Zhiyao Wang

Anaerobic ammonium oxidation (anammox) bacteria are key players in the global nitrogen cycle and are widely applied in energy-efficient nitrogen removal processes. However, their activity is often inhibited in saline and sulfate-rich environments. Here, we report the discovery and characterization of Candidatus Loostrechtia thiotolerans (HSAMX1), a novel nonmarine anammox species that became dominant under combined high salinity (3% by weight) and high sulfate concentrations (∼86 mM). Through integrated metagenomic and metatranscriptomic analyses, we reveal the physiological and molecular strategies enabling HSAMX1 to thrive under dual-stress conditions. In response to osmotic stress, HSAMX1 activated ion export systems and subsequently synthesized organic osmoprotectant solutes to maintain cellular homeostasis. It also encoded and strongly expressed the sulfide:quinone oxidoreductase (SQR) gene, which accounted for over 90% of the total community SQR transcription. Intriguingly, HSAMX1 did not emerge under either salinity or sulfate stress alone, suggesting a previously unrecognized niche shaped by the interactions of these two stressors. These findings expand our understanding of nonmarine anammox diversity and identify a promising candidate for nitrogen removal in sulfate-laden, saline wastewater.

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.

The granule-based anammox process is a promising biotechnology for nitrogen removal; however, it is challenging to achieve a high rate and efficiency simultaneously. In this study, we addressed this challenge by coupling two granule types with complementary kinetics in an anammox sludge bed reactor. Bright red granules (GR) exhibited higher maximum specific anammox activity (SAA) but lower substrate affinity; they predominantly occupied the reactor's lower section, where substrate concentrations are high, forming a high-capacity layer. Yellow-brown granules (GY) showed higher substrate affinity but lower maximum SAA; they mainly populated the upper low-substrate zone, forming an efficient substrate-scavenging layer. This self-organized granule stratification enabled both a high nitrogen removal rate (14.5 kg-N/(m³·d)) and high efficiency (90.2%). Physicochemical property and microbial community analyses of granules showed that the stratification of "affinity" was mainly governed by the granular structure and mass transfer rather than intrinsic microbial traits. By actively withdrawing excessive sludge from the fast-growing lower zone while preserving biomass in the upper zone, such stratification can be stably maintained. Collectively, these findings advance our understanding of granule heterogeneity in anammox sludge bed bioreactors and offer a practical strategy for performance optimization.

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.

Ammonia oxidation, a crucial part in nitrogen cycle, is thought to be jointly driven by ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and complete ammonia oxidation (comammox) in the ocean. However, the spatial distribution of these three ammonia-oxidizing microorganisms in the marine sediments, especially at the transcriptional level, remains underexplored. This study utilizes quantitative PCR and activity experiments to quantify the amoA gene of three ammonia oxidizers at both DNA and RNA levels, measure their potential nitrification rate, and assess their relative contribution to ammonia oxidation in the marine sediments in Bohai region in China. Further, we analyzed their correlations with key environmental factors. In the marine sediments of Bohai, the transcript abundance of AOA, AOB, and comammox amoA genes ranged from 7.31 × 10² to 9.82 × 10⁴, 5.77 × 10³ to 3.98 × 10⁴ and 1.07 × 10⁴ to 5.44 × 10⁴ copies g⁻¹ dry sediment, respectively. The results revealed that TN and TOC had significant effects on total amoA gene abundance and transcript abundance for all ammonia oxidizers. Besides, the relative contribution of AOB to ammonia oxidation was greater than that of AOA and comammox based on activity measurement, likely due to nitrate nitrogen and total nitrogen. Our study demonstrated that RNA-based amoA abundance and activity measurements can accurately reflect the spatial variations of ammonia oxidizers in Bohai sediments.

The granule-based anammox process has been reported to be more resistant to the stress of antibiotics; however, the underlying resistance mechanism is still not fully understood. In this study, we found that more microbubbles stably adhered to the surface layer of anammox granular sludge (AnGS, Gs) operating under long-term sulfamethoxazole (SMZ) stress of 2 mg/L, compared to that in the control reactor (Gc). The difference in covering content can be up to over three times (1.0 ± 0.1% vs 0.3 ± 0.0%). Batch tests showed that the coverage ratio of microbubbles on Gs reached approximately 32.5%, which significantly reduced SMZ transfer into AnGS due to the adsorption of SMZ by bubbles, thus alleviating the inhibition of anammox bacterial activity by 36.5%. The adhesion force between the microbubbles and the surface layer of Gs was found to be largely enhanced by 75.0% compared to that of Gc. The increased hydrophobicity of surface layer due to the increased extracellular polymer substance (EPS, mainly proteins) content, and the larger capillary force of surface layer, owing to the unique micronano structure, were identified as key factors responsible for the stable adhesion of microbubbles on the Gs. Consequently, this study demonstrated the vital roles of the surface-adhered microbubbles in resisting the stress of SMZ and shed light on the regulation and development of robust granule-based anammox processes.