Start-up of anammox in an up-flow anaerobic sludge bed reactor: bacterial community composition and antibiotic resistance genes

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In this study, synthetic wastewater was used to start an up-flow anaerobic sludge bed reactor with a starting temperature of 20–31℃, and subsequently, the activated sludge samples were used to analyze the changes in bacterial community and antibiotic resistance genes (ARGs) by metagenomic sequencing. The results showed that the reaction successfully started up after 132 days of cultivation, achieving NH 4 + -N and NO 2 − -N removal rates over 99.5%. Candidatus Kuenenia, an anammox bacterium, increased from 0.01 to 50.86%. The denitrifying bacteria Delftia , Acidovorax , Thauera and Alicycliphilus decreased from 18.70, 8.02, 4.94 and 4.59% to 7.01, 1.77, 3.06 and 3.96%, respectively. The ammonia-oxidizing bacterium Nitrosomonas decreased from 2.91 to 1.87%. After cultivation, the relative abundance of ARGs in sludge decreased from 90.23 to 64.29 ppm, with sulfonamide, macrolide-lincosamide-streptogramin, tetracycline, aminoglycoside and multidrug ARGs being the main types. Additionally, the ARG subtypes sul1 , msrE and tetX decreased, while ermF , sul2 and floR increased. These results contribute to knowledge of the nitrogen removal performance, changes in bacterial community composition and ARGs in an anammox reactor, providing the guidance for the removal of ARGs by anammox. Biological sciences/Microbiology/Bacteria Biological sciences/Microbiology/Communities Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction With extensive attention placed on eutrophication, nitrogen removal has become one of the important functions of wastewater treatment plants (WWTPs) [ 1 ] . Currently, most WWTPs use the principle of nitrification and denitrification to facilitate biological nitrogen removal [ 2 ] . One Study has shown that nearly 80% of WWTPs in China operate at a biochemical oxygen demand (five days incubation) to total nitrogen ratios of less than 3.6, which represents low carbon-nitrogen ratio wastewater [ 3 ] . WWTPs require additional carbon source for denitrification [ 4 ] , which increases their operating cost [ 5 ] . Therefore, the development of more energy efficient wastewater treatment technologies has a broad research interest [ 6 ] . Anaerobic ammonia oxidation (anammox), an autotrophic biological nitrogen removal process discovered in the 1990s, can utilize microbial action in anaerobic environments to achieve the simultaneous removal of ammonia nitrogen (NH 4 + -N) and nitrite nitrogen (NO 2 − -N), and is a research hotspot in the current wastewater treatment [ 1 , 7 ] . Different from traditional biological nitrogen removal processes, the anammox process can effectively reduce the aeration cost, energy consumption, sludge production, and demand for organic carbon sources [ 8 ] . Six genera, namely Candidatus Anammoxoglobus, Candidatus Anammoximicrobium, Candidatus Brocadia, Candidatus Jettenia, Candidatus Kuenenia and Candidatus Scalindua, harboring more than 20 species therein, have been reported to pose anammox function [ 9 ] . Due to the environmental sensitivity of anammox bacteria, pure cultures have yet to be cultivated [ 10 ] , and it is generally assumed that anammox is accomplished by a variety of bacteria performing biological nitrogen removal [ 11 ] . Thus, the bacterial community composition determines the efficiency of biological nitrogen removal. Antibiotic resistance genes (ARGs), as emerging pollutants [ 12 ] , pose a serious risk to public safety. They have attracted worldwide attention and are currently a topic of intense research to control environmental pollutants [ 13 ] . A previous study indicated that, with the help of mobile genetic elements such as plasmids, integrons and transposons, ARGs can be transferred horizontally among microorganisms, causing their transmission and diffusion within the environment [ 14 ] . The microbial community diversity and structure in the biological wastewater treatment system is conducive to the selection, transfer and transmission of ARGs across a variety of bacteria [ 11 ] . Recently, a growing number of studies have been conducted to reveal the influence of ARGs in different anammox reactors amended with antibiotics [ 15 , 16 , 17 , 18 ] . Wu et al . [ 15 ] found that an up-flow anaerobic sludge bed (UASB) reactor had good adaptability to low concentrations of spiramycin, and the relative abundance of ARG subtypes ermB , ermF , ermQ , ereA and mphA increased in activated sludge as the spiramycin concentration increased. Kong et al . [ 16 ] studied changes in ARGs in a UASB reactor amended with erythromycin, sulfamethoxazole and tetracycline antibiotics over 360 days of operation, and found that the main ARG subtype was tetW and ermF in the sludge. Zhang et al. [ 17 ] showed that after the addition of oxytetracycline in a UASB reactor, the relative abundance of ARG subtypes tetG and tetX increased. Fan et al . [ 18 ] found that when oxytetracycline was added to a UASB reactor, the relative abundance of ARG subtypes tetA , tetB and tetC in activated sludge increased. However, ARGs have rarely been studied in anammox reactors under non-antibiotic conditions. At present, the most common technique used in ARGs research is q-PCR which requires the use of a known single ARG sequence for detection [ 19 ] . Due to the limitations of ARG primers, q-PCR has the disadvantage of only detecting ARGs with specific primers [ 20 ] . With the development of metagenomic sequencing, complete microbial community and genetic information of the functional genes or enzymes can be obtained. This technique has been applied to study the selection of ARGs in wastewater systems, and can overcome the difficulties of culturing and separating specific microorganisms [ 21 , 22 ] . In this study, activated sludge was obtained from an oxidation ditch of a WWTP located in Urumqi City (Xinjiang, China). This activated sludge was then used as the seeding sludge to initiate the anammox reaction at temperatures ranging from 20 to 31°C (room temperature) with synthetic wastewater. Changes in bacterial community composition and ARGs in the seeding sludge and anammox sludge cultivated for 132 days under non-antibiotic conditions were systematically analyzed using metagenomic sequencing. These results provide a further understanding of the nitrogen removal performance, existence of nitrogen-removing bacteria and ARGs in an anammox reactor. Furthermore, the information collected from this study provides theoretical basis for improving the anammox process by better achieving the removal of this emerging pollutants - ARGs. Materials and Methods Anammox reactor set-up In this study, the effective volume of the plexiglass UASB reactor used was 3.2 L (Fig. 1 ). The anammox reactor surface was covered with an insulating layer to prevent light from adversely affecting the bacteria. The influent water was pumped into the bottom of the reactor by a peristaltic pump. The temperature of the mixed liquid in the reactor was maintained at 20–31°C, and the hydraulic retention time was 17.1–51.2 h. Synthetic wastewater and sample collection In this study, synthetic wastewater was used as the influent, and 50 mg·L − 1 NH 4 Cl and 70 mg·L − 1 NaNO 2 were added to the synthetic wastewater to serve as sources of NH 4 + -N and NO 2 − -N, respectively. No organic matter was added. Other components included NaHCO 3 (500 mg·L − 1 ), MgSO 4 (300 mg·L − 1 ), CaCl 2 (126 mg·L − 1 ) and KH 2 PO 4 (30 mg·L − 1 ). The trace elements stock solution, which was added at 1 mL·L − 1 , comprised FeSO 4 (5000 mg·L − 1 ), MnCl 2 ·H 2 O (990 mg·L − 1 ), ZnSO 4 ·7H 2 O (430 mg·L − 1 ), CuSO 4 ·H 2 O (250 mg·L − 1 ), CoCl 2 ·6H 2 O (240 mg·L − 1 ), NiCl 2 ·6H 2 O (190 mg·L − 1 ) and H 3 BO 4 (14 mg·L − 1 ), as per Chen et al [ 23 ] . Samples of the seeding sludge (from day 1 of reactor operation) and the sludge from day 132, representing stable anammox operation were collected and labeled A1 and A2, respectively. These activated sludge samples were kept at -80°C prior to being sent to Majorbio Bio-Pharmaceutical Technology Co. Ltd. (Shanghai, China) for DNA extraction and metagenomic sequencing. Analytical methods Influent and effluent water quality indicators were determined by the standard