ORIGINAL RESEARCH published: 19 March 2021 doi: 10.3389/fmicb.2021.647861 Nutrient Exposure Alters Microbial Composition, Structure, and Mercury Methylating Activity in Periphyton in a Contaminated Watershed Alyssa A. Carrell 1, Grace E. Schwartz 2,3, Melissa A. Cregger 1, Caitlin M. Gionfriddo 1,4, Dwayne A. Elias 1, Regina L. Wilpiszeski 1, Dawn M. Klingeman 1, Ann M. Wymore 1, Katherine A. Muller 5 and Scott C. Brooks 2* 1 2 Oak Ridge National Laboratory, Biosciences Division, Oak Ridge, TN, United States, Oak Ridge National Laboratory, 3 Edited by: Environmental Science Division, Oak Ridge, TN, United States, Department of Chemistry, Wofford College, Spartanburg, 4 5 Tamar Barkay, SC, United States, Smithsonian Environmental Research Center, Edgewater, MD, United States, Pacific Northwest Rutgers, The State University of New National Laboratory, Earth Systems Science Division, Richland, WA, United States Jersey, United States Reviewed by: The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern Chu-Ching Lin, in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but National Central University, Taiwan Benjamin Peterson, little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, University of Wisconsin-Madison, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and United States Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and *Correspondence: Scott C. Brooks Hg-methylating microorganisms in periphyton communities grown in a contaminated [email protected] watershed in East Tennessee (United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and Specialty section: This article was submitted to function. We found that bacterial/archaeal richness in experimental conditions decreased Microbiological Chemistry and in summer and increased in autumn relative to control treatments, while fungal diversity Geomicrobiology, generally increased in summer and decreased in autumn relative to control treatments. a section of the journal Frontiers in Microbiology Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed Received: 30 December 2020 by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential Accepted: 22 February 2021 correlated with numerous bacterial families that do not contain hgcAB, suggesting that Published: 19 March 2021 the overall microbiome structure of periphyton communities influences rates of Hg Citation: Carrell AA, Schwartz GE, transformation within these microbial mats. To further explore these complex community Cregger MA, Gionfriddo CM, interactions, we performed a microbial network analysis and found that the nitrate- Elias DA, Wilpiszeski RL, amended treatment resulted in the highest number of hub taxa that also corresponded Klingeman DM, Wymore AM, Muller KA and Brooks SC (2021) with enhanced Hg methylation potential. This work provides insight into community Nutrient Exposure Alters Microbial interactions within the periphyton microbiome that may contribute to Hg cycling and will Composition, Structure, and Mercury Methylating Activity in Periphyton in a inform future research that will focus on establishing mixed microbial consortia to uncover Contaminated Watershed. mechanisms driving shifts in Hg cycling within periphyton habitats. Front. Microbiol. 12:647861. doi: 10.3389/fmicb.2021.647861 Keywords: periphyton, mercury, microbiome, methylmercury, nutrient addition Frontiers in Microbiology | www.frontiersin.org 1 March 2021 | Volume 12 | Article 647861 Carrell et al. Nutrient Addition Alters Periphyton Microbiome INTRODUCTION periphyton microbial community could be a crucial determinant of MMHg production. The conversion of mercury (Hg) to monomethylmercury (MMHg) Therefore, the objective of this study was to quantify the is a critical area of concern in global Hg cycling. Once MMHg impacts of nitrate and phosphate addition on the diversity, is produced, it is bioaccumulated in organisms and biomagnifies community composition, and Hg methylation activity of through food webs, ultimately impacting humans through the periphyton microbiomes in a Hg-contaminated stream across consumption of fish with elevated levels of MMHg. The two seasons. Specifically, we hypothesized nutrient amendments transformation of inorganic Hg into MMHg is mediated by would increase microbial interactions and diversity and therefore anaerobic microorganisms (Parks et al., 2013; Bravo and Cosio, increase methylation potentials. To test this, nutrient diffusion 2020), predominantly from the Deltaproteobacteria, substrates (NDS) were used to provide an artificial substrate Methanomicrobia, and Firmicutes (Christensen et al., 2016, for in situ periphyton growth and to