The spatial distribution of a microbial community in an oligotrophic region of the Tamagawa River which developed on the surface of a slide glass submerged in the river was observed by microscopy along with the course of biofilm formation.Bacterial cells attached to the slide glass after one hour and increased in number on the 4th day of submergence.Algal cells began to adhere to the bacterial cells on the 8th day, increasing the thickness of the biofilm.However, the surface of the slide glass was not entirely covered with bacterial cells.In the process of biofilm formation each of the biofilm microbes was sketched and enumerated.The results showed that bacterial cells pioneered the colonization and made up the base of the biofilm.The frequency and abundance of bacterial cells in each square implied bacterial cell division on the slide glass.

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Denitrification by both free-living bacteria and those attached to particles larger than 1μm was examined by the acetylene blockage technique in the hypolimnion of Lake Kizaki, Japan, from August to November, 1995.Bottom sediment gases(Ar, O₂, N₂, CH₄ and CO₂)were also measured at the beginning of June.Denitrification activity was detected from September to November, and ranged from 0.026 to 0.238μM-N₂O day^-1.During this period, free-living bacteria accounted for 31 to 57% of total denitrification.The total number of bacterial cells ranged from 2.34×10⁶ to 1.24×10⁷ml^-1, of which free-living bacteria accounted for 46 to 87%.High levels of N₂ in the surface sediments from both littoral and deep zones of the lake were observed, suggesting high rates of denitrification.These results show that both attached and free-living bacteria showed significant denitrification activity, with attached bacteria composing a higher proportion of the population in September in the deepest layer(28m).Free-living denitrifiers seemed to increase from September to November with expansion upwards of the low oxygen layer.

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To enrich nitrite-oxidizing bacteria, an activated sludge sample was transferred serially into an inorganic nitrite medium.Following the transfer, the culture maintained nitrite-oxidizing activity for over three months.Nitrite-oxidizing bacteria slightly decreased, but maintained their number at 10⁴MPN/ml, and 10⁶-10⁷cfu/ml of heterotrophic bacteria were also detected.Random cloning and analysis of amplified 16S rDNA using a universal primer set for bacteria showed that a culturable Pseudomonas putida-related strain was dominant in the culture, though the bacterium did not oxidize nitrite.The most dominant bacterial group estimated from the proportion of clones that showed identical pattern of restriction fragment length polymorphism belonged to the γ-subdivision of Proteobacteria.This was partly consistent with the results from whole-cell hybridization using group-specific fluorescent probes.Further limiting dilutions of the enriched culture produced a nitrite-oxidizing system with low numbers of heterotrophs.Molecular analysis suggested that the members were different from those in the enriched culture, and several kinds of Proteobacteria belonging to the β-, γ-, and α-subdivision, as well as bacteria in the high G+C Gram-positive phylum, existed.But in 16S rDNA sequence, none showed close similarity to any known autotrophic nitrite oxidizers.These results indicated that the population in serially transferred culture and limiting dilution culture is rather diverse, with some heterotrophic bacteria, and suggested the occurrence of an unidentified species of nitrite-oxidizing bacteria.

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We isolated the 2, 4-D-catabolizing bacterial strain RD5-C2 from Japanese upland soil that had no history of 2, 4-D exposure.RD5-C2 could use 2, 4-D as the sole carbon and energy sources for their growth.Phylogenetic analyses based on 16S rDNA sequencing revealed that RD5-C2 was a member of the BANA(Bradyrhizobium-Agromonas-Nitrobacter-Afipia)cluster in the α subdivision of the class Proteobacteria.The gene sequence of RD5-C2 most closely matched that of Bradyrhizobium sp.and also closely related to those of 2, 4-D-catabolizing α-Proteobacteria previously isolated from pristine environments of Hawaii, northern Canada and central Chile.This is the first instance that 2, 4-D-catabolizing bacteria in that phylogenetic group was isolated from an arable soil environment.

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The sporulation ratios in two dominant Bacillus groups were estimated both in the surface and deep sedimentary layers of a shallow pond.More than 60% of the members of A-group, represented by Bacillus megaterium, existed as vegetative cells both in the surface and deep layers.Although they have been regarded as strict aerobes, they seemed to have grown very slowly in the anaerobic condition.In contrast, More than 60% of the members of B-group, mainly consisting of Bacillus thuringiensis, existed as spores in the surface layer.

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Information regarding the deep subsurface biosphere has recently been accumulated.Major inhabitants of the deep biosphere are thought to be prokaryotic microorganisms, bacteria and archaea.Although some may be dormant, a certain fraction of the microbial population is considered to actively metabolize organic and inorganic materials.Thus, they influence the geochemical processes in the deep subsurface.Trophic bases of the deep subsurface biosphere, which are most likely H₂-driven, should be unique and different from those of the surface biospheres.

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