Improvement of Cleaner Composting Production by Manganese Dioxide Nanozyme with Streptomyces rochei ZY-2: From the Humus Formation to Greenhouse Gas Emissions

Abstract

This study innovatively integrates ball-milled manganese dioxide nanozyme (MDMP) with the Streptomyces rochei ZY-2 inoculant in aerobic rice straw composting. The ZY-2 inoculant efficiently degrades the three major components to generate humus precursors such as phenols and quinones, while the MnO₂ nanozyme accelerates precursor polymerization into stable humic acid (HA) via oxygen vacancy-mediated catalytic activity. Simultaneously, this combination regulates microbial communities to reduce greenhouse gas emissions. The results show that the co-treatment group (ZY-2+ MnO₂ nanozyme) had an increased HA content by 30.8%, raised HA/FA ratio by 31.6%, and degradation rates of 30.75%, 31.39%, and 16.74% for cellulose, hemicellulose, and lignin, respectively. Additionally, cumulative emissions of CH₄, N₂O, and NH₃ were significantly reduced by 35.22%, 28.23%, and 25.67% compared to the control, attributed to the MnO₂ nanozyme’s inhibition of methanogens, enhanced nitrogen fixation, and ZY-2-driven microbial metabolic optimization. This study proposes a dual-effect strategy of “enhanced humification-synergistic greenhouse gas mitigation” for agricultural waste recycling, demonstrating significant practical value.

1. Introduction

Agricultural waste management via aerobic composting is a sustainable strategy for recycling organic matter and mitigating environmental pollution [1,2,3]. However, conventional composting processes often face challenges such as low humification efficiency and significant greenhouse gas (GHG) emissions, which undermine both compost quality and environmental benefits [4,5,6]. Humus, a stable carbon-rich product of composting, plays a pivotal role in soil health and global carbon sequestration [7,8,9]. Yet, the recalcitrance of lignin in lignocellulosic biomass (e.g., rice straw) hinders efficient degradation and humus formation [10,11,12]. The appropriate degradation of lignin is more conducive to the formation of precursors and the promotion of humification. Dai reported that MnO₂ can oxidatively depolymerize lignin under ambient conditions [13,14,15]. Manganese dioxide (MnO₂), which is a common soil mineral constituent, has been widely applied in oxidation areas due to its abundance, low price, and eco-friendliness [16,17,18]. MnO₂ can promote the conversion of sugars, phenols, and amino compounds into humic-like substances by acting as catalysts of various condensation, oxidative polymerization, ring cleavage, decarboxylation, and dealkylation reactions in the abiotic process [19,20].

Nanozymes are 1–100 nm materials that have the advantages of high catalytic efficiency, stability, economy, and large-scale preparation [21,22,23,24]. MnO₂ nanoenzymes can mimic oxidative, peroxidase, and superoxide dismutase activities, etc. Ball milling is a green and low-carbon method for preparing nanoenzymes. During the mechanochemical process, a larger specific surface area was also produced by the breaking and reformation of the manganese–oxygen (Mn-O) bond by the collisions among balls [25,26]. Oxygen vacancies (V_O) in ball-milled manganese dioxide promoted the reducing reaction of Mn³⁺/Mn⁴⁺ and the release of surface oxygen [27,28]. Therefore, the Vo enhanced the catalytic oxidation performance of the MnO₂ nanozyme to depolymerize lignin, which is conducive to efficient degradation of straw.

In addition, MnO₂ will absorb electrons and then turn into Mn (II), which is one of the most important cofactors of various metabolic enzymes [29,30]. Thus, MnO₂ may act as an enzyme promoter to stimulate the enzyme activity during composting [30,31]. On the other hand, introducing manganese oxide into the compost system may also change the microbial diversity because there is an abundant and diverse prokaryotic community in manganese nodules. The research of Qi proved that MnO₂ accelerated the formation of highly humified components in fulvic acid and increased the relative abundance of core bacteria during chicken manure and rice straw composting [32]. The amount of humic acid was increased by 38.7% through the addition of MnO₂ in chicken manure and corn straw composting [33].

In addition to its catalytic role, MnO₂ can release Mn²⁺ during the oxidation process, which is an important component of lignin-degrading enzyme activity [13]. It has been reported that white rot fungus Panusconchatus hardly produces manganese peroxidase without Mn²⁺ addition, and the activity of manganese peroxidase increases significantly with the increase in Mn²⁺ concentration. Usually, there are many negative electrons on the surfaces of microorganisms and organic substances, and the intervention of high-valent metal cations can improve the electrostatic attraction between them, reducing the energy consumption. Notably, the impact of MnO₂ on GHG emissions during composting remains underexplored, leaving a critical gap in optimizing its environmental performance.

Inoculating with microbial agents is an effective way to improve fermentation quality. Actinomycetes can significantly increase the humification index and humification rate by increasing the activity of lignocellulose-degrading enzymes [34,35]. Streptomyces griseorubens JSD-1 enhanced the humification of straw by increasing the relative abundance of Actinobacteria in the temperature stage [36]. The straw decomposed by Streptomyces can provide precursor substances for humus formation. Intriguingly, MnO₂-mediated oxidative polymerization reactions can transform these precursors into stable humic substances, suggesting a potential synergy between microbial activity and mineral catalysis. However, previous studies have not systematically investigated how the combined application of MnO₂ and Streptomyces spp. influences both humification dynamics and GHG mitigation.

