Biology subjects

Bott, C.

Publications and source records attributed to Bott, C..

10 recordsLinked to original sources

bioRxiv2026

Genome-resolved and kinetic evidence for low-DO comammox-anammox synergy and acetate-stimulated nitrate reduction in IFAS biofilms

Mainstream anammox implementation for nitrogen removal is constrained by unstable nitrite supply and organic carbon requirements for nitrate byproduct removal. This study evaluated integrated fixed-film activated sludge (IFAS) biofilms to enhance anammox activity by coupling low-oxygen ammonium oxidation with volatile fatty acid (VFA)-driven nitrate reduction. Nanopore long-read metagenomic assembly recovered a high-quality, circular single-contig Candidatus Brocadia sapporoensis metagenome-assembled genome (MAG) from full-scale IFAS biofilms. This MAG encodes complete anammox metabolism, dissimilatory nitrate reduction to ammonium (DNRA) and acetate/propionate carbon transformation pathways. Metatranscriptomics showed that low dissolved oxygen (DO) upregulated Ca. B. sapporoensis genes involved in anammox, nitrate reduction, and carbon metabolism. Microaerobic assays established a DO level of 0.7 mg/L as optimal for sustaining near-maximal ammonium oxidation alongside anammox-driven total inorganic nitrogen (TIN) loss. Anoxic tests conducted in secondary effluent indicated that external acetate amendment promoted greater partial nitrate reduction and TIN loss than additional propionate amendment. Integrating this dissolved oxygen concentration with external acetate amendment in a two-stage microaerobic-anoxic system successfully achieved sequential ammonium oxidation, partial nitrate reduction, and anammox-mediated TIN removal. Stage-specific expression suggested Ca. B. sapporoensis could contribute to nitrite self-supplementation via nxrAB-mediated nitrate reduction. Overall, microaerobic ammonium oxidation and Ca. B. sapporoensis-driven partial nitrate reduction jointly sustain mainstream anammox activity. Furthermore, this study demonstrates that successful metabolic synergy depends fundamentally upon precise dissolved oxygen control and effective external acetate amendment.

microbiology↗

bioRxiv2026

High-accuracy hierarchical rRNA operon profiling resolves genomovar-level taxonomy and microdiversity using Nanopore sequencing.

Genomovar-level and intragenomic diversity cannot be resolved by conventional amplicon sequencing due to the limitation of fragment lengths and read accuracy, while the application of metagenomic profiling to a large number of samples can be resource intensive. Here, we report UltraRes-rrn, an integrated wet-lab and computational workflow for high-accuracy rrn (i.e., 16S- ITS-23S rRNA) operon profiling using Nanopore sequencing. By integrating ultra-long DNA recovery, long-read amplicon sequencing, and unique molecular identifiers (UMI)-based consensus correction, UltraRes-rrn obtains full-length 16S-ITS-23S rRNA operon consensus sequences with mean accuracies exceeding 99.98%. To achieve higher resolution, we propose a hierarchical rRNA operon profiling strategy in which concatenated 16S+23S rRNA genes (16S23S) serve as a primary and the internal transcribed spacer (ITS) provides a secondary marker. The 16S23S marker achieved discrimination at the genomovar level compared to either 16S or 23S rRNA genes alone, which mitigates the ITS-driven over-splitting observed with the full-length rrn operon and allows for larger proportion of data being classified at higher confidence thresholds. Further, ITS variation was strongly structured by tRNA occurrence patterns, suggesting that ITS can capture taxon microdiversity missed by either 16S or 23S rRNA gene sequences alone. The UltraRes-rrn workflow was applied to full-scale nitrogen removal reactors, revealing intraspecies diversity variation driven by different carbon regimes, which would not have been possible with a shorter gene sequence. In summary, UltraRes-rrn enables cost-effective community profiling at genomovar-level resolution in complex ecosystems.

