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.     

A Statistical Approach for Optimization of Polyhydroxybutyrate Production by Bacillus sphaericus NCIM 5149 under Submerged Fermentation Using Central Composite Design

The aim of this work was to statistically optimize the cultural and nutritional parameters for the production of polyhydroxybutyrate (PHB) under submerged fermentation using jackfruit seed hydrolysate as the sole carbon source. On the basis of results obtained from “one variable at a time” experiment, inoculum age, jackfruit seed hydrolysate concentration, and pH were selected for response surface methodology studies. A central composite design (CCD) was employed to get the optimum level of these three factors to maximize the PHB production. The CCD results predicted that jackfruit seed hydrolysates containing 2.5% reducing sugar, inoculum age of 18 h, and initial medium pH 6 could enhance the production of PHB to reach 49% of the biomass (biomass 4.5 g/l and PHB concentration 2.2 g/l). Analysis of variance exhibited a high coefficient of determination (R ²) value of 0.910 and 0.928 for biomass and PHB concentration, respectively, and ensured that the quadratic model with the experimental data was a satisfactory one. This is the first report on PHB production by Bacillus sphaericus using statistical experimental design and RSM in submerged fermentation with jackfruit seed hydrolysate as the sole source of carbon.     

Amylase Production by Saccharomycopsis fibuligera A11 in Solid-State Fermentation for Hydrolysis of Cassava Starch

The optimization of process parameters for high amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation was carried out using central composite design. Finally, the optimal parameters obtained with the response surface methodology (RSM) were moisture 610.0 ml/kg, inoculum 30.0 ml (OD_600 nm = 20.0)/kg, the amount ratio of wheat bran to rice husk 0.42, cassava starch concentration 20.0 g/kg, temperature 28 °C, and natural pH. Under the optimized conditions, 4,296 U/g of dry substrate of amylase activity was reached in the solid-state fermentation culture of the yeast strain A11 within 160 h, whereas the predicted maximum amylase activity of 4,222 U/g of dry substrate of amylase activity was derived from the RSM regression. It was found that cassava starch can be actively converted into monosaccharides and oligosaccharides by the crude amylase.     

Exploration of a Cheaper Carbon Source for Extracellular β-glucosidase Synthesis from <Emphasis Type="Italic">Debaryomyces pseudopolymorphus</Emphasis> NRRL YB-4229

In the present work, interactions between common media components and fermentation conditions were explored to come up with a simple media recipe for extracellular β-glucosidase (Dβ-gl) synthesis from Debaryomyces pseudopolymorphus to substitute cellobiose, which is currently used as a sole carbon source. Taguchi L₂₅ orthogonal array design was used to screen factors influencing Dβ-gl synthesis (carbon, organic nitrogen, inorganic nitrogen, trace elements, inoculum volume, and fermentation time). A significant influence of xylose, peptone, and potassium nitrate as carbon, organic nitrogen, and inorganic nitrogen sources, respectively, on Dβ-gl synthesis was identified by Taguchi. These factors were further optimized using central composite rotatable design (CCRD) of response surface methodology (RSM). The results showed that in the range studied, potassium nitrate had insignificant effect while xylose, peptone, and xylose-peptone interaction had a significant effect on Dβ-gl synthesis. Peptone/xylose ratio of 1.33 was found to be an important parameter for inducing Dβ-gl synthesis. The regression coefficient (R ²) of 0.915 and P value of <0.0003 for the model indicated that it was highly significant. The maximum activity obtained after RSM (32.2 U/ml) was comparable with that obtained (68.8 U/ml) when cellobiose (20 g/l) was used as a sole carbon source. Considering the cost difference between xylose and cellobiose, a 16-fold cost reduction could be obtained for equivalent Dβ-gl yield. Fed-batch fermentations were carried out wherein peptone/xylose ratio of 1.33 was maintained and continuous Dβ-gl synthesis was observed.     

