BASALT refines binning from metagenomic data and increases resolution of genome-resolved metagenomic analysis.

Metagenomic binning is an essential technique for genome-resolved characterization of uncultured microorganisms in various ecosystems but hampered by the low efficiency of binning tools in adequately recovering metagenome-assembled genomes (MAGs). Here, we introduce BASALT (Binning Across a Series of Assemblies Toolkit) for binning and refinement of short- and long-read sequencing data. BASALT employs multiple binners with multiple thresholds to produce initial bins, then utilizes neural networks to identify core sequences to remove redundant bins and refine non-redundant bins. Using the same assemblies generated from Critical Assessment of Metagenome Interpretation (CAMI) datasets, BASALT produces up to twice as many MAGs as VAMB, DASTool, or metaWRAP. Processing assemblies from a lake sediment dataset, BASALT produces ~30% more MAGs than metaWRAP, including 21 unique class-level prokaryotic lineages. Functional annotations reveal that BASALT can retrieve 47.6% more non-redundant opening-reading frames than metaWRAP. These results highlight the robust handling of metagenomic sequencing data of BASALT.

A mixed blessing of influent leachate microbes in downstream biotreatment systems of a full-scale landfill leachate treatment plant.

In landfill leachate treatment plants (LLTPs), the microbiome plays a pivotal role in the decomposition of organic compounds, reduction in nutrient levels, and elimination of toxins. However, the effects of microbes in landfill leachate influents on downstream treatment systems remain poorly understood. To address this knowledge gap, we collected 23 metagenomic and 12 metatranscriptomic samples from landfill leachate and activated sludge from various treatment units in a full-scale LLTP. We successfully recovered 1,152 non-redundant metagenome-assembled genomes (MAGs), encompassing a wide taxonomic range, including 48 phyla, 95 classes, 166 orders, 247 families, 238 genera, and 1,152 species. More diverse microbes were observed in the influent leachate than in the downstream biotreatment systems, among which, an unprecedented ∼30 % of microbes with transcriptional expression migrated from the influent to the biological treatment units. Network analysis revealed that 399 shared MAGs across the four units exhibited high node centrality and degree, thus supporting enhanced interactions and increased stability of microbial communities. Functional reconstruction and genome characterization of MAGs indicated that these shared MAGs possessed greater capabilities for carbon, nitrogen, sulfur, and arsenic metabolism compared to non-shared MAGs. We further identified a novel species of Zixibacteria in the leachate influent with discrete lineages from those in other environments that accounted for up to 17 % of the abundance of the shared microbial community and exhibited notable metabolic versatility. Meanwhile, we presented groundbreaking evidence of the involvement of Zixibacteria-encoded genes in the production of harmful gas emissions, such as N2O and H2S, at the transcriptional level, thus suggesting that influent microbes may pose safety risks to downstream treatment systems. In summary, this study revealed the complex impact of the influent microbiome on LLTP and emphasizes the need to consider these microbial characteristics when designing treatment technologies and strategies for landfill leachate management.

An integrated meta-omics approach reveals the different response mechanisms of two anammox bacteria towards fluoroquinolone antibiotics.

The emerging fluoroquinolone antibiotics (FQs) are highly influential in nitrogen removal from livestock wastewater. However, beyond the capability of nitrogen removal, little is known about the molecular mechanisms (e.g., shift of core metabolism and energy allocation) of different anaerobic ammonium-oxidizing bacteria (AnAOB) under continuous FQ stress. This study investigated the effects of ciprofloxacin, ofloxacin and their mixture at concentrations detected in livestock wastewater on two key anammox species in membrane bioreactors. It was found 20 µg/L FQs promoted nitrogen removal efficiency and community stability, and42-51 % of FQs were removed simultaneously. Integrated meta-omics analysis revealed varied gene expression patterns between the two dominant AnAOB, Candidatus Brocadia sapporoensis (B AnAOB) and Candidatus Kuenenia stuttgartiensis (K AnAOB). The nitrogen metabolic processes were bolstered in B AnAOB, while those involved in anammox pathway of K AnAOB were inhibited. This difference was tentatively attributed to the up-regulation of reactive oxygen species scavenger genes (ccp and dxf) and FQ resistance gene (qnrB72) in B AnAOB. Importantly, most enhanced core biosynthesis/metabolism of AnAOB and close cross-feeding with accompanying bacteria were also likely to contribute to their higher levels of biomass yield and metabolism activity under FQ stress. This finding suggests that B AnAOB has the advantage of higher nitrogen metabolism capacity over K AnAOB in livestock wastewater containing FQs, which is helpful for efficient and stable nitrogen removal by the functional anammox species.

