1. Introduction
In the past few decades’ nitrogen has become a significant contributor to biochemical pollution and the contamination of water sources [1,2]. Biological nitrogen removal (BNR) processes are extensively used to remove nitrogen pollutants in wastewater [3]. BNR widely applied in wastewater treatment plants (WWTPs) includes conventional nitrification and denitrification [3,4]. Recent innovations in the past 30 years have resulted in novel BNR processes being identified, i.e., complete ammonium oxidation (comammox), nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO), heterotrophic nitrification–aerobic denitrification (HN-AD), anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA), which have emerged as cost- and energy-effective BNR methods [5,6,7,8,9]. Microbial ecology studies characterizing BNR microbial communities, their structure, and the diversity of genes involved in nitrogen bioremediation are essential for determining factors that influence the efficiency and stable long-term operation of BNR systems. The BNR process is improved by developing optimized biotechnological and novel strategies in nitrogen bioremediation [10]. Recently, researchers are making great efforts to apply novel strategies to achieve high nitrogen removal efficiency, for example, coupling anammox with partial-denitrification (PD-A) [11], enhancing the anammox process by switching between warm side-stream and cold mainstream wastewater flows [12], coupling partial nitrification with anammox (PN-A) [13], real-time oxidation reduction potential (ORP) decrease rate control in a PN-A system [14], and achieving simultaneously enhanced nitrogen removal efficiency and electricity generation by coupling bio-electrochemical system (BES) with PD-A [15]. Traditional microbial techniques applied in culturing of BNR microbes in the laboratory have contributed immensely to what is known about the biodiversity of BNR microbial communities in natural and engineered systems. However, these techniques are limited as the majority of microbes are unculturable [16]. The introduction of culture-independent approaches in the late 20th century has helped reveal many important microbial lineages associated with BNR. Culture-independent techniques that are used in microbial ecology studies of BNR microbial communities include 16S rRNA gene amplicon sequencing [17], fluorescent in situ hybridization (FISH) [18], quantitative polymerase chain reaction (qPCR) [19], shotgun metagenomics [20], metagenome-assembled genomes [21] and multi-omics approaches [22].
Advances in relatively cheap and ultra-high throughput next-generation sequencing (NGS) technologies have afforded unprecedented insights into microbial-mediated bioremediation in environmental microbiology studies [23]. Vast quantities of DNA and RNA sequences generated by NGS platforms are deposited into public databases [24]. However, public databases, for example, GenBank [25], UniProtKB [26], and KEGG [27], store a huge amount of biological data. These databases cover other biological aspects apart from BNR processes and hence require foreknowledge of metabolic pathways for accurate usage. The extraction of BNR-related gene datasets from complex public databases is time-consuming. In addition, decades of published scientific literature has produced a vast wealth of data documenting nitrogen bioremediation. BNR-associated datasets are distributed across the literature and public databases, for example, PubMed [25], ScienceDirect [28], and Google Scholar [29], randomly and in an unsystematic manner, making it a challenging task for researchers to extract and comparatively analyze BNR datasets. Recently, the MiDAS field guide database was developed to provide curated taxonomical and physiological information of important microbial communities found in sludge-activated chambers and anaerobic digester systems [30]. However, it only provides taxonomical information based on 16S rRNA gene analyses, and datasets are collected from only 2 types of resources related to wastewater treatment systems. Therefore, it is desirable to develop a knowledge repertoire of manually curated BNR datasets from diverse environmental sources, which contains related information (e.g., functional genes, enzyme functions, and BNR-related datasets) derived from the literature to advance nitrogen bioremediation research.
Herein, we report the development of a new Biological Nitrogen Removal Database (BNRdb), a manually curated platform which integrates various literature-derived BNR data. To our knowledge, this is the first BNR database that systematically integrates BNR data from archaeal, bacterial, and fungal microbial groups to support BNR functional studies. BNRdb strives to provide a freely accessible and user-friendly platform for not only professional BNR researchers but also the broader scientific community to further investigate the function and activity of BNR microbial communities.
