Biochar Blended with Nitrogen Fertilizer Promotes Maize Yield by Altering Soil Enzyme Activities and Organic Carbon Content in Black Soil

Biochar and nitrogen fertilizers are known to increase soil carbon storage and reduce soil nitrogen loss as amendments, suggesting a promising strategy for highly effectively increasing soil productivity. However, few studies have explored the mechanisms of their effects on crop yield in terms of active carbon fraction and enzyme activity, which ultimately limits the potential for the application of biochar in combination with nitrogen fertilizers. To evaluate the effect of biochar and nitrogen fertilizer on the improvement of black soils in northeast China, a field experiment was conducted in the black soil to compare and analyze the application methods on total organic carbon (TOC), total nitrogen (TN), enzyme activities, and maize yields. Biochar rates: CK, C1, C2, and C3 (0, 9.8, 19.6, and 29.4 Mg·ha−1); N fertilizer rates: N1/2 and N (30 and 60 kg·ha−1). Results indicated that biochar and N fertilizer amendments significantly ameliorated soil fertility, such as TOC and TN, compared to the unamended soil. The TOC levels in the C3 treatment increased by 35.18% and the TN levels by 23.95%. The improvement in TN is more significant when biochar is blended with N fertilizer. Biochar blended with N fertilizer increased maize cellulase, urease, and invertase activities by an average of 53.12%, 58.13%, and 16.54%, respectively. Redundancy analysis showed that TOC, TN, and MBN contributed 42%, 16.2%, and 22.2%, respectively, to the maize yield indicator. Principal component analysis showed that reduced N fertilizer was more effective in improving yields, with a maximum yield increase of 50.74%. Biochar blended with N fertilizer is an effective strategy to improve the fertility and productivity of black soils in northeast China, while nitrogen fertilizer reduction is feasible and necessary for maintaining grain yield.


Introduction
Biochar is considered a solution for improving the soil's physical structure, forming a highly stable carbon pool, and regulating global climate change [1,2]. The beneficial effects of biochar on soil fertility have been explored in the Brazilian Amazon since pre-Columbian times; most soils in the region are highly weathered and depleted oxidized soils [3]. These modified Terra Preta soils were characterized by 35% black carbon in the surface layer [4]. Additionally, Hu [5] proposed that the mineralization of soil organic carbon (SOC) showed a strong negative excitation effect following the biochar addition, which reduced the mineralization rate of the SOC. Song [6] also indicated that the addition of biochar could increase the content of the SOC, which could be used by microorganisms, thus inhibiting organic carbon mineralization. Furthermore, Luo [7] found that exogenous carbon input can improve soil cohesion, prevent soil erosion, and decrease soil nutrient loss. Compared with the SOC, active organic carbon components (extractable organic carbon

Site Description
The field study was conducted at the Northeast Institute of Geography and Agroecology, CAS, in Jilin Province, China (43 • 59 51" N, 125 • 24 5" E), 200 m above sea level, in a temperate continental climate characterized by monsoons with an annual average precipitation of 520 mm. The experimental surface soil pH was 6.06, TN was 1.26 g·kg −1 , available phosphorus was 26.78 mg·kg −1 , available potassium was 133.54 mg·kg −1 , and organic matter was 26.72 g·kg −1 . For many years, continuous corn cropping has been carried out in conventional tillage patterns. The biochar was derived from corn husk pyrolysis at 400-500 • C for 4 h under anaerobic conditions. The mean particle diameter, surface area per volume, and ash concentration of the biochar were 0.0003-3.5 mm, 0.7 m 2 ·g −1 , and 45%, respectively. The biochar had a pH of 9.16 (biochar to water of 1:10), a total carbon content of 62.64%, and a C/N ratio of 39.08. In addition, the N fertilizer was high-quality urea and was produced by Erdos Yi Ding Ecological Agriculture Development Co., Ltd., with a TN ≥ 46% and a particle size range of 1.18-3.35 mm.

