Proficient bioconversion of rice straw biomass to bioethanol using a novel combinatorial pretreatment approach based on deep eutectic solvent, microwave irradiation and laccase.

Current study is the first report of the combined application of chemical (deep eutectic solvent), physical (microwave irradiation) and biological (laccase) pretreatment strategies for enhancing the enzymatic digestibility of rice straw biomass. Pretreated rice straw biomass was saccharified by cellulase/xylanase from Aspergillus japonicus DSB2 to get a sugar yield of 252.36 mg/g biomass. Design of Experiment based optimization of pretreatment and saccharification variables increased the total sugar yield by 1.67 times (421.5 mg/g biomass, saccharification efficiency 72.6%). Sugary hydrolysate was ethanol-fermented by Saccharomyces cerevisiae and Pichia stipitis to achieve an ethanol yield of 214 mg/g biomass (bioconversion efficiency 72.5%). Structural/chemical aberrations induced in the biomass due to pretreatment were elucidated by X-ray diffraction, scanning electron microscopy, Fourier-transform infrared spectroscopy, and 1H nuclear magnetic resonance techniques to unravel the pretreatment mechanisms. Combined application of various physico-chemical/biological pretreatment may be a promising approach for proficient bioconversion of rice straw biomass.

An eco-friendly novel approach for bioconversion of Saccharum spontaneum biomass to biofuel-ethanol under consolidated bioprocess.

Quest for renewable/eco-friendly energy sources has received immense focus in recent years. Current study involved consolidated bioprocessing of Saccharum spontaneum biomass (SSB) for biofuel-ethanol generation in a 'one pot consolidated bioprocess' (OPCB). SSB was pretreated with protic ionic liquid, triethylamine-bisulfate ([TEA][HSO4]), saccharified in-situ with cellulase/xylanase enzymes, and the released sugars were fermented to ethanol. Pretreatment and saccharification processes were optimized under OPCB to achieve 2.70-fold increased sugar yield i.e. from 196.56 to 531.00 mg/g biomass. Fermentation of sugars yielded ethanol at 209.6 mg/g biomass at a bioconversion efficiency of 72.56 %. The pretreated SSB was comprehensively examined by/for XRD, NMR, SEM, FT-IR, and properties such as water retention capacity, surface area and cellulase adsorption ability to elucidate functional mechanisms of [TEA][HSO4] pretreatment.

Phytochemicals targeting JAK/STAT pathway in the treatment of rheumatoid arthritis: Is there a future?

Rheumatoid arthritis (RA) is a chronic autoimmune disorder and the treatment involves the use of traditional and biological disease modifying anti-rheumatic drugs (DMARDs). Recent studies have shown JAK/STAT signaling pathway as potential target for the treatment of RA. Novel JAK/STAT inhibitors viz tofacitinib and baricitinib have been recently approved by FDA for RA treatment and have attained substantial importance. However, the discernible risks of thromboembolism, gastrointestinal (GIT) perforations, hepatotoxicity and serious infections including tuberculosis, herpes zoster associated with their administration cannot be overlooked. Furthermore, these are highly expensive which limits their application for a broader use. These limitations provide the basis of exploring novel JAK/STAT inhibitors of natural origin with increased tolerability, safety and cost-effectiveness. In this review we confer an account of various natural compounds/phytochemicals that have proved to be beneficial in attenuating inflammation in RA via modulation of JAK/STAT signaling pathway. Some of these natural compounds including resveratrol have clearly indicated biochemical and clinically significant therapeutic effects in ameliorating RA both in vivo and in clinical settings. We further discuss the physicochemical challenges of poor solubility and absorption coupled with the use of natural JAK/STAT inhibitors. We thereafter discuss and summarize various drug delivery systems (DDS) to confront the physicochemical limitations of natural JAK/STAT inhibitors with the aim to enhance the therapeutic efficacy. Overall the review unveils the potential of natural JAK/STAT inhibitors as a cost-effective approach in ameliorating RA without incorporating the risks of adverse repercussions, thus setting the stage for clinical exploration of these compounds that may possibly complement the present RA therapy.

Nanobiocatalysts for efficacious bioconversion of ionic liquid pretreated sugarcane tops biomass to biofuel.

