1. Introduction
Microplastic (MP) pollution has gained increasing interest and is a key environmental challenge [
1]. Increasing evidence has shown that MPs have been detected in oceans, lakes, farmland, and groundwater [
2], [
3], [
4], where plastic waste can accumulate for a long time. MPs can also reach remote areas through atmospheric transport [
5] and may settle onto the surface environment through rainfall or snowfall. For instance, MPs occur in the atmosphere from urban to remote areas, and their abundance in different regions shows significant differences [
6]. Atmospheric MP deposition significantly contributes to MP pollution in urban waters [
7]. The main method for MPs to enter remote areas is atmospheric deposition, and once they are deposited onto snow and glaciers, they will be retained for a long time [
8]. The abundance of MP particles has been reported to range from 0.02 × 10
3 to 154 × 10
3 items·L
−1 in snow from the Alps to the Arctic [
9]. Airborne plastic fallouts are an important source of MP pollution that can be taken in by humans [
10], [
11]. Previous studies have shown that both human activities and industrial production are the main sources of MP pollution at high altitudes [
12], [
13]. However, in comparison with water, little attention has been given to the characteristics of MPs in snow.
Considerable research has indicated that MPs can adsorb environmental chemicals (e.g., heavy metals and organic contaminants) and pathogens [
14], [
15]. The dominant sorption mechanism of pollution onto MPs is surface complexation, electrostatic, and hydrophobic interactions [
16]. Moreover, biofilm-developed MPs can sorb more pollutants than virgin MPs [
17]. Adsorbed MPs can affect the growth, reproduction, and other essential biological functions of organisms [
18]. Researchers have isolated the bacterial fish pathogen
Aeromonas salmonicida from marine MP pollutants [
19] and detected two opportunistic human pathogens (
Pseudomonas monteilii and
Pseudomonas mendocina) and one plant pathogen (
Pseudomonas syringae) in an MP biofilm [
15]. Marine fish also ingest MPs and artificial cellulose particles [
20], among which man-made cellulose fibers are more common [
21]. Moreover, MP residues in the environment result in an altered microbiome, in which the addition of biodegradable plastics increases the specific microbial diversity and growth rate in soil [
22]. The presence of polypropylene (PP) residues decreases the community diversity and richness of soil bacteria [
23], and MP-associated microbiomes can alter nutrient cycles by stimulating decomposition and denitrification [
24], [
25]. In addition, MPs, as carriers of bacterial pathogens and antibiotic-resistance genes, pose potential harm to ecosystems [
15], [
26]. Notably, MPs can cause the spread of antibiotic-resistant bacteria through biological uptake, particularly to edible aquatic products, which increases the risks to human health [
27], [
28]. It has also been found that MPs have created a new ecological habitat for microbial communities, leading to some special microorganisms gradually becoming dominant species [
29], [
30].
As one of the main sources of MPs, human activities play an important role in their spread and settling into the environment. MP fragments in air samples (< 100 μm) were found in five Chinese megacities [
31]. Researchers have confirmed that Tibetan glacier contaminants, such as MPs, flow into lakes via glacial melting, and 37% of lightweight PP and small MPs were found in glacial runoff [
8], [
32]. However, these studies mainly determined the concentrations and morphologies of MPs in the environment without considering the effect of MPs on the microbial community structure in sedimentary snow. In particular, more than 2 × 10
11 nanoplastic particles per square meters are deposited on the surface of snow weekly [
33], which has focused increased attention on the possible toxicological effects of MPs in snow. Therefore, in this study, the Inner Mongolia Plateau was selected, and snow samples were collected from different functional areas of a city. The differences in the samples were analyzed by comparing the microbial community structures. The differences observed prompted us to better explore the features of the effect of MPs on the microbial community of snow and to provide better insights into the possibility of MPs spreading bacteria and fungi in plateau cities.
2. Materials and methods
2.1. Study sites and sample collection
Arongqi County (48°10.883′N, 123°22.699′E) is situated on the Inner Mongolian Plateau, China. This area has a warm, temperate, and semihumid continental monsoon climate and frequent windy periods during winter and spring. The annual average temperature is approximately −20 °C in winter and approximately 20 °C in summer, and the annual precipitation is 320.0 mm with a mean annual snowfall of 22.2 mm. In the past 40 years, the snow depth has increased in 54.9% of the areas, with the highest trend of 0.79 cm·a
−1; 45.1% of the areas showed a downward trend [
34]. These areas are mainly distributed northeast of Hulun Buir City. In this study, six snow sampling sites were selected, which were located in a farmland area, a residential area, a thermal power plant, a suburban area, a hospital, and a landfill. In each area, three sampling points were randomly selected. In February 2020, the surface of freshly deposited snow (10-15 cm depth) was collected into a stainless-steel container from different functional areas of Arongqi City using a stainless-steel shovel. Samples (
n = 18) were then transported to the laboratory at low temperature. The samples were processed under sterile conditions, and we avoided using plastic instruments and consumables.
