Fungi play a crucial role in sustaining microbial networks and accelerating organic matter mineralization and humification during thermophilic phase of composting.

Fungi play an important role in the mineralization and humification of refractory organic matter such as lignocellulose during composting. However, limited research on the ecological role of fungi in composting system hindered the development of efficient microbial agents. In this study, six groups of lab-scale composting experiments were conducted to reveal the role of fungal community in composting ecosystems by comparing them with bacterial community. The findings showed that the thermophilic phase was crucial for organic matter degradation and humic acid formation. The Richness index of the fungal community peaked at 1165 during this phase. PCoA analysis revealed a robust thermal stability in the fungal community. Despite temperature fluctuations, the community structure, predominantly governed by Pichia and Candida, remained largely unaltered. The stability of fungal community and the complexity of ecological networks were 1.26 times and 5.15 times higher than those observed in bacterial community, respectively. Fungi-bacteria interdomain interaction markedly enhanced network complexity, contributing to maintain microbial ecological functions. The core fungal species belonging to the family Saccharomycetaceae drove interdomain interaction during thermophilic phase. This study demonstrated the key role of fungi in the composting system, which would provide theoretical guidance for the development of high efficiency composting agents to strengthen the mineralization and humification of organic matter.

Characteristics of humification, functional enzymes and bacterial community metabolism during manganese dioxide-added composting of municipal sludge.

The aim of this study was to assess effects of MnO2 addition (CK-0%, T1-2% and T2-5%) on humification and bacterial community during municipal sludge (MS) composting. The results suggested that MnO2 addition inhibited the growth of Nitrospira but stimulated Nonomuraea, Actinomadura, Streptomyces and Thermopolyspora, facilitating the lignocellulose degradation and humification with the increase in organic matter degradation by 13.8%-19.2% and humic acid content by 10.9%-20.6%. Compared to CK, the abundances of exoglucanase (EC:3.2.1.91), endo-1,4-beta-xylanase (EC:3.2.1.136) and endomannanase (EC:3.2.1.78) increased by 88-99, 52-66 and 4-15 folds, respectively. However, 5%-MnO2 induced the enrichment of Mizugakiibacter that harms the environment of agricultural production. The addition of 2%-MnO2 was recommended for MS composting. Furthermore, metabolic function analysis indicated that MnO2 addition altered amino acid and carbohydrate metabolism, especially enhancing propanoate metabolism and butanoate metabolism but inhibiting citrate cycle. Structural equation modeling revealed that Nonomuraea and Actinomadura were the main drivers for lignocellulose degradation. This study provided theoretical guidance in regulating humification via MnO2 for MS composting.

The bioaugmentation effect of microbial inoculants on humic acid formation during co-composting of bagasse and cow manure.

The effective degradation of recalcitrant lignocellulose has emerged as a bottleneck for the humification of compost, and strategies are required to improve the efficiency of bagasse composting. Bioaugmentation is a promising method for promoting compost maturation and improving the quality of final compost. In this study, the bioaugmentation effects of microbial inoculants on humic acid (HA) formation during lignocellulosic composting were explored. In the inoculated group, the maximum temperature was increased to 72.5 °C, and the phenol-protein condensation and Maillard humification pathways were enhanced, thus increasing the HA content by 43.85%. After inoculation, the intensity of the microbial community interactions increased, particularly for fungi (1.4-fold). Macrogenomic analysis revealed that inoculation enriched thermophilic bacteria and lignocellulose-degrading fungi and increased the activity of carbohydrate-active enzymes and related metabolic functions, which effectively disrupted the recalcitrant structure of lignocellulose to achieve a high humification degree. Spearman correlation analysis indicated that Stappia of the Proteobacteria phylum, Ilumatobacter of the Actinomycetes phylum, and eleven genera of Ascomycota were the main HA producers. This study provides new ideas for bagasse treatment and recycling and realizing the comprehensive use of resources.

Effect of microbial inoculum on composting efficiency in the composting process of spent mushroom substrate and chicken manure.