methods; NH 4 + -N was determined by Nessler's reagent spectrophotometry; nitrate nitrogen (NO 3 − -N) and NO 2 − -N were determined by ultraviolet spectrophotometry. The water temperature was measured using a mercury thermometer. DNA extraction and metagenomic sequencing Total genomic DNA was extracted from the two sludge samples (A1 and A2) using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, USA) according to the manufacturer’s instructions. The extracted DNA was tested for concentration and purity by TBS-380 and NanoDrop2000 systems, respectively. The quality of the extracted DNA was checked via 1% agarose gel electrophoresis. An ultrasonic breaker Covaris M220 (Gene Company Limited, Hong Kong, China) was used to break the extracted DNA fragment to an average size of about 400 bp. After fragmentation, the metagenomic sequencing library was constructed using NEXTFLEX Rapid DNA-Seq Kit. Finally, 150-bp paired-end metagenomic sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, USA). The raw metagenomic sequencing data obtained in this study have been uploaded to the NCBI Sequence Read Archive (SRA) under accession number SRP274797. Bioinformatic analysis High-quality paired-end reads from the metagenomic data were obtained using fastp ( http://github.com/OpenGene/fastp/ ) by cutting the 3′ and 5′ ends of the original adapter sequence, and by discarding reads less than 50 bp long with quality values below 20 or containing N bases. After quality control, clean reads were assembled using MEGAHIT [ 24 ] , and contigs with a length ≥ 300 bp were retained for assembly. MetaGene [ 25 ] was used to predict open reading frames, select genes with sequences ≥ 100 bp and translate selected genes into amino acid sequences. CD-HIT [ 26 ] was used to construct a non-redundant gene catalog, with sequence identification and coverage rates up to 90%. SOAPaligner [ 27 ] was used to map the quality-controlled reads to the non-redundant gene catalog with 95% identity and assess the gene abundance of each sample. Kraken2 ( https://ccb.jhu.edu/software/kraken2 ) was used for metagenomic classification, after which Bracken ( https://github.com/jenniferlu717/Bracken ) was used to estimate species abundance. The obtained non-redundant gene set was compared with the CARD database ( https://card.mcmaster.ca ) through BLASTP. An ARG was considered when a sequence had an E-value ≤ 10 − 5 , identity ≥ 80%, alignment length ≥ 25 amino acids. Finally, the relative abundance of ARGs was expressed in ppm (representing the number of ARGs per million clean reads). Results and discussion Anammox reactor performance Based on the concentrations and removal rates of NH 4 + -N and NO 2 − -N, the anammox process was divided into four stages (Fig. 2 ). From days 1–11 (stage Ⅰ), the concentration of NH 4 + -N in the effluent was similar to that in the influent. From days 12–57 (stage Ⅱ), the removal rate of NH 4 + -N ranged from 13.9 to 70.3%. Moreover, the removal rate of NO 2 − -N showed a declining trend, decreasing from 47.9 to 8.4%. From days 58–122 (stage Ⅲ), the NH 4 + -N and NO 2 − -N removal rates increased continuously, reaching 99.8 and 98.5%, respectively. After 122 days of cultivation, NO 3 − -N production reached 17.8 mg·L − 1 . Therefore, the anammox activity at this stage increased continuously with the increasing nitrogen removal rate. From days 123–135 (stage Ⅳ), the removal rates of NH 4 + -N and NO 2 − -N were over 99.5%. In the effluent, the average concentrations of NH 4 + -N and NO 2 − -N were 0.1 and 0.2 mg·L − 1 , respectively. Additionally, the average production of NO 3 − -N was 15.5 mg·L − 1 . The corresponding molar ratio of NH 4 + -N consumption to NO 2 − -N consumption to NO 3 − -N production was 1.00:1.41:0.31, which is close to the reported value of 1:1.32:0.26 [ 28 ] . This indicates that anammox had become the dominant reaction and the reactor was in the stable operation stage of anammox. Therefore, this stage represents the stable anammox period and the sludge sample was collected on day 132 which was the anammox sludge. Bacteria community analysis at the phylum level 29 bacterial phyla were identified in the two sludge samples, but the top 10 phyla accounted for most of the communities (Fig. 3 ). In the seeding sludge (A1), Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the predominant bacterial phyla, with the relative abundance levels of 82.01, 8.00, 7.12 and 1.76%, respectively. In the anammox sludge (A2), the phylum Planctomycetes showed the highest relative abundance (50.99%), followed by Proteobacteria (40.25%), Chloroflexi (2.99%) and Actinobacteria (2.90%). For the anammox reaction, the bacterial community is vitally important to facilitate biological nitrogen removal [ 29 ] . Previous studies have shown that Planctomycetes is a phylum of autotrophic nitrogen-removing functional bacteria, with anammox bacteria belonging to this phylum [ 17 , 30 , 31 ] . With the successful start-up of anammox, the relative abundance of Planctomycetes significantly increased from 0.02 to 50.99%. This is consistent with other studies which indicate Planctomycetes has the highest relative abundance in different anammox reactors [ 32 , 33 , 34 ] . When seeding a UASB reactor with anammox sludge, Fu et al . [ 32 ] studied the succession of the bacterial community in sludge, revealing that after 220–280 days of operation, the relative abundance of Planctomycetes was higher than 50%, becoming the absolute dominant phylum. Using conventional sludge and anammox sludge successfully started an anammox reactor, Luo et al . [ 33 ] showed that the relative abundance of Planctomycetes gradually increased from 0.1% on day 1 to 22.96% on day 160. When comparing anaerobic seeding sludge and mature anammox sludge, Sobotka et al. [ 34 ] found that the relative abundance of Planctomycetes accounted for 15 and 43%, respectively. Chloroflexi, a phylum of heterotrophic bacteria, are commonly found in anammox reactors [ 30 , 35 ] . These bacteria can participate in autotrophic denitrification [ 36 ] . They can also consume metabolites produced by anammox bacteria, as well as dead cells in the reactor, thereby providing a stable environment for anammox bacteria [ 37 ] . Its relative abundance increased from 0.21 to 2.99% in this study. Furthermore, one previous study showed that the phylum Chloroflexi harbors a large number of filamentous bacteria that can provide a skeleton and be conducive to the formation and stability of anammox sludge [ 38 ] . Proteobacteria and Bacteroidetes are mostly comprised of heterotrophic bacteria, which need to use organic matter to satisfy their growth demand [ 39 ] . In this study, their relative abundances decreased. The former decreased from 82.01 to 50.99%, while the latter decreased from 8.00 to 0.33%. Common in wastewater treatment systems, some studies have reported that phylum Proteobacteria contains a variety of nitrogen-removing functional bacteria, such as nitrite-oxidizing bacteria (NOB) and denitrifying bacteria [ 8 , 40 ] , which are widely present in anammox systems, providing growth factors for anammox bacteria and playing important roles in nitrogen removal [ 18 , 41 ] . Moreover, Proteobacteria and Chloroflexi can cooperate with anammox bacteria in the nitrogen and amino acid cycles [ 42 ] . Bacteroidetes is also considered a common bacterial phylum in activated sludge and participates in partial denitrification to achieve nitrogen removal [ 43 ] . Similarly, Sobotka et al . [ 34 ] found that the relative abundance of Bacteroidetes significantly decreased from 5% in the anaerobic seeding sludge to 1% in the mature anammox sludge of an anammox system. Bacterial community analysis at the genus level In the two sludge samples, 1172 bacterial genera were identified. The bacterial genera with a relative abundance greater than 1% in the seeding sludge (A1) and anammox sludge (A2) are shown in Fig. 4 . Among these, the anammox bacterium Candidatus Kuenenia was detected, with a relative abundance of only 0.01% in the seeding sludge (A1); however, it was enriched to 50.86% in the anammox sludge (A2), becoming the absolute dominant bacterium. This result agrees with previous studies. Li et al . [ 44 ] started a UASB reactor with synthetic wastewater and observed that the relative abundance of Candidatus Kuenenia increased from 4.75 to 48.77%. Zhang et