2. Methodology
2.1. Overview of Database Construction and Content
The construction of the BNRdb database was based on a review of BNR-related scientific literatures and information extracted from public databases (Figure 1). The Biological Nitrogen Removal Database (BNRdb, freely available at
2.2. Data Collection
The criteria established in the collection of the dataset making up the BNRdb were based on the biochemical pathways involved in nitrogen removal: nitrification, comammox, denitrification, anammox, HN-AD, DNRA, and n-DAMO. The following was considered: (i) microbes verified through experiments to possess any one or more of the functional genes encoding enzymes associated with these biochemical pathways; (ii) microbial strains identified from natural or engineered systems that were experimentally validated to have the ability to perform in any one or more of the biochemical pathways in nitrogen removal; and (iii) set experimental parameters and results obtained from experimentally validated studies into BNR in natural and engineered systems. Data collected in the BNRdb database was obtained with these criteria and extensive literature searches in publicly accessible databases such as PubMed [25], Google Scholar [29], and Science Direct [28]. Searches for BNR-related literature were performed using keywords generated based on ‘BNR: nitrification, comammox, denitrification, anammox, HN-AD, DNRA, and n-DAMO’, looking for functional genes, enzymes, and biological treatment systems associated with these biochemical pathways. In an effort to generate more focused search results, the following Boolean search string was used: (‘nitrifiers’ OR ‘complete nitrifiers’ OR ‘denitrifiers’ OR ‘anammox bacteria’ OR ‘HN-AD bacteria’ OR ‘DNRA’ OR ‘n-DAMO’) for the fields [Title, Abstract, Keywords]. The search result returned over 10,000 indexed citations. De-duplication was performed, eliminating duplicate records of literature. Manual curation of the abstracts was performed to eliminate the non-related hits. The experimentally supported BNR events were then manually curated from this scientific literature by at least two researchers. BNR datasets in our database were established by functional validation via a systematic literature review of 1541 peer-reviewed articles. In the BNRdb database, the NCBI GenBank ID accession numbers of the organisms were used as the unique identifiers. However, there is a possibility that some BNR-related datasets (sequences and operational details) were unintentionally omitted. An interactive feature is incorporated in BNRdb, via which contributors can submit those missing BNR datasets and subsequently upload them in future database updates.
The functional annotations of entries in BNRdb were performed with NCBI GenBank [25], UniProt [26], NCBI Protein [25], KEGG [27], and BRENDA [31].
2.3. Data Management and Layout
For clarity, functional data associated with the seven biochemical processes involved in BNR was manually curated into six core categories: Genes, Strains, Reactions, Biological treatment systems, Metagenomics, and Bioreactor systems. Internal links were used to connect related datasets on one section of the website to a different section of the same website. For example, the genes section contains curated lists of microbial isolates from which the genes are extracted. For every distinct microbial isolate, their functional data were collected; each microorganism page comprises the reaction stage, encoding gene, protein name, GenBank, and UniProt accession numbers from NCBI [25], UniProtKB [26], external links to the cDNA/protein sequences, environmental source, and scientific references. Information, such as references to literature sources and other external references such as enzyme commission (EC) numbers, is provided as an external link facilitating access to the relevant online resources, extending and supplementing information in the database.
The strain section contains lists of microbial strains capable of nitrogen removal as indicated by the literature. The information about each entry includes the name of the strain, source of isolation, type of respiration, NCBI GenBank ID, substrates utilized, electron donors, electrons acceptors, length of the cDNA sequence, and external link to the scientific references. The annotation fields used for microbial strain entries in BNRdb are described (Supplementary Tables S1 and S2).
The reaction section displays the literature-derived information of the reactions associated with the biochemical processes of nitrogen removal: nitrification, denitrification, anammox, comammox, DNRA, and n-DAMO. Extracted information relevant to enzymes associated with the BNR biochemical processes from the BRENDA database. Each enzyme entry contains information such as enzyme name, gene cluster, EC number, KEGG orthology IDs, classification, substrates, products, genes, brief detail on the subunits that make up the functional enzymes, and scientific references. Peer-reviewed literature extracted from PubMed that studied the biochemistry, function, and mechanism of the enzymes is provided as external links. This section also contains pathway maps, which users can access the outlines of how the nitrification, denitrification, anammox, comammox, DNRA, and n-DAMO reactions proceed. The reaction pathways display the series of reactions involved, the enzymes at every stage in the reaction pathway, their EC numbers, the genes encoding the enzymes, reactants, and products of the BNR processes.