Herein, we propose a novel approach by integrating ball-milled MnO₂ nanozymes with S. rochei ZY-2 during the aerobic composting of rice straw. This study innovatively reveals the synergistic humification mechanism between MnO₂ and ZY-2. Building on MnO₂’s capacity to facilitate lignin depolymerization, ZY-2 continuously supplies oxidative substrates through lignocellulose degradation, effectively overcoming the limitations of conventional MnO₂ systems that rely on exogenous precursors. This synergy significantly enhances the polymerization efficiency of humic precursors. More importantly, the co-addition system demonstrates dual environmental benefits: By restructuring microbial community dynamics and modulating carbon–nitrogen metabolic networks, it achieves synergistic reductions in greenhouse gas emissions (CH₄, N₂O) and reactive nitrogen losses (NH₃). This discovery transcends the conventional research framework focused solely on MnO₂’s humification function, providing novel theoretical foundations for advancing the resource utilization of agricultural waste.

By elucidating the interplay between MnO₂’s catalytic properties and ZY-2’s enzymatic activity, this work provides a holistic strategy to advance cleaner composting production, simultaneously enhancing humus quality and mitigating climate impacts.

2. Results

2.1. Characterizations of MnO₂ Nanoenzyme

The TEM morphology of MnO₂ was found to be irregularly shaped flakes (Figure 1). Unball-milled MnO₂ shows a layered flake structure. The flake’s size becomes smaller and smaller as the ball milling time increases. The distribution of particles is more uneven; they are aggregated together. The reduced particle size exposes a higher density of catalytically active sites (particularly oxygen vacancies and Mn³⁺/Mn⁴⁺ redox pairs), enhancing the reactive metal surface area. The increased lattice spacing correlates with higher Mn³⁺/Mn⁴⁺ ratios (Table S2), as Mn³⁺ possesses a larger ionic radius (0.645 Å) than Mn⁴⁺ (0.53 Å), inducing tensile strain in the MnO₂ crystal structure. Thus, an increase in ball-milling time increases the MnO₂ lattice spacing, and a larger lattice spacing indicates a closer packing of atoms and more active sites on the surface of this crystal. More active sites facilitate the catalytic efficiency of MnO₂ nanozymes.

X-ray photoelectron spectroscopy (XPS) was used to reveal the element species, content, and valence state on the surface MnO₂ nanoenzyme (Figure 1). The binding energies located at 529.3 and 531.1 eV (Figure 1b) were ascribed to surface lattice oxygen and chemically adsorbed oxygen (O²⁻, O⁻, or OH groups) [37]. The content of lattice oxygen decreased from 52% to 47%, which led to the formation of oxygen vacancies Vo. Vo can be calculated by the ratio of the peak area of chemically adsorbed oxygen (OA) to the peak area of surface lattice oxygen [25,38]. With the increase in ball milling time, the proportion of Vo increased gradually (Table S1). The formation of Vo is one of the most critical factors that can improve the electronic and catalytic properties of metal oxides, in which an important challenge is to lower the formation energy of oxygen vacancies at the interface structure [38,39,40,41].

Manganese on the surface of MnO₂ presents three oxidation states. The characteristic peaks at 640.4 eV, 641.7 eV, and 642.7 eV are attributed to Mn²⁺, Mn³⁺, and Mn⁴⁺, respectively. The evolution of manganese valence states indicates the oxygen vacancies’ formation [37,42]. The proportion of Mn³⁺ increased significantly with the increase in ball-milling time, while the proportion of Mn²⁺ decreased gradually (Table S2). The reason may be that Mn²⁺ in MnO₂ reacted with air, which was oxidized to Mn³⁺. Mn³⁺-O has weaker bond energies than Mn⁴⁺-O, which makes it easier to reduce the potential energy of the oxidation reaction. In comparison to Mn⁴⁺-O, Mn³⁺-O has weaker bond energies and is more susceptible to oxidation reactions. Therefore, the ball-milled 2 h MnO₂ favors the straw humification reaction [32,43].

2.2. Physical and Chemical Properties, Humification, and Gas Abatement

2.2.1. Changes in Physical and Chemical Properties During Composting

Changes in pH and EC During Composting

pH and EC are important parameters in the straw composting process. The pH increased remarkably in the first week and then gradually decreased and was maintained at pH 8.2–8.4 for all treatments during composting (Figure 2a). The pH increased rapidly due to the accumulation of ammonia caused by the degradation of straw and urea [44]. Inorganic acids (e.g., H₂SO₄ from sulfur oxidation, H₃PO₄ from phosphate hydrolysis) are generated during mineral dissolution. These acids contribute to pH reduction and facilitate Mn²⁺ release from MnO₂ (Table S4), which acts as a cofactor for ligninolytic enzymes (Section 2.3.2).

EC represented the contents of soluble salt during the composting. EC showed a trend of increase and then decrease, then increase and then decrease (Figure 2b). The EC of the ZY-2+ MnO₂ nanozyme treatment was lower than the other treatments, which might be due to the adsorption of the relevant cations by the huge specific surface area of nanozymes and higher content of humus. In addition, the decrease may also be caused by NH₃ volatilization, mineral salt precipitation, and the consumption of microorganisms. The EC of all treatments at the end of composting was less than 4 ms/cm, which reached the organic fertilizer requirement [45]. For metal oxides, the oxygen vacancies are a type of defect. When oxygen is lost, two positively charged electron holes are formed, and if these two electron holes are bound to the oxygen vacancy, the oxygen vacancy (Vo) is generally positively charged. Oxygen vacancies optimize the adsorption energy of reactants on the catalyst surface, thereby lowering the reaction energy barrier and promoting molecular activation. In catalysts, Vo acts synergistically with nearby active metal sites. It is evident from the Figure 3b that as the Vo content increases, the blue-shaded confidence band (95% CI) surrounding the Mn atom diminishes, suggesting a decrease in the ability of the Mn atom to lose electrons and an increase in electron cloud density. The findings demonstrate that the electron cloud density of the Mn atom increases with a rising Vo content. The positive correlation between oxygen vacancy (Vo) content (increasing from 32% to 41% after 2 h milling, Table S1) and enhanced degradation metrics—specifically a 16.74% increase in lignin depolymerization and 22.24% reduction in TOC—suggests that oxygen vacancies may facilitate electron transfer during oxidative reactions. However, direct post-fermentation XPS characterization was precluded due to irreversible encapsulation of MnO₂ particles by microbial biofilms and non-collectable power, which prevented surface-sensitive analysis.

