Extracellular Enzyme Patterns Provide New Insights Regarding Nitrogen Transformation Induced by Alkaline Amendment of Acidic Soil

Abstract

Nitrogen (N) availability is generally a limiting factor in highly acidic soil, which could be improved by amending these soils with alkaline materials. Soil extracellular enzyme activity (EEA) plays an important role in N transformation; a current knowledge gap is how this occurs in acidic soils amended with alkaline material. The present 45-day incubation experiment was designed to examine the effects of different amounts of alkaline materials (urea and/or calcium–silicon–magnesium–potassium fertilizer (CSMP)) on N transformation. The results show that soil pH significantly increased after the CSMP amendment (1.2 units) and increased soil net N mineralization (R_min), net nitrification (R_nit) rates, and net ammonification (R_amo) rates. CSMP amendment changed the different soil EEA but with differing or opposing effects, e.g., R_nit was positively correlated with the activities of L-leucine aminopeptidase, β-xylosidase, α-glucosidase, and N-acetyl-β-glucosaminidase but negatively correlated with β-1, 4-glucosidase and β-cellobiosidase. A machine learning analysis indicated that the best predictor for R_min and R_amo was soil pH, and for R_nit, it was nitrate. The results of the present study improve our understanding of N availability in acidic soils amended with materials to control soil pH. Such knowledge could lead to more bespoke nutrient management planning at the field scale, leading to better agronomic and environmental outcomes.

1. Introduction

Approximately 40% of the world’s arable soils are acidic (i.e., pH < 5.5), and this has continued to increase in recent years [1,2,3]. There is widespread concern about acidified arable land [4], because soil acidification depletes soil fertility, and thus reduces crop productivity and nitrogen (N) supply [5]. N mineralization and nitrification are two of the main reactions related to soil N supply [6]. It was widely believed that nitrification was relatively low in acidic soils since the availability of the substrate ammonia (NH₃) for the ammonia monoxygenase enzyme of ammonia oxidizers would be limited [7,8]. Therefore, how to increase the N turnover in acidic soil has been the subject of considerable attention [9,10,11].

The application of alkaline amendments to acidic soils is a common approach to alleviate soil acidification by increasing pH and exchangeable base cation levels [12,13]. Recently, some researchers reported that alkaline slag (e.g., calcium–silicon–magnesium–potassium (CSMP) fertilizer) has been found to be a successful alternative to gypsum and lime for mitigating soil acidity and improving crop productivity [14,15]. Combining N fertilization with lime has been intensively proposed as a promising approach for increasing soil N use efficiency [16]. However, more convincing evidence is required to understand the effect of alkaline amendment on nitrogen transformation in acidic soils.

The organic form of N constitutes the majority of N in soil [17]. After organic N has been mineralized to inorganic N, it can be easily assimilated by plants [18]. As a result, the rate of organic N mineralization is a critical factor affecting ecosystem productivity [19]. Depolymerization, which breaks down large N-containing polymers into monomers such as amino acids and amino sugars, is an important process in N mineralization [20]. The next step is to convert those monomers to ammonium (NH₄⁺-N) through ammonification [20]. Soil extracellular enzymes derived from microorganisms, such as N-acetyl-β-glucosaminidase (NAG) and L-leucine aminopeptidase (LAP), participate in a series of biochemical reactions that catalyze the degradation of complex compounds [21]. Soil extracellular enzymes may not only affect the production of NH₄⁺-N in soil but also further affect the nitrification of NH₄⁺-N as a substrate. In addition, labile organic carbon (OC) is also a key factor affecting N transformation, and the changes in soil carbon (C) substrates can influence soil mineral N dynamics [22]. Evidence from field experiments with varied soil pH suggests that low soil pH limits microbial growth and extracellular enzyme activities [23]. It has been reported that the activities of phenol oxidase and dehydrogenase were increased by the addition of alkaline amendments, while catalase and urease activity was decreased [13]. A more comprehensive assessment of extracellular enzyme activity (EEA) may help to further understand N transformation under alkaline amendment.

