ChemRxiv preprint | deposited on May 4, 2020 Systems Microbiology and Engineering of Aerobic-Anaerobic Ammonium Oxidation David G. Weissbrodt1,*, George F. Wells2, Michele Laureni1, Shelesh Agrawal3, Ramesh Goel4, Giancarlo Russo5, Yujie Men6, David R. Johnson7, Magnus Christensson8, Susanne Lackner3, Adriano Joss7, Jeppe Lund Nielsen9, 7 7,10 Helmut Bürgmann and Eberhard Morgenroth 1Department of Biotechnology, Delft University of Technology, Delft, The Netherlands 2Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL, USA 3Technische Universität Darmstadt, Institute IWAR, Chair of Wastewater Engineering, Darmstadt, Germany 4Department of Civil and Environmental Engineering, The University of Utah, Salt Lake City, UT, USA 5Functional Genomics Center, ETH Zürich and University of Zürich, Zürich, Switzerland 6Department of Chemical and Environmental Engineering, University of California, Riverside, USA 7Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland 8Veolia Water Technologies, AnoxKaldnes, Lund, Sweden 9Department of Chemistry and Bioscience, Center for Microbial Communities, Aalborg University, Aalborg Denmark 10Institute of Environmental Engineering, ETH Zürich, Zürich, Switzerland Abstract: Aerobic and anaerobic oxidations of ammonium are core biological pro- cesses driving the nitrogen cycle in natural and engineered microbial ecosystems. These conversions are tailored in mixed-culture biotechnology to propel partial nitritation and anammox (PN/A) for a complete chemolithoautotrophic removal of nitrogen from wastewater at low resource and energey expenditures. Good practices of microbiome science and engineering are needed to design microbial PN/A systems and translate them to a spectrum of wastewater environments. Inter-disciplinary investigations of systems microbiology and engineering are par- amount to harness the microbial compositions and metabolic performance of complex microbiomes. We propose “process ecogenomics” as an integration ground to combine community systems microbiology and microbial systems en- Partial nitritation and anammox (PN/A) is attrac- gineering by establishing a synergy between the life and physical sciences. It tive for a complete chemolithoautotrophic re- drives a high-resolution analysis, engineering and management of microbial com- moval of nitrogen from wastewater. Mixed-cul- munities and their metabolic performance in mixed-culture systems. While ad- ture biotechnologies like PN/A rely on systems- dressing the key underpinnings of the science and engineering of aerobic-anaer- level understanding and management of complex obic ammonium oxidations, we advocate the need to formulate targeted re- microbial communities. “Process ecogenomics” is search questions in order to elucidate and manage microbial ecosystems in promoted to integrate community systems micro- wastewater environments. We propose a systems-level roadmap to investigate biology into process physical designs. Scientific and functional engineer technical microbiomes like PN/A, via: (i) quantitative bi- otechnological measurement of stoichiometry and kinetics of nitrogen turnovers; elucidation of microbial and metabolic networks of (ii) genome-centric metagenomic fingerprinting of the microbiome; (ii) ecophys- microbiomes should provide readily assimilable in- iological examination of the main metabolizing lineages; (iii) multi-omics elucida- formation to engineer them. Systems microbiology tion of expressed metabolic functionalities across the microbial network; and (iv) delivers thorough insights on the balance of meta- translation of microbial and functional ecology principles into physical designs. bolic interactions in microbiomes that should then Keywords: Mixed-culture biotechnology; Microbial resource management; Mi- be managed by engineering of process conditions. crobial community engineering; Systems microbiology; Process ecogenomics; Ni- tritation-anammox. Acronyms and abbreviations AMO: anaerobic ammonium-oxidizing organism; anammox: anaerobic ammonium oxidation; ANRA: assimilatory nitrite reduc- tion to ammonia; AOA: ammonium-oxidizing archaeum; AOB: ammonium-oxidizing bacterium; AOO: aerobic ammonium-oxi- dizing organism; ASV: amplicon sequence variants; BNR: biological nutrient removal; Ca.: Candidatus; CLSM: confocal laser * Correspondence: Prof. David Weissbrodt, Assistant Professor, TU Delft, TNW-BT-EBT, Building 58, van der Maasweg 9, NL-2629 HZ Delft, The Netherlands; Tel +31 15 278 1482; e-mail: [email protected] Weissbrodt, Wells, Laureni, Agrawal, Goel, Russo, Men, Johnson, Christensson, Lackner, Joss, Lund Nielsen, Bürgmann and Morgenroth (2020) Systems Microbiology and Engineering of Aerobic-Anaerobic Ammonium Oxidation | ChemRxiv preprint | 