bioRxiv preprint doi: https://doi.org/10.1101/703868; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 DNA and RNA-SIP reveal Nitrospira spp. as key drivers of nitrification in 2 groundwater-fed biofilters 3 4 Running title: Nitrospira drives nitrification in groundwater-fed biofilters 5 Authors: Arda Gülay1,4*, Jane Fowler1, Karolina Tatari1, Bo Thamdrup3, Hans-Jørgen Albrechtsen1, 6 Waleed Abu Al-Soud2, Søren J. Sørensen2 and Barth F. Smets1* 7 1 Department of Environmental Engineering, Technical University of Denmark, Building 113, Miljøvej, 2800 8 Kgs Lyngby, Denmark. Phone: +45 45251600. FAX: +45 45932850. e-mail: [email protected], jfow@ 9 env.dtu.dk, [email protected], [email protected]* 10 2 Department of Biology, University of Copenhagen, Universitetsparken 15, Building 1, 2100 Copenhagen, 11 Denmark. Phone: +45 35323710. FAX: +45 35322128. e-mail: [email protected], [email protected] 12 3 Nordic Center for Earth Evolution, Department of Biology, University of Southern Denmark, Campusvej 55, 13 5230 Odense, Denmark. Phone: +45 35323710. FAX: +45 35322128. e-mail: [email protected] 14 4 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, United States, 15 26 Oxford St, Cambridge, MA 02138, Phone: +1 (617)4951564. e-mail: [email protected] 16 17 *Corresponding authors 18 Keywords: Nitrification, comammox, Nitrospira, DNA SIP, RNA SIP 19 bioRxiv preprint doi: https://doi.org/10.1101/703868; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Abstract 21 Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes 22 and plays a central role in the global nitrogen cycle. Our earlier metagenomics, amoA-amplicon, and 23 amoA-qPCR based investigations of groundwater-fed biofilters indicated a consistently high 24 abundance of comammox Nitrospira, and we hypothesized that these non-classical nitrifiers drive 25 ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 26 16S rRNA amplicon sequencing to identify the active members in the biofilter community when + - 13 - 12 - 27 subject to a continuous supply of NH4 or NO2 in the presence of C-HCO3 (labelled) or C-HCO3 28 (unlabelled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- 29 and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira 30 dominated ammonium oxidation in the biofilter community. A total of 78 (8 in RNA-SIP and 70 in 31 DNA-SIP) and 96 (25 in RNA-SIP and 71 in DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA 32 sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We - 33 also detected significant HCO3 uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, 34 and Acidovorax under conditions that favoured ammonium oxidation. Canonical Nitrospira alone 35 drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia oxidizing taxa 36 was not detected in the SIP fractions. This study provides the first in-situ evidence of ammonia 37 oxidation by comammox Nitrospira in an ecologically relevant complex microbiome. 38 bioRxiv preprint doi: https://doi.org/10.1101/703868; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 39 Introduction - - 40 Nitrification, the stepwise oxidation of ammonia (NH3) to nitrite (NO2 ) and nitrate (NO3 ), supplies 41 the substrates for processes that initiate the loss of reactive nitrogen from the biosphere as N2. 42 Understanding the organisms and environmental controls that drive nitrification is important as it 43 controls global homeostasis of the N cycle. In engineered environments, complete nitrification is 44 often desired: this is essential when waters are prepared and distributed for human consumption. - 45 Residual NH3 or NO2 - the result of incomplete nitrification - renders the water biologically unstable 46 and unsafe for human consumption. Hence, biological systems for source water treatment are 47 contingent on nitrifying prokaryotes. Based on evolutionarily conserved taxonomic (small subunit, 48 16S rRNA) and functional (e.g. ammonia monooxygenase, amoA) gene surveys, Nitrosomonas (1– 49 4), Nitrosoarchaeum, and Nitrososphaera have been identified as the abundant ammonium oxidizing 50 prokaryotes (AOP) and Nitrospira (5, 6) as the abundant nitrite oxidizing prokaryotes (NOP) in 51 drinking water treatment systems, consistent with the classical assumption of division of labor in the 52 two nitrification steps. 53 Our previous studies on rapid gravity sand filters (RGSF), used in potable water preparation from 54 groundwater, revealed nitrifying microbial communities with Nitrospira far more abundant than 55 Nitrosomonas (7),with several Nitrospira genomes containing genes for ammonia oxidation (8), and 56 with an abundance of comammox (complete ammonia oxidizing) amoA over AOB amoA genes (9). 