Molecular insights into enhanced nitrogen removal induced by trace fluoroquinolone antibiotics in an anammox system.

It has been widely reported that fluoroquinolones (FQs) can affect the anaerobic ammonium oxidization (anammox) microorganisms, which interferes with the performance of nitrogen removal from wastewater. However, the metabolic mechanism of anammox microorganisms responding to FQs has rarely been explored. In this study, it was found that 20 µg/L FQs promoted the nitrogen removal performance of anammox microorganisms in batch exposure assays, and 36-51% of FQs were removed simultaneously. Combined metabolomics and genome-resolved metagenomic analysis revealed up-regulated carbon fixation in anammox bacteria (AnAOB), while purine and pyrimidine metabolism, protein generation and transmembrane transport were enhanced in AnAOB and symbiotic bacteria by 20 µg/L FQs. Consequently, hydrazine dehydrogenation, nitrite reduction, and ammonium assimilation were bolstered, improving the nitrogen removal efficiency of the anammox system. These results revealed the potential roles of specific microorganisms in response to emerging FQs and provided further information for practical application of anammox technology in wastewater treatment.

Sustainable treatment of nitrate-containing wastewater by an autotrophic hydrogen-oxidizing bacterium.

Bacteria are key denitrifiers in the reduction of nitrate (NO3 --N), which is a contaminant in wastewater treatment plants (WWTPs). They can also produce carbon dioxide (CO2) and nitrous oxide (N2O). In this study, the autotrophic hydrogen-oxidizing bacterium Rhodoblastus sp. TH20 was isolated for sustainable treatment of NO3 --N in wastewater. Efficient removal of NO3 --N and recovery of biomass nitrogen were achieved. Up to 99% of NO3 --N was removed without accumulation of nitrite and N2O, consuming CO2 of 3.25 mol for each mole of NO3 --N removed. The overall removal rate of NO3 --N reached 1.1 mg L-1 h-1 with a biomass content of approximately 0.71 g L-1 within 72 h. TH20 participated in NO3 --N assimilation and aerobic denitrification. Results from 15N-labeled-nitrate test indicated that removed NO3 --N was assimilated into organic nitrogen, showing an assimilation efficiency of 58%. Seventeen amino acids were detected, accounting for 43% of the biomass. Nitrogen loss through aerobic denitrification was only approximately 42% of total nitrogen. This study suggests that TH20 can be applied in WWTP facilities for water purification and production of valuable biomass to mitigate CO2 and N2O emissions.

Genome-centric metagenomics provides new insights into the microbial community and metabolic potential of landfill leachate microbiota.

