Effects of rice and corn straw biochar combined with microbial inoculant on soil health in protected vegetable production

Abstract

The biochar and microbial inoculant are commonly used amendments in soil improvement, but their effectiveness in protective agriculture is currently unclear. Based on this, this study takes the pepper vegetable greenhouse located in Zhuxi Town, Chongqing as the research object, and selects biochar prepared from corn straw and rice straw, as well as microbial inoculant, as the improvement materials. Five treatments were established, including corn straw biochar alone (CB), rice straw biochar alone (RB), microbial inoculant alone (CKM), combined corn straw biochar and microbial inoculant application (CBM), and combined rice biochar and microbial inoculant application (RBM), conventional fertilization was used as a control. This study explores the changes in soil physicochemical properties of protected vegetable production under different treatments at distinct time points (first, third, and fifth months after returning field) and soil depth (0–10 cm and 10–20 cm). The results demonstrated that the overall soil bulk density increased With the increase of returning time. There was no significant difference in the soil capillary porosity or mineral-associated organic carbon content (P > 0.05), whereas the other soil properties decreased with increasing return field time. In contrast, the soil bulk density (BD) increased with increasing soil depth. Except the capillary porosity, which exhibited no significant differences at different depths (P > 0.05), all the other soil properties demonstrated downward trends with increasing depth. After 5 months of transplantation, the soil carbon pool management index of RBM treatment was higher than that of CBM treatment, with a 4.35% increase. The soil health index was 14.51% lower than that of CBM. However, the differences in soil carbon pool management index and soil health index between the two treatments did not reach a significant level. Overall, the RBM and CBM showed better improvement effects on soil carbon pool management index and soil health status, making them the better ratios for vegetable greenhouse farmland soil improvement in this study.

Introduction

Protected vegetable land production in China continues to expand, driven by its advantages of high intensification, increased replanting index values, superior unit yields, enhanced economic efficiency, and minimal seasonal influences^1,2. Over the past decade, the scale of protected vegetable cultivation in China has remained stable at approximately 4 × 10⁶ ha, accounting for nearly one-third of the national total vegetable output³. Pepper is a key economic crop within protected cultivation systems and occupies a significant proportion of production areas, accounting for 26.6% of the overall protected vegetable acreage of China and positioning the country as a global leader in pepper production⁴. However, the long-term implementation of continuous cropping and intensive agricultural practices has resulted in severe soil degradation that poses a threat to the sustainability of protected pepper production systems^5,6.

The application of soil amendments represents a widely adopted strategy for ameliorating degraded soils and preventing further soil deterioration. Existing soil amendment technologies are primarily categorized into four main categories: (i) natural amendments, (ii) synthetic amendments, (iii) natural–synthetic copolymer amendments, and (iv) bioamendments⁷. Biochar, a commonly employed natural soil amendment, comprises primarily carbon. It features a well-developed pore structure with a substantial specific surface area and abundant oxygen-containing functional groups⁸. As an emerging multifunctional material, biochar is rapidly becoming a focal point of research globally because of its unique physical architecture, favorable surface properties, and significant environmental benefits^9,10. Research has demonstrated that biochar application significantly enhances the soil health^8,10. However, the improvement effect of biochar on soil after application will be influenced by many factors. Research has shown that there is an appropriate amount of biochar used to improve the weight diameter and geometric mean diameter of aggregates. When the appropriate amount is exceeded, its effect on improving the weight diameter and geometric mean diameter of aggregates weakens¹¹. Soil texture can also affect the improvement effect of biochar on soil. The addition of biochar increased enzyme activity in sandy clay loam, but had no effect in sandy loam. And the effectiveness of biochar as a soil amendment highly depends on the pyrolysis temperature¹². In addition, Wang Hedong’s research shows that the improvement effect of biochar on soil nutrients is closely related to the growth period of crops. In the early stage of crop growth after applying biochar, it can significantly increase the nitrogen content in the rhizosphere soil; however, in the later stage of crop growth, the improvement effect gradually weakens until it is no longer significant¹³.

