This full text version, available on TeesRep, is the post-print (final version prior to publication) of: Rahman, P. K. S. M. et. al. (2003) 'The potential of bacterial isolates for emulsification with a range of hydrocarbons', Acta Biotechnologica, 23 (4), pp.335345. For details regarding the final published version please click on the following DOI link: http://dx.doi.org/10.1002/abio.200390043 When citing this source, please use the final published version as above. This document was downloaded from http://tees.openrepository.com/tees/handle/10149/98675 Please do not use this version for citation purposes. All items in TeesRep are protected by copyright, with all rights reserved, unless otherwise indicated. TeesRep: Teesside University's Research Repository http://tees.openrepository.com/tees/ The potential of bacterial isolates for emulsification with a range of hydrocarbons K.S.M. Rahman1*, Thahira J. Rahman1, P. Lakshmanaperumalsamy2, R. Marchant1 and I.M. Banat 1 1. Biotechnology Research Group, School of Biological and Environmental Science, University of Ulster, Coleraine, County Londonderry, United Kingdom - BT52 1SA. 2. Department of Environmental Sciences, Bharathiar University, Coimbatore – 641 046, Tamilnadu, India. * Corresponding Author Present address: Dr Pattanathu K.S.M. Rahman Chemical and Bioprocess Engineering Group School of Science and Engineering Teesside University, Middlesbrough - TS1 3BA Teesvalley, United Kingdom. Tel: +44-1642-384669 Email: p.rahman@tees.ac.uk Running title: Emulsification of hydrocarbons by bacteria 1 Summary A study was undertaken to investigate the distribution of biosurfactant producing, crude oil degrading bacteria in the oil contaminated environment. Our research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10-129 produced maximum of 7.5 ± 0.4 g / l of biosurfactant with corresponding reduction in surface tension from 68 mN / m to 29.4 ± 0.7 mN / m at 84 h incubation. The isolates Micrococcus sp. GS2-22, Bacillus sp. DS6-86, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Pseudomonas sp. DS10-129, Pseudomonas sp. DS9-119 and Acinetobacter sp. DS5-74 emulsified xylene, benzene, n-hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had highest emulsification activity and crude oil degradation ability and they were selected for the preparation of mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and mixed bacterial consortium which have not been reported before. 2 Introduction Biosurfactants are surface-active substances synthesised by living cells. They have the properties of reducing surface tension, stabilising emulsions, promoting foaming and are generally non-toxic and biodegradable. Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, environmentally friendly nature, possibility of large-scale production, selectivity, performance under extreme conditions and potential applications in environmental protection (1, 2). Rosenberg and Ron (3) have extensively studied the nature of microbial biosurfactants. The use of chemicals for treatment of a hydrocarbon polluted site may contaminate the environment by their by-products, whereas biological treatment may efficiently destroy pollutants while being biodegradable themselves. Biosurfactants enhance emulsification of hydrocarbons, have the potential to solubilise hydrocarbon contaminants and increase their availability for microbial degradation. Hence, biosurfactant producing microorganisms may play an important role in the accelerated bioremediation of hydrocarbon contaminated sites (3-5). These compounds can also be used in enhanced oil recovery and may be considered for other potential applications in environmental protection (5, 6). Other applications include herbicides and pesticides formulations, detergents, health care and cosmetics, pulp and paper, coal, textiles, ceramic processing and food industries, uranium ore-processing and mechanical dewatering of peat (1, 2, 7). Several microorganisms are known to synthesize surface-active agents, most of them are bacteria and yeast (8, 9). When grown on hydrocarbon substrate as carbon source, these microorganisms synthesize a wide range of chemicals with surface activity such as glycolipid, phospholipid and others (10, 11). These chemicals are apparently synthesized to emulsify the hydrocarbon substrate and facilitate its transport into the cells. 