Increases of Temperature Response for CO 2 Emission in a Biochar-Amended Vegetable Field Soil

To explore the effects of biochar application on CO 2 and CH 4 emission as well as the temperature response of CO 2 emission, an one-year experiment was conducted with three treatments (Control; CF, chemical fertilizer only; BCF, biochar combined with chemical fertilizer) in a vegetable eld. Results shown that (1) compared with CF, short-term application of biochar signicantly enhancing the cumulative CO 2 emission by 27.5% from soil-plant system, via increasing the soil microbial biomass (e.g., MBC) and C substrates (e.g., SOC). (2) A lowest emission of CH 4 was observed in BCF treatment, and an increase CH 4 consumption and reduce competition with NH 4+ may be responsible for the signicant reduction in CH 4 source strength in biochar amended soil. (3) Activation energy (E a ) was identied as an important factor inuencing the temperature sensitivity (Q 10 ) of CO 2 emission. Fertilization (CF and BCF) reduced the average Q 10 and E a values of CO 2 emission by 9.0-26.7% and 23.5-10.1%, relative to the control, respectively. Besides, the average of E a value in BCF treatment (51.9 KJ mol -1 ) was signicantly higher than those in control and CF treatment. The increase in Q 10 and E a values following biochar application possibly contributed to the supplement of limit labile C and nutrient but highly resistant C following biochar application. Soil pH and crop cultivation may play key roles in inuencing the change of E a . Our study concludes that biochar amendment increased CO 2 emission and temperature response of CO 2 emission from soil-plant system, while reduced CH 4 emission. to the exogenous C, especially in the short-term time. In this study, we conducted a short-term experiment (about one year) in vegetable cultivation to investigate the response of bicohar amendment on the CO 2 and CH 4 emission as well as the temperature response of CO 2 emission. The objectives of this study were to (1) explore the effects of biochar amendment on the soil CO 2 and CH 4 emission; (2) determine the temperature response of CO 2 emission in biochar amended soil; and (3) trying to identify some factors that signicantly inuenced C emission and temperature response of CO 2 emission in short-term application of biochar.


Introduction
Biochar, as a soil amendment has been incorporation into soil to improve soil properties and soil structure, increase nutrient available and microbial activities ( Therefore, there has been growing call to add biochar into soil to promote C sequestration and improve soil quality. However, in short periods of time (i.e. months), biochar will undergo structural changes, primarily the oxidation of surface, and can be utilized by microbes as a C source (Cheng et al. 2006;Zavalloni et al. 2011). As a result, biochar could be an ecosystem C source, instead of sink, within a shortterm period in soil. For example, Ameloot et al. (2013) determined that the increase of short-term CO 2 and N 2 O emissions (117 days) were observed in biochar-amended soils due to the rapid degradation of labile compounds in the biochar (Zimmerman et al. 2011). Alternatively, new substrates addition would stimulate the "priming effects", de ned as the changes in the mineralization of native soil organic matter (Kuzyakov, et al. 2000;Kuzyakov, 2010). The negative priming effects, such as reduced N 2 O production and CH 4  biochar incorporation can increase the root biomass, net photosynthesis and grain yield, and then in uence the net CO 2 emission from the soil-plant system (Masto et al. 2013;Sun et al. 2017). Hence, the short-term response of greenhouse gas emission on the biochar application in agriculture systems should be received more attention.
Temperature plays a vital role in affecting soil organic C (SOC) mineralization and result in variability in the C pool (Criscuoli et al. 2019;Kan et al. 2020; Wang et al. 2019). The response to temperature changes, such as temperature sensitivity (Q 10 , de ned as the rate of change of soil CO 2 emission as a consequence of temperature increase of 10 ℃) (Kirschbaum 1995) and activation energy (E a , de ned as the necessary energy for reacting molecules to break and form new bonds after a collision) (Thiessen et al. 2013), could be used to evaluate the feedback intensity between CO 2 emission and global warming (Zhou et al. 2009), as well as the response of SOC to global warming (Fang et al. 2014). Exogenous C input (e.g. biochar) may alter the chemical recalcitrance of organic matter and environmental conditions, and resulted in changing the temperature response of CO 2 emission (Fang et al. 2014Wang et al. 2019). According to the fundamental enzymatic kinetic theory, organic compounds with higher molecule weights showed lower rates of decomposition, higher values of Q 10 and E a relative to organic compounds with lower molecule weights. However, the decreases and increases in the Q 10 (Conant et al. 2011). Biochar application in short-term period may introduce the more C including stable and labile C, which is related to the temperature response. However, there is a lack of knowledge about the temperature response of CO 2 emission from soil-plant systems in biochar amended vegetable eld soil.
Here, we hypothesized that biochar incorporated to soil would increase the C gas loss and enhanced the temperature response of CO 2 emission due to the exogenous C, especially in the short-term time. In this study, we conducted a short-term experiment (about one year) in vegetable cultivation to investigate the response of bicohar amendment on the CO 2 and CH 4 emission as well as the temperature response of CO 2 emission. The objectives of this study were to (1) explore the effects of biochar amendment on the soil CO 2 and CH 4 emission; (2) determine the temperature response of CO 2 emission in biochar amended soil; and (3) trying to identify some factors that signi cantly in uenced C emission and temperature response of CO 2 emission in short-term application of biochar.

