Mechanism of oligosaccharides on nitrogen and ammonia-oxidizing microbial communities in aerobic composting processes

Mechanism of oligosaccharides on nitrogen and ammonia-oxidizing microbial communities in aerobic composting processes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanism of oligosaccharides on nitrogen and ammonia-oxidizing microbial communities in aerobic composting processes Manli Duan, Mingxiu Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4486496/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract To explore the effects of oligosaccharides on nitrogen and ammonia-oxidizing microbial communities during aerobic composting of cattle manure and straw, this study conducted composting experiments with four concentrations of oligosaccharides: 0.1% (M0.1), 0.5% (M0.5), 1.0% (M1), and 2.0% (M2), along with a control group (CK). The results indicated that different concentrations of oligosaccharides increased the peak temperatures during the thermophilic phase of composting to above 60°C, higher than that of the CK (57.4°C), while ensuring that all treatments met the requirements for harmless disposal. Particularly, the GI value of the 0.5% oligosaccharide treatment reached 109.3%, demonstrating excellent treatment efficacy. The 0.5% oligosaccharide treatment significantly increased the NO 3 -N content in compost ( P < 0.05), thereby enhancing nitrogen content. AOB amoA functional gene detection identified two dominant ammonia-oxidizing bacteria, Nitrosomonas and Nitrosospira , with Nitrosomonas primarily present in the 0.5% oligosaccharide treatment, playing a crucial role in ammonia nitrogen fixation. SEM analysis showed a significant positive correlation between AOB amoA genes and NO 3 -N in the 0.5% oligosaccharide treatment, indicating effective promotion of nitrogen conversion by ammonia-oxidizing bacteria in the compost. In conclusion, the addition of 0.5% oligosaccharides can increase the dominance of AOB genera, enhance nitrogen transformation during composting, provide more available nitrogen sources for crops, and thereby improve nitrogen fertilizer utilization efficiency. Ammonia-oxidizing Composting Community structure Oligosaccharides Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction A large number of wastes are produced in agricultural production every year. These livestock manure and straw containing a large number of nutrients have been called "misplaced resources". Most of these wastes have not been treated as resources, harmless and reduced, causing serious environmental pollution and threats to human health. Aerobic composting technology has been widely used in the disposal of livestock manure, agricultural straw and other wastes due to its advantages of low cost, good deodorization effect and fertilizable products. Composting technology is to transform organic materials into mature organic fertilizer containing a large number of available nitrogens, phosphorus, potassium nutrients and humus which can be absorbed and utilized by plants through high-temperature fermentation under the action of microorganisms (Tian et al., 2017 ). It has good ecological, economic and social benefits. However, the transformation and nitrogen fixation in composting affect the quality and environmental impact of compost, hindering the popularization of composting technology (Awasthi et al., 2017 ). Nitrogen in compost can be released through processes such as ammonia volatilization and conversion (Fukumoto et al., 2003 ; Luo et al., 2014 ), as well as inorganic nitrogen transformation (Chang et al., 2019 ). It has been reported that nitrogen losses from composting are approximately 35–71% (Jiang et al., 2015 ; Chen et al., 2019a ). Nitrification controls the mineralization of nitrogen in the composting process. Mineralized nitrogen can be directly utilized by organisms and plays an important role in the process of nitrogen transformation. The ammoxidation process is the first and rate limiting step of nitrification, which determines the conversion balance between oxidized and reduced nitrogen during composting (Chen et al., 2020 ), and directly or indirectly affects composting efficiency, greenhouse gases and ecological environment (Wang and Zeng, 2017 ). The key mechanism of reducing nitrogen loss and improving fertilizer nutrients during composting is to improve the reproduction and activity of ammonia oxidizing microorganisms that can transform more ammonia oxidizing nitrogen into nitrite nitrogen and further oxidize to form nitrate nitrogen. Ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) are two major ammonia oxidizing microorganisms. Some scholars have indicated that AOA may be the dominant microorganism in ammonia oxidation process, and the amount of AOA in some environments (including the composting process) is higher than AOB, and even contributes more to nitrification than AOB (Yamamoto et al., 2010 ). However, previous studies have believed that AOA mostly exists in soil and marine systems with low oxygen content (Francis et al., 2005 ; Chen, 2019a). AOB is the dominant microorganism in aerobic composting of livestock manure (Yamada et al., 2013 ), in which genus Nitrosomonas and Nitrosospira are important bacteria involved in nitrification (Yin et al., 2016 ). Yamada et al. ( 2013 ) investigated population dynamics of ammonia-oxidizing bacteria (AOB) and archaea (AOA) during ammonia-rich livestock waste-composting processes. Quantitative PCR (Q-PCR) assays showed a high relative abundance of β-proteobacterial AOB during ammonia oxidation but did not detect AOA in any composting stage. Yan et al. ( 2016 ) analyzed the abundance, diversity and structure of AOB and AOA in response to different aeration rates during cattle manure composting by Q-PCR and high-throughput sequencing. The results revealed that oxygen was significantly ( P < 0.05) correlated with AOB diversity but not with AOA, and AOB were more important than AOA in nitrogen transformation. At the same time, AOB can encode amoA genes to facilitate nitrification (Jarvis et al., 2009 ; Zeng et al., 2011 ). Therefore, the study on the structure of ammonia oxidizing bacteria is helpful to reveal the internal mechanism of nitrification during composting. At present, the nitrogen loss during composting is mainly reduced by adjusting the composting parameters, adding biochar and exogenous nitrogen retention agents, and inoculating microbial agents (Yan et al., 2016 ; Mao et al., 2018 ; Wang et al., 2018 ; Guo et al., 2020 ). However, there are still some problems, such as insignificant effects, secondary pollution, and unclear mechanisms. Oligosaccharides are straight chain or branched polymer polymerized from 2–10 monosaccharides with glycosidic bond (Rastall, 2010 ), which are mainly used in food, medical treatment and livestock and poultry industry (Hafsa et al., 2021 ; Liu, et al., 2021 ; Flilkinger, et al., 2002). It is found that oligosaccharides have the characteristics of water solubility and structural stability, which are easy to be metabolized by bacteria, can reshape the microbial community structure, promote the proliferation and activity of beneficial bacteria (such as Bifidobacteria and Lactobacilli), and maintain the micro ecological balance (Ambrogi et al., 2021). Oligosaccharides have the ability to recognize, adhere to, and exclude pathogenic microorganisms (e.g., Staphylococcus aureus, Aspergillus Niger, and E. coli) (Zhang et al., 2022 ). Oligosaccharides can also promote crop growth (Amerany et al., 2020 ). However, oligosaccharides, as regulators of microbial community, have not been studied for their effect on the transformation of nitrogen during composting. In this study, oligosaccharides were selected to study their effects on loss and transformation of nitrogen during composting. Q-PCR and high throughput sequencing were conducted to detect the abundances of the bacterial amoA genes during composting, as well as the diversity and structure of the bacterial community that harbored amoA genes. The results obtained in this study may support great significance for taking appropriate measures to reduce nitrogen loss, improve nitrogen nitrification and increase the nutrients of composting products. 2. Materials and methods 2.1 Material characteristics The raw compost materials used in this study comprised cow manure and wheat straw. Fresh cow dung was collected from Shanxi Qinbao Animal Husbandry Co. Ltd and wheat straw was collected from a local farm in Yangling, China. The physicochemical properties of the cow manure and wheat straw after being air-dried and broken are shown in Table 1 . Oligosaccharides was purchased from Henan Wanbang Industrial Co. Ltd, China. It was extracted from natural ingredients and had good acid stability and thermal stability. Table 1 Physico-chemical features of compost sample Properties Water content (%) pH TOC g/kg C/N TN g/kg Cow manure 7.52 8.41 292 17.5 16.7 Wheat straw 5.88 7.61 414 60.0 6.9 2.2 Experimental setup and sample collection The composting experiment was conducted in the glasshouse at Xi’an University of Technology, Shanxi, China. The environmental temperature in glasshouse was controlled 30°C. The reactors comprised four insulated foam boxes with dimensions of: length = 60 cm, width = 58 cm, height = 57 cm, and thickness = 6 cm, which had two circular holes (2 × 2 cm) in the top, bottom, and four walls of the foam boxes to ensure ventilation and the supply of oxygen. Cow manure and wheat straw were mixed and adjusted to a C:N ratio of 25:1. The weight of each treated composting material is 9kg, straw 3kg and cow manure 6kg. Oligosaccharides was added at four different concentrations of 0 (CK), 0.1% (M0.1), 0.5% (M0.5), 1.0% (M1), and 2.0% (M2) relative to the dry weight of the compost. Oligosaccharides powder was mixed evenly with 5.4L deionized water and balanced for 2 hours, then the mixture was poured into the composting material and mixed evenly, adjusting the water content of the compost to 60%. The composting process was conducted for 26 days, and the compost reactors were turned over on days 2, 4, 7, 15, 20, and 21 to enhance aeration. Samples were collected in the initial phase (day 0), mesophilic phase (day 1), thermophilic phase (day 3), cooling phase (day 7), first maturation phase (day 14) and second maturation phase (day 26). According to the compost temperature, the samples were taken from the top, middle, and bottom of each reactor and mixed well, where a total of 900 g (fresh weight was collected. Each sample was divided into two parts. The first part was stored in a refrigerator at 4°C to determine the physical and chemical parameters. The second part was freeze dried at low temperature (Beijing Songyuan, China), crushed with a low temperature freeze-grinding machine (Retsch Z200, Germany), and sieved through a mesh of 0.5 mm, before storing at − 80°C for molecular biological experiments. 