A feasibility study was conducted to evaluate the capability of bacterial strains to degrade crude oil under in vitro conditions. Pseudomonas strain PS-I could ...
Indian Journal of Experimental Biology Vol. 47, September 2009, pp. 760-765
Isolation of hydrocarbon degrading bacteria from soils contaminated with crude oil spills Anupama Mittal* & Padma Singh Department of Microbiology, Kanya Gurukul Mahavidyalaya (Gurukul Kangri University), Jwalapur, Haridwar 249 407, India Received 24 December 2008; revised 19 May 2009 A feasibility study was conducted to evaluate the capability of bacterial strains to degrade crude oil under in vitro conditions. Pseudomonas strain PS-I could degrade alkanes (70.69%) and aromatics (45.37%). Alkanes and aromatic fractions separated by column chromatography were analyzed by gas chromatography. In case of Pseudomonas strain PS-I, nC17/Pr, nC18/Ph ratios decreased from 2.5100 to 0.1232 and from 7.2886 to 0.3853, respectively. It was concluded that out of the isolated strains, Pseudomonas strain PS-I, PS-II and PS-III were comparatively better and potent hydrocarbon degraders. Pseudomonas strain PS-I was almost comparable with standard strain of Acinetobacter calcoaceticus in crude oil biodegradation potency. Keywords: Acinetobacter calcoaceticus, Bioremediation, Crude Oil, Pseudomonas
Oil spills have become a global problem particularly in industrialized and developing countries. Attention has been focused on the marine environment, because oceans and estuaries have generally been the sites of the largest and most dramatic spills1. Apparently inevitable spillages, which occur during routine operations of crude oil production, refining, distribution and as a consequence of acute accidents, have generated continuous research interest in this field2. The parameters typically measured in laboratory tests of bioremediation efficacy include enumeration of microbial populations3-5, determination or fate of hydrocarbon degradation (disappearance of individual hydrocarbons and/or total hydrocarbons)6. Undoubtedly, the most direct measure of bioremediation efficacy is the monitoring of hydrocarbon disappearance rates7. Generally type and identity of fresh to biodegraded oils and petroleum products can be readily revealed their GCFID traces especially where the biodegradation of spilled oil or petroleum product is heavy and background hydrocarbon levels are low in an impacted environment. In addition to measuring TPH (Total petroleum hydrocarbons) in samples, GC-FID _____________________ *Address for correspondence: Department of Microbiology, Kanya Gurukul Mahavidylaya, Gurukul Kangri University, Haridwar 249407, India Telephone: (91) 9999882926 (Mobile)
chromatograms provide a distribution pattern of petroleum hydrocarbons {e.g., carbon range and profile of UCM (Unresolved complex mixture)}, fingerprints of the major oil components (e.g., individual resolved n-alkanes and major isoprenoids), and information on the biodegradation extent of the spilled oil. Comparing biodegradation indicators (such as nC17/Pristane nC18/Phytane) for the spilled oil to the source oil can also be used to monitor the effect of microbial degradation on the loss of hydrocarbons at the spill site8,9. The objective of the study is to assess the crude oil biodegradation potential of selected five bacterial strains under in vitro conditions. Degradation studies to be carried out with different isolates at varying interval of time will help to find out the most potent hydrocarbon degrading strains, which can be used for any bioaugmentation studies during bioremediation. Materials and Methods Soil samplesSamples (500g) contaminated with oil since 1986 (when oil production began in Lingala oil field, India) used for isolation of hydrocarbon utilizing microorganisms, were collected from oil production site of ONGC (oil wells from Lingala Oil field Project, India). Subsurface soil sample contaminated with refined product of crude oil viz. diesel and lubricating oil was collected from local
MITTAL & SINGH: ISOLATION OF HYDROCARBON DEGRADING BACTERIA
