Microbial mechanism on the turnover of soil organic carbon responded to litter C:N ratio and incubation temperature

Microbial mechanism on the turnover of soil organic carbon responded to litter C:N ratio and incubation temperature | 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 Microbial mechanism on the turnover of soil organic carbon responded to litter C:N ratio and incubation temperature Tian Li, Shujie Miao, Qiao Yunfa, Jie Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7570212/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and aims Soil organic carbon (SOC) turnover is closely linked to global carbon cycling, yet the microbial mechanisms underlying its response to warming and litter quality remain poorly understood. Therefore, this study aimed to explore the bacterial mechanisms driving SOC turnover in response to different incubation temperature and litter C:N ratio. Methods Here, a one-year incubation experiment was conducted with three types of ¹³C-labeled litter differing in C:N ratio (11.66, 27.77, and 35.81) under two incubation temperatures (23°C and 33°C). At the end of incubation, the soil properties were measured, and soil bacterial community properties were examined using the Illumina MiSeq sequencing method. Results SOC turnover was maximized, and SOC half-life minimized, under the medium C:N litter (27.77), indicating that a C:N ratio close to microbial demand favors carbon transformation. Warming significantly accelerated SOC turnover, particularly with medium and high C:N litter additions. Bacterial α-diversity increased with medium C:N litter but declined with warming. Elevated temperature reduced the abundance of copiotrophic taxa (e.g., Proteobacteria) and enhanced oligotrophic groups (e.g., Firmicutes). Path modeling revealed that DOC and pH positively, but available nitrogen negatively, regulated the incorporation of newly derived carbon into SOC. Conclusions Our findings highlight that SOC turnover is jointly controlled by litter quality and warming, with temperature exerting a stronger influence on bacterial communities. This study provides new insights into the microbial mechanisms linking substrate quality, warming, and SOC stability, with implications for predicting soil carbon dynamics under future climate change. Soil organic carbon SOC turnover Litter C:N ratio Warming Soil bacterial community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Soil organic carbon (SOC) pool is widely concerned due to its large C storage, a small change of which plays a key role in global climate change (Batjes 1996 ; Davidson and Janssens 2006 ). Thus, SOC turnover has become a hot research topic in recent years, where referred to the relationship between SOC turnover and carbon sequestration potential (Luo et al. 2003 ), and their factors, such as soil properties (Li et al. 2019 ; Varney et al. 2020 ; Huang et al. 2023 ), exogenous carbon (Qiao et al. 2015 ), and soil microorganisms (Don et al. 2017 ). However, the mechanism of SOC turnover has always been controversial, attributed to the variability and complexity of factors. Litter serves as a key precursor to SOC in terrestrial ecosystem (Kästner et al. 2021 ), and its decomposition is primarily regulated by its C:N ratio (Kuzyakov and Blagodatskaya 2015 ; Huang 2020 ). Further, the C:N ratio (quality) of litter affects the dynamics of SOC turnover (Qiao et al. 2015 ). In agricultural ecosystems, the SOC turnover rate is larger with soybean straw (high nitrogen content, high quality) addition compared to corn straw (low nitrogen content, low quality) (Huggins et al. 2007 ). Similarly, Qiao et al. ( 2015 ) observed that introducing wheat residues with a lower C:N ratio (higher quality) reduced the SOC half-life. This might be because the addition of energy-rich litter stimulates soil microbial activity and extracellular enzyme secretion (Cheng and Kuzyakov 2015 ; Sauvadet et al. 2018 ), which increases the fraction of newly derived soil C and accelerates the decomposition of native SOC. However, other studies have found that introducing high C:N ratio legumes into grazing systems, through biological nitrogen fixation and legume litter inputs, causes nitrogen accumulation in the soil, which slows down SOC decomposition and turnover, resulting in SOC sequestration (Conrad et al. 2017 ). Therefore, there is still an open to the SOC dynamic in response to different C:N ratio litter addition. Soil temperature is regarded as one of the factors affecting soil carbon emission and SOC turnover (Dong et al. 2023 ), which might be attributed to the changes in the soil microbial survival, community activity, and composition under different temperature (Zhou et al. 2012 ; Li et al. 2019 ; Stuble et al. 2019 ). Based on the natural 13 C isotope tracing method, Liu et al. ( 2023 ) discovered that warming increased the relative change in the turnover rate (RCT) of decadally cycling soil carbon (dSOC) in microbial biomass carbon (MBC). However, some studies indicated that warming reduced microbial substrate utilization efficiency, decreasing MBC (Bradford 2013 ). Incubation experiments also indicated that warming significantly boosted the substrate utilization efficiency of soil microbes (Frey et al. 2013 ) and enhanced soil enzyme activity, particularly in colder regions (D’Alò et al. 2021 ), leading to the reduction in soil carbon turnover time (τ s ) (Varney et al. 2020 ). Even with short-term warming, soil microbial communities quickly altered their composition to adapt to environmental changes rapidly (Zhang et al. 2016 ), thus influencing SOC turnover. To reveal the microbial mechanisms of SOC turnover in response to exogenous carbon quality and soil temperature, the diversity and composition of microbial communities will inevitably be addressed since many key functions of the soil ecosystem are ultimately driven by soil microbial community (Talbot et al. 2014 ). Although scholars have already studied the effects of temperature, litter addition, and their interactions on SOC turnover (Creamer et al. 2015 ; Phillips et al. 2019 ; Li et al. 2022 ), the impacts of warming and litter C:N ratio on the diversity and composition of soil bacterial communities, as well as their relationship with SOC turnover, remains to be explored. In this study, a 1-year incubation experiment was carried out with 3-C:N ratios 13 C-labelled litters added to soils under 23℃ and 33℃ dark conditions. We hypothesized that: i) litter with a C:N ratio close to microbial requirement would favor soil bacterial community, and further accelerated SOC turnover; ii) warming would be negative to the survival of soil bacteria due to the temperature-sensitivity of microorganisms (Frindte et al., 2019 ). iii) oligotrophic-associated bacteria might be higher adaptable to warming environments than copiotrophic-associated microorganisms because their relatively slow growth and less reactive to abrupt resource availability (Kumar et al. 2023 ). Materials and methods Soil and litter preparation The soil was collected from the Hailun Agroecological Experimental Station, Chinese Academy of Sciences (126°38′E, 47°26′N), Heilongjiang province, northeast China, in 2022. The soils of 0–20 cm surface layer were collected at selected sites that had never been cultivated and transported to the Nanjing University of Information Science & Technology. According to the USDA Taxonomy, the soil was classified as Pachic Haploborolls (Service and Department 2010). In the laboratory, the soil was through a 2 mm sieve to remove visible residues. A portion of the soil was air-dried for determination of basic properties and δ 13 C abundance (Table 1), and a portion of the soil was adjusted to 60% water holding capacity (WHC) and pre-incubated for 7 days at 23°C dark condition. Three types of litter with significantly different C:N ratios (Table 1) were introduced into the soil in this study. They were Oryza sativa (C:N ratio = 11.66, thereafter referred to as L1), leaves of Acronychia pedunculata (C:N ratio = 27.77, thereafter referred to as L2), and Cryptocarya chinensis (C:N ratio = 35.81, thereafter referred to as L3). All litters used in the experiment were pulse-labeled with 13 C-CO 2 gas (Qiao et al. 2014). Litter samples were washed, blanched at 105°C for 30 min, dried at 80°C to constant weight, ground into powder using a grinder, and sieved through a 0.25 mm mesh sieve. One part of the litter powder was used to determine basic properties and δ 13 C abundance (Table 1), and another part was used for subsequent incubation experiments. Table 1 Basic properties and δ 13 C abundance of soil and litters before incubation TC (g kg − 1 ) TN (g kg − 1 ) TP (g kg − 1 ) C:N ratio δ 13 C (‰) pH soil 41.46 ± 0.85 4.42 ± 0.05 1.01 ± 0.00 9.38 ± 0.15 –26.51 5.36 ± 0.01 L1 293.70 ± 2.54c 25.19 ± 0.19a 2.97 ± 0.01a 11.66 ± 0.17c 821.7 N/A L2 337.81 ± 7.10b 12.16 ± 0.07b 2.35 ± 0.11b 27.77 ± 0.53b –12.06 N/A L3 418.44 ± 3.20a 11.69 ± 0.21b 1.27 ± 0.05c 35.81 ± 0.87a –11.21 N/A Note, Data were mean of three replicated with standard deviation. The different letter in the same line means significant difference ( P < 0.05). TC, total carbon; TN, total nitrogen; TP, total phosphorus; N/A, not available. L1, L2, and L3 represent the litter of Oryza sativa , Acronychia pedunculata , and Cryptocarya chinensis , respectively. Soil incubation experiment A two-factor completely randomized experiment was designed with litter C:N ratio (3 levels) and incubation temperature (23°C and 33°C). The ‘no-litter’ soils were seen as control treatment. Each treatment was replicated three times. Briefly, litter-carbon as the rate of 5% of total SOC was added to 30 g (dry weight) of pre-incubated soil and then mixed homogeneously. The mixture was placed into1-L Mason jars, which were covered with a semi-permeable membrane to be breathable and minimize water evaporation. During the year-long incubation (March 2023-March 2024), soil moisture was kept by weighing and adding deionized water to 60% WHC every 7 days. All Mason jars were incubated in dark and constant temperature (23°C and 33°C) incubators (Shengyuan Instrument Co., Ltd., Zhengzhou, China), separately. Laboratory analyses Soil pH was measured with a pH meter (Mettler-Toledo FE28, Shanghai, China) with soil/water (1:2.5) suspensions. The DOC contents were measured using a TOC/TN analyzer (Multi N/C 3100, Jena, Germany). An elemental analyzer (VarioEL III, Elementar, Hanau, Germany) was used to analyze the concentrations of SOC, total soil N, total C and N in litters. The total P concentrations of soil and litters were measured with a spectrometer (UVmini-1285, Shimadzu, Kyoto, Japan) using the vanadomolybdate method. MBC were determined by the chloroform fumigation-extraction method (Vance et al., 1987). Both NH 4 + -N and NO 3 - -N were determined after 1 M KCl extraction (soil: solution = 1:10) by a continuous flow analyzer (San ++ Continuous Flow Analyzer, Skalar, Breda, Netherlands). An isotope ratio mass spectrometer “Deltaplus” (IRMS, Sercon Ltd., Cheshire, UK) was used to measure the δ 13 C abundance (‰) of the litters before incubation, and soil samples before and after incubation. At the end of incubation, soil (approximately 2 g) was removed from the Mason jar using a sterile spoon and placed into sterile test tubes, which were immediately stored on dry ice and transported at -80°C until Illumina MiSeq sequencing. Soil bacterial analysis was performed by Majorbio Bio-Pharm Technology Co., ltd. (Shanghai, China). Microbial DNA was extracted using the Fast DNA SPIN Kit for Soil (Qbiogene Inc., CA, USA). Universal primers (338F: ACTCCTACGGGAGGCAGCAG, 806R: GGACTACHVGGGTWTCTAAT) were used to amplify the 16S rRNA bacterial gene of the bacterial V4–V5 region in the thermocycler PCR system (GeneAmp PCR-Systme®9700, ABI, USA). Each sample was assayed three times and mixed to reduce the error in the DNA extraction process. