Biogeosciences, 17, 3203–3222, 2020 https://doi.org/10.5194/bg-17-3203-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. The contribution of microbial communities in polymetallic nodules to the diversity of the deep-sea microbiome of the Peru Basin (4130–4198 m depth) Massimiliano Molari1, Felix Janssen1,2, Tobias R. Vonnahme1,a, Frank Wenzhöfer1,2, and Antje Boetius1,2 1Max Planck Institute for Marine Microbiology, Bremen, Germany 2HGF MPG Joint Research Group for Deep-Sea Ecology and Technology, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany apresent address: UiT the Arctic University of Tromsø, Tromsø, Norway Correspondence: Massimiliano Molari ([email protected]) Received: 16 January 2020 – Discussion started: 3 February 2020 Revised: 27 April 2020 – Accepted: 15 May 2020 – Published: 25 June 2020 Abstract. Industrial-scale mining of deep-sea polymetal- tween the Clarion–Clipperton Fracture Zone (CCZ) and the lic nodules will remove nodules in large areas of the sea Peru Basin suggest that changes in environmental setting floor. The regrowth of the nodules by metal precipita- (e.g. sedimentation rates) also play a significant role in struc- tion is estimated to take millions of years. Thus, for fu- turing the nodule microbiome. ture mining impact studies, it is crucial to understand the role of nodules in shaping microbial diversity and function in deep-sea environments. Here we investigated microbial- community composition based on 16S rRNA gene sequences 1 Introduction retrieved from sediments and nodules of the Peru Basin (4130–4198 m water depth). The nodule field of the Peru Polymetallic nodules (or manganese nodules) occur in Basin showed a typical deep-sea microbiome, with domi- abyssal plains (4000–6000 m water depth) and consist pri- nance of the classes Gammaproteobacteria, Alphaproteobac- marily of manganese and iron as well as many other metals teria, Deltaproteobacteria, and Acidimicrobiia. Nodules and and rare earth elements (Crerar and Barnes, 1974; Kuhn et sediments host distinct bacterial and archaeal communities, al., 2017). Nodules are potato- or cauliflower-shaped struc- with nodules showing lower diversity and a higher propor- tures with typical diameters of 4–20 cm and are typically tion of sequences related to potential metal-cycling Bac- found at the sediment surface or occasionally buried in the teria (i.e. Magnetospiraceae, Hyphomicrobiaceae), bacterial uppermost 10 cm of the sediment horizon. The mechanisms and archaeal nitrifiers (i.e. AqS1, unclassified Nitrosomon- of nodule formation are not completely elucidated. The cur- adaceae, Nitrosopumilus, Nitrospina, Nitrospira), and bac- rent understanding is that they are formed via mineral pre- terial sequences found in the oceanic crust, nodules, hy- cipitation from bottom waters (hydrogenetic growth) or pore drothermal deposits, and sessile fauna. Sediment and nod- waters (diagenetic growth) involving both abiotic and mi- ule communities overall shared a low proportion of opera- crobiological processes (Crerar and Barnes, 1974; Riemann, tional taxonomic units (OTUs; 21 % for Bacteria and 19 % 1983; Halbach et al., 1988; Wang et al., 2009). The forma- for Archaea). Our results show that nodules represent a spe- tion of nodules is a slow process that is estimated to range cific ecological niche (i.e. hard substrate, high metal concen- between thousands and millions of years per millimetre of trations, and sessile fauna), with a potentially relevant role growth (Kerr, 1984; Boltenkov, 2012). in organic-carbon degradation. Differences in nodule com- Rising global demand for metals has renewed interests munity composition (e.g. Mn-cycling bacteria, nitrifiers) be- in commercial mining of deep-sea nodule deposits. Min- ing operations would remove nodules, disturb or erode the Published by Copernicus Publications on behalf of the European Geosciences Union. 3204 M. Molari et al.: Microbial communities in polymetallic nodules of the Peru Basin top decimetres of sediment, and create near-bottom sediment approximately 2 times higher than in the CCZ, resulting in plumes that would resettle and cover the sea floor (Miller higher content of organic carbon in the surface sediments et al., 2018). Although the first nodules were discovered in (> 1 % vs. 0.2 %–0.6 % in the CCZ) and a shallower oxic– the 1870s (Murray and Renard, 1891), only little is known sub-oxic front (10 cm vs. tens of metres of sediment depth about the biodiversity, biological processes, and ecological in the CCZ; Müller et al., 1988; Haeckel et al., 2001; Volz functions of the nodules and their surrounding sediments as a et al., 2018). As a consequence of differences in environ- specific deep-see habitat. Major questions remain, for exam- mental conditions (e.g. organic carbon flux, carbonate com- ple as to spatial turnover on local and global scales, the role pensation depth, sediment type, topography, and near-bottom of the microbial community in and around nodules, and the currents), the Peru Basin and