TL;DR: It is analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems and characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or meethanol-assimilating populations.
Abstract: Stable-isotope probing (SIP) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. A sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [13C]acetate or [13C]methanol as the main carbon source and nitrate as the electron acceptor. We analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems. Most of the acetate- or methanol-assimilating bacteria identified by SIP have been known as denitrifiers in wastewater treatment systems. When acetate was used as the carbon source, 16S rRNA gene sequences retrieved from 13C-labeled DNA were closely related to the 16S rRNA genes of Comamonadaceae (e.g., Comamonas and Acidovorax) and Rhodocyclaceae (e.g., Thauera and Dechloromonas) of the Betaproteobacteria, and Rhodobacteraceae (e.g., Paracoccus and Rhodobacter) of the Alphaproteobacteria. When methanol was used as the carbon source, 16S rRNA gene sequences retrieved from 13C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from 13C-DNA showed that the diversity levels for methanol-assimilating bacteria were considerably lower than those for acetate-assimilating bacteria. Furthermore, we characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or methanol-assimilating populations and detected the nirS or nirK sequence related to that of some known pure cultures, such as Alcaligenes, Hyphomicrobium, and Thauera. However, most of the nirS or nirK sequences retrieved from 13C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from 13C-DNA, these unidentified nir sequences might be identified by examining the nir gene in candidates for true denitrifiers (e.g., the families Comamonadaceae, Hyphomicrobiaceae, Methylophilaceae, and Rhodobacteraceae).
TL;DR: A comparative study of three denitrifying Hyphomicrobium species highlights the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.
Abstract: Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.
TL;DR: Both, oligocarbophilic growth and the formation of poly-β-hydroxybutyrat als Speicherprodukt wurden bei the Wachstumsbestimmungen berücksichtigt.
Abstract: Budding bacteria from aquatic or terrestrial habitats were found to accumulate ferric oxide hydrate (“ferric hydroxide”) on their cell surfaces. Metal paper clips served as the source of oxidizable iron. Pure cultures deposited ferric hydroxide during growth on sea water medium at a pH of 7.8, but not in a mineral salts medium of normal ionic strength, of pH 7.2, and without NaCl, although some active strains came from fresh water or soil.
TL;DR: A comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph.
Abstract: The yield of Hyphomicrobium EG on dimethyl sulphoxide, dimethyl sulphide and methylamine, considering the metabolic pathways of these compounds, suggested that the organism gained energy from the oxidation of the sulphur moiety of the former compounds. Indeed, a comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph. The apparent Ysulphide and Ythiosulphate were comparable, being 8–10 g dry weight/mol. In batch cultures thiosulphate concentrations up to 10 mM had a stimulatory effect on the growth rate of Hyphomicrobium EG, whereas higher concentrations increased the organisms doubling time. Enzyme- and respiration data showed that the organism had constitutive enzymes for the breakdown of dimethyl sulphoxide although they were clearly regulated to need. Addition of sulphide or thiosulphate to methylamine-limited chemostat cultures of Hyphomicrobium EG not only resulted in the induction of enzymes necessary for their breakdown, but also caused the enzymes for dimethyl sulphoxide metabolism, especially methyl mercaptan oxidase, to be induced. The formation of H2O2, a product of the latter enzyme, was reflected in the relatively high catalase activities during growth on dimethyl sulphoxide and in the organisms inability to grow on this compound in the presence of a catalase inhibitor.
TL;DR: An enrichment procedure for the isolation of stalked bacteria of the genus Hyphomicrobium is described, based on the use of an organic C1 compound as carbon and energy source for growth together with anaerobic incubation in the presence of nitrate as an electron acceptor.
Abstract: An enrichment procedure for the isolation of stalked bacteria of the genusHyphomicrobium is described. The method is based on the use of an organic C1 compound as carbon and energy source for growth together with anaerobic incubation in the presence of nitrate as an electron acceptor. Optimal conditions for the growth of a number ofHyphomicrobium isolates have been investigated. Applying these conditions,Hyphomicrobium spp. have been enriched from a wide range of natural habitats within 1–2 weeks.