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA). The amplicons were sequenced with the Illumina MiSeq Platform (Illumina, San Diego, USA) using the paired-ends model (PE300). All raw sequences were analyzed using the QIIME pipeline (version 1.17; http://qiime.org/). We assigned bacterial sequences with the same barcode to the same sample, and then removed the barcode and primer sequences. Sequences with similarity above 97% were used to yield one operational taxonomic unit (OTU). Calculation of SOC turnover rate The differences in δ 13 C abundance between litter and soil was used to calculate SOC turnover with mixing model. SOC turnover was expressed as the fraction of newly derived C in SOC ( f ) and half-life time of SOC ( T half ). $$\:\begin{array}{c}f=\:\frac{{\delta\:}^{13}{C}_{soil}-{\delta\:}^{13}{C}_{soil-CK}}{{\delta\:}^{13}{C}_{litter}-\:{\delta\:}^{13}{C}_{soil-CK}}\:\times\:\:100\#\left(1\right)\end{array}$$ where δ 13 C soil and δ 13 C soil-ck is the soil δ 13 C abundance after 1 year of incubation with and without litter, respectively; δ 13 C litter is the litter δ 13 C abundance. $$\:\begin{array}{c}{T}_{half}\:=\:-0.693\:\times\:\:\frac{Y}{ln\left(1-\:\frac{f}{100}\right)}\#\left(2\right)\end{array}$$ where 0.693 is the approximate value for ln2; Y is the incubation time (years, Y = 1 in this experiment); f is the fraction of newly derived C in SOC. Statistical analysis All data were analyzed using R (version 4.3.3, R Development Core Team). Two-way analysis of variance (ANOVA) was used to test the effects of litter and temperature, as well as their interactive effects on experimental indicators (SOC turnover, soil/microbial properties). Fisher's LSD multiple comparisons were performed (at P = 0.05) to test whether the effects of adding different litter on experimental indicators at the same incubation temperature were significant to each other. Student's T test was performed (at P = 0.05) to determine the effects of two incubation temperatures on experimental indicators under the premise of adding the same litter. The α-diversity indices of Chao 1, Richness and Shannon were calculated using the online Cloud Platform of Majorbio (Shanghai, China). Correlation heatmaps were used to display the relationship between soil variables and the relative abundance of bacteria at the phylum level. Non-metric multidimensional scaling analyses (NMDS) equipped with analysis of similarities (ANOSIM) tests were conducted to show the differences in soil bacterial communities related to experimental warming and microtopography. Redundancy analysis (RDA) was performed to investigate the relationship between soil bacterial communities and soil variables. Figures were created using the online Cloud Platform of Majorbio (Shanghai, China). Random forest analysis was performed to identify the properties significantly affecting the fraction of newly derived soil C using the ‘rfPermute’ package in R (v. 4.3.3). Finally, partial least squares path modeling (PLS-PM) was conducted to further explore the potential mechanisms underlying the formation of newly derived carbon in SOC, driven by soil substrates and bacterial communities. The models were constructed using the “plspm” package in R (v. 4.3.3). Results Variations in SOC turnover and available substrates After 1-year incubation, the newly derived C in SOC ranged from 1.04% to 6.23% across temperature and litter addition (Fig. 1 a). Except for L1 treatment, the percentage of newly derived C in SOC under 33°C was significantly higher by 139.38% and 248.91% in L2 and L3 treatments compared to 23°C ( P < 0.05). Under the same temperature condition, the L2 treatment significantly increased the percentage of newly derived C in SOC compared to L1 and L3 treatments ( P < 0.05). The L3 treatment showed higher newly derived C in SOC compared to L1 at 33°C ( P 0.05). Two-way ANOVA indicated a significant interaction between temperature and litter on the newly derived C in SOC ( P < 0.001). The effects of incubation temperature and litter C:N ratio on the half-life of SOC were highly significant (Fig. 1 b, P < 0.001). Increased temperature significantly shortened the half-life time of SOC with L2 and L3 additions ( P < 0.05), except for L1 treatment. Under the same incubation temperature, the half-life of SOC followed the order of L2 < L3 < L1 at 23°C ( P < 0.05). While at 33°C, no significant difference in half-life of SOC was observed between the L2 and L3 treatments. Two-way ANOVA revealed a significant interactive effect between incubation temperature and litter C:N ratio on half-life of SOC ( P < 0.001). From Tables 2 and 3 , at 23°C, the DOC content of L2 treatment was significantly lower than those of L1 and L3 ( P < 0.05), with the reductions of 25.03% and 11.51%, respectively. Similar results were observed in the changes in NO 3 − -N and TP contents. However, the pH of L2 treatment was higher than that of L1 and significantly higher than that of L3 and CK. The NH 4 + -N content showed a significant decrease with increasing litter C:N ratio ( P < 0.05). Compared with the CK without litter addition, the soil C:N ratio at 23°C significantly decreased after litter addition ( P < 0.05), with no significant differences among the litter treatments. At 33°C, the NO 3 − -N content in the L2 treatment was significantly decreased by 33.56% and 18.47%, respectively, compared with L1 and L3 ( P < 0.05). The TN content in the L2 treatment was also significantly lower than in L1 by 5.35% ( P < 0.05). However, soil pH in the L2 treatment at 33°C increased by 1.28% and 0.91% compared to L1 and L3, respectively ( P < 0.05). NH 4 + -N content was significantly reduced in the L3 treatment ( P < 0.05), by 34.85% and 33.62% compared to L1 and L2, respectively. Similar to the results at 23°C, soil C:N ratios decreased after litter addition at 33°C, but significant differences were only observed between CK and L1 ( P < 0.05). Warming significantly increased the concentrations of DOC and NH 4 + -N by 63.40%-129.63% and 102.35%-165.26% across litter additions, respectively ( P < 0.05). However, warming significantly reduced the MBC content by 54.37%, 57.17%, and 52.90% with the addition of L1, L2, and L3, respectively ( P < 0.05). Under the L3 treatment, soil warming significantly increased soil pH (Table 2 , 3 , P < 0.05). The NH 4 + -N and TP contents were significantly affected by the interaction of litter C:N ratio and incubation temperature (Table 3 , P < 0.05). Table 2 Soil properties were measured after 1-year incubation at two different incubation temperatures (23°C and 33°C) with the additions of three different C:N ratios of litters (L1, L2 and L3). Temperature (°C) Litter SOC TN TP DOC MBC NO 3 − -N NH 4 + -N pH Soil C:N ratio (g kg − 1 ) (mg kg − 1 ) 23 L1 34.62 ± 0.59Aa 4.00 ± 0.04Aa 1.05 ± 0.03Aa 182.96 ± 8.98Ab 501.66 ± 56.50Aa 131.38 ± 2.92Aa 34.95 ± 1.09Ab 5.45 ± 0.03ABa 8.66 ± 0.12Ba L2 35.11 ± 0.14Aa 3.98 ± 0.01Aa 0.77 ± 0.03Ba 137.17 ± 2.09Cb 563.84 ± 29.27Aa 85.18 ± 2.21Da 29.36 ± 0.36Bb 5.51 ± 0.03Aa 8.83 ± 0.01Ba L3 34.22 ± 0.93Aa 3.95 ± 0.10Aa 1.11 ± 0.03Aa 155.01 ± 3.04Bb 523.12 ± 16.60Aa 100.00 ± 0.88Ca 25.55 ± 0.52Cb 5.41 ± 0.04Bb 8.68 ± 0.21Ba CK 34.30 ± 0.74Aa 3.66 ± 0.01Ba 0.67 ± 0.07Bb 171.12 ± 1.52Ab 471.06 ± 32.05Aa 123.68 ± 1.38Ba 27.85 ± 1.62BCb 5.42 ± 0.01Ba 9.37 ± 0.17Aa 33 L1 33.65 ± 0.16Aa 4.11 ± 0.06Aa 0.93 ± 0.04Aa 298.95 ± 0.51Aa 228.90 ± 18.16Ab 127.44 ± 4.16Aa 79.36 ± 2.33Aa 5.47 ± 0.02BCa 8.19 ± 0.09Bb L2 34.11 ± 0.05Ab 3.89 ± 0.04Ba 0.93 ± 0.07Aa 314.98 ± 33.03Aa 241.51 ± 15.65Ab 84.67 ± 1.96Ca 77.88 ± 0.21Aa 5.54 ± 0.01Aa 8.76 ± 0.09ABa L3 34.68 ± 0.53Aa 3.90 ± 0.06Ba 1.01 ± 0.09Aa 289.05 ± 13.73Aa 246.41 ± 24.68Ab 103.85 ± 2.66Ba 51.70 ± 9.20Ba 5.49 ± 0.01Ba 8.89 ± 0.27Aa CK 34.13 ± 0.32Aa 3.69 ± 0.04Ca 0.95 ± 0.03Aa 306.78 ± 7.20Aa 193.08 ± 8.23Ab 121.47 ± 3.69Aa 61.27 ± 5.42ABa 5.44 ± 0.02Ca 9.26 ± 0.19Aa Note, Data were mean of three replicated with standard deviation. Different capital letters indicate significant differences ( P < 0.05) between the additions of three different litters at the same temperature, whereas different lowercase letters indicate significant differences ( P < 0.05) between two incubation temperatures when the same litter was added. SOC, soil organic carbon; DOC, dissolved organic carbon; MBC, microbial biomass carbon; NO 3 − -N, nitrate nitrogen; NH 4 + -N, ammonium nitrogen; TN, total nitrogen; TP, total phosphorus. Table 3 Two-way ANOVA ( F and P -values) for the response of pH, SOC content, DOC content, MBC content, NO 3 − -N content, NH 4 + -N content, TN content, TP content, and C:N ratio to the incubation temperature (T), litter C:N ratio (L), and their interaction (T × L). Temperature Litter T×L F P F P F P pH 5.923 0.027 6.077 0.006 0.679 0.578 SOC 1.307 0.270 0.348 0.791 0.894 0.466 DOC 222.352 < 0.001 0.982 0.426 1.921 0.167 MBC 200.192 < 0.001 2.214 0.126 0.331 0.803 NO 3 − -N 0.136 0.717 112.969 < 0.001 0.771 0.527 NH 4 + -N 188.203 < 0.001 9.245 0.001 3.377 0.044 TN 0.008 0.932 17.140 < 0.001 1.203 0.340 TP 2.311 0.148 8.796 0.001 6.191 0.005 Soil C:N ratio 0.867 0.366 9.858 0.001 1.420 0.274 Soil bacterial relative abundance and its relationships with soil variables A total of 22 bacterial phyla were identified in the soil samples. The bacterial communities were dominated by Actinobacteriota, Firmicutes, Proteobacteria, Chloroflexi, and Acidobacteriot. The five phyla together accounted for more than 90% of the total bacterial abundance (Fig. 2 a). The Kruskal-Wallis H test on the top 10 most abundant bacterial phyla indicated that, at the same incubation temperature, different C:N ratio litter additions did not significantly affect the relative abundance of these phyla (Fig. 2 b, c, P > 0.05). Student’s t-test results showed that higher incubation temperature led to an increased relative abundance of Firmicutes, while decreasing the relative abundances of other major phyla (Proteobacteria, Chloroflexi, Acidobacteriota, etc.) with the same litter addition (Fig. 3 , P < 0.05). Furthermore, it was observed that the relative abundance of Actinobacteriota showed an increasing trend following litter addition and higher incubation temperature, though the increase was not statistically significant (Fig. 3 b, c, d, P > 0.05). At 33 ℃, the relative abundance of Firmicutes, the representative oligotrophic phylum, showed the largest increase (25.92%) after L3 addition compared to 23 ℃. However, under high temperature conditions, there were no significant differences in the changes in the relative abundances of other oligotrophic bacterial phyla irrespective of litter C:N ratios. The relative abundance of representative copiotrophic bacterial phyla, such as Proteobacteria and Bacteroidota, was decreased significantly by warming, but the decrease was not significantly different across different C:N ratio litters. The correlation heatmap revealed that only Gemmatimonadota exhibited a significantly negative correlation with soil pH at 23°C (Fig. 4 a). Firmicutes showed a significantly negative correlation with soil MBC and NH 4 + -N content at 33°C (Fig. 4 b). Acidobacteriota and Gemmatimonadota exhibited significantly negative correlation with TN content, while positively correlated with soil C:N ratio. Myxococcota and Bacteroidota displayed significantly negative correlations with soil NO 3 − -N content. Diversity and structure of soil bacterial community, and their relationships with soil variables Two-way ANOVA revealed that both incubation temperature and the C:N ratio of litter significantly influenced the α-diversity of soil bacterial community (Fig. 5 a, b, c, P < 0.05). However, their interaction effect only was observed on the Richness index (Fig. 5 c, P < 0.05). Warming significantly reduced soil bacterial α-diversity (Fig. 5 a, b, c). At 23°C, the addition of L2 significantly increased the Shannon index compared to CK and L1, but no difference in the Chao 1 and Richness indices (Fig. 5 a, b, c). At 33°C, different litter C:N ratios did not affect the Shannon index (Fig. 5 b, P > 0.05), while the Chao1 and Richness indices were in the order of L2 > L1 > L3 > CK (Fig. 5 a, c, P < 0.05). The results of NMDS indicated that bacterial communities were distinctly separated by different