Potential Of Immobilized Microbial Consortium In Petroleum Contaminated Soil Rhizodegradation

Crude oil is one of the most important natural non-renewable energy sources. In the recentyears due to the advancement and industrialization, the more consumption of this natural resourceincreases its exploitation, processing and transportation and hence created environmentalcontamination problems. Therefore the uses of some advance low cost remediation technology areneeded for pollution control and environment health.In present study rhizosphere bioaugmentation was performed with free and immobilized oildegrading consortium, which was isolated from the same contaminated site of Yellow river delta ofShengli oil field. The soil sample was taken from the field of composted soil, field composting wasperformed in our previous lab work, initial biodegradation of crude oil was already done, whichdecreased the TPH concentration from18800mg kg-1to2541mg kg-1and the soil containedrecalcitrant fraction which needed to be remediated to Chinese government EPA acceptable limitof≤500mg kg-1. The size of contaminated site is very long so need of cost effective techniquewhich can remediate such large area. In the present study in the beginning soil was bioaugmentedwithout any other structural amendment to make the technique applicable in the large field area.Initially soil texture was found clayey which did not support the plant growth and ultimately nodegradation was seen in that case, so the texture amendments were performed along with Carbon,Nitrogen and Phosphorous maintained ratio.Local dominant species of Sesbania cannabina (Retzius) Poir plant was used in this studywhich has the abilities of good tolerance in adverse environment and pollution control. The effectof plant growth in bioaugmented soil on TPH degradation was investigated, different rootmorphological characteristics (root length, volume, surface area, number of root tips) werechecked along with root and shoot biomass with Epson scanning instrument equipped with WinRhizo pro. Root length was highest during all sampling time in the control plant and significancedifference (p <0.05) of root length was observed between free bacterial bioaugmented rhizosphere(FR) and un-inoculated control rhizosphere (CR) during90days while no difference amongtreatment was observed during rest of the time, root length and TPH concentration were significant negatively correlated (r=-0.85) and highest root length and root biomass also had a higher TPHdissipation rate. Higher root volume was found in case of CR which was obvious and consistentwith root length and surface area. The possible reason for lower root and shoot biomass in thebioaugmented treatment might be the inhibition of plant growth promoting bacteria due tocompetition between inoculated and indigenous bacteria which results in lower growth of Sesbaniaplant and hence lower TPH degradation. Overall, growth performance indicators suggested that S.cannabina is an ideal candidate for rhizoremediation application based on highest potential for rootdepth penetration, biomass production, relative growth rate (root biomass) and root to shoot ratiowhen grown in oil contaminated soil.In the present study effect of rhizosphere bioaugmentaion in TPH degradation on microbialcommunity number, species and functional diversity was assessed in order to find their role in TPHrhizodegradation and to determine the survival of introduced oil degrading consortium, total MPNof heterotrophs was checked. Significantly higher (P<0.05) MPN gm-1of dry soil was found after90days of plant growth and total microbial activity was also higher at that time. Significantcorrelation (r=0.79, n=18) between these two parameters assured the optimum conditionsprovided by the plant rhizosphere during that time, which gradually decreased with the maximumplant growth at120days. Pronounced rhizospheric effect observed. No significance differences ofMPN were noted between FR, immobilized bacterial inoculated rhizosphere (IR) and CR treatmentpots. TPH degrading bacterial communities were relatively stable during the course of study,averaging between103and104CFUs g-1soil.Physiological profile was assessed at90days of plant growth by using Biolog MicrostationSystem, indicated that lower diversity and higher abundance and evenness were found in CR andFR which interpreted that some specific bacterial species were present in higher abundanceresponsible for higher TPH degradation in these samples.To compare different treatments and control plant microbial community profile and toidentify the potential heterotrophic species in the rhizosphere of S. cannabina, DGGE profiles of16S rRNA amplified gene fragment and its subsequent band analysis was performed. Gel-Proanalyzed4.5software (Cybernetics, Inc) was used to check the band intensity and different indicesShannon–Weiner (H) diversity index, Simpson’s index and Evenness were calculated. On the basisof their high intensities,18distinguished bands were selected for sequence analysis. The potentialmicrobial community belongs to Flavobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Halomonadaceae, Rhodospirillaceae, Erythrobacteraceae families and Cyanobacteria. Presence ofSesbania plant was a key factor of banding pattern in the DGGE profile, neither bioaugmentationby free culture nor immobilized oil degrading culture. However, band17which belongs toFlavobacteriaceae was only found in CR and CN, these unidentified bacteria was the most activeTPH degraders due to its higher abundance in these samples with subsequent higher TPHdegradation. As no band were found related with the inoculated culture in FR and IR indicated theinhibition of was that culture.The crude oil degradation by plant and microorganisms were evaluated by Gaschromatograph-mass spectrometry (GC-MS) for extractable TPH, results showed thatbioaugmentation did not improve the TPH degradation. TPH concentration decreased from2541mg kg-1to673mg kg-1and867mg kg-1in rhizosphere of free (FR) and immobilized bacterialinoculated soil (IR) respectively at120days while in the rhizosphere of un-inoculated soil (CR)concentration decreased to679mg kg-1only at90days, showing higher and rapid rhizodegradationwith indigenous bacteria than bioaugmented bacterial culture.Therefore it is interpreted from the present research work that the less diverse and morerelative abundant hydrocarbon degraders species were under the influence of the S. cannabinawhich was found sufficient to degrade recalcitrant fraction of TPH and no further bioaugmentationwas seems to be effective. Moreover, plant rhizosphere immobilized bacterial community in itssurrounding therefore immobilized culture has no effect on petroleum degradation. It does notnecessary that microbial consortium used for TPH degradation must also facilitate plant growth, asroot and shoot biomass did not show any significance difference rather more in CR than inbioaugmented soil samples, bioaugmented culture has detrimental negative interactions with plantand indigenous microorganisms and with rhizospheric microflora.Further investigation are needed to apply the potential bacterial species isolated from the sameplant rhizosphere which may act simultaneously as plant growth promoting bacteria (PGP) andefficient TPH degrader, along with MPN of total oil degrading bacteria enumeration of fungishould be investigated in order to analyze the total microbial contribution and need of moresuitable inoculation technique and stable agent for immobilization of microbial consortium.