2.3. Analytical methods 2.3.1. Analysis of physicochemical parameters The temperature of the compost was monitored twice every day (9:00 and 19:00) at the top, core, and bottom of compost. The pH was measured with a digital pH meter after mechanically shaking the fresh sample as a suspension in water without CO 2 at a ratio of 1:10 (w/v) for 40 min at 200 rpm. The moisture content was measured as the loss after heating at 105°C for 8 h. The NH 4 + -N and NO 3 – -N contents of the samples were extracted with 2 mol/L KCl and determined by Discrete Chemistry Analyzer (SmartChem450, Italy). The C/N ratio was calculated as the total organic carbon (TOC)/ total nitrogen (TN). TOC and TN were determined using elemental analyzer (Elementar, Germany) after the inorganic carbon was fully removed by 1mol/L HCl. 2.3.2. DNA extraction and qPCR Composting samples (0.1g) were extracted using a DNeasy PowerSoil Kit (QIAGEN, Germany), according to the manufacturer’s instructions. The concentration and purity of the DNA samples were detected with an ultramicro spectrophotomete Spectrophotometer (NanoDrop2000, USA). The quality checked DNA samples were stored at − 20°C. The quantitative PCR (qPCR) was performed in triplicate on an iCycler IQ5 Thermocycler (Bio-Rad, USA) using primer sets amoA-1F(5’-GGGGTTTCTACTGGTGGT-3’) and amoA-2R (5’-CCCCTCKGSAAAGCCTTCTTC-3’) for bacterial amoA genes and Arch-amoAF(5’-STAATGGTCTGGCTTAGACG-3’) and ArchamoAR (5’-GCGGCCATCCATCTGTATGT-3’) for archaeal amoA genes (Rotthauwe et al., 1997 ). Each 20 mL of qPCR reaction contained 10 mL of 2 × SYBR Green PCR Master mix (Qiagen, Shanghai, China), 0.5 mL of each AOB or AOA primer (10 mM), and 1 mL of DNA template (20 ng/mL) and adjusted with sterile water to volume. The amoA gene amplification was conducted using an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 30 s at 95°C, 30 s at 55°C for AOB, 30 s at 72°C. Melting curve analysis was carried out to verify amplicon specificity. A negative control without DNA template was used in all of the qPCR amplifications. Standard curves were generated on the basis of serial tenfold dilution of plasmids containing cloned amoA genes and ranged from 1.0 × 10 3 to 1.0 × 10 8 copies per assay. Amplification efficiencies of 96.68% (AOB) and 104.5% (AOA) were obtained using the slopes − 3.41 and − 3.21 of standard curve, respectively. Linearity (r 2 ) for AOB and AOA were 0.999 and 0.998, respectively. 2.3.3. High-throughput sequencing analysis of bacterial amoA genes DNA sample of composts were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) for High throughput sequencing of bacterial functional gene amoA using Illumina Miseq sequencing platform. QIIME 1.8.0 software was used to filter, splice and remove chimera from the raw sequencing data. Operational Taxonomic Units (OTUs) were clustered using Usearch 7.1 based on cutoff of 3% dissimilarity. Microbial composition and the nearest relatives were obtained by comparing the sequences with FGR/ amoA database of GeneBank (Release7.3, http://fungene.cme.msu.edu/ ). To evaluate the community richness and diversity of Ammonia-oxidizing microbial community, ACE, Chao 1 index and Shannon diversity index were calculated by Mothur 1.30.1, respectively. 2.4. Statistical analysis All descriptive statistics for the raw data were obtained using Excel 2019 (Microsoft Office 2019, Microsoft, USA). SPSS 20.0 software was used to perform multiple comparative and Pearson correlation analysis of soil physical and chemical properties, AOB amoA gene copy Numbers (ANOVA, LSD, P < 0.05). Linear regression plots and bar charts were produced using SigmaPlot 14.0. Heatmap analysis was conducted with R3.1.0. A structural equation modeling (SEM) was constructed using IBM SPSS AMOS 22.0 in order to study the key factors influencing nitrogen changes during the composting process. 3. Results and discussion 3.1 The effect of adding oligosaccharides on composting temperature The temperature variation in composting is primarily attributed to the heat generated by microbial decomposition of soluble organic compounds such as proteins and lipids in the compost materials, providing a reference basis for the humification and stabilization of compost materials (Liu et al., 2020 ). Figure 1 depicts the temperature changes during the composting process in this study, showing a pattern of temperature elevation followed by decline, ultimately approaching ambient temperature for all treatments. By the second day of composting, except for the control group (CK), temperatures in all other treatments rose to above 50°C and remained in the thermophilic phase for 4–5 days, meeting the requirement stipulated in the "Sanitary standards for composting of excrement" (GB7959-87) that the highest temperature during composting should exceed 50–55°C and be maintained for 5–7 days. This high-temperature stage effectively eliminates most pathogenic microorganisms and pathogens. With the increase in oligosaccharide concentration, the rate of temperature rise in the compost pile accelerated. On the second day of composting, the maximum temperatures in M2, M1, M0.5, and M0.1 reached 63.4°C, 61.0°C, 63.2°C, and 62.8°C, respectively, all higher than the maximum temperature of CK at 57.4°C. This may be attributed to the high concentration of oligosaccharides serving as carbon nutrients, providing ample energy for microorganisms and accelerating the mineralization process of organic matter (Chen et al., 2024 ). As the composting enters the secondary maturation stage, the temperatures in M0.5 and M0.1 are significantly higher than those in M2 and M1 ( P < 0.05). This could be because the higher concentration of oligosaccharides is largely consumed in the early stage, leading to insufficient energy for microorganisms in the later stage and slowing down the humification process. However, the 0.5% and 0.1% oligosaccharides enhance the humification degree and organic matter stability in the later stage of composting, reducing the water-soluble nutrients available to microorganisms (Chang et al., 2019 ), resulting in a gradual decrease in temperature and stabilization. 3.2 The impact of adding oligosaccharides on the physicochemical properties of compost The physicochemical properties changes of CK, M0.1, M0.5, M1, and M2 are illustrated in Fig. 2 . From the pH variation, it can be observed that the pH of all treatments shows an initial increase followed by a decrease trend throughout the composting process. The pH variation in composting may be related to the generation of organic acids and the release of ammonia compounds from compost materials (Wang et al., 2016 ). In the early stage of composting, vigorous microbial metabolism, mineralization of nitrogenous organic matter, and continuous decomposition of organic acids lead to an increase in pH (Li et al., 2015 ). In the later stage of composting, pH gradually decreases until the end of composting when organic matter is almost completely decomposed, microbial activity declines, and pH decreases. Among them, the compost pH of the M0.5 treatment is the lowest, at 7.6. The E 4 /E 6 value can reflect the degree of humification and aromatization during composting, with lower values indicating higher aromaticity and more stable organic matter structure (Hadda et al., 2019 ). From the change in E 4 /E 6 values, the E 4 /E 6 values of CK, M0.1, M0.5, and M1 are lower, indicating higher humification degree. The E 4 /E 6 value of the M2 treatment is significantly higher than that of other treatments, indicating lower humification degree of the compost. Germination index (GI) is an important indicator for assessing compost toxicity, and throughout the composting process, the GI of all treatments shows an increasing trend. On the 26th day, the GI values of CK, M0.1, M0.5, M1, and M2 increased to 92.8%, 91.6%, 109.3%, 100.8%, and 89.9%, respectively, all exceeding 80%, which meets the harmless standard for composting (Li et al., 2020 ). The GI value of M0.5 is higher than that of other treatments, indicating higher safety and harmlessness of the compost. 3.3 The effect of adding oligosaccharides on ammonium nitrogen and nitrate nitrogen in composting During aerobic composting, the NH 4 + -N content initially increases and then decreases, as shown in Fig. 3 . Among all treatments, the NH 4 + -N content with the addition of 0.5% oligosaccharides exhibits the largest increase on the first day, significantly higher than that of the CK treatment by 3.5 times ( P < 0.05). This may be attributed to the provision of suitable nutritional conditions for microorganisms by 0.5% oligosaccharides in the early stage of composting, accelerating both the microbial ammonification and organic nitrogen mineralization processes. Consequently, the NH 4 + -N content continues to increase. However, in the later stage of composting, as a portion of NH 4 + -N is assimilated by microorganisms, some undergo nitrification to form NO 3 − -N, and some volatilize in gaseous form, leading to a decrease in NH 4 + -N content (Zhang et al., 2011 ). By the end of composting, the reduction in NH 4 + -N is the greatest in the M0.5 treatment, with the lowest content, indicating a conversion into other nitrogen forms. From Fig. 3 , it can be observed that during the temperature rise phase of composting, except for the continuous decrease in NO 3 − -N content in the M0.1 treatment, the NO 3 − -N content in other treatments shows an increasing trend. During the thermophilic phase of composting, the NO 3 − -N content in M0.5 and M1 is higher, exceeding that of the CK treatment by 2.56 and 3.10 mg/kg, respectively ( P < 0.05). This may be related to the higher initial substrate NO 3 − -N content in M0.5 and M1, leading to a higher NO 3 − -N content generated by nitrification. However, the increase in NO 3 − -N during the thermophilic phase is smaller than that during the initial phase of composting. This could be due to the excessive NO 3 − -N content and high temperatures, which inhibit the activity of nitrifying microorganisms to some extent, resulting in a slowdown in NO 3 − -N production (Zhang et al., 2011 ). By the end of composting, with no significant differences in NO 3 − -N content among treatments, the NO 3 − -N content in M0.5 and M1 is significantly higher than that in other treatments ( P < 0.05), and the NO 3 − -N content in M0.5 is significantly higher than that in M1 ( P < 0.05). The NO 3 − -N content in M0.5 is 8.53 times that of CK and 15.36 times that of M2. This indicates that a large amount of NH 4 + -N has been converted into NO 3 − -N, increasing the available nitrogen sources in the compost. Throughout the composting process, the NH 4 + -N and NO 3 − -N content in the CK and M0.1 treatments exhibit similar trends. By the end of composting, the NH 4 + -N and NO 3 − -N content in the M0.1 treatment are lower than those in the CK treatment, indicating greater nitrogen loss compared to the CK treatment. To further investigate the influence of ammonia-oxidizing bacteria genes and the diversity of ammonia-oxidizing bacterial communities on nitrogen changes in composting, this study focuses on the CK, M0.5, M1, and M2 treatments. 