incubation temperatures (stress = 0.032, P = 0.001, Fig. 5 d). The variation in bacterial communities could be explained by different C:N ratios litter at 23°C (stress = 0.047, P = 0.004) (Fig. 5 e), but could not at 33°C (stress = 0.039, P = 0.084, Fig. 5 f). The results of ANOSIM indicated that the effect of incubation temperature on the shift of soil bacterial communities was stronger than litter C:N ratio (Fig. 5 d, e, f). RDA results showed that TN content was the primary environmental factor significantly affecting bacterial communities at 23°C ( P = 0.002), followed by NH 4 + -N content ( P = 0.011) and soil C:N ratio (Fig. 6 a, P = 0.011). At 33°C, NH 4 + -N content was the significant environmental factor influencing bacterial communities (Fig. 6 b, P = 0.015). The remaining environmental factors had no significant effects on the soil microbial community ( P > 0.05). Pathways of SOC turnover regulated by soil variables and bacterial communities Random forest analysis (Fig. 7 a) indicated that soil NO 3 − -N, NH 4 + -N, DOC concentration, and pH were the primary soil factors regulating the fraction of newly derived C in SOC.The C:N ratio of exogenous litter and incubation temperature emerged as the dominant external drivers. In addition, Richness, Chao1 index, and bacterial community composition (NMDS 1) were identified as the critical biological factors influencing SOC turnover. The partial least squares path modeling (PLS-PM) was developed based on the key drivers identified by the random forest analysis. PLS-PM explained 75% of the total variance in the fraction of newly derived C in SOC (Fig. 7 b). Soil DOC and pH had significant positive effects on the fraction of newly derived C in SOC, with standardized coefficients of + 0.75 and + 0.30, respectively ( P < 0.05). In contrast, available nitrogen exhibited a strong negative effect on the fraction of newly derived C (coefficient = − 0.87, P < 0.001). The pathway from soil bacterial diversity to the fraction of newly derived C in SOC showed a non-significant positive effect. Incubation temperature indirectly affected newly derived C via its positive associations with carbon substrates and available nitrogen, with a total effect of + 0.52. Similarly, the litter C:N ratio had a direct negative effect on available nitrogen (coefficient = − 0.66, P < 0.001), thereby indirectly enhancing the fraction of newly derived C, with a total effect of + 0.48 (Fig. 7 a, b). In summary, elevated incubation temperature affected newly derived C in SOC by increasing carbon substrates and available nitrogen, as well as by altering bacterial community composition and diversity. In contrast, the litter C:N ratio primarily regulated newly derived C through its impact on available nitrogen. The total effects of soil bacterial community composition and diversity on the fraction of newly derived C in SOC were − 0.21 and + 0.34, respectively (Fig. 7 c). Discussion Bacterial community differed over litters and temperature The addition of different C:N ratio litters had a significant effect on the α-diversity and structure of bacterial community (Fig. 5 , Fig. 7 ). Given the absence of relative abundance of bacteria at phylum level (Fig. 2 ), the shift in community composition is most likely primarily a response to the C:N ratio in litters, which serves as basic substrate for microbial growth and metabolism (Zeba et al. 2024 ). Previous studies had shown the responses of microbial activity and community structure to litters (Wickings et al. 2012 ; Allison et al. 2013 ; Kästner et al. 2021 ). As our first hypothesis that the C:N ratio close to the requirement of microbial community would favor bacterial survival and activity, the higher Chao1, Shannon, and Richness indexes were observed in L2 treatment compared to L1 or L3 (Fig. 5 ). The L2 also decreased oligotrophic populations (e.g. Firmicutes), and increased copiotrophic populations (e.g. Bacteroidetes), which was supported by a meta-analysis on straw return (Zhang 2024 ). As litter was broken down, more available N (e.g., NO 3 − -N and NH 4 + -N) was released to soil (Fig. 7 ), which altered the chemical profile of soil over time and influenced soil bacterial community structure (Fig. 4 , Fig. 6 ). Previous results had supported the responses of soil microbial communities to external environmental conditions (Dong et al. 2023 ), and the correlation heatmap and RDA results in the present study (Fig. 4 , Fig. 6 ). All these indicated that soil bacterial community was more sensitive to change in around 25 C:N ratio litter, irrespective of its source. In line with these findings, we also expected that temperature negatively influenced bacterial survival and shift bacterial community composition. As our second expectation, soil bacterial composition was separated by temperature (Fig. 5 d, P < 0.05), which was due to the great sensitivity of soil bacteria to temperature variation (Biasi et al. 2005 ; Curiel Yuste et al. 2007 ; Frindte et al. 2019 ). It also modified the relative abundance of soil bacteria, with a notable increase in Firmicutes (Fig. 4 ). Firmicutes, as a typical oligotrophic group, exhibited slow growth but prioritize the mineralization recalcitrant SOC (Zhang 2024 ), which was reflected in the rapid increase in SOC turnover rate (Fig. 1 ). All these supported the third hypothesis. Moreover, the oligotrophic populations showed stronger adaptability under higher C:N ratio conditions, whereas the copiotrophic populations did not exhibit more tolerant under lower C:N ratio conditions (Fig. 3 ). This might because the 33℃ incubated temperature exceeded the tolerant limits of copiotrophic bacteria, thus temperature rather than litter quality controlled their relative abundances. However, the favorable C:N ratio around 25 litter addition reduced the negative impact of high temperature on soil bacterial alpha diversity (Fig. 5 a, c). These results suggested that the favorable litter would alter the impact of warming on soil bacterial communities, further influencing SOC turnover. Optimal litter C:N ratio favored to SOC turnover and associated with temperature L2 induced the largest SOC turnover rate (indicated by the fraction of newly-derived SOC or half-life time of SOC), followed by L3 and L1 (Fig. 1 a, b), which confirmed our first hypothesis that the C:N ratio of litters close to microbial requirement favors to SOC turnover. This result was supported by previous studies (Parton et al., 2007 ; Prescott, 2010 ; Zhou et al., 2019 ). The greatest SOC turnover with L2 addition is likely due to the C:N ratio of L2 is between 25–30, which aligns with the optimal average C:N requirement for microbial survival (Brady and Weil 2001 ; Lyu et al. 2023 ). This was strongly supported by the significant increase in the bacterial diversity with L2 addition (Fig. 5 a, b, c). Moreover, the activated bacterial community rapidly and extensively consumed available soil carbon and nitrogen for "microbial turnover" (Liang et al. 2017 ), converting exogenous carbon into microbial residues (Kallenbach et al. 2016 ), ultimately increased the fraction of newly derived C in SOC (Fig. 1 a). These could be explained by the lower soil DOC and NO 3 − -N contents with L2 addition than L1 or L3 (Table 2 ,Table 3 ). Moreover, L2 shift bacterial community towards copiotrophic types (Fig. 2 , Fig. 5 ), that preferentially utilizes labile substrates (Fierer et al. 2005 ). Copiotrophic bacteria, known for their high metabolic activity (Eilers et al. 2010 ; Ramirez et al. 2010 ), can secrete more extracellular enzymes (such as polyphenol oxidase and peroxidase) (Creamer et al. 2015 ; Guo et al. 2018 ; Yang et al. 2021 ), to decompose native SOC. The present results indicated that the enhanced SOC turnover might be due to the increased soil bacterial diversity driven by higher substrate availability (Fig. 7 ). Elevated temperature favored to SOC turnover with higher C:N ratio litters addition, which was supported by the increased the fraction of newly derived soil C and shortened the SOC half-life in the L2 and L3 treatments (Fig. 1 a, b), suggesting the positive influence of temperature on SOC turnover depended on exogenous C quality. This might be due to the activity and composition of microbial community derived by exogenous C addition as above mention. The activated microbial community significantly increased the decomposition rate of litter, which induced the increase in soil DOC and NH 4 + -N contents, but decreased MBC content (Table 2 , Table 3 ; Peplau et al., 2021 ). All these would meet the nutrient requirement of soil microbial to favor SOC turnover. Overall, the effects of elevated temperature on SOC turnover are likely related to changes in nutrient provision, including both C and N. In addition to C and N, elevated temperature accelerated the desorption rate of SOC-minerals to increase the availability of substrate (Ten Hulscher and Cornelissen, 1996; Conant et al., 2011 ). Here, we did not examine in detail, more research is needed to fully understand the effects of temperature on sorption-desorption of SOC minerals, and their relationships with exogenous C quality. Conclusion Litters, their C:N ratios close to microbial requirements, increased the diversity and richness of soil bacterial community, and shifted the bacterial community to copiotrophic lifestyles, which would enhance the mineralization of available substrates. Soil warming decreased soil bacterial diversity, but caused the increase in the relative abundance of the oligotrophic phylum, which would be benefit to recalcitrant material decomposition. Moreover, oligotrophic bacteria was observed stronger high-temperature adaptability with a higher C:N ratio litter addition. We also demonstrated the importance of temperature for the changes in the soil bacterial community, which is greater than the C:N ratio of litter. Declarations Acknowledgements The authors are grateful for the insightful comments suggested by the editor. Author contributions Tian Li: Conceptualization, Investigation, Formal analysis, Visualization, Writing - Original Draft; Shujie Miao: Methodology and Writing - Review & Editing; Yunfa Qiao: Conceptualization, Methodology and review; Jie Yu: Software and Conceptualization. Funding This research was supported by National Natural Science Foundation of China (42177279); Special Funds for Scientific and Technological Innovation of Jiangsu province, China (BE2023400-02). 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16:04:20","extension":"xml","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149855,"visible":true,"origin":"","legend":"","description":"","filename":"PLSOD25034470structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/ae8c440e2bcd63f53275be9c.xml"},{"id":92279163,"identity":"28384368-99c4-42b2-810d-973e8cb14b2a","added_by":"auto","created_at":"2025-09-26 15:56:20","extension":"html","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158523,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/5943f7a9dd7ddc2e523f1900.html"},{"id":92279414,"identity":"a01882f8-f180-48ec-a08e-3e5430dfbbf4","added_by":"auto","created_at":"2025-09-26 16:04:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":349863,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of litters with different C:N ratios at different incubation temperatures on the fraction of newly derived soil C (\u003cstrong\u003ea\u003c/strong\u003e) and the half-life time of SOC turnover (\u003cstrong\u003eb\u003c/strong\u003e) after 1 year of incubation. Different capital letters indicated significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) between the addition of three different litters under 23°C condition, different lowercase letters indicated significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) between the addition of three different litters under 33°C condition, while stars above the bars indicated significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) between two incubation temperatures with the same litter addition. Bars indicated standard errors of means (n = 3).