• Enzymatic Hydrolysis
    Tests at Celignis

Background on Enzymatic Hydrolysis

Enzymes are biocatalysts and play a key role in biorefinery processes for the biochemical/biological conversion of biomass to biofuels and biobased products.

First generation and second generation biofuels use enzymes for conversion of structural polymers to monomers so that these can then be fermented to the desired fuel by yeast or bacteria.

Starch Biorefinery Enzymes

actions of amylases

Amylases are key enzymes in starch biorefineries. There are two main kinds of amylases that are widely used in industrial applications:

Alpha-amylase

These enzymes are also known as Taka Amylase-A. They are endo-enzymes and are responsible for the initial hydrolysis of starch granules to soluble products. They act in random fashion on alpha (1-4) linkages of amylose and amylopectin, but cannot cleave the branched alpha (1-6) linkages. These enzymes release linear short chain dextrans, oligosachharides, maltose and glucose.

Glucoamylase

This enzyme, also known as amyloglucosidase, is an exo-acting amylase that can hydrolyse both alpha (1-4) and alpha (1-6) linkages. It works on non-reducing endings of the polysaccharide and releases glucose units.

Lignocellulose Biorefinery Enzymes


Plant biomass is a complex and recalcitrant matrix made of cellulose, hemicellulose and lignin. Lignocellulosic biomass typicaly needs a pre-treatment in order to make cellulose more accessible to enzymes.

The common class of enzymes that are used in lignocellulosic biorefineries are cellulases and xylanases. Lignin-degrading enzymes are also considered important in the biological and biochemical pre-treatment of biomass in order to avoid the formation of inhibitors.

Cellulase Enzymes

actions of cellulases

Cellulases are produced from both bacteria and fungi. Fungal cellulases are a complex of the following individual enzymes: Endoglucanase, exoglucanase and betaglucosidase. Fungal enzymes are the most commonly used cellulases in the advanced biofuels and biorefining industries because of the ability of fungi to produce these enzymes in high titres. The individual enzymes of cellulase act synergistically to breakdown cellulose to glucose:

 
  • Endoglucanases:

      These enzymes break down the cellulose fibers and expose reducing and non-reducing ends for the action of exoglucanases.

  • Exoglucanases:

      These enzymes further break down the cellulose into the disaccharide cellobiose.

  • Betaglucosidases:

      These enzymes take the cellobiose formed by the action of exoglucanases and break it up into glucose monomers.



  • The products of enzymatic hydrolysis can lead to feedback inhibition on the enzymes. For example, cellobiose has strong inhibition on endo and exo cellulose, but its accumulation beyond a certain concentration is avoided by the action of beta-glucosidase. However, beta-glucosidase itself is feedback-inhibited by glucose and hence cellobiose can accumulate, inhibiting exocellulases and endocellulases. To overcome this, enzyme industries produce mixes of cellulases and individual components of enzymes. In many cases, cellulases are supplemented with excess beta-glucosidases in order to avoid feedback inhibition. Substrate-specific and application-specific enzyme cocktails are developed by supplementing cellulase mixes with individual cellulase components.

    Xylanase Enzymes

    actions of xylanases

    Hemicellulose is a complex branched structure with different sugars and acidic compounds substituted at various positions on a backbone chain. As a result of this, multiple different enzymes are required for the complete hydrolysis of hemicellulose. Xylanases are mixtures of endoxylanases, beta-xylosidases, arabinofuranosidases, galactosidases, acetyl xylan esterases, and alpha-glucuronidases. The mix of xylanolytic enzymes act in synergy to convert xylan polymers to their monomeric constituents.

    Endoxylanases reduce the viscosity of xylan by cleaving the xylan to soluble fragments and beta-xylosidases act on the soluble fragments and xylobiose to release xylose. Arabinofuranosidases and alpha-glucuronidases act on sugar and uronic-acid side groups of xylan.


    Enzymatic Hydrolysis Tests at Celignis


    We are pleased to offer a variety of analysis packages for evaluating both the suitability of biomass for enzymatic digestion and the activities of selected enzymes.

    Evaluation of Biomass and Pre-Treated Biomass for Enzymatic Digestibility

    In analysis package P121 we add an enzyme solution to a sample and incubate it for 3 days, monitoring the sugars that are hydrolysed over time. Subsamples of the hydrolysate are collected after 0, 12, 24, 48, and 72 hours of digestion and are analysed for monosaccharides (glucose, xylose, mannose, arabinose, galactose, rhamnose) and cellobiose using our ion chromatography system. The resulting data allow us to plot a time-series of the concentrations of these sugars in solution and we also provide statistics for the conversion yield and conversion rate of cellulose and xylan.

    These enzymatic hydrolysis tests can be carried out on native biomass and on pre-treated samples and we can either use a commercial enzyme mix or you can supply your own enzymes.

    Evaluation of Pre-Treatment on Enzymatic Digestibility

    Lignocellulose is a recalcitrant feedstock, with cellulose protected by lignin and hemicellulose, so making it less accessible to enzymatic degradation and microbial attack.

    As a result, several pre-treatment strategies to increase the accessibility of cellulose and enhance the digestibility of lignocellulosic biomass by enzymes have been developed. An effective pre-treatment will result in increased accessibility of cellulose to enzymes and thus in high concentrations, yields and productivities of sugars in the enzymatic hydrolysate when compared to the un-treated biomass.

    At Celignis we can undertake the enzymatic hydrolysis of a pre-treated sample using analysis package P121 or we can undertake a more detailed study to evaluate the efficacy of pre-treatment. This study involves analysis package P121 being undertaken on the native (non-pretreated) sample with analysis package P122 used for the enzymatic hydrolysis of the pre-treated sample(s).

    This package allows for the generation of a number of additional statistics outlining the relative advantages of the pre-treated sample(s) as substrates for enzymatic hydrolysis compared with the original sample. These statistics include:

      - Increase in Cellulose Accessibility after Pre-Treatment



    - Percent Increase in Cellulose Conversion Efficiency


    - Percent Increase in Cellulose Conversion Rate



    Fermentation Inhibitors in Enzymatic Hydrolysate

    It should be noted that an increase in digestibility does not always directly result in an increase in fermentability. Inhibitory sugar-degradation products including organic acids (e.g. acetic acid and formic acid), and furans (e.g. furfural and hydroxymethylfurfural) that may be formed during the pre-treatment process can be partially bound to the substrate and may be subsequently released into the enzymatic hydrolysate.

    Based on their concentrations, these inhibitory compounds can negatively affect the fermentation process. For example, furans can inhibit cell replications at higher concentrations whilst weak acids can inhibit fermentation by accumulation of intracellular anions or by uncoupling of cell metabolism. Knowledge of the types of inhibitors in the enzyme hydrolysate and their concentrations will help in developing pre-treatment processes to avoid or reduce inhibitor formation and also to select suitable detoxification processes prior to fermentation.

    We therefore recommend that the analysis of these potentially inhibitory compounds be undertaken when undertaking enzymatic hydrolysis tests, particularly those concerning the evaluation of the efficiency of biomass pre-treatment processes. Our analysis package P124 will determine the concentrations of these important inhibitors in the hydrolysate produced in our enzymatic hydrolysis tests.


    Analysis of the Solid Residue after Enzymatic Hydrolysis

    In order to get the most detailed information about the efficacy of enzymatic digestion, we recommend that the solid residue is analysed for its lignocellulosic composition. The resulting data will show what amounts of the structural polysaccharides have not been hydrolysed and will also help to inform decisions regarding how this residue is valorised.

    In many biorefineries the solid residue after enzymatic hydrolysis is combusted or processed in other thermal conversion technologies (e.g. gasification or pyrolysis) in order to provide process heat and energy. If you would like to evaluate the hdyrolysis residue for this end-use then we recommend that analysis package P40 is also undertaken.


    Cellulase Saccharification Efficiency

    At Celignis we also offer a range of analysis packages for evaluating enzymes and enzyme cocktails. In analysis package P125 we evaluate the efficiency of your enzymes for hydrolysing cellulose. The ability of cellulases to breakdown cellulose to its monomers is called saccharification efficiency and it can vary based on the activity of individual cellulase components. The efficiency is measured by determining the percentage of cellulose converted to glucose under standard/optimum conditions of the enzyme supplied.

    Our cellulose saccharification test can be undertaken using pure cellulose or complex lignocellulosic substrates taken either from Celignis's repository or, alternatively, you can supply these substrates yourself.

    Xylanase Saccharification Efficiency

    The supplementation of xylanases with cellulases is an important strategy to improve the overall saccharification of lignocellulosic biomass. The ability of xylanase to hydrolyse the hemicellulose fraction of lignocellulose to xylose is called xylanase saccharification efficiency. It is measured by determining the percentage of xylan converted to xylose under standard/optimum conditions of the enzyme supplied. This test can be undertaken using pure xylan substrates or xylan-rich complex model substrates taken either from Celignis's repository or, alternatively, you can supply these substrates yourself.

    Amylase Saccharification Efficiency

    The efficiency of amylases to break down the starch polymer to its constituent monomeric sugars is called amylase saccharification efficiency. The level of efficiency and the extent of hydrolysis will depend on the activity of the enzymes alpha-amylase and glucoamylase in the amylase enzyme mix. In analysis package P127 the rate of hydrolysis of starch to glucose and percentage of starch converted to glucose are analysed and given in terms of conversion rate and conversion efficiency respectively.

    Cellulase Activity

    We measure the activity of cellulase enzymes by filter paper assay (FPA) and express the activity in filter paper units (FPU/ml). The conversion of filter paper cellulose to glucose by cellulases is not a linear reaction. Hence, the assay requires enzyme dilutions to release fixed amount of glucose. Because the FPA is based on concentration of product released in a certain time in a nonlinear reaction, a conversion factor is used to approximate a specific activity-type unit.

    Cellulolytic Enzymes Activity

    As described earlier, cellulases are a complex of endocellulases, exocellulases and beta-glucosidases. The total activity of cellulase depends on individual enzymes activities and their ability to act in synergy. In analysis package P129 we can determine the activities of these indivifual enzymes, an important test that will help in understanding the limiting components in the enzyme mix. The different tests undertaken in this analysis package are outlined below:

     
  • Endoglucanase Activity:

      Endo-b-1,4-D-glucanase (EC 3.2.1.4) randomly cleaves accessible intermolecular b-1,4-glucosidic bonds on the surface of cellulose. We employ the IUPAC-recommended endoglucanase (CMCase) assay which uses carboxymethyl cellulose (CMC) as a substrate and is a fixed conversion method based on 0.5 mg of glucose released under the reaction condition. It is expressed as CMC Units/ml or International Units (IU/ml).

  • beta-Glucosidase Activity:

      Beta-Glucosidase (BGL) acts on Beta-1,4-glucosidic bonds of cellobiose and cellodextrins with a degree of polymerisation (DP) ranging from 3 to 6. It also acts on chromogenic substrates such as para-nitrophenyl glucopyranoside (PNPG). BGL activity can be measured either by using cellobiose or PNPG as substrate. The PNPG assay considers the initial rate of reaction and the enzyme activity is calculated based on the linear range between absorbance and enzyme concentrations. The enzyme activity is expressed in International units (IU/ml).


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  • Xylanase Activity:

      D-xylanases are either exo or endo enzymes. Solubilised xylan is used as the substrate for measuring the xylanase activity. Total reducing sugar released by the enzyme is measured and xylanase activity is expressed as international units per ml (IU/ml) which indicates the number of umoles of reducing sugar released per minute per ml of enzyme used.


  • Amylolytic Enzymes Activity

    With analysis package P130 we can determine the activity of amylolytic enzymes. This package involves the following tests:

     
  • a-Amylase Activity:

      Alpha-amylase is an endo-enzyme and acts randomly on the alpha-1,4 linkages of starch and releases a combination of disaccharides (maltose) and oligosaccharides. One unit of amylase releases 1 mg of maltose in the presence of starch under standard assay conditions. The activity of the enzyme is reported in International units (IU/ml).



  •  
  • Glucoamylase Activity:

      Gluco-amylase is both exo-enzyme and acts on both alpha-1,4 and alpha-1,6 linkages of starch. It releases glucose as the final product. One unit of glucoamylase releases 1 mg of glucose in the presence of starch under standard conditions.


  • Summary of Analysis Packages for Enzymatic Hydrolysis and Enzyme Activities


    The analysis packages that we offer for enzymatic hydrolysis and enzyme activities, and the analytes that they determine, are listed below. Click on an entry for further details or go to the Celignis Database to place an order.

