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Hayes, D. J. M. (2013) Report on Optimal Use of DIBANET Feedstocks and Technologies, DIBANET WP5 Report84 pages

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The DIBANET process chain, as a result of its patented pre-treatment stage, has significantly increased the yields of levulinic acid, formic acid, and furfural beyond what was considered to be the state of the art. By fractionating lignocellulosic biomass into its three main polymers (cellulose, hemicellulose, lignin) it has also allowed for lignin to be recovered and sold as a higher-value product. These developments have meant that the amount of acid hydrolysis residues (AHRs) that have been produced are significantly (up to 88%) less than in the Biofine process. These AHRs are required to provide process heat for DIBANET. Direct combustion is the most efficient means for doing this. If such combustion does not occur and the AHRs are instead used in other processes, e.g. pyrolysis and gasification, then more biomass will need to be purchased to fuel the core DIBANET process. The AHRs have not been proven to be superior to virgin biomass when put through these thermochemical processes. Indeed, many of the results from DIBANET Work Package 4 indicate the opposite. Hence, given that DIBANET, and the modelling of its optimal configuration, is designed on the basis of an integrated process, centred on the core element of the acid hydrolysis of biomass, then combustion is the only viable end use for the AHRs. Given that realisation, the focus of this modelling Deliverable is on what the optimal configuration of the process chain would be regarding the three core stages (pretreatment, hydrolysis, and the esterification of levulinic acid with ethanol). It has been demonstrated that a scenario incorporating only the first stage can be profitable in its own right and allow for commercial development at much lower capital costs. In this instance bagasse is a much more attractive feedstock, compared with Miscanthus, due to its higher pentose content.

Integrating the second stage increases capital costs but improves the net present value. The esterification step is somewhat capital intensive but an integrated DIBANET biorefinery that incorporates all three stages can still be highly profitable providing the furfural is sold at its current market price and the lignin is sold rather than used as a fuel for process needs. Indeed, the DIBANET process should not be considered only in the context of biofuels but as a true biorefinery that produces lower value fuels (e.g. ethyl-levulinate) in addition to high value chemicals and bio-products (e.g. furfural and lignin).

The energy and carbon balances of the various DIBANET scenarios have been investigated and are highly positive with values significantly superior to those for the energy-intensive Biofine process. A socioeconomic survey has also been carried out and has shown that there can be a positive effect on employment, both direct and indirect, particularly when Miscanthus is used as the feedstock. The DIBANET integrated process also holds up well when its environmental and social performances are ranked for a range of important parameters.

The development of the core DIBANET IP towards commercial deployment appears to be warranted, based on data provided from the models developed. Indeed, these models present possible scenarios whereby even demonstration-scale DIBANET facilities could operate at significant profits and provide healthy returns on the capital invested.

Hayes, D. J. M. (2012) Review of Biomass Feedstocks and Guidelines of Best Practice, DIBANET WP2 Report150 pages

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This document is the result of the evaluation of biomass feedstocks, from Europe and Latin America, that took place as part of the DIBANET project. That project is co-financed from the 7 th Framework Programme for Research and Technological Demonstration of the European Union. (Title: Enhancing international cooperation between the EU and Latin America in the field of biofuels; Grant Agreement No: 227248-2).

The work in Task 2.1 of Work Package 2 (WP2) at DIBANET partners UL, CTC, and UNICAMP involved evaluating, on a number of levels, potential feedstocks for utilisation in the DIBANET acid-hydrolysis process (WP3). In the early stage of the project a wide number of feedstocks were examined and relevant secondary compositional data were sought from the literature. Selected feedstocks were analysed at the laboratories of UL, CTC, and UNICAMP and, from these, a limited number of feedstocks were subjected to more in-depth analysis/evaluation.

Work at UL focused on Miscanthus, cereal straws, and waste papers. The wet-chemical and spectroscopic analysis that was carried out on a wide number of Miscanthus samples have allowed for in-depth understandings to be reached regarding the changes in lignocellulosic composition, and potential biomass/biofuel yields that could be realised over the harvest window. Straws present much less chemical variation but have enough structural carbohydrates to warrant their processing in the DIBANET technology. Waste papers can have amongst the highest total carbohydrate contents of any of the feedstocks studied.

Work at CTC focused on the residues of the sugarcane industry - sugarcane bagasse and sugarcane trash (field residues from harvesting). A large number of samples were collected from a variety of sugar mills and plantations. It has been seen that there can be a significant variation in the composition of different bagasse samples, particularly with regards to the ash content. Sugarcane trash has lower total carbohydrates contents than bagasse but is still a suitable feedstock for DIBANET.

Work at UNICAMP focused on the evaluation of residues from the banana, coffee, and coconut industries. It was found that these also have potential for utilisation in the DIBANET process, however the value of the residues for this end-use is dependent on which part of the plant is utilised. For instance, coffee husks have sufficient structural carbohydrates to allow for decent yields of levulinic acid, formic acid, and furfural in DIBANET, however the leaves of the coffee plant do not. Leaves from the banana plant are also of less value for DIBANET than the other parts of the plant (e.g. stem).

A major output of this Deliverable is the downloadable electronic database that contains all of the WP2 analytical data obtained during the course of the project. It contains analytical data and predicted biorefining yields for a total of 1,281 samples. It can be obtained, free of charge, from the DIBANET website and will be a valuable tool for stakeholders in biorefining projects.

This document presents the data and evaluations that were made regarding biomass feedstocks, and also puts forward guidelines of best practice in terms of making the best use of these resources. A shortened version of this document can also be downloaded from the DIBANET website.

Hayes, D. J. M. (2011) Analysis of Lignocellulosic Feedstocks for Biorefineries with a Focus on The Development of Near Infrared Spectroscopy as a Primary Analytical Tool, PhD Thesis832 pages (over 2 volumes)

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The processing of lignocellulosic materials in modern biorefineries will allow for the production of transport fuels and platform chemicals that could replace petroleum-derived products. However, there is a critical lack of relevant detailed compositional information regarding feedstocks relevant to Ireland and Irish conditions. This research has involved the collection, preparation, and the analysis, with a high level of precision and accuracy, of a large number of biomass samples from the waste and agricultural sectors. Not all of the waste materials analysed are considered suitable for biorefining; for example the total sugar contents of spent mushroom composts are too low. However, the waste paper/cardboard that is currently exported from Ireland has a chemical composition that could result in high biorefinery yields and so could make a significant contribution to Ireland’s biofuel demands.

Miscanthus was focussed on as a major agricultural feedstock. A large number of plants have been sampled over the course of the harvest window (October to April) from several sites. These have been separated into their anatomical fractions and analysed. This has allowed observations to be made regarding the compositional trends observed within plants, between plants, and between harvest dates. Projections are made regarding the extents to which potential chemical yields may vary. For the DIBANET hydrolysis process that is being developed at the University of Limerick, per hectare yields of levulinic acid from Miscanthus could be 20% greater when harvested early compared with a late harvest.

The wet-chemical analysis of biomass is time-consuming. Near infrared spectroscopy (NIRS) has been developed as a rapid primary analytical tool with separate quantitative models developed for the important constituents of Miscanthus, peat, and (Australian) sugarcane bagasse. The work has demonstrated that accurate models are possible, not only for dry homogenous samples, but also for wet heterogeneous samples. For glucose (cellulose) the root mean square error of prediction (RMSEP) for wet samples is 1.24% and the R2 for the validation set ( ) is 0.931. High accuracies are even possible for minor analytes; e.g. for the rhamnose content of wet Miscanthus samples the RMSEP is 0.03% and the is 0.845. Accurate models have also been developed for pre-treated Miscanthus samples and are discussed. In addition, qualitative models have been developed. These allow for samples to be discriminated for on the basis of plant fraction, plant variety (giganteus/non-giganteus), harvest-period (early/late), and stand-age (one-year/older).

Quantitative NIRS models have also been developed for peat, although the heterogeneity of this feedstock means that the accuracies tend to be lower than for Miscanthus. The development of models for sugarcane bagasse has been hindered, in some cases, by the limited chemical variability between the samples in the calibration set. Good models are possible for the glucose and total sugars content, but the accuracy of other models is poorer. NIRS spectra of Brazilian bagasse samples have been projected onto these models, and onto those developed for Miscanthus, and the Miscanthus models appear to provide a better fit than the Australian bagasse models.

Publications 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.