This work aimed to study the hydrolysis of ionic liquid (IL) pretreated sugarcane tops (SCT) biomass with in-house developed IL-stable enzyme preparation, from a fungal isolate Aspergillus flavus PN3. Maximum reducing sugar yield (181.18 mg/g biomass) was obtained from tris (2-hydroxyethyl) methylammonium-methylsulfate ([TMA]MeSO4) pretreated biomass. Pretreatment parameters were optimized to attain enhanced sugar yield (1.57-fold). Functional mechanism of IL mediated pretreatment of SCT biomass was elucidated by SEM, XRD, FTIR and 1H NMR studies. Furthermore, nanobiocatalysts prepared by immobilization of enzyme preparation by covalent coupling on magnetic nanoparticles functionalized with amino-propyl triethoxysilane, were assessed for their hydrolytic efficacy and reusability. Nanobiocatalysts were examined by SEM and FTIR analysis for substantiation of immobilization. This is the first ever report of application of magnetic nanobiocatalysts for saccharification of IL-pretreated sugarcane tops biomass.

Phytochemical add-on therapy to DMARDs therapy in rheumatoid arthritis: In vitro and in vivo bases, clinical evidence and future trends.

The use of biologically active compounds derived from plants i.e. phytochemicals, have been known for ages for their pharmacological activities in the treatment of autoimmune disorders like rheumatoid arthritis (RA). Besides enormous scientific evidence, the therapeutic potential of phytochemicals is often undervalued. The treatment in RA involves the use of synthetic and biological disease modifying anti-rheumatic drugs (DMARDs). However, the long-term treatment in RA is associated with the risk of gastrointestinal, liver, pulmonary and renal toxicities and serious infections including latent tuberculosis, pneumococcus influenza, herpes zoster and hepatitis. These adverse effects sometimes lead to discontinuation of the therapy. A relatively new vision based on the combination of DMARDs with phytochemicals exhibiting anti-inflammatory, anti-arthritic, anti-oxidant, hepatoprotective and nephroprotective properties for the treatment of RA has achieved substantial importance in the last decade. From this perspective, the present review focuses on the combination of DMARDs (primarily MTX) with phytochemicals that have shown synergistic therapeutic effects while decreasing the toxic repercussions of current RA therapy. The review covers recent evidences of such combination studies that have shown promising results both in experimental arthritic models and clinical arthritis. Few of the combinations including resveratrol, sinomenine, coenzyme Q10 exhibited considerable interest because of their efficacy as an adjuvant to the MTX/standard DMARDs therapy in clinical trials. Besides giving an overview of such combination studies the review also critically discusses the limitations with the use of phytochemicals (e.g. solubility, permeability and bioavailability) compromising their clinical application. Additionally, it stresses upon the need of novel delivery systems and pharmaceutical technologies to increase the therapeutic efficacy of the combination therapy. Overall, the review unveils the potential of phytochemicals in combination with DMARDs with increased tolerability and superior efficacy in further refining the future of the RA therapy.

Chemo-enzymatic approaches for consolidated bioconversion of Saccharum spontaneum biomass to ethanol-biofuel.

A novel strategy involving sodium dodecylsulfate (SDS) (SDS assisted tris (2-hydroxyethyl) methyl- ammonium methyl sulphate ([TMA][MeSO4], ionic liquid) pretreatment of Saccharum spontaneum biomass (SSB) following its enzymatic saccharification, and conversion into ethanol-biofuel in a consolidated bioprocess (CBP) was developed. Ionic liquid stable enzyme preparation developed from Bacillus subtilis G2 was used for saccharification. Optimized pretreatment and saccharification variables enhanced the sugar yield (2.35-fold), which was fermented to ethanol content of 104.42 mg/g biomass with an efficiency of 35.73%. The pretreated biomass was examined for textural/ultrastructural alterations by scanning electron microscopy (SEM), 1H/13C nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), surface area measurements, water retention value, and cellulase adsorption isotherms. The combined [TMA][MeSO4] and SDS pretreatment disrupted the lignocellulosic microfibrils, and increased the porosity and surface area. The study provides new mechanistic insights on combined IL and surfactant pretreatment of biomass for its efficient conversion to biofuel.

Multifarious cholesterol lowering potential of lactic acid bacteria equipped with desired probiotic functional attributes.

