Effective Synergetic Biodegradation of Diesel oil by ... - URP Journals

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ISSN 2277–386X Original Article Effective Synergetic Biodegradation of Diesel oil by Bacteria Cherukupalle Bhuvaneswar, Gunduluru swathi, Baki Vijaya Bhaskar, Tirumalasetty Munichandrababu, Wudayagiri Rajendra* Division of Molecular Biology, Department of Zoology, Sri Venkateswara University, Tirupati -517502, A.P., India. Email: [email protected] Received 02 October 2012; accepted 23 October 2012 Abstract Oil spill pollution is considered to be one of the major environmental hazards. Biodegradation is a process by which the toxic complex compounds are converted into less toxic and/or nontoxic simpler compounds by the microorganisms. Bacteria can degrade the diesel oil and utilize the hydrocarbons as carbon source for their own energy needs, growth and reproduction. In the present study five bacterial strains were isolated from the diesel contaminated soil and two of them show maximum growth and were identified as Pseudomonas and Staphylococcus based on the morphological and biochemical characteristics. Hydrocarbon utilization test and Biodegradability test indicate that the mixed cultures of Pseudomonas and Staphylococcus synergetically can degrade the diesel oil more effectively when compared to the individual species. © 2012 Universal Research Publications. All rights reserved Key Words: Diesel; Synergetic degradation; Redox indicator; Biodegradation. INTRODUCTION Microbial application for the removal of contaminants/pollutants from the environment is one of the best techniques employed over the past few years. Normally pollutants are toxic either in their native form or modified to be toxic and hence there is an imperative need to remove these pollutants. Environmental damage due to spills during commercialization of diesel causes adverse effect both on target and non target species. Many oil producing communities have been suffering from the accidental discharge of oil spillage on soil or water surface. Diesel oil is a complex mixture of normal branched and cyclic alkanes and aromatic compounds which contains 2000 to 4000 hydrocarbons obtained from the middle distillate fraction during petroleum separation [1]. Although diesel oil is one of the major energy sources, it plays a key role in the global environmental pollution. Prolonged exposure and high oil concentration may cause kidney disease, possible damage to the bone marrow and an increased risk of cancer [2]. Diesel oil spills on agricultural land generally reduce plant growth. Suggested reasons for the reduced plant growth in diesel oil contaminated soils range from direct toxic effect on plants [3] to unsatisfactory soil condition due to insufficient aeration of the soil because of the displacement of air from the space between the soil particles by diesel oil [4]. The toxicity of aromatic

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compounds has been widely documented and their disastrous effect towards human and environment is greatly concerned. It causes negative effects to aquatic flora and fauna as well. Previous reports elucidated that the oil spilled area is rich with oil degrading bacteria include Psuedomonas, Micrococcus, Staphylococcus, Bacillus, Flavobacterium, Acromobacter, Klebsiella, Actinomycetes, Acetobacter, Rhodococcus etc which can utilize hydrocarbons as primary carbon source and hence they are called Hydrocarbon utilizing bacteria. Although there are several reports on bioremediation of pollutants by the action of different bacterial strains, only very few studies reported on the synergetic effect of consortium on biodegradation of diesel oil. Individual organisms often prefer to metabolize only a limited range of hydrocarbon substrates. The adopted microorganisms degrade diesel oil normally but rate of this action is critically depends on different environmental factors which include temperature, water availability, microbial composition, geology of polluted site, contaminant type and chemical conditions at the contaminated site. The bioremediation of petroleum products such as diesel oil has been tried successfully many a time at the commercial scale; however it is known that intrinsic bioremediation rates are typically slow, taking several years to fully