Gaurang Chaudhary, Vasudeo Zambare, Rasika Pawar (2018) Screening, isolation and characterization of probiotically safe lactic acid bacteria from human faecal for biofilm formation, International Journal of Research in BioSciences 7(2): 10-18

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Lactic acid bacteria (LAB), one of the most important human friendly bacteria found in the digestive tract (gut), due to their secretions that inhibit the pathogenic microbes. The present study was aimed at screening of such LAB from faecal samples for various characteristics, particularly in relation to the biofilm formation. Total 110 LAB isolates were obtained from infant and adults faecal samples. All isolates showed catalase negative and inability to lyse the human red blood cells (RBCs) hence considered as safe for humans. Among all 110 isolates, 38 isolates (44.44%) showed protease secretion and all isolates showed biofilm formation abilities. Protease secretion indicated major role in protein digestion in gut, however biofilm formation showed sticking ability to gut and inhibition of pathogenic microbes. Almost more than 80% of isolates were able to tolerate conditions that mimic the gastro intestinal tract i.e. bile salt concentration and acidic environment, which qualifies them to be used as potential probiotic organism. Isolate RP-29was the only isolate showed 82% bile tolerance at 1% concentration, 58% tolerance in acidic pH 2 and 95% biofilm formation. Biofilm formation means secretion of exopolysaccharide(EPS) and was enhanced by supplementation of glucose, MgSO4, MnSO4 and tween 80 in MRS medium. Using 16S rRNA sequencing, the isolate RP-29 was identified as Pediococcusacidilactici. Based on thebile-acid tolerance and biofilm formation activities, P. acidilacticifound as a potent probiotic strain and

Shweta Shinde, Archana Zambare, Vibha Parashere; Vasudeo Zambare (2018) In Vitro studies on antimicrobial activity and phytochemical analysis of Tinospora cordifolia and use as chocolate supplement, International Journal of Advanced Biotechnology and Research 9(4): 632-639

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Background and Objectives: Day by day the antibiotic resistance is increasing and become a serious problem. Hence it’s a today’s need to find some naturally occurring bioactive compounds such as plant bioactive compounds. The objective of the study was to evaluate the antimicrobial activity of Tinospora cordifolia root extracts against human pathogens and T. cordifolia extract were tested for phytochemical examination. A chocolate product development with T. cordifolia extract was also developed. Materials and Methods: Antimicrobial efficiency of Tinospora cordifolia, a medicinal plants were examined using isopropyl alcohol, water, methanol, and acetone, as solvents and tested against four human pathogens like Escherichia coli, Salmonella sp., Staphylococcus aureus, Pseudomonas auruginosa using agar well diffusion method and minimum inhibitory concentration. The phytochemical analysis carried out for qualitative testing methods and T. cordifolia water extract was used as a supplement in chocolate preparation. Results: Water extract showed more extraction yield of 2.68%, followed by methanol (1.6%), isopropyl alcohol (1.13%) and acetone (0.58%). All the extracts showed significant activity against all pathogens, but the isopropyl alcoholic extract of T. cordifolia showed maximum zone of inhibition against all the microorganisms. Also, S. aureaus was inhibited by all extracts. Acetone extract was less potent to inhibit pathogens except S. aureus. The minimum inhibitory concentration of isopropyl alcohol, water, methanol and acetone extracts showed 3.48 mg, 8.04 mg, 3.9 mg and 1.74 mg, respectively. The phytochemical analysis carried out revealed the presence of alkaloids, carbohydrates, terpenoids, proteins, flavonoids, steroids, glycosides in most of the extracts of T. cordifolia. A chocolate preparation containing water extract of T. cordifolia was developed and can be available as source of medication for kids. Conclusion: The Spectrum of activity observed in the present study may be indicative of the present study extracts of T. cordifolia plants could be a possible source to obtain new and effective herbal medicines to treat infections, hence justified the ethnic uses of T. cordifolia against various infectious diseases.

Pawar RS, Chaudhari GT and Zambare VP (2018) Pediococcus acidilactici strain RSP1 16S ribosomal RNA gene, partial sequence, GenBank: MG911001.1

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Haigh K.F, Petersen A.M, Gottumukkala, L, Mandegari M, Naleli, K, Gorgens J.F (2018) Simulation and comparison of processes for biobutanol production from lignocellulose via ABE fermentation, Biofuels, Bioproducts and Biorefining 12(6): 1023-1036

Six conceptual process scenarios for the production of biobutanol from lignocellulosic biomass through acetone?butanol?ethanol (ABE) fermentation, using reported data on process performances, were developed with ASPEN Plus® V8.2 software. The six scenarios covered three fermentation strategies, i.e. batch separate hydrolysis and fermentation (SHF), continuous SHF, and batch simultaneous saccharification and fermentation (SSF) integrated with gas stripping (GS). The two downstream processing options considered were double?effect distillation (DD) and liquid?liquid extraction and distillation (LLE&D). It was found that the SSF?GS/DD scenario was the most energy efficient with a liquid fuel efficiency of 24% and an overall efficiency of 31%. This was also the scenario with the best economic outcome, with an internal rate of return (IRR) of 15% and net present value (NPV) of US$387 million. The SSF?GS/DD scenario was compared to a similar molasses process, based on the product flow rates, and it was found that the molasses process was more energy efficient with a gross energy value (GEV) of 23?MJ?kg1 butanol compared to ?117?MJ?kg1 butanol for the lignocellulosic process. In addition, the molasses?based process was more profitable with an IRR of 36% compared to 21%. However, the energy requirements for the molasses process were supplied from fossil fuels, whereas for the lignocellulose processes a portion of the feedstock was diverted to provide process energy. Improved environmental performance is therefore associated with the lignocellulosic process.

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.

Hayes, D. J. M., Hayes, M. H. B., Leahy, J. J. (2017) Use of Near Infrared Spectroscopy for the Rapid Low-Cost Analysis of Waste Papers and Cardboards, Faraday Discussions 202: 465-482

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Analytical data and quantitative near infrared (NIR) spectroscopy models for various lignocellulosic components (including Klason lignin and the constituent sugars glucose, xylose, mannose, arabinose, galactose, and rhamnose), ash, and ethanol-soluble extractives, were obtained for 53 samples of paper and cardboard. These samples were mostly the type of materials typically found in domestic wastes (e.g. newspapers, printing paper, glossy papers, food packaging). A number of the samples (48) were obtained by separating a sample, after milling, into two particle size fractions. It was found that the fractions containing the smaller particles typically had higher ash and Klason lignin contents and lower glucose and xylose contents that the larger particle size fractions. Nevertheless, all of the sample types had attractive total sugars contents (>50%) indicating that these could be suitable feedstocks for the production of biofuels and chemicals in hydrolysis-based biorefining technologies. NIR models of a high predictive accuracy (R2 of > 0.9 for the independent validation set) were obtained for total sugars, glucose, xylose, Klason lignin, and ash and with values for the Root Mean Square Error of Prediction (RMSEP) of 2.36%, 2.64%, 0.56%, 1.98%, and 4.87%, respectively. Good NIR models (R2 of > 0.8) were also obtained for mannose, arabinose, and galactose. These results suggest that NIR is a suitable method for the rapid, low-cost, analysis of the major lignocellulosic components of waste paper/cardboard samples.

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.

Zambare,V.P., Zambare,A.V. and Thakare,S.R. (2017) Frateuria aurantia strain KSB-05 16S ribosomal RNA gene, partial sequence, GenBank: KY818293.1

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1 agagtttgat cctggctcag attgacgctg gcgtatgctt aacacatgca agtcgaacgg 61 cagcacagca gagcttgctc tgtgggtggc gagtggcgga cgggtgagta atacatcggg 121 acctacccag acgtggggga taacgtaggg aaacttacgc taataccgca tacgtctacg 181 ggagaaagca ggggatcttc ggaccttgcg cggttggacg gaccgatgtt cgattagctt 241 gttggtgagg taatggctca ccaaggcgac gatcgatagc tggtctgaga ggatgatcag 301 ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgg 361 acaatgggcg caagcctgat ccagcattac cgcgtgtgtg aagaaggcct tcgggttgta 421 aagcactttt atcaggaacg aaacgctgtc ggttaatacc cggcggaact gacggtacct 481 gaggaataag caccggctaa cttcgtgcca gcagccgcgg taatacgaag ggtgcaagcg 541 ttaatcggaa ttactgggcg taaagcgtgc gtaggcggtt tgttaagtct gttgtgaaat 601 ccccgggctc aacctgggaa tggcaatgga tactggcaag ctagagtgtg atagaggatg 661 gtggaattcc cggtgtagcg gtgaaatgcg tagagatcgg gaggaacatc agtggcgaag 721 gcggccatct ggatcaacac tgacgctgag gcacgaaagc gtggggagca aacaggatta 781 gataccctgg tagtccacgc cctaaacgat gcgaactgga tgttggtctc aactcggaga 841 tcagtgtcga agctaacgcg ttaagttcgc cgcctgggga gtacggtcgc aagactgaaa 901 ctcaaaggaa ttgacggggg cccgcacaag cggtggagta tgtggtttaa ttcgatgcaa 961 cgcgaagaac cttacctggc cttgacatgt ctccaatcct gtagagatat gggagtgcct 1021 tcgggaatca gaacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1081 ttaagtcccg caacgagcgc aacccttgtc cttagttgcc agcacgtaat ggtgggaact 1141 ctaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatggc 1201 ccttacggcc agggctacac acgtactaca atggtcggta cagagggttg caataccgcg 1261 aggtggagcc aatcccagaa agccgatccc agtccggatc gaagtctgca actcgacttc 1321 gtgaagtcgg aatcgctagt aatcgcggat cagctatgcc gcggtgaata cgttcccggg 1381 ccttgtacac accgcccgtc acaccatggg agtagctgct ccagaagccg ttagtctaac 1441 cgcaaggggg acgagcggac cggagtggtt catgactggg gtgaagtcgt aacaaggtag 1501 cgtgaagtcg taacaaggta gccgtattcg aaggtgcggc tggatcacct cctt

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.