This work aimed to investigate the microbial mechanisms for the improvement of composting efficiency driven by the compound microbial inoculum (MI) (Bacillus subtilis SL-44, Enterobacter hormaechei Rs-189 and Trichoderma reesei) during co-composting of spent mushroom substrate (SMS) and chicken manure (CM). The treatments used in the study were as follows: 1) MI (inoculation with microbial inoculum), 2) CI (inoculation with commercial microbial inoculum), and 3) CK (without inoculation). The results demonstrated that MI increased the seed germination index (GI) by 25.11%, and contents of humus, humic acid (HA) and available phosphorus (AP) were correspondingly promoted by 12.47%, 25.93% and 37.16%, respectively. The inoculation of MI increased the temperature of the thermophilic stage by 3-7 °C and achieved a cellulose degradation rate of 52.87%. 16S rRNA gene analysis indicated that Actinobacteria (11.73-61.61%), Firmicutes (9.46-65.07%), Proteobacteria (2.86-32.17%) and Chloroflexi (0.51-10.92%) were the four major phyla during the inoculation composting. Bacterial metabolic functional analysis revealed that pathways involved in amino acid and glycan biosynthesis and metabolism were boosted in the thermophilic phase. There was a positive correlation between bacterial communities and temperature, humification and phosphorus fractions. The average dry weight, fresh weight and seedling root length in the seedling substrates adding MI compost were 1.13, 1.23 and 1.06 times higher than those of the CK, respectively. This study revealed that biological inoculation could improve the composting quality and efficiency, potentially benefiting the resource utilization of agricultural waste resources.

Biochar improves the humification process during pig manure composting: Insights into roles of the bacterial community and metabolic functions.

Biochar could promote humification in composting, nevertheless, its mechanism has not been fully explored from the perspective of the overall bacterial community and its metabolism. This study investigated the effects of bamboo charcoal (BC) and wheat straw biochar (WSB) on the humic acid (HA) and fulvic acid (FA) contents during pig manure composting. The results showed that BC enhanced humification more than WSB, and significantly increased the HA content and HA/FA ratio. The bacterial community structure under BC differed from those under the other treatments, and BC increased the abundance of bacteria associated with the transformation of organic matter compared with the other treatments. Furthermore, biochar enhanced the metabolism of carbohydrates and amino acids in the thermophilic and cooling phases, especially BC. Through Mantel tests and network analysis, we found that HA was mainly related to carbon source metabolism and the bacterial community, and BC might change the interaction patterns among carbohydrates, amino acid metabolism, Bacillales, Clostridiales, and Lactobacillales with HA and FA to improve the humification process during composting. These results are important for understanding the mechanisms associated with the effects of biochar on humification during composting.

Co-composting of dewatered sludge and wheat straw with newly isolated Xenophilus azovorans: Carbon dynamics, humification, and driving pathways.

Composting is a biological reaction caused by microorganisms. Composting efficiency can be adequately increased by adding biochar and/or by inoculating with exogenous microorganisms. In this study, we looked at four methods for dewatered sludge waste (DSW) and wheat straw (WS) aerobic co-composting: T1 (no additive), T2 (5% biochar), T3 (5% of a newly isolated strain, Xenophilus azovorans (XPA)), and T4 (5% of biochar-immobilized XPA (BCI-XPA)). Throughout the course of the 42-day composting period, we looked into the carbon dynamics, humification, microbial community succession, and modifications to the driving pathways. Compared to T1 and T2, the addition of XPA (T3) and BCI-XPA (T4) extended the thermophilic phase of composting without negatively affecting compost maturation. Notably, T4 exhibited a higher seed germination index (132.14%). Different from T1 and T2 treatments, T3 and T4 treatments increased CO2 and CH4 emissions in the composting process, in which the cumulative CO2 emissions increased by 18.61-47.16%, and T3 and T4 treatments also promoted the formation of humic acid. Moreover, T4 treatment with BCI-XPA addition showed relatively higher activities of urease, polyphenol oxidase, and laccase, as well as a higher diversity of microorganisms compared to other processes. The Functional Annotation of Prokaryotic Taxa (FAPROTAX) analysis showed that microorganisms involved in the carbon cycle dominated the entire composting process in all treatments, with chemoheterotrophy and aerobic chemoheterotrophy being the main pathways of organic materials degradation. Moreover, the presence of XPA accelerated the breakdown of organic materials by catabolism of aromatic compounds and intracellular parasite pathways. On the other hand, the xylanolysis pathway was aided in the conversion of organic materials to dissolved organics by the addition of BCI-XPA. These findings indicate that XPA and BCI-XPA have potential as additives to improve the efficiency of dewatered sludge and wheat straw co-composting.

