Seafloor seeping methane bubbles were successfully sampled on the summit area of the Kuroshima Knoll (depth of ca. 640 m) using the gas-tight sampler WHATS attached to the Shinkai 2000. To evaluate the origin of the bubbles and verify that the dissociation of methane hydrate was actually in progress, the chemical and isotopic composition of the samples were analyzed. The major component of the gas bubbles was methane (C₁) with traces of CO₂ (67 ± 16 ppmv) and helium (11 ± 1 ppmv; ⁴He/²⁰Ne = 320) having a moderate ³He/⁴He ratio (0.44R_a). C₁ was enriched relative to other hydrocarbons (C₁/(C₂+C₃) > 3000). The δ¹³C values for C₁ (-40.1‰_VPDB), C₂ (-28.3‰_VPDB), and C₅ (-28.0‰_VPDB) were similar to those of hydrocarbons produced by thermal decomposition of organic matter. The contribution of the mantle-derived ³He-enriched component in coexisting helium also supports thermogenic generation. On the other hand, the other light hydrocarbons showed an unusual ¹³C-enrichment in C₃ (-19.1‰_VPDB), iso-C₄ (-22.4‰_VPDB), and n-C₄ (-19.9‰_VPDB). C₃ and C₄, had been fractionated both chemically and isotopically through subsequent microbial destruction during the long storage from Miocene in the gas reservoir. In addition, the anaerobic oxidation of CH₄ within shallow sediments removed about 20% of CH₄, until seepage into ocean water column. Regarding the contribution of gases originating from hydrate dissociation to the bubbles, observed helium amount in the bubbles suggests that methane hydrate is considered to be a minor contributor to the bubbles, at least at present. Direct leakage of gases from deep reservoirs is a more plausible for the source. Regarding the fate of the hydrocarbons in the bubbles in the water column, all rising bubbles at Kuroshima Knoll dissolved within 140 m of the seafloor. After the dissolution, the plume spreads horizontally along with the surface of equal density in the water column, while the concentrations decrease through dilution by eddy diffusion, rather than by oxidation.