和不同的发酵温度等条件对ZJ316生长和产细菌素的影响.[结果]最佳培养基为MRS培养基;优化后的培养基配方为葡萄糖10 g/L,麦芽糖10 g/L,酵母提取物10 g/L,蛋白胨10 g/L,柠檬酸三铵2 g/L,吐温80为1 Ml/L,K2HPO4·3H2O 6 g/L,乙酸钠5 g/L,硫酸镁0.2 g/L,硫酸锰0.05 g/L.培养基初始Ph6.5,30℃静置培养24 h.[结论]通过培养基成分和发酵条件的优化,细菌素产量提高了2.3倍,为进一步研究和规模化生产奠定基础.     

Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources

The effects of nitrate, ammonium, and urea as nitrogen sources on the heterotrophic growth of Chlorella protothecoides were investigated using flask cultures. No appreciable inhibitory effect on the algal growth was observed over a nitrogen concentration range of 0.85-1.7 g l(-)(1). In contrast, differences in specific growth rate and biomass production were found among the cultures with the various nitrogen compounds. The influence of different nitrogen sources at a concentration equivalent to 1.7 g l(-)(1) nitrogen on the heterotrophic production of biomass and lutein by C. protothecoides was investigated using the culture medium containing 40 g l(-)(1) glucose as the sole carbon and energy source in fermentors. The maximum biomass concentrations in the three cultures with nitrate, ammonium, and urea were 18.4, 18.9, and 19.6 g l(-)(1) dry cells, respectively. The maximum lutein yields in these cultures were between 68.42 and 83.81 mg l(-)(1). The highest yields of both biomass and lutein were achieved in the culture with urea. It was therefore concluded that urea was the best nitrogen source for the production of biomass and lutein. Based on the experimental results, a group of kinetic models describing cell growth, lutein production, and glucose and nitrogen consumption were proposed and a satisfactory fit was found between the experimental results and predicted values. Dynamic analysis of models demonstrated that enhancing initial nitrogen concentration in fermentor cultures, which correspondingly enhances cell growth and lutein formation, may shorten the fermentation cycle by 25-46%.     

Evaluation of parallel operated small-scale bubble columns for microbial process development using Staphylococcus carnosus

Shake flasks and pH-controlled small-scale bubble columns were compared with respect to their usefulness as a basic tool for process development for human calcitonin precursor fusion-protein production with Staphylococcus carnosus. Parallel control of the pH (and making use of the base addition data) is necessary to study the effects of medium composition, to identify pH-optima and to develop a medium, which minimizes the acid excretion of S. carnosus. This medium with glycerol as energy source and yeast extract as carbon and nitrogen source resulted in cell dry weight concentration in shake flasks of 5 g l(-1), which were thus improved by a factor of 10. Cell dry weight concentrations of up to 12.5 g l(-1) were measured in the batch process with pH-controlled small-scale bubble columns due to their higher oxygen transfer capability. In contrast to shake flasks it was demonstrated, that the batch process performance of recombinant S. carnosus secreting the human calcitonin precursor fusion-protein was identical within the estimation error in pH-controlled small-scale bubble columns compared to the stirred-tank reactor.