Effects of Mixed Addition of Fraxinus mandshurica Rupr. and Larix gmelinii (Rupr.) Kuzen. Litter on Nitrogen Mineralization in Dark Brown Soil of Northeast China

Abstract

The changes in soil nitrogen mineralization rate induced by litter input can determine the availability of nitrogen for plant growth in the soil. In forest ecosystems, the mixing of different species of litter can alter the chemical properties of the litter, ultimately affecting the rates of soil nitrogen transformation and cycling. In this study, litters with Fraxinus mandshurica Rupr. and Larix gmelinii (Rupr.) Kuzen. and mixed litter with Fraxinus mandshurica and Larix gmelinii were added to dark brown soil and incubated in the lab for 175 days at 25 °C. NH₄⁺-N and NO₃⁻-N contents and nitrogen mineralization rates were periodically measured to explore the effect of mixed litter addition on soil nitrogen mineralization. The results showed that compared to Larix gmelinii litter, Fraxinus mandshurica litter demonstrates higher carbon, nitrogen, and phosphorus contents while exhibiting lower lignin and cellulose contents and lower C/N and lignin/N ratios. Soil inorganic nitrogen content showed a trend of initial decrease followed by an increase. At the end of the incubation, soil NH₄⁺-N and NO₃⁻-N and the total inorganic nitrogen contents were 4.6–7.8 times, 2.2–3.4 times, and 2.9–4.3 times higher than the initial value, respectively. The soil nitrogen mineralization rate exhibited an initial rapid increase followed by stabilization. During days 7–28 of incubation, the nitrogen mineralization rates in litter addition treatments were lower than that in the control, while they were higher than that in the control during days 42–175. The soil nitrogen mineralization rate in the treatments with Fraxinus mandshurica litter and mixed litter were higher than those in the treatment with Larix gmelinii litter. The cumulative net nitrogen mineralization amounts in the Fraxinus mandshurica litter and mixed litter treatments were higher than those in the Larix gmelinii litter treatment, being 1.5 and 1.2 times those of the Larix gmelinii litter treatment, respectively. MBC and MBN presented a trend of first increasing and then decreasing, peaking on days 7 and 14 of incubation, respectively. Correlation analysis revealed that soil inorganic nitrogen content and nitrogen mineralization rate were positively correlated with the litter total nitrogen and soil microbial carbon and nitrogen and negatively correlated with litter C/N and lignin/N. The changes in soil inorganic nitrogen and nitrogen mineralization are primarily associated with soil microbial immobilization. Initially, in the treatments with litter addition, an increase in microbial biomass enhanced the immobilization of soil inorganic nitrogen. Subsequently, as litter mineralization progressed, the amount of litter decreased, leading to reduced microbial biomass and weakened immobilization. This study indicates that the interaction between litter types and soil microorganisms is the key factor affecting soil nitrogen mineralization process and soil mineral nitrogen content. These findings provide a scientific basis for soil fertility management in the forest ecosystems of Northeast China.

1. Introduction

As a major element of the matter cycle in terrestrial ecosystems, nitrogen (N) is also an essential nutrient for plant growth [1]. In forest ecosystems, soil N mainly comes from the decomposition of litter [2]. In the process of litter decomposition, most soluble compounds are leached firstly. Subsequently, the residues are fragmented by meso- and microfauna and decomposed by microorganisms, and ultimately organic nitrogen is converted into inorganic nitrogen that can be utilized by plants [3]. The biochemical process through which soil microorganisms convert organic nitrogen into plant-available inorganic forms is termed soil nitrogen mineralization. Therefore, the soil nitrogen mineralization induced by litter input directly affects the amount of soil inorganic nitrogen and determines the availability of nitrogen for plant growth in the soil [4]. For example, Xu et al. concluded that the removal of litter decreased total nitrogen and extractable inorganic nitrogen content in the soil, whereas the addition of litter had no effect in a meta-analysis [5]. Wang et al. [6] also found that the removal of litter decreased the net nitrogen mineralization rate of the soil in a Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) plantation, leading to a reduction in soil ammonium nitrogen content. However, the addition of litter increased both the net nitrogen mineralization rate and the soil ammonium nitrogen content. Therefore, the process in which litter input affects nitrogen mineralization in forest soils is of great significance for maintaining the productivity of forest ecosystems.

