In flooded rice fields, methanogenic archaea produce CH₄, while methanotrophic bacteria oxidize a part of the produced CH₄. Thus, the latter bacteria are considered as suitable organisms for controlling CH₄ emission from paddy fields. In this paper, the author demonstrates a case of organic matter application, enumeration and isolation of methanogenic archaea and methanotrophic bacteria in a subtropical paddy field. The rice rhizosphere is one of the typical areas where anaerobic and aerobic environments interface, methanogens produce CH₄ and methanotrophs utilize it for energy. Although how they interact in the anaerobic and aerobic interfaces is an attractive research area, it has not yet been fully elucidated, because a two-member co-culture of methanogen and methanotroph is not well developed. Co-culture of a strictly anaerobic methanogenic archaeon and an obligately aerobic methanotrophic bacterium using sterilized paddy soil was carried out. The rice root system affects CH₄ production and oxidation in the rice rhizosphere, and its influence varies with different rice cultivars. Rice cultivars with few unproductive tillers, a small root system, high root oxidative activity, and high harvest index are ideal for mitigating CH₄ emission in paddy fields.

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Using a halo-forming assay with polysaccharide-degrading enzymes encoded by Chlorella virus (chlorovirus) CVK2, several free-living Chlorella strains, especially those belonging to C. vulgaris, were found to be sensitive to the viral enzymes. Among these sensitive strains, a taxonomically established strain C. prototechoides 211-6 served as a new laboratory host for chloroviruses (NC64A-viruses). Many zoochlorella strains isolated from Paramecium were totally resistant to the enzymes. Attachment of CVK2 to the cells of both Chlorella strain NC64A and C. prototechoides 211-6 was markedly blocked by treatment of the cells with a mixture of the viral enzymes. The treatment with vAL-1 may destroy the virus receptor molecules.

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Four representative strains selected from algicidal filamentous bacteria isolated from shrimp aquacultural ponds and coastal seawater of Kagoshima Bay were tentatively identified as the genus Saprospira and divided into 4 taxonomic groups as described previously. The small subunit ribosomal DNA (SSU rDNA) sequences of the 4 representative strains were determined and applied to a phylogenetic analysis with the type species of the genus Saprospira (S. grandis ATCC23116 and ATCC23119). The results revealed a close relationship between the isolated strains and S. grandis which belongs to the Flexibacter-Bacteroides-Cytophaga (FBC) group. PCR products of intergenic spacer regions (IGS) between large and small subunit ribosomal RNA genes were shown to be composed of two or three fragments by agarose gel electrophoresis. Higher molecular weight fragments of PCR products for IGS included tRNA^Ile and tRNA^Ala genes but the similarity values of IGS sequences among test strains were less than 60%.

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In a design utilizing a sterilized soil-mediated co-culture of methanogenic archaeon and methanotrophic bacterium, the activities of both the methanogen and methanotroph were examined. It was confirmed that CH₄ was produced by the methanogen after methanotrophic inoculation and suggested that this CH₄ was oxidized by the methanotroph as the concentration of O₂ increased, although emission of CH₄ itself was indirect proof of CH₄-oxidizing activity. On the other hand, in the control treatment, methanogenic inoculation without methanotrophic inoculation at different levels of O₂, CH₄ was not emitted (there was no activity to produce CH₄). These results suggest a symbiotic relation in this co-culture system, because the methanogen provided CH₄ to the methanotroph, while inoculation of the methanotroph was necessary for subsequent production of CH₄ by the methanogen. The development of co-culture systems is essential for understanding the close relationship and interaction between methanogens and the methanotrophs in natural and agricultural environments.

