Petroleum Hydrocarbon Degradation Potential of Biosurfactant extracted from Bacteria isolated from Oil Contaminated Sites

The concept of plants and microbes utilization for remediation measure of pollutant contaminated soil is the newest development in term of petroleum waste management technique. The research objective was to obtain wild grass types and hydrocarbonoclastic bacteria which are capable to synergize in decreasing petroleum concentration within petroleum contaminated soil. This research was conducted by using randomized completely block design. This research was conducted by using randomized completely block design. The first factor treatments were consisted of without plant, Tridax procumbens grass and Lepironia mucronata grass. The second factor treatments were consisted of without bacterium, single bacterium of Alcaligenes faecalis, single bacterium of Pseudomonas alcaligenes, and mixed bacteria of Alcaligenes faecalis with P. alcaligenes. The results showed that mixed bacteria (A. faecalis and P. alcaligenes) were capable to increase the crown and roots dry weights of these two grasses, bacteria population, percentage of TPH (total petroleum hydrocarbon) decrease as well as TPH decrease and better pH value than that of single bacterium. The highest TPH decrease with magnitude of 70.1% was obtained on treatment of L. mucronata grass in combination with mixed bacteria.[How to Cite: Gofar N. 2013.Synergism of Wild Grass and Hydrocarbonoclastic Bacteria in Petroleum Biodegradation. J Trop Soils 18 (2): 161-168. Doi: 10.5400/jts.2013.18.2.161][Permalink/DOI: www.dx.doi.org/10.5400/jts.2013.18.2.161]REFERENCESBello YM. 2007. Biodegradation of Lagoma crude oil using pig dung. Afr J Biotechnol 6: 2821-2825.Gerhardt KE, XD Huang, BR Glick and BM Greenberg. 2009. Phytoremediation and rhizoremediation of organic soil contaminants: Potential and challenges. Plant Sci 176: 20-30.Glick BR. 2010. Using soil bacteria to facilitate phytoremediation. Biotechnol Adv 28: 367-374. Gofar N. 2011. Characterization of petroleum hydrocarbon decomposing fungi isolated from mangrove rhizosphere. J Trop Soils 16(1): 39-45. doi: 10.5400/jts.2011.16.1.39Gofar N. 2012. Aplikasi isolat bakteri hidrokarbonoklastik asal rhizosfer mangrove pada tanah tercemar minyak bumi. J Lahan Suboptimal 1: 123-129 (in Indonesian). Hong WF, IJ Farmayan, CY Dortch, SK Chiang and JL Schnoor. 2001. Environ Sci Technol 35: 1231.Khashayar T and T Mahsa. 2010. Biodegradation potential of petroleum hydrocarbons by bacterial diversity in soil. Morld App Sci J 8: 750-755.Lal B and S Khanna. 1996. Degradation of Crude Oil by Acinetobacter calcoaceticus and Alcaligenes odorans, J Appl Bacteriol 81: 355- 362.Mackova M, D Dowling and T Macek. 2006. Phytoremediation and rhizoremediation: Theoretical background. Springer, Dordrecht, Netherlands. 300 p. Malik ZA and S Ahmed. 2012. 