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Trophic Status and Local Conditions Affect Microbial Potential for Denitrification

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Trophic Status and Local Conditions Affect Microbial Potential for Denitrification bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452135; this version posted July 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 1 Trophic status and local conditions affect microbial potential for denitrification 2 versus internal nitrogen cycling in lake sediments 3 Baumann K.B.L.1,2, Thoma R.1,2, Callbeck C.M.1#, Niederdorfer R.1, Schubert C.J. 1,2, 4 Müller B.1, Lever M.A.2, Bürgmann H.1*¶ 5 *corresponding author 6 1) Eawag, Swiss Federal Institute for Aquatic Science and Technology, Department 7 of Surface Waters-Research and Management, 6047 Kastanienbaum, 8 Switzerland 9 2) ETH Zurich, Institute of Biogeochemistry and Pollutant Dynamics (IBP), 10 Universitätstrasse 16, 8092 Zurich, Switzerland 11 #current address: University of Basel, Biogeochemistry, Bernoullistrasse 30 12 CH-4056 Basel, [email protected] 13 Keywords: Metagenomics, microbial ecology, freshwater, porewater, DNRA, 14 Nitrification, Denitrification, Anammox, Comammox 15 Abstract 16 The nitrogen (N) cycle is of global importance as N is an essential element and a 17 limiting nutrient in terrestrial and aquatic ecosystems. Excessive anthropogenic N 18 fertilizer usage threatens sensitive downstream aquatic ecosystems. Although 19 freshwater lake sediments remove N through various microbial transformation 20 processes, few studies have investigated the microbial communities involved. In an 21 integrated biogeochemical and microbiological study on a eutrophic and oligotrophic 22 lake, we estimated N removal rates in the sediments from porewater concentration 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452135; this version posted July 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 23 gradients. Simultaneously, the abundance of different microbial N transformation 24 genes was investigated using metagenomics on a seasonal and spatial scale. We 25 observed that contrasting nutrient concentrations in the sediments were reflected in 26 distinct microbial community compositions and significant differences in the 27 abundance of various N transformation genes. Within each lake, we observed a 28 more pronounced spatial than seasonal variability. The eutrophic Lake Baldegg 29 showed a higher denitrification potential with higher nosZ gene (N2O reductase) 30 abundance and higher nirS:nirK (nitrite reductase) ratio, indicating a greater capacity 31 for complete denitrification. Correspondingly, this lake had a higher N removal 32 efficiency. The oligotrophic Lake Sarnen, in contrast, had a higher potential for DNRA 33 and nitrification, and specifically a high abundance of Nitrospirae, including some 34 capable of comammox. In general, the oligotrophic lake ecosystems had a higher 35 microbial diversity, thus acting as an important habitat for oligotrophic microbes. Our 36 results demonstrate that knowledge of the genomic N transformation potential is 37 important for interpreting N process rates and understanding the limitations of the N 38 cycle response to environmental drivers. 39 Importance¶ 40 Anthropogenic nitrogen (N) inputs can lead to eutrophication in aquatic systems, 41 specifically in N limited coastal ecosystems. Lakes act as N sinks by transforming 42 reactive N to N2 through denitrification or anammox. The N cycle in lake sediments is 43 mediated by microbial processes and affected by environmental drivers such as the 44 amount and quality of settling organic material or nitrate concentration. However, the 45 microbial communities mediating the different N transformation processes and their 46 impact on N removal in freshwater lake sediments remain largely unknown. We 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452135; this version posted July 14, 2021. 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 4.0 International license. 47 provide the first seasonally and spatially resolved metagenomic analysis of the N 48 cycle in the sediments of two lakes with different trophic states. We show that the 49 trophic state of lakes provokes other microbial communities with characteristic key 50 players and functional potential for N transformation. 51 Introduction 52 Nitrogen (N) is an essential nutrient, and microbes play central roles in the natural N 53 cycle. Only N-fixing microbes can convert di-nitrogen (N2) to reactive N (Nr; N - - + 54 compounds readily available for biological conversion; e.g., NO3 , NO2 , NH4 , N2O). 