Effect of nutrient supplements addition on ethanol production from cheese whey usingCandida pseudotropicalis under batch condition

Candida pseudotropicalis ATCC 8619 was selected among nine strains of lactose fermenting yeast for the production of ethanol from cheese whey. The effects of three nutrients (ammonium sulfate (NH₄)₂SO₄, dipotassium hydrogen phosphate K₂HPO₄, yeast extract, and a combination of them) on the ethanol yield from cheese whey were investigated. The results indicated that no addition of nutrient supplement is necessary to achieve complete lactose utilization during the cheese whey ethanol fermentation. However, addition of a small concentration (0.005% w/v) of these supplements reduced the lag period and the total fermentation time and increased the specific growth rate of the yeast. Higher concentrations (0.01 and 0.015% w/v) of ammonium sulfate and dipotassium hydrogen phosphate inhibited the cell growth and reduced lactose consumption. The highest ethanol (21.17 g/L) was achieved using yeast extract at a concentration of 0.01% w/v, given a conversion efficiency of 98.3%. No indication of alcohol inhibition was observed in this study.     

Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture

The production of pullulan from beet molasses by a pigment-free strain of Aureobasidium pullulans on shake-flask culture was investigated. Combined pretreatment of molasses with sulfuric acid and activated carbon to remove potential fermentation inhibitors present in molasses resulted in a maximum pullulan concentration of 24 g/L, a biomass dry wt of 14 g/L, a pullulan yield of 52.5%, and a sugar utilization of 92% with optimum fermentation conditions (initial sugar concentration of 50 g/L and initial pH of 7.0). The addition of other nutrients as carbon and nitrogen supplements (olive oil, ammonium sulfate, yeast extract) did not further improve the production of the exopolysaccharides. Structural characterization of the isolated polysaccharides from the fermentation broths by ¹³C-nuclear magnetic resonance spectroscopy and pullulanase digestion combined with size-exclusion chromatography confirmed the identity of pullulan and the homogeneity (>93% dry basis) of the elaborated polysaccharides by the microorganism. Using multiangle laser light scattering and refractive index detectors in conjunction with high-performance size-exclusion chromatography molecular size distributions and estimates of the molecular weight (M _w =2.1−4.1×10⁵), root mean square of the radius of gyration (R _g =30−38 nm), and polydispersity index (M _w /M _n =1.4−2.4) were obtained. The fermentation products of molasses pretreated with sulfuric acid and/or activated carbon were more homogeneous and free of contaminating proteins. In the concentration range of 2.8−10.0 (w/v), the solution’s rheologic behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s⁻¹ at 20°C); a slight shear thinning was observed at 10.0 (w/v) for the high molecular weight samples. Overall, beet molasses pretreated with sulfuric acid and activated carbon appears as an attractive fermentation medium for the production of pullulan by A. pullulans.     

Effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans in an airlift reactor

The effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans P56 in an airlift reactor was investigated. A maximum polysaccharide concentration (18.5 g/L), biomass dry weight (14.0 g/L), polysaccharide yield (38.5%), and sugar utilization (96%) was achieved at a shear rate of 42 s⁻¹. A. pullulans grown on beet molasses produced a mixture of pullulan and other polysaccharides. The highest value of pullulan proportion (30% of total polysaccharide) was obtained at a low shear rate (42 s⁻¹). The apparent viscosity of the fermentation broth increased as the shear rate increased up to 42 s⁻¹ and then decreased. On the other hand, the dissolved oxygen concentration and the volumetric mass transfer coefficient increased with the increase of the shear rate from 21 to 84 s⁻¹. The external addition of L-glutamic acid, olive oil, and Tween-80 improved significantly the production of crude polysaccharide (27.0 g/L), but the pullulan content of the polysaccharide was low (20%).     