57 Together with the concurrent discovery of comammox Nitrospira strains by others (10–12), this 58 suggested that comammox Nitrospira may drive ammonia oxidation in the examined groundwater- 59 fed RGSFs. In addition, like Nitrospira, several Acidobacterial, and γ- and α-proteobacterial taxa 60 were at consistently higher abundance than Nitrosomonas questioning their potential role in 61 nitrification, as NH3 is the primary growth substrate entering the filters (7, 13–15). Identifying the 62 active ammonia and nitrite oxidizing organisms is essential not only for making predictions for 63 engineering purposes, but also for understanding the niches and biodiversity of nitrifiers. There has 64 been a rapidly increasing documentation of global comammox Nitrospira occurrence across a myriad 65 of habitats ranging from the subsurface, to soils to sediments, and from groundwaters to source and 66 residual water treatment plants, but apparently excluding open oceanic waters (9, 16–19); with 67 occasional abundances that exceed those of canonical AOB (9, 20, 21). Nonetheless, it is yet to be 68 shown whether comammox Nitrospira truly drives ammonia oxidation in open oligotrophic 69 freshwater and soil environments, their presumed preferred habitat based on genomic and 70 physiological evidence (22, 23). bioRxiv preprint doi: https://doi.org/10.1101/703868; this version posted July 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 71 Here, we sought to identify the active ammonia and nitrite oxidizers in a groundwater-fed RGSF 72 using RNA and DNA stable isotope probing (SIP) coupled to 16S rRNA amplicon sequencing. Lab- 73 scale columns packed with filter material from a full-scale RGSF were fed with effluent water + - 13 74 amended with NH4 or NO2 and with C labelled or unlabelled HCO3- for 15 days in the presence 75 or absence of inhibitors of autotrophic ammonia and nitrite oxidation (24). Our findings indicate that 76 Nitrospira drive both ammonia and nitrite oxidation. In addition, several other taxa take up - 77 substantial HCO3 and their DNA and RNA increases in relative abundance when ammonia was the 78 only energy source provided. This study provides the first in-situ evidence of ammonia oxidation by 79 comammox Nitrospira in an ecologically relevant complex microbiome. 80 Materials and methods 81 Sampling sites and procedure 82 Filter material samples were collected from a rapid gravity sandfilter (biofilter) at Islevbro 83 waterworks (Rødovre, Denmark) in May 2013. The influent and effluent water quality is reported 84 elsewhere (13, 14, 25). Filter material was collected from three random horizontal locations of the 85 biofilter using a hand-pushed core sampler. From the extracted filter material core, the top 10 cm was 86 aseptically segregated on site and stored on ice for further use. A portion was frozen on-site in liquid 87 nitrogen for RNA extraction. 88 Column experiments and stable isotope labelling 89 Experiments were conducted using a continuous-flow lab-scale system consisting of glass columns 90 (2.6 cm diameter, 6 cm long) filled with parent filter material (26.5 cm3) as described previously 91 (14). Effluent water from the investigated waterworks was used as the medium in all experiments to 92 avoid interference of other autotrophic processes and approximate full scale conditions. 93 The experimental design consisted of 4 treatments applied to labeled and unlabeled control columns. 94 The experiments were organized in two phases of 4 columns each; filter material was sampled for 95 DNA and RNA extraction just before the onset of each experimental phase. In the 4 treatments, the + 96 influent waters were spiked with: (i) NH4 (NH4Cl at 1 mg/L N (71 M); Sigma-Aldrich, 254134), + 97 (ii) NH4 and ATU (N-Allylthiourea at 100 µM, Merck chemicals, 808158) , (iii) NO2ˉ (NaNO2 at 1 + 98 mg/L N (71 M)) Sigma-Aldrich, S2252), (iv) NH4 and ClO3ˉ (KClO3ˉ at 1 mM; 99%, Sigma- + 99 Aldrich, 12634) (Table 1) (26).(26). The applied flow rates (40 mL/hr) and influent (NH4 or NO2ˉ) + 3 100 concentrations were set to match the volumetric NH4 -N loading rates (approx. 1.5 g N/m /hr) 101 experienced by the full-scale parent biofilter (14). Test and control columns were operated for 15 102 days with continuous feeding to allow sufficient 13C label incorporation.