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Authors: Chhatre, Suneel
Issue Date: 1998
Abstract: Preamble Oil exploration, refining and transport activities have considerably increased over the past few decades due to increasing demand for petroleum crude world wide. This enhanced activity poses the problems related to oil pollution which are difficult to manage inview of recalcitrant and insoluble nature of oil. Accidental oil spills receive much attention and evoke public concern. Although oil spills occur rarely, they invariably lead to significant contamination of ocean and shoreline environment. Traditionally the oil spills are dealt with physical and chemical methods, which are well established [Ollis, 1995]. However, the biological methods, which are preferable due to ecological reasons, require considerable research endeavour [Prince, 1993]. A number of different technologies may fall into the category of biological methods including the use of straw or plant material as adsorbent for oil, addition of nutrients to enhance biodegradation of crude oil with indigenous microorganisms and addition of allochthonous organisms to the site (bioremediation). The last two procedures fall under programmed bioremediation [Swanell, 1996]. Exxon Valdez oil spill focused attention to alternative clean up methodologies [Pritchard & Costa, 1991]. For example, in physical methods, the use of sorbants is advisable as it facilitates a change of pollutant phase from liquid to semi-solid, which is easy to contain. While hydrophobicity and oleophilicity are primary determinants of successful sorbants, other important factors include retention of oil over time, amount of oil sorbed per unit weight of sorbent, and available specific surface area for sorption [Choi, 1992]. Among synthetic products, polypropylene and polyurethane foam are the most widely used sorbents in oil spill cleanup due to their high oleophilic and hydrophobic properties. Several reports are available on the degradability of hydrocarbon components of crude oil through biological methods. In Exxon Valdez oil spill, nitrogen and phosphorus containing fertilizers were applied in order to accelerate the biodegradation of the oil by indigenous microorganisms. Most of the bioremediation techniques currently in use are based on this principle of enhancing oil degrading capabilities of indigenous microbes by adding nutrients [Prince, 1993]. However, a substantial lag period is always observed in microbial utilization of crude oil. Although the utilization of microorganisms specialized in degradation of recalcitrant compounds has been proposed by several researchers, successful applications of exogenous microorganisms to clean up pollutants have not yet been reported [Swanell, 1996]. Since, crude oil is a multisubstrate system of more than 300 compounds, it is not amenable to complete biodegradation by a mono culture system. Mixed culture approach has been reported by several researchers which provides fairly good degradation. However, mixed culture approach has several disadvantages as a process particularly as the rate of biodegradation is a crucial factor in oil spill clean up. Since a multispecies system, like mixed culture, is not fully characterized, the organisms being present in varying ratios undergo uncontrolled selective population growth and ultimately inhibit the degradative activity leading to accumulation of various components of the multisubstrate crude oil system. Another pre-requisite to biotreatment of oil spills in on-site emulsification 'as the microorganisms effectively attack solubilized components. Further, the biotreatment of marine spills requires that the organisms are augmented with ability to tolerate high salinity. The present study explores the possibility of use of a two step treatment process for oil spill remediation, where in the first step a cost-effective sorbant is used to absorb crude oil, followed' by biodegradation of remaining crude oil by a designed and adapted bacterial consortium. Unlike the mixed population, the constituent of consortia have been synchronized to specific physiological conditions leading to effective biodegradation of crude oil. This biodegradation strategy could also be applied to the "seeding" of biotreatment processes for petrochemical wastewaters/ refinery wastewaters or hydrocarbon contaminated soils. The present study has the following objectives : • Physical removal of crude oil using alkali treated sawdust • Isolation and screening of hydrocarbonoclastic organisms from contaminated site(s) • Design of consortium for degradation of crude oil and model petroleum/aromatic mixture • Role of biosurfactant in crude oil degradation • Determination of the impact of various inducer compounds on the adaption of consortia in favour of degradation of aromatic fraction of crude oil Physical Skimming of Crude Oil Hydrophobicity and buoyancy are the main characteristics of successful sorbents. In present study, a biological matrix sawdust has been used as sorbent after alkali treatment. Raw sawdust has a tendency to sink when it is applied on water body. To increase the buoyancy property and specific surface area, sawdust was treated with 1% sodium hydroxide under high