microbiology↗

bioRxiv2025

Metagenomes and Metagenome-Assembled Genomes from Microbial Communities in a Biological Nutrient Removal Plant Operated at Hamptons Road Sanitation District (HRSD) with High and Low Dissolved Oxygen Conditions

Aeration in biological nutrient removal (BNR) systems constitutes one of the largest energy demands in water resource recovery facilities (WRRFs). Previous studies have shown that lowering dissolved oxygen (DO) concentrations can sustain effective nitrification and phosphorus removal while substantially reducing energy consumption. However, the microbial mechanisms enabling these low-DO processes remain poorly understood. In this study, we investigated microbial communities associated with reduced DO in a pilot-scale BNR system operated by the Hampton Roads Sanitation District (HRSD). DO was reduced over an 18-month period from 2.5 mg/L to 0.2 mg/L. Metagenomic DNA was obtained from samples from each DO condition then sequenced using PacBio HiFi technology. A total of 316 metagenome-assembled genomes were recovered and after dereplication, 207 were found to be unique. These data augment the metagenomic information related to wastewater treatment under low-DO conditions and provide valuable resources for understanding microbial adaptation to oxygen-limited BNR operation.

genomics↗

bioRxiv2025

Quantitative metagenomics using a portable protocol.

A field-deployable DNA sequencing approach for quantitative microbial community profiling can enable rapid responses for a range of applications in the water sector - from process control to wastewater surveillance. Current quantitative approaches have complex instrumentation requirements and long turnaround time for DNA recovery and absolute quantitation. In this study, we report a field-deployable rapid detection and absolute quantitation (rD+rQ) workflow that leverages the real-time sequencing capabilities of Nanopore sequencing for quantitative metagenomics. This workflow integrates a high-molecular-weight DNA recovery protocol for diverse environmental matrices of relevance to the water sector and multiplexed Nanopore sequencing with barcoded spike-in-based calibration (BSINC). BSINC using multi-species genomic spike-in controls exhibits significantly higher calibration accuracy compared to conventional approaches that utilize either single DNA fragment or single organism spike in controls. Dynamic detection and quantitation limits were established based on coverage fraction of sequenced genomes and the coefficient of variation of genome copy numbers across replicates to enhance the accuracy and precision of microbial quantitation. The rD+rQ workflow achieves species-level identification and absolute quantitative results comparable to digital PCR in environmental samples. This portable laboratory and easy-to-use rD+rQ workflow should facilitate rapid decision-making for the water industry.

microbiology↗

bioRxiv2024

Metatranscriptomic analysis reveals synergistic activities of comammox and anammox bacteria in full-scale attached growth nitrogen removal system.

0-Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is poor understanding of how operational parameters, in particular dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial communitys transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supply anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations. SynopsisComammox bacteria differentially regulate ammonia and nitrite oxidation in response to dissolved oxygen concentration suggesting dissolved oxygen dependence of their synergistic nitrogen removal with anammox bacteria in IFAS biofilms.

microbiology↗

bioRxiv2023

Revisiting the role of Acinetobacter sp. in EBPR systems

Side-stream Enhanced biological phosphorus removal (S2EBPR) has been incorporated with B-stage process to enable simultaneous phosphorus and nitrogen removal. However, the dominating phosphorus accumulating organisms (PAOs) in this novel configuration has not been evaluated. The dominance of Acinetobacter was confirmed by 16S sequencing. In addition, single cell Raman spectrum (SCRS) analysis in couple with in situ fluorescence in situ hybridization (FISH) was applied to obtain the feature spectrum and verify the phosphorus release/uptake activity of Acinetobacter spp. The phenotypic profiling further suggested the dominance of Acinetobacter-like organisms among all poly-phosphorus containing organisms and only certain phenotypic Acinetobacter (oligotype 1) contribution to P-removal in a unique HRAS-P(D)N-S2EBPR system. The findings suggest that Acinetobacter may outcompete other heterotrophic organisms in EBPR systems due to their sensitivity to operational conditions. However, stable P-removal was only observed during a specific section of the operation period, coinciding with an increase in the VFA/P ratio. Further research is needed to identify the phenotypes of Acinetobacter responsible for P-removal in EBPR systems. The study contributes to a better understanding of the microbial ecology and engineering aspects of EBPR systems and wastewater treatment in general.

