Urea Fertilization Buffered Acid-Inhibiting Effect on Litter Decomposition in Subtropical Plantation Forests of Southern China

Abstract

Acid deposition, a major environmental issue causing soil acidification and microbial suppression, impacts forest nutrient cycling. Meanwhile, nitrogen (N) fertilization is widely applied in subtropical forests, yet its interaction with acid deposition on litter decomposition is unclear. We conducted a field experiment using two common tree species, Cunninghamia lanceolata and Cinnamomum camphora, and applied three acid deposition levels (0, 0.25, and 0.50 g H⁺ m⁻² month⁻¹) and four N fertilization levels (0, 3, 6, and 9 g N m⁻² year⁻¹) in a factorial design. Our results showed that acid deposition alone significantly reduced litter decomposition rates, with maximum mass loss decreasing by 23.6% for Cunninghamia and 36.3% for Cinnamomum (p < 0.05). Urea fertilization alone also suppressed decomposition, reducing maximum mass loss by 27.3% for Cunninghamia and 37.3% for Cinnamomum (p < 0.05). However, when combined, urea fertilization mitigated the suppressive effect of acid deposition, particularly under severe acid conditions, where maximum mass loss increased by 18.5% for Cunninghamia and 43.1% for Cinnamomum (p < 0.05). Acid deposition reduced microbial respiration and enzyme activities related to carbon cycling, while urea fertilization showed both positive and negative effects depending on the acid levels (p < 0.05). Urea can enhance the litter layer’s acid-buffering capacity, offering potential management insights for acid deposition-affected forests. Further research on microbial mechanisms across ecosystems is recommended.

1. Introduction

Litter decomposition is governed by three interacting factors: physicochemical environment, substrate quality, and decomposer community composition [1,2]. Among these, anthropogenic environmental changes, including grazing, pollution (e.g., acid deposition, heavy metals), fertilization, and deforestation, exert profound effects on decomposition dynamics and associated microbial activity [3,4,5]. Understanding these impacts is critical for predicting carbon (C) balance and nutrient cycling in forest ecosystems [6], particularly under accelerating global change scenarios.

Acid deposition, a global environmental concern resulting from intensive industrialization [7], is particularly severe in China and is expected to worsen with ongoing economic development and increased fossil fuel consumption. At a nationwide scale, the average pH of precipitation in the 2010s was 4.70 ± 0.031 (SE) [8,9], showing a slight improvement from 4.59 ± 0.039 in the 1990s. This indicates that while precipitation acid deposition in China has not significantly deteriorated, it remains a critical environmental challenge. An increased influx of acid deposition results in a higher H⁺ load in soils, triggering acidification and suppressing soil microbial activity [10,11]. The H⁺ ions from acid deposition are toxic to soil microorganisms, impeding soil enzyme activity and heterotrophic respiration [12]. Soil acidification can undermine the structure and function of the soil ecosystem, diminishing its buffering capacity [13,14]. Moreover, a low pH can mobilize Al³⁺ from the soil, further intensifying the toxic effects on soil microorganisms [15].

Numerous studies have demonstrated that acid deposition generally inhibits forest litter decomposition and associated microbial activity, even in aquatic systems [16]. Lv et al. [17] further confirmed that regardless of its type (sulfuric, nitric, or mixed), acid rain significantly curtails both litter decomposition and microbial activity. Liang et al. [18] attributed this inhibition directly to system acidification, which reduces the biomass of decomposer microorganisms, respiration rates, enzyme activity, and microbial diversity, and alters the fungal-to-bacterial ratio, ultimately promoting carbon accumulation in soil ecosystems. However, these studies mainly focused on the impact of acid deposition as a single factor, with limited research on its interaction with other factors like nitrogen fertilization.

Nevertheless, the impacts of acidification can hinge on the surrounding biotic and abiotic environments. Bewley and Stotzky [19] reported that acidification can suppress soil respiration, yet this effect can be mitigated by the addition of clay minerals. Vanhala et al. [20] and Lieb et al. [13] suggested that nutrient-rich soil conditions can alleviate the inhibitory effects of acidification. Scheu and Wolters [21] discovered that saprophagous invertebrates can mitigate the inhibitory effects of acid deposition on the carbon mineralization of beech leaf litter by 65%–82%. Some studies also indicated that microbial communities at specific decomposition stages can modify the inhibitory effects of acid rain [22]. These findings underscore that certain environmental factors can modulate the inhibitory impacts of acid deposition.

Nitrogen (N) is another crucial factor influencing soil ecological processes. Nitrogen input into the soil modifies its chemical properties and significantly impacts litter decomposition in forest soils [23,24]. On one hand, nitrogen directly affects decomposition rates by influencing the decomposer community and enzyme activity. On the other hand, it indirectly affects decomposition by influencing forest plant growth, community structure, species composition, and litter chemical composition [24,25]. Nitrogen can have diverse effects on decomposition rates—accelerating, inhibiting, or having a neutral impact. These disparities may depend on the biotic and abiotic environments of the study site and the type of litter. Although subtropical forest ecosystems are not generally nitrogen-limited on a large scale, specific forests such as Cunninghamia lanceolata and Cinnamomum camphora plantations are nitrogen-limited due to their infertile soils, which are also prone to frequent precipitation erosion. In western Hunan Province, China, nitrogen fertilization is commonly employed to sustain the high productivity of plantations for timber and ecological purposes. This inevitably affects soil ecological processes and functions, including litter carbon release [26,27,28].

