The responses of soil organic carbon mineralization and microbial communities to fresh and aged biochar soil amendments

While biochar soil amendment has been widely proposed as a soil organic carbon (SOC) sequestration strategy to mitigate detrimental climate changes in global agriculture, the SOC sequestration was still not clearly understood for the different effects of fresh and aged biochar on SOC mineralization. In the present study of a two‐factorial experiment, topsoil samples from a rice paddy were laboratory‐incubated with and without fresh or aged biochar pyrolyzed of wheat residue and with and without crop residue‐derived dissolved organic matter (CRM) for monitoring soil organic matter decomposition under controlled conditions. The six treatments included soil with no biochar, with fresh biochar and with aged biochar treated with CRM, respectively. For fresh biochar treatment, the topsoil of a same rice paddy was amended with wheat biochar directly from a pyrolysis wheat straw, the soil with aged biochar was collected from the same soil 6 years following a single amendment of same biochar. Total CO2 emission from the soil was monitored over a 64 day time span of laboratory incubation, while microbial biomass carbon and phospholipid fatty acid (PLFA) were determined at the end of incubation period. Without CRM, total organic carbon mineralization was significantly decreased by 38.8% with aged biochar but increased by 28.9% with fresh biochar, compared to no biochar. With CRM, however, the significantly highest net carbon mineralization occurred in the soil without biochar compared to the biochar‐amended soil. Compared to aged biochar, fresh biochar addition significantly increased the total PLFA concentration by 20.3%–33.8% and altered the microbial community structure by increasing 17:1ω8c (Gram‐negative bacteria) and i17:0 (Gram‐positive bacteria) mole percentages and by decreasing the ratio of fungi/bacteria. Furthermore, biochar amendment significantly lowered the metabolic quotient of SOC decomposition, thereby becoming greater with aged biochar than with fresh biochar. The finding here suggests that biochar amendment could improve carbon utilization efficiency by soil microbial community and SOC sequestration potential in paddy soil can be enhanced by the presence of biochar in soil over the long run.