al . [ 45 ] reported that the genera Candidatus Kuenenia was enriched to 43.3% after 80 days of operation in a UASB reactor by seeding anammox sludge and using synthetic wastewater. Chen et al . [ 23 ] reported that Planctomycetes were highly enriched, increasing from 11.5 to 82% in a UASB biofilm reactor after seeding bulking sludge. It has been reported that Candidatus Kuenenia can easily carry out the anammox reaction under laboratory conditions [ 31 ] . Although its growth rate is low, it has a high affinity for substrate, and is a commonly detected bacterial genera in an anammox system [ 46 ] . Another study showed that Candidatus Kuenenia was usually the dominant bacterial genera detected during the treatment of low-concentration NH 4 + -N wastewater [ 47 ] . In this study, the influent had a low NH 4 + -N concentration, which is favorable for the successful enrichment of Candidatus Kuenenia within the reactor. In addition, a study has shown that the anammox bacteria are not always the same in different living environments, with only one species of anammox bacteria usually being dominant under a stable growing environment [ 48 ] . The relative abundance of the genera Delftia , Acidovorax , Thauera and Alicycliphilus , which are denitrifying bacteria, decreased from 18.70, 8.02, 4.94 and 4.59% to 7.01, 1.77, 3.06 and 3.96%, respectively. Although the relative abundance of denitrifying bacteria decreased, their roles in the process of nitrogen removal should not be ignored. A previous study showed that denitrifying and anammox bacteria coexisted in a system, denitrifying bacteria could use NO 2 − -N generated by anammox reaction, while the presence of denitrifying bacteria was beneficial to maintaining the anaerobic conditions in a reactor, which provided more NO 2 − -N for anammox bacteria [ 49 ] . Ammonia-oxidizing bacteria (AOB) and NOB can coexist with anammox bacteria in the natural environment, which participates in nitrogen removal [ 6 ] . AOB provides the substrate for anammox bacteria by oxidizing NH 4 + -N to NO 2 − -N, and anammox bacteria reduce inhibition of AOB activity by utilizing NH 4 + -N and NO 2 − -N [ 50 ] . Nitrosomonas , which is an AOB, can absorb oxygen fast and can exist for a long time in an anammox system, thereby enhancing the nitrifying process [ 51 ] . However, Nitrosomonas was inhibited during the reactor start-up, and its relative abundance decreased from 2.91 to 1.87%. Similarly, Li et al . [ 52 ] found a decrease in the relative abundance of Nitrosomonas from the seeding sludge (~ 10%) to the anammox sludge (~ 1%). Nitrospira was the only detected NOB, and the relative abundance decreased from 0.04 to 0.01% over the length of the experiment, indicating that the competitiveness of NOB for NO 2 − N decreased, which was beneficial to increasing the activity of anammox bacteria. It has been reported that Nitrospira can carry out the complete ammonia oxidation and participate in the nitrogen cycle [ 53 ] . Antibiotic resistance genes in the anammox reactor The types and relative abundance of ARGs were assessed in the two samples (Fig. 5 ). The total relative abundance of ARGs decreased from 90.23 to 64.29 ppm after the anammox reactor successfully started up. Macrolide-lincosamide-streptogramin (MLS), sulfonamide, aminoglycoside, multidrug and tetracycline were the main ARG types detected in both the seeding sludge (A1) and anammox sludge (A2). Among these, the relative abundance of aminoglycoside, tetracycline and multidrug decreased from 18.09, 14.84, and 13.96 ppm to 8.15, 6.58 and 8.50 ppm, respectively. Conversely, the relative abundance of sulfonamide and MLS increased from 14.66 and 15.73 ppm to 21.11 and 17.57 ppm, respectively. Different literatures have also reported on the detection of ARG types in the WWTP activated sludge and anammox sludge. For example, Guo et al . [ 54 ] detected beta-lactam, fluoroquinolone and sulfonamide as the main ARG types in aerobic sludge of a WWTP by using metagenomic sequencing. Yang et al . [ 55 ] found that aminoglycoside ARGs at the highest relative abundance, followed by tetracycline, sulfonamide and multidrug ARGs, in activated sludge of a WWTP in Hong Kong, also by metagenomic sequencing. Bi et al . [ 56 ] showed that elfamycin, sulfonamide, tetracycline and aminoglycoside ARGs are the main types in anammox sludge obtained after 90 days of operation in a UASB reactor. Therefore, different ARG types can be detected in different sludge samples. The relative abundance of dominant ARG subtypes were assessed in samples A1 and A2, with 123 and 79 ARG subtypes detected, respectively (Fig. 6 ). The decrease in the number of ARG subtypes may be attributed to the inhibition or death of ARG-carrying bacteria which could not be adapted to the anaerobic environment. The main subtypes in the seeding sludge (A1) were sul1 (10.44 ppm), msrE (8.86 ppm), ermF (7.47 ppm), tetX (5.85 ppm) and ANT(3'')-IIa (4.92 ppm). Wang et al . [ 57 ] found that sul , tet and erm were the most common ARG subtypes in Municipal WWTPs through a literature review of ARG statistics in WWTPs. In the anammox sludge (A2), ermF (11.64 ppm), sul2 (10.95 ppm), sul1 (9.81 ppm) and floR (3.53 ppm) were the main ARG subtypes. After successful start-up of anammox, the ARG subtype sul1 accounted for 10.44 ppm in the seeding sludge (A1) and 9.81 ppm in the anammox sludge (A2). Often located on large plasmids or small non-attached plasmids capable of spreading antibiotic resistance, sul1 can be transferred between bacteria through binding and transduction, thereby leading to the widespread presence of sulfonamide ARGs in the environment [ 58 ] . In this study, the relative abundance of ARG subtypes msrE and tetX decreased from 8.86 and 5.85 ppm to 0.70 and 1.92 ppm, respectively. msrE confers multidrug antibiotic and belongs to an ABC-F subfamily protein harbored on plasmid DNA and expressed by Klebsiella pneumoniae . tetX is an enzymatic modification gene, with its antibiotic resistance mechanism involving antibiotic inactivation. In contrast, the relative abundance of ARG subtypes ermF , sul2 and floR increased from 7.47, 3.76 and 1.35 ppm to 11.64, 10.95 and 3.53 ppm, respectively. Among them, ermF belongs to the MLS class, which has been detected in various environmental media, such as sludge and livestock manure [ 59 ] . sul2 is an ARG subtype has been detected at a high relative abundance in anaerobic sludge, and frequently in different WWTPs [ 22 ] . Conclusions The UASB reactor successfully started anammox over 132 days of operation, reaching NH 4 + -N and NO 2 − -N removal rates of over 99.5%. Metagenomic sequencing showed that Candidatus Kuenenia was the only anammox bacterium detected, registering a relative abundance of 0.01% in the seeding sludge and 50.86% in the anammox sludge. The denitrifying bacterial genera Delftia , Acidovorax , Thauera and Alicycliphilus decreased from 18.70, 8.02, 4.94 and 4.59% to 7.01, 1.77, 3.06 and 3.96%, respectively. The AOB Nitrosomonas decreased from 2.91 to 1.87%. After cultivation, the total relative abundance of ARGs decreased from 90.23 to 64.29 ppm, with MLS, sulfonamide, aminoglycoside, tetracycline and multidrug being the main ARG types in the two samples. sul1 , msrE and tetX were the main ARG subtypes in the seeding sludge, while ermF , sul2 , sul1 and flo R were the main subtypes in the anammox sludge. Among these, sul1 , msrE and tetX decreased, while ermF , sul2 , and floR increased after successful anammox start-up. Declarations Competing interests The authors declare no competing interests. Author Contribution X.L. is assigned to the writing and statistical analysis. J.Q.Y. designed the study and provided financial resources. J.Q.L. is involved in the statistical analysis. Y.Y.J. and Y.G.C. reviewed and revised the paper. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 52160005) and the Natural Science Foundation of Xinjiang of China (No. 2021D01C047). 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Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production. Environ. Microbio. 10, 3120–3129 (2008). Xu, L. Z. J. et al. Deciphering the microbial community and functional genes response of anammox sludge to sulfide stress. Bioresour. Technol. 302, 122885 (2020). Chen, H. et al. A critical review on microbial ecology in the novel biological nitrogen removal process: Dynamic balance of complex functional microbes for nitrogen removal. Sci. Total Environ. 