Lactic acid bacteria (LAB) isolates possessed functional probiotic attributes, such as high hydrophobicity and autoaggregation ability, coaggregation capability with bacterial pathogens, antimicrobial activity, antioxidant potential, and hypocholesterolemic effects. Selected potential probiotic LAB, i.e. Lactobacillus paracasei M3, L. casei M5, L. paracasei M7, and few others were studied for their ability to lower cholesterol using a number of methods viz. cholesterol assimilation, bile salt deconjugation, cholesterol co-precipitation, cholesterol adhesion to probiotic cell wall, and miceller sequestration of cholesterol. L. casei M5 showed maximum bile salt hydrolase (BSH) activity, and released 57.63 nmol of glycine/min, and was closely followed by LAB isolate M9 which generated 52.12 nmol of glycine/min. Sodium glycocholate was deconjugated by L. casei M5 to produce 27.77 µmol/mL of cholic acid, while other isolates produced 20-26 µmol/mL of cholic acid. Cholesterol was assimilated significantly by isolate M6 (82.15%) and L. casei M5 (76.51%). L. casei M5 showed higher cholesterol co-precipitation ability (50.16 µg/mL) as compared to other LAB isolates (33-44 µg/mL). Miceller cholesterol concentration was reduced maximally by LAB isolate M8 (87.5%), followed by isolates M5 (84.75%), M9 (84%), M10 (80%), and M37 (79%). Higher cell wall adhesion of cholesterol was realized by L. casei M5 (42.48 µg/mL) than other LAB isolates (30-40 µg/mL). Selected LAB probiotics demonstrated short chain fatty acid (acetate, propionate, and butyrate) producing ability, yet another way of probiotics-mediated cholesterol lowering.

Consolidated bioprocessing of surfactant-assisted ionic liquid-pretreated Parthenium hysterophorus L. biomass for bioethanol production.

The current study presents the first ever report of surfactant (Tween-20) assisted ionic liquid IL, (1-ethyl-3-methylimidazolium methane sulphonate [Emim][MeSO3]) pretreatment of Parthenium hysterophorus biomass, its saccharification by in-house developed enzyme cocktail from Aspergillus aculeatus PN14, and fermentation of sugars to bioethanol under consolidated bioprocess. Optimization of pretreatment process variables viz. biomass loading, temperature and time, resulted in enhanced sugar yield (40.1%) upon saccharification of pretreated biomass with IL-stable cellulase and xylanase enzymes from an IL-tolerant newly isolated fungus Aspergillus aculeatus PN14. Physicochemical analysis of surfactant assisted IL-pretreated biomass by SEM, FT-IR and XRD provided molecular insights into inter/intra molecular ultrastructural changes in the biomass that eased the saccharification. Thorough understanding of chemical/molecular structure of biomass may help developing customized pretreatment regimes of apt severity which might result in enhanced accessibility of enzymes to biomass, and hence more sugar content.

Ultrasound and surfactant assisted ionic liquid pretreatment of sugarcane bagasse for enhancing saccharification using enzymes from an ionic liquid tolerant Aspergillus assiutensis VS34.

Ionic liquid (IL) pretreatment represents an effective strategy for effective fractionation of lignocellulosic biomass (LB) to fermentable sugars in a biorefinery. Optimization of combinatorial pretreatment of sugarcane bagasse (SCB) with IL (1-butyl-3-methylimidazolium chloride [Bmim]Cl) and surfactant (PEG-8000) resulted in enhanced sugar yield (16.5%) upon enzymatic saccharification. The saccharification enzymes (cellulase and xylanase) used in the current study were in-house produced from a novel IL-tolerant fungal strain Aspergillus assiutensis VS34, isolated from chemically polluted soil, which produced adequately IL-stable enzymes. This is the first ever report of IL-stable cellulase/xylanase enzyme from Aspergillus assiutensis. To get the mechanistic insights of combinatorial pretreatment physicochemical analysis of variously pretreated biomass was executed using SEM, FT-IR, XRD, and 1H NMR studies. The combined action of IL, surfactant and ultrasound had very severe and distinct effects on the ultrastructure of biomass that subsequently resulted in enhanced accessibility of saccharification enzymes to biomass, and increased sugar yield.

Application of ionic liquid and alkali pretreatment for enhancing saccharification of sunflower stalk biomass for potential biofuel-ethanol production.