Study site description
The experiment was conducted in the National Monitoring Station of Soil Fertility and Fertilizer E ciency on Purple Soils (30°26′N, 106°26′E) in the Beibei district of Chongqing, southwestern China. The in-situ soil is classi ed as Regosol in the Food and Agriculture Organization classi cation scheme (FAO, 1988).
The details of this trail site were described in study of Huang et al. (2018Huang et al. ( , 2019. The basic property of soil was shown in Table 1.

Experimental design
Nine 2 m × 1 m plots were selected for this study from 2016-2017. Three treatments (one treatment per plot), including no fertilizer (control), chemical fertilizer only (CF), and biochar combined with chemical fertilizer (BCF), were arranged in a completely randomized design with three replicates (total 9 plots). The same amount of total nitrogen (N), phosphorus (P) and potassium (K) was applied in CF and BCF treatments. Chemical fertilizers were applied as urea (N-eq, 46%), single superphosphate (P 2 O 5 -eq, 12%) and muriate of potash (K 2 O-eq, 60%), respectively. Biochar derived from rape straw, was purchased from Sichuan Jiusheng Agricultural Technology Development Co. Lid., China. The property of biochar was given in Table 1. . Air and soil temperature (5 cm depth in soil) and soil moisture content were recorded at the beginning and the end of sampling, and average of the two values was calculated. Due to the greenhouse gas chamber measurement cannot exclude the CO 2 emission from plant roots, the CO 2 emission in this study was the net CO 2 emission from vegetable eld, which integrated soil respiration, belowground greenhouse gas emission and the CO 2 assimilated by plants.

Soil sampling and measurements
Topsoil (0-20 cm) were sampled on November 23, 2017. In each plot, ve soil cores were randomly sampled and mixed to form a pooled sample. The pooled samples were placed in the sterile plastic bags and transported to the laboratory. Meanwhile, soil bulk density was obtained via the cutting ring method.
Sampled soil was thoroughly mixed and passed through a 2-mm sieve after all the visible roots and stones had been removed. Fresh soil was used for the analysis of soil dissolved organic carbon (DOC) and microbial biomass carbon (MBC), and the nal concentrations of DOC and MBC were normalized by the dry mass of soil. The remaining soil was air-dried for measuring the total soil organic carbon (SOC) and other soil properties.
Soil water-lled pore space (WFPS) was calculated according to the following equation (Li et al. 2013

Temperature response
Temperature sensitivity (Q 10 ) and activation energy (E a ) of CO 2 emission were used to describe the relationship between temperature and CO 2 emission. Where, y is the ux of CO 2 over time (mg m −2 h −1 ), A is the constant, E a is the activation energy (J mol −1 ), R is the universal gas constant (8.314 J mol −1 K −1 ), T is the soil temperature in Kelvin (K). In chemical kinetics, E a is de ned as the necessary energy for reacting molecules to break and form new bonds after a collision. For calculating the daily E a , a maximum likelihood estimate of the slope of the linear regression of the natural logarithms of CO 2 ux against the reciprocal of absolute soil temperature. To estimate the average E a during the experiment period, we multiplied the slope values by the gas constant R.

Statistical analysis
The data were statistically analyzed using the SPSS 23.0 and Origin 8.5 software. The Kolmogorov-Smirnov test was used to test the normality of all data. Both parametric and non-parametric approaches were used to test the differences. For the normal distributed data, comparisons of data among treatments were performed by one-way analysis of variance analysis (ANOVA) in combination with the least signi cant difference (LSD) test. For non-normal distributed data, the comparisons of data were performed by Kruskal-Wallis test. The variables related to soil properties, Q 10 , E a and cumulative CO 2 , CH 4 emission were subjected to principal component analysis (PCA) to identify key factors for Q 10 , E a and cumulative CO 2 , CH 4 emission using Origin 8.5. Automatic linear modeling was performed at the 95% con dence level using SPSS 18.0. The Spearman's coe cient was used in the non-parametric correlation analysis. The statistical signi cance was determined at p = 0.05 and p = 0.01.

