Effects of Fertilization Regimes on Soil Organic Carbon Fractions and Its Mineralization in Tea Gardens

Abstract

Changes in the organic carbon fraction and mineralization of soil aggregates play an important role in the improvement of soil quality by organic fertilization. Nevertheless, the effects of organic fertilizer application on the organic carbon fraction and mineralization characteristics of soil aggregates are still unclear. This study carried out a 6-year field trial with five different fertilization treatments, namely, no fertilization (CK), chemical fertilization (CF), rapeseed cake (CR), chicken manure (CM), and a combination of chicken manure with biochar (CMB). The distribution, organic carbon density, and carbon mineralization characteristics in soil aggregate fractions were tested. The results showed that CR significantly increased the contents of large soil macroaggregates and enhanced the stability of soil aggregates of all sizes. CM increased the contents of macroaggregates and microaggregates but decreased the stability of the soil aggregates. CR, CM, and CMB increased the content of soil organic carbon and its components in the tea garden, while the contribution rate of organic carbon to large aggregates was the highest under the CR treatment. Organic fertilizer treatments had a positive effect on enhancing soil microbial biomass and carbon and nitrogen contents. The mineralization rate and accumulation of organic carbon in tea garden soil aggregates were increased under organic fertilizer treatments, of which CMB was the most significant. There was a high increase in tea yield and bud density under the CR treatment. In short, the application of organic fertilizers in tea garden soil is helpful to improve soil nutrients and soil fertility; plant- and animal-derived organic fertilizers are recommended for corporate application in agricultural production.

1. Introduction

Tea (Camellia sinensis) is an important economic crop in China, which holds 3.2 million hm² of tea gardens and produces 2.93 million tons of dry tea in 2022 [1]. However, the ecological sustainability of tea garden soil has become a major controversial concern because of its uneven nutrient ratios, alarming decrease in acidification, declining systemic productivity, and diminishing economic returns [2,3]. Due to the characteristics of chemical fertilizers, such as fast efficacy and easy absorption by crops, farmers are constantly increasing the input of chemical fertilizer in pursuit of higher yields, leading to a series of soil health problems—for example, soil hardening, acidification, decreased microbial activity, damage to the soil structure, and continuous decline in soil fertility levels [4,5,6]. Replacement of chemical fertilizer with organic fertilizer is believed to be helpful in mitigating the severe consequences of chemical fertilizer overuse in tea plantations [7], since organic fertilizers contain a variety of essential nutrients for crop growth [8], which can exert a positive influence on soil nutrition, increasing the soil organic matter and regulating soil acidity and alkalinity [9,10,11]. Nonetheless, the high concentration of organic components in organic fertilizers is advantageous to the rapid propagation of soil microorganisms [12,13]. However, combined application of chemical fertilizer and organic manure is generally used in agricultural production because of the slower release of nutrients from organic fertilizer, which cannot meet the nutrient needs of crops an a timely manner in critical periods.

As the fundamental unit of soil structure, soil aggregates mediate many physical and chemical processes in soils—such as soil compaction, soil nutrient recycling, soil erosion, root penetration, and crop yield [14]—and regulate the soil fertility and nutrient supply capacity [15]. Results from previous studies show that the content and stability of soil aggregates can be developed by applying organic fertilizers [16,17]. Furthermore, the application of organic fertilizers can also increase soil organic carbon (SOC) content [18]—an important indicator for soil quality, which has a close nexus with the formation of large soil aggregates [16] and plays a key role in maintaining soil’s nutrient status and structural stability [19]. When it reaches a certain level, SOC is mineralized, thereby furnishing a sustained release of available nutrients to plants, and its fractions have a vital impact on soil carbon cycling [20]. It was reported that the addition of organic fertilizer enhanced the stability of soil aggregates in tea gardens as the content and composition of SOC developed [21,22]. The most common plant-derived organic fertilizers in tea gardens are cake manure and green manure, while animal manure is the main source of animal-derived organic fertilizer, and both of these have a very detailed division [23].Research shows that the application of green manure produces a certain improvement effect on the soil quality of tea plantations [24]. The rapeseed cake fertilizer is popular among agricultural cultivators for its high concentrations of cellulose and polysaccharides, which can enhance the soil structure and fertility quality [25]. Song’s [26] study found that the soil bulk density of tea plantations was significantly reduced after the application of chicken manure fertilizer. The effect of applying pig manure as an organic fertilizer can adversely affect soil health due to its heavy metal contents exceeding the standard values [27,28]. Organic fertilizers from different sources differ greatly in terms of their physicochemical properties. Therefore, clarifying the mechanisms of different types of organic fertilizer to enhance soil structure and fertility in tea plantations is vitally important to the sustainable development of the tea industry in China.

We hypothesized that the application of organic fertilizers could increase soil aggregate and SOC contents and stimulate organic carbon mineralization in a tea garden. Therefore, the present research was conducted over a 6-year period to quantify the impacts of applying different organic fertilizers on soil aggregate contents and stability, SOC components, and mineralization characteristics in the tea gardens of China. In this way, the effects of different types of organic fertilizers on soil structure and soil fertility in tea gardens can be further explored.