International Journal of Environmental Biology 2012; 2(4): 195-199

cleanup sites with high levels of concentration of Hydrocarbons. Over the past few decades, pollution of the environment due to oil spillage has been increasing steadily. In spite of clean up of the diesel contaminated areas with the physical and chemical methods, it has become increasingly difficult to eliminate the accumulated diesel and their byproducts completely from the soil. Most of the researchers pay attention towards the ecofriendly techniques rather than physico-chemical processes due to their cost effective and laborious process. Hence, bioremediation, a technique based on the action of microorganisms, has become an attractive alternative to convert hazardous contaminants into nontoxic substances such as CO2, H2O and biomass. Although some work has been done related with the bioremediation of diesel oil by microorganisms, very few successive reports have been recorded with particular reference to synergetic degradation. The information that is available regarding this synergetic degradation of diesel oil is not much profound. Hence, keeping in view the importance of synergetic effect of degradation, the present work was carried out in such a way that helps to convert toxic complex compounds into less toxic and/or nontoxic simpler compounds by the combined effect of microorganisms. MATERIALS AND METHODS Collection of soil sample: Diesel spilled soil sample used in this experiment was collected from diesel filling station in Tirupati town, Chittoor district, A.P., India. Prior to testing, the soil sample was air dried and mixed thoroughly to increase homogenisity and shifted through 2mm (millimeter) sieve and stored at 40C for further study. Physico-chemical properties: The physico-chemical parameters for soil was analysed by using standard procedures. pH for 1:1 soil/water mixture was determined by using pH meter [5]. The temperature of soil sample was determined by using mercury thermometer. The total organic carbon and nitrogen were determined by the methods of Walkey and Black [6] and Microjeldhal [7] respectively. The phosphorous content was determined [8]. Electrical conductivity was estimated by the addition of 100ml of distilled water to 1gm of soil sample in Elico conductivity meter. Cent percent water holding capacity of soil sample was measured by finding out the amount of water added to get saturation point and then 60% water holding capacity was estimated [9]. Isolation and Enumeration of Bacteria: Isolation of bacteria from soil sample was performed by taking 1 gm of soil dissolved in 9ml of distilled water, followed by serial dilutions up to 10-8 . Different diluted suspensions such as 10-4, 10-5, and 10-6 were plated and spread with sterile spreader on nutrient agar (pH 7.4). After gentle spreading, the nutrient agar plates were incubated at 370C for 24h. After the incubation period, enumeration of different isolates was carried out by counting the colonies appeared on the agar surface by using colony counter. Selected isolated colonies of bacteria were maintained as pure cultures and stored at 40C for future studies. Characterization of isolates: The isolates were subjected to morphological and biochemical characterization. The

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morphological characters such as size, shape, elevation, motility were determined [10]. Biochemical characteristics of the isolates were characterized [11]. Different tests such as Gram’s staining, Indole test, Methyl red test, Vogus proskaur test, Citrate utilization, Catalase test, Endospore formation, Lactose fermentation were performed adopting standard procedures. Bergey’s Manual of Determinative Bacteriology was used as reference to identify the isolates. Hydrocarbon utilization test: The bacterial isolates were tested for their ability to utilize diesel oil as sole source of carbon and energy for their growth. The bacterial inoculated mineral salt media without hydrocarbons (diesel) served as control. The overnight cultures of the bacterial isolates were inoculated into mineral salt media with 10%v/v of diesel served as sole carbon source. The bacterial cultures were kept for 24-48 hrs incubation at 370C on mechanical shaker. Growth of the organisms was assayed at regular time intervals up to 15 days by measuring the optical density at 600nm. The bacterial growth determination at 10%v/v of diesel oil were measured and compared with control. Biodegradation test: The biodegradability test for the synergetic degradation of diesel oil by using mixed culture was verified by using redox indicator, 2, 6 DCPIP. 0.5 ml of each bacterial culture was added to the test tube that contained 10 ml of sterile Bushnell-Hass (BH) medium and 1%v/v of the diesel oil. BH medium which contains inoculum and DCPIP (2mg/ml) indicator but not hydrocarbons (diesel oil) was maintained as control. The tubes were kept under agitation at 80 rpm at 350 ± 20C for one week. RESULTS Physico-chemical properties of diesel contaminated soil: The various physico-chemical properties such as texture, pH, temperature, electrical conductivity, water holding capacity, mineral matter of soil sample was analysed and noted (Table1). Table 1: Physico-chemical properties S.No. Physico-chemical properties 1 Texture Sandy loam 2 Soil colour Thick brown 3 pH 7.7 4 Temperature 540C 5 Electrical Conductivity 2.80 2.6 ml for 100 % 6 Water holding capacity 1.56 ml for 60% 7 Organic carbon 0.35% 8 Total nitrogen 0.05% 9 Phosphorous 13 kg/Acre 10 Potassium 340 kg/Acre Isolation and screening of diesel degrading bacteria from diesel contaminated soil: Five bacterial strains were isolated from diesel contaminated soil samples by serial dilution technique. The isolated bacterial strains were identified based on their morphological and biochemical characteristics. Among the nine isolates, two strains were efficiently used diesel oil as sole carbon and energy source. Two of them were selected on the basis of their counts and