By changing soil nitrogen mineralization, litter quality and type can also affect the supply of available nitrogen in the soil [7,8,9]. Compared to broadleaf, coniferous litter generally decomposes more slowly in forest soils due to more lignin and cellulose [10], which can limit the effective supply of soil nitrogen. For example, compared to the addition of Schima superba litter, the ammonium nitrogen content was significantly lower in Chinese fir plantation soil with Chinese fir litter addition [11]. However, some researchers found different results. For instance, Chen et al. [12] found that the rates of soil nitrification, ammonification, and nitrogen mineralization increased in the soil of a Castanopsis platyacantha Rehder & E.H.Wilson.-Schima sinensis (Hance) Airy Shaw. forest with the addition of Cryptomeria fortunei Hooibrenk ex Otto & A.Dietr. litter. Therefore, the effects of litter addition from different tree species on soil nitrogen mineralization are currently inconsistent. The relationship between litter from different tree species and soil nitrogen mineralization needs to be further clarified.

In forest ecosystems, litter from different tree species is often mixed together. The litter mixture can increase nutrient heterogeneity and change the community structures and activities of soil fauna and microorganisms, thereby affecting the rates of soil nitrogen transformation and cycling [13]. For example, Wang et al. [13] found that the addition of peanut leaves and stems can increase the soil mineral nitrogen content in poplar (Populus × euramericana (Dode) Guinier. ‘Nanlin 95’) plantations. Gong et al. [14] also found that the addition of litter from natural forest can increase the rates of soil nitrification, ammonification, and nitrogen mineralization in Sassafras tzumu (Hemsl.) Hemsl. plantations and Cryptomeria fortunei plantations. Therefore, the addition of mixed litter can change the soil nitrogen mineralization process and affect the nitrogen availability in forests.

The forest area in Northeast China accounts for one-third of the total forest area in China and is an important timber production region in China [15]. Due to strong adaptability and rapid growth, Larix Mill. is naturally distributed in northeastern, northern, and southwestern China and Inner Mongolia. It is commonly used for large-scale artificial afforestation [16]. However, some research [17] has shown that Larix Mill. plantations can reduce N, P, K, and organic matter in the soil, leading to decreased soil fertility and hindering the development of Larix Mill. plantations. Fraxinus mandshurica Rupr., as a vital native tree species in northern China, is characterized by its rapid growth and strong adaptability. It is also an important precious timber species in the region [18]. It is often used as a mixed tree species in Larix gmelinii (Rupr.) Kuzen. plantations. When Larix gmelinii and Fraxinus mandshurica were planted together, the soil organic matter, total nitrogen, and ammonium nitrogen content increased in the plantation soil [19]. But, the control mechanism of the mixed litter from Fraxinus mandshurica and Larix gmelinii on soil nitrogen mineralization remains unclear. To explore the relationship between mixed litter decomposition and soil nitrogen mineralization, as well as analyze the factors affecting soil nitrogen mineralization after mixed litter addition, we added litters with Fraxinus mandshurica and Larix gmelinii and mixed litter with Fraxinus mandshurica and Larix gmelinii to dark brown soil for a 175-day constant-temperature incubation experiment. The hypotheses were as follows: (1) the mixed litter treatment exhibited a significantly higher soil nitrogen mineralization rate compared to the Larix gmelinii litter treatment; (2) the higher soil nitrogen mineralization rate observed in the mixed litter treatment was primarily attributed to the intrinsic properties of mixed litter, particularly its low lignin content and C/N ratio.