Many studies have looked into the separate effects of acid precipitation and nitrogen fertilization on forest litter decomposition. But so far, we still lack a good understanding of how acid deposition and nitrogen fertilization interactively affect litter decomposition in subtropical plantation forests, especially in the context of karst-developed infertile soils. Most existing research has only touched on the individual impacts of acid deposition or nitrogen fertilization. There’s a knowledge gap regarding how urea, a weakly alkaline and long-lasting nitrogen source, influences the litter decomposition process under acid deposition. This gap limits our comprehensive understanding of soil ecological processes in response to environmental changes and our ability to accurately predict carbon balance and nutrient cycling in forest ecosystems.

Furthermore, the impact of acid deposition on litter decomposition may involve the acidolysis of litter substrates. Acidolysis can break down easily hydrolyzable components and loosen the substrate structure. This process enhances the availability of the substrate, making it more accessible for microbial utilization. Specifically, it benefits the penetration ability of fungi, as they can more readily access and colonize the loosened substrate. The study area is characterized by karst-developed soils, which are generally infertile. In such nutrient-poor ecosystems, the addition of urea is expected to boost microbial activity. This is because urea supplies essential nitrogen, which is often in short supply in these soils. Both the increased substrate availability resulting from acidolysis and the enhanced microbial activity due to urea addition work in tandem to promote microbial action on litter decomposition. Microbes, with greater access to substrates and more energy resources from the added nitrogen, are able to produce more enzymes and allocate more energy to carbon-acquiring enzymes [29,30,31]. As a result, the overall decomposition capacity of microorganisms on plant litter can be improved.

Therefore, we hypothesize that in the litter layer of subtropical plantation forests, urea fertilization can mitigate the inhibitory effect of acid deposition on litter decomposition. Specifically, the nitrogen supplied by urea not only boosts microbial activity in the infertile soil but also synergizes with acidolysis to enhance the availability of litter substrates. Moreover, urea mitigates the stress imposed by acid deposition by neutralizing the H⁺ ions within it. Collectively, these mechanisms promote the microbial decomposition of litter.

The main objectives of this research are as follows: (1) to explore the interaction mechanism between acid deposition and urea fertilization in the litter decomposition process of subtropical plantation forests; (2) to evaluate the buffering capacity of urea fertilization against the inhibitory effect of acid deposition on litter decomposition; (3) to provide a scientific basis for the management of soil ecological processes under environmental changes to better maintain the carbon balance and nutrient cycling in forest ecosystems.

2. Materials and Methods

2.1. Site Description and Litter Collection

The experiment was conducted at two plantation forest sites in western Hunan Province, China. The first site was a coniferous forest dominated by Cunninghamia lanceolata, located in the suburbs of Jishou city (28°15′ N, 109°40′ E, elevation 254 m) with an area of 1500 m². The second site was a broad-leaved forest dominated by Cinnamomum camphora, situated near Jishou University (28°17′ N, 109°43′ E, elevation 258 m) with an area of 1000 m². The two sites were approximately 5 km apart. The climate of the region is characterized by an annual mean temperature of 16.6 °C, with the monthly mean temperature peaking at 29.2 °C in July and dropping to 5.2 °C in January. The annual precipitation is 1375.5 mm, and the rainy season typically commences in May. The underlying parent material of the soil at both sites is predominantly karst limestone, and the soil is classified as ultisol [32].

For the experiment, newly fallen C. lanceolata needle and C. camphora leaf litters were collected during the peak abscission period in April. To ensure the collection of litters with complete senescence, trees were shaken, and the fallen litters were gathered on a plastic sheet placed beneath the trees to prevent soil contamination. The collected litters were then transported to the laboratory and oven-dried at 50 °C to achieve a constant weight. Subsequently, 5 g of dried litter was placed into 1-mm mesh litterbags (20 cm × 20 cm, made of durable nylon net). These litterbags were designed to permit microbial colonization while preventing the litters from being consumed by large soil fauna. The initial size of the litters was sufficiently large to avoid loss through the net mesh.

2.2. Experimental Design

We conducted a factorial experiment using a completely randomized block design to investigate the impacts of acid deposition and nitrogen (N) fertilization on litter decomposition under natural conditions. According to the EANET report 2019 (https://www.eanet.asia/, accessed on 1 January 2020), wet deposition of sulfur and nitrogen in China corresponds to an H⁺ range of 0.01 to 0.20 mol H⁺ m⁻² month⁻¹. Additionally, data from the Jishou Environmental Protection Agency for 2020 showed a higher value of 0.12 mol H⁺ m⁻² month⁻¹. Based on this information, the three acid deposition levels in our experiment—0 mol H⁺ m⁻² month⁻¹ (A0), 0.25 mol H⁺ m⁻² month⁻¹ (A0.25), and 0.50 mol H⁺ m⁻² month⁻¹ (A0.5)—were selected to cover the observed deposition rates and create a gradient for evaluating ecological impacts. The moderate and severe levels (A0.25 and A0.5) go beyond the upper limit reported by EANET to simulate possible future scenarios of increased acid deposition, providing insights into ecosystem responses under different stress conditions. Based on background N deposition data from the Jishou Environmental Protection Agency, which indicates levels of approximately 5–10 kg N ha⁻¹ year⁻¹, we established four N fertilization levels: 0 g N m⁻² year⁻¹ (N0), 3 g N mol H⁺ m⁻² month⁻¹ (N3), 6 g N mol H⁺ m⁻² month⁻¹ (N6), and 9 g N mol H⁺ m⁻² month⁻¹ (N9). This resulted in a total of 12 treatments. The pH values of the undecomposed litters of C. lanceolata needles and C. camphora leaves were 5.4 ± 0.1 and 6.2 ± 0.1, respectively (unpublished data). Based on these pH values, the mean H⁺ contents per litterbag were calculated to be 1.98 × 10⁻⁷ mol for C. lanceolata and 3.16 × 10⁻⁸ mol for C. camphora.

