Population analysis of microbial consortia during oil degradation process at the in situ experiment field situated on a beach in Japan

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We constructed an open-environment experiment field on a sandy beach facing to Mazuru Bay in Japan, for the purpose of verifying the efficiency of newly developed fertilizer Z18 (Japan Patent Office P2001-87754A) on bioremediation of crude oil pollution. In the experiment field, periodical tidal movement supplied oxygen and several nutrients for heterotrophic bacteria from seawater, and we placed several small permeable bags with artificially oil-contaminated sand samples at the intertidal depth. On experimental days 7, 14, 28, 56 and 112, we analyzed the oil degradability, the number of oil-degrading bacteria (ODB) and bacterial population in each sand bag, and compared them between the fertilized and non-fertilized samples. The ODB numbers were determined by a MPN method and bacterial population was estimated by a culture-independent protocol, PCR-DGGE (denaturing gradient gel electrophoresis) targeting bacterial 16S rRNA genes. The nitrogen containing fertilizer Z18 excluded oil com … More ponents (saturated alkanes, aromatics, resins and asphaltenes) more effectively than non-fertilized samples. The growth of GOB and the biodegradability of oil (estimated from the survivability of normal alkanes relative to hopane) were also accelerated by Z18 addition. Contrarily, the bacterial population dynamics estimated by PCR-DGGE patterns during the oil-degrading process were not greatly different between fermented and non-fermented samples, suggesting that the fermentation of Z18 did not promote a particular ODB denitrifying bacteria belonging to alpha-proteobacteria subgroup. Unfortunately, dissolved inorganic nitrogen (DIN) had gradually accumulated in fertilized samples after 56 days of experiment, by which time almost normal alkanes were diminished, and there remained the problem of eutrophication of marine environments. However, we compared the effect of oil degradation and accumulation of DIN among the samples to which various forms, and concentrations of Z18 applied, and proposed an ingenious application of the new fertilizer for spilt oil treatment on seashore, without excess nitrogen accumulation. Less