2. Materials and Methods

2.1. Study Site

This study was conducted at the Maoershan Experimental Station (127°30′–127°34′ E, 45°21′–45°25′ N) of Northeast Forestry University in Shangzhi City, Heilongjiang Province, China. The study area is characterized by low mountains and hills, with an average altitude of 300 m and slopes generally ranging from 6° to 15°. The climate in this area is a temperate continental monsoon, with an annual precipitation of 723 mm, which is mainly concentrated from June to August [20,21]. The annual average temperature is 2.8 °C, and the growing season ranges from 120 to 140 days [22]. The zonal soil in this area is dark brown soil, which is classified as Luvisols according to the international classification system (WRB 2014). It is generally distributed in the low mountainous and hilly regions above 300 m.

2.2. Collection and Preparation of Experimental Materials

Litter preparation: In this study, we selected a 33-year-old mixed plantation of Larix gmelinii and Fraxinus mandschurica. In the autumn of 2020, 8 litter traps were placed, and all traps were collected after 4 days. Sufficient fresh fallen leaves (four times the amount required for the incubation experiment) were separately packed into labeled plastic bags and transported back to the lab. In the lab, intact leaves with similar color and wilting degree were selected. The leaves were rinsed with distilled water to remove impurities and then spread out to air dry in a shaded place at 20–25 °C with 40%–60% humidity for 8 days. During the drying period, the leaves were turned every 2 days to ensure uniform desiccation. After drying, the litter was cut into 1 cm × 1 cm pieces to eliminate the interference of needle and broadleaf shapes and mimic the fragmentation effect of soil fauna in the early stage of decomposition.

Soil sample collection: Typical dark brown soil from natural forests was chosen for the incubation experiment. Soil samples at a depth of 0–10 cm were randomly collected from 15 points in the natural forest, and the collected soils were mixed into a composite sample and transported to the laboratory in the low-temperature insulated box. In the lab, stones and roots were removed from the soil samples, which were then thoroughly mixed, sieved through 2 mm mesh, and air-dried in a shady place. The initial organic carbon content of the soil was measured to be 106.19 g/kg, the total nitrogen content was 9.71 g/kg, the total phosphorus content was 1.20 g/kg, the pH was 6.03, and the maximum water-holding capacity was 120.76%.

2.3. Incubation Experiment

The aerobic incubation method was used in this experiment. Three treatments of litter addition were set up as follows: Fraxinus mandshurica litter (F, 6 g), Larix gmelinii litter (L, 6 g), and a mixed litter of Fraxinus mandshurica and Larix gmelinii (FL, 3 g + 3 g). Meanwhile, a control (Ctrl) without the addition of litter was established. Each treatment contained 24 replicates. A total of 400 g of air-dried soil was placed in an incubation bottle (the PET plastic bottle with an inner diameter of 10 cm and a height of 15 cm). Soil water content was regulated to 60% of the soil’s maximum water-holding capacity determined via the ring knife method by spraying deionized water. The bottles were then covered with plastic wrap containing circular micropores (1 mm in diameter, the number of micropores accounting for 10% of total surface area) to maintain oxygen availability. Then, the bottles were placed in a 25 °C constant-temperature incubator for a 7-day pre-incubation (to restore soil microbial activity). After the pre-incubation, the litter samples, cut into 1 cm × 1 cm pieces and weighed based on oven-dried weight, were placed into litter bags (13 cm × 13 cm, with a mesh size of 0.25 mm) and buried at a depth of approximately 11 cm from the top of the bottle for a 175-day mineralization incubation. During the incubation period, the soil water content was maintained at 60% of the maximum water-holding capacity by supplementing with water every 3 days using the gravimetric method, and the incubation temperature was kept at 25 °C. Soil and litter samples were taken at 0, 7, 14, 28, 42, 70, 112, and 175 days of incubation, a total of eight times. Three samples were randomly selected from each litter treatment and the control. The litter bags were retrieved from the incubation bottles, rinsed quickly to remove the soil and mycelium on the surface, dried to constant weight at 65 °C, and then accurately weighed. Subsequently, the chemical properties of the litter were determined. The soil sample from the incubation bottle was thoroughly mixed, sieved, and measured for microbial biomass, ammonium nitrogen, nitrate nitrogen, and so on.

