Variation in Soil Extracellular Enzyme Activities and Stoichiometry During Biological Soil Crust Formation in the Loess Plateau

YAO Hongjia¹, WANG Baorong²,³, AN Shaoshan¹,², YANG E'nv¹, HUANG Yimei⁴

¹State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Northwest A&F University, Yangling 712100, Shaanxi, China
²State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling 712100, Shaanxi, China
³University of Chinese Academy of Sciences, Beijing 100049, China
⁴Key Laboratory of Plant Nutrition and the Agro-Environment in Northwest China, Ministry of Agriculture, College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, Shaanxi, China

Abstract

Biological soil crusts (BSCs) play an important role in enhancing soil resistance to water and wind erosion and improving soil nutrient conditions. Soil extracellular enzyme activity serves as a microbial indicator of soil biochemical reaction intensity, which is crucial for understanding nutrient cycling processes involving microorganisms in desert ecosystems. This study investigated soils from five biological crust development stages (bare sand, algal crust, algal-moss mixed crust, moss-algal mixed crust, and moss crust) in the Liudaogou watershed of Shenmu County on the Loess Plateau to characterize the variation in soil extracellular enzyme activities [β-1,4-glucosidase (BG), β-1,4-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and alkaline phosphatase (AP)] and their stoichiometry during biocrust formation. The results demonstrated that: (1) The activities of all four extracellular enzymes increased significantly along the biological crust developmental sequence, with moss crust soils exhibiting significantly greater activity than algal crust soils (P<0.05). (2) Extracellular enzyme activities were significantly higher in the BSC layer than in underlying soil layers, decreasing continuously with soil depth. (3) Nutrient contents and soil extracellular enzyme activities in crust-covered soils were significantly higher than in bare sand, with moss crust soils showing significantly higher values than algal crust soils. (4) Correlation analysis revealed that soil C, N, P, C:P, and N:P were all extremely significantly positively correlated with soil extracellular enzyme activities (P<0.01). (5) Standardized major axis estimation indicated that soil extracellular enzyme activities were significantly enhanced and exhibited homeostatic characteristics with BSC development. The slopes between N- and P-acquiring enzymes relative to C-acquiring enzymes showed isometric relationships, suggesting that soil microorganisms play an important role in nutrient cycling in arid and semi-arid ecosystems through homeostatic regulation of extracellular enzyme stoichiometry.

Keywords: biological soil crusts; developmental sequence; extracellular enzymes; stoichiometry; Loess Plateau

Introduction

Biological soil crusts (BSCs) are organic complexes formed by cyanobacteria, fungi, algae, lichens, mosses, and soil particles, covering approximately 12% of the global terrestrial surface area. They enhance soil aggregation and stability, improve soil aeration and porosity, promote vascular plant establishment, and increase microbial community relative abundance, thereby playing crucial ecological roles in desert ecosystems. Through microbial photosynthetic carbon fixation, BSCs improve soil quality and contribute significantly to global soil carbon cycling. As BSCs develop, the complexity of soil substrates increases, promoting enzyme substrate supply and enhancing soil enzyme activities.

Soil extracellular enzymes, primarily derived from the decomposition and release of soil fauna, flora, and microorganisms, degrade high-molecular-weight organic compounds into assimilable molecules, playing vital roles in regulating soil organic matter degradation and nutrient cycling. Soil enzyme stoichiometry refers to the ratios of enzyme activities involved in nutrient element cycling within ecosystems, including "C-acquiring enzymes" (such as hemicellulase, cellulase, and glucosidase), "N-acquiring enzymes" (such as urease, chitinase, and peptidase), and "P-acquiring enzymes" (such as phosphatase). The most common extracellular enzymes involved in C, N, and P cycling include β-1,4-glucosidase, β-1,4-N-acetylglucosaminidase, leucine aminopeptidase, and phosphatase.

Soil enzyme ecological stoichiometry reflects the biogeochemical equilibrium between microbial metabolic processes and nutrient demands, providing a theoretical framework for understanding soil nutrient limitation and cycling dynamics. Previous research has demonstrated that soil extracellular enzyme activities gradually decrease during desert grassland desertification. However, the effects of BSC formation on soil extracellular enzyme activities and stoichiometric characteristics remain poorly understood. During BSC development, soil properties and microbial communities exhibit coupling and synergistic changes. Because BSCs exist at the atmosphere-soil interface, microorganisms are more susceptible to disturbance, necessitating consideration of microbial homeostasis when studying extracellular enzyme activities.

BSCs have become widespread surface covers following the Grain for Green Project on the Loess Plateau, comprising various types including algal, lichen, moss, and mixed crusts. While numerous studies have documented the effects of BSC types and succession on soil nutrients, how BSCs regulate soil extracellular enzyme activities and physicochemical properties to influence enzyme stoichiometric characteristics requires further investigation. Therefore, this study examined changes in soil extracellular enzyme activities across BSC developmental sequences on the Loess Plateau to elucidate the effects of BSC formation on enzyme stoichiometric characteristics and to deepen understanding of BSCs' role in nutrient cycling in sandy ecosystems.