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Determination of total petroleum hydrocarbon (TPH) and some cations (Na+, Ca2+ and Mg2+) in a crude oil polluted soil and possible phytoremediation by Cynodon dactylon L (Bermuda grass). J Environ Earth Sci 2: 12-17.Pezeshki SR, MW Hester, Q Lin and JA Nyman. 2000. The effect of oil spill and clean-up on dominant US Gulf Coast Marsh Macrophytes: a review. Environ Pollution 108: 129-139.Pikoli MR, P Aditiawati and DI Astuti. 2000. Isolasi bertahap dan identifikasi isolat bakteri termofilik pendegradasi minyak bumi dari sumur bangko. Laporan Penelitian pada Jurusan Biologi, ITB, Bandung (unpublished, in Indonesian).Pilon-Smits E and JL Freeman. 2006. Environmental cleanup using plants: biotechnological advances and ecological considerations. Front Ecol Environ 4: 203-10. Rahman KSM, JT Rahman, P Lakshmanaperumalsamy, and IM Banat. 2002. Towards efficient crude oil degradation by a mixed bacterial consortium. Bioresource Technol 85: 257-261.Rossiana N. 2004. Oily Sludge Bioremediation with Zeolite and Microorganism and It’s Test with Albizia Plant (Paraserianthes falcataria) L (Nielsen). Laboratory of Environmental Microbiology, Department of Biology Padjadjaran University, Bandung (unpublished).Rossiana, N. 2005. Penurunan Kandungan Logam Berat dan Pertumbuhan Tanaman Sengon (Paraserianthes falcataria L (Nielsen) Bermikoriza dalam Media Limbah Lumpur Minyak Hasil Ekstraksi. Laboratorium Mikrobiologi dan Biologi Lingkungan Jurusan Biologi Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Padjajaran, Bandung (in Indonesian).Sathishkumar M, B Arthur Raj, B Sang-Ho, and Y Sei-Eok. 2008. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium isolated from hydrocarbon contaminated areas clean. Ind J Biotechnol 36: 92-96.Shirdam R, AD Zand, GN Bidhendi and N Mehrdadi. 2008. Phytoremediation of hydrocarbon-contaminated soils with emphasis on effect of petroleum hydrocarbons on the growth of plant species. Phytoprotection 89: 21-29.Singer AC, DE Crowley and IP Thompson. 2003. Secondary plant metabolites in phytoremediation and biotransformation. Trends Biotechnol 21: 123-130.Singh A and OP Ward. 2004. Applied Bioremediation and Phytoremediation. Springler, Berlin, 281p.Surtikanti H and W Surakusumah. 2004. Peranan Tanaman dalam Proses Bioremediasi Oli Bekas dalam Tanah Tercemar. Ekol Biodivers Trop 2: 48-52 (in Indonesian).Wenzel WW. 2009. Rhizosphere processes and management in plant-assisted bioremediation (phytoremediation) of soil. Plant Soil 321: 385-408.Widjajanti H, I Anas, N Gofar and MR Ridho. 2010. Screening of petroleum hydrocarbons degrading bacteria as a bioremediating agents from mangrove areas. Proceeding of International Seminar, workshop on integrated lowland development and management, pp. C7 1-9.Widjajanti H. 2012. 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Biodegradation of crude oil by Chelatococcus daeguensis HB-4 and its potential for microbial enhanced oil recovery (MEOR) in heavy oil reservoirs