55 The different Nr compounds are used as electron acceptors or donors in several 56 microbial metabolic pathways. Further, N can be a limiting nutrient in many terrestrial 57 and aquatic ecosystems, such as the coastal ocean 1–3. Thus, excessive 58 anthropogenic Nr input, e.g., from runoff of agricultural fertilizer, human wastewater, 59 and fossil fuel combustion, can lead to trophic changes in aquatic ecosystems and 60 the occurrence of harmful algal blooms 4,5. Switzerland is an essential headwater 61 system of European rivers and a non-negligible N source despite its small area. N 62 loads from atmospheric deposition (44 kt yr-1), mineral fertilizers (52 kt yr-1), and 63 sewage (43 kt yr-1) are about equally responsible for the N contamination of 64 ecosystems in Switzerland 6. Atmospheric deposition rates on the Swiss Plateau, 65 locally exceeding 40 kg N ha-1yr-1, are among the highest in the world 6,7. Today, 66 Switzerland exports 63 kt yr-1 dissolved N via the Rivers Rhine and Rhone (8, an 67 average of 1995-2013). This load corresponds to 1.3% of the total export of Europe 68 to the seas (4761 kt yr-1; 9) and has remained high over recent decades. 69 Lakes have been identified as important N sinks that convert up to 90% of reactive N 10 70 to less bioavailable N2 gas and thus substantially reduce N loads to oceans . N 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452135; this version posted July 14, 2021. 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 4.0 International license. 71 removal occurs through several microbially-mediated processes, mainly in the 72 sediment at the oxic-anoxic transition zone. The main N loss processes are 73 denitrification and anaerobic ammonia oxidation (anammox), both of which produce 11–17 74 N2 as an end product, or long-term burial (sequestration) . The effectiveness of 75 this removal process depends on interactions with other processes in the N cycle 76 that provide or compete for their substrates and various other environmental factors. 77 A better understanding of the environmental and ecological controls of these N 78 removal processes is thus necessary to understand and predict the N removal by 79 lakes today and under future conditions of global change. 80 Many different environmental drivers influence the N transformation processes in 81 lakes. Suitable growth conditions and N substrates are key factors for the removal - 82 process. Accordingly, parameters that influence N removal rates are NO3 83 concentration, organic matter (OM) input and remineralization, or redox conditions, 1,4,10,11,16,18–23 84 indicated e.g., by O2 concentrations, among others . Most of these 85 factors are intricately linked to the overall trophic status of lakes. For example, Finlay 86 et al. 12 reported seven-fold higher N removal rates in eutrophic than in oligotrophic 87 lakes. Other studies found that inter- and intra-lake variations in denitrification rates 88 were positively correlated with water residence times and N loads 5,24,25. 89 It is less well understood how these system observations are linked to the ecology of 90 the microbial communities that mediate these processes. By studying the diversity, 91 ecology, and identity of the microbial populations involved in the N transformation 92 processes, we better understand the mechanisms underlying efficient N removal in 93 lake ecosystems. Microbial ecology has made considerable progress over recent 94 decades on detecting, characterizing, and quantifying the key gene families encoding 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452135; this version posted July 14, 2021. 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 4.0 International license. 95 for central enzymatic systems of the N transformation pathways (N transformation 96 genes) in environmental systems using a variety of molecular tools 15,17,26–29. This 97 has improved our understanding of the microbial ecology of nitrification, complete 98 ammonia oxidation (comammox), assimilatory and dissimilatory nitrate reduction to 99 ammonia (ANRA and DNRA), denitrification, anammox, and N2 fixation 100 1,14,22,27,28,30,31. Metagenomic sequencing technology allows for a more holistic view 101 on microbial N cycling by characterization of multiple N gene families in parallel 32. 102 Several metagenomic studies focused on understanding the N cycle and the 103 microbes involved. 33, for example, found a significant change in the microbial 104 community and its metabolic function along a gradient of N availability in different 105 soils. Nelsen et al. 34 showed that the soil C and N content explained the N pathway 106 frequency and that N cycling specialists were encoding a few transformation 107 processes, as well as generalists were encoding all N transformation processes.
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