| INTRODUCTION
Biochar, a carbon (C)-rich solid produced from pyrolysis of biowastes in the absence of oxygen, is able to persist or remain deposited in soil for hundreds to thousands of years (Kuzyakov, Bogomolova, & Glaser, 2014;Lehmann et al., 2011). For this reason, biochar soil application has been widely accepted as an important strategy to increase soil organic carbon (SOC) sequestration (Sohi, 2012;Zheng, Han, et al., 2017). The impacts of biochar application on SOC dynamics have been reported in detail in the literatures (Ameloot et al., 2014;Jiang, Tan, Cheng, Haddix, & Cotrufo, 2019;Zheng et al., 2016). Previous studies showed that aging of biochar is a vital factor influencing SOC mineralization (Maestrini, Nannipieri, & Abiven, 2015;Wang, Xiong, & Kuzyakov, 2016). Lu et al. (2014) found that corn (C 4 plant) biochar addition had no effect on total soil CO 2 emissions but could significantly reduce CO 2 emissions by 64.9%-68.8% from native SOC in a cultivated sandy loam soil (C 3 soil) over an incubation period of 30 days. In another study, a 3.2 year incubation experiment conducted by Kuzyakov, Subbotina, Chen, Bogomolova, and Xu (2009) showed that fresh biochar addition had no effect on total SOC mineralization from two soil types within 2 months of incubation; however, a contrasting effect was found between the two soil types by biochar addition. CO 2 emission rates slightly increased to about 0.6 mg CO 2 -C kg −1 day −1 in the loamy Haplic Luvisol, while they significantly decreased to 0.2-1.5 mg CO 2 -C kg −1 day −1 in the loess. Recently, Zhao, Coles, and Wu (2015) compared the soil C mineralization rate between fresh and aged biochar amendments and observed a lower mineralization rate in aged biochar amendments. However, Spokas (2013) stated that CO 2 emission was enhanced two to eightfold under three weathered biochar additions in laboratory over a 100 day incubation. These results indicated that biochar addition in the short term (fresh biochar) and the long term (aged biochar) could influence the soil C mineralization intensity and direction with time through different mechanisms. Furthermore, these results suggest that aging of biochar in soil may alter SOC sequestration potential. To date, however, little information has been available on the response of organic C mineralization to biochar with aging in paddy soils.
Soil microbes play a key role in soil organic matter stabilization and nutrient cycling Watzinger et al., 2014). The effects of biochar on soil microbial community composition have been investigated using highthroughput sequencing or phospholipid fatty acid (PLFA) technology (Ameloot et al., 2014;Chen et al., 2017;Sheng & Zhu, 2018;Zhou et al., 2019). Xu et al. (2014) reported that highthroughput sequencing results in increased α-diversity significantly and the relative abundances of Flammeovirgaceae and Chitinophagaceae related to C cycling after 45 days following biochar addition in a pot trial. Similarly, Watzinger et al. (2014) revealed that biochar addition significantly increased Gram-negative bacteria and actinomycetes from temperate soil in short-term incubation using 13 C-PLFA. Chen et al. (2018) demonstrated that 3 year biochar soil amendment reduced the relative abundance of three dominant bacterial phyla related to C cycling, while increasing the abundance of Ascomycota, a key fungal community, and reducing the dependence of soil respiration on temperature. By contrast, a 6 year field experiment conducted by Tian et al. (2016) indicated that microbial metabolic activity strongly increased due to biochar amendment, but it did not change microbial community structure in paddy soil. These changes in soil would cause differences in exogenous organic C utilization (organic C sequestration) by microbial organisms. Although there were studies that reported that biochar amendment affected the soil microbial community composition in the short term or long term (Chen, Jiang, et al., 2019;Cheng, Hill, Bastami, & Jones, 2017;Sun, Meng, Xu, & Chen, 2016), the microbial usage of exogenous organic matter following the addition of fresh and aged biochar amendments is still unclear.
Paddy soil is one of the main soil types in agriculture systems in China, and it has a meaningful effect on soil C storage (Pan, Li, Wu, & Zhang, 2003). Crop residues are the main form of waste in agriculture production. In 2013, it was estimated that the total amount of crop residue production was approximately 3.8 × 10 9 Mg/year in global agricultural ecosystems (Thangarajan, Bolan, Tian, Naidu, & Kunhikrishnan, 2013), and most crop residues are returned to the soil. Meanwhile, approximately 5%-15% of total residues enter into the environment in the form of soluble C (Cleveland, Neff, Townsend, & Hood, 2004), which is a very reactive fraction of organic C and easily utilized by soil microbes in terrestrial ecosystems (De Troyer, Amery, Moorleghem, Smolders, & Merckx, 2011). A study by Wang et al. (2017) showed that the dissolved organic carbon (DOC) content is generally higher in paddy soil with biochar amendment. However, a converse community and SOC sequestration potential in paddy soil can be enhanced by the presence of biochar in soil over the long run.

K E Y W O R D S
dissolved organic matter, fresh/aged biochar, phospholipid fatty acid, SOC mineralization, soil microbial community composition finding was reported by Zheng et al. (2016), who found that DOC content was significantly lower after 4 years of biochar amendment in paddy soil. It was reported that the biochar's labile C may rapidly degrade within several days (Smith, Collins, & Bailey, 2010) or a few months (Kuzyakov et al., 2009) in soil. The biochar aging process develops a balance of chemical exchange and biological activity in the biochar-soil system, so the 'fresh' biochar effect on soil physicochemical and biological properties is likely different from the long-term effect of 'aged' biochar (Luo et al., 2017). Soil dissolved organic matter (DOM) could be adsorbed by the biochar due to its high porosity and large surface area (Smebye et al., 2016). Pan, Zhou, Zhang, Qiu, and Chu (2006) stated that SOC sequestration could play a key role in stabilizing and increasing rice productivity and in sequestering C for mitigation of CO 2 emissions in paddy soils. Water management as a necessary agricultural practice is one of the crucial measures for crop production, especially in paddy soils. Straw residues that are returned to paddy soil produce much of the DOM due to seasonal flooding of agricultural practices. For C sequestration, it is essential to understand the organic C dynamics under the interactive effect of biochar and crop-derived DOM in paddy soil.
In this study, we hypothesized that (a) lower organic C mineralization would occur in the soil with aged biochar amendment compared to the soil with fresh biochar addition due to labile C depletion, and (b) soil microbial community composition would be modified differently by the fresh and aged biochar soil amendment and would utilize exogenous organic C differently. The main aims of this study were to (a) determine the variation of organic C mineralization dynamics under fresh (short-term) and aged (long-term) biochar amendment in a paddy soil, and (b) investigate response of soil microbial community and organic C mineralization to fresh and aged biochar soil amendments followed by exogenous C addition.