The BNR treatment systems section displays relevant BNR experimental conditions and datasets in the removal of nitrogen from natural wetlands and engineered systems. Each entry contains various information extracted from literature: the influent source, type of system, bioreactor, bio-carrier medium, respiration, electron donor, electron acceptor, loading and removal rates of ammonium and nitrate, nitrogen removal efficiencies, and scientific references. Entries in this section were each assigned unique IDs for linking the data to external databases. The metagenomics section displays BNR-related multi-omics sequencing datasets of entire microbial communities isolated from diverse environments. Each entry contains information about the data type, library source, external links to the Sequence Read Archive database, and references to the published literature. The bioreactor section displays information about the types of bioreactors used in BNR in wastewater treatment systems. Information on the basic operation of commonly used biofilm carriers, variations of bioreactors developed based on the main types, the benefits, and limitations of each bioreactor system were collected. A general description of modification applied to the reactors for BNR in large scale community applications is also included.
The inter-connecting nature of BNRdb affords users the ease of navigating through the website, facilitating access to all the data within the database irrespective of the starting point.
2.4. Database Architecture and Web Interface Implementation
The backend framework was implemented in Django 2.0 using Python 3.6. The front-end framework was implemented using HTML5, CSS3, and JavaScript with Bootstrap 4. Data were stored and managed in MariaDB 5.5.56. Protein and nucleotide alignment were implemented with HMMER 3.2 [32] and BLAST [33], respectively. Multiple sequence alignment and phylogenetic tree generation were implemented with Clustal Omega [34] and PHYLIP [35] packages.
3. Results
3.1. Data Statistics
The current version of BNRdb contains information on 23,308 microbial strains, 46 gene families, 24 enzymes, 18 reactions, 301 BNR treatment systems datasets, 860 BNR-associated next-generation sequencing datasets, and 6 commonly bioreactors in BNR from wastewater plants and other environmental systems. There are 21,511 microbial strains for bacteria, 1117 for archaea, and 680 for fungi (Figure 2a). Of these, 75.67% (16,277 strains), 65.80% (735 strains), and 38.24% (260 strains) of the bacterial, archaeal, and fungal strains, respectively, were identified based on functional gene analysis (Figure 2b).
A total of 46 gene families were found through analyzing 6 BNR processes in the nitrogen cycling, namely, nitrification, comammox, denitrification, anammox, DNRA, and n-DAMO.
Nitrification: Sequences were collected from eight functional genes encoding the nitrification enzymes: amoA, amoB, amoC, hao, nxrA, nxrB, nxrC, and nxrD. The amoCAB operon is responsible for NH4+ oxidation to NH2OH, the hao gene is responsible for conversion of NH2OH to NO, and nxrABCD is responsible for NO2− oxidation into NO3−. The prokaryotic domains, Archaea and Bacteria, were regarded as two separate groups for the retrieval of sequences of amoA, amoB, and amoC gene families. A total of 694 and 1232 archaeal and bacterial sequences were collected, respectively. The amoA gene was the most prevalent in both archaeal and bacterial groups, accounting for 87.61% and 60.31% of the total entries, respectively.
Comammox: Comammox Nitrospira sequences were collected based on the amoA functional gene, which encodes the AmoA subunit of the ammonia monooxygenase enzyme. There are two distinct clades (clade A and B) of comammox Nitrospira formed by different amoA orthologs genes from comammox Nitrospira bacteria. A total of 632 bacterial sequences were collected.