3.4 The impact of adding oligosaccharides on the copy number of AOB amoA gene Ammonia-oxidizing bacteria (AOB) possess the amoA gene, which catalyzes the conversion of ammonium nitrogen to hydroxylamine, serving as a preliminary and rate-limiting step in nitrification, thereby driving the process forward (Guo et al., 2020 ). The variation in AOB amoA gene copies during composting is illustrated in Fig. 4 . The highest content of AOB amoA gene is found in composting raw materials, but as composting temperature increases, the abundance of amoA gene is rapidly inhibited. This inhibition could be attributed to the relatively high temperature and low oxygen conditions, which are unfavorable for the growth of ammonia-oxidizing bacterial communities (Reed et al., 2018 ). Previous studies have reported that the optimal temperature range for the growth and activity of nitrifying microorganisms is 15–25°C (Taylor et al., 2017 ). As the temperature decreases, the nitrifying microbial community gradually regains activity until composting enters the maturation stage, where the AOB amoA gene copies are more than five times higher than during the thermophilic phase of composting. The addition of 0.5% and 1% oligosaccharide treatments significantly increases the AOB amoA gene copies compared to other treatments ( P < 0.05), promoting the oxidation of NH 4 + -N by AOB amoA genes and thereby reducing ammonia emissions. However, as shown in Fig. 3 , the NO 3 − -N content remains high during the thermophilic phase, which may be related to the activity of ammonia-oxidizing archaea (AOA). Studies have reported that under high temperature and high NO 3 − -N concentration conditions, AOA may dominate (Wang and Zeng, 2017 ; Oishi et al., 2012 ; Zeng et al., 2011 ), contributing to organic nitrogen mineralization. Meanwhile, ammonia-oxidizing bacteria (AOB) may play a significant role in the maturation stage of composting (Yamamoto et al., 2010 ). 3.5 Ammonia-Oxidizing Bacterial Community Composition Analysis This study analyzed the community of ammonia-oxidizing bacteria (AOB) in CK, M0.5, M1, and M2 treatments, and assessed the similarity of bacterial communities between different treatments using Principal Coordinate Analysis (PCoA). In the PCoA plot, PC1 explained 31.06% of the variance, and PC2 explained 21.15%, indicating that these two principal axes effectively captured the differences in microbial community structures among samples. As shown in Fig. 5 , variations in the concentration of oligosaccharides during composting led to differences in bacterial community composition among treatments at different stages of composting. In the early stages of composting (days 3–7), except for the CK treatment, the distances between other treatments were relatively small, and there were no significant differences. This might be attributed to the presence of oligosaccharides in the early composting stages, promoting microbial metabolism and resulting in similar microbial community structures. However, as composting entered the secondary maturation stage (day 13), the distances between treatments increased, and the differences became significant ( P < 0.05). This indicated a significant change in microbial community composition under different treatments at this stage, likely due to the transformation of composting materials and microbial ecological succession. Finally, at the end of composting (day 26), the distances between CK, M0.5, M1, and M2 treatments gradually decreased, and the composition of microbial communities became more similar, indicating the stabilization of composting maturity. This further highlights the influence of oligosaccharides on microbial community structure. Understanding the dynamic changes in microbial communities during composting and the trend of composting maturation in the later stages is crucial for comprehending the composting process. 3.6 Variations in Ammonia-Oxidizing Bacteria Diversity Ammonia-oxidizing bacteria (AOB) are a strict aerobic autotrophic microorganism widely distributed in various natural environments. In the ninth edition of Bergey's Manual of Systematics of Archaea and Bacteria, they are classified into five genera: Nitrosomonas , Nitrosospira , Nitrosovibrio , and Nitrosococcus . Two dominant genera, Nitrosomonas and Nitrosospira , were detected using the AOB amoA functional gene. This is consistent with the findings of Shimaya et al. (2008), who identified Nitrosomonas and Nitrosospira as dominant AOB genera in compost from agricultural waste materials. During the initial stage of composting, when organic matter is relatively abundant, AOB grow well. As composting progresses and the temperature rises, the diversity of AOB from different genera changes. Nitrosomonas is a genus with strong tolerance, and previous studies have shown that it is a typical ammonia-oxidizing bacterium commonly found in systems with high denitrification rates. Nitrosomonas was significantly present throughout the composting process in the M0.5 treatment ( P < 0.05), with its abundance maintained at a high level during the high-temperature and maturation phases of composting. Chikako Shimaya and Tomoyoshi Hashimoto successfully enriched thermophilic AOB from compost at 50°C, indicating that AOB may play a role in nitrogen fixation as compost matures. 3.7 Correlation Analysis of the Influence of Ammonia-Oxidizing Bacteria amoA Gene on Nitrogen Changes in Composting Structural Equation Modeling (SEM) can reveal causal relationships between different variables by fitting data to a hypothesized model (Eisenhauer et al., 2015 ). In this experiment, structural correlations were constructed using physicochemical indicators (pH, GI, E 4 /E 6 ), AOB amoA gene, NH 4 + -N, and NO 3 − -N to evaluate the impact of adding different concentrations of oligosaccharides on nitrogen changes in composting, as shown in Fig. 7 . GI and E 4 /E 6 showed significant negative correlations with AOB amoA ( P < 0.001). This is consistent with the results in Figs. 2 and 4 , where during the high-temperature and cooling periods of composting, GI increased significantly while the copy number of AOB amoA gene remained low. During the maturation phase of composting, as compost entered the humification stage, the value of E 4 /E 6 gradually decreased, while the copy number of AOB amoA gene showed an increasing trend. This might be because ammonia-oxidizing bacteria are more concentrated during the maturation phase of composting, and the high-temperature environment is unfavorable for their survival (Yamamoto et al., 2010 ). The pH and E 4 /E 6 of M1 and M2 treatments showed a positive correlation with NO 3 − -N but not significant. The E 4 /E 6 of M0.5 treatment had a positive correlation effect on NH 4 + -N ( P < 0.05), with decreasing E 4 /E 6 values associated with decreased NH 4 + -N content. The E 4 /E 6 of M0.5 treatment had a significant negative correlation effect on NO 3 − -N ( P < 0.01), indicating that as the degree of humification of compost increased, more NH 4 + -N was converted into NO 3 − -N, increasing the NO 3 − -N content in compost. The AOB amoA gene of the CK treatment showed a positive correlation with NH 4 + -N and NO 3 − -N but no significant relationship. The AOB amoA gene of the M0.5 treatment showed a negative correlation with NH 4 + -N ( P < 0.05) and a significant positive correlation with NO 3 − -N ( P < 0.01), indicating that the appearance of ammonia-oxidizing bacteria during the maturation phase favored an increase in NO 3 − -N content in compost. The AOB amoA gene of M1 and M2 treatments showed a negative correlation with NO 3 − -N ( P < 0.05), suggesting that high concentrations of sugar inhibited the role of ammonia-oxidizing bacteria, affecting the nitrogen conversion process (Chen et al., 2024 ). It can be seen that adding different concentrations of oligosaccharides leads to differences in the physicochemical properties of compost materials. Changes in the physicochemical properties of compost materials, especially GI and E 4 /E 6 , affect the variation of AOB amoA gene, which further influences the types and community structure of ammonia-oxidizing bacteria, thereby affecting the nitrogen transformation process. Comparing different treatments, the 0.5% oligosaccharide concentration in the M0.5 treatment is conducive to the survival of ammonia-oxidizing bacteria, increases nitrogen conversion, allows more nitrogen to exist in the form of nitrate nitrogen in compost, reduces ammonia emissions, and improves compost quality. 4 Conclusion Adding oligosaccharides to composting can increase the peak temperature, effectively kill more potential pathogens, thereby enhancing the harmlessness of composting. In the M0.5 treatment, the dominant genera of ammonia-oxidizing bacteria accounted for a higher proportion, effectively playing the role of nitrogen fixation. Therefore, the addition of 0.5% oligosaccharide treatment has higher nitrogen fertilizer utilization value. Declarations CRediT authorship contribution statement Manli Duan: Conceptualization, Funding acquisition, Methodology, Investigation, Writing - review & editing. Mingxiu Li: Investigation, Data curation, Writing - original draft, Visualization. Zhenlun Qin: Investigation, Methodology, Formal analysis. Beibei Zhou: Investigation, Methodology. Quanjiu Wang: Conceptualization, Visualization, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This study was supported by the Major Special Science and Technology Project of Xinjiang Province (2023A02002-3), National Natural Science Foundation of China (52339003), Shanxi Science and Technology Program (2024SF-YBXM-588). We thank Dr. Duncan E. Jackson for language editing. References Awasthi, M.K., Wang, M., Chen, H., et al., 2017. Heterogeneity of biochar amendment to improve the carbon and nitrogen sequestration through reduce the greenhouse gases emissions during sewage sludge composting. Bioresour. Technol. 