2. Materials and Methods

2.1. Study Site

The study was carried out in the tea estate of the Jiangsu Polytechnic College of Agriculture and Forestry, which is located in Jurong, Jiangsu Province, China (31°55′ N, 119°15′ E). The region has a typical subtropical monsoon climate, with a mean annual temperature of 15.5 °C and a mean annual precipitation of 1019 mm. The average nighttime and daytime temperatures are 13 °C and 21 °C, respectively, and the relative humidity is 76%. The soil is classified as an Alfisol, with a sandy clay texture, which contains 17.3% clay, 18.8% silt, and 63.9% sand in the surface soil (0–20 cm). At the beginning of the experiment in 2015, the soil pH was 5.42, the organic matter content was 22.71 g kg⁻¹, the total nitrogen was 0.88 g kg⁻¹, the available phosphorus was 176.94 mg kg⁻¹, and the available potassium was 163.46 mg kg⁻¹.

2.2. Experimental Design

The field experiment started in October 2015. There were 5 different treatments: no fertilization (CK), chemical fertilization (CF), rapeseed cake (CR), chicken manure (CM), and a combination of chicken manure with biochar (CMB). The nutritional contents of the studied organic manures (i.e., CR, CM, and CMB) are shown in

Table 1. Nutritional contents of the organic fertilizers.

Manure	pH	C (%)	N (%)	C/N	P2O5 (%)	K2O (%)	MgO (%)
Rapeseed cake	5.41	43.2	6.30	6.85	2.53	0.98	0.85
Chicken manure	8.03	8.66	0.91	9.56	1.41	1.90	1.70
Biochar	10.5	51.3	1.09	47.06

. The nutrient dosages in CF were 300 kg N ha⁻¹, 150 kg P₂O₅ ha⁻¹, and 150 kg K₂O ha⁻¹, supplied by urea, calcium superphosphate, and potassium sulfate, respectively. The amount of biochar in CMB was 7.5 t ha⁻¹, and the biochar was made from corn straw at 450 °C. Except for CK, the other 4 treatments received the same amount of nitrogen (N), i.e., 300 kg N ha⁻¹, and no other chemical fertilizer was applied.

The fertilizers of each treatment were applied once in every October or November during the experiment. According to the conventional practice in tea plantation, all fertilizers and biochar were applied in ditches (~20 cm in depth and ~25 cm in width) between the plant rows. Each treatment was replicated with 4 plots (4 m × 6 m), and a total of 20 plots were arranged in a randomized complete block design. The tea variety in this study was Camellia sinensis (L.) O. Kuntze var. ‘Fuding Dahao’, which has been growing well under regular water and fertilizer management since it was planted in 2002. The distance between adjacent tea tree rows was 1.5 m. The plant density was about 40,000 plants ha⁻¹.

2.3. Soil Sampling

Soil samples were collected in June 2021. In each plot, surface soil (0–20 cm) samples were collected in five different positions with a 5 cm diameter stainless steel soil-sampling auger and then carefully mixed to form a composite. The soil was gently broken into small pieces of 10~12 mm in diameter along the natural structural plane. The coarse roots, stones, and litter residues was discarded. Soil samples weighing about 2 kg were collected from each plot and stored in a closed wooden box before being taken to the laboratory. All soil samples were kept in two parts: one part was air-dried for analysis, and the other part was stored at 4 °C in the refrigerator for further use.

2.4. Soil Aggregate Fractionation

According to the soil aggregate separation method shown in the work of Six [29], The XL.02-TTF-100 (Beijing Zhuochuan Electronic Science and Technology Co., Ltd., Beijing, China) was used to sieve soil particles of different sizes. In this process, the air-dried soil samples (100 g) were filtered using sieves with successive diameters of 2, 0.25, and 0.053 mm. Next, 20 min of vertical oscillation (along 5 cm amplitude) was achieved at a rate of 1 oscillation s⁻¹. Then, 4 types of aggregates were acquired: (i) large macroaggregates (>2 mm), (ii) small macroaggregates (2~0.25 mm), (iii) microaggregates (0.25~0.053 mm), and (iv) silt + clay fractions (<0.053 mm). After sieving, the smallest fractions (silt + clay fractions) were centrifuged for 10 min at 2500× g and the pellets were backwashed into an aluminum box and dried overnight at 50 °C together with the other aggregate sizes. The dried aggregate size fractions were weighed and stored in wide-mouth vials at room temperature.

2.5. Soil Analyses

Total organic carbon (TOC) and dissolved organic carbon (DOC) were determined following the method of Sun [30]. Accurately weighed 1 g of soil aggregate sample and put into a 50 mL beaker. We also weighed and recorded the quality of the beaker and the carbon-free filter paper. Next, 10 mL of 2 mol L⁻¹ HCl was added to the beaker and kept at room temperature for 24 h, stirring 3~5 times. Then, the acidified soil samples were leached with distilled water, and the samples were washed to neutrality. The filter paper was put into a beaker and dried at 55 °C for 24 h. The filter paper was then taken out and weighed, and the total amounts of filter paper and sample after drying were recorded. After complete acidification, 2 mg samples were packed, and the total organic carbon content in the soil was determined using a Vario TOC Cube Total Organic Carbon Analyzer (Elementar Trading Co., Ltd., Shanghai, China).

Next, 20 g soil samples with different particle sizes were weighed and extracted with 40 mL of 0.5 mol L⁻¹ potassium sulfate. The extraction solution was filtered through a 0.45 μm filter membrane. The filtrate was used to determine the soil soluble organic carbon using the same equipment as the TOC content testing machine.

