Globally environmental scientists have shown increased interest in promoting biological methods referred to as “Green technology” in the clean-up technologies for hydrocarbon-polluted sites. These methods are less expensive and do not introduce additional chemicals/pollutants into the environment. Compared to physico-chemical methods, bioremediation / biodegradation offer a feasible alternative for this purpose to the petroleum industry. One reason is that the majority of the molecules in the crude oil and refined products are biodegradable. We have reported the isolation of Hydrocarbon Utilizing Bacteria (HUB) from crude oil contaminated soil and Untreated Effluent water (UEW) samples collected samples from selected ONGC sites in Gujarat state. Screening for hydrocarbon utilizing capacity of these bacteria was carried out using Bushnell-Hass (BH) medium with crude oil (K#X) and redox indicator 2, 6 - dichlorophenol indophenol (DCPIP). Maximum utilization of hydrocarbons from crude oil was indicated by the total discoloration of DCPIP. In total five consortia (Hydrocarbon Utilizing Bacterial Consortium: HUBC) named HUBC-S, HUBC-W, HUBC, HUBC-Ank, and HUBC-AH were prepared to screen for the most efficient consortium for hydrocarbon degradation. Our results indicated that the consortium HUBC consisting of six isolates showed best results. Total discolouration of DCPIP by “HUBC” and its individual isolate was about 53 h and 120 h, respectively. The results of this study are presented here.

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Comparative Studies on Bacterial Consortiafor Hydrocarbon Degradation

Sixty nine hydrocarbon utilizing bacteria isolated from selected crude oil contaminated sites of ONGC (Gujarat) were screened for biosurfactant producing potential. VSHUB005 showing 28.59 ± 0.50% emulsification index, 69.73 ± 0.53 mN/m surface tension and 90.46 ± 0.08% interfacial tension reduction was considered as potential biosurfactant producer using primary (hemolytic activity and CTAB agar plate method) secondary (drop collapse, oil spread, emulsification index and surface tension) and tertiary (interfacial tension) screening methods. Very strong, negative correlation between surface tension measurement and interfacial tension reduction (rs= -0.961, AE = -1.000) was measured. VSHUB005 was identified as P. aeruginosa by 16S rDNA gene sequencing technique. The GenBank accession number for 16S rDNA sequence and NCIM culture deposition ID assigned are KC713611 and 5514, respectively

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Screening and identification of biosurfactant(bioemulsifier) producing bacteria from crude oilcontaminated sites of Gujarat, India

The purpose of this study was to analyze the potential of bacteria isolated from diesel-contaminated seawater for diesel biodegradation. Samples of diesel-contaminated seawater were collected from a ship dismantling facility in Madura Island, Indonesia. Bacteria were isolated using the serial dilution method. Bacteria with potential for diesel degradation were screened using nutrient agar and mineral salt medium (MSM) with 0%, 5%, 10%, and 15% (v/v) diesel as carbon source. Diesel degradation test was performed by inoculating potential bacterium into MSM with the optimum concentration selected from the screening stage. Diesel degradation percentage was analyzed by gas chromatography–mass spectrometry (GC–MS). In this research, six bacterial species were isolated and used in the screening stage. The optimum concentration was 10% diesel (v/v) based on the screening stage, and the bacteria coded as MFI5 exhibited the highest growth with 8.13 ± 0.19 log colony forming unit/mL after 7 days of incubation. The bacteria coded as MFI5 was identified as Vibrio alginolyticus. Results showed that V. alginolyticus was able to degrade diesel up to 26.78% after 14 days of incubation at 10% initial diesel concentration (v/v). V. alginolyticus can potentially degrade diesel in a contaminated environment with further investigation using the biostimulation and bioaugmentation techniques.

