Abiotic Nitrogen Mineralization of Peptone by γ-MnO₂: Effects of Dissolved Oxygen and pH

Abstract

Current research predominantly focuses on the microbial-driven processes of soil organic nitrogen mineralization, often overlooking the significant contributions of abiotic pathways mediated by reactive minerals. While manganese oxides are known to promote the abiotic mineralization of organic nitrogen, the influence of key environmental factors on this process remains poorly understood. This study established a simulated system to investigate the effects of dissolved oxygen and pH on the γ-MnO₂-mediated abiotic nitrogen mineralization of peptone. The results showed that under an air atmosphere at pH 5.0–8.0, the rate of nitrogen mineralization from peptone catalyzed by γ-MnO₂ initially increased and then subsequently decreased. Dissolved oxygen was identified as a major electron acceptor in the peptone nitrogen mineralization process, playing a critical role in its rate and extent. Direct oxidation by Mn (IV) and Mn (III) within γ-MnO₂ accounted for 72.5% of the peptone nitrogen mineralization, and reactive oxygen species generated on the mineral surface accounted for 27.5% through a catalytic oxidation mechanism. This study provides a preliminary analysis of how key environmental factors influence the abiotic mineralization of protein-derived organic nitrogen, which is expected to deepen the understanding of soil organic nitrogen mineralization mechanisms, enrich the knowledge of nitrogen cycling in agricultural ecosystems, and provide a theoretical basis for efficient nitrogen management in farmland.

1. Introduction

Nitrogen is a fundamental nutrient for crop growth and a primary limiting factor for agricultural productivity [1]. Many natural ecosystems, including agricultural systems, often exhibit a phenomenon termed a “missing nitrogen sink,” where the amount of bioavailable nitrogen stored in the soil exceeds the quantities predicted to be released by conventional mechanisms [2]. This disparity strongly suggests the existence of other nitrogen mineralization pathways that have yet to be fully elucidated. Current research on soil organic nitrogen mineralization has predominantly focused on microbial activities [3], largely overlooking the significant contributions from abiotic processes [2,4]. This oversight persists despite evidence from simulation experiments and molecular dynamics calculations demonstrating that common soil minerals, such as iron oxides, manganese oxides, and clay minerals, can abiotically hydrolyze or oxidatively cleave proteins into fragmented residues [5,6,7]. Among these reactive minerals, ubiquitous manganese oxides are of particular importance. However, while the ability of manganese oxides to mediate abiotic nitrogen mineralization is established [8], the specific impact of key environmental drivers, including pH and dissolved oxygen, on the activity of particular phases like γ-MnO₂ remains poorly understood.

Our previous work has established that γ-MnO₂ can facilitate the abiotic mineralization of organic nitrogen (e.g., soybean peptone) to inorganic nitrogen (primarily ammonium nitrogen (NH₄⁺-N)) under oxic conditions at pH 7.0. In this process, besides direct oxidation by Mn (IV) within the γ-MnO₂ mineral structure, reactive intermediates such as Mn (III) and reactive oxygen species (ROS), including O₂^•−, ^•OH, and H₂O₂, act as key oxidants for peptone mineralization [8]. Since the rate of organic nitrogen mineralization is a crucial parameter governing ecosystem productivity, elucidating its controlling factors is essential for improving soil nutrient management [9,10]. Key environmental factors are known to significantly regulate the rate of soil nitrogen mineralization [11]. For example, in the context of global warming, a high temperature sensitivity of nitrogen mineralization is projected to disproportionately increase soil nitrogen availability in colder, high-latitude regions compared to lower latitudes [12]. Similarly, seasonal changes in temperature in California soils exponentially accelerate nitrogen mineralization rates, with significant implications for cropping systems [13]. A meta-analysis of terrestrial ecosystem nitrogen mineralization studies revealed that net nitrogen mineralization rates increase with mean annual precipitation but decrease as soil pH rises [14]. Furthermore, research on the effects of drought on global natural ecosystems found that while drought increased mineral nitrogen pools (+31%), it concurrently reduced nitrogen mineralization rates (–5.7%) [15]. These findings collectively demonstrate that environmental factors exert critical control over soil organic nitrogen mineralization. Therefore, while the ability of manganese oxides to facilitate abiotic nitrogen mineralization is recognized, a significant research gap persists regarding how key environmental variables, such as pH and oxygen availability, regulate the rates and mechanisms of this process. Furthermore, the relative contributions of different oxidative pathways, specifically direct oxidation by the mineral versus catalytic oxidation involving reactive oxygen species, have not been quantified. This lack of mechanistic understanding hinders the integration of abiotic processes into soil nutrient models and limits our broader understanding of the nitrogen cycle.