Mineralized organic carbon (MOC) was measured using the method of Huang [31]. First, 8 g soil samples were put into 500 mL tissue incubation bottles, and deionized water was used to adjust the water content to 60% of the field capacity. The same amount of water was added to the samples of each component of the aggregates. Centrifugal tubes containing 10 mL of 0.5 mol L⁻¹ NaOH solution were suspended in tissue incubation flasks to absorb CO₂ released during incubation. Each treatment was repeated four times, and the blank control was set at the same time, where no other treatment steps for the soil samples were performed. The incubation experiment was carried out at 25 °C in darkness. On the 3rd, 6th, 9th, 12th, 15th, 18th, 21st, 24th, 27th, and 30th days, the test tubes in the tissue incubation bottles were transferred to 150 mL triangular bottles, and excess BaCl₂ was added. The CO₂ released in the culture process was determined via 0.2 mol L⁻¹ HCI titration. All bottles were aerated for 30 min at the sampling time to prevent hypoxia and put into a new 10 mL 0.5 mol L⁻¹ NaOH solution to absorb and release CO₂. At the same time, the tissue incubation bottles were adjusted to the initial weight with deionized water to keep the soil water content unchanged.

Resistant organic carbon (ROC) was measured via acid hydrolysis consisting of refluxing soil in 6 mol L⁻¹ HCl [32], and the testing equipment was the same as the TOC content testing machine. Easily oxidized organic carbon (EOC) was measured according to the 333 mmol L⁻¹ KMnO₄ digestion method, with a slight modification [33]. A 759s UV–Vis Spectrophotometer (Shanghai Lengguang Technology Co., Shanghai, China) was used to determine the extract concentration.

2.6. Calculations and Statistical Analyses

Four indicators were used to evaluate the soil aggregate stability: soil mean weight diameter (MWD, mm), geometric mean diameter (GMD, mm), >0.25 mm aggregate content (R_0.25), and fractal dimension D.

$$\mathrm{MWD}={{\displaystyle \sum }}_{i=1}^n\left(\overline{X_i}\times W_i\right)$$

(1)

$$\mathrm{GMD}=\mathrm{EXP}\left[\frac{{{\displaystyle \sum }}_{i=1}^n{M}_i\times \ln \overline{X_i}}{{{\displaystyle \sum }}_{i=1}^n{M}_i}\right]$$

(2)

R_0.25 = M_t>0.25/M_t

(3)

$$\mathrm{D}=3-\frac{l\mathrm{g}\left[\frac{W(\delta <\overline{X_i})}{W_t}\right]}{\lg \left(\frac{\overline{X_i}}{X_{\max }}\right)}$$

(4)

where $\overline{X_i}$ denotes the aggregate average diameter at the ith size (mm), W_i is the quality of level i aggregates. M_t > 0.25 is the mass of aggregates with particle size > 0.25 mm, M_t is the aggregates’ total mass, lg is a logarithmic function based on 10, $W(\delta <\overline{X_i})$ is the cumulative weight of soil particles with soil particle diameter <$\overline{X_i}$, W_t is the sum of the weight of soil particles and X_max is the maximum average aggregate diameter.

$$C=\frac{{\mathrm{SOC}}_i\times W_i}{{{\displaystyle \sum }}_{i=1}^n({\mathrm{SOC}}_i\times W_i)}$$

(5)

where C is the relative contribution rate of an organic carbon component to the ith particle level aggregate of soil (%), SOC_i is the organic carbon content at the ith particle level (g kg⁻¹), and W_i has the same meaning as above.

Soil organic carbon mineralization and organic carbon mineralization rates were calculated by the following formula:

MOC = C_HCl × (V₀ − V₁) × M_CO2/m

(6)

MR = CN/t

(7)

where MOC is the soil organic carbon mineralization content calculated based on CO₂ (g kg⁻¹), C_HCl is the hydrochloric acid concentration (mol L⁻¹); V₀ is the blank titration volume (mL), V₁ is the volume of hydrochloric acid consumed (mL), M_CO2 is the molar mass of CO₂ (44 g mol⁻¹); m is the soil quantity for incubation tests (g), MR is the soil organic carbon mineralization rate in CO₂ (g(kg d)⁻¹), C_N is the SOC mineralization amount during the culture time (g kg⁻¹), and t is the incubation time (d).

Soil microbial biomass carbon (MBC, mg kg⁻¹) and nitrogen (MBN, mg kg⁻¹) were determined by the chloroform fumigation extraction method [34]. The MBC and MBN contents of the extracts were determined using the Vario TOC Cube Total Organic Carbon Analyzer (Elementar Trading Co., Ltd., Shanghai, China) and SKALAR (San++) Continuous Flow Analyzer (Guangzhou Changlixin Scientific Instrument Co., Ltd., Guangzhou, China), respectively. We used the following formulae to obtain the results:

MBC = 2.64 Ec

MBN = Ec/0.54

(8)

where 2.64 and 0.54 are the proportions of carbon and nitrogen extracted by K₂SO₄ from microorganisms killed by chloroform fumigation, respectively, while Ec is the difference in the mass fractions of carbon in the fumigated and unfumigated extracts.

Bud density was determined by the sample frame survey method; the number of tea buds in the 0.35 m² sample frame was randomly counted, and each plot was replicated four times. All tea buds (one bud and one leaf) from each plot were picked manually at one time and weighed to determine the total tea yield. The dry weight of 100 tea buds (one bud and one leaf) was calculated as the 100-bud weight.