Microbial fertilizers, which are biological amendments created through fermentation by specific microbial strains, exhibit environmentally benign characteristics with neither toxicity nor pollution risks¹⁴. Research has indicated that microbial inoculants can effectively increase soil nutrient availability and improve the soil structure¹⁵. However, numerous studies have demonstrated that the effects of microbial fertilizers as soil amendments are significantly influenced by soil properties and management practices^16,17. In practical applications, microbial inoculants are frequently coapplied with other soil amendments. The diverse combinations of microbial inoculant types and amendment materials have led to unquantified variations in soil improvement outcomes, thus underscoring the need for systematic investigations^18,19.

As the global leader in rice and maize cultivation, China possesses abundant reserves of crop residues characterized by high availability and low cost¹⁸. However, the resource utilization of straw in China is still relatively low compared to developed countries. And due to the large amount of straw that cannot be utilized, the phenomenon of straw open-field burning cannot be ignored in China. According to the 2020 China Ecological Environment Status Bulletin, 7,635 active burning sites were documented nationwide that year. Notably, extensive open-field burning of crop residues results in substantial resource waste and severe ecological contamination^20,21. As an important base for pepper greenhouse vegetable production in China, the severe soil degradation in the Yangtze River Basin directly threatens the sustainable development of pepper greenhouse vegetables in the region. Therefore, using straw as raw material to improve the soil of pepper greenhouse vegetables in the region can not only broaden the channels for straw resource utilization and increase farmers’ income, but also improve the soil to promote sustainable agricultural development, thus achieving a win–win situation of economic and ecological benefits. Consequently, experimental plots were established in Chongqing Municipality, a primary pepper cultivation zone within the middle–lower reaches of the Yangtze River Basin. In this study, rice straw biochar (RB), corn straw biochar (CB), and microbial inoculant were employed as soil amendments to investigate their combined effects on (1) soil organic carbon fractions, (2) aggregate stability metrics, and (3) porosity characteristics. The evaluation of soil health and soil carbon pools under different soil amendment treatments was conducted to identify the most effective treatments for soil improvement, thereby providing a scientific basis for selecting appropriate soil amendments in pepper vegetable greenhouse cultivation.

Materials and methods

Study site description

The study sites are located in Zhuxi, Chongqing. Vegetable base greenhouse facilities were constructed in 2005, and this area exhibits a 20-year history to date and serves as a significant pepper cultivation base. Zhuxi features gently undulating terrain within a subtropical monsoon climate zone. Meteorological records indicate an average annual temperature of 15.4 °C, with frost-free periods spanning 245–305 d annually and precipitation levels ranging from 1,128 to 1,378 mm. The elevation ranges from its lowest point of 171 m to its highest peak of 1,171 m.

Experimental materials

In the experiment, Tiaojiao Pepper 118 was selected as the pepper cultivar. Biochar includes corn straw biochar and rice straw biochar, purchased from Zhengzhou Jinbang Environmental Protection Technology Co., Ltd. It uses a slow pyrolysis method to produce biochar, with a heating temperature of 450–520 °C, and its basic parameters are shown in Table 1. The microbial fertilizer applied was a Bacillus subtilis microbial inoculant, resulting in a viable count ≥ 50 billion colony-forming units (CFU)/g.

Table 1.

Fundamental properties of different biochar types.

	SOC (%)	SSA (m2 g−1)	pH	CT (°C)	N (%)
RB	78	683	9–11	450–520	1.5
CB	75	698	10.5–12	450–520	1.7

RB is rice straw biochar, CB is corn straw biochar, SOC is soil origan carbon, SSA is specific surface area, CT is carbonization temperature, and N is nitrogen, the same as below.

Experimental design

This experiment is based on previous experiments, using rice straw biochar, corn straw biochar, and microbial inoculant as improved materials. Five treatments were set up, including corn straw biochar alone, rice straw biochar alone, microbial inoculant alone, combined corn straw biochar and microbial inoculant application, and combined rice biochar and microbial inoculant application, with conventional fertilization as the control. The amounts of biochar and microbial inoculant used for different treatments are shown in Table 2. The amount of biochar refers to Nie et al. (2025), and the amount of microbial inoculant is recommended by the manufacturer. Each treatment was replicated three times, resulting in a total of 18 experimental plots (4 m × 16 m per plot). Biochar and microbial inoculant was applied to the soil surface and subsequently incorporated through tillage. The tillage depth is 0—20 cm, with fertilizer applied using an integrated water-fertilization system.

