Microbial Biogeochemical Impacts on Manganese Reduction in Abandoned Mine Drainage Passive Remediation Systems

Microbial Biogeochemical Impacts on Manganese Reduction in Abandoned Mine Drainage Passive Remediation Systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Microbial Biogeochemical Impacts on Manganese Reduction in Abandoned Mine Drainage Passive Remediation Systems Anna Vietmeier, Natalie Lamagna, Michelle Valkanas, Abigail Strassner, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9065662/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Passive remediation systems ( PRSs ) treat abandoned legacy mines to remove the contaminants in abandoned mine drainage ( AMD ), using settling ponds, limestone beds, and wetlands to precipitate contaminants onsite. These PRSs remove high levels of metals in AMD, including manganese ( Mn ). The impact of microbes that naturally colonize the PRSs on Mn solubilization is poorly understood. We sought to determine the microbial mechanisms contributing to Mn solubilization. We determined in circumneutral AMD, Mn reduction can be microbially facilitated through acidogenesis and sulfidogenesis, whereas in acidic AMD systems it is primarily geochemically driven by low pH. Within the circumneutral Wingfield Pines AMD PRS, spikes of Mn occurred in the wetland at the end of the system. Culturable acidogenic and sulfidogenic bacteria were enumerated from the Wingfield Pines PRS in multiple locations with spikes at the end of the system. Bacterial isolates were identified via 16S rRNA gene sequencing for acidogenesis as Bacillus spp. and Corynebacterium spp., and sulfidogenesis as Citrobacter sp., Aeromonas spp., and Shewanella spp. Whole community surveys of Wingfield Pines PRS by 16S rRNA analysis showed the presence of potentially acidogenic or sulfidogenic bacteria throughout the system, with an increase in relative abundance in the wetlands. We demonstrated microbial Mn resolubilization as acidogenesis and sulfidogenesis in the laboratory to determine potential in-field impacts. This establishes a critical basis for considering microbial processes in future PRS design for effective Mn remediation and potential recovery of Mn as a critical mineral. manganese cycling acidogenesis sulfidogenesis abandoned mine drainage passive remediation system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Legacy abandoned mine sites affect millions of Americans who live within one mile of an abandoned coal mine (Emili, Pizarchik et al. 2016 , USDOI 2022 ). In Pennsylvania ( PA ), there are ~ 11,000 abandoned coal mines that leach abandoned mine drainage ( AMD ), impacting 5,000 km of streams (Cravotta 2008 , OAI 2018 ). AMD starts acidic, however, dependent on the geochemistry of the region AMD can be classified as acidic or circumneutral at ground level (Roth, Gallo et al. 2019 ). AMD can have high levels of soluble metals, including manganese ( Mn ) and sulfuric acid (Cravotta 2008 , Roth, Gallo et al. 2019 ). Mn is usually soluble and mobile as reduced Mn(II) and insoluble as Mn(III)/Mn(IV) oxides (Gounot 1994 ). Acidification accelerates the release of Mn oxides as soluble Mn(II), which is thermodynamically favored at a low pH (< 8) (Fujimoto 1947 , Chao 1972 , Hem 1972 , Tebo, Bargar et al. 2004 , Neculita and Rosa 2019 , Zong, Li et al. 2023 ). High concentrations of Mn and sulfur can impact downstream waster use and must be treated before entering the watershed through precipitation and increasing the pH (Heizer, Sandler et al. 1997 , Gerber, Léonard et al. 2002 , Darbi, Viraraghavan et al. 2003 , Komarnisky, Christopherson et al. 2003 , O'Neal and Zheng 2015 , Singh and Yadav 2016 , Zakharcheva, Gening et al. 2017 , Hatat-Fraile and Barbeau 2019 , EPA 2023 ). Recovery of Mn precipitants from these PRSs is of interest as it is classified as a critical mineral and sequesters additional rare elements from solution. Passive remediation systems ( PRSs ) are an economical and low maintenance approach for remediation of AMD. PRSs are designed to geochemically increase the pH, reduce sulfate levels, and precipitate metals, e.g. Mn, on site (Cravotta 2008 , Muhammad, Kusin et al. 2015 , Roth, Gallo et al. 2019 ). Mn concentrations can be increased abiotically by the decrease in pH. However, there are some systems where the Mn dissolution appears to increase while the pH also increases, suggesting a more nuance reaction driving this; potentially microbial. Microbes naturally colonize AMD PRSs and are known to be the main catalysts for Mn redox cycling between soluble Mn(II) and precipitated Mn(III)/Mn(IV) (Burdige 1983 , Burdige and Nealson 1986 , Myers and Nealson 1988 , Gounot 1994 , Thamdrup, Rosselló-Mora et al. 2000 , Tebo, Bargar et al. 2004 , Das, Sukla et al. 2011 , Lee, Kennedy et al. 2011 , Luther, Thibault de Chanvalon et al. 2018 ). Microbially produced organic acids reduce Mn oxides, therefore increasing Mn(II) solubilization (Gounot 1994 , Tebo, Bargar et al. 2004 , Das, Sukla et al. 2011 ). Sulfide, a known microbial product can mobilize Mn oxides into Mn(II) without decreasing the pH (Gounot 1994 , Das, Sukla et al. 2011 , Lee, Kennedy et al. 2011 , Luther, Thibault de Chanvalon et al. 2018 ). Within the context of the PRS, we hypothesize microbes can contribute to Mn(II) solubilization and undo system remediation, increasing contamination levels exiting the system (White, Sayer et al. 1997 ). However, within a controlled context this microbial metabolism may be useful for the release and recovery of Mn precipitants and co-sequestered rare earths from AMD solids as critical minerals. Here we compare Mn(II) solubilization at an acidic PRS to a circumneutral PRS. Previous seasonal studies at the circumneutral pH Wingfield Pines PRS and acidic pH Boyce Park PRS surveyed the chemical and microbial 16S rRNA communities (Valkanas and Trun 2018 , Cochran 2019 , Valkanas, Rosso et al. 2021 , Vietmeier, Valkanas et al. 2025 ). The PRSs both do not meet the Mn drinking water standards. At the circumneutral PRS, there was an increased level of Mn at the end of the system in the wetlands, before entering the watershed (Valkanas and Trun 2018 ). The mechanisms that lead to Mn cycling in AMD systems are poorly understood, even more so the microbial role in this cycle. We believe at acidic PRSs the Mn(II) solubilization is abiotic based on geochemistry. In circumneutral AMD PRSs, we elucidate two mechanisms for microbial Mn solubilization as 1) acidogenesis and 2) sulfidogenesis. We determined these microbial metabolic reactions have the capacity to impact Mn remediation in the circumneutral Wingfield Pines PRS. Our work, furthers the knowledge of bacterial biogeochemical abiotic-biotic interactions within a PRS (Sturman, Stein et al. 2008 ). METHODS Sampling at Circumneutral and Acidic AMD PRSs The circumneutral Wingfield Pines AMD PRS was constructed in 2009 from an underground mine with five settling ponds (locations 1–5) and a wetland (locations 6, 7) at latitude 40° 20' 26.9988"N, longitude 80° 6' 34.9992"W (Fig. 1 ) (Roth, Gallo et al. 2019 , Valkanas, Rosso et al. 2021 , Datashed 2024 ). As the source of AMD into Wingfield Pines comes from a deep underground mine, it passed through naturally occurring limestone and neutralizing agents in the ground before reaching the surface at a circumneutral pH. The acidic Boyce Park AMD PRS (pH ~ 4) was constructed in 2008 with six settling ponds (ponds 1, 2, 3, 4, 6, 7), one limestone bed (pond 5), and a wetlands area (pond 8) at latitude 40° 27’ 51.9984”N, longitude 79° 44’ 56.0004”W ( SI Fig. 1 ) (Valkanas 2020 , Datashed 2024 , Vietmeier, Valkanas et al. 2025 ). AMD at Boyce Park is generated from a surface mine with multiple acidic inflows into the system that have not been naturally neutralized prior to entry. ArcGIS Pro software was used to visualize pH data collected by a YSI Professional Plus Series handheld with YSI Quatro ISE-ISE-DO-COND 18E100032 probe (Xylem Inc, Yellow Spring, OH, USA). Mn data was collected by Perkin Elmer NexION 300x Inductively Coupled Plasma Mass Spectrometry ( ICP-MS ) with Perkin Elmer S10 Autosampler and the NexION 300x ICP-MS software. Sulfate data collected with Dionex ICS Series ICS-1100 Ultimate 3000 Diode Array Ion Chromatography ( IC ) (Thermo Fisher Scientific, Waltham, MA, USA) (Valkanas and Trun 2018 , Cantlay, Bain et al. 2020 , Valkanas 2020 , Vietmeier, Valkanas et al. 2025 ). One liter of mud-water slurry samples (approximately equal parts soil and water) was collected from all ponds of the AMD system; the mixed microbial community from the mud-water slurry was used for metabolic testing and AMD was autoclaved at 121°C for 45-mins. to sterilize. Assessing Abiotic Geochemical Mn Reduction in Circumneutral and Acidic AMD PRSs The potential for geochemical (abiotic) Mn reduction in circumneutral Wingfield Pines PRS and acidic Boyce Park PRS was determined. Sterile Mn oxide (MnO 2 ; final concentration 50 mM) was independently added to sterile AMD from both systems. The levels of soluble Mn(II) was measured with the colorimetric formaldoxime ( FAD ) assay, the pH was measured with Corning Model 440 pH meter 3-in-1 combo with RJ pH electrode (Corning Incorporated, Corning, NY, USA), and the media sterility was determined by monitoring for microbial growth at OD 600 using a Jenway Genova Plus spectrophotometer (Bibby Scientific Ltd, Stone, Staffs, UK) (Brewer and Spencer 1971 ). Acidification of sterile circumneutral Wingfield Pines AMD with hydrochloric acid (HCl) was assessed for its ability to solubilize 50 mM MnO 2 with the FAD assay and concurrent pH and OD 600 measurements. Sodium sulfide was added to a final concentration of 10 mM, 30 mM, or 50 mM to sterile Wingfield Pines AMD with 50 mM MnO 2 , and the FAD assay was used to determine the Mn(II) in solution. Statistical analysis via an ANOVA was performed using Microsoft Excel v.16.92 and data was visualized with RStudio v.2021.09.01. Differential Mn reduction agar from the literature was modified into a broth (3 g peptone, 2.5 g glucose, 0.010 g FePO 4 , in 1,000 mL, pH 7.6 with NaOH autoclaved, cooled to 48°C, addition of 13 mL 2% potassium permanganate [KMnO 4 ]) and assessed for Mn reduction by the clearing of color for acidification with HCl or by the addition of sulfide (Krumbein and Altman 1973 ). Prevalence of Acidogenic Mn Reducers and Sulfidogenic Microbes in the Culturable Community at Wingfield Pines PRS To assess the prevalence of acidogenesis in the culturable mixotrophic microbial community at Wingfield Pines, non-sterile AMD slurries were serial diluted and plated for single colonies on R2A pH 7.0 plates at 30°C (Reasoner and Geldreich 1985 ). Single colonies were inoculated into individual wells of a sterile 96-well plate with 200 µL of differential Mn reduction broth, and incubated for 3 days at 30°C in a closed container lined with paper towels (wetbox) (Krumbein and Altman 1973 ). Wells were enumerated for clearing indicating Mn reduction. To confirm acid production, 20 µL of 0.2% phenol red was added to each well and examined for yellow color development indicating a ~ pH < 6 (acidic), that was then enumerated (Morgan, Babu et al. 2019 ). Two hundred eighty-five colonies (n = 285) from each sampling location were screened. Data was visualized with RStudio v.2021.09.01 Modified sulfide indole motility ( SIM ) medium developed for this study was used for the detection and enumeration of culturable sulfidogenic bacteria within the Wingfield Pines PRS (Waltman, Shotts et al. 1986, Lamagna 2023 ). Standard SIM medium was modified from a slant to 1.5% agar poured into standard petri plates. Wingfield Pines slurry samples were collected from the inflow of each component (locations 1–6) and diluted in 0.85% saline and mixed with 4 mL of molten top agar (0.7%). The top agar overlay was used to create a low oxygen environment for sulfidogenesis. Sulfidogenic colonies that grew under the top agar formed a black metal-sulfide precipitate around the colony. Plates were incubated at 30°C for single colonies to enumerate the prevalence of sulfidogenesis in the mixed microbial community. Data was visualized with RStudio v.2021.09.01Sulfide (S 2− ) can be microbially produced through the reduction of multiple sulfur chemical intermediates in the environment including thiosulfate (S 2 O 3 2− ), sulfate (SO 4 2− ), sulfite (SO 3 2− ), and/or elemental sulfur (S 0 ). Standard SIM media is formulated with thiosulfate as the source of sulfur. Alternative forms of sulfur compounds were substituted in the plates for additional sulfidogenesis screens including sulfate (S a IM), sulfite (S i IM), and elemental sulfur (S 0 IM). Characterizing Acidogenic Mn Reducers Bacterial isolates (AV20, AV21, AV22, AV23, AV24, KB7, JR07), all of which were positive for Mn reduction on differential Mn reduction agar plates (15 g/L agar), were single colony purified twice and inoculated into a modified differential Mn reduction broth that substituted KMnO 4 with 50 mM MnO 2 (Krumbein and Altman 1973 ). The FAD assay was performed with concurrent pH and OD 600 readings (Brewer and Spencer 1971 ). Sterile media was used as a control. Measurements were taken on day 0, 1, 2, and 3. A two-factor ANOVA with replication was performed using Microsoft Excel v16.92 and data was visualized with RStudio v.2021.09.01. The addition of cell-free 0.2 µM filter sterilized bacterially acidified spent media was added to 50 mM MnO 2 and measured with the FAD assay, pH and OD 600 to determine if bacterially produced acid can facilitate Mn(II) solubilization. Sanger sequencing of a portion of the 16S rRNA gene was used to identify isolates. DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA) from isolates grown in R2A pH 7.0 broth. The 16S rRNA gene primers 27F (5’AGAGTTTGATCMTGGCTCAG3’) and 518R (5’GTATTACCGCGGCTGCTGG3’) were used for AV20, AV21, AV22, AV23, and AV24 (Das, Dash et al. 2014 , Goolam Mahomed, Peters et al. 2021 ). PCR products were inserted into plasmid vector pCR2.1 and cloned in competent E. coli TOP10F’ cells using the Original TA Cloning kit and the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Transformed cells were grown on Luria-Bertani ( LB ) agar with 100 mM IPTG, 40 µg/mL X-gal, and 50 µg/mL Kanamycin ( Kan ) at 37°C. White colonies were purified three times by single colony purification on LB/IPTG/X-gal/Kan plates and grown in LB broth with 50 µg/mL Kan. Plasmid DNA was purified using the Plasmid Miniprep kit (Qiagen, Hilden, Germany) and was sent for Sanger sequencing at GeneWiz ( www.genewiz.com , Azenta Life Sciences, South Plainfield, NJ, USA). DNA sequences were analyzed in 4Peaks and with The National Center for Biotechnology Information ( NCBI ) Basic Local Alignment Search Tool ( BLAST ) using default settings (Vietmeier, Valkanas et al. 2025 ). DNA from KB7 and JR07 was sent for whole genome sequencing using the Illumina MiSeq at SeqCenter ( www.seqcenter.com , Pittsburgh, PA, USA). DNA sequences received were imported into KBase ( www.kbase.us ) and processed as previously done (Vietmeier, Valkanas et al. 2025 ). The 16S rRNA gene was recovered from the annotated genome and put through NCBI BLAST to identify. Characterizing Sulfidogenesis Microbes Ten of the sulfidogenic bacterial isolates (NML8, NML9, NML10, NML11, NML14, NML16, NML34, NML35, NML36, NML37) were single colony purified on R2A pH 7.0 plates a minimum of three times (Lamagna 2023 ). These ten isolates were grown in a Sulfidogen media, a modified version of Postgate media, that contains thiosulfate as the electron donor (0.225 g potassium phosphate dibasic, 0.225 g potassium phosphate monobasic, 0.46 sodium chloride, 0.225 g ammonium sulfate, 0.118 g magnesium sulfate, 1.0 g yeast extract, 3.74 mL 30% sodium lactate, 4.1 g sodium bicarbonate, 2.48 g sodium thiosulfate, 5 mL trace element mix, and 5 mL vitamin mix per liter) (Postgate 1963 ). Wheaton bottles were filled with 75 mL of Sulfidogen media that was gassed with nitrogen for 10-mins., followed by gassing the headspace with nitrogen for 2-mins. The bottles were immediately capped, crimped, and autoclaved at 121°C for 45-mins., cooled to room-temperature and inoculated for each of the ten isolates from overnight cultures. Isolates were grown for 2-weeks at 30°C. A sterile control was included. Microbial sulfidogenesis in the cultures was quantified from precipitates to control for potential thiosulfate interference with the colorimetric methylene blue assay (Cline 1969 ). Samples were centrifuged for 15-mins. to pellet sulfide solids, the supernatant was decanted. Solids were resuspended in 0.2 mL 9N HCl and diluted in 4.8 mL nanopure water before being mixed with 400 µL diamine reagent in tightly capped tubes. Samples were incubated for 20-mins., and absorbance was measured at OD 670 . Results of each isolate were analyzed with t-test compared to sterile control using Excel v16.92 and data was visualized with RStudio v.2021.09.01. OD 600 of sulfidogenic isolates growth was not recorded due to black color of media inhibiting this measurement. Five sulfidogenic bacteria (NML11, NML14, NML16, NML35, and NML36) were identified by their 16S rRNA gene by Sanger sequencing as described above. DNA was extracted using a Zymo Bacterial/Fungal DNA Extraction kit (Zymo Research, Irvine, CA, USA) and PCR amplification was carried out with primers 27F and 805R (5′GACTACHVGGGTATCTAATCC3′) (Tanner, Goebel et al. 1998 , Fujiyoshi, Muto-Fujita et al. 2020 ). Potential Acidogenic and Sulfidogenic Microbes at Wingfield Pines by 16S rRNA Sequencing Wingfield Pines PRS 16S rRNA gene analysis data that was previously generated by the Trun laboratory was mined for bacteria identified as acidogenic and sulfidogenic (Valkanas and Trun 2018 ). Bacteria included in the retrospective screen for acidogenic and sulfidogenic producers included those identified in this paper via their 16S rRNA gene and bacterial isolates previously identified in the literature (Burdige and Nealson 1986 , Myers and Nealson 1988 , Grupe and Gottschalk 1992 , Lovley and Phillips 1994 , Crescenzi, Crisari et al. 2006 , Zhao, Ren et al. 2008 , Kim, Hwang et al. 2010 , Lee, Kennedy et al. 2011 , Liu, Wang et al. 2012 , Zhang, Zhang et al. 2013 , Kushkevych 2014 , Grady, MacDonald et al. 2016 , Duan, Wang et al. 2018 , Henkel, Dellwig et al. 2019 , Sun, Li et al. 2020 , Tahir, Miran et al. 2020 , Zhang, Loh et al. 2020 , Valkanas, Rosso et al. 2021 ). Acidogenic bacteria include Bacillus sp., Bacteroides sp., Clostridium sp., Corynebacterium sp., Lactobacillus sp., Paenibacillus sp., Propionibacterium sp., Pseudomonas sp., Ruminococcus sp., Thiobacillus sp., Veillonella sp. ( SI Table 1 ) (Grupe and Gottschalk 1992 , Crescenzi, Crisari et al. 2006 , Zhao, Ren et al. 2008 , Kim, Hwang et al. 2010 , Liu, Wang et al. 2012 , Grady, MacDonald et al. 2016 , Duan, Wang et al. 2018 , Tahir, Miran et al. 2020 , Zhang, Loh et al. 2020 ). Sulfidogenic bacteria include Alteromonas sp., Shewanella sp., Sulfurimonas sp., Desulfuromonas sp., Desulfomicrobium sp., Desulfovibrio sp., Desulfobulbus sp., Sulfuricurvum sp., and Desulfomonile sp. ( SI Table 2 ) (Burdige and Nealson 1986 , Myers and Nealson 1988 , Lovley and Phillips 1994 , Zhao, Ren et al. 2008 , Lee, Kennedy et al. 2011 , Kushkevych 2014 , Henkel, Dellwig et al. 2019 , Tahir, Miran et al. 2020 , Valkanas, Rosso et al. 2021 ). Data was visualized with RStudio v.2021.09.01. Briefly, the previous methods used to collect 16S rRNA gene data was as follows: DNA was extracted from Wingfield Pines samples using the PowerSoil Kit (MoBio Laboratories Inc., Carlsbad, CA, USA). The 16S rRNA V4 region was amplified using the Illumina’s 515F and 806R primers (16S Illumina Amplicon Protocol, www.earthmicrobiome.org ) and were sent for sequencing at Wright Labs (Huntingdon, PA, USA). Sequences were trimmed to 252 bp, quality filtered, chimeras were removed, and operational taxonomic units ( OTUs ) assigned (DeSantis, Hugenholtz et al. 2006 , Edgar 2010 ). QIIME v1.9.0 was used to construct a table of OTUs with their taxonomic assignments (Caporaso, Kuczynski et al. 2010 , Caporaso, Lauber et al. 2011 ). The 16S rRNA gene profiles were successfully collected for locations 1, 3, 4, 5, 6, and 7. RESULTS Wingfield Pines PRS and Boyce Park PRS Chemical Data for Mn, Sulfate, and pH Seasonal changes are seen at Wingfield Pines PRS for Mn, sulfate, and pH (Fig. 1 ) (Valkanas and Trun 2018 ). For all four seasons, the level of Mn increases as AMD moves through the Wingfield Pines PRS, with the highest level of Mn found in the wetlands, the last component of the PRS before exiting into the stream (Valkanas 2020 ). The highest level of Mn exiting the Wingfield Pines system was in the Fall at 7.39 PPM. Seasonally, sulfate is detected within the Wingfield Pines system, with the highest level reported in Spring at 323 PPM in the wetlands. The sulfate levels remain high, with an average for all seasons > 490 PPM for all ponds. The lowest sulfate level is reported in pond 3 in winter at 168 PPM. For all seasons the Wingfield Pines system remains circumneutral at a pH between 6.8–7.5. At the Boyce Park PRS, seasonally the average Mn concentration stays > 0.20 PPM, with the greatest concentration in pond 4 in the summer at 1.52 PPM (Vietmeier, Valkanas et al. 2025 ). The pH at Boyce Park seasonally stays acidic, below 6.0 with an average pH of ~ 4 ( SI Fig. 1 ). Chemical Abiotic Mn Reduction Occurs in High Acid and Sulfide Conditions Samples collected from circumneutral Wingfield Pines PRS and acidic Boyce Park PRS were autoclaved and MnO 2 was added to 50 mM to assess chemical abiotic impacts on Mn. The data showed that the abiotic chemistry of the Wingfield Pines AMD PRS does not drive Mn solubilization to Mn(II), however it does at acidic Boyce Park (Fig. 2 A-C). There was a significant difference in the solubilization of Mn(II) from MnO 2 between sterilized acidic Boyce Park AMD samples and sterilized circumneutral Wingfield Pines AMD samples (p < 0.00001) (Fig. 2 A). Mn(II) solubilization did not occur in circumneutral Wingfield Pines AMD (Fig. 2 A). There is a significant difference in pH between circumneutral Wingfield Pines AMD and acidic Boyce Park AMD (p < 0.000001), and the pH of both Wingfield Pines and Boyce Park AMD systems does not change with the addition of MnO 2 in the lab (p = 0.12) (Fig. 2 B). The sterilized AMD from both Boyce Park and Wingfield Pines remained sterile as no bacterial growth was detected (OD 600 = 0.000) (Fig. 2 C). These findings allude to a chemical abiotic solubilization of MnO 2 to Mn(II) in acidic AMD, like Boyce Park, that is most likely facilitated by the low pH (Fig. 2 A-C). There is no observed chemical abiotic solubilization of Mn(II) in circumneutral Wingfield Pines, which indicates the potential for a microbially driven mechanism. Due to geochemical solubilization of Mn(II) in acidic AMD, and potential for microbial Mn(II) solubilization in circumneutral AMD, experiments moving forward were performed in circumneutral Wingfield Pines AMD. MnO 2 can be reduced to soluble Mn(II) in Wingfield Pines when AMD is acidified, or sulfide concentrations increase. Solubilization of Mn(II) from MnO 2 occurs when sterile AMD from Wingfield Pines is acidified (p = 0.00089) (Fig. 2 D-F). There is a significant difference between the pH of Wingfield Pines before and after acidification (p < 0.0000001), and no significant drift in the pH over time for either the sterile Wingfield Pines or sterile acidified Wingfield Pines AMD samples (p = 0.25) (Fig. 2 E). There is no significant difference in OD 600 between Wingfield Pines and acidified Wingfield Pines over time (p = 0.07) (Fig. 2 F). In sterile circumneutral Wingfield Pines AMD, MnO 2 is geochemically reduced to Mn(II) with increasing sulfide concentrations (p = 0.0018) (Fig. 2 G). The addition of sulfide without MnO 2 does not result in an increase of soluble Mn(II) ( SI Fig. 2 ). Both increasing acidity with HCl and increasing concentrations of sulfide results in increased chemical Mn reduction as noted by the change in color from deep gold to pale-yellow or white respectively in differential Mn reduction broth (Fig. 2 H). The clearing of color from the differential Mn reduction broth can visually be noted at pH 5.9 and becomes more prominent as the pH decreases, where at pH 3.6 the media is a pale yellow in appearance. The addition of 0.1 mM sulfide visually results in a more pale-yellow of the media and the increasing concentrations of sulfide results in a more prominent chemical reaction decreasing the deep gold color to a cloudy pale yellow (Fig. 2 H). Prevalence of Acidogenic Mn Reducers and Sulfidogenesis in the Culturable Community Within the mixed microbial community at Wingfield Pines PRS, bacteria cultured from every location produced acid capable of reducing Mn and generated sulfide. Across all the sampling locations at Wingfield Pines PRS, 285 bacterial isolates were screened for Mn reduction by acidogenesis in differential Mn broth with a pH indicator (Fig. 3 A, 3 B). Of the 1,995 bacteria screened (285 isolates from each of the 7 locations), Mn reduction and acidogenic metabolism were found in every location within the system. The percentage of isolates performing concurrent Mn reduction and acidogenesis shows the general trend of increasing as AMD moves through the PRS. The highest percentage of acidogenic isolates were found in location 7, the wetlands, at 80% (228/285). All sampling locations screened at Wingfield Pines also identified bacterial isolates capable of sulfidogenesis (Fig. 3 C). All plates for sulfidogenesis screening contained between 10–400 colonies, which appeared after 18–24 hours, with a black coloration of colonies appearing after ~ 18–72 hours. Sulfidogenesis was detected on thiosulfate SIM plates and elemental sulfur S 0 IM plates. The greatest percentage of isolates positive for sulfidogenesis on SIM media was in location 5 at 35% (218/615) and the lowest was in location 1 at 2% (3/129). Sulfidogenesis from elemental sulfur S 0 IM plates was only observed in location 1 at 3% (3/94) and in location 6 at 1% (1/117). Sulfidogenesis was not observed on sulfite S i IM and sulfate S a IM plates. Characterizing Acidogenic Mn Reducers Seven bacterial isolates (AV20, AV21, AV22, AV23, AV24, KB7, JR07) capable of KMnO 4 reduction on differential plates were identified and purified. These isolates reduce MnO 2 to soluble Mn(II) in a modified Mn reduction broth, substituting MnO 2 for KMnO 4 , as they grow and produce acid (Fig. 4 A-C). The soluble Mn(II) increased to ~ 20 PPM when the bacterial isolates grew (p = 0.003) (Fig. 4 A). There was a significant decrease in the pH over time when inoculated with the bacterial isolates (p < 0.00001) (Fig. 4 B). Microbial growth increased over time (p < 0.00001), alluding to a biotic role in acidic production and Mn reduction (Fig. 4 C). The addition of sterile cell-free microbially acidified spent media to MnO 2 results in the solubilization of Mn(II) (Fig. 4 D-F). Cell-free spent acidified media resulted in a significant increase of Mn(II) from MnO 2 (p < 0.001) (Fig. 4 D). There was a not significant change in the pH over time (p = 0.9), but there was a significant difference in pH between sterile spent media and spent media that was microbially acidified (p < 0.000001) (Fig. 4 E). Microbial growth was not detected over time (p = 0.9) indicating samples remained sterile (Fig. 4 F). Of the acidogenic isolates, five isolates (AV20, AV22, AV23, KB7, JR07) were identified as relatives of Bacillus spp. and two isolates (AV21, AV24) were identified as relatives of Corynebacterium spp. (Table 1 ). Characterizing Sulfidogenesis Microbes Of the 39 sulfidogenic bacteria purified from the thiosulfate SIM medium, 36 maintained their phenotype on thiosulfate SIM plates. Ten of the sulfidogenic isolates were chosen and grown in Sulfidogen medium for 2-weeks based on their formation of large black precipitates on SIM plates; all ten isolates produced greater than 8.3 PPM of sulfide during growth (Fig. 5 ). Black precipitates were visible in all isolates within 24 hours of growth and media turned black in color within 14 days of growth indicating sulfide production ( SI Fig. 3 ). The sterile control did not produce detectable levels of sulfide by the colorimetric assay. Results of statistical analysis showed all isolates were significant when compared to sterile control for sulfide production: NML10, NML34, NML35, and NML36 (p < 0.01); NML11 and NML14 (p < 0.001); NML8 and NML16 (p < 0.0001); NML9 and NML37 (p < 0.00001). The greatest amount of sulfidogenesis was measured in NML37 at 20.5 PPM sulfide, followed by NML36 at 20.4 PPM sulfide. Quantification of bacterial sulfidogenesis with the Sulfidogen media and colorimetric assay validated the use of the modified SIM plate overlay method to screen for sulfidogenic bacteria. Five sulfidogenic isolates were successfully identified by their 16S rRNA gene, NML11 was identified as Citrobacter freundii , both NML14 and NML16 were identified as Aeromonas veronii , NML35 was identified Shewanella baltica , and NML36 was identified Shewanella sp. (Table 1 ). Table 1 Identification of acidogenic and sulfidogenic bacterial isolates. Isolate 16S rRNA % identity Metabolism AV20 90% Bacillus cereus Acidogenesis AV21 98% Corynebacterium sp. Acidogenesis AV22 83% Bacillus sp. Acidogenesis AV23 90% Bacillus