Prokaryote growth temperatures in environmental samples are difficult to measure because it is hard to culture viable prokaryotes in natural environments. We comprehensively surveyed growth temperatures and 16S rRNA sequences of prokaryotes to estimate their growth temperatures based on guanine-plus-cytosine contents (P(GC)) of their 16S rRNA sequences. We focused on archaea because of the wide range of growth temperatures within this group. Their minimum (Tmin), optimum (Topt) and maximum (Tmax) growth temperatures correlated strongly with PGC of their 16S rRNA genes. Linear regression equations were established to approximate Tmin, Topt and Tmax from P(GC). We also established a linear regression equation for calculating P(GC) of 16S rRNA genes based on the melting temperatures (Tm) of PCR fragments, without using a clone library or sequencing. Environmental samples were obtained from a wide variety of microbial natural habitats. Tm of archaeal 16S rRNA genes amplified by real-time PCR were determined by melting curve analysis. Based on those values, P(GC) of 16S rRNA genes and mean Tmin, Topt and Tmax were calculated using the linear regression equations. These temperatures correlated strongly with the in situ temperatures. Tmax agreed particularly well with these temperatures, suggesting many archaea live at their maximum growth temperatures.

Sulfur-turf microbial mats develop in sulfide-containing hot spring water dominated by chemolithoautotrophic sulfur-oxidizing bacteria. The sulfur-turf mat that developed at a source of hot water (72°C, pH 6.8) exhibited a growth rate of 0.48±0.04 h −1 and biomass production of 4.6±1.0 mg of C h −1 . On a per-cell basis, this biomass production was at least an order of magnitude higher than the CO2 uptake rate calculated for a photosynthetic mat dominated by thermophilic Synechococcus spp. at 70°C. The sulfur-turf-associated microbial community likely contributes to carbon fixation and primary production in this geothermal habitat.

Microbial biomass production has been measured to investigate the contribution of planktonic bacteria to fluxations in dissolved organic matter in marine and freshwater environments, but little is known about biomass production of thermophiles inhabiting geothermal and hydrothermal regions. The biomass production of thermophiles inhabiting an 85 degrees C geothermal pool was measured by in situ cultivation using diffusion chambers. The thermophiles' growth rates ranged from 0.43 to 0.82 day(-1), similar to those of planktonic bacteria in marine and freshwater habitats. Biomass production was estimated based on cellular carbon content measured directly from the thermophiles inhabiting the geothermal pool, which ranged from 5.0 to 6.1 microg C l(-1) h(-1). This production was 2-75 times higher than that of planktonic bacteria in other habitats, because the cellular carbon content of the thermophiles was much higher. Quantitative PCR and phylogenetic analysis targeting 16S rRNA genes revealed that thermophilic H2-oxidizing bacteria closely related to Calderobacterium and Geothermobacterium were dominant in the geothermal pool. Chemical analysis showed the presence of H2 in gases bubbling from the bottom of the geothermal pool. These results strongly suggested that H2 plays an important role as a primary energy source of thermophiles in the geothermal pool.

The temperature ranges of growth of archaea are strongly correlated with the guanine-plus-cytosine (G+C) contents of their 16S rRNA sequences (P GC ). In order to estimate minimum (T min ), optimal (T opt ), and maximum (T max ) growth temperatures of uncultured archaea based on P GC , the 16S rRNA gene sequences of 207 archaeal species were collected from public databases, and their T min , T opt and T max were extracted from description papers and reviews. These values of growth temperatures were plotted againstP GC , and then the regression lines for estimating T min , T opt and T max were calculated. We PCR-amplified the archaeal 16S rRNA gene fragments from the hot water samples, cloned the fragments, and determined the sequences. Growth temperatures of environmental archaea were inferred from G+C content of the 16S rRNA gene sequences by the regression lines. In the terrestrial hot springs (74 • C and 85 • C), both estimated growth temperatures of archaea were higher than in situ temperatures of hot spring waters. Even from tepid hydrothermal fluid (40 • C) we obtained a significant number of archaeal genes indicating high growth temperatures. These results suggested that hot subsurface environments exist under those hydrothermal and geothermal regions. In this study, growth temperatures of uncultured archaea and in situ subsurface temperatures were roughly inferred from 16S rRNA gene sequences of archaea that were transported from the subsurface biosphere. This new method based on microbial molecular information may be applicable to temperature estimation of subsurface environments for We are grateful to the captains and crews of the R/V Yokosuka and Natsushima and to the operation teams of the Shinkai 6500 and Hyper-Dolphin for helping us to collect the deep-sea hydrothermal fluid samples. We thank Dr. Julia Maresca for comments on the manuscript.which it has been difficult to measure the actual temperature with appropriate instrumentation.