Performance and stability of a large-scale sulfate-reducing bioreactor with rice bran for passive treatment of acid mine drainage over two years

Performance and stability of a large-scale sulfate-reducing bioreactor with rice bran for passive treatment of acid mine drainage over two years | 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 Performance and stability of a large-scale sulfate-reducing bioreactor with rice bran for passive treatment of acid mine drainage over two years Masataka Kondo, Yuya Sato, Yusei Masaki, Hiroshi Habe, Takaya Hamai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9361529/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 A large-scale sulfate-reducing bioreactor, with a treatment flow rate of 100 L/min, was operated for approximately two years, from September 16, 2022 to September 9, 2024. Initially, 2 ton of a mixture of rice bran and rice husks was placed within the bioreactor in September 2022. After one year of operation, an additional 2 ton of rice bran was fed to the rice bran–rice husk mixture layer using heavy machinery for their mixing in September 2023. Both sulfate reduction and metal removal efficiencies were maintained at high levels, especially in the latter half of the period, without major clogging, even during the cold winter period. Consequently, the maximum concentration of zinc (the second most abundant metal in this AMD after iron) in the effluent remained below 0.3 mg/L (removal ratio: >98%). In the large-scale bioreactor, besides the family Desulfosarcinaceae (sulfate-reducing bacteria), the genus Berkelbacteria was predominant. The results of the large-scale passive treatment highlight the possibility of long-term continuous treatment with the addition of organic sources once a year, and the prevention of bioreactor clogging to assess operational stability and maintenance requirements. acid mine drainage large-scale bioreactor metal removal passive treatment sulfate-reducing bioreactor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Acid mine drainage (AMD) from metal and coal mines is a serious environmental issue worldwide. Various treatment methods have been employed for neutralizing and removing the metals (Younger et al. 2004 ; Nordstrom 2011 ). Japan has more than 5,000 abandoned or closed mines, and approximately 100 of these, require treatment of mine discharge (Ministry of Economy, Trade and Industry 2025 ; Doyama et al. 2025 ). The AMD treatment method in Japan involves adding alkaline reagents such as Ca(OH) 2 to neutralize acidity, precipitating dissolved metals as hydroxides, and subsequently removing them via solid–liquid separation in settling ponds before discharging into rivers or other receiving waters. However, its implementation incurs substantial costs, amounting to several billion yen annually; the heads of expenses include labor for inspection and monitoring of treatment facilities, electricity for agitators and other equipment, and chemicals, such as Ca(OH) 2 and coagulants. Considering that AMD treatment is expected to remain semi-permanent, a strong demand exists for reducing both economic and environmental burdens. Passive treatment represents a low-cost and environment-friendly alternative method for treating AMD, as it does not require external energy inputs such as electricity; instead, it relies on microbial activity and naturally occurring chemical reactions (e.g., Skousen et al. 2017 ). However, the applicability of passive treatment is constrained by the flow rate, acidity, and metal loading, and it is only suitable for AMD with relatively low acidity, low flow rates, and limited metal loads (Taylor et al. 2005 ; Eppink et al. 2020 ; Kleinmann et al. 2023 ). In Japan, most abandoned mines, where AMD treatment is practiced, have been closed for several decades, which has resulted in stable water quality and low discharge flow. Potential for passive treatment application at the domestic abandoned mine sites has been investigated by the Japan Organization for Metals and Energy Security (JOGMEC). Among the passive treatment technologies for removing metal ions, the one using sulfate-reducing bacteria (SRB), is prominent. SRB reduce sulfate ions in AMD to sulfide ions, which subsequently precipitate dissolved metals as insoluble metal sulfides (Habe et al. 2020 ; Mafane et al. 2025 ). Because SRB are heterotrophic microorganisms, they require a supply of organic nutrient sources, for example, manure, compost, wood chips, sawdust, and straw (Waybrant et al. 1998 ; Lefticariu et al. 2015 ). The high-molecular-weight organic matter contained in these nutrient sources is decomposed by microorganisms, and the resulting low-molecular-weight organics are utilized by the SRB as electron donors to reduce sulfate in mine drainage (Neculita et al. 2007 ). In anaerobic treatment with SRB, the hydraulic retention time (HRT) is typically several days, which often necessitates a large land area (Skousen et al. 2017 ; U.S. Army Corps of Engineers 2003 ). However, mine water treatment facilities in Japan are often located in mountainous terrain with limited land space and require compact processes with shorter HRTs for the installation of passive treatment. In the United Kingdom, which faces similar land constraints as Japan, laboratory-scale studies on sulfate-reducing bioreactors with short HRTs have been reported (Gandy et al. 2016 , 2023 ). However, a decrease in HRT can cause reactor clogging owing to reduced permeability. Additionally, in northern parts of Japan, AMD treatment must be sustained during winter, when the outside temperature falls below freezing, and stable performance under low-temperature conditions is a critical challenge. Microbial activity declines in low-water-temperature environments, thereby affecting the treatment efficiency (Harrington et al. 2015 ; Nielsen et al. 2018 ; Ben Ali et al., 2019 , 2020 ). Since 2007, JOGMEC has been conducting research on AMD treatment using a constructed sulfate-reducing bioreactor with shorter HRTs and high robustness against annual temperature fluctuations. In this process, rice bran and rice husk—agricultural byproducts generated during rice production—have been investigated as nutrient sources for microorganisms and packing materials for microbial attachment (Sato et al. 2018 ; Sato et al. 2019 , 2022 ). Rice is a major crop in regions such as East Asia and Latin America, and is readily available in Japan. Rice bran, produced during the polishing of rice, is a low-cost agricultural byproduct, which is rich in carbohydrates, proteins, and lipids (Sharif et al. 2014 ), and rice husk, the outer hull of the grain, is rich in silica and fiber. This process employs a vertical downflow system in which rice husk and limestone are used as filling materials, and Zn and other metals are removed from AMD mainly via precipitation as metal sulfides through SRB activity (Sato et al. 2018 ). In previous studies, continuous AMD flow tests using columns and box-type reactors in an abandoned mine site in northern Japan revealed that Zn and other metals could be treated stably for more than one year, confirming the usability of rice bran as an effective substrate for sulfate-reducing microbial consortia (Sato et al. 2018 ; Sato et al. 2019 , 2024 ). In this process, because the target AMD (approximately pH 3.5) contained Fe ions, a two-stage treatment system consisting of a Fe oxidation reactor (aerobic process) for removing Fe as a pretreatment, followed by a sulfate-reducing bioreactor (anaerobic process) with SRB, was used. Until now, regarding operational issues in the anaerobic process, metal removal efficiency has been found to decrease during the winter period when SRB activity declines, resulting in elevated Zn concentrations in the effluent. Another issue is clogging caused by biofilm formation in the upper part of the reactor, possibly because of the rice bran and the microorganisms utilizing it (Sato et al. 2023 ). Clogging necessitates frequent maintenance of the bioreactor. Similar challenges have been reported in other studies (Mayes et al. 2021 ; Tsukamoto et al. 2004 ). To advance the practical application of sulfate-reducing bioreactor for AMD treatment, a large-scale test with a treatment flow rate of 100 L/min has been conducted at the same abandoned mine site in Japan since 2020. After some preparation and preliminary test operations for the start of the bioreactor, full-fledged continuous operation of the bioreactor started in September 2022. In this study, the large-scale bioreactor operation was conducted outdoors for two years in the range of ambient temperature from − 10°C to 40°C. Both the sulfate-reduction activity and metal removal performance were continuously monitored to clarify seasonal variations in the treatment performance. In addition, SRB possibly involved in sulfate reduction within the large-scale bioreactor were analyzed using high-throughput Illumina sequencing. In the large-scale bioreactor operation, we focused on 1) long-term continuous treatment with lower maintenance costs (addition of rice bran once a year) and 2) prevention of bioreactor clogging to assess operational stability and maintenance requirements. Materials and Methods Chemical analyses Both influent and effluent water samples from the bioreactor (one per week) were obtained during the two-year operation period. The pH, oxidation–reduction potential (ORP), and water temperature dissolved oxygen (DO) were measured using a portable multi-water quality meter MM-42DP (DKK-TOA Corp., Tokyo, Japan). The DO was measured using a portable dedicated DO meter HQ1130 (Hach Company, Loveland, CO, USA). The metal concentrations were determined using an inductively coupled plasma optical emission spectrometer, Agilent 5110 ICP-OES (Agilent Technologies Inc., Santa Clara, CA, USA). Samples rich in organic content were digested using a mixture of HNO 3 and HClO 4 under heated wet conditions, and the levels of total organic carbon (TOC) and total inorganic carbon (TIC) were determined using a TOC analyzer, TOC-L (Shimadzu, Kyoto, Japan). The concentration of sulfate was determined using an ion chromatography system, Dionex ICS-1100, with Dionex IonPac columns (Thermo Fisher Scientific, Waltham, MA, USA), and the concentration of sulfide was determined with methylene blue (Reese et al. 2011 ) using a ratio beam spectrophotometer, U-5100 (Hitachi High-Tech Corp., Tokyo, Japan). The concentrations of organic acids (acetic acid and propionic acid) were estimated using a UPLC system, ACQUITY UPLC H-Class PLUS, with an ion exclusion column, IC-Pak (7 µm, 7.8 mm × 300 mm; Waters Corp., Milford, MA, USA). The detection limits of the chemicals were as follows: Zn, 8.0 × 10 − 5 mg/L; Cu, 1.8 × 10 − 4 mg/L; Cd, 6.0 × 10 − 5 mg/L; Fe 9.0 × 10 − 5 mg/L. Large-scale sulfate-reducing bioreactor system (i) Acid mine drainage and process flow The AMD from an abandoned mine site in northern Japan was used in this study. Through 2022 to 2023, the original AMD (pH 3.2–4.0) contained 249–334 mg/L SO 4 2− , 28–49 mg/L Fe, 15–20 mg/L Zn, 2–10 mg/L Cu, and 0.04–0.08 mg/L Cd. A photograph of the large-scale passive treatment system illustrating the process flow is shown in Fig. 1 A. To prevent the oxidation of ferrous iron by air, the AMD was conveyed from the mine tunnel through polyethylene pipes. As the mine water level was higher than the top level of the reactors, the mine water was supplied to the tanks under gravity flow (Fig. 1 B). Before entering the bioreactors, the AMD was first split and introduced into two iron oxidation reactor tanks with rice husk and limestone (designated tanks A and B, respectively) at 75 and 25 L/min, respectively. After Fe removal, the effluents from the two tanks were combined. The resultant effluents (pH 2.8–3.3) contained 257–328 mg/L SO 4 2− , 4–14 mg/L Fe, 15–20 mg/L Zn, 2–11 mg/L Cu, and 0.04–0.09 mg/L Cd