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/e0d0b3f801935293624757f1.png"},{"id":92279121,"identity":"644c49dd-ac92-44f6-9aa5-6bb98b4f3c04","added_by":"auto","created_at":"2025-09-26 15:56:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1160732,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of bacteria at the phylum level (\u003cstrong\u003ea\u003c/strong\u003e) and changes in relative abundance of the top 10 phyla across L1, L2, and L3 additions at 23 ℃ (\u003cstrong\u003eb\u003c/strong\u003e) and 33 ℃ (\u003cstrong\u003ec\u003c/strong\u003e). CK represents no litter addition.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/782d5711e6be3a63aa9823ac.png"},{"id":92279122,"identity":"9db189d1-b4ed-45e2-8e93-76a831e7daba","added_by":"auto","created_at":"2025-09-26 15:56:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2076064,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of the top 10 phyla between 23°C and 33°C treatment of CK (\u003cstrong\u003ea\u003c/strong\u003e), L1 (\u003cstrong\u003eb\u003c/strong\u003e), L2 (\u003cstrong\u003ec\u003c/strong\u003e), and L3 (\u003cstrong\u003ed\u003c/strong\u003e). Data are presented as a difference between proportions. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/55b5af6c7d753372a992ed5a.png"},{"id":92280631,"identity":"5e258c0f-9154-41fa-868a-4092980d82c8","added_by":"auto","created_at":"2025-09-26 16:12:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1082381,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of soil variables and the relative abundance of bacteria at the phyla level of 23°C (\u003cstrong\u003ea\u003c/strong\u003e) and 33°C (\u003cstrong\u003eb\u003c/strong\u003e). * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. SOC, soil organic carbon; DOC, dissolved organic carbon; MBC, microbial biomass carbon; NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, nitrate nitrogen; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, ammonium nitrogen; TN, total nitrogen; TP, total phosphorus.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/f2974c70f716dcc862809f37.png"},{"id":92279413,"identity":"16a12fee-c613-4e82-b33f-57560a4eebaa","added_by":"auto","created_at":"2025-09-26 16:04:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1149727,"visible":true,"origin":"","legend":"\u003cp\u003eSoil bacterial community diversity and composition. (\u003cstrong\u003ea\u003c/strong\u003e) Chao 1 index. (\u003cstrong\u003eb\u003c/strong\u003e) Shannon index. (\u003cstrong\u003ec\u003c/strong\u003e)Richness index. (\u003cstrong\u003ed\u003c/strong\u003e) Bacterial community composition of two incubation temperature indicated by the NMDS based on the Bray-Curtis dissimilarity. (\u003cstrong\u003ee\u003c/strong\u003e) Bacterial community composition under a 23°C incubation temperature indicated by NMDS under no-litter addition (CK), L1 addition, L2 addition, and L3 addition. (\u003cstrong\u003ef\u003c/strong\u003e) Bacterial community composition under a 33°C incubation temperature indicated by NMDS under no-litter addition (CK), L1 addition, L2 addition, and L3 addition.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/641d4160b8542089417a9e56.png"},{"id":92279418,"identity":"237743f2-068c-4794-a03e-226b8891ebcc","added_by":"auto","created_at":"2025-09-26 16:04:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1301747,"visible":true,"origin":"","legend":"\u003cp\u003eRDA analysis of the relationship between bacterial structure at the OTU level and soil variables of 23°C (\u003cstrong\u003ea\u003c/strong\u003e) and 33°C (\u003cstrong\u003eb\u003c/strong\u003e). SOC, soil organic carbon; DOC, dissolved organic carbon; MBC, microbial biomass carbon; NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, nitrate nitrogen; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, ammonium nitrogen; TN, total nitrogen; TP, total phosphorus.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/03e8c0284ccd74f4c3d05e39.png"},{"id":92279125,"identity":"74708c3e-eb20-4412-a71b-034fb1454384","added_by":"auto","created_at":"2025-09-26 15:56:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1814893,"visible":true,"origin":"","legend":"\u003cp\u003eRandom forest analysis indicating the effects of influencing factors for the fraction of newly derived C in SOC (\u003cstrong\u003ea\u003c/strong\u003e).Percentage increases in the MSE (mean squared error) of variables was used to estimate the importance of these predictors, and higher MSE% values implied more important predictors. Path analysis (Partial least squares path modeling, PLS-PM) of the effects of litter C:N and incubation temperature on the fraction of newly derived C in SOC (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e). Red and blue arrows indicate positive and negative flows of causality, respectively. Non-significant effects are represented by dashed arrows. “Composition” is indicated by NMDS 1. NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, nitrate nitrogen; DOC, dissolved organic carbon; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, ammonium nitrogen; MBC, microbial biomass carbon; SOC, soil organic carbon; Soil TN, soil total nitrogen; Soil TP, soil total nitrogen phosphorus. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/4a1f172943c99275489190fc.png"},{"id":93881224,"identity":"2a140359-a61e-4c04-9ac9-84bec851a14e","added_by":"auto","created_at":"2025-10-19 16:32:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10219272,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7570212/v1/f2e078f9-7996-437a-8480-dc10bf06c497.pdf"}],"financialInterests":"","formattedTitle":"Microbial mechanism on the turnover of soil organic carbon responded to litter C:N ratio and incubation temperature","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil organic carbon (SOC) pool is widely concerned due to its large C storage, a small change of which plays a key role in global climate change (Batjes \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Davidson and Janssens \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Thus, SOC turnover has become a hot research topic in recent years, where referred to the relationship between SOC turnover and carbon sequestration potential (Luo et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and their factors, such as soil properties (Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Varney et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), exogenous carbon (Qiao et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and soil microorganisms (Don et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the mechanism of SOC turnover has always been controversial, attributed to the variability and complexity of factors.\u003c/p\u003e\u003cp\u003eLitter serves as a key precursor to SOC in terrestrial ecosystem (K\u0026auml;stner et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and its decomposition is primarily regulated by its C:N ratio (Kuzyakov and Blagodatskaya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Huang \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Further, the C:N ratio (quality) of litter affects the dynamics of SOC turnover (Qiao et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In agricultural ecosystems, the SOC turnover rate is larger with soybean straw (high nitrogen content, high quality) addition compared to corn straw (low nitrogen content, low quality) (Huggins et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Similarly, Qiao et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) observed that introducing wheat residues with a lower C:N ratio (higher quality) reduced the SOC half-life. This might be because the addition of energy-rich litter stimulates soil microbial activity and extracellular enzyme secretion (Cheng and Kuzyakov \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sauvadet et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which increases the fraction of newly derived soil C and accelerates the decomposition of native SOC. However, other studies have found that introducing high C:N ratio legumes into grazing systems, through biological nitrogen fixation and legume litter inputs, causes nitrogen accumulation in the soil, which slows down SOC decomposition and turnover, resulting in SOC sequestration (Conrad et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, there is still an open to the SOC dynamic in response to different C:N ratio litter addition.\u003c/p\u003e\u003cp\u003eSoil temperature is regarded as one of the factors affecting soil carbon emission and SOC turnover (Dong et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which might be attributed to the changes in the soil microbial survival, community activity, and composition under different temperature (Zhou et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Stuble et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Based on the natural \u003csup\u003e13\u003c/sup\u003eC isotope tracing method, Liu et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) discovered that warming increased the relative change in the turnover rate (RCT) of decadally cycling soil carbon (dSOC) in microbial biomass carbon (MBC). However, some studies indicated that warming reduced microbial substrate utilization efficiency, decreasing MBC (Bradford \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Incubation experiments also indicated that warming significantly boosted the substrate utilization efficiency of soil microbes (Frey et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and enhanced soil enzyme activity, particularly in colder regions (D\u0026rsquo;Al\u0026ograve; et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), leading to the reduction in soil carbon turnover time (τ\u003csub\u003es\u003c/sub\u003e) (Varney et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Even with short-term warming, soil microbial communities quickly altered their composition to adapt to environmental changes rapidly (Zhang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), thus influencing SOC turnover.\u003c/p\u003e\u003cp\u003eTo reveal the microbial mechanisms of SOC turnover in response to exogenous carbon quality and soil temperature, the diversity and composition of microbial communities will inevitably be addressed since many key functions of the soil ecosystem are ultimately driven by soil microbial community (Talbot et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although scholars have already studied the effects of temperature, litter addition, and their interactions on SOC turnover (Creamer et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Phillips et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the impacts of warming and litter C:N ratio on the diversity and composition of soil bacterial communities, as well as their relationship with SOC turnover, remains to be explored.\u003c/p\u003e\u003cp\u003eIn this study, a 1-year incubation experiment was carried out with 3-C:N ratios \u003csup\u003e13\u003c/sup\u003eC-labelled litters added to soils under 23℃ and 33℃ dark conditions. We hypothesized that: i) litter with a C:N ratio close to microbial requirement would favor soil bacterial community, and further accelerated SOC turnover; ii) warming would be negative to the survival of soil bacteria due to the temperature-sensitivity of microorganisms (Frindte et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). iii) oligotrophic-associated bacteria might be higher adaptable to warming environments than copiotrophic-associated microorganisms because their relatively slow growth and less reactive to abrupt resource availability (Kumar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eSoil and litter preparation\u003c/p\u003e\n\u003cp\u003eThe soil was collected from the Hailun Agroecological Experimental Station, Chinese Academy of Sciences (126°38′E, 47°26′N), Heilongjiang province, northeast China, in 2022. The soils of 0–20 cm surface layer were collected at selected sites that had never been cultivated and transported to the Nanjing University of Information Science \u0026amp; Technology. According to the USDA Taxonomy, the soil was classified as Pachic Haploborolls (Service and Department 2010). In the laboratory, the soil was through a 2 mm sieve to remove visible residues. A portion of the soil was air-dried for determination of basic properties and δ\u003csup\u003e13\u003c/sup\u003eC abundance (Table 1), and a portion of the soil was adjusted to 60% water holding capacity (WHC) and pre-incubated for 7 days at 23°C dark condition.