An appraisal of the utility of biochar in a rotation involving tobacco–rice in southern China

The employment of biochar in crop production can not only improve soil quality, but also helps the field ecosystem to fix carbon and reduce emissions. Although the benefits of their application in crop production have been more and more confirmed, it is not clear when it comes to the acidic soil of tobacco and rice rotation. A tobacco–rice rotation experiment was conducted in southern China to probe the application value of biochar under these conditions. Three biochar application rates were employed in this experiment. BC0 (without biochar), BC25 (25 t ha−1), and BC50 (50 t ha−1). The findings show that biochar significantly boosted soil fertility and crop yields. Meanwhile, the soil organic carbon of tobacco rice rotation field with biochar increased by 31.76%. After a whole growth period of tobacco and rice, the cumulative emission reduction of CO2 and N2O from the soil by biochar were 15,944 kg ha−1 and 1810 g ha−1, respectively. The use of biochar not only significantly improved the bacterial diversity of tobacco and rice rotation soil, but also altered the original microbial community structure. The profusion of Proteobacteria and Acidobacteria was reduced and the abundance of Actinobacteria and Bacteroidetes was enhanced in the treatments with biochar. Among them, Sphingomonadales, Planctomycotes, and Ktedonobacteria, which are beneficial to plant growth and soil health, have become key phylotypes. The carbon balance analysis data show that the net carbon sequestration of the two treatments with biochar is positive, while that of the treatment without biochar is negative. In terms of economic benefit, the application of biochar increased the average of 2.055 CNY kg−1 consumed energy (CE) in the whole tobacco–rice rotation system. The ecological benefit was 0.51 kg C kg−1 CE. In conclusion, biochar can be effectively used in the practice of tobacco–rice rotation and acidic soil improvement in southern China.


| INTRODUCTION
China is the largest player in the world when it comes to the production and consumption of tobacco (Zou et al., 2018). The southern region is the main tobacco planting area in China. However, in southern China, the area dedicated to large-scale tobacco cultivation is limited, thus leading to several issues associated with the necessity of continual tobacco farming. For example, plant diseases and insect pests, the decline of soil fertility, tobacco leaf quality loss, continuous accumulation of toxic substances in soil, etc. (Ratnayake et al., 2017). Besides that, the tobacco soils in southern China are markedly acidic and have low fertility. To address this issue, local farmers employ the agricultural practice of tobacco-rice crop rotation (Hu et al., 2021). In addition to improving soil fertility and reducing crop damage from pests and diseases, the rotation pattern can also ameliorate soil structure and increases the yield and income obtained from the two crops (Hu et al., 2021). But this measure also has flaws, which is that it will lead to greater utilization of tobacco fields, resulting in a larger amount of carbon sources being released into the environment, such as from plant harvesting and greenhouse gas emissions. Reducing farmland greenhouse gas emissions and developing green and low-carbon agriculture are of great significance to mitigate global warming. Consequently, it is critical to embrace dependable strategies to lower carbon emissions in agricultural production, especially with regard to the 2030 carbon peak target suggested by China .
Biochar, created through the pyrolysis of organic biomass, is characterized by its plentiful recalcitrant organic carbon and its highly porous structure Liu, Wang, Tang, & Liu, 2021;Shaker et al., 2021). As a cost-effective and environmentally friendly soil amendment, biochar plays a huge role in improving soil nutrients conditions (Lian et al., 2020), enhancing crop growth and yield (Hale et al., 2020), and altering the composition of soil microbial. More importantly, an increasing number of research results confirm that biochar plays an undeniable role in reducing carbon emissions in agricultural production (Gao et al., 2023;Sun et al., 2021). Since Rondon et al. (2005) first reported the decrease in N 2 O emissions followed by modifying the biochar soil amendment in a greenhouse test, employing biochar as a greenhouse gas emission control strategy for agricultural soils has become more appealing. Although the emission reduction effects of different types of biochar are not the same (Wu et al., 2018), and biochar also has different emission reduction mechanisms for the three main greenhouse gases CO 2 , CH 4 , and N 2 O (Feng et al., 2012;Spokas, 2013;Yuan et al., 2019), it is still a consensus in most studies that adding biochar can effectively suppress soil greenhouse gas emissions. In addition to reducing greenhouse gas emissions, biochar can also retain carbon in field soil ecosystems by increasing the amount of soil organic carbon (Zhang et al., 2017).
At present, there are many research works going on the field application of biochar, but there are only a limited number of analyses on its comprehensive value from the perspective of economic and ecological benefits. In particular, it is even rarer to introduce biochar into the tobaccorice rotation system in southern China. In addition to Huang's research report that biochar can increase CH 4 uptake and reduce N 2 O emission in tobacco-rice rotation soil, no more practical cases have been reported for the time being. It is also still unclear whether the overall application value of biochar to tobacco-rice rotation system is positive or negative.
To find out whether biochar has a positive effect on crop rotation system of tobacco-rice, we performed a field experiment with tobacco-rice rotations in southern China. By comparing the influence of different biochar usage rates on SOC pool, greenhouse gas emission, and soil bacteria, combined with calculating the carbon footprint of farmland ecosystem using the life cycle assessment (LCA), we analyzed the application value of biochar in tobacco and rice rotation in southern China from both economic and ecological aspects. This study provides a theoretical basis and applied foundations for a better understanding of agricultural carbon peak and carbon neutralization targets.

