Microbial communities associated with sediments and 1 polymetallic nodules of the Peru Basin 2 3

Abstract. Industrial-scale mining of deep-sea polymetallic nodules will need to remove nodules in large areas of the seafloor. The regrowth of the nodules by metal precipitation is estimated to take millions of years. Thus for future 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 retrieved from sediments and nodules of the Peru Basin (> 4100 m water depth). The nodule field of the Peru Basin showed a typical deep-sea microbiome, with dominance of the classes Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, and Acidimicrobiia. Nodules and sediments host distinct bacterial and archaeal communities, with nodules showing lower diversity and a higher proportion of sequences related to potential metal-cycling bacteria (i.e. Magnetospiraceae, Hyphomicrobiaceae), bacterial and archaeal nitrifiers (i.e. AqS1, unclassified Nitrosomonadaceae, Nitrosopumilus, Nitrospina, Nitrospira), and bacterial sequences found in ocean crust, nodules, hydrothermal deposits and sessile fauna. Sediment and nodule communities overall shared a low proportion of Operational Taxonomic Units (OTU; 21 % for Bacteria and 19 % for Archaea). Our results show that nodules represent a specific ecological niche (i.e. hard substrate, high metal concentrations and sessile fauna), with a potentially relevant role in organic carbon degradation. Differences in nodule community composition (e.g. Mn-cycling bacteria, nitrifiers) between the Clarion-Clipperton Fracture Zone (CCZ) and the Peru Basin suggest that changes in environmental setting (i.e. sedimentation rates) play also a significant role in structuring the nodule microbiome.


. Similarly, contaminant sequences (as observed in the negative control) and 156 unspecific sequences (i.e., bacterial sequences in the archaeal amplicon dataset, and archaeal, 157 chloroplast, and mitochondrial sequences in the bacterial dataset) were removed from amplicon data 158 sets before the analysis ( Table 2). The dominant OTU sequences and OTU sequences highly abundant 159 in the nodules were subjected to BLAST search (BLASTn; Gene Bank nucleotide database

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The number of bacterial sequences retrieved from DNA extracted from sediments and nodules was on 195 average 5±5 and 25±14 times higher, respectively, than those obtained for archaea (t-test: p<0.001, 196 df=11, t=4.5).
197 Table 2 shows the statistics of sequence abundance and proportion of singletons and cosmopolitan 198 types. Sequence abundances of bacteria were comparable between sediments and nodules. OTUs (present exclusively in one station). For this analysis, the latter were calculated with sequence 207 re-sampling, to overcome differences in sequencing depth. Abundance-based coverage estimators, 208 exponential Shannon (Hill number q=1; H 1 ) and inverse Simpson (Hill number q=2; H 2 ), were also 209 calculated. The rarefaction curve indicates that the richness (H 0 ) of the less abundant and rare OTUs 210 was somewhat underestimated both in nodules and in sediments ( Figure S1 a-b). However, the 211 bacterial and archaeal diversity was well described for the abundant OTUs (H 1 and H 2 ; Figure S1 a-b); 212 with more than 90 % of the estimated diversity covered (Figure S1 c-d). Both in sediments and nodules 213 the alpha-diversity indices were higher for Bacteria than for Archaea (t-test: p<0.0001, df=12, 214 t=8.0−16.0), while the contribution of unique OTUs to the total number of OTUs was comparable 215 (Table 3). Bacterial communities in manganese nodules have lower Hill numbers and Chao1 indices 216 compared to those associated to sediments (Table 3, Figure 1a). Archaeal communities showed the 217 same patterns for diversity indices and unique OTUs, with exception for H 2 index that did not show 218 significant difference between nodules and sediments (Table 3, Figure 1b). The changes in microbial community structure at OTU level (beta-diversity) between substrates and 221 samples were quantified by calculating Bray-Curtis dissimilarities from CLR transformed OTU

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Microbial communities associated to nodules are generally less diverse than those in the sediments, and 368 the decrease in diversity was observed both in rare and abundant bacterial types (Figure 1 and S1). This

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(PERMANOVA) showed significant differences between nodule and sediment associated microbial 734 communities (for details see Table S1). Each sample (dot) is connected to the weighted averaged mean 735 of the within group distances. Ellipses represent one SD of the weighted averaged mean.         https://doi.org/10.5194/bg-2020-11 Preprint. Discussion started: 3 February 2020 c Author(s) 2020. CC BY 4.0 License.