2 of 34 scanning microscopy; DNA: deoxyribonucleic acid; DNRA: dissimilatory nitrate reduction to ammonia; DHO: heterotrophic denitrifying organism; EFM: epifluorescence microscopy; EPS: extracellular polymeric substances; FISH: fluorescence in situ hybridization; FLBA: fluorescence lectin-binding analysis; gDNA: genomic DNA; HRT: hydraulic retention time; MAR: micro- autoradiography; MCE: microbial community engineering; mRNA: messenger RNA; NanoSIMS: nanoscale secondary ion mass spectrometry; N-DAMO: nitrite-driven anaerobic methane oxidation; NOO: nitrite-oxidizing organism; OHO: ordinary hetero- trophic organism; OTU: operational taxonomic unit; PCR: polymerase chain reaction; PN/A: partial nitritation and anammox; qFISH: quantitative FISH; qPCR: quantitative real-time PCR; RNA: ribonucleic acid; rRNA: ribosomal RNA; RT: reverse tran- scription; SIP: stable isotope probing; SRT: sludge retention time. 1 Introduction Evolving concepts on the field of PN/A are illus- trated by Fig. 1. The biotechnology of the nitrogen cycle 1 is an at- tractive field under constant evolution and redis- Full-scale PN/A systems are increasingly imple- covery 2 to promote sustainable solutions for mented to treat high loads of nitrogen from concen- wastewater treatment and environmental services 3. trated streams such as anaerobic digester centrates Complex ecosystems like activated sludge remains (“side streams” on flow-schemes of wastewater a vast below 4. Discoveries of new organismal treatment plants, WWTPs), landfill leachates, live- functions promote new engineering designs 5. Sig- stock effluents, and source-separated urine 23-27. nificant breakthroughs in microbiological science Current incentives target PN/A implementation for 6, process engineering science 7, and analytical bi- the direct treatment of diluted municipal oscience 8 have led to better understand and make wastewaters (“main streams”) 28-35. Success in this use of metabolic interactions that govern nitrogen endeavor may constitute a major advance for the conversions. This microbial network comprises a environmental engineering sector, while techno- diversity of lithoautotrophic, organoheterotrophic logical challenges still needs to get overcome. An and nitrogen-fixing organisms that act in concert to efficient combination of several disciplines within metabolize nitrogen. environmental biotechnology is needed to this end 36. The completely autotrophic process of aerobic (i.e., nitrification) and anaerobic ammonium oxidation 1.2 Managing PN/A processes under the scope of (anammox) is economically and technologically systems microbiology appealing to remove nitrogen from wastewater at low energetic and resource expenditures 9-11. This PN/A processes harbor high potential to improve open mixed-culture biotechnology is referred to as the energy efficiency of WWTPs while achieving partial nitritation and anammox (PN/A) in engi- effective nitrogen removal 7,37. Engineering ambi- neering practice 12,13. It is one masterpiece of envi- tions and limitations have been formulated. Fol- ronmental biotechnology. lowing promising start-up behavior, longer-term PN/A operations have been related to process in- 1.1 An established potential from microbial process stabilities and operational challenges 23,38. Main- discovery to engineering innovation taining a reliable performance is not trivial 39 since PN/A systems should rely on a coordinated activity PN/A developments started from the postulation and cross-feeding between AOO and AMO guilds for and discovery of novel chemolithoautotrophic 40. PN/A biosystem performance and instability microorganisms involved in anammox from natu- should be elucidated on a continuum from process ral 14,15 and engineered settings 16,17. Anammox is engineering to environmental biotechnology and credited with massive transformations of nitrogen systems microbiology. Systems-level investiga- and production of about half of atmospheric dini- tions 41 should bridge process boundaries (i.e., trogen 18-21. Engineering efforts to metabolically macro scale), bioaggregate properties (i.e., meso combine guilds of anammox organisms (referred to scale), and microbial communities (i.e., micro as AMOs according to standardized notation by scale) from populations to their genomes and ex- Corominas, et al. 22) with their aerobic counterparts pressed metabolisms. (AOOs) has driven the attractiveness of PN/A on the biological wastewater treatment market 7. Weissbrodt, Wells, Laureni, Agrawal, Goel, Russo, Men, Johnson, Christensson, Lackner, Joss, Lund Nielsen, Bürgmann and Morgenroth (2020) Systems Microbiology and Engineering of Aerobic-Anaerobic Ammonium Oxidation | ChemRxiv preprint | 3 of 34 Figure 1. Evolution of the