2.2. Data collection and preprocessing
The bacterial and fungal populations of the 18 snow samples were analyzed using high-throughput sequencing. The snow sampling methodology and microbiological analysis have been described by Ref. [
35]. Briefly, samples were melted under dark conditions at room temperature, and 500 mL of solution was filtered through a 0.22 μm membrane once the snow samples were completely melted. Filters were stored in glass-bottomed culture dishes at 80 °C for sequencing and further analysis. DNA extraction kits (MP Biomedicals, USA) were used to extract total DNA from the membrane, and the quantity and quality of the DNA were confirmed using spectrophotometric analysis (NanoDrop-2000; Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis. The total bacterial 16S ribosomal deoxyribonucleic acid (rDNA) gene was amplified using the bacterial universal primer pair 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGAC-TACHVGGGTWTCTAAT-3′). The fungal forward primer ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and reverse primer ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) were used to analyze the fungal community.
Amplification of DNA fragments by PCR was performed using rTaq DNA polymerase (TaKaRa Bio Inc., Japan). The PCR products were purified using an Axy-Prep DNA gel extraction kit (Axygen Biosciences, USA). Amplicon sequencing was performed using the Illumina MiSeq platform (Majorbio BioPharm Technology Co., Ltd., China). Raw sequence reads were demultiplexed, filtered for quality, and merged using Trimmomatic and Flash (version 1.2.11). Sequences were grouped into operational taxonomic units (OTUs) based on a threshold of 97% sequence identity.
All laboratory materials were disinfected with ethanol and rinsed with sterile distilled water. Upon defrosting, 1 L of the sample was filtered through membranes using a vacuum filtration pump (Tianjin Jinteng Experiment Equipment Co., Ltd., China). The water sample (melted snow) at each sampling point was filtered through three filter membranes. MP analysis was performed using a laser micro-Raman spectrometer (SOE-066; Renishaw Co., Ltd., UK) equipped with wavelength lasers. All particles were used to isolate and count MPs and were analyzed using micro-Raman spectroscopy to determine the polymer composition [
9], [
13].
2.3. Statistical analysis
Ordinary one-way analysis of variance (ANOVA) was performed to analyze the MP concentration in the snow samples. Bacterial community composition variation was clustered using nonmetric multidimensional scaling (NMDS) performed on the Bray-Curtis distance of the OTUs. Significant differences in the bacterial and fungal communities were assessed using one-way ANOVA at the genus level. Functional prediction of bacteria and fungi was conducted using the phylogenetic investigation of communities by the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States algorithm (PICRUSt2).
3. Results
3.1. Morphology and composition characterization of MPs
The highest abundance of MPs (
Fig. 1(a)) was found in the thermal power plant ((199 ± 22) MPs·L
−1), followed by landfill ((173 ± 35) MPs·L
−1), residential area ((125 ± 21) MPs·L
−1), hospital ((119 ± 19) MPs·L
−1), and farmland ((76 ± 14) MPs·L
−1), while the lowest abundance was found in the suburban area ((68 ± 10) MPs·L
−1). The characteristics varied considerably, with nine common polymer types (e.g., PP and polyethylene terephthalate (PET)) found in total, ranging between five (landfill) and nine (residential area) types per sample (
Fig. 1(b)). The largest proportion of PP was found in snow samples from the thermal power plant (39.8%) and suburban area (33%). PET was the most significant component of MPs detected in the residential and hospital samples. The types of polymers per sample was lowest in the landfill, comprising mainly polyvinylchloride (PVC; 33.5%), PET (28.6%), and polyester (15.3%). The primary sample types of MP particles included fibers, fragments, and foam (
Fig. 2). In the thermal power plant, the main types of MPs detected in the snow samples were fibers (63.0%), fragments (11.6%), foam (18%), and others (7.4%). However, MPs in the form of foam were not detected in snow samples from the farmland or landfill (
Fig. 2).
3.2. Bacterial richness and diversity across sampling sites
MPs can act as biological carriers or as interferents that affect the microbial community structure, as their diverse characteristics affect environmental properties [
36], [
37]. Thus, to analyze the differences among the groups, NMDS analysis results based on Bray-Curtis similarity distances determined that farmland, residential, and hospital samples were tightly clustered together, while thermal power plant, landfill, and suburban samples were further apart from each other on the ordination (
Fig. 3). Analysis of similarities (ANOSIM) indicated significant variance in the microbiotic structure among all sampling sites (significant
P-value
= 0.002).