| Experimental design
The collected spent mushroom substrate after harvesting the mushroom was kept under dry conditions prior to conversion to biochar. The production process of the biochar refers to our previous research (Hu et al., 2022). The temperature for making biochar was set at 500°C. The reaction pressure was 50-60 kPa. The pyrolysis continued for 3 h. The ratio of the chemical elements constituting the biochar was measured to be 61.45% carbon, 2.4% nitrogen, 3.12% hydrogen, 0.59% sulfur, and 21.28% oxygen. The trial was conducted in Linli County, Hunan Province, southern of China (111°32′ E, 29°17′ N) in 2021, with an average annual temperature and precipitation of 17.3°C and 1400 mm, severally. Regarding the FAO classification, the soil of the shallow mound experimental field was paddy soil (Fao, 2014). The tobacco cultivar 'Yunyan 87' and rice cultivar 'Lingliangyou 268' were used in the present research. Prior to sowing, the top soil layer (0-20 cm) contained various nutrients including 15.7 g kg −1 organic matter, 1.6 g kg −1 total nitrogen, 13.8 g kg −1 total potassium, 2.6 g kg −1 total phosphorus, 110.4 mg kg −1 available nitrogen, 116.2 mg kg −1 available potassium, and 49.6 mg kg −1 available phosphorus. The cropping progression implemented at the experiment field was flue-cured tobacco season (TS), planted on March 20th and harvested on July 15th and rice season (RS), planted on July 20th and harvested on October 25th.
The tests were performed with two different rates of the applied biochar and a control with no biochar. The samples are named as follows: BC0: no biochar applied; BC25: biochar applied at the rate of 25 t ha −1 biochar; BC50: biochar applied at the rate of 50 t ha −1 biochar. The experiment was arranged in a block with a random design containing three replications and each plot had an area of 14 × 8 m. The biochar was applied before the transplantation of the tobacco seedlings. The total amounts of the fertilizers used in the tobacco-rice rotation are potassium fertilizer, superphosphate, and nitrogen fertilizer applied at the rates of 450 kg K 2 O ha −1 , P 2 O 5 of 115 kg ha −1 , and 280 kg N ha −1 , respectively. The crop management of different treatments and plots is consistent.