thuringiensis Acidogenesis AV24 98% Corynebacterium sp. Acidogenesis KB7 100% Bacillus pseudomycoides Acidogenesis JR07 100% Bacillus mycoides Acidogenesis NML11 97% Citrobacter freundii Sulfidogenesis NML14 98% Aeromonas veronii Sulfidogenesis NML16 99% Aeromonas veronii Sulfidogenesis NML35 97% Shewanella baltic Sulfidogenesis NML36 99% Shewanella sp. Sulfidogenesis 16S rRNA Gene Prevalence of Known Acidogenic and Sulfidogenic Bacteria in Wingfield Pines PRS 16S rRNA gene analysis of the mixed microbial community from a previous seasonal Wingfield Pines PRS publication was mined for species related to known acidogenic and sulfidogenic bacteria by OTUs using relative abundance ( SI Tables 1 & 2) (Valkanas and Trun 2018 ). Acidogenic bacteria predicted in the system include Bacillus sp. 0.01%, Clostridium sp. 0.12%, Bacteroides sp. 0.01%, Ruminococcus sp. 0.02%, Pseudomonas sp. 0.10%, Veillonella sp. 0.002%, Lactobacillus sp. 0.01%, Thiobacillus sp. 1.0%, Paenibacillus sp. 0.002%, Corynebacterium sp. 0.001%, and Propionibacterium sp. 0.0005%. The majority of the acidogenic bacteria were found in the wetlands, sampling location 6 (18,643 OTUs, 2.5%) and location 7 (8,985 OTUs, 3.4%) (Grupe and Gottschalk 1992 , Crescenzi, Crisari et al. 2006 , Zhao, Ren et al. 2008 , Kim, Hwang et al. 2010 , Liu, Wang et al. 2012 , Zhang, Zhang et al. 2013 , Grady, MacDonald et al. 2016 , Duan, Wang et al. 2018 , Tahir, Miran et al. 2020 , Zhang, Loh et al. 2020 ). In total, 24,106 OTUs from acidogenic bacterial sequences were predicted within the system (1.3% of total OTUs) based on isolates identified in this study and in the literature (Fig. 6 A, SI Table 1 ). Sulfidogenic bacteria predicted in the system include Alteromonas sp. (0.001%), Desulfacinum sp. (0.02%), Desulfarculus sp. (0.003%), Desulfobacca sp. (0.08%), Desulfobulbus sp. (0.77%), Desulfococcus sp. (0.56%), Desulfofrigus sp. (0.001%), Desulfomicrobium sp. (0.02%), Desulfomonile sp. (0.06%), Desulfotalea sp. (0.001%), Desulfovibrio sp. (0.03%), Desulfuromonas sp. (0.005%), Pelobacter sp. (0.0002%), Shewanella sp. (0.0003%), Sulfuricurvum sp. (2.2%), Sulfurimonas sp. (0.08%), and Syntrophobacter sp. (0.03%). Sulfidogenic bacteria were found in all locations of the system with the greatest amount of sulfidogenic bacteria in the wetlands at location 6 (37,270 OTUs, 9%) ( SI Table 2 ) (Burdige and Nealson 1986 , Myers and Nealson 1988 , Lovley and Phillips 1994 , Zhao, Ren et al. 2008 , Lee, Kennedy et al. 2011 , Kushkevych 2014 , Henkel, Dellwig et al. 2019 , Tahir, Miran et al. 2020 , Valkanas, Rosso et al. 2021 ). In total 71,633 unique OTUs of sequences matching sulfidogenic bacteria (3.8% of total OTUs) were predicted within the system (Fig. 6 B). The number of predicted acidogenic and sulfidogenic bacteria increases as AMD moves through the PRS with the highest numbers found in the wetlands of the system (location 6 and 7). DISCUSSION The biogeochemical mechanisms involved in the seasonal chemical fluctuations within circumneutral and acidic AMD PRS have not been uniformly well characterized (Fig. 1 , SI Fig. 1 ) (Valkanas and Trun 2018 , Ly, Wright et al. 2019 ). We sought to understand the potential of microbes that naturally colonize PRSs to metabolically impact Mn solubilization in circumneutral and acidic AMD PRSs (Burdige 1983 , Gounot 1994 , Tebo, Bargar et al. 2004 , Valkanas and Trun 2018 , Ly, Wright et al. 2019 ). At circumneutral Wingfield Pines PRS, for all seasons there is an increase in Mn within the wetlands of the system prior to entering the watershed (Fig. 1 ). Spikes in Mn in the wetlands of circumneutral Wingfield Pines PRS occurred concurrent with spikes in sulfate, which was not observed at acidic Boyce Park PRS (Fig. 1 , SI Fig. 1 ) (Cochran 2019 ). We sought to first understand the geochemical mechanisms driving Mn solubilization in circumneutral and acidic AMD PRSs. Sterile AMD from circumneutral Wingfield Pines PRS does not result in the chemical solubilization of Mn(II) when sterile MnO 2 is added (Fig. 2 A-C). However, when sterile Wingfield Pines AMD is acidified, it results in the abiotic solubilization of MnO 2 to Mn(II) (Fig. 2 D-F). The acidification of media facilitating the chemical speciation of Mn(II) is consistent with low pH reported in Pourbiax diagrams (Sun, Kitchaev et al. 2019 , Wang, Guo et al. 2020 ). Further, the addition of sterile MnO 2 to sterile acidic Boyce PRS AMD results in the increase of soluble Mn(II) (Fig. 2 A-C). This alludes to the solubilization of Mn(II) being chemically driven by low pH, which is further supported in reference to the phase stability of Mn speciation in acidic conditions reported in Pourbiax diagrams (Sun, Kitchaev et al. 2019 , Wang, Guo et al. 2020 ). At acidic Boyce Park PRS, it is likely Mn solubilization and mobilization is primarily geochemically driven based on low pH from our in-lab findings ( SI Fig. 1 , Fig. 2 A-C). We also noted that when increasing concentrations of sterile sodium sulfide is added to sterile Wingfield Pines AMD with sterile MnO 2 there is an increase of soluble Mn(II) proportional to the sulfide concentration added (Fig. 2 G). The impacts of acidification and high sulfide concentrations on Mn reduction was further elucidated in differential Mn reduction broth (Fig. 2 H, SI Fig. 2 ). Due to the solubilization of MnO 2 to Mn(II) in acidic conditions, we assessed the potential for biotically mediated Mn reduction within circumneutral Wingfield Pines PRS using both culture-dependent and culture-independent molecular approaches. In circumneutral Wingfield Pines AMD, culturable microbes capable of acidogenesis and sulfidogenesis are present. Acidogenic microbes that reduce Mn were found in every location of Wingfield Pines as determined with differential Mn reduction broth with the additional of a pH indicator (Fig. 3 A, 3 B) (Krumbein and Altman 1973 ). A general increase in acidogenic bacteria that reduce Mn is observed as AMD moves through the PRS with the greatest percentage of acidogenic bacteria in location 7 near the end of the system at (80% of all culturable bacteria metabolically screened) (Fig. 3 B). Sulfidogenic microbes that produce sulfide were found in all locations of Wingfield Pines with the modified thiosulfate SIM agar plates, with a general increase of sulfidogenic bacteria near the end of the system (Fig. 3 C). Seasonal chemical studies of Wingfield Pines show there is an increased availability of sulfate at the end of the system which could be utilized by sulfidogenic microbes to produce sulfide leading to increased soluble Mn(II) (Fig. 1 ). We suggest based on previous research the slow flow of AMD through the PRS creates an oxic to anoxic gradient that could facilitate microbial sulfate reduction to sulfide reactions, especially within the sediment of these systems (Valkanas and Trun 2018 ). It is possible additional microbes are present at Wingfield Pines that are capable of acidogenesis and sulfidogenesis that are difficult to culture or were not captured with these culturing methods that also contributing to Mn cycling. Which may be the case for the lack of reduction observed on the sulfate and sulfite plates, or due to suboxic culturing conditions from the low oxygen top agar overlay that are not fully anaerobic conditions. Alternatively, the reduction of sulfate, sulfite, and elemental sulfur to sulfide require additional microbial metabolic pathways not required for the reduction of thiosulfate to sulfide, suggesting these isolates do not encode those metabolic machineries. The frequency of bacteria that can facilitate Mn reduction by organic acid or sulfide byproducts at Wingfield Pines PRS can be inferred from the frequency of these cultured bacteria. Further, the increase of acidogenic and sulfidogenic metabolic phenotypes as AMD moves through the system with elevation of these phenotypes at the end of the system correspond to Mn spikes and potentially explain them (Fig. 1 , Fig. 3 ). Microbial isolates from the metabolic screens of acidogenesis and sulfidogenesis were further characterized. The seven acidogenic microbial isolates recovered reduce multiple Mn chemical species in different oxidation states including the reduction of KMnO 4 [Mn(VII)] and MnO 2 [Mn(IV)] (Fig. 4 A-C). We showed that the microbially produced organic acids facilitate the reduction of MnO 2 to Mn(II) through the addition of sterile cell-free spent media (Fig. 4 D-F). These findings show that Mn(II) can be released from MnO 2 without the presence of any cells, but rather their metabolic production of acid drives the solubilization of Mn(II). All ten of the sulfidogenic isolates recovered from thiosulfate SIM plates could produce quantifiable levels of sulfide (Fig. 5 ). As sulfidogenesis quantification was measured using only the sulfide precipitates due to thiosulfate interference in the colorimetric methylene blue assay, a greater amount of sulfide may have been produced in liquid and as hydrogen sulfide gas. Microbial reduction to sulfide can result in the reduction of MnO 2 (Burdige and Nealson 1986 , Lee, Kennedy et al. 2011 , Sun, Li et al. 2020 , Valkanas, Rosso et al. 2021 ). 16S rRNA analysis of acidogenic bacteria identified them as relatives to Bacillus spp., and Corynebacterium spp., and sulfidogenic bacteria as relatives of Citrobacter sp., Aeromonas spp., and Shewanella spp. which are all consistent with metabolisms reported in the literature for organic acid and sulfide production (Table 1 , SI Table 1 , SI Table 2 ) (Qiu, Zhao et al. 2009 , Kim, Hwang et al. 2010 , Lee, Kennedy et al. 2011 , Liu, Wang et al. 2012 , Wu, Li et al. 2015 , Grady, MacDonald et al. 2016 , Yan, Zhong et al. 2018 , Abedi and Hashemi 2020 , Sharma, Garg et al. 2020 , Wu 2020 , Zhang, Liu et al. 2021 , Semwal, Kumar et al. 2023 ). Laboratory results demonstrated that culturable microbes from the Wingfield Pines PRS can produce quantifiable levels of organic acids that solubilize Mn(II) and produce quantifiable levels of sulfide that can solubilize Mn(II). To relate this culture-based laboratory finding on microbial driven Mn(II) solubilization to the Wingfield Pines system, 16S rRNA data was mined for relatives to known acidogenic and sulfidogenic microbes. Known acidogenic and sulfidogenic bacteria were present in every location of Wingfield Pines as predicted by OTUs, with a general trend of increasing relative abundance as AMD moves through the system, with the highest number found in the wetlands (location 6 and 7; Fig. 6 ). The presence of phylogenetic relatives in the 16S rRNA gene sequence community alludes to a potential of these microbial metabolisms impacting Mn solubilization within the systems, consistent with seasonal increases of Mn seen before exiting the system (Fig. 1 ). As metabolic acidogenesis occurs in cultured isolates from the wetland and 16S rRNA relatives are predicted within the Wingfield Pines PRS, it is possible microbial acidification is happening on a local level leading to Mn(II) solubilization that is not being detected in large scale surveys of pH. Mn spikes are possibly also driven by sulfidogenesis as sulfide producers are present in the culturable community, sulfidogenic relatives are predicted in the 16S rRNA system surveys, and sulfate is available in increasing concentrations as seen in the seasonal chemical studies. It is more likely there is a complex combination of both acidogenesis and sulfidogenesis impacting biogeochemical cycling and remobilization of Mn in circumneutral systems. This study highlights the importance of considering the impact of microbial metabolism and placement of wetlands within PRS design (Neculita and Rosa 2019 ). This is especially true within circumneutral PRS AMD as microbial driven Mn(II) solubilization by acid and sulfide can potentially be found at other circumneutral systems, as similar spikes in Mn are seen in the wetlands in analogous circumneutral Lowber AMD PRS (Cochran 2019 ). Wetlands are carbon-rich environments designed to promote sulfate reduction; however, these conditions can lead to both the production of organic acids from carbon fermentation and the production of sulfide, both of which, from our findings, can reduce/re-mobilize Mn (Pester, Knorr et al. 2012 , Candry, Flinkstorm et al. 2024 ). This study highlights the interconnectivity of microbial metabolism and chemical cycling for multiple AMD contaminants including Mn and sulfate. Although remediation of sulfate is also a goal of AMD systems the reduction to sulfide can lead to complications with Mn mobilization (Heizer, Sandler et al. 1997 , Hallberg and Johnson 2005 , Hatat-Fraile and Barbeau 2019 , Ray and Dey 2020 , Sun, Li et al. 2020 , Valkanas, Rosso et al. 2021 , EPA 2022 , EPA 2023 ). We suggest that wetlands may not be the optimal choice to be the final component of the system and an additional settling pond specific for Mn removal may be beneficial. This study also attempted to address the knowledge gap of how microbes impact the remediation of Mn in AMD as it is likely these microbes are preventing the Wingfield Pines PRS from effectively remediating Mn to full optimization. As Mn is a critical mineral and known to sequester other minerals in its oxide form, microbial metabolism that results in Mn(II) solubilization with the context of the AMD PRS may be of use in biotechnology, specifically for the biomining of critical minerals. Declarations Author has no competing interests to disclose Acknowledgements We acknowledge Duquesne University School of Science and Engineering Department of Biological Sciences, The Bayer Research Fellowship, Duquesne University Women in STEM, American Society for Microbiology (ASM) Future Leaders Mentorship Fellowship (FLMF), The Allegheny Branch of ASM, Geological Society of American Research Grant through the National Science Foundation, National Association of State Land Reclaimationists, and American Society of Reclamation Sciences for their support. We also acknowledge the Oak Ridge Institute for Science and Education (ORISE), National Energy Technology Laboratory (NETL), Department of Energy (DOE), and the Gulliver Laboratory at NETL. We acknowledge the Stolz laboratory at Duquesne University and Dr. Tetiana Cantlay. 