Study of the Effects of Co-Application of Biochar and Biogas Slurry on Nitrogen Cycling Enzyme Activity and Nitrogen Use Efficiency

Abstract

This study investigated the effects of combined biochar and biogas slurry application on Acrisols in Hangzhou, Zhejiang Province. Biochar was applied at 0–2% (C0–C3) with or without slurry substitution (B0, B1) to evaluate changes in soil properties, nitrogen-cycle-related enzymes, and functional genes. The joint amendment significantly increased soil pH, reduced bulk density, and enhanced total nitrogen, with the C3B1 treatment showing marked improvements in ammonium, nitrate, and total nitrogen compared with sole chemical fertilization (p < 0.05). Co-application also suppressed ammonia volatilization and N₂O emissions, while stimulating nitrate reductase, nitrite reductase, urease activities, and gene abundances. Regression analysis identified nitrate reductase as the primary driver of nitrogen use efficiency, and correlation analysis indicated that ammonium and total nitrogen were strongly associated with enzyme activities. Overall, biochar–slurry integration improved soil fertility and nitrogen utilization while mitigating nitrogen losses.

1. Introduction

Nitrogen (N) is one of the most essential elements required for crop growth. After application of N fertilizers to the soil, some of the N is absorbed and utilized by crops during the growing season, while another portion is stored in the soil in the form of inorganic N or organically bound N for subsequent crop uptake [1,2]. However, a significant amount is lost to the environment through gaseous emissions (NH₃, N₂O, and NOₓ), leaching via irrigation and precipitation, and surface runoff. These losses reduce N-use efficiency and contribute to serious environmental issues such as air pollution, eutrophication of water bodies, and soil acidification [3,4]. Therefore, a comprehensive understanding of the processes and mechanisms of soil N-cycling is crucial for sustainable agriculture and ecological security.

Acrisols are widely distributed across 16 provinces in southern China, covering an area of 2.18 × 10⁸ hm², approximately 20% of the national territory. This region is rich in heat and water resources and serves as an important grain and forestry production base in China. However, these soils are often characterized by acidity, poor fertility, compaction, seasonal drought, and susceptibility to erosion. In particular, N retention in red soils is weak, with substantial N lost through nitrate leaching and gaseous emissions of N oxides [5,6,7]. The intensity and dynamics of N-cycling are key factors influencing the restoration and reconstruction of Soil nitrogen fertility. Investigating N forms and transformation pathways in Acrisols is therefore essential for developing strategies to mitigate N leaching and gaseous emissions. Soil enzymes, which play active roles in soil ecosystems, are involved in virtually all processes of carbon, N, and phosphorus cycling and nutrient transformation. Thus, enzyme activity can serve as an effective indicator of soil fertility.

Biochar and biogas slurry are typical soil amendments that can enhance soil organic carbon, supply carbon sources for N-cycling microorganisms, and directly or indirectly regulate the activities of enzymes involved in N transformations. These changes ultimately improve Soil N-use efficiency and crop yield. Biogas slurry, a liquid by-product remaining after the anaerobic digestion of livestock manure, is rich in N and other nutrients [8]. In large-scale livestock farms, biogas slurry is usually separated from biogas residues, and, once fully stabilized, it contains low levels of heavy metals and other potential contaminants. In recent years, it has been recognized as a high-quality alternative to chemical nitrogen fertilizers, contributing to integrated crop–livestock systems and circular agriculture [9,10]. It has been shown to increase crop yield and improve quality [11,12]. However, as a liquid organic fertilizer, its application may cause leaching losses and ammonia volatilization, leading to waste and pollution. Biochar, a carbon-rich material produced from biomass pyrolysis under limited or no oxygen, is chemically stable, highly aromatic, and recalcitrant [13]. Biochar influences soil nitrogen dynamics through a combination of physicochemical, biological, and environmental mechanisms. On the physicochemical level, the porous structure and abundant surface functional groups of biochar enhance the retention of ammonium and nitrate, while its alkalinity and high cation exchange capacity contribute to the amelioration of acidic soils and improvement of soil structure. Biologically, biochar provides a favorable microhabitat for nitrifiers, denitrifiers, and other functional microorganisms, and it can alter the activity of key nitrogen-cycling enzymes such as urease, nitrate reductase, and nitrite reductase. These changes not only regulate the rates of nitrification and denitrification but also affect microbial pathways responsible for nitrogen mineralization and immobilization. These effects alter soil microstructure and microecology, thereby regulating the activity of enzymes involved in N-cycling and improving N-use efficiency in agricultural systems [14,15,16,17]. From an environmental perspective, biochar application has been reported to suppress ammonia volatilization and nitrous oxide emissions, thereby reducing gaseous nitrogen losses and lowering the risk of greenhouse gas release.