Multi-stage aeration regime to regulate organic conversion toward gas alleviation and humification in food waste digestate composting.

Aerobic composting has been considered as a pragmatic technique to convert food waste digestate into high-quality biofertiliser. Nevertheless, massive gaseous emission and immature product remain the primary challenges in food waste digestate composting. Thus, the performance of multi-stage aeration regimes to improve gaseous emissions and organic humification during food waste digestate composting was investigated in this study. In addition to continuous aeration with a constant intensity of 0.3 L kg·dry mass (DM)-1·min-1, two multi-stage decreased aeration regimes were designed as "0.3-0.2-0.1" and "0.3-0.1-0.1" L·kg·DM-1·min-1 from the thermophilic to cooling and then mature stages, respectively. Results showed that the decreased aeration regimes could alleviate nitrous oxide (N2O) and ammonia (NH3) emission and slightly enhance humification during composting. The alleviated N2O and NH3 emission were mainly contributed by abiotically reducing gaseous release potential as well as biotically inactivating denitrifers (Pusillimonas and Pseudidiomarina) and proliferating Atopobium to reduce nitrate availability under lower aeration supply. The "0.3-0.2-0.1 L kg·DM-1·min-1" regime exhibited a more excellent performance to alleviate N2O and NH3 emission by 27.5% and 16.3%, respectively. Moreover, the decreased aeration regimes also favored the enrichment of functional bacteria (Caldicoprobacter and Syntrophomonas) to accelerate lignocellulosic biodegradation and thus humic acid synthesis by 6.5%-11.2%. Given its better performance to improve gaseous emissions and humification, the aeration regime of "0.3-0.2-0.1 L kg·DM-1·min-1" are recommended in food waste digestate composting in practice.

Optimization of organic solid waste composting process through iron-related additives: A systematic review.

Composting is an environmentally friendly method that facilitates the biodegradation of organic solid waste, ultimately transforming it into stable end-products suitable for various applications. The element iron (Fe) exhibits flexibility in form and valence. The typical Fe-related additives include zero-valent-iron, iron oxides, ferric and ferrous ion salts, which can be targeted to drive composting process through different mechanisms and are of keen interest to academics. Therefore, this review integrated relevant literature from recent years to provide more comprehensive overview about the influence and mechanisms of various Fe-related additives on composting process, including organic components conversion, humus formation and sequestration, changes in biological factors, stability and safety of composting end-products. Meanwhile, it was recommended that further research be conducted on the deep action mechanisms, biochemical pathways, budget balance analysis, products stability and application during organic solid waste composting with Fe-related additives. This review provided guidance for the subsequent targeted application of Fe-related additives in compost, thereby facilitating cost reduction and promoting circular economy objectives.

Spatial differences of dissolved organic matter composition and humification in an artificial lake.

The depth-dependent dynamics of dissolved organic matter (DOM) structure and humification in an artificial lake limits the understanding of lake eutrophication and carbon cycling. Using fluorescence regional integration (FRI) and parallel factor analysis (PARAFAC) models to analyze the 3D fluorescence spectroscopy dataset, we revealed the depth-dependent structure and vertical distribution of DOM in the estuarine and center regions of Lake Hongfeng. The percentage fluorescence response (Pi,n) showed humic acid is an important part of DOM in Lake Hongfeng. Fluorescence results show that the fulvic-like and protein-like materials in HF1-DOM located at the estuarine position showed greater variation in the middle stage, probably due to human influence and sediment suspension. Fluorescence index (PI+II+IV,n/PIII+V,n and FIC4/FIC3) can be used to indicate the degree of humification of DOM in artificial lakes. Results of each index show that the estuary is more affected by human activities, and the humification degree is significantly lower than that of the center of the lake. The evaluation index system of the humification degree of artificial lake established in this study can effectively predict the eutrophication state of the typical area of artificial lake and deeply understand the possible important influence of human activities on the carbon cycle of lake.