Table 2.

Experimental treatment.

	Biochar types	Biochar application rate (t ha−1)	Microbial inoculant application rate (t ha−1)
CBM	CB	3	0.015
CB	CB	3	–
RBM	RB	3	0.015
RB	RB	3	–
CKM	–	–	0.015
CK	–	–	–

CBM is CB + Microbial inoculant, RBM is RB + Microbial inoculant, CKM is CK + Microbial inoculant, the same as below.

Sampling collection and measurements

The experiment began in March 2024. Samples were collected in the field at 1 month after transplantation, at harvest (3 months after transplantation), and at the end of the study (5 months after transplantation). Each plot was sampled according to the five-point sampling method, and the five samples collected from each plot were mixed and transported to the laboratory in bags. Soil samples were obtained at depths ranging from 0–10 to 10–20 cm. The soil samples were divided into two parts: one part was stored in a refrigerator at a temperature of 4 °C for the determination of the amount of carbon microbial, and the other part was dried naturally and ground for the determination of other soil indicators. Undisturbed soil cores were also collected, passed through an 8-mm mesh, and dried naturally for soil aggregate stability analysis. The soil cores were collected using 100-cm³ stainless steel rings to determine the bulk density (BD) and porosity. The specific methods for soil sample determination are detailed in Table 3.

Table 3.

Specific methods for the determination of soil samples.

Item	Method	References
POC	Sodium hexametaphosphate dispersion method	22
ROC	Potassium permanganate (KMnO₄) oxidation method	23
DOC	Total Organic Carbon (TOC) analyzer	22
MBC	Chloroform fumigation method	25
MAOC	Sodium hexametaphosphate dispersion method	26
BD	Core sampling method	24
CP and NCP	Core sampling method	27
Ag	Wet sieving method	22

POC is particulate organic carbon; ROC is readily oxidizable organic carbon; DOC is dissolved organic carbon; MBC is microbial biomass carbon; MAOC is mineral-associated organic carbon; CP is capillary porosity; NCP is non-capillary pore, Ag is > 0.25 mm soil aggregates, the same as below

Index calculation

Soil health assessment

1. Indicator screening

Through the analysis of the coefficient of variation (CV), indicators with CV values exceeding 10% across treatments were selected.

• (2)Membership degree value

Continuous membership functions were applied to standardize the soil health indicators, with S-shaped (sigmoid) functions employed to calculate membership degree values.

The membership value of the S-shaped increasing function can be calculated as:

1

The membership value of the S-shaped decreasing function can be calculated as:

2

In this equation:

X_max denotes the maximum threshold value of a given soil health indicator; and.

X_min denotes the minimum threshold value of the soil health indicator.

• (3)Weighting coefficient

3

In this equation:

W_i denotes the weighting coefficient of the i-th indicator (the ratio of the average correlation coefficient between a certain indicator and other indicators to the sum of the average correlation coefficients of all evaluation indicators); and.

C_i denotes the communality of the i-th indicator.

• (4)Integrated soil health assessment model

4

In this equation:

SQI denotes the soil health index (integrated assessment score); and.

F(X_i) denotes the membership degree values of the i-th quality indicator.

Carbon pool index (CPI) calculation

With the CK treatment as the reference soil treatment, the following indices were calculated on the basis of soil organic carbon (SOC) and its fractions:

5

6

7

8

Note: CA represents soil carbon activity. CAI represents soil carbon activity index. CPI represents soil carbon pool index. CPMI represents soil carbon pool management index.

Data analysis

One-way analysis of variance (ANOVA) was employed to assess the differences in indicators across the various treatments and sampling periods, accompanied by Tukey’s honestly significant difference (HSD) post hoc testing (α = 0.05). Paired-sample t tests were used to investigate the variations in indicators with respect to the soil depth. Principal component analysis (PCA) was performed to calculate the communality of each indicator. Pearson’s correlation analysis was conducted to evaluate the relationships among the different indicators. All the statistical analyses were executed in SPSS version 20.0, while the graphs were generated in Origin version 8.0.