the CCZ host manganese nod- role of nodules as substrate for endemic species. Hence, there ules with different geological features (Kuhn et al., 2017): is the need to thoroughly characterise baseline conditions as (i) nodules from the Peru Basin are often larger, with a typi- a requirement for any mining operations as these will require cal cauliflower shape, compared to those in the CCZ, which assessments of impacts associated with mining. have a discoidal shape and a size of 2–8 cm (Kuhn et al., Extensive and dense nodule fields are found in different ar- 2017); (ii) the average nodule abundance in the Peru Basin eas of the Pacific and Indian oceans. Nodule accumulations is lower (10 kg m−2) than in the CCZ (15 kg m−2; Kuhn et of economic interest have been found in four geographical al., 2017); (iii) Mn nodules from the Peru Basin are thought locations: the Clarion–Clipperton Fracture Zone (CCZ) and to be mainly formed by sub-oxic diagenesis, whereas CCZ the Penrhyn Basin in the north-central and south-central Pa- nodules apparently exhibit a mixture of diagenetic and hy- cific Ocean, respectively; the Peru Basin in the south-eastern drogenetic origin (von Stackelberg, 1997; Chester and Jick- Pacific; and in the centre of the northern Indian Ocean (Miller ells, 2012); (iv) while Peru Basin and CCZ nodules consist et al., 2018). To our knowledge the Peru Basin is the only re- of the same type of mineral (disordered phyllomanganates), gion that does not have exploration activities and plans for they have a different metal content (Wegorzewski and Kuhn, mining so far. Previous work on the structure of microbial 2014; Wegorzewski et al., 2015). communities of nodule fields by 16S rRNA gene sequenc- An increasing number of studies and policy discussions ing has focused on the CCZ and the south-central Pacific address the scientific basis of ecological monitoring in deep- Ocean (Xu et al., 2007; Wu et al., 2013; Tully and Heidel- sea mining, highlighting the need to identify appropriate in- berg, 2013; Blöthe et al., 2015; Shulse et al., 2017; Lindh dicators and standards for environmental impact assessments et al., 2017). All studies showed that polymetallic nodules and ecological management. A key aspect is avoiding harm- harbour microorganisms that are distinct from the surround- ful effects to the marine environment, which will have to in- ing sediments and overlying water. They indicate that nod- clude loss of species and ecosystem functions. The primary ule communities show a pronounced spatial variability, but aims of this study were to assess the structure and similarity these results are so far not conclusive. Similar microbial com- of benthic microbial communities of nodules and sediments munities were observed in nodules collected at distances of of the Peru Basin nodule province and to compare them with 6000 and 30 km (Wu et al., 2013; Shulse et al., 2017), while those of other global deep-sea sediments and nodules in the Tully and Heidelberg (2013) found that nodule communities CCZ. The focus was on similarity comparisons in order to in- varied among sampling sites ( < 50 km). Besides, potential vestigate endemism and potential functional taxa that could Mn oxidisers and reducers such as Alteromonas, Pseudoal- be lost due to the removal of manganese nodules by mining teromonas, Shewanella, and Colwellia were proposed as a activities. To achieve this, the hypervariable 16S rRNA gene core of the nodule microbiome involved in the formation of regions V3–V4 for Bacteria and V3–V5 for Archaea were nodules (Wu et al., 2013; Blöthe et al., 2015), but they were amplified from DNA extracted from nodules and surround- not found in all nodules sampled so far (Tully and Heidel- ing sediments and sequenced using the Illumina paired-end berg, 2013; Shulse et al., 2017). The lack of knowledge on MiSeq platform. The hypotheses tested were that (i) nodules the diversity and composition of microbial assemblages of shape deep-sea microbial diversity and (ii) nodules host a other nodule provinces makes it difficult to assess whether specific microbial community compared to the surrounding observed differences within the CCZ may reflect regional dif- sediments. The secondary aim of this study was to investi- ferences in environmental conditions (e.g. input of organic gate the nodule features that may play a major role in shaping matter, bathymetry, topography, sediment type), in the abun- microbial-community composition and microbially mediated dance and morphology of nodules, or in the colonisation of functions. the nodules by epifauna and protozoans. In this study we investigated the diversity and composi- tion of bacterial and archaeal communities associated with manganese nodule fields of the Peru Basin. The Peru Basin is located about 3000 km off the coast of Peru and covers about half of the size of the CCZ, which is 5000–9000 km away. The present-day organic carbon flux in this area is Biogeosciences, 17, 3203–3222, 2020 https://doi.org/10.5194/bg-17-3203-2020 M.