    Examples of Cellulosic Biomass Suitable for Enzymatic Hydrolysis Tests

    Publications on Enzymes By The Celignis Team

    Bedzo, O.K.K, Trollope K, Gottumukkala L.D, Coetzee G, Gorgens J.F. (2019) Amberlite IRA 900 versus calcium alginate in immobilization of a novel, engineered ? fructofuranosidase for short?chain fructooligosaccharide synthesis from sucrose, Biotechnology progress 35(3)

    The immobilization of ? fructofuranosidase for short?chain fructooligosaccharide (scFOS) synthesis holds the potential for a more efficient use of the biocatalyst. However, the choice of carrier and immobilization technique is a key to achieving that efficiency. In this study, calcium alginate (CA), Amberlite IRA 900 (AI900) and Dowex Marathon MSA (DMM) were tested as supports for immobilizing a novel engineered ??fructofuranosidase from Aspergillus japonicus for scFOS synthesis. Several immobilization parameters were estimated to ascertain the effectiveness of the carriers in immobilizing the enzyme. The performance of the immobilized biocatalysts are compared in terms of the yield of scFOS produced and reusability. The selection of carriers and reagents was motivated by the need to ensure safety of application in the production of food?grade products. The CA and AI900 both recorded impressive immobilization yields of 82 and 62%, respectively, while the DMM recorded 47%. Enzyme immobilizations on CA, AI900 and DMM showed activity recoveries of 23, 27, and 17%, respectively. The CA, AI900 immobilized and the free enzymes recorded their highest scFOS yields of 59, 53, and 61%, respectively. The AI900 immobilized enzyme produced a consistent scFOS yield and composition for 12 batch cycles but for the CA immobilized enzyme, only 6 batch cycles gave a consistent scFOS yield. In its first record of application in scFOS production, the AI900 anion exchange resin exhibited potential as an adequate carrier for industrial application with possible savings on cost of immobilization and reduced technical difficulty.

    Vodonik M, Vrabec K, Hellwig P, Benndrof D, Sezun M, Gregori A, Gottumukkala L.D, Andersen R.C, Reichel U (2018) Valorisation of deinking sludge as a substrate for lignocellulolytic enzymes production by Pleurotus ostreatus, Journal of Cleaner production 197(1): 253-263

    Disposal of waste sludges produced in large amounts in the pulp and paper industry imposes significant environmental and economical problems. One strategy to address these issues involves revalorization of paper mill sludges by their application as substrates for microbial production of biotechnologically relevant enzymes. The application of lignocellulolytic enzymes in paper, textile and bioenergy industries is encouraged in order to decrease chemicals and energy consumptions. In the following work, deinking sludge was assessed as a substrate for production of lignocellulases. Based on the results of growth and activity screenings, Pleurotus ostreatus PLAB was chosen as the most promising candidate among 30 tested strains and its secretome was further studied by quantitative enzyme assays and mass spectrometry. While endoglucanase and xylanase activities detected in P. ostreatus secretome produced on deinking sludge were similar to activities of cultures grown on other lignocellulosic substrates, average laccase activity was significantly higher (46?000 U/kg DIS). Mass spectrometry identification of the most prominent proteins in the secretome of the target strain confirmed that significant amounts of different lignin-modifying oxidases were produced on this substrate despite its low lignin content, indicating the presence of other inducible compounds. The findings of this study suggest deinking sludge may represent a good substrate for fungal production of the aforementioned enzymes with broad biotechnological applications, including bioremediation, paper and bioenergy industries.

    Ahire V, Zambare A, Zambare V. (2017) Extraction, purification and characterization of protease from latex of Plumeria Sp, International Journal of Advanced Biotechnology and Research 8(2): 1349-1353

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    Protease was isolated from the latex of Plumeria sp. using ammonium sulphate precipitation (60% saturation) method and purified by a dialysis followed by DEAE cellulose column chromatography. DEAE Cellulose chromatographic method showed 9.86-fold purification which is about 57.36% yield as compared to crude latex protease. Purified plumerian protease was showing optimum activity at pH 7 and temperature 500C. It was activated by 10mM Calcium chloride and betamercaptoethanol, however inhibited by 10mM iodoacetamide, indicated the presence of sulfhydryl as an essential group for its activity. The enzyme kinetic with casein substrate showed km and Vmax of 1.66 mg/ml and 333U/mg, respectively. Plumeria sp. showed a single protein band on SDS-PAGE and molecular weight was of 80 kDa. Thus, protease from the latex of Plumeria sp. was purified and characterized and it may be explored further to study its impact in medical science as an effective anti-inflammatory agent.

    Gottumukkala L.D, Haigh K, Gorgens J (2017) Trends and advances in conversion of lignocellulosic biomass to biobutanol: microbes, bioprocesses and industrial viability, Renewable and Sustainable Energy Reviews 76: 963-973

    Biobutanol has gained attention as an alternative renewable transportation fuel for its superior fuel properties and widespread applications in chemical industry, primarily as a solvent. Conventional butanol fermentation has drawbacks that include strain degeneration, end-product toxicity, by-product formation, low butanol concentrations and high substrate cost. The complexity of Clostridium physiology and close control between sporulation phase and ABE fermentation has made it demanding to develop industrially potent strains. In addition to the isolation and engineering of superior butanol producing bacteria, the development of advanced cost-effective technologies for butanol production from feedstock like lignocellulosic biomass has become the primary research focus. High process costs associated with complex feedstocks, product toxicity and low product concentrations are few of the several bioprocess challenges involved in biobutanol production. The article aims to assess the challenges in lignocellulosic biomass to biobutanol conversion and identify key process improvements that can make biobutanol commercially viable.

    V. P. Zambare and S. S. Nilegaonkar (2016) Proteases in Leather Processing, Industrial Biotechnology, Sustainable Production and Bioresource Utilization, Apple Academic Press, CRC Press

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    Gottumukka L.D, Haigh K, Collard F.X, Van Rensburg E, Gorgens J (2016) Opportunities and prospects of biorefinery-based valorisation of pulp and paper sludge, Bioresource technology 215: 37-49

    The paper and pulp industry is one of the major industries that generate large amount of solid waste with high moisture content. Numerous opportunities exist for valorisation of waste paper sludge, although this review focuses on primary sludge with high cellulose content. The most mature options for paper sludge valorisation are fermentation, anaerobic digestion and pyrolysis. In this review, biochemical and thermal processes are considered individually and also as integrated biorefinery. The objective of integrated biorefinery is to reduce or avoid paper sludge disposal by landfilling, water reclamation and value addition. Assessment of selected processes for biorefinery varies from a detailed analysis of a single process to high level optimisation and integration of the processes, which allow the initial assessment and comparison of technologies. This data can be used to provide key stakeholders with a roadmap of technologies that can generate economic benefits, and reduce carbon wastage and pollution load.

    Boshoff A, Gottumukka L.D, Van Rensburg E, Gorgens J (2016) Paper sludge (PS) to bioethanol: Evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentationl, Bioresource technology 23: 103-111

    Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20 FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11 FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2 g/L at 66.9% and 45.5 g/L at 78.2%, respectively.

    Gottumukkala L.D. Gorgens J.F (2016) Biobutanol production from lignocellulosics, Biofuels Production and future perspectives, Taylor & Francis group

    Next-generation biofuels from renewable sources have gained interest among research investigators, industrialists, and governments due to major concerns on the volatility of oil prices, climate change, and depletion of oil reserves. Biobutanol has drawn signicant attention as an alternative transportation fuel due to its superior fuel properties over ethanol. e advantages of butanol are its high energy content, better blending with gasoline, less hydroscopic nature, lower volatility, direct use in convention engines, low corrosiveness, etc. Butanol production through (acetone, butanol, and ethanol) ABE fermentation is a well-established process, but it has several drawbacks like feedstock cost, strain degeneration, product toxicity, and low product concentrations. Lignocellulosic biomass is considered as the most abundant, renewable, low-cost feedstock for biofuels. Production of butanol from lignocellulosic biomass is more complicated due to the recalcitrance of feedstock and inhibitors generated during the pretreatment and hydrolysis process. Advanced fermentation and product recovery techniques are being researched to make biobutanol industrially viable.

    LP Christopher; VP Zambare, AV Zambare, H. Kumar, L Malek (2015) A thermo-alkaline lipase from a new extremophile Geobacillus thermodenitrificans AV5 with potential application in biodiesel production, Journal of Chemical Technology and Biotechnology 90(11): 2007-2016

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    BACKGROUND A thermophilic lipase?producing Geobacillus thermodenitrificans strain AV?5 was isolated from the Mushroom Spring of Yellowstone National Park in WY, USA and studied as a source of lipase for transesterification of vegetable oils to biodiesel. RESULTS A maximum activity of 330 U mL?1 was produced on 2% (v/v) waste cooking oil at 50 C, pH 8, aeration rate of 1 vvm and agitation speed of 400 rpm. However, the higher lipase productivity (14.04 U mL?1 h?1) was found at a volumetric oxygen transfer coefficient (kLa) value of 18.48 h?1. The partially purified lipase had a molecular weight, temperature and pH optimum of 50 kDa, 65 C and pH 9, respectively, and was thermo?alkali stable: at 70 C, it retained 81% activity and 45% stability; at pH 10 it lost only 15% and 2.6% of its maximum activity and stability, respectively. Enzyme kinetic studies with p?nitrophenyl laurate as substrate revealed high substrate specificity (km of 0.440 mmol L?1) and kinetic activity (vmax of 556 nmol mL min?1) of lipase. CONCLUSIONS The kLa was found to be highly dependent on aeration and agitation rates. Following optimization of fermentation medium and parameters, a 7.5?fold increase in lipase production by G. thermodenitrificans was attained. The lipase activity and substrate specificity (as km) are among the highest reported in the literature for bacterial lipases. It was demonstrated that the enzyme can produce biodiesel from waste cooking oil with a conversion yields of 76%. 2015 Society of Chemical Industry

    V. P. Zambare, S. S. Nilegaonkar and P. P. Kanekar (2014) Scale up production of protease using Pseudomonas aeruginosa MCM B-327 and its detergent compatibility, Journal of Biochemical Technology 5(2): 698-707

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    The Maximum Protease Activity Was Obtained From P. Aeruginosa MCM B-327 With Soybean Meal 1%, Tryptone 1%, Initial Medium PH 7, Agitation Rate 250 Rpm, Aeration Rate 0.75 Vvm And Fermentation Temperature 30 C, Under Submerged Fermentation Conditions (SmF). The Protease Productivity At 10 And 120L Fermenters Was Found To Be 16,021 And 9,975 UL-1h-1 Respectively. Kinetics Of Cell Growth Revealed That Specific Cell Growth Rate Was 0.025 H-1. Protease Was Active And Stable At Different PH, Temperatures, In Anionic, Cationic And Non-Ionic Detergent Additives, As Well As In Commercial Detergents. The Protease Exhibited Blood Stains Removing Performance Indicating Its Potential In Detergent Industry. The Dried Ammonium Sulphate Precipitated Protease Was Stable At Room Temperature For A Period Of One Year. The Protease Has Shown Properties Suitable For Its Application In Detergents. The Results Contribute To Basic Knowledge And Application Of Protease From P.Aeruginosa To Detergent Industry. The Studies Will Help To Optimize The Production Of This Protease For Biotechnological Applications.

    Lew P. Christopher, Hemanathan Kumar, Vasudeo P. Zambare (2014) Enzymatic biodiesel: Challenges and opportunities, Applied Energy 119: 497-520

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    The chemical-catalyzed transesterification of vegetable oils to biodiesel has been industrially adopted due to its high conversion rates and low production time. However, this process suffers from several inherent drawbacks related to energy-intensive and environmentally unfriendly processing steps such as catalyst and product recovery, and waste water treatment. This has led to the development of the immobilized enzyme catalyzed process for biodiesel production which is characterized by certain environmental and economical advantages over the conventional chemical method. These include room-temperature reaction conditions, elimination of treatment costs associated with recovery of chemical catalysts, enzyme re-use, high substrate specificity, the ability to convert both free fatty acids and triglycerides to biodiesel in one step, lower alcohol to oil ratio, avoidance of side reactions and minimized impurities, easier product separation and recovery; biodegradability and environmental acceptance. This paper provides a comprehensive review of the current state of advancements in the enzymatic transesterification of oils. A thorough analysis of recent biotechnological progress is presented in the context of present technological challenges and future developmental opportunities aimed at bringing the enzyme costs down and improving the overall process economics towards large scale production of enzymatic biodiesel. As the major obstacles that impede industrial production of enzymatic biodiesel is the enzyme cost and conversion efficiency, this topic is addressed in greater detail in the review. A better understanding and control of the underpinning mechanisms of the enzymatic biodiesel process would lead to improved process efficiency and economics. The yield and conversion efficiency of enzymatic catalysis is influenced by a number of factors such as the nature and properties of the enzyme catalyst, enzyme and whole cell immobilization techniques, enzyme pretreatment, biodiesel substrates, acyl acceptors and their step-wise addition, use of solvents, operating conditions of enzymatic catalysis, bioreactor design. The ability of lipase to catalyze the synthesis of alkyl esters from low-cost feedstock with high free fatty acid content such as waste cooking oil, grease and tallow would lower the cost of enzymatic biodiesel. Discovery and engineering of new and robust lipases with high activity, thermostability and resistance to inhibition are needed for the establishment of a cost-effective enzymatic process. Opportunities to create a sustainable and eco-friendly pathway for production of enzymatic biodiesel from renewable resources are discussed.