Microbial eco-physiological strategies for salinity-mediated crude oil biodegradation

The biodegradation of oil products in the environment is often limited by their low water solubility and dissolution rate. Rhamnolipids produced by Pseudomonas aeruginosa AT10 were investigated for their potential to enhance bioavailability and hence the biodegradation of crude oil by a microbial consortium in liquid medium. The characterization of the rhamnolipids produced by strain AT10 showed the effectiveness of emulsification of complex mixtures. The addition of rhamnolipids accelerates the biodegradation of total petroleum hydrocarbons from 32% to 61% at 10 days of incubation. Nevertheless, the enhancement of biosurfactant addition was more noticeable in the case of the group of isoprenoids from the aliphatic fraction and the alkylated polycyclic aromatic hydrocarbons (PHAS) from the aromatic fraction. The biodegradation of some targeted isoprenoids increased from 16% to 70% and for some alkylated PAHs from 9% to 44%.

The aim of this research was to study the influence of nanoparticles and the initial oil amount on the biodegradation of crude oil. Production of biosurfactants was also assessed. Crude oil-utilizing bacteria were isolated from oilfields via enrichment in chemically defined medium with crude oil a sole carbon source. The isolates could be affiliated to the genera Bacillus, Pseudomonas, Achromobacter, and Microbacterium by 16S rDNA sequencing and phylogenetic analysis. GC/FID analysis revealed 52 to 98% degradation of the oil saturate fraction within one month. Nanoparticles of ZnO inhibited growth and crude oil biodegradation by one isolate. (NBHCO4). Two strains, NBHCO2 and NCEOW, emulsified and utilized water-in-oil emulsions (chocolate mousse). Two bacterial strains, I-19 and NBHCO2 grew with crude oil in cultures containing up to 20% oil. Degradation extent in the I-19 culture increased as the oil amount increased. On the contrary, the NBHCO2 culture exhibited a decrease in degradation extent as the oil amount increased. Biosurfactant production in only one crude oil culture (I-19) could be confirmed by the observed reduction in surface tension. Some isolates produced biosurfactants from water-soluble substrates such as glucose. The NBHCO2 strain produced a lipopeptide biosurfactant which reduced the surface tension of the growth medium from 72 to 27mN/m. A gene of catechol dioxygenase was detected in the I-19, NBHCO4, and NCEOW isolates. In conclusion, metal oxide nanoparticles can interfere with crude oil biodegradation. Biosurfactants are not necessarily a prerequisite for crude oil biodegradation. The initial oil amount is a significant determinant of oil biodegradability. The isolates can by applied for bioaugmentation of petroleum-polluted soil and biosurfactants production.

Kinetic modeling and half life study on bioremediation of crude oil dispersed by Corexit 9500

Crude oil was treated with purified emulsan, the heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population as well as nine different pure cultures isolated from various sources was tested for biodegradation of emulsan-treated and untreated crude oil. Biodegradation was measured both quantitatively and qualitatively. Recovery of CO(2) from mineralized C-labeled substrates yielded quantitative data on degradation of specific compounds, and capillary gas chromatography of residual unlabeled oil yielded qualitative data on a broad spectrum of crude oil components. Biodegradation of linear alkanes and other saturated hydrocarbons, both by pure cultures and by the mixed population, was reduced some 50 to 90% after emulsan pretreatment. In addition, degradation of aromatic compounds by the mixed population was reduced some 90% in emulsan-treated oil. In sharp contrast, aromatic biodegradation by pure cultures was either unaffected or slightly stimulated by emulsification of the oil.

The biodegradation of five weathered crude oils by two species ofAeromonas, (B59-4 and E. BOB) was investigated in varying concentrations of sodium chloride. A minimal salts medium whose NaCl concentration increased serially by 0.5% w/v up to 1.5% w/v was used to investigate the growth of these strains in glucose, and their biodegradation of the crude oils. The latter was also investigated in fresh and aged sea water. Strain B59-4 was more potent than E. BOB in the degradation of all five crude oils and at all four levels of salt concentration tested. The amount of oil degraded by each strain increased initially to a maximum level at 0.5% w/v NaCl, but thereafter decreased with increasing salt concentration, and the patterns were similar to those of aged and fresh sea water, respectively. The Forties and Nigerian crude oils with lower specific gravity, were more readily degraded than the Libyan and Venezuelan with higher specific gravity. The growth of the two strains ofAeromonas in glucose and their biodegradation of crude oils was optimal at 0.5% w/v NaCl, and thereafter decreased with increasing salt concentration of the basal medium.

