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

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 | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results 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 Blaise M. Enuh , Kevin S. Myers , Charles Bott , Stephanie Klaus , Kester McCullough , Lilian McIntosh , Natalie Beach , Michelle Young , Timothy J. Donohue , Daniel R. Noguera doi: https://doi.org/10.1101/2025.11.10.687637 Blaise M. Enuh a Great Lakes Bioenergy Research Center, University of Wisconsin-Madison , Madison, Wisconsin, USA b Wisconsin Energy Institute, University of Wisconsin-Madison , Madison, Wisconsin, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kevin S. Myers a Great Lakes Bioenergy Research Center, University of Wisconsin-Madison , Madison, Wisconsin, USA b Wisconsin Energy Institute, University of Wisconsin-Madison , Madison, Wisconsin, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charles Bott c Hamptons Road Sanitation District , Virginia Beach, Virginia, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephanie Klaus c Hamptons Road Sanitation District , Virginia Beach, Virginia, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kester McCullough c Hamptons Road Sanitation District , Virginia Beach, Virginia, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lilian McIntosh c Hamptons Road Sanitation District , Virginia Beach, Virginia, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Natalie Beach d Carollo Engineers , Westminster, Colorado, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michelle Young d Carollo Engineers , Westminster, Colorado, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Timothy J. Donohue a Great Lakes Bioenergy Research Center, University of Wisconsin-Madison , Madison, Wisconsin, USA b Wisconsin Energy Institute, University of Wisconsin-Madison , Madison, Wisconsin, USA e Department of Bacteriology, University of Wisconsin-Madison , Madison, Wisconsin, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel R. Noguera a Great Lakes Bioenergy Research Center, University of Wisconsin-Madison , Madison, Wisconsin, USA b Wisconsin Energy Institute, University of Wisconsin-Madison , Madison, Wisconsin, USA f Department of Civil and Environmental Engineering, University of Wisconsin-Madison , Madison, Wisconsin, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: noguera{at}engr.wisc.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract 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. Background Aeration within biological nutrient removal (BNR) systems can account for a large fraction of the electricity costs at water resource recovery facilities (WRRF). Several studies have demonstrated that by reducing dissolved oxygen (DO) levels in BNR processes, it is possible to maintain efficient nitrification and phosphorus removal with lower energy consumption ( Fitzgerald et al., 2015 ; Keene et al., 2017 ; Stewart et al., 2021 ). Lowering DO in aerated sections of BNR plants leads to an adaptation of the microbial community to the new environmental conditions ( Fitzgerald et al., 2015 ; Park and Noguera, 2008 , 2004 ). The underlying dynamics of these microbial community changes when DO is reduced remain poorly understood. Here, we report on the metagenomes and metagenome-assembled genomes (MAGs) from a pilot plant operated by the Hamptons Road Sanitation District (HRSD) when the DO was reduced in the aerated portions of the treatment train. Activated sludge samples were collected from the pilot plant at the beginning of the operation when the target DO was high (2.5 mg/L), and at the end of the operation, 18 months after the target DO was lowered (0.2 mg/L). In total, 4 samples were analyzed, 2 obtained during high-DO operation and the other 2 from low-DO operation. Metagenomic DNA extraction, library preparation, sequencing, and analysis DNA was extracted using the DNeasy PowerSoil Kit using the published protocol (Qiagen, Germantown, MD), quantified using a Qubit fluorometer (Fisher Scientific, Waltham, MA), and stored at -20°C until sequencing. The quality of the extracted DNA was measured on a NanoDrop One (Fisher Scientific). Quantification of the extracted DNA was done using the Qubit dsDNA High Sensitivity kit following standard protocols (Fisher Scientific). The samples were sent to the University of Wisconsin-Madison Biotechnology Center (Madison, WI) for library preparation and sequencing. A HiFi library was prepared according to PN 102-166-600 Version 04 workflow (Pacific Biosciences, Menlo Park, CA) with standard parameters. Briefly, the procedure involved