Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans

Aureobasidium pullulans had a maximum yield coefficient of pullulan (Y _p/s=0.24) with an initial pH of the culture broth of 6.5 in a shake-flask culture. In a batch culture, the maximum pullulan yield coefficient of 0.30 was obtained at the aeration rate of 0.5 vvm. A yeast-like form and mycelial form of cells were found at the culture broth with pH controlled at 4.5 with a maximum yield coefficient of pullulan of 0.27. However, a high portion (35%) of high molecular weight pullulan (M _w>2 000 000) was produced at pH 6.5 with a yeast-like morphology of the cells.     

Application of data consistency tests and new parameter estimation methods to microbial growth on corn dust in batch culture

The microorganism Candida utilis was grown on both filtered and unfiltered substrate obtained from enzymatic hydrolysis of starch in corn dust. For growth on filtered substrate, the average integrated biomass energetic yield value based on biomass-substrate data was η = 0.55 and for growth on unfiltered substrate an average yield value of η = 0.59 was obtained. Material and energy balances showed that the presence of unfiltered corn residue in the media had no significant effect on the yields. Statistical methods were developed and used to obtain best estimates of the growth parameters. Values of the biomass energetic yield corrected for maintenance (η_max = 0.619) and the maintenance coefficient (m_e = 0.043) were obtained for growth on filtered substrate. Values of η_max = 0.741 and m_e = 0.142 were obtained for the growth on unfiltered substrate. The consistency of data and parameter estimates was relatively good for filtered substrate; however, parameter estimates for unfiltered substrate were not consistent. Growth experiments without filtration of the products of starch hydrolysis resulted in protein-enriched products with about 39.73% protein.     

Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1

Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µ_max = 0.050–0.055 h^–1) and a maximum value of the specific acetate consumption rate (q _infs ^supps ) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K _m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. Offprint requests to: R. Moletta     

Improved production of (R)-2-octanol via asymmetric reduction of 2-octanone with Oenococcus oeni CECT4730 in a biphasic system

Abstract

Oenococcus oeni CECT4730, which catalyses the asymmetric reduction of 2-octanone to (R)-2-octanol with high enantioselectivity, was further studied to exploit its potential for production of (R)-2-octanol in an aqueous/organic solvent biphasic system. Variables such as the volume ratio of aqueous to organic phase (V_a/V_o), buffer pH, reaction temperature, shaking speed, co-substrates and the ratio of biocatalyst to substrate were examined with respect to the molar conversion, the initial reaction rate and the product enantiomeric excess (e.e.). Under the optimized conditions (V_a/V_o=1:1 (v/v), buffer pH=8.0, reaction temperature=30°C, shaking speed=150 rev/min, ratio of glucose to biomass=5.4:l (w/w), ratio of biocatalyst to substrate=0.51:l (g/mol)), the highest space time yield of (R)-2-octanol, 24 mmol L^?1 per h, and >98% product e.e. were obtained at a substrate concentration close to 1.0 mol L^?1 after 24 h reduction.     

Expression and purification of ubiquitin-specific protease (UBP1) ofSaccharomyces cerevisiae in recombinantEscherichia coli

Truncated form of UBP1, an ubiquitin-specific protease ofSaccharomyces cerevisiae, was overexpressed inEscherichia coli. The hexahistidine residue (His₆) was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were 40°C and pH 8.0, respectively.     

Purification and Some Properties of Cyclomaltodextrin Glucanotransferase from Bacillus circulans

Cyclomaltodextrin glucanotransferase was purified from B. circulans C31 through two successive steps of starch and Biogel column chromatography. The enzyme was purified up to 90-fold with a 30% yield. Its molecular weight was around 103,000. The purified enzyme converted 28% of the soluble starch to β-cyclodextrin at pH 7.0 and a substrate concentration of 5%. The optimum pH for the enzyme was found to be 5.5. The optimum temperature was 60°C. The enzyme optimum was stable from pH 5.5~9.0 and up to 50°C.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

Fusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr^?1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr^?1 were obtained at μ = 0.205 hr^?1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr^?1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr^?1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr^?1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr^?1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr^?1 in order to maximize protein production.     