| Site description
The field experiment site was located in Jing-tang Village (31°24′N, 119°41′E) in Yixing Municipality, Jiangsu Province, China. The local climate is a subtropical humid monsoon climate with a mean annual temperature of 15.7°C and a mean annual precipitation of 1,177 mm. The soil type is a typical paddy soil and is classified as a Hydroagric Stagnic Anthrosol (Gong, 1999). The basic soil properties before biochar amendment were as follows: bulk density of 1.01 g/cm 3 , pH (H 2 O) of 5.36, SOC of 23.5 g/kg, total N (TN) of 2.37 g/kg, cation exchange capacity of 18.1 cmol (+)/kg, and clay content of 390 g/kg. The local traditional farming system is a typical rice-wheat rotation in this region.

| Field experiment treatment
The field experiment was set up in May 2009 after wheat harvest and included two treatments: without and with biochar soil amendment. To exaggerate the biochar's effect on soil properties, the biochar was applied only once to soil at a rate of 40 t/ha for biochar treatment. The biochar was spread on the soil surface and mixed homogeneously with soil before rice planting. Each plot size was 20 m 2 (4 m × 5 m) in area with three replicates and laid out in a randomized block design. Urea, calcium biphosphate, and KCl were used as N, P, and K fertilizers in the field following local agronomic practices at the rates of 300 kg N/ha, 89 kg P/ha, and 169 kg K/ha, respectively.

| Soil sampling and pretreatment
Soil samples from without and with biochar soil amendments for laboratory incubation were sampled randomly from the surface layer (0-15 cm) of each plot by using an Eijkelkamp soil core sampler (5 cm in diameter) after the rice harvest in 2015 and shipped to the laboratory. Then, the samples were air-dried after removing the roots from the soil. Prior to the incubation experiment, the soil samples were passed through a 2 mm sieve and homogenized. The topsoil basic properties are listed in Table 1.
To compare the response of C mineralization and soil microbial communities to aged and fresh biochar soil amendments, the 6 year soil with biochar amendment in the field was used for the aged biochar-amended treatment, and the soil with the fresh biochar from the same pyrolysis procedure was used for fresh biochar-amended treatment. rate of the fresh biochar was calculated according to the difference in C content between the field soil samples without and with biochar amendment. The biochar used in this study was produced commercially by Sanli New Energy Company in Henan Province, China. Wheat straw was pyrolyzed at a mid-temperature and slow carbonization in a vertical kiln at a temperature range of 350-550°C varying from the start to the endpoint. Basic properties of biochar were: organic C: 467.2 g/kg, total N: 5.90 g/kg, pH (H 2 O): 10.4, DOC: 530.3 g/kg, surface area: 8.92 m 2 /g, and ash content: 20.8%.

| Crop-derived dissolved organic matter preparation and addition rate
Wheat straw harvested from the field was air-dried and chopped prior to use. The crop residue was extracted for 24 hr by distilled water (1:40 by wt:vol). The supernatant solution was passed through a 0.45 μm filter membrane and immediately added to the soil. The amount of crop residuederived dissolved organic matter (CRM) was added at the rate of 400 ml solution/kg soil (equivalent to 320 mg C/kg soil) for the treatments with CRM addition.