    Thomas L, Joseph A, Gottumukkala L.D. (2014) Xylanase and cellulase systems of Clostridium sp.: an insight on molecular approaches for strain improvement, Bioresource technology 158: 343-350

    Bioethanol and biobutanol hold great promise as alternative biofuels, especially for transport sector, because they can be produced from lignocellulosic agro-industrial residues. From techno-economic point of view, the bioprocess for biofuels production should involve minimal processing steps. Consolidated bioprocessing (CBP), which combines various processing steps such as pretreatment, hydrolysis and fermentation in a single bioreactor, could be of great relevance for the production of bioethanol and biobutanol or solvents (acetone, butanol, ethanol), employing clostridia. For CBP, Clostridium holds best promise because it possesses multi-enzyme system involving cellulosome and xylanosome, which comprise several enzymes such as cellulases and xylanases. The aim of this article was to review the recent developments on enzyme systems of clostridia, especially xylanase and cellulase with an effort to analyse the information available on molecular approaches for the improvement of strains with ultimate aim to improve the efficiencies of hydrolysis and fermentation.

    Padul MV, Patil MT, Chouguale AD, Zambare VP, Patil RM, Ghule RB, Naikwade SV, Garad AS, Shaikh FK, Gadge PP, Shinde KD, Dama LB and Salve AN. (2013) In vitro screening of proteinase inhibitors (trypsin, chymotrypsin and helicoverpa gut proteinase inhibitors) in different plant tissue extracts, Trends in Biotechnology Research 1(1): 7-14

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    Most of the plant protection strategies are focused on selection and application of the natural proteinase inhibitors (PIs) against insect pests. In addition, PIs also play a vital role in medicine for treatment of immunity related diseases. PI activity exists mainly in seeds, leaves and flowers of plants. In search of novel PIs, 135 different plant tissue extracts (leaf, flower and seed) were screened for their PI (trypsin, chymotrypsin and Helicoverpa gut proteinase inhibitors) activities by using dot-blot assays. Most of the plant tissues screened revealed moderate PI activity, few showed low PI activity and very few of them showed strong PI activity against trypsin, chymotrypsin and Helicoverpa gut proteinases. The inhibitory potency of positive samples was further determined by solution assays. Five plants namely Arachis hypogaea, Vigna sinensis, Dolichos lablab, Phaseolus aureus and Cassia siamea showed higher activity which ranged from 22.91 to 58.33 %. Higher activities recorded in the seed as compare to leaf and flower tissues. Dolichos lablab showed highest PI activity (58.33 %) followed by Cassia siamea (52.08 %). PI activity was found to be distributed unequally in ammonium sulfate (NH 2 SO 4) fractions. INTRODUCTION Proteolytic enzymes catalyzing the hydrolytic cleavage of specific peptide bonds in target proteins are called as proteinases. These proteolytic enzymes are widely distributed in nearly all plants, animals and microorganisms (Christeller, 2005; Joanitti et al., 2006). In higher organisms proteinases play key roles in many biological processes. The proteolytic events catalyzed by these enzymes serve as mediators of signal initiation, transmission and termination in many of the cellular events such as inflammation, apoptosis, blood clotting and hormone processing pathways (Ivanov et al., 2006). But they may be potentially damaging when present in higher concentrations. For this reason their activities need to be strictly regulated and controlled.

    V. P. Zambare, S. S. Nilegaonkar and P. P. Kanekar (2013) Protease production and enzymatic soaking of salt-preserved buffalo hides for leather processing, IIAOB Letters 3(1): 1-4

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    Response surface methodological (RSM) optimization of protease by Pseudomonas aeruginosa MCM B327, increased 1.3-fold activity with 1% inoculum having cell density of 27.57 x 109 cells mL-1 at pH 7, 300C and 72 h of incubation. Protease enzyme recovered from P. aeruginosa showed characteristic activities against diverse proteins of hide. Enzyme was found to be active with substrates e.g. casein, Bovine serum albumin, gelatin, elastin, haemoglobin but inactive against keratin and collagen. During leather manufacturing, non-collagenase and non-keratinase activities have advantageous in a quality leather and hair saving process, respectively. Increased proteolytic enzyme concentration (0.1-0.5%) in soaking process showed increased water penetration because of hydrolysis of albumin and elastin proteins as indicated by opened fibers in histopathological sections. These findings suggest, protease secreted by P. aeruginosa may have application in soaking operation of leather processing for minimizing harmful deharing chemicals and processing time.

    Gottumukkala L.D, Parameswaran B, Valappil S.K, Mathiyazhakan, K (2013) Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01, Bioresource technology 145: 182-187

    Biobutanol from lignocellulosic biomass has gained much attention due to several advantages over bioethanol. Though microbial production of butanol through ABE fermentation is an established technology, the use of lignocellulosic biomass as feedstock presents several challenges. In the present study, biobutanol production from enzymatic hydrolysate of acid pretreated rice straw was evaluated using Clostridium sporogenes BE01. This strain gave a butanol yield of 3.43 g/l and a total solvent yield of 5.32 g/l in rice straw hydrolysate supplemented with calcium carbonate and yeast extract. Hydrolysate was analyzed for the level of inhibitors such as acetic acid, formic acid and furfurals which affect the growth of the organism and in turn ABE fermentation. Methods for preconditioning the hydrolysate to remove toxic end products were done so as to improve the fermentation efficiency. Conditions of ABE fermentation were fine tuned resulting in an enhanced biobutanol reaching 5.52 g/l.

    V.P. Zambare, Lew P. Christopher (2012) Optimization of enzymatic hydrolysis of corn stover for improved ethanol production, Energy Exploration & Exploitation 30(2): 193-205

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    Response surface methodology (RSM) was used to optimize the enzymatic hydrolysis of corn stover (CS), an abundant agricultural residue in the USA. A five-level, three-variable central composite design (CCD) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.16.0), solids loadings (6.623.4%), and enzyme loadings (6.6?23.4 FPU g?1 DM) on glucose yield from thermo-mechanically extruded CS. The extruded CS was first hydrolyzed with the crude cellulase of Penicillium pinophilum ATCC 200401 and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance (ANOVA) showed a coefficient of determination (R2 ) of 0.82. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU g?1 DM, the enzymatic hydrolysis of pretreated CS produced a glucose yield of 57.6% of the glucose maximum yield which was an increase of 10.4% over the non-optimized controls at zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges.

    Vasudeo Zambare, Archana Zambare, Debmalya Barh, Lew Christopher (2012) Optimization of enzymatic hydrolysis of prairie cordgrass for improved ethanol production, Journal of Renewable and Sustainable Energy 4(3): 1-8

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    Prairie cordgrass (PCG), Spartina pectinata, is considered an energy crop with potential for bioethanol production in North America. The focus of this study was to optimize enzymatic hydrolysis of PCG at higher solids loadings using a thermostable cellulase of a mutant Penicillium pinophilum ATCC 200401. A three variable, five-level central composite design of response surface methodology (RSM) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.16.0), solids loadings (6.6%23.4%), and enzyme loadings (6.623.4 FPU/g dry matter, DM) on glucose yield from a thermo-mechanically extruded PCG. The extruded PCG was first hydrolyzed with the crude P. pinophilum cellulase and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance showed a coefficient of determination (R2) of 0.89. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU/g DM, the enzymatic hydrolysis of pretreated PCG produced a glucose yield of 76.1% from the maximum yield which represents an increase of 15% over the non-optimized controls at the zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges. Furthermore, statistical optimization of solids and enzyme loadings of enzymatic hydrolysis of biomass may have important implications for reduced capital and operating costs of ethanol production.

    V. P. Zambare, S. S. Nilegaonkar and P. P. Kanekar (2012) Optimization of Nutritional Factors for Extracellular Amylase Production from Bacillus cereus MCM B-326 Using Response Surface Methodology, Research Journal of BioTechnology 7(4): 58-65

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    The optimization of nutritional factors and their concentrations for the amylase production by Bacillus cereus MCM B-326 in submerged fermentation was carried out using response surface methodology (RSM) based on the central composite design (CCD). The design contains a total of 20 experimental trials containing starch, soybean meal and CaCO3 as model factors for three levels. The mutual interaction between these variables resulted into 1.36 fold increase in amylase activity as compared to the mean predicted response at zero level of all variables. Amylase from B. cereus has approximate molecular weight of 40 kDa with optimum activity at pH 7.0 and temperature 30C.

    V.P. Zambare, Lew P. Christopher (2012) Biopharmaceutical potential of lichens, Pharmaceutical Biology 50(6): 778-798

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    Context: Lichens are composite organisms consisting of a symbiotic association of a fungus (the mycobiont) with a photosynthetic partner (the phytobiont), usually either a green alga or cyanobacterium. The morphology, physiology and biochemistry of lichens are very different from those of the isolated fungus and alga in culture. Lichens occur in some of the most extreme environments on the Earth and may be useful to scientists in many commercial applications. Objective: Over the past 2 decades, there has been a renewed and growing interest in lichens as a source of novel, pharmacologically active biomolecules. This review summarizes the past and current research and development trends in the characterization and use of lichens and their bioactive compounds in traditional medicine and other biopharmaceutical applications of commercial interest. Methods: The present review contains 10 illustrations and 188 references compiled from major databases including Science Direct, Chemical Abstracts, PubMed and Directory of Open Access Journals. Results: Lichen morphology, symbiosis, diversity and bioactivities including enzyme inhibitory, antimicrobial, antifungal, antiviral, anticancer, anti-insecticidal and antioxidant actions were reviewed and summarized. Recent progress in lichens and lichen-forming fungi was discussed with emphasis on their potential to accelerate commercialization of lichen-based products. Conclusions: Lichens are an untapped source of biological activities of industrial importance and their potential is yet to be fully explored and utilized. Lichen-derived bioactive compounds hold great promise for biopharmaceutical applications as antimicrobial, antioxidant and cytotoxic agents and in the development of new formulations or technologies for the benefit of human life.

    V.P. Zambare, A. Bhalla, K. Muthukumarappan, R. Sani, L. Christopher (2011) Bioprocessing of agricultural residues to ethanol utilizing a cellulolytic extremophile, Extremophiles 15: 611-618

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    A recently discovered thermophilic isolate, Geobacillus sp. R7, was shown to produce a thermostable cellulase with a high hydrolytic potential when grown on extrusion-pretreated agricultural residues such corn stover and prairie cord grass. At 70C and 1520% solids, the thermostable cellulase was able to partially liquefy solid biomass only after 36 h of hydrolysis time. The hydrolytic capabilities of Geobacillus sp. R7 cellulase were comparable to those of a commercial cellulase. Fermentation of the enzymatic hydrolyzates with Saccharomyces cerevisiae ATCC 24860 produced ethanol yields of 0.450.50 g ethanol/g glucose with more than 99% glucose utilization. It was further demonstrated that Geobacillus sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step that could facilitate the development of a consolidated bioprocessing as an alternative approach for bioethanol production with outstanding potential for cost reductions.

