The fate of pollutants in the co-composting of natural mineral biochar and animal manures in an intermittent aeration and mixing bioreactor (IAMB)

The fate of pollutants in the co-composting of natural mineral biochar and animal manures in an intermittent aeration and mixing bioreactor (IAMB) | 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 Article The fate of pollutants in the co-composting of natural mineral biochar and animal manures in an intermittent aeration and mixing bioreactor (IAMB) Mitra Mohammadi, Ali Almasi, Seyyed Alireza Mousavi, Mostafa Hadei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6743840/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Animal manures (AMs) are widely utilized as organic fertilizers but often contain significant levels of emerging pollutants, posing environmental and health risks. This experimental study investigated the impact of 650-million-year natural mineral biochar (NMB) and animal manure (CWM:PUM) on the fate of pollutants in the process of intermittent aeration and mixing composting. 12 treatments were monitored over 60 days to evaluate the fate of pollutants, nutrients, and environmental risks. The cumulative temperatures (°C) recorded for treatments T1–T12 were 897, 858, 836, 981, 926, 898, 1007, 999, 959, 1056, 1025, and 972, respectively. At the start of the process, the Zn concentrations in treatments T1, T4, T7, and T10 were 197.15, 336.32, 287.53, and 244.16 g/kg, respectively. By day 60, they had decreased to 185.78, 296.14, 197.52, and 129.58 g/kg, respectively. Cu concentration in mature compost was 228.31, 109.36, 76.11, and 131.5 g/kg. The removal efficiency rankings varied across treatments: in control: Zn > Cu > Cr, in 5% NMB: Cr > Cu > Zn, and in 10% and 15% NMB: Zn > Cr > Cu. Zn, Cu, and Cr showed significant reductions due to adsorption, surface complexation, and pH-mediated mechanisms, with final concentrations below the USEPA standards. This study highlights the efficacy of NMB in improving compost quality, reducing pollutant bioavailability, and mitigating environmental risks, underscoring its potential as a sustainable solution for managing animal manure. Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Health sciences/Risk factors Mineral Biochar Fate Ecological Risk Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Over the past few decades, animal food production in developing countries has increased rapidly, leading to large volumes of animal manure (AMs) being dumped near urban areas [ 1 ]. In Iran, the annual production of manure is estimated at 251,313 tons for livestock and 744.77 million tons for poultry (including chickens and turkeys). Recent studies have shown that AMs contain significant amounts of heavy metals (HMs) [ 2 ]. If not properly managed, these pollutants can accumulate in soil, increase their bioavailability, be absorbed by plants, contaminate ground and surface water, and pose risks to public health [ 3 ]. The presence of HMs in AMs is often linked to contamination in crops and animal feed [ 2 ]. Despite EPs' critical importance, there is limited information on their residues and fate in AMs in Iran. Investments in waste recycling and conversion, particularly the production of organic fertilizers, have shown significant long-term benefits and should be prioritized as part of sustainable development efforts [ 4 ]. Interestingly, Kerman, Iran, is home to the only biochar mine in the country, a prehistoric resource dating back 650 million years. This study represents the first global investigation into the use of this biochar as an organic, environmentally friendly material, comparing it with conventional biochar compounds in AMs, which are produced in exceptionally high volumes in Iran. 2. Material and Methods 2.1. Source and Characteristics of Compost Materials Pea straw (PS) was sourced from pea fields in Kermanshah city, Iran and crushed into 1–3 cm pieces. CWM and PUM were obtained from cow and poultry farms in the Kermanshah. The physicochemical properties of the raw materials are presented in Table 1. Powder of Natural mineral biochar (NMB) was sourced from a natural mine in Kerman, Iran, that is entirely natural and, according to geological studies, was formed through natural processes approximately 650 million years ago [ 5 ]. Table 1. Physicochemical properties of raw materials 2.2. Experimental Setup and Composting Process Composting was conducted using a galvanized bioreactor with intermittent aeration and mixing (IAMBR) over 60 days. The IAMBR had a vertical oval cross-section (H: 50 cm, L: 60 cm, W: 30 cm, freeboard: 15 cm) and was equipped with four sharp-edged agitators (L: 54 cm, W: 6 cm, RPM: 24). Ambient humidity and temperature, as well as chamber temperature, were monitored using an online thermometer and hygrometer kit (HTC-2, China) installed on the IAMB. Aeration was provided by an electromagnetic aeration pump (Aqua AP-9805, China) with a power rating of 6.5 W, a pressure of over 0.025 MPa, and an output flow rate of 5.5 L/min. The pump supplied air to 15 aeration diffusers installed along the bottom sides of the BR. Aeration was automatically controlled using a dial timer (Zhejiang, China) set to operate five times daily for 20 minutes. To homogenize the compost pile, mixing was performed daily using the four agitators. The pile's core temperature was measured three times daily before turning the pile using a digital thermometer (TP101, China). Compost turning was performed every two days, alternating between fresh and mature compost. Twelve treatments (T1–T12) (Table.2) were tested to evaluate the effects of NMB (0%, 5%, 10%, 15%) and CWM:PUM (1:1, 1:3, 3:1), as detailed in Table 2. The carbon-to-nitrogen ratio (C/N) and moisture content were adjusted to 24–25:1 and 60%, respectively. Samples (600 grams) were collected on days 0, 5, 10, 15, 20, 30, 40, 50, and 60 using a five-point sampling method to ensure representative samples from the early mesophilic, thermophilic, secondary mesophilic, and maturation phases. Table 2. Experimental Setup 2.3. Physicochemical Analysis The collected samples were dried, ground, and stored, then divided into two portions: one portion was used to determine the physicochemical properties, while the other was air-dried, ground, and stored at 4°C for HMs and chemical properties analysis. The pH was measured by mixing the samples in a 1:10 ratio with water and allowing the mixture to sit for 0.5 hours before using a pH meter. Total organic carbon (TOC) was determined using the modified Walkley-Black wet oxidation method [ 6 ]. Total nitrogen was analyzed through the Kjeldahl digestion method using an automatic Kjeldahl apparatus, while nitrate levels were measured following the method described by [ 7 ]. Heavy metals, including copper (Cu), chromium (Cr), and zinc (Zn), were extracted using acid digestion with sulfuric acid and quantified via atomic absorption spectrometry [ 8 ]. 2.4. Ecological Risk Assessment The ecological risk potential of Cu, Cr, and Zn was calculated following the approach outlined by Negahban et al.(2021) [ 9 ]. 2.5. Statistical analysis Data analysis was conducted using SPSS software, applying the Kolmogorov-Smirnov test to assess data normality, parametric and non-parametric tests, and Pearson correlation coefficients, with a significance level of 0.05. Microsoft Excel was used for data organization, Grapher for graphing, and Design-Expert software for response surface methodology analysis at three levels (-1, 0, + 1) for CWM/PUM (factor A) and NMB (factor B). 