224, 428–438. Ambrogi, V., Bottacini, F., O’Callaghan, J., et al., 2021.Infant-associated bifidobacterial β-galactosidases and their ability to synthesize galacto-oligosaccharides. Front. Microbiol. 12, 662959. Amerany, E.F., Rhazi, M., Wahbi, S., et al. 2020. The effect of chitosan, arbuscular mycorrhizal fungi, and compost applied individually or in combination on growth, nutrient uptake, and stem anatomy of tomato. Sci. Hortic. 261, 109015. 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Beneficial effects of bacterial agent/bentonite on nitrogen transformation and microbial community dynamics during aerobic composting of pig manure. Bioresour. Technol. 298, 122384. Hafsa, J., Smach, M.A., Mrid, R.B., et al., 2021. Functional properties of chitosan derivatives obtained through Maillard reaction: A novel promising food preservative. Food Chem. 349, 129072. Hadda., B.M., Imen, B.M., Rayda, C., et al., 2019. Change of soil quality based on humic acid with date palm compost incorporation. Hashimoto, T., Shimaya, C., 2005. Molecular diversity of ammonia-oxidizing bacteria in full-scale wastewater treatment plants. Water Res. 39(20), 4925–4935. Jiang, J., Liu, X., Huang, Y., et al., 2015. Inoculation with nitrogen turnover bacterial agent appropriately increasing nitrogen and promoting maturity in pig manure composting. Waste Manag. 39, 78–85. Jarvis, A., Sundberg, C., Milenkovski, S., et al., 2009. Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH 3 and N 2 O in a compost reactor treating organic household waste. J. Appl. Microbiol. 106(5), 1502–1511. Luo, W., Yuan, J., Luo, Y., et al., 2014. Effects of mixing and covering with mature compost on gaseous emissions during composting. Chemosphere. 117 (1), 14–19. Li, Q., Guo, X., Lu, Y., et al., 2016. Impacts of adding FGDG on the abundance of nitrification and denitrification functional genes during dairy manure and sugarcane pressmud co-composting. Waste Manage. 56, 63–70. Lu, Y., Gu, W., Xu, P., et al., 2018. Effects of sulphur and Thiobacillus thioparus 1904 on nitrogen cycle genes during chicken manure aerobic composting. Waste Manag. 80, 10–16. Li, H., Zhang, T., Tsang, D.C.W., et al., 2020. Effects of external additives: Biochar, bentonite, phosphate, on co-composting for swine manure and corn straw. Chemosphere. 248, 125927. Liu, T., Awasthi, S.K., Duan, Y., et al., 2020. Effect of fine coal gasification slag on improvement of bacterial diversity community during the pig manure composting. Bioresour. Technol. 304, 123024. Li, R., Wang, Q., Zhang, Z., et al., 2015. Nutrient transformation during aerobic composting of pig manure with biochar prepared at different temperatures. Environ. Technol. 36, 815–826. Liu, M., Cai, M., Ding, P., 2021. Oligosaccharides from traditional Chinese herbal medicines: A review of chemical diversity and biological activities. J. Chin. Med. 49(3), 577–608. Mao, H., Lv, Z., Li, R., et al., 2018. Improvement of biochar and bacterial powder addition on gaseous emission and bacterial community in pig manure compost. Bioresour. Technol. 258, 195–202. Oishi, R., Hirooka, K., Otawa, K., et al., 2012. Ammonia-oxidizing Archaea in laboratory-scale activated sludge systems for wastewater of low-or high-ammonium concentration. Anim. Sci. J. 83, 571–576. Rastall, R.A., 2010. Functional oligosaccharides: Application and manufacture. Annu. Rev. Food Sci. T. 1(1), 305–339. Reed, S., Knez, M., Uzan, A., et al., 2018. Alterations in the gut (Gallus gallus) microbiota following the consumption of zinc biofortified wheat (Triticum aestivum)-based diet. J. Agric. Food Chem. 66, 6291–6299. Rotthauwe, J.H., Witzel, K.P., Liesack, W., 1997. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl. Environ. Microbiol. 63, 4704–4712. Shimaya, C., Hashimoto, T., 2008. Improvement of media for thermophilic ammonia-oxidizing bacteria in compost. Soil Sci. Plant Nutr. 54(4), 529–533. Siripong, P., Rittmann, B.E., 2007. Diversity study of nitrifying bacteria in full-scale municipal wastewater treatment plants. Water Sci. Technol. 56(7), 65–73. Tian, X., Yang, T., He, J., et al., 2017. Fungal community and cellulose-degrading genes in the composting process of Chinese medicinal herbal residues. Bioresour. Technol. 241, 374–383. Taylor, A.E., Giguere, A.T., Zoebelein, C.M., et al., 2017. Modeling of soil nitrification responses to temperature reveals thermodynamic differences between ammonia-oxidizing activity of archaea and bacteria. ISME J. 11, 896–908. Wang, K., Wu, Y., Li, W., et al., 2018. Insight into effects of mature compost recycling on N 2 O emission and denitrification genes in sludge composting. Bioresour. Technol. 251, 320–326. Wang, Q., Wang, Z., Awasthi, M.K., et al., 2016. Evaluation of medical stone amendment for the reduction of nitrogen loss and bioavailability of heavy metals during pig manure composting. Bioresour. Technol. 220, 297–304. Wang, S., Zeng, Y., 2017. Ammonia emission mitigation in food waste composting: a review. Bioresour. Technol. 248. Yamada, T., Araki, S., Ikeda-Ohtsubo, W., et al., 2013. Community structure and population dynamics of ammonia oxidizers in composting processes of ammonia-rich livestock waste. Syst. Appl. Microbiol. 36(5), 359–367. Yin, Y., Song, W., Gu, J., et al., 2016. Effects of copper on the abundance and diversity of ammonia oxidizers during dairy cattle manure composting. Bioresour. Technol. 221, 181–187. Yan, L., Li, Z., Wang, G., et al., 2016. Diversity of ammonia-oxidizing bacteria and archaea in response to different aeration rates during cattle manure composting. Ecol. Eng. 93, 46–54. Yamamoto, N., Otawa, K., Nakai, Y., 2010. Diversity and abundance of ammonia-oxidizing bacteria and ammonia-oxidizing archaea during cattle manure composting. Microb. Ecol. 60, 807–815. Zhao, S., Schmidt, Susanne., Qin, W., et al., 2020. Towards the circular nitrogen economy-A global meta-analysis of composting technologies reveals much potential for mitigating nitrogen loss. Sci. Total Environ. 704, 135401. Zeng, G., Zhang, J., Chen, Y., et al., 2011. Relative contributions of archaea and bacteria to microbial ammonia oxidation differ under different conditions during agricultural waste composting. Bioresour. Technol. 102 (19), 9026–9032. Zeng, Y., De Guardia, A., Ziebal, C., et al., 2012. Nitrification and microbiological evolution during aerobic treatment of municipal solid wastes. Bioresour. Technol. 110, 144–152. Zhang, N., Jin, M., Wang, K., et al., 2022. Functional oligosaccharide fermentation in the gut: Improving intestinal health and its determinant factors-A review. Carbohydrate Polymers. 284, 119043. Zhang, J., Zeng, G., Chen, X., et al., 2011. Effects of physico-chemical parameters on the bacterial and fungal communities during agricultural waste composting. Bioresour. Technol. 102(3), 2950–2956. Supplementary Files GraphicalAbstract.doc Highlight.doc Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 16 Jun, 2024 Editor invited by journal 12 Jun, 2024 Editor assigned by journal 28 May, 2024 First submitted to journal 27 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4486496","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315012821,"identity":"3a4714f3-6629-41bc-a055-27d4860b93d9","order_by":0,"name":"Manli Duan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYDCCA8wNBz78sOHh528+AGQQpYWx8eDMnjQ5yRnHEoEM4rQ0H+ZhO2xscCDH+DAHGxE6+K4dbDjAw8Oc2HDgzIfDDDwM8vxiB/BrkbwNVCxhwZbY2Ny74XCBBYPhzNkJ+LUYgLQY8PAkNjOc3XB4Bg9DgsFtYrQksEkktjHkPAB6ilgtB9gMjHkYchiI0wLyy8HGngQ5CYljBsBAliDsF77byYc///nxn8f+fPPjD8A4leeXJqAFHUiQpnwUjIJRMApGAXYAABE9UshWTa2iAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0008-9779-2580","institution":"Xi'an University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Manli","middleName":"","lastName":"Duan","suffix":""},{"id":315012822,"identity":"98b72f6c-c5cd-40fb-b05e-b244c167c147","order_by":1,"name":"Mingxiu Li","email":"","orcid":"","institution":"Xi'an University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mingxiu","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-05-27 17:32:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4486496/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4486496/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59507599,"identity":"7652291f-5696-465a-b6fd-ac94239b6665","added_by":"auto","created_at":"2024-07-02 15:32:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23138,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature variation during composting in different treatments\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/35e60c22d9a8a42dbfca75f5.png"},{"id":59508588,"identity":"6563399e-102e-4471-9396-00d834c2a4ee","added_by":"auto","created_at":"2024-07-02 15:48:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25865,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in pH, E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e, and GI during composting in different treatments\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/94ad967758f344831774d60b.png"},{"id":59508102,"identity":"7c386897-d299-4980-b242-db44aad54374","added_by":"auto","created_at":"2024-07-02 15:40:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15201,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N during composting in different treatments\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/3fe0705fb1f12b6624ffb089.png"},{"id":59507600,"identity":"aba4e3a5-e283-45f8-9765-57f7b6b8d977","added_by":"auto","created_at":"2024-07-02 15:32:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18324,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in AOB \u003cem\u003eamoA\u003c/em\u003efunctional gene copy numbers in different treatments\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/da97437cb92e9124419d8e25.png"},{"id":59507606,"identity":"3428307d-ae23-4d37-8dac-31852ae5b8ae","added_by":"auto","created_at":"2024-07-02 15:32:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59824,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) of Ammonia-Oxidizing Bacterial Community\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/7a83d4aa23d3649826e9ea3e.png"},{"id":59507610,"identity":"7f3b92a4-752f-46f5-9326-58a01f8a8ddb","added_by":"auto","created_at":"2024-07-02 15:32:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53072,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in microbial diversity at the genus level of ammonia-oxidizing bacteria\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/18a49a4b1ec90ce5e9a237ab.png"},{"id":59507604,"identity":"ad3c7b93-e153-4bda-ac62-df11e0519f32","added_by":"auto","created_at":"2024-07-02 