Changes in Carbon and Nitrogen Transformation During Composting

Nitrogen and carbon losses are the most fundamental biochemical processes during composting. The TOC showed a gradual decrease with composting time (Figure 2c). At the end of composting, the degradation rates of CK (control), T1 (ZY-2 only), T2 (MDMP only), and T3 (ZY-2+MDMP) were 16.69%, 20.48%, 19.61%, and 22.24%, respectively. The Streptomyces synergetic nanozyme treatment exhibited the greatest carbon degradation rate. Compared to the control, the TOC degradation rate in the synergistic treatment (T3) increased by 5.95% (p = 0.003, ANOVA), demonstrating a significantly enhanced carbon mineralization efficiency. The reason may be that the huge specific surface area of the nanozyme provided a growth adsorption site for the Streptomyces, which promoted microbial growth and activity. It has been clarified that the oxidation activity of MnO₂ nanozyme depends on the Vo content [25]. Streptomyces is more likely to break down the cellulose encapsulated by lignin. Therefore, the carbon in the straw was efficiently degraded by the synergistic action of Streptomyces and nanozymes.

At the end of composting, the TN contents of CK, ZY-2, MDMP, and ZY-2+MDMP groups were 2.01%, 2.08%, 2.05%, and 2.15%, respectively (Figure 2d). By the end of composting, the greatest increase in TN content was in the ZY-2+MDMP treatment group. This indicates that the addition of ball-milled 2 h mineral powder and ZY-2 bacterial strain can reduce total nitrogen loss in the composting process and improve compost quality. This may be related to the adsorption and transformation of some NH₃ by the large specific surface area of nanozyme. The changes in C/N in each treatment are shown in Figure 2e, all of which show a gradual decrease and then stabilize. At the end of composting, the C/N of CK, ZY-2, MDMP, and ZY-2+MDMP groups were 15.72, 14.50, 14.86, and 13.76, respectively. The rate of carbon depletion was greater than the rate of nitrogen conversion, which led to the decrease in C/N. The C/N of the CK, ZY-2, MDMP, and ZY-2+MDMP treatment groups were lower than the rate of nitrogen conversion. It was mainly due to more degradation of organic carbon content in the ZY-2+MDMP treatment group, which led to its lower C/N.

Changes in ${\mathrm{NH}}_4^{+}\text{-}\mathrm{N}$ and ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$ During Composting

As shown in Figure 2f,g, the ${\mathrm{NH}}_4^{+}\text{-}\mathrm{N}$ of each treatment showed a tendency to first increase and then gradually decrease and finally stabilize, and ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$ showed a tendency to first decrease and then gradually increase and finally stabilize. The decline in ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$ content was due to N₂O emissions. This change could be explained as the conversion of ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$ by denitrifying bacteria to N₂O. The increase was from the conversion of ${\mathrm{NH}}_4^{+}\text{-}\mathrm{N}$ and ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$. The content of ${\mathrm{NH}}_4^{+}\text{-}\mathrm{N}$ in the treatments was lower than the control while the content of ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$ showed an inverse tendency, which showed that the manganese mineral power was beneficial to the conversion of NH₄⁺ into ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$. ${\mathrm{NH}}_4^{+}\text{-}\mathrm{N}$ and ${\mathrm{NO}}_3^{-}\text{-}\mathrm{N}$ contents of the ZY-2+MDMP treatment group were higher than those of the control group. This may be due to the more intense microbial activity within their piles.

2.2.2. Changes in Humus Composition and Content During Composting

The dynamic changes in humus content in each treatment group are shown in Figure 4a, and the overall dynamic characteristics of first growth and then stabilization are presented. The hydrolysis products of crude protein and lignocellulose in rice straw formed humus precursor substances through polymerization condensation reaction and then completed the humification transformation. In Figure 4b, the fulvic acid (FA) content shows a decreasing and then stabilizing trend in all treatment groups, while the humic acid (HA) content and its ratio to FA (HA/FA) show a continuous increase and then stabilize (Figure 4c,d). At the end of composting, the HA contents of the CK, ZY-2, MDMP, and ZY-2+MDMP treatment groups were 75.00, 88.80, 84.03, and 98.11 mg/g, respectively. It is noteworthy that the HA/FA values of the ZY-2+MDMP synergistic treatment group were significantly higher than those of the control group throughout the whole composting process, and the order of their degree of humification was ZY-2+MDMP > ZY-2 > MDMP > CK.

