Bioremediation of Petrol Engine Oil Polluted Soil Using Microbial Consortium and Wheat Crop

1Uttrakand State Council for Science & Technology. India. 2Department of Plant Pathology, G.B.P.U.A.T, Pantnagar, (U.K.), 263145, India. 3Department of Biochemistry, SDAU, Gujarat, India. 4Department of Microbiology, CBSH, G.B.P.U.A.T, Pantnagar (U.K.), India. 5Department of Environmental Microbiology BBAU, Lucknow (UP), India. 6Ecology, Climate change and forest influence Division, Forest Research Institute, Dehradun, India.

Petroleum contaminated soil causes severe pollution of ground water, threatens the safety of potable water, limits the use of ground water, and causes enormous economic losses and ecological disaster 1 .Pollution of soil by petroleum hydrocarbons causes major changes in soil water, soil air, exchangeable iron and available phosphorous and sulphate.These changes affect plant growth adversely.However, certain microorganisms acclimatized to grow and thrive in oil polluted environment and play an important role in the treatment of pollutant by degradation process.Plant microbe and soil interactions are known to convert certain toxic pollutant to non toxic forms [2][3][4] .One of the limiting factors in this process is the bioavailability (i.e the extent to which hydrophobic contaminant available to microorganisms) of some fractions of the oil.Hydrocarbon degrading microorganisms produce biosurfactants of diverse chemical nature and molecular size.Biosurfactant are environment friendly, biodegradable, less toxic and non-hazardous hence preferred over chemical surfactants 5 .Biosurfactant increases the surface area of hydrophobic water-insoluble substrates and improve their bioavailability, thereby enhancing the growth of bacteria and the rate of bioremediation through emulsification 6 .They have better foaming properties and higher selectivity.They are active at extreme temperatures, pH and salinity 7 .Biosurfactants can be produced from industrial wastes and their by-products.This feature favours their cheap production and also allows utilizing waste substrates and reducing their polluting effect at the same time [8][9][10][11] .Hydrophobic pollutants present in petroleum hydrocarbons, soil/ water environment require solubilisation before being degraded by microbial cells.Present study is based on the use of microbial consortia and hyper accumulator crop (bio-phytoremediation).Crops like mustard and wheat facilitates the growth of microorganism in the rhizospheric zone by excreting root exudates and the microorganism support plant growt by producing IAA, siderophore, P solubilisation and indirectly by producing HCN and antibiotics which inhibits the growth of phytopathogens 12 .Characterizations of bacterial isolates recovered from the oil contaminated sites were characterized on the basis of their emulsification potential for different oil.
Most important species of oil degrading bacteria belong to genera Pseudomonas .These microorganisms excrete a variety of biosurfactants which are biodegradable and consequently environmentally safe and help in solubilisation of xenobiotic compounds.Different groups of microorganisms plant growth promotory properties are reported to produce biosurfactants.

Strains and culture conditions
All the bacterial strains (H1A= Pseudomonas aeruginosa and P1A= P. plecoglossicida) used in this study were isolated from oil contaminated sites (Source: Haldia & Pantnagar (22 0 3' 37.65''N latitude, 88 0 06' 35.09''E longitude and 29 0 01' 20.00N latitude and 79 0 29' 15.00E longitude.These two strains were selected and characterized on the basis of 16SrDNA sequencing.These two bacterial strains (H1A and P1A) which were previously used for biodegradation of mobil oil hydrocarbon by 13 .

PGPR Properties
The plant growth promotory properties viz.phosphate solubilization, siderophore production, indole acetic acid production, ammonia production, hydrogen cyanide (HCN) production were tests performed for biosurfactant production and their characterization included turbidity test, foaming, cell biomass and emulsification index (mobil oil) 13 .

Consortia development
Based on PGPR properties bacterial strains were selected to develop consortium.Bacterial strains showing compatibility on kings B agar medium were selected for consortium.Compatibility among the strains was also checked in MSM broth medium in the presence and absence of hydrocarbons (petrol engine oil 2%).The absorbance was taken from 0 to 120h at an interval of 24h using a spectrophotometer (Lamda35, Perkin Elmer).

Pot Trial
The experimental soil was mixed, allowed to dry to get water content less than 1%, sieved with 2 mm sieve and autoclaved at 121°C for 1hr for three consecutive days.Autoclaved soil (1.5 kg) was transferred to each pot and mixed with 30g (2%) of petrol engine oil hydrocarbons.Microbial consortium were also added to the experimental pots (50 ml i.e. ~ 8 × 10 5 cells ml -1 ).Suitable sets without hydrocarbons and/or bacterial consortium were kept as control.Surface sterilized seeds of wheat (Triticum aestivum var.2565) were sown at a depth of 2.5 cm.Observation regarding germination (seedling germination percentage) and plant health parameters (i.e.seedling survival, average plant height, chlorophyll content using Spadmeter (optisciences 2000), plant biomass, average shoot height, average stem dry weight with fruit, average seed weight, average root weight and average yield per pot) (data not shown).This experiment was carried out for 120 days in poly house at room temperature.