15:32:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":73189,"visible":true,"origin":"","legend":"\u003cp\u003eStructural Equation Model Constructing Key Factors Influencing Nitrogen Changes\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/d2d7d28f5cf7f6d31e9da4af.png"},{"id":59509209,"identity":"effa1416-8af6-478f-b4f2-4b06560dcee1","added_by":"auto","created_at":"2024-07-02 15:56:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":844295,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/cf65241f-4b0c-4820-8daa-0fe449c9f38d.pdf"},{"id":59507625,"identity":"8cbf2c87-e25e-46d2-89cd-3ab28c8b6992","added_by":"auto","created_at":"2024-07-02 15:32:19","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14873600,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.doc","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/729e4c762c7acd544b14596e.doc"},{"id":59508104,"identity":"edb75f26-0223-4c3b-b650-dc485cd87e1a","added_by":"auto","created_at":"2024-07-02 15:40:18","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11776,"visible":true,"origin":"","legend":"","description":"","filename":"Highlight.doc","url":"https://assets-eu.researchsquare.com/files/rs-4486496/v1/05980b1eaff5ef216ba014d7.doc"}],"financialInterests":"","formattedTitle":"Mechanism of oligosaccharides on nitrogen and ammonia-oxidizing microbial communities in aerobic composting processes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA large number of wastes are produced in agricultural production every year. These livestock manure and straw containing a large number of nutrients have been called \u0026quot;misplaced resources\u0026quot;. Most of these wastes have not been treated as resources, harmless and reduced, causing serious environmental pollution and threats to human health. Aerobic composting technology has been widely used in the disposal of livestock manure, agricultural straw and other wastes due to its advantages of low cost, good deodorization effect and fertilizable products. Composting technology is to transform organic materials into mature organic fertilizer containing a large number of available nitrogens, phosphorus, potassium nutrients and humus which can be absorbed and utilized by plants through high-temperature fermentation under the action of microorganisms (Tian et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). It has good ecological, economic and social benefits. However, the transformation and nitrogen fixation in composting affect the quality and environmental impact of compost, hindering the popularization of composting technology (Awasthi et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Nitrogen in compost can be released through processes such as ammonia volatilization and conversion (Fukumoto et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e; Luo et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), as well as inorganic nitrogen transformation (Chang et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been reported that nitrogen losses from composting are approximately 35\u0026ndash;71% (Jiang et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen et al., \u003cspan class=\"CitationRef\"\u003e2019a\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eNitrification controls the mineralization of nitrogen in the composting process. Mineralized nitrogen can be directly utilized by organisms and plays an important role in the process of nitrogen transformation. The ammoxidation process is the first and rate limiting step of nitrification, which determines the conversion balance between oxidized and reduced nitrogen during composting (Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), and directly or indirectly affects composting efficiency, greenhouse gases and ecological environment (Wang and Zeng, \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The key mechanism of reducing nitrogen loss and improving fertilizer nutrients during composting is to improve the reproduction and activity of ammonia oxidizing microorganisms that can transform more ammonia oxidizing nitrogen into nitrite nitrogen and further oxidize to form nitrate nitrogen. Ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) are two major ammonia oxidizing microorganisms. Some scholars have indicated that AOA may be the dominant microorganism in ammonia oxidation process, and the amount of AOA in some environments (including the composting process) is higher than AOB, and even contributes more to nitrification than AOB (Yamamoto et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, previous studies have believed that AOA mostly exists in soil and marine systems with low oxygen content (Francis et al., \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Chen, 2019a). AOB is the dominant microorganism in aerobic composting of livestock manure (Yamada et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), in which genus \u003cem\u003eNitrosomonas\u003c/em\u003e and \u003cem\u003eNitrosospira\u003c/em\u003e are important bacteria involved in nitrification (Yin et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Yamada et al. (\u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) investigated population dynamics of ammonia-oxidizing bacteria (AOB) and archaea (AOA) during ammonia-rich livestock waste-composting processes. Quantitative PCR (Q-PCR) assays showed a high relative abundance of \u0026beta;-proteobacterial AOB during ammonia oxidation but did not detect AOA in any composting stage. Yan et al. (\u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) analyzed the abundance, diversity and structure of AOB and AOA in response to different aeration rates during cattle manure composting by Q-PCR and high-throughput sequencing. The results revealed that oxygen was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) correlated with AOB diversity but not with AOA, and AOB were more important than AOA in nitrogen transformation. At the same time, AOB can encode \u003cem\u003eamoA\u003c/em\u003e genes to facilitate nitrification (Jarvis et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zeng et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, the study on the structure of ammonia oxidizing bacteria is helpful to reveal the internal mechanism of nitrification during composting.\u003c/p\u003e\n\u003cp\u003eAt present, the nitrogen loss during composting is mainly reduced by adjusting the composting parameters, adding biochar and exogenous nitrogen retention agents, and inoculating microbial agents (Yan et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mao et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Guo et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, there are still some problems, such as insignificant effects, secondary pollution, and unclear mechanisms. Oligosaccharides are straight chain or branched polymer polymerized from 2\u0026ndash;10 monosaccharides with glycosidic bond (Rastall, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), which are mainly used in food, medical treatment and livestock and poultry industry (Hafsa et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu, et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Flilkinger, et al., 2002). It is found that oligosaccharides have the characteristics of water solubility and structural stability, which are easy to be metabolized by bacteria, can reshape the microbial community structure, promote the proliferation and activity of beneficial bacteria (such as Bifidobacteria and Lactobacilli), and maintain the micro ecological balance (Ambrogi et al., 2021). Oligosaccharides have the ability to recognize, adhere to, and exclude pathogenic microorganisms (e.g., Staphylococcus aureus, Aspergillus Niger, and E. coli) (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Oligosaccharides can also promote crop growth (Amerany et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, oligosaccharides, as regulators of microbial community, have not been studied for their effect on the transformation of nitrogen during composting.\u003c/p\u003e\n\u003cp\u003eIn this study, oligosaccharides were selected to study their effects on loss and transformation of nitrogen during composting. Q-PCR and high throughput sequencing were conducted to detect the abundances of the bacterial \u003cem\u003eamoA\u003c/em\u003e genes during composting, as well as the diversity and structure of the bacterial community that harbored \u003cem\u003eamoA\u003c/em\u003e genes. The results obtained in this study may support great significance for taking appropriate measures to reduce nitrogen loss, improve nitrogen nitrification and increase the nutrients of composting products.\u003c/p\u003e\n"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Material characteristics\u003c/h2\u003e\n \u003cp\u003eThe raw compost materials used in this study comprised cow manure and wheat straw. Fresh cow dung was collected from Shanxi Qinbao Animal Husbandry Co. Ltd and wheat straw was collected from a local farm in Yangling, China. The physicochemical properties of the cow manure and wheat straw after being air-dried and broken are shown in Table \u003cspan\u003e1\u003c/span\u003e. Oligosaccharides was purchased from Henan Wanbang Industrial Co. Ltd, China. It was extracted from natural ingredients and had good acid stability and thermal stability.