The bioavailability of organic contaminants to the degrading bacteria is a major limitation to efficient bioremediation of sites contaminated with hydrophobic pollutants. Such limitation of bioavailability can be overcome by steady-state biofilm-based reactor. The aim of this study was to examine the effect of such multicellular aggregation by naturally existing oil-degrading bacteria on crude oil degradation. Microorganisms, capable of utilizing crude oil as sole carbon source, were isolated from river, estuary and sea-water samples. Biochemical and 16S rDNA analysis of the best degraders of the three sources was found to belong to the Pseudomonas species. Interestingly, one of the isolates was found to be close to Pseudomonas otitidis family which is not reported yet as a degrader of crude oil. Biodegradation of crude oil was estimated by gas chromatography, and biofilm formation near oil-water interface was quantified by confocal laser scanning microscopy. Biofilm supported batches of the isolated Pseudomonas species were able to degrade crude oil much readily and extensively than the planktonic counterparts. Volumetric and topographic analysis revealed that biofilms formed in presence of crude oil accumulate higher biomass with greater thickness compared to the biofilms produced in presence of glucose as sole carbon source. 1. Introduction World marine ecosystem has been studied extensively since the second half of the last century. Oil spillage and oil pollution in marine environment have been a major threat to the ecosystem including the ocean life as well as to the human being through the transfer of toxic organic materials including polycyclic aromatic hydrocarbons (PAHs) into the food chain [1–3]. Presence of polycyclic aromatic hydrocarbons (PAHs) in soil and water is major problem as environmental contaminants and most of these PAHs are recalcitrant in nature. PAHs mean a potential risk to the marine animals as well as to the human health as many of them are carcinogenic [4]. Physical and chemical methods like volatilization, photooxidation, chemical oxidation, and bioaccumulation [5] are rarely successful in rapid removal and in cleaning up PAHs [6], and also these methods are not safe and cost effective when compared to microbial bioremediation. Bacteria have long been considered as one of the predominant hydrocarbon degrading agents found in the environment, which are free living and ubiquitous. Over twenty genera of bacteria of marine origin have been documented to be hydrocarbon degrading [7–9]. Bacteria belonging to subphyla α-, β-, and
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