Biorefining of lignocellulosic biomass to fuels/chemicals has recently gained immense research momentum. Current study reports sequential pretreatment of sunflower stalk (SFS) biomass in a combinatorial regime involving alkali (NaOH) and ionic liquid 1-butyl-3-methyl imidazolium chloride. The pretreatment enhanced the enzymatic digestibility, and resulted in increased sugar yield (163.42 mg/g biomass) as compared to standalone pretreatment using alkali (97.38 mg/g biomass) or ionic liquid (79.6 mg/g biomass). Ultrastructural and morphological analysis (FTIR and SEM) of pretreated biomass showed that the combined ionic liquid and alkali pretreatment causes more drastic alterations in the biomass ultrastructure as compared to alone ionic liquid or alkali pretreatment. Thus, combined pretreatment led to ease of enzymatic saccharification and consequent increased sugar yield, and this observation was corroborated by physicochemical analysis of the pretreated biomass. The pretreated SFS biomass was subjected to consolidated bioprocessing for its direct conversion to bioethanol in a single vessel.

Process desired functional attributes of an endoxylanase of GH10 family from a new strain of Aspergillus terreus S9.

Huge industrial application potential of xylanases is stalled due to lack of process suitable characteristics like thermostability, broad range pH stability, and high catalytic efficiency in the available enzymes. Current study presents the first ever report of a pH stable (pH 6-11) and thermostable (80-100 °C) xylanase from a novel strain of Aspergillus terreus S9. The xylanase was purified to homogeneity (6.67-fold) by ammonium sulphate precipitation, ion exchange chromatography, and molecular exclusion chromatography. SDS-PAGE analysis revealed an estimated molecular mass of ~33 kDa for the xylanase. Metal ions and surfactants such as K+, Ca2+, Mn2+, Mg2+, CTAB and Tween-80 enhanced the xylanase activity while Cu2+ and Hg2+ strongly inhibited the activity. Kinetic parameters i.e. Km, Vmax,Kcat and Kcat/Km of A. terreus S9 xylanase were 2.94 mg/ml, 285.71 µmol/min/mg, 1587.28 s-1and 539.89 ml/mg/s, respectively. The substrate specificity confirmed the true endoxylanolytic nature of xylanase. The conserved domain analysis, and Blastn and Blastx results showed that the xylanase belonged to GH10 family. A. terreus S9 xylanase may be used as model system for understanding the molecular basis of robust nature of enzymes, and the knowledge generated may help designing novel enzymes that are suitable for industrial applications.

Hypocholesterolemic and bioactive potential of exopolysaccharide from a probiotic Enterococcus faecium K1 isolated from kalarei.

Bioprospecting of novel probiotic strains especially from unexplored eco-niches has been a continuous practice. Enterococcus faecium K1, an isolate from indigenously fermented milk product kalarei possesses numerous desirable functional attributes. In current study, E. faecium K1 has been used for EPS production, and it yielded 355 ±â€¯0.019 mg/L EPS. EPS demonstrates remarkable hypocholesterolemic, antioxidant, antibiofilm, and emulsification characteristics. EPS is constituted of mannose, glucose and galactose. SEM analysis reveals flake like compact structure of EPS while TEM and X-ray diffractogram confirms the amorphous structure of EPS. FTIR substantiates the functional groups/bonds typical of polysaccharides. Thermal analysis indicates adequate stability of EPS at 237 °C with average weight loss of 22%. E. faecium K1 EPS possesses unique functional bioactivities and physicochemical characteristics, and may potentially be explored for applications in food/pharmaceutical industries.

Functional Characterization of Potential Probiotic Lactic Acid Bacteria Isolated from Kalarei and Development of Probiotic Fermented Oat Flour.

Considerable variations among probiotics with respect to their health benefitting attributes fuel the research on bioprospecting of proficient probiotic strains from various ecological niches especially the poorly unexplored ones. In the current study, kalarei, an indigenous cheese-like fermented milk product, and other dairy-based sources like curd and raw milk were used for isolation of lactic acid bacteria (LAB). Among 34 LAB isolates, 7 that could withstand simulated gastrointestinal (GI) conditions were characterized for functional probiotic attributes, viz. adhesion ability, aggregation and coaggregation, extracellular enzyme producing capability, antibacterial activity against pathogens and antibiotic resistance. The isolate M-13 (from kalarei) which exhibited most of the desirable probiotic functional properties was identified as Lactobacillus plantarum based on 16S ribosomal DNA sequence analysis and designated as L. plantarum M-13. The sequence was submitted to GenBank (accession number KT592509). The study presents the first ever report of isolation of potential probiotic LAB, i.e. L. plantarum M-13 from indigenous food kalarei, and its application for development of potential probiotic fermented oat flour (PFOF). PFOF was analysed for parameters like viability of L. plantarum M-13, acidity and pH. Results show that PFOF serves as a good matrix for potential probiotic L. plantarum M-13 as it supported adequate growth of the organism (14.4 log cfu/ml after 72 h of fermentation). In addition, appreciable acid production by L. plantarum M-13 and consequential pH reduction indicates the vigorous and active metabolic status of the potential probiotic organism in the food matrix. Thus, study shows that fermented oat flour may possibly be developed as a potential probiotic carrier especially in view of the problems associated with dairy products as probiotic vehicles.

Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media.

Intravascular thrombosis is one of the major causes of variety of cardiovascular disorders leading to high mortality worldwide. Fibrinolytic enzymes from microbial sources possess ability to dissolve these clots and help to circumvent these problems in more efficient and safer way. In the present study, fibrinolytic protease with higher fibrinolytic activity than plasmin was obtained from Serratia sp. KG-2-1 isolated from garbage dump soil. Response surface methodology was used to study the interactive effect of concentration of maltose, yeast extract + peptone (1:1), incubation time, and pH on enzyme production and biomass. Maximum enzyme production was achieved at 33 °C after 24 h at neutral pH in media containing 1.5% Maltose, 4.0% yeast extract + peptone and other trace elements resulting in 1.82 folds increased production. The enzyme was purified from crude extract using ammonium sulfate precipitation and DEAE-Sephadex chromatography resulting in 12.9 fold purification with 14.9% yield. The purified enzyme belongs to metalloprotease class and had optimal activity in conditions similar to physiological environment with temperature optima of 40 °C and pH optima of 8. The enzyme was found to be stable in various solvents and its activity was enhanced in presence of Na+, K+, Ba2+, Cu2+, Mn2+, Hg2+ but inhibited by Ca2+ and Fe3+. Hence, the obtained enzyme may be used as potential therapeutic agent in combating various thrombolytic disorders.

Phytase-Producing Potential and Other Functional Attributes of Lactic Acid Bacteria Isolates for Prospective Probiotic Applications.

Wide variations among multifaceted-health benefitting attributes of probiotics fueled investigations on targeting efficacious probiotics. In the current study, lactic acid bacteria (LAB) isolated from poultry gut, feces of rat, chicken, human infants, and fermented foods were characterized for desired probiotic functional properties including the phytase-producing ability which is one of the wanted characteristics for probiotics for potential applications for upgrading animal nutrition, enhancing feed conversion, and minimizing anti-nutritional properties. Among 62 LAB isolates Weissella kimchii R-3 an isolate from poultry gut exhibited substantial phytase-producing ability (1.77 U/ml) in addition to other functional probiotic characteristics viz. hydrophobicity, autoaggregation, coaggregation with bacterial pathogens, and antimicrobial activity against pathogens. Survival of W. kimchii R-3 cells (in free and calcium alginate encapsulated state) was examined sequentially in simulated gastric and intestinal juices. Encapsulated cells exhibited better survival under simulated gut conditions indicating that encapsulation conferred considerable protection against adverse gut conditions. Furthermore, simulated gastric and intestinal juices with pepsin and pancreatin showed higher survival of cells than the juices without pepsin and pancreatin. W. kimchii R-3 due to its significant functional probiotic attributes may have prospective for commercial applications in human/animal nutrition.

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation.

Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728 U ml(-1)), which was followed by gram husk (714 U ml(-1)), mustard cake (680 U ml(-1)), and soybean meal (653 U ml(-1)). Plackett-Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020 U ml(-1)). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.

Cost-effective-substrates for production of poly-ß-hydroxybutyrate by a newly isolated Bacillus cereus PS-10.