2.7. Statistical Analysis

All reported values are means of the three replicates of each sample. Statistical analysis was conducted using SPSS 22 (IBM, Chicago, IL, USA). One-way ANOVA was used to estimate the effects of the different fertilization treatments on soil organic carbon components and organic carbon mineralization characteristics in tea gardens. Duncan’s multiple range test was used to examine significant differences between treatments at a 5% confidence interval.

3. Results

3.1. Soil Aggregates and Stability

Aggregates with different sizes were significantly influenced by the application of different fertilizers. Large macroaggregates had the highest proportion, followed by small macroaggregates, under the three organic fertilization treatments (

Table 2. Percentage contents of water-stable soil aggregates of different sizes under different fertilization treatments (mean ± standard error) %.

Treatments	Water-Stable Soil Aggregates of Different Sizes
	>2	0.25~2	0.053~0.25	<0.053
	(mm)	(mm)	(mm)	(mm)
CK	54.66 ± 9.83 ab	23.49 ± 5.54 b	10.51 ± 5.6 ab	11.34 ± 0.92 ab
CF	22.80 ± 10.38 b	44.46 ± 6.93 a	19.15 ± 3.59 ab	13.59 ± 1.55 ab
CR	75.41 ± 7.49 a	12.55 ± 5.36 b	3.28 ± 1.67 b	8.76 ± 0.94 b
CM	37.56 ± 3.80 b	31.87 ± 1.14 ab	16.99 ± 2.09 a	13.58 ± 0.98 ab
CMB	42.46 ± 7.14 b	29.99 ± 3.52 ab	16.53 ± 2.55 a	11.02 ± 2.11 a

Values in a column followed by the same letter(s) are not significantly different at p < 0.05 between different treatments. CK, no fertilization; CF, chemical fertilization; CR, rapeseed cake; CM, chicken manure; CMB, combination of chicken manure with biochar.

). The proportion of large macroaggregates was profoundly increased by 20.75% compared to CK, while there were reductions in the percentages of small macroaggregates, microaggregates, and silt + clay fractions of 10.94%, 7.23%, and 2.58%, respectively, under the CR treatment. On the other hand, relative to CK, the percentages of small macroaggregates, microaggregates, and silt + clay fractions increased by 20.97%, 8.64%, and 2.25%, respectively, while the proportion of large macroaggregates decreased by 31.86%, under the CF treatment. The application of CM and CMB slightly increased the contents of small macroaggregates and microaggregates but, overall, the CM and CMB treatments led to a subtle impact on the proportions of the aggregates compared to the control, and there were no significant differences between them.

The larger the MWD, GMD, and R_0.25 values of aggregates, the smaller the fractal dimension D, and the more stable the aggregate structure [30]. The MWD, GMD, and R_0.25 values of soil aggregates increased by 12.5%, 27.36%, and 11.18% under the CR treatment compared with the control, respectively, and the D value decreased by 9.91% (

Table 3. Stability of water-stable soil aggregates of different sizes under different fertilization treatments (mean ± standard error).

Treatment	MWD	GMD	R0.25	D
	(mm)	(mm)	(%)
CK	1.92 ± 0.231 ab	1.06 ± 0.195 ab	76.21 ± 5.157 ab	2.12 ± 0.226 ab
CF	1.54 ± 0.238 b	0.85 ± 0.204 ab	74.38 ± 5.088 ab	2.49 ± 0.192 a
CR	2.16 ± 0.163 a	1.35 ± 0.187 a	84.96 ± 2.288 a	1.91 ± 0.234 b
CM	1.44 ± 0.100 b	0.66 ± 0.067 b	67.92 ± 2.891 b	2.56 ± 0.055 a
CMB	1.50 ± 0.174 b	0.72 ± 0.145 b	69.20 ± 4.048 b	2.50 ± 0.126 a

Values in a column followed by the same letter(s) are not significantly different at p < 0.05 between different treatments. MWD: mean weight diameter; GMD: geometric mean diameter; R_0.25: content of aggregates > 0.25 mm; D: fraction dimension. CK, no fertilization; CF, chemical fertilization; CR, rapeseed cake; CM, chicken manure; CMB, combination of chicken manure with biochar.

). However, the CF, CM, and CMB treatments reduced the MWD, GMD, and R_0.25 values of the soil aggregates, while the D value increased by 17.45%, 20.75%, and 17.92%, respectively, and there were no noteworthy differences between the treatments.

3.2. Soil Carbon Fractions’ Concentrations in Aggregates

3.2.1. Total Organic Carbon

The application of organic fertilizer to the tea plantation resulted in a significant increase in the TOC content of the soil aggregates (p < 0.05) compared to the control (

Table 4. Effects of soil aggregates’ organic carbon fractions under different fertilization treatments (mean ± standard error).

