Emulsan quantitation by Nile red quenching fluorescence assay

A Nile red fluorescent technique to quantify 20–200 g ml^–1 of emulsan was developed. Nile red dissolved in DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm^–1 M^–1. Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG-1.     

Novel polysaccharide–protein-based amphipathic formulations

Previous results showed that the cell-surface esterase from Acinetobacter venetianus RAG-1 enhances the emulsification properties of the polymeric bioemulsifier emulsan and its deproteinated derivative apoemulsan (Bach H, Berdichevsky Y, Gutnick D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl Environ Microbiol 69:2608–2615). Here we show that in the presence of the his-tagged recombinant esterase from RAG-1, 18 different polysaccharides from microbial, plant, insect and synthetic sources formed hexadecane-in-water emulsions. Emulsifying activities were distributed over a 13-fold range from over 4800 U/mg protein/mg polysaccharide in the case of apoemulsan to 370 U/mg protein/mg polysaccharide in the case of alginic acid. The stability of the emulsions ranged between 95 and 58%. Emulsions formed in the presence of seven of the polysaccharides exhibited stabilities of over 80%. The esterase from A. calcoaceticus BD4, which shows sequence homology to the RAG-1 esterase, was inactive in emulsification enhancement. The sequence of the RAG-1 esterase was shown to contain two conserved peptide sequences previously shown to be implicated in carbohydrate/polysaccharide binding. A hypothetical model illustrating a possible mode of interaction between the esterase, the apoemulsan and the oil droplet is presented. The complex is presumed to generate a series of “coated” oil droplets which are restricted in their ability to coalesce resulting in a relatively stable emulsion.     

Optimization of <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation on sugarcane bagasse

Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH₂PO₄ as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH₂PO₄ 1.63, K₂HPO₄ 0.325, and MgSO₄ 0.325. Under optimal conditions, spore production reached 1.1 × 10¹⁰ spores/g dried carrier, much higher than that on wheat bran (1.7 × 10⁹ spores/g initial dry matter).     

Medium optimization for keratinase production in hair substrate by a new <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KD-N2 using response surface methodology

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO₄ and K₂HPO₄ were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO₄ and K₂HPO₄ concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl₂ 0.05, KH₂PO₄ 0.7, sucrose 3, MgSO₄ 0.91, K₂HPO₄ 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.     

Construction of a chimeric gene cluster for the biosynthesis of apoemulsan with altered molecular weight

Acinetobacter venetianus RAG-1 produces an extracellular protein/high-molecular-weight (HMW) polysaccharide complex termed emulsan. As an emulsion stabilizer, emulsan has potential industrial applications. To control the molecular weight of the polymer, a stable chromosomal mutant was generated where RAG-1 wza, wzb, wzc genes were replaced by Escherichia coli homologs. The heterologous Wza, Wzb, Wzc proteins restored production of HMW polysaccharide. The polymer produced was of higher molecular weight than from the parent strain and with the cells exhibiting modified hydrophobicity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimization of the medium for glutathione production in Saccharomyces cerevisiae

As a powerful statistical experimental design, uniform design (UD) method has been successfully applied in various fields such as fermentation industry, pharmaceuticals, and others. In this paper, UD was applied to optimize the medium composition for glutathione production in shake-flask culture of Saccharomyces cerevisiae T65. The experiments of nine factors (glucose, yeast extract, peptone, malt extract, molasses, MgSO₄, ZnSO₄, (NH₄)₂HPO₄ and thiamine) and nine levels were carried out according to the uniform design table U₂₇(9⁹). The experimental data was analyzed to obtain the regression model and the optimal medium composition was achieved by optimization with UD 3.0 software. The optimal medium consisted of 70 g/L glucose, 3 g/L yeast extract, 5 g/L peptone, 70 g/L malt extract, 20 g/L molasses, 5.6 g/L MgSO₄, 16 mg/L ZnSO₄, 7 g/L (NH₄)₂HPO₄ and 0.2 mg/L thiamine. The GSH yield at the optimal point achieved 74.6 mg/L, which was 1.81 times higher than that of the control. The application of UD method resulted in enhancement in GSH production.     

Relationship between phage resistance and emulsan production,interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 10⁶) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.     

Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a′ using response surface methodology

The novel exopolysaccharide bioflocculant HBF-3 is produced by Halomonas sp. V3a′, which is a mutant strain of the deep-sea bacterium Halomonas sp. V3a. Response surface methodology (RSM) was employed to optimize the production medium for increasing HBF-3 production. Using a Plackett–Burman experimental design to aid in the first step of optimization, edible glucose, MgSO₄·7H₂O, and NH₄Cl were found to be significant factors affecting HBF-3 production. To determine the optimal concentration of each significant variable, a central composite design was employed. Based on response surface and canonical analysis, the optimum concentrations of the critical components were obtained as follows: edible glucose, 16.14 g/l; MgSO₄·7H₂O, 2.73 g/l; and NH₄Cl, 1.97 g/l. HBF-3 production obtained by using the optimized medium was 4.52 g/l, which was in close agreement with the predicted value of 4.55 g/l. By scaling up fermentation from flask to fermenter, HBF-3 production was further increased to 5.58 g/l.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

High cell density production of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> under optimized conditions

Deinococcus radiodurans is a bacterium being investigated for mechanisms of extreme radiation resistance and for bioremediation of environmental radioactive waste sites. In both fundamental and applied research settings, methods for large-scale production of D. radiodurans are needed. In this study, a systematic investigation was carried out to optimize D. radiodurans production at the 20-L fermentor scale. In defined medium, the phosphate buffer typically used was found to be inhibitory to D. radiodurans growth, and caused cell aggregation. Substitution of HEPES and MOPS buffers for phosphate buffer improved D. radiodurans growth characteristics. Several antifoaming agents were investigated to support large-scale production with submerged aeration, and the defoamer KFO 673 was chosen based on its ability to prevent foaming without affecting D. radiodurans growth. The conventional undefined rich medium tryptone/glucose/yeast extract (TGY) maximally supported D. radiodurans growth to an OD₆₀₀ of 10. Using a ‘design of experiments’ approach, we found glucose, Mg and Mn to be critical in supporting high-density growth of D. radiodurans. The optimal pH and temperature for D. radiodurans growth in large-scale preparations were 7.0 and 37°C, respectively. Growth was carried out in a 20-L fermentor using the newly developed media under the optimal conditions. With addition of 10 g/L glucose, 0.5 g/L MgSO₄ · 7H₂O, 5 μM MnCl₂ into TGY media, an OD₆₀₀ of 40 was achieved.     

Enhanced 2,3-butanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> SDM

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH₄)₂HPO₄ were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation. Cuiqing Ma and Ailong Wang contributed equally to this work.     

Medium optimization and structural characterization of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

In this study, response surface methodology was employed to optimize the medium compositions for the production of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3. Firstly, fractional factorial design was applied to evaluate the effects of different components in the medium. It was found that sucrose, yeast extract and CaCl₂ influenced significantly the production of EPS. Then, steepest ascent method and central composite design were used to optimize the concentrations of the three variables. As results, the optimal medium compositions were determined as following (g/L): sucrose 188.2, yeast extract 25.8, K₂HPO₄ 5 and CaCl₂ 0.34, with a corresponding yield of 35.26 g/L. In addition, both polysaccharide fractions (EPS-1 and EPS-2) from crude EPS were mainly composed of (2 → 6)-linked β-d-fructofuranosyl residues backbone with (2 → 1)-linked branches based on their structural characterization by FT-IR spectroscopy, methylation analysis and ¹³C NMR spectroscopy.     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.     

Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of exopolysaccharides

Optimization of medium composition for the production of exopolysaccharides (EPS) from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of EPS were carried out. Firstly, the medium components having significant effect on EPS production were screened out to be glucose, yeast extract and diammonium oxalate monohydrate by using a 2⁽⁷⁻³⁾ fractional factorial design. Secondly, the concentrations of the three factors were optimized using central composite design in response surface methodology. As results, a quadratic model was found to fit for EPS production, and the optimal medium composition was determined as following (g/l): 34.12 glucose, 4 peptone, 5.01 yeast extract, 0.88 diammonium oxalate monohydrate, 0.75 MgSO₄ and 1 KH₂PO₄ and 0.0075 thiamine (VB₁). A yield of 2.363 ± 0.04 g/l for EPS was observed in verification experiment. Finally, EPS from P. baumii Pilát was found to have direct immuno-stimulating activity in vitro on splenocyte proliferative response and acid phosphatase activity in peritoneal macrophages in a dose-dependent manner.     

Enhanced production of amidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.     