| Monitoring of soil greenhouse gas emissions
Static closed chamber/gas chromatography was used to measure greenhouse gas emission in tobacco-rice rotation soil during the experiment (Zou et al., 2005). The sampling box was made of stainless steel and consisted of a pedestal and a top box. The pedestal (50 cm × 50 cm cross section) was fixed in the field during the experiment. The pedestal was inserted into the soil for about 10 cm and there was a sealed water tank with a depth and width of 3 cm at the top of the pedestal. Before sampling, water should be injected into the tank to prevent air leakage at the joint of the pedestal and top box. The top box (50 cm× 50 cm× 50 cm) was covered with a thermal insulation material to reduce the influence of external temperature on trial results. Two small fans are installed inside the top box for mixing the gas in the box. The gas samples were gathered between 9 and 11 am in a 10-day hiatus in the course of the TS and RS. A syringe was employed to take gas samples at various time points of 0, 20, 40, and 60 min during the TS and at 0, 10, 20, and 30 min within the RS after closing the chamber. The concentrations of CO 2 and N 2 O gases were determined by gas chromatography (Agilent 7890A). Argon methane (Ar CH 4 ) was the carrier gas of N 2 O. The N 2 O detector is ECD (electron capture detector) and the detection temperature is 3000°C. The CO 2 detector is FID (flame ionization detector) and the detection temperature is 3000°C. The gas emission from soil was determined by Equation (1): where f is CO 2 (mg m −2 h −1 ) or N 2 O (μg m −2 h −1 ) emission flux. p represents the density of CO 2 and N 2 O under standard the conditions. v stands for sampling box's volume (m 3 ); s represents the surface area of the soil existing in the sampling pedestal. △c/△t is the gas emission rate. The temperature of the sampling box is shown with T (°C). The average emission flux during the whole crop growth period was weighed by the interval of each sampling time. Cumulative emissions during the growing season are expressed as a weighed sum of each emission flux.

| Soil sampling
Prior to the experiment, soil samples were gathered and employed for property analyses after harvesting. Samples were taken from five soil cores along two diagonal lines in each plot. They were further homogenized to produce a composite. Each specimen was categorized into two parts. One of the samples was dried with air and passed through a 2 mm sieve to determine the physicochemical features of the soil. The other specimen was kept at −80°C to perform the microbial analysis.

| Analysis of soil properties
SOC was analyzed by wet digestion with H 2 SO 4 -K 2 Cr 2 O 7 . Soil pH was employing a pH meter (soil to water ratio of 1:5). The quantities of NO 3 − − N, NH 4 + − N, K + , and HPO 4 2− were measured by a continuous flow analyser (SEAL-AA3) according to our previous method . Soil bulk density was analyzed via the cutting ring method. Dissolved organic carbon (DOC) is determined by a carbon analyzer (3100, Analytikjena). Soil microbial biomass carbon (MBC) was measured by the chloroform fumigation direct extraction technique (Beck et al., 1997). Readily oxidized organic carbon (ROC) was assessed using potassium permanganate colorimetry.

| Characterization of soil microbiota
The total soil bacterial DNA of each treatment group of samples was evaluated using DNA Extract All Reagents Kit (Thermo Fisher Scientific). Rhonin Bioscience Co., Ltd. amplified and characterized the 16S rDNA bacteria of every specimen. After raw data were obtained, the low-quality ones were filtered followed by assembling and re-filtering. To obtain operational taxonomic units (OTUs) results, Uparse software was employed in a default way to prepare 97% identity to bundle the sequences. Further analysis of the variations in microbial community structure and profusion in each treatment was based on the obtained OTUs.
The αdiversity and βdiversity of the soil bacteria analysis were conducted with R package v. 3.0.6 (Tao et al., 2023). The Bray-Curtis distance was calculated with the vegdits function of the Vegan package (Looft et al., 2012). Moreover, the permutational multivariate analysis of variance was employed to analyze the serious variations in communities between treatments. The statistically different biomarkers between different treatments were employed to evaluate linear discriminant analysis (LDA) combined with effect size analysis (LEfSe; Segata et al., 2011).