The top ten bacterial genera found in the snow samples are shown in
Fig. 4;
Blastococcus,
Rubellimicrobium, and
Janibacter were the predominant genera in snow.
Blastococcus is a Gram-positive, coccoid, aerobic genus of bacteria that is part of the Geodermatophilaceae family. According to 16S ribosomal ribonucleic acid (rRNA) gene-based phylogeny,
Rubellimicrobium belongs to the family Rhodobacteraceae.
Janibacter is a genus of Gram-positive and nonspore-forming bacteria that was originally isolated from sludge in a wastewater treatment plan [
38]. The relative abundances of
Blastococcus and
Rubellimicrobium in the thermal power plant were lower than those in other areas. The relative abundance of
Janibacter was higher in the hospital and landfill samples, accounting for 2.11% and 1.26%, respectively.
Changes in microbial communities may affect the diversity of metabolic functions [
36], [
39]. Data analysis was conducted to determine the functional traits of the different microbiota compositions using the PICRUSt2. A substantial decrease (46.81%-50.44%) was observed for the functional enzyme nicotinamide adenine dinucleotide hydrogen (NADH) in the high-density MP enrichment areas (thermal power plant and hospital) compared with low-density MP sites (suburban area) in
Fig. 5. Nicotinamide adenine dinucleotide (NAD
+) and its related metabolites are important regulators of physiological processes, enabling cells to adapt to changes in the external environment, including disturbances and toxic stresses [
40]. These effects are primarily achieved by driving the metabolic pathways of species and their associated effects, indicating that NAD
+ can be used as an important cofactor in physiological processes. Various NAD
+- dependent enzymes are involved in physiological metabolic processes, including the regulation of chemical modification of DNA, RNA, and proteins after synthesis and the transfer of the secondary messenger cyclic adenosine diphosphate (ADP)-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP
+) [
41]. MPs can adsorb chemical pollutants and carry pathogens, which have great biological toxicity [
29], [
42]. Disturbance of the physiological function related to NAD
+ metabolism leads to pathological changes in the organism and affects normal growth. Therefore, it is speculated from the functional prediction results that MPs may interfere with this physiological process.
The relative abundance of glutamine-hydrolyzing glutaminyl-transfer RNA (tRNA) synthetase and monosaccharide-transporting adenosine triphosphatase (ATPase) decreased by 23.73%-30.52% in the bacterial community from the thermal power plant compared with the suburban area one (
Fig. 5). Glutaminyl-tRNA synthetase and monosaccharide-transporting ATPase catalyze chemical reactions. Glutaminyl-tRNA synthetases belong to the family of ligases, particularly carbon-nitrogen ligases that use glutamine as the amino-N-donor to form carbon-nitrogen bonds [
43]. These enzymes are involved in the metabolism of glutamate, alanine, and aspartate. However, these enzymes are hydrolases that can act on acid anhydrides, catalyze the movement of substances across membranes, and participate in ATP-binding cassette (ABC) transporters [
44].
3.3. Fungal richness and diversity across sampling sites
To observe these differences, analyses among groups were further conducted in terms of fungal communities. The results of the NMDS ordination demonstrated that farmland and thermal power plant were further apart from each other in the ordination, while other sampling stations were clustered more closely together (
Fig. 6). In the ANOSIM,
P values demonstrating significant differences in the NMDS plot (
P = 0.001) were considered significant (
Fig. 6). Based on relative abundances of the top ten dominant orders,
Cladosporium,
Mrakia, and
Vishniacozyma were the dominant fungal genera in the snow (
Fig. 7).
Cladosporium is a fungus commonly found in outdoor air, and its spores can be transported by wind.
Mrakia and
Vishniacozyma are adaptable to low temperatures and are detected in extreme Antarctic environments [
45]. The relative abundances of
Mrakia and
Vishniacozyma were highest in the thermal power plant samples than in those from the other sites (
Fig. 7).
Aspergillus is defined as a group of fungi that are in an asexual state, and the relative abundance of
Aspergillus in the snow samples from the landfill was significantly increased (
Fig. 7).
The relative abundances of carbonyl reductase and NAD
+ ADP-ribosyltransferase decreased by 39.34%-41.22% in thermal power plant samples compared with those from the suburban area (
Fig. 8). Exo-alpha-sialidase is a glycoside hydrolase that cleaves the glycoside linkage of neuraminic acid and can cause microbial infections [
46]. A substantial increase (52.02%) was observed in the functional enzyme exo-alpha-sialidase in the high-density MP enrichment areas (thermal power plant) compared with the low-density MP area (suburban area in
Fig. 8). MPs can significantly alter the microbial community structure [
47], leading to changes in the characteristics of functional enzymes [
37]. For instance, the abundance of functional genes of human disease in the higher concentration MP group was significantly higher than that in the lower concentration MP group in sediment [
48].