In April 2023, at each site (coniferous and broad-leaved forests), we established 12 experimental plots within a close range of altitude in relatively flat fields, each measuring 5 m × 5 m. The 12 treatments were randomly assigned to the plots to ensure randomness and reduce potential bias. The distance between adjacent plots was approximately 10 m. Within each plot, three closely adjacent subplots (2 m × 2 m, serving as replicates) were randomly distributed for each treatment. Eighteen litterbags filled with the corresponding litters were placed onto the litter layer in each subplot to simulate natural decomposition conditions. In total, 1296 litterbags were used in the experiment: 9 litterbags per treatment × 3 acid deposition levels × 4 N fertilization levels × 6 sampling times × 2 litter types.

To simulate acid deposition, we prepared an acid solution using a 98% sulfuric acid (H₂SO₄) stock solution (w/w) to avoid any nitrogen influence. This stock solution was diluted with sterile water to achieve the desired acid levels and was sprayed onto the subplots every 15 days. For nitrogen fertilization, we applied a 1-L urea solution in three separate applications, following a 2:1:1 ratio, with the first application in May 2023, the second in October 2023, and the third in January 2024. This schedule aimed to mimic natural nitrogen input patterns and ensure a consistent nitrogen supply throughout the year. The plantation forest manager permitted only urea for fertilization to maintain its effectiveness.

From June 2023 to April 2024, we randomly selected three litterbags from each subplot every two months, totaling nine litterbags sampled for each treatment. This regular sampling allowed us to measure mass loss every two months to calculate the decomposition rate constant (k), as described in the Statistical Analyses section. It also enabled us to accumulate enzyme activity data across all sampling periods, providing a more stable and comprehensive measure of enzyme activity. Soil particles and fine roots were gently removed using a small brush and sterile tweezers. Three of the nine litterbags per treatment were oven-dried at 50 °C to constant weight for measuring litter dry mass, while the remaining six were stored at 4 °C for subsequent microbial activity analysis.

2.3. Measurements of H⁺ Content, Mass Loss, Water Content and Fungal Biomass

To determine the initial pH (mean ± SE) of undecomposed litters, we used a pH meter. Specifically, we prepared a solution by mixing litter powder (sieved through a 0.2-mm mesh) from each plant species with distilled water at a ratio of 1:10 (to facilitate pH measurement). This mixture was stirred for 1 h and then left undisturbed for another hour before measuring the pH. Based on the pH value, we calculated the H⁺ concentration and expressed the H⁺ content as mol H⁺ per gram of dry litter. The remaining H⁺ amount in each treatment, calculated as the difference between mol N and mol H⁺ after potential neutralization by urea, was expressed as mol H⁺ m⁻² year⁻¹ (

Table 1. The remaining amounts (mol H⁺∙m⁻²∙year⁻¹) of H⁺ after potential neutralization by urea.

N Fertilization Treatments	Acid Deposition Treatments
	A0	A0.25	A0.5
N0	1.44	3.00	6.00
N3	1.23	2.79	5.79
N6	1.01	2.57	5.57
N9	0.80	2.36	5.36

Note: The value of A₀ treatment (ambient treatment) was calculated at 1.44 mol H⁺∙m⁻²∙year⁻¹ (selecting a high value in study site because of close distance from city and industrial area; data from Jishou Environmental Protection Agency).

).

Litter mass loss was quantified as the percentage of dry mass loss relative to the initial dry mass. Water content was assessed for each litterbag by determining the difference in dry and wet mass on each sampling date after in situ exposure and was expressed as grams of H₂O per gram of dry litter.

Fungal biomass was measured as hyphal length density (m g⁻¹ litter) [33]. We milled 0.5 g of litter per litterbag and homogenized it in 100 mL distilled water in a laboratory blender for 5 min. An aliquot of this homogenate was then diluted fivefold. We passed 3 mL of the diluent through a cellulose nitrate membrane filter. For each diluent, we prepared three membranes for observation and measured hyphal length density using the gridline intercept method with a Motic microscope (BA 210-T, Motic China Group Co., Ltd., Xiamen, China).