| Incubation experiment
The incubation experiment was designed as a two-factorial of biochar and CRM, including six treatments with three replications: (a) soil without biochar (Control), (b) control with fresh biochar addition (BC F ), (c) soil with a 6 year biochar (aged) amendment in the field (BC A ), (d) Control with CRM addition (C-control), (e) BC F with CRM addition (C-BC F ), and (f) BC A with CRM addition (C-BC A ). One hundred grams of treated soil (dry weigh equivalent) was put into a 500 ml jar, and then, the prepared CRM solution was added into the designated jars at the rate mentioned above. Each treatment contained 24 jars, three of which were used to monitor the C mineralization, and the remaining ones were used to determine the DOC, microbial biomass C (MBC), and PLFA. All treatment samples were incubated at constant moisture (60% water holding capacity) and temperature (25 ± 1.0°C) in the incubator.
To maintain the soil moisture, deionized water was added to the jars by weight during the incubation. The respired CO 2 was sampled after 1, 2, 3,4,5,6,7,8,10,12,14,16,18,20,23,26,29,34,40,50, and 64 days of incubation by a 10 ml gas-tight locking syringe. The CO 2 concentration in a gas sample was analyzed by a gas chromatograph (Agilent 7890A) equipped with a flame ionization detector (FID, 250°C). Before sampling, the headspace air in the jar was completely replaced with pure air for 10 min at a rate of 300 ml/min. Then, the jar was covered immediately by a cap with a three-way valve for 4 hr. The concentration of CO 2 in the headspace was measured at 0 and 4 hr after closing the three-way valve. Three replicates in each treatment were used to measure the DOC content at 1, 4, 8, 14, 23, 34, and 64 days of incubation. At the end of incubation, MBC and PLFA were analyzed. The metabolic quotient (qCO 2 ) was calculated as the ratio of basal respiratory C (mg CO 2 -C kg −1 hr −1 ) to MBC (mg/kg).

| Data process
The cumulative amount of mineralized C (C (t) : mg CO 2 -C/ kg soil) was plotted against time (t), and a combined first-plus-zero-order kinetic model was fitted to the data using the Levenberg-Marquardt algorithm: where this model assumes an initial size of easily mineralizable C pool (C e ), which is diminished according to first-order kinetics (Stanford & Smith, 1972), and the more resistant fraction is mineralized by zero-order kinetics (Sleutel, Neve, Roibas, & Hofman, 2005). In Equation (1), k e is a rate constant for the easily mineralizable C pool (day −1 ), and k s is the slow C pool mineralization rate (mg CO 2 -C kg −1 soil day −1 ).
Net C mineralization (ΔC min ) of CRM addition was calculated as: where C min CRM is the total C mineralization of C-control, C-BC F, and C-BC A treatments, respectively; C min non-CRM is the total C mineralization of Control, BC F, and BC A treatments, respectively.
All data were expressed as the means of three replicates with 1 SD. One-way analysis of variance (ANOVA) was used to analyze the effect of the biochar. Two-way ANOVA was used to test the interaction effect of biochar and CRM. All statistical analyses were carried out using the JMP11.0 software (SAS Institute). The significant differences between the means were tested using Tukey's honestly signifcant difference at p < .05. All figures were plotted using Origin 8.0 software.