Landfills are important sources of microorganisms associated with anaerobic digestion. However, the knowledge on microbiota along with their functional potential in this special habitat are still lacking. In this study, we recovered 1168 non-redundant metagenome-assembled genomes (MAGs) from nine landfill leachate samples collected from eight cities across China, spanning 42 phyla, 73 classes, 114 orders, 189 families, and 267 genera. Totally, 74.1% of 1168 MAGs could not be classified to any known species and 5.9% of these MAGs belonged to microbial dark matter phyla. Two putative novel classes were discovered from landfill leachate samples. The identification of thousands of novel carbohydrate-active enzymes showed similar richness level compared to the cow rumen microbiota. The methylotrophic methanogenic pathway was speculated to contribute significantly to methane production in the landfill leachate because of its co-occurrence with the acetoclastic and hydrogenotrophic methanogenic pathways. The genetic potential of dissimilatory nitrate reduction to ammonium (DNRA) was observed, implying DNRA may play a role in ammonium generation in landfill leachate. These findings implied that landfill leachate might be a valuable microbial resource repository and filled the previous understanding gaps for both methanogenesis and nitrogen cycling in landfill leachate microbiota. Our study provides a comprehensive genomic catalog and substantially provides unprecedented taxonomic and functional profiles of the landfill leachate microbiota.

Specific Denitrifying and Dissimilatory Nitrate Reduction to Ammonium Bacteria Assisted the Recovery of Anammox Community From Nitrite Inhibition.

The anaerobic ammonium oxidation (anammox) by autotrophic anaerobic ammonia-oxidizing bacteria (AnAOB) is a biological process used to remove reactive nitrogen from wastewater. It has been repeatedly reported that elevated nitrite concentrations can severely inhibit the growth of AnAOB, which renders the anammox process challenging for industrial-scale applications. Both denitrifying (DN) and dissimilatory nitrate reduction to ammonium (DNRA) bacteria can potentially consume excess nitrite in an anammox system to prevent its inhibitory effect on AnAOB. However, metabolic interactions among DN, DNRA, and AnAOB bacteria under elevated nitrite conditions remain to be elucidated at metabolic resolutions. In this study, a laboratory-scale anammox bioreactor was used to conduct an investigation of the microbial shift and functional interactions of AnAOB, DN, and DNRA bacteria during a long-term nitrite inhibition to eventual self-recovery episode. The relative abundance of AnAOB first decreased due to high nitrite concentration, which lowered the system's nitrogen removal efficiency, but then recovered automatically without any external interference. Based on the relative abundance variations of genomes in the inhibition, adaptation, and recovery periods, we found that DN and DNRA bacteria could be divided into three niche groups: type I (types Ia and Ib) that includes mainly DN bacteria and type II and type III that include primarily DNRA bacteria. Type Ia and type II bacteria outcompeted other bacteria in the inhibition and adaptation periods, respectively. They were recognized as potential nitrite scavengers at high nitrite concentrations, contributing to stabilizing the nitrite concentration and the eventual recovery of the anammox system. These findings shed light on the potential engineering solutions to maintain a robust and efficient industrial-scale anammox process.

Highly efficient ammonium removal through nitrogen assimilation by a hydrogen-oxidizing bacterium, Ideonella sp. TH17.

Ideonella sp. TH17, an autotrophic hydrogen-oxidizing bacterium (HOB), was successfully enriched and isolated from activated sludge in a domestic wastewater treatment plant (WWTP). Batch experiments were conducted to identify the cell growth and ammonium (NH4+-N) removal, and to verify the pathways of nitrogen utilization under different conditions. At a representative NH4+-N concentration of 100 mg/L in domestic wastewater, it was the first time that a HOB strain achieved a nearly 100% ammonium removal. More than 90% of NH4+-N was assimilated to biomass nitrogen by strain TH17. Only a little of N2 (<10% of initial NH4+-N) was detected without N2O emission in aerobic denitrification process. Autotrophic NH4+-N assimilation contributed predominantly to biomass nitrogen production, supplemented by assimilatory nitrate (NO3--N) reduction under aerobic conditions. A total of 17 amino acids, accounting for 54.25 ± 1.98% of the dry biomass, were detected in the bacterial biomass harvested at 72 h. These results demonstrated that the newly isolated strain TH17 was capable of removing NH4+-N and recovering nutrients from wastewater efficiently. A new solution was thus provided by this HOB strain for ammonium treatment in sustainable WWTPs of future.