Item	Treatments	Aggregates’ Size
		>2	0.25~2	0.053~0.25	<0.053
		(mm)	(mm)	(mm)	(mm)
TOC g kg−1	CK	23.13 ± 0.38 c	35.30 ± 2.57 b	14.70 ±0.53 cd	12.20 ± 0.49 d
	CF	13.13 ± 0.27 d	17.00 ± 1.10 c	10.70 ± 0.79 d	12.73 ± 0.64 d
	CR	38.97 ± 2.44 b	22.13 ± 0.58 c	19.03 ± 0.30 c	17.87 ± 0.78 d
	CM	51.90 ± 2.21 a	51.43 ± 2.72 a	26.60 ± 1.60 b	24.37 ± 0.64 c
	CMB	52.90 ± 1.76 a	55.63 ± 2.35 a	32.60 ± 1.88 a	30.17 ± 2.49 c
DOC mg kg−1	CK	109.42 ± 1.71 d	96.17 ± 0.44 d	63.76 ± 0.54 c	82.08 ± 0.09 d
	CF	127.91 ± 2.21 c	115.74 ± 5.46 c	94.82 ± 5.39 b	90.93 ± 0.86 cd
	CR	175.73 ± 2.56 b	124.13 ± 4.13 c	125.73 ± 2.38 a	103.86 ± 4.99 c
	CM	168.41 ± 5.19 b	155.60 ± 2.86 b	116.66 ± 4.43 a	144.02 ± 2.70 b
	CMB	206.88 ± 2.85 a	182.79 ± 1.59 a	130.60 ± 0.65 a	164.77 ± 3.11 a
ROC g kg−1	CK	18.37 ± 0.74 c	28.63 ± 0.33 c	12.03 ± 0.38 cd	10.70 ± 0.36 d
	CF	11.00 ± 0.38 d	13.87 ± 0.58 a	9.47 ± 0.69 d	10.97 ± 0.33 d
	CR	35.83 ± 2.07 b	20.20 ± 0.40 a	13.63 ± 1.41 c	13.50 ± 0.70 c
	CM	36.40 ± 1.04 b	29.77 ± 1.67 c	17.90 ± 0.20 b	17.80 ± 0.70 b
	CMB	43.40 ± 0.70 a	38.53 ± 2.60 b	26.30 ± 0.67 a	32.00 ± 0.10 a
EOC g kg−1	CK	4.56 ± 0.69 bc	3.68 ± 0.35 bc	2.45 ± 0.13 b	1.77 ± 0.30 c
	CF	3.94 ± 0.47 c	2.65 ± 0.36 c	1.64 ± 0.17 b	1.27 ± 0.16 c
	CR	7.54 ± 0.38 ab	4.43 ± 0.54 b	2.79 ± 0.28 b	2.07 ± 0.14 bc
	CM	8.30 ± 1.09 a	5.13 ± 0.32 b	4.47 ± 0.14 a	3.01 ± 0.27 ab
	CMB	10.26 ± 0.18 a	8.69 ± 0.29 a	4.39 ± 0.59 a	3.74 ± 0.15 a

Values in a column followed by the same letter(s) are not significantly different at p < 0.05 between different treatments. CK, no fertilization; CF, chemical fertilization; CR, rapeseed cake; CM, chicken manure; CMB, combination of chicken manure with biochar.

). Except for the silt + clay fractions, the TOC contents in the aggregates of different grain classes were significantly lower under the CF treatment compared to the non-fertilizer treatment. The CR treatment increased the TOC content of large macroaggregates by 68.48% compared to the CK treatment, while the TOC level was reduced by 37.31% in microaggregates. The CM and CMB treatments increased the TOC contents by 128.71%, 124.38%, 45.69%, and 57.59% in large and small aggregates, respectively, and only in the microaggregates did the TOC content show significant differences between the two.

3.2.2. Dissolved Organic Carbon

Both organic and chemical fertilizer treatments increased the DOC contents in tea garden soil aggregates to varying degrees (Table 4), with the former having a more significant effect than the latter (p < 0.05). Compared to the non-fertilizer treatment, the DOC contents in the aggregates of different sizes were considerably higher under the CF treatment (except in the silt + clay particles). The aggregates’ DOC contents were higher under the CM treatment than the CR treatment, and the difference between the two was remarkable in the small aggregates and the silt + clay fractions. The application of CMB increased the DOC contents by 89.07%, 90.07%, 104.83%, and 100.74% in the four particle size aggregates in descending order, respectively. This was more significant than the CM treatment in the same particle size aggregates, apart from the silt + clay fractions.

3.2.3. Resistant Organic Carbon

The order of ROC content under the five fertilizer treatments was CMB > CM > CR > CK > CF and showed the same trend in the different particle size aggregates (Table 4). Organic fertilizer treatment increased the ROC contents in the soil aggregates, with the CMB treatment having the best effect, followed by CM and CR. The ROC contents in the large aggregates increased by 136.25%, 98.15%, and 95.05% under the CMB, CM, and CR treatments, respectively, compared to the CK. On the other hand, the ROC contents in the different sizes of aggregates under the CF treatment experienced a decline as compared to the control, which was significant in the >0.25 mm aggregates (p < 0.05).

3.2.4. Easily Oxidized Organic Carbon

As shown in Table 4, the EOC contents of the soil aggregates differed significantly (p < 0.05) with the different fertilizer treatments and decreased with the size of aggregates under the same fertilizer treatment. The EOC contents in the same-sized aggregates were in the following order: CMB > CM > CR > CK > CF. Under the CF treatment, the EOC contents in the different-sized aggregates declined compared to CK, but the decrease was not significant. The EOC contents increased by 82.02%, 39.40%, 82.45%, and 70.06% compared to CK for the soil aggregates in descending order of particle size under the CM treatment, while the CMB fertilizer exerted a further improvement in the soil EOC contents compared to the CM treatment. The EOC contents of aggregates of all sizes were increased under the CR treatment, but the discrepancy was not significant compared to the CK.