3. Results 3.1. Change in pH Initially, the pH of the compost was neutral but increased over time, reaching the alkaline range during the curing stage (Fig. 1and 2.a) ( P < 0.05). Treatments with NMB and a higher proportion of CWM exhibited relatively higher pH values compared to T1–T5 ( P 0.05), the final compost showed a greater influence of NMB ( P < 0.05) (Table.3). Among the treatments, T2 exhibited the smallest pH changes at the beginning and the end of the process, while T6 and T7 showed the largest variations. Table 3. ANOVA results by response surface methodology in ANBC-containing treatment 3.2. Change in Temperature The overall temperature trends in all treatments followed a similar pattern. Initially, the compost pile temperature ranged between 20–40°C. It then entered the thermophilic phase, reaching 40–60°C, before cooling down as mesophilic bacteria resumed activity, bringing the pile temperature closer to ambient levels (Fig. 2. and3). The retention time at high temperatures increased with higher NMB levels and lower CWM/PUM ratios ( P < 0.05). The cumulative temperatures (°C) recorded for treatments T1–T12 were 897, 858, 836, 981, 926, 898, 1007, 999, 959, 1056, 1025, and 972, respectively. The thermophilic phase lasted 14, 16, and 16 days for piles containing 75% PUM combined with 5%, 10%, and 15% NMB, respectively. The shortest thermophilic phase duration (9 days) was observed in T3, while the longest (16 days) occurred in T10 and T7 (Fig.3). 3.3. Fate of Carbon On the first day of composting, TOC concentrations ranged from 40% to 42.32% (Fig.4a). As the decomposition process progressed, TOC levels decreased significantly ( P < 0.05). NMB had a notable effect on TOC reduction ( P 0.05). However, higher CWM content was associated with smaller TOC reductions (Fig. 2.c and 4a). TOC levels in the control group differed significantly from those in the other treatments ( P < 0.05). During the stability phase, TOC reduction efficiencies were highest in T10, T11, and T12 at 39%, 38.51%, and 35.01%, respectively, and slightly lower in T7, T8, and T9 at 43.39%, 42.39%, and 41.84%, respectively. Treatments containing 15% NMB exhibited a lower rate of TOC reduction compared to those with 5% or 10% biochar ( P < 0.05). The piles with 15% NMB also had the highest initial organic carbon content. 3.4. Fate of Nitrogen Compounds 3.4.1. NH₄⁺-N In all treatments, the concentration of NH₄⁺-N gradually increased from 0.059–0.66% on day 1 to 0.1–0.18% on day 15 ( P < 0.05). The maximum increases in NH₄⁺-N concentration were observed in treatments T3–T5, with increases of 83.33%, 80.32%, and 69.49%, respectively, which were significantly lower than those in treatments T6–T12 ( P < 0.05) (fig.4,b). After day 15, NH₄⁺-N concentrations decreased rapidly, reaching 0.06–0.048% by day 60. On day 60, NH₄⁺-N levels in piles containing NMB were significantly lower than in those without NMB ( P 0.05). However, with increasing CWM at a constant volume, the rise in TOC during the thermophilic phase and the decrease by day 60 were less pronounced ( P < 0.05). 3.4.2. NH₄⁺-N / NO₃⁻-N The NH₄⁺-N / NO₃⁻-N ratio in treatments T1–T12 ranged between 1.96 and 1.58 (Fig. 2.d and 4b). Over time, the NH₄⁺-N / NO₃⁻-N ratio increased during the first 15 days and then began to decrease, with the most significant decrease observed in piles containing 15% NMB. A significant difference in nitrate levels was detected between treatments with 10% and 15% NMB compared to those with 5% NMB and between treatments with and without NMB ( P < 0.05). A significant negative correlation was observed between the decrease in the NH₄⁺-N / NO₃⁻-N ratio and NMB content ( P 0.05). 3.5. Fate of Heavy Metals (HMs) In this study, the changes in heavy metals were examined under the optimized CWM:PUM ratio of 1:3 and varying NMB levels (0%, 5%, 10%, and 15%) (Fig. 5). At the start of the process, the Zn concentrations in treatments T1, T4, T7, and T10 were 197.15, 336.32, 287.53, and 244.16 g/kg, respectively. By day 60, these concentrations had decreased to 185.78, 296.14, 197.52, and 129.58 g/kg, respectively. Similarly, Cu concentrations in T1, T4, T7, and T10 were initially 240.25, 127.63, 97.4, and 189.55 g/kg, respectively, and declined to 228.31, 109.36, 76.11, and 131.5 g/kg in the final compost. Cr concentrations in the control treatment began at 26.12 g/kg and remained largely unchanged in the mature compost. However, in treatments containing 5%, 10%, and 15% NMB, Cr concentrations decreased to 6.76, 12.15, and 7.05 g/kg, respectively, representing a significant reduction. The removal efficiency rankings varied across treatments: in control: Zn > Cu > Cr, in 5% NMB: Cr > Cu > Zn, and in 10% and 15% NMB: Zn > Cr > Cu. A positive correlation was observed between pH and HM removal efficiency, with increasing pH enhancing the removal efficiency from the beginning to the end of the process. ANOVA results confirmed that removal efficiency differences between the control and other treatments were statistically significant ( P < 0.05). NMB had a direct and significant positive effect on the removal of Zn, Cu, and Cr ( P < 0.05). HMs exhibited a positive correlation with humification (Fig. 6). 4. Discussion pH is a key indicator in composting as it affects the survival of microorganisms. With aeration and the improved decomposition of organic matter, microbial activity intensified, reducing the environment's acidity and increasing the pH. Chen et al.(2021) [ 10 ] reported that under favorable conditions, organic acids are completely decomposed, causing compost to transition from an acidic to a neutral range, aligning with the findings of this study. Biochar was also observed to increase pH due to the availability of mineral nutrients, which is consistent with the findings of Choudhary et al.(2021) [ 11 ]. Research suggests that pH values in the range of 5.5–8.0 are optimal for composting [ 12 ], matching the results of this study. Similarly, Awasthi et al.(2020) [ 13 ] found that using bamboo biochar (2–10%) in composting sheep manure led to a pH increase, further corroborating these findings. The final pH of the compost in this study complied with international standards [ 14 ]. In general, temperature showed a significant positive correlation with NMB and a significant negative correlation with CWM/PUM. Treatments containing NMB exhibited a relatively higher heating rate and an extended thermophilic phase, indicating improved mineralization and compost maturity [ 15 ]. Maintaining temperatures above 55°C for at least 3 days is crucial for eliminating pathogens and parasites in compost [ 16 ]. In this study, all treatments met standard hygiene requirements. The findings of Afriliana et al.(2021) [ 17 ] align with the current study, reporting a maximum composting temperature of 60°C. The increased temperatures with higher NMB levels may be attributed to NMB's high surface area and porosity, which facilitate oxygen transport and improve microbial proliferation conditions [ 18 ]. In contrast, Abd El-Rahim et al.(2021) [ 19 ] observed a maximum temperature of 73.5°C, with slower temperature increases. This difference might be due to cooler ambient conditions during autumn and winter. Wang et al.(2021) [ 20 ] reported that biochar addition resulted in higher temperatures for a shorter duration, with compost entering the cooling phase more quickly, which contradicts the results of this study. The duration of the thermophilic phase was also influenced by the CWM/PUM ratio. Higher PUM levels enhanced temperature retention and duration in the thermophilic phase, likely due to the nutrient content in PUM, which supports microbial activity and heat production. Total organic carbon (TOC) is a critical parameter for evaluating compost quality. Various studies have reported similar trends, indicating an increase in TOC during the early stages of composting when NMB is introduced, despite the lower carbon content of natural biochar compared to other mineral biochar [ 21 , 22 ]. For instance, Awasthi et al.(2020) [ 13 ] observed an increase in TOC from 45.83–49.97% with the addition of biochar. During composting, some carbon is consumed and released as CO₂, while the remainder contributes to cellular structure formation alongside nitrogen [ 23 ]. TOC reductions occur due to mineralization and the formation of humic substances. In a study by Biyada et al.