Nitrogen-induced acidification plays a vital role in driving microbial carbon cycles: Insights from a 10-year experiment in a semi-arid steppe

Anthropogenic nitrogen (N) deposition and increased precipitation are known to alter soil microbial communities. However, the interactions between N deposition and increased precipitation on soil microbial composition and the microbial-mediated carbon (C) and N cycling functions remain unclear. A 10-year experiment involving N addition (0, 10g N m–2 year–1) and water addition (0%, 30% of the average annual precipitation) were conducted on the Mongolian Plateau to assess abiotic (e.g., soil nutrients and pH) and biotic (e.g., biological community composition) effects on bacterial community structure and C and N cycling functions. We found that long-term N deposition significantly altered the composition of soil bacteria community, specifically increasing the relative abundance of Firmicutes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, while decreasing the abundance of Acidobacteria and Chloroflexi. Additionally, N deposition decreased the bacterial network degree, the number of edges, as well as overall network complexity, and stimulated soil C metabolic functions through reduced soil pH. Water addition raised the relative abundance of C metabolic functions (e.g., chitin and nitrogen fixation). Our findings underscore that N deposition will increase the risk of C loss, and also emphasize the importance of pH and soil nutrients in regulating microbial nutrient cycles. This study provides new experimental insights into the potential mechanisms by which nutrient inputs and precipitation changes affect microbial community structure and functions of the C and N cycling.

Keywords: nitrogen deposition, water addition, bacterial community composition, C and N cycling, bacterial function prediction

2. Materials and methods

2.1. Experimental site and design

We conducted field experiments at the Inner Mongolia Grassland Ecosystem Research Station (42°01′N, 116°16′E) in China. The climate of the region is semiarid temperate continental climate which has a dry summer and cold winter. Mean annual temperature is 1.4 °C, and precipitation is 380 mm.  In the semi-arid steppe, the dominant plant species are perennial grasses (Stipa krylovii, Leymus chinensis) and forbs (Artemisia frigida, Potentilla acaulis). The type of soil is classified as Haplic Calcisols according to the Food and Agriculture Organization of the United Nations. Initial soil physical and chemical characters were as follows: soil pH, 7.16; organic matter content, 18.53 g kg–1; total N content, 1.75 g kg–1; available N content, 15.37 mg kg–1.

In May 2013, a randomized block design was used with four treatments, including: C (without N addition and water addition); N (application of 10 g N m–2 year–1); W (water addition at rate of 30% average annual precipitation); WN (water addition at a rate of 30% average annual precipitation + application of 10 g N m–2 year–1). The C, N, W, and WN are used to refer the four treatments in the following text. Each treatment was set up four repetitions with an area of 4 × 4 m2 per plot, total of 16 plots. The plots within each block were separated by a 1 m wide buffer strip to minimize disturbance from neighboring plots. Nitrogen treatments (NH4NO3) were added in solution twice per year, half in mid-June and half in mid-July. Water addition treatment was carried out in July and August every year, with a total rainfall enhancement of 126 mm per year, divided into two months. Each month's precipitation treatment is carried out four times, with an increase of 15 mm each time.

2.2. Sampling and measurements

Four soil cores (7 cm in diameter and 10 cm in depth) were randomly drilled and pooled together to form one composite sample per plot in August 2023. The soil samples were passed through a 2 mm sieve to remove plant roots and stones. Soil samples were stored at 4 °C before soil physicochemical property measurements and –80 °C before DNA extraction.

Fresh soil was dried at 105 °C for 24 h to estimate soil moisture. Soil pH was measured on soil slurry at 1:2.5 soil to water (m: v) ratio using a glass electrode. Soil nitrate nitrogen (NO3–-N) and soil ammonia nitrogen (NH4+-N) were extracted with 2 M KCl and analyzed by an automatic nutrient analyzer (SmartChem 200, Alliance, France).  Soil organic carbon (SOC) and total nitrogen (TN) contents was measured by a total organic carbon analyzer (Elementar vario TOC, Elementar Co., Germany).