before June 2023. After July 2023, the effluent (pH 3.1–4.1) contained 253–308 mg/L SO 4 2− , 1–8 mg/L Fe, 15–18 mg/L Zn, 2–8 mg/L Cu, and 0.04–0.07 mg/L Cd. The combined effluents were then evenly divided at 50 L/min into two sulfate-reducing bioreactors (designated tanks A and B). The treated water from the two bioreactors was merged at the bioreactor outlet and discharged from the system. (ii) Bioreactor The concept of a sulfate-reducing bioreactor using rice bran has been described previously at the laboratory and pilot scales (Sato et al. 2018 , 2019 ). This anaerobic treatment was conducted using two vertical flow type rectangular concrete bioreactors [16 m (wide), 5 m (long), and 3.5 m (deep)]. A cross-sectional view of the bioreactor is shown in Fig. 2 , and the specifications of the reactor packing materials are mentioned in Table 1. To mitigate the drop in water temperature during winter, 3.2 m of the reactor was embedded underground. At the bottom of each reactor, drainage pipes made of polyvinyl chloride (PVC) were installed, and to protect these pipes, a 0.15 m layer of limestone (particle size: 20–40 mm) was laid over them (bottom layer in Fig. 2 ). A 1.0 m thick mixture of rice husk and limestone (weight ratio, 1:4) was placed directly above this limestone layer (layer 3 in Fig. 2 ), and it was followed by a 0.5 m thick layer of a mixture with a weight ratio of 1:16 (layer 2 in Fig. 2 ). Rice husk serves as a scaffold material for the colonization of SRB and captures precipitated metal sulfides such as ZnS. The limestone acts to neutralize the effluent from iron oxidation reactor tanks (pH 2.8–4.1), bringing it to circumneutral pH, in which the activity of SRB is enhanced. Furthermore, as a substrate for anaerobes in the bioreactor, a 0.3 m thick surface layer composed of a mixture of 2 tons of rice bran and rice husk was placed on the 0.5 m thick layer (layer 1 in Fig. 2 ); on top of this layer, a mine water layer with a thickness of approximately 0.3 m was maintained as a water-sealing layer (Fig. 2 ). The AMD was continuously supplied to each bioreactor at a flow rate of 50 L/min to achieve an HRT of 22.5 h at the rice husk–limestone mixture layer. Sampling ports made of PVC pipes were installed on both the upstream and downstream sides at depths of 0, 0.3, 0.4, 0.8, 1.3, and 1.8 m from the top of the rice bran–rice husk layer (layer 1), and at approximately 2 m from the center of the bioreactor. Water samples were collected via suction using a drill pump connected to hoses at the ground surface. Additionally, PVC pipes were installed at the boundary depths of each layer (0.3, 0.8, and 1.8 m) to monitor clogging within the bioreactor. (iii) Rice bran, rice husk, and their feeding The composition of rice bran, rice husk, and their mixture is listed in Table 2. Rice bran contained high levels of oil (19.7%), protein (13.6%), and nitrogen-free extract (34.8%), whereas rice husk contained high levels of fiber (15.4%) and ash (31.7%). In contrast, the mixture of rice bran and rice husk contained protein (9.0%), oil (0.8%), fiber (24.6%), ash (14.9%), and soluble inorganic nitrogen (35.7%). The large-scale sulfate-reducing bioreactor was operated for two years, from September 16, 2022 to September 9, 2024. Initially, 2 ton of a mixture of rice bran and rice husk was placed within the bioreactor in September 2022. After one year of operation, an additional 2 ton of rice bran was fed to the rice bran–rice husk mixture layer (layer 1) using heavy machinery for mixing in September 2023. The mine water level shifted approximately 0.7 m lower than usual during mixing. Amplicon sequencing of 16S rRNA gene From a total of 40 bioreactor samples (eight time points [November 2022, February 2023, May 2023, August 2023, November 2023, February 2024, May 2024, and August 2024] and five sampling ports [port 1, 3, 5, 7, and outlet]), DNA was extracted using a direct lysis protocol (Noll et al. 2005 ) with bead beating. The extracted DNA was purified using phenol-chloroform extraction, isopropanol precipitation, RNase treatment (Type II-A; Sigma-Aldrich, St. Louis, MO, USA), polyethylene glycol precipitation, and ethanol precipitation, and the purified DNA was quantified using a NanoDrop Lite (Thermo Fisher Scientific). Using polymerase chain reaction (PCR) with high-fidelity DNA polymerase (Q5; New England Biolabs, Ipswich, MA, USA), the V4 region of the 16S rRNA gene was amplified with the universal primers 515F and 806R; both the primers were modified to contain an Illumina adapter region, and 806R contained a 12-basepair barcode for multiplex sequencing (Caporaso et al. 2012 ). The amplicons were purified using the AMPure XP Kit (Beckman Coulter, Brea, CA, USA), Wizard SV GEL, and PCR Clean-up System (Promega, Madison, WI, USA). The barcode-encoded DNA library and the initial control (PhiX; Illumina, San Diego, CA, USA) were subjected to paired-end sequencing using a 300-cycle MiSeq Reagent kit (Illumina) on a MiSeq sequencer (Illumina). The 16S rRNA gene sequence data were analyzed using QIIME 2 (Bolyen et al. 2019 ). From the obtained paired-end reads, those with more than two expected errors were discarded (default maxEE = 2), and chimeric sequences were identified and removed using the “consensus” method inherent to the DADA2 pipeline (Callahan et al. 2016 ). The obtained nucleotide sequence data were deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive (DRA), European Nucleotide Archive (ENA), and Sequence Read Archive (SRA) databases under accession number PRJDB40188 (DRR901113–DRR901152). Results and Discussion Seasonal changes in pH, ORP, temperature, and DO during the large-scale bioreactor operation over two years The large-scale sulfate-reducing bioreactor was operated from September 16, 2022 to September 9, 2024, and the former half (September 2022 to September 2023) and latter half (October 2023 to September 2024) periods were designated Phase 1 and Phase 2, respectively. The ambient air temperature varied between − 10 and 40°C throughout the year, with average winter temperatures (December to March) of − 0.1 and 0.2°C during Phase 1 and Phase 2, respectively (Fig. 3 A). Therefore, the influent and effluent water temperatures of the bioreactor varied in response to changes in the ambient temperature (Fig. 3 B). The temperature fluctuations of influent AMD, which was the effluent from the preceding iron oxidation reactor with a short HRT of 2 h, were relatively small, ranging from 8 to 18°C. In contrast, because the anaerobic bioreactor had a longer HRT of 22.5 h, the effluent water temperature fluctuations were a little larger, ranging from 5 to 20°C. As approximately 90% of the anaerobic bioreactor was buried underground, the use of ground temperature was allowed to buffer against ambient temperature effects, although the ambient temperature fell below 0°C in winter. Consequently, the minimum effluent temperature never fell below 4°C. Additionally, although partial surface freezing of the water-seal layer occurred during certain winter periods, freezing was limited to the surface and had a negligible impact on the inside of the bioreactor. The pH values of both influent and effluent water remained stable throughout the year, ranging from 2.9 to 4.1 for influent and from 6.5 to 7.3 for effluent regardless of water temperature. As shown in Fig. 3 C, the increase in influent pH to approximately 4 in July 2023 was observed owing to the feeding of additional limestone to the preceding iron oxidation reactor at that time. A similar pH increase occurred in July 2024, when additional limestone was added to replenish the consumed portion. This increase in influent pH had little effect on the effluent pH in the anaerobic bioreactor (Fig. 3 C). In addition, we observed that the effluent pH dropped to approximately 6.5 in September 2022, September 2023, and July 2023; at the former two time points, (additional) rice bran was fed to the bioreactor as a nutrient source, and in the latter, the HRT in the bioreactor temporarily increased four-fold (to 90 h). This increase in effluent pH was considered to have resulted from a temporary increase in the concentrations of organic acids derived from rice bran (Fig. S1 ) and a sharp increase in the efficiency of sulfate reduction (Fig. 4 C), which accelerated the proton release associated with sulfate reduction and ionization of hydrogen sulfide ions (Eqs. 1 and 2). These effects temporarily outpaced the rate of pH increase owing to limestone dissolution. 2CH 2 O + SO 4 2- → 2HCO 3 ། + H 2 S (1) HS → HS + H (2) The ORP values for effluent remained consistently less than − 200 mV for most of the year, except during the winter period; in the winter of 2022, the effluent ORP increased to a maximum of − 179 mV (Fig. 3 D). In contrast, during the winter of 2023, the highest recorded value was − 252 mV in February 2024, indicating that a stable reducing environment was maintained even under low-temperature conditions. This stability of the ORP values was considered to have resulted from sufficient microbial consumption activity following the addition of rice bran in September 2023, when the bioreactor already contained a partially established microbial community capable of utilizing rice bran. Furthermore, the temporary extension of the HRT in July 2023 described above (prior to rice bran addition) may have contributed to the stabilization of the microbial community within the bioreactor. Since the initial installation of the rice bran–rice husk mixture layer in Phase 1, the DO values of effluent remained below 1.0 mg/L, which was indicative of the maintenance of anaerobic conditions in the bioreactor regardless of seasonal change. The same trend was observed in Phase 2, with stable DO levels in the effluent. The increase in the DO of the effluent water on June 10, 2024, during Phase 2, was due to the replacement of the sensor cap of the DO meter (Fig. 3 E). Sulfate reduction efficiency during the large-scale bioreactor operation Throughout the two-year bioreactor operation period, the sulfate concentration in the influent ranged from 253 to 308 mg/L (Fig. 4 A). In contrast, the sulfate concentration in the effluent was less than 276 mg/L during this period, which was the maximum value recorded on March 22, 2023. The sulfate reduction rate (represented by vertical bars in Fig. 4 A) was calculated as the percentage decrease in the sulfate concentration between the influent and effluent water in the bioreactor. Approximately one month after the addition of rice bran, the maximal sulfate reduction rates in Phase 1 and Phase 2 reached 47% and 94%, respectively, indicating an increase in sulfate reduction associated with a temporary increase in the availability of carbon source due to the addition of rice bran. However, subsequently, the sulfate reduction rate declined alongside a decrease in water temperature (Fig. 3 B), with both Phase 1 and Phase 2 exhibiting lower rates during the winter season. The average sulfate reduction rates in winter were 7.7% and 27.7%, with that in Phase 2 being 20% higher than that in Phase 1. Both sulfate reduction rate (Fig. 4 A) and effluent S² − concentration (Fig. 4 B) showed trends similar to those for the sulfate reduction efficiency. The average winter sulfate reduction efficiency in Phase 1 and Phase 2 was 0.14 and 0.52 mol-SO 4 /day/m³, respectively (Fig. 4 C). In Phase 1, the effluent S² − concentrations during most of the winter period were below the detection limit, whereas in Phase 2, the average during the winter was 5.0 mg/L (Fig. 4 B). These results suggested that in Phase 2, a part of the organic matter contained in the rice bran-rice husk mixture layer from Phase 1 remained, resulting in relatively higher organic carbon supply. In addition, the microbial community capable of utilizing rice bran had already been established in Phase 1, which led to higher