bioengineering↗

bioRxiv2023

Comammox and Unknown Candidate AOBs Contribute to Nitrite Accumulation in An Integrated A-B stage process that Incorporates Side-stream EBPR (S2EBPR)

A novel integrated pilot-scale A-stage high rate activated sludge, B-stage short-cut biological nitrogen removal and side-stream enhanced biological phosphorus removal (A/B-shortcut N- S2EBPR) process for treating municipal wastewater was demonstrated with the aim to achieve simultaneous and carbon- and energy-efficient N and P removal. In this studied period, an average of 7.62 {+/-} 2.17 mg-N/L nitrite accumulation was achieved through atypical partial nitrification without canonical known NOB out-selection. Network analysis confirms the central hub of microbial community as Nitrospira, which was one to two orders of magnitude higher than canonical aerobic oxidizing bacteria (AOB) in a B-stage nitrification tank. The contribution of comammox Nitrospira as AOB was evidenced by the increased amoB/nxr ratio and higher ammonia oxidation activity. Furthermore, oligotyping analysis of Nitrospira revealed two dominant sub-clusters (microdiveristy) within the Nitrospira. The relative abundance of oligotype II, which is phylogenetically close to Nitrospira_midas_s_31566, exhibited a positive correlation with nitrite accumulation in the same operational period, suggesting its role as comammox Nitrospira. Additionally, the phylogenetic investigation suggested that heterotrophic organisms from the family Comamonadacea and the order Rhodocyclaceae embedding ammonia monooxygenase and hydroxylamine oxidase may function as heterotrophic nitrifiers. This is the first study that elucidated the impact of integrating the S2EBPR on nitrifying populations with implications on short-cut N removal. The unique conditions in the side-stream reactor, such as low ORP, favorable VFA concentrations and composition, seemed to exert different selective forces on nitrifying populations from those in conventional biological nutrient removal processes. The results provide new insights for integrating EBPR with short-cut N removal process for mainstream wastewater treatment.

bioengineering↗

bioRxiv2023

Molecular Evidence of Internal Carbon-Driven Partial Denitrification Annamox (PdNA) in a mainstream Pilot A-B System Coupled with Side-stream EBPR treating municipal wastewater

Achieving mainstream short-cut nitrogen removal via nitrite has been a carbon and energy-efficient goal which wastewater engineers are dedicated to explore. This study applied a novel pilot-scale A-B-S2EBPR system process integrated with sidestream enhanced biological phosphorus removal) to achieve the nitrite accumulation and downstream anammox for treating municipal wastewater. Nitrite accumulated to 5.5 {+/-} 0.3 mg N/L in the intermittently aerated tanks of B-stage with the nitrite accumulation ratio (NAR) of 79.1 {+/-} 6.5%. The final effluent concentration and removal efficiency of total inorganic nitrogen (TIN) were 4.6 {+/-} 1.8 mg N/L and 84.9 {+/-} 5.6%, respectively. Batch nitrification/denitrification activity tests and functional gene abundance of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) suggested that the nitrite accumulation was mostly caused by partial denitrification without NOB- selection. The unique features of S2EBPR (longer anaerobic HRT/SRT, lower ORPs, high and more complex VFAs etc.) seemed to impact the nitrogen microbial communities: the conventional AOB kept at a very low level of 0.13 {+/-} 0.13% during the operation period, and the dominant candidate internal carbon-accumulating heterotrophic genera of Acinetobacter (17.8 {+/-} 15.5)% and Comamonadaceae (6.7 {+/-} 3.4%) were highly enriched. Furthermore, the single-cell Raman spectroscopy-based intracellular polymer analysis revealed the dominate microorganisms that could utilize polyhydroxyalkanoates (PHA) as the potential internal carbon source to drive partial denitrification. This study provides insights and a new direction for implementing the mainstream PdNA short-cut nitrogen removal via incorporating S2EBPR into sustainable A-B process.

bioengineering↗

bioRxiv2022

Co-occurrence and cooperation between comammox and anammox bacteria in a full-scale attached growth municipal wastewater treatment process.