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"Microbial solubilization and immobilization of toxic metals: key biogeochemical processes for treatment of contamination." FEMS Microbiology Reviews 20 : 503-516. Wu, G. (2020). "Hydrogen sulfide-producing kinetics of Shewanella oneidensis in sulfite and thiosulfate respiration." Process Biochemistry 93 : 21-27. Wu, G., N. Li, Y. Mao, G. Zhou and H. Gao (2015). "Endogenous generation of hydrogen sulfide and its regulation in Shewanella oneidensis." Front Microbiol 6 : 374. Yan, J., K. Zhong, S. Wang, Z. Chen, H. Hu, Z. Jian, H. Wen and H. Zhang (2018). "Carbon metabolism and sulfate respiration by a non-conventional Citrobacter freundii strain SR10 with potential application in removal of metals and metalloids." International Biodeterioration & Biodegradation 133 : 238-246. Zakharcheva, K. A., L. V. Gening, K. Y. Kazachenko and V. Z. Tarantul (2017). "Cells Resistant to Toxic Concentrations of Manganese Have Increased Ability to Repair DNA." Biochemistry (Mosc) 82 (1): 38-45. Zhang, J., Y. Zhang, J. Chang, X. Quan and Q. Li (2013). "Biological sulfate reduction in the acidogenic phase of anaerobic digestion under dissimilatory Fe (III)--reducing conditions." Water Res 47 (6): 2033-2040. Zhang, L., K. C. Loh, Y. Dai and Y. W. Tong (2020). "Acidogenic fermentation of food waste for production of volatile fatty acids: Bacterial community analysis and semi-continuous operation." Waste Manag 109 : 75-84. Zhang, Y., Z. Liu, Y. Tang, X. Ma, H. Tang, H. Li and Z. Liu (2021). "Cbl upregulates cysH for hydrogen sulfide production in Aeromonas veronii." PeerJ 9 : e12058. Zhao, Y., N. Ren and A. Wang (2008). "Contributions of fermentative acidogenic bacteria and sulfate-reducing bacteria to lactate degradation and sulfate reduction." Chemosphere 72 (2): 233-242. Zong, Y., Z. Li, R. Gui, D. Chen, M. Yuan, Y. Chai, S. Shan and M. H. Wong (2023). "Manganese losses induced by severe soil acidification in the extensive Lei bamboo (Phyllostachys violascens) plantation stands in Eastern China." Chemosphere 339 : 139669. Supplementary Files SUPPLEMENTARYINFORMATION.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 29 Mar, 2026 Editor assigned by journal 16 Mar, 2026 First submitted to journal 08 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9065662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":613997932,"identity":"effd8328-00c1-48ee-a66b-b7d903548fdd","order_by":0,"name":"Anna Vietmeier","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACZjApAcSMjQ+AJA8fEVoYGyBamJsNQFrYiLAHpAUE2NtAGhkIajE4znz80Y0KCznz9oNtlV9z7GTYGJgfPrqBT8thtsTmnDMSxjJnEttuy25LBjqMzdg4B48WyWYew+bcNonEGQxALZLbmIFaeNik8Wvh/9ic+0+ifgb/w7ZiyW31hLXwM/MwNuc2SCRISCS2MX7cdpgYLWyGs3OOSRjOkHjYLM247TgPGzMBv7DxH37wOaemTl6CP/3hx5/bqu352ZsfPsanBQUw84BJYpWDAOMPUlSPglEwCkbBiAEAn4pArF8hRrQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2610-0654","institution":"Duquesne University","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"","lastName":"Vietmeier","suffix":""},{"id":613997933,"identity":"31a206f2-2291-437d-83ef-53bbc7ee1647","order_by":1,"name":"Natalie Lamagna","email":"","orcid":"","institution":"Duquesne University","correspondingAuthor":false,"prefix":"","firstName":"Natalie","middleName":"","lastName":"Lamagna","suffix":""},{"id":613997934,"identity":"35592e02-4817-406a-bb8d-77495462d514","order_by":2,"name":"Michelle Valkanas","email":"","orcid":"","institution":"PennWest California: Pennsylvania Western University California","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Valkanas","suffix":""},{"id":613997935,"identity":"2ccbff4c-12cd-43ea-b8d1-b3c93583c194","order_by":3,"name":"Abigail Strassner","email":"","orcid":"","institution":"Duquesne University","correspondingAuthor":false,"prefix":"","firstName":"Abigail","middleName":"","lastName":"Strassner","suffix":""},{"id":613997936,"identity":"2f8002f4-63b5-4882-a8da-d5efc4fa10fa","order_by":4,"name":"Kayla Brennan","email":"","orcid":"","institution":"Duquesne University","correspondingAuthor":false,"prefix":"","firstName":"Kayla","middleName":"","lastName":"Brennan","suffix":""},{"id":613997937,"identity":"39b928e1-8eb6-42d7-8204-6027aea458f9","order_by":5,"name":"Lance Daley","email":"","orcid":"","institution":"Duquesne University","correspondingAuthor":false,"prefix":"","firstName":"Lance","middleName":"","lastName":"Daley","suffix":""},{"id":613997938,"identity":"66fbf877-4cdf-4531-9a33-593a9a10e70f","order_by":6,"name":"Samuel Flett","email":"","orcid":"","institution":"National Energy Technology Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Flett","suffix":""},{"id":613997939,"identity":"cb444c7c-3efa-4d5d-a7b9-ded2551e1622","order_by":7,"name":"Djuna Gulliver","email":"","orcid":"","institution":"National Energy Technology Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Djuna","middleName":"","lastName":"Gulliver","suffix":""},{"id":613997940,"identity":"7a50f6cd-bda8-4964-9d8f-34bfed5c06cf","order_by":8,"name":"Nancy Trun","email":"","orcid":"","institution":"Duquesne University","correspondingAuthor":false,"prefix":"","firstName":"Nancy","middleName":"","lastName":"Trun","suffix":""}],"badges":[],"createdAt":"2026-03-08 16:54:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9065662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9065662/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105999498,"identity":"f2225073-8587-4a10-8274-f78a7b111528","added_by":"auto","created_at":"2026-04-02 09:44:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSeasonal changes in Wingfield Pines PRS for manganese, sulfate, and pH. \u003c/strong\u003eFlow and numbers of the system components are shown on the left. The key for chemical levels is shown on the right.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/9774bd964e1e8e4ecba4d584.jpg"},{"id":105999600,"identity":"035d478d-0bd7-4524-bcaf-894f1defe479","added_by":"auto","created_at":"2026-04-02 09:44:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185711,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/b04bae8bca331c326baf3cf9.jpg"},{"id":105999471,"identity":"8b9ea572-e5a3-4a27-8010-807ccd7c93ee","added_by":"auto","created_at":"2026-04-02 09:43:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe prevalence of acidogenic facilitated Mn reduction and sulfidogenic microbes in the culturable community from Wingfield Pines PRS. \u003c/strong\u003eThe sampling location shown corresponds to the annotated system in \u003cstrong\u003eFig 1\u003c/strong\u003e. Culturable bacterial isolates from the mixed microbial community capable of Mn reduction (\u003cstrong\u003eA\u003c/strong\u003e), acidogenesis (\u003cstrong\u003eB\u003c/strong\u003e), and sulfidogenesis (\u003cstrong\u003eC\u003c/strong\u003e), in general increase as AMD moves through the Wingfield Pines system.\u003cstrong\u003e \u003c/strong\u003eBoth \u003cstrong\u003eA \u003c/strong\u003eand \u003cstrong\u003eB \u003c/strong\u003eare \u003cem\u003en = 285\u003c/em\u003efor each location. \u003cstrong\u003eC\u003c/strong\u003e \u003cem\u003en = varied\u003c/em\u003e based on number of single colonies on the plates.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/81340a8bf405ffb982913daf.jpg"},{"id":105999461,"identity":"b617054e-107c-4744-a149-7a9a6e0f1cc9","added_by":"auto","created_at":"2026-04-02 09:43:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":195495,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/b8f6573e0323c270124f116d.jpg"},{"id":105999601,"identity":"85fc7c43-b74f-4f20-84a0-99cc79aa25c1","added_by":"auto","created_at":"2026-04-02 09:44:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSulfidogenesis by bacterial isolates from Wingfield Pines PRS. \u003c/strong\u003eSulfide produced by each of the 10 analyzed isolates grown in Sulfidogen media after two weeks.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/9c6c9696fcd4756fb5246d6b.jpg"},{"id":105999463,"identity":"f6f9e5b3-5708-4a45-b076-898aafb1272b","added_by":"auto","created_at":"2026-04-02 09:43:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":66614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe prevalence of bacteria known to be acidogenic (A) and sulfidogenic (B) at Wingfield Pines PRS based on 16S rRNA\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene sequencing of the mixed microbial community based on relative abundance. \u003c/strong\u003eThe sampling location corresponds to the annotated maps of the system in \u003cstrong\u003eFig 1\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/a38a2c4f94e8977fbc5cf565.jpg"},{"id":105999602,"identity":"df72b901-7358-4e5f-b886-c7a7a99cd940","added_by":"auto","created_at":"2026-04-02 09:44:10","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":51935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrobial Mn reduction can be facilitated by acidogenesis or sulfidogenesis\u003c/strong\u003e. In these processes a microbial produced byproduct of organic acids or sulfide facilitates the reduction of Mn to soluble Mn(II). These microbial mechanisms can contribute to the increase of soluble Mn(II) within circumneutral pH AMD PRSs.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/d1fcc3d74a0a5ae5e52056c2.jpg"},{"id":105999766,"identity":"887500d3-1c03-449b-b92c-885bc87dd3b6","added_by":"auto","created_at":"2026-04-02 09:44:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2287442,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/4b885c17-6227-4093-8ae6-6d850053d5c8.pdf"},{"id":105999472,"identity":"c88f03db-335c-4bd7-b24a-fb6b2febf0f3","added_by":"auto","created_at":"2026-04-02 09:43:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4134150,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYINFORMATION.docx","url":"https://assets-eu.researchsquare.com/files/rs-9065662/v1/429d04ba0896f260aaae750f.docx"}],"financialInterests":"","formattedTitle":"Microbial Biogeochemical Impacts on Manganese Reduction in Abandoned Mine Drainage Passive Remediation Systems","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLegacy abandoned mine sites affect millions of Americans who live within one mile of an abandoned coal mine (Emili, Pizarchik et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, USDOI \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In Pennsylvania (\u003cb\u003ePA\u003c/b\u003e), there are ~\u0026thinsp;11,000 abandoned coal mines that leach abandoned mine drainage (\u003cb\u003eAMD\u003c/b\u003e), impacting 5,000 km of streams (Cravotta \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, OAI \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). AMD starts acidic, however, dependent on the geochemistry of the region AMD can be classified as acidic or circumneutral at ground level (Roth, Gallo et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). AMD can have high levels of soluble metals, including manganese (\u003cb\u003eMn\u003c/b\u003e) and sulfuric acid (Cravotta \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Roth, Gallo et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Mn is usually soluble and mobile as reduced Mn(II) and insoluble as Mn(III)/Mn(IV) oxides (Gounot \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Acidification accelerates the release of Mn oxides as soluble Mn(II), which is thermodynamically favored at a low pH (\u0026lt;\u0026thinsp;8) (Fujimoto \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1947\u003c/span\u003e, Chao \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1972\u003c/span\u003e, Hem \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1972\u003c/span\u003e, Tebo, Bargar et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Neculita and Rosa \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Zong, Li et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). High concentrations of Mn and sulfur can impact downstream waster use and must be treated before entering the watershed through precipitation and increasing the pH (Heizer, Sandler et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Gerber, L\u0026eacute;onard et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Darbi, Viraraghavan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Komarnisky, Christopherson et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, O'Neal and Zheng \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Singh and Yadav \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Zakharcheva, Gening et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Hatat-Fraile and Barbeau \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, EPA \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recovery of Mn precipitants from these PRSs is of interest as it is classified as a critical mineral and sequesters additional rare elements from solution.\u003c/p\u003e \u003cp\u003ePassive remediation systems (\u003cb\u003ePRSs\u003c/b\u003e) are an economical and low maintenance approach for remediation of AMD. PRSs are designed to geochemically increase the pH, reduce sulfate levels, and precipitate metals, e.g. Mn, on site (Cravotta \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Muhammad, Kusin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Roth, Gallo et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Mn concentrations can be increased abiotically by the decrease in pH. However, there are some systems where the Mn dissolution appears to increase while the pH also increases, suggesting a more nuance reaction driving this; potentially microbial. Microbes naturally colonize AMD PRSs and are known to be the main catalysts for Mn redox cycling between soluble Mn(II) and precipitated Mn(III)/Mn(IV) (Burdige \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1983\u003c/span\u003e, Burdige and Nealson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Myers and Nealson \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, Gounot \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Thamdrup, Rossell\u0026oacute;-Mora et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Tebo, Bargar et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Das, Sukla et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Lee, Kennedy et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Luther, Thibault de Chanvalon et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Microbially produced organic acids reduce Mn oxides, therefore increasing Mn(II) solubilization (Gounot \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Tebo, Bargar et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Das, Sukla et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Sulfide, a known microbial product can mobilize Mn oxides into Mn(II) without decreasing the pH (Gounot \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Das, Sukla et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Lee, Kennedy et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Luther, Thibault de Chanvalon et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Within the context of the PRS, we hypothesize microbes can contribute to Mn(II) solubilization and undo system remediation, increasing contamination levels exiting the system (White, Sayer et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). However, within a controlled context this microbial metabolism may be useful for the release and recovery of Mn precipitants and co-sequestered rare earths from AMD solids as critical minerals.