3 In this paper we described the isolation and identification of several bacterial cultures from oil contaminated sites, capable of growing on hydrocarbon containing media. We also investigated the relationship between biosurfactant production and emulsification activity for various hydrocarbons. Materials and Methods Screening of samples Soil samples were collected from gasoline spill (GS) and diesel fuel spill (DS) in gas station soil and wastewater (WW) samples from service stations for the isolation of oil utilizing microorganisms. Enrichment and isolation of oil degrading bacterial cultures were done using mineral salts medium (12) with Bombay High (BH) crude oil as substrate and a serial dilution agar plate technique on nutrient agar medium (Himedia, Mumbai, India). Characterization of bacteria The isolates were grouped to various genera as per Bergey's Manual of Determinative Bacteriology (13). These cultures were characterized depending on their morphology, gram staining, spore staining, motility, oxidase, catalase, oxidation, fermentation, gas production, ammonia formation, nitrate and nitrite reduction, indole, methyl-red, Voges-Proskauer, citrate utilization, utilization of mannitol and urea, hydrolysis of casein, gelatin, starch and lipid (14). Growth of bacteria on BH crude oil The bacterial cultures isolated from oil spill environment were inoculated in mineral salts medium with 1% BH crude oil as carbon source. It was kept in the shaker at 200 rpm at 30°C for a period of 4 seven days. The broth culture was kept undisturbed for an hour to separate the emulsion formed with crude oil at the top of the medium. The culture without oil droplets was used for bacterial growth estimation. The growth was recorded and categorized spectrophotometrically as low growth with optical density (OD) in the range 0.21-0.4, moderate growth (0.41-0.6 OD), high growth (0.61-0.8 OD) and excellent growth (0.81-1.0 OD) all measured at 620nm (15). Selection of bacteria for surfactant production Among oil degrading isolates 26 isolates showed biosurfactant production and therefore selected for further study (Tab. 1). They belonged to Acinetobacter (1), Alcaligenes (1), Bacillus (4), Corynebacterium (9), Flavobacterium (1), Micrococcus (1), Moraxella (1) and Pseudomonas (8). A consortium consisting of a mixture of five isolates (Micrococcus sp. GS2-22, Bacillus sp. DS686, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73 and Pseudomonas sp. DS10-129) was also prepared and used for comparison. Bacterial growth and biosurfactant production A series of 500 ml flasks containing 200 ml of sterile mineral salts medium with 1% Glucose as substrate were prepared and the pH was maintained at 7.5. Each of the individual bacterial cultures and the mixed bacterial consortium were inoculated and the flasks were incubated at 30°C in a shaker at 200 rpm followed by addition of 1 % glycerol after 24 h. At every 12 h interval, biomass, biosurfactant production, surface tension and emulsification activity were measured. 