## 3 Results
### Metagenomic Pathway Enrichment  
The microbial genetic pathways for each of the 126 metagenomic samples were summarized by MetaPhlAn3. The relative pathway abundances for each of the silver treatments (31 ppb, 125 ppb, 500 ppb silver) were compared to the relative pathway abundances of the control (0 ppb silver) samples within the arcillite and hydroponics setups. Each treatment type has a replicate of at least three samples (Supplemental Table S2). The differentially expressed pathways in the silver treatments of the arcillite setup illustrate pathway commonalities (Supplemental Table S3). All arcillite-grown silver treatment samples show a greater than 2.5 log2 FC for ECASYN-PWY: enterobacterial common antigen biosynthesis. In addition, the bacteria represented in the 31 ppb and 125 ppb treatments share an enrichment in pathways PWY-8173, PWY-8174, and PWY-8175 associated with branched-chain fatty-acid biosynthesis. The pathway common to the 125 ppb and 500 ppb treatments includes a decrease in NAD-BIOSYNTHESIS-II: NAD salvage pathway III (to nicotinamide riboside), commonly affected by carbon and nitrogen availability. Furthermore, the 31 ppb and 500 ppb treatments both hold in common an enrichment of the PWY-5896 superpathway of menaquinol-10 biosynthesis involved in the generation of vitamin K2. Figure 2: (**A**) Image of arcillite-grown plants at 28 DAP. (**B**) Harvest metrics for arcillite grown lettuce averaged for each of the three treatment grow outs. ns is not significant. Figure 3: (**A**) Image of hydroponic-grown plants at 28 DAP. (**B**) Harvest metrics for hydroponic-grown lettuce results averaged for each of the three treatment grow outs. ******* is \(p\leq 0.0001\), *** is \(p\leq 0.001\), ns is not significant. Figure 4: Lettuce leaf elemental content of arcillite-grown plants at 28 DAP average for each of the three treatment grow-outs. No silver was detected. ns is not significant. Figure 5: Lettuce leaf silver and elemental content of hydroponically grown plants at 28 DAP averaged for each of the three treatment grow-outs. *** is \(p\leq 0.001\); *** is \(p\leq 0.01\); * is \(p\leq 0.05\); ns is not significant. Figure 6: Boxplots of Shannon index alpha diversity metrics for silver-treated: (**A**) arcillite roots; (**B**) arcillite water; (**C**) arcillite shoots; (**D**) hydroponic roots; (**E**) hydroponic water, each at 28 DAP; and (**F**) weekly hydroponic water sampling averaged for all replicates over the three grow-outs; pairwise \(p\)-values. ** is \(p\leq 0.01\); * is \(p\leq 0.05\); ns is not significant. In the 31 ppb hydroponic-grown root samples, no bacterial genomic pathways were enriched as compared to the control hydroponic root sample (Figure 8A). However, thirteen differentially enriched pathways for the 125 ppb silver-treated hydroponic root samples overlapped with the differentially enriched pathways of the 500 ppb silver-treated hydroponic root samples when compared to the control hydroponic root samples (Figure 8A). These enriched pathways are present in the genomes of microbes, which increased in both diversity and abundance within the plant roots as the silver concentration increased in the hydroponic tray water. Such enriched pathways include BRANCHED-CHAIN-AA-SYN-PWY: branched-chain amino acid biosynthesis encompassing valine (VALSYN-PWY) and isoleucine (ILEUSYN-PWY) biosynthesis as well as fatty-acid biosynthesis (PWY-7664: oleate biosynthesis IV (anaerobic), PWY0-862: (5Z)-dodecenoate biosynthesis I, and PWY-6282: palmitoelastic biosynthesis I (from (5Z)-dodec-5-enoate)) and elongation. This is accompanied by enrichment in the ability to utilize the glyoxylate cycle (GLYOXYALTE-BYPASS: glyoxylate cycle) and access substrates such as acetyl-CoA, fatty acids, alcohols, esters, waxes, alkenes, and methylated compounds. PWY66-389: phytol degradation is also enriched, likely allowing for this plant-derived compound to be metabolized by the microbe (Figure 8B).