2.4. Analytical Methods

Analytical methods of litter properties: After drying, the litter samples were ground using a ball mill (Retsch, MM400, Hann, Germany) and sieved through a 0.149-mm sieve. Total carbon (TC) and total nitrogen (TN) were analyzed using an elemental analyzer (Vario MACRO CN, Elementar, Langenselbold, Germany) [23]. The samples were digested using a concentrated sulfuric acid/hydrogen peroxide (H₂SO₄-H₂O₂) digestion method [23]. Total phosphorus (TP) was analyzed by using a flow injection analyzer (Seal, AA3, Madison, WI, USA). The Acid Detergent Fiber (ADF) method was selected to determine cellulose and lignin contents [24]. The initial chemical properties of the litter are shown in

Table 1. The initial chemical properties of the litter.

Litter	C (g/kg)	N (g/kg)	P (g/kg)	Lignin (%)	Cellulose (%)	C/N	Lignin/N
Fraxinus mandshurica	422.75	22.27	2.34	11.52	16.89	18.98	0.52
Larix gmelinii	357.68	10.08	1.75	11.54	24.42	35.55	1.14

.

Analytical methods of soil properties: The soil water-holding capacity was determined by using the ring knife method [25]. Five grams of fresh soil sample was weighed and placed into a 150 mL Erlenmeyer flask. Fifteen mL of 1 mol/L KCl solution was added for extraction, and the extract was analyzed using a flow injection analyzer (Seal, AA3) to determine the contents of nitrate nitrogen (NO₃⁻-N) and ammonium nitrogen (NH₄⁺-N) [26]. Two aliquots of fresh soil (40 g each) were weighed. One aliquot was subjected to chloroform fumigation (24 h at 25 °C in the dark), and the other served as a non-fumigated control. Subsequently, both samples were extracted with 0.5 mol/L K₂SO₄ solution. Soil microbial biomass carbon (MBC) and soil microbial biomass nitrogen (MBN) in the extracts were determined using a total organic carbon analyzer (multi N/C 2100, Analytik Jena, Jena, Germany) [27,28].

Calculation of soil nitrogen mineralization:

TIN (mg·kg⁻¹) = NH₄⁺-N (mg·kg⁻¹) + NO₃⁻-N (mg·kg⁻¹)

(1)

CNA (mg·kg⁻¹) = _afterNH₄⁺-N − _beforeNH₄⁺-N

(2)

CNN (mg·kg⁻¹) = _afterNO₃⁻-N − _beforeNO₃⁻-N

(3)

CNM (mg·kg⁻¹) = _afterNH₄⁺-N + _afterNO₃⁻-N − _beforeNH₄⁺-N − _beforeNO₃⁻-N

(4)

NAR = (_afterNH₄⁺-N − _beforeNH₄⁺-N)/t

(5)

NNR = (_afterNO₃⁻-N − _beforeNO₃⁻-N)/t

(6)

NMR = (_afterNH₄⁺-N + _afterNO₃⁻-N − _beforeNH₄⁺-N − _beforeNO₃⁻-N)/t

(7)

where TIN is the soil total inorganic nitrogen content, CNA is the soil cumulative net ammonification amount, CNN is the soil cumulative net nitrification amount, CNM is the soil cumulative net nitrogen mineralization amount. NAR is the net ammonification rate of the soil, NNR is the net nitrification rate of the soil, NMR is the net nitrogen mineralization rate of the soil. _afterNH₄⁺-N is the NH₄⁺-N content after incubation, _afterNO₃⁻-N is the NO₃⁻-N content after incubation, _beforeNH₄⁺-N is the NH₄⁺-N content before incubation, _beforeNO₃⁻-N is the NO₃⁻-N content before incubation, and t represents the number of days of incubation.