\u003c/p\u003e\n \u003cdiv\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003ePhysico-chemical features of compost sample\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003eProperties\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eWater content (%)\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003epH\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eTOC g/kg\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eC/N\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eTN g/kg\u003cbr\u003e\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eCow\u003cbr\u003emanure\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e7.52\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e8.41\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e292\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e17.5\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e16.7\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eWheat straw\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e5.88\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e7.61\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e414\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e60.0\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e6.9\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\u003cbr\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Experimental setup and sample collection\u003c/h2\u003e\n \u003cp\u003eThe composting experiment was conducted in the glasshouse at Xi\u0026rsquo;an University of Technology, Shanxi, China. The environmental temperature in glasshouse was controlled 30\u0026deg;C. The reactors comprised four insulated foam boxes with dimensions of: length\u0026thinsp;=\u0026thinsp;60 cm, width\u0026thinsp;=\u0026thinsp;58 cm, height\u0026thinsp;=\u0026thinsp;57 cm, and thickness\u0026thinsp;=\u0026thinsp;6 cm, which had two circular holes (2 \u0026times; 2 cm) in the top, bottom, and four walls of the foam boxes to ensure ventilation and the supply of oxygen. Cow manure and wheat straw were mixed and adjusted to a C:N ratio of 25:1. The weight of each treated composting material is 9kg, straw 3kg and cow manure 6kg. Oligosaccharides was added at four different concentrations of 0 (CK), 0.1% (M0.1), 0.5% (M0.5), 1.0% (M1), and 2.0% (M2) relative to the dry weight of the compost. Oligosaccharides powder was mixed evenly with 5.4L deionized water and balanced for 2 hours, then the mixture was poured into the composting material and mixed evenly, adjusting the water content of the compost to 60%. The composting process was conducted for 26 days, and the compost reactors were turned over on days 2, 4, 7, 15, 20, and 21 to enhance aeration.\u003c/p\u003e\n \u003cp\u003eSamples were collected in the initial phase (day 0), mesophilic phase (day 1), thermophilic phase (day 3), cooling phase (day 7), first maturation phase (day 14) and second maturation phase (day 26). According to the compost temperature, the samples were taken from the top, middle, and bottom of each reactor and mixed well, where a total of 900 g (fresh weight was collected. Each sample was divided into two parts. The first part was stored in a refrigerator at 4\u0026deg;C to determine the physical and chemical parameters. The second part was freeze dried at low temperature (Beijing Songyuan, China), crushed with a low temperature freeze-grinding machine (Retsch Z200, Germany), and sieved through a mesh of 0.5 mm, before storing at \u0026minus;\u0026thinsp;80\u0026deg;C for molecular biological experiments.\u003c/p\u003e\n \u003ch2\u003e2.3. Analytical methods\u003c/h2\u003e\n \u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.3.1. Analysis of physicochemical parameters\u003c/h2\u003e\n \u003cp\u003eThe temperature of the compost was monitored twice every day (9:00 and 19:00) at the top, core, and bottom of compost. The pH was measured with a digital pH meter after mechanically shaking the fresh sample as a suspension in water without CO\u003csub\u003e2\u003c/sub\u003e at a ratio of 1:10 (w/v) for 40 min at 200 rpm. The moisture content was measured as the loss after heating at 105\u0026deg;C for 8 h. The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e-N contents of the samples were extracted with 2 mol/L KCl and determined by Discrete Chemistry Analyzer (SmartChem450, Italy). The C/N ratio was calculated as the total organic carbon (TOC)/ total nitrogen (TN). TOC and TN were determined using elemental analyzer (Elementar, Germany) after the inorganic carbon was fully removed by 1mol/L HCl.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.3.2. DNA extraction and qPCR\u003c/h2\u003e\n \u003cp\u003eComposting samples (0.1g) were extracted using a DNeasy PowerSoil Kit (QIAGEN, Germany), according to the manufacturer\u0026rsquo;s instructions. The concentration and purity of the DNA samples were detected with an ultramicro spectrophotomete Spectrophotometer (NanoDrop2000, USA). The quality checked DNA samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C. The quantitative PCR (qPCR) was performed in triplicate on an iCycler IQ5 Thermocycler (Bio-Rad, USA) using primer sets amoA-1F(5\u0026rsquo;-GGGGTTTCTACTGGTGGT-3\u0026rsquo;) and amoA-2R (5\u0026rsquo;-CCCCTCKGSAAAGCCTTCTTC-3\u0026rsquo;) for bacterial amoA genes and Arch-amoAF(5\u0026rsquo;-STAATGGTCTGGCTTAGACG-3\u0026rsquo;) and ArchamoAR (5\u0026rsquo;-GCGGCCATCCATCTGTATGT-3\u0026rsquo;) for archaeal amoA genes (Rotthauwe et al., \u003cspan\u003e1997\u003c/span\u003e). Each 20 mL of qPCR reaction contained 10 mL of 2 \u0026times; SYBR Green PCR Master mix (Qiagen, Shanghai, China), 0.5 mL of each AOB or AOA primer (10 mM), and 1 mL of DNA template (20 ng/mL) and adjusted with sterile water to volume. The \u003cem\u003eamoA\u003c/em\u003e gene amplification was conducted using an initial denaturation step at 95\u0026deg;C for 10 min, followed by 40 cycles of 30 s at 95\u0026deg;C, 30 s at 55\u0026deg;C for AOB, 30 s at 72\u0026deg;C. Melting curve analysis was carried out to verify amplicon specificity. A negative control without DNA template was used in all of the qPCR amplifications. Standard curves were generated on the basis of serial tenfold dilution of plasmids containing cloned \u003cem\u003eamoA\u003c/em\u003e genes and ranged from 1.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e to 1.0 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e copies per assay. Amplification efficiencies of 96.68% (AOB) and 104.5% (AOA) were obtained using the slopes \u0026minus;\u0026thinsp;3.41 and \u0026minus;\u0026thinsp;3.21 of standard curve, respectively. Linearity (r\u003csup\u003e2\u003c/sup\u003e) for AOB and AOA were 0.999 and 0.998, respectively.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e2.3.3. High-throughput sequencing analysis of bacterial \u003cem\u003eamoA\u003c/em\u003e genes\u003c/h2\u003e\n \u003cp\u003eDNA sample of composts were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) for High throughput sequencing of bacterial functional gene \u003cem\u003eamoA\u003c/em\u003e using Illumina Miseq sequencing platform. QIIME 1.8.0 software was used to filter, splice and remove chimera from the raw sequencing data. Operational Taxonomic Units (OTUs) were clustered using Usearch 7.1 based on cutoff of 3% dissimilarity. Microbial composition and the nearest relatives were obtained by comparing the sequences with FGR/\u003cem\u003eamoA\u003c/em\u003e database of GeneBank (Release7.3, \u003cspan\u003e\u003cspan\u003ehttp://fungene.cme.msu.edu/\u003c/span\u003e\u003c/span\u003e). To evaluate the community richness and diversity of Ammonia-oxidizing microbial community, ACE, Chao 1 index and Shannon diversity index were calculated by Mothur 1.30.1, respectively.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll descriptive statistics for the raw data were obtained using Excel 2019 (Microsoft Office 2019, Microsoft, USA). SPSS 20.0 software was used to perform multiple comparative and Pearson correlation analysis of soil physical and chemical properties, AOB \u003cem\u003eamoA\u003c/em\u003e gene copy Numbers (ANOVA, LSD, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Linear regression plots and bar charts were produced using SigmaPlot 14.0. Heatmap analysis was conducted with R3.1.0. A structural equation modeling (SEM) was constructed using IBM SPSS AMOS 22.0 in order to study the key factors influencing nitrogen changes during the composting process.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 The effect of adding oligosaccharides on composting temperature\u003c/h2\u003e\n \u003cp\u003eThe temperature variation in composting is primarily attributed to the heat generated by microbial decomposition of soluble organic compounds such as proteins and lipids in the compost materials, providing a reference basis for the humification and stabilization of compost materials (Liu et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the temperature changes during the composting process in this study, showing a pattern of temperature elevation followed by decline, ultimately approaching ambient temperature for all treatments. By the second day of composting, except for the control group (CK), temperatures in all other treatments rose to above 50\u0026deg;C and remained in the thermophilic phase for 4\u0026ndash;5 days, meeting the requirement stipulated in the \u0026quot;Sanitary standards for composting of excrement\u0026quot; (GB7959-87) that the highest temperature during composting should exceed 50\u0026ndash;55\u0026deg;C and be maintained for 5\u0026ndash;7 days. This high-temperature stage effectively eliminates most pathogenic microorganisms and pathogens. With the increase in oligosaccharide concentration, the rate of temperature rise in the compost pile accelerated. On the second day of composting, the maximum temperatures in M2, M1, M0.5, and M0.1 reached 63.4\u0026deg;C, 61.0\u0026deg;C, 63.2\u0026deg;C, and 62.8\u0026deg;C, respectively, all higher than the maximum temperature of CK at 57.4\u0026deg;C. This may be attributed to the high concentration of oligosaccharides serving as carbon nutrients, providing ample energy for microorganisms and accelerating the mineralization process of organic matter (Chen et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). As the composting enters the secondary maturation stage, the temperatures in M0.5 and M0.1 are significantly higher than those in M2 and M1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This could be because the higher concentration of oligosaccharides is largely consumed in the early stage, leading to insufficient energy for microorganisms in the later stage and slowing down the humification process. However, the 0.5% and 0.1% oligosaccharides enhance the humification degree and organic matter stability in the later stage of composting, reducing the water-soluble nutrients available to microorganisms (Chang et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), resulting in a gradual decrease in temperature and stabilization.