Enhanced Pharmaceutically Active Compounds Productivity from Streptomyces SUK 25: Optimization,Characterization, Mechanism and Techno-Economic Analysis

The present research aimed to enhance the pharmaceutically active compounds’ (PhACs’) productivity from Streptomyces SUK 25 in submerged fermentation using response surface methodology (RSM) as a tool for optimization. Besides, the characteristics and mechanism of PhACs against methicillin-resistant Staphylococcus aureus were determined. Further, the techno-economic analysis of PhACs production was estimated. The independent factors include the following: incubation time, pH, temperature, shaker rotation speed, the concentration of glucose, mannitol, and asparagine, although the responses were the dry weight of crude extracts, minimum inhibitory concentration, and inhibition zone and were determined by RSM. The PhACs were characterized using GC-MS and FTIR, while the mechanism of action was determined using gene ontology extracted from DNA microarray data. The results revealed that the best operating parameters for the dry mass crude extracts production were 8.20 mg/L, the minimum inhibitory concentrations (MIC) value was 8.00 µg/mL, and an inhibition zone of 17.60 mm was determined after 12 days, pH 7, temperature 28 °C, shaker rotation speed 120 rpm, 1 g glucose /L, 3 g mannitol/L, and 0.5 g asparagine/L with R² coefficient value of 0.70. The GC-MS and FTIR spectra confirmed the presence of 21 PhACs, and several functional groups were detected. The gene ontology revealed that 485 genes were upregulated and nine genes were downregulated. The specific and annual operation cost of the production of PhACs was U.S. Dollar (U.S.D) 48.61 per 100 mg compared to U.S.D 164.3/100 mg of the market price, indicating that it is economically cheaper than that at the market price.     

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.     

Experimental Investigation and Optimization of Process Variables Affecting the Production of Extracellular Lipase by Kluyveromyces marxianus IFO 0288

In this study, the production and optimization of extracellular lipase from Kluyveromyces marxianus IFO 0288 was investigated by using optimized nutritional and cultural conditions in a yeast medium containing glucose as the carbon source in fully aerobic batch fermentation (150?rpm). The influence of four fermentation parameters (type of lipidic source, initial culture pH, temperature, and length of fermentation) on growth and lipase production was investigated and evaluated using the conventional ??one variable at a time?? approach and response surface methodology. An 18-fold increase in lipase production during 65?h of fermentation was obtained with optimized nutritional (0.5?% olive oil) and cultivation (pH?6.5, 35?°C) conditions by employing the conventional optimization method. By applying the response surface methodology technique the initial pH value of 6.4 and temperature of 32.5?°C were identified as optimal and led to further improvements (up to 18-fold) of extracellular lipase production. The results provide, for the first time, evidence that K. marxianus has the potential to be used as an efficient producer of extracellular lipase with prospective application in a variety of industrial and biotechnological areas.     

Rhamnolipid Production by <Emphasis Type="Italic">Pseudomonas Aeruginosa</Emphasis> GIM 32 Using Different Substrates Including Molasses Distillery Wastewater

A rhamnolipid production strain newly isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa GIM32 by its morphology and 16S rDNA sequence analysis. The effect of carbon source and carbon to nitrogen (C/N) ratio on rhamnolipids production was investigated. Palm oil was favorable as a carbon source for rhamnolipid production. The maximum biomass and rhamnolipid concentration were 8.24 g/L and 30.4 g/L, respectively, with an optimization medium containing 50 g/L palm oil and 5 g/L sodium nitrate. Molasses distillery wastewater as an unconventional substrate for rhamnolipid production was investigated. It was found that 2.6 g/L of rhamnolipids was produced; this amount was higher than that of past reports using wastewater as a substrate. In addition, 44% of the chemical oxygen demand of wastewater was removed at the same time under the optimization condition. Eleven kinds of different molecular weight rhamnolipid homologues were identified in the rhamnolipids obtained from molasses distillery wastewater by P. aeruginosa GIM32 by LC–MS analysis.     

High-Yield Hypocrellin A Production in Solid-State Fermentation by <Emphasis Type="Italic">Shiraia</Emphasis> sp. SUPER-H168

Hypocrellin A production by Shiraia sp. SUPER-H168 was studied under solid-state fermentation. Corn was found to be the best substrate after evaluating eight kinds of agro-industrial crops and residues. The optimized solid-state fermentation conditions were as follows: inoculum size 3 × 10⁶ spores, substrate particle size 0.8–1 mm, initial moisture content 50%, and temperature 30 °C. Six kinds of external carbon source and seven kinds of external nitrogen source were evaluated, respectively, for HA production. Glucose and NaNO₃ were the best. The combination of them was optimized by the response surface method. The optimum compositions of the supplementary glucose and NaNO₃ were 1.65 g/100 g and 0.43 g/L, respectively. Hypocrellin A production reached 4.7 mg/g.     