utilization efficiency of the rice bran newly added as an organic carbon source in Phase 2. Furthermore, the average effluent temperature in Phase 2 winter was almost the same with that in Phase 1 (7.6°C), which might not have affected the maintenance of microbial activity. Therefore, it can be concluded that sufficient sulfate-reducing capacity was maintained in Phase 2, even during winter. In July 2023 (Phase 1), the HRT increased four-fold to 90 h, resulting in a maximum sulfate reduction rate of 80% in Phase 1, which confirmed that extension of the HRT led to an increase in sulfate reduction. This indicated that even nine months after rice bran addition, a sufficient amount of organic matter remained, and that prolonging the HRT accelerated its decomposition. In contrast, in September 2024, approximately one year after rice bran addition at the beginning of Phase 2, the sulfate reduction rates declined to levels lower than those observed in winter, suggesting that without the addition of organic matter once per year in the warmer summer season, sulfate reduction would be insufficient for the upcoming winter. However, in winter, organic matter consumption slows, and a greater amount is required; therefore, under the relatively short HRT of this process (22.5 h), the addition of organic matter prior to the winter was identified as an appropriate operational strategy. Metal removal performance in the large-scale bioreactor during seasonal operation The Zn concentrations in the effluent were sufficiently reduced to < 0.1 mg/L throughout most of the operational period outside winter (Fig. 5 A). In Phase 1, an increase in total Zn concentration was observed around December 2022 as the water temperature declined, with an average of 0.84 mg/L and a maximum of 1.05 mg/L during winter. In contrast, in Phase 2, only a minor increase in total Zn concentration was observed in winter, with an average of 0.04 mg/L and a maximum of 0.27 mg/L. The concentrations of 1.95 and 1.01 mg/L were recorded in December 2022 (Phase 1) and in July 2024 (Phase 2), respectively; however, these values were considered to have resulted from sampling errors, in which deposits adhered to the inner surface of the piping were dislodged during sample collection. During the entire operational period, the Zn concentration in the effluent never exceeded the effluent standard in Japan (2.0 mg/L), revealing a stable treatment performance over 725 consecutive days. Sufficient sulfate reduction achieved using SRB within the bioreactor likely led to the precipitation and immobilization of Zn ions as sulfide minerals, including that in the winter. In both Phase 1 and Phase 2, rice bran was added in September when the water temperature began to decrease, and both the timing and dosage of rice bran were considered appropriate for Zn removal during the winter period. The concentrations of Cu and Cd never exceeded the effluent standards in Japan (3 mg/L and 0.03 mg/L, respectively) during the entire period, and their removal was consistently stable (Fig. 5 B and 5 C). The inlet Fe concentrations were reduced to < 10 mg/L by the preceding iron oxidation reactor throughout the operational period (Fig. 5 D). However, an increase in the effluent Fe concentration was observed in Phase 1, along with a decline in sulfate reduction (Fig. 4 C), reaching a maximum of 7.4 mg/L. In Phase 2, only a slight increase in the effluent Fe concentration was detected, with a maximum concentration of 0.3 mg/L (Fig. 5 D). At approximately pH 7, the solubility product of the respective metal sulfides follows the order CuS < CdS < ZnS < FeS. Therefore, when the sulfate-reducing activity in the bioreactor decreased and the resultant sulfide supply became insufficient, the effluent metal concentrations were expected to increase. During the winter period in Phase 1, 13 days after the effluent Fe concentration began to increase, the Zn concentrations in the effluent also started to increase, with maximum values of 7.4 mg/L for Fe and 1.0 mg/L for Zn. In contrast, only slight increases in the effluent Fe and Zn concentrations were observed in Phase 2. If all divalent metal ions (Fe, Zn, Cd, and Cu) in the influent were precipitated as their metal sulfides, the sulfate required for their precipitation, as calculated using the sulfate reduction reaction (Eq. 3), would be approximately 30 mg/L. M + HS → MS + 2H (3) As shown in Fig. 4 A, the difference in sulfate concentration between the inlet and effluent fell below 30 mg/L only during the 78-day period from November 2022 to April 2023 in Phase 1, whereas sufficient sulfate reduction was achieved during all other periods. Hence, in November 2022, the sulfate reduction efficiency had already become insufficient (Fig. 4 C), and approximately 14 days later, an increase in the total Zn concentration was observed (Phase 1, Fig. 5 A). In contrast, the difference in sulfate concentration in Phase 2 did not fall below the threshold (30 mg/L) during the entire operational period. Clogging in the large-scale bioreactor during the operation In both Phase 1 and Phase 2, a gradual increase in the water level within the tank was observed over time owing to reduced permeability, with annual increases of 0.21 and 0.11 m, respectively (Fig. S2 ). The clogging rates were 0.12 and 0.06 m/y, which were one-fourth and one-eighth of the rates observed under the initial rice bran–only conditions when the facility was first commissioned in 2020 (unpublished data). Mixing rice husk with rice bran reduced the clogging rate, and as a result, no maintenance was required for approximately one year until the next rice bran addition. Similar to manure or compost, rice bran, which has a small particle size, tends to cause clogging (Botes et al. 2018 ). Hence, the addition of rice husk, which has a larger particle size, higher fiber and ash content, and is more resistant to degradation, was considered to have reduced clogging. Although the exact cause of clogging was not determined, it is likely influenced by the accumulation of biofilms and precipitation of metal sulfides within the bioreactor. Analysis of the microbial community in the large-scale bioreactor At eight time points during the two-year operation (November 2022, February 2023, May 2023, August 2023, November 2023, February 2024, May 2024, and August 2024), class- and genus-level phylogenetic analyses of sequence data were performed using QIIME2 to evaluate differences in microbial compositions (Fig. 6 A, B). In the large-scale bioreactor, besides Bacteroidia , Berkelbacteria and Parcubacteria also became dominant. Bacteroidia are capable of degrading a wide range of organic compounds and are therefore considered to be involved in the decomposition of organic substrates such as rice bran. In addition, Berkelbacteria and Parcubacteria , belonging to a bacterial supergroup known as Candidate Phyla Radiation (CPR) bacteria (including a wide variety of uncultured organisms), are dominant constituents of biofilms in sulfide-rich springs, although their physiological functions remain largely unknown. However, among these, Berkelbacteria are presumed to be involved in sulfur metabolism and reduction of elemental sulfur by putative sulfhydrogenases (Valentin-Alvarado et al. 2024 ). As for SRB, non-spore-forming members of the family Desulfosarcinaceae (gram-negative bacteria, the class Desulfobacteria ; Waite et al. 2020 ) were the most dominant taxa. In addition, Desulfofarcimen , an endospore-forming gram-positive bacterium reclassified from Desulfotomaculum sp. (the class Clostridia ; Watanabe et al. 2018 ), was detected in the downstream section of the reactor (Fig. 6 C). Because of adjustment and preliminary operation (October 2020 to September 2022) over two years prior to the start of the large-scale experiment in September 2022, sufficient anaerobic conditions had already been established. In addition, the reactor was amended with a mixture of rice bran (as a carbon source) and rice husk (as a microbial carrier), which minimized reactor clogging. Consequently, strictly anaerobic members of the family Desulfosarcinaceae were predominant throughout the operational period and were believed to be responsible for sulfate reduction. The relative abundance of Desulfosarcinaceae increased in August 2023 compared with that in May 2023, partly due to a temporary increase in the HRT in July 2023. Following the additional rice bran input in September 2023, the relative abundance of Desulfosarcinaceae further increased in November 2023 (Fig. 6 C). Hence, the fact that the relative abundance of Desulfosarcinaceae was higher in Phase 2 than in Phase 1 may have enabled stable sulfate reduction, even during the cold winter period. Similarly, after the addition of rice bran in September 2023, the microbial community analysis in November 2023 showed a substantial increase in the relative abundance of Berkelbacteria , which has been suggested to be involved in sulfur reduction (Fig. 6 A, B; Valentin-Alvarado et al. 2024 ). Therefore, these bacteria may play important roles in the complex sulfur transformation network within the mixed layer of rice bran and rice husk. Conclusion A large-scale sulfate-reducing bioreactor using rice bran (treatment flow rate: 100 L/min) was evaluated for the treatment of metal-containing AMD over two years. By incorporating a mixture of rice bran and rice husk into the nutrient layer (Fig. 2 ), biofilm-induced clogging in the upper part of the bioreactor was mitigated, enabling minimal maintenance with rice bran being replenished only once annually. Consequently, the bioreactor performance for sulfate reduction was effectively sustained for 725 days. The concentrations of Fe, Zn, Cu, and Cd in the treated AMD satisfied the standard effluent concentrations throughout the testing period. Using high-throughput Illumina sequencing of 16S rRNA genes, the family Desulfosarcinaceae was found to be the predominant SRB. To further broaden the scope of the findings, future investigations should focus on determining the optimal quantity of rice bran and evaluating the long-term sustainability of the system, in preparation for its potential deployment in actual operational settings. Declarations Competing Interests: The authors declare that they have no competing financial or non-financial interests. Acknowledgments We thank Yuki Watanabe for providing technical assistance. This work was supported in part by JSPS KAKENHI (grant number 24K03111). We would like to thank Editage ( www.editage.jp ) for English language editing. References Ben Ali HE, Neculita CM, Molson JW, Maqsoud A, Zagury GJ (2019) Performance of passive systems for mine drainage treatment at low temperature and high salinity: A review. Min Eng 134:325–344. https://doi.org/10.1016/j.mineng.2019.02.010 Ben Ali HE, Neculita CM, Molson JW, Maqsoud A, Zagury GJ (2020) Salinity and low temperature effects on the performance of column biochemical reactors for the treatment of acidic and neutral mine drainage. 