| LCA method
The baseline scenario for this study was set as the background emissions without the application of biochar. The system boundary of the LCA includes two stages of biochar production and agricultural application, excluding the production of agricultural machinery, transport of agricultural materials, and consumer consumption. The key steps of LCA measurement include the following: (1) emissions from biochar production, (2) emissions of biochar during transport to farmland, (3) field seeding emissions of biochar, (4) increase in soil carbon sink, and (5) greenhouse gas emissions during the crop growing season. The environmental impact indices of energy production and use processes in this study refer to Liang's (Liang, 2009) study.

| Carbon balance calculation
The net carbon balance was evaluated by combining the total carbon analysis and the biomass-emission calculation of the soil-crop system.
The increment of soil carbon storage was calculated using Equation (2), as follows: where △SOC (t hm −2 ) is soil carbon increment. SOC i (g kg −1 ) is the quantity of the organic carbon of soil at harvest. SOC 0 (g kg −1 ) is the quantity of the organic carbon of soil before the experiment. The bulk density is depicted by BD (g cm −3 ). H is the tillage depth (20 cm).
Net primary productivity was calculated using Equation (3), as follows: where C NNP is the net primary productivity of carbon. C crop is the carbon fixation of crops (including the aboveground grains, straws, and roots of crops). C litter is the field litter carbon.
Agricultural cost carbon emission was calculated using Equation (4), as follows: where C e is the total carbon emissions and C r is the carbon emission from soil respiration. C pesticide , C fertilizer , C irrigation , C plowing , C harvesting , and C labor , respectively, accounted for carbon emissions from pesticides, fertilizers, irrigation, machine farming, harvesting, and production management labor. The carbon emission conversion coefficients are shown in Table 1 (Lal, 2004).
Net carbon sequestration was calculated using Equation (5), as follows: where C s is net carbon sequestration and C i is the carbon input to the field through biochar. Moreover, C h is the carbon contained in biomass removed from crops after harvest.

| Carbon efficiency evaluation of tobacco-rice rotation ecosystem
The carbon efficiency assessment in this study includes carbon productivity, carbon economic, and ecological efficiency. Carbon productivity (C P ) is the economic yield of crops produced per unit of carbon emissions. C P = C yield / C i . Carbon economic efficiency (C J ) is the ratio of the economic output value of crops to carbon input. C J = C value / C i . Carbon ecological efficiency (C E ) is the ratio of C NNP to carbon input. C E = C NNP /C i .

| Statistical analysis
The data demonstrated in the tables and figures of this study are shown as the means of all replicates ± standard deviation (SD). Data variance analysis was performed employing SAS software version 9.2 (SAS Institute). The least significant difference test was employed to compare the average values. The differences were considered to be important at p < 0.05.

| Soil properties and crop yields
Table 2 reveals that the effects of using biochar on soil nutrient conditions and crop growth. Compared with the treatment without biochar, the soil organic matter of TS and RS increased by 25.06% and 23.25%, respectively. In addition, biochar also significantly increased the available nitrogen and potassium as well as the crop yield of rotation soil and reduced the bulk density. Meanwhile, employing biochar also increased the average crop output value of the year by 87,149,717 ¥ (Chinese currency) per hectare. It was noteworthy that the pH value of the treatment using biochar in TS is remarkably above that without biochar, but it did not change considerably in RS.

| Soil organic carbon pool
Figure 1 displays that the application of biochar not only significantly increased the total storage capacity of soil organic carbon pool in tobacco and rice rotation, but also significantly raised the amount of each organic carbon pool component. Compared with the treatment without biochar, the SOC of BC25 and BC50 increased by 30.16% and 33.36%, respectively. In addition, the application of biochar had the most positive effect on MBC. The MBC of BC25 and BC50 treatments increased 88.46% and 195.67%, respectively, compared with the BC0. For different biochar application rates, the difference between BC50 and BC25 treatment was significant only in MBC and ROC.