Extraction of Lepidium apetalum seed oil using supercritical carbon dioxide and anti-oxidant activity of the extracted oil

The supercritical fluid extraction (SFE) of Lepidium apetalum seed oil and its anti-oxidant activity were studied. The SFE process was optimized using response surface methodology (RSM) with a central composite design (CCD). Independent variables, namely operating pressure, temperature, time and flow rate were evaluated. The maximum extraction of Lepidium apetalum seed oil by SFE-CO? (about 36.3%) was obtained when SFE-CO? extraction was carried out under the optimal conditions of 30.0 MPa of pressure, 70 °C of temperature, 120 min of extraction time and 25.95 L/h of flow rate. GC-MS analysis showed the presence of four fatty acids in Lepidium apetalum seed oil, with a high content (91.0%) of unsaturated fatty acid. The anti-oxidant activity of the oil was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay and 2,2'-azino- bis(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) test. Lepidium apetalum seed oil possessed a notable concentration-dependent antioxidant activity, with IC?? values of 1.00 and 3.75 mg/mL, respectively.     

Optimization of a Natural Medium for Cellulase by a Marine Aspergillus niger Using Response Surface Methodology

The components of a natural medium were optimized to produce cellulase from a marine Aspergillus niger under solid state fermentation conditions by response surface methodology. Eichhornia crassipes and natural seawater were used as a major substrate and a source of mineral salts, respectively. Mineral salts of natural seawater could increase cellulase production. Raw corn cob and raw rice straw showed a significant positive effect on cellulase production. The optimum natural medium consisted of 76.9?% E. crassipes (w/w), 8.9?% raw corn cob (w/w), 3.5?% raw rice straw (w/w), 10.7?% raw wheat bran (w/w), and natural seawater (2.33 times the weight of the dry substrates). Incubation for 96?h in the natural medium increased the biomass to the maximum. The cellulase production was 17.80?U/g the dry weight of substrates after incubation for 144?h. The natural medium avoided supplying chemicals and pretreating substrates. It is promising for future practical fermentation of environment-friendly producing cellulase.     

Optimization of Levan Production by Cold-Active <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> ANT 179 and Fructooligosaccharide Synthesis by Its Levansucrase

Fructooligosaccharides (FOS) and levan attract much attention due to a wide range of applications in food technology and pharmaceutical and cosmetic industry. Bacillus licheniformis ANT 179, isolated from Antarctica soil, produced levansucrase and levan in a medium containing sucrose as carbon substrate. In this study, characterization of levansucrase and production of short-chain FOS and levan were investigated. Temperature and pH optimum of the enzyme were found to be 60 °C and pH 6.0, respectively. The optimization of fermentation conditions for levan production using sugarcane juice by response surface methodology (RSM) was carried out. Central composite rotatable design was used to study the main and the interactive effects of medium components: sugarcane juice and casein peptone concentration on levan production by the bacterium. The optimized medium with sugarcane juice at 20 % (v/v) and casein peptone at 2 % (w/v) was found to be optimal at an initial pH of 7.0 and incubation temperature of 35 °C for 48 h. Under these conditions, the maximum levan concentration was 50.25 g/L on wet weight basis and 16.35 g/L on dry weight basis. The produced inulin type FOS (kestose and neokestose) and levan were characterized by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) analysis. The study revealed that the levansucrase could form FOS from sucrose. The locally available low-cost substrate such as sugarcane juice in the form of a renewable substrate is proposed to be suitable even for scale-up production of enzyme and FOS for industrial applications. The levan and FOS synthesized by the bacterium are suitable for food applications and biomedical uses as the bacterium has GRAS status and devoid of endotoxin as compared to other Gram-negative bacteria.     