Denitrification: Bacterial sequences were retrieved from 17 gene families: narG, narH, narJ, narI, narZ, narY, narV, narW, napA, napB, napC, napD, nirK, nirS, norB, norC, and nosZ. The genes in clusters napEDABC, narGHJI, and narZYVW were handled as individual entities. During the denitrification, narGHJI, narZYVW, and napEDABC gene clusters mediate the reduction of NO3− to NO2−, nirK and nirS genes are associated with the reduction of NO2− to NO, norCB genes are responsible for the reduction of NO to N2O, and the nosZ genes are associated with the reduction of N2O to N2. The BNRdb contains 10,247 denitrifier sequences, with the majority being the nirK and nirS genes sequences, encoding the copper and cytochrome cd1 type forms of the metalloenzyme nitrite reductase. The genes encoding the nitrite reductase account for 86.69% of the database’s bacterial denitrification genes.
Fungal denitrification: Fungal sequences of two gene families, nirK and p450nor, were retrieved. The nirK and p450nor genes are genes associated with reducing NO2− to NO and NO to N2O, respectively. A total of 260 fungal sequences were collected.
Anammox: Anammox bacterial sequences were collected from eight gene families: hzsA, hzsB, hzsC, hzo, hdh, ccsA, ccsB, and ccsX. The hzsCBA gene cluster is responsible for coupling NH4+ and NO producing hydrazine (N2H4), and the hzo and hdh genes are associated with N2H4 oxidation to N2. The ccsA, ccsB, and ccsX gene families included in this database are of interest to the scientific community as they are key to the unique anammox metabolism. The BNRdb database contains a total of 2553 anammox bacteria sequences. The hzsCBA gene cluster accounts for 48.69% of the BNRdb’s anammox gene sequences.
DNRA: Sequences were collected from seven functional genes encoding the enzymes associated with the DNRA process: nrfA, nrfB, nrfC, nrfD, nrfH, nirB, and nirD. The nrfABCDEFG and nirBD gene clusters are associated with the reduction of NO2− to NH4+ without any nitrogenous intermediates being produced. A total of 267 bacterial sequences were collected.
n-DAMO: Sequences were collected from three functional genes encoding the n-DAMO enzymes: mcrA, nod, and pmoA. The mcrABG gene cluster is associated with the anaerobic metabolism of methane, the pmoCAB gene cluster is associated with methane oxidation to methanol, and the nod gene is responsible for the direct disproportion of NO into N2 and O2. A total of 41 and 1346 archaeal and bacterial sequences were collected, respectively.
BNRdb has 6036 microbial strains collected from the literature based on 16S and 18S rRNA gene analysis (see
3.2. Search Tools
The BNRdb database (
A set of bioinformatic utilities for the characterization and taxonomic placement of novel BNR associated microbial strains were also included. Users can use the BLAST and HMMER alignment modules implemented in BNRdb to query the database. Users can submit either their nucleotide or protein bacterial query sequence in FASTA format in the BLAST and HMMER search tools, respectively, to detect nucleotide or protein sequence homologs from the manually curated, high-quality sequences in the BNRdb. The Clustal O alignment module implemented in BNRdb allows users to perform multiple sequence alignments, which reveal subtle similarities between sequences. Phylotree.js was implemented to explore an interactive analysis of the phylogeny of BNR microbial strains. This platform facilitates the interactive probing and manipulation of the phylogenetic tree, such as selecting nodes and swapping branches.
3.3. Usage and Functionalities
The database provides example sequences to guide users on using the analytical tools and performing bioinformatic analysis.
For example, selecting the “Nuc_GenBankID” filter in the search bar and inputting NCBI GenBank accession number “DQ518208.1” as the keyword (Figure 3a), the query result is displayed, with the features of reaction stage, encoding genes, enzyme names, microorganisms, accession IDs, references, and a link for detailed information (Figure 3b). By clicking the “Link” tab, the detailed information for the denitrifying isolate is shown (Figure 3c). The information presented on this page comprises the microorganism’s taxonomy, isolation source, gene information, protein information, and information about the article. The gene nucleotide sequences, protein sequences, and references to the research articles are also provided as external links.
