Identification of Acetate- or Methanol-Assimilating Bacteria under Nitrate-Reducing Conditions by Stable-Isotope Probing

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 [(13)C]acetate or [(13)C]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 (13)C-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 (13)C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from (13)C-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 (13)C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from (13)C-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).

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria

“…Recently, Arcobacter strains were isolated from the graphite electrode of a microbial fuel cell inoculated with marine sediment and fed with acetate (Fedorovich et al, 2009) (Figure 5), indicating that Arcobacter species are able to use acetate and transfer electrons onto solid-phase electron acceptors. In a recent cultivation-independent SIP study with estuarine sediment, Arcobacter-related bacteria incorporated 13 C into their DNA from acetate and glucose under various terminal electron acceptor conditions (aerobic, nitrate-, sulfate-reducing, methanogenic) (Osaka et al, 2006;Webster et al, 2010). Under sulfatereducing conditions with 13 C-labeled acetate, Arcobacter became enriched fast and although the authors suggested that Arcobacter was involved in sulfur cycling, iron and manganese reduction during the sediment incubations were indicated by increasing concentrations of dissolved manganese and iron (Webster et al, 2010).…”

Sludge bulking impact on relevant bacterial populations in a full-scale municipal wastewater treatment plant

“…M. parvicella was believed not to reduce nitrite to nitrogen gas [22]. However, many other bacteria could reduce nitrite, such as the members of Rhodobacteraceae (accounting for 8.1 and 5.2% respectively for the ASB and ISB libraries) and Hyphomicrobium (accounting for 2.0 and 2.1% respectively for the ASB and ISB libraries) [32]. Thus the denitrification performance was not adversely affected, either, as indicated by the equivalent TN removals (68.5-83.6% during sludge bulking period vs. 66.5-75.5% during non-bulking period).…”

Assessing genetic structure, diversity of bacterial aerosol from aeration system in an oxidation ditch wastewater treatment plant by culture methods and bio-molecular tools

“…Previous research isolated TB127-79 from activated sludge in a circulation flush toilet (Hoshino et al 2006), while BXHB27 has been found in activated sludge from a wastewater treatment plant. The 12C-M17 and PHOS-HE19 have been identified from microbial communities in nitratereducing (Osaka et al 2006) and phosphorus-removal ecosystems (Dabert et al 2001), respectively. These four bacteria are frequently found in aerobic activated sludge or domestic wastewater treatment equipment.…”