3.2.5. Relative Contribution Rate of Organic Carbon Fractions in Aggregates

The contribution rate of large aggregates to soil organic carbon was significantly higher than that of microaggregates (Figure 1), and the contribution rate of >0.25 mm aggregates to soil organic carbon and its fractions exceeded 70% in all fertilizer treatments. Under the same fertilization treatments, the contribution of organic carbon decreased with decreasing aggregate size and showed the same trend in all organic carbon fractions (except for CF). The relative contribution rate of large macroaggregates under the CR treatment was the highest among the four carbon fractions compared to other treatments. Meanwhile, small aggregates had a higher contribution rate to the four organic carbon fractions under the CF treatment. There was no considerable difference in the relative contribution rate of organic carbon in the four carbon fractions by small aggregates and silt + clay fractions under the different fertilizer treatments.

3.3. Soil Microbial Biomass Carbon and Nitrogen

The obtained results showed that both the organic and inorganic fertilizer treatments enhanced the soil MBC and MBN contents under the applied fertilization regimes (

Table 5. Effects of microbial biomass carbon and nitrogen under the different fertilization treatments (mean ± standard error).

Treatment	MBC (mg kg−1)	MBN (mg kg−1)	MBC/MBN
CK	112.22 ± 3.85 d	33.73 ± 3.99 c	3.40 ± 0.32 bc
CF	171.56 ± 21.14 c	39.13 ± 1.89 c	4.45 ± 0.73 ab
CR	278.90 ± 10.21 a	55.30 ± 0.89 b	5.04 ± 0.11 a
CM	218.48 ± 4.82 b	69.50 ± 3.54 a	3.16 ± 0.15 c
CMB	134.33 ± 5.20 d	60.11 ± 3.24 ab	2.25 ± 0.19 d

Values in a column followed by the same letter(s) are not significantly different at p < 0.05 between different treatments. CK, no fertilization; CF, chemical fertilization; CR, rapeseed cake; CM, chicken manure; CMB, combination of chicken manure with biochar.

). The highest amount of MBC was produced in the CR fertilizer treatment, followed by CM, which caused increases of 148.53% and 94.696%, respectively, compared to CK. Meanwhile, the highest MBN content from the different fertilization regimes showed the following trend: CM (69.50 mg kg⁻¹), CMB (60.11 mg kg⁻¹), CR (55.30 mg kg⁻¹), CF (39.13 mg kg⁻¹), and CK (33.73 mg kg⁻¹). Moreover, the soil MBC/MBN values increased by 48.25% under the CR treatment compared to the control and, conversely, decreased by 33.82% under the CMB treatment.

3.4. Mineralization Characteristics of Soil Aggregates

3.4.1. Variation of Cumulative Mineralization of Aggregates

The application of organic fertilizers significantly (p < 0.05) improved the MOC contents of soil aggregates compared with CK, and the difference between the CF and CK treatments was not noteworthy (Figure 2). The MOC of the different particle sizes under the same treatments showed the same trend: large macroaggregates > small macroaggregates > silt + clay fractions > microaggregates. The order of MOC content in large macroaggregates from largest to smallest was CMB > CR ≈ CM > CK > CF. The CMB treatment was most effective in increasing the MOC contents of soil aggregates at all particle sizes. Significant differences existed between the CMB and CM treatments in terms of elevating the MOC contents of the soil aggregates, while there were no differences between the CM and CR treatments.

3.4.2. Variation of Cumulative Mineralization of Aggregates with Incubation Time

The changes in MOC with incubation time can reflect the process of soil organic carbon mineralization dynamically. The CO₂ accumulation increased with culture time under the different fertilization treatments, but the intensity of release gradually decreased (Figure 3). On the 30th day of incubation, the CO₂ release of large macroaggregates was in the following order: CMB > CR > CM > CK > CF. Under the CMB treatment, the CO₂ release strength of aggregates with different sizes maintained a high development, and the CO₂ release content of large macroaggregates was 16.56% higher than that of the control, while the CF treatment resulted in a 6.39% decrease. Throughout the incubation, the intensity of CO₂ release under the CR and CM treatments was higher than that of CK and showed the same trend in soil aggregates of different sizes. The intensity of CO₂ release in the silt + clay fraction was essentially equivalent under the CMB and CM treatments.

3.4.3. Mineralization Rate of SOC

MR changes were roughly divided into two phases throughout the incubation, with a rapid decline in MR in the first 20 days, followed by a slower decline (Figure 4). Compared with CK and CF, the organic fertilizer treatments were able to significantly increase the rate of organic carbon mineralization in soil aggregates (p < 0.05), and MR was always maintained at the highest level under the CMB treatment, followed by CM. The initial MR values of the soil aggregates under the different fertilizer treatments were different, with the CMB treatment showing a higher MR value than the other treatments and, conversely, CF having a lower MR.

3.5. Tea-Yield-Related Indices

Variations were observed for indices related to tea yield under the applied treatments (

Table 6. Effects of tea-yield-related indices under different fertilization treatments (mean ± standard error).