The complex interactions between minerals and organic matter, including adsorption, catalysis, and oxidation, are themselves profoundly influenced by environmental factors [16]. pH and dissolved oxygen, as two of the most important environmental variables, markedly affect the transformation of soil organic matter. For instance, the oxidation rate of organic matter by manganese oxides increases with decreasing pH due to a higher redox potential at lower acidity [16]. One study found that birnessite-mediated degradation of prion protein exhibited a strong pH dependence [17], while another showed that the adsorption and reaction rates of Gb1 protein on birnessite were highly sensitive to pH due to charge effects [6]. Oxygen also plays a pivotal role in certain mechanisms of organic matter degradation [18]. Recent research suggests that oxygen may inhibit the adsorption of fulvic acid onto birnessite, thereby suppressing electron transfer between Mn (IV) and the fulvic acid and impeding the regeneration of reactive sites on the MnO₂ surface [19]. Consequently, under prolonged reaction conditions, oxygen might actually slow down the mineralization of fulvic acid mediated by MnO₂ [19]. ROS are exceptionally important intermediate oxidants that drive the oxidation of organic matter—ranging from partial oxidation to low-molecular-weight organic acids to complete mineralization—with reaction conditions such as pH playing a decisive role [16,20,21]. Therefore, it is of great significance to assess the effects of key environmental factors like pH and dissolved oxygen on the abiotic nitrogen mineralization mediated by γ-MnO₂.

This study established a simulated mineralization system using proteinaceous nitrogen as a representative of the soil’s largest single organic nitrogen pool [22,23]. The primary objectives are to (i) investigate the impacts of varying pH and oxygen levels on the γ-MnO₂-mediated abiotic mineralization of peptone and (ii) quantify the contributions of different oxidants to this process. The findings of this research will provide initial clarification on the environmental controls governing abiotic nitrogen mineralization and will contribute to a better understanding of the global nitrogen geochemical cycle.

2. Materials and Methods

2.1. Reagents and Materials

Soy protein isolate (SPI, BR), soy peptone (SP, BR), thimerosal (AR, 98%), manganese dioxide (AR, ≥97.5%), sodium hydroxide (AR, ≥96.0%), and hydrochloric acid (AR, 36.0–38.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. High-purity argon gas (99.999%) was obtained from Wuhan Runhua Hui Gas Cylinder Inspection Co., Ltd., Wuhan, China. All solutions were prepared using sterile deionized water (18.2 MΩ·cm) filtered through 0.22 μm membranes. The crystalline phase of MnO₂ was previously confirmed as γ-MnO₂ through atomic/molecular-scale characterization [8].

2.2. Experimental Design

Given that proteins constitute the largest single organic nitrogen pool in soils [22,23], plant-derived SPI and SP were selected as representative soil organic nitrogen sources. Batch experiments were designed to simulate mineralization in systems containing γ-MnO₂, a ubiquitous reactive mineral, and SPI/SP (at initial concentrations of 0.0 or 0.25 g L⁻¹) at 25 °C with magnetic stirring for 60 h. It is important to note that the selected reactant concentrations and experimental duration were chosen to represent a simplified model system. This approach was designed to elucidate the fundamental mechanisms and the influence of key environmental factors on abiotic nitrogen mineralization under controlled laboratory conditions. For aerobic experiments, which were conducted under an air atmosphere, SPI/SP and γ-MnO₂ (at initial concentrations of 0.0 or 1.0 g L⁻¹) were added to sterile 500 mL reaction vessels. To inhibit microbial growth, 0.20 g L⁻¹ of thimerosal was added to each vessel [7]. The systems were then filled to a final volume of 300 mL with sterile deionized water. To eliminate any potential photochemical effects, the vessels were wrapped in aluminum foil. The pH range of 5.0–8.0 was selected to represent the conditions of diverse and widespread agricultural soils. This range encompasses, for instance, the acidic soils (pH 5.0–6.5) of major rice-producing regions in Southern China, as well as the neutral to alkaline soils (pH 7.0–8.0) of primary wheat and maize belts in Northern China. During the experiments, the pH was continuously maintained at 5.0, 6.0, 7.0, or 8.0 using 0.05–0.1 mol L⁻¹ NaOH or HCl solutions. At predetermined time intervals, 20 mL of the suspension was withdrawn and filtered through 0.22 μm membranes to collect both the liquid and solid phases. The collected solids were subjected to an extraction procedure with 2 mol L⁻¹ KCl via a 1 h oscillation in a sealed thermostatic shaker, followed by centrifugation, washing, and collection of the resulting filtrate for analysis.