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Water Res 140:268–279. https://doi.org/10.1016/j.watres.2018.04.035 Noll M, Matthies D, Frenzel P, Derakshani M, Liesack W (2005) Succession of bacterial community structure and diversity in a paddy soil oxygen gradient. Environ Microbiol 7:382–395. https://doi.org/10.1111/j.1462-2920.2005.00700.x Nordstrom D (2011) Mine waters: Acidic to circumneutral. Elements 7:393–398. https://doi.org/10.2113/gselements.7.6.393 Reese BK, Finneran DW, Mills HJ, Zhu M-X, Morse JW (2011) Examination and refinement of the determination of aqueous hydrogen sulfide by the methylene blue method. Aquat Geochem 17:567–582. https://doi.org/10.1007/s10498-011-9128-1 Sato Y, Hamai T, Hori T, Habe H, Kobayashi M, Sakai T (2018) Year-round performance of a passive sulfate-reducing bioreactor that uses rice bran as an organic carbon source to treat acid mine drainage. Mine Water Environ 37:586–594. https://doi.org/10.1007/s10230-017-0489-6 Sato Y, Hamai T, Hori T, Aoyagi T, Inaba T, Kobayashi M, Habe H, Sakata T (2019) Desulfosporosinus spp. were the most predominated sulfate-reducing bacteria in pilot- and laboratory-scale passive bioreactors for acid mine drainage treatment. Appl Microbiol Biotechnol 103:7783–7793. https://doi.org/10.1007/s00253-019-10063-2 Sato Y, Hamai T, Hori T, Aoyagi T, Inaba T, Hayashi K, Kobayashi M, Sakata T, Habe H (2022) Optimal start-up conditions for the efficient treatment of acid mine drainage using sulfate-reducing bioreactors based on physicochemical and microbiome analyses. J Hazard Mater 423:127089. https://doi.org/10.1016/j.jhazmat.2021.127089 Sato Y, Hamai T, Hori T, Aoyagi T, Inaba T, Habe H (2023) Biofilm microbiomes present in pilot- and laboratory-scale sulfate-reducing bioreactors for acid mine drainage treatment. J Environ Biotechnol 23:53–62. https://doi.org/10.50963/jenvbio.23.1_53 Sato Y, Hamai T, Masaki Y, Aoyagi T, Inaba T, Hori T, Habe H (2024) Replacing rice bran with low-molecular-weight substrates affected the performance and metabolic feature of sulfate-reducing bioreactors treating acid mine drainage. J Environ Chem Eng 12:112118. https://doi.org/10.1016/j.jece.2024.112118 Sharif MK, Butt MS, Anjum FM, Khan SH (2014) Rice bran: A novel functional ingredient. Crit Rev Food Sci Nutr 54:807–816. http://doi.org/10.1080/10408398.2011.608586 Skousen J, Zipper CE, Rose A, Ziemkiewicz PF, Nairn R, McDonald LM, Kleinmann RL (2017) Review of passive systems for acid mine drainage treatment. Mine Water Environ 36:133–153. http://doi.org/10.1007/s10230-016-0417-1 Taylor J, Pape S, Murphy N (2005) A summary of passive and active treatment technologies for acid and metalliferous drainage (AMD). Proc, 5th Australian Workshop on Acid Drainage, Fremantle, Western Australia, p 1–49 Tsukamoto TK, Killion HA, Miller GC (2004) Column experiments for microbiological treatment of acid mine drainage: low-temperature, low-pH and matrix investigations. Water Res 38:1405–1418. https://doi.org/10.1016/j.watres.2003.12.012 U.S. Army Corps of Engineers (2003) Passive and semi-active treatment of acid rock drainage from metal mines-state of the practice. URS Corporation Valentin-Alvarado LE, Fakra SC, Probst AJ, Giska JR, Jaffe AL, Oltrogge LM, West-Roberts J, Rowland J, Manga M, Savage DF, Greening C, Baker BJ, Banfield JF (2024) Autotrophic biofilms sustained by deeply sourced groundwater host diverse bacteria implicated in sulfur and hydrogen metabolism. Microbiome 12:15. http://doi.org/10.1186/s40168-023-01704-w Waite DW, Chuvochina M, Pelikan C, Parks DH, Yilmaz P, Wagner M, Loy A, Naganuma T, Nakai R, Whitman WB, Hahn MW, Kuever J, Hugenholtz P (2020) Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities. Int J Syst Evol Microbiol 70:5972–6016. https://doi.org/10.1099/ijsem.0.004213 Watanabe M, Kojima H, Fukui M (2018) Review of Desulfotomaculum species and proposal of the genera Desulfallas gen. nov., Desulfofundulus gen. nov., Desulfofarcimen gen. nov. and Desulfohalotomaculum gen. nov. Int J Syst Evol Microbiol 68:2819–2899. http://doi.org/10.1099/ijsem.0.002915 Waybrant KR, Blowes DW, Ptacek CJ (1998) Selection of reactive mixtures for use in permeable reactive walls for treatment of mine drainage. Environ Sci Technol 32:1972–1979. https://doi.org/10.1021/es9703335 Younger PL, Wolkersdorfer C, ERMITE-Consortium (2004) Mining impacts on the fresh water environment: Technical and managerial guidelines for catchment scale management. Mine Water Environ 23:s2–s80. https://doi.org/10.1007/s10230-004-0028-0 Tables Table 1. Specifications of the packing materials of the biochemical reactor Layer 1 Layer 2 Layer 3 Bottom layer Height 0.30 m 0.50 m 1.00 m 0.15 m Volume 24.0 m 3 40.0 m 3 80.0 m 3 12.0 m 3 Media Mixture of rice bran and rice husk Rice husk +Limestone 40/20 Rice husk +Limestone 40/20 Limestone 40/20 Weight Rice bran 2000 kg +Rice husk 2400 kg Rice husk 3500 kg +Limestone 56000 kg (weight ratio of 1:16) Rice husk 9000 kg +Limestone 36000 kg (weight ratio of 1:4) 2500 kg Porosity - 45.2% 51.7% 42.9% Table 2. Composition of rice bran, rice husk, and mixtures Moisture (wt%) Protein (wt%) Oil (wt%) Ash (wt%) Fiber (wt%) Nitrogen-free extract (wt%) Rice bran 14.4 13.6 19.7 8.2 9.4 34.8 Rice husk 28.1 1.6 0.7 31.7 15.4 22.6 Mixture of rice bran and rice husk (layer 1) 15.1 9.0 0.8 24.6 14.9 35.7 Supplementary Files FigS1.tif Fig. S1. Seasonal changes in carbon source of the sulfate-reducing bioreactor during Phase 1 (September 2022 to September 2023) and Phase 2 (October 2023 to September 2024). (A) Inorganic carbon, (B) Acetate, (C) Lactate, (D) Formate. Symbols: blue circle, effluent. FigS2.tif Fig. S2. Seasonal changes in water head difference of the sulfate-reducing bioreactor. Symbols: red line, rice bran layer (layer1, 0–0.3 m); green line, upper rice husk and limestone layer (layer 2, 0.3–0.8 m); blue line, lower rice husk and limestone layer (layer 3, 0.8–1.8 m). Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 21 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 14 Apr, 2026 First submitted to journal 08 Apr, 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9361529","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626931916,"identity":"9609b680-7cc4-4102-b43b-e0f2bbf3922d","order_by":0,"name":"Masataka Kondo","email":"data:image/png;base64,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","orcid":"","institution":"Japan Organization for Metals and Energy Security Technology \u0026 Research Center: Dokuritsu Gyosei Hojin Energy Kinzoku Kobutsu Shigen Kiko Gijutsu Center","correspondingAuthor":true,"prefix":"","firstName":"Masataka","middleName":"","lastName":"Kondo","suffix":""},{"id":626931917,"identity":"2bd017db-05c4-4513-829a-0d19a28ae5ca","order_by":1,"name":"Yuya Sato","email":"","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology Environment Management Research Institute: Sangyo Gijutsu Sogo Kenkyujo Kankyo Sosei Kenkyu Bumon","correspondingAuthor":false,"prefix":"","firstName":"Yuya","middleName":"","lastName":"Sato","suffix":""},{"id":626931918,"identity":"2ad948e4-d302-42c4-9d9f-54415860f216","order_by":2,"name":"Yusei Masaki","email":"","orcid":"","institution":"Japan Organization for Metals and Energy Security Metals Environment Management Department: Dokuritsu Gyosei Hojin Energy Kinzoku Kobutsu Shigen Kiko Kinzoku Kankyo Jigyobu","correspondingAuthor":false,"prefix":"","firstName":"Yusei","middleName":"","lastName":"Masaki","suffix":""},{"id":626931919,"identity":"e6140ff9-e8af-4fd7-b5d9-dbd44b91dd65","order_by":3,"name":"Hiroshi Habe","email":"","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology Environment Management Research Institute: Sangyo Gijutsu Sogo Kenkyujo Kankyo Sosei Kenkyu Bumon","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Habe","suffix":""},{"id":626931920,"identity":"3ac53f6f-f22b-425b-af98-822341c54ab4","order_by":4,"name":"Takaya Hamai","email":"","orcid":"","institution":"Japan Organization for Metals and Energy Security Metals Environment Management Department: Dokuritsu Gyosei Hojin Energy Kinzoku Kobutsu Shigen Kiko Kinzoku Kankyo Jigyobu","correspondingAuthor":false,"prefix":"","firstName":"Takaya","middleName":"","lastName":"Hamai","suffix":""}],"badges":[],"createdAt":"2026-04-08 23:55:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9361529/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9361529/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108109066,"identity":"e4a2d633-193a-4583-95e1-9176ded7b769","added_by":"auto","created_at":"2026-04-29 12:36:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1350561,"visible":true,"origin":"","legend":"\u003cp\u003ePhotograph (A) and schematic diagram (B) of the large-scale passive treatment system combined with iron-oxidation and sulfate-reducing processes. The acid mine drainage (AMD) from an abandoned mine divided at 100 L/min and installed at the treatment site through polyvinyl chloride pipe. AMD was first split and introduced into two iron-oxidation reactor tanks (designated tank A and B) at 75 and 25 L/min, respectively. Effluent from these two tanks were combined in the mixing tank and then divided evenly at 50 L/min into two sulfate-reducing bioreactors (designated tank A and B). The treated water from the two bioreactors was merged and discharged from the system.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/6981988a76416ed602b7e38b.png"},{"id":108109068,"identity":"0fe3b028-a9be-4a04-bece-94c06bad0d56","added_by":"auto","created_at":"2026-04-29 12:36:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92198,"visible":true,"origin":"","legend":"\u003cp\u003eCross-sectional schematic diagram of the large-scale sulfate-reducing bioreactor installed at an abandoned mine site.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/08e0fc8cbc0dd3caf0b23862.png"},{"id":108182164,"identity":"5c41b97e-fe16-4553-9392-a161f826798a","added_by":"auto","created_at":"2026-04-30 08:59:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83375,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal changes in on-site parameters of influent and effluent water from the sulfate-reducing bioreactor during Phase 1 (September 2022 to September 2023) and Phase 2 (October 2023 to September 2024). (A) Air temperature, (B) water temperature, (C) pH, (D) oxidation–reduction potential (ORP), and (E) dissolved oxygen (DO). Symbols: black circles, air temperature; gray square, influent; blue circle, effluent; dark gray circle, water seal layer.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/10cca2c2e8a6b5b81d1f5ca8.png"},{"id":108109070,"identity":"fedc9c99-4ee9-4654-bc39-19f8f7d53d84","added_by":"auto","created_at":"2026-04-29 12:36:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52119,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal changes in chemical parameters of influent and effluent water from the sulfate-reducing bioreactor during Phase 1 (September 2022 to September 2023) and Phase 2 (October 2023 to September 2024). (A) Sulfate concentration/sulfate reduction rate, (B) sulfide concentration, and (C) sulfate reduction efficiency. Symbols: gray square, influent; blue circle, effluent; yellow line, sulfate reduction ratio from influent to effluent.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/a96da6b85f173adb0311db2f.png"},{"id":108490950,"identity":"50797600-ddcc-4764-b017-ce5bf570a2bc","added_by":"auto","created_at":"2026-05-05 09:50:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63694,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal changes in metal removal of the sulfate-reducing bioreactor during Phase 1 (September 2022 to September 2023) and Phase 2 (October 2023 to September 2024). (A) Zn, (B) Cu, (C) Cd, and (D) Fe. Symbols: gray square, influent; blue circle, effluent.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/42210957979e4a24c6e1f874.png"},{"id":108109073,"identity":"9e80f533-e608-4e88-9e82-fdd4ea7e3820","added_by":"auto","created_at":"2026-04-29 12:36:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":678167,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in microbial composition during two-year operation of the large-scale sulfate-reducing bioreactor (November 2022 to August 2024). (A) The class-level microbial composition. (B) The genus-level microbial composition. (C) The relative abundance of the known sulfate-reducing bacteria. Sampling ports 1, 3, 5, 7, and out are shown in Fig. 2. The phylogenetic groups are indicated by colors and their taxonomies are shown in the upper section of the graph.