Isolation and characterization of Lipolytic microorganisms from oil contaminated soil.

Transcription

1 Isolation and characterization of Lipolytic microorganisms from oil contaminated soil. Veeranna S. Hombalimath, Basavaraj B. Udapudi Laxmikant R. Patil, Anil R. Shet, Deepak A. Yaraguppi, Gururaj Tennalli, Department of Biotechnology, B.V. Bhoomaraddi College of Engineering and Technology, Hubli, Karnataka , India; ABSTRACT Enzymes are biocatalyst, produced by living systems, are proteineous in nature with catalytic properties Lipases (triacylglycerol acylhydrolases, EC ) are a class of hydrolyses that are primarily responsible for the hydrolysis of acylglycerides. Lipases are produced by various microbes, such as bacteria, fungi, yeast, and also in the pancreas of mammals, like pigs and humans. Commercially useful lipases are usually obtained from microorganisms that produce a wide variety of extracellular lipases. Only about 2% of world s microorganisms have been tested as enzyme sources. In the present study different oil contaminated soils were taken for isolation of lipolytic organisms. Different samples were collected and isolation of lipolytic microorganisms is done by standard plate assay method. This methodology helps in getting cultures with less contamination and abundance of bacteria can be minimized by using antibiotics in culture medium Totally seven strain were isolated, out of seven strains collected two isolates were primarily identified as Aspergillus flavus and two strains identified as Micrococcus. The bacterial strain I3 showed maximum lipase activity of 0.032mg/ml/min at 72 hours of incubation. The bacterial strain shown the maximum activity at 48 hours of incubation was mg/ml/min Keywords: Lipase. Tributyrine. Extracellular. Oil contaminated soil. Standard plate assay method. INTRODUCTION Lipases are produced by animals, plants, and microorganisms. Microbial lipases have gained special industrial attention due to their stability, selectivity, and broad substrate specificity [1] Many microorganisms are known as potential producers of extra cellular lipases, including bacteria, yeast, and fungi [2]. Fungal species are preferably cultivated in solid-state fermentation (SSF), while bacteria and yeast are cultivated in submerged fermentation [3]. Taxonomically close strains may produce lipases of different types. There are many microorganisms known to produce different lipases [4]. Microbial lipases processing either high alkalophilic or hemophilic properties have been reported from Alcaligenes [6] Pseudomonas fragi [6] P. nitroreducens [7]. When screening bacteria for lipase production both culture ph and assay ph are important parameters. The stability depends upon the presence of substrate [8]. Almost all microbial lipases can be regarded as acid lipases or neutral lipases if they are classified by their optimum ph value for the lipolytic activity [9]. The industrial demand for new sources of lipases with different catalytic characteristics stimulates the isolation and selection of new strains [10]. Lipase-producing microorganisms have been found in different habitats such as industrial wastes, vegetable oil processing factories, dairy plants, and soil contaminated with oil and oilseeds among others. Lipases are produced by various microbes, such as bacteria, fungi, yeast, and also in the pancreas of mammals, like pigs and humans. Commercially useful lipases are usually obtained from microorganisms that produce a wide variety of extracellular lipases. Only about 2% of world s microorganisms have been tested as enzyme sources. Fungal Lipase is a specific enzyme that digests fat and is characterized by its ability to hydrolyze fat over a wide range of temperatures and ph. Bacteria produce different classes of lipolytic enzyme, including carboxylesterases, which hydrolyze small ester containing molecules at least partly soluble in water, true lipases, [11] which display maximal activity towards water insoluble long-chain triglycerides, and various types of phospholipids The fat decomposition for the microorganisms is a source of carbon and it begins through lipase enzyme acting on the fats and is accompanied by the formation of glycerol, fatty acids. Mode of action of lipases as a biocatalyst is shown below: Fats (or) Oils + Water Lipase Fatty acids + Glycerol ISSN : X Vol. 2 No. 3 Aug-Oct