Potential of bacteria isolated from diesel-contaminated seawater in diesel biodegradation

Petroleum hydrocarbons pollutants, such as diesel fuel, have caused ecosystem damage in terrestrial and aquatic habitats. They have been recognized as one of the most hazardous wastes. This study was designed to optimize the effect of Tween 80 concentration, nitrogen (N)/phosphorus (P) ratio and salinity level on diesel biodegradation by Vibrio alginolyticus (V. alginolyticus). Response surface methodology with Box-Behnken design was selected with three factors of Tween 80 concentration (0, 5, 10 mg/L), N/P ratio (5, 10, 15) and salinity level (15‰, 17.5‰, 20‰) as independent variables. The percentage of diesel degradation was a dependent variable for 14 d of the remediation period. The results showed that the percentages of diesel degradation generally increased with an increase in the amount of Tween 80 concentration, N/P ratio and salinity level, respectively. The optimization condition for diesel degradation by V. alginolyticus occurred at 9.33 mg/L of Tween 80, 9.04 of N/P ratio and 19.47‰ of salinity level, respectively, with percentages of diesel degradation at 98.20%. The statistical analyses of the experimental results and model predictions (R = 0.9936) showed the reliability of the regression model and indicated that the addition of biostimulant can enhance the percentage of diesel biodegradation.

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Optimization of diesel biodegradation by Vibrio alginolyticus using Box-Behnken design

One of the primary issues noted worldwide in the marine environment is contamination by crude oil and it's by products. Contrasted with conventional methods, bioremediation offer a possible alternative to remove hydrocarbon pollution from the environment. The present study was undertaken to examine the capability of biosurfactant producing Pseudomonas aeruginosa (DKB1) on recovery of hydrocarbon contaminated soil. Initially Bacterial cell surface hydrophobicity (BATH) of the selected organism towards various hydrocarbons was observed in the following order; hexadecane (91.44%), sunflower oil (83.21%), olive oil (81.86%), hexane (74.78%), toluene (73.99%), diesel (72.60%) and a lower affinity was found against crude oil (46.98%). Further the biosurfactant produced by the candidate strain was extracted and the toxicity effect of the produced biosurfactant was tested against the micro crustacean, brine shrimp Artemia salina nauplii (Cytotoxicity) and the plants like cauliflower, cabbage, rice and green gram (Phytotoxicity). The result revealed that there was no toxicity observed in the tested A. salina and also in the tested plants. The biodegradation efficacy of the potent strain was screened by DCPIP (2, 6- Dichlorophenol indophenol) method using various hydrocarbons (kerosene, benzene, toluene and diesel). The result revealed that the selected organism has high hydrocarbon degrading potential and it turns the medium to become colorless. The crude oil biodegradation by the selected strain was evaluated by gravimetric analysis and also optimized the crude oil degradation potential. Finally the bioremediation of the crude oil contaminated soil was performed in situ laboratory scale experiment using the potent strain and the result indicated that there was up to 63.38% degradation of crude oil after 60 days of incubation. FTIR analysis result of the treated and untreated crude oil was confirmed that the candidate strain has the ability to recover the hydrocarbon polluted environment.

Studies on reclamation of crude oil polluted soil by biosurfactant producing Pseudomonas aeruginosa (DKB1)

In the present work we have studied degradation of diesel oil in oil-polluted soil. Oil-polluted soil culturing showed presence of variety of microorganisms. Hydrocarbon degrading potential of these microbes was confirmed and was identified. Acinetobacter sp, Bacillus sp., Micrococci sp., Pseudomonas sp. Streptomyces sp., and fungi Aspergillus sp, Trichoderma sp, Penicillin sp.were found part of hydrocarbon degrading microbial consortium. Analysis of extracted diesel oil was done using FTIR, HPLC, 1H NMR and GCMS techniques. It confirmed biodegradation of diesel oil confirming naphthalene and it’s derivatives as major pollutants which are able to dissolve in water. This further confirmed by hydrocarbon dominated water table.

https://www.imedpub.com/articles/bioremediation-of-complex-hydrocarbons-using-microbial-consortium-isolatedfrom-diesel-oil-polluted-soil.pdf

Pramod N. Kamblea

Papers

Bioremediation of complex hydrocarbons using microbial consortium isolated from diesel oil polluted soil.