Nevertheless, most current studies have focused on biochar combined with inorganic fertilizers or biogas slurry combined with inorganic fertilizers, while research on the synergistic application of biochar and biogas slurry remains limited. Therefore, this study aims to explore the effects of combined application of biochar and biogas slurry on soil physicochemical properties and nitrogen cycle regulation. Specifically, we hypothesize that:

• Due to the large specific surface area and high porosity of biochar, as well as the abundance of organic matter in biogas slurry, their combined application alters soil physicochemical properties and improves soil quality.
• The improvement of soil quality through the co-application of biochar and biogas slurry helps to reduce gaseous emissions, decrease nitrogen losses, and enhance both the abundance of nitrogen-cycling-related functional genes and the activities of key enzymes.
• These changes induced by the combined application ultimately contribute to higher crop yields and improved nitrogen use efficiency.

The results aim to provide theoretical support for improving soil properties, enhancing N-use efficiency, and mitigating agricultural pollution caused by excessive fertilizer application.

2. Materials and Methods

2.1. Experimental Site

The experiment was conducted at Zhejiang University of Science and Technology, Hangzhou, Zhejiang Province, China (30°13′ N, 120°1′ E). The soil type at the site is red-yellow soil, and the area is classified as a subtropical monsoon climate, with a mean annual temperature of 17.8 °C, average annual sunshine duration of 1970 h, and mean annual precipitation of 1573 mm. The experimental soil is typical southern red soil with the following basic physicochemical properties: pH 5.44, bulk density (Bd) 1.45 g·cm⁻³, total nitrogen (TN) content 0.6 g·kg⁻¹, nitrate nitrogen (NO₃⁻-N) content 5.72 mg·kg⁻¹, ammonium nitrogen (NH₄⁺-N) content 7.91 mg·kg⁻¹. The test crop was cabbage (Brassica oleracea var. capitata).

2.2. Experimental Materials

Swine manure biochar was produced through slow pyrolysis at 350’C in a vertical kiln reactor (finguo Company, Jinhua China) under oxygen-limited conditions for 4 h. The biochar was ground to pass through a 2 mm sieve and stored in sealed containers before application. Biogas slurry was obtained from anaerobic digestion of pig farm wastewater at Shunkang pig farm (Kaihua County, Zhejiang Province) with a hydraulic retention time of 30 days at mesophilic conditions (35–40 °C). The pig manure biochar had the following properties: pH 8.5, TN content 18.95 g·kg⁻¹, NO₃⁻-N content 11.37 mg·kg⁻¹, NH₄⁺-N content 15 mg·kg⁻¹, and organic carbon (OC) content 605 g·kg⁻¹. The biogas slurry was derived from the anaerobic fermentation of wastewater from the pig farm of Shunkang Animal Husbandry in Kaihua County, Zhejiang Province. The experimental station was equipped with a dedicated storage and regulation pool for standardized treatment of the slurry, ensuring that its quality met the farmland irrigation water standard (GB5084-2005). The properties of the biogas slurry were as follows: NO₃⁻-N 10 mg·L⁻¹, NH₄⁺-N 220 mg·L⁻¹, TN 2.95 g·L⁻¹, and pH 8.5.

2.3. Experimental Design

The experiment started in April 2022 and adopted a full factorial design with biochar and biogas slurry as factors. Four levels of biochar were set at 0%, 0.5%, 1%, and 2%, while three levels of biogas slurry were set at 0%, 50%, and 100% (see

Table 1. Test design scheme.

Treatment	Biochar Level (%)	Biogas Slurry Level (%)	Treatment	Biochar Level (%)	Biogas Slurry Level (%)	Treatment	Biochar Level (%)	Biogas Slurry Level (%)
C0B0	0	0	C0B1	0	50	C0B2	0	100
C1B0	0.5	0	C1B1	0.5	50	C1B2	0.5	100
C2B0	1	0	C2B1	1	50	C2B2	1	100
C3B0	2	0	C3B1	2	50	C3B2	2	100

B2 corresponds to 100% biogas sludge. The percentage of biochar application represents the percentage of the total weight of residential tillage soil; the percentage of biogas slurry application represents the percentage of total biogas slurry applied for the TN applied. According to the residential area and TN content of biogas slurry, The application amount of 0% biochar was 0 kg. The application amount of 0.5% biochar was 13.5 kg. The application amount of 1% biochar was 27 kg. The application amount of 2% biochar was 54 kg, the 0% biogas slurry application amount was 0 L, the 50% biogas slurry application amount was 22.7 L and the 100% biogas slurry application amount was 45.4 L.