5 Biomass estimation The culture broth was filtered using GF/C filters, The filters were kept at 110°C for 24h. Then they were taken out and weighed. To find the net biomass, the filters were once again burnt in a furnace at 550°C and weighed. The net biomass was calculated as the difference between the two. Biosurfactant extraction Surface active compounds were extracted by liquid-liquid extraction (16) from 10 ml of the cell free culture broth previously acidified with 1N HCl to pH 2. Supernatant fluid was mixed with an equal volume of a chloroform: methanol (2:1) mixture. The organic extracts were concentrated by overnight drying in drying chamber at the temperature of 44 ° C and the mass of the biosurfactant was measured. Surface tension Surface tension was measured by drop weight method (17). A vertical fine capillary tube having round tapered nozzle was used. The liquid was drawn and passed slowly to make a fine drop, which hangs by its own weight and then falls down by gravity. The mass of a single drop from cell free culture broth was measured by the average mass of 200 drops for each sample. The following empirical formula was applied to calculate the surface tension in mN / m. ST = mxg 3.8 x r Where, m = mass of single drop of liquid (mg) r = radius of the nozzle (m) g = gravitational force 6 Determination of Emulsification activity Emulsification activity (E24) was determined by the addition of the respective hydrocarbon (xylene, benzene, n-hexane, BH crude oil, kerosene, gasoline, diesel fuel and olive oil) to the same volume of cell free culture broth, mixing with a vortex for 2 minutes and leaving to stand for 24 h. The emulsification activity was determined as the percentage of height of emulsified layer (mm) divided by total height of the liquid column (mm) (18). Results and Discussion The enrichment and isolation procedure resulted in 130 pure bacterial cultures able to grow in mineral salts medium (MSM) with BH crude oil as carbon source. Out of 130 isolates, 50.77%, 24.61%, 20.77% and 3.85% showed low growth (0.21-0.4 OD), moderate growth (0.41-0.6 OD), high growth (0.61-0.8 OD) and excellent growth (0.81-1.0 OD) at 620nm respectively (Tab. 1). The isolated crude oil degraders belonged to the genera Micrococcus, Corynebacterium, Bacillus, Enterobacteriaceae, Pseudomonas, Alcaligenes, Flavobacterium, Moraxella, Aeromonas, Acinetobacter and Vibrio. The biota reflects the typical heterotrophic bacteria present in soil and native genera seem to be crude oil utilizers. However, the dominant strains belonged to Corynebacterium, Bacillus, Micrococcus and Pseudomonas. The ecological studies of MarquezRocha et al. (19) also identified the above genera among hydrocarbon degrading microorganisms. The addition of hydrocarbons to an ecosystem, as a result of an oil spillage, may selectively increase or decrease the size of microbial population depending upon the chemical composition of the contaminating hydrocarbons and the species of microorganisms present within the microbial community of the particular ecosystem (20). Such an event may enrich primarily for microorganisms capable of utilising the hydrocarbons and secondarily for microorganisms 7 capable of utilising metabolites produced by the hydrocarbon-utilising micro-organisms resulting in an increased numbers of hydrocarbon-utilising micro-organisms and associated secondary colonisers. There are numerous reports of such increases in microbial numbers following addition of hydrocarbons to a variety of microbial communities (12, 21). Isolates Micrococcus sp. GS2-22, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Bacillus sp. DS6-86 and Pseudomonas sp. DS10-129 had the highest growth at 30oC in mineral salts medium with 1% glucose and 1% glycerol as substrates. Among these genera Pseudomonas sp. DS10-129 produced maximum biosurfactant of 7.5 ± 0.4 g / l at 84 h with a biomass concentration of 7.1 ± 0.6 g / l in 1% glucose + 1% glycerol as substrates and surface tension was reduced from 68 to 29.4 ± 0.7 mN / m (Tab. 2). About 0.97 - 2.7 g / l of