PRODUCTION OF EXTRACELLULAR PIGMENT FROM

International Journal on Applied Bioengineering, Vol 9, Issue 2, July 201523PRODUCTION OF EXTRACELLULAR PIGMENT FROM MICROBES AND ITSAPPLICATIONV.Navin raju, T.Radha.Department of Biochemistry, KonguArts and Science College, Erode, Tamilnadu.Abstract—Vegetable sample procured from local markets of Tamil Nadu were used in this study to isolate pigments producing bacteria.Red pigment bacteria were isolated and developed into nutrient media. Morphological observations revealed that the isolatedbacteria are Gram Negative, cocci and biochemical characterization was carried out and isolated are identified asRhodotorula sp. Maximum production of pigments was observed at 35 C and pH 7. Pigment was extracted from the isolatedmicroorganism and it was dried and powdered. Extracted pigment was check for the microbial action and it was used todyeing the cloth.Key words : Biocolours, Bacteria, Microbial action, Dyeing.INTRODUCTIONNatural colours are generally extracted from fruits,vegetables, roots and microorganisms and are oftencalled “biocolours” because of their biological origin (Pattnaik et al., 1997).Heath environmental concerns due to unmonitoredutilization of synthetic colorants revived interest in naturaldyes as they are safer, healthier, biodegradable, andexhibit higher compatibility with the environment ( Fatimashatila et al., 2013 )Many artificial synthetic colorants are widely used infood stuff, dye, cosmetic and pharmaceuticalmanufacturing processes. The synthetic pigments whichare used produce harmful effect to human and pollutewater and soil. In textile industry during manufacturingand usage approximately 10-15% of the dye is lostdirectly to wastewater and pollute the environment (Palanivel velmurugan et al., 2009 ).Natural pigments can be obtained from two majorsources namely plants and microorganisms. Theaccessible authorized natural pigments from plants havenumerous drawbacks such as instability against light,heat, or adverse pH and low water solubility.The advantage of pigment production frommicroorganisms include easy and fast growth in thecheap culture media. Microoganisms produce variouspigment like carotenoids, melanins, flavones, quinines.The various types of microorganisms like bacteria, fungi,algae, yeast are present in different colours. The colouredmicroorganisms are isolated from various samples andpigment was extracted from the microorganisms and itused for industry ( Sahara sayeed khan et al., 2013 ).Different pigmented microorganisms were isolatedfrom the vegetable effects on temperature and pH forpigment production was identified. The particular strainwas identified by using different biochemical test. Hencemicrobial pigment production is one of the emerging fieldsof research to demonstrate its potential for variousindustrial application.There is an increasing demand for natural colour inthe food, pharmaceutical, cosmetics, textile, printing anddye industry.MATERIALS AND METHODSCollection of Samples:Vegetable (beetroot) sample was collected fromlocal market in Erode, Tamil Nadu, India.Sample preparation:1gm of vegetable (beetroot) was mashed with 10 mlof distilled water and it was used.Isolation and screening of pigmentedmicroorganisms:The sample was serially diluted up to 10-3 and 0.1ml of the diluted sample was spread on nutrient agarplate. Incubated for 24hrs at 300C and it was checked for

24International Journal on Applied Bioengineering, Vol 9, Issue 2, July 2015the pigmented production. The pigment producedcolonies were used for the future studies.Purification of culturesPigmented Bacterial isolates was purified bystreaking onto nutrient agar plate and it was incubated for24hrs at 300C.Maintenance of culturePigmented Bacterial cultures were grown on nutrientagar and it was maintained at 2-40C temperature inrefrigerator and sub cultured into respective medium.Characterization of isolated pigmentedmicroorganisms:Gram stain:The stain makes use of the differing membranestructures between Gram positive (single cell membranewith a tough outer cell wall of peptidoglycan), and Gramnegative organisms (have two layers of membranes, witha thin layer of peptidoglycan sandwiched between them).Prepare a bacterial smear and heat fixed on a slide,pour a few drops of crystal violet on a smear waite for 1minute and wash with water. Now fixed the smear withGram’s iodine for 1 minute and wash again with waterand decolourize the stain with 95% ethyl alcoholdropwise, wash with water and conter stain with safranine( 45 sec ) and again wash with water. After dryingexamine under oil immersion.hydrogenlyase. Red color developed indicates Positiveresult and yellow color developed indicates Negativeresult.Voges‐ Proskauer TestIt is used to determine the ability of an organism toproduce acetoin; 2,3 butanediol; and ethanol whichcauses lowering of the pH than the methyl red positiveorganisms. VP test detects the presence of acetoin,which is a precursor to 2,3 butanediol.Citrate UtilizationIt is used to determine if an organism is capable ofusing citrate as the sole source of carbon with productionof the enzyme citratase. The media contains sodiumcitrate as the carbon source, and ammonium salts as thenitrogen source, with bromothymol blue as the pHindicator. An organism that uses citrate breaks down theammonium salts to ammonia, which creates an alkalinepH. Alkaline pH causes media to change from green toPrussian blue shows positive result and no color changein the medium denotes negative result.Catalase TestVarious Biochemical test which was performed aslisted below:It is used to test for the presence of enzymecatalase. Hydrogen peroxide (H2O2) is formed as an endproduct of the aerobic breakdown of sugars. When H2O2accumulates, it becomes toxic to the organism. Catalasedecomposes H2O2 and enables the organism to survive.Only obligate anaerobes lack this enzyme. Bubbling (O2gas is liberated from the H2O2) shows positive result andno bubbling denotes negative result.Indole test:Nitrate Reductase TestUsed to determine the ability of an organism to splitindole from the amino acid tryptophan using the enzymetryptophanase. Red layer formed on surface of the mediashowed Positive result and yellow layer showed negativeresult.It is used to determine the ability of an organism toreduce nitrate (NO3) to nitrite (NO2) or nitrogen gas (N2)by the production of the enzyme nitratase. The reductionof nitrate to nitrite or nitrogen gas takes place underanaerobic conditions in which an organism derives itsoxygen from nitrate. Appearance of red color denotes thatnitrate is reduced to nitrite and it is a positive result. Ifthere is no color change the confirmation test was doneby adding a small pinch of zinc powder.Methyl Red TestIt is used to determine the ability of an organism toproduce mixed acid end products from glucosefermentations. Some organisms produce large amountsof various acids (lactic, acetic, succinic, formic) plus H2and CO2. The large amounts of acids lower the pH tolower than 5.0. These organisms also produce greatamounts of gas due to the presence of the enzyme formicUrease TestUsed to determine the ability of an organism to spliturea to form ammonia (an alkaline end product) by theaction of the enzyme urease. Media also contains the pH