temperature (120CC) and pressure (15 lb). The treatment increased the specific area of sawdust from 42 to 96 m2/g and resulted in pore formation, which was confirmed by scanning electron micrographs. This was further supported by mercury porosimeter studies. The application of alkali treated sawdust yielded 90-95% removal of floating crude oil from water surface. Isolation and Screening of Hydrocarbonoclastic Organisms for Design of Consortium A semicontinuous batch fed reactor was used to enrich 5 It. of activated sludge with crude oil. After six months the chemical oxygen demand of the effluent was reduced to 220 mg/L from 572 mg/L of initial value. At this stage, organisms were isolated by serial dilution plating and subjected to a three tier screening process. Primary screening was based on the morphological characteristics of organisms (colony appearance, optimal temperatur, incubation time etc.). Antibiotic sensitivity was the criterion for seconary screening. Six antibiotics were used for the in test. Tertiary screening involved hydrocarbon utilization potential of organisms. Five representative hydrocarbons (catechol, phenanthrene, dodecane, eicosane and octacosane) were used for final screening, on the basis of degradative charcteristics three bacterial isolates were selected alongwithn P.putida MTCC* 102 in the design of consortium. All the three selected isolates were subjected to a battery of biochemical tests to characterize their metabolic potential. Presence of certain key dissimilatory enzymes (phenol monooxygenase, catechol 2,3 dioxygenase, alk B) was checked by PCR. Bacterial Consortium On the basis of three tier screening; three isolates, viz. NCC.DSS3, GSS6 , DSS8 and a procured strain of Pseudomonas putida MTCC" 102 were used in the design of the consortium for crude oil degradation NCC.GSS3 : gram negative, oxidase and catalase positive, utilises catechol and phenanthrene efficiently NCC.DSS6 : gram negative, oxidase and catalase positive, produces a biosurfactant NCC.DSSo : gram negative, oxidase and catalase positive, capable of utilising long chain aliphatics (octacosane and tetracosane) P.putida MTCC'102 : Capable of consuming down stream metabolites formed in aromatic degradation processes. The designed consortium was adapted on catechol/ benzoate/ salicylate for 48 hours, and then used for degradation of a model petroleum. Gas chromatographic analysis demonstrated that catechol adapted consortium performed better. Therefore, for crude oil, catechol was selected for preculturing. Capillary gas chromatographic and gravimetric analysis have shown that 65-70% of crude oil is degraded by the designed consortium in 72 hours. Consortium grown on nutrient rich media (Luria Bertani Broth) could not degrade crude oil as efficiently as the adapted one. Similarly the individual organisms also did not exhibit the dissimilation property as iv they demonstrated when inoculated together. The efficacy of consortium for crude oil degradation in soil was also tested where black cotton soil was spiked with crude oil. IOCL, Mathura soil, contaminated and weathered, was also subjected to bioremediation successfully. The aromatics present in soil were identified by GC-MS. Osmotolerance & Hydrocarbon Degrading Potential Though the designed consortium had metabolic flexibility to attack crude oil components effectively in normal conditions, these soil isolates were unable to tolerate high salt conditions. Therefore the consortium members were imparted with 'U' operon, subcloned in a broad host range vector (pMMB 206). The expression of pro'U' operon in selected transformants was evident from their ability to tolerate and grow under high salt concentration. Since the ptac, a strong promoter, is used in the pMMB 206 to express the operon, its basal expression is sufficient to provide desired level of osmo-protection. The degradation potential of genetically engineered consortium was tested with model petroleum (a mixture of representative molecules present in crude oil) and crude oil under 0.7 M NaCI (4% w/v) condition. It was observed that the addition of salt tolerance phenotype did not hamper the hydrocarbon utilizing capability of the members of the consortium. Biosurfactant Production by DSS6 and its Characterization Biosurfactants are amphibolic molecules synthesized by many oil-degrading bacteria and include glycolipids, lipopetides and polysaccharide-protein complexes [Fiechter, 1992]. NCC. DSS6 a member of consortium produced a biosurfactant, which was identified as rhamnolipid by Infrared Spectroscopy. The produced biosurfactant lowered the surface tension of liquid media from 72 to 37 dynes/cm in 96 hours. NCC. DSS6 harbours a plasmid which was proved by alkaline lysis, boiling prep and Qiagen Column methods. The 20 kb plasmid was subjected to restriction digestion to confirm the size. To study the role of plasmid in biosurfactant production, the plasmid was cured by acridine orange. The cured NCC. DSS6 could not lower the surface tension of liquid media. The same plasmid was mobilized into Pseudomonas putida MTCC'102 by electroporation where it could not impart the property of surfactant production. Retransformation of the cured NCC. DSS6 with the same plasmid again