Increased Replication Rates of Dissimilatory Nitrogen-Reducing Bacteria Leads to Decreased Anammox Reactor Performance

bioRxiv preprint doi: https://doi.org/10.1101/534925; this version posted January 30, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 Title: Increased Replication Rates of Dissimilatory Nitrogen-Reducing Bacteria Leads to Decreased 2 Anammox Reactor Performance 3 4 Authors: Ray Keren1*, Jennifer E. Lawrence1*, Weiqin Zhuang1,2, David Jenkins1, Jillian F. Banfield3, 5 Lisa Alvarez-Cohen1, Lijie Zhou1,4+, Ke Yu1,5+ 6 *These authors contributed equally to the work 7 +Corresponding authors 8 9 Affiliations 10 1- Department of Civil and Environmental Engineering, University of California Berkeley, 11 Berkeley, CA, USA 12 2- Department of Civil and Environmental Engineering, The University of Auckland, 13 Auckland, New Zealand 14 3- Earth and Planetary Sciences, University of California Berkeley, Berkeley, CA, USA 15 4- College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 16 China 17 5- School of Environment & Energy, Peking University, Beijing, China 18 19 20 Abstract 21 Anaerobic ammonium oxidation (anammox) is a biological process employed to remove reactive nitrogen 22 from wastewater. While a substantial body of literature describes the performance of anammox 23 bioreactors under various operational conditions and perturbations, few studies have resolved the 24 metabolic roles of community members. Here, we use metagenomics to study the microbial community 25 within a laboratory-scale anammox bioreactor from inoculation, through performance destabilizations, to 1 bioRxiv preprint doi: https://doi.org/10.1101/534925; this version posted January 30, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 26 stable steady-state. Metabolic analyses reveal that dissimilatory nitrogen reduction to ammonium 27 (DNRA) is the primary nitrogen removal pathway that competes with anammox in the bioreactor. 28 Increased replication rates of bacteria capable of DNRA leads to out-competition of annamox bacteria, 29 which is the key source of fixed carbon, and the loss of reactor performance. Ultimately, our findings 30 underline the importance of metabolic interdependencies related to carbon and nitrogen-cycling within 31 anammox bioreactors and highlight the potentially detrimental effects of bacteria that are otherwise 32 considered to be core community members. 33 34 35 36 Main Text 37 Anammox bacteria obtain energy from the conversion of ammonium and nitrite into molecular 38 nitrogen gas1. The only currently known bacteria to catalyze this process are Planctomycetes2,3, none of 39 which have yet been isolated3,4. In practice, anammox bacteria are employed in combination with the 40 partial nitritation (PN) process to remove ammonium from wastewaters or anaerobic digestor though side- 41 streams. First, in PN, approximately half of the ammonium in solution is aerobically oxidized to nitrite. 5,6 42 Second, in anammox, both ammonium and nitrite are anaerobically converted to N2 . PN/anammox is 43 beneficial because it consumes 60% less energy, produces 90% less biomass, and emits a significantly 44 smaller volume of greenhouse gases than conventional nitrogen removal by nitrification and 45 denitrification processes7. 46 Even though over 100 full-scale PN/anammox processes have been installed across the globe at 47 municipal and industrial wastewater treatment plants8 anammox bacteria have very low growth rates 48 within engineered environments and are easily inhibited by a variety of factors, including fluctuating 49 substrate and metabolite concentrations9,10. Furthermore, recovery from an inhibition event can take up to 50 six months, which is unacceptably long for municipalities who must meet strict nitrogen discharge 51 limits11. These problems are compounded by what is currently only a cursory understanding of the 2 bioRxiv preprint doi: https://doi.org/10.1101/534925; this version posted January 30, 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. 52 microbial communities responsible for stable, robust anammox performance. The broad application of 53 PN/anammox in wastewater treatment processes requires a more comprehensive understanding of the 54 complex interactions between the numerous bacterial species within the bioreactors. 