Roles of Natural Phenolic Compounds in Polycyclic Aromatic Hydrocarbons Abiotic Attenuation at Soil-Air Interfaces through Oxidative Coupling Reactions.

Little information is available on the roles of natural phenolic compounds in polycyclic aromatic hydrocarbons (PAHs) attenuation at dry soil-air interfaces. The purpose of this study was to determine the roles of model phenolic constituents of soil organic matter (SOM) on the abiotic attenuation of PAHs. The phenolic compounds can significantly change the attenuation rates of PAHs, among which hydroquinone was the most effective in promoting anthracene and benzo[a]anthracene attenuation. Product identification and sequential extraction experiments revealed hydroquinone enhanced the formation of oxidative coupling products and promoted the incorporation of PAHs into humic analogues, thereby reducing potential risks to humans and ecosystems. Electron paramagnetic resonance spectroscopy analyses showed both PAHs and phenolic compounds could donate electrons to Lewis acid sites of soil minerals, resulting in the generation of persistent free radicals (PFRs). PFRs could promote the generation of ·OH to enhance PAH oxidation and could cross-couple with PAHs, resulting in high-molecular-weight oxidative coupling products. This study revealed for the first time the reaction mechanism between PAHs and phenolic components of SOM under relatively dry conditions and provided new insights into promoting PAHs detoxification in soils but also a potential strategy to increase the organic carbon sequestration.

Novelty three stages for humification of sewage sludge during hyperthermophilic aerobic fermentation.

Compared with conventional aerobic fermentation (CAF), there is limited knowledge of how hyperthermophilic aerobic fermentation (HAF) enhances the humification of sewage sludge. This study compared three novel stages of organic degradation, precursors, functional groups, bacterial community, and humus synthesis mechanism in HAF with CAF. The results showed that organic matter (OM) degraded rapidly, and 68% of the degradation could be completed of stage I in HAF. Compared with the initial stage, ammonium nitrogen (NH4+-N), water-soluble organic carbon, and water-soluble total nitrogen increased by 2.83 times, 40.5 times, and 33.5 times, respectively. Cellulose and hemicellulose decreased by 29.22% and 21.85%, respectively. These results suggested that temperature (>80 °C) and Bacillus dominated accelerate the humification process by rapidly improving OM degradation. Compared with the initial value of HAF, the maximum increment of reducing sugar at stage II was 297%, and the degradation rate of cellulose was effectively increased by 21.03% compared with that of CAF. The precursors such as reducing sugars and amino acids formed humus at stage II. The content of Aryl C increased significantly during the HAF process, the degree of polymerization of humus and the aromatization degree of HA and FA increased significantly, and complex organic macromolecular material polymers were formed at stage III. The sugar-amine condensation was the mechanism of humification in the sludge HAF process. This investigation provided three new stages of insights into the synthesis of humification during the HAF process and extended the current mechanism of humification in the HAF process.

The dominant role of cooperation in fungal community drives the humification process of chicken manure composting under addition of regulatory factors.

This study aimed to explore the action mechanism of fungal community on the enhancement of humification during chicken manure composting by regulating the core pathway of carbon metabolism - the tricarboxylic acid cycle. Regulators adenosine triphosphate (ATP) and malonic acid were added at the beginning of composting. The analysis of changes in humification parameters showed that the humification degree and stability of compost products were improved by adding regulators. Compared with CK, the humification parameters of adding regulators group increased by 10.98% on average. Meanwhile, adding regulators not only increased key nodes, but also strengthened the positive correlation between fungi, and network relationship was closer. Moreover, core fungi associated with humification parameters were identified by constructing OTU networks, and the division and cooperation mechanism of fungi were confirmed. Ultimately, the functional role of the fungal community acting on humification was confirmed by statistical means, that was, the fungal community promoting humification was the main group of composting process. And the contribution was more obvious in ATP treatment. This study was helpful to gain insight into the mechanism of regulators addition to advance the humification process, and provided new ideas for the safe, efficient and harmless disposal of organic solid waste.

