• Fermentation Tests
    At Celignis Analytical

Fermentation is a well-known industrial process; however, development of fermentation processes requires knowledge of an array of important fators including: biomass, the microbes used for fermentation, nutrient media, and fermentation conditions.

Here at Celignis our team is highly experienced in numerous types of fermentations and can help you determine and optimise the potenital yields of an array of different fermentation products. If you already have a technology, e.g. pre-treatment and/or hydrolysis, that produces a sugars-containing liquid then we can undertake fermentation tests directly on that. Alternatively, if you are starting with a feedstock and looking for the best approach to get your targetted fermentation product(s) in high yield then we can help you optimise the whole process, covering pre-treatment, hydrolysis and the subsequent fermentation.

Types of Fermentations We Undertake


Fermentation can be divided into three types:

Bacterial Fermenation

Yeast and Fungal Fermenation

Algal Fermenation

1. Bacterial Fermentation

Bacteria are mainly used to produce organic acids and alcohols by anaerobic fermentation and enzymes by aerobic fermentation processes. Very well-known natural fermenters are lactic acid bacteria (LAB) for lactic acid production and Bacillus species such as B. subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium etc. for the production of enzymes, antibiotics, surfactins, and biopolymers.

Get more info...Bacterial Fermentation









We have expertise in the following bacterial fermentations:

Lactic Acid Fermentation - At Celignis we have expertise and experience in screening lactic acid bacteria for the selection of substrate- and product-tolerant strains. We can also develop: fed-batch strategies to achieve high cell mass, and in situ product recovery techniques to separate lactic acid from the fermentation broth. We will work with you and develop bespoke lactic acid fermentation methods for your feedstock or industrial waste streams.
Get more info...Lactic Acid Fermentation



Propionic Acid Fermentation - We can perform anaerobic fermentations and develop fermentation strategies to achieve high cell mass and in situ product recovery techniques. We can screen your feedstock for propionic acid production, adapt the strain to any inhibitors present in the feed, and develop bespoke fermentation and product recovery processes.
Get more info...Propionic Acid Fermentation



Butyric Acid Fermentation - We have strong expertise in Clostridial fermentation. We can isolate and or adapt the strains that are suitable for your feedstock and can develop fermentation strategies to reduce substrate and product inhibition. We will innovate with you for you.
Get more info...Butyric Acid Fermentation



Butanol Fermentation - At Celignis, we have considerable expertise in Clostridial fermentation and especially butanol fermentation. Our Chief Innovation Officer Dr Lalitha Gottumukkala has extensively worked in this area and has isolated novel strains and developed novel methods for non-acetogenic butanol fermentation as part of her PhD.
Get more info...Butanol Fermentation



1,3-PDO Fermentation - At Celignis, we have expertise and experience in performing anaerobic fermentations and developing fermentation strategies to achieve high cell mass and in situ product recovery techniques. We can screen your feedstock for 1,3 PDO production, adapt the strain to any inhibitors present in the feed and develop bespoke fermentation and product recovery process.
Get more info...1,3-PDO Fermentation



Polyhydoxy Alkanoates (PHAs) Production - We have experience in enrichment of desired microorganisms, fed-batch and continuous fermentations with cell-recycling. We can design and develop the most suitable process for your feedstock by using mixed or mono-culture fermentations. We can also develop cost-efficient downstream processing steps for efficient PHA extraction by using non-toxic and environmentally friendly techniques.
Get more info...PHAs Production





2. Yeast and Fungal Fermentation

Yeast fermentation is one of the oldest fermentations and is used in everyday life to produce a variety of commodity products including bread, beer, wine, cheese, and soy sauce. A few decades ago, yeast gained popularity as an industrial strain for biorefinery and biofuel applications.

Get more info...Yeast Fermentation









We have expertise in the following yeast and fungal fermentations:

Ethanol Production - We can use a variety of yeast types to produce bioethanol. We can ferment the liquids obtained in a prior hydorlysis step (separate hydrolysis and fermentation, SHF) or we can combine the hydrolysis and fermentation stages usingf simultaneous saccharification and fermentaiton (SSF) or simultaneous saccharification and co-fermentation (SSCF) processes.
Get more info...Ethanol Production



Glycerol Production - Bio-glycerol can be produced in high glucose concentrations using osmo-tolerant yeasts.
Get more info...Glycerol Production



Single Cell Oils (SCOs) Production - We can use selected yeast and fungal strains to produce lipids in a variety of substrates such as different types of sugars, glycerol, etc.
Get more info...Single Cell Oils Production



Emulsifiers Production - For this application it is important to optimise the process for product quality and quantity. This is only possible through detailed understanding of microbial strain, its metabolism and needs. We can guide you through and optimise the process.
Get more info...Emulsifiers Production





3. Microalgal Fermentation

Algal cultivation is complicated and requires optimisation to achieve high biomass yields. Algal biomass production depends on nutrient uptake and other environmental conditions such as temperature, pH, salt concentration etc. It is important to select the strain based on the type of production (open ponds, photobioreactors), feedstock and application. We have particular expertise in the evaluation and optimisation of algae thorugh our Chief Innovation Officer, Lalitha, who is currently undertaking a Marie-Curie funded project at Celignis on this topic.

Get more info...Microalgae Fermentation






Contact Us For Further Details

We are available to answer any questions you may have on how to get high value chemicals and biofuels from biomass through fermentation processes. Just get in touch with us by sending us an email info@celignis.com, giving us a call at (+353) 61 371 725, or through our contact form.

Get more info...Get in Touch





Publications on Fermentation By The Celignis Team

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

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.

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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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.

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.

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.

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.

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

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

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

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

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
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.

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.

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.

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

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

Vasudeo Zambare, Archana Zambare, Lew Christopher (2011) Enzymatic hydrolysis of hemicellulose from corn stover and prairie cordgrass, Advances in Biotech Research, Microbiologist Society and Education Book Publisher & Distributor
Singhania R.R, Sukumaran R.K, Rajasree K.P, Mathew A, Gottumukkala L.D, Pandey A (2011) Properties of a major ?-glucosidase-BGL1 from Aspergillus niger NII-08121 expressed differentially in response to carbon sources, Process Biochemistry 46(7): 1521-1524

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

V. P. Zambare (2010) 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.

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.

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.

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

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

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

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

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

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

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

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