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

ABSTRACT: Recently discovered microorganisms affiliated to the bacterial phylum NC10, named "Candidatus Methylomirabilis oxyfera", perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were screened for the endogenous presence of M. oxyfera using molecular diagnostic methods. We could identify NC10 bacteria with 98% similarity to M. oxyfera in nine out of ten WWTPs tested. Sludge from one selected WWTP was used to start a new enrichment culture of NC10 bacteria. 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.

SUBMITTER: Luesken FA

PROVIDER: S-EPMC3198195 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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

Luesken Francisca A FA van Alen Theo A TA van der Biezen Erwin E Frijters Carla C Toonen Ger G Kampman Christel C Hendrickx Tim L G TL Zeeman Grietje G Temmink Hardy H Strous Marc M Op den Camp Huub J M HJ Jetten Mike S M MS

Applied microbiology and biotechnology 20110611 4

Recently discovered microorganisms affiliated to the bacterial phylum NC10, named "Candidatus Methylomirabilis oxyfera", perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were ...[more]

PMID: 21667086

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Project description:Anaerobic wastewater treatment offers several advantages; however, the effluent of anaerobic digesters still contains high levels of ammonium and dissolved methane that need to be removed before these effluents can be discharged to surface waters. The simultaneous anaerobic removal of methane and ammonium by denitrifying (N-damo) methanotrophs in combination with anaerobic ammonium-oxidizing (anammox) bacteria could be a potential solution to this challenge. After a molecular survey of a wastewater plant treating brewery effluent, indicating the presence of both N-damo and anammox bacteria, we started an anaerobic bioreactor with a continuous supply of methane, ammonium, and nitrite to enrich these anaerobic microorganisms. After 14 months of operation, a stable enrichment culture containing two types of 'Candidatus Methylomirabilis oxyfera' bacteria and two strains of 'Ca. Brocadia'-like anammox bacteria was achieved. In this community, anammox bacteria converted 80% of the nitrite with ammonium, while 'Ca. Methylomirabilis' contributed to 20% of the nitrite consumption. The analysis of metagenomic 16S rRNA reads and fluorescence in situ hybridization (FISH) correlated well and showed that, after 14 months, 'Ca. Methylomirabilis' and anammox bacteria constituted approximately 30 and 20% of the total microbial community. In addition, a substantial part (10%) of the community consisted of Phycisphaera-related planctomycetes. Assembly and binning of the metagenomic sequences resulted in high-quality draft genome of two 'Ca. Methylomirabilis' species containing the marker genes pmoCAB, xoxF, and nirS and putative NO dismutase genes. The anammox draft genomes most closely related to 'Ca. Brocadia fulgida' included the marker genes hzsABC, hao, and hdh. Whole-reactor and batch anaerobic activity measurements with methane, ammonium, nitrite, and nitrate revealed an average anaerobic methane oxidation rate of 0.12 mmol h-1 L-1 and ammonium oxidation rate of 0.5 mmol h-1 L-1. Together, this study describes the enrichment and draft genomes of anaerobic methanotrophs from a brewery wastewater treatment plant, where these organisms together with anammox bacteria can contribute significantly to the removal of methane and ammonium in a more sustainable way. KEY POINTS: • An enrichment culture containing both N-damo and anammox bacteria was obtained. • Simultaneous consumption of ammonia, nitrite, and methane under anoxic conditions. • In-depth metagenomic biodiversity analysis of inoculum and enrichment culture.

Project description:Methane is a very potent greenhouse gas and can be oxidized aerobically or anaerobically through microbe-mediated processes, thus decreasing methane emissions in the atmosphere. Using a complementary array of methods, including phylogenetic analysis, physiological experiments, and light and electron microscopy techniques (including electron tomography), we investigated the community composition and ultrastructure of a continuous bioreactor enrichment culture, in which anaerobic oxidation of methane (AOM) was coupled to nitrate reduction. A membrane bioreactor was seeded with AOM biomass and continuously fed with excess methane. After 150 days, the bioreactor reached a daily consumption of 10 mmol nitrate · liter-1 · day-1 The biomass consisted of aggregates that were dominated by nitrate-dependent anaerobic methane-oxidizing "Candidatus Methanoperedens"-like archaea (40%) and nitrite-dependent anaerobic methane-oxidizing "Candidatus Methylomirabilis"-like bacteria (50%). The "Ca Methanoperedens" spp. were identified by fluorescence in situ hybridization and immunogold localization of the methyl-coenzyme M reductase (Mcr) enzyme, which was located in the cytoplasm. The "Ca Methanoperedens" sp. aggregates consisted of slightly irregular coccoid cells (∼1.5-μm diameter) which produced extruding tubular structures and putative cell-to-cell contacts among each other. "Ca Methylomirabilis" sp. bacteria exhibited the polygonal cell shape typical of this genus. In AOM archaea and bacteria, cytochrome c proteins were localized in the cytoplasm and periplasm, respectively, by cytochrome staining. Our results indicate that AOM bacteria and archaea might work closely together in the process of anaerobic methane oxidation, as the bacteria depend on the archaea for nitrite. Future studies will be aimed at elucidating the function of the cell-to-cell interactions in nitrate-dependent AOM.IMPORTANCE Microorganisms performing nitrate- and nitrite-dependent anaerobic methane oxidation are important in both natural and man-made ecosystems, such as wastewater treatment plants. In both systems, complex microbial interactions take place that are largely unknown. Revealing these microbial interactions would enable us to understand how the oxidation of the important greenhouse gas methane occurs in nature and pave the way for the application of these microbes in wastewater treatment plants. Here, we elucidated the microbial composition, ultrastructure, and physiology of a nitrate-dependent AOM community of archaea and bacteria and describe the cell plan of "Ca Methanoperedens"-like methanotrophic archaea.

Dataset Information

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

Publications

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

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