Results

Variations in soil physical properties

With increasing return field time, all the treatments exhibited decreasing trends in the proportion of > 0.25 mm soil aggregates (Fig. 1a) and noncapillary porosity (NPC) (Fig. 1d), whereas the soil BD increased (Fig. 1b). At pepper harvest (5 months after transplantation), compared with those at 1 month after transplantation, the proportion of > 0.25 mm aggregates decreased by 2.10–14.97%, the NCP decreased by 23.93–45.65%, and the BD increased by 2.78–10.99%. No significant temporal variations were observed in the capillary porosity (Fig. 1c, P > 0.05).

Fig. 1.

Characteristics of the changes in the soil physical properties with depth and sampling points. Note: (a) Ag represents > 0.25 mm soil aggregates. (b) BD represents soil bulk density. (c) CP represents capillary pores. (d) NCP represents non-capillary pore. The capital and lowercase nonitalicized letters denote the differences in various indicators at 0–10 cm under the different treatments and returning field time, respectively. The uppercase and lowercase italicized letters indicate the differences in the different indicators at 10–20 cm under the various treatments and returning field time, respectively. T and t denote the differences with depth for the various indicators and treatments, respectively, with T > t; Ag denotes > 0.25 mm aggregates; and 1–0–10 denotes the properties of the soil samples collected at depths from 0 to 10 cm during the first period. And so on.

At pepper harvest, compared with the CK treatment, the CB, RB, CBM, and RBM treatments consistently provided higher proportions of > 0.25 mm soil aggregates, greater NCP values, and lower BD values. Compared with those under the CK treatment, the proportions of > 0.25 mm soil aggregates under the CBM and CB treatments were 35.81% and 29.81% greater, respectively (Fig. 2a), the NCP values were 157.35% and 98.15% greater, respectively (Fig. 4d), and the BD values were 10.77% and 10.01% lower (Fig. 2b), respectively. Compared with those under the CK treatment, the proportions of > 0.25 mm soil aggregates under the RBM and RB treatments were 31.53% and 21.78% higher, respectively, the NCP values were 149.77% and 82.13% greater, respectively, and the bulk weight values were 9.87% and 8.34% lower, respectively.

Fig. 2.

Mean value distribution characteristics of soil physical properties under different treatments. Note: (a) Ag represents > 0.25 mm soil aggregates. (b) BD represents soil bulk density. (c) CP represents capillary pores. (d) NCP represents non-capillary pore. The capital and lowercase letters denote the differences in various indicators under the different treatments and returning field time, respectively.

Fig. 4.

Characteristics of changes in mean soil chemical properties under different treatments. Note: (a) MBC represents microbial biomass carbon. (b) DOC represents dissolved organic carbon. (c) ROC represents readily oxidizable organic carbon. (d) MAOC represents mineral-associated organic carbon. (e) POC represents particulate organic carbon. The meanings of different letters are consistent with Fig. 2.

Across the different treatments, with increasing soil depth, the proportion of > 0.25 mm soil aggregates decreased, the BD increased, and the capillary porosity ‌did not significantly differ. The NCP decreased with depth but without significant differences. Overall, the combined application of microbial inoculant and biochar demonstrated superior effectiveness in increasing the proportion of > 0.25 mm soil aggregates, BD, and NCP. Specifically, the CBM treatment outperformed the RBM treatment in terms of the proportion of > 0.25 mm aggregates, BD, and NCP (P > 0.05). Across the different treatments, with increasing soil depth, the content of > 0.25 mm soil aggregates decreased, the soil BD increased, and the capillary porosity did not significantly differ. The number of noncapillary pores in the amended soil decreased with increasing depth, but there was no significant difference.

Variations in soil chemical properties

With increasing return field time, all the organic carbon fractions exhibited decreasing trends across the treatments (Fig. 3a, b, c, e), whereas the mineral-associated organic carbon content did not significantly differ (Fig. 3d). Compared with those at 1 month after transplantation, the MBC, DOC, ROC, and POC contents decreased by 8.76–19.50%, 47.99–78.40%, 13.09–18.96%, 2.42%, and 30.21%, respectively, under the different treatments compared with those at one month after transplanting.

Fig. 3.

Changes in soil chemical properties with depth and sampling points. Note: (a) MBC represents microbial biomass carbon. (b) DOC represents dissolved organic carbon. (c) ROC represents readily oxidizable organic carbon. (d) MAOC represents mineral-associated organic carbon. (e) POC represents particulate organic carbon. The meanings of different letters are consistent with Fig. 1.