Oxygen, as one of the most common oxidants and electron acceptors in nature, plays a vital role in numerous biochemical reactions in the soil. To investigate the effects of dissolved oxygen, an anoxic experimental system was established. First, sterile deionized water was boiled for 15 min. To prevent the re-dissolution of oxygen during the cooling process, it was purged with high-purity argon gas for 1 h while cooling. A continuous flow of high-purity argon was maintained in the anoxic system throughout the experiments. The dissolved oxygen concentration in this system was confirmed to be below 0.1 mg L⁻¹ using a JPB-607A dissolved oxygen meter (Leici, Shanghai, China). To ensure the accuracy and reliability of the experimental data, all treatments were performed in triplicate.

2.3. Analysis Methods

The concentration of NH₄⁺-N in the samples was quantified using the indophenol blue colorimetric method at a wavelength of 625 nm with a UV-visible spectrophotometer (UV-6000, Shanghai Metash Instruments Co., Ltd., Shanghai, China) [24]. The specific procedure involved: diluting the sample to 10 mL in a 25 mL colorimetric tube; adding 5 mL of phenol solution (10.0 g L⁻¹ containing 0.1 g L⁻¹ sodium nitroprusside) and 5 mL of alkaline sodium hypochlorite solution (10.0 g L⁻¹ NaOH, 7.06 g L⁻¹ Na₂HPO₄∙7H₂O, 31.8 g L⁻¹ Na₃PO₄∙12H₂O, 0.525 g L⁻¹ NaClO); allowing the reaction to proceed for 1 h at room temperature; adding 1 mL of masking agent (200.0 g L⁻¹ potassium sodium tartrate, 50.0 g L⁻¹ EDTA-2Na, 2.0 g L⁻¹ NaOH); diluting to a final volume of 25 mL with deionized water; and finally, measuring the absorbance at 625 nm. Nitrate nitrogen (NO₃⁻-N) concentration was determined using a dual-wavelength UV method [25]. Absorbance was measured at both 220 nm and 275 nm to correct for the interference of dissolved organic matter, with the final absorbance calculated as A = A₂₂₀ − 2A₂₇₅. The concentrations of Mn (II) in the samples were analyzed using an atomic absorption spectrometer (PinAAcle 900, PerkinElmer, Waltham, MA, USA).

To understand the influence of pH on the γ-MnO₂-mediated abiotic mineralization rate of peptone, the zeta potentials of both peptone and γ-MnO₂ were measured across different pH values using a Nano ZS90 Zetasizer (Malvern Panalytical, Malvern, Worcestershire, UK). Prior to measurement, samples were diluted, filtered through a 0.45 μm microporous membrane, sonicated for 5 min to degas, and the pH was adjusted using 0.1 mol L⁻¹ HCl or NaOH solutions. After a 15 min equilibration period with magnetic stirring, measurements were performed following a 30 min instrument warm-up. Each sample measurement was repeated in triplicate. The analytical methods employed are standard procedures with high precision, and the relative standard deviations for all measurements were consistently below 5%, ensuring the reliability of the data.

3. Results

3.1. Interaction Between γ-MnO₂ and SPI

Figure 1 presents the concentrations of dissolved NH₄⁺-N, NO₃⁻-N, and Mn (II) in both the single system containing SPI (0.25 g L⁻¹) and the mixed system containing SPI (0.25 g L⁻¹) and γ-MnO₂ (1.0 g L⁻¹) under air atmosphere at pH 7.0. In the single SPI system, the concentrations of NH₄⁺-N and NO₃⁻-N showed no significant changes over the reaction period. Similarly, in the mixed SPI-γ-MnO₂ system, the NO₃⁻-N concentration remained largely unchanged after 60 h of reaction compared to its initial level. In contrast, the NH₄⁺-N concentration increased markedly by 380.89%. Concurrently, a significant increase of 269.42% was observed in the Mn (II) concentration in the solution. These results collectively demonstrate that γ-MnO₂ can directly mediate the mineralization of soybean protein isolate, leading to the production of inorganic nitrogen species.

