Comammox Nitrospira and Ammonia-Oxidizing Archaea Are Dominant Ammonia Oxidizers in Sediments of an Acid Mine Lake Containing High Ammonium Concentrations.

Exploring nitrifiers in extreme environments is vital to expanding our understanding of nitrogen cycle and microbial diversity. This study presents that complete ammonia oxidation (comammox) Nitrospira, together with acidophilic ammonia-oxidizing archaea (AOA), dominate in the nitrifying guild in sediments of an acid mine lake (AML). The lake water was characterized by acidic pH below 5 with a high ammonium concentration of 175 mg-N/liter, which is rare on the earth. Nitrification was active in sediments with a maximum nitrate production potential of 70.5 µg-N/(g-dry weight [dw] day) for mixed sediments. Quantitative PCR assays determined that in AML sediments, comammox Nitrospira and AOA amoA genes had relative abundances of 52% and 41%, respectively, among the total amoA genes. Further assays with 16S rRNA and amoA gene amplicon sequencing and metagenomics confirmed their dominance and revealed that the comammox Nitrospira found in sediments belonged to comammox Nitrospira clade A.2. Metagenomic binning retrieved a metagenome-assembled genome (MAG) of the comammox Nitrospira from sediments (completeness = 96.76%), and phylogenomic analysis suggested that it was a novel comammox Nitrospira. Comparative genomic investigation revealed that this comammox Nitrospira contained diverse metal resistance genes and an acidophile-affiliated F-type ATPase. Moreover, it had a more diverse genomic characteristic on nitrogen metabolism than the AOA in sediments and canonical AOB did. The results suggest that comammox Nitrospira is a versatile nitrifier that can adapt to acidic environments even with high ammonium concentrations. IMPORTANCE Ammonia-oxidizing archaea (AOA) was previously considered the sole dominant ammonia oxidizer in acidic environments. This study, however, found that complete ammonia oxidation (comammox) Nitrospira was also a dominant ammonia oxidizer in the sediments of an acidic mine lake, which had an acidic pH < 5 and a high ammonium concentration of 175 mg-N/liter. In combination with average nucleotide identity analysis, phylogenomic analysis suggested it is a novel strain of comammox Nitrospira. Moreover, the adaption of comammox Nitrospira to the acidic lake had been comprehensively investigated based on genome-centric metagenomic approaches. The outcomes of this study significantly expand our understanding of the diversity and adaptability of ammonia oxidizers in the acidic environments.

Long-Term Low Dissolved Oxygen Operation Decreases N2O Emissions in the Activated Sludge Process.

Nitrous oxide (N2O) is an important greenhouse gas and a dominant ozone-depleting substance. Nitrification in the activated sludge process (ASP) is an important N2O emission source. This study demonstrated that a short-term low dissolved oxygen (DO) increased the N2O emissions by six times, while long-term low DO operation decreased the N2O emissions by 54% (P < 0.01). Under long-term low DO, the ammonia oxidizer abundance in the ASP increased significantly, and thus, complete nitrification was recovered and no NH3 or nitrite accumulated. Moreover, long-term low DO decreased the abundance of ammonia-oxidizing bacteria (AOB) by 28%, while increased the abundance of ammonia-oxidizing archaea (AOA) by 507%, mainly due to their higher oxygen affinity. As a result, AOA outnumbered AOB with the AOA/AOB amoA gene ratio increasing to 19.5 under long-term low DO. The efficient nitrification and decreased AOB abundance might not increase N2O production via AOB under long-term low DO conditions. The enriched AOA could decrease the N2O emissions because they were reported to lack canonical nitric oxide (NO) reductase genes that convert NO to N2O. Probably because of AOA enrichment, the positive and significant (P = 0.02) correlation of N2O emission and nitrite concentration became insignificant (P = 0.332) after 80 days of low DO operation. Therefore, ASPs can be operated with low DO and extended sludge age to synchronously reduce N2O production and carbon dioxide emissions owing to lower aeration energy without compromising the nitrification efficiency.

Effects of inorganic particles and their interactions with biofilms on dynamic membrane structure and long-term filtration performance.

In present study, the effects of inorganic particles and their interaction with biofilms on the filtration behavior of dynamic membrane bioreactor (DMBR) were investigated. When no inorganic particles were included in the simulated domestic wastewater, a porous biofilm DM was formed on support materials. As a result, the transmembrane pressure (TMP) did not increase (< 10 Pa) during the 97 days' experiment and the effluent turbidity was consistently lower than 1.0 NTU. When sands (1.3-69.2 µm; 50 mg/L) were the only inorganic particles contained in wastewater, the effluent turbidity became instable and ranged from 0.31 to 3.88 NTU, probably because the DM structures were disturbed by sand scouring. The natural clays (0.5-2.7 µm) in wastewater were very liable to deposit on the support materials of DMBRs to form thick and compact DMs with greater contents of biomass and EPS, especially co-existing with sands. Due to the existence of natural clays, the DM porosity decreased significantly and rapid rising in TMP occurred frequently. This study demonstrated that pure biofilms without containing inorganic particles were ideal materials for DMs, which could achieve long-term stable operation with low effluent turbidity (< 1 NTU) and low TMP (< 10 Pa), while inorganic particles with any size could deteriorate the filtration performance. Therefore, removing the inorganic particles in wastewater as many as possible prior DMBR is critically important for achieving long-term stable operation.

Characterizing the biofilm stoichiometry and kinetics on the media in situ based on pulse-flow respirometer coupling with a new breathing reactor.

Biofilm based systems and the hybrid between activated sludge and biofilms have been popularly applied for wastewater treatment. Unlike the suspended biomass, the biofilm concentration and kinetics on the media cannot be easily measured. In this study, a novel and easy-to-use approach has been developed based on pulse-flow respirometer to characterize the biofilm stoichiometry and kinetics in situ. With the new designed breathing reactor, the mutual interference between the magnetic stirring and biofilm media that happened in the conventional breathing reactor was solved. Moreover, Microsoft Excel based programs had been developed to fit the oxygen uptake rate curves with dynamic nonlinear regression. With this new approach, the yield coefficient, maximum oxidation capacity, and half-saturation constant of substrate for the heterotrophic biofilms in a fix bed reactor were determined to be 0.46 g-VSS/g-COD, 67.0 mg-COD/(h·L-media), and 4.4 mg-COD/L, respectively. Those parameters for biofilm ammonia oxidizers from a moving bed biofilm reactor were determined to be 0.17 g-VSS/g-N, 18.6 mg-N/(h·L-media), and 1.2 mg-N/L, respectively, and they were 0.11 g-VSS/g-N, 20.9 mg-N/(h·L-media), and 0.98 mg-N/L for nitrite oxidizers in the same biofilms. This study also found that the maximum specific substrate utilization rate for detached biofilms increased by 3.2 times, indicating that maintaining biofilm integrity was very important in the kinetic tests. Using this approach, the biofilm kinetics on the media can be regularly measured for treatment optimization.