Robus C.L.L, Gottumukkala, L.D, Van Rensburg E, Gorgens J.F. (2016) Feasible process development and techno-economic evaluation of paper sludge to bioethanol conversion: South African paper mills scenario, Renewable energy 92: 333-345

Paper sludge samples collected from recycling mills exhibited high ash content in the range of 54.59%–65.50% and glucose concentrations between 21.97% and 31.11%. Washing the sludge reduced the total ash content to between 10.7% and 19.31% and increased the concentration of glucose, xylose and lignin. Samples were screened for ethanol production and fed-batch simultaneous saccharification and fermentation (SSF) was optimised for the washed samples that resulted in highest and lowest ethanol concentrations. Maximum ethanol concentrations of 57.31 g/L and 47.72 g/L (94.07% and 85.34% of the maximum theoretical yield, respectively) was predicted for high and low fermentative potential samples, respectively, and was experimentally achieved with 1% deviation. A generic set of process conditions were established for the conversion of high ash-containing paper sludge to ethanol. Techno-economic analysis based on three different revenue scenarios, together with Monte Carlo analysis revealed 95% probability of achieving IRR values in excess of 25% at a paper sludge feed rate of 15 t/d. Feed rates of 30 t/d and 50 t/d exhibited a cumulative probability of 100%. This study presents the technical feasibility and economic viability of paper mills expansion towards bioethanol production from paper sludge.

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.

Wnetrzak, R., Hayes, D. J. M., Jensen, L. S., Leahy, J. J., Kwapinski, W. (2015) Determination of the higher heating value of pig manure, Waste and Biomass Valorization 6(3): 327-333

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The ability of using novel method of near infrared (NIR) spectra to predict the composition and higher heating value (HHV) of dry pig manure was examined. Number of pig manure solid fractions variously pre-treated samples were collected in Denmark, from different pig slurry treatment plants (using mechanical or chemical-mechanical separation) and then analysed for their energy values. These values were determined by conventional method using bomb calorimetry and also calculated based on ultimate analysis. NIR spectra method was successfully applied and reasonable R2 values were obtained for the independent prediction set for nitrogen, ash, and the HHV. NIR also showed ability for predicting which type of treatment plants the samples came from. In addition, new empirical equations, based on ultimate analyses of pig manure solids used for prediction of the HHV was established.

Hayes, D. J. M., Hayes, M. H. B., Leahy, J. J. (2015) Analysis of the lignocellulosic components of peat samples with development of near infrared spectroscopy models for rapid quantitative predictions, Fuel 150: 261-268

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Analytical data and quantitative near infrared (NIR) spectroscopy models for various lignocellulosic components (including Klason lignin and the constituent sugars glucose, xylose, mannose, arabinose, galactose, and rhamnose), moisture, and ash were obtained for 53 peat samples. These included samples with high, medium, and low degrees of humification. Klason lignin was the main constituent and was greatest in the samples classified as being highly humified, with structural sugars the lowest in this class. The total sugars contents of all samples were considered to be insufficient to allow for their use in biorefining hydrolysis processes for the production of chemicals and biofuels. NIR models were developed for spectral datasets obtained from the samples in their unprocessed (wet), dry and unground, and dry and ground states. Typically the most accurate models were based on the spectra of dry and ground samples. However the NIR models for the wet samples still offered reasonable predictive capabilities. All models were suitable at least for sample screening, with the models for total sugars, glucose, xylose, galactose, and moisture suitable for quantitative analyses.

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

Sajna K.P, Sukumaran R.K, Gottumukkala L.D, Pandey A (2015) Crude oil biodegradation aided by biosurfactants from Pseudozyma sp. NII 08165 or its culture broth, Bioresource technology 191: 133-139

The aim of this work was to evaluate the biosurfactants produced by the yeast Pseudozyma sp. NII 08165 for enhancing the degradation of crude oil by a model hydrocarbon degrading strain, Pseudomonas putida MTCC 1194. Pseudozyma biosurfactants were supplemented at various concentrations to the P. putida culture medium containing crude oil as sole carbon source. Supplementation of the biosurfactants enhanced the degradation of crude oil by P. putida; the maximum degradation of hydrocarbons was observed with a 2.5 mg L?1 supplementation of biosurfactants. Growth inhibition constant of the Pseudozyma biosurfactants was 11.07 mg L?1. It was interesting to note that Pseudozyma sp. NII 08165 alone could also degrade diesel and kerosene. Culture broth of Pseudozyma containing biosurfactants resulted up to ?46% improvement in degradation of C10–C24 alkanes by P. putida. The enhancement in degradation efficiency of the bacterium with the culture broth supplementation was even more pronounced than that with relatively purer biosurfactants.

Gottumukkala L.D, Sukumaran R.K. Mohan S.V. Valappil S.K. Sarkar O, Pandey A (2015) Rice straw hydrolysate to fuel and volatile fatty acid conversion by Clostridium sporogenes BE01: bio-electrochemical analysis of the electron transport mediators involved, Green chemistry 17(5): 3047-3058

Clostridium sporogenes BE01, a non-acetone forming butanol producer, can produce hydrogen and volatile fatty acids (VFAs) during butanol fermentation from rice straw hydrolysate. Bio-electrochemical analysis revealed the changes that occurred in the redox microenvironment and electron transport mediators during fermentation at different pH and CaCO3 concentrations. CaCO3 played a very important role in enhancing the production of hydrogen, volatile fatty acids and solvents by stimulating the changes in the electron transport system. The electron transport system mediated by NAD/NADH, flavins, Fe–S clusters, protein bound FAD, and cytochrome complex in C. sporogenes BE01 was analysed by cyclic voltammetry (CV). Electrokinetic analysis revealed that the favorability for redox reactions increased with an increase in pH, and the polarization resistance reduced significantly with CaCO3 supplementation.

Sajna K.V, Gottumukkala L.D, Sukumaran R.K, Pandey A. (2015) White biotechnology in cosmetics, Industrial Biorefineries & White Biotechnology, Elsevier
M. F. Md. Din, M. Ponraj, M. Van Loosdrecht, Z. Ujang, S. Chelliapan, V. Zambare (2014) Utilization of palm oil mill effluent for polyhydroxyalkanoate production and nutrient removal using statistical design, International Journal of Environmental Science and Technology 11(3): 671-681

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he optimization for poly-?-hydroxyalkanoate production was carried out with nutrient removal efficiency for total organic carbon (TOC), phosphate, and nitrate from palm oil mill effluent waste. The experiment was conducted in a fabricated fed-batch reactor and the data obtained was analyzed using central composite rotatable design and factorial design for response surface methodology as a systematic approach for designing the experiment statistically to obtain valid results with minimum effort, time, and resources. The analysis of numerical optimization with propagation of error showed that 66 % of poly-?-hydroxyalkanoate production can be obtained with nutrient removal of TOC and nitrate by 19 and 3 %, respectively. However, phosphate removal efficiency was not found to be much effective. More over, the chemical oxygen demand: nitrogen phosphate (509 g/g N), chemical oxygen demand: phosphate (200 g/g P), air flow rate (0.59 L/min), substrate feeding rate (20 mL/min), and cycle length (20 h) were the optimized variables for maximum poly-?-hydroxyalkanoate production and nutrient removal.

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.