    V. P. Zambare, S. S. Nilegaonkar, P. P. Kanekar (2011) Production optimization and purification of a novel extracellular protease from Pseudomonas aeruginosa MCM B-327, New Biotechnology 28(2): 173-181

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    The focus of this study was on production, purification and characterization of dehairing protease from Pseudomonas aeruginosa MCM B-327, isolated from vermicompost pit soil. Optimum protease activity, 395 U mL?1, was observed in the medium containing soybean meal and tryptone, at pH 7 and 30C. The crude enzyme exhibited dehairing activity. As compared to chemical method, enzymatic method of dehairing showed reduction in COD, TDS and TSS by 34.28%, 37.32% and 51.58%, respectively. Zymogram of crude enzyme on native-PAGE presented two bands with protease activity of molecular weights of 56 and 67 kDa. Both proteases showed dehairing activity. Out of these, 56 kDa protease (PA02) was purified 3.05-folds with 2.71% recovery. The enzyme was active in pH range 79 and temperature 2050C with optimum pH of 8 and temperature 35C. Moreover, the enzyme activity of PA02 protease was not strongly inhibited by specific inhibitor showing the novel nature of enzyme compared to serine, cysteine, aspartyl and metalloproteases. Kinetic studies indicated that substrate specificity of PA02 protease was towards various natural and synthetic proteolytic substrates but inactive against collagen and keratin. These findings suggest protease secreted by P. aeruginosa MCM B-327 may have application in dehairing for environment-friendly leather processing.

    Zambare, V. P., Zambare, A. V., Muthukumarappan, K., Christopher, L. P. (2011) Potential of thermostable cellulases in the bioprocessing of switchgrass to ethanol, BioResources 6(2): 2004-2020

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    Switchgrass (Panicum virgatum), a perennial grass native to North America, is a promising energy crop for bioethanol production. The aim of this study was to optimize the enzymatic saccharification of thermo-mechanically pretreated switchgrass using a thermostable cellulase from Geobacillus sp. in a three-level, four-variable central composite design of response surface methodology. Different combinations of solids loadings (5 to 20%), enzyme loadings (5 to 20 FPU g-1 DM), temperature (50 to 70 oC), and time (36 to 96 h) were investigated in a total of 30 experiments to model glucose release from switchgrass. All four factors had a significant impact on the cellulose conversion yields with a high coefficient of determination of 0.96. The use of higher solids loadings (20%) and temperatures (70 oC) during enzymatic hydrolysis proved beneficial for the significant reduction of hydrolysis times (2.67-times) and enzyme loadings (4-times), with important implications for reduced capital and operating costs of ethanol production. At 20% solids, the increase of temperature of enzymatic hydrolysis from 50 oC to 70 oC increased glucose concentrations by 34%. The attained maximum glucose concentration of 23.52 g L-1 translates into a glucose recovery efficiency of 46% from the theoretical yield. Following red yeast fermentation, a maximum ethanol concentration of 11 g L-1 was obtained, accounting for a high glucose to ethanol fermentation efficiency of 92%. The overall conversion efficiency of switchgrass to ethanol was 42%.

    V. P. Zambare, S. S. Nilegaonkar, P. P. Kanekar (2011) Use of agroresidues for protease production and application in degelatinazation, Research Journal of BioTechnology 6(2): 62-65
    Vasudeo Zambare, Archana Zambare, Kasiviswanathan Muthukumarappan, Lew Christopher (2011) Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing, International Journal of Energy and Environment 2(1): 99-112

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    A thermophilic microbial consortium (TMC) producing hydrolytic (cellulolytic and xylanolytic) enzymes was isolated from yard waste compost following enrichment with carboxymethyl cellulose and birchwood xylan. When grown on 5% lignocellulosic substrates (corn stover and prairie cord grass) at 600 C, the thermophilic consortium produced more xylanase (up to 489 U/l on corn stover) than cellulase activity (up to 367 U/l on prairie cord grass). Except for the carboxymethyl cellulose-enriched consortium, thermo-mechanical extrusion pretreatment of these substrates had a positive effect on both activities with up to 13% and 21% increase in the xylanase and cellulase production, respectively. The optimum temperatures of the crude cellulase and xylanase were 600 C and 700 C with half-lives of 15 h and 18 h, respectively, suggesting higher thermostability for the TMC xylanase. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the crude enzyme exhibited protein bands of 25-77 kDa with multiple enzyme activities containing 3 cellulases and 3 xylanases. The substrate specificity declined in the following descending order: avicel>birchwood xylan>microcrystalline cellulose>filter paper>pine wood saw dust>carboxymethyl cellulose. The crude enzyme was 77% more active on insoluble than soluble cellulose. The Km and Vmax values were 36.49 mg/ml and 2.98 U/mg protein on avicel (cellulase), and 22.25 mg/ml and 2.09 U/mg protein, on birchwood xylan (xylanase). A total of 50 TMC isolates were screened for cellulase and xylanase secretion on agar plates. All single isolates showed significantly lower enzyme activities when compared to the thermophilic consortia. This is indicative of the strong synergistic interactions that exist within the thermophilic microbial consortium and enhance its hydrolytic capabilities. It was further demonstrated that the thermostable enzyme-generated lignocellulosic hydrolyzates can be fermented to bioethanol by a recombinant strain of Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.

    Vasudeo Zambare (2011) Optimization of amylase production from Bacillus sp. using statistics based experimental design, Emirates Journal of Food and Agriculture 23(1): 37-47

    Production of amylase under submerged fermentation Bacillus sp. was investigated using wheat bran, soybean meal and CaCO3 (WSC) medium. Response surface methodology (RSM) was used to evaluate the effect of the main variables, i.e., pH (11.35), temperature (35.16C) and inoculum size (2.95%) on amylase production by applying a full factorial central composite design (CCD). The mutual interaction between these variables resulted into 4.64 fold increase in amylase activity as compared to the non-optimized environmental factors in the basal medium.

    V.P. Zambare, Lew P. Christopher (2011) Statistical analysis of cellulase production in Bacillus amyloliquefaciens UNPDV-22, Extreme Life, Biospeology & Astrobiology 3(1): 38-45

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    The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology (RSM). Central composite design (CCD) was used to study the interactive effect of fermentation medium components (wheat bran, soybean meal, and malt dextrin) on cellulase activity. Results suggested that wheat bran, soybean meal, and malt dextrin all have significant impact on cellulase production. The use of RSM resulted in a 70% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 11.23 U/mL was obtained in a fermentation medium containing wheat bran (1.03%, w/v), soybean meal (2.43%, w/v), and malt dextrin (2.95%, w/v).

    V.P. Zambare, Lew P. Christopher (2011) Optimization of culture conditions for cellulase production from thermophilic Bacillus strain, Journal of Chemistry and Chemical Engineering 5(7): 521-527

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    The production of cellulase in Bacillus amyloliquefaciens UNPDV-22 was optimized using response surface methodology (RSM). Central composite design (CCD) was used to study the interactive effect of culture conditions (temperature, pH, and inoculum) on cellulase activity. Results suggested that temperature and pH all have significant impact on cellulase production. The use of RSM resulted in a 96% increase in the cellulase activity over the control of non-optimized basal medium. Optimum cellulase production of 13 U/mL was obtained at a temperature of 42.24 C, pH of 5.25, and inoculum size of 4.95% (v/v) in a fermentation medium containing wheat bran, soybean meal and malt dextrin as major nutritional factors.

    M. Ponraj, P. Jamunarani, V. Zambare (2011) Isolation and optimization of conditions for decolorization of true blue by textile dye decolorizing fungi, Asian Journal of Experimental and Biological Sciences 2(2): 270-277

