Enhanced Production of Laccase from Coriolus versicolor NCIM 996 by Nutrient Optimization Using Response Surface Methodology

Plackett and Burman design criterion and central composite design were applied successfully for enhanced production of laccase by Coriolus versicolor NCIM 996 for the first time. Plackett and Burman design criterion was applied to screen the significance of ten nutrients on laccase production by C. versicolor NCIM 996. Out of the ten nutrients tested, starch, yeast extract, MnSO(4), MgSO(4) x 7H(2)O, and phenol were found to have significant effect on laccase production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Plackett-Burman design. The optimized medium composition for production of laccase was (g/l): starch, 30.0; yeast extract, 4.53; MnSO(4), 0.002; MgSO(4) x 7H(2)O, 0.755; and phenol, 0.026, and the optimum laccase production was 6,590.26 (U/l), which was 7.6 times greater than the control.     

Solid-state Fermentation for Enhanced Production of Laccase using Indigenously Isolated <Emphasis Type="Italic">Ganoderma</Emphasis> sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25°C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.     

Xylose reductase production by candida guilliermondii

The effect of pH, time of fermentation, and xylose and glucose concentration on xylitol production, cell growth, xylose reductase (XR), and xylitol dehydrogenase (XD) activities ofCandida guilliermondii FTI 20037 were determined. For attaining XR and XD activities of 129-2190 U/mg of protein and 24-917 U/mg of protein, respectively, the cited parameters could vary as follows: initial pH: 3.0-5.0; xylose: 15-60 g/L; glucose: 0-5 g/L; and fermentation time: 12-24 h. Moreover, the high XR and XD activities occurred when the xylitol production by the yeast was less than 19.0 g/L.     

Fed-Batch production of glucose 6-Phosphate dehydrogenase using recombinant saccharomyces cerevisiae

The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC 181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in the formation of glucose 6-phosphate dehydrogenase (EC.1.1.1.49). Two liters of culture medium (10.0 g/L glucose, 3.7 g/L yeast nitrogen broth (YNB), 0.02 g/L L-tryptophan, 0.02 g/L L-histidine, 0.02 g/L uracil, and 0.02 g/L adenine) were inoculated with 1.5 g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30 degrees C, and agitation of 400 rpm. After glucose concentration in the medium was lower than 1.0 g/L, the cell culture was fed with a solution of glucose (10.0 g/L) or micronutrients (L-tryptophan, L-histidine, uracil, and adenine each one at a concentration of 0.02 g/L) following the constant, linear, or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2 L up to 3 L during 5 h. The highest glucose 6-phosphate dehydrogenase activity (350 U/L; 1 U=1 micromol of NADP/min) occurred when the glucose solution was fed into the fermenter through the decreasing linear mode.     

Processing Parameters Matching Effects upon Rhizobium tropici Biopolymers’ Rheological Properties

The combined effects of the processing parameters upon rheological properties of biopolymers produced by Rhizobium tropici were studied as a function of the Ca(+2) ions' concentration variation, yeast extract concentration added to the medium, aeration, and agitation, maintaining the mannitol concentration in 10 g/L. The experiments were carried out using a fermenter with 20-L capacity as a reactor. All processing parameters were monitored online. The temperature [(30 +/- 1) degrees C] and pH values (7.0) were kept constant throughout the experimental time. As a statistical tool, a complete 2(3) factorial design with central point and response surface was used to investigate the interactions between relevant variables of the fermentation process: calcium carbonate concentration, yeast extract concentration, aeration, and agitation. The processing parameter setup for reaching the maximum response for rheological propriety production was obtained when applying mannitol concentration of 10.0 g/L, calcium carbonate concentration 1.0 g/L, yeast extract concentration 1.0 g/L, aeration 1.30 vvm, and agitation 800 rpm. The viscosimetric investigation of polysaccharide solutions exposed their shear-thinning behavior and polyelectrolytic feature.     

Bioconversion of fumarate to succinate using glycerol as a carbon source

In this study, a facultative bacterium that converts fumarate to succinate at a high yield was isolated. The yield of biocon version was enhanced about 1.2 times by addition of glucose into culture medium at an initial concentration of 6 g/L. When the initial cell density was high (2 g/L), the succinate produced at pH 7.0 for initial fumarate concentrations of 30, 50, 80, and 100 g/L were 29.3, 40.9, 63.6, and 82.5 g/L, respectively, showing an increase with the initial fumarate concentration. The high yield of 96.8%/mole of fumarate in just 4 h was obtained at the initial fumarate concentration of 30 g/L. Comparing these values to those obtained with low cell culture (0.2 g/L), we found that the amount of succinate produced was similar, but the production rate in the high cell culture was about three times higher than was the case in the low cell culture. This strain converted fumarate to succinate at a rate of 3.5 g/L·h under the sparge of CO₂.     