Gottumukkala L.D, Parameswaran B, Valappil S.K, Pandey A (2014) Growth and butanol production by Clostridium sporogenes BE01 in rice straw hydrolysate: kinetics of inhibition by organic acids and the strategies for their removal, Biomass Conversion and Biorefinery 4(3): 277-283

Growth inhibition kinetics of a novel non-acetone forming butanol producer, Clostridium sporogenes BE01, was studied under varying concentrations of acetic and formic acids in rice straw hydrolysate medium. Both the organic acids were considered as inhibitors as they could inhibit the growth of the bacterium, and the inhibition constants were determined to be 1.6 and 0.76 g/L, respectively, for acetic acid and formic acid. Amberlite resins—XAD 4, XAD 7, XAD 16, and an anion exchange resin—Seralite 400 were tested for the efficient removal of these acidic inhibitors along with minimal adsorption of sugars and essential minerals present in the hydrolysate. Seralite 400 was an efficient adsorbent of acids, with minimal affinity towards minerals and sugars. Butanol production was evaluated to emphasize the effect of minerals loss and acids removal by the resins during detoxification.

Hayes, D. J. M. (2013) Mass and Compositional Changes, Relevant to Biorefining, in Miscanthus x giganteus Plants over the Harvest Window, Bioresource Technology 142: 591-602

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Miscanthus plants were sampled from several plantations in Ireland over the harvest window (October-April). These were separated into their anatomical components and the loss of leaves monitored. Three distinct phases were apparent: there was minimal loss in the "Early" (October to early December) and "Late" (March and April) phases, and rapid leaf loss in the interim period. Samples were analysed for constituents relevant to biorefining. Changes in whole-plant composition included increases in glucose and Klason lignin contents and decreases in ash and arabinose contents. These changes arose mostly from the loss of leaves, but there were some changes over time within the harvestable plant components. Although leaves yield less biofuel than stems, the added biomass provided by an early harvest (31.9-38.4%) meant that per hectare biofuel yields were significantly greater (up to 29.3%) than in a late harvest. These yields greatly exceed those from first generation feedstocks.

Hayes, D. J. M. (2013) Second-generation biofuels: why they are taking so long, Wiley Interdisciplinary Reviews: Energy and Environment 2(3): 304-334

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There has been a significant degree of hype regarding the commercial potential of second-generation biofuels (2GBs; biofuels sourced from lignocellulosic materials). In 2007, ambitious targets for the mass substitution of fossil-fuel-derived transport fuels by 2GBs were put forward in the United States and similar targets exist for other countries. However, as of May 2012, no commercial-scale 2GB facilities are currently operating. The technical and financial obstacles that have delayed the deployment of these facilities are discussed, as are recent advancements in research that may help to overcome some of these. There are six commercial-scale facilities currently (May, 2012) in construction and many more are planned in the near term. The prospects for 2GBs are more promising now than in the past but the delays in getting to this point mean that the ambitious targets of several years ago are unlikely to be reached in the near term.

Hayes, D.J.M. (2013) Biomass composition and its relevance to biorefining, The Role of Catalysis for the Sustainable Production of Biofuels and Bio-chemicals, Elsevier B. V.27-65

Biomass feedstocks for the production of biofuels and chemicals vary greatly in their chemical compositions. These differences affect which technologies are used for processing. First generation technologies focus on the conversion of sugars, starches, and oils whilst second generation technologies process lignocellulose. While the conversion in first generation processes is relatively facile, the processing of lignocellulose is hindered by the complexity of the biomass matrix. Lignocellulosic feedstocks, however, tend to be significantly less costly, in economic, environmental, and energy terms, to produce. The effects of the various constituents on the conversion of biomass by either hydrolytic or thermochemical means are discussed, as are the logistical considerations needed when sourcing feedstocks. Biomass can be classified as a specifically grown energy crop, an agricultural residue, or a waste resource. Several examples of lignocellulosic feedstocks are discussed for each of these types and representative chemical data for a variety of materials presented.

Hayes, D. J. M. (2013) Report on Optimal Use of DIBANET Feedstocks and Technologies, DIBANET WP5 Report84 pages

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The DIBANET process chain, as a result of its patented pre-treatment stage, has significantly increased the yields of levulinic acid, formic acid, and furfural beyond what was considered to be the state of the art. By fractionating lignocellulosic biomass into its three main polymers (cellulose, hemicellulose, lignin) it has also allowed for lignin to be recovered and sold as a higher-value product. These developments have meant that the amount of acid hydrolysis residues (AHRs) that have been produced are significantly (up to 88%) less than in the Biofine process. These AHRs are required to provide process heat for DIBANET. Direct combustion is the most efficient means for doing this. If such combustion does not occur and the AHRs are instead used in other processes, e.g. pyrolysis and gasification, then more biomass will need to be purchased to fuel the core DIBANET process. The AHRs have not been proven to be superior to virgin biomass when put through these thermochemical processes. Indeed, many of the results from DIBANET Work Package 4 indicate the opposite. Hence, given that DIBANET, and the modelling of its optimal configuration, is designed on the basis of an integrated process, centred on the core element of the acid hydrolysis of biomass, then combustion is the only viable end use for the AHRs. Given that realisation, the focus of this modelling Deliverable is on what the optimal configuration of the process chain would be regarding the three core stages (pretreatment, hydrolysis, and the esterification of levulinic acid with ethanol). It has been demonstrated that a scenario incorporating only the first stage can be profitable in its own right and allow for commercial development at much lower capital costs. In this instance bagasse is a much more attractive feedstock, compared with Miscanthus, due to its higher pentose content.

Integrating the second stage increases capital costs but improves the net present value. The esterification step is somewhat capital intensive but an integrated DIBANET biorefinery that incorporates all three stages can still be highly profitable providing the furfural is sold at its current market price and the lignin is sold rather than used as a fuel for process needs. Indeed, the DIBANET process should not be considered only in the context of biofuels but as a true biorefinery that produces lower value fuels (e.g. ethyl-levulinate) in addition to high value chemicals and bio-products (e.g. furfural and lignin).

The energy and carbon balances of the various DIBANET scenarios have been investigated and are highly positive with values significantly superior to those for the energy-intensive Biofine process. A socioeconomic survey has also been carried out and has shown that there can be a positive effect on employment, both direct and indirect, particularly when Miscanthus is used as the feedstock. The DIBANET integrated process also holds up well when its environmental and social performances are ranked for a range of important parameters.

The development of the core DIBANET IP towards commercial deployment appears to be warranted, based on data provided from the models developed. Indeed, these models present possible scenarios whereby even demonstration-scale DIBANET facilities could operate at significant profits and provide healthy returns on the capital invested.

Gottumukkala, L. D, Valappi, S. 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.

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.

Artur Silva, Rommel Ramos, Adriana Carneiro, Sint ia Almeida, Vinicius De Abreu, Anderson Santos Siomar Soares, Anne Pinto, Luis Guimarães, Eudes Barbosa, Paula Schneider, Vasudeo Zambare, Debmalya Barh, Anderson Miyoshi, and Vasco Azevedo (2013) , OMICS: Applications in Biomedical, Agriculture and Environmental Sciences , CRC Press, Taylor and Francis

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Debmalya Barh, Vasudeo Zambare, Vasco Azevedo (2013) , OMICS: Applications in Biomedical, Agriculture and Environmental Sciences, CRC Press, Taylor and Francis

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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.

Sajna K.V, Sukumaran R.K, Gottumukkala L.D, Jayamurthy H, Dhar K.S (2013) Studies on structural and physical characteristics of a novel exopolysaccharide from Pseudozyma sp. NII 08165, International Journal of Biological Macromolecules 59: 84-89
Hayes, D. J. M. (2012) Development of near infrared spectroscopy models for the quantitative prediction of the lignocellulosic components of wet Miscanthus samples, Bioresource Technology 119: 393-405

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Miscanthus samples were scanned over the visible and near infrared wavelengths at several stages of processing (wet-chopped, air-dried, dried and ground, and dried and sieved). Models were developed to predict lignocellulosic and elemental constituents based on these spectra. The dry and sieved scans gave the most accurate models; however the wet-chopped models for glucose, xylose, and Klason lignin provided excellent accuracies with root mean square error of predictions of 1.27%, 0.54%, and 0.93%, respectively. These models can be suitable for most applications. The wet models for arabinose, Klason lignin, acid soluble lignin, ash, extractives, rhamnose, acid insoluble residue, and nitrogen tended to have lower R(2) values (0.80+) for the validation sets and the wet models for galactose, mannose, and acid insoluble ash were less accurate, only having value for rough sample screening. This research shows the potential for online analysis at biorefineries for the major lignocellulosic constituents of interest.

Hayes, D. J. M. (2012) Review of Biomass Feedstocks and Guidelines of Best Practice, DIBANET WP2 Report150 pages

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This document is the result of the evaluation of biomass feedstocks, from Europe and Latin America, that took place as part of the DIBANET project. That project is co-financed from the 7 th Framework Programme for Research and Technological Demonstration of the European Union. (Title: Enhancing international cooperation between the EU and Latin America in the field of biofuels; Grant Agreement No: 227248-2).