(2021) [ 24 ], TOC followed a similar trend, decreasing from 32.64% at the start of composting to 23.53% in mature compost. These findings suggest that treatments with higher biochar content experience reduced carbon losses because biochar's carbon is resistant to decomposition and highly stable. Conversely, higher CWM/PUM ratios resulted in lower TOC reduction efficiency, likely due to decreased microbial activity associated with increased CWM content, which in turn affected carbon breakdown. The conversion of organic nitrogen to inorganic nitrogen during composting is a potential mechanism for inhibiting the growth of host bacteria. The large surface area and high adsorption capacity of NMB facilitated nitrogen fixation, resulting in increased uptake of organic nitrogen ions by NMB and reduced nitrogen loss. However, nitrogen reduction trends were similar between biochar and non-biochar treatments, indicating a compensatory effect of biochar addition. The increase in NH₄⁺-N observed until day 15, concurrent with rising temperatures, may be attributed to the rapid decomposition of organic nitrogen compounds and relatively weak nitrification activity by nitrifying bacteria. This aligns with the findings of Li et al. (2020) [ 25 ], who reported a significant increase in NH₄⁺-N in all treatments during the initial stages of composting as temperatures increased. A similar result was observed by Rong et al.(2019) [ 26 ], who co-composted chicken manure and corn leaves. The mechanisms underlying biochar-assisted reductions in NH₄⁺-N emissions may include the high adsorption capacity of NMB. This capacity is attributed to the large specific surface area and internal pore volume of NMB, as well as the presence of surface acidic functional groups, cation exchange sites, and micropores, which collectively limit the release of NH₄⁺-N during composting [ 18 ]. Agyarko-Mintah et al.(2017) [ 27 ] demonstrated that NH₃ adsorption primarily occurs within the pore spaces of biochar. Additionally, NMB enhances aerobic conditions, supports the proliferation of aerobic nitrifying bacteria, and provides a habitat for microorganisms, further aiding in NH₄⁺-N retention and overall compost efficiency [ 28 ]. During the bio-oxidation stage, the NH₄⁺-N / NO₃⁻-N ratio fluctuates due to competition between ammonification and nitrification [ 29 ]. The decrease in NH₄⁺-N and the simultaneous increase in NO₃⁻-N are attributed to the rapid conversion of NH₄⁺-N to NO₃⁻-N by nitrifying microorganisms [ 30 ]. Proteins, amino acids, and other organic materials are initially used by microorganisms as energy and nitrogen sources. Once these materials are depleted, the NH₄⁺-N formed during microbial deamination becomes unstable, evaporates as ammonia gas, and is partially converted to NO₃⁻-N by ammonia-oxidizing microbes [ 31 ]. Since nitrifying bacteria cannot grow at temperatures above 40°C, nitrification primarily occurs during the cooling and compost maturation stages. Bernal et al.(2009) [ 32 ] reported that an NH₄⁺-N / NO₃⁻-N ratio below 0.16 is an indicator of compost maturity. The National Standards Organization of Iran defines an acceptable NH₄⁺-N / NO₃⁻-N ratio range of 0.5 to 3.0 for grades 1 and 2 compost. Shan et al., (2021) observed a slight reduction in total nitrogen with increasing NMB levels [ 33 ]. Similarly, Steiner et al.(2010) [ 34 ] found comparable nitrogen losses in PUM composting with 5% or without NMB, but in piles with 20% biochar, nitrogen loss was significantly reduced by up to 52%. The study of HMs in AMs provides valuable insights into their bioavailability and environmental pollution potential. HMs are non-degradable and can persist in manure and compost, leading to their accumulation with long-term application [ 2 ]. NMB reduces HM bioavailability through adsorption and fixation. By adsorbing nutrients in its micropores, biochar reduces the efficiency of organic pollutant degradation, blocking microbial contact and enhancing HM removal [ 35 ]. Another mechanism contributing to the inactivation of HMs is biochar adsorption, which involves physical trapping in micropores and the formation of complexes between Zn, Cu, and Cr and the functional groups on biochar, such as –OH, –COOH, and C–O [ 36 ]. For cations, the primary mechanisms include surface precipitation with anions on NMB, the substitution of exchangeable cations, and the formation of surface complexes with –OH groups or delocalized π electrons from biochar [ 37 ]. The pH changes in compost with biochar influence the complexation behavior of functional groups such as –OH, –COOH, and –NH₂, which play a role in metal adsorption. Electrostatic interactions and cation exchange are identified as the dominant mechanisms of metal adsorption by biochar [ 38 ]. Increasing NMB from 5–15% significantly enhanced the immobilization of HMs. A one-sample t-test revealed that the concentrations of Zn, Cr, and Cu in the final compost were significantly lower than the standards set by the US Environmental Protection Agency (EPA) [ 39 ]. Humification is a critical process in reducing the bioavailability of HMs during aerobic composting. In this process, metal cations combine with organic acids and polymerize into stable humic substances [ 40 ], supporting the findings of this study. Humic molecules are rich in carboxyl and hydroxyl groups, which form stable complexes with metal cations. During aerobic composting, the mineralization and humification of organic carbon regulate the bioavailability of HMs. Metal cations released through organic carbon mineralization can react with organic acids, forming more stable complexes [ 41 ]. Similarly, during dehumidification, metal cations readily combine with organic acids to form stable complexes, as adsorbed inorganic cations reduce competition with metal cations. In summary, biochar enhances the deactivation of Zn, Cu, and Cr primarily through surface adsorption mechanisms, significantly contributing to the reduction of their bioavailability. The contamination factor (CF) for Zn was highest in T4 and lowest in T10. In general, the CF of Cr was much lower than that of Cu and Zn (CF: Cu > Zn > Cr). An examination of the contamination degree (Cdegree) revealed that treatments T4, T7, and T10 exhibited very low contamination (Cdegree < 1.5), while the control treatment (T1) showed low contamination (1.5 < Cdegree < 2) [ 42 ]. The ecological risk (ER) and risk index (RI) parameters indicate that the studied heavy metals, both individually and collectively, pose a low risk in compost piles and do not threaten the environment. Conclusion This study demonstrates the significant role of NMB in optimizing the composting process of AMs, particularly cow and poultry manure. The addition of NMB significantly enhanced the removal efficiencies of heavy metals (Zn, Cu, and Cr), reducing their bioavailability and mitigating associated environmental risks. Treatments containing higher NMB levels (10–15%) exhibited the most effective pollutant immobilization and nutrient stabilization, while meeting international safety standards, including those set by the US EPA. The findings emphasize that NMB improves compost quality by facilitating microbial activity, enhancing adsorption, and stabilizing nutrients through mechanisms such as surface complexation, electrostatic interactions, and pH regulation. Environmental risk assessments confirmed that composting, particularly with NMB, transitions raw manure with medium to high risks into safer compost products with low or risk-free classifications. These results highlight the potential of NMB as a sustainable and eco-friendly solution for managing AMs while addressing emerging pollutants. Incorporating NMB into composting systems can contribute to better resource utilization, reduced environmental pollution, and the promotion of circular agricultural practices. Further research is recommended to explore the long-term impacts of NMB-amended compost in real-world applications and its interactions with diverse environmental and soil conditions. Declarations Declaration of Competing Interest The authors declare they have no known competing financial interests or personal relationships. Funding Kermanshah University of Medical Sciences. Author Contribution M.M: Methodology, software, validation, formal analysis, investigation, resources, data curation, writing—original draft preparation; A.A: conceptualization, supervisor, project administration; S.A.M: Advisor; M.H: writing—original draft preparation in English Acknowledgement The authors thank the Kermanshah University of Medical Sciences for its financial support (No. 4010775). Data Availability Data is provided within the manuscript files. References Quaik, S. et al. Veterinary antibiotics in animal manure and manure laden soil: Scenario and challenges in Asian countries. J. King Saud University-Science . 