Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing

Abstract

Methane is used as an alternative carbon source in the denitrification of wastewater lacking organic carbon sources because it is nontoxic and may be efficiently produced by anaerobic biological processes. Methane-dependent denitrification (MDD) in the presence of oxygen requires the co-occurrence of methanotrophy and denitrification. Activated sludge was incubated with 13C-labeled methane in either a nitrate-containing medium or a nitrate-free medium. Then, bacterial and methanotrophic populations were analyzed by cloning analysis and terminal restriction fragment length polymorphism analysis targeting 16S rRNA gene and cloning analysis targeting pmoA genes. DNA-based stable-isotope probing (DNA-SIP) analysis of the 16S rRNA gene revealed an association of the Methylococcaceae and the Hyphomicrobiaceae in a MDD ecosystem. Furthermore, supplementation of nitrate stimulated methane consumption and the activity of methanotrophic populations (i.e. the stimulation of uncultivated relatives of distinct groups of the Methylococcaceae). In particular, uncultured type-X methanotrophs of Gammaproteobacteria were dominant when nitrate was added, i.e. in the MDD incubations. On the other hand, most methanotrophs (types I, II, and X methanotrophs) were found to have been labeled with 13C under nitrate-free conditions. This DNA-SIP study identifies key bacterial populations involved in a MDD ecosystem.

Linking microbial community structure to membrane biofouling associated with varying dissolved oxygen concentrations

“…5, Table 3). At low DO (0.5 mg L À1 ), the diversity of predominant microbial populations decreased, and two obvious band, band 14 and 24, became obvious also at this period, which were similar with uncultured denitrifying bacterium clone H2SRC121X and R1-44 based on sequence alignment (Osaka et al, 2008;Maestre et al, 2009). Hence, the emergence of denitrifying bacteria might well indicate membrane fouling.…”

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria

“…Consequently, full-scale implementation of heterotrophic denitrification is still guided by a somewhat empirical understanding of its microbial ecology and biokinetics, which in turn limits efforts to optimize denitrification reactor design, monitoring and modeling. Although biokinetic parameters for methylotrophic denitrification in activated sludge have been extensively reported (dos Santos et al, 2004;Gauntlett, 1979;Her and Huang, 1995;Janning et al, 1995;Lee and Welander, 1996;Louzeiro et al, 2002;Mulcahy et al, 1981;Purtschert and Gujer, 1999), the actual abundance and diversity of organisms in activated sludge that actually metabolize methanol and alternate carbon and electron sources has only recently been identified (Ginige et al, 2004(Ginige et al, , 2005Osaka et al, 2006Osaka et al, , 2008. The reason for such sparse data on the identity of bacterial populations denitrifying using specific electron donors is that unlike nitrifying bacteria, which are phylogenetically closely related (Purkhold et al, 2000), denitrifying bacteria are distributed widely across taxonomic groups (Zumft, 1997).…”

Identification and isolation of active N2O reducers in rice paddy soil

“…Using the SIP approach, we can analyze the 16S rRNA and functional gene diversities of microbial populations involved in specific functions. Previously, 13 C-assimilating populations under denitrifying conditions were analyzed by DNAbased SIP analysis (Ginige et al, 2004;Osaka et al, 2006Osaka et al, , 2008Saito et al, 2008). However, microbial populations responsible for N 2 O reduction have not been examined to date.…”