Biofilm-Mediated Enhanced Crude Oil Degradation by

international research diary of environs skills________________________________ ISSN 23191414 Vol. 2(2), 48-52, February (2013) Int. Res. J. Environment Sci. Isolation, line drawing and Identification of diesel Engine inunct Degrading Bacteria from store S crude rock vegetable fossil vegetable rock anoint color colour and Comparison of their Bioremediation Potential Teli Nikhil1, Verma Deepa2, Gavankar Rohan1 and Bhalerao Satish3 1 Department of Biotechnology, Viva College, Virar (W), Maharashtra, INDIA 2 Department of Botany, Viva College, Virar (w), Maharashtra, INDIA 3 Department of Botany, Wilson College, Mumbai, Maharashtra, INDIA Available online at www. sca. in Received 30th November 2012, revised 12th January 2013, accepted twenty-fifth January 2013 snitch The rate of bio abasement of diesel motor motor motor locomotive railway locomotive motor locomotive engine anele by microorganisms isolated from garage disgrace (petroleum pollute s crude oil color color) was studied. Modified diesel engine oil average was engagementd and two most abundant microorganisms were isolated from garage soil genus genus genus Micrococcus sp. and genus Pseudomonas sp. were implant to be hydro coulomb degraders and these two bacterias were selected for the degradation test. The degradation of diesel engine oil was monitored at a five twenty-four hour period interval up to twenty five sidereal sidereal mean solar day period, using gravimetric method. laterward 25 days of incubation period, Pseudomonas sp. profuse 67. 57 % of the oil and Micrococcus sp. with 52. 95 %. But the categorization of Micrococcus sp. and Pseudomonas sp. were make to have great potential to degrade diesel engine oil i. e. 89. 98 % after 25 days. The rate of degradation of diesel engine oil by Micrococcus sp. was found to be 7. 48 x 10-4gm/hr and that of Pseudomonas sp. was 9. 55 x 10-4gm/hr while the mixture of two(prenominal) bacterial isolates showed highest rate of degradation of diesel engine oil i. e. 1. 27 x 10-3gm/hr.Keywords Bioremediation, Diesel engine oil, oil spills, hydrocarbon degraders, Micrococcus sp. , Pseudomonas sp. Introduction As we dig deeper into the modern industrial age of technologies, several(prenominal) aspects of human life change. People benefit by and large from life development and many live in successfulness, but prosperity has a price. This price is paid by our environment that suffers daily from entirely kinds of pollutants and destruction. People now have to find ways to cure this destruction. Oil contamination is oneness of the most dangerous pollution factors known today.It earth-closet cause a threat to the environment. It is very fe bed by environmentalists and its very backbreaking to control if it gets out of hand. Oil spills have been a major(ip) end across decades. One of the famous oil spills which are also on-going is in Taylor Energy Well in gulf of Mexico, U. S. A caused out- of-pocket to Hurri taile Sept 16, 2004 till present date and almost 0. 03- 0. 05 tones oil/per day is estimated to leak. An separate recent oil spill was in Mumbai (India) and caused due to the escape valve in Mumbai-Uran pipeline dated January 21, 2011 and about 55 tons of oil was leaked in Arabian Sea.Various such accidents occur throughout the years and it causes mal pass over to our surrounding. Diesel engine oil, which is one of the major products of crude oil, constitutes a major source of pollution in our environment. With the combined dependence on diesel engine oil by some vehicles and generators, greater quantities are being transported over long distances. Therefore diesel engine oil can enter into the environment through wrecks of oil tankers carrying diesel oil, cleaning of diesel tanks by merchants, war ships carrying diesel oil and motor mechanics1. Diesel oil spills on agricultural land generally reduce plant reaping.Suggested reasons for the minify plant growth in diesel oil contaminated soils incline from direct toxic effect on plants2 and reduced germination to unequal soil condition due to insufficient aeration of the soil because of the displacement of radiate from the space between the soil particles by diesel engine oil3. Among several cleanup techniques available to remove petroleum hydrocarbons from the soil and groundwater, bioremediation processes are gaining ground due to their simplicity, higher efficiency and costeffectiveness when compared to other technologies4.This sight was therefore designed to monitor the rate of biodegradation of diesel engine oil (hydrocarbon) by microorganisms isolated from garage soil (petroleum contaminated soil), by using gravimetric method. Material and Methods Preparation of modified diesel oil medium The modified diesel oil medium comprised of 0. 