Anaerobic wastewater treatment sludge represents part of the natural microbial population found in various anaerobic ecosystems. The sludge drives the degradation of organic pollutants in wastewater, generally converting complex organic compounds into carbon dioxide and methane. This anaerobic conversion (methanogenesis) relies greatly on close interaction among a variety of different trophic groups of anaerobes within the sludge. Recent culture-independent molecular inventories of the microbial community of anaerobic (methanogenic) sludges revealed a vast diversity of microbes, the majority of which have not yet been cultivated and hence whose ecophysiological functions remain largely unknown. Two ongoing challenges in this field are to explain the in situ functions of these uncultured organisms and to establish how they interact with other organisms to make the methanogenic degradation of complex organic matter possible. In this review, recent progress in the study of the composition of microbial communities and the functions of individual populations in anaerobic sludge is summarized, with a special emphasis placed on the ecology and function of yet-to-be cultured microbes. In addition, it is also emphasized that the granular sludge in upflow anaerobic sludge blanket (UASB) reactors is an ideal model ecosystem with which to explore the functions of uncultured lineages of anaerobes and to elucidate the interaction of such organisms with other trophic groups of microbes in situ.

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Bacterial attachments often cover the entire surface of flagellated protists in the guts of termites. Based on PCR-amplified 16S rRNA gene sequences, we investigated the phylogenetic positions of the rod-shaped bacteria (ectosymbionts) attached to the protist Oxymonas sp. in the termite Neotermes koshunensis. Two distinct and unique lineages of the ectosymbionts within the order Bacteroidales were identified, each belonging to a cluster exclusively comprised of the sequences from termite gut. We designed two oligonucleotide probes specific for the two lineages, and successfully detected the ectosymbionts, each of which distributed over the entire surface of Oxymonas sp. However, few cells of Oxymonas sp. simultaneously harbored both lineages of the ectosymbionts.

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To examine whether plant-derived clostridia and their consortium fix nitrogen in plants, an inoculation system was developed using the grass Miscanthus sinensis under aseptic conditions. Among 13 clostridial strains previously isolated from M. sinensis, Clostridium sp. strain Kas107-1 was selected as the best colonizer in the plant with the non-diazotrophic bacterium Entrobacter sp. strain B901-2. Nitrogen-fixing (acetylene-reducing) activity was not observed in the plants inoculated with Kas107-1 without a carbon source. On the other hand, nitrogen-fixing activity was detected when carbon sources were supplied to the roots. To confirm the endophytic nitrogen-fixing activity, we cloned nifH genes and monitored their expression in strain Kas107-1. Although this bacterium possessed at least two copies of nifH (nifH1 and nifH2), the nifH1 transcript was exclusively detected in free-living cells and endophytic cells in the plants by reverse transcription (RT)-PCR analysis. RT-PCR analysis of ribosomal RNA suggested the endophytic colonization of the plants by Kas107-1. These results indicate that Clostridium sp. strain Kas107-1 can potentially fix nitrogen in plants. A RT-PCR analysis targeting both functional gene transcripts and the ribosomal RNA molecule is useful for researching endophyte ecology, because their function and colonization in plants can be examined simultaneously with a single preparation of RNA.

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SEp22, identical to Salmonella Dps, is a pathogenicity-related protein which we have isolated from the virulent Salmonella enterica subsp. enterica serovar Enteritidis (SE) from poultry farms. SEp22 is produced at the stationary phase, and rapidly lost upon incubation in fresh medium in vitro¹⁾. In this study, we examined the effects of nutrients in the medium on the regulation of SEp22 expression. While Luria-Bertani (LB) medium induced the production of a large amount of SEp22 during culture overnight, M9 minimal medium had little or no effect. Addition of LB to M9 dose-dependently increased the amount of SEp22 produced, showing the requirement of certain nutrients in LB medium for the induction. It also suggests that simply entering the stationary phase from a growth phase is insufficient for the expression of SEp22 in a nutrient-poor medium like M9, contrary to the induction of Dps in nutritionally starved Escherichia coli (E. coli). A similar requirement of LB nutrients was observed during the rapid production of SEp22 in a logarithmic phase of growth in Salmonella treated with H₂O₂. On the other hand, no difference was observed between M9 and LB medium in the effect to decrease the amount of SEp22 accumulated during reincubation. These results show the importance of culture nutrients not in the breakdown but in inducing the expression of SEp22 in pathogenic SE.

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Two bacterial consortia, K-3 and No. 22, capable of degrading aromatic hydrocarbons in crude petroleum at high rates were screened from crude petroleum-contaminated soil. The K-3 consortium required saturated hydrocarbons (4 g/l) fractionated from crude petroleum for the efficient degradation (20%) of aromatics (4 g/l) within seven days, whereas the No. 22 consortium degraded 66% of aromatics (4 g/l) without supplementation with saturates in fourteen days. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and a colony isolation procedure gave five and fourteen DNA bands, and six and three different shaped colonies, respectively from the K-3 and No. 22 communities. Among the strains isolated from the K-3 consortium, Pseudomonas aeruginosa was the predominant species and decomposed aromatic hydrocarbons in the presence of saturates, while among the pure cultures from the No. 22 community, Burkholderia multivorans strain Y4 and Pandoraea sp. strain Y1 degraded aromatics at high rates without saturates. However, in a mixed culture of the strains, the degradation of aromatic hydrocarbons by a consortium of three strains, Hyphomicrobium facile strain Y3, strains Y1 and Y4, was nearly as efficient as that by the No. 22 community.

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The fenitrothion hydrolase gene encoded on the plasmid pNF2 in Burkholderia sp. strain NF100 was cloned and sequenced. The 6.5-kbp EcoRI fragment contained three ORFs (ORF1 to 3) the deduced amino acid sequences of which shared no significant homology with any sequence present in databases. The results of frameshift mutation showed that ORF1 and ORF2 were essential for full enzymatic activity, and ORF3 was not involved in the degradation of fenitrothion. Fenitrothion hydrolase was located in the cell membrane fraction. The DNA region containing the fenitrothion hydrolase gene was spontaneously lost from pNF2.

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The diversity of fenitrothion-degrading bacteria active in soils from several distant locations in Japan was analyzed on the basis of 16S rRNA gene sequences. One hundred seventy fenitrothion-degrading isolates from four locations were assigned to five genera of the phylum Proteobacteria: Bartonella, Rhizobium, Burkholderia, Cupriavidus, and Pseudomonas. Bartonella, Cupriavidus, and Rhizobium strains were shown to degrade organophosphorus pesticides for the first time. Burkholderia strains were dominant in all soils. Bartonella strains degraded fenitrothion cometabolically, while all other strains utilized the pesticide, indicating that a potential for both complete and partial degradation of fenitrothion by bacteria exists in soil in Japan. Soil microcosms were prepared and exposed to repeated applications of fenitrothion. In each microcosm, one single Burkholderia strain was favored by this treatment and subsequently dominated the fenitrothion-degrading bacterial population.

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