32 (2), 1300–1305 (2020). Zheng, X. et al. Review on fate and bioavailability of heavy metals during anaerobic digestion and composting of animal manure. Waste Manage. 150 , 75–89 (2022). Liu, X. et al. Frontiers in Environmental Cleanup: Recent Advances in Remediation of Emerging Pollutants from Soil and Water. J. Hazard. Mater. Adv. , : p. 100461. (2024). 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Additives for reducing nitrogen loss during composting: A review. J. Clean. Prod. 307 , 127308 (2021). Steiner, C. et al. Reducing nitrogen loss during poultry litter composting using biochar. J. Environ. Qual. 39 (4), 1236–1242 (2010). Gholizadeh, M. & Hu, X. Removal of heavy metals from soil with biochar composite: A critical review of the mechanism. J. Environ. Chem. Eng. 9 (5), 105830 (2021). Vithanage, M. et al. Kinetics, thermodynamics and mechanistic studies of carbofuran removal using biochars from tea waste and rice husks. Chemosphere 150 , 781–789 (2016). Yang, X. et al. Preparation and modification of biochar materials and their application in soil remediation. Appl. Sci. 9 (7), 1365 (2019). Liu, C. & Zhang, H. X. Modified-biochar adsorbents (MBAs) for heavy-metal ions adsorption: A critical review. J. Environ. Chem. Eng. 10 (2), 107393 (2022). Agency, U. E. P. Acid digestion of sediments, sludges, and soils. USEPA Washington, DC. (1996). Shan, G. et al. The transformation of different dissolved organic matter subfractions and distribution of heavy metals during food waste and sugarcane leaves co-composting. Waste Manage. 87 , 636–644 (2019). Piccolo, A. et al. Soil washing with solutions of humic substances from manure compost removes heavy metal contaminants as a function of humic molecular composition. Chemosphere 225 , 150–156 (2019). Urrutia-Goyes, R. et al. Insights on trace metal enrichments in tourists beaches of Santa Elena Province, Ecuador. Reg. Stud. Mar. Sci. 73 , 103452 (2024). Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table.docx Cite Share Download PDF Status: Published Journal Publication published 26 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviews received at journal 12 Jun, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviewers agreed at journal 09 Jun, 2025 Reviewers agreed at journal 09 Jun, 2025 Reviewers invited by journal 08 Jun, 2025 Editor assigned by journal 03 Jun, 2025 Submission checks completed at journal 29 May, 2025 First submitted to journal 29 May, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6743840","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468666514,"identity":"e5316e49-354c-4bb2-93a1-a5476644a082","order_by":0,"name":"Mitra Mohammadi","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mitra","middleName":"","lastName":"Mohammadi","suffix":""},{"id":468666517,"identity":"abb701a0-491f-441e-8475-4b4aa00f678e","order_by":1,"name":"Ali Almasi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYDACdsYGhgdAmo0HxKsAYmbmBvxamIFaEuBazkBF8GsBYpAWBpAWxjYQi4AW/mbmxg+JOXb2fDxnTDf+nFcbzd8O1PKjYhtOLRKHGZslErclJ7bx9pjd5t12PHfGYcYGxp4zt3FbA1QA1MKcwMbPY3abcdux3AagCDNjG24t8kBbfiRuq7cHabn5c86x3PmEtBgcZmwD2gIkgQ67wdtQk7uBkBZDoGKLxG3HE9t4jpXd5jl2IHcjUMtBfH6RO97++MbHbdX28j3J227+qKnLnXf+8MEHPyrweB8NHAaTB4hWDwR1pCgeBaNgFIyCEQIAsaZbojbHcekAAAAASUVORK5CYII=","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"","lastName":"Almasi","suffix":""},{"id":468666518,"identity":"2c8e9a04-3cad-4cf8-ac60-154464bd58af","order_by":2,"name":"Seyyed Alireza Mousavi","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Seyyed","middleName":"Alireza","lastName":"Mousavi","suffix":""},{"id":468666520,"identity":"7407b01a-11c0-4909-925c-de9001fd7a03","order_by":3,"name":"Mostafa Hadei","email":"","orcid":"","institution":"Tehran University Of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Hadei","suffix":""}],"badges":[],"createdAt":"2025-05-25 13:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6743840/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6743840/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18122-7","type":"published","date":"2025-09-26T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84377642,"identity":"b3ae1dc1-dd8b-42b4-8bf7-aab8ad0b5f1f","added_by":"auto","created_at":"2025-06-11 08:38:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":277993,"visible":true,"origin":"","legend":"\u003cp\u003epH changes under different treatments\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/dd962e5a6a8a302a43f649ca.jpg"},{"id":84376867,"identity":"29f6c81e-bb08-4cdd-98f9-76140f4ea25e","added_by":"auto","created_at":"2025-06-11 08:30:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134290,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional (3D) plots examining a) pH b) cumulative temperature c) TOC, d) NH4-N/NO3-N, e) Cu , f) Zn, and g) Cr in NMB-containing treatments\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/d49629beca6d0df0384415a4.jpg"},{"id":84376862,"identity":"dc441035-367d-4cc0-bbfe-ac27a2d7e5d7","added_by":"auto","created_at":"2025-06-11 08:30:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73879,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in a) treatments temperature and b) ambient temperature during the animal manure composting process.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/8bc02004f119b342f930d42d.jpg"},{"id":84377652,"identity":"fe53c95c-9284-4c1c-b1ea-816219fd0d87","added_by":"auto","created_at":"2025-06-11 08:38:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70672,"visible":true,"origin":"","legend":"\u003cp\u003eChange of a)TOC, b)NH4-N/NO3-N in different treatment\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/9c29093905ffef7241e89cfc.jpg"},{"id":84378042,"identity":"c7dd5add-02d6-4ee9-9ad2-36c944710207","added_by":"auto","created_at":"2025-06-11 08:46:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29027,"visible":true,"origin":"","legend":"\u003cp\u003eChange of heavy metals in AMs composting process\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/3b4e893995c1867df5180784.jpg"},{"id":84376872,"identity":"24843f33-e770-4956-8861-17f41a591ed0","added_by":"auto","created_at":"2025-06-11 08:30:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30764,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between Humification and removal a) Zn b) Cu and c) Cr in different treatments\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/8da200edad90b886c17ef8e4.jpg"},{"id":92430955,"identity":"bae8b268-fc3c-4233-b128-ce2f04837597","added_by":"auto","created_at":"2025-09-29 16:08:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1300863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/e0dfcb23-d554-4428-a016-971decba5c8c.pdf"},{"id":84378041,"identity":"ca5abba6-4506-4134-b917-abe9fa6b9d87","added_by":"auto","created_at":"2025-06-11 08:46:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16844,"visible":true,"origin":"","legend":"","description":"","filename":"table.docx","url":"https://assets-eu.researchsquare.com/files/rs-6743840/v1/613b890e563650ddb80f72f6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The fate of pollutants in the co-composting of natural mineral biochar and animal manures in an intermittent aeration and mixing bioreactor (IAMB)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOver the past few decades, animal food production in developing countries has increased rapidly, leading to large volumes of animal manure (AMs) being dumped near urban areas [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Iran, the annual production of manure is estimated at 251,313 tons for livestock and 744.77\u0026nbsp;million tons for poultry (including chickens and turkeys). Recent studies have shown that AMs contain significant amounts of heavy metals (HMs) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. If not properly managed, these pollutants can accumulate in soil, increase their bioavailability, be absorbed by plants, contaminate ground and surface water, and pose risks to public health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The presence of HMs in AMs is often linked to contamination in crops and animal feed [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite EPs' critical importance, there is limited information on their residues and fate in AMs in Iran. Investments in waste recycling and conversion, particularly the production of organic fertilizers, have shown significant long-term benefits and should be prioritized as part of sustainable development efforts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, Kerman, Iran, is home to the only biochar mine in the country, a prehistoric resource dating back 650\u0026nbsp;million years. This study represents the first global investigation into the use of this biochar as an organic, environmentally friendly material, comparing it with conventional biochar compounds in AMs, which are produced in exceptionally high volumes in Iran.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Source and Characteristics of Compost Materials\u003c/h2\u003e \u003cp\u003ePea straw (PS) was sourced from pea fields in Kermanshah city, Iran and crushed into 1\u0026ndash;3 cm pieces. CWM and PUM were obtained from cow and poultry farms in the Kermanshah. The physicochemical properties of the raw materials are presented in Table\u0026nbsp;1. Powder of Natural mineral biochar (NMB) was sourced from a natural mine in Kerman, Iran, that is entirely natural and, according to geological studies, was formed through natural processes approximately 650\u0026nbsp;million years ago [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;1. Physicochemical properties of raw materials\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental Setup and Composting Process\u003c/h2\u003e \u003cp\u003eComposting was conducted using a galvanized bioreactor with intermittent aeration and mixing (IAMBR) over 60 days. The IAMBR had a vertical oval cross-section (H: 50 cm, L: 60 cm, W: 30 cm, freeboard: 15 cm) and was equipped with four sharp-edged agitators (L: 54 cm, W: 6 cm, RPM: 24). Ambient humidity and temperature, as well as chamber temperature, were monitored using an online thermometer and hygrometer kit (HTC-2, China) installed on the IAMB.\u003c/p\u003e \u003cp\u003eAeration was provided by an electromagnetic aeration pump (Aqua AP-9805, China) with a power rating of 6.5 W, a pressure of over 0.025 MPa, and an output flow rate of 5.5 L/min. The pump supplied air to 15 aeration diffusers installed along the bottom sides of the BR. Aeration was automatically controlled using a dial timer (Zhejiang, China) set to operate five times daily for 20 minutes.\u003c/p\u003e \u003cp\u003eTo homogenize the compost pile, mixing was performed daily using the four agitators. The pile's core temperature was measured three times daily before turning the pile using a digital thermometer (TP101, China). Compost turning was performed every two days, alternating between fresh and mature compost.\u003c/p\u003e \u003cp\u003eTwelve treatments (T1\u0026ndash;T12) (Table.2) were tested to evaluate the effects of NMB (0%, 5%, 10%, 15%) and CWM:PUM (1:1, 1:3, 3:1), as detailed in Table\u0026nbsp;2. The carbon-to-nitrogen ratio (C/N) and moisture content were adjusted to 24\u0026ndash;25:1 and 60%, respectively.\u003c/p\u003e \u003cp\u003eSamples (600 grams) were collected on days 0, 5, 10, 15, 20, 30, 40, 50, and 60 using a five-point sampling method to ensure representative samples from the early mesophilic, thermophilic, secondary mesophilic, and maturation phases.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;2. Experimental Setup\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Physicochemical Analysis\u003c/h2\u003e \u003cp\u003eThe collected samples were dried, ground, and stored, then divided into two portions: one portion was used to determine the physicochemical properties, while the other was air-dried, ground, and stored at 4\u0026deg;C for HMs and chemical properties analysis. The pH was measured by mixing the samples in a 1:10 ratio with water and allowing the mixture to sit for 0.5 hours before using a pH meter. Total organic carbon (TOC) was determined using the modified Walkley-Black wet oxidation method [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Total nitrogen was analyzed through the Kjeldahl digestion method using an automatic Kjeldahl apparatus, while nitrate levels were measured following the method described by [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Heavy metals, including copper (Cu), chromium (Cr), and zinc (Zn), were extracted using acid digestion with sulfuric acid and quantified via atomic absorption spectrometry [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Ecological Risk Assessment\u003c/h2\u003e \u003cp\u003eThe ecological risk potential of Cu, Cr, and Zn was calculated following the approach outlined by Negahban et al.(2021) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eData analysis was conducted using SPSS software, applying the Kolmogorov-Smirnov test to assess data normality, parametric and non-parametric tests, and Pearson correlation coefficients, with a significance level of 0.05. Microsoft Excel was used for data organization, Grapher for graphing, and Design-Expert software for response surface methodology analysis at three levels (-1, 0, +\u0026thinsp;1) for CWM/PUM (factor A) and NMB (factor B).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Change in pH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, the pH of the compost was neutral but increased over time, reaching the alkaline range during the curing stage (Fig. 1and 2.a) (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). Treatments with NMB and a higher proportion of CWM exhibited relatively higher pH values compared to T1\u0026ndash;T5 (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). While the initial pH was not significantly affected by the amount of NMB and the CWM/PUM ratio (\u003cstrong\u003eP\u003c/strong\u003e \u0026gt; 0.05), the final compost showed a greater influence of NMB (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05) (Table.3). Among the treatments, T2 exhibited the smallest pH changes at the beginning and the end of the process, while T6 and T7 showed the largest variations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3. ANOVA results by response surface methodology \u0026nbsp;in ANBC-containing treatment\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Change in Temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe overall temperature trends in all treatments followed a similar pattern. Initially, the compost pile temperature ranged between 20\u0026ndash;40\u0026deg;C. It then entered the thermophilic phase, reaching 40\u0026ndash;60\u0026deg;C, before cooling down as mesophilic bacteria resumed activity, bringing the pile temperature closer to ambient levels (Fig. 2. and3). The retention time at high temperatures increased with higher NMB levels and lower CWM/PUM ratios (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). The cumulative temperatures (\u0026deg;C) recorded for treatments T1\u0026ndash;T12 were 897, 858, 836, 981, 926, 898, 1007, 999, 959, 1056, 1025, and 972, respectively. The thermophilic phase lasted 14, 16, and 16 