Diversity Of Biofilm-Forming Filamentous Fungi From Sundarban Mangrove Ecosystem and Sequestration Of Chromium By Fungal Biofilm and Biosynthesized Iron Oxide Nanoparticles

Abstract

This thesis illustrates the potential of biofilm-forming manglicolous filamentous fungi in hexavalent chromium [Cr(VI)] sequestration and green synthesis of iron oxide nanoparticles. In this study, sediment and water samples were collected from three sites of Indian Sundarban mangrove ecosystem, i.e., Jharkhali, Bali, and Canning. A total of sixty three fungal isolates were obtained from both sediment (fifty isolates) and water samples (thirteen isolates) of these three sites, out of which Bali island possessed the highest fungal population, i.e., 1.5×105 CFU/g in sediment and 3.2×104 CFU/ml in the water sample. Among sixty three isolates, twelve isolates exhibited strong adherence to the glass surface, indicating potential biofilm-forming ability. Colony morphology and microscopic analysis of fungal isolates revealed that fungal isolates belonged to genus Aspergillus, Penicillium, Fusarium, and Trichoderma. Out of twelve fungal isolates, Aspergillus niger BSC-1 showed the highest biofilm-forming ability and Cr tolerance capacity. Further, the present work probes various stages of biofilm formation by Aspergillus niger BSC-1. Scanning electron micrograph and confocal micrograph depicted the development of fungal biofilm comprised of six stages, i.e., (i) adsorption, (ii) active attachment, (iii) germling and monolayer formation, (iv) hyphal development and formation of ECM, (v) maturation of ECM, and (vi) dispersal of spores. At the maturation stage (36 h), the thickness of biofilm was observed up to ~15 μm. Further, ATR-FTIR spectroscopic analysis exhibited peaks at 3398 cm-1, 2930 cm-1, 1571 cm-1, 1391 cm-1, 1092 cm-1, and 977 cm-1, which confirmed the presence of protein, carbohydrate, and lipid in the biofilm-associated matrix. To determine the efficacy of filamentous fungal biofilm in heavy metal detoxification, two morphologically diverse biosorbents, such as fungal pellet, i.e., planktonic phase and fungal biofilm were prepared. The removal of Cr(VI) using both the biosorbents revealed that fungal biofilm demonstrated significantly (P<0.05) higher Cr(VI) removal efficiency, i.e., 97.81±2.5% than the fungal pellet. The batch adsorption study showed that the highest Cr(VI) removal was attained at 40°C and pH 3 with 3 g/L of fungal biofilm. The adsorption isotherms, kinetic model, and thermodynamic analysis suggested endothermic and monolayer chemisorption of Cr(VI) onto the surface of the fungal biofilm. The biosorption process was optimized using response surface methodology (RSM) having Central Composite Design (CCD) interface, which suggested the quadratic model as the most suitable model for viii Cr adsorption having high significance (P<0.0001). The 3D surface plots among three key variables, i.e., pH, temperature, and Cr concentration, revealed that maximum sequestration, i.e., 92.39 %, was achieved at 40°C, pH 3, and 30 mg/L metal concentration using fungal biofilm. Regeneration study demonstrated that fungal biofilm retained 87.19±3.23 % of Cr(VI) removal efficiency with no significant loss after five successive adsorption/desorption cycles. Additionally, twelve fungal isolates were utilized for extracellular synthesis of iron oxide nanoparticles (IONPs) which were characterized using by UV-Vis spectroscopy, ATR-FTIR spectroscopy, Raman spectroscopy, XRD, electron microscopy (TEM and FESEM), zeta sizer and VSM. It was found that only A. niger BSC-1 was able to synthesize crystalline superparamagnetic IONPs (Fe3O4) of 20-40 nm size, which was utilized for the removal of Cr(VI) from aqueous solution. The batch experiment revealed that significantly (P<0.05) high Cr(VI) removal was at 40°C and pH 3 with 2.5 g/L of IONPs dose. Similar to biosorption, the adsorption isotherm and kinetic model suggested monolayer adsorption of Cr onto the IONPs surface. The presence of coexisting cations and anions did not exhibit any competitive effect on Cr(VI) removal revealing the selectivity of mycosynthesized IONPs towards Cr(VI). Regeneration study confirmed that IONPs conserved their Cr(VI) removal efficiency with minimal loss (19.3%) after five adsorption/desorption cycles. Further, X-ray photoelectron spectroscopy (XPS) indicated that both adsorption and redox reactions were involved in Cr(VI) detoxification. To understand the genetic features of biofilm formation and metal fungi interaction, whole genome sequencing of A. niger BSC-1 was performed, which revealed the presence of rodA, exgA, and Ags genes which are involved in initial adhesion to surface and matrix formation, respectively, during biofilm growth. Semi-quantitative qRT-PCR analysis confirmed the role of Ags1 and P-type ATPase in biofilm formation and Cr resistance, respectively. Several other genes encoding metalloprotease, copper and zinc binding proteins, and NADH-dependent oxidoreductase were also found in the genome of A. niger BSC-1. These proteins are also involved in heavy metal tolerance and nanofabrication