Field Experimental Design
The biochar and N fertilizer were applied to the black soil in April 2021, and the corn was sown in May 2021. The field experiment had a split zone design with four biochar input gradients: 0 Mg·ha −1 (CK, 0%), 9.8 Mg·ha −1 (C1, 0.4%), 19.6 Mg·ha −1 (C2, 0.75%), and 29.4 Mg·ha −1 (C3, 1.5%). Also, N was applied in the form of basal fertilizer at rates of 300 kg N ha −1 (N1/2) and 600 kg N ha −1 (N) ( Table 1). Each treatment was applied to a plot of 3.9 × 6.5 m 2 and was separated by a row to avoid contamination. There were three individual treatment plots per treatment, and they were organized in a randomized block design.

Sample Collection and Determination
The soil samples were collected after the corn harvest was completed on 29 October 2021, and the "five-point sampling method" was used to sample 1 kg of soil in each plot. Soil drills were used to collect the 0-20 cm surface soil, and it was put into self-sealing bags. The animal and plant residues were removed, and the soil samples were immediately stored at 4 • C until the determination of the soil MBC, MBN, and enzyme activity [26]. Then, part of the soil was naturally air-dried, and the undisturbed soil was wet sieved to determine the soil aggregates. Another part of the soil samples was screened using a 2 mm screen to test the physical and chemical soil properties [27].
The potassium dichromate oxidation-external heat approach was used to determine the soil TOC [28]. Then, the TN and MBN contents were measured using the Kjeldahl method [29]. Also, the DOC, EOC, and MBC were determined by UP water concussion extraction (TOC), 333 mmol·L −1 potassium sulfate oxidation-colorimetry, and chloroform fumigation-K 2 SO 4 extraction [26].
The soil invertase activity was measured using the 3,5-dinitrosalicylic acid colorimetric method [25,30]: The 5 g of soil sample was weighed and added to a 50 mL triangular flask. Then 15 mL of sucrose solution at a concentration of 8%, 5 mL of phosphate buffer (pH 5.5), and 5 drops of toluene were added to the triangular flask. Mixed, shaken thoroughly on a shaker, and incubated at 37 • C in a thermostat for 24 h. After incubation, the sample was placed in a centrifuge, centrifuged, and filtered; 1 mL of the upper filtrate was placed in a 50-mL volumetric flask, and 3 mL of the reducing agent 3,5-dinitrosalicylic acid was added to the filtrate. Placed in a thermostatic water bath and heated at 100 • C for 5 min, removed, and cooled for 3 min, the filtrate was fixed to 50 mL with distilled water and determined colorimetrically at 508 nm using a UV-Vis spectrophotometer. Cellulase activity was measured using the 3,5-dinitrosalicylic acid colorimetric method. The 10 g of soil sample was weighed, added to a 50 mL triangular flask, and then 5 mL of 1% carboxymethyl cellulose solution, 5 mL of phosphate buffer (pH 5.5), and 5 drops of toluene were added. After mixing, shake well on a shaker and incubate at 37 • C for 72 h in a thermostat. At the end of the incubation, 1 mL of the filtrate was filtered and measured colorimetrically at 549 nm using a UV-Vis spectrophotometer [31]. Urease activity was determined for each sample by measuring the formation of the product, NH4 + [32]: The 5 g of soil sample was weighed and added to a 50 mL triangular flask. 1 mL of toluene was added to the mouth of the flask and shaken gently for 15 min. 5 mL of 10% urea and 10 mL of citrate buffer pH 6.7 were added to the flask and mixed, then incubated at 37 • C for 2 h. After incubation, the sample was diluted to the mark with distilled water at 38 • C, shaken, and filtered. Dilute 1 mL in a 50-mL volumetric flask with distilled water to 10 mL, add 4 mL of sodium phenol solution, and 3 mL of sodium hypochlorite solution, and allow to settle to the mark after 20 min. The absorbance value was measured at a wavelength of 578 nm.
After harvest, a representative 10 m 2 sample area was taken back to the laboratory to measure the yield; the yield of 14% water content was converted, and the spike length, spike weight, grain number per spike, and 100-grain weight and yield of each pot were statistically analyzed [33].

Statistical Analysis
IBM Statistics SPSS 22.0 software was used to test data normality, homogeneity, and principal component analysis (PCA). The ANOVA was performed to determine the significant differences between treatments in R (p < 0.05). If the data did not meet the criteria, a nonparametric Kruskal-Wallis test was performed to determine statistical significance. Fitting and mapping were done with Origin Pro 9.0.