2.4. Determination of Microbial Respiration and C-Cycling Extracellular Enzyme Activity

Microbial respiration was assessed by quantifying CO₂ release at field moisture levels using a two-phase titration method [34]. For this measurement, 0.5 g of litter was incubated in sterile, airtight flasks for 48 h at approximately 25 °C in the dark. CO₂ released during incubation was absorbed by a 0.5 M NaOH solution and subsequently determined via two-phase titration with 0.05 M HCl. Each treatment was replicated three times, and litter CO₂ release was expressed as µmol g⁻¹ dry litter h⁻¹.

Extracellular enzyme activities involved in labile C decomposition, including cellulolytic enzymes [exo-1,4-β-glucanase (EC 3.2.1.91), carboxymethyl cellulase (EC 3.2.1.4), β-glucosidase (EC 3.2.1.21)], and amylases (β-amylase and α-amylase), were measured optically at 540 nm using the DNS (3,5-dinitrosalicylic acid) method according to Miller [35]. Enzyme activity was expressed as units equivalent to μmol of glucose released per gram of dry litter mass per hour. Enzyme activities involved in recalcitrant C decomposition, including laccase (EC 1.10.3.2) and peroxidase (EC 1.11.1.7), were measured following the protocol of Fioretto et al. [36]. Activity was calculated as μmol of tolidine oxidized per minute, using a molar extinction coefficient of 6340. All enzyme assays were conducted in triplicate for each treatment. For detailed information, please refer to the Supplementary Materials.

2.5. Statistical Analyses

The litter decomposition rate (k, month⁻¹) was calculated by the negative exponential model:

X_t/X₀ = e^−k·t

where X_t/X₀ is the fraction of mass remaining at time t (month), X_t the mass remaining at time t, and X₀ the original mass [37].

The effects size of acid deposition and N fertilization were assessed by the response ratio (RR): RR = ln(X_T/X_C) = ln(X_T) − ln(X_C), where X_T are the mean values in treatment groups, including acid deposition and N fertilization, and X_C is control. The variance (ν) of each RR was calculated as below:

$$V={\displaystyle \frac{S_T^2}{n_T{X}_T^2}}+{\displaystyle \frac{S_C^2}{n_C{X}_C^2}}$$

where n_T and n_C are the number of replicates; and S_T and S_C are the standard deviations of means in treatments and control, respectively. A bootstrapping technique (sample size corresponded with the real data structure, 1000 permutations) was used to estimate the uncertainty of k value and effect size.

Data were checked for deviations from normality and homogeneity of variance before analysis. We analyzed the effect of acid deposition and N fertilization and their interactions on cumulative mass loss, fungal biomass, microbial respiration, and enzyme activity using a factorial mixed-model ANOVA with plot as the random effect. Significant differences among treatments were compared according to one-way ANOVA followed by Duncan’s test at p < 0.05. The relationship between environmental variables (acid deposition and N fertilization) and response variables including mass loss, CO₂ release, fungal biomass, water content, and enzyme activity was analyzed by canonical correspondence analysis (CCA). We also employed piecewise structural equation modeling (piecewiseSEM) to evaluate the causal relationships among variables. We initially constructed a prior model that included all hypothesized pathways and iteratively simplified the model by removing non-significant pathways until the final model was achieved. The suitability of the final model was evaluated utilizing Fisher’s C statistic, as implemented in the piecewiseSEM 2.3.0 package. All the aforementioned statistical analyses were performed using R version 4.3.3.

3. Results

3.1. Effect on Litter Decomposition

The mixed-model ANOVA revealed that the fixed and/or interactive effects of nitrogen (N) fertilization and acid deposition were significant in most cases (p < 0.05,

Table 2. Analyses of the fixed and interactive effects of the N fertilization (N) and acid deposition (A) treatments of the litters of C. lanceolata and C. camphora.

Variables	Parameter	C. lanceolata				C. camphora
		Intercept	N	A	N × A	Intercept	N	A	N × A
Mass loss	F	4188.2	4.4	0.4	25.7	9962.1	3.2	26.4	59.5
	p	<0.01	<0.05	>0.05	<0.01	<0.01	<0.05	<0.01	<0.01
Water content	F	3455.9	2.7	81.1	4.1	6818.6	9.6	46.8	1.8
	p	<0.01	>0.05	<0.01	<0.01	<0.01	<0.01	<0.01	>0.05
Fungal biomass	F	7982.4	130.7	4.5	8.3	5298.6	79.6	5.8	2.6
	p	<0.01	<0.01	<0.05	<0.01	<0.01	<0.01	<0.01	<0.05
CO2 release	F	3156.2	1.9	0.1	7.1	14,849.1	28.6	18.7	73.5
	p	<0.01	>0.05	>0.05	<0.01	<0.01	<0.01	<0.01	<0.01
Exo-1,4-β-glucanase	F	27,388.5	20.3	710.1	157.8	85,751.2	340.5	414.3	177.3
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
Carboxymethyl cellulase	F	12,619.2	99.1	832.0	71.3	76,322.1	556.4	10.91	1855.2
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
β-Glucosidase	F	53,945.2	32.4	106.3	228.1	16,502.2	5.0	62.6	200.0
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
α-Amylase	F	9584.9	33.8	7.3	24.8	43,911.6	73.9	33.7	150.5
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
β-Amylase	F	45,016.8	107.1	119.9	7.4	17,462.4	25.4	83.7	23.2
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
Laccase	F	23,057.5	193.7	842.9	493.2	14,434.5	319.7	285.6	286.8
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
Peroxidase	F	4467.9	70.1	184.1	147.4	10,732.9	204.7	151.3	24.3
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