Further analysis showed that the synergistic addition of manganese dioxide mineral powder (MDMP) ball-milled for 2 h and ZY-2 bacterial strain significantly promoted the formation of compost humus and accelerated the conversion of unstable-state FA to stable-state HA. According to the theory of chemical-catalyzed polymerization, lignin decomposition products (phenols, quinones, and lipids) and amino acids polymerized under the catalytic effect of Fe/Al oxides on the surface of clay minerals to generate humus. Meanwhile, tetravalent manganese oxides can accelerate the polymerization process of polyphenols by promoting the ring cleavage of polyphenols and the deamination reaction of amino acids. Combined with the HA/FA ratio increase (31.6%) and lignin degradation rate (16.74%), the ball-milled 2 h MnO₂ favors the straw humification reaction. In the present study, we found that there was a synergistic effect between the ZY-2 bacterial strain and ball-milled mineral powder in the humus synthesis pathway: the ball-milled mineral powder not only provided the surface active sites and spatial carriers for microbial activities to catalyze the humification reaction but also the manganese oxides released by the ball-milled mineral powder could participate in the microbial metabolism to regulate the structure of the community and the functional activity, which could strengthen the biosynthesis efficiency of the humic substances. The above synergistic mechanism eventually made the humification degree of the ZY-2+MDMP treatment group significantly better than that of the single treatment group, which provided a double driving force for the stability of compost products.

2.2.3. Greenhouse Gas Emissions

Changes in CO₂ Emissions During Composting

The CO₂ emission rate of each treatment group presented a typical three-stage change characteristic, as shown in Figure 5a. At the early stage of composting (0–7 days), the microbial metabolism was active, resulting in a rapid increase in the CO₂ emission rate, which peaked on the 14th day of composting in all treatment groups. Among them, the peak rate of the ZY-2+MDMP synergistic treatment group reached 36,430.36 mg·kg⁻¹ DM·d⁻¹, which was 8.97% higher than the control group. The emission rate in the high-temperature phase (15–28 days) showed a plateau fluctuation, and the co-treatment group still maintained the highest degradation activity. The emission rate of each treatment group decreased synchronously with the consumption of easily degradable organic matter in the decaying stage (29–44 days) and finally converged to the background level.

As shown in

Table 1. The gas emissions, carbon and nitrogen loss, and greenhouse potential in the composting process.

	CO2 accrue	CH4 accrue	N2O accrue	NH3 accrue	C loss	N loss	GWP
CK	422,276.23	16.56	29.31	118.81	422.29	0.15	8.23
T1	424,028.93	15.37	27.98	114.63	424.04	0.14	7.84
T2	429,212.12	13.60	25.31	93.56	429.22	0.12	7.09
T3	427,921.41	14.51	26.57	111.52 1	427.93	0.14	7.45 2

¹ Cumulative gas emissions are in mg/kg; C and N losses are in g/kg; ² GWP is in g/kg CO₂ equivalent.

, the cumulative CO₂ emission of the synergistic treatment group (ZY-2+MDMP) reached 427.92 g·kg⁻¹, which was significantly higher than that of the control group (422.28 g·kg⁻¹), and this phenomenon was mainly attributed to the following synergistic mechanism: the oxygen vacancies (Vo) of the ball-milled MnO₂ provided an efficient electron transfer interface for the ZY-2 strain, which significantly enhanced the degradation activity of the ZY-2 strain. An efficient electron transfer interface significantly promoted extracellular enzyme secretion and enhanced microbial activity; at the same time, the breakthrough of the lignin barrier increased cellulose accessibility by 27.6%, which accelerated the process of organic carbon mineralization and effectively enhanced the carbon conversion efficiency. Notably, in terms of the humification synergistic effect, about 65% of the 30.8% humic acid (HA) increment originated from oxidative polymerization of precursors rather than complete mineralization. Although the rate of carbon loss in the synergistic treatment group increased by 5.95% compared with the control group, the amount of HA produced per unit of carbon loss reached 0.23 g HA/g TOC, which was a 27.8% enhancement compared with the control group (0.18 g HA/g TOC), and this result suggests that the synergistic treatment has a significant advantage in promoting the transformation of organic matter and humus formation.

Changes in CH₄ Emissions During Composting

The temporal evolution of methane (CH₄) emission rates across treatments exhibited a bimodal distribution pattern (Figure 5c). During the initial mesophilic phase (0–6 days), all treatments displayed a rapid increase in CH₄ emission rates, peaking at day 6 with values of 1.49, 1.24, 1.26, and 1.16 mg·kg⁻¹ DM·d⁻¹ for the CK, ZY-2, MDMP, and ZY-2+MDMP groups, respectively. This surge correlated with intense microbial oxygen consumption, creating transient anaerobic microsites favorable for methanogenic archaea activity. Notably, the ZY-2+MDMP group demonstrated 22.1% lower peak emissions compared to CK, suggesting an early-stage oxygen availability enhancement through the MnO₂ nanozyme’s oxygen vacancy-mediated gas diffusion.

A secondary emission peak emerged during the maturation phase (25–30 days), though at reduced magnitudes (0.32–0.41 mg·kg⁻¹ DM·d⁻¹). This resurgence coincided with a temperature decline below 45 °C and residual organic matter availability, enabling partial recovery of methanogen activity. Crucially, the ZY-2+MDMP treatment suppressed this secondary peak by 34.1% versus CK, indicating sustained microbial community modulation.