FT-ICR-MS combined with fluorescent spectroscopy reveals the driving mechanism of the spatial variation in molecular composition of DOM in 22 plateau lakes.

Dissolved organic matter (DOM) is the largest carbon pool and directly affects the biogeochemistry in lakes. In the current study, fourier transform ion cyclotron mass spectrometry (FT-ICR-MS) combined with fluorescent spectroscopy was used to assess the molecular composition and driving mechanism of DOM in 22 plateau lakes in Mongolia Plateau Lakes Region (MLR), Qinghai Plateau Lakes Region (QLR) and Tibet Plateau Lakes Region (TLR) of China. The limnic dissolved organic carbon (DOC) content ranged from 3.93 to 280.8 mg L-1 and the values in MLR and TLR were significantly higher than that in QLR. The content of lignin was the highest in each lake and showed a gradually decreasing trend from MLR to TLR. Random forest model and structural equation model implied that altitude played an important role in lignin degradation while the contents of total nitrogen (TN) and chlorophyll a (Chl-a) have a great influence on the increase of DOM Shannon index. Our results also suggested that the inspissation of DOC and the promoted endogenous DOM production caused by the inspissation of nutrient resulted in a positive relationship between limnic DOC content and limnic factors such as salinity, alkalinity and nutrient concentration. From MLR to QLR and TLR, the molecular weight and the number of double bonds gradually decreased but the humification index (HIX) also decreased. In addition, from the MLR to the TLR, the proportion of lignin gradually decreased, while the proportion of lipid gradually increased. Both above results suggested that photodegradation was dominated in lakes of TLR, while microbial degradation was dominated in lakes of MLR.

Mechanism analysis of humification coupling metabolic pathways based on cow dung composting with ionic liquids.

This study focused on how adding ionic liquids (IL) affects composting humification. During the warming and thermophilic phases, addition of IL increased precursors content, and increased the polymerization of humus (HS) at later stages. Furthermore, the final HS and humic acid (HA) content of experimental groups (T) groups 129.79 mg/g and 79.91 mg/g were higher than in control group (CK) 118.57 mg/g and 74.53 mg/g, respectively (p < 0.05). IL up-regulated the gene abundance of metabolism for carbohydrate and amino acid (AA), and promoted the contributions of Actinobacteria and Proteobacteria, which affected humification. The redundancy analysis (RDA) results showed that the citrate-cycle (TCA cycle)(ko0020), pentose phosphate pathway (ko00030), pyruvate metabolism (ko00620), glyoxylate and dicarboxylate metabolism (ko00630), propanoate metabolism (ko00640), butanoate metabolism (ko00650) positively correlated with HA and HI. HA and humification index (HI) positively correlated with AA metabolic pathways, and fulvic acid (FA) was negatively correlated with these pathways. Overall, metabolism for carbohydrate and AA metabolism favored compost humification. ILs improved metabolism for carbohydrate and amino acid metabolism, thus enhancing humification.

Insight into humification of mushroom residues under addition of Rich-N sources: Comparing key molecular evolution processes using EEM-PARAFAC and 2D-FTIR-COS analysis.

Accelerating the humification of organic solid waste is one of the most important issues in composting. This present study aims to study and compare the humification process of different rich-N sources (chicken manure, cattle manure, and urea) addition during the composting of mushroom residues, from macro physicochemical properties to micro humic molecular structure evolution process. The physicochemical elements and humic components were determined for evaluating the compost quality and humification degree as composting proceed. The coupled analysis of excitation-emission matrix with parallel factor analysis (EEM-PARAFAC) and two-dimensional correlation with Fourier transform infrared spectrum (2D-FTIR-COS) were used to characterize the functional molecular structure evolution of dissolved organic matter during humification process. The results indicated that the rank order for humification level were the treatments of chicken manure (HM), urea (UM), cattle manure (CM), and single mushroom residue treatment (CK), with their humification index of 22.18%, 22.05%, 18.47%, and 16.52%, respectively. Humic substance, humic acid, and fulvic acid were obtained the highest in HM treatment with contents of 35.41 ± 0.86%, 23.32 ± 1.57%, and 10.97 ± 0.52%, respectively. The rich-N source addition enhanced the degradation of protein-like and polysaccharides-like substances in dissolved organic matter, thus accelerating the humification process of mushroom residues. The key structure evolution of dissolved organic matter in the HM treatment, in which the CO and CC stretching of quinone, amide, or ketone, and the C-O stretching of polysaccharides may be responsible for the faster formation of humus compared to the other nitrogen treatments. In this study, redundancy analysis indicated that the total nitrogen (TN) and nitrate nitrogen (NO3--N) may be the potential indicators for determining the humification level as composting proceed. The result provides significant insight into the humification mechanism of mushroom residue under different types of nitrogen sources at the molecular level, and will be reference for improving the composting technique in practical field.