biosurfactant production by different strains of Pseudomonas aeruginosa using glucose and waste fry oil as carbon source had been reported (2, 22). When compared to earlier reports Pseudomonas sp. DS10-129 showed higher quantity of biosurfactant production. Among the Corynebacterium strains isolated, GS5-66 produced the maximum amount of biosurfactant (4.1 ± 0.6 g / l at 48 h) in glucose + glycerol and surface tension was reduced to 36.4 ± 0.2 mN / m. Similarly, Haferburg et al. (23) reported biosurfactant production by Corynebacterium fascians in media supplemented with yeast extract + hexadecane and kerosene, and observed a reduction in surface tension to 27.5 and 33 mN / m respectively. Bacillus sp. DS686 produced the maximum quantity of biosurfactant (2.1 ± 0.3 g / l at 48 h) with the reduction of surface tension to 31.6 ± 0.9 mN / m. Heba et al. (22) reported the production of lipopeptide biosurfactant by Bacillus subtilis ATCC 6633 with a reduction in surface tension of the medium to 39 mN / m. Similar reduction in surface tension was observed by Jenny et al. (24) by the lipopeptide type of biosurfactant produced by Bacillus licheniformis. Several authors have reported 8 similar activity of the biosurfactant produced by Bacillus sp. (25, 26). The Acinetobacter sp. DS574 produced 1.9 ± 0.2 g / l of biosurfactant in 96 h with the reduction in surface tension to 33.7 ± 0.9 mN / m. Heba et al. (22) reported lipoprotein type of biosurfactant produced by Acinetobacter calcoaceticus CECT 441 on olive oil and sunflower oil with reduction of surface tension to 42.5 and 38 mN / m respectively. In the earlier studies, several authors reported about the biosurfactant produced by Acinetobacter sp. (27). About 2.4 ± 0.1 g / l of biosurfactant was produced by Alcaligenes sp. GS4-49 at 72 h with reduction in surface tension from 72 mN/m to 46.2 ± 0.7 mN / m. Dixon (28) reported that Alcaligenes sp. strain MM-1 produced biosurfactant similar to our findings. The production of 1.3 ± 0.2 g / l of biosurfactant by Micrococcus sp. GS2-22 that reduced the surface tension to 32.9 ± 0.7 mN / m was recorded at 72 h of incubation. Gutnick (29) reported the production of phospholipids and fatty acids/neutral lipid type of surfactant by Micrococcus sp. Other coccal forms such as Streptococcus thermophilus (30) produced biosurfactant, which are applied in fouling control of heat exchanger plates. Biosurfactant production by Moraxella sp. DS1-13 and Flavobacterium sp. DS5-73 was 1.3 ± 0.1 g / l and 1.3 ± 0.7 g / l respectively. However we could not find any report on the production of biosurfactant by Moraxella and Flavobacterium in published literature. The mixed bacterial consortium produced about 4.9 ± 0.8 g / l of biosurfactant at 84 h incubation with biomass of 6.5 ± 0.4 g / l and surface tension was reduced to 34.1 ± 0.3 mN / m. When oil degraders were introduced individually, the amount of surfactant production was more when compared to the production of surfactant by mixed bacterial consortium. This may be due to the competition between the bacteria for nutrient substrate. However, biosurfactant production by mixed bacterial consortium was not reported earlier. 9 Petroleum hydrocarbon compounds generally bind to soil particles and are difficult to remove or degrade mainly due to limited availability to micro-organisms (31). Hence for efficient degradation, hydrocarbons should be solubilized prior to microbial degradation (32). Surfactants can emulsify hydrocarbons, thus enhancing their dispersion in water through reduction of surface tension and increased displacement of oily substances from soil particles (3, 33). Hydrocarbon contaminants may be nonavailable because of their hydrophobic nature and sorption to soil. Oberbremer et al. (34) showed that both the rate and extent of hydrocarbon