Production and spillage of petroleum hydrocarbons which is the most versatile energy resource causes disastrous environmental pollution. Elevated oil degrading performance from microorganisms is demanded for successful microbial remediation of those toxic pollutants. The employment of biosurfactant-producing and hydrocarbon-utilizing microbes enhances the effectiveness of bioremediation as biosurfactant plays a key role by making hydrocarbons bio-available for degradation. The present study aimed the isolation of a potent biosurfactant producing indigenous bacteria which can be employed for crude oil remediation, along with the characterization of the biosurfactant produced during crude oil biodegradation. A potent bacterial strain Pseudomonas aeruginosa PG1 (identified by 16s rDNA sequencing) was isolated from hydrocarbon contaminated soil that could efficiently produce biosurfactant by utilizing crude oil components as the carbon source, thereby leading to the enhanced degradation of the petroleum hydrocarbons. Strain PG1 could degrade 81.8% of total petroleum hydrocarbons (TPH) after 5 weeks of culture when grown in mineral salt media (MSM) supplemented with 2% (v/v) crude oil as the sole carbon source. GCMS analysis of the treated crude oil samples revealed that P. aeruginosa PG1 could potentially degrade various hydrocarbon contents including various PAHs present in the crude oil. Biosurfactant produced by strain PG1 in the course of crude oil degradation, promotes the reduction of surface tension (ST) of the culture medium from 51.8 to 29.6 mN m−1, with the critical micelle concentration (CMC) of 56 mg L−1. FTIR, LC-MS, and SEM-EDS studies revealed that the biosurfactant is a rhamnolipid comprising of both mono and di rhamnolipid congeners. The biosurfactant did not exhibit any cytotoxic effect to mouse L292 fibroblastic cell line, however, strong antibiotic activity against some pathogenic bacteria and fungus was observed.

To determine the type and maturity of normal and biodegraded crude oil and condensates, 13 samples from diverse types of Tertiary and Cretaceous reservoirs from Alabama, California, Texas, and Venezuela were characterized utilizing pulsed laser fluorescence microscopy (developed by Borst for coal characterization). Parameters such as fluorescence lifetime in nanoseconds (ns) for three resolved fluorophores and percent contribution of each fluorophore were correlated with organic geochemical and bulk chemical data on crude oils and condensates. The fluorescence lifetime (ns) of the intermediate component has been correlated with ratios of aromatics to the heterocomponents (fluorescence emitters). This relationship, when compared with the ratio of aromatics to asphaltenes (fluorescence quenchers), revealed characteristic distribution of oils derived from two different reservoirs: (1) marine source-derived oil from Lovetts Creek field (Smackover), Alabama, and (2) terrestrial source-derived oil from Charamousca field (Wilcox), south Texas. These data are supported by geochemical fingerprinting. The above-mentioned ratios and the ratios of the fluorescence lifetimes of two individual fluorophores also show characteristic patterns for biodegraded (alteration in reservoir) and nonbiodegraded crude oils from the Wilcox, Duval County, south Texas, and the Oficina reservoir, Venezuela. These ratios also suggest the possible maturity of two biodegraded crude oils from the Wilcox,more » south Texas, that coincides with the results of a biomarker study. The above parameters indicate the similarity in origin of two crude oils and one condensate from the Travis Peak Formation, Chapel Hill field, east Texas.« less

Crude oil biodegradation by bacterial strains isolated from oil contaminated soil samples, Oman, were performed and its potential applications in crude oil waste management were analyzed. Accidental and occasional crude oil spills, treatment of produced water containing hydrocarbons and oil, and waste management are a major concern for petroleum industries. Various techniques such as, chemical, physical, biological and thermal treatments, are reported for treating spills and wastes on-site. We analyzed crude oil biodegradation by selected bacterial isolates from Oman, under reservoir conditions. Four potential bacterial isolates were selected, characterized by MALDI-Biotyper, and studied for crude oil biodegradation at 40 °C. The isolates were studied morphologically and by scanning electron microscope (SEM), and any changes in surface tension (biosurfactant production), during growth on crude oil as the only carbon source. Crude oil characteristics before and after biodegradation were analyzed by Gas chromatography-Mass specrtrometry (GC-MS). The bacterial strains were identified as Pseudomonas mendocina, Pseudomonas putida, and Brevibacillus agri. During the course of crude oil biodegradation, bacterial isolates showed growth, as analyzed by optical density measurement at 660 nm and cellular protein estimation; no changes were observed in surface tension values, and alteration in the cell morphology in presence of crude oil was observed. All four isolates showed oil clearing zones on agar plates coated with crude oil. Crude oil degradation was analyzed by GC-MS with respect to carbon numbers from C12 −C30. P. mendocina II, P. putida and B. agri showed reduction in all the compounds, but P. mendocina I showed very little degradation of hydrocarbons. Maximum crude oil biodegradation (~50%) was observed by P. mendocina II. It can be concluded that the present findings indicate the application potential of these bacterial isolates in the crude oil biodegradation. This could be the ideal solution to treat the contaminated soil and water, which can also be applied for the bioremediation of oil spills and water bodies as a cost effective and environmental friendly approach.