best growths on the mineral salt medium were identified as Staphylococcus and Pseudomonas based on their morphological and biochemical characteristics and the results were presented in Table 2. Table 2: Morphological & Biochemical Characteristics Morphological & Biochemical Characteristics Shape Motility Grams staining Indole test Methyl red test Vogus proskaur test Citrate utilization Catalase Endospore formation Lactose fermentation

S.No. 1 2 3 4 5 6 7 8 9 10

Pseudomonas

Staphylococcus

Rod Motile – – +

Spherical Non motile + – –

+

–

– +

+ +

–

_

–

+

Hydrocarbon utilization test: The biodegradation assays were carried out with 10% v/v of diesel oil for the two selected strains over a period of 15 days. The hydrocarbon utilization test was performed by determining the bacterial growth by using 10% v/v diesel oil as sole carbon source. Fig 1 showed the pattern of growth of two isolates by using diesel oil as the sole carbon source. The graph clearly indicates that both the isolates were effectively utilized diesel oil as the sole carbon source and energy.

Fig. 1: Pattern of growth of isolates with diesel oil as the sole carbon source Biodegradability test: The results obtained with the biodegradability test using the redox indicator DCPIP were tabulated in table 3. The microbial consortium consisting of two bacterial strains is effective in degrading the diesel oil synergetically within less period of time. The individual isolates respond less quickly to the biological oxidation of diesel oil as evidenced by longer time period taken for decolourization of DCPIP indicator. Table 3: Time taken for biodegradability S.No.

Type of Bacterial culture

1 2

Pseudomonas Staphylococcus Consortium (Pseudomonas + Staphylococcus)

3

197

Decolourisation time (in hrs) 72 96 48

DISCUSSION Biodegradability is essential for bioremediation of pollutants. Microorganisms isolated from environment with a history of pollution by petroleum hydrocarbons have higher ability to degrade such pollutants. [12,13]. In nature, the diversity in microorganisms and energy sources make it possible to breakdown a large number of different chemicals. Sometimes the target pollutant is a complex molecule or a mixture of compounds that can only be broken down by a very specific combination of microorganisms usually referred as consortium. Microbial strains belonging to various genera have been detected in petroleum -contaminated soil or water [14, 15, 16]. This strongly supports that each strain or genera have their roles in the hydrocarbon transformation processes. The diesel oil was used as the sole source of carbon and energy, tested the diesel oil biodegradation ability of cultures isolated from Arabian Sea sediments. [17]. Different microorganisms have the ability to degrade diesel oil depending upon the nature and concentration of contaminant and metabolic needs such as energy, growth and reproduction. Diesel contains a large amount of alkanes (hydro carbon chains from C10-C20) lacking oxygen, demands adapted microorganisms that produce enzymes which recognize these molecules. Individual microorganisms cannot mineralize most hazardous substances completely. Co-metabolism is one form of secondary substrate transformation in which enzymes produced for primary substrate oxidation are capable of degrading the secondary substrate fortuitously, even though the secondary substrates do not provide sufficient energy to sustain the microbial population. It is thus explained as degradation of a compound only in presence of other organic compound that serves as a primary energy source. The growth dynamics of the organisms was determined by their optical densities and the total viable count. Table-3 in the results showed that the mixed culture can degrade diesel oil and utilize better than the individual isolates. An increase in oil degradation was corresponding to an increase in cell number which can be detected by optical density. The rate of biodegradation by individual organisms has been the limiting factor and hence the need for microbial seedling. Despite the majority of hydrocarbon constituents of diesel fuels are biodegraded by several microorganisms commonly occurring in soil each capable of breaking down a specific group of molecules [18]. It is possible that one species removes the toxic metabolites of the species preceding it and the second species is able to degrade compounds that the first is able to degrade only partially [19] Besides, mixed bacterial culture offers an economically feasible bioremediation process. Pseudomonas species are most widely detected microbes in the contaminated sites due to their extensive biodegradation capacities. Pseudomonas and Actinobacter species are the most common bacterial hydrocarbon degraders reported in literature [20]. In the present work, similar results were obtained with the contamination of soil from a diesel station. The advantages of employing mixed cultures as opposed to pure cultures in bioremediation have been