degradation in soil slurry were enhanced by biosurfactants. Hence treating soil with biosurfactants will increase the availability of hydrocarbon to the degrading microorganisms, thus stimulating organic biodegradation in the soil. The emulsification activity is an extensively used method to identify and quantify biosurfactants produced by microbial cultures. Bacillus sp. DS6-86 showed maximum emulsification activity on xylene (87 ± 3 %). Banat et al. (35) reported the emulsification activity on xylene during batch fermentation of pet 1006 strain in modified basal salts medium. In the earlier study Pseudomonas sp. MR-3 emulsified xylene to the level of 78.13% (17). Pseudomonas sp. DS10-129 showed 93 ± 9 % of emulsification activity on benzene and mixed bacterial consortium emulsified olive oil at the maximum of 47 ± 4 %. Heba et al. (22) reported about 61.3% emulsification activity by the glycolipid biosurfactant produced by Pseudomonas sp. 55T1 on olive oil. Bacillus sp. DS2-24 showed 87 ± 6 % of emulsification activity on n-hexane. BH crude oil was emulsified to the maximum of 73 ± 6 % by Pseudomonas sp. DS10-129. A different strain of the same genera (Pseudomonas sp. MR-3) emulsified 31.70% (17). Rosenberg et al. (36) also recorded similar findings with Arthrobacter RAG1. Iqbal et al. (18) reported about 70% of emulsification activity on BH crude oil by Pseudomonas aeruginosa strain S-8. Kerosene was emulsified to 96 ± 2 % by Pseudomonas sp. DS4-55, while other isolates showed lesser activity, which is higher when 10 compared to the emulsification activity of Arthrobacter RAG1 (36). In our previous work, about 71.23% of kerosene was emulsified by Pseudomonas sp. MR-3 (17). Allen et al. (37) reported weaker emulsification activity on kerosene by some microbial isolates from subsurface soil. Johnson et al. (38) isolated Rhodotorula glutunis capable of producing extracellular emulsifying agent on glucose in fed batch fermentation, which emulsified n-hexadecane, xylene, kerosene and gas oil. Muriel et al. (10) observed 55% emulsification of kerosene by the cladosan biosurfactant produced by Cladosporium resinae. The experimental values obtained in the present investigation were higher when compared to all the earlier reports. Microbes isolated from gasoline contaminated areas showed emulsification activity when overlaid with gasoline (37). Abu-Ruwaida et al. (39) reported the highest emulsion value (water in oil) of about 78% using Kuwait motor oil. Moreover, in the present study about 79 ± 7 % of emulsification activity on gasoline was showed by the surfactants produced by Pseudomonas sp. DS10-129. Sixty two percent of emulsification activity was observed for diesel fuel by Corynebacterium sp. GS4-48. Willumsen and Karlson (40) found that 67% of bacterial isolates taken from polyaromatic hydrocarbon (PAH) contaminated soil were able to form detectable emulsion with diesel fuel, whereas the report of Allen et al. (37) also showed weaker emulsification activity by cultures with diesel fuel. Members of various genera found to be capable of producing surfactants showed emulsification activity on various hydrocarbons. Flavobacterium sp. DS5-73 and Micrococcus sp. GS2-22 produced surfactants which emulsified all the hydrocarbons tested. Allen et al. (37) found that all microbial isolates from subsurface soil contaminated with unleaded gasoline showed emulsification activity when overlaid with gasoline, whereas emulsification activity by microbial cultures overlaid 11 with kerosene and diesel fuel were weaker. Willumsen and Karlson (40) found that 67% of their isolates were able to form detectable emulsions with diesel fuel. One might speculate that this relatively low percentage of emulsifiers among isolates from soil contaminated with PAH as opposed to soil contaminated with