Impact of compost methods on humification and heavy metal passivation during chicken manure composting.

Livestock manure is one of the main sources of heavy metals (HMs) in agricultural soil. So, it is necessary to reduce its bioavailability before used as organic fertilizer. In this study, the passivation effect of HMs and the evolution of dissolved organic matter (DOM) during four composting processes were explored. Results showed that different composting methods had a great effect on HMs passivation rate and humification degree. HMs were released during the thermophilic phase, and were bound by resynthesized humus during the cooling period. The best passivation effect of HMs was found in FV + T treatment, the passivation rate of Cu, Zn, Cd and Pb reached 63.80%, 34.07%, 86.54% and 45.14%, respectively, then followed by the treatment of NV + T and SC. UV-Vis spectra and excitation-emission matrix (EEM) spectra indicated that humus precursors were produced during thermophilic phase and the accumulation of humus mainly occurred in cooling period. This study can be used as a theoretical support for the safe utilization livestock manure.

Synergistic effect triggered by Fe2O3 and oxygen-induced hydroxyl radical enhances formation of amino-phenolic humic-like substance.

Metal oxides play a promising role in the transformation of polyphenols and amino acids involved in naturally occurring humification. The objective of this study was to explore the synergistic interactions between Fe2O3 and O2 in the formation of humic substances under a controlled O2 atmosphere (0%, 21% and 40% O2 levels). The results indicate that an O2 level of 21% with Fe2O3 was optimal for humic acid (HA) production. Hydroxyl radicals (∙OH) formed and promoted the formation of HA in the presence of O2, and O2 improved the enhancing capacity of Fe2O3 by oxidizing Fe(II) to Fe(III). Moreover, the combination of these processes resulted in a synergistic improvement in humification. The evolution of functional groups in HA suggested that O2 promoted the formation of oxygen-containing groups such as lipids, and Fe2O3 was conducive to the formation of dark-coloured polymers during the darkening process of humification. Furthermore, the O2 level of 40% inhibited the formation of HA by reducing the transformation from Fe(III) to Fe(II). The XRD results showed few changes in the composition of Fe2O3 before and after humification, which indicated that Fe2O3 was a catalyst and an oxidant. The heterospectral UV-Vis/FTIR results suggested that ∙OH attacked phenolic rings to form the aromatic ring skeleton of HA and benefit the ring-opening copolymerization of humic precursors. In addition, structural equation modelling demonstrated that dissolved Fe was the key parameter affecting the HA yield. These findings provide new insights into the synergism of O2-mediated ∙OH production associated with metal oxide-facilitated humification.

Production of artificial humic acid from corn straw acid hydrolysis residue with biogas slurry impregnation for fertilizer application.

This study investigated hydrothermal humification of corn straw acid hydrolysis residue with biogas slurry impregnation, aiming at producing water-soluble artificial humic acid fertilizer for fertilizer application and soil remediation. Hydrothermal humification parameters, including potassium hydroxide concentration (1-3 mol/L), retention time (2-6 h), and temperature (140-180 °C), were investigated using water as the liquid phase. The selected hydrothermal humification condition was 1.5 mol/L potassium hydroxide at 180 °C for 4 h. Moreover, biogas slurry impregnation (0-30 days) was evaluated to improve humic acid yield without introducing additional chemicals or energy input. Biogas slurry as the liquid phase increased the humic acid production by 73.24% with 5 days of impregnation compared to the control due to the alkalinity. The humic acid concentration was sufficient for China's national standard of water-soluble humic acid fertilizers in such conditions. The organic components in biogas slurry were involved in artificial humification as a precursor, forming C-N bonds with humic acid. The product with fortified nitrogen-containing functional groups enhanced the nutrient slow-release characteristics and water retention capabilities. The pot experiment further confirmed that artificial humic acid prepared in this study not only promoted the growth of plants but also achieved soil remediation.