The online BLAST and HMMER interfaces can be used to input the bacterial query sequence(s) and search against all the manually curated high-quality nucleotide or protein sequences in our database (Supplementary Figure S1a,b). Users have the option to download their sequence homologs results report for further analysis. The BLAST and HMMER reports consist of three main components: the header comprises text indicating the nature of the query sequence and the target sequence database. There is a section for one-line descriptions of scores for sequences matching the query sequence for a quick overview of browsing. The third component is for domain annotations for each of the database sequence alignment matches compared to the query sequence. For the first hit in the list, the accession number is DQ420239.1; the definition is Pseudomonas grimontii isolate PD 9 nitric oxide reductase; the score is 484; and the E-value is 2 × 10−137. All of the hits in this BLAST report have very low E-values (much less than 1), meaning that these alignments are more precise and truly meaningful (Supplementary Figure S2).
Users can construct the phylogenetic tree diagrams showing evolutionary relationships between their query sequences and those sequences found in the BNRdb database (Supplementary Figure S1c,d). The example sequences were used to infer the phylogenetic tree of denitrifying isolates that possess the nitric oxide reductase encoding gene (Supplementary Figure S3). The Clustal Omega package with 1000 cycles of iterative refinement facilitated the alignment of the protein sequences. Clustal O uses seeded guide trees and hidden Markov models (HMMs) profile-profile techniques to generate multiple alignments [34]. Simple phylogeny provides rapid phylogenetic tree reconstruction using the Approximate Maximum Likelihood method of PHYLIP from the choice of either nucleotide or protein alignment [38]. The tree is displayed in Phylotree.js, a JavaScript library for working with phylogenetic trees [35].
The BNRdb database presents experimental datasets of previous and current studies into BNR treatment systems from lab scale to full-scale applications. Several parameters presented in this section, such as feed nitrate, nitrogen loading rate, carbon, and energy source, nitrogen removal rate, and nitrogen removal efficiencies, are instrumental in helping scientists determine the appropriate wastewater treatment technology and bioreactor technology and configuration (Supplementary Figure S4).
In the BNRdb database, the six commonly used bioreactors configurations for maintaining BNR microbes are included. Users can access information on the basic operation, biofilm carriers used, variations of the bioreactors, and the benefits and limitations associated with each of these bioreactor configurations, which is helpful for formulating an effective nitrogen removal strategy for wastewater treatment and improving the cultivation of slow-growing BNR microbes (Supplementary Figure S5).
3.4. Technical Validation and Updating of the Database
Submission of data—An interactive data submission feature is incorporated into the BNRdb to generate clean and uniform data sets that can be useful to other users (Supplementary Figure S6). Contributors who wish to submit new data sets related to BNR, which are not already included in the database, can use this feature following the submission guidelines. The administrator/curator may then contact the authors who submitted a new dataset to be the editor of that particular dataset and engage in reviewing any inaccurate or potentially missing data during the validation stage. The curator of the database will then upload the new dataset after its validation.
Download page—A download page is provided, enabling users to download the gene sequences, strains information, BNR treatment systems, and bioreactor data tables. By properly citing the original work, users are allowed to redistribute and reproduce in any form.
Database update—BNRdb is in its release 1.0 and will continually be updated to accommodate new literature entries that are continuously updated on the NCBI and other public repositories. Manual curation of data will be carried out to detect and remove multiple data entries when conflicting datasets are detected.
Users’ feedback—Any queries or data issues concerning the BNRdb can be reported via the contact feature incorporated in the BNRdb. The administrator will respond to their queries.
4. Discussion
BNR microbial communities and BNR treatment experimental datasets are important for the optimization of conventional WWTPs and the development of novel BNR treatment technologies in order to meet the stringent discharging standards. To achieve this purpose, information sharing is essential for the development of BNR research. We developed BNRdb, a database for exploring the various BNR-related data of BNR microbial communities manually curated from published literature.