Inulinase synthesis from a mesophilic culture in submerged cultivation

A newly isolated mesophilic bacterial strain from dahlia rhizosphere, identified as Staphylococcus sp. and designated as RRL-M-5, was evaluated for inulinase synthesis in submerged cultivation using different carbon sources individually or in combination with inulin as substrate. Inulin appeared as the most favorable substrate at a 0.5–1.0% concentration. Media pH influenced the enzyme synthesis by the bacterial strain, which showed an optimum pH at 7.0–7.5. Supplementation of fermentation medium with external nitrogen (organic and inorganic) showed a mixed impact on bacterial activity of enzyme synthesis. The addition of soybean meal and corn steep solid resulted in about an 11% increase in enzyme titers. Among inorganic nitrogen sources, ammonium sulfate was found to be the most suitable. Maximum enzyme activities (446 U/L) were obtained when fermentation was carried out at 30°C for 24 h with a medium containing 0.5% inulin as a sole carbon source and 0.5% soybean meal as the nitrogen source. Bacterial inulinase could be a good source for the hydrolysis of inulin for the production of d-fructose.     

Optimization of Influential Parameters for Extracellular Keratinase Production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (MTCC9102) in Solid State Fermentation Using Horn Meal—A Biowaste Management

A Bacillus subtilis (MTCC9102) isolate was shown to produce significant amount of keratinase under optimized conditions in solid-state fermentation using Horn meal as a substrate. Optimized value for moisture, inoculum, and aeration were found to be 100% (v/w), 50% (v/w), and 150% (w/w), respectively, and the optimum nitrogen source was peptone and carbon source was dextrose. Maximum keratinolytic activity was observed at 48 h after incubation, and the optimum age (24 h) of inoculum was significant. The influence of cultivation temperature and initial pH of the medium on keratinase production revealed the optimum values for the temperature and pH as 37 °C and 7, respectively. Maximum keratinase activity of the crude extract was 15,972 U/mg/ml. These results indicate that this bacterial strain shows a high biotechnological potential for keratinase production in solid-state fermentation, and use of the horn meal as the substrate can be implemented for keratinous solid wastes management.     

Optimization of Enzymatic Clarification of Sapodilla Juice: A Statistical Perspective

Response surface methodology (RSM) was employed to establish optimum conditions for enzymatic clarification of sapodilla juice. Polygalacturonase obtained from Streptomyces lydicus had been purified to homogeneity and was used for the treatment. The independent variables were temperature (30–45 °C), enzyme concentration (0.5–1.5 U), and treatment time (30–90 min), whose effects on viscosity and clarity of the juice were evaluated using a Box–Behnken design. Significant regression models describing the changes of viscosity and clarity with respect to the independent variables were obtained, with the coefficient of determination, R ² , greater than 0.8. Based on response surfaces and contour plots, the optimum conditions for clarifying sapodilla juice were enzyme concentration 1.15 U, incubation temperature 34 °C, and incubation time 70 min.     

Statistical optimization of conditions for protease production fromBacillus sp. and its scale-up in a bioreactor

A statistical approach, response surface methodology (RSM), was used to study the production of extracellular protease fromBacillus sp., which has properties of immense industrial importance. The most influential parameters for protease production obtained through the method of testing the parameters one at a time were starch, soybean meal, CaCl₂, agitation rate, and inoculum density. This method resulted in the production of 2543 U/mL of protease in 48 h fromBacillus sp. Based on these results, face-centered central composite design falling under RSM was employed to further enhance protease activity. The interactive effect of the most influential parameters resulted in a 1.50-fold increase in protease production, yielding 3746 U/mL in 48 h. Analysis of variance showed the adequacy of the model and verification experiments confirmed its validity. On subsequent scale-up in a 30-L bioreactor using conditions optimized through RSM, 3978 U/mL of protease was produced in 18 h. This clearly indicated that the model remained valid even on a large scale. RSM is a quick process for optimization of a large number of variables and provides profound insight into the interactive effect of various parameters involved in protease production.     

Production of Acid-Stable and High-Maltose-Forming α-Amylase of Bacillus acidicola by Solid-State Fermentation and Immobilized Cells and Its Applicability in Baking

Among matrices used for immobilizing Bacillus acidicola cells [calcium alginate, chitosan + alginate, scotch brite, and polyurethane foam (PUF)], ??-amylase production was highest by PUF-immobilized cells (9.1?U?ml^?1), which is higher than free cells (7.2?U?ml^?1). The PUF-immobilized cells could be reused over seven cycles with sustained ??-amylase production. When three variables (moisture, starch, and ammonium sulfate), which significantly affected enzyme production in solid-state fermentation (SSF), were optimized using response surface methodology, 5.6-fold enhancement in enzyme production was attained. The enzyme production in SSF is 3.8-fold higher than that in submerged fermentation. The bread made by supplementing dough with ??-amylase of B. acidicola scored better than those with the xylanase of Bacillus halodurans and thermostable ??-amylase of Geobacillus thermoleovorans.     