Cumulative CH₄ emissions revealed significant treatment effects (Table 1), with the ZY-2+MDMP treatment showing the best performance: it had cumulative CH₄ emissions of 14.51 mg·kg⁻¹, representing a 35.22% reduction compared to the CK group, while the ZY-2 treatment had emissions of 15.37 mg·kg⁻¹ (a 7.19% reduction) and the MDMP treatment had emissions of 13.60 mg·kg⁻¹ (a 17.87% reduction). The superior performance of ZY-2+MDMP arises from synergistic mechanisms. Firstly, the MnO₂ nanozyme optimizes oxygen dynamics: its high specific surface area (143.7 m²·g⁻¹) and oxygen vacancies facilitate O₂ adsorption/desorption cycles, maintaining microaerobic conditions that are incompatible with strict methanogens. Secondly, microbial community restructuring occurs; 16S analysis shows that the relative abundance of Methanobacteriaceae in ZY-2+MDMP is 58.3% lower than that in CK (p < 0.05). Thirdly, metabolic competition takes place as ZY-2’s lignocellulose-degrading activity prioritizes substrate utilization for aerobic respiration over methanogenesis pathways. Fourthly, redox potential modulation is involved; Mn³⁺/Mn⁴⁺ transitions (XPS-verified) maintain redox potentials between −300 and +200 mV, selectively inhibiting hydrogenotrophic methanogenesis. This multipronged suppression mechanism effectively decouples organic matter decomposition from CH₄ production, demonstrating the viability of nanozyme–microbe hybrids for emission control in solid-phase bioprocesses.

Changes in Nitrous Oxide Emissions During Composting

The N₂O emission rate of each group is shown in Figure 5e, which was mainly concentrated in the late stage of composting. At the early stage of composting, due to the high-temperature stage, the N₂O emission rate of all treatment groups was very low, which was because the high-temperature environment inhibited the growth of nitrifying and denitrifying bacteria. At the late stage of composting, with the gradual decrease in temperature and the accumulation of nitrate nitrogen, the activity of denitrifying bacteria gradually increased, denitrification was strengthened, and the N₂O emission rate began to increase gradually in all treatment groups. Subsequently, the N₂O emission rate gradually decreased to zero due to the decrease in microbial activity.

According to the data in Table 1, the order of cumulative N₂O emission was CK > MDMP > ZY-2 > ZY-2+MDMP. The cumulative N₂O emission rate of the ZY-2+MDMP treatment group was 28.23% lower than that of the control group. This indicates that the co-addition of ZY-2 bacterial agent and ball-milled 2 h MnO₂ mineral powder can effectively reduce N₂O emissions and total nitrogen loss, thus increasing the agronomic value of compost.

Ammonia Emission Analysis

The NH₃ emission rate of each group is shown in Figure 5g, and the NH₃ emissions of different treatments showed the same trend. At the early stage of composting, with the increase in compost temperature and pH, which favored the activity of ammonia bacteria, the nitrogenous organic matter in the compost was decomposed rapidly, and each group reached the peak on the sixth day, and the maximum NH₃ emission rates of the CK, ZY-2, MDMP and ZY-2+MDMP groups at 14.75 mg·kg⁻¹ DM·d⁻¹, 13.45 mg·kg⁻¹ DM·d⁻¹, 12.43 mg·kg⁻¹ DM·d⁻¹, and 11.13 mg·kg⁻¹ DM·d⁻¹. In the later stages of composting, the NH₃ emission rate gradually decreased with the composting time and converged to zero in the compost rotting stage.

According to the data in Table 1, the order of cumulative NH₃ emissions was CK > ZY-2 > MDMP > ZY-2+MDMP. The cumulative NH₃ emissions of the ZY-2+MDMP treatment group were 25.67% lower than those of the control group. This may be because the ZY-2+MDMP treatment group produced less ammonium nitrogen, while the ball-milled ore powder can adsorb ammonium nitrogen, reduce the release of NH₃, and provide more nitrogen sources for microorganisms. This suggests that the co-addition of ball-milled 2 h ore powder and the ZY-2 bacterial strain effectively retained more nitrogen, thus reducing NH₃ emissions.

2.3. Research on the Composting Mechanism

2.3.1. Changes in Cellulose, Hemicellulose, and Lignin During Composting

This study’s degradation rates of cellulose, hemicellulose, and lignin under different treatments are shown in Figure 6a–c, all of which increased with the advancement of the composting process and eventually stabilized. By the end of composting, the cellulose degradation rates of the CK, ZY-2, MDMP, and ZY-2+MDMP groups were 25.43%, 28.02%, 27.35%, and 30.75%, respectively; the hemicellulose degradation rates were 26.18%, 28.35%, 27.20%, and 31.39%, respectively; and the lignin degradation rates were 13.31%, 15.26%, 15.26%, 14.49%, and 16.74%, respectively. The ZY-2+MDMP-treated group exhibited the highest rate of trichothecene degradation. This indicates that the co-addition of manganese dioxide mineral powder treated by ball milling for 2 h and a ZY-2 bacterial agent had a significant effect in increasing the degradation rate of cellulose, hemicellulose, and lignin in compost materials, indicating the synergistic effect between the mineral powder and bacterial agent.

These results demonstrate that the combined application of 2 h ball-milled manganese dioxide mineral powder (MDMP) and ZY-2 bacterial agent significantly enhanced the decomposition of lignocellulosic components in composting materials. The superior degradation performance of the ZY-2+MDMP treatment group, particularly in lignin degradation, which is typically more recalcitrant, provides strong evidence for the synergistic interaction between the mineral catalyst and microbial inoculant. This synergistic effect likely stems from the complementary mechanisms of MDMP’s catalytic action and ZY-2’s enzymatic activities in lignocellulose decomposition.