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/5a41dc0da40944d48b54bb30.png"},{"id":108494719,"identity":"d4649249-629d-4842-915d-64f09d0404d7","added_by":"auto","created_at":"2026-05-05 10:06:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2683939,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/db45ffa1-f625-4016-a9f1-ccff2c4cd1a6.pdf"},{"id":108182192,"identity":"4fb258d6-c717-422d-bf6a-690b9918ce5e","added_by":"auto","created_at":"2026-04-30 08:59:12","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":487926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e Seasonal changes in carbon source of the sulfate-reducing bioreactor during Phase 1 (September 2022 to September 2023) and Phase 2 (October 2023 to September 2024). (A) Inorganic carbon, (B) Acetate, (C) Lactate, (D) Formate. Symbols: blue circle, effluent.\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/98dcc266256baf70dbd290f1.tif"},{"id":108109069,"identity":"d449c52f-e4bf-48f4-bca8-f9a77214cf2d","added_by":"auto","created_at":"2026-04-29 12:36:36","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":257002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2.\u003c/strong\u003e Seasonal changes in water head difference of the sulfate-reducing bioreactor. Symbols: red line, rice bran layer (layer1, 0–0.3 m); green line, upper rice husk and limestone layer (layer 2, 0.3–0.8 m); blue line, lower rice husk and limestone layer (layer 3, 0.8–1.8 m).\u003c/p\u003e","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-9361529/v1/1aab09fa2e4742e9b39ea74c.tif"}],"financialInterests":"","formattedTitle":"Performance and stability of a large-scale sulfate-reducing bioreactor with rice bran for passive treatment of acid mine drainage over two years","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcid mine drainage (AMD) from metal and coal mines is a serious environmental issue worldwide. Various treatment methods have been employed for neutralizing and removing the metals (Younger et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nordstrom \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Japan has more than 5,000 abandoned or closed mines, and approximately 100 of these, require treatment of mine discharge (Ministry of Economy, Trade and Industry \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Doyama et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The AMD treatment method in Japan involves adding alkaline reagents such as Ca(OH)\u003csub\u003e2\u003c/sub\u003e to neutralize acidity, precipitating dissolved metals as hydroxides, and subsequently removing them via solid\u0026ndash;liquid separation in settling ponds before discharging into rivers or other receiving waters. However, its implementation incurs substantial costs, amounting to several billion yen annually; the heads of expenses include labor for inspection and monitoring of treatment facilities, electricity for agitators and other equipment, and chemicals, such as Ca(OH)\u003csub\u003e2\u003c/sub\u003e and coagulants. Considering that AMD treatment is expected to remain semi-permanent, a strong demand exists for reducing both economic and environmental burdens.\u003c/p\u003e \u003cp\u003ePassive treatment represents a low-cost and environment-friendly alternative method for treating AMD, as it does not require external energy inputs such as electricity; instead, it relies on microbial activity and naturally occurring chemical reactions (e.g., Skousen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the applicability of passive treatment is constrained by the flow rate, acidity, and metal loading, and it is only suitable for AMD with relatively low acidity, low flow rates, and limited metal loads (Taylor et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Eppink et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kleinmann et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In Japan, most abandoned mines, where AMD treatment is practiced, have been closed for several decades, which has resulted in stable water quality and low discharge flow. Potential for passive treatment application at the domestic abandoned mine sites has been investigated by the Japan Organization for Metals and Energy Security (JOGMEC).\u003c/p\u003e \u003cp\u003eAmong the passive treatment technologies for removing metal ions, the one using sulfate-reducing bacteria (SRB), is prominent. SRB reduce sulfate ions in AMD to sulfide ions, which subsequently precipitate dissolved metals as insoluble metal sulfides (Habe et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mafane et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Because SRB are heterotrophic microorganisms, they require a supply of organic nutrient sources, for example, manure, compost, wood chips, sawdust, and straw (Waybrant et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Lefticariu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The high-molecular-weight organic matter contained in these nutrient sources is decomposed by microorganisms, and the resulting low-molecular-weight organics are utilized by the SRB as electron donors to reduce sulfate in mine drainage (Neculita et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In anaerobic treatment with SRB, the hydraulic retention time (HRT) is typically several days, which often necessitates a large land area (Skousen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; U.S. Army Corps of Engineers \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, mine water treatment facilities in Japan are often located in mountainous terrain with limited land space and require compact processes with shorter HRTs for the installation of passive treatment. In the United Kingdom, which faces similar land constraints as Japan, laboratory-scale studies on sulfate-reducing bioreactors with short HRTs have been reported (Gandy et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, a decrease in HRT can cause reactor clogging owing to reduced permeability. Additionally, in northern parts of Japan, AMD treatment must be sustained during winter, when the outside temperature falls below freezing, and stable performance under low-temperature conditions is a critical challenge. Microbial activity declines in low-water-temperature environments, thereby affecting the treatment efficiency (Harrington et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nielsen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ben Ali et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince 2007, JOGMEC has been conducting research on AMD treatment using a constructed sulfate-reducing bioreactor with shorter HRTs and high robustness against annual temperature fluctuations. In this process, rice bran and rice husk\u0026mdash;agricultural byproducts generated during rice production\u0026mdash;have been investigated as nutrient sources for microorganisms and packing materials for microbial attachment (Sato et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sato et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Rice is a major crop in regions such as East Asia and Latin America, and is readily available in Japan. Rice bran, produced during the polishing of rice, is a low-cost agricultural byproduct, which is rich in carbohydrates, proteins, and lipids (Sharif et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and rice husk, the outer hull of the grain, is rich in silica and fiber. This process employs a vertical downflow system in which rice husk and limestone are used as filling materials, and Zn and other metals are removed from AMD mainly via precipitation as metal sulfides through SRB activity (Sato et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In previous studies, continuous AMD flow tests using columns and box-type reactors in an abandoned mine site in northern Japan revealed that Zn and other metals could be treated stably for more than one year, confirming the usability of rice bran as an effective substrate for sulfate-reducing microbial consortia (Sato et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sato et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this process, because the target AMD (approximately pH 3.5) contained Fe ions, a two-stage treatment system consisting of a Fe oxidation reactor (aerobic process) for removing Fe as a pretreatment, followed by a sulfate-reducing bioreactor (anaerobic process) with SRB, was used. Until now, regarding operational issues in the anaerobic process, metal removal efficiency has been found to decrease during the winter period when SRB activity declines, resulting in elevated Zn concentrations in the effluent. Another issue is clogging caused by biofilm formation in the upper part of the reactor, possibly because of the rice bran and the microorganisms utilizing it (Sato et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Clogging necessitates frequent maintenance of the bioreactor. Similar challenges have been reported in other studies (Mayes et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tsukamoto et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo advance the practical application of sulfate-reducing bioreactor for AMD treatment, a large-scale test with a treatment flow rate of 100 L/min has been conducted at the same abandoned mine site in Japan since 2020. After some preparation and preliminary test operations for the start of the bioreactor, full-fledged continuous operation of the bioreactor started in September 2022. In this study, the large-scale bioreactor operation was conducted outdoors for two years in the range of ambient temperature from \u0026minus;\u0026thinsp;10\u0026deg;C to 40\u0026deg;C. Both the sulfate-reduction activity and metal removal performance were continuously monitored to clarify seasonal variations in the treatment performance. In addition, SRB possibly involved in sulfate reduction within the large-scale bioreactor were analyzed using high-throughput Illumina sequencing. In the large-scale bioreactor operation, we focused on 1) long-term continuous treatment with lower maintenance costs (addition of rice bran once a year) and 2) prevention of bioreactor clogging to assess operational stability and maintenance requirements.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eChemical analyses\u003c/p\u003e\n\u003cp\u003eBoth influent and effluent water samples from the bioreactor (one per week) were obtained during the two-year operation period. The pH, oxidation\u0026ndash;reduction potential (ORP), and water temperature dissolved oxygen (DO) were measured using a portable multi-water quality meter MM-42DP (DKK-TOA Corp., Tokyo, Japan). The DO was measured using a portable dedicated DO meter HQ1130 (Hach Company, Loveland, CO, USA). The metal concentrations were determined using an inductively coupled plasma optical emission spectrometer, Agilent 5110 ICP-OES (Agilent Technologies Inc., Santa Clara, CA, USA). Samples rich in organic content were digested using a mixture of HNO\u003csub\u003e3\u003c/sub\u003e and HClO\u003csub\u003e4\u003c/sub\u003e under heated wet conditions, and the levels of total organic carbon (TOC) and total inorganic carbon (TIC) were determined using a TOC analyzer, TOC-L (Shimadzu, Kyoto, Japan). The concentration of sulfate was determined using an ion chromatography system, Dionex ICS-1100, with Dionex IonPac columns (Thermo Fisher Scientific, Waltham, MA, USA), and the concentration of sulfide was determined with methylene blue (Reese et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e) using a ratio beam spectrophotometer, U-5100 (Hitachi High-Tech Corp., Tokyo, Japan). The concentrations of organic acids (acetic acid and propionic acid) were estimated using a UPLC system, ACQUITY UPLC H-Class PLUS, with an ion exclusion column, IC-Pak (7 \u0026micro;m, 7.8 mm \u0026times; 300 mm; Waters Corp., Milford, MA, USA). The detection limits of the chemicals were as follows: Zn, 8.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mg/L; Cu, 1.8 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e mg/L; Cd, 6.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mg/L; Fe 9.