\u003c/p\u003e\n\u003cp\u003eThree types of litter with significantly different C:N ratios (Table 1) were introduced into the soil in this study. They were \u003cem\u003eOryza sativa\u003c/em\u003e (C:N ratio = 11.66, thereafter referred to as L1), leaves of \u003cem\u003eAcronychia pedunculata\u003c/em\u003e (C:N ratio = 27.77, thereafter referred to as L2), and \u003cem\u003eCryptocarya chinensis\u003c/em\u003e (C:N ratio = 35.81, thereafter referred to as L3). All litters used in the experiment were pulse-labeled with \u003csup\u003e13\u003c/sup\u003eC-CO\u003csub\u003e2\u003c/sub\u003e gas (Qiao et al. 2014). Litter samples were washed, blanched at 105°C for 30 min, dried at 80°C to constant weight, ground into powder using a grinder, and sieved through a 0.25 mm mesh sieve. One part of the litter powder was used to determine basic properties and δ\u003csup\u003e13\u003c/sup\u003eC abundance (Table 1), and another part was used for subsequent incubation experiments.\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eBasic properties and δ\u003csup\u003e13\u003c/sup\u003eC abundance of soil and litters before incubation\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTC (g kg\u003csup\u003e− 1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTN (g kg\u003csup\u003e− 1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTP (g kg\u003csup\u003e− 1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC:N ratio\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eδ\u003csup\u003e13\u003c/sup\u003eC (‰)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.46 ± 0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.42 ± 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01 ± 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.38 ± 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e–26.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.36 ± 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e293.70 ± 2.54c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.19 ± 0.19a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.97 ± 0.01a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.66 ± 0.17c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e821.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e337.81 ± 7.10b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.16 ± 0.07b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.35 ± 0.11b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.77 ± 0.53b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e–12.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e418.44 ± 3.20a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.69 ± 0.21b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.27 ± 0.05c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.81 ± 0.87a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e–11.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eNote, Data were mean of three replicated with standard deviation. The different letter in the same line means significant difference (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). TC, total carbon; TN, total nitrogen; TP, total phosphorus; N/A, not available. L1, L2, and L3 represent the litter of \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eAcronychia pedunculata\u003c/em\u003e, and \u003cem\u003eCryptocarya chinensis\u003c/em\u003e, respectively.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003eSoil incubation experiment\u003c/p\u003e\n\u003cp\u003eA two-factor completely randomized experiment was designed with litter C:N ratio (3 levels) and incubation temperature (23°C and 33°C). The ‘no-litter’ soils were seen as control treatment. Each treatment was replicated three times. Briefly, litter-carbon as the rate of 5% of total SOC was added to 30 g (dry weight) of pre-incubated soil and then mixed homogeneously. The mixture was placed into1-L Mason jars, which were covered with a semi-permeable membrane to be breathable and minimize water evaporation. During the year-long incubation (March 2023-March 2024), soil moisture was kept by weighing and adding deionized water to 60% WHC every 7 days. All Mason jars were incubated in dark and constant temperature (23°C and 33°C) incubators (Shengyuan Instrument Co., Ltd., Zhengzhou, China), separately.\u003c/p\u003e\n\u003cp\u003eLaboratory analyses\u003c/p\u003e\n\u003cp\u003eSoil pH was measured with a pH meter (Mettler-Toledo FE28, Shanghai, China) with soil/water (1:2.5) suspensions. The DOC contents were measured using a TOC/TN analyzer (Multi N/C 3100, Jena, Germany). An elemental analyzer (VarioEL III, Elementar, Hanau, Germany) was used to analyze the concentrations of SOC, total soil N, total C and N in litters. The total P concentrations of soil and litters were measured with a spectrometer (UVmini-1285, Shimadzu, Kyoto, Japan) using the vanadomolybdate method. MBC were determined by the chloroform fumigation-extraction method (Vance et al., 1987). Both 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 were determined after 1 M KCl extraction (soil: solution = 1:10) by a continuous flow analyzer (San\u003csup\u003e++\u003c/sup\u003e Continuous Flow Analyzer, Skalar, Breda, Netherlands). An isotope ratio mass spectrometer “Deltaplus” (IRMS, Sercon Ltd., Cheshire, UK) was used to measure the δ\u003csup\u003e13\u003c/sup\u003eC abundance (‰) of the litters before incubation, and soil samples before and after incubation.\u003c/p\u003e\n\u003cp\u003eAt the end of incubation, soil (approximately 2 g) was removed from the Mason jar using a sterile spoon and placed into sterile test tubes, which were immediately stored on dry ice and transported at -80°C until Illumina MiSeq sequencing. Soil bacterial analysis was performed by Majorbio Bio-Pharm Technology Co., ltd. (Shanghai, China). Microbial DNA was extracted using the Fast DNA SPIN Kit for Soil (Qbiogene Inc., CA, USA). Universal primers (338F: ACTCCTACGGGAGGCAGCAG, 806R: GGACTACHVGGGTWTCTAAT) were used to amplify the 16S rRNA bacterial gene of the bacterial V4–V5 region in the thermocycler PCR system (GeneAmp PCR-Systme®9700, ABI, USA). Each sample was assayed three times and mixed to reduce the error in the DNA extraction process. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA). The amplicons were sequenced with the Illumina MiSeq Platform (Illumina, San Diego, USA) using the paired-ends model (PE300). All raw sequences were analyzed using the QIIME pipeline (version 1.17; http://qiime.org/). We assigned bacterial sequences with the same barcode to the same sample, and then removed the barcode and primer sequences. Sequences with similarity above 97% were used to yield one operational taxonomic unit (OTU).\u003c/p\u003e\n\u003cp\u003eCalculation of SOC turnover rate\u003c/p\u003e\n\u003cp\u003eThe differences in δ\u003csup\u003e13\u003c/sup\u003eC abundance between litter and soil was used to calculate SOC turnover with mixing model. SOC turnover was expressed as the fraction of newly derived C in SOC (\u003cem\u003ef\u003c/em\u003e) and half-life time of SOC (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003ehalf\u003c/em\u003e\u003c/sub\u003e).\u003c/p\u003e\n\u003cdiv id=\"Equa\"\u003e\n \u003cdiv id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:\\begin{array}{c}f=\\:\\frac{{\\delta\\:}^{13}{C}_{soil}-{\\delta\\:}^{13}{C}_{soil-CK}}{{\\delta\\:}^{13}{C}_{litter}-\\:{\\delta\\:}^{13}{C}_{soil-CK}}\\:\\times\\:\\:100\\#\\left(1\\right)\\end{array}$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cem\u003e13\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003esoil\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cem\u003e13\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003esoil-ck\u003c/em\u003e\u003c/sub\u003e is the soil δ\u003csup\u003e13\u003c/sup\u003eC abundance after 1 year of incubation with and without litter, respectively;\u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cem\u003e13\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003elitter\u003c/em\u003e\u003c/sub\u003e is the litter δ\u003csup\u003e13\u003c/sup\u003eC abundance.\u003c/p\u003e\n\u003cdiv id=\"Equb\"\u003e\n \u003cdiv id=\"FileID_Equb\" name=\"EquationSource\"\u003e$$\\:\\begin{array}{c}{T}_{half}\\:=\\:-0.693\\:\\times\\:\\:\\frac{Y}{ln\\left(1-\\:\\frac{f}{100}\\right)}\\#\\left(2\\right)\\end{array}$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere 0.693 is the approximate value for ln2; \u003cem\u003eY\u003c/em\u003e is the incubation time (years, \u003cem\u003eY\u003c/em\u003e = 1 in this experiment); \u003cem\u003ef\u003c/em\u003e is the fraction of newly derived C in SOC.\u003c/p\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll data were analyzed using R (version 4.3.3, R Development Core Team). Two-way analysis of variance (ANOVA) was used to test the effects of litter and temperature, as well as their interactive effects on experimental indicators (SOC turnover, soil/microbial properties). Fisher's LSD multiple comparisons were performed (at \u003cem\u003eP\u003c/em\u003e = 0.05) to test whether the effects of adding different litter on experimental indicators at the same incubation temperature were significant to each other. Student's T test was performed (at \u003cem\u003eP\u003c/em\u003e = 0.05) to determine the effects of two incubation temperatures on experimental indicators under the premise of adding the same litter.\u003c/p\u003e\n \u003cp\u003eThe α-diversity indices of Chao 1, Richness and Shannon were calculated using the online Cloud Platform of Majorbio (Shanghai, China). Correlation heatmaps were used to display the relationship between soil variables and the relative abundance of bacteria at the phylum level. Non-metric multidimensional scaling analyses (NMDS) equipped with analysis of similarities (ANOSIM) tests were conducted to show the differences in soil bacterial communities related to experimental warming and microtopography. Redundancy analysis (RDA) was performed to investigate the relationship between soil bacterial communities and soil variables. Figures were created using the online Cloud Platform of Majorbio (Shanghai, China). Random forest analysis was performed to identify the properties significantly affecting the fraction of newly derived soil C using the ‘rfPermute’ package in R (v. 4.3.3). Finally, partial least squares path modeling (PLS-PM) was conducted to further explore the potential mechanisms underlying the formation of newly derived carbon in SOC, driven by soil substrates and bacterial communities. The models were constructed using the “plspm” package in R (v. 4.3.3).