). Our results indicated that at the end of incubation acid deposition alone significantly reduced litter decomposition rates (p < 0.05), with maximum mass loss decreasing by 23.6% for C. lanceolata (Figure 1a) and 36.3% for C. camphora (Figure 1b). Similarly, urea fertilization alone also significantly suppressed decomposition (p < 0.05), reducing maximum mass loss by 27.3% for C. lanceolata (Figure 1a) and 37.3% for C. camphora (Figure 1b). However, when combined, urea fertilization mitigated the suppressive effect of acid deposition, particularly under severe acid conditions, where maximum mass loss increased by 18.5% for C. lanceolata (Figure 1a) and 43.1% for C. camphora (Figure 1b).

Prior to conducting one-way ANOVA, all data underwent normality tests, which confirmed that the data conformed well to normality (p > 0.05). Additionally, tests for homogeneity of variance among the different treatment groups revealed that the variances were homogeneous (p > 0.05). Both acid deposition and urea fertilization significantly affected the litter decomposition rate of C. lanceolata and C. camphora (p < 0.05, Figure 2a,b). Without acid deposition, urea fertilization exhibited a significant concentration effect (p < 0.05) on the litter decomposition of C. lanceolata needles (Figure 2a) and C. camphora leaves (Figure 2b), with the decomposition rate (k) decreasing from 0.06 month⁻¹ to 0.02 month⁻¹ for C. lanceolata and from 0.12 to 0.05 month⁻¹ for C. camphora as the N level increased. Under moderate acid conditions, the decomposition rate was lowest at 3 or 9 g N m⁻² year⁻¹ for C. lanceolata litters (0.04 month⁻¹, Figure 2a), and at 6 g N m⁻² year⁻¹ for C. camphora litters (0.06 month⁻¹, Figure 2b). Under severe acid conditions, the litter decomposition rate significantly increased with N level, irrespective of litter species (p < 0.05), rising from 0.03 to 0.05 month⁻¹ for C. lanceolata and from 0.05 to 0.15 month⁻¹ for C. camphora. Acid deposition significantly increased the decomposition rate only when the N level was above 6 g N m⁻² year⁻¹ for C. lanceolata (from 0.02 to 0.05 month⁻¹), while for C. camphora, this effect was observed at an N level of 3 g N m⁻² year⁻¹ (from 0.05 to 0.15 month⁻¹). This suggests that the decomposition of C. camphora leaves was more sensitive to N addition than that of C. lanceolata needles.

Figure 3a shows that, compared to the control group, all treatments generally had inhibitory effects, except for the N9-A0.5 treatment, which exhibited an accelerating effect. Additionally, it reveals that C. camphora litter (response ratio (RR) ranging from −0.84 to 0.25) was more sensitive to acid deposition and nitrogen fertilization treatments than C. lanceolata litter (RR ranging from −0.85 to −0.25). In the presence of urea fertilization, acid deposition had contrasting effects on the two litter types under low N level (3 g N m⁻² year⁻¹), with a negative effect (RR value < 0) on C. lanceolata and a positive effect (RR value > 0) on C. camphora (p < 0.05, Figure 3b). Interestingly, under medium and high N levels (6 and 9 g N m⁻² year⁻¹), both moderate and severe acid deposition showed improving effects (RR value > 0) on decomposition, regardless of litter species (p < 0.05, Figure 3b). The RR values also demonstrated the negative effects (RR value < 0) of urea fertilization on decomposition under low acid levels for both litter species (p < 0.05, Figure 3c). However, under high acid levels, urea fertilization reversed the negative impact of acid deposition on decomposition for both litter species. Notably, the magnitude of the increase in decomposition rate (k) by 9 g N m⁻² year⁻¹ urea fertilization was substantial (0.15 month⁻¹), significantly higher than the control (p < 0.05, Figure 3c).

3.2. Effect on Labile and Recalcitrant C Decomposition Enzyme Activity

Acid deposition generally decreased the activities of cellulolytic enzymes in C. lanceolata litters, with carboxymethyl cellulase activity declining by 74%, exo-1,4-β-glucanase by 70%, and β-glucosidase by 61% (Figure 4a,c,e). However, the effects of acid deposition on enzyme activities in C. camphora litters varied with nitrogen (N) addition treatments. Specifically, under ambient N conditions, enzyme activities were negatively affected, while at 3 g N m⁻² year⁻¹, a bell-shaped effect was observed, and at 6 and 9 g N m⁻² year⁻¹, enzyme activities were positively affected (Figure 4b,d,f). Urea fertilization had a negative impact on cellulolytic enzyme activities in C. lanceolata needles under ambient and moderate acid deposition levels, with decreases of 40%, 36%, and 47% for the three cellulolytic enzymes, respectively. In contrast, under severe acid deposition, enzyme activities increased by 41%, 57%, and 65%. For C. camphora litters, urea fertilization typically exhibited a single-peak effect on cellulolytic enzymes, with maximum activities of 595, 12, and 491 μmol glucose g⁻¹ h⁻¹ for the three enzymes at 6 g N m⁻² year⁻¹.

