The mean and standard deviation of the yield of prokaryotic DNA, Tm and reproducibility for success in DNA extraction are shown in Fig. 1a and Table S1. It was confirmed that prokaryotic DNA was not extracted when the sediment was incubated at 94 °C for 30, 40 or 90 min. Although DNA was repeatedly extracted when the sediment was heated at 94 °C RG 7204 for 50 min, prolonged heat incubation reduced the reproducibility of DNA extraction. Especially, prokaryotic DNA

was obtained with 80-min incubation from one of four extractions. It was also evident that the mean and standard deviation of the yield of prokaryotic DNA were relatively high when DNA was extracted with the prolonged incubation at 94 °C. To optimize this method, we tested other extraction conditions from the consolidate sediment sample in terms of incubation temperature (65 °C) and NaOH concentration (0.07 N) (Table S1). Akt inhibitor However, prokaryotic DNA were not extracted from the consolidate sediment sample

under the other extraction conditions. We also applied all extraction conditions tested for the sediment sample to 1.5 × 108 cells of P. stutzeri (Fig. 1b, Table S1). In sharp contrast to the sediment sample, DNA was extracted from P. stutzeri under all tested conditions. Assuming that P. stutzeri have four copies of 16S rRNA gene in its genome (Yan et al., 2008), recovery rate of PCR-amplifiable DNA was calculated. The highest recovery rate (76.7%) was obtained by the heat treatment at 65 °C for 50 min with 0.33 N NaOH. Heat treatment at 94 °C for 90 min with 0.33 N NaOH resulted in the lowest recovery rate (0.6%). DNA recovery decreased with increasing incubation time, temperature and NaOH concentration. Owing to concerns that the heat treatment under alkaline

conditions might cause severe fragmentation of DNA, length of DNA extracted from P. stutzeri cells was visualized by agarose gel electrophoresis (Fig. 2). Fragmentation of extracted DNA was more pronounced when the cells were these incubated in 0.33 N NaOH solution at 94 °C for longer incubation times. Prokaryotic DNA primarily composed of the aforementioned phylotypes was likely extracted from prokaryotic populations indigenous to the consolidated sediment, rather than contaminant prokaryote. This is because the main contaminant DNA from drilling fluid and from laboratory air and apparatuses was supposed to be extracted under the conditions with high recovery of DNA from P. stutzeri. The mechanism of the DNA extraction from the consolidated sediment could be attributed to the dissolution of silica minerals and subsequent release of DNA. To investigate this possibility, X-ray diffraction pattern analysis of the sediment sample was conducted for different incubation times (0, 30, 50, 70 and 90 min).