An Evaluation Method of Bacterial Flora in Contaminated Soils －For the Purpose of a Countermeasure against H<SUB>2</SUB>S Gas Production－

Abstract

Gas productions in illegal dumping sites and waste landfills have caused serious problems. The gas production was induced by bacterial flora inhabited soils. In order to construct a culture independent evaluation system of the soil bacteria, bacterial communities were analyzed quantitatively and qualitatively, about 16 soil samples at 4 sites, both using culture and culture-independent methods. The real time PCR method was developed for counting total bacterial number. Sequencing analysis of 16S rDNA amplified by a direct PCR method revealed that non-spore forming sulfate reducing bacteria and sulfur-oxidizing bacteria were detected at a similar frequency at an illegal dumping site near mountains. On the other hand, spore-forming sulfate reducing bacteria and Clostridium sp. were mainly detected in deep samples at reclaimed landfills from the sea, whereas sulfur-oxidizing bacteria was hardly detected. The result regarding sulfate-reducing bacteria was confirmed also by an anaerobic culture method. Culture-independent molecular analyses of soil bacteria would give us useful information for prediction of gas production and for the evaluation of soil equilibrium.

Bacterial Hazards of Sludge Brought Ashore by the Tsunami after the Great East Japan Earthquake of 2011

“…Aerobic bacteria were counted after incubation on standard agar medium (yeast extract, 0.5 g/l; peptone, 1.0 g/l; glucose, 0.2 g/l; agar, 15 g/l) at 30°C for 6 days 4,5) . Samples were also cultivated on bromothymol blue (BTB) lactose agar medium, thiosulfate− citrate−bile salts−sucrose (TCBS) agar medium, SSB agar medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and Wadowsky−Yee−Okuda agar medium supplemented with α -ketoglutarate (WYOα ) (Eiken Chemical Co., Ltd., Tokyo, Japan) for at least 48 h at 37°C under aerobic conditions.…”

“…Cell concentrations (cells/ml (water) or g (soil)) were determined by epifluorescence staining using ethidium bromide [5][6][7] . Briefly, 100 µ l samples were added to 900 µ l of ethidium bromide solution (100 µ g/ml) and left to stand for 10 min at room temperature, after which the mixture (1.0 ml) was passed through a 0.2 µm pore filter (Millipore, Bedford, MA, USA).…”

“…Fukuda et al analyzed sediment samples collected from Doukai Bay in Kitakyushu using a culture-independent clone library method and reported that the genera Vibrio, Staphylococcus, and Mycobacterium were present 4) . Using the same molecular method, Taniguchi et al identified sulfate-reducing bacteria that could produce hydrogen sulfide, which is very harmful 5) . To control The aim of this study was to identify bacteria in sludge brought by the 2011 tsunami in Japan to determine the necessary precautions for workers who handle the sludge.…”

Isolation and Identification of Mycobacteria from Soils at an Illegal Dumping Site and Landfills in Japan

“…In Japan, the production of toxic gases such as H 2 S and CH 4 at waste disposal fields and illegal dumping sites is a serious social problem. In our laboratory, we have developed culture-independent or nucleic acids-based methods for studying the relation between microbial flora and H 2 S gas production in polluted soils (37), because it is well known that soil bacteria participate in the material cycle of atmospheric gases and in redox-transitions of metal. In the study, we found the 16S rRNA genes of mycobacteria in the polluted soils (unpublished data).…”