Linking above and belowground carbon sequestration, soil organic matter properties, and soil health in Brazilian Atlantic Forest restoration.

Forest restoration mitigates climate change by removing CO2 and storing C in terrestrial ecosystems. However, incomplete information on C storage in restored tropical forests often fails to capture the ecosystem's holistic C dynamics. This study provides an integrated assessment of C storage in above to belowground subsystems, its consequences for greenhouse gas (GHG) fluxes, and the quantity, quality, and origin of soil organic matter (SOM) in restored Atlantic forests in Brazil. Relations between SOM properties and soil health indicators were also explored. We examined two restorations using tree planting ('active restoration'): an 8-year-old forest with green manure and native trees planted in two rounds, and a 15-year-old forest with native-planted trees in one round without green manure. Restorations were compared to reformed pasture and primary forest sites. We measured C storage in soil layers (0-10, 10-20, and 20-30 cm), litter, and plants. GHG emissions were assessed using CH4 and CO2 fluxes. SOM quantity was evaluated using C and N, quality using humification index (HLIFS), and origin using δ13C and δ15N. Nine soil health indicators were interrelated with SOM attributes. The primary forest presented the highest C stocks (107.7 Mg C ha-1), followed by 15- and 8-year-old restorations and pasture with 69.8, 55.5, and 41.8 Mg C ha-1, respectively. Soil C stocks from restorations and pasture were 20% lower than primary forest. However, 8- and 15-year-old restorations stored 12.3 and 28.3 Mg ha-1 more aboveground C than pasture. The younger forest had δ13C and δ15N values of 2.1 and 1.7‰, respectively, lower than the 15-year-old forest, indicating more C derived from C3 plants and biological N fixation. Both restorations and pasture had at least 34% higher HLIFS in deeper soil layers (10-30 cm) than primary forest, indicating a lack of labile SOM. Native and 15-year-old forests exhibited higher soil methane influx (141.1 and 61.9 µg m-2 h-1). Forests outperformed pasture in most soil health indicators, with 69% of their variance explained by SOM properties. However, SOM quantity and quality regeneration in both restorations approached the pristine forest state only in the top 10 cm layer, while deeper soil retained agricultural degradation legacies. In conclusion, active restoration of the Atlantic Forest is a superior approach compared to pasture reform for GHG mitigation. Nonetheless, the development of restoration techniques to facilitate labile C input into deeper soil layers (>10 cm) is needed to further improve soil multifunctionality and long-term C storage.

Insights into the Applications of Extracellular Laccase-Aided Humification in Livestock Manure Composting.

Traditional composting is a well-suited biotechnology for on-farm management of livestock manure (LM) but still leads to the release of toxic micropollutants and imbalance of nutrients. One in situ exoenzyme-assisted composting has shown promise to ameliorate the agronomical quality of end products by improving humification and polymerization. The naturally occurring extracellular laccase from microorganisms belongs to a multicopper phenoloxidase, which is verified for its versatility to tackle micropollutants and conserve organics through the reactive radical-enabled decomposition and polymerization channels. Laccase possesses an indispensable relationship with humus formation during LM composting, but its potential applications for the harmless disposal and resource utilization of LM have until now been overlooked. Herein, we review the extracellular laccase-aided humification mechanism and its optimizing strategy to maintain enzyme activity and in situ production, highlighting the critical roles of laccase in treating micropollutants and preserving organics during LM composting. Particularly, the functional effects of the formed humification products by laccase-amended composting on plant growth are also discussed. Finally, the future perspectives and outstanding questions are summarized. This critical review provides fundamental insights into laccase-boosted humification that ameliorates the quality of end products in LM composting, which is beneficial to guide and advance the practical applications of exoenzyme in humification remediation, the carbon cycle, and agriculture protection.