The MBC, DOC, and POC contents were greater under the CB, RB, CBM, and RBM treatments than under the CK treatment. After the harvesting of peppers, in the treatment of returning corn straw biochar to the field, the MBC of CB and CBM was 10.53% and 22.47% higher than that of CK, respectively (Fig. 4a); The DOC increased by 18.80% and 26.74% respectively (Fig. 4b); The POC was 34.45% and 50.30% higher, respectively (Fig. 4e). Under the rice straw biochar amendment treatment, the MBC, DOC, and POC of RB and RBM were increased by 14.04% and 19.88%, 6.95% and 5.24%, 15.21% and 3.28%, respectively, compared with CK. Considering the content of readily oxidizable organic carbon (Fig. 4c), only two treatments, i.e., the CBM and RBM treatments, provided higher values than the CK treatment, which were 12.15% and 11.51% higher, respectively. The content of mineral-bound organic carbon (Fig. 4d) was also greater than that under the CK treatment, but the difference was not significant.

With increasing soil depth, the content of each organic fraction under the different treatments decreased. Overall, the combination of microbial inoculant and biochar treatment (CBM, RBM) has a better effect on improving soil organic carbon components than the single application of straw biochar treatment (CB, RB) and single application of microbial inoculant treatment (CKM). The MBC, DOC, POC, MAOC, and ROC contents were significantly greater under the CBM treatment than under the RBM treatment.

Variations in soil carbon pools and soil health

The carbon activity (CA), carbon activity index (CAI), and soil carbon pool management index (CPMI) of different treatments showed an overall increasing trend with the extension of returning field time (Fig. 5a, c, d), while the soil carbon pool index (CPI) showed an overall decreasing trend (Fig. 5b). After five months of transplanting peppers, the CPMI and CPI of different treatments were higher than those of CK. Among the two indicators of CA and CAI, except for the CKM treatment, the CA and CAI of the other treatments were still lower than CK, but did not reach a significant level.

Fig. 5.

Characteristics of soil carbon pool changes under different treatments. Note: (a) CA represents soil carbon activity. (b) CPI represents soil carbon pool index. (c) CAI represents soil carbon activity index. (d) CPMI represents soil carbon pool management index. The meanings of different letters are consistent with Fig. 2.

In soil health evaluation, all metrics except the BD, capillary porosity, and easily oxidizable organic carbon content exhibited CV values exceeding 10%. Consequently, these higher-variability parameters were incorporated into the comprehensive soil health evaluation index system. As shown in Fig. 6, the biochar with microbial inoculant treatment consistently demonstrated superior soil health across all the sampling periods but exhibited an overall decreasing trend in the soil health with increasing transplanting duration. Overall, both the CBM and RBM treatments resulted in higher soil CPMI and soil health levels (Fig. 6). After 5 months of transplantation, the CPMI of RBM treatment was higher than that of CBM treatment, with a 4.35% increase. The soil health index was 14.51% lower than that of CBM. However, the differences in CPMI and soil health between the two treatments did not reach a significant level (Table 4).

Fig. 6.

Soil health Assessment. Note: The meanings of different letters are consistent with Fig. 2.

Table 4.

Variation analysis of soil health evaluation indicators.

	MAX	MIN	Mean value	SD	CV(%)
Aggregate	68.36	39.98	55.43	7.47	13.48
BD	1.62	1.317	1.42	0.07	4.73
MBC	376.97	196.22	279.27	40.63	14.55
DOC	141.95	44.85	84.70	26.21	30.95
ROC	4.08	2.53	3.30	0.29	8.79
POC	1.41	0.44	0.76	0.20	26.14
MAOC	6.66	4.35	5.56	0.62	11.08
CP	45.63	34.94	40.314	2.48	6.14
NCP	13.29	2.17	6.63	2.46	37.15