area, Haridwar, India. The soil samples were collected in pre-sterilized sample bottles and also in whirl pack bags following aseptic conditions. The samples duly labeled were stored at - 4°C for further analysis. Crude oil contentsPhysico-chemical properties of crude oil of Lingala well # X were - depth (m), 1893.5-95; Formation, Sand stone; Type, Oil; API Gravity, 35.6; Saturates, 67.3%; Aromatic, 21.2%; NSO (Nitrogen, Sulphur, Oxygen containing compounds), 11.2%; Asphaltenes, 0.3%; Sat/Aro, 3.17; HC/NSO, 7.7; Pr/Ph, 4.3; Pr/nC17, 1.693; Ph/nC18, 0.266; Pr+nC17/ Ph+nC18, 1.434; nC21+nC22/ nC28+nC29, 1.219. Medium used and maintenance of microorganismsSoil sample (1 g) from each source was suspended and vortexed with distilled water (10 mL). The suspension was allowed to settle down and supernatant (5 mL) was used as inoculum, in 100 mL of Luria Bertani broth containing crude oil (1%) in an Erlenmeyer flask. The flask was incubated for 48 h at 37°C on a rotary shaker at 100 rpm. Three successive subculturing were done on the same medium containing crude oil (1%). After three subculturing steps broth was centrifuged at 5,000 rpm for 10 min and the cell pellets were obtained. The cell pellets were washed with 0.1M phosphate buffer solution (pH-6.8) twice, after suspending in mineral salt medium, cell pellets were inoculated in 100 mL of mineral salt medium in an Erlenmeyer flask, containing 1% crude oil as a single source of carbon and energy. The mineral salt medium10 contained (g): KNO3, 1.0; MgSO4.7H2O, 1.0; CaCl2.6H2O, 0.1; FeSO4, 0.05; trace element sol, 250 mL; phosphate buffer (1M; pH-6.8), 20 mL; and distilled water, 980 mL. Trace element solution11comprised(g) - SnCl2, 0.05; KI, 0.05; LiCl, 0.05; MnSO4.4H2O, 0.08; HBO3, 0.50; ZnSO4.7H2O, 0.10; CoCl2.6H2O, 0.10; NiSO4.6H2O, 0.10; BaCl2, 0.05; Ammonium molybdate, 0.05; and distilled water, 1000 mL (All salts were dissolved in defined sequence only). Pure hydrocarbon degrading strains were isolated on petroleum agarose plates. In the preparation of petroleum agarose plates 1-2 drops of sterile crude oil was evenly spread with glass spreader, so that a film of crude oil got absorb over the entire agarose surface of mineral medium in the petriplates and then inoculum was spread with spreader on the medium. The plates were incubated at 37°C for one week in an incubator. Pure and representative colonies were transferred to slants for preservation.
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Preservation and subculture of the strainsThe Isolated strains were preserved in 25% v/v glycerol solution at -70°C. For day-to-day experimentation strains were maintained on nutrient agar slants at 4°C in refrigerator and sub-cultured at an interval of 30 days. Biodegradation studiesLaboratory biodegradaation studies were carried out under optimized conditions as described elsewhere12 for assessing the hydrocarbon degradation potential of isolated strains. Mineral salt medium (200mL) in each, was prepared in three sets of 24 Erlenmeyer flasks (1000mL capacity) and autoclaved. These 24 Erlenmeyer flasks were grouped in sets of six flasks and subjected to treatment for 15, 30, 45, and 60 days Sterile crude oil (2.4 g, sterilized by filtration, Millipore size, 0.25 mm) of Lingala well # X was added in each flask. One mL inoculum (Bacterial number was adjusted to give initial cell number 1 × 108 CFU ml-1) of each of hydrocarbon degraders, standard strain (Acinetobacter calcoaceticus) and test isolates (Pseudomonas strains PS-I, PS-II, PS-III, PS-IV and Bacillus) was added in sets of six flasks, respectively. Flasks were incubated in incubator shaker at 30°C, at 100 rpm. After desired interval of time, the flasks were taken out and bacterial activities were stopped by adding 1% 1NHCl. For extraction of crude oil, 50 mL of culture broth was mixed with 50 mL petroleum ether : acetone (1:1) in a separating funnel and was shaken vigorously to get a single emulsified layer. Acetone was then added to it and shaken gently to break the emulsification, which resulted in three layers. Top layer was a mixture of petroleum ether, crude oil and acetone; clumping cells make the middle layer and the bottom aqueous layer contains acetone, water and biosurfactant in soluble form. The lower two layers were separated out while top layer containing petroleum ether mixed with crude oil and acetone was taken out in a clean beaker. The extracted oil was passed through anhydrous sodium sulphate to remove moisture. The petroleum ether and acetone was evaporated on a water bath. The gravimetric estimation of residual oil left after biodegradation was made by weighing the quantity of oil in a tared vial. The biodegraded crude oils were further fractioned for their gross and molecular composition. Column chromatography Saturates, aromatics and NSO fractions were fractionated quantitatively by column chromatography13. Silica gel (60-120 mesh) and alumina neutral were activated at 150°C for 24 h