857, 159462 (2023). Cho, S. J., Kambey, C. & Nguyen, V. K. Performance of Anammox Processes for Wastewater Treatment: A Critical Review on Effects of Operational Conditions and Environmental Stresses. Water 12, 1 (2019). Li, Y. Q. et al. Response mechanism of a highly efficient partial nitritation-anammox (PN/A) process under antibiotic stress: Extracellular polymers, microbial community, and functional genes. Environ. Res. 251, 118575 (2024). Clagnan, E. et al. Conventional activated sludge vs. photo-sequencing batch reactor for enhanced nitrogen removal in municipal wastewater: Microalgal-bacterial consortium and pathogenic load insights. Bioresour. Technol. 401, 130735 (2024). Guo, J. H., Li, J., Chen, H., Bond, P. L. & Yuan, Z. G. Metagenomic analysis reveals wastewater treatment plants as hotspots of antibiotic resistance genes and mobile genetic elements. Water Res. 123, 468–478 (2017). Yang, Y., Li, B., Ju, F. & Zhang, T. Exploring Variation of Antibiotic Resistance Genes in Activated Sludge over a Four-Year Period through a Metagenomic Approach. Environ. Sci. Technol. 47, 10197–10205 (2013). Bi, Z., Song, G. & Sun, X. M. Deciphering antibiotic resistance genes and microbial community of anammox consortia under sulfadiazine and chlortetracycline stress. Ecotoxicology and Environmental Safety 234, 113343 (2022). Wang, J. L., Chu, L. B., Wojnarovits, L. & Takacs, E. Occurrence and fate of antibiotics, antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) in municipal wastewater treatment plant: An overview. Sci. Total Environ. 744, 140997 (2020). Li, N. et al. Distribution and major driving elements of antibiotic resistance genes in the soil-vegetable system under microplastic stress. Sci. Total Environ. 906, 167619 (2024). Koniuszewska, I. et al. The occurrence of antibiotic-resistance genes in the Pilica River, Poland. Ecohydrol. Hydrobiol. 20, 1–11 (2020). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4502825","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":313522674,"identity":"f35aa3d1-21ac-4f12-b6e1-7004396f243c","order_by":0,"name":"Xin Li","email":"","orcid":"","institution":"Xinjiang University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Li","suffix":""},{"id":313522675,"identity":"e2019d0d-b11b-492a-b9f4-761de3a195a7","order_by":1,"name":"Junqin Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYJACAyDmYWBvADMYG4jXwnOABC0QIJEApghrMTh++EAx7w5rGXPJNwbFPAw2shsOMD97gFfLmbQEY94z6TyWs4EMHoY04w0H2MwN8Go5kGNgzNt2mMfgdvIBoJbDiRsO8LBJ4NVy/g1Uy82DDUAt/4nQcgNmyw1mkC0HCGuRvPEswXBuWzoPyFOGcwySjWceZjPDq4XvfPIxg7dt1vYGx8+YGbypsJPtO978DK8WhQMMbMDwYQaxgQxQUDHjUw8E8g0MzA+gykCMUTAKRsEoGAWYAACWN0aIvPxfXQAAAABJRU5ErkJggg==","orcid":"","institution":"Xinjiang University","correspondingAuthor":true,"prefix":"","firstName":"Junqin","middleName":"","lastName":"Yao","suffix":""},{"id":313522676,"identity":"4c2c0704-4e9d-48f4-a4a7-c6fc9564cea5","order_by":2,"name":"Yangyang Jia","email":"","orcid":"","institution":"Xinjiang University","correspondingAuthor":false,"prefix":"","firstName":"Yangyang","middleName":"","lastName":"Jia","suffix":""},{"id":313522677,"identity":"3988f894-f5ce-40ce-89fd-04ec1d3228e2","order_by":3,"name":"Jiaqi Liu","email":"","orcid":"","institution":"Xinjiang University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Liu","suffix":""},{"id":313522679,"identity":"80aa0c34-8336-49d3-878a-ed3f05d69a95","order_by":4,"name":"Yinguang Chen","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Yinguang","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-05-30 11:51:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4502825/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4502825/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58296150,"identity":"72f19032-6b27-4b87-95d1-bdde3383f8cf","added_by":"auto","created_at":"2024-06-13 14:46:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":376226,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the reactor.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4502825/v1/bddf3a0778963b6a168f9b4c.png"},{"id":58296692,"identity":"853e2131-2717-4276-aef6-6b68275133a5","added_by":"auto","created_at":"2024-06-13 14:54:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":167846,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations and removal rates of ammonia nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) and nitrite nitrogen (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4502825/v1/1be6ed7d826b0d563036122f.png"},{"id":58296149,"identity":"2a9b7579-e3c3-4c03-a417-8d40cd6f61b2","added_by":"auto","created_at":"2024-06-13 14:46:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84870,"visible":true,"origin":"","legend":"\u003cp\u003eThe bacterial community at the phylum level.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4502825/v1/d7067301aeac722178b07bf4.png"},{"id":58296148,"identity":"75db5269-f09e-48f0-8091-54a280d83807","added_by":"auto","created_at":"2024-06-13 14:46:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148454,"visible":true,"origin":"","legend":"\u003cp\u003eThe bacterial community at the genus level.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4502825/v1/5a47006a32b575d26821fe3d.png"},{"id":58296146,"identity":"c828970f-b65e-4507-9008-3019a22dc63d","added_by":"auto","created_at":"2024-06-13 14:46:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85788,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of antibiotic resistance genes (ARGs) in the two sludge samples.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4502825/v1/f921bf1af8d9b04d7528627e.png"},{"id":58296151,"identity":"dcbbd4ea-4a31-449f-b4a5-346b4cf222bd","added_by":"auto","created_at":"2024-06-13 14:46:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":173734,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of the top 15 antibiotic resistance gene (ARG) subtypes in the two sludge samples.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4502825/v1/b1b7517cc3a7a003ea11908b.png"},{"id":61089256,"identity":"d40a5dc5-a150-49c1-be8e-1d419b2d7124","added_by":"auto","created_at":"2024-07-25 12:44:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1689107,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4502825/v1/c5f540be-18ee-4d13-a95e-82e777d51280.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Start-up of anammox in an up-flow anaerobic sludge bed reactor: bacterial community composition and antibiotic resistance genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith extensive attention placed on eutrophication, nitrogen removal has become one of the important functions of wastewater treatment plants (WWTPs)\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Currently, most WWTPs use the principle of nitrification and denitrification to facilitate biological nitrogen removal\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. One Study has shown that nearly 80% of WWTPs in China operate at a biochemical oxygen demand (five days incubation) to total nitrogen ratios of less than 3.6, which represents low carbon-nitrogen ratio wastewater\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. WWTPs require additional carbon source for denitrification\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, which increases their operating cost\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, the development of more energy efficient wastewater treatment technologies has a broad research interest\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Anaerobic ammonia oxidation (anammox), an autotrophic biological nitrogen removal process discovered in the 1990s, can utilize microbial action in anaerobic environments to achieve the simultaneous removal of ammonia nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) and nitrite nitrogen (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N), and is a research hotspot in the current wastewater treatment\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDifferent from traditional biological nitrogen removal processes, the anammox process can effectively reduce the aeration cost, energy consumption, sludge production, and demand for organic carbon sources\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Six genera, namely \u003cem\u003eCandidatus\u003c/em\u003e Anammoxoglobus, \u003cem\u003eCandidatus\u003c/em\u003e Anammoximicrobium, \u003cem\u003eCandidatus\u003c/em\u003e Brocadia, \u003cem\u003eCandidatus\u003c/em\u003e Jettenia, \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia and \u003cem\u003eCandidatus\u003c/em\u003e Scalindua, harboring more than 20 species therein, have been reported to pose anammox function\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Due to the environmental sensitivity of anammox bacteria, pure cultures have yet to be cultivated\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, and it is generally assumed that anammox is accomplished by a variety of bacteria performing biological nitrogen removal\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Thus, the bacterial community composition determines the efficiency of biological nitrogen removal.