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    Isolation and identification of dye decolourizing fungal isolates from textile dye effluent was carried out. The isolates of Aspergillus flavus, Aspergillus niger, Helminthosporium sp., Mucor sp. and Penicillium sp. were isolated from the textile effluent samples collected from Elampillai Jalagandapur. Different parameters were used for optimizing. Conditions for maximum decolourization effect by the fungal isolates. The optimization conditions used to decolorize textile dye true blue were different carbon, nitrogen, temperature and pH. Among the carbon sources, A. niger showed maximum dye decolourization (96.75%) with maltose as a carbon source. Among the nitrogen sources used, A. flavus showed maximum dye decolourization (97.82%) with yeast extract as a nitrogen source. Among the different temperatures used. A. niger showed maximum dye decolourization (97.26%) with room temperature.Among the differentpH used, A. niger showed maximum dye decolourization (97.52%) with pH 6. It is clearly evident from the results that fungal isolates were able to decolourizing the textile dye (true blue) efficiently. Dye decolourization with microorganisms is low cost effective and environmentally friendly and the only way for ultimate controlling of pollution generated by textile and dyestuff industries. However, more and more research and development works are needed to develop a viable alternative process for the treatment of textile effluent. Discover the world's research 15+ million members 118+ million publications 700k+ research projects Join for free Figures - uploaded by Jamunarani Pachamuthu Author content Content may be subject to copyright. .Effecttoffnitrogennsourcessonndecolorizationnofftrueeblueetextileedye .Effecttoffnitrogennsourcessonndecolorizationnofftrueeblueetextileedye .Effecttoffcarbonnsourcessonndecolorizationnofftrueeblueetextileedye .Effecttoffcarbonnsourcessonndecolorizationnofftrueeblueetextileedye .EffecttoffpHHonndecolorizationnofftrueeblueetextileedye .EffecttoffpHHonndecolorizationnofftrueeblueetextileedye .Effecttofftemperaturessonndecolorizationnofftrueeblueetextileedye .Effecttofftemperaturessonndecolorizationnofftrueeblueetextileedye Content uploaded by Jamunarani Pachamuthu Author content Content may be subject to copyright. Download full-text PDF Other full-text sources Society of Applied SciencesIsolation?and?Optimization?of?Culture?Conditions?For Decolorization?ofTrue?Blue?Using?Dye?Decolorizing?FungiM.?Ponraj?,1P.?Jamunarani?, V.?ZambareINTRODUCTION1 2*125Department?of?Microbiology, Vivekanandha?College?of Arts?and?Science?for Women,??Namakkal?Dist, TN?India.South?Dakota?School?of?Mines?and Technology,?Rapid?City,?SD?57701,?USADyes are intensely colored organic compounds which have widespread application. Dmetric tons worldwide are commercially available and are intensively used in textileprocessing, paper printing, pharmaceutical, food and other industries. Amongst various applications of synthetic dyesabout 30,000 tons of different dye stuffs are used per year for textile dyeing operations, thus dye houses are the majorconsumers of synthetic dyes and consequently are the main cause of water pollution and imposes severe damage to thequality of the soil [1]. The textile industry in India alone consumes up to 80% of the total dyestuffs produced [2].Textile dyes are classified as azo, diazo, cationic, basic, anthraquinone base, metal complex dyes based on the natureof their chemical structure. Synthetic dyes such as azo dyes, xanthenes dyes and anthraquinone dyes are very toxic toliving organisms The largest group of all synthetic dyes represented 70% of all organic dyes used by the textileindustry [3]. Azo dyes constitute a major class of environmental pollutants. Some of the azo dyes or their breakdownproducts also have a strong toxic and mutagenic influence on the living organisms [4].Textile industries generate waste water with different characteristics. The waste water characteristics vary accordingto the process employed [5]. Various waste liquor coming out of the operations in wet processing such as desizing,scouring, bleaching, mercerizing, dyeing, printing and finishing [6]. These concentration of dye contained in theyes are used in textile industriesfor dyeing nylon, polyacylonitrile modified nylon wool, silk cotton etc Around 10,000 different dyes with an annualproduction of more than 7?10, ..ASIAN?J.?EXP.?BIOL.?SCI.?VOL 2(2)?2011:?270-277ORIGINAL ARTICLEABSTRACT:Isolation and identification of dye decolourizing fungal isolates from textile dye effluent was carried out. The isolates ofAspergillus flavus, Aspergillus niger, Helminthosporium sp., Mucor sp. and Penicillium sp. were isolated from the textileeffluent samples collected from Elampillai Jalagandapur. Different parameters were used for optimizing. Conditions formaximum decolourization effect by the fungal isolates. The optimization conditions used to decolorize textile dye true bluewere different carbon, nitrogen, temperature and pH. Among the carbon sources, A. niger showed maximum dyedecolourization (96.75%) with maltose as a carbon source. Among the nitrogen sources used, A. flavus showed maximum dyedecolourization (97.82%) with yeast extract as a nitrogen source. Among the different temperatures used. A. niger showedmaximum dye decolourization (97.26%) with room temperature. Among the different pH used, A. niger showed maximum dyedecolourization (97.52%) with pH 6. It is clearly evident from the results that fungal isolates were able to decolourizing thetextile dye (true blue) efficiently. Dye decolourization with microorganisms is low cost effective and environmentally friendlyand the only way for ultimate controlling of pollution generated by textile and dyestuff industries. However, more and moreresearch and development works are needed to develop a viable alternative process for the treatment of textile effluent.True blue, decolorization, textile effluent, decolorizing fungiKeywordsASIAN?J.?EXP.?BIOL.?SCI. VOL 2(2)??2011270 effluent varies depending on the dyeing process, but it is generally in the range of 10-200 mg/L [7]. Many dyes andpigments are hazardous and toxic at the concentration discharged to receiving water for human as well as aquatic life.The water pollution caused by the textile mill effluent in hazardous for aquatic Eco system [8]. The high concentrationof dyes causes many water born diseases and increase BOD of the receiving water because of their complex structureand largest molecular size. Dyes present in the water on contact can causes ulceration of skin, and mucous membrane,dermatitis, perforation of nasal septum and severer irritation of respiratory track. Its injection may cause omitting,pain, haemorrhage and sharp diarrhoea [8].Dyes used in the textile industry are difficult to remove by conventional waste water treatment methods since they arestable to light and oxidizing agents and are resistant to aerobic digestion. Parameters that affect water quality aretemperature, turbitidity, pH, alkalinity, acidity, BOD, COD and color. Most of the dyes are toxic in nature and theirhigh concentration causes many water borne diseases and increases the problem. The presence of carcinogens has alsobeing reported in combined waste water of dyeing and printing units [9]. As the dyes present in waste waterdecompose very slowly in normal conditions, a treatment method to remove the dyes has to the employed [5]The solution to the environmental problems caused by the textile dye effluent is being sought by physical, chemicaland biological treatment processes. The physico-chemical methods include adsorption, chemical precipitation,flocculation, electro floatation, oxidation via chlorine, peroxide, electrolysis and ozone treatment, reduction,electrochemical destruction and ion-pair extraction [10]. Biological methods of removal involve the use of microorganism such as bacteria and fungi to turn these pollutants into non-toxic harmless substances. Biological processesconvert organic compounds completely into water and carbon dioxide, have low cost and are easy to use [11]. Variousbacteria and fungi are effective in decolorization; and in many cases, adsorption of dyes to the microbial cell surface isthe primary mechanism for decolorization [12].The microbial degradation and decolorization of dyes have received considerable attention from the viewpoint oftreating industrial wastewater containing dyes. Azo dyes are the largest class of dyes, which are not readily degradedby microorganisms. Microorganisms those are able to degrade azo dyes anaerobically, have been isolated [13].However aromatic amines produced by all these anaerobic microorganisms may be toxic and carcinogenic.Wastewater treatment facilities are often unable to completely remove commercial dyestuffs, thus contributing to thepollution of aqueous habitats.This study aims to investigate the potential of fungal cultures isolated from industrial dye effluent for decolorizationof a textile dye, True blue. Dye decolorization by fungal cultures was optimized with respect to various nutritionalsources (carbon and nitrogen), environmental parameters (temperature, pH ).All chemicals used in this experiment were ofAR grade. The dye true blue was collected from a dye industry located atJalagandapuram, Tamilnadu. Carbon and nitrogen sources used were purchased from Himedia Laboratories(Mumbai, India).The dyeing industry effluent sample was collected from a dyeing industry located at Jalagandapuram, Tamilnadu. Theeffluent temperature and other physical characteristics were examined at site and in laboratory, respectively. One mlof effluent was transferred into 9 ml of distilled water in sterile test tubes. Serial dilution was done up to 10 bythorough mixing. 0.1 ml of sample from each dilution was spread on potato dextrose agar (PDA) plates containingchloramphenicol with the help of L-rod. The petridishes were incubated at room temperature for 5 days. A plug ofmycelium of the fungal isolate was placed on a clean slide containing a drop of Lactophenol Cotton Blue (LCB)solution. The mycelium was spread using a sterile needle and a clean cover slip was placed above the preparation andobserved under the light microscope for the identification of fungal isolate.Pure fungal isolates were obtained on the PDA plates; these isolates were further sub-cultured on PDA slants andincubated at room temperature. After sufficient growth was obtained, the slants were stored in refrigerator and servedas stock cultures. Subcultures were routinely made every 30 to 60 days.A mycelium disc of 1.2 cm diameter obtained from a 4 to 5 days old culture plates of fungus were transferred to 25 mlPDAin a 250 ml conical flask and incubated at room temperature for 4 to 5 days. At the end of the incubation period 30.Isolation, screening and identification of dye degrading fungiPreservation and maintenanceSpore suspension preparationMaterials and MethodsChemicals and media-7Isolation?and?Optimization?of?Culture?Conditions?For Decolorization?Of True?Blue?Using?Dye?Decolorizing?Fungi?............................M.?Ponraj et?al.ASIAN?J.?EXP.?BIOL.?SCI. VOL 2(2)??2011271 ml sterile water was added to each culture and the flasks were shaken with shaker. Then the content of each conicalflasks were filtered through glass wool. The filtrate contained spores and were used for spore count on PDA. The samespore suspension was used in the experiments described below.Decolorization activity in terms of percent decolorization was determined by following method described by(Moorthi [14]. 10 ml of sample was centrifuged at 1000 rpm for 10 minutes. Spectrophotometer was used forabsorbance measurement. The decrease in absorbance was monitored at 450nm for true blue. Decolorization activitywas calculated according to the following formula [14].where,?D,?decolourization; A?,?initial?absorbance; A?,?final?absorbanceDecolorization of true blue textile dye (0.02g) in SD broth by all five isolates was optimized with respect to the effectof 1%, carbon sources (maltose, fructose, sucrose), 0.25%, nitrogen sources (beef extract, yeast extract, peptone), pH(4-8) and temperature (4, 27, 37?C). All experiments were carried out with 1%, (v/v) inoculum of 10?spores/ml and SDbroth without culture was served as control. All the flasks were incubated at 27?C under shaking conditions for 21days.The?data?were?analyzed?as?mean?of?triplicates??standard?deviation?(SD).Microbial decolorization of true blue has not been investigated so far. Decolorization of textile dye effluent is seriousenvironmental problem, which is evident from the magnitude of research done in this field in the last decade.Treatment of textile dye effluent by physical and chemical methods have a high cost potential and a high sludgeproblem, whereas biological process convert organic compounds completely into water and carbon dioxide, have lowcost and are easy to use [7]. In the present study microbial decolourization of textile dye effluent was carried out usingthe fungal isolates obtained from the textile dye effluent. Textile dye effluent samples were collected from the disposalsite of effluent for screening efficient microorganisms, (fungi).Isolation of fungi capable of decolorizing textiles dye effluent was carried out on SD broth from textile effluent samplecollected from textile industry. Different types of fungal isolates were obtained from the textile dye effluent sample.All fungi were identified on the basis of morphological, microscopic observations and cultural characteristics (Table -1). The solates 1, 2, 3, 4, and 5 obtained were further identified as , sp.,sp. and sp., respectively. The fungal isolates were mainly identified based on their microscopicappearance and cultural characteristics. Based on that was found to have culture character with colonies ofrapidly growing, green color, septate hyphae, conidiopore hyline, globose or elliptical vessicle, both monoseriate andbiseriate stericmata. aseptate, short conidiophores and terminal globose vesicle, doubled sterigmatacovered with vesicle and cottony growth with green or yellow color covered with black spores. sp.was of whitish-black colony, spindle shaped olivaceous brown, 3-8 septate conidia with a prominent hilum. sp.had characterstic microscopic morphology with the absence of rhizodes, branched sporangiophores, whitish graycottony growth. sp. was found to have cultural character with initially white and fluffy, later producedpigmented spores turn into green or bluish green. From the literature survey various fungus such asandare reported for decolorization of textile dye [14].Screening of decolorizing fungiDecolorization assayDye?decolorization?optimizationStatistical?analysisIsolation and identification of fungi from effluent dyeiet al.D=?[A?-A?)/A?]?x?100A. flavus A. niger, Helminthosporium MucorPenicilliumA. flavusA.niger wasHelminthosporiumMucorPeniciliumAltermariaaltermata, Chaetomium globosum, Fusarium oxysporum, Mucor mucedo, Penicillium notatum Trichodermaviride0 1 00 10 5oResults?and?DiscussionAll the isolates were selected for screening of decolorizing activity of dye. Inoculums (10 spores/ml) of each isolatewere added to 100 ml of Sabouraud dextrose (SD) broth supplemented with 10% dye effluent and incubated at 27?Cfor 21 days.After 21 days, effective decolorization was seen visually. Those isolates showing decolorization of textiledye effluent were selected for further studies with decolorization of synthetic dye true blue. Five fungal strains werefound to be potential in dye decolorization and were identified on the basis of their microscopic observations.-5oIsolation?and?Optimization?of?Culture?Conditions?For Decolorization?Of True?Blue?Using?Dye?Decolorizing?Fungi?............................M.?Ponraj et?al.ASIAN?J.?EXP.?BIOL.?SCI. VOL 2(2)??2011272 Table?1?Identification?of?fungal?strain?on?the?basis?of?microscopic?observations?and?cultural?characteristicsScreening?of?dye?decolorizationOptimization of dye decolorizationEffect of carbon sourcesEffect of nitrogen sourcesFor the maximization of decolourization of the textile dye (true blue) by the selected fungal isolates, experiments wereconducted for optimization of carbon source, nitrogen source, pHand temperature.All the fungal isolates showed higher percent decolorization than control showing that all the three sugars could beutilized effectively as carbon source by these isolates. The range of activity on decolorization of true blue withfructose was 94.76%, 88.48%, 76.01%, 90.92% and 94.02% with , sp, sp.and sp., respectively. Maximum decolorization was observed by . (Fig.1). The range of activityon decolourization of true blue with maltose was 94.40%, 76.84%, 89.58%, 71.40% and 80.34% with ,sp, sp. and sp., respectively. The range of activity on decolourization oftrue blue with sucrose was 93.11%, 91.45%, 72.44%, 92.28% and 94.74% with ,sp, sp. and sp., respectively. spp was found to be the most effective decolorizer. Fromthe study was found to be the most effective decolorizing fungi among all 5 isolates and among the threesugars used fructose was the most effective carbon source for maximum decolorization of true dye accounting 94.76% decolorization of the textile dye. Nosheen [10] used glucose and starch as carbon sources for optimizing themaximum decolorization of azo dyes Reactive Black B and Reactive Orange 16. Also Parshetti [16] usedmolasses and sucrose as carbon sources for decolorization of Malachite Green (91%) using MTCC1532.The range of % decolourization of true blue with beef extract was 94.40%, 76.84%, 89.58%, 71.40% and 80.34% with, sp, sp. and sp., respectively. was found to be themost effective decolorizer (Fig.2). The range of decolorization activity of true blue with peptone was 78.58%,94.28%, 72.14%,A. flavus A. niger, Helminthosporium MucorPenicillium A. flavusA. flavus A.niger, Helminthosporium Mucor PenicilliumA. flavus A. niger, HelminthosporiumMucor Penicillium PenicilliumA. flavuset al.et al.Kocuria roseaA. flavus A. niger, Helminthosporium Mucor Penicillium A. flavusThe obtained spore suspension showed spore count of 10 spores/ml was obtained. In SD broth, all fungal isolatesshowed high decolorization of true dye (0.02%, w/v) after 21days of incubation at 27?C under shaking. Only the rate ofdecolorization of dye and final percent color removal varied for each isolates. In the present investigation the rate ofcolor removed increased with incubation periods. This was confirmed with the earlier findings of Nehra [7],Moorthi [14] and Spadaro [15].5oet al.et al. et al.IsolateNo.Microscopic observations Cultural characteristics Organismsidentified1 Septate hyphae, conidiospores are hyline,thick walled fine to coarsely roughed vesicle,may be globose, sub globose or elliptical.sterigmata are single,both monosteriate andbisteriate sterigmataColonies are rapidly growing.Velvet in texture, yellowishgreen in colour.Aspergillusflavus2 Aseptate, short conidiophores and terminallywith globose vesicle. Sterigmata are doubledand covered with entire vesicle.Cottony growth with green oryellow colour covered withblack spores.Aspergillusniger3 Thick walled, spindle shaped olivaceousbrown, 3-8 septate conidia with a prominenthilum.The colonies are black colour atthe base and top producewhitish black colour.Helminthosporium sp.4 Do not produce rhizoids. Sporangiophoresare branched, dark brown or black.Whitish grey cottony coloniesand grow rapidly.Mucor sp.5 Hyline and septate hyphae. conidiopores arelong.they are branch and give the brush likeappearance, sterigmata are long and producechain of conidia. Conidia are spherical oroval.Initially white and fluffy, laterproduced pigmented spores turninto green or bluish green.Penicillium sp.Isolation?and?Optimization?of?Culture?Conditions?For Decolorization?Of True?Blue?Using?Dye?Decolorizing?Fungi?............................M.?Ponraj et?al.ASIAN?J.?EXP.?BIOL.?SCI. VOL 2(2)??2011273 Fig.?1.?Effect?of?nitrogen?sources?on?decolorization?of?true?blue?textile?dyeFig.?2.?Effect?of?carbon?sources?on?decolorization?of?true?blue?textile?dyeF u n g a l I s o la t e sAspergillusflavusAspergillusnigerHelminthosporiumsp.MucorspPenicilliumsp% D e c o lo r iz a ti o n02 04 06 08 01 0 01 2 0B e e f e x t r a c tY e a s t e x tr a c tP e p to n e%?DecolorizationPenicillium sp.Aspergillus flavusAspergillus nigerhelminthosporium sp.Mucur?sp.F u n g a l I s o l a te sAspergillusflavusAspergillusnigerHelminthosporiumspMucorspPenicilliumsp% D e c o lo r iz a t i o n02 04 06 08 01 0 01 2 0M a lto s eF r u c to s eS u c ro s e%?DecolorizationPenicillium sp.Aspergillus flavusAspergillus nigerhelminthosporium sp.Mucur?sp.Isolation?and?Optimization?of?Culture?Conditions?For Decolorization?Of True?Blue?Using?Dye?Decolorizing?Fungi?............................M.?Ponraj et?al.ASIAN?J.?EXP.?BIOL.?SCI. VOL 2(2)??2011274 91.22 % and 90.24% with , sp, sp. and sp., respectively. Therange of decolorization activity of true blue with yeast extract was 97.82%, 57.09%, 60.05%, 60.06% and 66.85%with , sp, sp. and sp., respectively. In few isolates, nitrogensources inhibited the decolorization efficiency which are on exact line of Zhang [17], Tatarko and Bumpus [18]and Sanghi [19]. was found to be the most effective decolourizer and yeast extract was the mosteffective nitrogen source for maximum decolorization (97.82 %) of true dye. Nosheen [20] used urea andammonium nitrate as inorganic nitrogen sources for optimizing the maximum decolorization of azo dyes ReactiveBlack B and Reactive Orange 16. Similar to this urea a nitrogen source was used to decolorize Malachite Green (91%)using MTCC 1532 [16]. Shahvali [21] studied various environmental parameters ondecolorization of textile wastewater using and found 35?C as optimum temperature formaximum decolorization.The different pH used for the dye decolorization of true blue by the five selected fungal isolates was pH 4, 6 and 8respectively. The maximum decolorization observed in pH 6, 4 and 8 is shown in (Fig. 3). The range of activity ondecolorization of true blue with pH 4 was 92.52%, 93.40%, 81.33%, 95.92% and 88.23% with ,sp, sp. and sp., respectively. The range of decolorization activity of true bluewith pH 6 was 94.71%, 97.52%, 87.12%, 84.87% and 95.58% with , sp,sp. and sp., respectively. The range of decolorization activity of true blue with pH 8 was 92.88%, 95.52%,82.64%, 86.24% and 94.00% with , sp, sp. and sp.,respectively. was found to be the most effective decolorizer at pH 6 and sp. showed maximumdecolorization at pH 4. Whereas at pH 8, was to be found as the most effective decolorizer.Among these threepH used, maximum decolorization of 97.52 % was achieved at pH 6 by Khan and Husain [22] reportedptimum pH 3 for decolorization of reactive blue 4 using immobilized polyphenol oxidase.Fig.?3.?Effect?of?pH?on?decolorization?of?true?blue?textile?dyeA. flavus A. niger, Helminthosporium Mucor PenicilliumA. flavus A. niger, Helminthosporium Mucor Penicilliumet al.et al. A. flavuset al.Kocuria rosea et al.Phanerochaete chrysosporiumA. flavus A. niger,Helminthosporium Mucor PenicilliumA. flavus A. niger, Helminthosporium MucorPenicilliumA. flavus A. niger, Helminthosporium Mucor PenicilliumA. niger MucorA. nigerA. niger.ooEffect of pHF u n g a l I s o l a te sAspergi l lusflavusAspergi l lusnigerHel mi nt hosporiumspMucorspPeni ci l l iumsp% D e c o l o r i z a t i o n02 04 06 08 01 0 01 2 0p H 4p H 6p H 8%?DecolorizationIsolation?and?Optimization?of?Culture?Conditions?For Decolorization?Of True?Blue?Using?Dye?Decolorizing?Fungi?............................M.?Ponraj et?al. Effect of temperaturesCONCLUSIONDifferent temperatures used were as refrigerator temperature (4?C), room temperature (27?C) and incubatortemperature (37?C). The maximum decolorization was observed 27?C followed by 37?C and 4?C (Fig. 4). Therange of decolorization activity of true blue with refrigerator temperature was 82.70%, 84.58%, 56.76%, 78.58% and80.89% with , sp, sp. and sp., respectively The range ofdecolorization activity of true blue with room temperature was 91.02%, 97.26%, 80.71%, 64.22% and 91.21% with, sp, sp. and sp., respectively. The range of activity ondecolorization of true blue with incubator temperature was 95.84%, 83.93%, 74.18%, 93.28% and 86.27% with, sp, sp. and sp., respectively. and sp. were foundto be the most effective decolourizer at 27 and 37?C, respectively, whereas at 4?C the maximum decolorization wasshown by . Remazole Black B was decolorized with mix culture with optimum decolorizing temperature of30?C [23].Elisangela [24] successfully decolorize four different azo dyes under microaerophilic conditions(decolourization percentage >97%) using strain VN-11. The present study is thus an effort todevelop a potential fungal isolate as an effective decolorizer of textile dye true blue. More research on thedecolorization of dye industry effluents and bioremediation of dye contaminated soil using efficient strains of fungalisolates are under progress.Fig.?4.?Effect?of?temperatures?on?decolorization?of?true?blue?textile?dyeThe true blue dye is degradable under aerobic conditions with a concerted effort of fungi isolated from textile dyeeffluent. Nutrients (carbon and nitrogen sources) and physical parameters (pH and temperature) had significant effecton dye decolorization. showed highest decolorization of true blue effectively during optimization butpredominantly showed consistent decolorization of true blue dye.Mr. B.T. Sureshkumar Dr. S. Murugesh,Dr. M. Karunanithi, Chairman Secretaryo oo o o oo ooA. flavus A. niger, Helminthosporium Mucor Penicillium .A.flavus A. niger, Helminthosporium Mucor PenicilliumA.flavus A. niger, Helminthosporium Mucor Penicillium A. niger MucorA.nigeret al. dStaphylococcus arlettaeA. flavusA. nigerACKNOWLEDGEMENTAuthors are very much thankful to , Head of the Department, Microbiology, Principal,and and who gave an ideal opportunity and facilities to do this work.atF u n g al Iso latesAspergillusflavusAspergillusnigerHelminthosporiumspMucorspPenicilliumsp% D e c o lo riz a tio n02 04 06 08 01 0 01 2 04oC2 7oC3 7oC%?DecolorizationIsolation?and?Optimization?of?Culture?Conditions?For Decolorization?Of True?Blue?Using?Dye?Decolorizing?Fungi?............................M.?Ponraj et?al. REFERENCES[1]. Nigam, P., Banat, IM, Singh, D. & Marchant, R. (1996). Microbial process for the decolourization of textile effluent containing azo, diazoand reactive dyes. 31: 435-442.[2]. Nupur, M. & Pradeep, B. (2007). Mutagenicity assessment of textile dyes from Sanganer. 28 123-126.[3]. Khadijah, O., Lee, KK., Faiz, M. & Abdullah, F. (2009). Isolation, screening and development of local bacterial consortia with azo dyesdecolourising capability. 5 25-32.[4]. Olukanni, OD., Osuntoki, AA. & Gbenle, GO. (2009). Decolourization of azo dyes by a strain of isolated from a refuse dumpsoil. 8: 442-448.[5]. Verma, V?K. & Mishra,A?K. (2005). Dye removal by sawdusd waste adsorbent. 24 97-99.[6]. Sharma, J?K. &Arora, M?K. (2001). Environmental friendly processing for textile. ., 20: 447-451.[7]. Nehra, K. Meenakshi. A. & Malik. K. (2008). Isolation and optimization of conditions for maximum decolourization by textile dyedecolourizing bacteria. 27 257-264.[8]. Deshmukh, S?K. & Rathod A?P. (2008). Adsorption of dyes from waste water using coconut shell as bio-adsorbent. 27 569-573.[9]. Mathur, N., Bhatnagar, P. & Bakre, P. (2005). Assessing mutagenesity of textile dyes from Pali (Rajasthan) using ames bioassay.4 111-118.[10]. Nosheen, S?, Nawaz, H., & Rehman, K?U. (2000). Physico-chemical characterization of effluents of local textile industries ofFaisalabadPakistan. 2 232-233.[11]. Ali, H., Ahmad, W. & Taqweemul, H. (2009). Decolorization and degradation of malachite green by and. 8 1574-1576.[12]. Selvam, K., Swaminathan, K. & Chae, KS. (2003). Microbial decolorization of azo dyes and dye industry effluent by .19 591-593.[13]. Growther, L. & Meenakshi, M. (2009). Biotechnological approaches to combat textile effluents. 7 1-7.[14]. Moorthi, SP, Selvam, PS, Sasikalaveni, Murugesan AK. & Kalaichelvan, PT. (2007). Decolorization of textile dyes and their effluentsusing white rot fungi. 6 424-429.[15]. Spadaro, J?T., Michael, H?G. & Renganathan, V. (1992). Degradation of azo dyes by the lignin degrading fungus. ., 58: 2397-2401.[16]. Parshetti, G?, Kalme, S, Saratale, G. & Govindwar, S. (2006). Biodegradation of Malachite Green by MTCC 1532., 53: 492498.[17]. Zhang, F., Knapp J?S. & Tapley, K?N. (1999). Development of bioreactor systems for decolourization of orange II using white rot fungus.., 24: 4853.[18]. Tatarko, M. & Bumpus, J?A. (1998). Biodegradation of Congo Red by Phanerochaete chrysosporium. ., 32: 17131717.[19]. Sanghi, R., Dixit, A. & Guha, S. (2006). Sequential batch culture studies for the decolorization of reactive dye by .., 97: 396400.[20]. Nosheen, S., Nawaz, R., Arshad, M. & Jamil, A. (2010). Accelerated biodecolorization of reactive dyes with added nitrogen and carbonsources. , 12: 426430.[21]. Shahvali, M., Assadi, M.M. & Rostami, K. (2000). Effect of environmental parameters on decolorization of textile wastewater using. 23 721-726.[22]. Khan, A?A, & Husain, Q. (2007). Decolorization and removal of textile and non-textile dyes from polluted wastewater and dyeing effluentby using potato (Solanum tuberosum) soluble and immobilized polyphenol oxidase. 98 1012-1019.[23]. Leena, R. & Raj, S?D. (2008). Bio-decolorization of textile effluent containing Reactive Black-B by effluent-adapted and non-adaptedbacteria. 7 3309-3313.[24]. Elisangela, F., Andrea, Z, Fabio, D?J., Cristiano, R. de M., Regina, D?L. & Artur, CP. (2009). Biodegradation of textile azo dyes by afacultative strain VN-11 using a sequential microaerophilic/aerobic process. 63:280288.Process Biochem.,J. Environ. Biol.,Mal. J. Microbiol.,MicrococcusBiotechnol.,. . Poll. Res.,. . Poll. ResPoll Res.. . Poll ResAppl.Ecol. Environ.,. .Int. J. Agri. Biol.,Aspergillus flavus Alternariasolani Afr. J. Biotechnol.,Fomes lividus WorldJ. Microbiol. Biotechnol.,The Internet J. Microbiol.,Afr. J. Biotechnol.,. . Phanerochaetechryosporium Appl. Enviro. Microbial. Kocuria rosea ActaChim. Slov., . .Enzyme Microb. Technol. Water ResCoriolus versicolorBioresour. TechnolInt. J. Agric. Biol.Phanerochaete chrysosporium Bioprocess Biosyst. Eng.,.Bioresour. Technol.,Afr. J. Biotechnol.,. .Staphylococcus arlettae Int. Biodeterior. Biodeg.,(1):(1):(1):(2):(3):(1):(3):(8):(6):(1):(4):(6):(5):.(18):.Corresponding Author: Vasudeo?Zambare ,?South?Dakota?School?of?Mines?and Technology,?Rapid?city?,SD?57701,USAEmail?:?vasuzambare@gmail.comIsolation?and?Optimization?of?Culture?Conditions?For Decolorization?Of True?Blue?Using?Dye?Decolorizing?Fungi?............................M.?Ponraj et?al. Citations (13) References (23) ... Under these conditions, glucose oxidase catalyses the oxidation of glucose and provide required substrate (H 2 O 2 ) for peroxidases ( Lan et al., 2006). A previous study reported that fungal dye decolorization properties are connected with the type of substrate (inorganic, organic) and the dose of nitrogen ( Abedin, 2008;Ponraj et al., 2011;Shedbalkar et al ... ... SQ01 decolorized Remazol Brilliant Blue R solution the fastest at pH 3.0 and 4.5 ( Sarnthima, 2009;Yang, 2009). In turn, microscopic fungi effectively decolorized textile dyes, when pH of the starting dye solution was close to neutral ( Abedin, 2008;Ponraj et al., 2011;Shedbalkar et al., 2008). Ponraj et al. (2011 showed that fungi of the genus Aspergillus sp. and Penicillium sp. ... ... It was shown that Lentinus polychrous and Trametes. sp. SQ01 decolorized Remazol Brilliant Blue R solution the fastest at pH 3.0 and 4.5 ( Sarnthima, 2009;Yang, 2009). In turn, microscopic fungi effectively decolorized textile dyes, when pH of the starting dye solution was close to neutral ( Abedin, 2008;Ponraj et al., 2011;Shedbalkar et al., 2008). Ponraj et al. (2011 showed that fungi of the genus Aspergillus sp. and Penicillium sp. exhibited the most effective decolorization of the dye (azo-dye) at pH 6.0. Other authors ( Abedin, 2008;Shedbalkar et al., 2008) determined that the MTCC 517 strain of Penicillium ochrochloron and Fusarium solani required pH = 6.5 and 7.0, respectively, for the maximum ... Comparative study of eco- and cytotoxicity during biotransformation of anthraquinone dye Alizarin Blue Black B in optimized cultures of microscopic fungi Article Full-text available Jan 2018ECOTOX ENVIRON SAFE Kamila Rybczy?ska-Tkaczyk Agata ?wi?ci?o Konrad Andrzej SzychowskiTeresa-Korni??owicz-Kowalska View Show abstract ... Reverse multistage coagulation followed by ozonation was shown to be superior to their sequential application of single pass. The advantages of this application in several steps was more convincing if the wastewater is with recalcitrant composition [116][117][118][119][120]. ... Industrial Wastewater Treatment: A Challenging Task in the Industrial Waste Management Article Jan 2017 Shah MP View Show abstract ... Textile effluent is generated through printing, dying, bleaching, sourcing and mercerizing (Ponraj et al., 2011;Gupta et al., 2003). Dye concentration in effluent varies between 10-200mg/ml, depending on the dye processing and it is estimated that, around 10-15% of dyes are lost in effluent during the dye process ( Kumar et al., 2006). ... Biodegradation and Decolorization of Azo Dye (Deep Red Glx) by Alkaliphilic Bacilluscereus Strain BPL Isolated from Textile Effluent Contaminant Soil Article Full-text available Aug 2016 Indrani Jadhav Kapilesh JadhavRoshan Vasniwal Divya Shrivastava View ... There has been an enormous increase in the volume of wastewater generation due to industrialization, leading to a decline in the quality and availability of natural water in the environment. It is estimated that about 100 L of water is required to process 1 kg of textile material [1] while the textile industry consumes about two-third of the more than 7 10 5 tonnes of a wide variety of synthetic dyes produced annually [2][3][4]. Azo dyes are the largest and most versatile class of dyes. With more than 3000 different ones currently used for different dyeing purposes, they constitute 60-70% of dyes use in textile processing. ... Kinetics of the decolourization of a dyehouse effluent by Providencia rettgeri ODO Article Aug 2013Int J Environ Stud Akinniyi Osuntoki Ayodeji Awotula Olumide Olukanni View Show abstract ... Aspergillus sp. and Penicillium sp. from wastes of potato and its epidermis showed ability to decolourize Isolan Red and coloured waste water by exhibiting oxidase activity [13]. A. flavus and A. niger effectively decolourizes the azo dyes when compared to the other fungal genus viz., Mucor and Penicillium sp. Aspergillus niger showed decolourization activity, with a range of 89 to 97% after 15 days of incubation was reported in similar study by Ponraj et al.,2011 [14]. In this study, the effectiveness of colour removal was shown to increase with increase in incubation time and this was subsequently found to be in agreement with the study documented by Spadaro et al., 1992 [15], where maximum decolourization was obtained after 15 days of incubation. A. niger showed to possess potential decolourization capacity of Orange 2R. ... ECO-FRIENDLY COLOUR REMOVAL POTENTIAL OF FUNGAL SPECIES ISOLATED FROM VEGETABLE AND FRUIT WASTES Article Full-text available Dec 2014 Palani Velan. RP.M. Ayyasamya Subashni BhoopathySuresh S S Raja View Show abstract ... The decolorisation efficacy was highly reduced when the pH was raised to pH 7 0.2 (62%). It has already been reported that dye decolorization of true blue by A. flavus (94.71%) was best at the pH 6 [14] . The same trend was observed in the case of degradation of a reactive dye, Orange M2R by A.flavus that yielded a maximum value of 83.36% at pH 5and its activity got reduced when the pH was raised to 7 [15] . ... DEGRADATION OF TRIPHENYLMETHANE DYE: MALACHITE GREEN BY ASPERGILLUS FLAVUS W WO OR RL LD D J JO OU UR RN NA AL L O OF F P PH HA AR RM MA AC CY Y A AN ND D P PH HA AR RM MA AC CE EU UT TI IC CA AL L S SC CI IE EN NC CE ES S *Correspondence for Author Article Full-text available Jul 2014 Janakiraman Subramanian Ramesh ThangaveluMurugaiyan KalaiselvamTamilnadu India View Show abstract Comparative study of eco- and cytotoxicity during biotransformation of anthraquinone dye Alizarin Blue Black B in optimized cultures of microscopic fungi Article Sep 2017 Kamila Rybczy?ska-Tkaczyk Agata ?wi?ci?o Konrad Andrzej SzychowskiTeresa Korni??owicz-Kowalska View Show abstract Biotransformation and ecotoxicity evaluation of alkali lignin in optimized cultures of microscopic fungi Article Full-text available Feb 2017INT BIODETER BIODEGR Kamila Rybczy?ska-TkaczykTeresa Korni??owicz-Kowalska View Show abstract Microbe-Mediated Degradation of Synthetic Dyes in Wastewater Article Oct 2015 Maulin P Shah View Show abstract Mycoremediation of bismarck brown y by indigenous fungal isolate alternaria brassicae tsf -07 and optimization of cultural conditions to enhance its decolourization Article Jan 2013IJPBS K.P. ShindeP.R. Thorat View Show abstract Show more