| Soil CO 2 release dynamics and variation in the mineralizable C pool under different treatments
Overall, the CO 2 release dynamics showed a similar trend under different treatments (Figure 1a,b). CO 2 release sharply declined in the first 6 days, and then gradually stabilized with time during the whole incubation period for all treatments. The fresh biochar addition significantly increased CO 2 efflux. The mean CO 2 release rate increased by 20.9%-53.9% in the BC F than in the Control within 1-8 days (p < .05). In contrast, the consistently lower CO 2 release rate in aged biochar-amended soil was observed across the whole incubation period. With CRM addition, the CO 2 release rate increased by 26.8%-79.5%, 16.4%-43.2%, and 20.3%-86.3% in the C-control, C-BC F, and C-BC A treatments, respectively, within 1-8 days compared with the corresponding biochar treatment without CRM addition. The cumulative C mineralization was extreme significantly affected by the biochar amendment and CRM addition individually (p < .01), but no interaction effect (biochar × CRM) was found (p > .05; Figure 2; Table S1). (1) (2) ΔC min = C min CRM − C min non -CRM , F I G U R E 1 The total C mineralization rate (a, b) during the 64 day incubation under different treatments. Control, soil without biochar; BC F , Control with fresh biochar addition; BC A , the soil with biochar applied in the field (a); C-control, Control with CRM addition; C-BC F , BC F with CRM addition, C-BC A , BC A with CRM addition (b). Error bars show the standard deviation (n = 3) F I G U R E 2 Cumulative C mineralization under different treatments during the 64 day incubation. BC A , the soil with biochar applied in the field; BC F , Control with fresh biochar addition; C-BC A , BC A with CRM addition; C-BC F , BC F with CRM addition; C-control, Control with CRM addition; Control, soil without biochar. Error bars show the standard deviation (n = 3). Different letters indicate significant difference between the treatments at p < .05. '**' indicates significant effects of crop-derived DOM or biochar addition at p < .01, and 'ns' indicates no significanance (p > .05) of their interaction Compared to the Control, cumulative C mineralization amount significantly increased by 28.9% in BC F , whereas a significantly low cumulative C mineralization was observed in the BC A . Similarly, for the CRM addition treatments, the highest cumulative C mineralization occurred in C-BC F , followed by Control and C-BC A . The cumulative C mineralization amount increased by 24.3%, 15.2%, and 16.3% in the C-control, C-BC F, and C-BC A treatments, respectively, due to addition of CRM, compared with the corresponding biochar treatment without CRM addition (Figure 2).
The easily mineralizable C pool (C e ), mineralization rate of the slow C pool (k s ), and the k e were extremely and significantly affected by the biochar amendment and CRM addition (p < .01; Table 2; Table S2). The size of C e significantly increased by 45.9%-79.6% under addition of CRM compared to the treatments without CRM addition. For the treatments without CRM addition, the C e sizes were calculated in the following order: BC F (238.8 mg C/kg) > Control (169.8 mg C/kg) > BC A (125.6 mg C/kg). Similarly, the k s significantly increased by 27.9% in the BC F compared to the Control. By contrast, the k s significantly decreased by 40.1% in the BC A compared to the Control.

| Dynamics of DOC under different treatments
The dynamics of DOC content in different treatments exhibited a similar trend as the CO 2 release rates during the whole incubation period (Figure 3a,b). There was a significant decrease in the DOC content, from 282.0 mg/kg to 39.7 mg/kg and from 346.9 mg/kg to 43.8 mg/kg in the treatment without and with CRM addition, respectively, in the first 8 days. A slow decrease was observed in the later incubation period. The lowest DOC occurred in aged biochar soil during the whole incubation period. The DOC was significantly higher in the BC F than in the Control within the first 8 days, but there was no significant difference between the BC F and the Control from 14 to 64 days. Meanwhile, the DOC of all treatments increased by the CRM addition, especially in the early incubation period (i.e., <8 days). However, there were no significant differences in DOC between the C-control and the C-BC F .

| The variation in MBC and microbial qCO 2 under different treatments
Biochar amendment significantly affected the MBC content, while the CRM addition had no effect on the content of MBC (Table 3; Table S3). Compared to the Control, soil MBC content significantly increased by 63.1% and 67.9% under the BC F and BC A , respectively. However, there was no significant difference (p > .05) in MBC content between the BC F and BC A . For the treatments with CRM addition, compared to C-control, the MBC content significantly increased by 35.5% and 41.7% for the C-BC F and C-BC A , respectively.
As shown, qCO 2 decreased by 21.5% and 63.9% in the BC F and BC A , respectively, compared to the Control (Table 3). In addition, qCO 2 decreased by 11.8% and 58.9% in the C-BC F and C-BC A , respectively, compared to the C-control. However, there were no significant differences (p > .05) in qCO 2 between the Control and C-control, BC F and C-BC F , or BC A and C-BC A .