\u003c/p\u003e \u003cp\u003eHere we compare Mn(II) solubilization at an acidic PRS to a circumneutral PRS. Previous seasonal studies at the circumneutral pH Wingfield Pines PRS and acidic pH Boyce Park PRS surveyed the chemical and microbial 16S rRNA communities (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Cochran \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Valkanas, Rosso et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Vietmeier, Valkanas et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The PRSs both do not meet the Mn drinking water standards. At the circumneutral PRS, there was an increased level of Mn at the end of the system in the wetlands, before entering the watershed (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The mechanisms that lead to Mn cycling in AMD systems are poorly understood, even more so the microbial role in this cycle. We believe at acidic PRSs the Mn(II) solubilization is abiotic based on geochemistry. In circumneutral AMD PRSs, we elucidate two mechanisms for microbial Mn solubilization as 1) acidogenesis and 2) sulfidogenesis. We determined these microbial metabolic reactions have the capacity to impact Mn remediation in the circumneutral Wingfield Pines PRS. Our work, furthers the knowledge of bacterial biogeochemical abiotic-biotic interactions within a PRS (Sturman, Stein et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eSampling at Circumneutral and Acidic AMD PRSs\u003c/h2\u003e\n \u003cp\u003eThe circumneutral Wingfield Pines AMD PRS was constructed in 2009 from an underground mine with five settling ponds (locations 1\u0026ndash;5) and a wetland (locations 6, 7) at latitude 40\u0026deg; 20\u0026apos; 26.9988\u0026quot;N, longitude 80\u0026deg; 6\u0026apos; 34.9992\u0026quot;W (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Roth, Gallo et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Valkanas, Rosso et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Datashed \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As the source of AMD into Wingfield Pines comes from a deep underground mine, it passed through naturally occurring limestone and neutralizing agents in the ground before reaching the surface at a circumneutral pH. The acidic Boyce Park AMD PRS (pH\u0026thinsp;~\u0026thinsp;4) was constructed in 2008 with six settling ponds (ponds 1, 2, 3, 4, 6, 7), one limestone bed (pond 5), and a wetlands area (pond 8) at latitude 40\u0026deg; 27\u0026rsquo; 51.9984\u0026rdquo;N, longitude 79\u0026deg; 44\u0026rsquo; 56.0004\u0026rdquo;W (\u003cstrong\u003eSI\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Valkanas \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Datashed \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Vietmeier, Valkanas et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). AMD at Boyce Park is generated from a surface mine with multiple acidic inflows into the system that have not been naturally neutralized prior to entry. ArcGIS Pro software was used to visualize pH data collected by a YSI Professional Plus Series handheld with YSI Quatro ISE-ISE-DO-COND 18E100032 probe (Xylem Inc, Yellow Spring, OH, USA). Mn data was collected by Perkin Elmer NexION 300x Inductively Coupled Plasma Mass Spectrometry (\u003cstrong\u003eICP-MS\u003c/strong\u003e) with Perkin Elmer S10 Autosampler and the NexION 300x ICP-MS software. Sulfate data collected with Dionex ICS Series ICS-1100 Ultimate 3000 Diode Array Ion Chromatography (\u003cstrong\u003eIC\u003c/strong\u003e) (Thermo Fisher Scientific, Waltham, MA, USA) (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Cantlay, Bain et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Valkanas \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Vietmeier, Valkanas et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). One liter of mud-water slurry samples (approximately equal parts soil and water) was collected from all ponds of the AMD system; the mixed microbial community from the mud-water slurry was used for metabolic testing and AMD was autoclaved at 121\u0026deg;C for 45-mins. to sterilize.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAssessing Abiotic Geochemical Mn Reduction in Circumneutral and Acidic AMD PRSs\u003c/h3\u003e\n\u003cp\u003eThe potential for geochemical (abiotic) Mn reduction in circumneutral Wingfield Pines PRS and acidic Boyce Park PRS was determined. Sterile Mn oxide (MnO\u003csub\u003e2\u003c/sub\u003e; final concentration 50 mM) was independently added to sterile AMD from both systems. The levels of soluble Mn(II) was measured with the colorimetric formaldoxime (\u003cstrong\u003eFAD\u003c/strong\u003e) assay, the pH was measured with Corning Model 440 pH meter 3-in-1 combo with RJ pH electrode (Corning Incorporated, Corning, NY, USA), and the media sterility was determined by monitoring for microbial growth at OD\u003csub\u003e600\u003c/sub\u003e using a Jenway Genova Plus spectrophotometer (Bibby Scientific Ltd, Stone, Staffs, UK) (Brewer and Spencer \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Acidification of sterile circumneutral Wingfield Pines AMD with hydrochloric acid (HCl) was assessed for its ability to solubilize 50 mM MnO\u003csub\u003e2\u003c/sub\u003e with the FAD assay and concurrent pH and OD\u003csub\u003e600\u003c/sub\u003e measurements. Sodium sulfide was added to a final concentration of 10 mM, 30 mM, or 50 mM to sterile Wingfield Pines AMD with 50 mM MnO\u003csub\u003e2\u003c/sub\u003e, and the FAD assay was used to determine the Mn(II) in solution. Statistical analysis via an ANOVA was performed using Microsoft Excel v.16.92 and data was visualized with RStudio v.2021.09.01. Differential Mn reduction agar from the literature was modified into a broth (3 g peptone, 2.5 g glucose, 0.010 g FePO\u003csub\u003e4\u003c/sub\u003e, in 1,000 mL, pH 7.6 with NaOH autoclaved, cooled to 48\u0026deg;C, addition of 13 mL 2% potassium permanganate [KMnO\u003csub\u003e4\u003c/sub\u003e]) and assessed for Mn reduction by the clearing of color for acidification with HCl or by the addition of sulfide (Krumbein and Altman \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1973\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrevalence of Acidogenic Mn Reducers and Sulfidogenic Microbes in the Culturable Community at Wingfield Pines PRS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the prevalence of acidogenesis in the culturable mixotrophic microbial community at Wingfield Pines, non-sterile AMD slurries were serial diluted and plated for single colonies on R2A pH 7.0 plates at 30\u0026deg;C (Reasoner and Geldreich \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Single colonies were inoculated into individual wells of a sterile 96-well plate with 200 \u0026micro;L of differential Mn reduction broth, and incubated for 3 days at 30\u0026deg;C in a closed container lined with paper towels (wetbox) (Krumbein and Altman \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). Wells were enumerated for clearing indicating Mn reduction. To confirm acid production, 20 \u0026micro;L of 0.2% phenol red was added to each well and examined for yellow color development indicating a\u0026thinsp;~\u0026thinsp;pH\u0026thinsp;\u0026lt;\u0026thinsp;6 (acidic), that was then enumerated (Morgan, Babu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Two hundred eighty-five colonies (n\u0026thinsp;=\u0026thinsp;285) from each sampling location were screened. Data was visualized with RStudio v.2021.09.01\u003c/p\u003e\n\u003cp\u003eModified sulfide indole motility (\u003cstrong\u003eSIM\u003c/strong\u003e) medium developed for this study was used for the detection and enumeration of culturable sulfidogenic bacteria within the Wingfield Pines PRS (Waltman, Shotts et al. 1986, Lamagna \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Standard SIM medium was modified from a slant to 1.5% agar poured into standard petri plates. Wingfield Pines slurry samples were collected from the inflow of each component (locations 1\u0026ndash;6) and diluted in 0.85% saline and mixed with 4 mL of molten top agar (0.7%). The top agar overlay was used to create a low oxygen environment for sulfidogenesis. Sulfidogenic colonies that grew under the top agar formed a black metal-sulfide precipitate around the colony. Plates were incubated at 30\u0026deg;C for single colonies to enumerate the prevalence of sulfidogenesis in the mixed microbial community. Data was visualized with RStudio v.2021.09.01Sulfide (S\u003csup\u003e2\u0026minus;\u003c/sup\u003e) can be microbially produced through the reduction of multiple sulfur chemical intermediates in the environment including thiosulfate (S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), sulfite (SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), and/or elemental sulfur (S\u003csub\u003e0\u003c/sub\u003e). Standard SIM media is formulated with thiosulfate as the source of sulfur. Alternative forms of sulfur compounds were substituted in the plates for additional sulfidogenesis screens including sulfate (S\u003csub\u003ea\u003c/sub\u003eIM), sulfite (S\u003csub\u003ei\u003c/sub\u003eIM), and elemental sulfur (S\u003csub\u003e0\u003c/sub\u003eIM).\u003c/p\u003e\n\u003ch3\u003eCharacterizing Acidogenic Mn Reducers\u003c/h3\u003e\n\u003cp\u003eBacterial isolates (AV20, AV21, AV22, AV23, AV24, KB7, JR07), all of which were positive for Mn reduction on differential Mn reduction agar plates (15 g/L agar), were single colony purified twice and inoculated into a modified differential Mn reduction broth that substituted KMnO\u003csub\u003e4\u003c/sub\u003e with 50 mM MnO\u003csub\u003e2\u003c/sub\u003e (Krumbein and Altman \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). The FAD assay was performed with concurrent pH and OD\u003csub\u003e600\u003c/sub\u003e readings (Brewer and Spencer \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Sterile media was used as a control. Measurements were taken on day 0, 1, 2, and 3. A two-factor ANOVA with replication was performed using Microsoft Excel v16.92 and data was visualized with RStudio v.2021.09.01. The addition of cell-free 0.2 \u0026micro;M filter sterilized bacterially acidified spent media was added to 50 mM MnO\u003csub\u003e2\u003c/sub\u003e and measured with the FAD assay, pH and OD\u003csub\u003e600\u003c/sub\u003e to determine if bacterially produced acid can facilitate Mn(II) solubilization. Sanger sequencing of a portion of the 16S rRNA gene was used to identify isolates. DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA) from isolates grown in R2A pH 7.0 broth. The 16S rRNA gene primers 27F (5\u0026rsquo;AGAGTTTGATCMTGGCTCAG3\u0026rsquo;) and 518R (5\u0026rsquo;GTATTACCGCGGCTGCTGG3\u0026rsquo;) were used for AV20, AV21, AV22, AV23, and AV24 (Das, Dash et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Goolam Mahomed, Peters et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). PCR products were inserted into plasmid vector pCR2.1 and cloned in competent \u003cem\u003eE. coli\u003c/em\u003e TOP10F\u0026rsquo; cells using the Original TA Cloning kit and the manufacturer\u0026rsquo;s instructions (Invitrogen, Carlsbad, CA, USA). Transformed cells were grown on Luria-Bertani (\u003cstrong\u003eLB\u003c/strong\u003e) agar with 100 mM IPTG, 40 \u0026micro;g/mL X-gal, and 50 \u0026micro;g/mL Kanamycin (\u003cstrong\u003eKan\u003c/strong\u003e) at 37\u0026deg;C. White colonies were purified three times by single colony purification on LB/IPTG/X-gal/Kan plates and grown in LB broth with 50 \u0026micro;g/mL Kan. Plasmid DNA was purified using the Plasmid Miniprep kit (Qiagen, Hilden, Germany) and was sent for Sanger sequencing at GeneWiz (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.genewiz.com\u003c/span\u003e\u003c/span\u003e, Azenta Life Sciences, South Plainfield, NJ, USA). DNA sequences were analyzed in 4Peaks and with The National Center for Biotechnology Information (\u003cstrong\u003eNCBI\u003c/strong\u003e) Basic Local Alignment Search Tool (\u003cstrong\u003eBLAST\u003c/strong\u003e) using default settings (Vietmeier, Valkanas et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). DNA from KB7 and JR07 was sent for whole genome sequencing using the Illumina MiSeq at SeqCenter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.seqcenter.com\u003c/span\u003e\u003c/span\u003e, Pittsburgh, PA, USA). DNA sequences received were imported into KBase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.kbase.us\u003c/span\u003e\u003c/span\u003e) and processed as previously done (Vietmeier, Valkanas et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The 16S rRNA gene was recovered from the annotated genome and put through NCBI BLAST to identify.