Linnea K. Honeker*, Julia W. Neilson, Jon Chorover, Raina M. Maier Effect of Re-Acidification on Buffalo Grass Rhizosphere and Bulk Microbial Communities During Phytostabilization of Metalliferous Mine Tailings Linnea K. Honeker*, Julia W. Neilson, Jon Chorover, Raina M. Maier Department of Soil, Water, and Environmental Science, The University of Arizona, Tucson, AZ *correspondence: linneah@email.arizona.edu Introduction Results (cont.) Results Results (cont.) The legacy mine tailings at Iron King Mine and Humboldt Smelter Superfund Site (IKMHSS) in Dewey Humboldt, AZ poses a significant threat to surrounding communities due to wind erosion of contaminated tailings which contain up to 3,000 mg kg-1 of As and Pb. Sample Phase pH TOC (g kg-1) TN EC (dS m-1) Plot24b 1 2.35 47.6 3.60 10.20 Plot19b 2.47 51.1 3.46 4.46 Plot5a 2.88 35.2 3.07 3.42 Plot19a 44.6 3.66 2.97 Plot24a 3.49 57.1 3.76 2.93 Plot5b 39.6 3.29 3.17 Plot10b 5.38 49.4 4.50 5.47 Plot10a 6.01 72.2 5.22 7.94 Row2b 3 2.56 47.7 4.52 14.56 Row4b 4.71 48.7 5.78 5.76 Row2c 51.9 5.62 4.30 Row6a 6.54 99.2 9.04 8.10 Row6b 7.11 68.4 7.13 9.46 Row4c 7.72 126.8 9.54 9.83 Row2a 7.73 153.6 9.53 4.10 Row4a 7.76 140.8 8.68 8.86 Fig. 5. LEfSe analysis of R vs. B compartments of plants growing in high pH and low pH. At low pH, over 100 taxa were identified as differentially abundant in the rhizosphere (more than shown here). At high pH, much less taxa were differentially abundant in the R. Table 1. Physicochem-ical properties of surface samples collected next to each plant. Phase 3 pH was higher than Phase 1. pH, TOC, and TN are all positively correlated. Fig. 8. Bubble plot depicting relative abundance (size) and activity (color) of the most abundant putative Fe-cycling/S-oxidizing and PGPB/N-fixing OTUs. Overall patterns reveal that Fe-cycling/S-oxidizing bacteria are more abundant at low pH, and PGPB/N-fixing bacteria are more abundant at high pH in both the B and R. It is notable that at low pH, even though PBPB/N-fixing bacteria are essentially absent in the B, some PGPB/N-fixing bacteria persist in the R. pH 4 is indicated by vertical dashed line. Low pH (<4) High pH (>4) Phytostabilization is being studied as a remediation strategy at IKMHSS assisted by the addition of a compost-amendment1. Microbial communities in the rhizosphere and Bulk (pH ) Rhizosphere (pH ) Fig. 1. Mine Tailing wind erosion bulk compartments, such as plant growth-promoting bacteria (PGPB), can play an important role in plant survival in extreme mine tailing conditions (acidic pH, low nutrients and carbon, predominance of autotrophic Fe/S oxidizers, and high metal content)2,3. Due to erosion over time of compost-amendment, re-acidification of tailings has occurred in certain areas, creating high heterogeneity in pH and potential shifts in bulk and rhizosphere microbial communities. Fig. 3. Phylogenetic profiles for rhizosphere (R) and bulk (B) substrates from plants in Phase 1 and Phase 3 in order of ascending pH. Fig. 6. Beta diversity displayed as PCoA plot of unweighted UNIFRAC distances. The beta diversity of R and B from high pH are much less divergent than at low pH. Clusters outlined are significantly different (p<0.05, PERMANOVA). Phase 1 Low pH (R) High pH (R and B) Research Goals Relative Abundance (%) Relative abundance RNA/DNA ratio (activity) Identify pH threshold critical for maintaining the beneficial microbial community. Compare microbial communities and diversity between rhizosphere and bulk compartments. Characterize abundance and activity of PGPB and Fe/S cycling species in rhizosphere and bulk. 5 10 15 20 25 0 1 2 ≥3 Conclusions pH 4 is a critical point characterized by a shift in microbial community and diversity in the buffalo grass rhizosphere and bulk compartments. At low pH, there is an apparent difference between rhizosphere and bulk communities and diversity that is not observed at high pH, as evidenced by LEfSe, alpha diversity, beta diversity, and phylogenetic profile data. The observed increase in Fe-cycling and S-oxidizing bacteria in the rhizosphere and bulk at low pH will contribute to acidifying conditions. Because the bulk is the source from where the rhizosphere recruits bacteria, a drastic depletion in this compartment of PGPB/N-fixing bacteria may be detrimental for long-term continued buffalo grass growth. Phase 3 Low pH (B) Methods Rhizosphere (R) and bulk (B) samples were collected from buffalo grass plants across a range of pH (2.35-7.76) for DNA/RNA extractions. A surface sample was collected next to each plant for physico-chemical analysis. Relative Abundance (%) Fig. 7. Alpha diversity displayed as observed species (species richness). Species richness is higher at high pH and is similar between the R and B, a pattern indicative of a typical soil4. In contrast, at low pH, species richness of R is significantly greater than for B (p<0.05). 16S rRNA amplicon sequencing was performed on RNA and DNA samples. Sequences were processed and analyzed using QIIME v.1.9. As a proxy for activity, rRNA: rRNA gene ratios were calculated. Phase 1 (2010): Plots 5, 10, 19, 24 Fig. 4. Phylogenetic profiles of R and B representing averages from plants growing in low pH ( < 4) and high pH ( > 4) conditions. Greater variation evident between R and B of low pH compared to high pH. References Gil-Loaiza et al., 2016, Sci. Tot. Environ., 565: 451-461. Grandlic et al., 2008, Environ. Sci. Tech., 42: 2079-2084. De-Bashan et al., 2010, Microb. Ecol., 60: 915-927. Edwards et al., 2015, PNAS, E920. Relative Abundance (%) Phase 3 (2012): Rows 2, 4, 6 Fig. 2. Satellite view of IKMHSS phyto-stabilization study . Phase 1 is an older side initiated in 2010. Phase 3 was initiated in 2012. Acknowledgements This research material is based on work supported by the NIEHS SRP Grant P42 ES04940 and R01 ES17079 and the National Science Fellowship Program Grant DGE-1143953