2.5. Statistical Analysis

The experimental data were organized and initially processed using Microsoft Excel 2021. Differences in NH₄⁺-N, NO₃⁻-N, soil total inorganic nitrogen, CAN, CNN, CNM, NAR, NNR, NMR, MBC, and MBN among soils were determined by one-way and multi-way analysis of variance followed by Least Significant Difference tests to compare means among soils. The correlation analysis was conducted using the Pearson’s two-tailed test. Significant differences were evaluated at the 0.05 level. All the statistical analyses were performed using IBM SPSS Statistics 26.0, and all the figures were drawn using Origin 2021.

3. Results

3.1. Soil Inorganic Nitrogen Under Different Litter Addition Treatments

The species of litter and incubation time significantly affected soil NH₄⁺-N, NO₃⁻-N, and inorganic nitrogen content (

Table 2. Two-way analysis of variance on soil inorganic nitrogen and soil nitrogen mineralization rate during the incubation period.

Factor	F Value
	NH4+	NO3−	TIN	NAR	NNR	NMR
Litter	54.78 *	164.92 *	188.30 *	3.02 *	9.12 *	10.02 *
Time	1598.08 *	1330.38 *	2197.34 *	164.01 *	120.98 *	186.59 *
Litter × Time	21.15 *	24.84 *	31.01 *	2.53 *	4.00 *	4.89 *

* Significant (p < 0.05). Litter denotes the main effect of litter treatment. Time signifies the main effect of the incubation time. Litter × Time represents the interaction effect between litter treatment and incubation time.

). During the incubation period, soil NH₄⁺-N, NO₃⁻-N, and total inorganic nitrogen contents of all treatments decreased initially, reached the lowest values on day 7, and then gradually increased and reached the maximum values at the end of the incubation (Figure 1). At the end of the incubation, soil NH₄⁺-N content was 4.6–7.8 times higher than the initial value, soil NO₃⁻-N content was 2.2–3.4 times higher than the initial value, and the total inorganic nitrogen content was 2.9–4.3 times higher than the initial value. From day 7 to 28 of incubation, the control treatment had the highest soil NH₄⁺-N, NO₃⁻-N, and total inorganic nitrogen contents. However, from day 42 to 175 of incubation, the contents of NO₃⁻-N and total inorganic nitrogen contents in the litter addition treatment were significantly higher than those in the control (p < 0.05). From day 70 to 175 of incubation, the contents of NH₄⁺-N, NO₃⁻-N, and total inorganic nitrogen in the Larix gmelinii litter treatment were significantly lower than those in the Fraxinus mandshurica and mixed litter treatments. (p < 0.05).

3.2. Soil Nitrogen Mineralization Rate Under Different Litter Addition Treatments

The species of litter and incubation time significantly affected soil net ammonification rate (NAR), net nitrification rate (NNR), and net nitrogen mineralization rate (NMR) (Table 2). During the first 42 days of incubation, NAR, NNR, and NMR all increased gradually with incubation time. Subsequently, NAR tended to stabilize (Figure 2a), while NNR and NMR decreased slightly at day 70 and then tended to stabilize (Figure 2b,c). From day 7 to 28 of incubation, the control treatment had the highest NAR, NNR, and NMR. On day 7 of incubation, the control was significantly higher than the mixed litter treatment (p < 0.05). From day 42 to 175 of incubation, the control treatment had the lowest NNR and NMR, which were significantly lower than those of the Fraxinus mandshurica litter and mixed litter treatments (p < 0.05). NAR in the control treatment was only the lowest on day 42 of incubation. From day 70 to 175 of incubation, NAR, NNR, and NMR in the Larix gmelinii litter treatment were significantly lower than those in the other litter treatments (p < 0.05).