Production of Raw Starch-Saccharifying Thermostable and Neutral Glucoamylase by the Thermophilic Mold <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> in Submerged Fermentation

Among physical and nutritional parameters optimized by “one variable at a time” approach, four cultural variables (sucrose, MgSO₄ ^.7H₂O, inoculum size, and incubation period) significantly affected glucoamylase production. These variables were, therefore, selected for optimization using response surface methodology. The p-values of the coefficients for linear effect of sucrose and inoculum size were less than 0.0001, suggesting them to be the key experimental variables in glucoamylase production. The enzyme production (34 U/ml) attained under optimized conditions (sucrose, 2%; MgSO₄ ^.7H₂O, 0.13%; yeast extract, 0.1%; inoculum size, 5 × 10⁶ spores per 50 ml production medium; incubation time, 48 h; temperature, 40°C; and pH 7.0) was comparable with the value predicted by polynomial model (34.2 U/ml). An over all 3.1-fold higher enzyme titers were attained due to response surface optimization. The experimental model was validated by carrying out glucoamylase production in shake flasks of increasing capacity (0.25–2.0 l) and 22-l laboratory bioreactors (stirred tank and airlift), where the enzyme production was sustainable. Furthermore, the fermentation time was reduced from 48 h in shake flasks to 32 h in bioreactors.     

Hexavalent Molybdenum Reduction to Molybdenum Blue by <Emphasis Type="Italic">S. Marcescens</Emphasis> Strain Dr. Y6

A molybdate-reducing bacterium has been locally isolated. The bacterium reduces molybdate or Mo⁶⁺ to molybdenum blue (molybdate oxidation states of between 5+ and 6+). Different carbon sources such as acetate, formate, glycerol, citric acid, lactose, fructose, glucose, mannitol, tartarate, maltose, sucrose, and starch were used at an initial concentration of 0.2% (w/v) in low phosphate media to study their effect on the molybdate reduction efficiency of bacterium. All of the carbon sources supported cellular growth, but only sucrose, maltose, glucose, and glycerol (in decreasing order) supported molybdate reduction after 24 h of incubation. Optimum concentration of sucrose for molybdate reduction is 1.0% (w/v) after 24 h of static incubation. Ammonium sulfate, ammonium chloride, valine, OH-proline, glutamic acid, and alanine (in the order of decreasing efficiency) supported molybdate reduction with ammonium sulfate giving the highest amount of molybdenum blue after 24 h of incubation at 0.3% (w/v). The optimum molybdate concentration that supports molybdate reduction is between 15 and 25 mM. Molybdate reduction is optimum at 35 °C. Phosphate at concentrations higher than 5 mM strongly inhibits molybdate reduction. The molybdenum blue produced from cellular reduction exhibits a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as Serratia marcescens Strain Dr.Y6 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny.     

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.     

Effect of media on spore yield and thermal resistance of Bacillus stearothermophilus

The interference of eight components in the yield of sporulation and thermal resistance to moist heat (121°C) of Bacillus stearothermophilus spores suspended in 0.02 M calcium acetate solution and inoculated on paper strips previously treated with calcium acetate/calcium hydroxide was studied. The spore yield of 1.0×10⁸/mL was developed at 62°C in 17 media containing different concentrations of d-glucose, sodium chloride, l-glutamic acid, yeast extract, peptone, manganese sulfate, potassium phosphate, and ammonium phosphate. The combined effects of yeast extract, peptone, and glucose contributed positively to the spore yield and to the stability of the thermal resistance of both spores in suspension and on strips.     

Response Surface Optimization of Medium Components for Naringinase Production from <Emphasis Type="Italic">Staphylococcus xylosus MAK2</Emphasis>

Response surface methodology was used to optimize the fermentation medium for enhancing naringinase production by Staphylococcus xylosus. The first step of this process involved the individual adjustment and optimization of various medium components at shake flask level. Sources of carbon (sucrose) and nitrogen (sodium nitrate), as well as an inducer (naringin) and pH levels were all found to be the important factors significantly affecting naringinase production. In the second step, a 22 full factorial central composite design was applied to determine the optimal levels of each of the significant variables. A second-order polynomial was derived by multiple regression analysis on the experimental data. Using this methodology, the optimum values for the critical components were obtained as follows: sucrose, 10.0%; sodium nitrate, 10.0%; pH 5.6; biomass concentration, 1.58%; and naringin, 0.50% (w/v), respectively. Under optimal conditions, the experimental naringinase production was 8.45 U/mL. The determination coefficients (R ²) were 0.9908 and 0.9950 for naringinase activity and biomass production, respectively, indicating an adequate degree of reliability in the model.     