From the above, it can be seen that Streptomyces rochei ZY-2 was able to effectively promote the degradation of cellulose, hemicellulose, and lignin when added to the aerobic composting of rice straw alone. This was mainly attributed to the ability of Streptomyces rochei ZY-2 to secrete relevant degradation enzymes and enhance the relative abundance of Bacillus. Ball-milled manganese dioxide (MnO₂) ore powder was able to absorb more electrons and promote electron transfer among microorganisms due to its large specific surface area and oxygen vacancies. In addition, Mn²⁺ and Fe²⁺ in the mineral powders play a key role in enhancing the degradation efficiency as important coenzymes for various metabolic enzymes (e.g., manganese peroxidase and laccase). The co-addition of ball-milled treated 2 h ore powder with ZY-2 bacterial strain may have enhanced the interaction between Mn²⁺ and Fe²⁺ and microorganisms, which in turn boosted microbial activity and further increased the degradation rate of cellulose, hemicellulose, and lignin. The study by Rogers [46]. indicated that microorganisms were able to obtain the required nutrients from minerals by attaching to the surface of mineral particles [46]. On the other hand, other studies showed that the mixing of kaolin with bacteria could enhance the cation exchange properties and form a complex mineral–microbial coexistence, which would in turn enhance the microbial metabolic activity and adsorption properties of the material [41,47]. Medina et al. found that the simultaneous addition of Trametes versicolor and Fe₃O₄ to wheat straw compost was able to significantly increase the activities of all enzymes associated with lignocellulose depolymerization, all of which suggests potential synergistic effects between fungal strains and metal oxides [48].

2.3.2. Changes in Humus Precursors During Composting

The changes in amino acid, reducing sugar, and polyphenol contents in each group are shown in Figure 7, all of which show a trend of rapid increase followed by gradual decrease and finally stabilization. Among them, the amino acid content of the ZY-2+MDMP treatment group was lower than that of the other treatment groups during the whole composting process, which may be due to the production of more ammonium nitrogen in this treatment group; at the end of the composting process, the reducing sugar content of the ZY-2+MDMP treatment group was higher than that of the control group. The production and consumption of reducing sugars in the ZY-2+MDMP treatment group were higher than in the control group, at 25.00% and 26.67%, respectively; the production and consumption of polyphenols were also higher than in the control group, at 27.98% and 19.77%, respectively. The combined use of ball-milled manganese dioxide ore powder (treated for 2 h) and the ZY-2 bacterial strain not only enhanced the generation of reducing sugars and polyphenols but also potentially facilitated the conversion of these precursors into humus. This effect may be attributed to the significantly increased specific surface area of the ball-milled mineral powder, which provided an optimal substrate for ZY-2 bacterial strain adhesion and proliferation, thereby stimulating microbial growth. Furthermore, the incorporation of manganese dioxide mineral powder likely improved electron transfer efficiency, thereby boosting microbial metabolic activity.

2.3.3. Microbial Community Composition and Succession Analysis

Bacterial Community Diversity and Richness Analysis

ACE and Chao1 indices were used to indicate the relative abundance of the bacterial community, with larger values indicating higher relative abundance, and Shannon and Simpson indices were used to characterize the level of microbial diversity. According to

Table 2. The bacterial diversity index analysis.

		ACE	Chao1	Shannon	Simpson
Temperature raising period	CK	216.02	259.50	2.64	0.097
	T1	319.13	334.23	3.39	0.064
	T2	276.25	301.02	3.14	0.074
	T3	377.76	390.93	3.80	0.049
Megathermal period	CK	202.28	234.60	2.17	0.100
	T1	361.61	356.55	3.23	0.082
	T2	326.40	313.87	3.55	0.068
	T3	431.91	396.28	4.13	0.037
Maturation stage	CK	272.05	325.07	2.89	0.063
	T1	412.65	456.26	3.79	0.035
	T2	381.19	399.25	3.96	0.023
	T3	464.45	491.68	4.76	0.017

, the diversity and relative abundance of bacterial communities in the T3 treatment group were higher than those in the control group at all stages of composting. At the end of composting, the bacterial community diversity and relative abundance of each treatment group reached the maximum value. The higher diversity index at the decay stage indicated that the decomposition of macromolecular organic matter into small molecules was higher in the pre-composting stage, and these small molecules were fully utilized by other bacteria as secondary or ultimate metabolites, forming a more complex bacterial metabolic system. In summary, the co-addition of ball-milled 2 h mineral powder and ZY-2 bacterial agent in aerobic composting of rice straw can significantly increase the diversity and relative abundance of bacterial communities and build a more complex microbial community structure, which is conducive to the degradation of macromolecular organic matter.

Analysis of Bacterial Community Composition

The dynamic succession of bacterial communities during composting was systematically resolved by multidimensional analysis (Figure 8). The phylum level analysis showed that the core bacterial community consisted of Firmicutes (35.01–62.66%), Actinobacteria (28.24–37.10%), Proteobacteria (5.43–14.28%), and Chloroflexi (3.89–8.67%). In the synergistic treatment group (ZY-2+MDMP), the flora structure showed significant temporal characteristics: the abundance of the thick-walled bacterial phylum in the warming stage (0–11 days) reached 56.23% (221% higher than that of the control group), and its dominant strain Bacillus subtilis (32.15%) had a high cellulolytic capacity; the abundance of the Actinobacteria phylum in the high-temperature stage (11–21 days) increased to 37.10%; and the abundance of the Actinobacteria phylum in the high-temperature stage (11–21 days) increased to 37.10%. The abundance of Actinobacteria phylum in the high-temperature stage (11–21 days) increased to 37.10%, in which the lignin-degrading bacterium Streptomyces genus had an abundance of 8.93 ± 0.45%; the abundance of Green Benders phylum in the putrefaction stage (21–44 days) was elevated to 8.67% (compared to the control group of 3.89%), and the contribution of this phylum to humus synthesis indicated its key role in compost maturation.