Poly-ß-hydroxybutyrate (PHB) may serve as one of the imperative substitutes for petroleum derived plastics because of their close functional analogy and biodegradation quality. In the present study, PHB producing ability of bacterial isolates was examined on low-cost agro industrial residues. Isolate PS-10 from domestic waste landfills, identified as Bacillus cereus PS-10 produced and accumulated appreciable amount of PHB. Bacillus cereus PS-10 was capable of using a wide variety of agro-based residues viz. maize bran, rice husk, wood waste, molasses, whey etc. as cost-effective carbon sources for PHB production. Molasses at 3% (w/v) supported maximum PHB production (9.5 gl(-1)) and was followed by glycerol (8.9 gl(-1)) at 2% (w/v). Certain carbon sources like almond shell powder and walnut shell powder are being reported for the first time for PHB production and supported reasonable PHB yield i.e. 6.6 and 4.6 gl(-1), respectively. Different cost-effective nitrogen sources like corn steep liquor, chick pea bran, soy bean meal, mustard cake etc. were used for PHB production. Highest PHB production was observed at pH 7 (9.6 gl(-1)) after 48 hrs of fermentation, although B. cereus PS-10 grew and produced PHB over pH range of 5-9. Optimum inoculum level for maximum PHB production was found to be 5% v/v (A600 0.9; approximately 10(8) cfu ml(-1)). Fourier transform infrared spectroscopy (FT-IR) analysis of the extracted PHB showed characteristic peaks (1721.95, 1632.19 and 2926.43 cm(-1)) similar to standard PHB. Melting point of PHB was found to be 185°C. Bacillus cereus PS-10 may be a sound PHB producer, especially by exploiting low cost substrates.

Production and characterization of poly-3-hydroxybutyrate from Bacillus cereus PS 10.

Usage of renewable raw materials for production of fully degradable bioplastics (bacterial poly-3-hydroxybutyrate, PHB) has gained immense research impetus considering recalcitrant nature of petroleum based plastics, dwindling fossil fuel feed stocks, and associated green house gas emissions. However, high production cost of PHB is the major bottleneck for its wide range industrial applications. In current study, Bacillus cereus PS 10, a recent isolate, efficiently utilized molasses, an abundantly available by-product from sugar industries as sole carbon source for growth and PHB production. Most influential bioprocess variables i.e. molasses, pH and NH4Cl were identified based on Plackett-Burman-designed experiments. Design of experiment approach (response surface methodology) was further employed for optimization of these bioprocess variables, and an enhanced PHB yield (57.5%) was obtained. PHB produced by Bacillus cereus PS 10 was investigated using various physico-chemical approaches viz. thermogravimetric analysis, proton and carbon NMR ((1)H and (13)C) spectroscopy, melting point, elemental analysis and polarimetry for its detail characterization, and assessment for industrial application potential.

Production of poly-ß-hydroxybutyrate by Bacillus cereus PS 10 using biphasic-acid-pretreated rice straw.

Poly-ß-hydroxybutyrate (PHB) has attracted a great deal of attention in recent years due to its potential use for production of fully degradable bioplastics, however, high cost of PHB production is the major bottleneck for its wide range industrial applications. In the current study rice straw hydrolysate (RSH) was employed as a cost-effective substrate for PHB production. RSH was prepared based on biphasic acid-pretreatment of rice straw i.e. first phase treatment with 1% sulphuric acid at 121 °C for 45 min, followed by second phase treatment using 5% sulphuric acid at 121 °C for 60 min (solid:liquid ratio, 1:10). RSH turned out be an efficient substrate for PHB production from a recently isolated Bacillus cereus PS 10, and yielded higher PHB amount than that obtained with glucose (8.6g/L in glucose based medium vs 10.61 g/L in RSH based medium) after response surface methodology (RSM) based optimization. Design of experiments based on RSM was used to optimize three process variables i.e. amount of RSH and NH4Cl, and medium pH, and enhanced PHB yield (23.3%) was obtained. PHB produced was investigated by differential scanning calorimetry and X-ray diffraction powder analysis.

Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues

The aim of this work was to study the production of fibrinolytic protease by Bacillus subtilis I-2 on agricultural residues. Molasses substantially enhanced (63%) protease production (652.32 U/mL) than control (398.64 U/mL). Soybean meal supported maximum protease production (797.28 U/mL), followed by malt extract (770.1 U/mL), cotton cake (761.04 U/mL), gelatin (742.92 U/mL) and beef extract (724.8 U/mL). Based on the Plackett-Burman designed experiments, incubation time, soybean meal, mustard cake and molasses were identified as the significant fermentation parameters. Ammonium sulfate precipitation and DEAE sephadex chromatography resulted 4.8-fold purification of protease. Zymography showed the presence of three iso-forms in the partially purified protease preparation, which was confirmed by the SDS-PAGE analysis (42, 48, 60 kDa). Protease exhibited maximum activity at 50oC and at pH 8.0. Significant stability was observed at 30-50oC and at pH 7.0-10.0. Mg2+, Zn2+, Co2+, Ca2+, Mn2+ and Cu2+,EGTA, EDTA and aprotinin severely decreased the enzyme activity.