The BNRdb database incorporates BNR-associated genes/strains, BNR treatment experimental datasets and metagenomic datasets from BNR engineered systems and diverse environments. Natural environments such as intertidal zones, where the oxic/anoxic conditions vary through time, are valuable microbial sources for discovering novel BNR metabolic pathways and microbial functions [39,40]. Users can thus access the BNR information from natural environments held within the BNRdb to evaluate the real applicability of novel BNR microbial strains in the optimization of BNR treatment systems. Understanding the syntrophic interactions that occur within microbial communities and dynamics in BNR treatment systems is crucial for the efficient and stable removal of nitrogen from WWTPs and extending knowledge of microbe-driven biogeochemical cycles [22,41]. Therefore, literature-derived (meta-) genomic and multi-omics datasets within the BNRdb are helpful to promote research to gain new insights into the metabolic functions of BNR microbial communities. The elucidation of novel metabolic pathways will flourish the development of simpler, cheaper, and greener biological wastewater treatment technologies [42]. Achieving efficient BNR treatment system performance is determined by process control and optimizing environmental conditions to ensure optimal functional diversity within the BNR treatment system [43]. BNRdb provides summary information about BNR experimental datasets and system operational conditions. Users can thus perform comparative analysis studies with the experimental datasets held within BNRdb in order to determine the optimal conditions for enhancing the survival and performance of BNR microbial communities in the removal of nitrogen from WWTPs.
5. Conclusions and Future Directions
This study presents the BNRdb, a publicly available resource entirely dedicated to BNR. The BNRdb integrates data extracted from publicly accessible data repositories and scientific literature to provide insightful information about BNR, which would foster studies of nitrogen removal microbial communities and the development of technologies for the efficient removal of nitrogen pollutants. It is expected that the BNRdb database will be proven to be a valuable resource for professional BNR researchers and the broader scientific community working to advance our understanding of BNR systems.
With advances in NGS technologies developing exponentially, the number of multi-omics approaches towards the functional analysis of BNR communities is expected to keep increasing. Hence, efforts will be made to periodically update the BNRdb database with the intention of including the most recent BNR related research. A future step in the development of BNRdb will be dedicated to improving the information available in BNRdb, such as collecting detailed experimental details, cataloging BNR-related function genes, and incorporating web-accessible bioinformatics tool for analyzing and visualizing BNR-related high throughput NGS data. We plan to integrate 16S and functional gene information with experimental data. We believe that this correlation function can offer valuable insights into how the inherent genes or strains contribute to the BNR efficiency. We will look to construct an additive database on syntrophic microbes which contribute to the BNR processes. This will help in extending our knowledge of syntrophic associations of the microbial community in WWTPs, electron transfer modes of microbes, and understanding the ecosystem function within BNR treatment systems.
T.R.N.: Conceptualization, methodology, software, data curation, writing—original draft, Writing—review and editing, visualization; P.Z.: methodology, validation, software, investigation, data curation; H.Z.: conceptualization, supervision, writing—review and editing, funding acquisition. All authors have read and agreed to the published version of the manuscript.
Financial support was provided by the National Key Research and Development Program of China [2019YFA0905500] and the National Natural Science Foundation of China [Grant No. 31670793].
The datasets associated with this article are available in the Github repository (
The authors declare no conflict of interest.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Enlarge this image.Figure 1. Construction, content, and interface of BNRdb. Data were collected from published literature and databases and curated into six categories. BNRdb provides a user-friendly interface to query, browse, analyze, and download detailed information about these data sets.
Enlarge this image.Figure 2. Statistics of nitrogen-removing microbes in BNRdb. (a) The distribution of microbial strains (see in Table S3) in BNRdb. (b) Distribution of microbial strains identified based on functional gene analysis. (c) Distribution of microbial strains identified based on either 16S rRNA gene analysis or functional screening studies.
Enlarge this image.Figure 3. Overview of the BNRdb user interface to access data. (a) Users can input the GenBank ID accession number for querying and filtering. For example, accession “DQ518208.1”. (b) The returned search result can be further explored by clicking the link to access the detailed information. (c) The comprehensive information of the denitrifying microbial strain.
Supplementary Materials
The following supporting information can be downloaded at:
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; Zeng, Peiji; Zhang, Houjin