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

为了验证新开发的肥料Z18（日本专利局P2001-87754A）对原油污染的生物修复效果，我们在日本面向马祖鲁湾的沙滩上建立了一个开放环境的试验场。在试验田，周期性的潮汐运动为海水中的异养细菌提供了氧气和几种营养物质，我们在潮间带深度放置了几个装有人工油污染砂样的小透水袋。在试验第7、14、28、56和112天，对每个沙袋的油降解能力、油降解细菌（ODB）数量和细菌数量进行了分析，并对施肥和未施肥样品进行了比较。通过MPN法测定ODB数量，通过针对细菌16S rRNA基因的非培养方案PCR-DGGE（变性梯度凝胶电泳）估计细菌数量。含氮肥料Z18比未施肥的样品更有效地排除了更多的组分（饱和烷烃、芳烃、树脂和沥青质）。Z18的加入也加速了GOB的生长和油的生物降解性（从正构烷烃相对于藿烷的生存能力来估计）。相反，通过PCR-DGGE模式估计的细菌种群动态在发酵和未发酵的样品中没有太大差异，这表明Z18的发酵没有促进属于α -变形菌亚群的特定ODB反硝化细菌。不幸的是，经过56天的试验，施肥样品中溶解的无机氮（DIN）逐渐积累，此时几乎正常的烷烃都减少了，海洋环境的富营养化问题仍然存在。然而，我们比较了施用不同形式和浓度的Z18对样品的油降解和DIN积累的影响，并提出了一种巧妙的新肥料用于海滨溢油处理，没有过量的氮积累。少

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