In this study, net N mineralization and nitrification rates and the activities of six soil enzymes involved in C and N cycling were examined in highly acidic soil with different amounts of CSMP and urea treatments. It was hypothesized that the net N mineralization and nitrification rates of the acidic soil would be accelerated due to the shift in EEA after CSMP amendments.

2. Materials and Methods

2.1. Soil and Alkaline Mineral Amendment Properties

Topsoil (0–20 cm) was collected from a paddy field (27°49′53″ N, 113°24′55″ E) in Zhuzhou City, Hunan province, China. The field is located in a subtropical humid monsoon climate zone, with annual rainfall ranging from 1200 to 1600 mm and an annual average temperature of 18 °C. The soil has an organic carbon content of 26.6 g kg⁻¹, a pH of 4.80, and total nitrogen of 2.42 g kg⁻¹. Further information on soil parameters can be found in Table S1. For the incubation experiments, the sampled soil was sieved through a 2 mm mesh and air-dried. Oven-dried soil mass was used for all measurements and calculations in this study.

The alkaline mineral amendment calcium–silicon–magnesium–potassium (CSMP) fertilizer, which is a mixture of phosphorus tailings and insoluble potassium-containing rocks, was obtained from a commercial source (Kingenta Ecological Engineering Group Co., Ltd., Linyi, China). The acid buffering capacity of the amendment was 618 kg t⁻¹ with a pH of 10.3. Table S2 contains details of the chemical composition of the amendment.

2.2. Soil Incubation Experiment

The soil microcosm was established in 0.7 L cylindrical incubation containers (80 mm diameter × 88 mm height) with 100 g of soil samples. The incubation experiment consisted of six treatments (three replicates each): (i) control (CK, no mineral amendments or urea); (ii) urea at 60 mg N kg⁻¹ soil (U); (iii) 1 g CSMP kg⁻¹ soil (1 CSMP); (iv) urea at 60 mg N kg⁻¹ + 1 g CSMP kg⁻¹ soil (U+1 CSMP); (v) 10 g CSMP kg⁻¹ soil (10 CSMP); and (vi) 60 mg N kg⁻¹ + 10 g CSMP kg⁻¹ soil (U+10 CSMP). The moisture content (60% of the water holding capacity (WHC)) was replenished every 2 to 3 days by adding deionized water. The containers were covered with perforated parafilm and incubated at 25 °C for 45 days in the dark. Afterwards, the soil pH, mineral N content, and enzyme activities were measured on day 2, 7, 15, 30, and 45.

2.3. Physicochemical Analysis

The soil pH was measured using a pH meter (MP522 version 3, SANXIN, China) with a 10 g: 25 mL dry soil: distilled water slurry. Soil nitrate (NO₃⁻-N) and NH₄⁺-N concentrations (extracted with 1M KCl) were measured via a continuous flow analyzer (AA3, Seal Analytical, Norderstedt, Germany). Soil was extracted using a 0.5 M K₂SO₄ solution for dissolved organic carbon (DOC) analysis, and the extract was filtered through a 0.45 μm membrane and measured using a TOC/TN analyzer (TOC-VCSH, Shimadzu, Kyoto). The N transformation rates were based on the proportion of NO₃⁻ and NH₄⁺ using the following Equations (1)–(3) [24]:

R_min = [(NH₄⁺-N_i+1 − NH₄⁺-N_i) + (NO₃⁻-N_i+1 − NO₃⁻-N_i)]/(t_i+1 − t_i)

(1)

R_nit = (NO₃⁻-N_i+1 − NO₃⁻-N_i)/(t_i+1 − t_i)

(2)

R_amo = (NH₄⁺-N_i+1 − NH₄⁺-N_i)/(t_i+1 − t_i)

(3)

where R_min and R_nit are soil net N mineralization and net nitrification rates, respectively; t_i and t_i+1 are the beginning and end dates of each incubation period, respectively. NH₄⁺-N_i, NO₃⁻-N_i and NH₄⁺-N_i+1, NO₃⁻-N_i+1 were the concentrations of soil NH₄⁺-N and NO₃⁻-N in the initial and incubated samples, respectively.