The work in Task 2.1 of Work Package 2 (WP2) at DIBANET partners UL, CTC, and UNICAMP involved evaluating, on a number of levels, potential feedstocks for utilisation in the DIBANET acid-hydrolysis process (WP3). In the early stage of the project a wide number of feedstocks were examined and relevant secondary compositional data were sought from the literature. Selected feedstocks were analysed at the laboratories of UL, CTC, and UNICAMP and, from these, a limited number of feedstocks were subjected to more in-depth analysis/evaluation.

Work at UL focused on Miscanthus, cereal straws, and waste papers. The wet-chemical and spectroscopic analysis that was carried out on a wide number of Miscanthus samples have allowed for in-depth understandings to be reached regarding the changes in lignocellulosic composition, and potential biomass/biofuel yields that could be realised over the harvest window. Straws present much less chemical variation but have enough structural carbohydrates to warrant their processing in the DIBANET technology. Waste papers can have amongst the highest total carbohydrate contents of any of the feedstocks studied.

Work at CTC focused on the residues of the sugarcane industry - sugarcane bagasse and sugarcane trash (field residues from harvesting). A large number of samples were collected from a variety of sugar mills and plantations. It has been seen that there can be a significant variation in the composition of different bagasse samples, particularly with regards to the ash content. Sugarcane trash has lower total carbohydrates contents than bagasse but is still a suitable feedstock for DIBANET.

Work at UNICAMP focused on the evaluation of residues from the banana, coffee, and coconut industries. It was found that these also have potential for utilisation in the DIBANET process, however the value of the residues for this end-use is dependent on which part of the plant is utilised. For instance, coffee husks have sufficient structural carbohydrates to allow for decent yields of levulinic acid, formic acid, and furfural in DIBANET, however the leaves of the coffee plant do not. Leaves from the banana plant are also of less value for DIBANET than the other parts of the plant (e.g. stem).

A major output of this Deliverable is the downloadable electronic database that contains all of the WP2 analytical data obtained during the course of the project. It contains analytical data and predicted biorefining yields for a total of 1,281 samples. It can be obtained, free of charge, from the DIBANET website and will be a valuable tool for stakeholders in biorefining projects.

This document presents the data and evaluations that were made regarding biomass feedstocks, and also puts forward guidelines of best practice in terms of making the best use of these resources. A shortened version of this document can also be downloaded from the DIBANET website.

Syed Hassan, Maria Paula Schneider, Rommel Thiago Ramos, Adriana Carneiro, Alex Ranieri Lima, Luis Carlos Guimarães, Amjad Ali, Syeda Bakhtiar, Ulisses Pereira, Anderson Santos, Siomar de Castro Soares, Fernanda Dorella, Anne Pinto, Dayana Ribeiro, Maria Silvanira Barbosa, Sintia Almeida, Vinicius Augusto Abreu, Flavia Aburjaile, Karina Kelly Fiaux, Eudes Guilherme Barbosa, Carlos Diniz, Flavia Rocha, Rashmi Saxena, Sandeep Tiwari, Vasudeo Zambare, Preetam Ghosh, Luis Gustavo Pacheco, Christopher Dowson, Anil Kumar, Debmalya Barh, Anderson Miyoshi, Vasco Azevedo, and Artur Silva (2012) Whole-genome sequence of Corynebacterium pseudotuberculosis strain 162, isolated from Camel, Journal of Bacteriology 194(20): 5718-5719

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Corynebacterium pseudotuberculosis is a pathogen of great veterinary and economic importance, since it affects livestock, mainly sheep and goats, worldwide, together with reports of its presence in camels in several Arabic, Asiatic, and East and West African countries, as well as Australia. In this article, we report the genome sequence of Corynebacterium pseudotuberculosis strain Cp162, collected from the external neck abscess of a camel in the United Kingdom.

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 (4.1–6.0), solids loadings (6.6–23.4%), 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 (4.1–6.0), solids loadings (6.6%–23.4%), and enzyme loadings (6.6–23.4 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.

Axel Hollmann, Mariano Saviello, Lucrecia Delfederico, Tessália Diniz Luerce Saraiva, Debmalya Barh, Neha Jain, Sandeep Tiwari, Sudha Chandra, Krishnakant Gupta, Vasudeo Zambare, Anil Kumar, Lew Christopher, Vasco Azevedo, Liliana Semorile, Anderson Miyoshi (2012) Tight controlled expression and secretion of Lactobacillus brevis SlpA in Lactococcus lactis, Biotechnology Letters 34(7): 1275-1281

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Prokaryotes commonly present outer cell wall structures composed of a crystalline array of proteinaceous subunits, known as surface layers (S-layers). The ORF encoding the S-layer protein (SlpA) of Lactobacillus brevis was cloned into Lactococcus lactis under the transcriptional control of the xylose-inducible expression system (XIES). SlpA was secreted into the extracellular medium, as determined by immunoblotting, and assays on the kinetics of SlpA production revealed that repression of the system with glucose did not require the depletion of xylose from the medium that allows transitory ORF expression. The successful use of XIES to express S-layer proteins in the versatile and generally recognized as safe species L. lactis opens new possibilities for an efficient production and isolation of SlpA S-layer protein for its various applications in biotechnology and importantly as an antigen-carrying vehicle.

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 30°C.

Din M.F.M., Ponraj M., Zaini U., Van Loosdrecht, M.C.M., Salmiati Y., Chelliapan S., Zambare V., G. Olsen (2012) Development of Bio-PORecŽ System for polyhydroxyalkanoates (PHA) production and its storage in mixed cultures of palm oil mill effluent (POME), Bioresource Technology 124: 208-216

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High PHA production and storage using palm oil mill effluent (POME) was investigated using a laboratory batch Bio-PORec® system under aerobic-feeding conditions. Results showed that maximum PHA was obtained at a specific rate (qp) of 0.343 C-mol/C-mol h when air was supplied at 20 ml/min. The PHA yield was found to be 0.80 C-mol/C-mol acetic acid (HAc) at microaerophilic condition and the mass balance calculation showed that PHA production increased up to 15.68 ± 2.15 C-mmol/cycle. The experiments showed that short feeding rate, limited requirements for electron acceptors (e.g. O2, NO3) and nutrients (N and P) showed lower tendency of glycogen accumulation and contributed more to PHA productivity.

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.

Hemaiswarya S., Raja R., Carvalho Isabel S., Ravikumar R., Vasudeo Zambare and Debmalya Barh (2012) An Indian scenario on renewable and sustainable energy sources with emphasis on algae, Applied Microbiology and Biotechnology 96: 1125-1135

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India is the fifth largest primary energy consumer and fourth largest petroleum consumer after USA, China, and Japan. Despite the global economic crisis, India’s economy is expected to grow at 6 to 8 %/year. There is an extreme dependence on petroleum products with considerable risks and environmental issues. Petroleum-derived transport fuels are of limited availability and contribute to global warming, making renewable biofuel as the best alternative. The focus on biogas and biomass-based energy, such as bioethanol and biohydrogen, will enhance cost-effectiveness and provide an opportunity for the rural community. Among all energy sources, microalgae have received, so far, more attention due to their facile adaptability to grow in the photobioreactors or open ponds, high yields, and multiple applications. Microalgae can produce a substantial amount of triacylglycerols as a storage lipid under photooxidative stress or other adverse environmental conditions. In addition to renewable biofuels, they can provide different types of high-value bioproducts added to their advantages, such as higher photosynthetic efficiency, higher biomass production, and faster growth compared to any other energy crops. The viability of first-generation biofuels production is, however, questionable because of the conflict with food supply. In the future, biofuels should ideally create the environmental, economic, and social benefits to the communities and reflect energy efficiency so as to plan a road map for the industry to produce third-generation biofuels.