). For the 0% biogas slurry treatment, an equal volume of ultrapure water was applied instead. According to local cultivation practices, the optimal fertilizer rate was: urea (equivalent to 90 kg N·hm⁻²), potassium chloride (195 kg·hm⁻²), and calcium-magnesium phosphate fertilizer (375 kg·hm⁻²). Each treatment was replicated three times, with a control (CK) without biochar or biogas slurry, resulting in a total of 39 plots. Each plot covered 3 m² (2 m × 1.5 m). Cabbage seedlings were first raised in a nursery and then transplanted into the experimental plots at a row spacing of 40 cm and plant spacing of 20 cm. Two weeks before the start of the experiment, pig manure biochar was applied uniformly to the soil surface and manually incorporated into the 15–30 cm soil layer, with no further application thereafter. The CK treatment was tilled but received neither biochar nor biogas slurry. The crop cultivation practices followed local standards. Biogas slurry and all chemical fertilizers were applied as basal fertilizers one week before cabbage transplantation, using irrigation and surface broadcasting methods, and they were reapplied before each subsequent crop season. Additionally, biogas slurry was applied once a month during the growing season, with a total of three applications per season.

2.4. Soil Sampling and Determination

Soil samples were collected during four key growth stages of cabbage: seedling, vegetative growth, maturity, and harvest. Samples were taken using an S-shaped sampling method, with five cores collected from each plot, thoroughly mixed, and stored at 4 °C before being transported to the laboratory. Within two days, plant debris, stones, and sand particles were removed, and each composite sample was divided into three subsamples.

One subsample was air-dried for the determination of basic soil physicochemical properties following the methods described in Methods of Soil Agro-Chemical Analysis [18]; another was refrigerated for the determination of Soil nitrate-N (NO₃⁻-N) and ammonium-N (NH₄⁺-N); and the third was stored at –80 °C for soil DNA extraction. Soil bulk density was measured using the core-ring method, soil pH was determined with a pH meter (soil:water = 1:5), NO₃⁻-N and NH₄⁺-N contents were determined via KCl extraction followed by flow injection analysis (SEAL, AA3, Europe), and TN was measured by concentrated H₂SO₄ digestion followed by flow injection analysis (SEAL, AA3, Europe). At harvest, the number of cabbages per plot was recorded and converted to yield per hectare. Plant samples were oven-dried at 105 °C to constant weight, ground, sieved through a 100-mesh screen, and digested with H₂SO₄–H₂O₂ prior to TN determination. N-use efficiency was calculated according to Yang et al. [19]. Agronomic N-use efficiency (AEN, kg·kg⁻¹) was calculated as:

$$\mathrm{AEN}={\displaystyle \frac{\mathrm{Yield}\mathrm{in}\mathrm{N}\mathrm{treatment}-\mathrm{Yield}\mathrm{in}\text{no-N}\mathrm{treatment}}{\mathrm{Amount}\mathrm{of}\mathrm{N}\mathrm{applied}}}$$

2.5. Measurement of Soil Ammonia and Nitrous Oxide Emissions

The ammonia volatilization in field soil was monitored using the closed chamber method [20], with N₂O gas in the rice paddy collected using a static dark chamber and measured via gas chromatography [21].

2.6. Determination of Soil Enzyme Activities

Three soil enzyme activities related to N-cycling were selected: nitrate reductase, nitrite reductase, and urease. Nitrate reductase and nitrite reductase activities were determined using commercial assay kits (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China). Urease activity was determined using the phenol-sodium hypochlorite colorimetric method. Briefly, 5 g of air-dried soil (sieved to 2 mm) was placed in a 50 mL Erlenmeyer flask, and 1 mL of toluene was added and mixed thoroughly. After 15 min, 10 mL of 10% urea solution and 20 mL of citrate buffer (pH 6.7) were added, shaken well, and incubated at 37 °C for 24 h. After incubation, the mixture was filtered, and 1 mL of the filtrate was transferred into a 50 mL volumetric flask, followed by the addition of 4 mL phenol-sodium solution and 3 mL sodium hypochlorite solution (mixed immediately). The solution was left to develop color for 20 min, then diluted to volume. Absorbance was measured within 1 h at 578 nm using a spectrophotometer (Cary 5000, Agilent, Shimazu, Japan), with indophenol blue remaining stable for up to 1 h. A standard curve was prepared using N standards. To eliminate background nitrogen interference, a substrate-free control was set for each soil sample, and a soil-free control was included for the entire experiment. Urease activity (Ure) was expressed as the amount of NH₃-N (mg) produced per gram of soil within 24 h (mg·g⁻¹·24 h⁻¹).