Discussion

Effects of biochar application on soil physicochemical properties

At pepper harvest, the content of > 0.25 mm soil aggregates, NCP, and organic carbon fractions were greater in both the single and combined biochar applications than in the CK treatment, whereas the BD was lower. The capacity of biochar to greatly improve soil physicochemical properties may be attributed to its high organic matter content, substantial specific surface area, and porous structure^28,29. Following biochar application, the inherent soil porosity and high specific surface area contribute to a reduction in the soil BD, an increase in the soil porosity, and an increase in the adsorption capacity for nutrients and water. These increases lead to increased soil porosity and nutrient‒moisture availability levels, and the abundant organic matter within biochar creates favorable habitat conditions for microbial activity^30,31. Consequently, microbial activity is significantly stimulated, which likely accounts for the higher MBC content observed in the biochar-treated soils than in the CK soils in this study. Furthermore, the increased POC content can be attributed primarily to direct biochar incorporation³². The increase in the DOC content originates from two pathways: direct leaching of soluble organic compounds from biochar upon application^18,33 and microbial transformation of biochar constituents into soluble fractions through biochemical processes^34–36. Although the levels of ROC and MAOC were higher in the application of biochar than in the CK treatment, the difference was not significant. This may be due to the short transplantation time of biochar in this study, which has limited time for soil improvement. The conversion rate of biochar into ROC and MAOC under microbial action is relatively slow, resulting in the accumulation of organic carbon not yet significantly different from the control treatment³⁷. The absence of significant differences in the organic carbon fractions between the microbial inoculant treatments and the CK treatment could be attributed to carbon source limitations. Despite the introduction of microbial inoculants, the insufficient availability of carbon substrates impedes microbial survival and functionality, thereby compromising the effectiveness of soil improvement^32,38.

Although the DOC and MBC contents under the biochar–microbial inoculant co-application treatments exceeded those under the biochar-only treatments, the differences were not statistically significant (P > 0.05). This may occur because although the straw microbial–fertilizer application treatment provided the introduction of microbial inoculant, resulting in a greater microbial population and activity in the short term and enabling more efficient transformation of biochar³⁹, both treatments involved the use of identical substrate materials. Consequently, the total carbon available to microorganisms remained relatively consistent. As the energy and material foundation required for microbial growth, the availability of carbon sources is the main limiting factor in determining the final biomass of microbial communities. Thus, owing to the limitation in the total carbon source, the final MBC and DOC under the two treatments converged³⁸. The higher soil ROC content under the straw–microbial inoculant treatment than under the single biochar application treatment might be attributed to the fact that the soil readily oxidizable organic carbon fraction is governed primarily by the balance between microbial use and decomposition of soil organic carbon⁴⁰. At the early stage, the straw–microbial inoculant treatment exhibited greater MBC levels. Although the MBC content reached levels similar to those under the single microbial inoculant treatment by the end of the incubation period, it remained generally higher than that under the single biochar application treatment. Consequently, this elevated MBC content likely accelerated the conversion of SOC into ROC^16,40, thereby resulting in a relatively high ROC content compared with that under the single biochar application treatment. The higher POC content under the straw–microbial inoculant treatment may be attributed to its greater macroaggregate content, which provides enhanced physical protection for POC, thereby preventing its rapid microbial decomposition^41,42.

The content of > 0.25 mm soil aggregates treated with biochar combined with microbial inoculant is relatively high, which may be due to the increase in various organic carbon components in the soil after the application of microbial inoculant and biochar. Most studies have shown that the composition and content of soil organic carbon have a significant impact on the content of soil macroaggregates. Therefore, in this study, the relatively high content of > 0.25 mm soil aggregates treated with straw combined with microbial inoculant may be due to the high content of different organic carbon components.^42,43. A relatively high macroaggregate content in soil is typically associated with an improved soil pore structure and reduced BD values. This may explain why biochar–microbial inoculant treatment resulted in greater NPC and lower BD values⁴⁴. The absence of significant changes in soil capillary porosity may be attributed to the predominant influence of the soil texture. While soil organic matter can increase the capillary porosity, this process requires relatively long-term accumulation, and the short returning field time of the current experiment likely contributed to the observed stability⁴⁵.