| CO 2 and N 2 O emissions
The continuous monitoring of greenhouse gas emissions in the soil of tobacco rice rotation showed that the application of biochar significantly inhibited the escape of soil CO 2 and N 2 O to the atmosphere during the whole growth period of the crops. The CO 2 emission in TS has a large range of changes, which is shown in the pattern of initial increment followed by a decreasing trend (Figure 2). This is consistent with the change in soil temperature. Correspondingly, the CO 2 emissions change slightly in RS, and the total emissions are far lower than in TS. In terms of cumulative CO 2 emissions, treatments with biochar were significantly lower in both TS and RS than in the treatment without it. The cumulative CO 2 emission of BC0 was 28.4% higher in TS and 22.5% higher in RS than in the treatment with biochar. However, the cumulative amount of CO 2 emissions between the treatments with dissimilar biochar application rates did not differ significantly. The limiting effect of biochar on N 2 O emission from tobacco and rice rotation soil showed a similar pattern to that of CO 2 (Figure 3). BC0 treatment showed the highest emission rate of N 2 O during the whole experiment. It is worth noting that the cumulative amount of N 2 O emission in TS is 12.32-12.83 kg ha −1 , which is about six times that of RS. At the same time, the difference in the accumulation of N 2 O emission among all treatments in TS was negligible, but the treatment using biochar in RS was significantly lower than that without using biochar.

| Composition of soil microbial communities
By analyzing the αand βdiversities of the bacteria in the soil samples, this study obtained some information about the influence of the used biochar on the diversity and affluence of soil bacteria. According to Figure 4a, the number of OTUs of BC0 was less than BC25 and BC50. At the same time, the results of the rarefaction curves show that

F I G U R E 2
Dynamic changes in CO 2 release rate (A) and cumulative emissions (B) in various processes in the course of tobacco and rice growth period. The period from 3/20 (MM/DD) to 7/15 was the tobacco growth season (TS), and the period from 7/20 to 10/25 was the rice growth season (RS). Different lowercase letters mark significant discrepancies in RS and TS (p < 0.05), respectively.
there was no increase in the number of the detected OTUs along with the enhancement of the sample size and sequencing depth, suggesting the accuracy and reliability of the sequencing results ( Figure 4a). Figure 4b,d shows that the Chao1 and Shanon indexes of the treatments with biochar were significantly higher than those of BC0 treatments (27.58% and 16.04%, respectively), indicating that both the richness and diversity of soil bacteria treated with biochar were increased. The principal coordinates analysis (PCoA) scores plot exhibited a distinguishable clustering between soil bacteria compositions obtained from dissimilar samples. These data demonstrated the obvious difference between each treatment from the others (Figure 5a), revealing a clear distinction between their soil bacteria. The specimens within each group clustered together. A multivariate investigation based on the Bray-Curtis method showed that BC50, BC25, and BC0 samples were significantly separated (Figure 5b), which is in agreement with the results of PCoA. Figure 6a displays the variations in soil bacteria at the phylum level. Four prevailing bacteria in the BC0 treatment were Acidobacteria (19.02%), Rokubacteria (7.88%), Actinobacteria (5.31%), and Proteobacteria (41.63%). In comparison with the BC0 treatment, the profusion of Actinobacteria in BC50 and BC25 treatments increased significantly. Simultaneously, the average number of Proteobacteria and Acidobacteria decreased by 14.13% and 5.02%, respectively. Figure 6b displays the heatmap clustering analysis of the soil bacterial community relative abundance at the genus level in each treatment. The columns indicate the various treatment groups, whereas the lines show various bacteria strains. In addition, the color of the box was representative of the abundance of bacteria kinds. The results of cluster characterization display a statistical separation between the microbial communities among the treatment groups. Furthermore, the dominant soil microbiota was inconsistent, while the samples of the same group were basically clustered together.
In addition to αand βdiversity studies, one can further investigate the community composition by distinguishing specialized bacterial communities of different groups. To this end, we employed the LEfSe tool to detect biomarkers from the phylum to the family level. Cladograms shown in Figure 7a illustrate the various groups and LDA scores of 4 or more affirmed by LEfSe (Figure 7b). A total number of 34 OTUs have been identified as essential phylotypes that can be used to differentiate BC0(10), BC25(12), and BC50(12). Regarding Figure 7a, the main flora of bacteria enriched by different treatments varied greatly, suggesting the change in the soil microbial composition as a result of using biochar. BC50 was mainly enriched in Proteobacteria (including Sphingomonadales, Sphingomonadaceae, Rhizobiales), Bacteroidetes (including Cytophagales, Microscillaceae), and Planctomycotes (including Planctomycetacia, Pirellulales, Pirellulaceae). However, the relative abundance of the phylum Acidobacteria (including Blastocatellia, Pyrinomonadales, Pyrinomonadaceae), and Rokubacteria (including Rokubacteriales) was significantly higher in BC0 than in the other treatments.