\u003c/p\u003e \u003cp\u003eAntibiotic resistance genes (ARGs), as emerging pollutants\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, pose a serious risk to public safety. They have attracted worldwide attention and are currently a topic of intense research to control environmental pollutants\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. A previous study indicated that, with the help of mobile genetic elements such as plasmids, integrons and transposons, ARGs can be transferred horizontally among microorganisms, causing their transmission and diffusion within the environment\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The microbial community diversity and structure in the biological wastewater treatment system is conducive to the selection, transfer and transmission of ARGs across a variety of bacteria\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, a growing number of studies have been conducted to reveal the influence of ARGs in different anammox reactors amended with antibiotics\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Wu \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e found that an up-flow anaerobic sludge bed (UASB) reactor had good adaptability to low concentrations of spiramycin, and the relative abundance of ARG subtypes \u003cem\u003eermB\u003c/em\u003e, \u003cem\u003eermF\u003c/em\u003e, \u003cem\u003eermQ\u003c/em\u003e, \u003cem\u003eereA\u003c/em\u003e and \u003cem\u003emphA\u003c/em\u003e increased in activated sludge as the spiramycin concentration increased. Kong \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e studied changes in ARGs in a UASB reactor amended with erythromycin, sulfamethoxazole and tetracycline antibiotics over 360 days of operation, and found that the main ARG subtype was \u003cem\u003etetW\u003c/em\u003e and \u003cem\u003eermF\u003c/em\u003e in the sludge. Zhang \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e showed that after the addition of oxytetracycline in a UASB reactor, the relative abundance of ARG subtypes \u003cem\u003etetG\u003c/em\u003e and \u003cem\u003etetX\u003c/em\u003e increased. Fan \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e found that when oxytetracycline was added to a UASB reactor, the relative abundance of ARG subtypes \u003cem\u003etetA\u003c/em\u003e, \u003cem\u003etetB\u003c/em\u003e and \u003cem\u003etetC\u003c/em\u003e in activated sludge increased. However, ARGs have rarely been studied in anammox reactors under non-antibiotic conditions. At present, the most common technique used in ARGs research is q-PCR which requires the use of a known single ARG sequence for detection\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Due to the limitations of ARG primers, q-PCR has the disadvantage of only detecting ARGs with specific primers\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. With the development of metagenomic sequencing, complete microbial community and genetic information of the functional genes or enzymes can be obtained. This technique has been applied to study the selection of ARGs in wastewater systems, and can overcome the difficulties of culturing and separating specific microorganisms\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, activated sludge was obtained from an oxidation ditch of a WWTP located in Urumqi City (Xinjiang, China). This activated sludge was then used as the seeding sludge to initiate the anammox reaction at temperatures ranging from 20 to 31\u0026deg;C (room temperature) with synthetic wastewater. Changes in bacterial community composition and ARGs in the seeding sludge and anammox sludge cultivated for 132 days under non-antibiotic conditions were systematically analyzed using metagenomic sequencing. These results provide a further understanding of the nitrogen removal performance, existence of nitrogen-removing bacteria and ARGs in an anammox reactor. Furthermore, the information collected from this study provides theoretical basis for improving the anammox process by better achieving the removal of this emerging pollutants - ARGs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnammox reactor set-up\u003c/h2\u003e \u003cp\u003eIn this study, the effective volume of the plexiglass UASB reactor used was 3.2 L (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The anammox reactor surface was covered with an insulating layer to prevent light from adversely affecting the bacteria. The influent water was pumped into the bottom of the reactor by a peristaltic pump. The temperature of the mixed liquid in the reactor was maintained at 20\u0026ndash;31\u0026deg;C, and the hydraulic retention time was 17.1\u0026ndash;51.2 h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSynthetic wastewater and sample collection\u003c/h2\u003e \u003cp\u003eIn this study, synthetic wastewater was used as the influent, and 50 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NH\u003csub\u003e4\u003c/sub\u003eCl and 70 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eNaNO\u003csub\u003e2\u003c/sub\u003e were added to the synthetic wastewater to serve as sources of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, respectively. No organic matter was added. Other components included NaHCO\u003csub\u003e3\u003c/sub\u003e (500 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), MgSO\u003csub\u003e4\u003c/sub\u003e (300 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CaCl\u003csub\u003e2\u003c/sub\u003e (126 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (30 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The trace elements stock solution, which was added at 1 mL\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, comprised FeSO\u003csub\u003e4\u003c/sub\u003e (5000 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), MnCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO (990 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (430 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO (250 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CoCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (240 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), NiCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (190 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e4\u003c/sub\u003e (14 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), as per Chen \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSamples of the seeding sludge (from day 1 of reactor operation) and the sludge from day 132, representing stable anammox operation were collected and labeled A1 and A2, respectively. These activated sludge samples were kept at -80\u0026deg;C prior to being sent to Majorbio Bio-Pharmaceutical Technology Co. Ltd. (Shanghai, China) for DNA extraction and metagenomic sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical methods\u003c/h2\u003e \u003cp\u003eInfluent and effluent water quality indicators were determined by the standard methods; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N was determined by Nessler's reagent spectrophotometry; nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N were determined by ultraviolet spectrophotometry. The water temperature was measured using a mercury thermometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and metagenomic sequencing\u003c/h2\u003e \u003cp\u003eTotal genomic DNA was extracted from the two sludge samples (A1 and A2) using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, USA) according to the manufacturer\u0026rsquo;s instructions. The extracted DNA was tested for concentration and purity by TBS-380 and NanoDrop2000 systems, respectively. The quality of the extracted DNA was checked via 1% agarose gel electrophoresis. An ultrasonic breaker Covaris M220 (Gene Company Limited, Hong Kong, China) was used to break the extracted DNA fragment to an average size of about 400 bp. After fragmentation, the metagenomic sequencing library was constructed using NEXTFLEX Rapid DNA-Seq Kit. Finally, 150-bp paired-end metagenomic sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, USA). The raw metagenomic sequencing data obtained in this study have been uploaded to the NCBI Sequence Read Archive (SRA) under accession number SRP274797.