EFFECT OF SAMPLE HANDLING ON ESTIMATION OF BACTERIAL DIVERSITY IN MARINE-SEDIMENTS BY 16S RIBOSOMAL-RNA GENE SEQUENCE-ANALYSIS

The diversity of bacterial communities in deep marine sediments, up to 503 metres below the sea floor of the Japan Sea, was investigated by sequence analysis of amplified 16S rRNA genes. The use of different sa mple handling procedures greatly affected the types and diversity of s equences obtained. DNA from sediment samples stored aerobically for up to 24 h before freezing was dominated by sequences belonging to the b eta- and gamma-proteobacteria, many of which appeared to originate fro m aerobic bacteria. Sub-samples equilibrated anaerobically at 16 degre es C, were then injected with a radiotracer and immediately frozen, to simulate the conditions of a typical control sample from a radiotrace r based activity assay, contained mostly alpha-proteobacterial sequenc es. Pristine sediment samples taken anaerobically and frozen within 2 h contained the widest diversity of sequences from alpha-, gamma-, del ta-proteobacteria and Gram-positive bacteria, which appeared to have o riginated from predominantly anaerobic or facultative bacteria. It was clear that both samples that were not frozen immediately (within 2 h) showed signs of enrichment of specific bacterial groups. Our results strongly suggest that immediate freezing should always be employed whe n sediment samples are to be used to assess bacterial diversity by mol ecular methods.