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Degradation of Fats, Oil and Grease (FOG) By Using Lipase Producing Microorganism

Introduction Fats, oil and grease (FOG) in wastewater create problems including the blocked of sewage system and may interfere with the operation of wastewater treatment plants. A wide variety of industries, such as the restaurant trade, the dairy industry and food processing produce effluents rich in FOG. Fats often solidify causing pipes and sewage system to become blocked. Lipids can form oil films on the surface of activated sludge flocs, preventing from the diffusion of oxygen and causing problems in the pumping and aeration systems combined with development of filamentous microorganisms. An alternative needs to be invented to tackle this unsolved problem. Bioremediation of lipid rich wastes, either aerobically or anaerobically have been investigated. Industrial scale extraction of lipase is carried out in bacteria, fungi actinomycetes and culture of plants and animal cells. Among them, microbial lipase are metabolically versatile and hence have advantage in many industrial process. Enzymatic treatment technique has gained more attention because of stringent environmental regulations and clean and friendly applications of enzymes. Lipase are serine hydrolyses of considerable physiological significance and industrial potential that can catalyze numerous reactions such as hydrolysis, interestification, alcoholysis and aminolysis. The purpose of this study is to investigate what type of lipase producing microorganism that able to degrade FOG.

Methodology Isolation of lipase producing microorganism 1. Fat and oil rich wastewater samples were collected from palm oil mill, dairy industry slaughter house, soap industry and domestic water in sterile container. 2. 1.0 g of soil sample were collected from different sites and was stirred in 100ml of double distilled water for the isolation of lipolytic microbes. 3. The serially diluted (10-1 to 10-6 ) samples were plated on tributyrin agar plates. 4. The formation of clear zone around the colony on the plate was considered as lipolytic microbes. 5. Microbes which formed large clear zone around the colony were identified based on morphological, biochemical and physiological characters according to Bergeys manual of determinative bacteriology. 6. Pure cultures of these organism were maintained on nutrient agar slant supplemented with 1% olive oil.

Preparation of BOD test 1. The wastewater sample (5L) taken in 10L container was inoculated with 1% (v/v) bacterial culture (O.D. at 600~2). 2. After vigorous shaking, it was divided with 12 portions of 250ml each in 500ml flasks and rest of the samples were divided into two portions of 1L each in 2L flasks. 3. All the cultures were incubated at 30oC at 200 rpm. 4. Samples were taken at regularly intervals of 48h from 2L flasks for BOD analysis and from two 500ml flasks for the determination of lipid content.

BOD determination 1. BOD bottle containing 300ml diluted sample of sterile distilled water was taken, sterile air was blown for 10 minutes and incubated in dark at 20oC for 5 day prior to test. 2. 2ml of MnSO4 and 2ml of alkaline iodine-sodium azide solution was added to each BOD bottle. 3. Stoppers were placed and air bubbles expelled by inverting bottles several time. 4. Bottles were then left for precipitation. 5. 2ml of H2SO4 was added and mixed by inverting the bottles until iodine was uniformly distributed. 6. Starch indicator (2-3drops) was added to 2ml sample and then titrated with 0.025N Na2S2O3 until blue colour disappeared. 7. Volume of Na2S2O3 was used to calculate the BOD.

Determination of lipid content (using partition-gravimetric method of Kirschman and Pomeroy) 1. 250 ml sample was taken and acidified with 1:1 diluted HCL up to pH 2. 2. Lipid was extracted repeatedly with 30ml portions of 1,1,2-trichloro-trifluoroethane (Freon) until the aqueous phase showed no oil layer and the solvent phase became clear. 3. All the solvents extracts were combined and evaporated at 70oC until dried. 4. The dry weight obtained indicated the amount of oil and grease present in the sample.

COD test 1. Single culture of microorganism that have been identified was grown in wastewater from bakery industry.

2. pH of wastewater was adjusted to 7.0, then 5% of each inoculum was isolated or single total mixed culture were added and shaken at 250 rpm, at 30oC. 3. Sampling were carried out every 24 hours for 7 days 4. COD, fat and oil contents were analysed by standard method (APHA, 1989) presented as percent degradation.