Acid deposition generally decreased the activities of two amylases in C. lanceolata and C. camphora litters when N ≤ 6 g m⁻² year⁻¹ but increased the activities of amylases in most cases when N was 9 g m⁻² year⁻¹. In most cases, urea fertilization significantly enhanced the activities of α-amylase and β-amylase in degrading litters of C. lanceolata needle, reaching the maximum values in the moderate or high N treatments (p < 0.05; Figure 5a,c). However, for C. camphora litters, N fertilization had a reducing effect on α-amylase under ambient and moderate acid deposition treatments, and a single-peak effect occurred in the severe acid treatment (Figure 5b). With respect to β-amylase, a single-peak type effect of N fertilization was observed in the ambient and moderate acid treatments for C. camphora litters, while in the severe acid treatment N fertilization inhibited this enzyme (Figure 5d).

Urea fertilization reduced the activities of ligninolytic enzymes in degrading litters of C. lanceolata under ambient acid conditions, with laccase activity decreasing by 48% and peroxidase by 52%. In contrast, under moderate or severe acid conditions, enzyme activities increased, with laccase activity rising from 109 to 332 μmol o-tolidine g⁻¹ h⁻¹ and peroxidase from 36 to 298 μmol o-tolidine g⁻¹ h⁻¹ (Figure 6a,c). For C. camphora litters, N fertilization enhanced laccase activity in the ambient acid environment, produced a single-peak effect under moderate acid conditions, and had a reducing effect under severe acid conditions (Figure 6b). Urea fertilization consistently decreased peroxidase activity, regardless of acid deposition level (Figure 6d). Acid deposition lowered laccase activities in C. lanceolata litters when the N fertilization rate was ≤6 g N m⁻² year⁻¹, but a single-peak effect was observed at a fertilization rate of 9 g N m⁻² year⁻¹ (Figure 6a). For C. camphora litters, acid deposition significantly diminished laccase activities in the ambient environment (p < 0.05), produced a single peak at N fertilization rates of 3 or 6 g N m⁻² year⁻¹, and had a negative effect at 9 g N m⁻² year⁻¹ (Figure 6b). Acid deposition reduced peroxidase activities in C. lanceolata litters in the ambient N environment, but the addition of N led to increased activities (Figure 6c). For C. camphora litters, acid deposition decreased peroxidase activities, except in the ambient N treatment (Figure 6d).

3.3. Effect on Microbial Respiration, Fungal Biomass and Water Content

Acid deposition generally suppressed microbial respiration under lower urea fertilization levels but had a stimulating effect under high urea fertilization levels. Specifically, urea fertilization reduced microbial respiration in C. lanceolata litters under ambient and moderate acid deposition levels but enhanced it under severe acid deposition (Figure 7a). For C. camphora litters, microbial respiration was decreased by urea fertilization under ambient acid deposition, with the highest respiration rate observed under severe acid deposition combined with 6 g N m⁻² year⁻¹ urea fertilization (Figure 7b).

Acid deposition decreased fungal biomass (hyphal length density) when nitrogen (N) fertilization was ≤3 g m⁻² year⁻¹, but this suppressive effect diminished at higher N levels (6 and 9 g N m⁻² year⁻¹). Urea fertilization significantly increased fungal biomass in the degrading litters of both C. lanceolata and C. camphora across all treatments (p < 0.05, Figure 7c,d).

Acid deposition, particularly at high levels, significantly reduced litter water content in most cases (p < 0.05, Figure 7e,f).

3.4. Relationship of Decomposition Function to Acid Deposition and N Fertilization

For both litter species, nitrogen (N) fertilization generally exhibited approximate orthogonality with acid deposition, despite the proportion of total variance explained by canonical correspondence analysis (Figure 8).

For C. lanceolata litters, the first two canonical axes accounted for 34.52% and 11.96% of the total variability, respectively, with the permutation test indicating statistical significance for the first canonical axis (Figure 8a). Acid deposition was positively associated with mass loss, CO₂ release, and peroxidase activity, while N fertilization was positively related to fungal biomass, α-amylase, β-amylase, and laccase. Cellulolytic enzyme activities and litter water content showed varying degrees of negative correlation with either N fertilization or acid deposition. Similar to the CCA analysis, piecewise structural equation modeling (SEM) analysis revealed significant positive correlations between acid deposition and both decomposition rate and peroxidase activity, whereas N fertilization showed significant positive associations with β-amylase and laccase (Figure 9a).

For C. camphora litters, the first two canonical axes explained 27.01% and 9.91% of the total variability, respectively, with the permutation test indicating statistical significance for the first canonical axis (Figure 8b). Acid deposition had positive correlations with mass loss, exo-1,4-β-glucanase, carboxymethyl cellulase, β-glucosidase, and α-amylase, while N fertilization had positive correlations with laccase, fungal biomass, and β-amylase. Peroxidase activity showed clear negative correlations with both N fertilization and acid deposition. Piecewise SEM analysis revealed significant inhibitory effects of acid deposition on laccase and β-amylase, while N fertilization demonstrated significant promoting effects on the decomposition rate (Figure 9b). Water content had significant positive effects on the decomposition of both types of forest litter.