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and cooled in desiccators. The glass column (Internal diameter 1.1 cm, length-65 cm and reservoir capacity 60-100 mL) was packed by placing a thin cotton plug at the bottom. The slurry of 20 g activated alumina was filled, followed by 20 g silica gel. The column was washed with petroleum ether. The oil sample (approx. 100 mg/known weight) was dissolved in chloroform, adsorbed on silica gel and evaporated the excess chloroform at 80°C. The adsorbed sample was charged at the top and eluted saturates with 60 mL of petroleum ether (40°-60°C), aromatics with 90 mL benzene and NSO with 60 mL methanol, respectively. The solvents were evaporated in rotary vacuum evaporator at 60°C. Each fraction was transferred in tared sample vials, dried and weighed. Gas chromatographic analysisChemito-1000 gas chromatograph was used for gas chromatographic analysis of whole oils, saturate and aromatic fractions on a FID detector under the conditions mentioned below. Conditions for saturate (alkanes)Column, BP-5 (30 m × 0.25 mm); Initial temperature, 80°C; final temperature, 300°C; ramp rate, 4°C per min; carrier gas, nitrogen (40 mL/min); injection temperature, 310°C. Conditions for aromatic analysisColumn, BP-5 (60 m X 0.25 mm); Initial temperature, 80°C; final temperature, 300°C; ramp rate, 3°C per min; carrier gas, nitrogen (30 mL/min); injection temperature, 310°C. Identification of isolated potent hydrocarbon degraderAfter examining the chromatograms, a greater decrease in front-end boiling range and residual carbon of the oil samples degraded by Pseudomonas strain PS-I suggested that this strain was responsible for the breakdown of higher molecular weight constituents of the oil compared to two Pseudomonas strain PS-II, PS-III. Therefore, identification of Pseudomonas strain PS-I (the potent strain) was carried out by biochemical test as well as by 16Sr-DNA genes sequencing from The Energy and Resources Institute (TERI), New Delhi, India. 16S rDNA sequencing of PS-I−for molecular characterizationBacterial strain PS-I was identified by 16S rDNA sequence structure. The 500 bases sequence of 16S rDNA was obtained by sequencing. The electrophoregram of partial sequence was aligned with blast search of NCBI, RDP and microseqTM databases. The sequence aligned gave 97% similarity with Pseudomonas putida. Out of 474 bases matched
with databases, 468 bases gave homology with Pseudomonas putida. Therefore, bacterial strain PS-I was identified as Pseudomonas putida. Results and Discussion Test microorganismsTwenty microbial strains capable of using hydrocarbon as sole source of carbon, were isolated of which 11 (A1-A11) were recorded from the soil sample of oil production site of Lingala oil field and other 9 (B1-B9) from oil contaminated soil of a nearby garage of Hardwar. Similarly, in preliminary study 57 oil degrading bacteria were isolated from oil impregnated soil in search of potent hydrocarbon degrader14. These isolates were purified from soil samples on the basis of their colony morphology, texture, growth, temperature and hydrocarbon utilization ability. Preliminary screening and laboratory biodegradation studyThe pure hydrocarbon utilizing bacterial strains were isolated first in mineral salt broth containing 1% crude oil (Lingala well # X) and later on petroleum agarose plates. The isolated strains were examined further for substrate utilization. Over the year, substrate specificity in newly isolated organisms has been a routine diagnostic test for speciation and identification15. Microbes isolated on petroleum agarose plate have been tested for its