Soil extracellular enzyme activities were measured with fluorogenically labeled artificial substrates according with a multimode reader (Varioskan flash, Thermo Scientific, Waltham, MA, USA) [25]. Fluorogenic 4-methylumbelliferone (MUB)-based substrates were used to determine the activities of β-1,4-glucosidase (BG), β-cellobiosidase (CBH), N-acetyl-β-glucosaminidase (NAG), β-xylosidase (XYL), and α-glucosidase (AG). Fluorogenic 7-amino-4-methylcoumarin (AMC)-based substrate was used to determine the activity of L-leucine aminopeptidase (LAP).

2.4. Statistical Analysis

Statistical analysis was conducted using Excel 2016 and SPSS 20.0 (IBM, USA). Analysis of variance (ANOVA) was used to verify the differences between the means in the results. Prior to ANOVA, all variables were tested for normality, and data were log transformed as needed. The Least Significant Difference (LSD) test was used to evaluate the significance of the results, and all statistical tests were performed with a significance level of p < 0.05. SigmaPlot (version 12.5) and R (version 4.1.3) were used to draw graphs. The best predictors were identified using a machine learning approach implemented in the randomForest package. Random forest regression uses bootstrap sampling (bootstrap sampling; 500 trees; random seed = 123) [26].

3. Results

3.1. Soil pH

As shown in Figure 1, the soil pH of each treatment increased in the first 15 days and decreased thereafter. During the first 30 days, the soil pH in the treatments with urea application (U, U+1 CSMP, and U+10 CSMP) was higher than in the corresponding treatments without urea application (CK, 1 CSMP, and 10 CSMP), especially the pH of the U treatment was significantly higher than CK (p < 0.01). In comparison to CK and U, the low amount (1 g kg⁻¹) of amendment (1 CSMP and U+1 CSMP) had small effects on soil pH, while the high amount (10 g kg⁻¹) of amendment (10 CSMP and U+10 CSMP) significantly increased the soil pH to >6 (p < 0.05).

3.2. Soil Mineral Nitrogen

In all treatments, concentrations of soil NH₄⁺-N significantly increased for the first 15 days and then decreased until the end of the incubation period (Figure 2). The application of urea increased soil NH₄⁺-N concentrations. With the addition of amendment at 10 g kg⁻¹ (10 CSMP), the NH₄⁺-N content increased in the first 15 days and then significantly decreased compared to the other treatments (p < 0.05). The same result occurred in the addition of amendment at 1 g kg⁻¹ (CSMP), but its effect was far less than 10 CSMP.

The effects of different amendment dosages on the change in NO₃⁻-N concentration in the acidic soil are shown in Figure 2. During the whole incubation period, the soil NO₃⁻-N content of each treatment showed no significant change in the first seven days. The soil NO₃⁻-N content began to increase continuously on day 15 with the addition of amendment at 10 g kg⁻¹ treatment (10 CSMP and U+10 CSMP), while the NO₃⁻-N contents of other treatments increased significantly on day 30 (p < 0.05). After 45 days of incubation, the accumulation of NO₃⁻-N in 10 CSMP (163 mg N kg⁻¹) and U+10 CSMP (220 mg N kg⁻¹) treatments increased by 4.58 and 2.75 times compared with CK (29.3 mg N kg⁻¹) and U (58.7 mg N kg⁻¹), respectively.

3.3. Dissolved Organic Carbon

The DOC contents of soils were influenced by the CSMP addition of 10 g kg⁻¹ and fluctuated over the course of the incubation period (Figure 3). In comparison with CK (609 mg kg⁻¹) and U (675 mg kg⁻¹), the DOC content was significantly decreased to 380 mg kg⁻¹ in 10 CSMP and 346 mg kg⁻¹ in U+10 CSMP treatments on day 7 (p < 0.05). On day 15, the DOC contents were significantly higher in the 10 CSMP (434 mg kg⁻¹) and U+10 CSMP (483 mg kg⁻¹) treatments than in the CK (304 mg kg⁻¹) and U (293 mg kg⁻¹) treatments (p < 0.05).