Soil Organic Carbon and Nitrogen Contents
After continuous cultivation, the initial soil (CK treatment) had no external carbon input, the soil fertility was lacking, and at this time the TOC content was 7.42 g·kg −1 . The TOC level in the C3 treatment was 35.18% greater than the control. There was a significant positive correlation between biochar addition and soil TOC level (p < 0.05) ( Figure 1). There was no significant difference between a combined application and a single application of TOC. Under the C3 treatment, the soil's TN content reached 1.93 g·kg −1 , which was 23.95% greater than that of the CK treatment. The TN content of the C3N1/2 treatment increased to 2.44 g·kg −1 , which was 56.18% greater than that of the CK. The C/N ratio followed the same trend as TOC, peaking at C3 and C3N.

Soil Active Organic Carbon Components
The dynamic changes in the soil's active organic carbon fractions are sho 2. The effect of the single addition of biochar on the EOC level was not sig 0.05). The EOC level in the bulk soil under the C3N1/2 treatment was remarka by 31.71% relative to the CK treatment and increased by 28.41% relative treatment. Biochar increased the soil DOC content by 7.70% on average. dynamic changes in the MBC, the C3 and C3N1/2 treatments had the best eff which were 42.09% and 44.65% greater than the CK treatment, respectively. T application of biochar and full nitrogen fertilizer had unsatisfactory improve on MBC and MBN, with an average reduction of 12.50% and 44.62%. Further soil active organic carbon fraction, EOC was the main soil component, ac 23.69-62.91% of TOC, followed by DOC and MBC, accounting for 1.45-2.60 2.93% of TOC, respectively (Table 3).

Soil Active Organic Carbon Components
The dynamic changes in the soil's active organic carbon fractions are shown in Table 2. The effect of the single addition of biochar on the EOC level was not significant (p > 0.05). The EOC level in the bulk soil under the C3N1/2 treatment was remarkably elevated by 31.71% relative to the CK treatment and increased by 28.41% relative to the C3N treatment. Biochar increased the soil DOC content by 7.70% on average. Among the dynamic changes in the MBC, the C3 and C3N1/2 treatments had the best effect (p < 0.05), which were 42.09% and 44.65% greater than the CK treatment, respectively. The combined application of biochar and full nitrogen fertilizer had unsatisfactory improvement effects on MBC and MBN, with an average reduction of 12.50% and 44.62%. Furthermore, for the soil active organic carbon fraction, EOC was the main soil component, accounting for 23.69-62.91% of TOC, followed by DOC and MBC, accounting for 1.45-2.60% and 1.04-2.93% of TOC, respectively (Table 3).

Enzyme Activity
The application of biochar and nitrogen fertilizer considerably raised the soil enzyme activity ( Figure 2). The C1N treatment had the best effect, which increased cellulase activity by 98.29%. Biochar significantly increased urease activity, with an average increase of 51.36%. Biochar blended with N fertilizer was more effective in enhancing urease and cellulose. Soil convertase activity was highest in black soils, with biochar and nitrogen fertilizer increasing convertase activity by 5.43-25.23%.

Enzyme Activity
The application of biochar and nitrogen fertilizer considerably raised the soil enzyme activity (Figure 2). The C1N treatment had the best effect, which increased cellulase activity by 98.29%. Biochar significantly increased urease activity, with an average increase of 51.36%. Biochar blended with N fertilizer was more effective in enhancing urease and cellulose. Soil convertase activity was highest in black soils, with biochar and nitrogen fertilizer increasing convertase activity by 5.43-25.23%.
The PCA method was used to visualize and analyze the effect of soil carbon and nitrogen fractions on enzyme activity under different treatment conditions (Figure 3). The first and second principal components (PC1 and PC2) accounted for 28.3% and 17.4% of the variance, respectively, and the treatments were significantly separated from the CK treatment. The principal component analysis (PCA) showed that biochar with nitrogen fertilizer differed significantly from the CK treatment and had the best improvement effect. There is a positive correlation between TN, TOC, and DOC and between cellulase and sucrase.  The PCA method was used to visualize and analyze the effect of soil carbon and nitrogen fractions on enzyme activity under different treatment conditions ( Figure 3). The first and second principal components (PC1 and PC2) accounted for 28.3% and 17.4% of the variance, respectively, and the treatments were significantly separated from the CK treatment. The principal component analysis (PCA) showed that biochar with nitrogen fertilizer differed significantly from the CK treatment and had the best improvement effect. There is a positive correlation between TN, TOC, and DOC and between cellulase and sucrase.