fragmentation with g-TUBEs (Covaris, Woburn, MA) according to the shearing protocol for large genomes (2,164 x g) and size selection using diluted AMPurePB beads (Pacific Biosciences) as detailed in the procedure-checklist. Library integrity was evaluated on the FemtoPulse System (Agilent, Santa Clara, CA). The library was quantified using the Qubit dsDNA High Sensitivity kit and sequenced on a Sequel II using Sequel Polymerase Binding Kit 2.2 following the standard protocol (Pacific Biosciences). Default parameters for all programs were used unless otherwise stated. Separate sequencing runs were done at each sampling period for the two DO end points. The Circular Consensus Sequence (CCS) reads were assembled utilizing either metaFLYE (v2.9-b1768) ( Kolmogorov et al., 2020 ) polished with racon (v1.4.20) ( Vaser et al., 2017 ) for the high-DO samples, or metaMDBG (v0.3) ( Benoit et al., 2024 ) with a racon module (v1.4.20) ( Vaser et al., 2017 ) for the low-DO samples. Subsequently, the reads were mapped onto the assemblies using minimap2 (v2.22-r1101) ( Li, 2018 ). Binning of the assemblies was conducted with metaBAT2 (v2:2.15) ( Kang et al., 2019 ). Identification of contaminated contigs within each bin was done with ProDeGe (v2.3) ( Tennessen et al., 2016 ) and with custom scripts used for tetranucleotide frequency analysis (run.GC.sh, Calculating_TF_Correlations.R; https://github.com/GLBRC/metagenome_analysis ). Contigs identified as contaminated through both methods were removed from the final MAG assemblies. All MAGs were evaluated for quality using CheckM (v1.2.2) ( Parks et al., 2015 ) and the taxonomy of each MAG was determined using GTDB-Tk (v2.1.0) database release 09-RS214 ( Chaumeil et al., 2020 ). Functional annotations for each MAG were assigned using Bakta (v1.9.1) ( Schwengers et al., 2021 ). RAxML-NG adaptive (v1.2.1) ( Kozlov et al., 2019 ) with default settings was used to infer the best phylogenetic tree by maximum likelihood estimation based on the multiple sequence alignment of 120 bacterial marker genes generated by GTDB-Tk. The resulting tree was visualized in TreeViewer (v2.2.0) ( Bianchini and Sánchez-Baracaldo, 2024 ) and annotated in Inkscape (v1.2.2) (The Inkscape team, 2025). The finalized tree is shown in Figure 1 . Download figure Open in new tab Figure 1. Phylogenetic map showing an overview of community structure based on the 207 unique MAGs from the HRSD plant assembled from both low and high DO samples. From the innermost to the outermost ring: The innermost ring represents phylogenetic clustering of MAGs. The names of the phylum are colored according to the clusters they represent. The next ring in orange points to MAGs that were assembled from the high DO samples while the blue ring points to MAGs that were assembled from the low DO samples. In total, we obtained 316 MAGs from the 4 samples (Supplementary File S1), with 278 MAGs from the low DO samples and 38 from the high DO samples. These MAGs were dereplicated with dRep (v0.6.1) ( Olm et al., 2017 ), resulting in a set of 207 unique MAGs with over 75% completeness and less than 10% contamination ( Figure 1 ). These data augment the metagenomic information related to wastewater treatment under low-DO conditions. The MAGs obtained and their characteristics are provided in the Supplementary File S1. Data availability Fastq files for the 4 metagenomes have been deposited in the NCBI database under Bio project number PRJNA1156690 . The MAGs and their annotation dataset can be accessed on Figshare ( 10.6084/m9.figshare.30389092 ). The custom scripts used in these analyses are available at the GLBRC GitHub repository ( https://github.com/GLBRC/metagenome_analysis ). Acknowledgments This work was supported by funding from the U.S. Department of Energy (DOE), Office of Energy Efficiency & Renewable Energy, under award DE-EE0009509, and partially based upon work at the Great Lakes Bioenergy Research Center supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research under Award Number DE-SC0018409. DNA sequencing was done at the UW Biotechnology Center’s DNA Sequencing Facility (Research Resource Identifier – RRID:SCR_017759). Funder Information Declared United States Department of Energy, https://ror.org/01bj3aw27 , DE-EE0009509 , DE-SC0018409 Footnotes https://figshare.com/articles/dataset/Metagenome-Assembled_Genomes_from_Microbial_Communities_in_the_Hamptons_Road_Sanitation_District_HRSD_Biological_Nutrient_Removal_Pilot_Plants_Operated_with_High_and_Low_Dissolved_Oxygen_Conditions_/30389092 References ↵ Benoit , G. , Raguideau , S. , James , R. , Phillippy , A.M. , Chikhi , R. , Quince , C. , 2024 . High-quality metagenome assembly from long accurate reads with metaMDBG . Nat. Biotechnol . 