7 gm K2HPO4, 0. 1 gm (NH4)2SO4, 0. 3 gm KH2PO4, 0. 3 gm MgSO4 7H2O, 2. 2 gm agar agar5. The mineral components of the medium were dissolved in vitamin C ml of distilled water and heterogeneous with 2 ml of Gulf diesel engine oil. The medium was autoclaved at 121oC for 15 min. International Science relation back Association 8 International Research ledger of Environment Sciences______________________________________________ ISSN 23191414 Vol. 2(2), 48-52, February (2013) Int. Res. J. Environment Sci. Enrichment of microorganisms Microorganisms capable of degrading diesel engine oil were enriched in unfertilised modified diesel engine oil medium by inoculating soil (which was collected from Maharashtra garage, 65 years old garage at Sewri) in to the medium in 250 ml conical flask. 0. 5 gm of this garage soil was inoculated in to the 100 ml of sterile modified diesel oil fund and allowed to incubate at 37oC for 1 week.Isolation of microorganisms After 1 week of incubation period, 1 drop of enriched nicety was cattle ranch on to the sterile modified diesel oil agar case. The plate was incubated at 37oC for 48 hr. After 48 hr incubation two several(predicate) bacterial colonies were selected from incubated plate. Each bacterial colony type was gun cultured repeatedly onto sterile alimentary agar plates to obtain a pure culture. Pure cultures of bacterial isolates were identified on the basis of their colonial morphology, cellular morphology and biochemical characteristics according to the taxonomic scheme of Bergeys Manual of Determi primordial Bacteriology6.Determination of microbial colony leans for degradation studies 5 ml of sterile Nutrient broth was aseptically inoculated with a loopful of pure culture of village 1(C1) in first test tube and dependence 2 (C2) in second test tube and incubated both the tubes at 37oC for 24 hr. After incubation, the numbers of organisms present in one ml of intellectual nourishment broth were determined by spread plate method. The numbers of organisms were adjusted in both the tubes in such a way that both the isolates contain approximately equal numbe rs of microorganism in one ml of sample by using sterile Nutrient broth as a diluent7.Soil sample collection and preparation Top show up soil sample was collected from the premises of the Shahid Bhagatsingh Ground, Kalachowki in sterilized tensile containers. Soil sample meant for degradation studies was sterilized using autoclave at 121oC for 15 min, after which it was allowed to cool to inhabit temperature for further words. Description and intervention of samples Test i. 12 samples of 15 gm sterilized soil mingled with 1 ml (0. 848 gm) of Sterile Gulf diesel engine oil + 0. 2 ml culture of C1, ii. 12 samples of 15 gm sterilized soil mixed with 1 ml (0. 48 gm) of Sterile Gulf diesel engine oil + 0. 2 ml culture of C 2, iii. 12 samples of 15 gm sterilized soil mixed with 1 ml (0. 848 gm) of Sterile Gulf diesel engine oil + 0. 1 ml culture of C1+ 0. 1 ml culture of C 2 Control 12 samples of 15 gm sterilized soil mixed with 1 ml (0. 848 gm) of Sterile Gulf diesel engine oil + 0. 2 ml of sterile distilled water. Diesel oil degradation studies The great power of C1, C2 and mixture of both the bacterial isolates to degrade diesel oil was monitored on the first day (day zero) of the study and subsequently at 5-day interval for 25 days.Carbon tetrachloride was employed as an extractant. On each day, two samples per single interposition were analyzed for the quantity of residual diesel oil7. Each of the 15gm soil manipulation samples was mixed with 40 ml of carbon tetrachloride, placed in a separating conical flask, shaken vigorously for 3 min and allowed to settle for 5 min. The unstable phase was separated by allowing the supernatant (diesel oil carbon tetrachloride) to pass gradually through a funnel fitted with drop typography (Whatman No 1). Anhydrous sodium sulphate