ability to utilize the major classes of hydrocarbons viz., gaseous alkanes, n-paraffins, aromatics etc., which has been described elsewhere12. After substrate utilization study, it was concluded that out of 20, only five strains (A2, A7, B1, A9, B5) were potent hydrocarbon degraders and capable of degrading practically most of the representative fractions of petroleum. After morphological identification and number of biochemical tests, these isolates (A2, A7, B1, A9, and B5) were identified as Pseudomonas strains PS-I, PS-II, PS-III, PS-IV and Bacillus, respectively. Pseudomonas spp. comprised the first most numerous group than other taxa among petroleum degraders isolated from an oilcontaminated site in colgate creek in Baltimore Harbor. It was 27% of the total isolates identified16. Pseudomonas is most frequently reported and, so far, most studied of hydrocarbon degradation genus17-19. Laboratory biodegradation studies of crude oil (whole oil) have been conducted up to a period of 60 days, under optimized conditions of pH, temperature and crude oil concentration on Lingala well # X crude oil by deploying standard strain of Acinetobacter
MITTAL & SINGH: ISOLATION OF HYDROCARBON DEGRADING BACTERIA
calcoaceticus as well as isolated Pseudomonas strains PS-I, PS-II, PS-III, PS-IV and Bacillus. The biodegradation effect was seen after every 15 days. The concentration of oil before and after biodegradation has been estimated gravimetrically. The six different strains under study have shown varying biodegradation potential (Table 1). Out of four Pseudomonas strains, PS-I proved to be the most potent hydrocarbon degrader and crude oil degradation gradually increased from 29.77% in 15 days to 57.13% in 60 days. The study indicates that this strain was more or less as potent hydrocarbon degrader the standard strain. Gas chromatographic (GC) analysis of the crude oil and its fractions (saturate and aromatic) has been used to determine the biodegradability of crude oil20. Effect of biodegradation on fractions of crude oilEffect of biodegradation on alkane, aromatics and NSO + asphaltene fractions by standard strain and various isolated strains has been studied for 60 days. Effect was seen at time interval of 15 days (Table 2). Alkanes and aromatic fractions separated by column chromatography were analyzed by gas chromatography. Gas chromatogram of alkane fraction of crude oil, biodegraded by standard strain Acinetobacter calcoaceticus revealed extensive biodegradation in 60 days. Most of the lighter alkane fractions were metabolized. The most commonly used biodegradation parameters i.e. nC17/Pr, nC18/Ph and Pr/Ph were calculated from the gas chromatograms (Table 3). The ratios of Pr/Ph represent isoprenoidal alkanes and are considered as recalcitrant and therefore, are also referred to as biomarker parameter9,21. The visual examination of gas chromatogram of alkane fraction of crude oil biodegraded by isolated Pseudomonas strain PS-I
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indicated extensive biodegradation in 60 days, which was almost comparable with standard strain, Acinetobacter calcoaceticus. The results clearly revealed the extent of biodegradation as nC17/Pr ratio decreased from 2.5100 to 0.1232 in Pseudomonas strain PS-I in 60 days. Similarly, nC18/Ph ratio also decreased from 7.2886 to 0.3853. The ratio of Pr/Ph indicated meager degradation of pristane. Index used to monitor the progress of biodegradation is the rate of decrease in the ratios of nC17/Pristane and nC18/Phytane. The decreasing rate in the ratios of nC17/Pristane and nC18/Phytane has been used to monitor the progress of biodegradation during the EPA Alaska oil spill bioremediation project22. The application of this concept was based on the principle that during biodegradation, decrease of total oil residues could occur because of other nonbiological processes. Thus, changes in hydrocarbon composition that are indicative of biodegradation must be measured accurately and this is done historically by examining the weight ratio between hydrocarbons known to be readily biodegradable, such as the C17 and C18 alkanes, and those that biodegrade slowly, such as the branched alkanes (Pristane and Phytane), but with very close chromatographic behavior6. The two isolates (Acinetobacter calcoaceticus and Pseudomonas strain PS-I) showed a similar trend in the decreasing ratios of nC17/Pristane and nC18/Phytane up till the stage that the recalcitrant Pristane and Phytane were degraded. Gas chromatogram of alkane fraction of crude oil biodegraded by isolated Pseudomonas strain PS-II, Pseudomonas strain PS-III indicated moderate biodegradation and Pseudomonas strain PS-IV, Bacillus showed less biodegradation in 60 days. Nevertheless, other parameters indicated progressive