Optimization of the 11α-hydroxylation of steroid DHEA by solvent-adapted Beauveria bassiana

To extend the use of Beauveria bassiana for commercial applications, the optimization of reaction conditions and accurate prediction of biotransformation products are necessary. This work enhances the selective hydroxylation capacity of strain ATCC 7159, resulting in a cost effective and eco-friendly process for the synthesis of valuable 11α-hydroxy steroids. Our work establishes the biochemical pathway of DHEA to hydroxylated intermediates with strain ATCC 7159, and distinguishes the optimum conditions for reactor arrangements, substrate concentration, reaction temperature, and pH. Higher substrate conversion, selectivity, and yield of desired product was achieved with “resting cells.” Addition of higher volumes of growing medium relative to reaction buffer increases the reaction rate. When a diluted amount of substrate is used, a higher yield of 11α-hydroxy steroids is achieved. Also, reactions at 26?°C with pH ranges between 6.0 and 7.0 result in the highest conversion (70%) and the higher product yield (45.8%). B. bassiana has the capacity to metabolize DHEA and similar steroids in different reaction schemes, and has a promising future as biocatalyst to be used in the production of drug metabolites.     

Optimization of process parameters for the continuous production of alcohol with a cell immobilized bioreactor

The optimum values of substrate concentration, pH and temperature for higher yields of alcohol in cell immobilized bioreactor using alginate entrapped cells of yeasts for continuous fermentation of alcohol were obtained employing full factorial search. The results indicate that the yield of alcohol is predominantly influenced by the substrate concentration and temperature, both individually and in combination. The pH, on the other hand, has no significant influence. The path of steepest ascent method has been used to optimise the alcohol yield. A best alcohol yield has been obtained with 23.5% substrate concentration, 30°C temperature and at pH 5.0.     

Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

Continuous production of urocanic acid by immobilized Achromobacter liquidum cells

Several microorganisms having higher L -histidine ammonia-lyase activity were immobilized into polyacrylamide gel lattice. The yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667. As A. liquidum has urocanase activity, the cells were heat-treated at 70°C for 30 min to inactivate the urocanase. Enzymatic properties of the immobilized A. liquidum cells were investigated and compared with those of the intact cells. No difference was observed between the pH activity curve and optimal temperature for the intact and immobilized cells. The permeability of substrate or product through the cell wall was increased by immobilization of the cells. When an aqueous solution of 0.25M L -histidine (pH 9.0) containing 1mM Mg²⁺ was passed through a column packed with the immobilized A. liquidum cells at a flow rate of SV = 0.06 at 37°C, L -histidine was completely converted to urocanic acid. The L -histidine ammonia-lyase activity of the immobilized cell column was stable over 40 days at 37°C. From the effluent of the immobilized cell column, Urocanic acid was easily obtained in a good yield.     

Production of cellulase-free thermostable xylanases by an isolated strain of Aspergillus niger PPI, utilizing various lignocellulosic wastes

A strain of Aspergillus niger PPI having prolific xylanolytic potential was isolated and the optimum conditions for maximum xylanase production was studied, resulting in the following: 4% substrate concentration, 10% v/v inoculum size, 72 h of incubation and pH 3.5–4.5 at 28 °C. The production profile of xylanase was examined with various lignocellulosics and maximum yield was achieved with oat. The hemicellulose content of wastes was also determined and oatmeal was found to have maximum hemicellulose content followed by wheat straw, sugarcane bagasse, rice husk and gram residue respectively. The enzyme showed maximum activity at pH 4 and temperature 60 °C. However, maximum stability was achieved at pH 3.5 and temperature 55 °C. Cellulase activity was found altogether absent in the enzyme broth.     

Different kinetic patterns in the α-chymotrypsin-catalysed hydrolysis of synthetic ester substrates

The reaction of α-chymotrypsin with AcTyr-OEt and with AcTrp-OEt at pH 7.0 and 7.8 was studied over a wide range of substrate concentrations. The reaction with AcTyr-OEt at pH 7.8 was shown to be nonhyperbolic using a variety of criteria whereas those at pH 7.0 with the same substrate and at both pH values with AcTrp-OEt were hyperbolic. The non-hyperbolicity of the reaction with AcTyr-OEt at pH 7.8 followed a pattern of negative cooperativity with a Hill coefficient for the high substrate concentration range of 0.48. Although other explanations are possible, the pH dependence of the reaction with AcTyr-OEt could be related to the slow transition of the two known forms of the enzyme.Negative cooperativityNonhyperbolic kineticsα-ChymotrypsinHyperbolic kineticsHill coefficientSlow transition     

Oxidation of stibnite byThiobacillus ferrooxidans

Optimum pH, temperature and pulp density for microbiological leaching of museum-grade stibnite mineral has been investigated using a stibnite-adapted strain ofThiobacillus ferrooxidans. Optimum conditions were found to be pH 1.75, 35 C and 12 g solid substrate per 100 ml of basal salts medium as the initial dose. The energy of activation was determined to be 16.8 kcal per mole, and the temperature coefficient 2.2. The highest total dissolved-antimony concentration, [Sb_t] = [Sb⁺³] + [Sb⁺⁵] + [SbO⁺] + [SbO₂ ⁺], was about 1400 mg/litre, due to relatively low solubility of (SbO)₂SO₄ and (SbO₂)₂SO₄.     

