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Remediation of Hydrocarbon-contaminated soils through indigenous microbial biosurfactants

Thivaharan, Varadavenkatesan (2014) Remediation of Hydrocarbon-contaminated soils through indigenous microbial biosurfactants. Phd. Thesis thesis, Manipal Institute of Technology, Manipal.

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Abstract

The current research deals with the remediation of hydrocarbon-contaminated soils through the use of biosurfactants, produced by bacterial isolates. The applicability of the extracted biosurfactants for enhanced oil recovery operations and biodegradation of polycyclic aromatic hydrocarbons is tested. The bacteria were isolated from different hydrocarbon-contaminated zones. Serial dilution and plating led to the isolation of 23 different bacterial isolates. The isolates were screened for the production of biosurfactants through a series of qualitative tests like oil spreading technique, blood agar haemolysis test, drop collapse test, CTAB agar plate test, tilted glass slide test, emulsification index and determination of surface activity. The tests showed that seven of the isolates exhibited significant biosurfactant production. The morphological features of the strains were observed and biochemical tests were performed. Sequencing using 16S rRNA technique led to the complete identification of the seven isolates as Acinetobacter calcoaceticus, Bacillus subtilis, Pseudomonas aeruginosa, Rhodococcus terreus, Pseudomonas aeruginosa, Bacillus siamensis and Bacillus subtilis subsp. inaquosorum. Of these, B.siamensis has been reported to produce biosurfactant for the first time, to the best of knowledge. Similarly, the subsp. inaquosorum of B.subtilis is also noted for the first time, in literature. The role of plasmid DNA, if at all, in the production of biosurfactants was verified. The study implied that the genes responsible for biosurfactant synthesis were present in the chromosomal DNA, itself, where it is thought to be more stable. Three microbial consortia were developed comprising B.siamensis and B.subtilis subsp. inaquosorum and P.aeruginosa. The consortium CONS-1 was composed of B.siamensis and B.subtilis subsp. inaquosorum. While the consortium CONS-2 contained B.siamensis and P.aeruginosa. B.subtilis subsp. inaquosorum, the group CONS-3 had all the three isolates. An attempt was made to use domestic wastes and used engine oil as a sole source of carbon to be included in the production medium for the extracellular secretion of biosurfactants by the three selected isolates. Four novel substrates were used in fermentation experiments: garlic peel, jackfruit seed coat, plantain pith and used engine oil. The reduction of surface tension using garlic peel (72%), jackfruit seed coat (67%), plantain pith (43%) and used engine oil (40%) was comparable to reduction induced by glucose (58%), orange peel (46%) and apple pomace (55%). When the consortium, CONS-3, was grown using garlic peel as the sole carbon source, the amount of biomass and biosurfactant produced was found to be higher than that produced by the individual isolates using the same carbon source. The three isolates were grown in MSM supplemented with the selected cheaper carbon source and the appearance of the biosurfactant, extracted from the broth, ranged from an oily appearance to a viscous yellow product to a viscose honey-brown colored material. Growth and biosurfactant production kinetics was studied using Logistic Model and Logistic-incorporated Leudeking-Piret Model, respectively. When P.aeruginosa was grown in the presence of used engine oil as the sole carbon source, the parameters determined using Logistic Model were: coefficient of carrying capacity (μm) = 0.0792 /hr, initial biomass concentration (X0) = 0.1299 g/L and maximum biomass concentration (Xm) = 3.0025 g/L. The Leudeking-Piret Model estimated values of the parameters as α = 0.9045 g / g and β = 0.0033 g / g. hr. For B.siamensis grown using powdered garlic peel, the estimated values of the parameters using Logistic Model were: μm = 0.0882 /hr, X0 = 0.149 g/L and Xm = 3.0794 g/L. Using Leudeking-Piret Model, the estimated values of the parameters were: α = 0.8988 g / g and β = 0.2322 g / g. hr. B.subtilis subsp. inaquosorum was also grown using powdered garlic peel. Using the Logistic Model, the estimated values of the parameters were: μm = 0.0645 /hr, X0 = 0.2343 g/L and Xm = 5.298 g/L. Leudeking-Piret Model estimated the values of the parameters as: α = 1.0034 g / g and β = 0.3565 g / g. hr. The biosurfactants extracted from the three isolates were characterized using thin layer chromatography, FTIR spectroscopy and NMR spectroscopy. Stability studies were conducted to check the performance of the biosurfactant at elevated and reduced physical parameters. The effect of temperature (4˚C to 121˚C) on the surface activity and emulsification index was studied. Similar studies were performed to understand the effect of pH (2 to 12) and salinity (0% to 20%). The effect of chemical and biosurfactants on remediation of laboratory-made contaminated soil were compared by employing sand-pack column. The efficiency of the isolated biosurfactant in releasing the residual (commercial) engine oil was determined. To assess the influence of temperature on biosurfactant-induced oil recovery, the entire sequence of experiments was carried out at 30ºC, 50ºC and 70ºC. It was noted that higher temperatures led to better recovery of residual oil from the column.Three PAHs were chosen to study their extent of degradation – Naphthalene, Anthracene and Pyrene. Since the biosurfactants secreted by the isolates help in increasing the bioavailability of the PAH, they were added as the sole carbon source during degradation studies. Biodegradation of the three PAHs were found to follow first-order kinetics and the biodegradation rate constants were determined. It was found that the consortium, CONS-3 was invariably, the most effective in degrading the three selected PAHs, compared against individual isolates and the proven PAH degrader, B.subtilis BMT4i (MTCC 9447).

Item Type: Thesis (Phd. Thesis)
Uncontrolled Keywords: Biosurfactant, Polycyclic Aromatic Hydrocarbon, Biodegradation, Bacillus siamensis, Bacillus subtilis subsp. inaqousorum, Pseudomonas aeruginosa, Sand-pack, Microbial Consortium, Naphthalene, Anthracene, Pyrene
Subjects: Engineering > MIT Manipal > Biotechnology
Depositing User: MIT Library
Date Deposited: 12 Nov 2014 10:41
Last Modified: 12 Nov 2014 10:41
URI: http://eprints.manipal.edu/id/eprint/141022

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