days for piles containing 75% PUM combined with 5%, 10%, and 15% NMB, respectively. The shortest thermophilic phase duration (9 days) was observed in T3, while the longest (16 days) occurred in T10 and T7 (Fig.3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Fate of Carbon\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn the first day of composting, TOC concentrations ranged from 40% to 42.32% (Fig.4a). As the decomposition process progressed, TOC levels decreased significantly (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). NMB had a notable effect on TOC reduction (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05), while the CWM/PUM ratio did not significantly influence TOC reduction (\u003cstrong\u003eP\u003c/strong\u003e\u0026gt; 0.05). However, higher CWM content was associated with smaller TOC reductions (Fig. 2.c and 4a). TOC levels in the control group differed significantly from those in the other treatments (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). During the stability phase, TOC reduction efficiencies were highest in T10, T11, and T12 at 39%, 38.51%, and 35.01%, respectively, and slightly lower in T7, T8, and T9 at 43.39%, 42.39%, and 41.84%, respectively. Treatments containing 15% NMB exhibited a lower rate of TOC reduction compared to those with 5% or 10% biochar (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). The piles with 15% NMB also had the highest initial organic carbon content.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Fate of Nitrogen Compounds\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1. NH₄⁺-N\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn all treatments, the concentration of NH₄⁺-N gradually increased from 0.059\u0026ndash;0.66% on day 1 to 0.1\u0026ndash;0.18% on day 15 (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). The maximum increases in NH₄⁺-N concentration were observed in treatments T3\u0026ndash;T5, with increases of 83.33%, 80.32%, and 69.49%, respectively, which were significantly lower than those in treatments T6\u0026ndash;T12 (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05) (fig.4,b).\u003c/p\u003e\n\u003cp\u003eAfter day 15, NH₄⁺-N concentrations decreased rapidly, reaching 0.06\u0026ndash;0.048% by day 60. On day 60, NH₄⁺-N levels in piles containing NMB were significantly lower than in those without NMB (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). Overall, NH₄⁺-N concentrations decreased by 32.3\u0026ndash;22.3% on day 60 compared to day 1, signaling compost maturation. The CWM/PUM ratio did not significantly affect NH₄⁺-N levels (\u003cstrong\u003eP\u003c/strong\u003e \u0026gt; 0.05). However, with increasing CWM at a constant volume, the rise in TOC during the thermophilic phase and the decrease by day 60 were less pronounced (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2. NH₄⁺-N / NO₃⁻-N\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NH₄⁺-N / NO₃⁻-N ratio in treatments T1\u0026ndash;T12 ranged between 1.96 and 1.58 (Fig. 2.d and 4b). Over time, the NH₄⁺-N / NO₃⁻-N ratio increased during the first 15 days and then began to decrease, with the most significant decrease observed in piles containing 15% NMB. A significant difference in nitrate levels was detected between treatments with 10% and 15% NMB compared to those with 5% NMB and between treatments with and without NMB (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). A significant negative correlation was observed between the decrease in the NH₄⁺-N / NO₃⁻-N ratio and NMB content (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). However, the CWM/PUM ratios of 1:3, 3:1, and 1:1 showed no significant effect on this ratio (\u003cstrong\u003eP\u003c/strong\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Fate of Heavy Metals (HMs)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the changes in heavy metals were examined under the optimized CWM:PUM ratio of 1:3 and varying NMB levels (0%, 5%, 10%, and 15%) (Fig. 5).\u003c/p\u003e\n\u003cp\u003eAt the start of the process, the Zn concentrations in treatments T1, T4, T7, and T10 were 197.15, 336.32, 287.53, and 244.16 g/kg, respectively. By day 60, these concentrations had decreased to 185.78, 296.14, 197.52, and 129.58 g/kg, respectively. Similarly, Cu concentrations in T1, T4, T7, and T10 were initially 240.25, 127.63, 97.4, and 189.55 g/kg, respectively, and declined to 228.31, 109.36, 76.11, and 131.5 g/kg in the final compost.\u003c/p\u003e\n\u003cp\u003eCr concentrations in the control treatment began at 26.12 g/kg and remained largely unchanged in the mature compost. However, in treatments containing 5%, 10%, and 15% NMB, Cr concentrations decreased to 6.76, 12.15, and 7.05 g/kg, respectively, representing a significant reduction. The removal efficiency rankings varied across treatments: in control: Zn \u0026gt; Cu \u0026gt; Cr, in 5% NMB: Cr \u0026gt; Cu \u0026gt; Zn, and in 10% and 15% NMB: Zn \u0026gt; Cr \u0026gt; Cu.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA positive correlation was observed between pH and HM removal efficiency, with increasing pH enhancing the removal efficiency from the beginning to the end of the process. ANOVA results confirmed that removal efficiency differences between the control and other treatments were statistically significant (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). NMB had a direct and significant positive effect on the removal of Zn, Cu, and Cr (\u003cstrong\u003eP\u003c/strong\u003e \u0026lt; 0.05). HMs exhibited a positive correlation with humification (Fig. 6).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003epH is a key indicator in composting as it affects the survival of microorganisms. With aeration and the improved decomposition of organic matter, microbial activity intensified, reducing the environment's acidity and increasing the pH. Chen et al.(2021) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] reported that under favorable conditions, organic acids are completely decomposed, causing compost to transition from an acidic to a neutral range, aligning with the findings of this study. Biochar was also observed to increase pH due to the availability of mineral nutrients, which is consistent with the findings of Choudhary et al.(2021) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Research suggests that pH values in the range of 5.5\u0026ndash;8.0 are optimal for composting [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], matching the results of this study. Similarly, Awasthi et al.(2020) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] found that using bamboo biochar (2\u0026ndash;10%) in composting sheep manure led to a pH increase, further corroborating these findings. The final pH of the compost in this study complied with international standards [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn general, temperature showed a significant positive correlation with NMB and a significant negative correlation with CWM/PUM. Treatments containing NMB exhibited a relatively higher heating rate and an extended thermophilic phase, indicating improved mineralization and compost maturity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Maintaining temperatures above 55\u0026deg;C for at least 3 days is crucial for eliminating pathogens and parasites in compost [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, all treatments met standard hygiene requirements.\u003c/p\u003e \u003cp\u003eThe findings of Afriliana et al.(2021) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] align with the current study, reporting a maximum composting temperature of 60\u0026deg;C. The increased temperatures with higher NMB levels may be attributed to NMB's high surface area and porosity, which facilitate oxygen transport and improve microbial proliferation conditions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, Abd El-Rahim et al.