| PLFA analysis
Regardless of CRM addition, compared to soil without the biochar-amended treatment, the total PLFA concentration T A B L E 2 Parameters of the firstplus-zero-order kinetic model fitted with the cumulative C mineralization data (mean ± SD, n = 3) and individual PLFA markers of fungi, G + -bacteria, and G − -bacteria significantly increased by 19.0%-40.2% in BC F , and 19.5%-29.7% in C-BC F (p < .05; Figure 4a). However, there was no significant difference in the total PLFA concentration among the aged biochar-amended soil and without biochar-amended soil (p > .05). With the CRM addition, the total PLFA concentration was significantly increased by 20.4% in the C-control than in the Control. On the contrary, there was no difference (p > .05) in the total PLFA concentration between the treatments with and without CRM addition under fresh or aged biochar soil amendments, indicating a weak response of PLFA to CRM addition. The ratio of G + -bacteria/G − -bacteria and fungi/ bacteria was significantly affected by biochar and CRM, and there was a synergistic effect between biochar and CRM on the G + -bacteria/G − -bacteria ratio (Figure 4b,c; Table S4). The aged biochar soil amendment significantly increased the ratio of G − -bacteria/G + -bacteria and fungi/ bacteria by 32.2%-55.0% and 13.1%-36.5%, respectively, compared to the without and with fresh biochar addition under the same CRM addition treatments. Microbial community structure was altered by biochar addition (Figure 5). The first two principal components correlated meaningfully with the individual PLFA (loading plot in Figure 6). High positive loadings on the PC1 axis, such as in the BC A and C-BC A , correlated with higher mole percentages of 16:1ω7c and 18:1ω7c (G − -bacteria) and 18:2ω6,9c (fungi), with negative loadings from i16:0 and a17:0 (G + -bacteria) mole percentages. In contrast, PC2 had mainly positive loadings from 17:1ω8c (G − -bacteria) and i17:0 (G + -bacteria) mole percentages, with negative loadings from i14:0 and a15:0 (G + -bacteria) mole percentages.

| Decomposition of C under fresh/aged biochar soil amendments and its response to crop-derived DOM addition
Some laboratory incubation studies showed an immediate or short-term increase in total CO 2 emissions following the addition of fresh biochar (Yousaf et al., 2017;Zhao et al., 2015). In agreement with these results, our present study showed that fresh biochar significantly increased the total CO 2 release, especially in the initial days of incubation (Figure 1). The primary reason was that relatively small portion of labile C in F I G U R E 4 Absolute concentration (nmol/g) of the phospholipid fatty acid assigned to different microbial groups (a), G + -bacteria/G −bacteria ratio (b), and fungi/bacteria ratio (c) after 64 day incubation. BC A , the soil with biochar applied in the field; BC F , Control with fresh biochar addition; C-BC A , BC A with CRM addition; C-BC F , BC F with CRM addition; C-control, Control with CRM addition; Control, soil without biochar. Error bars show the standard deviation (n = 3). Different letters indicate significant difference between the treatments at p < .05. '*', '**', and 'ns' indicate significant levels at p < .05, p < .01, and not significant (p > .05), respectively F I G U R E 5 Principal component analysis of the percentage molar abundance of phospholipid fatty acid of individual soil sample. BC A , the soil with biochar applied in the field; BC F , Control with fresh biochar addition; C-BC A , BC A with CRM addition; C-BC F , BC F with CRM addition; C-control, Control with CRM addition; Control, soil without biochar. Error bars show the standard deviation (n = 3)