3.4. Soil Extracellular Enzyme Activity

The activities of extracellular enzymes NAG and LAP, which are involved in organic N decomposition, were significantly increased in the 10 CSMP and U+10 CSMP treatments compared to CK (p < 0.05) (Figure 4). Both NAG and LAP activities peaked on day 45 and day 15, respectively.

The activities of extracellular enzyme involved in the OC decomposition were influenced by soil amendment, but trends relating to BG and CBH activities were not consistent. For example, the addition of 10 g kg⁻¹ CSMP showed negative effects on BG and CBH activities. The activities of BG and CBH were lower in the 10 CSMP and U+10 CSMP treatments than in the other treatments, especially with 2 days of incubation, and the effect decreased with incubation days. In addition, the XYL and AG activities were significantly increased in the 10 CSMP and U+10 CSMP treatments compared with CK (p < 0.05).

3.5. Soil Nitrogen Transformation Rate

The effects of soil amendment on R_min, R_nit, and R_amo are shown in Figure 5. In the whole incubation period, R_min and R_amo decreased first and then stabilized with the increase in incubation time. The difference between treatments was mainly manifested between day 2 and 7, in which the R_min of the U+10 CSMP treatment was the highest (17.9 mg N kg⁻¹ d⁻¹), followed by the 10 CSMP treatment. The results show an upward trend in the R_min and R_amo of the corresponding N application treatments compared with the three treatments without urea application. The R_nit of the 10 CSMP and U+10 CSMP treatments were higher than those of the other treatments, and the effects peaked on day 30 of incubation. The R_nit of the other treatments, although only slight, increased continuously throughout the incubation period.

The contributions of soil variables to the dissimilarities of the N transformation rate are shown in Figure 6. The R_nit (91% explanation) showed stronger environmental associations than R_min (57.5% explanation) and R_min (67.3% explanation). Soil pH, NO₃⁻−N, NH₄⁺-N, and AG activity were strong predictors of R_min and R_amo. R_nit was mainly explained by soil NO₃⁻-N concentration and LAP activity, with a significant positive relationship. In addition, R_nit was significantly positively correlated with the activities of LAP (R = 0.770 **), XYL (R = 0.605 **), AG (R = 0.569 **), and NAG (R = 0.533 **), and was significantly negatively correlated with BG (R = −0.601 **) and CBH (R = −0.515 **).

4. Discussion

4.1. Nitrogen Transformation Regulation by Alkaline Amendment

In this study, the soil pH of all treatments increased initially and then decreased (Figure 1). The increase in soil pH in the initial period of incubation could be due to the combined effect of organic N mineralization and decarboxylation of organic anions [27], and then pH decreased due to the release of H⁺ by soil nitrification and the buffering effect of soil [28]. Compared with CK, 10 CSMP significantly increased the soil pH from 4.8 to more than 6.0, which confirmed the positive effect of amendment addition in reducing soil acidity (Figure 1). The basic cations were found to be the main contributor to soil pH increases, and their concentrations were significantly correlated with pH [29]. Composed of potassium gypsum, dolomite and calcium magnesium silicate minerals carrying salt-based ions, CSMP amendments can decrease exchangeable acids and reduce soil acidity [30]. In addition, it contains large amounts of carbonates and silicates that can neutralize H⁺ and Al³⁺ in soil by hydrolysis or specific adsorption and increase soil pH [31]. The improved effect of amendments on soil acidity was also related to the application amounts [32]. The results show that a lower dosage of amendment (1 g kg⁻¹) had no significant effect on soil pH. A field experiment showed that, when the amount of amendment is low, it needs to be applied repeatedly to significantly decrease soil acidity [33].