aliphatic hydrocarbons might indicate that growth on PAH does not require emulsification to the same extent as growth on aliphatic hydrocarbons. Alternatively, some essential growth factors for emulsification may have been lacking in their study. Conclusion Among the 130 bacterial isolates screened, 32 were efficient oil degraders, 80% of them were found to produce biosurfactants. Maximum of 7.5 ± 0.4 g / l of surfactant was produced by Pseudomonas sp. DS10-129 and minimum of 0.3 ± 0.1 g / l was produced by Corynebacterium sp. GS5-72. Surfactant production, biomass and emulsification activity reached the maximum at or before 96 h and was stable thereafter. No single isolate produced surfactant with maximum emulsification activity on all individual hydrocarbons tested. Biosurfactants produced by isolates such as Micrococcus sp. GS2-22, Bacillus sp. DS6-86, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73 and Pseudomonas sp. DS10-129, Acinetobacter sp. DS5-74, Pseudomonas sp. GS9-119 and mixed bacterial consortium showed broad spectrum of emulsification activity with all the hydrocarbons tested. Among the biosurfactant producers, all the isolates were able to emulsify xylene and benzene. However, BH crude oil was emulsified by 88% of the isolates, n-hexane and diesel fuel by 65% and kerosene, gasoline and olive oil by 73% of them. Among the emulsifiers, more than 70% of the isolates were members of Corynebacterium, Pseudomonas and Bacillus. Our findings showed that the above oil degrading bacteria are efficient biosurfactant producers and hydrocarbon emulsifiers. 12 Acknowledgements KSM Rahman wishes to thank the Council of Scientific and Industrial Research, New Delhi, India for the award of Senior Research Fellowship. Thanks also to Environment and Heritage Service, DOE for FRDF financial support under the Northern Ireland Single Programme (Ref. WM47/99). Reference [1] BANAT, I.M., MAKKAR, R.S., CAMEOTRA, S.S.: Potential commercial applications of microbial surfactants. Appl. Microbiol. Biotechnol. 53 (2000), 495-508. [2] RAHMAN, K.S.M., THAHIRA-RAHMAN, J., MCCLEAN, S., MARCHANT, R., BANAT, I.M.: Rhamnolipid biosurfactants production by strains of Pseudomonas aeruginosa using low cost raw materials. Biotechnol Prog. 18 (2002), 1277-1281. 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Lett. 14 (1992), 487-490. 15 [39] ABU-RUWAIDA, A.S., BANAT, I.M., HADITIRTO, S., SALEM, A., KADRI, M.: Isolation of biosurfactant - producing bacteria - product characterization and evaluation. Acta Biotechnol. 11 (1991), 315-324. [40] WILLUMSEN, P.A., KARLSON, U.: Screening of bacteria isolated from PAH contaminated soils for production of biosurfactants and bioemulsifiers. Biodegradation 7 (1997), 415-423. 16 Tab. 1 Distribution of bacteria showing different levels of growth on BH crude oil Growth of bacteria on BH crude oil Genera Total No. Optical Density at 620nm* 0.21-0.4 0.41 – 0.6 0.61 – 0.8 0.81 – 1.0 Micrococcus sp. 17 10 6 - 1 Corynebacterium sp. 45 17 17 10 1 Bacillus sp. 13 8 1 3 1 Enterobacteriaceae sp. 6 4 2 - - Pseudomonas sp. 16 4 2 9 1 Alcaligenes sp. 8 7 - 1 - Flavobacterium sp. 9 7 - 1 1 Moraxella sp. 7 5 1 1 - Aeromonas sp. 4 2 1 1 - Acinetobacter sp. 2 1 - 1 - Vibrio sp. 3 1 2 - - Total 130 66 32 27 5 Percentage 100 50.77 24.61 20.77 3.85 * 0.21 - 0.4 = Low growth 0.41 - 0.6 = Moderate growth 0.61 - 0.8 = High growth 0.81 - 1.0 = Excellent growth 17 Tab. 2. Maximum biosurfactant production, growth and surface tension of oil degraders S.No. Bacteria Incubation Biosurfactant (h) (g / l) Net biomass Surface tension (g/l) (mN / m)* Growth on 1% BH crude oil 1 Acinetobacter sp.DS5-74 96 1.9a ± 0.2b 4.1 ± 0.5 33.7 ± 0.9 H 2 Alcaligenes sp.GS4-49 72 2.4 ± 0.1 1.8 ± 0.2 46.2 ± 0.7 H 3 Bacillus sp.DS1-12 48 1.8 ± 0.4 2.3 ± 0.1 33.1 ± 0.9 H 4 Bacillus sp.DS2-24 48 1.6 ± 0.2 