Bioconversion of Sodium Dodecyl Sulphate to Rhamnolipid by Pseudomonas aeruginosa: A Novel and Cost-Effective Production Strategy

The utility of rhamnolipids in industry is currently limited due to the high constraints in its economic production. In this scenario, the novel utility of sodium dodecyl sulphate (SDS) as carbon source could serve as promising cost-effective strategy. Screening of effective SDS biodegraders led to the isolation of Pseudomonas aeruginosa S15 capable of concomitant SDS degradation and biosurfactant synthesis. SDS-based rhamnolipid production was proved on SDS minimal agar plates using cetyl trimethylammonium bromide–methylene blue method and optimised in SDS-based minimal salt (SBS) medium. SDS proved to be an ideal carbon source for rhamnolipid synthesis with a high substrate to product conversion rate yielding 6.9 g/l of rhamnolipids from 1 g/l SDS in 5 days. Fast atom bombardment mass spectroscopy analysis of the purified biosurfactant proved the presence of mono- and di-rhamnolipids, viz., Rha-C₁₀-C₁₀, Rha-C₁₀-C₁₂ and Rha-Rha-C₁₀-C₁₀ with surface active properties. The secreted rhamnolipids were not utilised by S15 as a carbon source, but it caused a dispersion of bacterial biofilms in SBS medium. To the best of our knowledge, this is the first report on bioconversion of synthetic detergent to biodetergent. This SDS-based novel methodology presents a more economised mode of rhamnolipid synthesis utilising SDS as sole carbon source.     

Application of Response Surface Methodology for Maximizing Dextransucrase Production from Leuconostoc mesenteroides NRRL B-640 in a Bioreactor

The production of dextransucrase from Leuconostoc mesenteroides NRRL B-640 was investigated using statistical approaches. Plackett-Burman design with six variables, viz. sucrose, yeast extract, K(2)HPO(4), peptone, beef extract, and Tween 80, was used to screen the nutrients that significantly affected the dextransucrase production. 2(4)-Central composite design with four selected variables (sucrose, K(2)HPO(4), yeast extract, and beef extract) was used for response surface methodology (RSM) for optimizing the enzyme production. The culture was grown under flask culture with 100 ml optimized medium containing 30 g/l sucrose, 18.5 g/l yeast extract, 15.3 g/l K(2)HPO(4), and 5 g/l beef extract at 25 degrees C and shaking at 200 rpm gave dextransucrase with specific activity of 0.68 U/mg. Whereas the same optimized medium in a 3.0-l bioreactor (1.4 l working volume) gave an experimentally determined value of specific activity of 0.70 U/mg, which was in perfect agreement with the predicted value of 0.65 U/mg by the statistical model.     

Urea enzymatic hydrolysis reaction: optimization by response surface methodology based on potentiometric measurements

The enzymatic hydrolysis reaction of urea by urease is optimized in this work by the chemometric response surface methodology (RSM), based on an initial rate potentiometric measurement using an NH(4)(+) ion-selective electrode (ISE). In this investigation, the ranges of critical variables determined by a preliminary "one at a time" (OVAT) procedure were used as input for the subsequent RSM chemometric analysis. The RSM quadratic response was found to be quite appropriate for modeling and optimization of the hydrolysis reaction as illustrated by the relatively high value of the determination coefficient (R(2)=90.1%), along with the satisfactory results obtained by the analysis of variance (ANOVA). All the evaluated analytical characteristics of the optimized method such as: the linear calibration curve, the upper and lower detection limits, the within-day precisions at low and at high levels, the assay recovery in pool serum media, along with the activation kinetic parameters, were also reported. Further, in order to check the quality of the optimization and the validity of the model, the assay of urea, both in aqueous laboratory and human serum samples, were performed. It has to be noted that the kinetic initial rate measurement method used in this work, permitted to overcome the general problem of NH(4)(+) ISE low selectivity against Na(+) and K(+) interfering ions in real samples.