(2021) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] observed a maximum temperature of 73.5\u0026deg;C, with slower temperature increases. This difference might be due to cooler ambient conditions during autumn and winter.\u003c/p\u003e \u003cp\u003eWang et al.(2021) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported that biochar addition resulted in higher temperatures for a shorter duration, with compost entering the cooling phase more quickly, which contradicts the results of this study. The duration of the thermophilic phase was also influenced by the CWM/PUM ratio. Higher PUM levels enhanced temperature retention and duration in the thermophilic phase, likely due to the nutrient content in PUM, which supports microbial activity and heat production.\u003c/p\u003e \u003cp\u003eTotal organic carbon (TOC) is a critical parameter for evaluating compost quality. Various studies have reported similar trends, indicating an increase in TOC during the early stages of composting when NMB is introduced, despite the lower carbon content of natural biochar compared to other mineral biochar [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For instance, Awasthi et al.(2020) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] observed an increase in TOC from 45.83\u0026ndash;49.97% with the addition of biochar.\u003c/p\u003e \u003cp\u003eDuring composting, some carbon is consumed and released as CO₂, while the remainder contributes to cellular structure formation alongside nitrogen [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. TOC reductions occur due to mineralization and the formation of humic substances. In a study by Biyada et al.(2021) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], TOC followed a similar trend, decreasing from 32.64% at the start of composting to 23.53% in mature compost.\u003c/p\u003e \u003cp\u003eThese findings suggest that treatments with higher biochar content experience reduced carbon losses because biochar's carbon is resistant to decomposition and highly stable. Conversely, higher CWM/PUM ratios resulted in lower TOC reduction efficiency, likely due to decreased microbial activity associated with increased CWM content, which in turn affected carbon breakdown.\u003c/p\u003e \u003cp\u003eThe conversion of organic nitrogen to inorganic nitrogen during composting is a potential mechanism for inhibiting the growth of host bacteria. The large surface area and high adsorption capacity of NMB facilitated nitrogen fixation, resulting in increased uptake of organic nitrogen ions by NMB and reduced nitrogen loss. However, nitrogen reduction trends were similar between biochar and non-biochar treatments, indicating a compensatory effect of biochar addition.\u003c/p\u003e \u003cp\u003eThe increase in NH₄⁺-N observed until day 15, concurrent with rising temperatures, may be attributed to the rapid decomposition of organic nitrogen compounds and relatively weak nitrification activity by nitrifying bacteria. This aligns with the findings of Li et al. (2020) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], who reported a significant increase in NH₄⁺-N in all treatments during the initial stages of composting as temperatures increased. A similar result was observed by Rong et al.(2019) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], who co-composted chicken manure and corn leaves.\u003c/p\u003e \u003cp\u003eThe mechanisms underlying biochar-assisted reductions in NH₄⁺-N emissions may include the high adsorption capacity of NMB. This capacity is attributed to the large specific surface area and internal pore volume of NMB, as well as the presence of surface acidic functional groups, cation exchange sites, and micropores, which collectively limit the release of NH₄⁺-N during composting [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAgyarko-Mintah et al.(2017) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] demonstrated that NH₃ adsorption primarily occurs within the pore spaces of biochar. Additionally, NMB enhances aerobic conditions, supports the proliferation of aerobic nitrifying bacteria, and provides a habitat for microorganisms, further aiding in NH₄⁺-N retention and overall compost efficiency [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring the bio-oxidation stage, the NH₄⁺-N / NO₃⁻-N ratio fluctuates due to competition between ammonification and nitrification [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The decrease in NH₄⁺-N and the simultaneous increase in NO₃⁻-N are attributed to the rapid conversion of NH₄⁺-N to NO₃⁻-N by nitrifying microorganisms [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Proteins, amino acids, and other organic materials are initially used by microorganisms as energy and nitrogen sources. Once these materials are depleted, the NH₄⁺-N formed during microbial deamination becomes unstable, evaporates as ammonia gas, and is partially converted to NO₃⁻-N by ammonia-oxidizing microbes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Since nitrifying bacteria cannot grow at temperatures above 40\u0026deg;C, nitrification primarily occurs during the cooling and compost maturation stages. Bernal et al.(2009) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] reported that an NH₄⁺-N / NO₃⁻-N ratio below 0.16 is an indicator of compost maturity. The National Standards Organization of Iran defines an acceptable NH₄⁺-N / NO₃⁻-N ratio range of 0.5 to 3.0 for grades 1 and 2 compost.\u003c/p\u003e \u003cp\u003eShan et al., (2021) observed a slight reduction in total nitrogen with increasing NMB levels [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, Steiner et al.(2010) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] found comparable nitrogen losses in PUM composting with 5% or without NMB, but in piles with 20% biochar, nitrogen loss was significantly reduced by up to 52%.\u003c/p\u003e \u003cp\u003eThe study of HMs in AMs provides valuable insights into their bioavailability and environmental pollution potential. HMs are non-degradable and can persist in manure and compost, leading to their accumulation with long-term application [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NMB reduces HM bioavailability through adsorption and fixation. By adsorbing nutrients in its micropores, biochar reduces the efficiency of organic pollutant degradation, blocking microbial contact and enhancing HM removal [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother mechanism contributing to the inactivation of HMs is biochar adsorption, which involves physical trapping in micropores and the formation of complexes between Zn, Cu, and Cr and the functional groups on biochar, such as \u0026ndash;OH, \u0026ndash;COOH, and C\u0026ndash;O [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For cations, the primary mechanisms include surface precipitation with anions on NMB, the substitution of exchangeable cations, and the formation of surface complexes with \u0026ndash;OH groups or delocalized π electrons from biochar [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pH changes in compost with biochar influence the complexation behavior of functional groups such as \u0026ndash;OH, \u0026ndash;COOH, and \u0026ndash;NH₂, which play a role in metal adsorption. Electrostatic interactions and cation exchange are identified as the dominant mechanisms of metal adsorption by biochar [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Increasing NMB from 5\u0026ndash;15% significantly enhanced the immobilization of HMs.\u003c/p\u003e \u003cp\u003eA one-sample t-test revealed that the concentrations of Zn, Cr, and Cu in the final compost were significantly lower than the standards set by the US Environmental Protection Agency (EPA) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Humification is a critical process in reducing the bioavailability of HMs during aerobic composting. In this process, metal cations combine with organic acids and polymerize into stable humic substances [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], supporting the findings of this study.