The Effects of the Biochar and Nitrogen Fertilizer Treatments on Maize Yield
The findings demonstrated that the quantity of biochar had a favorable impact on the ear length, ear weight, quality, and yield of the maize. The C3 treatment increased the corn yield by 22.91%. Furthermore, the effect of the C3N1/2 treatment was the best, and the yield reached 14,052.62 kg·hm 2 and increased by 50.97%. The yield difference was not obvious due to the difference in the N application rate (Table 4). RDA analysis showed the response of maize yield to soil environmental factors ( Figure 4). TOC, TN, and MBN were significantly correlated (p < 0.05) with maize yield indicators, with contributions of 42%, 16.2%, and 22.2%, respectively. The contributions of sucrase, cellulase, and urease were 6.8%, 4.4%, and 3.7%, respectively.

The Effects of the Biochar and Nitrogen Fertilizer Treatments on Maize Yield
The findings demonstrated that the quantity of biochar had a favorable impact on the ear length, ear weight, quality, and yield of the maize. The C3 treatment increased the corn yield by 22.91%. Furthermore, the effect of the C3N1/2 treatment was the best, and the yield reached 14,052.62 kg·hm 2 and increased by 50.97%. The yield difference was not obvious due to the difference in the N application rate (Table 4). RDA analysis showed the response of maize yield to soil environmental factors ( Figure 4). TOC, TN, and MBN were significantly correlated (p < 0.05) with maize yield indicators, with contributions of 42%, 16.2%, and 22.2%, respectively. The contributions of sucrase, cellulase, and urease were 6.8%, 4.4%, and 3.7%, respectively.  The PCA was used to evaluate the effects of different treatments on black soil traits and yields in northeast China ( Table 5). The results display that the cumulative variance contribution rate was 88.04%, which can explain the variation well. The higher the F value, the better the improvement effect, and the C3N1/2 treatment was optimal.
The expression of the principal component is:   The PCA was used to evaluate the effects of different treatments on black soil traits and yields in northeast China ( Table 5). The results display that the cumulative variance contribution rate was 88.04%, which can explain the variation well. The higher the F value, the better the improvement effect, and the C3N1/2 treatment was optimal. The expression of the principal component is: where X1-X14 represent EOC, DOC, MBC, MBN, TN, TOC, cellulase activity, urea activity, invertase activity, and yield, respectively.

The Biochar Effect on the Soil TOC and TN
The carbon and nitrogen content of the soil is an indicator of the strength of the soil's nutrient supply, and both biochar and N fertilizer applications are effective in increasing the medium carbon and nitrogen stocks. The results are in line with the findings by Zhang et al. (2018), showing that biochar application increased the TOC content in calcareous soils [34]. The reason for these is that biochar is a stable carbon-rich product with a carbon content of approximately 60% produced by pyrolysis of biomass under conditions of oxygen deprivation or limited oxygen supply [35]. Soil organic matter acts as a cementing substance to promote the formation of soil agglomerates, and good agglomerates promote the storage of soil organic carbon [36]. Biochar can enhance the ability of soil water and fertilizer conservation and inhibit the leaching of soil carbon and N [15]. Shi et al. proposed that the cumulative mineralization rate of TOC was reduced by 0.6-1.1% after biochar was applied with nitrogen fertilizer in loamy rice soils, which facilitated carbon aggregation and fixation [18]. In addition, Lu [37] suggested that biochar has a negative stimulatory effect on soil carbon mineralization due to the toxic substances released by biochar that may inhibit microbial activity or the preferential use of exogenous nutrients such as C and N by microorganisms, which leads to soil carbon sequestration. In this experiment, biochar and nitrogen fertilizer were applied to the soil in May. This was followed by a gradual increase in temperature and an average annual precipitation of 520 mm, concentrated in the months of June to August. The increase in soil fertility also stems from the response of urease and sucrase activity to changes in temperature and humidity, with warm and moist soil conditions favoring increased soil enzyme activity and facilitating the humification process [38]. The addition of N fertilizer alleviated the carbon limitation of soil microorganisms, supplied sufficient nutrients to plants, and increased the input of above-ground apoplankton and dead roots while improving crop yields. The results of this study showed that biochar applied with total N fertilizer (C2N and C3N treatments) reduced soil MBC and MBN content, and soil microbial biomass turnover was significantly reduced at high N addition. This may be explained by the fact that soil acidification, reduced cations, and inhibition of extracellular enzyme activity caused by N addition reduce microbial biomass, which in turn leads to reduced carbon capture and use by microorganisms [39]. According to Devereux [40], biochar has a high porosity, a hydrophilic structure, and a larger specific surface area, allowing it to retain more water and supply it to microorganisms and crops. Additionally, the role of N fertilizer is to improve the soil nutrient content, promote crop growth, increase the crop biomass, increase the return of the crop stubble and root exudates, and improve the soil TOC [12]. Song [6] discovered that the synergistic interaction between biochar and N fertilizer could significantly improve the carbon and N levels, water retention capacity, and soil nutrient availability in alkaline calcareous soils. However, it had no significant effect on the C/N or MBN. In comparison to their findings, our findings clearly showed that biochar-amended soil, particularly at higher rates (C3) or for biochar mixed with N fertilizer (C3N1/2, C3N), significantly improved the soil C/N (p < 0.05).