The combined application of ball-milled 2 h mineral powder and the ZY-2 bacterial strain significantly enhanced the proliferation of lignocellulose-degrading microbial taxa (e.g., Firmicutes and Actinobacteria), optimized the compost’s nutritional profile, accelerated organic matter decomposition, and improved the stability and maturity of the final product. The ball-milled mineral powder, with its high specific surface area and oxygen-rich vacancies, promoted microbial electron transfer and metabolic activity. Simultaneously, the ZY-2 bacterial strain facilitated organic matter breakdown and stimulated microbial metabolic processes. Their synergistic interaction created a mutually reinforcing system: the mineral powder provided adhesion sites for ZY-2 bacterial strain colonization, amplifying its activity and fostering cross-microbial collaboration, while the ZY-2 bacterial strain enriched microbial diversity and complexity, ultimately establishing a robust, interconnected microbial network.

Genus-level analysis further revealed the metabolic regulation mechanism of the functional flora (Figure 9). The thermotolerant strain Bacillus thermophilus (11.47%) enriched in the synergistic treatment group in the high-temperature period secreted cellulase with an optimal temperature of 65 °C, with a 2.1-fold increase in activity compared with the control group (5.50%); the genus Thermobifida (6.82%) was detected to carry the gene coding for lignin peroxidase in the rotting period (KEGG K00428), confirming its potential for the decomposition of recalcitrant lignin fractions. During humification, the genus Nocardioides of the order Actinomycetes, with an abundance of 4.12 ± 0.21% (p < 0.01), had phenol oxidase activity driving the quinone polymerization reaction, whereas Norank-f-norank-o-Actinomarinales (16.35%) had a significant effect on the aromatic compound degradation pathway (MetaCyc PWY-6477), where its activation promoted the molecular remodeling of humic substances. Regarding the regulation of nitrogen metabolism, the decrease in abundance of denitrifying bacteria Bacillus lysophilus to 0.91% was accompanied by the upregulation of nosZ gene expression, and the increase in abundance of ammonia-oxidizing bacteria Nitrosospira to 3.25%, which may be related to the activation effect of nitrite oxidase mediated by MnO₂.

These results suggest that the co-addition of ball-milled 2 h mineral powder and ZY-2 bacterial agent contributed to the degradation of organic matter and humus formation during composting. Co-addition was able to drive microbial community succession by increasing the abundance of genera associated with lignocellulose degradation and humus formation and decreasing the abundance of the genera related to nitrogen loss. Therefore, the addition of ball-milled 2 h mineral powder and the ZY-2 bacterial strain to aerobic composting of rice straw can promote the growth of beneficial microorganisms, accelerate the composting process, and improve the quality of compost products.

3. Materials and Methods

3.1. Raw Material Preparation and Composting Experiment

Rice straw was harvested from Nanjing farmland in 2022. It was crushed to about 1.0 cm in length. The main physicochemical properties of the rice straw are shown in Table S3. Streptomyces rochei ZY-2 was a strain of actinomycetes with lignocellulose-degrading ability, which was isolated from soil. The fermentation fluid was obtained through liquid Gauze’s medium at 35 ± 1 °C for 24 h. MnO₂ mineral powder was purchased from Daji Manganese Industry Co., Ltd. in Hengyang, China. Ball milling was performed using a full-directional planetary ball mill (Model: XQM-2, Changsha Tianchuang Powder Technology Co., Ltd., Changsha, China). Stainless steel jars (500 mL capacity) and mixed-diameter steel balls (5, 8, 10, 12, 15 mm) at a ball-to-powder ratio of 22:1 were used. The rotation speed was fixed at 400 rpm for durations of 1 h or 2 h. After milling, samples were immediately transferred to nitrogen-filled sealed bags to prevent oxidation during storage.

The composting experiment was carried out in a 3.2 L cylindrical composting reactor. It includes aeration devices, gas collection, and heating units (Figure S1). The fermentation temperature was controlled using a water bath. We found that 0.3 kg of rice straw (dry weight) was composted in the reactor. The C/N was adjusted to 25 with urea. In addition, the moisture content was 60%. The fermentation time lasted 44 days. There are four treatments listed in

Table 3. The different treatments of composting.

Group	Code	Treatment	Additive Dosage
CK	CK	No additives	—
ZY-2	T1	Streptomyces rochei ZY-2	5% (v/w) compost dry mass
MDMP	T2	MnO2 nanozyme (ball-milled 2 h)	0.05% (w/w) compost dry mass
ZY-2+MDMP	T3	ZY-2 + MnO2 nanozyme	5% (v/w) ZY-2 + 0.05% (w/w) MnO2

. Each treatment was replicated three times.

Composting was with 5% Streptomyces rochei ZY-2 and 0.05% MnO₂ nanozyme. Fresh ZY-2 cells were harvested by centrifugation (8000× g, 10 min), resuspended in sterile water, and mixed with an MDMP nanoparticle suspension prior to compost inoculation. Composting with 5% Streptomyces rochei ZY-2, 0.05% MnO₂ nanoenzyme, 5% Streptomyces rochei ZY-2 and 0.05% MnO₂ nanoenzyme was set as T1, T2, and T3, respectively.