Navin Raju.V et. al. : Production of Extracellular Pigment from Microbes.indicator phenol red, which turns an intense pink atalkaline pH. Intense pink/red color indicates positiveresult and no color change denotes negative result.Triple sugar iron agar test (TSI)This test is specifically used for the identification ofenteric bacteria, but can also be used for otherorganisms. This agar contained 0.1 % glucose, 1.0%lactose, and 1.0% sucrose in one tube. Along with thethree sugars, phenol red was also present to verify iffermentation occurred.Effect of different pH & Temperature on the pigmentcolorSelection of pH & temperature for pigment producingmicroorganisms.Table 1 Red pigment microorganism was terated with different pH &TemperatureColourLipid hydrolysisIt is used to determine the ability of an organism toproduce the enzyme lipase which hydrolyzes fat. Lipasesplits fats into glycerol and fatty acids that can be usedfor anabolism or energy production.Extraction of pigmentsDifferent solvents like ethyl acetate, methanol,acetone, hexane was used to check for the maximumsolubility of pigments. And the solvent was selected bychecking the maximum solubility of pigment in it. Afterincubation the bacterial cells were washed with methanoland it was transferred to centrifuge tube. The tube wascentrifuged at 5000 rpm for 15 minutes. The coloredsupernatant and pellet was separated. The coloredsupernatant was used directly and pellet was transferredto evaporating dish for one day and kept at lourchange2Light pink25No4Light pink40No7Red55No8Violet60No10VioletAbove 60PinkWhen the Red pigment producing colonies aretreated with different pH, different coloured pigments areproduced. Table 1 shows the various colours producedwhen pH & Temperature is changed ( Ahmad et al.,2012 ). When the temperature is changed from 250C to600C there was no change but above 600C the Redcolour colonies were changed to pink colony.RESULTS AND DISCUSSIONFig.1. Isolation of pigmented microorganism from sampleFrom the vegetable (beetroot) extract, one pigmentedmicroorganism was isolated.Fig.2Violet colour microorganism ( pH & 10 )

26International Journal on Applied Bioengineering, Vol 9, Issue 2, July 2015Table 2. Biochemical characterization of microorganismFig.3 Pink color microorganism ( Tem: above 600C )Biochemical test resultsThe biochemical results for the Red pigmentedmicroorganism was shown in the following Table 2. Thetable shows that the Red pigment producingmicroorganism can split indole & can produce mixed acidend products from glucose fermentations with productionof citrate, catalase, nitrate reductase & urease enzymes,TSI test shows that this organism can ferment glucoseonly. These organism can also hydrolyse lipidsFig4.Indole testVoges‐ Proskauer TestCitrate UtilizationBiochemical TestRedIndolePositiveMethyl redPositiveVoges tarate reductasePositiveUreasePositiveTSI TestGlucoseLipid hydrolysisPositive.Catalase Test

Navin Raju.V et. al. : Production of Extracellular Pigment from Microbes.Fig5.Nitrate Reductase TestUrease TestTriple sugar iron agar test27Lipid hydrolysisExtraction and drying of pigmentPigments were extracted from the isolatedmicroorganisms. Methanol was used for the pigmentextraction and the extracted pigment was dried and itwas powdered.Fig7.Application of extracted pigmentFig.6.Microbial action of extracted pigmentsPigments that are isolated from the microorganismswas checked for the microbial action. Extracted pigmentwas streaked on to the nutrient agar plate and it wasincubated at 300C for 24 hrs. After incubation there wasno microbial growth.Extracted extracellular pigment was used for dyeingthe cloth. These microbial pigments can also used forcoloring candles, paper, soap, pencil, and high lighterpen.Fig8.Identification of microorganismsMicroorganisms weremicroorganisms.identifiedusingknown