The Biochar Effect on the Carbon Fractions
The DOC and EOC contents, which are both useful indicators of the quality and function of the soil, reflect the capacity for the decomposition of organic matter and the release of nutrients [36,41]. However, the increase in the trend of the soil EOC and DOC was not significant after biochar application ( Table 2). The reason for this may be that biochar improves soil porosity and aggregation, enhances soil water and air permeability, stimulates soil biological activity, and increases the soil mineralization rate [37]. Furthermore, many scholars have shown that the soil DOC can be accumulated due to the decomposition process of soil animals and plants or microbial residues and can also be reduced due to microbial consumption [42]. The EOC is mainly derived from crop roots, plant residues, and soil microbial residues. The interaction between biochar and the total-N fertilizer (CN treatments) slowed the continuous development trend of the EOC. This may be due to the fact that organic fertilizers provide a source of nutrients for microorganisms, stimulate microbial activity, increase soil respiration intensity, and soil EOC is gradually consumed and mineralized by soil microorganisms, thus reducing the soil EOC/TOC ratio [43]. The lower the soil EOC/TOC, the greater the soil carbon stability [44]. The MBC and MBN increased at a faster rate in this study than the EOC and DOC (Table 2); thus, we concluded that the MBC and MBN were more susceptible to the soil charcoal and N fertilizer additions [45]. Biochar had little effect on MBC without N fertilizer, while in the case of N fertilizer matching, biochar application was highly significant and positively correlated with the increase in the soil MBC (p < 0.01) up to 44.65%. The half-N fertilizer treatments (CN1/2 treatments) had a greater impact on the soil MBC, possibly because N fertilizer provides a source of nutrients for microorganisms, stimulates their activity, promotes the carbon consumption of microorganisms, and decreases the ratio of the soil EOC/TOC [43,46]. Soil MBN did not change much or even tend to decrease in the total-N fertilizer treatment (CN treatments), either because of the slow and intricate response of soil microbial biomass to N application or because there was no significant microbial N fixation after biochar application.