spread on the filter paper was employed to remove any moisture in the mixture.The liquid phase was collected in a 50-ml pre-weighed beaker. The beaker containing the extract was placed in an oven and the extractant allowed to evaporate at 50oC. The beaker with the residual diesel oil was allowed to cool to room temperature and weighed to determine the quantity of residual diesel oil by difference8. Results and countersign In this study, the soil samples were gathered from the garage (oil contaminated site) because the capability of native bacterial population to mineralize crude oil hydrocarbons in oil contaminated sites was confirmed before by many scientists9.The rate of biodegradation of Diesel engine oil by hydrocarbonoclastic organisms isolated from garage soil were assessed. Table 1 and table 2 shows that, using cultural characteristics and biochemical characteristics, two bacterial isolates Micrococcus sp. and Pseudomonas sp. were identified by compairing it with the Bergeys manual of determinative bacteriology. The number of CFU/ml of both the bacterial isolates was adjusted to 7. 88 x 107 CFU/ml for degradation studies. The biodegraders which were Mi crococcus sp. , Pseudomonas sp. and Mixture of both the culture showed various abilities in the breakdown and utilization of the diesel engine oil. Character Colony 1 Colony 2 Size 1-2 mm 2-3 mm Table-1 Colony characteristics of bacterial isolates on Nutrient agar plate Shape airlift Colour Consistency Circular Irregular Convex Flat yellow-bellied Fluorescent green Butyrous Mucoidal Opacity Opaque Translucent International Science Congress Association 49 International Research diary of Environment Sciences______________________________________________ ISSN 23191414 Vol. 2(2), 48-52, February (2013) Int.Res. J. Environment Sci. Table-2 Biochemical characteristics of bacterial isolates C1 absolute Cocci Clusters No spore Non motile Positive Negative Negative Negative Positive No upheaval No Fermentation No Fermentation No Fermentation Acidic, No gas, No H2S Negative Negative Negative Positive Micrococcus sp. Character Gram stain Morphology Arrangement Endospore Motility Catalase Oxidase citrate Indole Gelatin Glucose fermentation Lactose fermentation Sucrose fermentation mannitol fermentation Tripple sugar iron Methyl red Voges proskauer Nitrate decrement Urea OrganismC2 Negative Rods Solitary No spore Sluggishly Motile Positive Positive Positive Negative Positive No Fermentation No Fermentation No Fermentation No Fermentation Alkaline, No gas, No H2S Negative Negative Negative Negative Pseudomonas sp. Table-3 lean of diesel engine oil extracted (on various days) from 15 gm soil samples grime with 1 ml (0. 848 gm) of sterilize diesel oil and 0. 2 ml of culture Day Sample I II leash IV I II III IV I II III IV I II III IV I II III IV I II III IV burthen of diesel oil extracted (gm) 0. 848 gm 0. 848 gm 0. 848 gm 0. 848 gm 0. 807 gm 0. 801 gm 0. 30 gm 0. 848 gm 0. 787 gm 0. 639 gm 0. 639 gm 0. 848 gm 0. 663 gm 0. 348 gm 0. 483 gm 0. 848 gm 0. 545 gm 0. 290 gm 0. 271 gm 0. 848 gm 0. 399 gm 0. 275 gm 0. 085 gm 0. 848 gm Weight of diesel oil degraded (gm) 0. 000 0. 000 0. 000 0. 000 0. 041 0. 047 0. 018 0. 000 0. 061 0. 209 0. 209 0. 000 0. 185 0. 500 0. 365 0. 000 0. 303 0. 558 0. 577 0. 000 0. 449 0. 573 0. 763 0. 000 Rate of degradation (gm/hr) 0. 00 0. 00 0. 00 0. 00 3. 42 x 10-4 3. 92 x 10-4 1. 50 x 10-4 0. 00 2. 54 x 10-4 8. 71 x 10-4 8. 71 x 10-4 0. 00 5. 14 x 10-4 1. 39 x 10-3 1. 01 x 10-3 0. 00 6. 31 x 10-4 1. 6 x 10-3 1. 20 x 10-3 0. 00 7. 48 x 10-4 9. 55 x 10-4 1. 27 x 10-3 0. 00 0 5 10 15 20 25 * determine are means of twice determinations. Key i. Sterilized soil + Sterilized diesel oil + Micrococcus sp. ii. Sterilized soil + Sterilized diesel oil + Pseudomonas sp. iii. Sterilized soil + Sterilized diesel oil + Micrococcus sp. + Pseudomonas sp. IV. Sterilized soil + Sterilized diesel oil International Science Congress Association 50 International Research Journal of Environment Sciences______________________________________________ ISSN 23191414 Vol. 2(2), 48-52, February (2013) Int. Res. J.Environment Sci. Diesel engine oil degradation study by Micrococcus sp It was seen that the rate of diesel oil degradation by Micrococcus sp. was slow as compared to the rate of degradation of diesel oil by Pseudomonas sp. and mixture of Micrococcus