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eVariations in SOC turnover and available substrates\u003c/p\u003e\u003cp\u003eAfter 1-year incubation, the newly derived C in SOC ranged from 1.04% to 6.23% across temperature and litter addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Except for L1 treatment, the percentage of newly derived C in SOC under 33\u0026deg;C was significantly higher by 139.38% and 248.91% in L2 and L3 treatments compared to 23\u0026deg;C (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under the same temperature condition, the L2 treatment significantly increased the percentage of newly derived C in SOC compared to L1 and L3 treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The L3 treatment showed higher newly derived C in SOC compared to L1 at 33\u0026deg;C (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but no significance between them at 23\u0026deg;C (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Two-way ANOVA indicated a significant interaction between temperature and litter on the newly derived C in SOC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eThe effects of incubation temperature and litter C:N ratio on the half-life of SOC were highly significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Increased temperature significantly shortened the half-life time of SOC with L2 and L3 additions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), except for L1 treatment. Under the same incubation temperature, the half-life of SOC followed the order of L2\u0026thinsp;\u0026lt;\u0026thinsp;L3\u0026thinsp;\u0026lt;\u0026thinsp;L1 at 23\u0026deg;C (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). While at 33\u0026deg;C, no significant difference in half-life of SOC was observed between the L2 and L3 treatments. Two-way ANOVA revealed a significant interactive effect between incubation temperature and litter C:N ratio on half-life of SOC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFrom Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, at 23\u0026deg;C, the DOC content of L2 treatment was significantly lower than those of L1 and L3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the reductions of 25.03% and 11.51%, respectively. Similar results were observed in the changes in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and TP contents. However, the pH of L2 treatment was higher than that of L1 and significantly higher than that of L3 and CK. The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content showed a significant decrease with increasing litter C:N ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the CK without litter addition, the soil C:N ratio at 23\u0026deg;C significantly decreased after litter addition (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no significant differences among the litter treatments. At 33\u0026deg;C, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content in the L2 treatment was significantly decreased by 33.56% and 18.47%, respectively, compared with L1 and L3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The TN content in the L2 treatment was also significantly lower than in L1 by 5.35% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, soil pH in the L2 treatment at 33\u0026deg;C increased by 1.28% and 0.91% compared to L1 and L3, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content was significantly reduced in the L3 treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), by 34.85% and 33.62% compared to L1 and L2, respectively. Similar to the results at 23\u0026deg;C, soil C:N ratios decreased after litter addition at 33\u0026deg;C, but significant differences were only observed between CK and L1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eWarming significantly increased the concentrations of DOC and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N by 63.40%-129.63% and 102.35%-165.26% across litter additions, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, warming significantly reduced the MBC content by 54.37%, 57.17%, and 52.90% with the addition of L1, L2, and L3, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under the L3 treatment, soil warming significantly increased soil pH (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and TP contents were significantly affected by the interaction of litter C:N ratio and incubation temperature (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSoil properties were measured after 1-year incubation at two different incubation temperatures (23\u0026deg;C and 33\u0026deg;C) with the additions of three different C:N ratios of litters (L1, L2 and L3).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTemperature (\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLitter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSOC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDOC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSoil\u003c/p\u003e\u003cp\u003eC:N ratio\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003e(g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e\u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.62\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.59Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.04Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.05\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.03Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e182.96\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;8.98Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e501.66\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;56.50Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e131.38\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;2.92Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e34.95\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;1.09Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.45\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.03ABa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e8.66\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.12Ba\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.11\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.14Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.98\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.01Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.03Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e137.17\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;2.09Cb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e563.84\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;29.27Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e85.18\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;2.21Da\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e29.36\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.36Bb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.51\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.03Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e8.83\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.01Ba\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.22\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.93Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.95\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.10Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.03Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e155.01\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;3.04Bb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e523.12\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;16.60Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.88Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e25.55\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.52Cb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.41\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.04Bb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e8.68\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.21Ba\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.30\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.74Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.66\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.01Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.07Bb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e171.12\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;1.52Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e471.06\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;32.05Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e123.68\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;1.38Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e27.85\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;1.62BCb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.42\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.01Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e9.37\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.17Aa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33.65\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.16Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.11\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.06Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.04Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e298.95\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.51Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e228.90\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;18.16Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e127.44\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;4.16Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e79.36\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;2.33Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.47\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.02BCa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e8.19\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.09Bb\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.11\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.05Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.89\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.04Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.07Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e314.98\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;33.03Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e241.51\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;15.65Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84.67\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;1.96Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e77.88\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.21Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.54\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.01Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e8.76\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.09ABa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.68\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.53Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.90\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.06Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.09Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e289.05\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;13.73Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e246.41\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;24.68Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e103.85\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;2.66Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e51.70\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;9.20Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.49\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.01Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e8.89\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.27Aa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.13\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.32Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.69\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.04Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.95\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.03Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e306.78\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;7.20Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e193.08\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;8.23Ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e121.47\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;3.69Aa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e61.27\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;5.42ABa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.44\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.02Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e9.26\u003c/p\u003e\u003cp\u003e\u0026plusmn;\u0026thinsp;0.19Aa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003eNote, Data were mean of three replicated with standard deviation. Different capital letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the additions of three different litters at the same temperature, whereas different lowercase letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between two incubation temperatures when the same litter was added. SOC, soil organic carbon; DOC, dissolved organic carbon; MBC, microbial biomass carbon; NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, nitrate nitrogen; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, ammonium nitrogen; TN, total nitrogen; TP, total phosphorus.