Treatment	Bud Density (A m−2)	100-Bud Weight DW (g)	Tea Yield DW (kg hm−2)
CK	408.75 ± 31.13 b	15.18 ± 0.20 ab	110.56 ± 5.62 bc
CF	443.13 ± 30.08 b	12.64 ± 0.28 c	94.72 ± 7.04 c
CR	683.33 ± 58.82 a	15.69 ± 0.80 a	135.83 ± 3.33 a
CM	416.88 ± 17.40 b	14.64 ± 0.59 ab	107.50 ± 8.22 bc
CMB	470.63 ± 38.46 b	15.18 ± 0.31 ab	119.17 ± 2.55 ab

Values in a column followed by the same letter(s) are not significantly different at p < 0.05 between different treatments. CK, no fertilization; CF, chemical fertilization; CR, rapeseed cake; CM, chicken manure; CMB, combination of chicken manure with biochar.

). The highest bud density and tea yield were observed for CR fertilization, which caused increases of 67.18% and 22.86%, respectively, compared to CK. Conversely, the CF treatment lowered the values of 100-bud weight and tea yield, reducing them to 83.27% and 85.67%, respectively, as compared to the control. The increase in tea yield under the different fertilization treatments was found to be in the following order: CR > CMB > CK > CM > CF. Additionally, there were no significant differences in the values of the bud density and 100-bud weight indicators between the treatments (except for CR).

4. Discussion

4.1. Soil Aggregates’ Composition and Stability

The promotion or inhibition of soil aggregate formation by inorganic fertilizers is still controversial. Munkholm’s [35] study showed that the application of inorganic fertilizer improved crop root exudates and microbial activities, promoting the formation of soil aggregates. However, excessive application of nitrogen fertilizer with high ammonium concentration led to dispersion of soil clay minerals and disintegration of aggregates’ structure [36]. In this experiment, chemical fertilizer treatment led to a decrease in the content of large soil macroaggregates, and the stability of soil aggregates in the tea garden experienced a decline, which may be attributable to a decline in soil organic matter content as ammonia volatilization and leaching of nitrogen, phosphorus, and potassium resulted in the loss of soil nutrients [37,38].

Studies have shown that plant-derived organic fertilizers are excellent for increasing soil organic matter contents, and that the application of plant-derived organic fertilizers can increase the contents of soil water-stable macroaggregates (R_0.25) by increasing soil organic matter content compared with the application of chemical fertilizers [39,40,41], which is consistent with the results of this experiment. Rapeseed cake fertilizer significantly increased the large macroaggregate contents compared with the control in the tea garden soil [42], and the possible reasons for this result are as follows: (i) Applying rapeseed cake fertilizer greatly increased the contents of soil organic carbon and other organic substances, which promoted the transformation of small soil macroaggregates to large macroaggregates through cementation [43,44]. (ii) The effect of fertilization on organic carbon in soil aggregates tended to be greater than that in microaggregates [45]. (iii) The use of organic fertilizers can accelerate the turnover of soil carbon, mainly through soil aggregates of different sizes [46]. Fu’s study showed that soil nutrient contents and microbial numbers were significantly increased under the combined application of rapeseed cake and green fertilizer [25]. Zhou’s [47] study indicated that the release rate and amount of nitrogen nutrients from animal-derived organic fertilizer were higher than those from plant-derived organic fertilizer. In the composting process of animal-derived organic fertilizer, crop straw and other auxiliary materials were added to improve the C/N ratio [48]. Therefore, animal-derived organic fertilizer is believed to exert a better impact in promoting soil aggregate formation. Yang et al. [49] found that the application of chicken manure increased the proportion of aggregates > 0.25 mm and improved the soil’s structural stability. However, the effect of chicken manure application on the enhancement of soil aggregates was not significant, which may be related to the amount of fertilizer applied and the soil texture; thus, further study is warranted of the interaction between soil properties and the impact of applying organic fertilizers in tea gardens.

The stability of soil aggregates determines the ability of the aggregates to resist exogenic action when exposed to changes in their external environment [50]. The present study demonstrated that the application of rapeseed cake fertilizer significantly enhanced the stability of soil aggregates in tea plantations, because the high amount of organic matter in the fertilizer improved the cementation effect between soil microaggregates [51,52]. Soil aggregates’ stability decreased under the chicken manure fertilizer treatment, which may be attributable to the acidic condition of tea garden soil. Acidic conditions can accelerate the transformation of free iron and aluminum ions into amorphous and complex states in the soil [53,54], enhancing the stability of the aggregates. However, the application of chicken manure may cause an increase in the pH value of tea garden soil, decreasing the stability of the aggregates [55].

4.2. Soil Carbon Fractions’ Concentrations in Aggregates

Our study found that organic fertilizer treatments dramatically improved the contents of soil organic carbon and its components, which is consistent with the results of Wang [56]. The reason for this was that the application of organic fertilizers not only directly increased the soil organic carbon content and soil microbial biomass, but also stimulated the mineralization of primary soil organic carbon, accelerating the decomposition of plant residues and organic matter by soil microorganisms [57,58]. The physical protection of the organic carbon by soil aggregates is also responsible for the improvement in organic carbon content [59]. Compared to the rapeseed cake fertilizer, chicken manure fertilizer was more effective in enhancing the soil organic carbon content, probably due to the fact that animal-derived organic fertilizers have higher nutrient contents and nutrient release rates compared to plant-derived organic fertilizers [47]. Moreover, there was a significant positive correlation between soil carbon content and exogenous carbon input in Zhang’s research [60]. The present study also found that soil organic carbon and its components were reduced (except DOC) after the application of chemical fertilizers in tea plantations, partially due to the long-term application of chemical fertilizers reducing the soil humic acids content [61], which is the main component of soil organic matter and plays an important role in soil carbon fixation and soil nutrient release. On the other hand, a large number of rapidly available nutrients in chemical fertilizers are prone to nutrient loss through surface runoff and leaching [37]. The observed increase in DOC content under chemical fertilizer treatments, which is contrary to the findings of Guo and Kou [62,63], may be related to the tea garden soil properties. The specific reasons need to be further investigated in the future.

