**1. Introduction**

As the highest geographical unit in the world, the Qinghai-Tibet Plateau (QTP) has a particular and representative ecological environment. The QTP is of great significance to global climate change, carbon cycle, and biological germplasm resources [1–4]. As a fragile ecosystem, the QTP ecosystem is particularly vulnerable to human activities [5,6]. In recent years, with the rapid economic development of the Qinghai-Tibet region, road transportation has also continued to develop [7,8]. The total length of highways in the Tibet Autonomous Region increased from 15,852 km in 1978 to 117,000 km in 2021 [9]. Highway traffic inevitably disturbs natural grasslands and has a series of negative influences on the ecosystem, including soil erosion, vegetation destruction, and water quality deterioration [10–13]. The sharp increase in negative impacts has resulted in imbalances in the self-regulation of the grassland ecosystem, and varying degrees of degradation of grasslands along highways [14].

As an important part of the ecosystem, soil microorganisms participate in the degradation of organic matter, biogeochemical cycles, and the maintenance of soil structure and

**Citation:** Liu, Z.; Yang, Y.; Ji, S.; Dong, D.; Li, Y.; Wang, M.; Han, L.; Chen, X. Effects of Elevation and Distance from Highway on the Abundance and Community Structure of Bacteria in Soil along Qinghai-Tibet Highway. *Int. J. Environ. Res. Public Health* **2021**, *18*, 13137. https://doi.org/10.3390/ ijerph182413137

Academic Editors: Roberto Alonso González Lezcano, Francesco Nocera and Rosa Giuseppina Caponetto

Received: 5 November 2021 Accepted: 4 December 2021 Published: 13 December 2021

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are closely related to the degradation of grassland ecosystems [15]. In natural grassland, vegetation growth, soil nutrients, moisture, pH value, etc. determine the community composition and structure of soil microorganisms [16,17]. In addition, it changes with depth due to factors such as redox conditions and soil nutrients. In the QTP, soil microorganisms are mainly distributed in the topsoil (0–20 cm) [18]. Many scholars point out that soil bacterial communities are extremely sensitive to human activities [19]. However, the response of soil bacterial communities to environmental changes resulting from road traffic is currently unclear.

Road traffic and construction will produce different types of heavy metal pollutants [20,21]. The components, fuels, and lubricants of vehicles and paving materials may contain heavy metals such as copper, copper, lead, zinc, and cadmium [22]. The burning of liquid fuels, the use of lubricating oil, the wear of vehicles and the loss of pavement will bring in heavy metal ion particles. These particles enter the ecosystem on both sides of the road, possibly through sedimentation pavement runoff and splashing [23]. Studies have shown that the concentrations of heavy metals in soil were significantly affected by road construction and traffic. The degree of impact is inversely proportional to the distance from the road [24,25]. Many studies have shown that the deposition of heavy metal pollutants in soil will affect the composition and structure of soil bacterial communities [26,27]. Additionally, soil nutrient content is another important factor affecting soil bacterial communities. Road construction and traffic drainage will cause varying degrees of soil erosion, topsoil stripping, and other changes. Many studies have shown that soil erosion can change physical and chemical properties such as soil density, soil moisture, and pH, leading to soil nutrient loss [28,29]. As the mediator of more than 90% of the energy and material exchange in the soil ecosystem, the bacterial community structure is bound to be affected to varying degrees [30].

In general, exploration of the influences of soil nutrient changes and heavy metal on the structure of soil bacteria communities can provide data support for grassland ecological restoration along the highway, and can also provide a decision for road construction in the QTP. The Qinghai-Tibet Highway (QTH) provides favorable conditions for exploring the relationship between the highway and soil microorganisms [25]. The grassland ecosystems in most areas along the route are well preserved, are very sensitive to external influences, and have poor natural recovery capabilities [21]. As an uninhabited area and nature reserve, there is almost no human disturbance in this area except for traffic [31,32]. The Golmud-Lhasa section of the QTH passes through areas of different altitudes, climates, soil types, and vegetation types. We assume that the QTH has an impact on the structure of the grassland soil bacterial community by changing soil and heavy metals factors. In this study, treatments at different distances from the highway and altitude were selected, and the bacterial communities along the QTH were determined by high-throughput sequencing technology. The results showed that: (1) The soil bacterial community structure changes with the distance from the QTH; (2) In high altitude areas, the soil bacterial community is more susceptible to highway impacts; (3) Soil nutrients are the main driving factor of soil bacterial community structure change; (4) Within a certain distance from the QTH, the heavy metal content was correlated with soil bacterial community diversity.

#### **2. Materials and Methods**

#### *2.1. Study Area and Sampling Lines*

This study selects the QTH (G109 National Highway) Golmud to Lhasa section as the research object. The region where the QTH passes through has a temperate/sub-frigid continental climate, with long and cold winters, strong winds, little rain in summer, and short spring and autumn. This section of the highway crosses two major soil types: alpine prairie soils (Cryuborolls) and alpine meadow soils (Cryaquet) and a small portion of graybrown desert soils (Gypsic Haplosalid). In this area, the highway passes through the Hoh Xil National Nature Reserve and the Sanjiangyuan Nature Reserve. There is no large-scale agriculture, animal husbandry, and manufacturing industries, and the natural grassland is

less affected except for the highway. The effect of traffic activity on the deposition of soil pollutants was stronger closer to the road and weakened with the increase of distance from the road [25,32]. Therefore, a plot 400 m from the highway was collected as a control in each area that may not be affected by traffic activity.

More than 91% of the studied sections have an altitude of >4000 m, and the highest altitude is 5231 m, which is located at Tanggula Mountain Pass. In this study, 3 sampling zones at 4000 m, 4600 m, and 5231 m were selected, and one sampling zone was set in an area within 400 m of each altitude where there were no other human influence factors except roads (sampling zone 35◦47 N, 94◦20 E; sampling line 2 at 34◦27 N, 92◦44 E; sampling zone 3 at 32◦53 N, 91◦55 E). The sampling lines were perpendicular to the highway and is 400 m long and 10 m wide (Figure 1). The altitude drop in the sampling zone is less than 30 m. For each sampling zone, 4 plots were set at a distance of 5 m, 20 m, 50 m, and 100 m away from the highway shoulder, and 1 plot was set at a distance of 400 m from the shoulder, which was used as a control plot. Four samples of 1 m × 1 m are randomly set for each plot.

**Figure 1.** Sampling sites along the Qinghai–Tibet highway, China.