Statistical optimization of medium components for avilamycin production by <Emphasis Type="Italic">Streptomyces viridochromogenes</Emphasis> Tü57-1 using response surface methodology

A fermentation medium for avilamycin production by Streptomyces viridochromogenes Tü57-1 has been optimized. Important components and their concentrations were investigated using fractional factorial design and Box–Behnken Design. The results showed that soybean flour, soluble starch, MgSO₄·7H₂O and CaCl₂·2H₂O are important for avilamycin production. A polynomial model related to medium components and avilamycin yield had been established. A high coefficient of determination (R ² = 0.92) was obtained that indicated good agreement between the experimental and predicted values of avilamycin yield. Student’s T-test of each coefficient showed that all the linear and quadratic terms had significant effect (P > |T| < 0.05) on avilamycin yield. The significance of tested components was related to MgSO₄·7H₂O (0.37 g/L), CaCl₂·2H₂O (0.39 g/L), soybean flour (21.97 g/L) and soluble starch (37.22 g/L). The yield of avilamycin reached 88.33 ± 0.94 mg/L (p < 0.05) that was 2.8-fold the initial yield.     

Development of medium for enhanced production of glutaminase-free <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase from <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> MTCC 1428

Glutaminase-free l-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of l-asparaginase using the Plackett–Burman experimental design. The medium components, viz., glucose, l-asparagine, KH₂PO₄, and MgSO₄·7H₂O, were screened based on their high confidence levels (P < 0.04). The optimum levels of glucose, l-asparagine, KH₂PO₄, and MgSO₄·7H₂O were found to be 2.076, 5.202, 1.773, and 0.373 g L⁻¹, respectively, using the central composite experimental design. The maximum specific activity of l-asparaginase in the optimized medium was 27.88 U mg⁻¹ of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.     

A unique polypeptide from the C-terminus of the exocellular esterase of Acinetobacter venetianus RAG-1 modulates the emulsifying activity of the polymeric bioemulsifier apoemulsan

An exocellular esterase from the oil-degrading Acinetobacter venetianus RAG-1 was previously shown to enhance the emulsification and emulsion stabilization properties of the amphipathic, aminopolysaccharide bioemulsifier, emulsan [Bach H, Berdichevsky Y, Gutnick D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl Environ Microbiol 69:2608–15]. This enhancement was specific for the RAG-1 esterase and was independent of catalytic activity. In this report, fragments from both the N′- and C′-termini were cloned as fusions to the C-terminus of the maltose-binding protein (MBP) and were tested for enhancement activity in the presence of the deproteinated form of emulsan, apoemulsan. The activity could be localized to the C-terminal third of the protein which exhibited the same activity as the intact enzyme. MBP itself was completely inactive and could be cleaved from the fusion without affecting the subsequent emulsification. However, the enhancement completely depended on the presence of a unique C-terminal 20 amino acid peptide not found in any other protein in the databases. In addition, progressive removal of amino acids from the N-terminus of the active MBP polypeptide resulted in a concomitant loss of activity, indicating that enhancement is also proportional to the size of the peptide fragment. The middle third and the C-terminal third of the enzyme each contained a copy of the conserved Cardin–Weintraub consensus sequence for protein binding to heparin. These sequences were not detected in homologous esterases from a closely related strain, Acinetobacter calcoaceticus BD413.     

Optimization of fermentation conditions for production of anti-TMV extracellular ribonuclease by Bacillus cereus using response surface methodology

Bacillus cereus ZH14 was previously found to produce a new type of antiviral ribonuclease, which was secreted into medium and active against tobacco mosaic virus. In order to enhance the ribonuclease production, in this study the optimization of culture conditions using response surface methodology was done. The fermentation variables including culture temperature, initial pH, inoculum size, sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃ were considered for selection of significant ones by using the Plackett–Burman design, and four significant variables (sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃) were further optimized by a 2⁴ factorial central composite design. The optimal combination of the medium constituents for maximum ribonuclease production was determined as 8.50 g/l sucrose, 9.30 g/l yeast extract, 2.00 g/l MgSO₄·7H₂O, and 0.62 g/l KNO₃. The enzyme activity was increased by 60%. This study will be helpful to the future commercial development of the new bacteria-based antiviral ribonuclease fermentation process.     

Optimization of nutrient parameters for lovastatin production by <Emphasis Type="Italic">Monascus purpureus</Emphasis> MTCC 369 under submerged fermentation using response surface methodology

Lovastatin, an inhibitor of HMG-CoA reductase, was produced by submerged fermentation using Monascus purpureus MTCC 369. Five nutritional parameters screened using Plackett–Burman experimental design were optimized by Box–Behnken factorial design of response surface methodology for lovastatin production in shake flask cultures. Maximum lovastatin production of 351 mg/l were predicted in medium containing 29.59 g/l dextrose, 3.86 g/l NH₄Cl, 1.73 g/l KH₂PO₄, 0.86 g/l MgSO₄·7H₂O, and 0.19 g/l MnSO₄·H₂O using response surface plots and point prediction tool of DESIGN EXPERT 7.0 (Statease, USA) software.