CO 2 and CH 4 emission
As shown in Fig. 1a, there were two peaks of CO 2 ux during the experimental period, which were observed in April and August, respectively. The highest CO 2 ux with the value of 3254.8 and 3201.9 mg m −2 h −1 were both found in the BCF treatment on April 13 and August 9, respectively. Compared with the control, fertilization (CF and BCF) increased the ux of CO 2 , except for the period of higher air temperature (from July to August). The higher CO 2 uxes were observed in the BCF treatment, relative to CF treatment, when the air temperature was over 18 ℃. Additionally, the second peak of CO 2 ux in the BCF treatment (on August 9) was later than that in the CF treatment (on July 26). During the experimental period (Fig.  1b), BCF signi cantly increased the cumulative CO 2 emission by 27.5% and 37.1%, relative to the control and CF treatment, respectively.
Different from CO 2 ux, variation of CH 4 ux during the experiment period was not signi cant (Fig. 1c).
However, after the application of biochar, the signi cant uctuation of CH 4 ux was observed, especially after the second time of biochar application. Compared with control, CF and BCF both reduced the cumulative CH 4 emission, and the cumulative CH 4 emission in the BCF treatment was -1.09 kg hm −2 (Fig.   1d).

Temperature sensitivity (Q 10 ) and activation energy (E a )of CO 2 emission
Because of the negative value of CH 4 ux, only temperature sensitivity (Q 10 ) and activation energy (E a ) of CO 2 emission were calculated in this study. The ux of CO 2 has an exponential relationship with the soil temperature ( Fig. S1a-c). The dynamic of Q 10 Fig. 2b, the lowest value of average Q 10 was observed in CF treatment, which signi cantly reduced by 29.2% relative to the control. However, there were no signi cant difference between CF and BCF treatments, even if the higher value of average Q 10 (Q 10 = 2.1) was observed in the BCF treatment.
Similar to the Q 10 dynamic of CO 2 emission, peaks of E a value were all found in each vegetable growing season, especially in the initial time of vegetable growing (Fig. 2c). Compared with the CF, BCF increased the E a values by 33.7-49.5%, regardless of times of biochar application. Besides, the average of E a value in BCF treatment (51.9 KJ mol −1 ) was signi cantly higher than those in control (60.4 KJ mol −1 ) and CF (36.2 KJ mol −1 ) treatments (Fig. 2d).

Soil property
Compared with CF, BCF increased the contents of DOC, MBC and SOC by 800.7% (p<0.05), 33.3% (p<0.05) and 68.9% (p>0.05), respectively ( Table 2). In addition, the highest values of soil pH and WFPS were both found in the control, following by those in BCF treatment. Numbers represent mean ± standard error (n = 3); different lowercase letters within the same column indicate significant differences (P < 0.05). Control, no fertilizer; CF, chemical fertilizer only; BCF, biochar combined with chemical fertilizer; DOC, dissolved organic carbon; MBC, microbial biomass carbon; SOC, soil organic carbon; WFPS, soil water-filled pore space.

Correlation of soil properties, Q 10 , E a and carbon emission
The rst two principal components (PC1 and PC2) accounted for 50.0% and 31.3% of the total variation in principle component analysis (PCA), respectively (Fig. 3). Variation of cumulative CO 2 emission has a positive relationship with SOC, but a negative relationship with the cumulative CH 4 emission (Fig. 3). Soil DOC was the key factor in uencing the variation of Q 10 and E a according to the results of PCA analysis.
Correlations among soil properties, Q 10 , E a and carbon emission (CO 2 and CH 4 ) were listed in Table S2.
The cumulative CO 2 and CH 4  and WFPS (r = 0.792). Besides, automatic linear modeling revealed that soil SOC, together with MBC were the primary factors associated with the cumulative CO 2 emissions, as well as SOC and pH associated with the cumulative CH 4 emissions (Fig. 3). Activation energy (E a ) and soil DOC was the key factor in uencing the Q 10 and E a, respectively.