2.5 ± 0.3 35.0 ± 0.2 H 5 Bacillus sp.DS6-86 48 2.1 ± 0.3 2.1 ± 0.2 31.6 ± 0.9 E 6 Bacillus sp.GS3-34 48 1.8 ± 0.1 3.7 ± 0.4 35.2 ± 0.4 H 7 Corynebacterium sp.DS3-37 120 2.6 ± 0.7 4.3 ± 0.6 39.7 ± 0.6 H 8 Corynebacterium sp.DS3-39 96 1.9 ± 0.2 3.9 ± 0.4 43.1 ± 0.3 H 9 Corynebacterium sp.GS5-66 48 4.1 ± 0.6 4.2 ± 0.3 36.4 ± 0.2 E 10 Corynebacterium sp.GS4-48 24 2.1 ± 0.3 2.5 ± 0.2 41.2 ± 0.1 H 11 Corynebacterium sp.GS4-52 96 2.4 ± 0.1 2.8 ± 0.3 39.3 ± 0.8 H 12 Corynebacterium sp.DS5-72 96 0.3 ± 0.1 2.9 ± 0.3 52.5 ± 0.1 H 13 Corynebacterium sp.WW1-46 24 1.4 ± 0.7 2.0 ± 0.2 45.4 ± 0.7 H 14 Corynebacterium sp.WW4-87 48 1.7 ± 0.2 3.5 ± 0.4 43.7 ± 0.9 H 15 Corynebacterium sp.WW4-92 72 1.2 ± 0.3 2.9 ± 0.2 46.8 ± 0.4 H 16 Flavobacterium sp.DS5-73 72 1.3 ± 0.7 3.3 ± 0.3 36.1 ± 0.2 E 17 Micrococcus sp.GS2-22 72 1.3 ± 0.2 1.2 ± 0.1 32.9 ± 0.7 E 18 Moraxella sp.DS1-13 72 1.3 ± 0.1 4.8 ± 0.5 39.5 ± 0.9 H 19 Pseudomonas sp.DS10-129 84 7.5 ± 0.4 7.1 ± 0.6 29.4 ± 0.7 E 20 Pseudomonas sp.DS1-11 48 1.7 ± 0.6 3.2 ± 0.3 38.2 ± 0.2 H 21 Pseudomonas sp.DS1-19 48 3.1 ± 0.8 4.5 ± 0.5 32.5 ± 0.6 H 22 Pseudomonas sp.DS3-38 48 2.1 ± 0.2 5.7 ± 0.6 34.2 ± 0.4 H 23 Pseudomonas sp.DS4-55 96 4.7 ± 0.7 4.2 ± 0.4 32.2 ± 0.2 H 24 Pseudomonas sp.GS4-51 72 2.6 ± 0.4 3.0 ± 0.2 32.7 ± 0.5 H 25 Pseudomonas sp.GS8-104 72 2.4 ± 0.1 4.4 ± 0.3 32.4 ± 0.7 H 26 Pseudomonas sp.GS9-119 96 4.3 ± 0.3 5.3 ± 0.5 30.6 ± 0.9 H 27 Mixed bacterial consortium 84 4.9 ± 0.8 6.5 ± 0.4 34.1 ± 0.3 E * = Initial surface tension value 68 mN / m, H = High growth (0.61 - 0.8 OD at 620 nm), a b =average value, = standard error E = Excellent growth (0.81 - 1.0 OD at 620 nm) 18 Tab. 3. Percentage of emulsification activity of the selected oil degraders on various hydrocarbons S.No Bacteria Xylene Benzene n-Hexane BH crude oil Kerosene Gasoline Diesel fuel Olive oil 1 Acinetobacter sp.DS5-74 a 51± 4 48 ± 4 37 ± 3 6±1 9±1 4±1 2 Alcaligenes sp.GS4-49 34 ± 3 51 ± 5 Bacillus sp.DS1-12 4±1 7±1 21 ± 3 15 ± 1 NE 24 ± 3 NE 9±1 NE 8±1 3 27 ± 2 NE 26 ± 2 NE 13 ± 2 11 ± 1 83 ± 6 87 ± 6 36 ± 2 NE NE NE 21 ± 1 5±1 NE 26 ± 7 b 4 Bacillus sp.DS2-24 5 Bacillus sp.DS6-86 87 ± 3 37 ± 2 11 ± 1 4±2 58 ± 5 Bacillus sp.GS3-34 74 ± 5 80 ± 6 20 ± 1 NE 62 ± 5 6 29 ± 4 7 Corynebacterium sp.DS3-37 58 ± 4 21 ± 1 NE 28 ± 2 11 ± 1 NE 5±1 NE 4±1 NE NE 8 Corynebacterium sp.DS3-39 84 ± 4 71 ± 5 NE 43 ± 3 NE NE NE 8±1 74 ± 3 61 ± 4 70 ± 6 59 ± 4 54 ± 3 27 ± 1 39 ± 3 16 ± 3 NE 9 Corynebacterium sp.GS5-66 10 Corynebacterium sp.GS4-48 68 ± 2 64 ± 6 Corynebacterium sp.GS4-52 9±1 33 ± 3 73 ± 3 NE 12 ± 1 11 29 ± 3 73 ± 4 NE 48 ± 3 NE 62 ± 4 NE 4±1 12 Corynebacterium sp.DS5-72 19 ± 2 19 ± 1 37 ± 1 11 ± 1 29 ± 2 NE 31 ± 3 13 Corynebacterium sp.WW1-46 48 ± 4 61 ± 3 7±1 29 ± 4 6±1 4±1 14 Corynebacterium sp.WW4-87 69 ± 6 79 ± 6 52 ± 4 56 ± 4 NE 35 ± 5 NE 33 ± 3 15 Corynebacterium sp.WW4-92 61± 5 67 ± 7 7±2 4±2 14 ± 1 29 ± 1 NE 7±1 19 ± 1 NE 16 Flavobacterium sp.DS5-73 3 ±1 26 ± 2 42 ± 6 68 ± 6 10 ± 1 8±1 24 ± 2 21 ± 1 17 Micrococcus sp.GS2-22 28 ± 2 34 ± 2 26 ± 2 46 ± 3 35 ± 6 27 ± 2 30 ± 3 18 Moraxella sp.DS1-13 14 ± 1 8±1 19 ± 2 NE 21 ± 1 19 ± 1 6±1 24 ± 1 12 ± 1 19 Pseudomonas sp.DS10-129 74 ± 3 93 ± 9 73 ± 6 89 ± 3 79 ± 7 71 ± 6 27 ± 3 29 ± 2 30 ± 2 48 ± 4 NE 4±1 7±1 9±1 8±1 31 ± 3 NE 43 ± 4 4±1 4±1 NE 27 ± 3 NE 6±1 NE 1±0 NE NE 14 ± 1 10 ± 2 NE 78 ± 4 NE 43 ± 2 20 Pseudomonas sp.DS1-11 21 Pseudomonas sp.DS1-19 71 ± 4 82 ± 6 17 ± 3 22 Pseudomonas sp.DS3-38 78 ± 9 32 ± 1 23 Pseudomonas sp.DS4-55 75 ± 4 70 ± 6 18 ± 1 NE 5±1 24 Pseudomonas sp.GS4-51 9±1 7±1 NE 47 ± 4 96 ± 2 NE 25 Pseudomonas sp.GS8-104 37 ± 2 63 ± 5 76 ± 3 36 ± 3 49 ± 3 26 Pseudomonas sp. GS9-119 52 ± 4 63 ± 4 72 ± 6 58 ± 5 84 ± 7 48 ± 3 24 ± 4 26 ± 2 44 ± 3 53 ± 1 66 ± 8 NE = No emulsification detected 67 ± 6 52 ± 4 31± 2 5±1 47 ± 4 27 Mixed bacterial consortium a b = Average value, = Standard error 51 ± 4 24 ± 2 19 20