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTwo-way ANOVA (\u003cem\u003eF\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e-values) for the response of pH, SOC content, DOC content, MBC content, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content, TN content, TP content, and C:N ratio to the incubation temperature (T), litter C:N ratio (L), and their interaction (T \u0026times; L).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLitter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eT\u0026times;L\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.027\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.077\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.578\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSOC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.307\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.270\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.348\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.791\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.894\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.466\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDOC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e222.352\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.982\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.426\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.921\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.167\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e200.192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.214\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.331\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.803\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.136\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.717\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e112.969\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.771\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.527\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e188.203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.377\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.044\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.340\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.311\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.796\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.005\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoil C:N ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.867\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.858\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.274\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSoil bacterial relative abundance and its relationships with soil variables\u003c/p\u003e\u003cp\u003eA total of 22 bacterial phyla were identified in the soil samples. The bacterial communities were dominated by Actinobacteriota, Firmicutes, Proteobacteria, Chloroflexi, and Acidobacteriot. The five phyla together accounted for more than 90% of the total bacterial abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The Kruskal-Wallis H test on the top 10 most abundant bacterial phyla indicated that, at the same incubation temperature, different C:N ratio litter additions did not significantly affect the relative abundance of these phyla (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eStudent\u0026rsquo;s t-test results showed that higher incubation temperature led to an increased relative abundance of Firmicutes, while decreasing the relative abundances of other major phyla (Proteobacteria, Chloroflexi, Acidobacteriota, etc.) with the same litter addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, it was observed that the relative abundance of Actinobacteriota showed an increasing trend following litter addition and higher incubation temperature, though the increase was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c, d, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eAt 33 ℃, the relative abundance of Firmicutes, the representative oligotrophic phylum, showed the largest increase (25.92%) after L3 addition compared to 23 ℃. However, under high temperature conditions, there were no significant differences in the changes in the relative abundances of other oligotrophic bacterial phyla irrespective of litter C:N ratios. The relative abundance of representative copiotrophic bacterial phyla, such as Proteobacteria and Bacteroidota, was decreased significantly by warming, but the decrease was not significantly different across different C:N ratio litters.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe correlation heatmap revealed that only Gemmatimonadota exhibited a significantly negative correlation with soil pH at 23\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Firmicutes showed a significantly negative correlation with soil MBC and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content at 33\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Acidobacteriota and Gemmatimonadota exhibited significantly negative correlation with TN content, while positively correlated with soil C:N ratio. Myxococcota and Bacteroidota displayed significantly negative correlations with soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N content.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDiversity and structure of soil bacterial community, and their relationships with soil variables\u003c/p\u003e\u003cp\u003eTwo-way ANOVA revealed that both incubation temperature and the C:N ratio of litter significantly influenced the α-diversity of soil bacterial community (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, c, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, their interaction effect only was observed on the Richness index (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Warming significantly reduced soil bacterial α-diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, c). At 23\u0026deg;C, the addition of L2 significantly increased the Shannon index compared to CK and L1, but no difference in the Chao 1 and Richness indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, c). At 33\u0026deg;C, different litter C:N ratios did not affect the Shannon index (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the Chao1 and Richness indices were in the order of L2\u0026thinsp;\u0026gt;\u0026thinsp;L1\u0026thinsp;\u0026gt;\u0026thinsp;L3\u0026thinsp;\u0026gt;\u0026thinsp;CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, c, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eThe results of NMDS indicated that bacterial communities were distinctly separated by different incubation temperatures (stress\u0026thinsp;=\u0026thinsp;0.032, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The variation in bacterial communities could be explained by different C:N ratios litter at 23\u0026deg;C (stress\u0026thinsp;=\u0026thinsp;0.047, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), but could not at 33\u0026deg;C (stress\u0026thinsp;=\u0026thinsp;0.039, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.084, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). The results of ANOSIM indicated that the effect of incubation temperature on the shift of soil bacterial communities was stronger than litter C:N ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e, f).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRDA results showed that TN content was the primary environmental factor significantly affecting bacterial communities at 23\u0026deg;C (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), followed by NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011) and soil C:N ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, P\u0026thinsp;=\u0026thinsp;0.011). At 33\u0026deg;C, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content was the significant environmental factor influencing bacterial communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, P\u0026thinsp;=\u0026thinsp;0.015). The remaining environmental factors had no significant effects on the soil microbial community (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePathways of SOC turnover regulated by soil variables and bacterial communities\u003c/p\u003e\u003cp\u003eRandom forest analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) indicated that soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, DOC concentration, and pH were the primary soil factors regulating the fraction of newly derived C in SOC.The C:N ratio of exogenous litter and incubation temperature emerged as the dominant external drivers. In addition, Richness, Chao1 index, and bacterial community composition (NMDS 1) were identified as the critical biological factors influencing SOC turnover.\u003c/p\u003e\u003cp\u003eThe partial least squares path modeling (PLS-PM) was developed based on the key drivers identified by the random forest analysis. PLS-PM explained 75% of the total variance in the fraction of newly derived C in SOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Soil DOC and pH had significant positive effects on the fraction of newly derived C in SOC, with standardized coefficients of +\u0026thinsp;0.75 and +\u0026thinsp;0.30, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, available nitrogen exhibited a strong negative effect on the fraction of newly derived C (coefficient = \u0026minus;\u0026thinsp;0.87, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The pathway from soil bacterial diversity to the fraction of newly derived C in SOC showed a non-significant positive effect. Incubation temperature indirectly affected newly derived C via its positive associations with carbon substrates and available nitrogen, with a total effect of +\u0026thinsp;0.52. Similarly, the litter C:N ratio had a direct negative effect on available nitrogen (coefficient = \u0026minus;\u0026thinsp;0.66, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), thereby indirectly enhancing the fraction of newly derived C, with a total effect of +\u0026thinsp;0.48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). In summary, elevated incubation temperature affected newly derived C in SOC by increasing carbon substrates and available nitrogen, as well as by altering bacterial community composition and diversity. In contrast, the litter C:N ratio primarily regulated newly derived C through its impact on available nitrogen. The total effects of soil bacterial community composition and diversity on the fraction of newly derived C in SOC were \u0026minus;\u0026thinsp;0.21 and +\u0026thinsp;0.34, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBacterial community differed over litters and temperature\u003c/p\u003e\u003cp\u003eThe addition of different C:N ratio litters had a significant effect on the α-diversity and structure of bacterial community (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Given the absence of relative abundance of bacteria at phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the shift in community composition is most likely primarily a response to the C:N ratio in litters, which serves as basic substrate for microbial growth and metabolism (Zeba et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Previous studies had shown the responses of microbial activity and community structure to litters (Wickings et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Allison et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; K\u0026auml;stner et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As our first hypothesis that the C:N ratio close to the requirement of microbial community would favor bacterial survival and activity, the higher Chao1, Shannon, and Richness indexes were observed in L2 treatment compared to L1 or L3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The L2 also decreased oligotrophic populations (e.g. Firmicutes), and increased copiotrophic populations (e.g. Bacteroidetes), which was supported by a meta-analysis on straw return (Zhang \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As litter was broken down, more available N (e.g., NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) was released to soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), which altered the chemical profile of soil over time and influenced soil bacterial community structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Previous results had supported the responses of soil microbial communities to external environmental conditions (Dong et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and the correlation heatmap and RDA results in the present study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). All these indicated that soil bacterial community was more sensitive to change in around 25 C:N ratio litter, irrespective of its source.