GCMS analysis
Approximately 5g of samples from petrol engine oil contaminated soil was taken initially (0 days, control) and after cultivation of wheat at 120 days.To each of the soil samples, 15 ml of acetone was added followed by 1hr shaking.Obtained residue was filtered and washed twice with acetone, followed by filtration and concentration on a rotatory vaccum evaporator at 50°C and analyzed for the presence of residual hydrocarbons using FTIR.For Gas Chromatography Mass Spectrophotometry (GC-MS) analysis of the extracted residual hydrocarbons sample was dissolved in 1 ml hexane.The quantification of hydrocarbon compound was done by high resolution gas chromatography using QP-2010 gas chromatograph (Shimadzu) equipped with a HP-DB 5MS column (60 m × 0.25 mm, 0.25 ¼m film thickness) coupled with mass spectrometer detector (HP 5972) at AIRF, JNU, New Delhi (India).

RESULTS AND DISCUSSION
These bacterial strains were found rod shaped and Gram -and showed homology with Pseudomonas (homology 97-99%).
All the morphological and Plant growth promotory traits of the recovered test strains were shown in Table 1 &2 13 .Application of these bacterial strains can help in solubilizing and sequester iron from soil and provide it to plant cells under stress.Once these Sid + strains bind iron and make microbial iron siderophores availability of complex soluble Fe ++ become carry to plants 14,15 .Indole acetic acid (IAA) production were best shown by H1A> H1B which can be used to enhance various stages of plant roots and different availability 13 .Biosurfactant, produced by these organisms, could be rhamnolipid as evident by 16 .Similar kind of study has been reported by 17 .
Consortia are better degraders of hydrocarbons as compared to single organism.So based on the emulsification index, compatibility studies and plant growth promotory properties bacterial consortium were developed in which both organisms were compatible with each other.Under in vitro conditions consortium (C2) utilized petrol engine oil as carbon source and showed enhanced exponential growth as compared to control in the absence of any carbon source.
Consortia are better degraders of hydrocarbon as compared to single organism 18 .Based on emulsification index, compatibility studies and plant growth promotory properties microbial consortia were developed showed compatible or better growth when combined together as compare to single organism (data not shown).
In order to investigate the optimum condition for the biodegradation of petroleum hydrocarbons soil was spiked with 2% petrol engine oil and bacterized with efficient consortium.In situ experiment was performed to assess the The GC-MS analysis showed that the compounds appearing in the control sample at 0 DAS in different retention time were absent from the samples after 120 days of treatment (Fig. 2).It indicates that compounds were degraded by the developed bacterial consortium and utilized as carbon and energy sources.The metabolic capability and cell growth were also observed at different time intervals from 0 to 120 days after sowing (DAS).However, during the course of 120 DAS consortium C2 & wheat crop have managed to reduce or mitigate the concentration of petrol engine oil hydrocarbon in polluted soil, as indicated by the increased the biodegradation potential cell growth during the incubation period at different time (0, 20, 40, 60, 80 and 120 DAS) intervals (Table 1).Further, the peaks were characterized for the type of metabolite produced during the biodegradation of petrol engine oil hydrocarbons in wheat rhizospheric soil.In wheat with consortium C2, new peak was observed at retention time (RT) 23.652 & 24.345 and identified as octanoic acid-2-ethylhexyl ester and 1, 2-benzenedicarboxylic acid.(Fig 2).In addition to this plant play significant effect on pollution removal as the wheat crop has significant contribution for mobil oil biodegradation 13 .The mass fragment and structure of new intermediate products were shown at Fig 3 .The average% degradation of petrol engine oil (2%) contaminated soil in C2PE2W (C2= consortia, PE2= 2% petrol engine oil, W= wheat plant) was 56.14 % after 120 DAS (Table 1).Similar type of study, at 50 days, methyl ester dodecanoic acid and methyl tetradecanoate etc. were detected from petroleum products hydrocarbon 19 .
After the development of consortium for petrol engine oil (C2= Pseudomonas aeruginosa & P. plecoglossicida).Consortium was capable of degradation of 2% petrol engine oil hydrocarbons in wheat (Triticum aestivum var.2565) rhizosphere.Consortium C2 degraded PEO hydrocarbons in wheat rhizosphere with a potential of 56.14% in 120 DAS.Various plants can be used for remediation but owing to the importance of plants and especially wheat (Triticum aestivum var.2565) the plant was selected for studying the interaction with biosurfactant producing plant growth promoting rhizobacteria (PGPR) in petroleum products contaminated soil anticipating that this study will be helpful in use of oil contaminated sites for farming and better understanding of remediation mechanism of such sites.

Fig. 1 .
Fig. 1.GC/MS spectrum of petrol engine oil contaminated soil at 0 DAS and 120 DAS

Fig. 2 .
Fig. 2. New peak observed in C2PE2W treatments and its mass spectrum

Table 1 .
Metabolic capability and growth (cell count) of microbial consortium.

Table 2 .
All plant health parameters of control and treated samples DAS: Days after sowing; RW: Root weight; SPW: Shoot+ pods weight; SW: Seed weight