Silva,A., Ramos,R., Carneiro,A., Almeida,S., Barbosa,S.,Pinto,A.C., Cerdeira,L., Santos,A., Soares,S., Guimaraes,L., Barbosa,E., Figueira,F., Souza,F., Abreu,V.C., Dorella,F., Pacheco,L., Ghosh,P., Zambare,V., Barve,N., Tiwari,S., Barh,D., Miyoshi,A., Schneider,M.P. and Azevedo,V. (2012) Corynebacterium pseudotuberculosis 316, complete genome, GenBank: CP003077.1

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Silva,A., Cerdeira,L., Bol,E., Barbosa,M.S., Muller,B., Helden,P.V., Santos,A.R., Ramos,R.T.J., Carneiro,A.R., Guimaraes,L.C., Aburjaile,F.F., Padua,U.D., Barbosa,E., Fiaux,K.K., Diniz,C.A.A., Soares,S.C., Pinto,A.C., Almeida,S.S., Abreu,V.A.C., Hassan,S.S., Khatri,G., Rahangdale,S., Gupta,K., Verma,A.S., Ghosh,P., Zambare,V., Kumavath,R.N., Barh,D., Miyoshi,A., Schneider,M.P.C. and Azevedo,V. (2012) Corynebacterium pseudotuberculosis P54B96, complete genome, GenBank: CP003385.1

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Silva,A., Lopes,T., Ramos,R.T.J., Carneiro,A.R., Barbosa,M.S., Santos,A.R., Guimaraes,L.C., Aburjaile,F.F., Padua,Ud., Barbosa,E.,Fiaux,K.K., Diniz,C.A.A., Soares,S.C., Pinto,A.C., Almeida,S.S., Abreu,V.A.C., Hassan,S.S., Khatri,G., Rahangdale,S., Gupta, K., Verma,A., Kumavath, R.N., Ghosh,P., Zambare,V., Barh,D., Miyoshi,A., Schneider,M.P.C. and Azevedo,V. (2012) Corynebacterium pseudotuberculosis 267, complete genome, GenBank: CP003407.1

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Hassan,S.S., Schneider,M.P., Ramos,R.T., Carneiro,A.R., Ranieri,A.,Guimaraes,L.C., Ali,A., Bakhtiar,S.M., Pereira Ude,P., Dos Santos,A.R., Soares Sde,C., Dorella,F., Pinto,A.C., Ribeiro,D., Barbosa,M.S., Almeida,S., Abreu,V., Aburjaile,F., Fiaux,K., Barbosa,E., Diniz,C., Rocha,F.S., Saxena,R., Tiwari,S., Zambare,V., Ghosh,P., Pacheco,L.G., Dowson,C.G., Kumar,A., Barh,D., Miyoshi, A., Azevedo,V. and Silva,A. (2012) Corynebacterium pseudotuberculosis Cp162, complete genome, GenBank: CP003652.1

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Silva,A., Saleh,M., Selim,S.A., Cerdeira,L., Ramos,R.T., Carneiro,A.R., Barbosa,S., Santos,A., Soares,S., Guimaraes,L., Diniz,C.A., Fiaux,K., Hassan,S.S., Ali,A., Barbosa,E., Figueira,F., Pinto,A.C., Almeida,S., Abreu,V.C., Dorella,F., Pacheco,L., Souza,F., Ghosh,P., Zambare,V., Barh,D., Miyoshi,A., Schneider,M.P. and Azevedo,V. (2012) Corynebacterium pseudotuberculosis 31, complete genome, GenBank: CP003421.1

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Silva,A., Saleh,M., Selim,S.A., Cerdeira,L., Ramos,R.T., Carneiro,A.R., Barbosa,S., Santos,A., Soares,S., Guimaraes,L., Diniz,C.A., Fiaux,K., Hassan,S.S., Ali,A., Barbosa,E., Figueira,F., Pinto,A.C., Almeida,S., Abreu,V.C., Dorella,F., Pacheco,L., Souza,F., Ghosh,P., Zambare,V., Barh,D., Miyoshi,A., Schneider,M.P. and Azevedo,V. (2012) Corynebacterium pseudotuberculosis 258, complete genome, GenBank: CP003540.1

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Hayes, D. J. M. (2011) Analysis of Lignocellulosic Feedstocks for Biorefineries with a Focus on The Development of Near Infrared Spectroscopy as a Primary Analytical Tool, PhD Thesis832 pages (over 2 volumes)

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The processing of lignocellulosic materials in modern biorefineries will allow for the production of transport fuels and platform chemicals that could replace petroleum-derived products. However, there is a critical lack of relevant detailed compositional information regarding feedstocks relevant to Ireland and Irish conditions. This research has involved the collection, preparation, and the analysis, with a high level of precision and accuracy, of a large number of biomass samples from the waste and agricultural sectors. Not all of the waste materials analysed are considered suitable for biorefining; for example the total sugar contents of spent mushroom composts are too low. However, the waste paper/cardboard that is currently exported from Ireland has a chemical composition that could result in high biorefinery yields and so could make a significant contribution to Ireland’s biofuel demands.

Miscanthus was focussed on as a major agricultural feedstock. A large number of plants have been sampled over the course of the harvest window (October to April) from several sites. These have been separated into their anatomical fractions and analysed. This has allowed observations to be made regarding the compositional trends observed within plants, between plants, and between harvest dates. Projections are made regarding the extents to which potential chemical yields may vary. For the DIBANET hydrolysis process that is being developed at the University of Limerick, per hectare yields of levulinic acid from Miscanthus could be 20% greater when harvested early compared with a late harvest.

The wet-chemical analysis of biomass is time-consuming. Near infrared spectroscopy (NIRS) has been developed as a rapid primary analytical tool with separate quantitative models developed for the important constituents of Miscanthus, peat, and (Australian) sugarcane bagasse. The work has demonstrated that accurate models are possible, not only for dry homogenous samples, but also for wet heterogeneous samples. For glucose (cellulose) the root mean square error of prediction (RMSEP) for wet samples is 1.24% and the R2 for the validation set ( ) is 0.931. High accuracies are even possible for minor analytes; e.g. for the rhamnose content of wet Miscanthus samples the RMSEP is 0.03% and the is 0.845. Accurate models have also been developed for pre-treated Miscanthus samples and are discussed. In addition, qualitative models have been developed. These allow for samples to be discriminated for on the basis of plant fraction, plant variety (giganteus/non-giganteus), harvest-period (early/late), and stand-age (one-year/older).

Quantitative NIRS models have also been developed for peat, although the heterogeneity of this feedstock means that the accuracies tend to be lower than for Miscanthus. The development of models for sugarcane bagasse has been hindered, in some cases, by the limited chemical variability between the samples in the calibration set. Good models are possible for the glucose and total sugars content, but the accuracy of other models is poorer. NIRS spectra of Brazilian bagasse samples have been projected onto these models, and onto those developed for Miscanthus, and the Miscanthus models appear to provide a better fit than the Australian bagasse models.

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 70°C and 15–20% 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 0.45–0.50 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 30°C. 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 7–9 and temperature 20–50°C with optimum pH of 8 and temperature 35°C. 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 (35.16°C) 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, Gokila, V. Zambare (2011) Bacterial decolorization of textile dye (Orange 3R), International Journal of Advanced Biotechnology and Research 2(1): 168-177

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The dye decolorizing isolates, Bacillus sp., Klebsiella sp. Salmonella sp. and Pseudomonas sp. were isolated from the textile effluent samples collected from Elampillai, Tamil Nadu. Different parameters such as various carbon source, nitrogen source, temperature, pH and inoculum size were optimized for decolorization of Orange 3R by using bacterial isolates. Pseudomonas sp. and Bacillus sp. showed maximum dye decolorization of 89% at the end of 144h under optimum condition. But the Bacillus sp. was found to be more efficient in dye decolorization. All parameters studied in this paper were found to be effective for all isolates. The results reported here warrant further investigation to establish the usefulness of these isolates for bioremediation and biodegradation application such as waste water treatment. High decolorization extent and facile conditions show the potential for this bacterial strain to be used in the biological treatment of dyeing mill effluents.

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). 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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 Â 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

Louise Teixeira Cerdeira, Anne Cybelle Pinto, Maria Paula Cruz Schneider, Sintia Silva de Almeida, Anderson Rodrigues dos Santos, Eudes Guilherme Vieira Barbosa, Amjad Ali, Maria Silvanira Barbosa, Adriana Ribeiro Carneiro, Rommel Thiago Jucá Ramos, Rodrigo Oliveira dos Santos, Debmalya Barh, Neha Barve, Vasudeo Zambare, Silvia Estevão Belchior, Luis Carlos Guimarães, Siomar de Castro Soares, Vinicius Augusto Carvalho de Abreu, Andreas Tauch, Eva Trost, Anderson Miyoshi, Vasco Azevedo, Artur Silva (2011) Whole genome sequence of Corynebacterium pseudotuberculosis PAT10 of strain isolated from sheep in Patagonia, Argentine, Journal of Bacteriology 193(22): 6420-6421

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In this work, we report the complete genome sequence of a Corynebacterium pseudotuberculosis PAT10 isolate, collected from a lung abscess in an Argentine sheep in Patagonia, whose pathogen also required an investigation of its pathogenesis. Thus, the analysis of the genome sequence offers a means to better understanding of the molecular and genetic basis of virulence of this bacterium.