| Carbon budget of soil ecosystem under tobacco-rice rotation
The data in Table 3 show the carbon balance of the tobacco-rice rotation field ecosystem under different treatments. Compared with the BC0, C i , C NNP , and △SOC F I G U R E 3 Dynamic changes in N 2 O release rate (A) and cumulative emissions (B) in various processes while tobacco and rice growth. The period from 3/20 (MM/DD) to 7/15 was the tobacco growth season (TS), and the period from 7/20 to 10/25 was the rice growth season (RS). Different lowercase letters mark significant discrepancies in RS and TS (p < 0.05), respectively. were all increased in treatments with biochar. On the other hand, their C e and C h values also increased, and the only carbon sink/source term that decreased was C r . The two treatments using biochar become carbon sinks, while the unused ones become carbon sources. From the perspective of the carbon budget, the C NNP of the field ecosystem is the most important component of the system's carbon sink. Furthermore, the C h and C e are the main carbon sources of the system. Figure 8 reveals that the application of biochar can comprehensively improve the C P , C J , and C E of the field in the tobacco-rice rotation system. The treatment without biochar was 30.39%, 24.97%, and 35.91% lower in C P , C J , and C E than the treatment with biochar. However, as for the treatment with different biochar application rates, there was no significant difference in these indicators.

| Effects of biochar on soil properties and organic carbon pool
Many researchers have reported that biochar as a soil additive can have beneficial effects on soil. For instance, biochar is able to enhance soil water capacity, as well as its structure and fertility (Gaskin et , 2002;Lehmann et al., 2003). In this study, soil nutrient and crop yields were significantly increased under the treatment of biochar application. This is consistent with previous research. Acidic soil fertility in south China is usually poor (Yang et al., 2022). It is a simple and effective agricultural production measure to improve soil fertility by adjusting soil pH (Tibbett et al., 2019). In this study, the soil pH of the two biochar treatments was nearly 0.5 above that of the treatment without biochar in TS. This indicates that alkalescence of biochar is especially suitable for improving the acidic environment of soil, which can significantly boost the productivity of the field.
The transformation of the organic carbon pool in the soil is tightly associated with the cycle of other nutrients in soil and affects its physical and chemical features. Therefore, the content and composition of SOC can determine soil fertility . Consistent with our conjecture, using biochar in soil not only greatly enhanced SOC, but also improved each component of the organic carbon pool in this study. However, the most substantial rise of the soil organic carbon pool is attributed to MBC (Figure 1), thereby demonstrating that soil microorganisms are the primary source of the increase in soil organic carbon pool capacity from biochar. It has been suggested that this result occurs because biochar can provide a habitat for plant roots and microbial communities, making soil-root-biochar interactions more active (Lehmann et al., 2015). Although the use of biochar significantly increased the content of organic carbon components in this part of the soil, there was no significant difference in the content of active organic carbon components between different biochar application rates, indicating that the influence of biochar on these active organic carbon components was not realized in a direct form. Unfortunately, the data in this study do not explain why this is the case. In future studies, the incubation experiment of soil organic carbon mineralization process will help to answer this question.

