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Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge.

Luesken FA, van Alen TA, van der Biezen E, Frijters C, Toonen G, Kampman C, Hendrickx TL, Zeeman G, Temmink H, Strous M, Op den Camp HJ, Jetten MS - Appl. Microbiol. Biotechnol. (2011)

Bottom Line: This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes.After 112 days, the enrichment consumed up to 0.4 mM NO(2)(-) per day.The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.

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a Phylogenetic tree of 16S rRNA gene sequences of the NC10 phylum, including sequences of nine different WWTPs located in The Netherlands (bold). The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. All sequences found are represented in group a of the NC10 phylum (group a, b, c, and d are described in Ettwig et al. 2009). b Phylogenetic tree of pmoA sequences including clones from inoc Lieshout, enr Lieshout, enr 332 day Lieshout, and WWTP Lieshout. The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. The clones indicated with “a” were obtained with the primers A189_b and 682R, clones indicated with “b” were obtained with a nested approach using primers A189_b and cmo682 as a template for primers cmo182 and cmo568. Clones obtained with a direct PCR using the primers A189_b and cmo682 are indicated with “c”. Clones obtained with primers cmo182 and cmo568 are indicated with “d”
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Fig1: a Phylogenetic tree of 16S rRNA gene sequences of the NC10 phylum, including sequences of nine different WWTPs located in The Netherlands (bold). The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. All sequences found are represented in group a of the NC10 phylum (group a, b, c, and d are described in Ettwig et al. 2009). b Phylogenetic tree of pmoA sequences including clones from inoc Lieshout, enr Lieshout, enr 332 day Lieshout, and WWTP Lieshout. The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. The clones indicated with “a” were obtained with the primers A189_b and 682R, clones indicated with “b” were obtained with a nested approach using primers A189_b and cmo682 as a template for primers cmo182 and cmo568. Clones obtained with a direct PCR using the primers A189_b and cmo682 are indicated with “c”. Clones obtained with primers cmo182 and cmo568 are indicated with “d”

Mentions: To find suitable inocula to start pilot scale tests for application of nitrite-dependent anaerobic methane oxidation, ten WWTPs in The Netherlands were screened with molecular tools (Table 1). All WWTPs treat wastewater originating from both domestic and industrial sources, except the WWTP in Lieshout. This plant treats only industrial (brewery) water at moderate temperatures. Relatively long sludge retention times and low BOD/N ratios were used as criteria to select the WWTPs for screening. These criteria in combination with alternating oxic–anoxic conditions present in all WWTPs might reflect the niche of M. oxyfera bacteria where both methane and oxidized nitrogen compounds are present at the same time. A molecular survey was conducted to screen for M. oxyfera-type bacteria after DNA extraction from the sludge samples of these WWTPs. To detect NC10 bacteria, a direct PCR with NC10 specific primer 202F and general primer 1545R targeting the 16S rRNA gene was performed on one of the WWTP sludge samples (Lieshout). This resulted in a PCR product of the right size, but this product was hardly visible with gel electrophoresis (data not shown). Therefore, a nested PCR approach was performed on all WWTP sludge samples. In nine out of ten WWTPs tested, M. oxyfera-type bacteria could be detected (Fig. 1a). M. oxyfera-type bacteria were not detected in the WWTP in Varsseveld. Recently, PCR primer sets for detection of M. oxyfera pmoA genes were published (Luesken et al. 2011). These sets were used in the later stage of this study (see below) complementary to the 16S rRNA approach.Fig. 1


Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge.

Luesken FA, van Alen TA, van der Biezen E, Frijters C, Toonen G, Kampman C, Hendrickx TL, Zeeman G, Temmink H, Strous M, Op den Camp HJ, Jetten MS - Appl. Microbiol. Biotechnol. (2011)

a Phylogenetic tree of 16S rRNA gene sequences of the NC10 phylum, including sequences of nine different WWTPs located in The Netherlands (bold). The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. All sequences found are represented in group a of the NC10 phylum (group a, b, c, and d are described in Ettwig et al. 2009). b Phylogenetic tree of pmoA sequences including clones from inoc Lieshout, enr Lieshout, enr 332 day Lieshout, and WWTP Lieshout. The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. The clones indicated with “a” were obtained with the primers A189_b and 682R, clones indicated with “b” were obtained with a nested approach using primers A189_b and cmo682 as a template for primers cmo182 and cmo568. Clones obtained with a direct PCR using the primers A189_b and cmo682 are indicated with “c”. Clones obtained with primers cmo182 and cmo568 are indicated with “d”
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Related In: Results  -  Collection

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Fig1: a Phylogenetic tree of 16S rRNA gene sequences of the NC10 phylum, including sequences of nine different WWTPs located in The Netherlands (bold). The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. All sequences found are represented in group a of the NC10 phylum (group a, b, c, and d are described in Ettwig et al. 2009). b Phylogenetic tree of pmoA sequences including clones from inoc Lieshout, enr Lieshout, enr 332 day Lieshout, and WWTP Lieshout. The phylogenetic tree was constructed using neighbor-joining and bootstrap analysis of 1,000 replicates. The clones indicated with “a” were obtained with the primers A189_b and 682R, clones indicated with “b” were obtained with a nested approach using primers A189_b and cmo682 as a template for primers cmo182 and cmo568. Clones obtained with a direct PCR using the primers A189_b and cmo682 are indicated with “c”. Clones obtained with primers cmo182 and cmo568 are indicated with “d”
Mentions: To find suitable inocula to start pilot scale tests for application of nitrite-dependent anaerobic methane oxidation, ten WWTPs in The Netherlands were screened with molecular tools (Table 1). All WWTPs treat wastewater originating from both domestic and industrial sources, except the WWTP in Lieshout. This plant treats only industrial (brewery) water at moderate temperatures. Relatively long sludge retention times and low BOD/N ratios were used as criteria to select the WWTPs for screening. These criteria in combination with alternating oxic–anoxic conditions present in all WWTPs might reflect the niche of M. oxyfera bacteria where both methane and oxidized nitrogen compounds are present at the same time. A molecular survey was conducted to screen for M. oxyfera-type bacteria after DNA extraction from the sludge samples of these WWTPs. To detect NC10 bacteria, a direct PCR with NC10 specific primer 202F and general primer 1545R targeting the 16S rRNA gene was performed on one of the WWTP sludge samples (Lieshout). This resulted in a PCR product of the right size, but this product was hardly visible with gel electrophoresis (data not shown). Therefore, a nested PCR approach was performed on all WWTP sludge samples. In nine out of ten WWTPs tested, M. oxyfera-type bacteria could be detected (Fig. 1a). M. oxyfera-type bacteria were not detected in the WWTP in Varsseveld. Recently, PCR primer sets for detection of M. oxyfera pmoA genes were published (Luesken et al. 2011). These sets were used in the later stage of this study (see below) complementary to the 16S rRNA approach.Fig. 1

Bottom Line: This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes.After 112 days, the enrichment consumed up to 0.4 mM NO(2)(-) per day.The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.

Show MeSH