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mg/L.\u003c/p\u003e\n\u003cp\u003eLarge-scale sulfate-reducing bioreactor system\u003c/p\u003e\n\u003cp\u003e(i) Acid mine drainage and process flow\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003cp\u003eThe AMD from an abandoned mine site in northern Japan was used in this study. Through 2022 to 2023, the original AMD (pH 3.2\u0026ndash;4.0) contained 249\u0026ndash;334 mg/L SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, 28\u0026ndash;49 mg/L Fe, 15\u0026ndash;20 mg/L Zn, 2\u0026ndash;10 mg/L Cu, and 0.04\u0026ndash;0.08 mg/L Cd. A photograph of the large-scale passive treatment system illustrating the process flow is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. To prevent the oxidation of ferrous iron by air, the AMD was conveyed from the mine tunnel through polyethylene pipes. As the mine water level was higher than the top level of the reactors, the mine water was supplied to the tanks under gravity flow (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Before entering the bioreactors, the AMD was first split and introduced into two iron oxidation reactor tanks with rice husk and limestone (designated tanks A and B, respectively) at 75 and 25 L/min, respectively. After Fe removal, the effluents from the two tanks were combined. The resultant effluents (pH 2.8\u0026ndash;3.3) contained 257\u0026ndash;328 mg/L SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, 4\u0026ndash;14 mg/L Fe, 15\u0026ndash;20 mg/L Zn, 2\u0026ndash;11 mg/L Cu, and 0.04\u0026ndash;0.09 mg/L Cd before June 2023. After July 2023, the effluent (pH 3.1\u0026ndash;4.1) contained 253\u0026ndash;308 mg/L SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, 1\u0026ndash;8 mg/L Fe, 15\u0026ndash;18 mg/L Zn, 2\u0026ndash;8 mg/L Cu, and 0.04\u0026ndash;0.07 mg/L Cd. The combined effluents were then evenly divided at 50 L/min into two sulfate-reducing bioreactors (designated tanks A and B). The treated water from the two bioreactors was merged at the bioreactor outlet and discharged from the system.\u003c/p\u003e\n\u003cp\u003e(ii) Bioreactor\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eThe concept of a sulfate-reducing bioreactor using rice bran has been described previously at the laboratory and pilot scales (Sato et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). This anaerobic treatment was conducted using two vertical flow type rectangular concrete bioreactors [16 m (wide), 5 m (long), and 3.5 m (deep)]. A cross-sectional view of the bioreactor is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, and the specifications of the reactor packing materials are mentioned in Table\u0026nbsp;1. To mitigate the drop in water temperature during winter, 3.2 m of the reactor was embedded underground. At the bottom of each reactor, drainage pipes made of polyvinyl chloride (PVC) were installed, and to protect these pipes, a 0.15 m layer of limestone (particle size: 20\u0026ndash;40 mm) was laid over them (bottom layer in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). A 1.0 m thick mixture of rice husk and limestone (weight ratio, 1:4) was placed directly above this limestone layer (layer 3 in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), and it was followed by a 0.5 m thick layer of a mixture with a weight ratio of 1:16 (layer 2 in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Rice husk serves as a scaffold material for the colonization of SRB and captures precipitated metal sulfides such as ZnS. The limestone acts to neutralize the effluent from iron oxidation reactor tanks (pH 2.8\u0026ndash;4.1), bringing it to circumneutral pH, in which the activity of SRB is enhanced. Furthermore, as a substrate for anaerobes in the bioreactor, a 0.3 m thick surface layer composed of a mixture of 2 tons of rice bran and rice husk was placed on the 0.5 m thick layer (layer 1 in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e); on top of this layer, a mine water layer with a thickness of approximately 0.3 m was maintained as a water-sealing layer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe AMD was continuously supplied to each bioreactor at a flow rate of 50 L/min to achieve an HRT of 22.5 h at the rice husk\u0026ndash;limestone mixture layer. Sampling ports made of PVC pipes were installed on both the upstream and downstream sides at depths of 0, 0.3, 0.4, 0.8, 1.3, and 1.8 m from the top of the rice bran\u0026ndash;rice husk layer (layer 1), and at approximately 2 m from the center of the bioreactor. Water samples were collected via suction using a drill pump connected to hoses at the ground surface. Additionally, PVC pipes were installed at the boundary depths of each layer (0.3, 0.8, and 1.8 m) to monitor clogging within the bioreactor.\u003c/p\u003e\n\u003cp\u003e(iii) Rice bran, rice husk, and their feeding\u003c/p\u003e\n\u003cp\u003eThe composition of rice bran, rice husk, and their mixture is listed in Table\u0026nbsp;2. Rice bran contained high levels of oil (19.7%), protein (13.6%), and nitrogen-free extract (34.8%), whereas rice husk contained high levels of fiber (15.4%) and ash (31.7%). In contrast, the mixture of rice bran and rice husk contained protein (9.0%), oil (0.8%), fiber (24.6%), ash (14.9%), and soluble inorganic nitrogen (35.7%).\u003c/p\u003e\n\u003cp\u003eThe large-scale sulfate-reducing bioreactor was operated for two years, from September 16, 2022 to September 9, 2024. Initially, 2 ton of a mixture of rice bran and rice husk was placed within the bioreactor in September 2022. After one year of operation, an additional 2 ton of rice bran was fed to the rice bran\u0026ndash;rice husk mixture layer (layer 1) using heavy machinery for mixing in September 2023. The mine water level shifted approximately 0.7 m lower than usual during mixing.\u003c/p\u003e\n\u003cp\u003eAmplicon sequencing of 16S rRNA gene\u003c/p\u003e\n\u003cp\u003eFrom a total of 40 bioreactor samples (eight time points [November 2022, February 2023, May 2023, August 2023, November 2023, February 2024, May 2024, and August 2024] and five sampling ports [port 1, 3, 5, 7, and outlet]), DNA was extracted using a direct lysis protocol (Noll et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e) with bead beating. The extracted DNA was purified using phenol-chloroform extraction, isopropanol precipitation, RNase treatment (Type II-A; Sigma-Aldrich, St. Louis, MO, USA), polyethylene glycol precipitation, and ethanol precipitation, and the purified DNA was quantified using a NanoDrop Lite (Thermo Fisher Scientific). Using polymerase chain reaction (PCR) with high-fidelity DNA polymerase (Q5; New England Biolabs, Ipswich, MA, USA), the V4 region of the 16S rRNA gene was amplified with the universal primers 515F and 806R; both the primers were modified to contain an Illumina adapter region, and 806R contained a 12-basepair barcode for multiplex sequencing (Caporaso et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). The amplicons were purified using the AMPure XP Kit (Beckman Coulter, Brea, CA, USA), Wizard SV GEL, and PCR Clean-up System (Promega, Madison, WI, USA). The barcode-encoded DNA library and the initial control (PhiX; Illumina, San Diego, CA, USA) were subjected to paired-end sequencing using a 300-cycle MiSeq Reagent kit (Illumina) on a MiSeq sequencer (Illumina).\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene sequence data were analyzed using QIIME 2 (Bolyen et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). From the obtained paired-end reads, those with more than two expected errors were discarded (default maxEE\u0026thinsp;=\u0026thinsp;2), and chimeric sequences were identified and removed using the \u0026ldquo;consensus\u0026rdquo; method inherent to the DADA2 pipeline (Callahan et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The obtained nucleotide sequence data were deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive (DRA), European Nucleotide Archive (ENA), and Sequence Read Archive (SRA) databases under accession number PRJDB40188 (DRR901113\u0026ndash;DRR901152).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eSeasonal changes in pH, ORP, temperature, and DO during the large-scale bioreactor operation over two years\u003c/p\u003e \u003cp\u003eThe large-scale sulfate-reducing bioreactor was operated from September 16, 2022 to September 9, 2024, and the former half (September 2022 to September 2023) and latter half (October 2023 to September 2024) periods were designated Phase 1 and Phase 2, respectively. The ambient air temperature varied between \u0026minus;\u0026thinsp;10 and 40\u0026deg;C throughout the year, with average winter temperatures (December to March) of \u0026minus;\u0026thinsp;0.1 and 0.2\u0026deg;C during Phase 1 and Phase 2, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Therefore, the influent and effluent water temperatures of the bioreactor varied in response to changes in the ambient temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The temperature fluctuations of influent AMD, which was the effluent from the preceding iron oxidation reactor with a short HRT of 2 h, were relatively small, ranging from 8 to 18\u0026deg;C. In contrast, because the anaerobic bioreactor had a longer HRT of 22.5 h, the effluent water temperature fluctuations were a little larger, ranging from 5 to 20\u0026deg;C. As approximately 90% of the anaerobic bioreactor was buried underground, the use of ground temperature was allowed to buffer against ambient temperature effects, although the ambient temperature fell below 0\u0026deg;C in winter. Consequently, the minimum effluent temperature never fell below 4\u0026deg;C. Additionally, although partial surface freezing of the water-seal layer occurred during certain winter periods, freezing was limited to the surface and had a negligible impact on the inside of the bioreactor.\u003c/p\u003e \u003cp\u003eThe pH values of both influent and effluent water remained stable throughout the year, ranging from 2.9 to 4.1 for influent and from 6.5 to 7.3 for effluent regardless of water temperature. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the increase in influent pH to approximately 4 in July 2023 was observed owing to the feeding of additional limestone to the preceding iron oxidation reactor at that time. A similar pH increase occurred in July 2024, when additional limestone was added to replenish the consumed portion. This increase in influent pH had little effect on the effluent pH in the anaerobic bioreactor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, we observed that the effluent pH dropped to approximately 6.5 in September 2022, September 2023, and July 2023; at the former two time points, (additional) rice bran was fed to the bioreactor as a nutrient source, and in the latter, the HRT in the bioreactor temporarily increased four-fold (to 90 h). This increase in effluent pH was considered to have resulted from a temporary increase in the concentrations of organic acids derived from rice bran (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and a sharp increase in the efficiency of sulfate reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which accelerated the proton release associated with sulfate reduction and ionization of hydrogen sulfide ions (Eqs.\u0026nbsp;1 and 2). These effects temporarily outpaced the rate of pH increase owing to limestone dissolution.