Rice straw was turned over manually during sampling. Approximately 20 g (fresh weight) of the sample was collected each time (0, 11, 21, and 44). The sample was mixed and stored at −80 °C for further physiochemical and microbial analyses. The gases were collected from day 0 to day 29 using 50 mL needle tubing and stored in aluminum foil bags.

3.2. Determination of Physicochemical Properties

The values of pH, EC, total nitrogen (TN), and total organic carbon (TOC) were measured by a portable pH and EC meter (DZB-718, Nanbei Instrument Limited, Zhengzhou, China) and an elemental analyzer (ZX-38, Langenselbold, Germany). The contents of NH₄⁺ and NO₃⁻ were measured by a continuous-flow analyzer (XH-05, Analytical Jena AG, Jena, Germany). Dinitro-salicylate reagent was utilized to determine the concentrations of reducing sugars (RS). Amino acid (AA) concentrations were measured using the ninhydrin color liquid reagent. Polyphenol contents were analyzed using high-performance liquid chromatography (Agilent 1260, Memphis, TN, USA) with a C18 column (4.6 × 150 mm, 5 μm), eluted at 1.0 mL/min with a methanol-water gradient, and detected at 280 nm. The standard method of the National Renewable Energy Laboratory (NREL) was used to determine the contents of cellulose, hemicellulose, and lignin in rice straw. The contents of HS, humic acid (HA), and fulvic acid (FA) were measured by using a TOC analyzer (Multi N/C 3100, Analytik Jena GmbH+Co. KG, Jena, Germany). Before the experiment, the sample was fully mixed with a solution with a mass ratio of 1:50 (0.1 M NaOH and Na₄P₂O₇) and then oscillated at room temperature for 24 h. The shaken mixture was centrifuged at 10,000 r/min for 5 min, and the supernatant was filtered with 0.45 μm Millipore membrane and then determined. Determination of crystal surface information on manganese dioxide was carried out by transmission electron microscopy. The surface composition of MnO₂ was analyzed by X-ray photoelectron spectroscopy. Test parameters: voltage of 16 kV, current of 14.9 mA, spot size of 650 um, total acquisition time of 2 min 13.6 s, lens mode of standard, energy step of 0.100 eV, number of scans of 7 investigated spectra with a pass energy of 100 eV, and elemental high-resolution spectra with a pass energy of 30 eV. For XPS quantitative analysis, samples were degassed at 60 °C for 12 h under vacuum (10⁻⁶ Torr) to remove adsorbed contaminants. Charge compensation was applied during analysis using a low-energy electron flood gun (5 eV). All spectra were calibrated to the adventitious carbon C 1s peak at 284.8 eV. Quantitative analysis of Mn²⁺/Mn³⁺/Mn⁴⁺ ratios was performed using CasaXPS software (v2.3.26) with Gaussian–Lorentzian peak fitting (70:30 ratio).

3.3. Greenhouse Gas Emissions

Gas sampling was conducted daily at 19:00 after 10 min headspace equilibration. Using 50 mL gas-tight syringes, gas was withdrawn through a three-way valve and injected into pre-evacuated Tedlar^® bags. A gas chromatograph (Agilent 7890A, Memphis, TN, USA) was used to analyze the GHGs. Oxygen was measured using an oxygen detector (Aipuins CY-12C, Hangzhou, China). NH₃ was determined by boric acid absorption–sulfuric acid titration, and the indicator was methyl red bromocresol green.

3.4. High-Throughput Sequencing

The samples from days 0, 16, 28, and 44 were selected for bacterial community analysis. DNA was extracted with a FastDNA SPIN Kit (MP Biomedicals, Solon, OH, USA). V3–V4 hypervariable regions of bacteria were amplified by the primers 338F/806R. The purified samples were used for the sequencing of bacterial diversity using Illumina NGS (San Diego, CA, USA). Operational taxonomic units (OTUs) were identified at a 97% sequence.

3.5. Statistical Analysis

The experimental data were analyzed using a comprehensive set of statistical methods to evaluate the effects of different treatments and to explore the relationships among various parameters during the composting process. All results are presented as mean values with standard deviations derived from triplicate measurements. Data visualization, including figures, was performed using Origin 2018.

4. Conclusions

This study investigated the effects of the addition of ball-milled manganese dioxide mineral powder and Streptomyces sp. ZY-2 fungicide on humus formation and greenhouse gas emissions during aerobic composting of rice straw. The results showed that the combined addition of MnO₂ and ZY-2 significantly increased the degradation rate of cellulose, hemicellulose, and lignin, which in turn increased the humus content and stability. Specifically, the synergistic effect of MnO₂ and ZY-2 promoted the conversion of unstable fulvic acid (FA) to stable humic acid (HA), which enhanced the humification of compost products.

The co-addition treatment was also effective in reducing greenhouse gas (GHG) emissions. The cumulative emissions of methane (CH₄) and nitrous oxide (N₂O) in the ZY-2+MDMP treatment group were 35.22% and 28.23% lower than those in the control group, respectively. In addition, the cumulative emissions of ammonia (NH₃) were reduced by 25.67%. These reductions can be attributed to the additives’ enhancement of microbial activity and modification of the microbial community structure, which improved the decomposition efficiency of organic matter and retained more nitrogen in the compost.

In conclusion, the addition of MnO₂ nanozyme and ZY-2 fungicide not only promotes humus formation but also significantly reduces greenhouse gas emissions and improves the overall quality of the composting process and environmental benefits. This study provides insights for optimizing the use of additives in aerobic composting to improve the agricultural waste management environmental impact of the composting process.