Louise Teixeira Cerdeira, Maria Paula Cruz Schneider, Anne Cybelle Pinto, Sintia Silva de Almeida, Anderson Rodrigues dos Santos, Eudes Guilherme Vieira Barbosa, Amjad Ali, Fla´via Figueira Aburjaile, Vinicius Augusto Carvalho de Abreu, Luis Carlos Guimara˜es, Siomar de Castro Soares, Fernanda Alves Dorella, Fla´via Souza Rocha, Erick Bol, Pablo Henrique Caracciolo Gomes de Sa´, Thiago Souza Lopes, Maria Silvanira Barbosa, Adriana Ribeiro Carneiro, Rommel Thiago Juca´ Ramos, Nilson Antoˆnio da Rocha Coimbra, Alex Ranieri Jeroˆnimo Lima, Debmalya Barh, Neha Jain, Sandeep Tiwari, Rathinam Raja, Vasudeo Zambare, Preetam Ghosh, Eva Trost, Andreas Tauch, Anderson Miyoshi, Vasco Azevedo, and Artur Silva (2011) Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain CIP 52.97, Isolated from a Horse in Kenya, Journal of Bacteriology 193(24): 7025-7026

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In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism.

V.P. Zambare, Lew P. Christopher (2011) Statistical optimization of process parameters for cellulase production by thermophilic Bacillus amyloliquefaciens, International Journal of Food and Fermentation Technology 1(1): 63-68

Statistical optimization of process parameters for cellulase production by thermophilic Bacillus amyloliquefaciens. International Journal of Food and Fermentation Technology

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 24°C. 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 µm 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
V. P. Zambare (2011) , Plant Beverages: antioxidant and antimicrobial approach, Lambert Academic Publishing

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Archana Zambare, Vasudeo Zambare, Lew Christopher (2011) Geobacillus thermodenitrificans strain AV-5 16S ribosomal RNA gene, partial sequence, GenBank: JN837496.1

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1 tggagagttt gatcctggct caggacgaac gctggcggcg tgcctaatac atgcaagtcg 61 agcggaccga acgagagctt gctcttgtty ggtcagcggc ggacgggtga gtaacacgtg 121 ggcaacctgc ccgcaagacc gggataactc cgggaaaccg gagctaatac cggataacac 181 caaagaccgc atggtctttg gttgaaaggc ggcttcggct gtcacttgcg gatgggcccg 241 cggcgcatta gctagttggt gaggtaacgg ctcaccaagg cgacgatgcg tagccggcct 301 gagagggtga ccggccacac tgggactgag acacggccca gactcctacg ggaggcagca 361 gtagggaatc ttccgcaatg gacgaaagtc tgacggagcg acgccgcgtg agcgaagaag 421 gccttcgggt cgtaaagctc tgttgtgagg gacgaaggag cgccgtttga ataaggcggc 481 gcggtgacgg tacctcacga gaaagccccg gctaactacg tgccagcagc cgcggta

Cerdeira L, Schneider MPC, Barbosa MS, Ramos RTJ, Carneiro AR, Santos RS, Lima M, DAFonseca V, Almeida SS, Santos AR, Soares SC, Pinto AC, Ali A, Barbosa E, Dorella FA, Rocha FS, Guimaraes LG, Belchior SB, Ghosh P, Zambare V, Barve N, Tiwari S, Barh D, Miyoshi A, Azevedo V, Silva A (2011) Corynebacterium pseudotuberculosis PAT10, complete genome, GenBank: CP002924.1

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Cerdeira LT, Schneider MPC, Pinto AC, de Almeida SS, dos Santos AR, Barbosa EGV, Ali A, Aburjaile FF, de Abreu VAC, Guimara˜es LC, de Castro Soares S, Dorella FA, Rocha FS, Bol E, de Sa´ PHCG, Lopes TS, Barbosa MS, Carneiro AR, Ramos RTJ, Coimbra NAR, Lima ARJ, Barh D, Jain N, Tiwari S, Raja R, Zambare V, Ghosh P, Trost E, Tauch A, Miyoshi A, Azevedo V and Silva A (2011) Corynebacterium pseudotuberculosis CIP 52.97, complete genome, GenBank: CP003061.1

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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.

Sukumaran R.K, Gottumukkala L.D, Rajasree K.P, Alex D, Pandey A (2011) Butanol fuel from biomass: Revisiting ABE fermentation, Biofuels, Elsevier

ABE (Acetone-Butanol-Ethanol) fermentations were next only to ethanol fermentations and used to be a major industry until 1960s. Later, biological route for butanol production lost its importance owing to competition from petrochemical route, and today there is a renewed interest in ABE fermentation due to increased concerns over petroleum depletion and the increased pollution due to burning of petroleum fuels. Though the ABE fermentation process used to be operational decades back, the same technologies are not applicable today due to the lack of cost effectiveness and the nonavailability of conventional raw materials. The most feasible feedstock for butanol seems to be lignocellulose, but the problems plaguing bioethanol are also applicable for biobutanol. However, the future for biobutanol seemsbright since the Clostridia that produce ABE are capable of utilizing a range of carbon sources for growth and solvent production and also are not inhibited by the sugar degradation products generated during biomass pretreatment are being developed. Meanwhile, in the short term, advanced fermentation technologies are being developed by the expert groups which tackle problems such as low cell density, viability, and solvent sensitivity by modulations in the methods of carbon feeding, mode of culture, and in situ removal and recovery of solvents. These efforts may be developed into commercially viable technologies.

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 (3.775±0.13U/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 µmoles 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 1,325±7.1 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, Archana Zambare, Lew Christopher (2010) Antioxidant and antibacterial activity of extracts from lichen Xanthoparmelia somloensis, native to the Black Hills, South Dakota, USA, International Journal Medical Science and Technology 3(7): 46-51

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The present study was carried out to evaluate the antioxidant and antibacterial activity of lichen Xanthoparmelia somloensis, native to the Black Hills in South Dakota, USA. The antioxidant activity of lichen extracts was assessed using the 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. The lipid peroxidation reaction of acetone and methanol extracts was inhibited 85% and 81%, respectively A free radical scavenging activity of 77% (acetone extract) and 65% (methanol extract) was determined. The antibacterial activity was assayed against four clinical strains using the agar well diffusion method. Except for Escherichia coli, both extracts were found inhibitory to Streptomyces aureus, Streptococcus pyogenes,and Steptococcus agalactiae with minimum inhibitory concentration values of 0.7-0.9 mg/ml. It was demonstrated that both the antioxidant and antibacterial activities correlated well with the protein to polysaccharide ratio rather than the polyphenol content of the lichen extracts. To the best of our knowledge, this is the first literature report on antibacterial activity from the lichen X.somloensis. The results reported here warrant further investigations to establish the usefulness of X.somloensis in biomedical applications such as treatment of respiratory and urinary tract infections.

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.

V.P. Zambare (2010) Bacillus amyloliquefaciens strain UNPDV-22 16S ribosomal RNA gene, partial sequence, GenBank: HQ005305.1

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1 acgctggcgg cgtgcctaat acatgcaagt cgagcggaca gatgggagct tgctccctga 61 tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc 121 cgggaaaccg gggctaatac cggatgcttg tttgaaccgc atggttcaga cataaaaggt 181 ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 241 ctcaccaagg caacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 301 acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 361 tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 421 gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg 481 gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 541 gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 601 gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 661 tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 721 gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 781 cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 841 acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 901 ggggccccgc acaagcggtg gagcatgtgg tttaattgga accaacgcga agaacatttc 961 caggtcatga catcctctga caatcctaga gataggacgt cccgttcggg ggcagagcga 1021 caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1081 agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt 1141 gacaaaccgg aggaaggtgg ggaagacgtc aaatcatcat gccccttatg acctgggcta 1201 cacacgtgct acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca 1261 caaatctgtt ctcagttcgg atcccagtct gcaactcgac tgcgtgaagc tggattcgct 1321 agaaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac actccgcccg 1381 tcacaccacg agagtttgta acacccgaag tcggtgaggt aacctttatg gatccatacg 1441 ctgaaggtgg gactagagtg

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 baker’s 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 30°C, respectively. The partially purified protease was stable at temperature range of 30-40°C and pH 6-7. However, protease was maximum stable at 30°C 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.

V. P. Zambare (2009) Antibiotic resistance and sensitivity pattern of pathogenic bacteria isolated from urinary tract infected sample, Biomedical and Pharmacology Journal 2(1): 137-140

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Urinary tract infection (UTI) represents one of the most common diseases encountered in medical practice today and occurring from the neonate to the geriatric age group. Despite the widespread availability of antibiotics, it remains the most common bacterial infection in the human being. Four s pathogenic bacteria as Escherichia coli, Proteus sp Shigella sp. and Klebsiella sp. were isolated from urine samples collected from pathology laboratory. Identification of these cultures was done o the basis of morphological and biochemical characteristics. E. coli, Proteus sp., Shigella sp. and Klebsiella sp. were resistance to 59, 68, 14 and 32% respectively.