brought back the biosurfactant production property. These experiments prove that DSS6 plasmid carries certain essential genetic determinants for biosurfactant operon, while rest of the genetic information is the on chromosome of NCC. DSS6. Crude oil is a mixture of several hydrophobic compounds. For effective biodegradation, availability of these compounds to bacteria is an essential prerequisite. Biosurfactants lower the surface tension of water, and facilitate the formation of oil-in-water emulsion which is followed by crude oil biodegradation process. It was observed that without NCC. DSS6l a biosurfactant producing organism, the designed and pre-adapted consortium lost the emulsifying potential and could not degrade oil rapidly. Utilization of Various Fractions of Crude Oil Crude oil is a heterogeneous mixture of unidentified compounds. Its components are broadly grouped into four classes according to their differential solubility in organic solvents, viz. the saturates (n- and branched-chain alkanes and cycloparaffins), the aromatics (mono-, di-, and polynuclear aromatic compounds containing side chains and/or fused cycloparaffinic rings), the resins (aggregates with a multitude of building blocks such as pyridines, aminolines, carbazoles, thiophenes), and the asphaltenes (aggregate of extended polyaromatics, naphthenic acids, sulphides, polyhydric phenols) [Leahy &Colwell, 1990]. The saturates present in crude oil are more amenable to biodegradation, and degrade rapidly while aromatic and polynuclear aromatic hydrocarbons are less susceptible. The resins and asphaltenes are known to be recalcitrant. To study the utilization of various fractions degraded by the designed consortium, the experimental samples were subjected to silica gel vi column chromatography. Consumption of saturates/ aliphatic fraction was 81%, aromatic degradation 63%, while asphalt fraction did not achieve any substantial degradation. The results were confirmed by gas chromatographic profile of saturates and aromatic fraction before and after degradation. Role of Inducer/ Adapter on Aromatic Degradation The aromatic and polynuclear aromatic hydrocarbons (PAHs) are less biodegradable in nature. In the experiments with crude oil, the designed consortium degraded aromatic fraction but to a lesser extent. To increase the rate and extent of aromatic degradation, Pseudomonas putida ppG7 harbouring NAH plasmid was substituted in the consortium instead of P. putida MTCC'102 as the former is capable of degrading napthalene and similar compounds. Adaption on different intermediate/ inducer molecules other than catechol (or cross acclimation) was tried. Benzoate and salicylate were used in the experiments which are known inducers for NAH operon. A standard mixture of eleven aromatic compounds, representing different classes of aromatics (monocyclic, dicyclic, hetero cyclic etc.), was used as substrate. Again it was observed that catechol adapted consortia yielded better results compared to salicylate and benzoate as in case of model petroleum. The rationale behind the use of salicylate/ benzoate/ catechol as inducer was that, in multisubstrate-multispecies system like the consortium and aromatic mixture, the lower molecular weight aromatics would be degraded at a higher rate, and that eventually the down stream metabolites of these compounds in turn activate other operons required for the degradation of the consistuents of crude oil. This cross acclimation was observed in experiments with model petroleum and model aromatic mixture. The consortium was grown on citrate for control experiments. The protein profiles were prepared using SDS-PAGE from the consortium members grown on the inducers (catechol, benzoate, salicylate) and citrate. However, twodimensional gels did not show any significant change. Conclusion Oil spill remediation has been attempted with various physico-chemical methodologies resulting in phase transfer of the hydrocarbons. The decontamination has been addressed with the supply of the nutrients, resulting in eutrophication or bioaugmentation as shown in Exxon valdez sea shore pollution studies. The present study deals with the problem through a combination of physico-chemical and biological methods. In the first step, the alkali treated sawdust removes 90-95% of crude oil from the water surface. The remaining oil is subjected to biodegradation by a designed bacterial consortium. The pre-adapted bacterial consortium has specialized organisms to perform different physiological functions for crude oil degradation, and synthetic petroleum wastewater treatment. The genetic modification for osmotolerance (imparting Pro 'If operon) extends the use of the consortium to high salt conditions. Biosurfactant induced emulsification enhances the flexibility of the consortium to attack various hydrophobic contaminants. The results from the study suggest that there is a need to evaluate the role of inducer(s) in aromatic degradation and the specificity of certain key enzymes. This may enable complete mineralization of the crude oil fractions to the final end product, viz. Carbondioxide.
Other Identifiers: Ph.D
Appears in Collections:DOCTORAL THESES (Bio.)

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