Budding methylotrophic bacteria resembling Hyphomicrobium spp. were counted for 12 months in a German sewage treatment plant by most-probable-number (MPN) methods. Influent samples contained up to 2 ؋ 10 4 cells ml ؊1 , activated sludge consistently contained 1 ؋ 10 5 to 5 ؋ 10 5 cells ml ؊1 , and the effluent contained 1 ؋ 10 3 to 4 ؋ 10 3 cells ml ؊1 . The receiving lake had only 2 to 12 cells ml ؊1 . Six morphological groups with different growth requirements could be observed among 1,199 pure cultures that had been isolated from MPN dilutions. With dot blot DNA hybridizations, 671 isolates were assigned to 30 hybridization groups (HGs) and 84 could not be classified. Only HG 22 hybridized with a known species, Hyphomicrobium facilis IFAM B-522. Fourteen HGs (HGs 8 to 20 and HG 22) were specific for the lake; most others occurred only in the treatment plant. HGs 1, 3, and 26 were found in the activated sludge tank throughout the year, and HGs 27 and 28 were found for most of the year. In summary, it was demonstrated that bacteria with nearly identical and specific morphologies and nutritional types showed a high level of genetic diversity, although they were isolated under the same conditions and from the same treatment plant or its receiving lake. A directional exchange of these genetically different populations was possible but less significant, as was shown by the establishment of distinct populations in specific stations.

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Hyphomicrobium spp. were counted and isolated for 12 months in a sewage treatment plant with a combination of simultaneous and intermittent nitrification and denitrification using Most-Probable-Number methods. Genomic DNA of these hyphomicrobia was investigated by Southern or dot blot hybridizations with gene probes specific for genes of dissimilatory nitrate reduction (nitrate reductase, narG; cytochrome c,d-containing nitrite reductase, nirS; Cu-containing nitrite reductase, nirK; nitrous oxide reductase, nosZ), nitrification (ammonia monooxygenase, amoA) and N2-fixation (nitrogenase, nifH). In particular, the Hyphomicrobium DNA/DNA-hybridization group HG 27 constituted one of the dominant denitrifying Hyphomicrobium populations in the activated sludge of this sewage treatment plant. A species-specific gene probe (Hvu-1) for HG 27 was generated from a transposon Tn5-132 insertion mutant defective in methanol oxidation using the inverse polymerase chain reaction. With this probe the abundance of this group in activated sludge of the sewage treatment plant and its receiving lake was determined as a subfraction of the total cultivable hyphomicrobia. Fragments of the mxaF gene encoding for the α-subunit of the methanol dehydrogenase of Hyphomicrobium spp. were amplified by PCR and analysed by denaturing gradient gel electrophoresis (DGGE). The DGGE analysis pattern showed a substantial separation of these fragments according to their nucleic acid sequences.

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