demonstrated earlier [21]. The cultivation of a consortium using diesel oil as the only source of carbon and energy and reported degradation of 90% of the fuel oil after 50 days of cultivation [22]. The bioremediation of soils and sites contaminated by diesel fuels is often limited by the poor diversity of indigenous micro flora and/or the scarcity of indigenous specialized microbes with the complementary substrate specificity required degrading the different hydrocarbons occurring at the contaminated site [19, 23]. Mixed population of bacteria is usually required to provide all the metabolic capabilities for complete degradation of complex mixtures of hydrocarbons. Further evidence for the cooperation of mixed cultures in biodegradation is apparent when the scientists observed a sequential change of the composition of oil-degrading bacteria over a period of time in oil-contaminated soil samples [16]. In bioremediation processes carried out by mixed cultures, commensalism plays an important role since each species may have a specific function in the enzymatic reaction sequences, responsible for the breakdown of more complex molecules. CONCLUSION We report here that the mixed bacteria utilize diesel oil as sole source of carbon and energy. Our findings demonstrated that the synergetic degradation is faster when compared to the individuals. Finally, we conclude that the consortium has less difficulty in biodegrading diesel. This fact is in agreement with the results obtained by the biodegradability test. ACKNOWLEDGEMENTS The authors are thankful to Prof. D.V.R. Saigopal, Coordinator, DST-PURSE (Promotion of University Research and Scientific Excellence) Centre, Sri Venkateswara University, Tirupati for providing laboratory facilities. CB is grateful to University Grants Commission, New Delhi for the financial assistance with BSR Fellowship. REFERENCES 1. J.L.R. Gallego, J. Loredo, J.F. Llamas, F. Vazquez, J.Sanchez, Bioremediation of diesel contaminated soils: Evaluation of potential insitu techniques by study of bacterial degradation, Biodegradation. 12 (2001) 325-335. 2. S. Mishra, J. Jyot, R.C. Kuhad, B. Lal, Evaluation of inoculum addition to stimulate insitu Bioremediation of oily-sludge-contaminated soil, Appl. Env. Microbiol. 67 (2001) 1675-1681. 3. K.H. Baker, D.S. Herson, Bioremediation, McGrawHill, New York, Inc. (1999) pp: 375. 4. L.A. Nwaogu, C.S. Alisi, C.U. Igwe, C.O. Ujowundu, A comparative study of the antimicrobial properties of the ethanolic extracts of Landolphia owariensis leaf and root, Afr. J. Biotechnol. 7 (2008) 368-372. 5. G.W. Thomas, Soil pH and soil acidity. In:Sparks, D.L.(Ed), Methods of soil analysis, Soil science society of America Book Series, American Society of agronomy and soil science society of America, Madison, Wisconsin, 5 (1996) 475-490. 6. D.W. Nelson, L.E. Sommers, Total carbon, Organic and organic matter. In: Sparks, D.L. (Ed.), Methods of

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Source of support: Nil; Conflict of interest: None declared

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