| Effects of biochar on N 2 O and CO 2 emissions from soil
The mineralization and decomposition of SOC and respiration of soil organisms are the sources of CO 2 emission, and N 2 O comes from nitrification and denitrification in   the soil. It has already been discovered that using biochar with a small C/N ratio (C/N < 30) may increase soil N 2 O emissions caused by microbial nitrogen fixation. (Baggs et al., 2000;Cayuela et al., 2010). Spent mushroom substrate biochar (C/N < 30) used in this study obviously has no ability to reduce N 2 O emissions from tobacco-rice rotation soil, but this was not consistent with our experiment results. Such a result may be due to the reduction of nitrification activity in acidic soil conditions, resulting in the reduction of microbial nitrogen fixation capacity. This is consistent with experimental results obtained on paddy soils in southern China. The use of biochar reduces the emission of N 2 O from the soil (Huang et al., 2019). Although using biochar can reduce soil CO 2 emission in both TS and RS, the emission reduction effect is more obvious in TS. Meanwhile, it can be seen from Figure 2 that CO 2 emission is highly correlated with soil temperature, indicating that microbial activities influenced by soil temperature play a dominant role in greenhouse gas emissions. As for the difference in cumulative emissions of CO 2 and N 2 O between tobacco and rice, it is mainly caused by soil temperature and the resulting activity of denitrification bacteria (Bateman & Baggs, 2005). In addition to N 2 O and CO 2 , CH 4 is also an important source of greenhouse gases in agricultural ecosystems. Different from N 2 O and CO 2 , CH 4 emission in saturated soil is higher than that in unsaturated soil. In other words, CH 4 is the main greenhouse gas source during rice production. The effect of biochar on CH 4 emission under tobacco rice rotation will be another important target for future research on this tillage practice.

| Effects of biochar on the diversity and richness of soil bacteria
The interaction of biochar with soil microorganisms is controversial. It is generally accepted that biochar can enhance the abundance and variety of soil bacteria (Yan et al., 2020;Zhang et al., 2021); however, certain reports suggested that the toxic properties of biochar (e.g., polycylic aromatic hydrocarbons, polar pyrolysis condensates) can suppress the activity of soil microorganisms . The results of this research exhibited that the application of biochar could significantly enhance the diversity of soil bacteria. Therefore, the application of biochar in tobaccorice rotation soil is still considered to be of positive value. As for the physiological toxicity of biochar, more of it comes from specific raw materials and the pyrolysis process (Jia et al., 2020). In addition, it can be seen from Figure 6 that the application of biochar also significantly changed the structure of the microbial community in the tobacco-rice rotation soil. The dominant bacteria in the treatment of BC0 were Proteobacteria and Acidobacteria. In the treatment with biochar, the abundance of Actinobacteria and Bacteroidetes increased, while that of Acidobacteria and Proteobacteria decreased. Bacteroidetes can degrade polymer organic compounds in soil and are the main decomposers of organic matter and humus in soil (Larsbrink & Mckee, 2020). At the same time, studies have shown that crop root diseases can be greatly relieved or eliminated after inoculation with Actinobacteria in the soil. Actinobacteria can also secrete cytokinin, which can significantly improve the biomass and yield of crop roots, stems, and leaves, and enhance the resistance against crop diseases (Babu F I G U R E 8 Analysis of ecological and economic benefits of the biochar application on tobacco-rice rotation field. Bao et al., 2021). The soil with a high abundance of Acidobacteria is generally characterized by acidic and low nutrient content. The decrease in Acidobacteria abundance after biochar application was also consistent with our previous results on the increase in soil pH (Table 2). It can also be seen from the biomarkers analyzed by the LefSe tool that the number of beneficial microorganisms in the soil with biochar has been increased (Figure 7). For example, the key phylotype Sphingomonadales belonging to BC50 has been proven to be PGPR (plant growth promoting rhizobacteria). These bacteria can produce GAs and IAA and induce root growth (Ke et al., 2021;Wang et al., 2009). Planctomycetes, belonging to BC25, is a typical bacterial population that can obtain energy by using NH 4 + and NO 3 − , and has a strong nitrogen fixation ability (Ali et al., 2015).