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis\u003c/h2\u003e \u003cp\u003eHigh-quality paired-end reads from the metagenomic data were obtained using fastp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://github.com/OpenGene/fastp/\u003c/span\u003e\u003cspan address=\"http://github.com/OpenGene/fastp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e by cutting the 3\u0026prime; and 5\u0026prime; ends of the original adapter sequence, and by discarding reads less than 50 bp long with quality values below 20 or containing N bases. After quality control, clean reads were assembled using MEGAHIT\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and contigs with a length\u0026thinsp;\u0026ge;\u0026thinsp;300 bp were retained for assembly. MetaGene\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e was used to predict open reading frames, select genes with sequences\u0026thinsp;\u0026ge;\u0026thinsp;100 bp and translate selected genes into amino acid sequences. CD-HIT\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003ewas used to construct a non-redundant gene catalog, with sequence identification and coverage rates up to 90%. SOAPaligner\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e was used to map the quality-controlled reads to the non-redundant gene catalog with 95% identity and assess the gene abundance of each sample. Kraken2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccb.jhu.edu/software/kraken2\u003c/span\u003e\u003cspan address=\"https://ccb.jhu.edu/software/kraken2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e was used for metagenomic classification, after which Bracken (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/jenniferlu717/Bracken\u003c/span\u003e\u003cspan address=\"https://github.com/jenniferlu717/Bracken\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e was used to estimate species abundance. The obtained non-redundant gene set was compared with the CARD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://card.mcmaster.ca\u003c/span\u003e\u003cspan address=\"https://card.mcmaster.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e through BLASTP. An ARG was considered when a sequence had an E-value\u0026thinsp;\u0026le;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, identity\u0026thinsp;\u0026ge;\u0026thinsp;80%, alignment length\u0026thinsp;\u0026ge;\u0026thinsp;25 amino acids. Finally, the relative abundance of ARGs was expressed in ppm (representing the number of ARGs per million clean reads).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnammox reactor performance\u003c/h2\u003e \u003cp\u003eBased on the concentrations and removal rates of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, the anammox process was divided into four stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). From days 1\u0026ndash;11 (stage Ⅰ), the concentration of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N in the effluent was similar to that in the influent. From days 12\u0026ndash;57 (stage Ⅱ), the removal rate of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N ranged from 13.9 to 70.3%. Moreover, the removal rate of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N showed a declining trend, decreasing from 47.9 to 8.4%. From days 58\u0026ndash;122 (stage Ⅲ), the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N removal rates increased continuously, reaching 99.8 and 98.5%, respectively. After 122 days of cultivation, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N production reached 17.8 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Therefore, the anammox activity at this stage increased continuously with the increasing nitrogen removal rate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom days 123\u0026ndash;135 (stage Ⅳ), the removal rates of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N were over 99.5%. In the effluent, the average concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N were 0.1 and 0.2 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Additionally, the average production of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N was 15.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The corresponding molar ratio of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N consumption to NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N consumption to NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N production was 1.00:1.41:0.31, which is close to the reported value of 1:1.32:0.26\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. This indicates that anammox had become the dominant reaction and the reactor was in the stable operation stage of anammox. Therefore, this stage represents the stable anammox period and the sludge sample was collected on day 132 which was the anammox sludge.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBacteria community analysis at the phylum level\u003c/h2\u003e \u003cp\u003e29 bacterial phyla were identified in the two sludge samples, but the top 10 phyla accounted for most of the communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the seeding sludge (A1), Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the predominant bacterial phyla, with the relative abundance levels of 82.01, 8.00, 7.12 and 1.76%, respectively. In the anammox sludge (A2), the phylum Planctomycetes showed the highest relative abundance (50.99%), followed by Proteobacteria (40.25%), Chloroflexi (2.99%) and Actinobacteria (2.90%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the anammox reaction, the bacterial community is vitally important to facilitate biological nitrogen removal\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that Planctomycetes is a phylum of autotrophic nitrogen-removing functional bacteria, with anammox bacteria belonging to this phylum\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. With the successful start-up of anammox, the relative abundance of Planctomycetes significantly increased from 0.02 to 50.99%. This is consistent with other studies which indicate Planctomycetes has the highest relative abundance in different anammox reactors\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. When seeding a UASB reactor with anammox sludge, Fu \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e studied the succession of the bacterial community in sludge, revealing that after 220\u0026ndash;280 days of operation, the relative abundance of Planctomycetes was higher than 50%, becoming the absolute dominant phylum. Using conventional sludge and anammox sludge successfully started an anammox reactor, Luo \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e showed that the relative abundance of Planctomycetes gradually increased from 0.1% on day 1 to 22.96% on day 160. When comparing anaerobic seeding sludge and mature anammox sludge, Sobotka \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e found that the relative abundance of Planctomycetes accounted for 15 and 43%, respectively.