Optimization of cyclodextrin glucanotransferase production from Bacillus clausii E16 in submerged fermentation using response surface methodology

Cyclodextrin glucanotransferase production from Bacillus clausii E16, a new bacteria isolated from Brazilian soil samples was optimized in shake-flask cultures. A 2(4) full-factorial central composite design was performed to optimize the culture conditions, using a response surface methodology. The combined effect among the soluble starch concentration, the peptone concentration, the yeast extract concentration, and the initial pH value of the culture medium was investigated. The optimum concentrations of the components, determined by a 2(4) full-factorial central composite design, were 13.4 g/L soluble starch, 4.9 g/L peptone, 5.9 g/L yeast extract, and initial pH 10.1. Under these optimized conditions, the maximum cyclodextrin glucanotransferase activity was 5.9 U/mL after a 48-h fermentation. This yield was 68% higher than that obtained when the microorganism was cultivated in basal culture medium.     

Optimization of L-Lactic acid production from glucose by Rhizopus oryzae ATCC 52311

The effect of nutrients on L(+)-lactic acid production from glucose was investigated using Rhizopus oryzae ATCC 523 11. From the shake-flask experiments, the optimal medium composition was defined for improved lactic-acid production. In order to enhance lactic-acid production rate and product yield, controlled aeration in a bubble column was conducted under optimal conditions. Results showed a maximum lactic-acid production rate of 2.58 g/L/h was obtained with an initial glucose concentration of 94 g/L. Finallactic-acid concentration of 83 g/L was achieved after 32 h of fermentation with a weight of 0.88 glactic acid/g glucose consumed.     

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A.     

Overproduction of riboflavin by anArthrobacter sp. mutant resistant to 5-fluorouracil

Effects of pH and dissolved oxygen concentration on batchwise riboflavin production by a 5-fluorouracil (5-FU)-resistant mutant ofArthrobacter sp. were investigated. The reaction was carried out in a jar fermentor. The optimal pH of culture medium was around 7.3. Dissolved oxygen concentration was almost constant during fermentation at 600 rpm of agitation rate. Production of riboflavin reached a maximum of 160 mg/L after 70 h fermentation under the agitation rate of 600 rpm, aeration rate of 1.0 L/min, and pH 7.0.     

Production of l-malic acid via biocatalysis employing wild-type and respiratory-deficient yeasts

Succinic acid was produced efficiently from fumaric acid by a recombinantE. coli strain DH5αt/pGC1002 containing multicopy fumarate reductase genes. The effects of initial fumaric acid and glucose concentration on the production of succinic acid were investigated. Succinic acid reached 41 to over 60 g/L in 48.5 h starting with 50 to 64 g/L fumaric acid. Significant substrate inhibition was observed at initial fumaric acid concentration of 90 g/L. L-Malic acid became the major fermentation product under these conditions. Provision of glucose (5-30 g/L) to the fermentation medium stimulated the initial succinic acid production rate over two folds.     

Optimization of beta-carotene production from synthetic medium by Blakeslea trispora: a mathematical modeling

The effect of inoculum, pH, carbon and nitrogen source, natural oils, fatty acids, antioxidant, and precursors on beta-carotene production by Blakeslea trispora in shake-flask culture was investigated. The highest concentration of beta-carotene was obtained in the medium (pH 7.0) inoculated with one loop of each culture. Sucrose, glycerol, cornmeal, soy protein acid hydrolysate, and distiller's solubles did not improve the production of beta-carotene. By contrast, glucose, corn steep liquor, antioxidant, olive oil, soybean oil, cottonseed oil, oleic and linoleic acids, and kerosene significantly increased the beta-carotene production. A central composite design was employed to determine the maximum beta-carotene production at optimum values for the process variables (linoleic acid, kerosene, and antioxidant). The fit of the model was found to be good. Linoleic acid, kerosene, and antioxidant had a strong linear effect on beta-carotene production. The concentration of beta-carotene was significantly affected by linoleic acid-kerosene and linoleic acid-antioxidant interactions as well as by the negative quadratic effects of these variables. The interaction between kerosene and antioxidant had no significant linear effect. The maximum beta-carotene concentration (2.88 g/L) was obtained at concentrations of 17.15 g/L of linoleic acid, 39.25 g/L of kerosene, and 9.04 g/L of antioxidant.     