Biochar application in uencing the carbon emission
Biochar, as a soil amendment, plays a key role in C utilization as well as in decreasing the greenhouse gasses emissions. In general, bichar reduces the CO 2 emission through the expansion of soil C pool (Kavitha et al. 2018). In the present study, however, biochar application increased the CO 2 emission from soil-plant system during short-term experiment, relative to the no-biochar (control and CF) treatments (Fig. 1b).  (Table 2). Besides, the result of automatic linear modeling also vertify that the enhanced microbial biomass (e.g. MBC) and C substrates (e.g. SOC) in soils may lead to the greater CO 2 emission (Fig. 3). It is worth noting that CO 2 emission in this study was the net CO 2 emission from soil-plant system, which integrated soil respiration, root respiration and the CO 2 assimilated by plants.
The signi cant negative relationship between total vegetable yield and cumulative CO 2 emission may index the key roles of root respiration and plant photosynthesis in CO 2 emission (Table S2), especially for the root respiration. Additionally, biochar application obtained high total vegetable yields than no-biochar (Table S3). Therefore, short-term biochar and N combined application cannot offset, at least partly, the negative effect of biochar or plant photosynthesis on the CO 2 emission.
It is well known that dryland soil in an oxic condition has a capacity of CH 4  , biochar application in this study signi cantly reduced the cumulative CH 4 emissions relative to the control and CF treatments (Fig. 1d). A potential explanation is the fact that the enhanced soil aeration would increase the activity of methanotrophs due to the biochar's large surface area and pore volume , which supported by the negative relationship of cumulative CH 4 emission and CO 2 emission ( Fig. 3 and Table S2). This result suggested the increased soil CH 4 consumption rather than decreased CH 4 production dominated the in uence of biochar in mitigating CH 4 emission from dryland soil-plant system. Another potential explanation, as discussed above, is that the progressive protection of biochar may prevent SOC from being used by methanogens (Zimmerman et al. 2011), resulting in the decreased CH 4 production. The higher contents of SOC observed in BCF treatment may be attributed by the protection of biochar in this study (Table 2). 4.2 Biochar application in uencing the temperature response of CO 2 emission In this study, fertilization incorporation reduced the temperature response of CO 2 emission (expressed as Q 10 or E a ), compared to the control (Fig. 2a-b). It may be caused by the fact that nutrients (e.g. N, P) from fertilizers changed the substrate C quality, which is linked to the soil C emission (Guo et al. 2017). Previous studies determined that the N addition potentially increased those microbial abundance using labile C and elevated the cellulose-decomposing enzymes activity (Carreiro et al. 2000;Keeler et al. 2009). Thus, increased Q 10 was observed following fertilization or arti cial N deposition in the previous studies  Table S2) possibly supported the enzyme kinetic hypothesis.
Therefore, the reduced Q 10 under short-term fertilizer inputs may be well explained by the lower E a in CF and BCF treatment. More biochar incorporated into soil can increase the non-biochar labile dissolvable C of native soil, which would be entrapped in the porous structure of biochar (Bending et al. 2014). While the co-location of microorganisms and entrapped C, as mentioned above, may enhance the availability of soil decomposable C, thus reducing the Q 10 values (Pei et al. 2017). Though the higher DOC content was observed in soil treated with biochar ( and E a (Table S1 and Fig. 3). Acidifying soil causing by fertilization is characterized with high osmotic pressures, low soil minerals and high aluminum toxicity, which would reduce the microbial activity and consequently decreasing the temperature response (Treseder 2008; Liu and Greaver 2010). Thus, the higher soil pH in BCF treatment may be partly responsible for the higher temperature response, relative to the CF. Besides, the peak of E a with time was observed within one week of crop transplant in each grow season, regardless of treatments (Fig. 2c). We speculate that the crop cultivation measures may in uence the E a possibly due to inducing the change of the external and/or direct factors (e.g., root biomass).
Unfortunately, the soil indexes with time have not been detected in this study. However, the signi cant relationship of E a and vegetable yields may index the important effect of vegetable cultivation on the temperature response of CO 2 emission (Table S2). As mention above, biochar application may impact the CO 2 emission due to the root respiration. Overall, short-term application of biochar increased the temperature response of CO 2 emission in the soil-plant system.

Conclusion
Short-term application of biochar signi cantly increased the CO 2 emission from soil-plant system.
However, biochar addition showed a signi cant reduction in CH 4 source strength in dryland soil, possibly via increasing CH 4 consumption and reducing competition with NH 4 + . Fertilization reduced the temperature sensitivity (Q 10 ) of CO 2 emission through decreasing activation energy (E a ). Besides, biochar signi cantly increased the temperature response (Q 10 and E a ) of CO 2 emission, relative to solely chemical fertilizer application, which is related to the supplement of limit labile C and nutrient but highly resistant C following biochar application. External factors (e.g. pH, crop cultivation) play key roles in in uencing the change of E a . Thus, our study suggests that short-term response of biochar on C gas emission and temperature should be obtained attention to better understand the long-term effect of biochar on C release and sequestration.