3.2. Effect of Dissolved Oxygen on Abiotic Nitrogen Mineralization

SP, the enzymatic hydrolysate of soybean protein, was selected for subsequent experiments as a representative of complex, high-molecular-weight organic nitrogen compounds. Its structure closely resembles the proteinaceous components ubiquitous in natural environments, such as soil organic matter and biological residues. The aforementioned results confirm that γ-MnO₂ promotes the abiotic nitrogen mineralization of such complex organic nitrogen. However, considering that the large molecular weight and relatively limited solubility of SPI could lead to slower reaction kinetics, SP was uniformly employed in the subsequent experimental systems. SP is rich in small peptides and amino acids, possessing superior solubility and significantly enhanced reactivity, which greatly improves experimental efficiency and data throughput [26].

Previous studies have indicated that dissolved oxygen can influence the oxidative degradation of organic matter mediated by manganese oxides [19]. To elucidate the role of dissolved oxygen in the abiotic mineralization of peptone within the mixed SP-γ-MnO₂ system, the interaction was investigated under air and argon atmospheres at pH 7.0 (Figure 2). Dissolved NO₃⁻-N concentrations showed no significant difference across the various reaction systems under either air or argon atmospheres (Figure 2b). In the single SP system, the concentration of dissolved NH₄⁺-N remained largely unchanged under an argon atmosphere, showing no significant difference compared to that under an air atmosphere. However, in the mixed SP-γ-MnO₂ system, the generation rate of dissolved NH₄⁺-N was markedly lower in the argon atmosphere compared to the air atmosphere. After 60 h of reaction, the dissolved NH₄⁺-N concentration in the argon system was 1.98 mg L⁻¹, a reduction of 12.0% compared to the concentration in the air atmosphere (2.25 mg L⁻¹) (Figure 2a). Concurrently, the release rate of dissolved Mn (II) was also significantly lower in the argon atmosphere (Figure 2c). After 60 h, the Mn (II) concentration in the mixed system under argon was reduced by 56.73% relative to that under air. These results therefore demonstrate that oxygen actively participates in the γ-MnO₂-mediated abiotic mineralization of SP.

3.3. Effect of pH on Abiotic Nitrogen Mineralization

Figure 3 presents the concentrations of dissolved NO₃⁻-N in the single peptone (SP) system, the single γ-MnO₂ system, and the mixed SP–γ-MnO₂ system under different pH conditions in an air atmosphere. In both the single SP and mixed SP–γ-MnO₂ systems, dissolved NO₃⁻-N concentrations showed no significant change over the reaction period at pH 5.0, 6.0, or 8.0, which aligns with previous findings at pH 7.0. Therefore, it can be concluded that within the pH range of 5.0–8.0, the inorganic nitrogen mineralized during the reaction of γ-MnO₂ and peptone is not predominantly NO₃⁻-N.

The concentrations of Mn (II) in the single SP system, the single γ-MnO₂ system, and the mixed SP–γ-MnO₂ system under different pH conditions in an air atmosphere were further measured (Figure 4). At pH 5.0, the Mn (II) concentrations in both the single γ-MnO₂ system and the mixed SP–γ-MnO₂ system exhibited a gradual increasing trend. After 60 h of reaction, the concentrations were 20.47 mg L⁻¹ and 23.79 mg L⁻¹, respectively. In contrast, at pH 6.0, the Mn (II) concentrations in both systems initially increased and then stabilized, a trend also seen at pH 7.0 [8]. After 60 h, their equilibrium concentrations were 1.67 mg L⁻¹ and 8.28 mg L⁻¹, respectively. At pH 8.0, the trend of Mn (II) concentration change over time in the single γ-MnO₂ and mixed SP–γ-MnO₂ systems was consistent with that at pH 5.0. After 60 h, their equilibrium concentrations were 7.59 mg L⁻¹ and 8.57 mg L⁻¹, respectively. A comparison of the mixed and single γ-MnO₂ systems after 60 h revealed that the Mn (II) concentrations in the mixed systems were 16.2% (pH 5.0), 395.8% (pH 6.0), 204.9% (pH 7.0), and 12.9% (pH 8.0) higher than in their corresponding single-system counterparts. These results collectively suggest that the abiotic peptone mineralization rate, as reflected by the Mn (II) generation rate, exhibited a trend of initially increasing with rising pH from 5.0 to 7.0 and then decreasing at pH 8.0.