4. Discussion

4.1. Acid Deposition Response

Our research findings substantiate the suppressive effect of acid deposition on the decomposition of both C. lanceolata needle and C. camphora leaf litter, echoing the results of prior studies [38,39]. A critical factor to consider when assessing the impact of acid deposition on litter decomposition is the potential neutralization or amplification of acidity stemming from the litter’s inherent pH. In our study, the annual H⁺ deposition per plot was 12 mol (0.25 mol H⁺ m⁻² month⁻¹ × 12 months × 4 m²), which vastly surpasses the H⁺ content within the litter. Consequently, the acidity of the litter itself has a negligible influence on the decomposition process, and the observed inhibitory effects can be predominantly ascribed to acid deposition. This inhibition occurs irrespective of whether the litter originates from coniferous or broad-leaved species. Furthermore, the degree of acid-induced inhibition was significant, resulting in approximately 24% and 36% reductions in mass loss for C. lanceolata needle and C. camphora leaf litter, respectively, by the end of the incubation period. This indicates that long-term acid deposition has a more substantial impact on decomposition than factors such as litter quality or specific microbial communities associated with the litter at a fine scale [16,19,40,41]. This observation appears to be consistent with the findings of Wei et al. [42], who reported significant differences in litter quality between species under acid deposition treatments, implying that litter quality is also an important factor governing decomposition. It is plausible that soil and litter quality may influence the stress response to acid deposition. Wei et al. [43] posited that low-quality soils might experience greater stress under acid deposition compared to high-quality soils. Given that the study area features karst soil and the decomposing substrates of C. lanceolata and C. camphora are categorized as low-quality [39], it is conceivable that these low-quality organic substrates are more susceptible to the pronounced inhibitory effects of acid deposition.

Acid deposition induces alterations in litter quality through the mechanism of H⁺ acidolysis. Elevated concentrations of H⁺ facilitate the chemical acidolysis of easily hydrolyzable polysaccharides such as starch and cellulose, which have a higher water retention capacity compared to other organic constituents. Notably, acid deposition reduced the water content of decomposing C. lanceolata needle and C. camphora leaf litter by 10% to 50%, irrespective of the quantity of nitrogen added. This indicates that acid deposition brings about changes in the organic chemical composition of litter. Moreover, the loss of readily hydrolyzable components leads to a loosening of the physical structure of the litter, which facilitates the penetration of decomposers, particularly filamentous fungi, into the inner substrate [39,44]. While such physicochemical changes might theoretically accelerate decomposition by enhancing fungal access [44], our data reveal a net inhibition, implying a decoupling between structural degradation and biological mineralization. This implies that the inhibitory influence of acid deposition on decomposition is predominantly associated with the response of decomposer activity within the litter habitat [45,46,47]. Additionally, this observation indicates that microbial activity exhibits a more variable response to acid deposition than mass loss. Hence, assessing the response of decomposition to environmental factors through microbial activity may yield more precise results than relying solely on mass loss [48].

As anticipated, microbial activities, encompassing respiration, enzymatic activity, and fungal biomass, generally exhibited negative responses to acid deposition. Analogous suppressive effects of acid deposition on microbial respiration and enzymatic activities have been documented in previous studies [17,18,49,50,51,52,53,54]. The decline in microbial activity resulting from acid deposition is predominantly attributed to the toxic effects of high H⁺ loads in soil ecosystems [12,55].

4.2. N Fertilization Altered the Acid Deposition Response

Our study explored how nitrogen fertilization influences the response to acid deposition. Urea fertilization slowed down the decomposition process. This is consistent with many studies on the effects of nitrogen inputs [56,57,58,59,60]. Several potential reasons may explain this observation. Firstly, the alkaline nature of urea may play a role. Generally, the pH value of a system closely influences microbial activity and corresponding functions. While the weak alkalinity of urea might have a limited effect on litter decomposition, laboratory measurements of urea-saturated solutions at 25 °C showed a pH of 7.3–7.5. However, the subsequent nitrification of ${\mathrm{N}\mathrm{H}}_4^{+}$—formed via urea hydrolysis catalyzed by urease produced by various prokaryotic and eukaryotic microbes—could potentially suppress litter decomposition through acidification [61,62]. Beyond the direct properties of urea, the response of microbial activities to nitrogen fertilization may influence ecosystem functioning [63,64]. At our study site, the two types of litter, rich in recalcitrant carbon, may have developed specific microbial decomposers according to the “home-field advantage” for litter decomposition [44,65,66,67]. Some studies suggest that lower-latitude soils, such as those at our study site, may host more recalcitrant carbon-decomposing microbes compared to higher-latitude soils [68]. One plausible explanation, supported by prior research, is that recalcitrant carbon-decomposing microbes may prefer inorganic nitrogen over organic nitrogen for producing extracellular enzymes [69]. Since urea is an organic nitrogen form, it may not effectively support the production of extracellular enzymes necessary for breaking down recalcitrant carbon compounds. Lin et al. [70] also reported that urea fertilization inhibited soil C cycling-related enzyme activities. The observed suppression of lignocellulolytic enzymes in response to urea fertilization in our study aligns with this hypothesis. We acknowledge that without direct evidence from microbial community data or nitrogen form utilization analysis, these explanations remain speculative. Future studies should consider incorporating such analyses to provide a more comprehensive understanding of the mechanisms underlying the observed effects.