\u003c/p\u003e\n\u003ch3\u003eCharacterizing Sulfidogenesis Microbes\u003c/h3\u003e\n\u003cp\u003eTen of the sulfidogenic bacterial isolates (NML8, NML9, NML10, NML11, NML14, NML16, NML34, NML35, NML36, NML37) were single colony purified on R2A pH 7.0 plates a minimum of three times (Lamagna \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These ten isolates were grown in a Sulfidogen media, a modified version of Postgate media, that contains thiosulfate as the electron donor (0.225 g potassium phosphate dibasic, 0.225 g potassium phosphate monobasic, 0.46 sodium chloride, 0.225 g ammonium sulfate, 0.118 g magnesium sulfate, 1.0 g yeast extract, 3.74 mL 30% sodium lactate, 4.1 g sodium bicarbonate, 2.48 g sodium thiosulfate, 5 mL trace element mix, and 5 mL vitamin mix per liter) (Postgate \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1963\u003c/span\u003e). Wheaton bottles were filled with 75 mL of Sulfidogen media that was gassed with nitrogen for 10-mins., followed by gassing the headspace with nitrogen for 2-mins. The bottles were immediately capped, crimped, and autoclaved at 121\u0026deg;C for 45-mins., cooled to room-temperature and inoculated for each of the ten isolates from overnight cultures. Isolates were grown for 2-weeks at 30\u0026deg;C. A sterile control was included. Microbial sulfidogenesis in the cultures was quantified from precipitates to control for potential thiosulfate interference with the colorimetric methylene blue assay (Cline \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1969\u003c/span\u003e). Samples were centrifuged for 15-mins. to pellet sulfide solids, the supernatant was decanted. Solids were resuspended in 0.2 mL 9N HCl and diluted in 4.8 mL nanopure water before being mixed with 400 \u0026micro;L diamine reagent in tightly capped tubes. Samples were incubated for 20-mins., and absorbance was measured at OD\u003csub\u003e670\u003c/sub\u003e. Results of each isolate were analyzed with t-test compared to sterile control using Excel v16.92 and data was visualized with RStudio v.2021.09.01. OD\u003csub\u003e600\u003c/sub\u003e of sulfidogenic isolates growth was not recorded due to black color of media inhibiting this measurement. Five sulfidogenic bacteria (NML11, NML14, NML16, NML35, and NML36) were identified by their 16S rRNA gene by Sanger sequencing as described above. DNA was extracted using a Zymo Bacterial/Fungal DNA Extraction kit (Zymo Research, Irvine, CA, USA) and PCR amplification was carried out with primers 27F and 805R (5\u0026prime;GACTACHVGGGTATCTAATCC3\u0026prime;) (Tanner, Goebel et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Fujiyoshi, Muto-Fujita et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePotential Acidogenic and Sulfidogenic Microbes at Wingfield Pines by 16S rRNA Sequencing\u003c/h3\u003e\n\u003cp\u003eWingfield Pines PRS 16S rRNA gene analysis data that was previously generated by the Trun laboratory was mined for bacteria identified as acidogenic and sulfidogenic (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Bacteria included in the retrospective screen for acidogenic and sulfidogenic producers included those identified in this paper via their 16S rRNA gene and bacterial isolates previously identified in the literature (Burdige and Nealson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Myers and Nealson \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, Grupe and Gottschalk \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Lovley and Phillips \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Crescenzi, Crisari et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Zhao, Ren et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Kim, Hwang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Lee, Kennedy et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Liu, Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Zhang, Zhang et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Kushkevych \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Grady, MacDonald et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Duan, Wang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Henkel, Dellwig et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Sun, Li et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Tahir, Miran et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhang, Loh et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Valkanas, Rosso et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Acidogenic bacteria include \u003cem\u003eBacillus\u003c/em\u003e sp., \u003cem\u003eBacteroides\u003c/em\u003e sp., \u003cem\u003eClostridium\u003c/em\u003e sp., \u003cem\u003eCorynebacterium\u003c/em\u003e sp., \u003cem\u003eLactobacillus\u003c/em\u003e sp., \u003cem\u003ePaenibacillus\u003c/em\u003e sp., \u003cem\u003ePropionibacterium\u003c/em\u003e sp., \u003cem\u003ePseudomonas\u003c/em\u003e sp., \u003cem\u003eRuminococcus\u003c/em\u003e sp., \u003cem\u003eThiobacillus\u003c/em\u003e sp., \u003cem\u003eVeillonella\u003c/em\u003e sp. (\u003cstrong\u003eSI\u003c/strong\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Grupe and Gottschalk \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Crescenzi, Crisari et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Zhao, Ren et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Kim, Hwang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Liu, Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Grady, MacDonald et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Duan, Wang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Tahir, Miran et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhang, Loh et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Sulfidogenic bacteria include \u003cem\u003eAlteromonas\u003c/em\u003e sp., \u003cem\u003eShewanella\u003c/em\u003e sp., \u003cem\u003eSulfurimonas\u003c/em\u003e sp., \u003cem\u003eDesulfuromonas\u003c/em\u003e sp., \u003cem\u003eDesulfomicrobium\u003c/em\u003e sp., \u003cem\u003eDesulfovibrio\u003c/em\u003e sp., \u003cem\u003eDesulfobulbus\u003c/em\u003e sp., \u003cem\u003eSulfuricurvum\u003c/em\u003e sp., and \u003cem\u003eDesulfomonile\u003c/em\u003e sp. (\u003cstrong\u003eSI Table\u0026nbsp;2\u003c/strong\u003e) (Burdige and Nealson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Myers and Nealson \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, Lovley and Phillips \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Zhao, Ren et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Lee, Kennedy et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Kushkevych \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Henkel, Dellwig et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tahir, Miran et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Valkanas, Rosso et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Data was visualized with RStudio v.2021.09.01. Briefly, the previous methods used to collect 16S rRNA gene data was as follows: DNA was extracted from Wingfield Pines samples using the PowerSoil Kit (MoBio Laboratories Inc., Carlsbad, CA, USA). The 16S rRNA V4 region was amplified using the Illumina\u0026rsquo;s 515F and 806R primers (16S Illumina Amplicon Protocol, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.earthmicrobiome.org\u003c/span\u003e\u003c/span\u003e) and were sent for sequencing at Wright Labs (Huntingdon, PA, USA). Sequences were trimmed to 252 bp, quality filtered, chimeras were removed, and operational taxonomic units (\u003cstrong\u003eOTUs\u003c/strong\u003e) assigned (DeSantis, Hugenholtz et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Edgar \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). QIIME v1.9.0 was used to construct a table of OTUs with their taxonomic assignments (Caporaso, Kuczynski et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Caporaso, Lauber et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The 16S rRNA gene profiles were successfully collected for locations 1, 3, 4, 5, 6, and 7.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWingfield Pines PRS and Boyce Park PRS Chemical Data for Mn, Sulfate, and pH\u003c/h2\u003e \u003cp\u003eSeasonal changes are seen at Wingfield Pines PRS for Mn, sulfate, and pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For all four seasons, the level of Mn increases as AMD moves through the Wingfield Pines PRS, with the highest level of Mn found in the wetlands, the last component of the PRS before exiting into the stream (Valkanas \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The highest level of Mn exiting the Wingfield Pines system was in the Fall at 7.39 PPM. Seasonally, sulfate is detected within the Wingfield Pines system, with the highest level reported in Spring at 323 PPM in the wetlands. The sulfate levels remain high, with an average for all seasons\u0026thinsp;\u0026gt;\u0026thinsp;490 PPM for all ponds. The lowest sulfate level is reported in pond 3 in winter at 168 PPM. For all seasons the Wingfield Pines system remains circumneutral at a pH between 6.8\u0026ndash;7.5. At the Boyce Park PRS, seasonally the average Mn concentration stays\u0026thinsp;\u0026gt;\u0026thinsp;0.20 PPM, with the greatest concentration in pond 4 in the summer at 1.52 PPM (Vietmeier, Valkanas et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The pH at Boyce Park seasonally stays acidic, below 6.0 with an average pH of ~\u0026thinsp;4 (\u003cb\u003eSI\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemical Abiotic Mn Reduction Occurs in High Acid and Sulfide Conditions\u003c/h3\u003e\n\u003cp\u003eSamples collected from circumneutral Wingfield Pines PRS and acidic Boyce Park PRS were autoclaved and MnO\u003csub\u003e2\u003c/sub\u003e was added to 50 mM to assess chemical abiotic impacts on Mn. The data showed that the abiotic chemistry of the Wingfield Pines AMD PRS does not drive Mn solubilization to Mn(II), however it does at acidic Boyce Park (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). There was a significant difference in the solubilization of Mn(II) from MnO\u003csub\u003e2\u003c/sub\u003e between sterilized acidic Boyce Park AMD samples and sterilized circumneutral Wingfield Pines AMD samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Mn(II) solubilization did not occur in circumneutral Wingfield Pines AMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). There is a significant difference in pH between circumneutral Wingfield Pines AMD and acidic Boyce Park AMD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000001), and the pH of both Wingfield Pines and Boyce Park AMD systems does not change with the addition of MnO\u003csub\u003e2\u003c/sub\u003e in the lab (p\u0026thinsp;=\u0026thinsp;0.12) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The sterilized AMD from both Boyce Park and Wingfield Pines remained sterile as no bacterial growth was detected (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.000) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings allude to a chemical abiotic solubilization of MnO\u003csub\u003e2\u003c/sub\u003e to Mn(II) in acidic AMD, like Boyce Park, that is most likely facilitated by the low pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). There is no observed chemical abiotic solubilization of Mn(II) in circumneutral Wingfield Pines, which indicates the potential for a microbially driven mechanism. Due to geochemical solubilization of Mn(II) in acidic AMD, and potential for microbial Mn(II) solubilization in circumneutral AMD, experiments moving forward were performed in circumneutral Wingfield Pines AMD.\u003c/p\u003e \u003cp\u003eMnO\u003csub\u003e2\u003c/sub\u003e can be reduced to soluble Mn(II) in Wingfield Pines when AMD is acidified, or sulfide concentrations increase. Solubilization of Mn(II) from MnO\u003csub\u003e2\u003c/sub\u003e occurs when sterile AMD from Wingfield Pines is acidified (p\u0026thinsp;=\u0026thinsp;0.00089) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). There is a significant difference between the pH of Wingfield Pines before and after acidification (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0000001), and no significant drift in the pH over time for either the sterile Wingfield Pines or sterile acidified Wingfield Pines AMD samples (p\u0026thinsp;=\u0026thinsp;0.25) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). There is no significant difference in OD\u003csub\u003e600\u003c/sub\u003e between Wingfield Pines and acidified Wingfield Pines over time (p\u0026thinsp;=\u0026thinsp;0.07) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In sterile circumneutral Wingfield Pines AMD, MnO\u003csub\u003e2\u003c/sub\u003e is geochemically reduced to Mn(II) with increasing sulfide concentrations (p\u0026thinsp;=\u0026thinsp;0.0018) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The addition of sulfide without MnO\u003csub\u003e2\u003c/sub\u003e does not result in an increase of soluble Mn(II) (\u003cb\u003eSI\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Both increasing acidity with HCl and increasing concentrations of sulfide results in increased chemical Mn reduction as noted by the change in color from deep gold to pale-yellow or white respectively in differential Mn reduction broth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). The clearing of color from the differential Mn reduction broth can visually be noted at pH 5.9 and becomes more prominent as the pH decreases, where at pH 3.6 the media is a pale yellow in appearance. The addition of 0.1 mM sulfide visually results in a more pale-yellow of the media and the increasing concentrations of sulfide results in a more prominent chemical reaction decreasing the deep gold color to a cloudy pale yellow (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence of Acidogenic Mn Reducers and Sulfidogenesis in the Culturable Community\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eWithin the mixed microbial community at Wingfield Pines PRS, bacteria cultured from every location produced acid capable of reducing Mn and generated sulfide. Across all the sampling locations at Wingfield Pines PRS, 285 bacterial isolates were screened for Mn reduction by acidogenesis in differential Mn broth with a pH indicator (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Of the 1,995 bacteria screened (285 isolates from each of the 7 locations), Mn reduction and acidogenic metabolism were found in every location within the system. The percentage of isolates performing concurrent Mn reduction and acidogenesis shows the general trend of increasing as AMD moves through the PRS. The highest percentage of acidogenic isolates were found in location 7, the wetlands, at 80% (228/285). All sampling locations screened at Wingfield Pines also identified bacterial isolates capable of sulfidogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). All plates for sulfidogenesis screening contained between 10\u0026ndash;400 colonies, which appeared after 18\u0026ndash;24 hours, with a black coloration of colonies appearing after ~\u0026thinsp;18\u0026ndash;72 hours. Sulfidogenesis was detected on thiosulfate SIM plates and elemental sulfur S\u003csub\u003e0\u003c/sub\u003eIM plates. The greatest percentage of isolates positive for sulfidogenesis on SIM media was in location 5 at 35% (218/615) and the lowest was in location 1 at 2% (3/129). Sulfidogenesis from elemental sulfur S\u003csub\u003e0\u003c/sub\u003eIM plates was only observed in location 1 at 3% (3/94) and in location 6 at 1% (1/117). Sulfidogenesis was not observed on sulfite S\u003csub\u003ei\u003c/sub\u003eIM and sulfate S\u003csub\u003ea\u003c/sub\u003eIM plates.