Figure 5 displays the concentrations of dissolved NH₄⁺-N in the single SP system, the single γ-MnO₂ system, and the mixed SP–γ-MnO₂ system under different pH conditions in an air atmosphere. Within the pH range of 5.0–8.0, the dissolved NH₄⁺-N concentrations in the single SP system showed no significant changes as the reaction proceeded. Conversely, in the mixed SP–γ-MnO₂ system, the dissolved NH₄⁺-N concentrations consistently showed a phenomenon of an initial slow increase followed by a plateau. After 60 h of reaction at pH 5.0, 6.0, and 8.0, the dissolved NH₄⁺-N concentrations in the mixed system reached 1.67, 2.39, and 2.43 mg L⁻¹, respectively. These values represent increases of 24.6%, 53.2%, and 53.8% compared to the corresponding concentrations in the single SP system (1.34, 1.56, and 1.58 mg L⁻¹). Previously, at pH 7.0, the NH₄⁺-N concentration in the mixed system increased by 82.4% relative to the single SP system (Figure 2) [8]. Therefore, integrating the trends observed for dissolved NH₄⁺-N concentrations in the mixed system across different pH conditions in this study, it can be further concluded that the abiotic mineralization rate of peptone initially increased and subsequently decreased with rising pH.

To further understand the reaction process between γ-MnO₂ and peptone under different pH conditions, the concentrations of NH₄⁺-N adsorbed onto the mineral surfaces were measured at different time intervals (Figure 6). As the reaction proceeded, the concentrations of adsorbed NH₄⁺-N gradually decreased under all tested pH conditions. Notably, the maximum adsorbed NH₄⁺-N concentrations were substantially lower than the maximum dissolved NH₄⁺-N concentrations released during the reaction. These results indicate that within the pH range of 5.0–8.0, the inorganic nitrogen mineralized during the reaction between γ-MnO₂ and peptone existed predominantly as NH₄⁺-N released into the solution, with only a minor fraction adsorbed onto the mineral surface.

To explore the mechanism underlying the pH-dependent influence on the γ-MnO₂-mediated abiotic mineralization rate of peptone, the zeta potentials of both peptone and γ-MnO₂ were determined across different pH values (Figure 7). The zeta potential of γ-MnO₂ remained negative across the entire pH range of 1.0–9.0, signifying a negatively charged surface under these conditions. In contrast, the zeta potential of peptone reached zero at approximately pH 1.9. This indicates that the peptone surface carried a positive charge below pH 1.9 and a negative charge above pH 1.9.

4. Discussion

4.1. Effect of pH

Environmental conditions, such as pH, the presence of coexisting cations and anions, and dissolved organic matter, can significantly influence the rate at which manganese oxides oxidize organic compounds [27]. The oxidation rate of most organic compounds by manganese oxides typically decreases as pH increases. The observed increase in organic oxidation rates under acidic conditions may be attributed to several factors: (1) more favorable adsorption of many organic compounds onto manganese oxide surfaces at lower pH due to changes in the surface speciation of both the organic reductants and the manganese oxides [28]; (2) the dependence of the redox potential of manganese oxides on pH, with studies indicating, for example, that the redox potential of MnO₂ can decrease linearly from 0.99 V at pH 4.0 to 0.76 V at pH 8.0 [29]; (3) the fact that electron transfer is more facile at lower pH because the reduction of MnO₂ requires protons [30,31]; and (4) the decrease in the surface charge density of MnO₂ as pH increases [27,32]. However, some studies have shown that the oxidation rate of certain organic compounds by manganese oxides does not always decrease with increasing pH. For instance, the oxidation rate of pentachlorophenol was found to be highest at pH 5.0. At higher pH values, the oxidation rate of pentachlorophenol decreased due to a lower affinity of the anion for the mineral surface, while at lower pH values, the rate was also reduced compared to pH 5.0 because pentachlorophenol had to compete with H⁺ for surface sites [33]. Another study found that the oxidation rate of 17β-estradiol by manganese oxides accelerated with increasing pH in the range of 5.1–7.8. This was attributed to the fact that the oxidation process of 17β-estradiol releases protons, and the redox reaction with the manganese oxides is primarily controlled by the oxidation of 17β-estradiol rather than the reduction of the manganese oxide [34]. These findings suggest that under certain reaction conditions, the speciation of the organic reductant can strongly influence the reaction rate of its interaction with manganese oxides [27].