In the present study, additional application of biochar along with chicken manure led to an increase in the contents of soil organic carbon and its components, which is consistent with the findings of previous studies [64,65]. A possible reason for this is that biochar interacts with the soil to form a protective matrix that inhibits the oxidation of soil organic matter and promotes the accumulation of soil organic matter [66,67]. Hernandez [68] studied the effects of biochar on soil C composition and stability using a new technique of combined spectroscopy–microscopy, and the results showed that using biochar to reduce soil C metabolism is an effective way to improve C stability in soil. Nearly 90% of soil organic carbon exists in aggregates [69]. Organic carbon and its components’ contents declined with the decreasing particle size of the aggregates because the separation effect of large aggregates on microaggregates slowed down the decomposition of organic carbon and resulted in the formation of large aggregates containing more organic carbon [70].

4.3. Soil MBC and MBN

The soil MBC and MBN contents are closely related to soil microbial activity and nutrient supply capacity [71,72]. In this study, the organic fertilizer (i.e., CR, CM) treatments resulted in higher soil MBC and MBN contents compared to the control, because organic fertilizer input both replenished the soil with carbon sources and enhanced the number and activity of microorganisms, promoting the conversion of SOC to MBC in the fertilizers [73]. Meanwhile, the higher microbial activity promoted the transformation of inorganic N to MBN and other organic N forms, allowing more ammoniacal N to enter the soil’s active organic N pool [74,75]. The same findings were obtained by Cheng et al. [76]. A lower MBC/MBN ratio indicates high biological effectiveness of soil nitrogen [77]. The biological effectiveness of soil nitrogen was improved under the chicken manure fertilizer treatment; this may have been closely related to the differences in fertilizer properties.

4.4. Mineralization Characteristics of Soil Aggregates

Soil organic carbon mineralization directly affects the maintenance of soil fertility, the supply and release of nutrient elements, and the emission of greenhouse gases [78]. Studies have shown that the application of a single organic fertilizer could significantly increase the MOC content in soil, because separate application of organic fertilizers would force soil microorganisms to decompose large amounts of organic matter before obtaining the nutrient elements for their own reproduction [79]. In this experiment, the application of CR and CM resulted in an increase in the MOC in soil aggregates, which supports this view. The MOC content of large macroaggregates was higher than that of other particle size aggregates, mainly because large aggregates have a higher content of organic carbon available for microbial decomposition. On the other hand, large macroaggregates contain large amounts of reactive organic carbon, which is more easily mineralized, while organic carbon in microaggregates mostly exists as highly humified inert fractions [80].

In present study, the MR was high at the beginning of the incubation period because there were more carbohydrates in the soil that could be directly transformed and used by microorganisms, which is conducive to the rapid reproduction of soil microorganisms. The carbohydrate content was consumed and decreased with the increase in the incubation time, limiting the microbial activity and, thus, leading to a lower MR [81]. Compared with the CM and CMB treatments, it was not difficult to find that biochar could promote soil organic carbon mineralization in tea plantations. Luo [82] reported that the application of biochar increased soil organic carbon mineralization and showed a “positive excitation effect”, while biochar could effectively inhibit soil organic carbon mineralization due to the adsorption and sequestration of soil organic carbon by the high surface energy of biochar in Zhang’s [32] study. It has also been suggested that biochar acts as a positive promoter or a negative inhibitor depending on the level of active organic carbon in the soil [83]. The differences between the results of this study and those of previous studies may be due to differences in soil texture and biochar type. The application of chemical fertilizers led to a further decrease in soil pH in the tea plantation, which was not conducive to the proliferation of soil microorganisms, so the soil aggregates’ MR was lower under the CF treatment [84,85].

4.5. Tea-Yield-Related Indices

In the present trial, the tea yield and bud density were considerably higher under the rapeseed fertilizer (CR), which is consistent with the trends of soil structure and nutrient indicators mentioned above. This shows that organic fertilizers are able to improve the soil structure and nutrient contents of tea plantations which, in turn, improves tea yield, and is consistent with the findings of Song [26]. The application of chemical fertilizers resulted in lower 100-bud weight compared to CK, owing to the high amounts of fast-acting nutrients in the chemical fertilizers, which allowed the tea plants to produce a larger number of fresh leaves, resulting in a smaller proportion of nutrients in the tea buds. In addition, organic fertilizers have slow and long-lasting effects; the longer the fertilization time, the more conducive to the improvement of tea yield and quality [86].

5. Conclusions

This study established that the application of organic fertilizers in tea garden soil is helpful to improve soil nutrients and fertility. Plant-derived organic fertilizer (rapeseed cake) significantly increased the content of large macroaggregates and enhanced the stability of aggregates of all sizes in the tea garden soil. The application of animal-derived organic fertilizer (chicken manure) was more effective than plant-derived organic fertilizer (rapeseed cake) in improving the contents of organic carbon and its components, as well as promoting the mineralization of organic carbon, while it also led to a decrease in the stability of soil aggregates in the tea garden. Therefore, the application of these two types of organic fertilizers in combination is recommended for agricultural production.