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 The impact of adding oligosaccharides on the physicochemical properties of compost\u003c/h2\u003e\n \u003cp\u003eThe physicochemical properties changes of CK, M0.1, M0.5, M1, and M2 are illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. From the pH variation, it can be observed that the pH of all treatments shows an initial increase followed by a decrease trend throughout the composting process. The pH variation in composting may be related to the generation of organic acids and the release of ammonia compounds from compost materials (Wang et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the early stage of composting, vigorous microbial metabolism, mineralization of nitrogenous organic matter, and continuous decomposition of organic acids lead to an increase in pH (Li et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the later stage of composting, pH gradually decreases until the end of composting when organic matter is almost completely decomposed, microbial activity declines, and pH decreases. Among them, the compost pH of the M0.5 treatment is the lowest, at 7.6. The E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e value can reflect the degree of humification and aromatization during composting, with lower values indicating higher aromaticity and more stable organic matter structure (Hadda et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). From the change in E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e values, the E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e values of CK, M0.1, M0.5, and M1 are lower, indicating higher humification degree. The E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e value of the M2 treatment is significantly higher than that of other treatments, indicating lower humification degree of the compost. Germination index (GI) is an important indicator for assessing compost toxicity, and throughout the composting process, the GI of all treatments shows an increasing trend. On the 26th day, the GI values of CK, M0.1, M0.5, M1, and M2 increased to 92.8%, 91.6%, 109.3%, 100.8%, and 89.9%, respectively, all exceeding 80%, which meets the harmless standard for composting (Li et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The GI value of M0.5 is higher than that of other treatments, indicating higher safety and harmlessness of the compost.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 The effect of adding oligosaccharides on ammonium nitrogen and nitrate nitrogen in composting\u003c/h2\u003e\n \u003cp\u003eDuring aerobic composting, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content initially increases and then decreases, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Among all treatments, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content with the addition of 0.5% oligosaccharides exhibits the largest increase on the first day, significantly higher than that of the CK treatment by 3.5 times (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This may be attributed to the provision of suitable nutritional conditions for microorganisms by 0.5% oligosaccharides in the early stage of composting, accelerating both the microbial ammonification and organic nitrogen mineralization processes. Consequently, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content continues to increase. However, in the later stage of composting, as a portion of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N is assimilated by microorganisms, some undergo nitrification to form NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and some volatilize in gaseous form, leading to a decrease in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). By the end of composting, the reduction in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N is the greatest in the M0.5 treatment, with the lowest content, indicating a conversion into other nitrogen forms.\u003c/p\u003e\n \u003cp\u003eFrom Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, it can be observed that during the temperature rise phase of composting, except for the continuous decrease in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in the M0.1 treatment, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in other treatments shows an increasing trend. During the thermophilic phase of composting, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in M0.5 and M1 is higher, exceeding that of the CK treatment by 2.56 and 3.10 mg/kg, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This may be related to the higher initial substrate NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in M0.5 and M1, leading to a higher NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content generated by nitrification. However, the increase in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N during the thermophilic phase is smaller than that during the initial phase of composting. This could be due to the excessive NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content and high temperatures, which inhibit the activity of nitrifying microorganisms to some extent, resulting in a slowdown in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N production (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). By the end of composting, with no significant differences in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content among treatments, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in M0.5 and M1 is significantly higher than that in other treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in M0.5 is significantly higher than that in M1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in M0.5 is 8.53 times that of CK and 15.36 times that of M2. This indicates that a large amount of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N has been converted into NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, increasing the available nitrogen sources in the compost.\u003c/p\u003e\n \u003cp\u003eThroughout the composting process, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in the CK and M0.1 treatments exhibit similar trends. By the end of composting, the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in the M0.1 treatment are lower than those in the CK treatment, indicating greater nitrogen loss compared to the CK treatment. To further investigate the influence of ammonia-oxidizing bacteria genes and the diversity of ammonia-oxidizing bacterial communities on nitrogen changes in composting, this study focuses on the CK, M0.5, M1, and M2 treatments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 The impact of adding oligosaccharides on the copy number of AOB \u003cem\u003eamoA\u003c/em\u003e gene\u003c/h2\u003e\n \u003cp\u003eAmmonia-oxidizing bacteria (AOB) possess the \u003cem\u003eamoA\u003c/em\u003e gene, which catalyzes the conversion of ammonium nitrogen to hydroxylamine, serving as a preliminary and rate-limiting step in nitrification, thereby driving the process forward (Guo et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The variation in AOB \u003cem\u003eamoA\u003c/em\u003e gene copies during composting is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The highest content of AOB \u003cem\u003eamoA\u003c/em\u003e gene is found in composting raw materials, but as composting temperature increases, the abundance of \u003cem\u003eamoA\u003c/em\u003e gene is rapidly inhibited. This inhibition could be attributed to the relatively high temperature and low oxygen conditions, which are unfavorable for the growth of ammonia-oxidizing bacterial communities (Reed et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Previous studies have reported that the optimal temperature range for the growth and activity of nitrifying microorganisms is 15\u0026ndash;25\u0026deg;C (Taylor et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). As the temperature decreases, the nitrifying microbial community gradually regains activity until composting enters the maturation stage, where the AOB \u003cem\u003eamoA\u003c/em\u003e gene copies are more than five times higher than during the thermophilic phase of composting. The addition of 0.5% and 1% oligosaccharide treatments significantly increases the AOB \u003cem\u003eamoA\u003c/em\u003e gene copies compared to other treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), promoting the oxidation of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N by AOB \u003cem\u003eamoA\u003c/em\u003e genes and thereby reducing ammonia emissions. However, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content remains high during the thermophilic phase, which may be related to the activity of ammonia-oxidizing archaea (AOA). Studies have reported that under high temperature and high NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N concentration conditions, AOA may dominate (Wang and Zeng, \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oishi et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zeng et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e), contributing to organic nitrogen mineralization. Meanwhile, ammonia-oxidizing bacteria (AOB) may play a significant role in the maturation stage of composting (Yamamoto et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Ammonia-Oxidizing Bacterial Community Composition Analysis\u003c/h2\u003e\n \u003cp\u003eThis study analyzed the community of ammonia-oxidizing bacteria (AOB) in CK, M0.5, M1, and M2 treatments, and assessed the similarity of bacterial communities between different treatments using Principal Coordinate Analysis (PCoA). In the PCoA plot, PC1 explained 31.06% of the variance, and PC2 explained 21.15%, indicating that these two principal axes effectively captured the differences in microbial community structures among samples. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, variations in the concentration of oligosaccharides during composting led to differences in bacterial community composition among treatments at different stages of composting. In the early stages of composting (days 3\u0026ndash;7), except for the CK treatment, the distances between other treatments were relatively small, and there were no significant differences. This might be attributed to the presence of oligosaccharides in the early composting stages, promoting microbial metabolism and resulting in similar microbial community structures. However, as composting entered the secondary maturation stage (day 13), the distances between treatments increased, and the differences became significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicated a significant change in microbial community composition under different treatments at this stage, likely due to the transformation of composting materials and microbial ecological succession. Finally, at the end of composting (day 26), the distances between CK, M0.5, M1, and M2 treatments gradually decreased, and the composition of microbial communities became more similar, indicating the stabilization of composting maturity. This further highlights the influence of oligosaccharides on microbial community structure. Understanding the dynamic changes in microbial communities during composting and the trend of composting maturation in the later stages is crucial for comprehending the composting process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Variations in Ammonia-Oxidizing Bacteria Diversity\u003c/h2\u003e\n \u003cp\u003eAmmonia-oxidizing bacteria (AOB) are a strict aerobic autotrophic microorganism widely distributed in various natural environments. In the ninth edition of Bergey\u0026apos;s Manual of Systematics of Archaea and Bacteria, they are classified into five genera: \u003cem\u003eNitrosomonas\u003c/em\u003e, \u003cem\u003eNitrosospira\u003c/em\u003e, \u003cem\u003eNitrosovibrio\u003c/em\u003e, and \u003cem\u003eNitrosococcus\u003c/em\u003e. Two dominant genera, \u003cem\u003eNitrosomonas\u003c/em\u003e and \u003cem\u003eNitrosospira\u003c/em\u003e, were detected using the AOB \u003cem\u003eamoA\u003c/em\u003e functional gene. This is consistent with the findings of Shimaya et al. (2008), who identified \u003cem\u003eNitrosomonas\u003c/em\u003e and \u003cem\u003eNitrosospira\u003c/em\u003e as dominant AOB genera in compost from agricultural waste materials.