Abstract
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.
Highlights
Anaerobic treatment systems offer many advantages such as biogas production in the form of methane and low sludge production
Nitrate- and nitrite-dependent anaerobic oxidation of methane (N-damo) was discovered a decade ago, but the ecophysiological characterization of the microorganisms involved has been hindered by the slow growth of the responsible organisms (Raghoebarsing et al 2006; Ettwig et al 2010; Haroon et al 2013)
Previous enrichments were fed with mixtures of nitrite and nitrate without ammonium, to ensure that anammox bacteria would not outcompete ‘Ca. Methylomirabilis’ bacteria for nitrite (Luesken et al 2011b; Shi et al 2013; Hu et al 2015; Ding et al 2017; Vaksmaa et al 2017b)
Summary
Anaerobic treatment systems offer many advantages such as biogas production in the form of methane and low sludge production. Because of this production of both nitrite and ammonium, the ‘Ca. Methanoperedens’ archaea are very useful partners for both ‘Ca. Methylomirabilis’ and anammox bacteria (Haroon et al 2013; Shen et al 2014; Meng et al 2016; Arshad et al 2017; Xu et al 2017; Fu et al 2019; Nie et al 2019, 2020) This is supported by studies showing these anaerobic methane- and ammonium-oxidizing microorganisms living together in oxygen-limited ecosystems (reviewed in Welte et al 2016). The blueprint of anammox bacteria contains many interesting and diagnostic features including hydrazine synthase (hzsA) and hydrazine dehydrogenase, multiheme protein complexes, and nitrite and nitrate reductases (Strous et al 2006; Kartal et al 2011)
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