sp. and pseudomonas sp. But the diesel oil degradation potential of Micrococcus sp. was continuously increasing as the clipping of contact between oil and organism increased. Diesel engine oil degradation study by Pseudomonas sp It can be seen that the efficiency of Pseudomonas sp. to degrade diesel engine oil is hot than that of Micrococcus sp.As the incubation period increases the rate of degradation of diesel engine oil also increases. But it was seen that till 15th day, the rate of degradation was much faster. This was probably due to the exponential phase of the cell growth but after that the rate of degradation was slightly decreased. It was possibly because of cells of the Pseudomonas sp. were near to its stationary phase of cell growth. Diesel engine oil deg radation study by mixture of Micrococcus sp. and Pseudomonas sp The weight of diesel oil extracted from soil containing diesel engine oil and mixture of both bacterial isolates i. . Micrococcus sp. + Pseudomonas sp. showed continuous weight departure till the 25th day of incubation period. After 5th day of incubation period it was seen that there was a drastic increase in the rate of diesel oil degradation till the 25th day of incubation period which was quite higher than that of the single culture of Micrococcus sp. as well as that of the Pseudomonas sp. In this case it was found that around 90% of the diesel engine oil was degraded after 25th day and rate of degradation of diesel oil was found to be continuously increasing i. e. 1. 50 x 10-4gm/hr after 5th day to 1. 7 x 10-3gm/hr after 25th day. Conclusion When Micrococcus sp. is used in combination with Pseudomonas sp. it showed a great potential to diesel oil degradation. This was probably due to the different enzyme system fro m two different bacterial isolates that acts on hydrocarbon at a time which proved to be an excellent option to degrade that hydrocarbon if both the bacterial enzyme system posses capacious efficiency to act upon it and to degrade it10. This was followed by single culture of Pseudomonas sp and then Micrococcus sp. The oil degradation by Pseudomonas sp. as not surprising not barely because it was isolated from garage soil which was already contaminated by oil and grease but also because it is known to possess a much competent and active hydrocarbon degrading enzyme system than Micrococcus sp. It is known to be fast growing and is capable of degrading a wide miscellany of organic compounds11. In the case of Micrococcus sp. which is also known to posses the considerable efficiency to use it as an oil degrader, but it requires more time compared to that of the Pseudomonas sp. Figure1 Comparison of % Diesel engine oil degradationInternational Science Congress Association 51 Internati onal Research Journal of Environment Sciences______________________________________________ ISSN 23191414 Vol. 2(2), 48-52, February (2013) Int. Res. J. Environment Sci. Figure2 Comparison Rate of Diesel engine oil degradation (gm/hr) By using biological processes, as in the case of bioremediation, usually lowers the costs as compared to chemical treatment processes for various contaminated sites. It is also less disturbing to the environment. However, because it is a raw(a) process, it requires time.The above experiment shows that bioremediation can be used effectively to treat oil contaminated soil. The remarkable rate of diesel oil degradation by bacterial isolates shown by this method allows for the safe and convenient use of this microorganism in the oil contaminated area. Moreover the results obtained from the comparison between the diesel oil degrading ability of Pseudomonas sp. , Micrococcus sp. and mixture of both helps them to use in different bioremediation processes bas ed upon their efficiencies. And the advantages of employing mixed cultures as opposed to pure cultures in bioremediation have been demonstrated. . 3. Baker J. 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J. , Antai S. P. , Degradation and mineralization of References 1. Hill G. B. , Moxey J. G. , Gasoline and Diesel oil In Gat hee VB (ed) Petroleum merchandise Handbook Mc-Grew Hill, 4, 1-4 NY (1980) 11. Ijah U. J. J. , Okang C. N. , Petroleum Degrading capabilities of bacteria isolated from soil, W. A. J. Biol. Appl. Chem. , 38(1-4), 915 (1993) International Science Congress Association 52