1 – 6 . doi: 10.1038/s41587-023-01983-6 OpenUrl CrossRef ↵ Bianchini , G. , Sánchez-Baracaldo , P. , 2024 . TreeViewer: Flexible, modular software to visualise and manipulate phylogenetic trees . Ecol. Evol . 14 , e10873 . doi: 10.1002/ece3.10873 OpenUrl CrossRef PubMed ↵ Chaumeil , P.-A. , Mussig , A.J. , Hugenholtz , P. , Parks , D.H. , 2020 . GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database . Bioinformatics 36 , 1925 – 1927 . doi: 10.1093/bioinformatics/btz848 OpenUrl CrossRef PubMed ↵ Fitzgerald , C.M. , Camejo , P. , Oshlag , J.Z. , Noguera , D.R. , 2015 . Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen . Water Res . 70 , 38 – 51 . doi: 10.1016/j.watres.2014.11.041 OpenUrl CrossRef ↵ Kang , D.D. , Li , F. , Kirton , E. , Thomas , A. , Egan , R. , An , H. , Wang , Z. , 2019 . MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies . PeerJ 7 , e7359 . doi: 10.7717/peerj.7359 OpenUrl CrossRef PubMed ↵ Keene , N.A. , Reusser , S.R. , Scarborough , M.J. , Grooms , A.L. , Seib , M. , Santo Domingo , J. , Noguera , D.R. , 2017 . Pilot plant demonstration of stable and efficient high rate biological nutrient removal with low dissolved oxygen conditions . Water Res . 121 , 72 – 85 . doi: 10.1016/j.watres.2017.05.029 OpenUrl CrossRef ↵ Kolmogorov , M. , Bickhart , D.M. , Behsaz , B. , Gurevich , A. , Rayko , M. , Shin , S.B. , Kuhn , K. , Yuan , J. , Polevikov , E. , Smith , T.P.L. , Pevzner , P.A. , 2020 . metaFlye: scalable long-read metagenome assembly using repeat graphs . Nat. Methods 17 , 1103 – 1110 . doi: 10.1038/s41592-020-00971-x OpenUrl CrossRef PubMed ↵ Kozlov , A.M. , Darriba , D. , Flouri , T. , Morel , B. , Stamatakis , A. , 2019 . RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference . Bioinformatics 35 , 4453 – 4455 . doi: 10.1093/bioinformatics/btz305 OpenUrl CrossRef PubMed ↵ Li , H. , 2018 . Minimap2: pairwise alignment for nucleotide sequences . Bioinformatics 34 , 3094 – 3100 . doi: 10.1093/bioinformatics/bty191 OpenUrl CrossRef PubMed ↵ Olm , M.R. , Brown , C.T. , Brooks , B. , Banfield , J.F. , 2017 . dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication . ISME J . 11 , 2864 – 2868 . doi: 10.1038/ismej.2017.126 OpenUrl CrossRef PubMed ↵ Park , H.-D. , Noguera , D. , 2008 . Nitrospira community composition in nitrifying reactors operated with two different dissolved oxygen levels . J. Microbiol. Biotechnol . 18 , 1470 – 4 . OpenUrl PubMed Web of Science ↵ Park , H.-D. , Noguera , D.R. , 2004 . Evaluating the effect of dissolved oxygen on ammonia-oxidizing bacterial communities in activated sludge . Water Res . 38 , 3275 – 3286 . doi: 10.1016/j.watres.2004.04.047 OpenUrl CrossRef PubMed ↵ Parks , D.H. , Imelfort , M. , Skennerton , C.T. , Hugenholtz , P. , Tyson , G.W. , 2015 . CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes . Genome Res . 25 , 1043 – 1055 . doi: 10.1101/gr.186072.114 OpenUrl Abstract / FREE Full Text ↵ Schwengers , O. , Jelonek , L. , Dieckmann , M.A. , Beyvers , S. , Blom , J. , Goesmann , A. , 2021 . Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification . Microb. Genomics 7 , 000685 . doi: 10.1099/mgen.0.000685 OpenUrl CrossRef PubMed ↵ Stewart , R.D. , Bashar , R. , Amstadt , C. , Uribe-Santos , G.A. , McMahon , K.D. , Seib , M. , Noguera , D.R. , 2021 . Pilot-scale comparison of biological nutrient removal (BNR) using intermittent and continuous ammonia-based low dissolved oxygen aeration control systems . Water Sci. Technol . 85 , 578 – 590 . doi: 10.2166/wst.2021.630 OpenUrl CrossRef Stewart , R.D. , Myers , K.S. , Amstadt , C. , Seib , M. , McMahon , K.D. , Noguera , D.R. , 2024 . Refinement of the “Candidatus Accumulibacter” genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration . mSystems 9 , e01188 – 23 . doi: 10.1128/msystems.01188-23 OpenUrl CrossRef PubMed ↵ Tennessen , K. , Andersen , E. , Clingenpeel , S. , Rinke , C. , Lundberg , D.S. , Han , J. , Dangl , J.L. , Ivanova , N. , Woyke , T. , Kyrpides , N. , Pati , A. , 2016 . ProDeGe: a computational protocol for fully automated decontamination of genomes . ISME J . 10 , 269 – 272 . doi: 10.1038/ismej.2015.100 The Inkscape team, 2025. Inkscape project. OpenUrl CrossRef PubMed ↵ Vaser , R. , Sović , I. , Nagarajan , N. , Šikić , M. , 2017 . Fast and accurate de novo genome assembly from long uncorrected reads . Genome Res . 27 , 737 – 746 . doi: 10.1101/gr.214270.116 OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted November 12, 2025. 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