test the response of the 2018). While significant work has examined Hg transformations periphyton microbiome to nitrate and phosphate additions in aquatic sediments (Bae et al., 2019; Branfireun et al., 2020; across two seasons. Xiang et al., 2020), less research has focused on aquatic microbial biofilms like periphyton. Recent work demonstrates that these biofilms may harbor significant amounts of MMHgMcDowell ( MATERIALS AND METHODS et al., 2020), but little is known about the community of microorganisms mediating Hg transformations. NDS cups were prepared to assess the impact of nitrate and Periphyton is a complex consortium of microorganisms phosphate exposure on periphyton microbial community including algae, fungi, bacteria, and archaea that are attached composition and Hg methylation and MMHg demethylation. to inorganic and organic substrates in aquatic environments. Periphyton biofilm growth, potential function, and community Mercury methylation in periphyton has been shown in many composition on the substrates were characterized with different ecosystems and has been attributed to sulfate-reducing measurements of biofilm mass; chlorophylla , b, and c; total bacteria (SRB) and methanogenic archaea that occupy anoxic ambient Hg and MMHg; Hg methylation and demethylation niches within the periphyton biofilm structure (Cleckner et al., potential; and amplicon sequencing of the ITS2 region and 1999; Achá et al., 2011; Hamelin et al., 2011). To our knowledge, 16S rRNA and hgcAB genes. there is only one study that has identified Hg-methylating microorganisms in periphyton using the Hg methylation gene cluster, hgcAB, as a biomarker (Bae et al., 2019). Bae et al. (2019) Nutrient Diffusion Substrate Preparation found that periphyton and flocculant material from the Florida NDS cups (Costello et al., 2016) were constructed using black, Everglades contained a diverse assemblage of hgcAB-containing 1-ounce plastic cups (#1 Poly-Cons®) with 2.5-cm holes drilled microorganisms, including Deltaproteobacteria, Chloroflexi, in the center of the lids (Supplementary Figure S1). The Firmicutes, and Methanomicrobia. In addition to harboring cups were filled to the top with a 2% agar solution Hg-methylating microorganisms, periphyton consortia can produce (bacteriological grade, VWR; control and single nutrient photosynthetic by-products and organic molecules that, in some treatments) or 3% agar solution (nitrate + phosphate). Four instances, increase Hg bioavailability and MMHg production nutrient treatments were prepared: control, nitrate (0.5 M (Mangal et al., 2019a,b; Branfireun et al., 2020). Thus, overall NaNO3), phosphate (0.5 M KH2PO4), and nitrate + phosphate MMHg production may be influenced by the interactions of (0.5 M NaNO3 + 0.5 M KH2PO4). For the nitrate treatment, the entire microbiome in the biofilm (Francoeur and Biggs, 2006; the nitrate salt was dissolved in Milli-Q water, which was Lázaro et al., 2013, 2018). brought to boiling before adding the agar. The same method Productivity of the periphyton microbiome is linked to was attempted with the phosphate-containing treatments, but abiotic factors such as temperature, light, stream velocity, and we found that the agar would not solidify when the phosphate dissolved oxygen (Biggs et al., 1999; Hill et al., 2001; Beck salt was added prior to dissolving the agar. So, for the et al., 2017). Nutrient types and concentration also play an phosphate and nitrate + phosphate treatments, Milli-Q water important role in the periphytic algal composition and periphyton was brought to a boil, the agar was added and allowed to productivity (Hoellein et al., 2010). Often nitrogen and dissolve, and then the nutrient salts were added to the hot phosphorus are limiting nutrients for periphyton growth in agar solution and mixed well. This preparation method should lotic freshwater systems (Beck et al., 2017; Beck and Hall, not have resulted in any biocidal by-products as no reducing 2018). Despite the role of periphyton in Hg cycling, the effect sugars were added (Tanaka et al., 2014). The hot agar was of nutrient addition on overall periphyton microbiome structure poured into the plastic cups and allowed to cool and solidify. is not well characterized, and the impact of nutrient addition The solidified agar was topped with a porous ceramic crucible on the Hg-methylating microbial community is unknown. The cover disc (EA Consumables), and the cup cap was closed single study of hgcAB in periphyton biofilms found that the and sealed with hot glue. A total of 160 NDS cups were abundance of hgcAB sequences was positively correlated with prepared for