F I G U R E 6
The loading plot of two components from the principal component analysis on the phospholipid fatty acid composition fresh biochar was decomposed by microorganisms during the early stages of incubation. More labile C fractions (containing higher H/C) existed in the fresh biochar, especially for biochar that was made at lower pyrolysis temperatures (Bian et al., 2016;Liu et al., 2019;Mukherjee, Zimmerman, & Harris, 2011). They can be utilized more easily by soil microorganisms over short periods . Consequently, a significant increase in CO 2 release was found over short time, because an abundance of nutrients contained in the biochar boosted microbial growth and reproduction ( Figure 4a) and accelerated organic matter decomposition (Sheng & Zhu, 2018;Singh, Cowie, & Smernik, 2012). In comparison, most of the degradable fractions in aged biochar that had been incorporated in the soil for several years were preferentially consumed by the soil microorganisms. A large amount of aromatic C remaining in the aged biochar increased C stability .We found that aged biochar soil amendment suppressed the total C mineralization ( Figure 2). Zimmerman, Gao, and Ahn (2011) reported that addition of biochar derived from grasses increased the C mineralization during the early period of incubation (first 90 days), whereas the addition of biochar derived from grasses at high temperatures (525 and 650°C) decreased to a lower CO 2 release during the later incubation period (250-500 days), compared to the soil without biochar treatment. In the present study, the size of easily mineralizable C pool and CO 2 release rate were significantly lower in the aged biochar-amended soil than in the fresh biochar-amended soil, suggesting the SOC in aged biochar-amended soil shifted to a more recalcitrant form against microbial degradation (Six, Frey, Thiet, & Batten, 2006). This result was also supported by Ameloot et al. (2014), who found mineralization rates of the slow C pool were lower in the biochar-amended plot than in Control plot in clay loam soils. The remaining stable C portion of aged biochar could interact with soil and improve soil aggregation, which reduces microbial access to organic matter and consequently limits organic C decomposition (Fang, Singh, Matta, Cowie, & Zwieten, 2017). The incorporation of biochar into soils could affect the plant-derived C (e.g., crop residues, DOM derived from straw, root exudates, etc.) decomposition. For example, Keith, Singh, and Singh (2011) found addition of sugar cane residue to soil reduced its C mineralization in the presence of biochar using labelled 13 C isotope. Although the CO 2 source from CRM and soil native C cannot be clearly distinguished in our study, we found a lower net C mineralization in biocharamended soils (C-BC F minus BC F and C-BC A minus BC A ), especially in aged biochar-amended soil, compared to no biochar-amended soil (C-control minus Control; Figure 7). We deduced that biochar amendment could modify C cycling and microbial growth and metabolism in soil. For one thing, biochar has higher porosity and large surface area/volume ratio (Zimmerman et al., 2011), which could increase its absorption ability and fix organic compounds to prevent biological decomposition (Smebye et al., 2016). Owing to the adsorption of dissolved organic compounds to the biochar surface or micropores, the bioavailability of labile organic compounds could be limited and reduce the exogenous C mineralization under biochar addition. In addition, the effect of microbial community change on CRM decomposition under biochar amendment will be discussed in the below section.