2.7. Determination of N Cycle-Related Functional Genes

Total microbial DNA from soil samples was extracted using the TIANNAMP Soil DNA Kit (TianGen, Beijing, China). The concentration and purity of the extracted DNA were measured using a TBS-380 fluorometer and a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA)spectrophotometer, respectively. The abundances of soil amoA and nosZ genes were determined by real-time quantitative PCR (qPCR) using the StepOnePlus™ Real-Time PCR System (Thermo, MA, USA).

2.8. Data Analysis

All statistical analyses were performed using SPSS Statistics 26. Data normality and homoscedasticity were tested using Shapiro–Wilk and Levene’s tests, respectively. Two-way ANOVA was used to assess the effects of biochar application, biogas slurry, as well as their interactions on measured variables, Pearson correlation analysis of soil environmental factors and enzyme activities related to soil nitrogen cycling.

3. Results and Discussion

3.1. Effects of Biochar–Biogas Slurry Co-Application on Basic Soil Physicochemical Properties

Table 2. Physicochemical properties of the soil under different biochar and biogas slurry treatments.

Treatment		pH	UW/ (g·cm−3)	NO3N/ (mg·kg−1)	NH4+N/ (mg·kg−1)	TN/ (g·kg−1)	SOM/ (g·kg−1)
	CK	5.31 ± 0.16c	1.45 ± 0.01a	3.54 ± 0.74c	0.79 ± 0.10d	0.6 ± 0.01c	8.31 ± 0.97d
C0	B0	5.15 ± 0.17c	1.43 ± 0.01ab	4.13 ± 1.37c	2.37 ± 0.52c	0.5 ± 0.01c	7.46 ± 0.64d
	B1	5.31 ± 0.07c	1.49 ± 0.01a	4.58 ± 0.07c	3.39 ± 0.10c	0.5 ± 0.01c	7.48 ± 0.33d
	B2	5.17 ± 0.09b	1.47 ± 0.02a	4.37 ± 0.93d	4.04 ± 0.81c	0.9 ± 0.06cd	7.17 ± 0.19d
C1	B0	6.28 ± 2.8b	1.25 ± 0.01c	8.68 ± 1.33b	3.40 ± 0.58c	1.4 ± 0.03b	19.78 ± 1.18c
	B1	7.14 ± 0.20b	1.28 ± 0.02b	3.51 ± 0.02c	4.11 ± 0.24c	1.3 ± 0.00b	20.55 ± 1.15c
	B2	7.38 ± 0.04a	1.29 ± 0.01c	8.02 ± 0.86c	4.88 ± 0.46c	1.7 ± 0.05bc	17.29 ± 1.77c
C2	B0	7.15 ± 0.34b	1.41 ± 0.02ab	14.78 ± 2.07a	7.44 ± 0.97b	1.9 ± 0.06b	27.35 ± 2.08b
	B1	7.35 ± 0.06ab	1.43 ± 0.06b	16.24 ± 5.75b	11.16 ± 1.46b	2.3 ± 0.006a	30.65 ± 1.05a
	B2	7.37 ± 0.16a	1.45 ± 0.04ab	15 ± 0.51b	13.54 ± 1.22b	2.7 ± 0.04b	23.62 ± 1.20b
C3	B0	7.49 ± 0.1a	1.36 ± 0.01b	15.02 ± 1.25a	15.72 ± 1.13a	4.5 ± 0.04a	38.67 ± 0.90a
	B1	7.39 ± 0.23a	1.42 ± 0.06b	28.34 ± 1.19a	18.66 ± 0.61a	2.2 ± 0.016a	24.51 ± 0.59b
	B2	7.46 ± 0.05a	1.36 ± 0.02bc	27.9 ± 1.8a	19.46 ± 1.00a	5.2 ± 0.09a	39.75 ± 0.28a

pH—soil pH, UW—soil bulk density, NO₃N—soil nitrate-N content, NH₄⁺N—soil ammonia N content, TN—total soil nitrogen, SOM—soil organic matter content. Different lowercase letters after different processing of the same column in the table indicate significant differences between treatments (p < 0.05).