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCharacterizing Acidogenic Mn Reducers\u003c/h2\u003e \u003cp\u003eSeven bacterial isolates (AV20, AV21, AV22, AV23, AV24, KB7, JR07) capable of KMnO\u003csub\u003e4\u003c/sub\u003e reduction on differential plates were identified and purified. These isolates reduce MnO\u003csub\u003e2\u003c/sub\u003e to soluble Mn(II) in a modified Mn reduction broth, substituting MnO\u003csub\u003e2\u003c/sub\u003e for KMnO\u003csub\u003e4\u003c/sub\u003e, as they grow and produce acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). The soluble Mn(II) increased to ~\u0026thinsp;20 PPM when the bacterial isolates grew (p\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). There was a significant decrease in the pH over time when inoculated with the bacterial isolates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Microbial growth increased over time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001), alluding to a biotic role in acidic production and Mn reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The addition of sterile cell-free microbially acidified spent media to MnO\u003csub\u003e2\u003c/sub\u003e results in the solubilization of Mn(II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Cell-free spent acidified media resulted in a significant increase of Mn(II) from MnO\u003csub\u003e2\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). There was a not significant change in the pH over time (p\u0026thinsp;=\u0026thinsp;0.9), but there was a significant difference in pH between sterile spent media and spent media that was microbially acidified (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Microbial growth was not detected over time (p\u0026thinsp;=\u0026thinsp;0.9) indicating samples remained sterile (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Of the acidogenic isolates, five isolates (AV20, AV22, AV23, KB7, JR07) were identified as relatives of \u003cem\u003eBacillus\u003c/em\u003e spp. and two isolates (AV21, AV24) were identified as relatives of \u003cem\u003eCorynebacterium\u003c/em\u003e spp. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCharacterizing Sulfidogenesis Microbes\u003c/h2\u003e \u003cp\u003eOf the 39 sulfidogenic bacteria purified from the thiosulfate SIM medium, 36 maintained their phenotype on thiosulfate SIM plates. Ten of the sulfidogenic isolates were chosen and grown in Sulfidogen medium for 2-weeks based on their formation of large black precipitates on SIM plates; all ten isolates produced greater than 8.3 PPM of sulfide during growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Black precipitates were visible in all isolates within 24 hours of growth and media turned black in color within 14 days of growth indicating sulfide production (\u003cb\u003eSI\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The sterile control did not produce detectable levels of sulfide by the colorimetric assay. Results of statistical analysis showed all isolates were significant when compared to sterile control for sulfide production: NML10, NML34, NML35, and NML36 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); NML11 and NML14 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001); NML8 and NML16 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001); NML9 and NML37 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001). The greatest amount of sulfidogenesis was measured in NML37 at 20.5 PPM sulfide, followed by NML36 at 20.4 PPM sulfide. Quantification of bacterial sulfidogenesis with the Sulfidogen media and colorimetric assay validated the use of the modified SIM plate overlay method to screen for sulfidogenic bacteria. Five sulfidogenic isolates were successfully identified by their 16S rRNA gene, NML11 was identified as \u003cem\u003eCitrobacter freundii\u003c/em\u003e, both NML14 and NML16 were identified as \u003cem\u003eAeromonas veronii\u003c/em\u003e, NML35 was identified \u003cem\u003eShewanella baltica\u003c/em\u003e, and NML36 was identified \u003cem\u003eShewanella\u003c/em\u003e sp. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentification of acidogenic and sulfidogenic bacterial isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16S rRNA % identity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMetabolism\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAV20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90% \u003cem\u003eBacillus cereus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAV21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98% \u003cem\u003eCorynebacterium\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAV22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83% \u003cem\u003eBacillus\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAV23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90% \u003cem\u003eBacillus thuringiensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAV24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98% \u003cem\u003eCorynebacterium\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKB7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% \u003cem\u003eBacillus pseudomycoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eJR07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% \u003cem\u003eBacillus mycoides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNML11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97% \u003cem\u003eCitrobacter freundii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSulfidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNML14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98% \u003cem\u003eAeromonas veronii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSulfidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNML16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99% \u003cem\u003eAeromonas veronii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSulfidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNML35\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97% \u003cem\u003eShewanella baltic\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSulfidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNML36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99% \u003cem\u003eShewanella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSulfidogenesis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e16S rRNA Gene Prevalence of Known Acidogenic and Sulfidogenic Bacteria in Wingfield Pines PRS\u003c/b\u003e \u003c/p\u003e \u003cp\u003e16S rRNA gene analysis of the mixed microbial community from a previous seasonal Wingfield Pines PRS publication was mined for species related to known acidogenic and sulfidogenic bacteria by OTUs using relative abundance (\u003cb\u003eSI\u003c/b\u003e Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; 2) (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Acidogenic bacteria predicted in the system include \u003cem\u003eBacillus\u003c/em\u003e sp. 0.01%, \u003cem\u003eClostridium\u003c/em\u003e sp. 0.12%, \u003cem\u003eBacteroides\u003c/em\u003e sp. 0.01%, \u003cem\u003eRuminococcus\u003c/em\u003e sp. 0.02%, \u003cem\u003ePseudomonas\u003c/em\u003e sp. 0.10%, \u003cem\u003eVeillonella\u003c/em\u003e sp. 0.002%, \u003cem\u003eLactobacillus\u003c/em\u003e sp. 0.01%, \u003cem\u003eThiobacillus\u003c/em\u003e sp. 1.0%, \u003cem\u003ePaenibacillus\u003c/em\u003e sp. 0.002%, \u003cem\u003eCorynebacterium\u003c/em\u003e sp. 0.001%, and \u003cem\u003ePropionibacterium\u003c/em\u003e sp. 0.0005%. The majority of the acidogenic bacteria were found in the wetlands, sampling location 6 (18,643 OTUs, 2.5%) and location 7 (8,985 OTUs, 3.4%) (Grupe and Gottschalk \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Crescenzi, Crisari et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Zhao, Ren et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Kim, Hwang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Liu, Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Zhang, Zhang et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Grady, MacDonald et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Duan, Wang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Tahir, Miran et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhang, Loh et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In total, 24,106 OTUs from acidogenic bacterial sequences were predicted within the system (1.3% of total OTUs) based on isolates identified in this study and in the literature (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cb\u003eSI\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSulfidogenic bacteria predicted in the system include \u003cem\u003eAlteromonas\u003c/em\u003e sp. (0.001%), \u003cem\u003eDesulfacinum\u003c/em\u003e sp. (0.02%), \u003cem\u003eDesulfarculus\u003c/em\u003e sp. (0.003%), \u003cem\u003eDesulfobacca\u003c/em\u003e sp. (0.08%), \u003cem\u003eDesulfobulbus\u003c/em\u003e sp. (0.77%), \u003cem\u003eDesulfococcus\u003c/em\u003e sp. (0.56%), \u003cem\u003eDesulfofrigus\u003c/em\u003e sp. (0.001%), \u003cem\u003eDesulfomicrobium\u003c/em\u003e sp. (0.02%), \u003cem\u003eDesulfomonile\u003c/em\u003e sp. (0.06%), \u003cem\u003eDesulfotalea\u003c/em\u003e sp. (0.001%), \u003cem\u003eDesulfovibrio\u003c/em\u003e sp. (0.03%), \u003cem\u003eDesulfuromonas\u003c/em\u003e sp. (0.005%), \u003cem\u003ePelobacter\u003c/em\u003e sp. (0.0002%), \u003cem\u003eShewanella\u003c/em\u003e sp. (0.0003%), \u003cem\u003eSulfuricurvum\u003c/em\u003e sp. (2.2%), \u003cem\u003eSulfurimonas\u003c/em\u003e sp. (0.08%), and \u003cem\u003eSyntrophobacter\u003c/em\u003e sp. (0.03%). Sulfidogenic bacteria were found in all locations of the system with the greatest amount of sulfidogenic bacteria in the wetlands at location 6 (37,270 OTUs, 9%) (\u003cb\u003eSI Table\u0026nbsp;2\u003c/b\u003e) (Burdige and Nealson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Myers and Nealson \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, Lovley and Phillips \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Zhao, Ren et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Lee, Kennedy et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Kushkevych \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Henkel, Dellwig et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tahir, Miran et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Valkanas, Rosso et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In total 71,633 unique OTUs of sequences matching sulfidogenic bacteria (3.8% of total OTUs) were predicted within the system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The number of predicted acidogenic and sulfidogenic bacteria increases as AMD moves through the PRS with the highest numbers found in the wetlands of the system (location 6 and 7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe biogeochemical mechanisms involved in the seasonal chemical fluctuations within circumneutral and acidic AMD PRS have not been uniformly well characterized (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSI\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Ly, Wright et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We sought to understand the potential of microbes that naturally colonize PRSs to metabolically impact Mn solubilization in circumneutral and acidic AMD PRSs (Burdige \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1983\u003c/span\u003e, Gounot \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Tebo, Bargar et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Ly, Wright et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). At circumneutral Wingfield Pines PRS, for all seasons there is an increase in Mn within the wetlands of the system prior to entering the watershed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Spikes in Mn in the wetlands of circumneutral Wingfield Pines PRS occurred concurrent with spikes in sulfate, which was not observed at acidic Boyce Park PRS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSI\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Cochran \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe sought to first understand the geochemical mechanisms driving Mn solubilization in circumneutral and acidic AMD PRSs. Sterile AMD from circumneutral Wingfield Pines PRS does not result in the chemical solubilization of Mn(II) when sterile MnO\u003csub\u003e2\u003c/sub\u003e is added (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). However, when sterile Wingfield Pines AMD is acidified, it results in the abiotic solubilization of MnO\u003csub\u003e2\u003c/sub\u003e to Mn(II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). The acidification of media facilitating the chemical speciation of Mn(II) is consistent with low pH reported in Pourbiax diagrams (Sun, Kitchaev et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Wang, Guo et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Further, the addition of sterile MnO\u003csub\u003e2\u003c/sub\u003e to sterile acidic Boyce PRS AMD results in the increase of soluble Mn(II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). This alludes to the solubilization of Mn(II) being chemically driven by low pH, which is further supported in reference to the phase stability of Mn speciation in acidic conditions reported in Pourbiax diagrams (Sun, Kitchaev et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Wang, Guo et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). At acidic Boyce Park PRS, it is likely Mn solubilization and mobilization is primarily geochemically driven based on low pH from our in-lab findings (\u003cb\u003eSI\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). We also noted that when increasing concentrations of sterile sodium sulfide is added to sterile Wingfield Pines AMD with sterile MnO\u003csub\u003e2\u003c/sub\u003e there is an increase of soluble Mn(II) proportional to the sulfide concentration added (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The impacts of acidification and high sulfide concentrations on Mn reduction was further elucidated in differential Mn reduction broth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, \u003cb\u003eSI\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Due to the solubilization of MnO\u003csub\u003e2\u003c/sub\u003e to Mn(II) in acidic conditions, we assessed the potential for biotically mediated Mn reduction within circumneutral Wingfield Pines PRS using both culture-dependent and culture-independent molecular approaches.