Shihu Hu

Selective suppression of nitrite-oxidizing bacteria (NOB) is essential for stable partial nitritation (PN), yet NOB often persist or recover under engineered suppression conditions. This study investigated the kinetic, microbial, and genomic responses of NOB and ammonia-oxidizing bacteria (AOB) in a PN system subjected to low pH and high free nitrous acid (FNA) sidestream treatment. A sequencing batch reactor was operated for 283 days under varying treatment intensities. Long-term operation selectively enriched a Candidatus nitrotogafabula-related MAG (bin.69), characterized by a relatively high apparent growth rate (∼1.3 d–1) and low substrate affinity (Ks = 2.3 ± 0.4 mg NO2–-N L–1; Ko = 1.1 ± 0.3 mg O2 L–1). Comparative genomic analyses identified stress-tolerance-associated traits related to proton transport, ion homeostasis, protein stabilization, and cellular stress responses. Proteome isoelectric (pI) point analysis further showed a lower genomic pI profile than other known NOB, potentially consistent with persistence under acidic and FNA stress. These findings provide genome-informed insights into NOB persistence under long-term suppression and implications for improving PN stability.

Anaerobic methanotrophic ‘Candidatus Methanoperedenaceae’ play a key role mitigating methane emissions from freshwater sediments. Members of the family are metabolically versatile, with species coupling anaerobic oxidation of methane (AOM) to the reduction of nitrate, metal oxides and/or humic compounds. Metagenomic analyses have indicated further uncharacterised metabolic diversity, including the potential for AOM coupled to arsenate reduction (As-AOM). While As-AOM has been confirmed as an important methane sink and contributor to arsenic mobilisation in freshwater reservoirs, the underlying microbiological mechanisms remain to be empirically demonstrated. In this study stoichiometric coupling of dissimilatory arsenate reduction to methane oxidation was demonstrated using enrichment cultures of the uncultured type species ‘Ca. Methanoperedens nitroreducens’. Meta-omics revealed that arsenate oxidoreductases were exclusively encoded by ‘Ca. M. nitroreducens’ and upregulated under As-AOM conditions, confirming this species was directly responsible. These findings expand our understanding of ‘Ca. Methanoperedenaceae’ physiology and demonstrate microbial mechanisms underpinning arsenic mobilisation in freshwater systems, with important implications for climate and public health.

Acid-tolerant ammonia-oxidizing bacteria (AOB)-based technologies show promise for sustainable wastewater treatment. Membrane bioreactors (MBRs) are suitable for retaining acid-tolerant AOB, but membrane fouling under acidic conditions remains poorly understood. To address this knowledge gap, an acidic MBR and a neutral MBR were operated in parallel for one year. The acidic MBR sustained ∼2 mg N/L in situ free nitrous acid (FNA) and an average nitrite accumulation ratio of 92.7%. Results showed comparable membrane fouling propensity between acidic and neutral MBRs under pristine membrane, NaClO chemical cleaning, and physical cleaning. Mechanistic experiments further indicated dual effects of in situ FNA on membrane fouling. Specifically, FNA facilitated soluble protein and humic substance production in acidic sludge, increasing pore blockage potential. However, due to the sludge concentration reduction, the volumetric concentrations of soluble polysaccharides and bound extracellular polymeric substances in acidic MBR were lower than in the neutral MBR. Moreover, in situ FNA altered sludge surface functional groups, reduced membrane adhesion propensity, and enriched genera with hydrolytic capabilities and putative quorum-quenching activity, resulting in reduced fouling layer accumulation compared to the neutral system. Overall, these findings extend understanding of FNA-mediated membrane fouling and support the application of acidic wastewater treatment.

Twenty years ago, an archaeal lineage capable of catalyzing nitrate-dependent anaerobic methane oxidation (n-damo) was identified. This archaeal lineage was originally grouped within the ANME-2d cluster, and later renamed as the Methanoperedenaceae family. Since then, researchers worldwide have advanced our understanding of the biology of this archaeal group by describing its phylogenetic diversity, versatile metabolic functions, unique kinetic and physiological properties, special growing patterns, extracellular electron transfer capability, and extrachromosomal genetic elements. In addition, the potential engineering and industrial applications of Methanoperedenaceae have been explored broadly, employing granular and biofilm platforms for wastewater treatment and other purposes. In this Review, we reflect on the history of Methanoperedenaceae research, covering both the biology and the potential applications, summarize main achievements and challenges, and propose avenues for future research and applications.

Aerobic digestion is commonly used for waste activated sludge stabilization in small-to-medium wastewater treatment plants (WWTPs), but the long retention time (15-30 days) required for stability severely limits process intensification. Acidic conditions maintained by nitrification can enhance digestion efficiency, but at short hydraulic retention time (HRT), nitrifiers are washed out. Here, we utilized a biofilm-based solution to retain nitrifiers and produce in situ free nitrous acid (HNO₂) to expedite WAS stabilization and reduce pH. Two laboratory-scale sludge digesters were operated for over one year to evaluate this approach. This process reduced pathogen levels to well below the Class A biosolids standard within an extraordinarily short HRT of 1.75 days. Furthermore, it achieved a high volatile solids (VS) reduction rate of 0.87 ± 0.13 kg/m³/d. Model-based analysis indicated the biofilm-based digester achieved both a higher hydrolysis rate (k) (0.58 ± 0.07 d^-1 vs 0.51 ± 0.13 d^-1) and a greater extent of VS degradability (f_d) (31 ± 2% vs 21 ± 2%) compared to the suspended sludge digester. Microbial community analysis revealed that biofilms enriched ammonia oxidizers and acid-tolerant heterotrophs, underpinning enhanced HNO₂ production and sludge degradation. Overall, this process provides a promising sludge management strategy for small-scale WWTPs.