Use of glucose oxidase in a membrane reactor for gluconic acid production

This article aims at the evaluation of the catalytic performance of glucose oxidase (GO) (EC.1.1.3.4) for the glucose/gluconic acid conversion in the ultrafiltration cell type membrane reactor (MB-CSTR). The reactor was coupled with a Millipore ultrafiltration-membrane (cutoff of 100 kDa) and operated for 24 h under agitation of 100 rpm, pH 5.5, and 30 degrees C. The experimental conditions varied were the glucose concentration (2.5, 5.0, 10.0, 20.0, and 40.0 mM), the feeding rate (0.5, 1.0, 3.0, and 6.0/h), dissolved oxygen (8.0 and 16.0 mg/L), GO concentration (2.5, 5.0, 10.0, and 20.0 U(GO)/mL), and the glucose oxidase/catalase activity ratio (U(GO)/U(CAT))(1:0, 1:10, 1:20, and 1:30). A conversion yield of 80% and specific reaction rate of 40 x 10(-4) mmol/h x U(GO) were attained when the process was carried out under the following conditions: D =3.0/h, dissolved oxygen =16.0 mg/L, [G] =40 mM, and (U(GO)/U(CAT)) =1:20. A simplified model for explaining the inhibition of GO activity by hydrogen peroxide, formed during the glucose/gluconic acid conversion, was presented.     

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×10⁵ Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.     

Laccase immobilized on mesoporous SiO<Subscript>2</Subscript> and its use for degradation of chlorophenol pesticides

In this paper, mesoporous silica with large specific surface area was used to immobilize laccase by the glutaraldehyde cross-linking method, and after screening and optimization experiments, the best enzyme immobilization process conditions were found (25°C, pH 5.4, 4% glutaraldehyde and 0.2 g/L laccase, treatment time 6 h). After that, the removal and degradation ratio of 2,4-dichlorophenol (abbreviated as DCP) under different conditions were also studied. After the degradation process was performed for 6 h at 30°C, pH 5.4, and DCP initial concentration of 50 mg/L in the presence of 0.1 g of immobilized laccase, the removal ratio and the degradation ratio were 42.28 and 15.93%, respectively. Compared with free laccase, the reusability of immobilized laccase is significantly improved.     

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).     

Enzymatic hydrolysis optimization to ethanol production by simultaneous saccharification and fermentation

There is tremendous interest in using agro-industrial wastes, such as cellulignin, as starting materials for the production of fuels and chemicals. Cellulignin are the solids, which result from the acid hydrolysis of the sugarcane bagasse. The objective of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cellulignin, and to study its fermentation to ethanol using Saccharomyces cerevisiae. Cellulose conversion was optimized using response surface methods with pH, enzyme loading, solid percentage, and temperature as factor variables. The optimum conditions that maximized the conversion of cellulose to glucose, calculated from the initial dried weight of pretreated cellulignin, (43 degrees C, 2%, and 24.4 FPU/g of pretreated cellulignin) such as the glucose concentration (47 degrees C, 10%, and 25.6 FPU/g of pretreated cellulignin) were found. The desirability function was used to find conditions that optimize both, conversion to glucose and glucose concentration (47 degrees C, 10%, and 25.9 FPU/g of pretreated cellulignin). The resulting enzymatic hydrolyzate was fermented yielding a final ethanol concentration of 30.0 g/L, in only 10 h, and reaching a volumetric productivity of 3.0 g/L x h, which is close to the values obtained in the conventional ethanol fermentation of sugar cane juice (5.0-8.0 g/L x h) in Brazil.     

Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A

The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH7.0) at 37°C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100–180 U/L) and process productivity 4.8-fold (1.4–6.7 U/[L·h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.     

Fermentation of xylose into acetic acid by Clostridium thermoaceticum

For optimum fermentation, fermenting xylose into acetic acid by Clostridium thermoaceticum (ATCC 49707) requires adaptation of the strain to xylose medium. Exposed to a mixture of glucose and xylose, it preferentially consumesxylose over glucose. The initial concentration of xylose in the medium affects the final concentration and the yield of acetic acid. Batch fermentation of 20 g/L of xylose with 5g/L of yeast extract as the nitrogen source results in a maximum acetate concentration of 15.2 g/L and yield of 0.76 g of acid/g of xylose. Corn steep liquor (CLS) is a good substitute for yeast extract and results in similar fermentation profiles. The organism consumes fructose, xylose, and glucose from a mixture of sugars in batch fermentation. Arabinose, mannose, and galactose are consumed only slightly. This organism loses viability on fed-batch operation, even with supplementation of all the required nutrients. In fed-batch fermentation with CSL supplementation, d-xylulose (an intermediate in the xylose metabolic pathway) accumulates in large quantities.     

Carob pod: A new substrate for citric acid production by Aspergillus niger

The production of citric acid from carob pod extract byA. niger in surface fermentation was investigated. A maximum citric acid concentration (85.5 g/L), citric acid productivity (4.07 g/L/d), specific citric acid production rate (0.18 g/g/d), and specific sugar uptake rate (0.358 g/g/d) was achieved at an initial sugar concentration of 200 g/L, pH of 6.5, and a temperature of 30°C. Other kinetic parameters, namely, citric acid yield, biomass yield, specific biomass production rate, and fermentation efficiency were maximum at pH 6.5, temperature 30°C, and initial sugar concentration 100 g/L. The external addition of methanol into the carob pod extract at a concentration up to 4% (v/v) improved the production of citric acid.