2 Lipases from fungi are find use in diverse range of industries like detergents, pharmaceuticals, beverages, dairy etc. which makes them commercially important enzyme and further these are involved in the breakdown and mobilization of lipids within the cells of an individual as well as transfer of lipids from one organism to another. Lipases can catalyze esterification, MATERIALS AND METHODS: All the media ingredients and chemicals required for isolation of bacteria were procured from Hi Media Lab (Mumbai) Ltd. Collection of Soil Sample: Soil samples rich in lipolytic content were collected from surrounding of A.J.Muddod and channabasweshvar oil mill at Hubli Karnataka, India Isolation and screening of Lipolytic microorganisms: Five bacterial and two fungal cultures were isolated by spread plate method on nutrient agar medium (0.5 % Peptone, 1% tributyrin, 0.3 % yeast extract, 2 % agar, 0.5% NaCl ) from soil sample collected. They were identified on the basis of morphological, cultural and microscopic characteristics. In this test all the bacterial and fungal culture were separately plated on tributary in agar plate and incubated at room temperature and after 72 hrs the zone of clearance was recorded. The lipase activity was detected due to occurrence of zone of clearance around the colony and subsequent formation of white precipitate of calcium monolaurate around the colony [12]. Pure cultures of isolates were maintained on minimal media agar slants (Yeast extract, Nacl, ph 7.0 and 2% agar) were subculture for every 20 days. Screening of isolates for the Lipase activity: Lipolytic organisms were screened by qualitative plate assay. Isolates were grown on tributyrin agar base plates and incubated at 36 0 C for two days. Zone of clearance was observed due to hydrolysis of tributyrin. Culturing and characterization of the isolates: The isolates showing the maximum zone of clearance here by referred as I2 is selected for further analysis.morphological and biochemical characterization of isolates have been studied for the identification of the isolates. Lipase production by Submerged Fermentation: The selected Lipase bacteria were tested for yield of lipase e production by submerged fermentation. Fermentation Medium used: Nutrient broth supplemented with used as a Production medium. [13] Inoculums preparation: Loopful of culture was inoculated into 5ml of production medium and incubated at room temperature (27 o + 2 o c) in a rotary shaker with continuous shaking at 200 rpm for 24hrs. After this 250μl of the incubated medium was transferred to 5ml of fresh production medium and was kept in rotary shaker at 200 rpm for 6 hrs at room temperature (27 o + 2 o c). This was used as inoculums for submerged fermentation process. [14] Submerged fermentation protocol: 1ml of the inoculums was inoculated into 50ml of the production medium. This inoculated medium was then incubated up to 72 hrs at room temperature agitating at 200 rpm in rotary shaker. The fermented broth was centrifuged at 10,000 rpm for 20 min; the supernatant was separated and used as crude enzyme source for the assay of protease produced[18] [19] Lipase assay: Lipase assay was performed by simple titration method using Tween 20 as a substrate. Briefly assay mixture contained 0.1ml of tween-20 and crude enzyme 1ml 2 drops of Phenolphthalein indicator in 250ml conical flask. The fatty acid released during the incubation was determined by titration with 0.1N NaOH (alkali) using Phenolphthalein indicator this was performed with all the three isolates and lipase activity was calculated. [15] One unit of lipase activity was defined as the amount of enzyme releasing in one mole of free fatty acids in one minute under standard assay condition. Lipase activity = (Volume of alkali consumed) (Normality of NaOH) (Time of incubation) (Volume of enzyme solution) Results and Discussion Isolation of bacteria The naturally occurring environments provide good protein source for microorganisms. Isolations and screening of bacteria from these natural environments can be supposed to be useful for obtaining bacterial species with potential of producing Lipase enzyme. So in this study isolation of lipase producing bacteria was aimed from soil source contaminated with oils and fats waste which acts as a protein source. The soil sample for this purpose was collected from surroundings of Karnataka Milk Federation (KMF) Dharwad Karnataka, India. The bacterial species were isolated by culturing on Nutrient agar. Five bacterial colonies and two fungal colonies appeared on the plate which was further streaked on nutrient agar plate to get pure culture. All the bacterial isolates were further processed for lipolytic activity. ISSN : X Vol. 2 No. 3 Aug-Oct

3 Screening of isolates for the Lipase activity by tributyrin Agar plate assay All the isolated bacterial colonies were screened on tributyrin agar plate for their lipolytic activity. For this a loopful of each bacterial isolatess were separately inoculated on lipolytic agar plate and incubated at room temperature (27 o + 2 o C) for about 72 hrs. After incubation we found thatt six isolates out of seven isolates showed considerablee lipolytic activity by distinctly forming clear zone around the colonies in tributyrin agar plate (fig.1& fig.2). Of all the cultures I7 showed highest zone of clearance of 36mm diameter (fig.3), followed by I5 whichh showed 31mm diameter (fig.4). Fig.1. Nutrient agar plate containing isolate I3 of 10-3 dilution Fig.2.Nutrienagar plate containing isolate I5 of 10-5 dilution Fig.3 Isolate I7of 10-4 dilution showing white Precipitate Fig.4. Isolate I5of 10-5 dilution showing white precipitate Fig.5. Gram s staining of I3 (gram negative rod). Fig.6.Gram s staining of I5 (gram positive rod) ISSN : X Vol. 2 No. 3 Aug-Oct

