ABSTRACT
Phosphate accumulating organisms (PAOs) stained with 4',6-diamidino-2-phenylindol dihydrochloride (DAPI) at polyphosphate probing concentration were sorted from enhanced biological phosphorus removal (EBPR) sludge by flow cytometric sorting. All the genome DNA was extracted from the sorted bacteria and the 16S rDNA genes were cloned. Cloned 16S rDNA was PCR-amplified and analyzed by restriction fragment length polymorphism (RFLP) analysis. Eighty eight clones were analyzed and the RFLP patterns which appeared more than twice were classified into seven groups. The most dominant group (Group 1) contained four clones and accounted for 4.5% of the total clones. Four groups (from Group 2 to Group 5) contained three clones. Group 6 and 7 consisted of two clones. Sixty-eight clones gave unique RFLP patterns. By sequencing 16S rDNA in seven groups, Group 1, 2 and 5 were Rhodocyclus relatives (11%, 10/88). Rhodocyclus relatives were suggested to be one of the bacteria responsible for EBPR in this sludge. Groups 6 and 7 were related to b- or g-Proteobacteria. Group 4 belonged to e-Proteobacteria. Group 3 was related to green nonsulfur bacteria. Considering the complex RFLP pattern and the existence of the groups not related to Rhodocyclus by sequence analysis, in this EBPR system, together with Rhodocyclus relatives, some other bacteria might also play a role as PAOs.
Subject(s)
Phosphorus/metabolism , Polymorphism, Restriction Fragment Length , Proteobacteria/physiology , Sewage/microbiology , Flow Cytometry , Polyphosphates/analysis , Polyphosphates/metabolism , Population Dynamics , Proteobacteria/genetics , Proteobacteria/isolation & purification , Waste Disposal, FluidABSTRACT
The microbial community structures of anaerobic-aerobic activated sludges, which are dominated with polyphosphate-accumulating bacteria (PABs) and glycogen-accumulating bacteria (GABs), were investigated by quinone profile method and dual staining DAPI (4',6-diamidino-2-phenylindol dihydrochloride) with fluorescent in situ hybridization (FISH). For both activated sludges, the predominant bacterial groups were the beta subclass of the Proteobacteria that contained mainly Q-8 as analyzed by the quinone profile method and gram-positive bacteria with a high DNA G+C content based on FISH. A 16S rRNA-targeted oligonucleotide probe (MP2) specific for M. phosphovorus, a known polyphosphate-accumulating bacterium, was also applied to PABs and GABs-enriched activated sludge to evaluate their possible role in the enhanced biological phosphorus removal (EBPR) process. Interestingly, the bacteria that hybridized with the probe specific to M. phosphovorus in the sludge dominated with PABs represent about 6.9% of the total microorganisms. In the sludge dominated with GABs, these bacteria were about 1.9%. These results suggest its possible involvement in the EBPR process.
Subject(s)
Bacteria, Aerobic/physiology , Bacteria, Anaerobic/physiology , Glycogen/metabolism , Polyphosphates/pharmacokinetics , Sewage/microbiology , In Situ Hybridization, Fluorescence , Phosphorus/metabolism , Population DynamicsABSTRACT
The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F420 in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium ruminantium and mobile, Methanobrevibacter smithii. Epifluorescence microscopy (F420 autofluorescence) and in situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6% of total ruminal microorganisms and approximately 54% of the total methanogens were M. mobile.
Subject(s)
Euryarchaeota/genetics , Methanomicrobiaceae/isolation & purification , Rumen/microbiology , Animals , DNA, Ribosomal/chemistry , Ecosystem , Euryarchaeota/classification , Gene Library , In Situ Hybridization, Fluorescence/veterinary , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , SheepABSTRACT
Polyphosphate-accumulating bacteria that were previously isolated from activated sludge and exhibited high phosphate removal activity were studied taxonomically and phylogenetically. These organisms were gram-positive, coccus-shaped, aerobic chemoorganotrophs that had a strictly respiratory type of metabolism in which oxygen was a terminal electron acceptor. They accumulated large amounts of polyphosphate under aerobic conditions. The major quinone was menaquinone MK-9(H4). The cell wall peptidoglycan contained LL-diaminopimelic acid. The guanine-plus-cytosine content of the DNA was 67.9 mol%. Our isolates were similar phenotypically and chemotaxonomically to Luteococcus japonicus, which was proposed recently as a new genus and species. However, our isolates differed from L. japonicus in cellular fatty acid composition and some other traits. A phylogenetic analysis based on 16S rRNA sequences showed that our isolate differ from the genus Luteococcus and other genera belonging to the high-G+C-content gram-positive group. Accordingly, we concluded that our strain NM-1T (T = type strain) should be assigned to a new genus and species, for which we propose the name Microlunatus phosphovorus.