3.3. Soil Cumulative Nitrogen Mineralization Amount Under Different Litter Addition Treatments

The species of litter affected the soil cumulative net ammonification (CNA), nitrification (CNN), and nitrogen mineralization (CNM) amount (p < 0.05). Among all treatments, the order of CNA was F > FL > Ctrl > L. The treatment with the Fraxinus mandshurica litter was significantly higher than the other treatments (p < 0.05), being 1.4 times that of SL, 1.4 times that of Ctrl, and 1.7 times that of L. The mixed litter treatment was significantly higher than the Larix gmelinii litter treatment (p < 0.05). CNN and CNM showed the same order: F > F L > L > Ctrl. The Fraxinus mandshurica litter treatment was also significantly higher than the other treatments (p < 0.05). CNN was 1.2 times that of FL, 1.4 times that of L, and 1.6 times that of Ctrl. CNM was 1.3 times that of FL, 1.5 times that of L, and 1.6 times that of Ctrl. The mixed litter treatment was significantly higher than the Larix gmelinii litter treatment and the control treatment (p < 0.05). CNN was 1.2 times that of L and 1.3 times that of Ctrl. CNM was 1.2 times that of L and 1.2 times that of Ctrl (

Table 3. Soil cumulative net nitrogen mineralization under four different treatments.

Indicator	Ctrl	F	L	FL
CNA	108.45 ± 1.18 B	152.09 ± 2.8 A	89.85 ± 2.36 C	110.75 ± 5.34 B
CNN	166.65 ± 4.26 C	273.18 ± 5.1 A	191.73 ± 7.54 C	223.31 ± 14.92 B
CNM	275.1 ± 4.24 C	425.26 ± 7.33 A	281.58 ± 9.48 C	334.06 ± 19.41 B

Data in the table are mean ± standard error. Different letters mean significant differences among different treatments in the same index at a 0.05 level.

).

3.4. Soil Microbial Biomass Carbon and Nitrogen Under Different Litter Addition Treatments

During the incubation period, soil microbial biomass carbon (MBC) in all treatments showed similar trends (Figure 3a). MBC increased rapidly and reached its peak on day 7 of incubation. Then, MBC decreased rapidly from day 7 to 42. It increased slightly on day 70 (except for Ctrl). Finally, it declined slowly from day 112 to 175 and reached its minimum value at the end of the incubation. Except for day 14 and day 112, MBC followed the order of F > FL > L > Ctrl. Specifically, on day 7 of incubation, the treatments with litter addition were significantly higher than the control (p < 0.05), being 1.5 times (F), 1.3 times (FL), and 1.2 times (L) that of the control, respectively. The change in soil microbial biomass nitrogen (MBN) in the control was consistent with that of MBC. However, the treatments with litter addition showed different trends, reaching the maximum values on day 14 (Figure 3b). On day 14 of incubation, the Fraxinus mandshurica litter treatment and the mixed litter treatment were significantly higher than the control (p < 0.05), being 1.2 times (F) and 1.1 times (FL) that of the control, respectively. After day 14 of incubation, the control was consistently lower than the litter addition treatments, and the Larix gmelinii litter treatment was also consistently lower than both the Fraxinus mandshurica litter and the mixed litter treatments.

3.5. Factors Affecting Soil Inorganic Nitrogen and Net Nitrogen Mineralization

Across incubation dates, the correlation analysis among litter N, litter lignin, litter cellulose, litter lignin/N, litter C/N, soil microbial biomasses, soil mineral nitrogen contents, and net nitrogen mineralization rates showed similar results. Especially on day 175 (

Table 4. The chemical properties of the litter after incubation.

Litter	N (g/kg)	Lignin (%)	Cellulose (%)	C/N	Lignin/N
Fraxinus mandshurica	30.31	28.08	6.02	13.54	9.27
Larix gmelinii	12.36	48.59	17.81	33.18	39.39
mixed litter	22.94	39.56	13.81	17.47	17.25

and

Table 5. Correlation analysis of soil inorganic N and N mineralization rate.