Materials and Methods

1.1 Study Area Overview

The Liudaogou watershed is located in the northern Loess Plateau within Shenmu County, Shaanxi Province (110°21′~110°23′E, 38°46′~38°51′N). The watershed covers approximately 6 km² and features a semi-arid temperate climate characterized by dry, windy winters and springs. Elevations range from 1094.0 to 1273.9 m, with an average annual temperature of 8.4°C and average annual precipitation of 408.5 mm exhibiting high interannual variability. Heavy rainfall is concentrated in summer and autumn, accounting for 70%-80% of annual precipitation, with annual potential evaporation reaching 1000 mm. Following years of afforestation and grassland restoration, BSCs are widely distributed throughout the area, primarily including algal crusts, mixed crusts, and moss crusts with coverage typically ranging from 60% to 70%. These BSCs are mainly distributed on hill slopes and summits, occupying 60.7% of the watershed area.

1.2 Soil Sample Collection

Five BSC development stages were selected in Liudaogou watershed: bare sand (CK), algal crust (dominated by cyanobacteria, A), algal-moss mixed crust (with greater algal crust coverage, AM), moss-algal mixed crust (with greater moss crust coverage, MA), and moss crust (dominated by Didymodon vinealis, M). Three representative plots were established at each stage with consistent slope aspect and position and similar gradients. Each plot measured 40 m × 40 m, with distances exceeding 100 m between plots. Within each plot, 12-15 sampling points were selected using an S-shaped random sampling pattern. Soil samples from these points were combined to obtain approximately 2 kg of representative material. Sampling was conducted in July 2019, yielding 75 representative samples (5 stages × 3 replicates × 5 soil layers). Samples included the BSC layer (0-2 cm) and underlying soil layers (2-10 cm, 10-20 cm). The BSC layer comprised cryptogams and associated soil microorganisms cemented with surface soil particles. BSC thickness averaged 11.1±1.9 mm for algal crust, 10.9±1.8 mm for algal-moss mixed crust, 7.2±2.2 mm for moss-algal mixed crust, and 9.6±1.9 mm for moss crust. After carefully removing moss tissues and residues, samples were placed in foam boxes with ice packs and transported to the laboratory within four hours. Soil samples were divided into two portions: one for immediate extracellular enzyme activity analysis within one week, and the other air-dried and passed through a 0.15 mm sieve for physicochemical property analysis.

1.3 Soil Sample Analysis

Soil organic carbon (SOC) content was determined by the potassium dichromate volumetric method. Soil total nitrogen (TN) content was measured using the Kjeldahl method with H₂SO₄ mixed accelerator digestion. Soil total phosphorus (TP) content was determined by the H₂SO₄-HClO₄ digestion-molybdenum blue colorimetric method.

Soil extracellular enzyme activities were measured using a multifunctional microplate reader (Tecan Infinite M200 PRO, Switzerland) with microplate fluorometric methods. The assay principle employs fluorescent substances (4-methylumbelliferone, MUB; 7-amino-4-methylcoumarin, AMC) conjugated to specific substrates. Four C-cycling enzymes (β-1,4-glucosidase, BG), two N-cycling enzymes (β-1,4-N-acetylglucosaminidase, NAG; leucine aminopeptidase, LAP), and one P-cycling enzyme (alkaline phosphatase, AP) were measured. Enzyme activities were expressed as nmol substrate per gram dry soil per hour (nmol·g⁻¹·h⁻¹).

1.4 Data Analysis

Microsoft Excel 2010 was used for data organization and calculation. Soil and enzyme stoichiometry were calculated using mass ratios. Data were log-transformed prior to analyzing linear relationships between soil C, N, P contents and their acquiring enzymes. Statistical analyses were performed using SPSS 25.0. One-way ANOVA was employed to test the effects of BSC formation stage, soil layer depth, and their interactions on soil C, N, P contents, stoichiometry, and extracellular enzyme activities. Origin 2018 and R 3.5.1 were used for graphical presentation. Redundancy analysis (RDA) was conducted to examine relationships between soil C, N, P contents, stoichiometry, and enzyme activities. Standardized major axis (SMA) estimation using SMATR 2.0 software was applied to explore proportional relationships between enzyme activities involved in C, N, and P cycling. The model log(y) = a + b·log(x) was used, where x and y represent enzyme activities, b is the slope, and a is the intercept. When the slope was not significantly different from 1.0, the relationship was described as isometric.

Results

2.1 Variation Characteristics of Soil C, N, and P Contents

Across different BSC development stages, SOC, TN, and TP contents all decreased gradually with soil depth (Table 3). Significant differences existed among development stages (P<0.05). In the 0-2 cm layer, SOC and TN contents followed the order: moss crust > moss-algal mixed crust > algal-moss mixed crust > algal crust > bare sand, with significant differences between moss crust and bare sand (P<0.05). TP content showed no significant variation among development sequences. Throughout the 0-20 cm profile, SOC and TN contents were significantly higher in crust-covered soils than in bare sand, and significantly higher in moss crust than in algal crust (P<0.05).