shows the physicochemical properties of the soil under different biochar and biogas slurry treatments. This study demonstrated that the application of biochar, biogas slurry, and their combined application significantly reduced soil bulk density (UW) while enhancing soil N content. Compared with the unfertilized control (CK), the UW, pH, NO₃N, TN, and soil organic matter content (SOM) under chemical fertilizer alone (C0B0) showed no significant differences (p > 0.05). Sole biochar application markedly increased soil pH; for instance, under the C3B0 treatment, soil pH was elevated by 45.43% compared with C0B0. A greater increase was observed under the combined biochar–biogas slurry treatment (C3B2), where soil pH increased by 44.85% relative to C0B0. Both biochar and biogas slurry applications reduced soil bulk density, with C1B0, C1B1, and C1B2 treatments showing reductions of 13.79%, 11.72%, and 11.03%, respectively, compared with C0B0. The most pronounced decrease occurred under C1B0 (0.5% biochar and 0% biogas slurry substituting chemical fertilizer), where UW was significantly reduced by 13.79% (p < 0.05). The highest improvement in Soil N content was observed in the combined treatment C3B2, which significantly enhanced nitrate and ammonium-N compared with C0B0 and exceeded the effects of sole biochar application (C3B0). Specifically, nitrate-N content in C3B2 increased by 86%, and ammonium-N by 23.79%, relative to C3B0. the combined application of biochar and digestate exerts remarkable effects on soil physicochemical properties and nutrient cycling. Biochar is weakly alkaline and enriched with oxygen-containing functional groups such as carboxyl and hydroxyl groups, which can neutralize hydrogen ions in the soil, thereby reducing acidity and elevating pH [22,23]. In addition, the porous and loose structure of biochar decreases bulk density, improves porosity, and enhances the soil’s capacity to adsorb and retain small molecules [24,25,26]. Biochar application is also closely linked with soil aggregate formation and stability, which increase with its appropriate addition [27]. Well-structured aggregates provide a stable environment for water and nutrient exchange, support microbial habitats, reduce carbon losses, and ultimately improve soil productivity [15]. Since biochar can adsorb nutrients while digestate is rich in organic matter and primarily contains N in inorganic forms, their combined use significantly enhances Soil nitrate-N and organic matter contents. Humic acids in digestate can form complexes with N compounds, reducing ammonium loss. Furthermore, as a carbon source, digestate stimulates microbial activity and increases the pool of available nutrients [28].

3.2. Two-Way ANOVA of Biochar–Biogas Slurry Co-Application on Soil Physicochemical Properties

A two-way analysis of variance was conducted to assess the effects of biochar and biogas slurry on soil physicochemical properties (

Table 3. Double factor analysis of soil physical and chemical properties under the influence of biochar and biogas slurry.

	pH	UW	SOM	NO3—N	NH4+-N	TN
C	0.052	0.035	0.021	0.025	0.047	0.008
B	0.29	0.15	0.34	0.02	0.26	0.1
B × C	0.005	0.012	0.04	0.04	0.01	0.046

Note: Numbers are shown as p values; p > 0.05 represents no significant difference; p < 0.05 represents significant difference; C: biochar; B: biogas slurry.

). The results showed that sole biochar application significantly affected SOM, nitrate-N, and TN (p < 0.05). Sole biogas slurry application exerted the most pronounced effect on soil nitrate-N (p < 0.05). Moreover, the combined application of biochar and biogas slurry significantly influenced soil pH and UW (p< 0.05), effectively mitigating soil acidification, increasing soil porosity, and enhancing microbial abundance.

3.3. Effects of Biochar–Biogas Slurry Co-Application on Total Ammonia and Nitrous Oxide Emissions

The results demonstrated that the application of biochar and biogas slurry effectively reduced the total emissions of ammonia (NH₃) and nitrous oxide (N₂O). As shown in Figure 1, both the sole application of biogas slurry (C0B2) and that of biochar (C3B0) decreased NH₃ volatilization compared with the CK treatment. Notably, the combined application of biochar and biogas slurry (C3B2) achieved the greatest reduction, with NH₃ volatilization being 43.4% lower than that of the CK treatment. In terms of N₂O emissions, both the sole application of biochar (C3B0) and the combined application (C3B2) reduced emissions relative to CK, by 28.8% and 33.7%, respectively. These findings suggest that biochar–biogas slurry co-application is particularly effective in mitigating gaseous N losses. This study further reveals that biochar and digestate have distinct yet complementary effects on N loss pathways, particularly in ammonia volatilization and nitrous oxide emissions. Digestate, as a by-product of anaerobic fermentation, contains high levels of NH₄⁺-N and is generally alkaline. When applied to soil surfaces, it facilitates NH₄⁺ conversion to NH₃, leading to substantial volatilization, especially under high temperatures or aerated conditions. By contrast, biochar, with its large surface area and porosity, adsorbs NH₄⁺ and releases it slowly, thereby lowering instantaneous concentrations in soil and mitigating NH₃ loss. Moreover, biochar alters soil pH and cation exchange capacity, which helps regulate the chemical form of NH₄⁺ and further suppress volatilization. Regarding N₂O emissions, digestate provides ample N and soluble organic carbon, intensifying nitrification and denitrification, particularly under high moisture conditions where anaerobic microsites favor denitrification. Conversely, biochar generally alleviates N₂O emissions by improving soil structure and aeration, thereby reducing accumulation during denitrification, and by shaping microbial community composition to promote the reduction of N₂O to N₂. When applied together, biochar’s adsorption of NH₄⁺ mitigates digestate-induced ammonia volatilization, while its improvement of soil physicochemical and microbial conditions further suppresses N₂O release. Thus, the co-application enhances N use efficiency while achieving both emission reduction and sustainability goals.