The Biochar Effects on the Activity of Enzymes
The mineralization of SOM is essential and is the process where carbon, N, and other nutrients are changed from an organic form to an inorganic one with the help of microorganisms [37]. Enzymes in the soil can stimulate the degradation of SOM and supply useful dissolved chemicals for development and metabolism in the soil [47]. Biochar can mitigate the negative effects of soil structure and function degradation on soil enzyme activity, particularly when applied in conjunction with nitrogen fertilizer, according to the findings of Song [6]. Lammirato [3] investigated the effect of activated carbon on the extracellular enzyme reaction of cellulose degradation, and the strong adsorption characteristics of biochar were mentioned. Therefore, the increase in enzyme activity may be due to activated carbon particles inhibiting the contact between the substrate and enzyme and the enzyme and substrate adsorbing onto different particles, thus avoiding enzymolysis in the adsorption process. However, Dempster [48] introduced jarrah biochar to coarse soil and discovered that biochar hindered the decomposition of soil organic matter and N mineralization, leading to a decrease in the activity of the soil microbial population. This also occurred in the present study, where urease and cellulase showed negative correlations with the MBC and MBN, respectively. Zimmerman [49] explained this phenomenon: when the soil has a higher biochar and organic matter content, the soil carbon mineralization is stronger. Or maybe the promoting effect of biochar on soil carbon mineralization does not affect its carbon sequestration effect. Principal component analysis showed that the activities of urease and sucrase showed significant positive correlations with the TOC and TN (p < 0.05), respectively. This suggests that biochar can provide direct nutritional value to microorganisms or can help to improve nutrient use efficiency [50].
The improvement in soil enzyme activity was more stable with the combined application treatment compared with biochar application alone. This might be due to the increased N sources that N fertilization offered for microbial decomposition and utilization of organic materials, and the soil carbon to N ratio increased, which enhanced the soil biochemical reactions and promoted material cycling, thus increasing the soil enzyme activity [47,51]. Therefore, the activity of soil enzymes was kept in check by biochar, but the addition of nitrogen was also a significant element that influenced soil enzyme activity. However, excessive N fertilization restricted the enhancement of sucrase activity.

The Biochar Effect on Maize Yield
Our study found that the yield of maize with the same management measures showed a large regional difference after the addition of different amounts of biochar and combined application with N fertilizer [52]. After continuous tillage in the CK treatment, there was no external carbon input in the soil, the yield was relatively low, the residual stubble of the corn crops was especially low, the number of organic materials that were returned to the soil was less, and the content of the SOC decreased year on year [53]. In addition, our research revealed that biochar greatly boosts grain production and yield stability. Since the mid-1990s, the soil and fertilizer station in Tonghua City, Jilin Province, has carried out a study on reducing the N application for corn and has achieved obvious results [54][55][56]. Using dark brown soil, alluvial soil, and black soil as examples, the yield of reduced N application was found to increase by up to 10% compared to conventional N application [57,58]. At the Lausanne Experimental Station in the UK, when the rate of N application was reduced to 150 kg·hm 2 and 180 kg·hm 2 , the maize yield was still the highest [59]. In this study, compared with the control treatment, the combination of biochar and reduced nitrogen fertilizer had obvious advantages in improving grain quality and yield. The corn grain yield of the C3N1/2 treatment was the highest, with an increase of 49.13%. The amount of biochar and the SOC content had a substantial positive connection. Under the treatment of the combined application of biochar and N fertilizer, the level of the SOC increased significantly by 20.08-35.58%. This conclusion was also supported by Xu [60], who proposed that the effect of an enhanced crop yield was mostly explained by soil fertility and that the change in wheat yield was positively linked with the SOC and TN contents. To improve the shortage of carbon and N in the soil, the use of biochar and N fertilizer has the potential to increase soil fertility and supply additional nutrients for crop growth [61,62]. The gradual increase in enzyme activity also accelerated the soil nutrient conversion cycle, thus improving soil fertility and productivity [63].
Biochar is expected to increase soil enzyme activity, accelerate carbon humification, and promote nutrient release [64]. RDA showed that the increase in maize yield was attributed to the improvement of soil carbon and nitrogen concentrations and microbial activity ( Figure 4). Therefore, it is necessary to apply biochar and nitrogen fertilizer to degraded black soils to improve soil fertility and create a favorable environment for microorganisms to survive.

Conclusions
In our study, we found a positive effect of both biochar and N fertilizer on soil C and N nutrients. Biochar significantly increased C-pool saturation by 35.18% compared to the control. There was a synergistic effect of biochar with nitrogen fertilizer to increase soil N, with a maximum increase of 56.18% in TN. The PCA showed that soil nutrient content was the main driver of enhanced soil enzyme activity. The input of exogenous organic matter promoted the decomposition and conversion of organic matter by soil enzymes and improved the soil carbon and nitrogen cycles. Significant correlations (p < 0.05) were found between soil organic carbon, total nitrogen content, and maize yield. Biochar-blend N fertilizer was more effective in improving maize yield than biochar alone. The best improvement was achieved with C3N1/2 treatment, with a 50.97% yield increase. In summary, rational application is the best strategy to increase soil organic carbon stocks, reduce N loss, optimize soil microstructure, and promote sustainable agricultural production.