Table 3 Variation characteristics of soil C, N, and P contents at different formation stages (mean ± SD)

Stage Depth (cm) SOC (g·kg⁻¹) TN (g·kg⁻¹) TP (g·kg⁻¹) CK 0-2 1.29±0.28Ab 0.10±0.01Ba 0.19±0.02ABd 2-10 1.28±0.43Ac 0.09±0.02Bb 0.19±0.06ABc 10-20 1.10±0.27Ba 0.08±0.01Bc 0.16±0.01Bb A 0-2 15.2±3.08Ba 0.46±0.14Ab 0.40±0.03Ba 2-10 1.72±0.44Ac 0.18±0.05Ac 0.32±0.03Ab 10-20 1.61±0.37Ac 0.17±0.05Ac 0.32±0.04Ab AM 0-2 17.8±3.20ABa 0.40±0.04ABa 0.34±0.05Ab 2-10 1.47±0.47Ac 0.16±0.03Ac 0.31±0.03Ab 10-20 1.38±0.16Aa 0.15±0.03Ac 0.27±0.12Ab MA 0-2 20.0±2.20Aa 0.45±0.06Aa 0.39±0.03Ba 2-10 1.30±0.26ABa 0.18±0.05Ac 0.32±0.04Ab 10-20 1.15±0.23Ba 0.16±0.05Ac 0.23±0.03ABc M 0-2 19.8±3.97Aa 0.49±0.14Ab 0.40±0.03Ba 2-10 1.72±0.44Ac 0.18±0.05Ac 0.32±0.04Ab 10-20 1.61±0.37Ac 0.17±0.05Ac 0.32±0.04Ab

Note: Different uppercase letters indicate significant differences among development stages within the same soil layer, while different lowercase letters indicate significant differences among soil layers within the same development stage (P<0.05). The same below.

2.2 Variation in Soil Extracellular Enzyme Activities and Relationships with Soil C, N, and P

Extracellular enzyme activities differed significantly among development stages (P<0.05) (Table 4). In the 0-2 cm layer, enzyme activities followed the order: moss crust > moss-algal mixed crust > algal-moss mixed crust > algal crust > bare sand, with all crust stages showing significant increases compared to bare sand (P<0.05). The algal-moss mixed crust and moss crust stages differed significantly from bare sand (P<0.05), while enzyme activities did not vary significantly with development sequence in the 2-20 cm layer.

Linear regression analysis revealed that C-, N-, and P-cycling enzyme activities were extremely significantly positively correlated with soil C, N, and P contents (P<0.001) (Fig. 2). Standardized major axis analysis indicated that the slope between NAG+LAP and BG activities was greater than 1.0 and statistically significant in the algal-moss mixed crust stage (P<0.001), while slopes in other BSC development stages were not significantly different from 1.0, demonstrating constrained relationships (Table 5). Additionally, slopes between SOC, TP contents and BG, NAG+LAP activities were significantly greater than 1.0.

Table 4 Variation characteristics of soil extracellular enzyme activities at different formation stages (mean ± SD)

Stage Depth (cm) BG (nmol·h⁻¹·g⁻¹) NAG (nmol·h⁻¹·g⁻¹) LAP (nmol·h⁻¹·g⁻¹) AP (nmol·h⁻¹·g⁻¹) CK 0-2 80.1±10.5ABa 19.5±1.65Ba 167±13.5Aa 145±12.3Aa 2-10 71.6±9.81Ba 41.5±6.83ABb 67.7±5.23Ab 60.8±8.08Bb 10-20 117±6.89Aa 26.9±5.11ABb 42.6±5.68Ab 42.4±7.69Ab A 0-2 111±19.3Aa 36.5±12.5ABb 61.0±8.45Aa 86.2±10.31Ab 2-10 13.7±1.95Ab 15.9±3.21Ac 12.3±1.60Ab 12.5±3.90Ab 10-20 12.2±2.51Ab 13.6±3.14Ab 12.5±3.90Ab 9.68±1.74Ab AM 0-2 6.73±1.97Ab 5.45±1.24Ab 6.14±2.76Ab 5.80±1.97Ab 2-10 2.44±0.29Ba 0.46±0.14Ab 1.53±0.37Bb 2.44±0.65Ac 10-20 2.27±0.46ABc 0.40±0.04ABa 2.09±0.69ABc 2.27±0.46ABc MA 0-2 5.80±1.97Ab 6.14±2.76Ab 1.53±0.37Bb 2.41±0.69Ac 2-10 2.44±0.65Ac 0.40±0.04ABa 2.09±0.69ABc 2.27±0.46ABc 10-20 2.27±0.46ABc 0.34±0.05Ab 2.41±0.69Ac 2.27±0.46ABc M 0-2 20.0±2.20Aa 0.45±0.06Aa 0.49±0.14Ab 0.40±0.03Ba 2-10 1.30±0.26ABa 0.18±0.05Ac 1.15±0.23Ba 0.32±0.04Ab 10-20 1.15±0.23Ba 0.16±0.05Ac 1.38±0.16Aa 0.32±0.04Ab

2.3 Variation in Soil Stoichiometry and Enzyme Stoichiometry

In the BSC layer, C:N, C:P, and N:P ratios increased gradually with development sequence: A