\u003c/p\u003e \u003cp\u003e2CH\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e \u0026rarr; 2HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e།\u003c/sup\u003e + H\u003csub\u003e2\u003c/sub\u003eS (1)\u003c/p\u003e\n\u003ch3\u003eHS → HS + H (2)\u003c/h3\u003e\n\u003cp\u003eThe ORP values for effluent remained consistently less than \u0026minus;\u0026thinsp;200 mV for most of the year, except during the winter period; in the winter of 2022, the effluent ORP increased to a maximum of \u0026minus;\u0026thinsp;179 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In contrast, during the winter of 2023, the highest recorded value was \u0026minus;\u0026thinsp;252 mV in February 2024, indicating that a stable reducing environment was maintained even under low-temperature conditions. This stability of the ORP values was considered to have resulted from sufficient microbial consumption activity following the addition of rice bran in September 2023, when the bioreactor already contained a partially established microbial community capable of utilizing rice bran. Furthermore, the temporary extension of the HRT in July 2023 described above (prior to rice bran addition) may have contributed to the stabilization of the microbial community within the bioreactor.\u003c/p\u003e \u003cp\u003eSince the initial installation of the rice bran\u0026ndash;rice husk mixture layer in Phase 1, the DO values of effluent remained below 1.0 mg/L, which was indicative of the maintenance of anaerobic conditions in the bioreactor regardless of seasonal change. The same trend was observed in Phase 2, with stable DO levels in the effluent. The increase in the DO of the effluent water on June 10, 2024, during Phase 2, was due to the replacement of the sensor cap of the DO meter (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSulfate reduction efficiency during the large-scale bioreactor operation\u003c/h2\u003e \u003cp\u003eThroughout the two-year bioreactor operation period, the sulfate concentration in the influent ranged from 253 to 308 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, the sulfate concentration in the effluent was less than 276 mg/L during this period, which was the maximum value recorded on March 22, 2023. The sulfate reduction rate (represented by vertical bars in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) was calculated as the percentage decrease in the sulfate concentration between the influent and effluent water in the bioreactor. Approximately one month after the addition of rice bran, the maximal sulfate reduction rates in Phase 1 and Phase 2 reached 47% and 94%, respectively, indicating an increase in sulfate reduction associated with a temporary increase in the availability of carbon source due to the addition of rice bran. However, subsequently, the sulfate reduction rate declined alongside a decrease in water temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), with both Phase 1 and Phase 2 exhibiting lower rates during the winter season. The average sulfate reduction rates in winter were 7.7% and 27.7%, with that in Phase 2 being 20% higher than that in Phase 1.\u003c/p\u003e \u003cp\u003eBoth sulfate reduction rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and effluent S\u0026sup2;\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) showed trends similar to those for the sulfate reduction efficiency. The average winter sulfate reduction efficiency in Phase 1 and Phase 2 was 0.14 and 0.52 mol-SO\u003csub\u003e4\u003c/sub\u003e/day/m\u0026sup3;, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In Phase 1, the effluent S\u0026sup2;\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations during most of the winter period were below the detection limit, whereas in Phase 2, the average during the winter was 5.0 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results suggested that in Phase 2, a part of the organic matter contained in the rice bran-rice husk mixture layer from Phase 1 remained, resulting in relatively higher organic carbon supply. In addition, the microbial community capable of utilizing rice bran had already been established in Phase 1, which led to higher utilization efficiency of the rice bran newly added as an organic carbon source in Phase 2. Furthermore, the average effluent temperature in Phase 2 winter was almost the same with that in Phase 1 (7.6\u0026deg;C), which might not have affected the maintenance of microbial activity. Therefore, it can be concluded that sufficient sulfate-reducing capacity was maintained in Phase 2, even during winter.\u003c/p\u003e \u003cp\u003eIn July 2023 (Phase 1), the HRT increased four-fold to 90 h, resulting in a maximum sulfate reduction rate of 80% in Phase 1, which confirmed that extension of the HRT led to an increase in sulfate reduction. This indicated that even nine months after rice bran addition, a sufficient amount of organic matter remained, and that prolonging the HRT accelerated its decomposition. In contrast, in September 2024, approximately one year after rice bran addition at the beginning of Phase 2, the sulfate reduction rates declined to levels lower than those observed in winter, suggesting that without the addition of organic matter once per year in the warmer summer season, sulfate reduction would be insufficient for the upcoming winter. However, in winter, organic matter consumption slows, and a greater amount is required; therefore, under the relatively short HRT of this process (22.5 h), the addition of organic matter prior to the winter was identified as an appropriate operational strategy.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMetal removal performance in the large-scale bioreactor during seasonal operation\u003c/h3\u003e\n\u003cp\u003eThe Zn concentrations in the effluent were sufficiently reduced to \u0026lt;\u0026thinsp;0.1 mg/L throughout most of the operational period outside winter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In Phase 1, an increase in total Zn concentration was observed around December 2022 as the water temperature declined, with an average of 0.84 mg/L and a maximum of 1.05 mg/L during winter. In contrast, in Phase 2, only a minor increase in total Zn concentration was observed in winter, with an average of 0.04 mg/L and a maximum of 0.27 mg/L. The concentrations of 1.95 and 1.01 mg/L were recorded in December 2022 (Phase 1) and in July 2024 (Phase 2), respectively; however, these values were considered to have resulted from sampling errors, in which deposits adhered to the inner surface of the piping were dislodged during sample collection. During the entire operational period, the Zn concentration in the effluent never exceeded the effluent standard in Japan (2.0 mg/L), revealing a stable treatment performance over 725 consecutive days. Sufficient sulfate reduction achieved using SRB within the bioreactor likely led to the precipitation and immobilization of Zn ions as sulfide minerals, including that in the winter. In both Phase 1 and Phase 2, rice bran was added in September when the water temperature began to decrease, and both the timing and dosage of rice bran were considered appropriate for Zn removal during the winter period.\u003c/p\u003e \u003cp\u003eThe concentrations of Cu and Cd never exceeded the effluent standards in Japan (3 mg/L and 0.03 mg/L, respectively) during the entire period, and their removal was consistently stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The inlet Fe concentrations were reduced to \u0026lt;\u0026thinsp;10 mg/L by the preceding iron oxidation reactor throughout the operational period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). However, an increase in the effluent Fe concentration was observed in Phase 1, along with a decline in sulfate reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), reaching a maximum of 7.4 mg/L. In Phase 2, only a slight increase in the effluent Fe concentration was detected, with a maximum concentration of 0.3 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eAt approximately pH 7, the solubility product of the respective metal sulfides follows the order CuS\u0026thinsp;\u0026lt;\u0026thinsp;CdS\u0026thinsp;\u0026lt;\u0026thinsp;ZnS\u0026thinsp;\u0026lt;\u0026thinsp;FeS. Therefore, when the sulfate-reducing activity in the bioreactor decreased and the resultant sulfide supply became insufficient, the effluent metal concentrations were expected to increase. During the winter period in Phase 1, 13 days after the effluent Fe concentration began to increase, the Zn concentrations in the effluent also started to increase, with maximum values of 7.4 mg/L for Fe and 1.0 mg/L for Zn. In contrast, only slight increases in the effluent Fe and Zn concentrations were observed in Phase 2. If all divalent metal ions (Fe, Zn, Cd, and Cu) in the influent were precipitated as their metal sulfides, the sulfate required for their precipitation, as calculated using the sulfate reduction reaction (Eq.\u0026nbsp;3), would be approximately 30 mg/L.\u003c/p\u003e\n\u003ch3\u003eM + HS → MS + 2H (3)\u003c/h3\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the difference in sulfate concentration between the inlet and effluent fell below 30 mg/L only during the 78-day period from November 2022 to April 2023 in Phase 1, whereas sufficient sulfate reduction was achieved during all other periods. Hence, in November 2022, the sulfate reduction efficiency had already become insufficient (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and approximately 14 days later, an increase in the total Zn concentration was observed (Phase 1, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, the difference in sulfate concentration in Phase 2 did not fall below the threshold (30 mg/L) during the entire operational period.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClogging in the large-scale bioreactor during the operation\u003c/h2\u003e \u003cp\u003eIn both Phase 1 and Phase 2, a gradual increase in the water level within the tank was observed over time owing to reduced permeability, with annual increases of 0.21 and 0.11 m, respectively (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The clogging rates were 0.12 and 0.06 m/y, which were one-fourth and one-eighth of the rates observed under the initial rice bran\u0026ndash;only conditions when the facility was first commissioned in 2020 (unpublished data). Mixing rice husk with rice bran reduced the clogging rate, and as a result, no maintenance was required for approximately one year until the next rice bran addition. Similar to manure or compost, rice bran, which has a small particle size, tends to cause clogging (Botes et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Hence, the addition of rice husk, which has a larger particle size, higher fiber and ash content, and is more resistant to degradation, was considered to have reduced clogging. Although the exact cause of clogging was not determined, it is likely influenced by the accumulation of biofilms and precipitation of metal sulfides within the bioreactor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the microbial community in the large-scale bioreactor\u003c/h2\u003e \u003cp\u003eAt eight time points during the two-year operation (November 2022, February 2023, May 2023, August 2023, November 2023, February 2024, May 2024, and August 2024), class- and genus-level phylogenetic analyses of sequence data were performed using QIIME2 to evaluate differences in microbial compositions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). In the large-scale bioreactor, besides \u003cem\u003eBacteroidia\u003c/em\u003e, \u003cem\u003eBerkelbacteria\u003c/em\u003e and \u003cem\u003eParcubacteria\u003c/em\u003e also became dominant. \u003cem\u003eBacteroidia\u003c/em\u003e are capable of degrading a wide range of organic compounds and are therefore considered to be involved in the decomposition of organic substrates such as rice bran. In addition, \u003cem\u003eBerkelbacteria\u003c/em\u003e and \u003cem\u003eParcubacteria\u003c/em\u003e, belonging to a bacterial supergroup known as Candidate Phyla Radiation (CPR) bacteria (including a wide variety of uncultured organisms), are dominant constituents of biofilms in sulfide-rich springs, although their physiological functions remain largely unknown. However, among these, \u003cem\u003eBerkelbacteria\u003c/em\u003e are presumed to be involved in sulfur metabolism and reduction of elemental sulfur by putative sulfhydrogenases (Valentin-Alvarado et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs for SRB, non-spore-forming members of the family \u003cem\u003eDesulfosarcinaceae\u003c/em\u003e (gram-negative bacteria, the class \u003cem\u003eDesulfobacteria\u003c/em\u003e; Waite et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) were the most dominant taxa. In addition, \u003cem\u003eDesulfofarcimen\u003c/em\u003e, an endospore-forming gram-positive bacterium reclassified from \u003cem\u003eDesulfotomaculum\u003c/em\u003e sp. (the class \u003cem\u003eClostridia\u003c/em\u003e; Watanabe et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), was detected in the downstream section of the reactor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Because of adjustment and preliminary operation (October 2020 to September 2022) over two years prior to the start of the large-scale experiment in September 2022, sufficient anaerobic conditions had already been established. In addition, the reactor was amended with a mixture of rice bran (as a carbon source) and rice husk (as a microbial carrier), which minimized reactor clogging. Consequently, strictly anaerobic members of the family \u003cem\u003eDesulfosarcinaceae\u003c/em\u003e were predominant throughout the operational period and were believed to be responsible for sulfate reduction.