Modelling of the enzymatic kinetically controlled synthesis of cephalexin: influence of diffusion limitation

In this study the influence of diffusion limitation on enzymatic kinetically controlled cephalexin synthesis from phenylglycine amide and 7-aminodeacetoxycephalosporinic acid (7-ADCA) was investigated systematically. It was found that if diffusion limitation occurred, both the synthesis/hydrolysis ratio (S/H ratio) and the yield decreased, resulting in lower product and higher by-product concentrations. The effect of pH, enzyme loading, and temperature was investigated, their influence on the course of the reaction was evaluated, and eventually diffusion limitation was minimised. It was found that at pH >or=7 the effect of diffusion limitation was eminent; the difference in S/H ratio and yield between free and immobilised enzyme was considerable. At lower pH, the influence of diffusion limitation was minimal. At low temperature, high yields and S/H ratios were found for all enzymes tested because the hydrolysis reactions were suppressed and the synthesis reaction was hardly influenced by temperature. The enzyme loading influenced the S/H ratio and yield, as expected for diffusion-limited particles. For Assemblase 3750 (the number refers to the degree of enzyme loading), it was proven that both cephalexin synthesis and hydrolysis were diffusion limited. For Assemblase 7500, which carries double the enzyme load of Assemblase 3750, these reactions were also proven to be diffusion limited, together with the binding-step of the substrate phenylglycine amide to the enzyme. For an actual process, the effects of diffusion limitation should preferably be minimised. This can be achieved at low temperature, low pH, and high substrate concentrations. An optimum in S/H ratio and yield was found at pH 7.5 and low temperature, where a relatively low reaction pH can be combined with a relatively high solubility of 7-ADCA. When comparing the different enzymes at these conditions, the free enzyme gave slightly better results than both immobilised biocatalysts, but the effect of diffusion limitation was minimal.     

Optimization of Citric Acid Production from Glucose by Yarrowia lipolytica

Citric acid (CA) is mainly produced in a biotechnological process using Aspergillus niger. In this process, large amounts of wastes have to be removed. Since the use of Yarrowia lipolytica for CA production is an environmental compatible alternative method, the CA production was optimized in regard to growth temperature and pH as well as substrate and product inhibition. The highest value of the maximum specific growth rate at pH 6.5 was found to be μ_max = 0.192 h^–1, whereas the largest amount of CA of 24.91 g/L as well as the highest selectivity of the bioprocess (89.9 % CA) and the maximum yield (0.22 g_CA/g_Glucose) were obtained at pH 6.0. During the growth phase, the temperature optimum was found to be in the range of 30–34 °C (μ_max = 0.132 h^–1). Nevertheless, the highest concentration of CA during the production phase was obtained at 30 °C (41 g/L CA, 93.1 % CA, 0.55 g_CA/g_glucose). In studying the substrate inhibition of the process, a clear tendency of decrease in the maximum specific growth rate was detected when the initial glucose concentration was increased from 50 g/L (μ_max = 0.17 h^–1) to 200 g/L (μ_max = 0.055 h^–1). The addition of 120 g/L CA to the culture broth at the start of the production phase reduced the production of CA from 32.1 g/L to 7.4 g/L.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Optimizing medium constituents and fermentation conditions for citric acid production from palmyra jaggery using response surface method

The quantitative effects of pH, temperature, time of fermentation, sugar concentration, nitrogen concentration and potassium ferrocyanide on citric acid production were investigated using a statistical experimental design. It was found that palmyra jaggery (sugar syrup from the palmyra palm) is a suitable substrate for increasing the yield of citric acid using Aspergillus niger MTCC 281 by submerged fermentation. Regression equations were used to model the fermentation in order to determine optimum fermentation conditions. Higher yields were obtained after optimizing media components and conditions of fermentation. Maximum citric acid production was obtained at pH 5.35, 29.76 °C, 5.7 days of fermentation with 221.66 g of substrate/l, 0.479 g of ammonium nitrate/l and 2.33 g of potassium ferrocyanide/l.