Zambare V. P, Bhoyte S. A. (2009) Antibacterial activity of tea, Biomedical and Pharmacology Journal 2(1): 173-175

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Antibacterial activities of tea extracts in various solvents were tested against six organisms, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Serracia sp., and Bacillus subtilis using agar-well method. Petroleum ether and chloroform extracts of tea showed strong antibacterial activities against P. aeruginosa and B. subtilis while other extracts were less active. The minimum inhibitory concentration (MIC) of chloroform extract of tea was found to be 25µg/mL. This study may establish the need for daily use of this product for medicinal purposes.

M. V. Padul and V. P. Zambare (2008) In vitro antibacterial activity of Enicostema littorale plant extracts, Journal of Pure and Applied Microbiology 2(1): 245-247

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The antibacterial activity of aqueous, ethanolic, methanolic, ether, acetone, chloroform and hexane extracts from Enicostema littorale plant has been evaluated, in vitro, against Serrasia sp:, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. All extracts of Enicostema littorale exhibited highest antibacterial activity against Sarrasia sp and P. aeruginosa followed by very less activity against E. coli and S. aureus. The results indicate that Enicostema littorale plant may be a good candidate as antimicrobial agent.

V. P. Zambare and A. A. Bhole (2008) In vitro antioxidant activity of Plumbago zeylanica, Research Journal of BioTechnology 3(3): 36-37

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The total antioxidant activity, total phenolic content and reducing power of ethanol, methanol and water extracts of roots of plant Plumbago zeylanica were determined in vitro. Ethanol, methanol and water extracts of P. zeylanica roots showed the good antioxidant activity. However, there was no correlation between antioxidant activity and total phenolic content of the extracts. Although the methanol extract of P. zeylanica had the highest total phenolic contents, it exhibited low antioxidant activity. In contrast, there was a strong correlation between reducing power and total antioxidant activity of the extracts. The highest reducing power was determined for the methanol extract of Plumbago zeylanica.

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

Zambare V.P. and Bhoyte S. A (2008) Antibacterial activity of ginger (Zingiber officinalis), Journal of Pure and Applied Microbiology 2(2): 591-593

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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): 1569–1574

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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 25–35 °C. Enzymatic dehairing may be a promising shift towards an environment-friendly leather processing method.

V. P. Zambare, P. S. Kothari, M. V. Kulkarni (2007) Plasmid curing of plumbagin and its application in bacterial antibiotic resistance, Journal of Pure and Applied Microbiology 1(2): 285-288

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Plumbago zeylanica, a medicinal plant, contain plumbagin a naphthoquinonic compound. Partially purified plumbagin was tested for its plasmid curing activity. E. coli, isolated from pathological samples showed multiple antibiotic resistance. Plumbagin showed antibacterial activity at MIC value; 80 ?g ml -1. Antibiotics and plumbagin together decreased the antibiotic resistance pattern, suggesting plasmid curing by plumbagin. Thus, plumbagin can be used as an adjuvant to antibiotic therapy for treating infectious diseases caused by antibiotic resistant organisms.

V. P. Zambare and A. A. Bhole (2007) In vitro antifungal activity of Plumbago zeylanica, Journal of Pure and Applied Microbiology 1(2): 325-326

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In vitro activity of ethanolic and methanolic plant extracts of Plumbago zeylanica was investigated against Aspergillus sp., Penicillium, Aspergillus sp. and Fusarium sp. The ethanolic extract exhibited significant activity against Aspergillus, Penicillium sp. and Fusarium sp. Aqueous and acetone extracts were showed moderate activity followed by petroleum ether extract.

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.

P. P. Kanekar, S. S. Nilegaonkar, V.P. Zambare, P.K. Dhakephalkar and S.S. Sarnaik (2006) Bacillus cereus strain MCM B-326 16S ribosomal RNA gene, partial sequence, GenBank: DQ479314.1

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1 ctnnnnncnt gcctatactg cagtcnagcg atggattant tnttgctctt atgaagttag 61 cggcggacgg gtgagtaaca cgtgggtaac ctgcccataa gactgggata actccgggaa 121 accggggcta ataccggata acattttgaa ccgcatggtt cgaaattgaa aggcggcttc 181 ggctgtcact tatggatgga cccgcgtcgc attagctagt tggtgaggta acggctcacc 241 aaggcaacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 301 cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 361 agcaacgccg cgtgagtgat gaaggctttc gggtcgtaaa actctgttgt tagggaagaa 421 caagtgctag ttgaataagc tggcaccttg acggtaccta accagaaagc cacggctaac 481 tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggaat tattgggcgt 541 aaagcgcgcg caggtggttt cttaagtctg atgtgaaagc ccacggctca ccgtggaggg 601 tcatnggaan ctgggagact tgagtgcaaa aaagaaagtg gattccntgt gtagcggtga 661 atgcgtaaaa tntggaggaa cccngtggcn angcnatttc tgtctgtact gacctgagnc 721 nnaaagntgg ggagaaaaag attanatccc tggtatcccn cctaacnaaa nnntaatgtt 781 aangntcccc nttatgtnaa tacccntaac ncccngggga ncgcccagnt aaannaanat 841 tngggnccc

S. S. Nilegaonkar, V.P. Zambare, P.P. Kanekar and P.K. Dhakephalkar (2006) Pseudomonas aeruginosa strain MCM B-327 16S ribosomal RNA gene, partial sequence, GenBank: DQ473435.1

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1 atggctcaga ttgaacgctg gcggcaggcc taacacatgc aagtcgagcg gatgaaggga 61 gcttgctcct ggattcagcg gcggacgggt gagtaatgcc taggaatctg cctggtagtg 121 ggggataacg tccggaaacg ggcgctaata ccgcatacgt cctgagggag aaagtggggg 181 atcttcggac ctcacgctat cagatgagcc taggtcggat tagctagttg gtggggtaaa 241 ggcctaccaa ggcgacgatc cgtaactggt ctgagaggat gatcagtcac actggaactg 301 agacacggtc cagactccta cgggaggcag cagtggggaa tattggacaa tgggcgaaag 361 cctgatccag ccatgccgcg tgtgtgaaga aggtcttcgg attgtaaagc actttaagtt 421 gggaggaagg gcagtaagtt aataccttgc tgttttgacg ttaccaacag aataagcacc 481 ggctaacttc gtgccagcag ccgcggtaat acgaagggtg caagcgttaa tcggaattac 541 tgggcgtaaa gcgcgcgtag gtggttcagc aagttggatg tgaaatcccc gggctcaacc 601 tgggaactgc atccaaaact actgagctag agtacggtag agggtggtgg aatttcctgt 661 gtagcggtga aatgcgtaga tataggaagg aacaccagtg gcgaaggcga ccacctggac 721 tgatactgac actgaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 781 ccacgccgta aacgatgtcg actagccgtt gggatccttg agatcttagt ggcgcagcta 841 acgcgataag tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa atgaattgac 901 gggggcccgc acaagcggtg gagcatgtgg ttta

S. S. Nilegaonkar, V. P. Zambare and P. P. Kanekar (2004) Antibacterial activity in vitro of plumbagin isolated from Plumbago zeylanica, Biotechnological Approaches for Sustainable Development, Allied Publishing Pvt. Ltd.

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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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V.P. Zambare, M. Ponraj, V. Rubeni () Prevalence and identification of toxic shock syndrome toxin (TSST) producing wound isolates of Staphylococcus aureus from Namakkal District of Tamil Nadu, BioChemistry: An Indian Journal 5(2): 1-9

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Nine- skin infection, eight- burn and nine- accident wound samples were analyzed for the isolation of Staphylococcus aureus on selective Mannitol salt agar (MSA) media and identified by biochemical tests. Out off 31 samples, 10 samples showed presence of S. aureus and out of which burn samples had highest (50%) prevalence of S. aureus. All S. aureus showed multiple antibiotic resistance especially against penicillin (100%), vanco- mycin (90%), methicillin (80%) and oxacillin (60%). The lowest resistance (30%) was against chloramphenical, erythromycin and trimethoprim. High- est antibiotic resistance of 74, 56.6 and 33.3% were obtained from accident wound samples, burn samples and skin infection isolates, respectively. Among the 10 isolates, 5 isolates produced the toxic shock syndrome toxin (TSST) gene and was confirmed by PCR amplification. TSST gene was present only in skin infection samples and few burn samples but not in accidental wound samples.All 5 isolates with TSST gene were also producing toxin TSST and was confirmed by SDS-PAGE. To the best of our knowl- edge this is the first report on PCR detection of the TSST-1 gene in S. aureusfromNamakkal District of Tamil Nadu, India.



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