Variation characteristics of soil indicators with soil depth and biochar returning field time

With the increase of returning time, all the organic carbon fractions except the mineral-bound organic carbon fraction exhibited a decreasing trend. This phenomenon may arise from two mechanisms: on the one hand, prolonged return enhances the level of microbial decomposition, leading to the consumption of various soil organic carbon components and a consequent reduction in their content²⁴. On the other hand, the photosynthetic capacity of pepper plants decreases with increasing return period, resulting in reduced secretion of organic substances from roots into the soil, which in turn contributes to the decrease in soil organic carbon fractions. The absence of significant changes in soil mineral-bound organic carbon may be attributed to its high stability and resistance to microbial transformation and use^46,47. With increasing return duration, the content of > 0.25 mm soil aggregates and NPC generally exhibited a decreasing trend across the different treatments, whereas the soil BD progressively increased. This phenomenon may be attributed to changes in the soil organic carbon fraction. Numerous studies have demonstrated that organic carbon, which serves as a crucial cementing material for macroaggregate formation, is typically significantly positively correlated with the content of large aggregates. Therefore, the reduced organic carbon fractions likely contributed to the decrease in the content of macroaggregates in this study. Consequently, the decreased macroaggregate content leads to fewer macropores and denser soil packing, thereby decreasing the NPC while increasing the BD^42,43.

The soil BD increased with increasing soil depth, whereas the NCP exhibited a progressive decrease. This pattern may be attributed to the overburden pressure exerted by the upper soil layers⁴⁸. The reduction in macropores compromises soil aeration in subsurface layers, thereby inhibiting microbial activity and weakening the decomposition and transformation of biochar. Consequently, the lower soil horizons contain reduced contents of organic carbon fractions and a decreased content of > 0.25 mm aggregates^49,50. The POC content in subsurface soils was lower than that in surface layers, likely because of the surface application and subsequent incorporation of biochar through tillage in this study. The implementation of practices led to the concentration of biochar in surface horizons, resulting in an elevated particulate carbon content in topsoil compared with subsurface layers.

This study demonstrated that the co-application of biochar and microbial inoculant significantly improved the soil physicochemical properties within five months after transplanting. However, there was no significant difference in soil health index and soil carbon pool management index between the RBM and the CBM treatment. Most studies suggest that the effectiveness of biochar in improving soil is influenced by the duration of field application. Given the relatively short amendment period in this study, it is likely that differential effects among the various biochar‒biofertilizer combinations on soil improvement could not yet be detected^32,37. Moreover, the limited number of evaluation metrics used for assessing the soil health resulted in the incomplete characterization of soil conditions, leading to a partial interpretation of treatment outcomes on the basis of existing parameters^51,52. Therefore, future research should focus on investigating the influence of the returning field time on soil property enhancement and should aim to adopt more comprehensive evaluation metrics to ensure that the findings provide robust guidance for improving soils in protected cultivation systems.

Conclusion

This study aimed to investigate the effects of the combined application of different types of biochar and microbial inoculant on soil physicochemical properties, carbon pools, and soil health. The principal findings are as follows: With the increase of soil depth, in different treatments, the content of > 0.25 mm soil aggregates, NCP, and soil organic carbon components showed a decreasing trend, while the BD showed an increasing trend, while there was no significant difference in CP. After five months of transplanting peppers, the CPMI and CPI of different treatments were higher than those of CK. The combination of biochar and microbial inoculant treatment resulted in relatively good soil health and CPMI. Among them, the CPMI of RBM treatment was higher than that of CBM treatment, with a 4.35% increase. The soil health index was 14.51% lower than that of CBM. However, the differences in CPMI and soil health index between the two treatments did not reach a significant level. Overall, the RBM and CBM treatment showed better ratios for vegetable greenhouse farmland soil improvement in this study. The research results can provide certain guidance for soil improvement in local vegetable greenhouses farmland soil.

Author contributions

R.S.W designed and performed the experiment, analyzed the data, and wrote the manuscript, Y. R., L. Z., L. Y. H. participated in sample collection and measurement, Y. Y., J. G. processed the data and analyzed the results; R.S.W., Y.R., L. Z., and Y.M. L. performed writing—review and edited final manuscript. All authors reviewed the article and approved the submitted version.

Funding

We would like to thank the reviewers and the editor for their constructive comments and suggestions. This research was supported by grants from the National Natural Science Foundation of China (CSTB2024JXJL-YFX0009, KYLX2024050301, and KYLX20250600355.).

Data availability

Data sets generated during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.