\u003c/p\u003e\u003cp\u003eIn line with these findings, we also expected that temperature negatively influenced bacterial survival and shift bacterial community composition. As our second expectation, soil bacterial composition was separated by temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which was due to the great sensitivity of soil bacteria to temperature variation (Biasi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Curiel Yuste et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Frindte et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It also modified the relative abundance of soil bacteria, with a notable increase in Firmicutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Firmicutes, as a typical oligotrophic group, exhibited slow growth but prioritize the mineralization recalcitrant SOC (Zhang \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which was reflected in the rapid increase in SOC turnover rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All these supported the third hypothesis. Moreover, the oligotrophic populations showed stronger adaptability under higher C:N ratio conditions, whereas the copiotrophic populations did not exhibit more tolerant under lower C:N ratio conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This might because the 33℃ incubated temperature exceeded the tolerant limits of copiotrophic bacteria, thus temperature rather than litter quality controlled their relative abundances. However, the favorable C:N ratio around 25 litter addition reduced the negative impact of high temperature on soil bacterial alpha diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, c). These results suggested that the favorable litter would alter the impact of warming on soil bacterial communities, further influencing SOC turnover.\u003c/p\u003e\u003cp\u003eOptimal litter C:N ratio favored to SOC turnover and associated with temperature\u003c/p\u003e\u003cp\u003eL2 induced the largest SOC turnover rate (indicated by the fraction of newly-derived SOC or half-life time of SOC), followed by L3 and L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b), which confirmed our first hypothesis that the C:N ratio of litters close to microbial requirement favors to SOC turnover. This result was supported by previous studies (Parton et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Prescott, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The greatest SOC turnover with L2 addition is likely due to the C:N ratio of L2 is between 25\u0026ndash;30, which aligns with the optimal average C:N requirement for microbial survival (Brady and Weil \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Lyu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This was strongly supported by the significant increase in the bacterial diversity with L2 addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, c). Moreover, the activated bacterial community rapidly and extensively consumed available soil carbon and nitrogen for \"microbial turnover\" (Liang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), converting exogenous carbon into microbial residues (Kallenbach et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), ultimately increased the fraction of newly derived C in SOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). These could be explained by the lower soil DOC and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N contents with L2 addition than L1 or L3 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e ,Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, L2 shift bacterial community towards copiotrophic types (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), that preferentially utilizes labile substrates (Fierer et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Copiotrophic bacteria, known for their high metabolic activity (Eilers et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ramirez et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), can secrete more extracellular enzymes (such as polyphenol oxidase and peroxidase) (Creamer et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), to decompose native SOC. The present results indicated that the enhanced SOC turnover might be due to the increased soil bacterial diversity driven by higher substrate availability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eElevated temperature favored to SOC turnover with higher C:N ratio litters addition, which was supported by the increased the fraction of newly derived soil C and shortened the SOC half-life in the L2 and L3 treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b), suggesting the positive influence of temperature on SOC turnover depended on exogenous C quality. This might be due to the activity and composition of microbial community derived by exogenous C addition as above mention. The activated microbial community significantly increased the decomposition rate of litter, which induced the increase in soil DOC and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N contents, but decreased MBC content (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Peplau et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). All these would meet the nutrient requirement of soil microbial to favor SOC turnover. Overall, the effects of elevated temperature on SOC turnover are likely related to changes in nutrient provision, including both C and N. In addition to C and N, elevated temperature accelerated the desorption rate of SOC-minerals to increase the availability of substrate (Ten Hulscher and Cornelissen, 1996; Conant et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Here, we did not examine in detail, more research is needed to fully understand the effects of temperature on sorption-desorption of SOC minerals, and their relationships with exogenous C quality.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLitters, their C:N ratios close to microbial requirements, increased the diversity and richness of soil bacterial community, and shifted the bacterial community to copiotrophic lifestyles, which would enhance the mineralization of available substrates. Soil warming decreased soil bacterial diversity, but caused the increase in the relative abundance of the oligotrophic phylum, which would be benefit to recalcitrant material decomposition. Moreover, oligotrophic bacteria was observed stronger high-temperature adaptability with a higher C:N ratio litter addition. We also demonstrated the importance of temperature for the changes in the soil bacterial community, which is greater than the C:N ratio of litter.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThe authors are grateful for the insightful comments suggested by the editor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp; Tian Li: Conceptualization, Investigation, Formal analysis, Visualization, Writing - Original Draft; Shujie Miao: Methodology and Writing - Review \u0026amp; Editing; Yunfa Qiao: Conceptualization, Methodology and review; Jie Yu: Software and Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis research was supported by National Natural Science Foundation of China (42177279); Special Funds for Scientific and Technological Innovation of Jiangsu province, China (BE2023400-02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial interests or personal relationships that could influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe datasets generated during and/or ana- lysed during the current study are available from the corre- sponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllison SD, Lu Y, Weihe C, et al (2013) Microbial abundance and composition influence litter decomposition response to environmental change. 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Nature Clim Change 2:106–110. https://doi.org/10.1038/nclimate1331\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Soil organic carbon, SOC turnover, Litter C:N ratio, Warming, Soil bacterial community","lastPublishedDoi":"10.21203/rs.3.rs-7570212/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7570212/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and aims\u003c/h2\u003e\u003cp\u003eSoil organic carbon (SOC) turnover is closely linked to global carbon cycling, yet the microbial mechanisms underlying its response to warming and litter quality remain poorly understood. Therefore, this study aimed to explore the bacterial mechanisms driving SOC turnover in response to different incubation temperature and litter C:N ratio.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eHere, a one-year incubation experiment was conducted with three types of \u0026sup1;\u0026sup3;C-labeled litter differing in C:N ratio (11.66, 27.77, and 35.81) under two incubation temperatures (23\u0026deg;C and 33\u0026deg;C). At the end of incubation, the soil properties were measured, and soil bacterial community properties were examined using the Illumina MiSeq sequencing method.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSOC turnover was maximized, and SOC half-life minimized, under the medium C:N litter (27.77), indicating that a C:N ratio close to microbial demand favors carbon transformation. Warming significantly accelerated SOC turnover, particularly with medium and high C:N litter additions. Bacterial α-diversity increased with medium C:N litter but declined with warming. Elevated temperature reduced the abundance of copiotrophic taxa (e.g., Proteobacteria) and enhanced oligotrophic groups (e.g., Firmicutes). Path modeling revealed that DOC and pH positively, but available nitrogen negatively, regulated the incorporation of newly derived carbon into SOC.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings highlight that SOC turnover is jointly controlled by litter quality and warming, with temperature exerting a stronger influence on bacterial communities. This study provides new insights into the microbial mechanisms linking substrate quality, warming, and SOC stability, with implications for predicting soil carbon dynamics under future climate change.\u003c/p\u003e","manuscriptTitle":"Microbial mechanism on the turnover of soil organic carbon responded to litter C:N ratio and incubation temperature","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 15:56:14","doi":"10.21203/rs.3.rs-7570212/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"059ccdf4-3e94-4919-bc8b-41d2222db913","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-19T16:23:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 15:56:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7570212","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7570212","identity":"rs-7570212","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}