\u003c/p\u003e \u003cp\u003eIn circumneutral Wingfield Pines AMD, culturable microbes capable of acidogenesis and sulfidogenesis are present. Acidogenic microbes that reduce Mn were found in every location of Wingfield Pines as determined with differential Mn reduction broth with the additional of a pH indicator (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) (Krumbein and Altman \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). A general increase in acidogenic bacteria that reduce Mn is observed as AMD moves through the PRS with the greatest percentage of acidogenic bacteria in location 7 near the end of the system at (80% of all culturable bacteria metabolically screened) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Sulfidogenic microbes that produce sulfide were found in all locations of Wingfield Pines with the modified thiosulfate SIM agar plates, with a general increase of sulfidogenic bacteria near the end of the system (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Seasonal chemical studies of Wingfield Pines show there is an increased availability of sulfate at the end of the system which could be utilized by sulfidogenic microbes to produce sulfide leading to increased soluble Mn(II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We suggest based on previous research the slow flow of AMD through the PRS creates an oxic to anoxic gradient that could facilitate microbial sulfate reduction to sulfide reactions, especially within the sediment of these systems (Valkanas and Trun \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is possible additional microbes are present at Wingfield Pines that are capable of acidogenesis and sulfidogenesis that are difficult to culture or were not captured with these culturing methods that also contributing to Mn cycling. Which may be the case for the lack of reduction observed on the sulfate and sulfite plates, or due to suboxic culturing conditions from the low oxygen top agar overlay that are not fully anaerobic conditions. Alternatively, the reduction of sulfate, sulfite, and elemental sulfur to sulfide require additional microbial metabolic pathways not required for the reduction of thiosulfate to sulfide, suggesting these isolates do not encode those metabolic machineries. The frequency of bacteria that can facilitate Mn reduction by organic acid or sulfide byproducts at Wingfield Pines PRS can be inferred from the frequency of these cultured bacteria. Further, the increase of acidogenic and sulfidogenic metabolic phenotypes as AMD moves through the system with elevation of these phenotypes at the end of the system correspond to Mn spikes and potentially explain them (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicrobial isolates from the metabolic screens of acidogenesis and sulfidogenesis were further characterized. The seven acidogenic microbial isolates recovered reduce multiple Mn chemical species in different oxidation states including the reduction of KMnO\u003csub\u003e4\u003c/sub\u003e [Mn(VII)] and MnO\u003csub\u003e2\u003c/sub\u003e [Mn(IV)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). We showed that the microbially produced organic acids facilitate the reduction of MnO\u003csub\u003e2\u003c/sub\u003e to Mn(II) through the addition of sterile cell-free spent media (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). These findings show that Mn(II) can be released from MnO\u003csub\u003e2\u003c/sub\u003e without the presence of any cells, but rather their metabolic production of acid drives the solubilization of Mn(II). All ten of the sulfidogenic isolates recovered from thiosulfate SIM plates could produce quantifiable levels of sulfide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As sulfidogenesis quantification was measured using only the sulfide precipitates due to thiosulfate interference in the colorimetric methylene blue assay, a greater amount of sulfide may have been produced in liquid and as hydrogen sulfide gas. Microbial reduction to sulfide can result in the reduction of MnO\u003csub\u003e2\u003c/sub\u003e (Burdige and Nealson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Lee, Kennedy et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Sun, Li et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Valkanas, Rosso et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 16S rRNA analysis of acidogenic bacteria identified them as relatives to \u003cem\u003eBacillus\u003c/em\u003e spp., and \u003cem\u003eCorynebacterium\u003c/em\u003e spp., and sulfidogenic bacteria as relatives of \u003cem\u003eCitrobacter\u003c/em\u003e sp., \u003cem\u003eAeromonas\u003c/em\u003e spp., and \u003cem\u003eShewanella\u003c/em\u003e spp. which are all consistent with metabolisms reported in the literature for organic acid and sulfide production (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSI\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSI Table\u0026nbsp;2\u003c/b\u003e) (Qiu, Zhao et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Kim, Hwang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Lee, Kennedy et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Liu, Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Wu, Li et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Grady, MacDonald et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Yan, Zhong et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Abedi and Hashemi \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Sharma, Garg et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Wu \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhang, Liu et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Semwal, Kumar et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Laboratory results demonstrated that culturable microbes from the Wingfield Pines PRS can produce quantifiable levels of organic acids that solubilize Mn(II) and produce quantifiable levels of sulfide that can solubilize Mn(II).\u003c/p\u003e \u003cp\u003eTo relate this culture-based laboratory finding on microbial driven Mn(II) solubilization to the Wingfield Pines system, 16S rRNA data was mined for relatives to known acidogenic and sulfidogenic microbes. Known acidogenic and sulfidogenic bacteria were present in every location of Wingfield Pines as predicted by OTUs, with a general trend of increasing relative abundance as AMD moves through the system, with the highest number found in the wetlands (location 6 and 7; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The presence of phylogenetic relatives in the 16S rRNA gene sequence community alludes to a potential of these microbial metabolisms impacting Mn solubilization within the systems, consistent with seasonal increases of Mn seen before exiting the system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As metabolic acidogenesis occurs in cultured isolates from the wetland and 16S rRNA relatives are predicted within the Wingfield Pines PRS, it is possible microbial acidification is happening on a local level leading to Mn(II) solubilization that is not being detected in large scale surveys of pH. Mn spikes are possibly also driven by sulfidogenesis as sulfide producers are present in the culturable community, sulfidogenic relatives are predicted in the 16S rRNA system surveys, and sulfate is available in increasing concentrations as seen in the seasonal chemical studies. It is more likely there is a complex combination of both acidogenesis and sulfidogenesis impacting biogeochemical cycling and remobilization of Mn in circumneutral systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis study highlights the importance of considering the impact of microbial metabolism and placement of wetlands within PRS design (Neculita and Rosa \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This is especially true within circumneutral PRS AMD as microbial driven Mn(II) solubilization by acid and sulfide can potentially be found at other circumneutral systems, as similar spikes in Mn are seen in the wetlands in analogous circumneutral Lowber AMD PRS (Cochran \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Wetlands are carbon-rich environments designed to promote sulfate reduction; however, these conditions can lead to both the production of organic acids from carbon fermentation and the production of sulfide, both of which, from our findings, can reduce/re-mobilize Mn (Pester, Knorr et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Candry, Flinkstorm et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This study highlights the interconnectivity of microbial metabolism and chemical cycling for multiple AMD contaminants including Mn and sulfate. Although remediation of sulfate is also a goal of AMD systems the reduction to sulfide can lead to complications with Mn mobilization (Heizer, Sandler et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Hallberg and Johnson \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Hatat-Fraile and Barbeau \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Ray and Dey \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Sun, Li et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Valkanas, Rosso et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, EPA \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, EPA \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We suggest that wetlands may not be the optimal choice to be the final component of the system and an additional settling pond specific for Mn removal may be beneficial. This study also attempted to address the knowledge gap of how microbes impact the remediation of Mn in AMD as it is likely these microbes are preventing the Wingfield Pines PRS from effectively remediating Mn to full optimization. As Mn is a critical mineral and known to sequester other minerals in its oxide form, microbial metabolism that results in Mn(II) solubilization with the context of the AMD PRS may be of use in biotechnology, specifically for the biomining of critical minerals.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003eAuthor has no competing interests to disclose\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe acknowledge Duquesne University School of Science and Engineering Department of Biological Sciences, The Bayer Research Fellowship, Duquesne University Women in STEM, American Society for Microbiology (ASM) Future Leaders Mentorship Fellowship (FLMF), The Allegheny Branch of ASM, Geological Society of American Research Grant through the National Science Foundation, National Association of State Land Reclaimationists, and American Society of Reclamation Sciences for their support. We also acknowledge the Oak Ridge Institute for Science and Education (ORISE), National Energy Technology Laboratory (NETL), Department of Energy (DOE), and the Gulliver Laboratory at NETL. We acknowledge the Stolz laboratory at Duquesne University and Dr. Tetiana Cantlay. Acknowledge Alexa Lovelace and Michaela Bosworth for early experiments with manganese reduction, Joshua Robinson for isolation of JR07, and introduction of the differential manganese reduction agar by Raegen Esenwein.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbedi, E. and S. M. B. Hashemi (2020). \u0026quot;Lactic acid production - producing microorganisms and substrates sources-state of art.\u0026quot; \u003cu\u003eHeliyon\u003c/u\u003e \u003cstrong\u003e6\u003c/strong\u003e(10): e04974.\u003c/li\u003e\n\u003cli\u003eBrewer, P. G. and D. W. 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Wong (2023). \u0026quot;Manganese losses induced by severe soil acidification in the extensive Lei bamboo (Phyllostachys violascens) plantation stands in Eastern China.\u0026quot; \u003cu\u003eChemosphere\u003c/u\u003e \u003cstrong\u003e339\u003c/strong\u003e: 139669.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mine-water-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mwen","sideBox":"Learn more about [Mine Water and the Environment](http://link.springer.com/journal/10230)","snPcode":"10230","submissionUrl":"https://www.editorialmanager.com/mwen/default2.aspx","title":"Mine Water and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"manganese cycling, acidogenesis, sulfidogenesis, abandoned mine drainage, passive remediation system","lastPublishedDoi":"10.21203/rs.3.rs-9065662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9065662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePassive remediation systems (\u003cb\u003ePRSs\u003c/b\u003e) treat abandoned legacy mines to remove the contaminants in abandoned mine drainage (\u003cb\u003eAMD\u003c/b\u003e), using settling ponds, limestone beds, and wetlands to precipitate contaminants onsite. These PRSs remove high levels of metals in AMD, including manganese (\u003cb\u003eMn\u003c/b\u003e). The impact of microbes that naturally colonize the PRSs on Mn solubilization is poorly understood. We sought to determine the microbial mechanisms contributing to Mn solubilization. We determined in circumneutral AMD, Mn reduction can be microbially facilitated through acidogenesis and sulfidogenesis, whereas in acidic AMD systems it is primarily geochemically driven by low pH. Within the circumneutral Wingfield Pines AMD PRS, spikes of Mn occurred in the wetland at the end of the system. Culturable acidogenic and sulfidogenic bacteria were enumerated from the Wingfield Pines PRS in multiple locations with spikes at the end of the system. Bacterial isolates were identified via 16S rRNA gene sequencing for acidogenesis as \u003cem\u003eBacillus\u003c/em\u003e spp. and \u003cem\u003eCorynebacterium\u003c/em\u003e spp., and sulfidogenesis as \u003cem\u003eCitrobacter\u003c/em\u003e sp., \u003cem\u003eAeromonas\u003c/em\u003e spp., and \u003cem\u003eShewanella\u003c/em\u003e spp. Whole community surveys of Wingfield Pines PRS by 16S rRNA analysis showed the presence of potentially acidogenic or sulfidogenic bacteria throughout the system, with an increase in relative abundance in the wetlands. We demonstrated microbial Mn resolubilization as acidogenesis and sulfidogenesis in the laboratory to determine potential in-field impacts. This establishes a critical basis for considering microbial processes in future PRS design for effective Mn remediation and potential recovery of Mn as a critical mineral.\u003c/p\u003e","manuscriptTitle":"Microbial Biogeochemical Impacts on Manganese Reduction in Abandoned Mine Drainage Passive Remediation Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-02 09:41:28","doi":"10.21203/rs.3.rs-9065662/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-07T08:11:32+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T10:41:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-16T06:39:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mine Water and the Environment","date":"2026-03-08T12:52:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mine-water-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mwen","sideBox":"Learn more about [Mine Water and the Environment](http://link.springer.com/journal/10230)","snPcode":"10230","submissionUrl":"https://www.editorialmanager.com/mwen/default2.aspx","title":"Mine Water and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e31fd2b6-4265-46dc-b63f-b2fb06e55e32","owner":[],"postedDate":"April 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T09:41:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-02 09:41:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9065662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9065662","identity":"rs-9065662","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}