Cooperation between comammox and anammox bacteria for nitrogen removal has been recently reported in laboratory-scale systems including synthetic community construct; however, there are no reports of full-scale municipal wastewater treatment systems with such cooperation. Here, we report intrinsic and extant kinetics as well as genome-resolved community characterization of a full-scale integrated fixed film activated sludge (IFAS) system where comammox and anammox bacteria co-occur and appear to drive nitrogen loss. Intrinsic batch kinetic assays indicated that majority of the aerobic ammonia oxidation was driven by comammox bacteria (1.75 {+/-} 0.08 mg-N/g TS-h) in the attached growth phase with minimal contribution by ammonia oxidizing bacteria. Interestingly, a portion of total inorganic nitrogen ([~]8%) was consistently lost during these aerobic assays. Aerobic nitrite oxidation assays eliminated the possibility of denitrification as a cause of nitrogen loss, while anaerobic ammonia oxidation assays resulted in rates consistent with anammox stoichiometry. Full-scale experiments at different dissolved oxygen (DO = 2-6 mg/L) set points indicated persistent nitrogen loss that was partly sensitive to DO concentrations. Genome-resolved metagenomics confirmed high abundance (relative abundance 6.53 {+/-} 0.34%) of two Brocadia-like anammox populations while comammox bacteria within the Ca. Nitrospira nitrosa cluster were lower in abundance (0.37% {+/-} 0.03%) and Nitrosomonas-like ammonia oxidizers even lower (0.12% {+/-} 0.02%). Collectively, our study reports for the first time the co-occurrence and co-operation of comammox and anammox bacteria in a full-scale municipal wastewater treatment system. SynopsisComammox and anammox cooperation resulted in dissolved oxygen concentration dependent nitrogen loss in municipal wastewater treatment system.

microbiology↗

bioRxiv2020

Modeling Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) System Using Agent-based Model with Adaptive Maintenance, Decay and TCA Metabolism

Side-stream enhanced biological phosphorus removal process (S2EBPR) has been demonstrated to improve performance stability and offer a suite of advantages compared to conventional EBPR design. Design and optimization of S2EBPR require modification of the current EBPR models that were not able to fully reflect the metabolic functions of and competition between the polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) under extended anaerobic conditions as in the S2EBPR conditions. In this study, we proposed and validated an improved model (iEBPR) for simulating PAO and GAO competition that incorporated heterogeneity and versatility in PAO sequential polymer usage, staged maintenance-decay, and glycolysis-TCA pathway shifts. The iEBPR model was first calibrated against bulk batch testing experiment data, being proved to perform better than the previous EBPR model for predicting the soluble orthoP, ammonia, biomass glycogen, and PHA temporal profiles in a batch starvation testing under prolonged anaerobic conditions. We further validated the model with another independent set of batch anaerobic batch testing data that included high-resolution single-cell and specific population level intracellular polymer measurements enabled by the single-cell Raman micro-spectroscopy technique. The model accurately predicted the temporal changes in the intracellular polymers at cellular and population levels within PAOs and GAOs, and further confirmed the proposed mechanism of sequential polymer utilization, and polymer availability-dependent and staged maintenance-decay in PAOs. These results indicate that under extended anaerobic phases as in S2EBPR, the PAOs may gain competitive advantages over GAOs due to the possession of multiple intracellular polymers and the adaptive switching of the anaerobic metabolic pathways that consequently lead to the later and slower decay in PAOs than GAOs. The iEBPR model can be applied to facilitate and optimize the design and operations of S2EBPR for more reliable nutrient removal and recovery from wastewater.

microbiology↗