Interaction of Extracellular Polymeric Substances of Biofilm forming Marine Bacteria with Petroleum Hydrocarbons for the Emulsification and Biodegradation

Abstract

This thesis illustrates the biofilm-forming potential and petroleum hydrocarbons (PHs) degradation efficiency of marine bacteria. Sediment samples were collected from the Paradip and Gopalpur ports of coastal regions of Odisha, India. A total of 50 marine bacterial strains were isolated following selective enrichment with 2% (v/v) crude oil. Biofilm-forming potential was observed in 45 marine bacterial isolates, of which 12 were strong, 9 were moderate, and 24 were weak biofilm formers. Further biofilm screening in the presence of crude oil resulted in the selection of 10 marine bacterial isolates having the potential to form biofilm at the oil-water interface. 16S rRNA gene sequencing revealed that all the potent biofilm-forming marine bacterial isolates belonged to Gammaproteobacteria class, out of which 6 isolates were Pseudomonas, 3 isolates were Acinetobacter, and 1 isolate was from Zobellella genera. Scanning electron micrographs (SEM) analysis revealed that isolates belonging to Pseudomonas genus were short rods clustered together in a thick layer of extracellular polymeric substances (EPS). However, isolates under the Acinetobacter genus appeared as coccobacilli in shape with compact aggregation, while the isolate under Zobellella genus appeared as long rods scattered in the EPS matrix. Confocal laser scanning micrographs (CLSM) revealed different biofilm components, and community statistics (COMSTAT) analysis revealed different biofilm parameters. A. junii PPS-3, P. mendocina PPS-8, P. guguanensis PPS-12, A. junii PPS-16, and P. furukawaii PPS-19 showed high specific growth rate and yield coefficient in the presence of crude oil. Biofilm-forming marine bacterial isolates showed significantly higher utilization of crude oil in the biofilm than in the planktonic mode of growth (P<0.05). P. furukawaii PPS-19 showed the highest utilization of crude oil and significantly reduced 38% (v/v) crude oil in the biofilm than planktonic mode of growth (61.3%) (P<0.001). Colorimetric analysis revealed polysaccharides were the major components of EPS of P. furukawaii PPS-19. The architecture of purifed EPS was studied through field emission scanning electron microscopy (FESEM), which exposed its porous and three-dimensional flakes-like structure. The structural characterization by Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) revealed that EPS was composed of primary alkane, amines, halide, hydroxyl groups, uronic acid, and saccharides. The X-ray diffractogram (XRD) profile exhibited an amorphous phase of the EPS with a crystallinity index of 0.336. The EPSa showed three-step thermal decomposition and thermal stability up to 600°C, as confirmed by thermogravimetric analysis (TGA). Differential scanning calorimetry (DSC) thermogram revealed one crystallization temperature at 69.18°C with 343.15 mJ latent energy and two melting temperatures at 424.76°C and 556.90°C with 211.73 mJ and 2030.44 mJ latent energies, respectively. EPS produced by the marine bacterium P. furukawaii PPS-19 was good bioemulsifer and showed the highest emulsifying activity of 66.23% on petrol. The emulsifying ability of the EPS was superior to the commercial polymer xanthan. The emulsion also showed high stability with time and temperature exposure. EPS interaction with crude oil and pyrene showed n→π* and π→π* transition in UV-Vis spectra, reflecting the change in energy of bond orbital of aromatic groups in EPS. Shifting of polysaccharides and protein-associated bonds after interaction with PHs was observed. Circular dichroism (CD) spectra presented conformational changes in the protein secondary structure of EPS treated with PHs. X-ray photoelectron spectroscopy (XPS) spectra exhibited changes in major element composition and percentage of different functionalities of EPS. X-ray diffraction (XRD) profile showed complete disruption of crystalline peaks in EPS treated with crude oil and n-Dodecane, while with pyrene, CIXRD changed from 0.3 to 0.8. Pore size of the purified EPS increased considerably after treatment with crude oil and n-Dodecane. Surface electronegativity and hydrodynamic diameter of EPS were increased, indicating the formation of the conglomerate. EPS was quenched in the presence of PHs, establishing a static mechanism, and the maximum binding was obtained with n-Dodecane (1.88 L/mol, - 1.57 kJ/K/mol). alg8 gene was identified in the bacterium, and it showed significantly increased expression in the presence of n-Dodecane (6.31 fold) (P<0.05). The n-Dodecane and pyrene bioadsorption capacity of EPS-alginate was significantly higher (356.5 and 338.2 mg/g, respectively) (P<0.001) than calcium-alginate. EPS-alginate also showed high n- Dodecane and pyrene bioadsorption capacity under various pH and salinity stress. The n- Dodecane and pyrene bioadsorption rate by the developed bioadsorbents fits into the pseudo- second-order kinetic model. P. furukawaii PPS-19 showed strong cell surface hydrophobicity (CSH) under different physicochemical stressors, such as pH and salinity. Strong aggregation of P. furukawaii PPS-19 was observed at hydrophobic interfaces of n-Dodecane and crude oil, while uptake of pyrene resulted in blue fluorescence of the bacterium. Changes in biofilm microcolonies were observed under different physicochemical stressors with maximum biofilm thickness of 15.15 μm and 15.77 μm at pH 7% and 1% salinity, respectively. Relative expression analysis of alkB2 gene revealed the maximum expression in n-Dodecane (10.5 fold) at pH 7 (1 fold) and 1% salinity (8.3 fold). 3D homology modeling followed by validation revealed a high-quality model with 93.7% residues in the favored zone of Ramachandran plot. Molecular docking study displayed a substantial binding affinity of n- Dodecane (-8.050 kcal/mol) with amino acid residues at the active binding site of AlkB2, indicating a strong interaction. During the degradation process, a significant drop in surface tension resulted in increased emulsification activity. P. furukawaii PPS-19 showed the respective n-Dodecane and pyrene degradation of 94.3% and 81.5% at pH 7% and 94.5% and 83% at 1% salinity. A significant positive correlation was obtained between CSH, biofilm formation, and PHs degradation (P<0.05) under all the physicochemical stressors, with the highest value at pH 7% and 1% salinity. Analysis of metabolites indicated that mono-terminal oxidation and multiple pathways were followed for n-Dodecane and pyrene biodegradation, respectively. Thus, P. furukawaii PPS-19 is an efficient hydrocarbonoclastic bacterium that may be exploited for large-scale oil pollution abatement. Moreover, EPS of P. furukawaii PPS-19 could also be used as a bioadsorbent material for biodegradation of PHs.