\u003c/p\u003e \u003cp\u003eChloroflexi, a phylum of heterotrophic bacteria, are commonly found in anammox reactors\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. These bacteria can participate in autotrophic denitrification\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. They can also consume metabolites produced by anammox bacteria, as well as dead cells in the reactor, thereby providing a stable environment for anammox bacteria\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Its relative abundance increased from 0.21 to 2.99% in this study. Furthermore, one previous study showed that the phylum Chloroflexi harbors a large number of filamentous bacteria that can provide a skeleton and be conducive to the formation and stability of anammox sludge\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProteobacteria and Bacteroidetes are mostly comprised of heterotrophic bacteria, which need to use organic matter to satisfy their growth demand\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In this study, their relative abundances decreased. The former decreased from 82.01 to 50.99%, while the latter decreased from 8.00 to 0.33%. Common in wastewater treatment systems, some studies have reported that phylum Proteobacteria contains a variety of nitrogen-removing functional bacteria, such as nitrite-oxidizing bacteria (NOB) and denitrifying bacteria\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, which are widely present in anammox systems, providing growth factors for anammox bacteria and playing important roles in nitrogen removal\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Moreover, Proteobacteria and Chloroflexi can cooperate with anammox bacteria in the nitrogen and amino acid cycles\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Bacteroidetes is also considered a common bacterial phylum in activated sludge and participates in partial denitrification to achieve nitrogen removal\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Similarly, Sobotka \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e found that the relative abundance of Bacteroidetes significantly decreased from 5% in the anaerobic seeding sludge to 1% in the mature anammox sludge of an anammox system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBacterial community analysis at the genus level\u003c/h2\u003e \u003cp\u003eIn the two sludge samples, 1172 bacterial genera were identified. The bacterial genera with a relative abundance greater than 1% in the seeding sludge (A1) and anammox sludge (A2) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Among these, the anammox bacterium \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia was detected, with a relative abundance of only 0.01% in the seeding sludge (A1); however, it was enriched to 50.86% in the anammox sludge (A2), becoming the absolute dominant bacterium. This result agrees with previous studies. Li \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e started a UASB reactor with synthetic wastewater and observed that the relative abundance of \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia increased from 4.75 to 48.77%. Zhang \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e reported that the genera \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia was enriched to 43.3% after 80 days of operation in a UASB reactor by seeding anammox sludge and using synthetic wastewater. Chen \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e reported that Planctomycetes were highly enriched, increasing from 11.5 to 82% in a UASB biofilm reactor after seeding bulking sludge. It has been reported that \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia can easily carry out the anammox reaction under laboratory conditions\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Although its growth rate is low, it has a high affinity for substrate, and is a commonly detected bacterial genera in an anammox system\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Another study showed that \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia was usually the dominant bacterial genera detected during the treatment of low-concentration NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N wastewater\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. In this study, the influent had a low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration, which is favorable for the successful enrichment of \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia within the reactor. In addition, a study has shown that the anammox bacteria are not always the same in different living environments, with only one species of anammox bacteria usually being dominant under a stable growing environment\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative abundance of the genera \u003cem\u003eDelftia\u003c/em\u003e, \u003cem\u003eAcidovorax\u003c/em\u003e, \u003cem\u003eThauera\u003c/em\u003e and \u003cem\u003eAlicycliphilus\u003c/em\u003e, which are denitrifying bacteria, decreased from 18.70, 8.02, 4.94 and 4.59% to 7.01, 1.77, 3.06 and 3.96%, respectively. Although the relative abundance of denitrifying bacteria decreased, their roles in the process of nitrogen removal should not be ignored. A previous study showed that denitrifying and anammox bacteria coexisted in a system, denitrifying bacteria could use NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N generated by anammox reaction, while the presence of denitrifying bacteria was beneficial to maintaining the anaerobic conditions in a reactor, which provided more NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N for anammox bacteria\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmmonia-oxidizing bacteria (AOB) and NOB can coexist with anammox bacteria in the natural environment, which participates in nitrogen removal\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. AOB provides the substrate for anammox bacteria by oxidizing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N to NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and anammox bacteria reduce inhibition of AOB activity by utilizing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eNitrosomonas\u003c/em\u003e, which is an AOB, can absorb oxygen fast and can exist for a long time in an anammox system, thereby enhancing the nitrifying process\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. However, \u003cem\u003eNitrosomonas\u003c/em\u003e was inhibited during the reactor start-up, and its relative abundance decreased from 2.91 to 1.87%. Similarly, Li \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e found a decrease in the relative abundance of \u003cem\u003eNitrosomonas\u003c/em\u003e from the seeding sludge (~\u0026thinsp;10%) to the anammox sludge (~\u0026thinsp;1%). \u003cem\u003eNitrospira\u003c/em\u003e was the only detected NOB, and the relative abundance decreased from 0.04 to 0.01% over the length of the experiment, indicating that the competitiveness of NOB for NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003eN decreased, which was beneficial to increasing the activity of anammox bacteria. It has been reported that \u003cem\u003eNitrospira\u003c/em\u003e can carry out the complete ammonia oxidation and participate in the nitrogen cycle\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic resistance genes in the anammox reactor\u003c/h2\u003e \u003cp\u003eThe types and relative abundance of ARGs were assessed in the two samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The total relative abundance of ARGs decreased from 90.23 to 64.29 ppm after the anammox reactor successfully started up. Macrolide-lincosamide-streptogramin (MLS), sulfonamide, aminoglycoside, multidrug and tetracycline were the main ARG types detected in both the seeding sludge (A1) and anammox sludge (A2). Among these, the relative abundance of aminoglycoside, tetracycline and multidrug decreased from 18.09, 14.84, and 13.96 ppm to 8.15, 6.58 and 8.50 ppm, respectively. Conversely, the relative abundance of sulfonamide and MLS increased from 14.66 and 15.73 ppm to 21.11 and 17.57 ppm, respectively. Different literatures have also reported on the detection of ARG types in the WWTP activated sludge and anammox sludge. For example, Guo \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e detected beta-lactam, fluoroquinolone and sulfonamide as the main ARG types in aerobic sludge of a WWTP by using metagenomic sequencing. Yang \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e found that aminoglycoside ARGs at the highest relative abundance, followed by tetracycline, sulfonamide and multidrug ARGs, in activated sludge of a WWTP in Hong Kong, also by metagenomic sequencing. Bi \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e showed that elfamycin, sulfonamide, tetracycline and aminoglycoside ARGs are the main types in anammox sludge obtained after 90 days of operation in a UASB reactor. Therefore, different ARG types can be detected in different sludge samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative abundance of dominant ARG subtypes were assessed in samples A1 and A2, with 123 and 79 ARG subtypes detected, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The decrease in the number of ARG subtypes may be attributed to the inhibition or death of ARG-carrying bacteria which could not be adapted to the anaerobic environment. The main subtypes in the seeding sludge (A1) were \u003cem\u003esul1\u003c/em\u003e (10.44 ppm), \u003cem\u003emsrE\u003c/em\u003e (8.86 ppm), \u003cem\u003eermF\u003c/em\u003e (7.47 ppm), \u003cem\u003etetX\u003c/em\u003e (5.85 ppm) and \u003cem\u003eANT(3'')-IIa\u003c/em\u003e (4.92 ppm). Wang \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e found that \u003cem\u003esul\u003c/em\u003e, \u003cem\u003etet\u003c/em\u003e and \u003cem\u003eerm\u003c/em\u003e were the most common ARG subtypes in Municipal WWTPs through a literature review of ARG statistics in WWTPs. In the anammox sludge (A2), \u003cem\u003eermF\u003c/em\u003e (11.64 ppm), \u003cem\u003esul2\u003c/em\u003e (10.95 ppm), \u003cem\u003esul1\u003c/em\u003e (9.81 ppm) and \u003cem\u003efloR\u003c/em\u003e (3.53 ppm) were the main ARG subtypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter successful start-up of anammox, the ARG subtype \u003cem\u003esul1\u003c/em\u003e accounted for 10.44 ppm in the seeding sludge (A1) and 9.81 ppm in the anammox sludge (A2). Often located on large plasmids or small non-attached plasmids capable of spreading antibiotic resistance, \u003cem\u003esul1\u003c/em\u003e can be transferred between bacteria through binding and transduction, thereby leading to the widespread presence of sulfonamide ARGs in the environment\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. In this study, the relative abundance of ARG subtypes \u003cem\u003emsrE\u003c/em\u003e and \u003cem\u003etetX\u003c/em\u003e decreased from 8.86 and 5.85 ppm to 0.70 and 1.92 ppm, respectively. \u003cem\u003emsrE\u003c/em\u003e confers multidrug antibiotic and belongs to an ABC-F subfamily protein harbored on plasmid DNA and expressed by \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003etetX\u003c/em\u003e is an enzymatic modification gene, with its antibiotic resistance mechanism involving antibiotic inactivation. In contrast, the relative abundance of ARG subtypes \u003cem\u003eermF\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e and \u003cem\u003efloR\u003c/em\u003e increased from 7.47, 3.76 and 1.35 ppm to 11.64, 10.95 and 3.53 ppm, respectively. Among them, \u003cem\u003eermF\u003c/em\u003e belongs to the MLS class, which has been detected in various environmental media, such as sludge and livestock manure\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003esul2\u003c/em\u003e is an ARG subtype has been detected at a high relative abundance in anaerobic sludge, and frequently in different WWTPs\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe UASB reactor successfully started anammox over 132 days of operation, reaching NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N removal rates of over 99.5%. Metagenomic sequencing showed that \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia was the only anammox bacterium detected, registering a relative abundance of 0.01% in the seeding sludge and 50.86% in the anammox sludge. The denitrifying bacterial genera \u003cem\u003eDelftia\u003c/em\u003e, \u003cem\u003eAcidovorax\u003c/em\u003e, \u003cem\u003eThauera\u003c/em\u003e and \u003cem\u003eAlicycliphilus\u003c/em\u003e decreased from 18.70, 8.02, 4.94 and 4.59% to 7.01, 1.77, 3.06 and 3.96%, respectively. The AOB \u003cem\u003eNitrosomonas\u003c/em\u003e decreased from 2.91 to 1.87%. After cultivation, the total relative abundance of ARGs decreased from 90.23 to 64.29 ppm, with MLS, sulfonamide, aminoglycoside, tetracycline and multidrug being the main ARG types in the two samples. \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003emsrE\u003c/em\u003e and \u003cem\u003etetX\u003c/em\u003e were the main ARG subtypes in the seeding sludge, while \u003cem\u003eermF\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e and \u003cem\u003eflo\u003c/em\u003eR were the main subtypes in the anammox sludge. Among these, \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003emsrE\u003c/em\u003e and \u003cem\u003etetX\u003c/em\u003e decreased, while \u003cem\u003eermF\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e, and \u003cem\u003efloR\u003c/em\u003e increased after successful anammox start-up.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.L. is assigned to the writing and statistical analysis. J.Q.Y. designed the study and provided financial resources. J.Q.L. is involved in the statistical analysis. Y.Y.J. and Y.G.C. reviewed and revised the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 52160005) and the Natural Science Foundation of Xinjiang of China (No. 2021D01C047).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw data generated from metagenomic sequencing has been deposited in the NCBI Sequence Read Archive (SRA) and is available via the BioProject SRP274797.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHu, K. Y. et al. Novel biological nitrogen removal process for the treatment of wastewater with low carbon to nitrogen ratio: A review. J. Water Process Eng. 53, 103673 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBegmatov, S. et al. 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Hydrobiol. 20, 1\u0026ndash;11 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4502825/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4502825/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnaerobic ammonia oxidation (anammox) is considered a high-efficiency and low-consumption biological nitrogen removal process. In this study, synthetic wastewater was used to start an up-flow anaerobic sludge bed reactor with a starting temperature of 20\u0026ndash;31℃, and subsequently, the activated sludge samples were used to analyze the changes in bacterial community and antibiotic resistance genes (ARGs) by metagenomic sequencing. The results showed that the reaction successfully started up after 132 days of cultivation, achieving NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N removal rates over 99.5%. \u003cem\u003eCandidatus\u003c/em\u003e Kuenenia, an anammox bacterium, increased from 0.01 to 50.86%. The denitrifying bacteria \u003cem\u003eDelftia\u003c/em\u003e, \u003cem\u003eAcidovorax\u003c/em\u003e, \u003cem\u003eThauera\u003c/em\u003e and \u003cem\u003eAlicycliphilus\u003c/em\u003e decreased from 18.70, 8.02, 4.94 and 4.59% to 7.01, 1.77, 3.06 and 3.96%, respectively. The ammonia-oxidizing bacterium \u003cem\u003eNitrosomonas\u003c/em\u003e decreased from 2.91 to 1.87%. After cultivation, the relative abundance of ARGs in sludge decreased from 90.23 to 64.29 ppm, with sulfonamide, macrolide-lincosamide-streptogramin, tetracycline, aminoglycoside and multidrug ARGs being the main types. Additionally, the ARG subtypes \u003cem\u003esul1\u003c/em\u003e, \u003cem\u003emsrE\u003c/em\u003e and \u003cem\u003etetX\u003c/em\u003e decreased, while \u003cem\u003eermF\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e and \u003cem\u003efloR\u003c/em\u003e increased. These results contribute to knowledge of the nitrogen removal performance, changes in bacterial community composition and ARGs in an anammox reactor, providing the guidance for the removal of ARGs by anammox.\u003c/p\u003e","manuscriptTitle":"Start-up of anammox in an up-flow anaerobic sludge bed reactor: bacterial community composition and antibiotic resistance genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-13 14:46:34","doi":"10.21203/rs.3.rs-4502825/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aabe2adf-779f-459c-b565-6fa7f4cfba2a","owner":[],"postedDate":"June 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33148781,"name":"Biological sciences/Microbiology/Bacteria"},{"id":33148782,"name":"Biological sciences/Microbiology/Communities"}],"tags":[],"updatedAt":"2024-07-25T12:36:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-13 14:46:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4502825","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4502825","identity":"rs-4502825","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}