    Gadge PP, Madhikar SD, Yewle JN, Jadhav UU, Chougale AD, Zambare VP, Padul MV (2011) Biochemical Studies of Lipase from Germinating Oil Seeds (Glycine max), American Journal of Biochemistry and Biotechnology 7(3): 141-145

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    Problem statement: Lipase is one of the important enzymes in food, pharmaceutical, detergent and biofuels industries. Search for the lipase with distinct features, possibly from germinating seeds, is of interest for industrial applications. Approach: The lipase produced by soybean oil seeds was partially purified and characterized in terms of the optimal pH and temperature for activity as well as substrate specificity. Results: The lipase was extracted and partially purified from germinating soybean seeds using chilled acetone and ammonium sulfate precipitation. Partially purified and dialyzed enzyme profile was observed on native-Polyacrylamide Gel Electrophoresis (PAGE). The lipase was optimally active at pH 8 and temperature of 24C. In the presence of Ca2+ and Mg2+ enhance the activity at low concentration, while the Hg2+ and Ethylene Diaminotetracetic Acid (EDTA) showed inhibitory effect. The enzyme was found to be metalloenzyme. Enzyme kinetics with olive oil emulsion substrate showed km and vmax of 7.67 mg and 0.0125 m mL min-1, respectively. Conclusion: The mettaloenzyme enzyme was able to attack specifically on oil in seeds to generate free fatty acids as the major end product. This understanding may help in devising efficient methods to overcome the problem of soybean seed oil in stability.

    Madhikar SD, Gadge PP, Yewle JN, Jadhav UU, Chougale AD, Zambare VP, Padul MV (2011) Isolation, partial purification and characterization of lipase from sunflower germinating oil seeds , International Journal of Biotechnology and Bioscience 1(4): 410-415
    Vasudeo Zambare, Archana Zambare, Lew Christopher (2011) Enzymatic hydrolysis of hemicellulose from corn stover and prairie cordgrass, Advances in Biotech Research, Microbiologist Society and Education Book Publisher & Distributor
    Singhania R.R, Sukumaran R.K, Rajasree K.P, Mathew A, Gottumukkala L.D, Pandey A (2011) Properties of a major ?-glucosidase-BGL1 from Aspergillus niger NII-08121 expressed differentially in response to carbon sources, Process Biochemistry 46(7): 1521-1524

    Aspergillus niger NII-08121/MTCC 7956 exhibited differences in expression of ?-glucosidase (BGL) in response to carbon sources provided in the medium. Activity staining with methyl umbelliferyl ?-d-glucopyranoside (MUG) indicated that four different isoforms of BGL were expressed when A. niger was grown under submerged fermentation with either lactose or cellulose, whereas only two were expressed when wheat bran or rice straw was used as the carbon source. Among the four isoforms of BGL expressed during lactose supplementation, two were found to retain 92% and 82% activity respectively in presence of 250 mM glucose in the MUG assay. The major ?-glucosidase (BGL1) was purified to homogeneity by electro elution from a Native PAGE gel. The purified 120 kDa protein was active at 50 C and was stable for 48 h without any loss of activity. The optimum pH and temperature were 4.8 and 70 C respectively.

    V. P. Zambare (2010) Solid state fermentation production of cellulase from Bacillus sp, International Journal of BioScience, Agriculture and Technology 2(1): 1-6

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    Bacillus sp. was cultured in solid-state fermentation (SSF) of wheat straw to produce cellulase. The fermented biomass was harvested after 36 h of SSF at pH 8 and temperature 400C. It was filtered and centrifuged at 10,000 rpm at 4 0C and supernatant was collected as crude enzyme extract. Maximum activity of cellulase (3.7750.13U/ml) was obtained after fermentation of wheat straw (10g) medium containing 0.2g soybean meal, 0.04g corn steep liquor (CSL), 80% moisture content (mineral salt medium, pH 8), 2-mL inoculum, and temperature 40 0C. SSF was found to be more productive than submerged fermentation (SmF) in terms of cellulase yields. The partial purification of cellulase was carried out through (NH4)2SO4 precipitation. The partially purified enzyme produced under SSF had molecular weight of 35 and 45kDa. It was active in a broad pH (4-10) and temperature range (25-550C). The optimum, pH and temperature of Bacillus cellulase were pH 5 and 450C, respectively. At 500C and 600C, the half lives of the partially purified cellulase were 194 and 163 min, respectively. All the results indicated that the Bacillus sp. had a promising application of treatment of agro-wastes and cellulase from Bacillus sp. could be potentially used in biofuel industries.

    Vasudeo Zambare (2010) Solid state fermentation of Aspergillus oryzae for glucoamylase production on agro residues, International Journal of Life Sciences 4: 16-25

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    Glucoamylase is a well recognized amylolytic enzyme used in food industry, which is generally produced by Aspergillus genus under solid-state fermentation (SSF). This study presents production of glucoamylase by Aspergillus oryzae on the solid surface of rice husk, wheat bran, rice bran, cotton seed powder, corn steep solids, bagasse powder, coconut oil cake, and groundnut oil cake as substrates. Optimization of the SSF media and parameters resulted in a 24% increase in the glucoamylase activity. Optimum glucoamylase production (1986 moles of glucose produced per minute per gram of dry fermented substrate) was observed on wheat bran supplemented with 1%, (w/w) starch, 0.25%, (w/w) urea at pH 6, 100%, (v/w) initial moisture and 300C after incubation 120 hrs. Therefore, A. oryzae can be useful in bioprocessing application for saccharification of agro-residues.