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Changes in methanogenic activity were determined in slurries of soil collected from a paddy field. Acetate or H₂ added as a methanogenic substrate stimulated methane production in most samples. In soil collected when the field was continuously flooded, the two substrates affected production similarly, while in the samples collected when the field had been drained, H₂ stimulated production more strongly than acetate. The effect of temperature on methane production in paddy soil was also investigated. In continuously flooded soil, the temperature optimum for production was 40°C, however, this shifted to 45°C during a period of intermittent irrigation accompanied by a marked decrease in activity. The temperature optimum during the non-cropping season was also 45°C. It was suggested that the dominant methanogens in the drained paddy field are H₂-utilizers, different from the dominant groups in flooded paddy soil.

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The red tide dinoflagellate Heterocapsa circularisquama Horiguchi has intracellular bacteria. The isolation and cultivation of these bacteria were attempted for three-clonal strains of H. circularisquama (HY9423, HA92-1, and HU9433) using various culture media containing cell components of the host alga prepared by homogenization and heat killing, in addition to different organic materials (peptone and extracts). Only one intracellular bacterium of the bacterial population UBb in H. circularisquama strain HU9433 was successfully cultivated on many kinds of culture media. The intracellular bacterial populations of Yb and Ab in the algal clones HY9423 and HA92-1, respectively, could not grow in the culture media used. The presence of live algal cells is thought to be essential for the growth and survival of the bacterial populations Yb and Ab. These results indicate that the degree of dependence of the intracellular bacteria on the host algal cells differs greatly among the algae-bacteria sets of strains.

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A conventional enrichment culture on branched nonylphenol (NP) with diluted nutrient broth as an additional source of organic nutrients yielded a bacterial strain able to degrade branched NP. The isolate (designated YT) was identified as Sphingomonas sp. based on an analysis of its 16S ribosomal RNA genes and cellular lipids. The degradation of NP by strain YT occurred primarily during the exponential phase of cell growth in cultures on a yeast extract-mineral salts medium. The degree of degradation was directly proportional to the amount of yeast extract present in the medium and no significant growth occurred when NP was the sole source of carbon and energy. Gas chromatography-mass spectrometry (GC-MS) of resting cell suspensions incubated with branched NP revealed that the degradation did not yield any metabolites containing aromatic residues but only branched alcohols. When a linear NP was used as the target substrate, GS-MS of the suspensions indicated the appearance of a hydroxylated linear NP as an intermediate during the degradation. Strain YT is expected to attack NP by an initial oxidative cleavage of the phenol ring.

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Prodigiosin is a red pigment characterizing S. marcescens and produced in abundance by the bacterium on peptone-glycerol (PG) agar plates incubated at 30°C. In a PG liquid culture, however, S. marcescens was unable to produce prodigiosin. On addition of silica gel (8.0 mg/ml) to the liquid medium, S. marcescens became able to proliferate better and produced prodigiosin and serrawettins (biosurfactant) profusely. The effect of the silica gel on the production of exolipids was dependent on source (manufacturers). By microscopic examination, sessile bacterial populations were recognized on the silica particles active in the promotion of prodigiosin production, but not on the particles unable to promote the production. Prodigiosin and serrawettins are structurally unrelated but seem to have the same production control systems responding to temperature (T. Matsuyama, et al., J. Gen. Microbiol., 132, 865-875, 1986) and silica gel.

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We developed a simple method for absorbance spectrophotometry to identify photosynthetic pigments of microbial mats using a portable spectrophotometer in the field. This method was very efficient for the identification of mat-forming phototrophs and estimations of their mixing ratios in the field. It was also applied to describe the structure of hot spring microbial mats developed at the Nakabusa Hot Spring, Nagano Prefecture. The microbial mats consisted of cyanobacteria and Chloroflexus, and their distribution depended on the water temperature. The ratio of these two bacteria determined by absorbance spectra was constant at temperatures ranging from 45 to 60°C, and the spectral mixing ratios of Chloroflexus were about 40%. That ratio increased with temperatures in the range of 60 to 70°C; above 70°C, only Chloroflexus was observed. The spectrophotometry also found a pink bacterial mat which had distinctive absorption peaks at 801 and 878 nm. These peaks strongly suggested that the organism was a novel phototrophic bacterium of a new taxon.

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