\u003c/p\u003e \u003cp\u003eThe relative abundance of \u003cem\u003eDesulfosarcinaceae\u003c/em\u003e increased in August 2023 compared with that in May 2023, partly due to a temporary increase in the HRT in July 2023. Following the additional rice bran input in September 2023, the relative abundance of \u003cem\u003eDesulfosarcinaceae\u003c/em\u003e further increased in November 2023 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Hence, the fact that the relative abundance of \u003cem\u003eDesulfosarcinaceae\u003c/em\u003e was higher in Phase 2 than in Phase 1 may have enabled stable sulfate reduction, even during the cold winter period. Similarly, after the addition of rice bran in September 2023, the microbial community analysis in November 2023 showed a substantial increase in the relative abundance of \u003cem\u003eBerkelbacteria\u003c/em\u003e, which has been suggested to be involved in sulfur reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B; Valentin-Alvarado et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, these bacteria may play important roles in the complex sulfur transformation network within the mixed layer of rice bran and rice husk.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA large-scale sulfate-reducing bioreactor using rice bran (treatment flow rate: 100 L/min) was evaluated for the treatment of metal-containing AMD over two years. By incorporating a mixture of rice bran and rice husk into the nutrient layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), biofilm-induced clogging in the upper part of the bioreactor was mitigated, enabling minimal maintenance with rice bran being replenished only once annually. Consequently, the bioreactor performance for sulfate reduction was effectively sustained for 725 days. The concentrations of Fe, Zn, Cu, and Cd in the treated AMD satisfied the standard effluent concentrations throughout the testing period. Using high-throughput Illumina sequencing of 16S rRNA genes, the family \u003cem\u003eDesulfosarcinaceae\u003c/em\u003e was found to be the predominant SRB. To further broaden the scope of the findings, future investigations should focus on determining the optimal quantity of rice bran and evaluating the long-term sustainability of the system, in preparation for its potential deployment in actual operational settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing financial or non-financial interests.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank Yuki Watanabe for providing technical assistance. This work was supported in part by JSPS KAKENHI (grant number 24K03111). We would like to thank Editage (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.editage.jp\u003c/span\u003e\u003c/span\u003e) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBen Ali HE, Neculita CM, Molson JW, Maqsoud A, Zagury GJ (2019) Performance of passive systems for mine drainage treatment at low temperature and high salinity: A review. 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Mine Water Environ 23:s2\u0026ndash;s80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10230-004-0028-0\u003c/span\u003e\u003cspan address=\"10.1007/s10230-004-0028-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Specifications of the packing materials of the biochemical reactor\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.1196%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 17.2757%;\"\u003e\n \u003cp\u003eLayer 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003eLayer 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003eLayer 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4286%;\"\u003e\n \u003cp\u003eBottom layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.1196%;\"\u003e\n \u003cp\u003eHeight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.2757%;\"\u003e\n \u003cp\u003e0.30 m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003e0.50 m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003e1.00 m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4286%;\"\u003e\n \u003cp\u003e0.15 m\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.1196%;\"\u003e\n \u003cp\u003eVolume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.2757%;\"\u003e\n \u003cp\u003e24.0 m\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003e40.0 m\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003e80.0 m\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4286%;\"\u003e\n \u003cp\u003e12.0 m\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.1196%;\"\u003e\n \u003cp\u003eMedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.2757%;\"\u003e\n \u003cp\u003eMixture of rice bran and rice husk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003eRice husk\u003c/p\u003e\n \u003cp\u003e+Limestone 40/20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003eRice husk\u003c/p\u003e\n \u003cp\u003e+Limestone 40/20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4286%;\"\u003e\n \u003cp\u003eLimestone 40/20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.1196%;\"\u003e\n \u003cp\u003eWeight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.2757%;\"\u003e\n \u003cp\u003eRice bran 2000 kg\u003c/p\u003e\n \u003cp\u003e+Rice husk 2400 kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003eRice husk 3500 kg\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e+Limestone 56000 kg (weight ratio of 1:16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003eRice husk 9000 kg\u003c/p\u003e\n \u003cp\u003e+Limestone 36000 kg (weight ratio of 1:4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4286%;\"\u003e\n \u003cp\u003e\u0026nbsp;2500 kg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.1196%;\"\u003e\n \u003cp\u003ePorosity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.2757%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003e45.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.588%;\"\u003e\n \u003cp\u003e51.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.4286%;\"\u003e\n \u003cp\u003e42.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Composition of rice bran, rice husk, and mixtures\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22.8188%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003eMoisture (wt%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003cp\u003e(wt%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003eOil\u003c/p\u003e\n \u003cp\u003e(wt%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003eAsh\u003c/p\u003e\n \u003cp\u003e(wt%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003eFiber\u003c/p\u003e\n \u003cp\u003e(wt%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.7785%;\"\u003e\n \u003cp\u003eNitrogen-free extract (wt%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22.8188%;\"\u003e\n \u003cp\u003eRice bran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e13.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e19.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e8.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.7785%;\"\u003e\n \u003cp\u003e34.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22.8188%;\"\u003e\n \u003cp\u003eRice husk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e31.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e15.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.7785%;\"\u003e\n \u003cp\u003e22.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22.8188%;\"\u003e\n \u003cp\u003eMixture of rice bran and rice husk (layer 1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e15.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e24.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0805%;\"\u003e\n \u003cp\u003e14.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.7785%;\"\u003e\n \u003cp\u003e35.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\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":"acid mine drainage, large-scale bioreactor, metal removal, passive treatment, sulfate-reducing bioreactor","lastPublishedDoi":"10.21203/rs.3.rs-9361529/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9361529/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA large-scale sulfate-reducing bioreactor, with a treatment flow rate of 100 L/min, was operated for approximately two years, from September 16, 2022 to September 9, 2024. Initially, 2 ton of a mixture of rice bran and rice husks was placed within the bioreactor in September 2022. After one year of operation, an additional 2 ton of rice bran was fed to the rice bran\u0026ndash;rice husk mixture layer using heavy machinery for their mixing in September 2023. Both sulfate reduction and metal removal efficiencies were maintained at high levels, especially in the latter half of the period, without major clogging, even during the cold winter period. Consequently, the maximum concentration of zinc (the second most abundant metal in this AMD after iron) in the effluent remained below 0.3 mg/L (removal ratio: \u0026gt;98%). In the large-scale bioreactor, besides the family \u003cem\u003eDesulfosarcinaceae\u003c/em\u003e (sulfate-reducing bacteria), the genus \u003cem\u003eBerkelbacteria\u003c/em\u003e was predominant. The results of the large-scale passive treatment highlight the possibility of long-term continuous treatment with the addition of organic sources once a year, and the prevention of bioreactor clogging to assess operational stability and maintenance requirements.\u003c/p\u003e","manuscriptTitle":"Performance and stability of a large-scale sulfate-reducing bioreactor with rice bran for passive treatment of acid mine drainage over two years","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 12:36:31","doi":"10.21203/rs.3.rs-9361529/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-21T22:59:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T09:04:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T14:10:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mine Water and the Environment","date":"2026-04-08T19:55:06+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":"10e32ec2-0ca6-4528-9d6a-a40ff891a5d2","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T12:36:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 12:36:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9361529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9361529","identity":"rs-9361529","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}