On the first 7 days of the whole incubation, a 10 g kg⁻¹ CSMP application could significantly promote the accumulation of NH₄⁺-N in acidic soil, especially when it was combined with urea (Figure 2). At the same time, the 10 g kg⁻¹ CSMP treatment was able to significantly improve R_min and R_amo, but had little effect on R_nit (Figure 5). The mineralization of soil organic N may be the primary source of N transformation in early incubation. Furthermore, the hydrolysis of urea accelerates with the alkaline amendment. As the incubation went on, it could be observed that the R_nit under 10 g kg⁻¹ CSMP application increased rapidly, and the peak value was significantly higher than that of CK (Figure 5). At the same time, the NH₄⁺-N content increased significantly and the NO₃⁻-N content decreased significantly (Figure 2). This result may be due to the 10 CSMP treatment increasing the R_amo, thereby increasing the NH₄⁺-N content in the soil, thus providing a reaction substrate for soil nitrification.

4.2. N Transformation and EEA

A protease LAP can release amino compounds by the hydrolysis of proteins [34]. During the whole incubation period, the LAP activity of the 10 CSMP treatment was significantly higher than that of CK, indicating that 10 CSMP can promote the hydrolysis of macromolecular organic N. At the same time, 10 CSMP also significantly increased the NAG activity in acidic soil, and its activity increased as incubation time went on. NAG is involved in the degradation of amino acids and is one of the ways for microorganisms to obtain N sources [35]. It indicates that 10 CSMP promoted the stepwise mineralization of macromolecular organic N to mineral N in soil by increasing the activities of LAP and NAG. The increase in LAP and NAG activities by 10 CSMP provides a substrate for nitrification.

The activities of enzymes mainly related to C decomposition is very important to maintain labile C and energy sources in soil and is closely related to the abundance of microorganisms [36]. The DOC contents were significantly influenced by 10 g kg⁻¹ CSMP application (Figure 3); the decrease and increase effect may be due to the macromolecule OC polymerization and DOC decomposition. The results of this study show that 10 CSMP significantly increased the activities of AG and XYL in soil, while it significantly decreased the activities of BG and CBH. The role of BG and CBH in soil were to catalyze the decomposition of cellobiose and cellulose, respectively [37]. A similar result reported by [23] shows a decrease in BG activity in response to increased soil pH. In general, microorganisms give priority to utilize easily decomposed C sources in soil [38]. For example, 10 CSMP may promote the increase in available C sources for microorganisms, resulting in the decrease in cellulose utilization and enzyme secretion, thereby reducing related enzyme activity. In addition, increased soil pH may often enhance the solubility and degradability of soil organic matter (SOM) by increasing the number of negative charges in the soil components and the repulsive forces between molecules [39]. Substrate diffusion, an important factor regulating the reaction rates between extracellular enzymes and organic substrates, would be less limited with increased SOM solubility, resulting in a smaller investment of resources from soil microorganisms to extracellular enzymes [23]. It is known that AG and XYL release glucose by participating in the metabolic hydrolysis of starch, glycogen, and xylan, respectively. The results herein suggest that the utilization of soil C was achieved by secretion of AG and XYL, thereby increasing microbial activity. This process mainly affected N transformation but had no significant effect on R_nit. The mineralization of soil organic N may be the primary source of N transformation in early incubation. As the incubation went on, it could be observed that the R_nit of the 10 CSMP treatment increased rapidly, and the peak value was significantly higher than that of CK. At the same time, the NH₄⁺-N content increased significantly and the NO₃⁻-N content decreased significantly. This result may be due to the 10 CSMP treatment increasing the R_min, thereby increasing the NH₄⁺-N content in the soil, thus providing a reaction substrate for soil nitrification.

5. Conclusions

The present study shows that 10 g kg⁻¹ CMSP amendment increased soil pH, which in turn accelerated the N supply and maintained the mineral N content in acidic soil. Soil mineralization, ammonification, and nitrification rates increased with increased NAG, ALP, XYL, and AG, and decreased BG and CBH under the 10 g kg⁻¹ CMSP amendment. Such knowledge should inform N availability in acidic soils and aid growers with respect to better agronomic and environmental outcomes.