Table 1Laboratory biodegradation studies of crude oil (whole oil) [Values are the mean ± SE of 3 replications] Strains
Acinetobacter calcoaceticus
Incub.Period(d) 0d [Initial conc. (g)] 15d
F 2.463
D -
Pseudomonas Strain-I F 2.489
Strain-II D -
F 2.474
Bacillus
Strain-III D -
F 2.467
Strain-IV D -
F 2.49
1.626±0.54 33.98 1.748±0.80 29.77 1.956±0.75 20.94 2.074±0.75 15.93 2.348±0.75
D -
F 2.454
D -
5.7
2.289±0.75
6.72
30d
1.162±0.67 52.82 1.313±0.56 47.25 1.743±0.45 29.56 1.799±0.69 27.07 2.201±0.59 11.61 2.088±0.76 14.91
45d
1.063±0.89 56.84 1.202±0.66 51.71 1.566±0.74 36.7 1.687±0.37 31.62 1.964±0.23 21.24 2.005±0.69
60d
0.966±0.32 60.78 1.067±0.78 57.13 1.383±0.23 44.1 1.473±0.49 40.29 1.814±0.53 27.15 1.930±0.82 21.35
F-Final Conc. (g); D−degradation (%); d-days
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Table 2Effect of biodegradation on various fractions of crude oil [Values are the mean ± SE of 3 replications] Strain
Treatment period (days)
Alkane R 1.658±0.34 0.897±0.45 0.494±0.23 0.453±0.12 0.413±0.09 1.675±0.78 0.985±0.48 0.624±0.33 0.539±0.17 0.491±0.20 1.675±0.67 1.189±0.84 1.013±0.53 0.896±0.08 0.752±0.30 1.660±0.96 1.285±0.73 1.073±0.74 0.977±0.56 0.838±0.48 1.676±0.77 1.561±0.56 1.419±0.62 1.229±0.45 1.103±0.38 1.652±0.92 1.489±0.43 1.317±0.33 1.247±0.52 1.204±0.21
0 15 30 45 60 Pseudomonas strain – I 0 15 30 45 60 Pseudomonas strain – II 0 15 30 45 60 Pseudomonas strain – III 0 15 30 45 60 Pseudomonas strain – IV 0 15 30 45 60 Bacillus 0 15 30 45 60 R−remaining Conc. (g); D−degradation (%) Acinetobacter calcoaceticus
Aromatic R 0.522±0.45 0.440±0.56 0.376±0.34 0.338±0.28 0.273±0.22 0.528±0.34 0.471±0.14 0.414±0.09 0.365±0.12 0.289±0.04 0.524±0.24 0.491±0.15 0.441±0.19 0.396±0.05 0.342±0.08 0.523±0.15 0.492±0.23 0.451±0.08 0.412±0.05 0.366±0.04 0.528±0.25 0.498±0.28 0.485±0.18 0.461±0.13 0.430±0.05 0.520±0.32 0.504±0.16 0.594±0.13 0.474±0.21 0.448±0.16
D 45.89 70.21 72.68 75.09 41.19 62.75 67.82 70.69 28.59 38.16 46.19 54.83 22.59 35.36 41.14 49.52 6.86 15.33 26.67 34.19 9.87 20.28 24.52 27.12
D 15.7 27.96 35.24 47.7 10.80 21.59 30.87 45.37 6.30 15.84 24.43 34.73 5.93 13.77 21.22 30.02 5.68 8.14 12.69 18.56 3.08 5.00 8.85 13.85
NSO+Asphalt R 0.283±0.65 0.289±0.35 0.292±0.30 0.272±0.17 0.280±0.18 0.286±0.12 0.292±0.07 0.275±0.11 0.298±0.13 0.287±0.06 0.285±0.07 0.276±0.03 0.289±0.14 0.274±0.03 0.289±0.11 0.284±0.14 0.279±0.07 0.275±0.05 0.298±0.11 0.269±0.03 0.286±0.25 0.289±0.18 0.297±0.02 0.274±0.09 0.281±0.07 0.282±0.07 0.296±0.11 0.270±0.07 0.284±0.04 0.278±0.08
Table 3Biodegradation indices Acinetobacter calc. Parameters nC17/Pr nC18/Ph Pr/Ph
Initial (0d) 30d 2.510 0.140 7.289 0.433 2.891 2.885
60d 0.120 0.350 2.620
Pseudomonas PS-II PS-III
PS-I 30d 0.143 0.424 2.860
60d 0.123 0.385 2.694
30d 0.182 0.665 2.809
bacterial attack upon the crude oil. The ratios of isoprenoids, pristane and phytane increase to North West suggesting depletion of normal paraffins 23. Gas chromatograms of standard strain and isolated Pseudomonas strains PS-I, PS-II, PS-III revealed that the lower aromatic fractions were
60d 0.143 0.427 2.722
30d 0.185 0.706 2.802
60d 0.151 0.436 2.763
PS-IV 30d 0.272 0.881 2.883
60d 0.199 0.493 2.871
Bacillus 30d 0.363 1.932 2.891
60d 0.262 1.185 2.890
removed over a period of time and higher aromatic hydrocarbon abundance was seen. This clearly indicated that Pseudomonas strains PS-I, PS-II and PS-III were comparatively better and potent aromatic hydrocarbon degraders. Out of these Pseudomonas strains PS-I was almost comparable with standard strain of Acinetobacter calcoaceticus.
MITTAL & SINGH: ISOLATION OF HYDROCARBON DEGRADING BACTERIA
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