\u003c/p\u003e \u003cp\u003eHumic molecules are rich in carboxyl and hydroxyl groups, which form stable complexes with metal cations. During aerobic composting, the mineralization and humification of organic carbon regulate the bioavailability of HMs. Metal cations released through organic carbon mineralization can react with organic acids, forming more stable complexes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Similarly, during dehumidification, metal cations readily combine with organic acids to form stable complexes, as adsorbed inorganic cations reduce competition with metal cations. In summary, biochar enhances the deactivation of Zn, Cu, and Cr primarily through surface adsorption mechanisms, significantly contributing to the reduction of their bioavailability. The contamination factor (CF) for Zn was highest in T4 and lowest in T10. In general, the CF of Cr was much lower than that of Cu and Zn (CF: Cu\u0026thinsp;\u0026gt;\u0026thinsp;Zn\u0026thinsp;\u0026gt;\u0026thinsp;Cr). An examination of the contamination degree (Cdegree) revealed that treatments T4, T7, and T10 exhibited very low contamination (Cdegree\u0026thinsp;\u0026lt;\u0026thinsp;1.5), while the control treatment (T1) showed low contamination (1.5\u0026thinsp;\u0026lt;\u0026thinsp;Cdegree\u0026thinsp;\u0026lt;\u0026thinsp;2) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The ecological risk (ER) and risk index (RI) parameters indicate that the studied heavy metals, both individually and collectively, pose a low risk in compost piles and do not threaten the environment.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eThis study demonstrates the significant role of NMB in optimizing the composting process of AMs, particularly cow and poultry manure. The addition of NMB significantly enhanced the removal efficiencies of heavy metals (Zn, Cu, and Cr), reducing their bioavailability and mitigating associated environmental risks. Treatments containing higher NMB levels (10\u0026ndash;15%) exhibited the most effective pollutant immobilization and nutrient stabilization, while meeting international safety standards, including those set by the US EPA.\u003c/p\u003e \u003cp\u003eThe findings emphasize that NMB improves compost quality by facilitating microbial activity, enhancing adsorption, and stabilizing nutrients through mechanisms such as surface complexation, electrostatic interactions, and pH regulation. Environmental risk assessments confirmed that composting, particularly with NMB, transitions raw manure with medium to high risks into safer compost products with low or risk-free classifications.\u003c/p\u003e \u003cp\u003eThese results highlight the potential of NMB as a sustainable and eco-friendly solution for managing AMs while addressing emerging pollutants. Incorporating NMB into composting systems can contribute to better resource utilization, reduced environmental pollution, and the promotion of circular agricultural practices. Further research is recommended to explore the long-term impacts of NMB-amended compost in real-world applications and its interactions with diverse environmental and soil conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare they have no known competing financial interests or personal relationships.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eKermanshah University of Medical Sciences.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.M: Methodology, software, validation, formal analysis, investigation, resources, data curation, writing\u0026mdash;original draft preparation; A.A: conceptualization, supervisor, project administration; S.A.M: Advisor; M.H: writing\u0026mdash;original draft preparation in English\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors thank the Kermanshah University of Medical Sciences for its financial support (No. 4010775).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQuaik, S. et al. 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Sci.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e, 103452 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mineral Biochar, Fate, Ecological Risk","lastPublishedDoi":"10.21203/rs.3.rs-6743840/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6743840/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnimal manures (AMs) are widely utilized as organic fertilizers but often contain significant levels of emerging pollutants, posing environmental and health risks. This experimental study investigated the impact of 650-million-year natural mineral biochar (NMB) and animal manure (CWM:PUM) on the fate of pollutants in the process of intermittent aeration and mixing composting. 12 treatments were monitored over 60 days to evaluate the fate of pollutants, nutrients, and environmental risks. The cumulative temperatures (\u0026deg;C) recorded for treatments T1\u0026ndash;T12 were 897, 858, 836, 981, 926, 898, 1007, 999, 959, 1056, 1025, and 972, respectively. At the start of the process, the Zn concentrations in treatments T1, T4, T7, and T10 were 197.15, 336.32, 287.53, and 244.16 g/kg, respectively. By day 60, they had decreased to 185.78, 296.14, 197.52, and 129.58 g/kg, respectively. Cu concentration in mature compost was 228.31, 109.36, 76.11, and 131.5 g/kg. The removal efficiency rankings varied across treatments: in control: Zn\u0026thinsp;\u0026gt;\u0026thinsp;Cu\u0026thinsp;\u0026gt;\u0026thinsp;Cr, in 5% NMB: Cr\u0026thinsp;\u0026gt;\u0026thinsp;Cu\u0026thinsp;\u0026gt;\u0026thinsp;Zn, and in 10% and 15% NMB: Zn\u0026thinsp;\u0026gt;\u0026thinsp;Cr\u0026thinsp;\u0026gt;\u0026thinsp;Cu. Zn, Cu, and Cr showed significant reductions due to adsorption, surface complexation, and pH-mediated mechanisms, with final concentrations below the USEPA standards. This study highlights the efficacy of NMB in improving compost quality, reducing pollutant bioavailability, and mitigating environmental risks, underscoring its potential as a sustainable solution for managing animal manure.\u003c/p\u003e","manuscriptTitle":"The fate of pollutants in the co-composting of natural mineral biochar and animal manures in an intermittent aeration and mixing bioreactor (IAMB)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 08:30:37","doi":"10.21203/rs.3.rs-6743840/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-17T10:08:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"143238218707911430540980046530422774498","date":"2025-06-17T04:03:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T10:35:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143435648024274521180262522228076656394","date":"2025-06-11T07:24:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308771690560742397963585006966959703833","date":"2025-06-09T14:04:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284908552349453235491170286353811821351","date":"2025-06-09T05:37:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-09T03:34:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-03T09:52:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-30T03:57:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-30T03:54:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d83ac460-aeca-4070-902a-a09d47846309","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49757441,"name":"Earth and environmental sciences/Ecology"},{"id":49757442,"name":"Earth and environmental sciences/Environmental sciences"},{"id":49757443,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2025-09-29T16:07:16+00:00","versionOfRecord":{"articleIdentity":"rs-6743840","link":"https://doi.org/10.1038/s41598-025-18122-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-26 15:57:21","publishedOnDateReadable":"September 26th, 2025"},"versionCreatedAt":"2025-06-11 08:30:37","video":"","vorDoi":"10.1038/s41598-025-18122-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18122-7","workflowStages":[]},"version":"v1","identity":"rs-6743840","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6743840","identity":"rs-6743840","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}