    Vasudeo Zambare (2010) Strain improvement of alkaline protease from Trichoderma reesei MTCC-3929 by physical and chemical mutagen, The IIOAB Journal 1(1): 25-28

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    The purpose of the present investigation is to enhance alkaline protease production by subjecting indigenous protease producing strain Trichoderma reesei MTCC-3929 to improvement by random mutagenesis by ultra-violet (UV) irradiation and N-Methyl-N-nitro-N-nitroso guanidine (NTG) treatment. Mutants were screened as protease producers on the basis of zone of clearance on skimmed milk agar plates. UV-8 mutant showed 9 mm clear zone diameter and activities of 199.6 and 552.6 U/ml for submerged fermentation (Smf) and solid state fermentation (SSF), respectively. UV-8 further mutated by NTG to produced NTG-17 mutant with zone of clearance 13mm diameter. Compared to wild strain, NTG-17 mutant was found to produce 2.6 and 2.2-fold more activities in SmF and SSF, respectively. Thus these findings have more impact on enzyme economy for biotechnological applications of microbial proteases.

    V. P. Zambare (2010) Optimization of nutritional parameters for extracellular protease production from Bacillus sp. using response surface resistance, International Journal of BioEngineering and Technology 1(1): 43-47

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    The optimization of nutritional parameters and concentrations for the protease production by Bacillus sp. in submerged fermentation was carried out using response surface methodology (RSM) based on the central composite design (CCD). The design contains a total of 20 experimental trials containing starch, soybean meal and CaCO3 as model factors. The mutual interaction between these variables resulted into 1.48 fold increase in protease activity as compared to the mean observed response at zero level of all variables.

    V. P. Zambare, Lew P. Christopher (2010) Solid state fermentation and characterization of a cellulase enzyme system from Aspergillus niger SB-2, International Journal of Biological Sciences and Technology 2(3): 22-29

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    The focus of this study was on the solid state fermentation (SSF) of cellulase enzymes produced by Aspergillus niger SB-2 utilizing lignocellulosic agricultural waste as carbon and energy source. Optimization of the SSF media and parameters resulted in a 32% increase in the cellulase activity. Maximum enzyme production of 1,3257.1 IU/g dry fermented substrate was observed on wheat bran and rice bran supplemented with malt dextrin and soybean meal at pH 6 and 300C after incubation for 120 h. The cellulase activities presented here appear to be among the highest reported in literature for A. niger to date. The A. niger SB-2 cellulase was partially purified and characterized. Zymogram analysis of the sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed two bands of cellulase activity with molecular weights of 30 and 45 kDa. To the best of our knowledge, a 45 kDa cellulase from A. niger has not been previously described in literature. The enzyme was active in a broad pH (4-7) and temperature (30-550C) range with a pH optimum of 6 and a temperature optimum of 450C. At 50 and 600C, the cellulase half life was 12.4 and 4.1 h, respectively. Dithiothreitol, iodoacetamide and Mg+2 acted as activators of cellulase activity. Kinetics studies indicated that the substrate specificity of A. niger SB-2 cellulase was 18% higher on insoluble cellulose than on soluble cellulose. Therefore, the cellulase complex of A. niger SB-2 would be useful in bioprocessing applications where efficient saccharification of lignocellulosic biomass is required.

    Vasudeo Zambare, Smita Nilegaonkar, Pradnya Kanekar (2010) Application of protease from Bacillus cereus MCM B-326 as a bating agent in leather processing, The IIOAB Journal 1(3): 18-21

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    Laboratory scale experiments were carried out to test the efficiency of the extracellular protease from Bacillus cereus MCM B-326; cattle dung and commercial bate powder (ComBate) as bating agents on delimed buffalo hide. Protease treated pelt was free from scud and pigments, clean and fine grain, white, smooth and silkier with loosen fat. Histological sections of bated pelts showed greater opening up of collagen fibers with Bacillus protease. The studies indicated potential importance of Bacillus protease as effective bating agent in leather processing.

    Vasudeo Zambare (2010) Purification and characterization of neutral serine protease from Bacillus sp., Asiatic Journal of Biotechnology Resources 3: 183-192

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    A neutral protease was purified from Bacillus sp. to electrophoretic homogeneity by using ammonium sulphate precipitation and 2-step-column chromatography. The purified protease expressed its maximum activity 40 o C and pH value of 7. It was stable up to 10-40 o C for 30 min of incubation and retained 80 and 65% of its activity at 50 and 60 o C, respectively. Ions of Ca and Na and showed a stimulatory effect and ions of K and Fe had no effect, ions of Mg, Cu, Zn and Mn showed an inhibitory effect. Moreover, ions of Hg showed strong inhibitory effect on the purified protease activity. Neutral protease was found to be a serine protease and confirmed by enzyme inhibition using phenylmethylsulfonylfluoride (PMSF). The enzyme has high affinity towards casein followed by Bovine serum albumin (BSA) and gelatin. Molecular weight of the purified NP was found to be 35kDa on SDS-PAGE.

    Parameswaran, B, Raveendran S, Singhania, R.R, Surender V, L Devi, Nagalakshmi S, Kurien N, Sukumaran R.K, Pandey A. (2010) Bioethanol production from rice straw: an overview, Bioresource technology 101(13): 4767-4774

    Rice straw is an attractive lignocellulosic material for bioethanol production since it is one of the most abundant renewable resources. It has several characteristics, such as high cellulose and hemicelluloses content that can be readily hydrolyzed into fermentable sugars. But there occur several challenges and limitations in the process of converting rice straw to ethanol. The presence of high ash and silica content in rice straw makes it an inferior feedstock for ethanol production. One of the major challenges in developing technology for bioethanol production from rice straw is selection of an appropriate pretreatment technique. The choice of pretreatment methods plays an important role to increase the efficiency of enzymatic saccharification thereby making the whole process economically viable. The present review discusses the available technologies for bioethanol production using rice straw.

    Aswathy U.S, Sukumaran R.K, Devi G.L, Rajasree K.P, Singhania R.R. (2010) Bio-ethanol from water hyacinth biomass: an evaluation of enzymatic saccharification strategy, Bioresource technology 101(3): 925-930

    Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and ?-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common bakers yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.

    Rasika Pawar, Vasudeo Zambare, Siddhivinayak Barve, Govind Paratkar (2009) Application of protease isolated from Bacillus sp.-158 in enzymatic cleaning of contact lenses, Biotechnology 8(2): 276-280

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    A neutral protease, isolated from Bacillus sp. 158 was used for removing protein deposits from contact lenses. Partial purification of the protease was carried out using ammonium sulphate and factors affecting the enzyme activity, such as assay temperature and assay pH were characterized. The optimum pH and temperature for protease were found to be pH 7.0 and 30C, respectively. The partially purified protease was stable at temperature range of 30-40C and pH 6-7. However, protease was maximum stable at 30C and pH 7.0. The enzyme could be effectively used to remove protein deposit from contact lenses indicating its potential to increase in transmittance of lenses.

    Zambare V. P. , M. V. Padul, Yadav A. A, Shete T. B. (2008) Vermiwash: biochemical and microbiological approach as eco-friendly soil conditioner, ARPN Journal of Agricultural and Biological Sciences 3(4): 1-5

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    Vermiwash was found to contain enzyme cocktail of proteases, amylases, urease and phosphatase. Microbiological study of vermiwash revealed that it contains nitrogen-fixing bacteria like Azotobactrer sp., Agrobacterium sp. and Rhizobium sp. and some phosphate solublizing bacteria. Laboratory scale trial showed effectiveness of vermiwash on Cowpea plant growth

    Vasudeo Zambare (2008) Biotechnological applications of proteases in leather processing: a green technology, Proceedings of National Conference on Green Technology (NCGT-2008), Government Polytechnic
    S. S. Nilegaonkar, V. P. Zambare, P. P. Kanekar, P. K. Dhakephalkar and S. S. Sarnaik (2007) Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326, Bioresource Technology 98(6): 1238-1245

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    Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87+/-1.32 U ml(-1)) in starch soybean meal medium of pH 9.0, at 30 degrees C, under shake culture condition, with 2.8 x 10(8) cells ml(-1) as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25-65 degrees C and pH 6-12, with maximum activity at 55 degrees C and pH 9.0. The enzyme required Ca(2+) ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry

    V. P. ZambareS. S. NilegaonkarP. P. Kanekar (2007) Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent, World Journal of Microbiology and Biotechnology 23(11): 15691574

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    The present investigation describes microbial production of an alkaline protease and its use in dehairing of buffalo hide. Bacillus cereus produced extracellular protease when grown on a medium containing starch, wheat bran and soya flour (SWS). The ammonium sulphate precipitated (ASP) enzyme was applied for dehairing of buffalo hide. Microscopic observation of longitudinal section of buffalo hide revealed that the epidermis was completely removed and hair was uprooted leaving empty follicles in the hide. The ASP enzyme was stable for one month at ambient temperature between 2535 C. Enzymatic dehairing may be a promising shift towards an environment-friendly leather processing method.

    S. S. Nilegaonkar, V. P. Zambare, P. P. Kanekar, P. K. Dhakephalkar, S. S. Sarnaik, N. K. Chandrababu, Rama Rajaram, B. Ramanaiah, T. Ramasami, Y. K. Saikumari and P. Balaram (2007) A novel protease for industrial application, German Patent Patent NO. 102007013950.2

    The present invention relates to an extracellular enzyme protease obtained by growing the culture of Pseudomonas aeruginosa MCM B-327 isolated from vermiculture pit soil and deposited in MTCC, IMTECH, Chandigarh with designation MTCC 5270, in production medium of pH 7.0; containing soybean meal and tryptone as raw materials, at 30 C. for 72 h. The organism was also able to produce protease using different agricultural products/byproducts as protein sources. The partially purified non-collagenolytic, calcium independent protease with molecular weight 60 kDa has activity in pH range of 6.0-11.0 and temperature range of 25-65 C.; stability in pH range of 6.0-10.0 and temperature 25-45 C. The protease activity was retained for 8 months when stored at ambient temperature. Ammonium sulphate precipitated enzyme was able to completely dehair animal skins and hides without chemicals like lime, sodium sulphide and calcium.

    S. S. Nilegaonkar, V. P. Zambare, P. P. Kanekar, P. K. Dhakephalkar, S. S. Sarnaik, N. K. Chandrababu, Rama Rajaram, B. Ramanaiah, T. Ramasami, Y. K. Saikumari and P. Balaram (2007) A novel protease for industrial application, US Patent Patent No US20080220499A1

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    The present invention relates to an extracellular enzyme protease obtained by growing the culture of Pseudomonas aeruginosa MCM B-327 isolated from vermiculture pit soil and deposited in MTCC, IMTECH, Chandigarh with designation MTCC 5270, in production medium of pH 7.0; containing soybean meal and tryptone as raw materials, at 30 C. for 72 h. The organism was also able to produce protease using different agricultural products/byproducts as protein sources. The partially purified non-collagenolytic, calcium independent protease with molecular weight 60 kDa has activity in pH range of 6.0-11.0 and temperature range of 25-65 C.; stability in pH range of 6.0-10.0 and temperature 25-45 C. The protease activity was retained for 8 months when stored at ambient temperature. Ammonium sulphate precipitated enzyme was able to completely dehair animal skins and hides without chemicals like lime, sodium sulphide and calcium.

    S. S. Nilegaonkar, V. P. Zambare, P. P. Kanekar, P. K. Dhakephalkar, S. S. Sarnaik, N. K. Chandrababu, Rama Rajaram, B. Ramanaiah, T. Ramasami, Y. K. Saikumari and P. Balaram (2006) A novel protease for industrial application, Indian Patent Patent NO. 2471DEL2006

    The present invention relates to an extracellular enzyme protease obtained by growing the culture of Pseudomonas aeruginosa MCM B-327 isolated from vermiculture pit soil and deposited in MTCC, IMTECH, Chandigarh with designation MTCC 5270, in production medium of pH 7.0; containing soybean meal and tryptone as raw materials, at 30 C. for 72 h. The organism was also able to produce protease using different agricultural products/byproducts as protein sources. The partially purified non-collagenolytic, calcium independent protease with molecular weight 60 kDa has activity in pH range of 6.0-11.0 and temperature range of 25-65 C.; stability in pH range of 6.0-10.0 and temperature 25-45 C. The protease activity was retained for 8 months when stored at ambient temperature. Ammonium sulphate precipitated enzyme was able to completely dehair animal skins and hides without chemicals like lime, sodium sulphide and calcium.

    S. S. Nilegaonkar, V. P. Zambare and P. P. Kanekar (2004) Extracellular protease from Bacillus sp. BSA-26: application in dehairing of buffalo hide, Biotechnological Approaches for Sustainable Development, Allied Publishing Pvt. Ltd.

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