55 Previous research suggests that a core microbial community exists within anammox bioreactors12– 56 16. In the majority of anammox bioreactors, besides Planctomycetes, uncultured members of the phyla 57 Bacteroidetes, Chloroflexi, Ignavibacteria, and Proteobacteria have been identified. Since these phyla 58 have primarily been identified through 16S rRNA studies, their interplay on anammox performance have 59 yet to be elucidated12–16. While anammox bacteria are responsible for the key nitrogen transformation 60 processes, additional groups of anaerobic nitrogen-cycling bacteria cooperate to transform and remove 61 nitrate, a product of anammox metabolism17–19. 62 Here, we illuminate the metabolic relationships between the anammox bacteria and its supporting 63 community members during the start-up and operation of a laboratory-scale anammox bioreactor. We 64 used genome-centric metagenomics to recover 337 draft genomes from six time-points spanning 440 days 65 of continuous bioreactor operation. Additionally, we combined our reconstruction of the microbial 66 community’s dynamic metabolic potential with bioreactor performance data and relative abundance 67 profiles based on both metagenomic and 16S rRNA sequencing. As a result, we were able to identify core 68 metabolic activities and potential interdependencies that inform the performance and stability of the 69 anammox bioreactor. We found that certain metabolic interactions between anammox bacteria and 70 associated community members may be responsible for the destabilization of anammox bioreactors. To 71 our knowledge, this is the first time-series-based study to link anammox metagenomic insights and 72 community composition to bioreactor functionality20. Our findings bolster the fundamental, community- 73 level understanding of the anammox process. Ultimately, these results will enable better understanding of 74 this important microbial process and a more comprehensive control of this promising technology that 75 should help facilitate its widespread adoption at wastewater treatment plants. 76 3 bioRxiv preprint doi: https://doi.org/10.1101/534925; this version posted January 30, 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. 77 Bioreactor performance. The performance of a laboratory-scale anaerobic membrane bioreactor 78 (described in methods) was tracked for 440 days from initial inoculation, through several performance 79 crashes, to stable and robust anammox activity (Figure 1). Performance was quantified in a variety of 80 ways, including by its nitrogen removal rate (NRR, g-N L-1 d-1). Bioreactor performance generally 81 improved over the first 103 days of operation. At this point, the hydraulic residence time was reduced 82 from 48 to 12 hours and influent concentrations were reduced to maintain a stable loading rate. 83 Additional biomass from a nearby pilot-scale PN/anammox process was added on Day 145 and reactor 84 performance improved enabling influent ammonium and nitrite concentrations to be steadily increased 85 until the NRR approached 2 g-N L-1 d-1. On Day 189 the bioreactor experienced a technical malfunction 86 and subsequent performance crash, identified by a rapid decrease in the NRR and the effluent quality. On 87 Day 203, the bioreactor was again amended with a concentrated stock of biomass and the NRR quickly 88 recovered. Influent ammonium and nitrite concentrations were again increased until the NRR reached 2 g- 89 N L-1 d-1. 90 The bioreactor subsequently maintained steady performance for approximately 75 days, until Day 91 288, when effluent concentrations of ammonium and nitrite unexpectedly began to increase and nitrate 92 concentrations disproportionately decreased. Seven days later, the NRR rapidly plummeted and had since 93 no technical malfunctions had occurred this indicated that a destabilized microbial community may have 94 been responsible for the performance crash. At that time, the cause of the performance decline was not 95 understood, so the bioreactor was not re-seeded with biomass. After 50 days of limited performance, 96 concentrations of copper, iron, molybdenum, and zinc in the bioreactor influent were increased21–24 and 97 the NRR rapidly recovered. Stable and robust bioreactor performance was subsequently maintained. 98 99 Metagenomic sequencing and binning. Whole community DNA was extracted and sequenced at six 100 time-points throughout the study: Day 0 (D0), for inoculant composition; Day 82 (D82), during nascent, 101 positive anammox activity; Day 166 (D166), three weeks after an

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