3.4. Effects of Biochar–Biogas Slurry Co-Application on Soil Nitrate Reductase Activity

The experiment showed that sole chemical fertilizer application increased soil nitrate reductase activity, while biochar application significantly enhanced this activity across all growth stages of cabbage (seedling, vegetative, maturity, and harvest) (Figure 2). Compared with the control (CK), the C0B0 treatment (chemical fertilizer alone) significantly increased nitrate reductase activity (p < 0.05). Biochar application (C3B0) further elevated nitrate reductase activity, with increases of 77.9% and 39.6% relative to C0B0 (chemical fertilizer alone) and C0B2 (biogas slurry alone), respectively (p < 0.05). Moreover, the combined biochar–biogas slurry treatment (C3B2) enhanced nitrate reductase activity by 30.9% compared with C0B2 and by 12.9% compared with C3B0. During the cabbage harvest stage, the largest increase in nitrate reductase activity was observed under C0B0, with a maximum rise of 65%. The application of biogas slurry not only enriches the soil with nitrate nitrogen, thereby ensuring sufficient substrates for the denitrification process, but also contributes to the accumulation of soil organic matter. The presence of additional organic compounds stimulates microbial activity by enhancing the functionality of denitrifying enzymes. In parallel, biochar, with its porous structure and strong adsorption capacity, can retain ammonium nitrogen and prolong its persistence in the soil environment. This extended retention not only minimizes nutrient loss but also creates favorable microhabitats and carbon sources that support microbial colonization. Consequently, the proliferation of diverse microbial communities is promoted, accompanied by an increase in nitrate reductase activity, which further facilitates denitrification.

3.5. Effects of Biochar–Biogas Slurry Co-Application on Soil Nitrite Reductase Activity

Changes in soil nitrite reductase activity during the seedling, vegetative, maturity, and harvest stages of cabbage are shown in Figure 3. Compared with the control (CK), chemical fertilizer alone (C0B0) significantly increased nitrite reductase activity (p < 0.05). Prior to harvest, both sole biochar application (C3B0) and sole biogas slurry application (C0B2) markedly enhanced nitrite reductase activity relative to C0B0, with increases of 94.0% and 94.1%, respectively; however, the difference between C3B0 and C0B2 was not significant. In the later harvest stage, the combined biochar–biogas slurry treatment (C3B2) further elevated nitrite reductase activity by 97.4%, 94.6%, and 5.7% compared with CK, C0B0, and C0B2, respectively. Moreover, relative to C0B2 and C3B0, the C3B2 treatment exerted the greatest enhancement of nitrite reductase activity across both pre- and post-harvest stages. The combined application of biogas slurry markedly enhances the concentration of nitrate nitrogen in soils, thereby providing a sufficient substrate pool to sustain denitrification processes. Beyond nutrient enrichment, the incorporation of slurry contributes to the accumulation of soil organic matter, which plays a crucial regulatory role by stimulating the catalytic activity of denitrifying enzymes. In addition, the amendment substantially reshapes the composition of soil microbial communities, offering an abundant supply of readily available carbon sources that facilitate the proliferation of denitrifying bacteria. Collectively, these synergistic effects culminate in a pronounced increase in nitrite reductase activity within the soil matrix, ultimately strengthening the overall efficiency of denitrification.

3.6. Effects of Biochar–Biogas Slurry Co-Application on Soil Urease Activity

Changes in soil urease activity during the seedling, vegetative, maturity, and harvest stages of cabbage are presented in Figure 4. During the growth and harvesting periods of cabbage, compared with the control (CK), chemical fertilizer alone (C0B0) significantly increased urease activity (p < 0.05). Relative to CK, C0B2 (biogas slurry alone), and C3B0 (biochar alone), the combined biochar–biogas slurry treatment (C3B2) showed slightly higher urease activity before and after harvest; however, the differences were not statistically significant. Biochar contains a certain proportion of soluble nitrogen, which can enrich the nitrogen pool in soils and thereby stimulate urease activity. In addition, its well-developed porous network allows for the adsorption of reaction substrates, further promoting the efficiency of urease-mediated processes. Application of biochar also increases soil microbial biomass nitrogen, a parameter that exhibits a strong positive relationship with urease performance. Beyond nutrient retention, biochar functions as a carbon donor for microbial populations, while the simultaneous application of biogas slurry serves as a complementary nitrogen source. This dual provision of essential resources establishes a favorable ecological niche for microbial proliferation, ultimately resulting in a pronounced enhancement of soil urease activity.