4 Graph 1. Enzyme activity of Isolate I3 Incubation Time in Hrs Volume of NaOH(0.1N) consumed (ml) Lipase activity (µ g/ml/min) Vol.of NaOH consumed/lipase activity Graph 2.Enzyme activity of isolate I Incubation Time in Hrs Volume of NaOH(0.1N) consumed (ml) Lipase activity (µg/ml/min) Vol.Of NaOH consumed/ Lipase activity Growth Measurement: The incubated plates were examined for the growth of the culture. The growth increments are measured at intervals of 24, 48,72hrs in all the plates. Growth responsee is recordedd in terms of horizontal zone of clearance. Two isolates I3 and I7 of the oil contaminated soil sample are grown on lipase assay media and observed for the growth increment in terms of clear zone diameter formed around colony. Isolate Number Incubation Zone of clearance (mm) I I Two isolates I 5 and I 7 of the oil mill waste dumped soil are grown on lipase assay media and observed for the growth increment in terms of clear zone diameter formed around colony. ISSN : X Vol. 2 No. 3 Aug-Oct

5 Isolate Number Incubation Zone of clearance (mm) I5 I Conclusion: Lipase enzymes are most important industrial enzymes, accounting a major volume of total worldwide enzyme sales. In the present study three best strains were isolated and characterized for their lipolytic activity. These three isolates I3, I5 and I7 strains shown highest activity at 96 hrs of incubation under submerged fermentation. Further these three strains can be exploited for large scale lipase production References: [1] Lomthaisong K,Buranarom A and Niamsup H. Journal of biological science.2012,12(3): [2] Lau1H.L, Ariff A. African Journal of Biotechnology.2011, Vol. 10(36): [3] [3] Treichel H, Débora De Oliveira, Marcio A Mazutti, Marco Di Luccio, J Vladimir Oliveira. Food and BioprocessTechnology.2009, 3(2): [4] Guranalaxmi K,Kumar sahu,sivakumar K,Tangaradjou T,sudha S and Kannan.Research journal of microbiology.2008, 3(2):73-81 [5] Vakhlu J, Kour A. Journal of Biotechnology.2006,Vol.9 No.1, Issue of January 15 [6] Walterl RP,Douglas B,Gareth J,James R and Dams J. Biotechnology Techniques.1989, Vol.3(2): [7] Rajan A,Soban kumar DR, Nair AJ. International journal of biological chemistry.2011,5(2): [8] Wood RG, Burger M,Anee Bevan and Beacham R. Microbiology. (2001), 147, [9] Baharum SN, Salleh AB, Razak CAN,Bastri M. Annals of microbiology,2003, 53:75-83 [10] Salihul A, Alam Z,Ismail M and Hamzah M. African Journal of Biotechnology,2011, Vol. 10(11): [11] Sharma R,Chisti Y, Banerjee UC. Biotenol Adv, 2001,(19): [12] Sirisha E,Rajshsekar N, Narsu L,Advances in Biological Research,2010,4(5): [13] Simons JW, Van Kampen MD, Riel S,Egmond MR,Verheji HM, Journal of Biochemistry,1998,253: [14] Chaturvedi M,Singh M, Chugh M, Rishi and Rahulkumar,Jornal of Microbiology,2004, [15] Ashok P,Biochemical Engineering Journal,2003,(13):81-84 [16] Sharma R,Christ Y, and Banrjee UC, Biotechnology Advances,2001,19(8): [17] T Nakashima,HFukudo,Skyotani,M Morkawa, Culture conditions for intracellular lipase production by Rhizopus chinesis J. Ferment.Technol. 1988, (66): [18] A B Shalleh,R Musami, MBasri,K Ampon, W M Z Yunus. Extra and intracellular lipase thermophillic R Oreyzae Can J Microbiol, 1993,(39): [19] H.Narhar, Y.Koyama,T Yosida,S.Pichangkuru.R Ueda,H.Teguchi, Growth and enzyme production in SSF by Aspergillus nigar, Ferment Technol (60): [20] R Sharma Y. Chisti, U.C.Banerjee, production and purification of application of lipases, Biotenol, Advan.2001,(19): [21] D Nutan, S. P Ulka,K.B. Bastawade, J.M. Khir, D.V. Ghokhale. Production of acid lipase By Aspergillium Nigeria,in SSF, Process Biochemistry.2002,(33): [22] N Pal, S.Das. A.K Kundu, Influence of culture and nutrinal conditions on production of lipase By submerged culture of Aspergillus niger ISSN : X Vol. 2 No. 3 Aug-Oct