Litter decomposition is a complex process governed by the interplay of biotic and abiotic factors, including climate, microbes, and soil properties. The magnitude and direction of changes in litter decomposition reflect the decomposition system’s sensitivity to environmental stressors and its response pattern. While numerous studies have examined the individual effects of nitrogen input and acid deposition on ecological processes, few have explored their combined interactions. For instance, Zhang et al. [56], Růžek et al. [57], and Xiao et al. [58] found that nitrogen addition and acid deposition each individually reduced soil respiration, a result consistent with ours. However, when both factors were applied together, their combined effect was inhibitory, contrasting with our findings. This discrepancy may arise from differences in nitrogen forms, acid deposition components, or study site characteristics across different studies. In our study, when urea fertilization was combined with acid deposition, decomposition accelerated regardless of the litter type (leaf or needle). Several potential reasons could explain this reversal. Firstly, despite its weak alkalinity, urea can partially neutralize H⁺ from acid deposition. In our study, this neutralization reduced the H⁺ amount by approximately 1/5 and 1/10 in the moderate and severe acid deposition treatments, respectively. Thus, urea fertilization may neutralize a considerable amount of H⁺, thereby alleviating the suppressive effect of acid deposition on litter decomposition. Secondly, in acidic soils, urea can release ${\mathrm{N}\mathrm{H}}_4^{+}$ and CO₂ through neutralization reactions, increasing inorganic nitrogen. This increase may benefit recalcitrant carbon users in degrading litter [64]. While our results suggest that urea fertilization can mitigate the inhibitory effects of acid deposition on litter decomposition, our attribution of this effect to the neutralization of H⁺ and the formation of ${\mathrm{N}\mathrm{H}}_4^{+}$ is based on chemical assumptions rather than direct measurements. Future studies should include measurements of pH, ion concentrations, and nitrogen transformations to provide empirical support for these mechanisms. Additionally, increased acid deposition may lead to litter acidolysis, loosening the litter structure and enhancing its biodegradability due to improved penetrative growth of filamentous fungi, further promoting litter decomposition [44]. Thus, although soil acidification may seem excessive and could be intensified by the nitrification of ${\mathrm{N}\mathrm{H}}_4^{+}$ released from neutralization reactions, these positive factors—including reduced H⁺ toxicity and sufficient nitrogen and carbon sources—may sufficiently stimulate the degrading activities of decomposer communities in infertile karst soil. The fact that urea fertilization enhanced CO₂ release and most extracellular enzyme activities in the severe acid deposition treatment supports this speculation. Therefore, urea fertilization appears to enhance the acid-buffering capacity of the litter layer. Some studies have also shown that acid deposition did not significantly suppress microbial respiration, enzymatic activity or microbial composition in decomposing litter or soil [71,72,73,74,75,76], and even improved litter decomposition in some cases [77]. Scheu and Wolters [21] suggested that these discrepancies might result from the counteracting effects of certain soil physicochemical and biological characteristics against acidification. Other studies have indicated that factors such as plant species combination, rhizosphere soil, rich nutrient availability, and saprophagous invertebrates can enhance the acid-buffering capacity of soil and litter layers [19,20,21,78,79].

5. Conclusions

In our study, urea fertilization was found to potentially enhance the acid-buffering capacity of the litter layer regarding litter decomposition and related microbial activity. This effect is context-dependent, influenced by acid levels, litter type, and fertilization dosage. The primary contributor to this enhancement is likely the urea-to-${\mathrm{N}\mathrm{H}}_4^{+}$ transformation through H⁺ action. Organic N can be hydrolyzed into ${\mathrm{N}\mathrm{H}}_4^{+}$ via both acid chemical reactions and microbial enzyme activity, leading to different microbial community compositions. The microbial community assembly history’s impact on species interactions may also play a role in the observed effects. However, our study has limitations, such as the absence of amplicon sequencing for microbial communities of litter decomposers, which hinders a comprehensive understanding of the specific microbial mechanisms involved.

While our findings indicate that urea fertilization could be a potential measure for restoring ecosystems damaged by acid deposition, we caution against overgeneralizing these results. Our study was conducted in a specific forest ecosystem, and the observed effects may differ across ecosystems due to variations in soil properties, vegetation composition, and climate conditions. Additionally, it is crucial to consider the potential negative side effects of urea fertilization, such as nitrogen leaching and eutrophication, which were not comprehensively addressed in our study. Further research is needed to provide clearer guidance on optimal N levels for different ecosystems.

Future research should address unresolved questions and test the generality of our findings. We suggest conducting studies in various forest ecosystems to assess the broader applicability of urea fertilization for enhancing acid buffering and restoring ecological function. Including amplicon sequencing of microbial communities would provide deeper insights into the decomposer communities and their responses to urea fertilization and acid deposition. Determining the quantitative relationship between acid deposition and urea fertilization in decomposition across ecosystems is also crucial for practical applications. Additionally, experiments manipulating different nitrogen forms and acid deposition components could clarify their interactive effects on litter decomposition and microbial communities. Such research would improve our understanding of nitrogen and acid deposition interactions in forest ecosystems and support more effective forest management and policy decisions.