3.7. Effects of Combined Biochar and Biogas Slurry Application on Soil N-Cycling Functional Genes

The effects of combined biochar and biogas slurry application on the soil ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes are shown in Figure 5. Compared with the control (CK), all treatments except the sole biochar application (C3B0) increased the abundance of the soil amoA gene, with the sole biogas slurry treatment (C0B2) showing the highest increase, reaching 65.5% above CK. For the nosZ gene, sole biogas slurry application (C0B2) increased its abundance by 8.2% compared with CK, while the combined biochar and biogas slurry treatment (C3B2) resulted in the greatest enhancement of nosZ gene abundance. At the microbial functional gene level, biochar and digestate significantly but differently influenced amoA and nosZ gene abundances. Digestate, which is rich in ammonium, provided direct substrates for nitrifiers, thereby generally increasing amoA abundance. The influence of biochar on amoA was context dependent: while enhanced aeration and favorable microhabitats promoted ammonia oxidizers, strong NH₄⁺ adsorption could reduce its availability and suppress amoA expression under certain conditions. This suggests that biochar’s regulation of nitrifying communities is shaped by factors such as the feedstock type, pyrolysis temperature, and soil characteristics. For nosZ, both amendments exerted positive effects. Digestate promoted denitrifiers by supplying N and soluble organic carbon, whereas biochar stimulated nosZ abundance by restructuring microbial communities, supplying electron donors, and improving aeration, thereby enhancing the reduction of N₂O to N₂. Importantly, their co-application had a synergistic effect: digestate supplied abundant substrates for denitrification, while biochar optimized the soil environment, resulting in a greater increase in nosZ abundance than either amendment alone.

3.8. Effects of Biochar–Biogas Slurry Co-Application on Cabbage Yield and Agronomic N-Use Efficiency

Cabbage yield and N-use efficiency (NUE) under different treatments are shown in Figure 6. Compared with the control (C0B0), chemical fertilizer alone (C3B0) significantly increased cabbage yield by 80.31 t·hm⁻² (p < 0.05). Sole biochar application further enhanced yield, with C3B0 reaching 111.80 t·hm⁻², representing a 46.35% increase relative to C0B0. The highest yield was observed under the combined biochar–biogas slurry treatment (C3B2), which was 61.46% higher than C0B0. In addition, NUE was improved by sole biochar application, with C3B2 achieving the greatest enhancement—23.40% higher than C0B0 (p < 0.05). N is indispensable for protein synthesis in both plants and animals and thus remains a critical input for improving crop yield and quality. With biochar and digestate application, biochar’s large surface area allowed retention of considerable ammonium and nitrate, thereby enhancing soil nutrient-holding capacity and reducing N loss [29]. Previous studies also confirmed that biochar improves crop N use efficiency [30]. Digestate, being rich in nutrients and vitamins, provided additional nutrient input to the soil. Their combined application enhanced soil fertility and crop productivity, achieving a synergistic effect of “reduced fertilizer input with increased efficiency”.

3.9. Two-Factor Analysis of Different Biochar and Biogas Slurry Application Rates on Gas Volatilization, Nitrogen-Cycle-Related Functional Genes and Enzymes, Crop Yield, and Nitrogen Utilization Efficiency

The results of the two-way ANOVA for varying application rates of biochar and biogas slurry (

Table 4. Two-factor analysis of different biochar and biogas slurry application rates on gas volatilization, nitrogen-cycle-related functional genes and enzymes, crop yield, and nitrogen utilization efficiency.

	NH3	N2O	NR	NiR	Urea	amoA	nosZ	COP	NUE
B	0.006 **	0.013 *	0.04 *	0.08 ns	0.02 *	0.03 *	0.25 ns	0.0016 **	0.012 *
C	0.48 ns	0.81 ns	0.01 *	0.42 ns	0.07 ns	0.05 ns	0.003 **	0.049 *	0.007 **
B × C	0.005 **	0.05 ns	0.47 ns	0.33 ns	0.35 ns	0.01 *	0.60 ns	0.41 ns	0.005 **

NH₃—ammonia, N₂O—nitrous oxide, NR—nitrate reductase, NiR—nitrite reductase, Urea—Urease, COP—crop yield, NUE—nitrogen use efficiency. Note: Numbers are shown as p values, ns represents no significant effect, * represents significant level (p < 0.05), and ** represents extremely significant level (p < 0.01). C: biochar; B: biogas slurry.