\u003c/p\u003e\n \u003cp\u003eDuring the initial stage of composting, when organic matter is relatively abundant, AOB grow well. As composting progresses and the temperature rises, the diversity of AOB from different genera changes. \u003cem\u003eNitrosomonas\u003c/em\u003e is a genus with strong tolerance, and previous studies have shown that it is a typical ammonia-oxidizing bacterium commonly found in systems with high denitrification rates. \u003cem\u003eNitrosomonas\u003c/em\u003e was significantly present throughout the composting process in the M0.5 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with its abundance maintained at a high level during the high-temperature and maturation phases of composting. Chikako Shimaya and Tomoyoshi Hashimoto successfully enriched thermophilic AOB from compost at 50\u0026deg;C, indicating that AOB may play a role in nitrogen fixation as compost matures.\u003c/p\u003e\n \u003cp\u003e3.7 Correlation Analysis of the Influence of Ammonia-Oxidizing Bacteria \u003cem\u003eamoA\u003c/em\u003e Gene on Nitrogen Changes in Composting\u003c/p\u003e\n \u003cp\u003eStructural Equation Modeling (SEM) can reveal causal relationships between different variables by fitting data to a hypothesized model (Eisenhauer et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this experiment, structural correlations were constructed using physicochemical indicators (pH, GI, E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e), AOB \u003cem\u003eamoA\u003c/em\u003e gene, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to evaluate the impact of adding different concentrations of oligosaccharides on nitrogen changes in composting, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e. GI and E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e showed significant negative correlations with AOB \u003cem\u003eamoA\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This is consistent with the results in Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, where during the high-temperature and cooling periods of composting, GI increased significantly while the copy number of AOB \u003cem\u003eamoA\u003c/em\u003e gene remained low. During the maturation phase of composting, as compost entered the humification stage, the value of E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e gradually decreased, while the copy number of AOB \u003cem\u003eamoA\u003c/em\u003e gene showed an increasing trend. This might be because ammonia-oxidizing bacteria are more concentrated during the maturation phase of composting, and the high-temperature environment is unfavorable for their survival (Yamamoto et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe pH and E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e of M1 and M2 treatments showed a positive correlation with NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N but not significant. The E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e of M0.5 treatment had a positive correlation effect on NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with decreasing E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e values associated with decreased NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content. The E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e of M0.5 treatment had a significant negative correlation effect on NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that as the degree of humification of compost increased, more NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N was converted into NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, increasing the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in compost.\u003c/p\u003e\n \u003cp\u003eThe AOB \u003cem\u003eamoA\u003c/em\u003e gene of the CK treatment showed a positive correlation with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N but no significant relationship. The AOB \u003cem\u003eamoA\u003c/em\u003e gene of the M0.5 treatment showed a negative correlation with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a significant positive correlation with NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that the appearance of ammonia-oxidizing bacteria during the maturation phase favored an increase in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in compost. The AOB \u003cem\u003eamoA\u003c/em\u003e gene of M1 and M2 treatments showed a negative correlation with NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that high concentrations of sugar inhibited the role of ammonia-oxidizing bacteria, affecting the nitrogen conversion process (Chen et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIt can be seen that adding different concentrations of oligosaccharides leads to differences in the physicochemical properties of compost materials. Changes in the physicochemical properties of compost materials, especially GI and E\u003csub\u003e4\u003c/sub\u003e/E\u003csub\u003e6\u003c/sub\u003e, affect the variation of AOB \u003cem\u003eamoA\u003c/em\u003e gene, which further influences the types and community structure of ammonia-oxidizing bacteria, thereby affecting the nitrogen transformation process. Comparing different treatments, the 0.5% oligosaccharide concentration in the M0.5 treatment is conducive to the survival of ammonia-oxidizing bacteria, increases nitrogen conversion, allows more nitrogen to exist in the form of nitrate nitrogen in compost, reduces ammonia emissions, and improves compost quality.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eAdding oligosaccharides to composting can increase the peak temperature, effectively kill more potential pathogens, thereby enhancing the harmlessness of composting. In the M0.5 treatment, the dominant genera of ammonia-oxidizing bacteria accounted for a higher proportion, effectively playing the role of nitrogen fixation. Therefore, the addition of 0.5% oligosaccharide treatment has higher nitrogen fertilizer utilization value.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManli Duan: Conceptualization, Funding acquisition, Methodology, Investigation, Writing - review \u0026amp; editing. Mingxiu Li: Investigation, Data curation, Writing - original draft, Visualization. Zhenlun Qin: Investigation, Methodology, Formal analysis. Beibei Zhou: Investigation, Methodology. Quanjiu Wang: Conceptualization, Visualization, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Major Special Science and Technology Project of Xinjiang Province (2023A02002-3), National Natural Science Foundation of China (52339003), Shanxi Science and Technology Program (2024SF-YBXM-588). We thank Dr. Duncan E. Jackson for language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAwasthi, M.K., Wang, M., Chen, H., et al., 2017. 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Technol. 102(3), 2950\u0026ndash;2956.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ammonia-oxidizing, Composting, Community structure, Oligosaccharides","lastPublishedDoi":"10.21203/rs.3.rs-4486496/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4486496/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo explore the effects of oligosaccharides on nitrogen and ammonia-oxidizing microbial communities during aerobic composting of cattle manure and straw, this study conducted composting experiments with four concentrations of oligosaccharides: 0.1% (M0.1), 0.5% (M0.5), 1.0% (M1), and 2.0% (M2), along with a control group (CK). The results indicated that different concentrations of oligosaccharides increased the peak temperatures during the thermophilic phase of composting to above 60\u0026deg;C, higher than that of the CK (57.4\u0026deg;C), while ensuring that all treatments met the requirements for harmless disposal. Particularly, the GI value of the 0.5% oligosaccharide treatment reached 109.3%, demonstrating excellent treatment efficacy. The 0.5% oligosaccharide treatment significantly increased the NO\u003csub\u003e3\u003c/sub\u003e-N content in compost (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), thereby enhancing nitrogen content. AOB \u003cem\u003eamoA\u003c/em\u003e functional gene detection identified two dominant ammonia-oxidizing bacteria, \u003cem\u003eNitrosomonas\u003c/em\u003e and \u003cem\u003eNitrosospira\u003c/em\u003e, with \u003cem\u003eNitrosomonas\u003c/em\u003e primarily present in the 0.5% oligosaccharide treatment, playing a crucial role in ammonia nitrogen fixation. SEM analysis showed a significant positive correlation between AOB \u003cem\u003eamoA\u003c/em\u003e genes and NO\u003csub\u003e3\u003c/sub\u003e-N in the 0.5% oligosaccharide treatment, indicating effective promotion of nitrogen conversion by ammonia-oxidizing bacteria in the compost. In conclusion, the addition of 0.5% oligosaccharides can increase the dominance of AOB genera, enhance nitrogen transformation during composting, provide more available nitrogen sources for crops, and thereby improve nitrogen fertilizer utilization efficiency.\u003c/p\u003e","manuscriptTitle":"Mechanism of oligosaccharides on nitrogen and ammonia-oxidizing microbial communities in aerobic composting processes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-02 15:32:13","doi":"10.21203/rs.3.rs-4486496/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2024-06-16T12:43:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2024-06-12T22:14:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-28T11:52:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2024-05-27T13:32:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d4ca45bf-af27-4561-8791-bcbb61046531","owner":[],"postedDate":"July 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-02T15:32:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-02 15:32:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4486496","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4486496","identity":"rs-4486496","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}