	MBC	MBN	N	Lignin	Cellulose	Lignin/N	C/N
NH4+	0.896 **	0.825 **	0.946 **	−0.949 **	−0.953 **	−0.874 **	−0.839 **
NO3−	0.868 **	0.835 **	0.940 **	−0.942 **	−0.909 **	−0.873 **	−0.843 **
TIN	0.883 **	0.834 **	0.946 **	−0.949 **	−0.924 **	−0.877 **	−0.844 **
NAR	0.912 **	0.833 **	0.942 **	−0.954 **	−0.945 **	−0.870 **	−0.833 **
NNR	0.869 **	0.828 **	0.896 **	−0.891 **	−0.837 **	−0.834 **	−0.805 **
NMR	0.869 **	0.838 **	0.924 **	−0.927 **	−0.891 **	−0.858 **	−0.825 **

** Extremely significant (p < 0.01).

), soil NH₄⁺-N, NO₃⁻-N, inorganic nitrogen content, NAR, NNR, and NMR were all positively correlated with TN in litter, soil MBC, and MBN (p < 0.05) and negatively correlated with lignin, cellulose, lignin/N, and C/N in litter (p < 0.05).

4. Discussion

4.1. Effects of Litter Addition on Soil Inorganic Nitrogen

In this study, nitrate nitrogen was the predominant form of inorganic nitrogen, accounting for 71% to 77% of the inorganic nitrogen in the soil of natural forests in Northeast China, which is consistent with other research [29,30]. During the incubation period, the changing trends of NH₄⁺-N, NO₃⁻-N, and total inorganic nitrogen content in the litter addition treatments were consistent, which initially decreased and then increased. The reason is that with the addition of litter, a large amount of carbon enters the soil, which stimulates the rapid growth and reproduction of soil microorganisms, thus resulting in a short-term surge in microbial biomass [31]. However, since the nitrogen amount provided by the litter cannot meet the growth needs of the microorganisms, they need to absorb and immobilize the inorganic nitrogen in the soil to maintain the carbon/nitrogen balance of their own growth and reproduction, leading to a significant decrease in the soil inorganic nitrogen content for some time after the addition of litter [30]. The increasing trend in microbial carbon and nitrogen contents during the early stage validated this view (Figure 3). Meanwhile, Bonanomi et al. [32] also demonstrated that organic amendments with high carbon but low nitrogen content can rapidly immobilize soil nitrogen in the short term. This also explains why the contents of NH₄⁺-N, NO₃⁻-N, and total inorganic nitrogen content in the litter addition treatment were lower than those in the control in the early stages of incubation. However, as the mineralization of litter progress, carbon is continuously released and reduced. The amount of immobilized inorganic nitrogen decreases, and the soil mineral nitrogen content gradually increases. These results aligned with the findings of Gong [14] and Ge [30].

In the later weeks of incubation, the inorganic nitrogen content in the Larix gmelinii litter treatment was significantly lower than that in the Fraxinus mandshurica litter treatment and the mixed litter treatment. This may be attributed to the higher C/N and lignin/N ratios of Larix gmelinii litter, making it more recalcitrant to decomposition compared to other types of litter. Consequently, after adding an equivalent amount of litter, it takes a longer time for the soil to return to the normal C/N ratio, and microorganisms immobilize the soil inorganic nitrogen for a longer period. As a result, the inorganic nitrogen content in the soil treated with Larix gmelinii litter was lower than that in the other litter treatments. Correlation analysis between the chemical properties of the litter and soil inorganic nitrogen content revealed a significant positive correlation between soil inorganic nitrogen and total nitrogen in the litter, as well as significant negative correlations with lignin, lignin/N, and C/N, supporting the aforementioned argument (Table 5). Furthermore, Hadas [33] suggested that the cellulose-like pool with a moderate decomposition rate has a significant impact on soil available nitrogen concentrations in the short and medium term. Specifically, a higher proportion of the cellulose-like pool enhances the immobilization of soil inorganic nitrogen by microorganisms. The correlation analysis in this study also demonstrated a significant negative relationship between cellulose and inorganic nitrogen content (Table 5), which aligned with the findings of the aforementioned research.