) demonstrate that biochar plays a crucial role in regulating soil nitrogen dynamics. Specifically, biochar application significantly enhanced nitrate reductase activity, increased the abundance of functional genes associated with nitrogen cycling, and improved crop yield. These findings suggest that biochar provides a favorable soil environment that stimulates microbial processes and accelerates nitrogen transformations. In contrast, both biogas slurry and the interaction between biochar and slurry had pronounced effects on ammonia volatilization and nitrogen use efficiency. This indicates that, while biogas slurry alone supplies readily available nitrogen and organic substrates, its interaction with biochar further stabilizes nitrogen in the soil system, reduces gaseous losses, and prolongs nitrogen availability in the rhizosphere. Importantly, the synergistic effect of biochar and slurry on nitrogen use efficiency exceeded the influence of either amendment when applied independently. This outcome highlights the complementary roles of biochar in nutrient retention and of slurry in nutrient supply, suggesting that their combined use represents an effective management strategy for enhancing nitrogen utilization and crop productivity in agricultural systems.

3.10. N-Cycling Enzymes and N-Use Efficiency

To explore the relationship between agronomic N-use efficiency (NUE) and the activities of nitrate reductase, nitrite reductase, and urease, a linear regression model was established, where Y represents NUE, X₁ nitrate reductase activity, X₂ nitrite reductase activity, and X₃ urease activity. The direct effects of X₁, X₂, and X₃ on NUE were P₁y = 1.139, P₂y = 0.046, and P₃y = –0.0393, respectively. These results indicate that nitrate reductase and nitrite reductase activities were positively correlated with NUE, with nitrate reductase showing the strongest response, suggesting that it is the key enzymatic factor regulating N use efficiency in farmland soils.

Pearson correlation analysis between N-cycling enzymes and environmental factors (Figure 7) further revealed that soil bulk density was negatively correlated with enzyme activities. Nitrate reductase activity exhibited positive correlations with most environmental factors, notably with ammonium-N (r = 0.924, p < 0.05) and TN (r = 0.883, p < 0.05). Nitrite reductase activity also showed a strong correlation with ammonium-N. Urease activity was significantly correlated with soil organic matter (p < 0.05) and displayed moderate positive correlations with multiple environmental factors.

Soil is a multi-enzyme system, and enzyme activities reflect variations in soil quality, fertility, and environmental conditions [31,32,33]. Environmental factors influence enzyme activities directly, for instance, through conformational shifts, binding with soil particles, or altered diffusion rates [34], and indirectly through changes in substrate solubility, concentration, accessibility, and microbial survival strategies [35]. Bulk density broadly indicates soil structural status; lower values imply looser, better-structured soils. In this study, N cycling-related enzyme activities were negatively correlated with bulk density, indicating that biochar and digestate together improved soil structure, lowered bulk density, and promoted N cycling enzymes. Nitrate reductase reduces nitrate to nitrite, which nitrite reductase then converts into ammonium, directly available to plants. Correlation analysis revealed positive associations between nitrate/nitrite reductase activity and soil ammonium as well as TN. Digestate, rich in organic matter and nutrients, stimulated microbial activity and accelerated N transformations, while biochar adsorbed nutrients from digestate and released them gradually through its porous structure. Urease activity was strongly and positively associated with soil organic matter, suggesting that the joint application enhanced soil structure, promoted microbial metabolism, facilitated nutrient turnover, and thereby improved urease activity and soil fertility [36].

4. Conclusions

A field experiment was carried out to investigate the combined application of biochar and biogas slurry at different rates, with particular attention to soil physicochemical characteristics, gaseous nitrogen losses, functional gene abundance, enzyme activities involved in nitrogen cycling, crop yield, and nitrogen use efficiency (NUE). The results showed that biochar alleviated soil acidification and reduced bulk density, while biogas slurry increased soil nitrogen content and enhanced fertility. Their combined application effectively decreased ammonia volatilization and nitrous oxide emissions. Both sole biochar and sole biogas slurry significantly stimulated soil nitrogen cycling enzyme activities, with the combined treatment exhibiting the strongest effect. Additionally, co-application increased the abundance of key nitrogen cycling functional genes (amoA and nosZ), improved soil fertility, and enriched the nitrogen-cycling microbial community, thereby promoting cabbage yield and nitrogen agronomic efficiency.

These findings suggest that partially substituting chemical fertilizers with biochar and biogas slurry offers a sustainable and environmentally friendly fertilization strategy tailored to local cropping systems. Subsequent analyses will focus on different forms of nitrogen content, nitrogen cycling functional genes, microbial communities, and nitrogen use efficiency, to clarify the overall impact of the combined application of biochar and biogas slurry on the nitrogen cycle.