Propyl Gallate Synthesis Using Acidophilic Tannase and Simultaneous Production of Tannase and Gallic Acid by Marine <Emphasis Type="Italic">Aspergillus awamori</Emphasis> BTMFW032

Marine Aspergillus awamori BTMFW032, recently reported by us, produce acidophilic tannase as extracellular enzyme. Here, we report the application of this enzyme for synthesis of propyl gallate by direct transesterification of tannic acid and in tea cream solubilisation besides the simultaneous production of gallic acid along with tannase under submerged fermentation by this fungus. This acidophilic tannase enabled synthesis of propyl gallate by direct transesterification of tannic acid using propanol as organic reaction media under low water conditions. The identity of the product was confirmed with thin layer chromatography and Fourier transform infrared spectroscopy. It was noted that 699 U/ml of enzyme could give 60% solubilisation of tea cream within 1 h. Enzyme production medium was optimized adopting Box–Behnken design for simultaneous synthesis of tannase and gallic acid. Process variables including tannic acid, sodium chloride, ferrous sulphate, dipotassium hydrogen phosphate, incubation period and agitation were recognized as the critical factors that influenced tannase and gallic acid production. The model obtained predicted 4,824.61 U/ml of tannase and 136.206 μg/ml gallic acid after 48 h of incubation, whereas optimized medium supported 5,085 U/ml tannase and 372.6 μg/ml of gallic acid production after 36 and 84 h of incubation, respectively, with a 15-fold increase in both enzyme and gallic acid production. Results indicated scope for utilization of this acidophilic tannase for transesterification of tannic acid into propyl gallate, tea cream solubilisation and simultaneous production of gallic acid along with tannase.     

Improvement of Ethanol Production Using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by Enhancement of Biomass and Nutrient Supplementation

Optimization of ethanol production through addition of substratum and protein-lipid additives was studied. Oilseed meal extract was used as protein lipid supplement, while rice husk was used as substratum. The effect of oil seed meal extract and rice husk was observed at varying concentration of medium sugar from 8% to 20%. Of the three oil seed meal extracts used, viz. groundnut, safflower, and sunflower, safflower was found to be most efficient. The use of oilseed meal extract at 4% was found to enhance ethanol production by almost 50% and enhanced sugar tolerance from 8% to 16%. A further increase of almost 48% ethanol was observed on addition of 2 g of rice husk per 100 ml of medium. An increase in cell mass with better sugar attenuation was observed. Further optimization was sought through use of sugarcane juice as the sugar source. While 8.9% ethanol yield with 75% sugar attenuation was observed at 20% sucrose concentration, it was found to increase to 12% (v/v) with almost complete utilization of medium sugar when sugarcane juice was used. Cell weight was also observed to increase by 26%.     

Process optimisation for the biosynthesis of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3 using submerged fermentation

This study deals with optimisation of cultural conditions for enhanced production of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3. Influence of different variables including incubation time, temperature, inoculum size, pH, nitrogen sources and metal ions has been studied. The optimum conditions for cellulase production were incubation period of 72 h, inoculum size 4% incubation temperature 37°C, pH 7, 0.25% ammonium sulphate, 0.2% peptone as inorganic and organic nitrogen source in case of Bacillus PC-BC6. In case of mutant Bacillus N3, optimal conditions were incubation period of 48 h, incubation temperature 37°C, inoculum size 3%, pH 7, 0.2% ammonium chloride and 0.15% yeast extract. Presence of MnSO₄ and CaCl₂ enhances the enzyme production by Bacillus PC-BC6 and mutant Bacillus N3, respectively. This study was innovative and successful in producing cellulase economically by using cheap indigenous substrate Saccharum spontaneum.     