Laboratory core studies were conducted to determine sediment oxygen demand (SOD) in petroleum hydrocarbon contaminated salt marshes. Natural degradation processes in salt marshes such as aerobic respiration, nitrification and sulfate reduction were investigated to quantify SOD. Oxygen demand in oiled/fertilized cores was greater than control and fertilized cores due to increased oxygen consumption by oil degradation and oxidation of reduced chemicals; sulfide and ammonium. SOD under non-flooded condition was greater than flooded condition due to an increased area of oxic-anoxic interface during air exposure. This may indicate that significant biodegradation of crude oil only occurs when the surface of the salt marsh is exposed to the atmosphere. Simultaneous measurements of total CO2 production and 35SO4 2- reduction were performed to partition carbon flow pathways between aerobic respiration and sulfate reduction in crude oil contaminated salt marsh sediment. Crude oil and fertilizer stimulated sulfate reduction rates and highest sulfate reduction rates were observed in the top 2-cm depth. Sulfate reduction was a major sink of oxygen demand. Mineralization kinetics of 14C-hexadecane and -phenanthrene were determined from core studies. The obtained zero-order rate constants at different level of nitrogen loading rates indicate that biodegradation of crude oil was enhanced when ammonia nitrogen concentration in pore water was maintained above 200 ppm. A SOD model was successfully applied to estimate SOD in oil contaminated salt marshes. Carbonaceous and nitrogenous sediment oxygen demand models were calibrated through a non-linear regression technique. Oil sediment oxygen demand (OSOD) model which simulates oxygen uptake, cell growth and oil degradation simultaneously was solved numerically and compared with experimental data. Field study was conducted to measure sediment oxygen demand, sulfate reduction and oil sediment oxygen demand over six months. SOD (flooded and non-flooded) in control, oiled, and oiled/fertilized (ammonia nitrate treated) salt marsh soils was measured. Higher SOD under oiled and oiled/fertilized conditions indicates that aerobic biodegradation of crude oil is occurring and thus increasing O2 demand. Although sulfate reduction consumes a significant amount of oxygen demand, major portion of the demand is caused by aerobic respiration due to crude oil degradation.

Bacterium Ralstonia pickettii has ability to survive and thrive in low nutrient condition as well as a capability to remediate some pollutants and using them as carbon and energy source. In this study, the ability of R. pickettii on biodegradation of crude oil under high salinity medium was investigated. R. pickettii was pre-incubated in nutrient broth (NB) medium and then, washed and transferred to artificial seawater medium. Crude oil was added to each culture and incubated for 7 and 14 days. The biodegradation of crude oil was analysed using Gas chromatography mass spectrometry (GC-MS). The result showed that R. pickettii had successfully degraded the crude oil in the high salinity artificial seawater. The incubation on 7 and 14 days did not show a significant effect on the number of the degraded compounds. The optimum recovery percent was obtained from the derivation of 2,6,10,14-tetramethyl hexadecane with the recovery percentage of 12.7% and 16.0% for 7 and 14 days respectively. This study indicates that R. picketti can be potentially used for bioremediation of crude oil under high salinity environments.

The contribution of chemical dispersants and biosurfactants on crude oil biodegradation by Pseudomonas sp. LSH-7′

The origin of the oil sand bitumens of Alberta: a chemical and a microbiological simulation study