| The effect of fresh/aged biochar soil amendments on microbial community composition and its implications for C sequestration
A lower qCO 2 was observed in biochar-amended soil compared to the no biochar-amended soil (Table 3). It suggested that biochar amendment could enhance the C utilization efficiency of microorganisms (Chen et al., 2018). This finding was also supported by Zheng et al. (2016), who reported that biochar decreased the microbial metabolic quotient 4 years after a single incorporation in a slightly acidic rice paddy. Similarly, Zhou et al. (2017) found an overall decrease of 13% in qCO 2 due to biochar addition by meta-analysis. Lehmann et al. (2011) demonstrated that biochar particles may generate microhabitats in the soil. The nutrients, substrates, and microorganisms were co-localized on biochar surfaces, thus mediating higher microbial biomass but lower C mineralization. The decreases in qCO 2 , especially in aged biochar-amended soils, may be attributed to the improved microbe-suitable habitats because biochar could provide moisture and nutrients and increase water availability for microbial growth and F I G U R E 7 The net C mineralization at 64 days of incubation under different treatments. ΔBC A , C-BC A minus BC A ; ΔBC F , C-BC F minus BC F ; ΔControl, C-control minus Control. Error bars show the standard deviation (n = 3). Different letters indicate significant difference between the treatments at p < .05 protect microorganisms from predators Lehmann et al., 2011). As fresh biochar addition increased the total C mineralization, a more significant increase of MBC content was observed in the fresh biochar amendment, compared to the no biochar-amended soil (Table 3). Soil pH was considered to be a dominant factor to control the microbial biomass. Increase of soil pH may stimulate microbial reproduction rate due to the liming potential (Ameloot et al., 2013). Lower qCO 2 in biochar-amended soils suggested that the microorganisms seemed to produce more cell mass per unit of C decomposition in the biocharamended soils than in the no biochar-amended soil and, therefore, improved the C sequestration in soil.
Microorganisms preferentially utilize the bioavailable fraction over the recalcitrant fraction of organic matter. It is difficult to use the substances in biochar for microorganisms, because most of the C fractions in biochar are recalcitrant (Singh et al., 2012). However, fresh biochar also contained soluble C fractions, which were likely derived from the bio-oil absorbed to the biochar during cooling (Smith et al., 2010). Our results revealed that total PLFA concentration and bacteria/fungi ratio were increased by fresh biochar addition, compared to the Control (Figure 4a,c). It could be inferred that labile C compound in the fresh biochar provided the C resources and energy to promote the growth of soil microorganisms, especially bacterial in the short-term incubation period. Meanwhile, biochar amendment could change the physical and chemical properties of the soil and the ecology of the system (Whitman et al., 2016). The different functions of fresh and aged biochar in altering the soil environment (e.g., available C, pH, nutrients, and water content) might cause some microorganisms to become competitively dominant by inhibiting other microbial groups. Changes in microbial biomass were not equally distributed across different functional groups, resulting in changes in microbial community composition (Luo et al., 2017). Shifts in microbial community composition due to biochar addition may in turn affect the C mineralization (Jiang, Denef, Stewart, & Cotrufo, 2016). The G + -bacteria are important consumers of recalcitrant C. Thus, they could benefit from the addition of aromatic C, such as biochar C (Jiang et al., 2016), and the G + -bacteria were significantly more by the addition of fresh biochar (Figure 4a). Gomez, Denef, Stewart, Zheng, and Cotrufo (2014) reported that the soil microbial community shifts toward G − -bacteria over other types of microorganisms with oak-derived biochar addition to agricultural soils. The present study also showed the aged biochar amendment significantly decreased the G + -bacteria/G − -bacteria ratio. These results are also supported by Watzinger et al. (2014), who demonstrated that a higher concentration of G − -bacteria was found after wheat husk biochar amendment in agricultural soils, while little change in G + -bacteria was observed. The promotion of G − -bacteria concentration by biochar addition might be attributed to the improved nutritional and physical conditions of biochar-amended soil (Watzinger et al., 2014).
Crop residue-derived dissolved organic matter addition, as labile C source, exerted different effects on soil microbial composition between biochar and no biochar soil amendments in this study. The fungi/bacteria ratio was significantly decreased by CRM addition under the no biochar treatment. However, there was no significant effect on the bacteria/fungi ratio in the fresh and aged biochar treatments. These findings suggest that the increase of available C substrate stimulated the reproduction and microbial activity of bacteria due to exogenous C addition in the short term. By contrast, lower molecular organic C would be absorbed by biochar via high porous structure and reduced the accessibility to bacteria. Moreover, it is different for survival strategies of bacteria and fungi. With fast high growth rates but few protective and/or structural compounds, soil bacteria typically act as r-strategists. By comparison, soil fungi usually behave as k-strategists, with slow growth rates but high C use efficiency (Six et al., 2006). The increase of bacteria/fungi ratio was limited due to CRM addition in the biochar soil amendments. Therefore, the biochar-amendment could increase the efficiency of microbial utilization for exogenous C and reduce net C mineralization, especially in the aged biochar-amended soil.
Long-term (aged) biochar amendment significantly decreased total C mineralization in soil, while it was enhanced under the presence of short-term (fresh) biochar. Furthermore, with the addition of exogenous C, its decomposition could be suppressed due to biochar-amendment, especially in the aged biochar soil amendment. Fresh biochar addition can increase the microbial biomass, while the aged biochar-amendment had no effect on the total PLFA concentration. Microbial community composition was altered mainly by an increase in the proportion of bacteria (17:1ω8c and i17:0) in fresh biochar amendment and fungi (18:2ω6, 9c) in aged biochar amendment. Moreover, biochar amendment, particularly aged, significantly decreased the microbial metabolic quotient, suggesting that biochar amendment could enhance the microbial use efficiency of C and the C sequestration potential of paddy soils.