<Emphasis Type="Italic">Hypericum japonicum:</Emphasis> a Double-Headed Sword to Combat Vector Control and Cancer

Weissella cibaria RBA12 produced a maximum of 9 mg/ml dextran (with 90% efficiency) using shake flask culture under the optimized concentration of medium components viz. 2% (w/v) of each sucrose, yeast extract, and K₂HPO₄ after incubation at optimized conditions of 20 °C and 180 rpm for 24 h. The optimized medium and conditions were used for scale-up of dextran production from Weissella cibaria RBA12 in 2.5-l working volume under batch fermentation in a bioreactor that yielded a maximum of 9.3 mg/ml dextran (with 93% efficiency) at 14 h. After 14 h, dextran produced was utilized by the bacterium till 18 h in its stationary phase under sucrose depleted conditions. Dextran utilization was further studied by fed-batch fermentation using sucrose feed. Dextran on production under fed-batch fermentation in bioreactor gave 35.8 mg/ml after 32 h. In fed-batch mode, there was no decrease in dextran concentration as observed in the batch mode. This showed that the utilization of dextran by Weissella cibaria RBA12 is initiated when there is sucrose depletion and therefore the presence of sucrose can possibly overcome the dextran hydrolysis. This is the first report of utilization of dextran, post-sucrose depletion by Weissella sp. studied in bioreactor.     

Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO₄·5H₂O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.     

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).     

Optimization of inulinase production by Kluyveromyces marxianus using factorial design

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2^5–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K₂HPO₄), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K₂HPO₄ had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K₂HPO₄. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.     

Yeast Biomass Production in Brewery’s Spent Grains Hemicellulosic Hydrolyzate

Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery’s spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h⁻¹, 0.61 g g⁻¹, and 0.56 g l⁻¹ h⁻¹, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.     

Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3

Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 °C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process.     

Continuous fermentation studies with xylose-utilizing recombinant Zymomonas mobilis

This study examined the continuous cofermentation performance characteristics of a dilute-acid “prehydrolysate-adapted” recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D _max), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D _max from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D _max was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D _max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D _max for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L·h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell·h).     

Xylanase production by Penicillium canescens 10–10c in solid-state fermentation

Filamentous fungi have been widely used to produce hydrolytic enzymes for industrial applications, including xylanases, whose levels in fungi are generally much higher than those in yeast and bacteria. We evaluated the influence of carbon sources, nitrogen sources, and moisture content on xylanase production by Penicillium canescens 10–10c in solid-state fermentation. Among agricultural wastes tested (wheat bran, untreated wheat straw, treated wheat straw, beet pulp, and soja meal), untreated wheat straw gave the highest production of xylanase. Optimal initial moisture content for xylanase production was 83%. The addition of 0.4 g of xylan or easily metabolizable sugar, such as glucose and xylose, at a concentration of 2% to wheat straw enhanced xylanase production. In solid-state fermentation, even at high concentrations of glucose or xylose (10%), catabolic repression was minimized compared to the effect observed in liquid culture. Yeast extract was the best nitrogen source among the nitrogen sources investigated: peptone, ammonium nitrate, sodium nitrate, ammonium chloride, and ammonium sulfate. A combination of yeast extract and peptone as nitrogen sources led to the best xylanase production.     

Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography

Establishment of hydrophilic organic/salt-containing aqueous two-phase system and purification of salvianolic acid B from crude extract of S. miltiorrhiza by counter-current chromatography with said system were studied. Ethanol and n-propanol were selected to constitute biphasic systems with ammonia sulphate, sodium chloride and phosphate separately, and related system characteristics including phase diagrams, phase ratio, separation time were tested. The partition coefficient of crude salvianolic acid B was also tested in above systems and further finely adjusted by altering the constitution of phosphate in n-propanol/phosphate system. Salvianolic acid B was purified to 95.5% purity by counter-current chromatography in 36% (w/w) n-propanol/8% (w/w) phosphate system with the ratio between dipotassium hydrogen phosphate and sodium dihydrogen phosphate of 94:6. One hundred and eight milligrams of salvianolic acid B was purified from 285 mg crude extract with the recovery of 89%.