Metalloenzymes play major roles to achieve high-rate nitrogen removal in N-damo communities: Lessons from metaproteomics.

Nitrite-driven anaerobic methane oxidation (N-damo) is a promising biological process to achieve carbon-neutral wastewater treatment solutions, aligned with the sustainable development goals. Here, the enzymatic activities in a membrane bioreactor highly enriched in N-damo bacteria operated at high nitrogen removal rates were investigated. Metaproteomic analyses, with a special focus on metalloenzymes, revealed the complete enzymatic route of N-damo including their unique nitric oxide dismutases. The relative protein abundance evidenced that "Ca. Methylomirabilis lanthanidiphila" was the predominant N-damo species, attributed to the induction of its lanthanide-binding methanol dehydrogenase in the presence of cerium. Metaproteomics also disclosed the activity of the accompanying taxa in denitrification, methylotrophy and methanotrophy. The most abundant functional metalloenzymes from this community require copper, iron, and cerium as cofactors which was correlated with the metal consumptions in the bioreactor. This study highlights the usefulness of metaproteomics for evaluating the enzymatic activities in engineering systems to optimize microbial management.

How efficiently does a metabolically enhanced system with denitrifying anaerobic methane oxidizing microorganisms remove antibiotics?

In this work, the novel N-damo (Nitrite dependent anaerobic methane oxidation) process was investigated at high biomass activities for its potential to remove simultaneously nitrite and methane, as well as selected antibiotics commonly found in sewage in trace amounts. For this purpose, two MBRs were operated at three high nitrite loading rates (NLRs), namely 76 ± 9.9, 161.5 ± 11.4 and 215.2 ± 24.2 mg N-NO⁻2 L-1 d-1, at long-term operation. The MBRs performance achieved a significantly high nitrite removal activity for an N-damo process (specific denitrifying activity of up to 540 mg N-NO⁻2 g-1 VSS d-1), even comparable to heterotrophic denitrification values. In this study, we have implemented a novel operational strategy that sets our work apart from previous studies with similar bioreactors. Specifically, we have introduced Cerium as a trace element in the feeding medium, which serves as a key differentiating factor. It allowed maintaining a stable reactor operation at high NLRs. Microbial community composition evidenced that both MBRs were dominated with N-damo bacteria (67-87% relative abundance in period III and I, respectively). However, a decrease in functional N-damo bacteria (Candidatus Methylomirabilis) abundance was observed during the increase in biomass activity and concentration, concomitantly with an increase of the other minor families (Hypomicrobiaceae and Xanthobacteraceae). Most of the selected antibiotics showed high biotransformation such as sulfamethoxazole, trimethoprim, cefalexin and azithromycin, whereas others such as roxithromycin and clarithromycin were only partially degraded (20-35%). On the contrary, ciprofloxacin showed almost no removal. Despite the metabolic enhancement, no apparent increase on the antibiotic removal was observed throughout the operation, suggesting that microbiological composition was of greater influence than its primary metabolic activity on the removal of antibiotics.

Non-target screening reveals the mechanisms responsible for the antagonistic inhibiting effect of the biocides DBNPA and glutaraldehyde on benzoic acid biodegradation.

The desalination and reuse of discharged cooling tower water (CTW) as feed water for the cooling tower could lower the industrial fresh water withdrawal. A potential pre-treatment method before CTW desalination is the use of constructed wetlands (CWs). Biodegradation is an important removal mechanism in CWs. In the present study, the impact of the biocides 2,2-dibromo-2-cyanoacetamide (DBNPA) and glutaraldehyde on the biodegradation process by CW microorganisms was quantified in batch experiments in which benzoic acid was incubated with realistic CTW biocide concentrations. DBNPA had a stronger negative impact on the biodegradation than glutaraldehyde. The combination of DBNPA and glutaraldehyde had a lower impact on the biodegradation than DBNPA alone. UHPLC-qTOF-MS/MS non-target screening combined with data-analysis script 'patRoon' revealed two mechanisms behind this low impact. Firstly, the presence of glutaraldehyde resulted in increased DBNPA transformation to the less toxic transformation product 2-bromo-2-cyanoacetamide (MBNPA) and newly discovered 2,2-dibromopropanediamide. Secondly, the interaction between glutaraldehyde and DBNPA resulted in the formation of new products that were less toxic than DBNPA. The environmental fate and toxicity of these products are still unknown. Nevertheless, their formation can have important implications for the simultaneous use of the biocides DBNPA and glutaraldehyde for a wide array of applications.