In this study, zeta potential measurements indicated that across a pH range of 5.0–8.0, the surfaces of both peptone and γ-MnO₂ carried a net negative charge (Figure 7), suggesting the presence of electrostatic repulsion. However, the observed reaction demonstrates that this repulsion was overcome by other dominant interaction forces. The pH-dependent mineralization rate is therefore more likely governed by pH-induced changes within the peptone constituents themselves, possibly through alterations in the protonation state of functional groups in the peptide and amino acid structures [35]. Under near-neutral pH conditions, the conformation of peptide and amino acid molecules may favor the exposure of amide (-CONH-), carboxyl (-COO⁻), and amino (-NH₃⁺) groups, which can subsequently form hydrogen bonds with the surface hydroxyls (Mn-OH) on γ-MnO₂ [36,37]. These high-affinity, short-range interactions are sufficiently strong to overcome long-range electrostatic repulsion, allowing the peptone molecules to effectively adsorb onto the mineral surface and creating the necessary conditions for subsequent electron transfer and oxidative mineralization. Conversely, at lower or higher pH values, the formation of these hydrogen bonds may be inhibited, thereby reducing the overall mineralization rate.

4.2. Contribution of Reactive Intermediates to γ-MnO₂-Mediated Peptone Nitrogen Mineralization

Manganese dioxide possesses a high redox potential, with the standard electrode potential of the Mn(IV)O₂/Mn²⁺ couple (1.230 V) being close to that of O₂/H₂O (1.229 V). However, due to its strong adsorption capacity and high surface activity, manganese dioxide is more likely to participate in environmental redox reactions than oxygen, serving as a powerful solid-phase oxidant in soils [27,38]. Beyond the direct oxidation mediated by Mn (IV), the presence of Mn (III) and ROS endows manganese dioxide with excellent catalytic oxidation activity [39,40]. In particular, oxygen vacancies on the mineral surface facilitate the exposure of Mn (III), which provides more active sites for peptone transformation [41]. At the same time, these vacancies play a key role in the generation of ROS on the manganese dioxide surface. Mn (III) can also donate an electron to be captured by O₂, forming O₂^•−. Based on previous research, which has confirmed the presence of reactive intermediates such as Mn (III) and ROS in the mixed system, the process of γ-MnO₂ promoting peptone nitrogen mineralization can be attributed to direct oxidation by Mn (IV)/Mn (III) and catalytic oxidation by ROS; however, the contribution of each oxidation pathway to this process remains unclear [8].

The experimental design, which utilized both air (oxic) and argon (anoxic) atmospheres, enabled the quantification of these distinct pathways. Under anoxic conditions, the ROS-mediated pathway is suppressed due to the absence of O₂, which serves as the ultimate electron acceptor for ROS generation. Consequently, the nitrogen mineralization observed under argon can be primarily attributed to direct oxidation by Mn (IV)/Mn (III). The data presented in Figure 2 show that after 60 h of reaction, the dissolved NH₄⁺-N concentrations in the peptone-only and mixed systems under the argon atmosphere were 1.24 and 1.98 mg L⁻¹, respectively. In contrast, under the air atmosphere, these concentrations were 1.23 and 2.25 mg L⁻¹. These results quantitatively demonstrate that at pH 7.0, direct oxidation by Mn (IV) and Mn (III) is the dominant pathway, contributing 72.5% to the abiotic nitrogen mineralization of peptone, while the ROS-mediated catalytic oxidation pathway accounts for the remaining 27.5% (Figure 8). Notably, this attribution, derived from the difference between oxic and anoxic conditions, represents a preliminary estimation of the overall contribution from oxygen-dependent pathways. While this method effectively quantifies the integral role of dissolved oxygen, it does not resolve the specific contributions of individual reactive oxygen species. Future research employing more direct methods, such as the use of specific ROS scavengers (e.g., tert-butanol for ^•OH or superoxide dismutase for O₂^•−), is required to definitively dissect the roles of these reactive intermediates in the mineralization process.