4.2. Effects of Litter Addition on Soil Nitrogen Mineralization

In the early stages of incubation, the net ammonification rate, net nitrification rate, and net nitrogen mineralization rate of all treatments were negative and lower than those of the control. The reason is that the addition of litter stimulated microbial growth (Figure 3a), which led to immobilization of more soil inorganic nitrogen [34]. Compared to the litter addition treatment, the control exhibited lower microbial biomass, leading to reduced soil nitrogen immobilization and consequently a higher nitrogen mineralization rate. As the incubation progressed, the carbon provided by the litter gradually decreased, and microbial biomass also declined (Figure 3a), leading to a corresponding decrease in the demand for inorganic nitrogen. Correlation analysis also showed a significant positive relationship between microbial biomass carbon and nitrogen and soil nitrogen mineralization rates (Table 5), which aligns with other findings that nitrogen mineralization generally increases with higher microbial biomass carbon and nitrogen [35,36].

This study found that soil nitrogen mineralization rate was significantly positively correlated with litter total nitrogen content and significantly negatively correlated with lignin, lignin/N, and C/N (Table 5), which was consistent with the following: lower litter quality (high lignin/N ratio) could limit nitrogen mineralization, and nitrogen mineralization would increase rapidly when the lignin/N ratio decreases to a relatively low value [37]. In the later stages of cultivation, the soil net ammonification rate, net nitrification rate, and net nitrogen mineralization rate in the treatment with Larix gmelinii litter addition were significantly lower than those in the treatments with Fraxinus mandshurica and mixed litter addition. This can be attributed to the lower nitrogen content and higher lignin/N and C/N ratio [38,39,40] of Larix gmelinii litter. The reason is that soil inorganic nitrogen is more limited than carbon. When fresh organic matter of different qualities is input, the efficiency and total amount of C provided to soil microorganisms are also different, which affects the competition of microbial populations for nutrients, especially nitrogen [41]. When the C/N ratio of the input litter is relatively low, the nitrogen available to microorganisms is relatively sufficient, resulting in less immobilization of soil inorganic nitrogen. Conversely, when nitrogen is scarce, the mineralized nitrogen is rapidly immobilized by the microorganisms themselves [42].

The cumulative net nitrification and cumulative net nitrogen mineralization of soil treated with total Larix gmelinii litter addition in this study were not significantly different from those of control but significantly lower than those of mixed litter and Fraxinus mandshurica litter addition, which indicated that Larix gmelinii litter addition had little effect on soil nitrogen supply capacity, which was consistent with Liu et al. [43], who observed no significant difference in net nitrogen mineralization between Pinus massoniana plantations and bare land. However, mixing Larix gmelinii and Fraxinus mandshurica litter significantly improved soil inorganic nitrogen supply, suggesting that mixed plantations of Larix gmelinii and Fraxinus mandshurica can increase soil inorganic nitrogen content and forest productivity. This is in line with the results of Wu et al. [44], who found that mixed coniferous and broadleaf forests generally increase nitrogen supply capacity in the rhizosphere soil of coniferous trees.

5. Conclusions

In this study, the key findings include the following: The soil nitrogen mineralization rate and cumulative net nitrogen mineralization amount in the treatments with Fraxinus mandshurica litter and mixed litter were higher than those in the treatment with Larix gmelinii litter. This suggests that mixing Fraxinus mandshurica and Larix gmelinii can promote the soil nitrogen mineralization and increase the inorganic nitrogen content in the soil and improve soil fertility, which has the potential to promote plant growth. Correlation analysis showed that soil mineral nitrogen content and nitrogen mineralization rate were positively correlated with litter total nitrogen, microbial carbon, and nitrogen contents and negatively correlated with litter C/N and lignin/N, which indicated that the soil nitrogen cycle was regulated by litter chemical properties and the soil microbial community. These suggest that the addition of Fraxinus mandshurica litter and the mixed litter with Fraxinus mandshurica and Larix gmelinii can enhance soil fertility by improving the nitrogen supply and regulating the soil microbial community. These changes can improve soil quality and promote the growth of forest trees.