| Positive benefits of biochar to the tobacco-rice rotation system
On the one hand, the field fixes CO 2 existing in the atmosphere into plants through the photosynthesis of crops, which is the carbon sink of greenhouse gases. On the other hand, it also emits greenhouse gases into the atmosphere through soil respiration and microorganism, which is the carbon source of greenhouse gases (Cayuela et al., 2014). Although in the previous results we found that returning biochar to the field limited the emission of soil CO 2 and N 2 O, the emission reduction of a single sink did not represent the performance of the whole system (Schlesinger, 2010). Therefore, the analysis of the whole system from the perspective of "net carbon balance" can more objectively evaluate the application value of biochar in the soil of tobacco-rice rotation, which is one of the main purposes of this study. It can be seen from the data in Table 3 that the net carbon sequestration of the treatment with the application of biochar was positive, while that of the treatment without it was negative. This shows that, although the application of biochar, an additional agricultural management measure, increases the C e , it generally turns the field ecosystem, originally used as a carbon source, into a carbon sink. In addition, although the C e of BC50 is far greater than that of BC25, the final C s value is still the highest. This shows that the carbon sequestration effect of biochar is obvious, which will be significant for the carbon mitigation of the agricultural production system. As can be seen from Figure 8, although biochar increases the C h and C r of the system, it also promotes the improvement of carbon productivity. Compared to the treatment without biochar, the addition of biochar increased the C NNP of tobacco-rice rotation soil by an average value of 16.2%. In terms of economic benefit, the application of biochar increased the average of 2.055 CNY kg −1 CE in the whole tobacco-rice rotation system. The ecological benefit was 0.51 kg C kg −1 CE. These results indicated that the biochar returning to the field had a positive effect on the whole tobacco-rice rotation system, both on economic and ecological aspects.
It is worth noting that, although the residual benefits are decreasing significantly in the long run, the carbon sink effect of biochar is still obvious . However, specifically for the tobacco rice rotation system, the ecological and economic benefits of applying biochar over a longer time scale still require long-term experimental results to verify.

| CONCLUSION
This study explored the response of soil properties, organic carbon pool, greenhouse gas emissions, and microbial community composition to different biochar application rates through a 1-year tobacco and rice rotation experiment. At the same time, based on the experimental results and LCA, the application value of biochar in the tobacco-rice rotation system was comprehensively evaluated. The results showed that biochar application not only effectively alleviated the poor fertility of acidic soil, but also increased the crop yield and output value of the current season by 13.65% and 14.27%, respectively. With the strong carbon sequestration capacity of biochar, the greenhouse gas emissions from tobacco-rice rotation soil have been significantly reduced. Results from 16S rDNA analysis indicated that the diversity of microorganisms presents in the soil was significantly enhanced due to the introduction of biochar, which also resulted in an increment in the number of bacteria beneficial to both plant growth and soil organic matter mineralization. Furthermore, LCA exhibits that biochar can also convert the field under tobaccorice rotation from carbon source to carbon sink and improve its economic and ecological benefits. Therefore, we believe that biochar has considerable application prospects in local tobacco production. Our findings will contribute to the realization of agricultural carbon peak and carbon neutral goal in southern China.

ACKNOWLEDGMENTS
This study was supported by Chongqing Technology Innovation and Application Development Project (CSTB2022TIAD-CUX0020).

FUNDING INFORMATION Chongqing Technology Innovation and Application Development
Project, Grant/Award Number: CSTB2022TIAD-CUX0020.