4.3. Biogeochemical Significance and Implications for the Nitrogen Cycle

Extrapolating laboratory rates to complex soil environments requires caution. The rates observed in this controlled system are intended to elucidate the fundamental mechanism and controlling factors (pH, O₂) rather than to serve as a direct 1:1 prediction for in situ field rates. In natural soils, factors such as mineral-organic ratios, competitive adsorption from diverse organic matter, and mineral coatings will influence the apparent kinetics. The true significance of this pathway, as discussed, is its role as a ‘slow but persistent’ abiotic N source, which becomes substantial when integrated over biogeochemical timescales in open, dynamic soil systems. Current biogeochemical models predominantly attribute soil nitrogen mineralization to microbial activities [3]. The findings of this study, however, provide quantitative evidence that abiotic processes mediated by common soil minerals like γ-MnO₂ can be a substantial contributor to the bioavailable nitrogen pool. This offers a tangible, mineral-driven mechanism that may help explain the long-standing “missing nitrogen sink” phenomenon observed in many ecosystems [2].

The absolute inorganic nitrogen concentrations released in the 60 h, closed-system experiments may appear modest. The environmental significance of this abiotic pathway, however, should not be judged solely by its short-term yield, but rather by its potential for persistence and longevity within open, dynamic soil systems. In a true soil environment, newly formed NH₄⁺-N is subject to continuous consumption by plant roots and microbes. This constant product removal would perpetually drive the forward mineralization reaction, allowing the process to function as a persistent source of inorganic N.

Therefore, the core significance of this study is the mechanistic identification of a long-overlooked, widespread abiotic pathway. The two main reactants—manganese oxides and proteinaceous material—are ubiquitous at the Earth’s surface. Proteinaceous materials constitute the largest single pool of organic nitrogen in soils [22,23], and manganese oxides are among the most common and reactive minerals [19]. While the reactant concentrations (1.0 g L⁻¹ γ-MnO₂ and 0.25 g L⁻¹ peptone) were selected to elucidate the fundamental mechanism under controlled conditions, they remain environmentally relevant. The mineral concentration employed (approx. 0.1% w/w) is at the lower end of the range commonly reported for agricultural soils, and the peptone concentration can be considered representative of transient, nutrient “hot-spots,” such as the immediate vicinity of decomposing roots or microbial necromass.

Unlike microbial activity, which is often episodic and constrained by specific environmental conditions, this chemical process can function as a “slow but persistent” background source of bioavailable nitrogen. When this abiotic flux is integrated over the vast expanse of global topsoils and over relevant biogeochemical timescales (e.g., months to years), its cumulative contribution to the N budget—a contribution currently unaccounted for in most models—has the potential to be substantial.

The present study provides an essential mechanistic framework for expanding the structure of future soil nitrogen cycling models, which currently lack modules for such abiotic processes. This work provides the initial basis and identifies key controlling factors (pH, O₂) for the development and integration of an “abiotic N mineralization submodule.” Incorporating such processes is a critical step toward closing the gap between model predictions and field observations of soil N availability. These findings suggest that the role of mineral-organic matter interactions in soil fertility and nutrient dynamics is more critical than previously appreciated.

5. Conclusions

This study provides a comprehensive investigation into the effects of key environmental factors, namely pH and dissolved oxygen, on the abiotic nitrogen mineralization of peptone mediated by γ-MnO₂. The key findings demonstrate that this abiotic process is highly sensitive to environmental conditions. Within the typical soil pH range of 5.0–8.0, the nitrogen mineralization rate exhibited a non-linear trend, initially increasing and then subsequently decreasing with rising pH. Dissolved oxygen was identified as a critical electron acceptor that significantly influences the mineralization rate. Differentiation of the reaction pathways quantitatively established that under neutral pH and aerobic conditions, direct oxidation by Mn (IV)/Mn (III) is the dominant pathway, contributing 72.5% to the process, while catalytic oxidation mediated by ROS contributes a significant 27.5%.

The findings of this research carry broader implications for soil science and geochemistry. By elucidating the mechanisms and environmental controls of a key abiotic pathway, this work underscores that mineral-mediated processes are a non-negligible component of the soil nitrogen cycle, which is often viewed through a predominantly biological lens. This provides a potential explanation for the “missing nitrogen sink” observed in many ecosystems and suggests that global nitrogen cycle models would be improved by incorporating such abiotic transformations. This work suggests that agricultural practices such as liming (for pH regulation), tillage (which impacts soil oxygenation), or the application of Mn-based amendments could have unappreciated effects on this abiotic N pathway, offering new leverage points for precise soil fertility management.