4. Discussion
Regarding the concentration status (68-199 MPs·L
−1) of MPs in the Inner Mongolia Plateau, China, the data found are comparable to those measured for the surface snow ((131 ± 24) MPs·L
−1) of a glacier in the tropical Andes [
13]. However, the MP concentrations in Arctic (0-14.4 × 10
3 MPs·L
−1) and European (0.19 × 10
3-154 × 10
3 MPs·L
−1) snow were markedly higher because MPs are transported over long distances under wind-driven transport [
9]. Although human production and living activities may be the initial sources of MPs, the atmosphere can retain suspended particles for a long time, allowing MPs to be transported over long distances [
49]. For instance, because MPs from European urban areas can lead to the formation of nanoplastics during atmospheric transport, the average concentration of nanoplastics (size < 1 μm) was 46.5 ng·mL
−1 of melted surface snow in the high-altitude Alps [
33].
Owing to their low density, MPs are transported to the upper layer of the atmosphere by wind and then settle in high-altitude or remote areas because of weather activities (e.g., snow and rainfall). In particular, rainfall promotes the deposition of fibrous and small MPs across urban areas in summer and winter, and the number of MPs accumulated in the urban environment is 1.7-12 times that of wastewater discharge [
7]. Consequently, environmental risks have increased accordingly, for instance, carrying pathogens for long-distance transmission [
6], [
8]. Atmospheric deposition is the dominant pathway through which MPs enter remote and high-altitude mountainous lake basins in the Tibetan Plateau [
8]. The pollution characteristics of MPs are also related to human activities; for example, the proportion of MPs released by human activities, such as local sources of contamination (i.e., skiing), is greater than that of atmospheric transport [
50]. Similarly, substantial accumulation of MPs was found in the eastern snowpack of the Mongolia Plateau in China, and atmospheric transport may play a crucial role in the spread of MPs. However, plastic waste buried in landfills is gradually broken down into small-sized MPs under the action of physics, chemistry, and biology, which increases the environmental risks to the development and utilization of waste resources [
51]. In addition, incineration cannot completely eliminate plastic waste, and the remaining bottom ash still releases MPs into the environment [
52]. Therefore, the irrational disposal of garbage is also a typical source of MPs in cities.
The identification of the physical and chemical characteristics of MPs is a critical step in MP investigations, as it can help identify the potential sources and transmission routes of MPs in the environment. Because of the different sources in cities, suburbs, and rural areas, the size distribution and chemical composition of MPs in the atmosphere differ [
5], [
53]. Fibers were found to be the main MP type, with the highest detection rate in snow samples. Owing to the large surface area to volume ratio, the air resistance is increased, and the settling speed is reduced, leading to a high deposition proportion of fiber at high-altitude deposition areas [
13], [
49]. The physical and chemical characteristics of MPs in snow are related to urban release sources, season, population density, and other factors. For instance, there were substantial differences and uniqueness in the shape and composition of MPs in the living (residential area and hospitals) and production (thermal power plant) areas of the sampled city. Transportation activities lead to a high concentration of MP pollution, which is subsequently scavenged by falling snow [
9]. Materić et al. [
33] found that densely populated urban areas might be hotspots of nanoplastic emissions, particularly during dry periods. Thus, MPs can easily reach snow at different altitudes via atmospheric transport, spread into terrestrial and water systems, and ultimately affect the structure and function of ecosystems.
This study demonstrated that the presence of plastic residues may change the bacterial and fungal community structures in snow. MPs can be used as a new habitat for microorganisms (e.g., bacteria, plankton, and algae) [
29], [
30], resulting in variations in the abundance of certain species. MPs in the environment can be easily colonized by microorganisms to form biofilms [
17]. For instance, distinct community structures and biofilm compositions have been observed between MP-associated and natural communities in aquatic ecosystems [
54]. MPs can also indirectly affect the composition and function of microbial communities by altering the physicochemical properties of the environmental media. For instance, polystyrene and PVC MPs significantly decreased the total P content in soil, and the total P content was an important factor that shaped bacterial communities [
23]. Fragments of polyamide might selectively enrich functional microorganisms and genes in a habitat-dependent manner in aquaculture pond waters [
55]. Therefore, MPs can alter the microbial diversity of water, sediment, and soil, and this study confirms that MPs can also alter the composition of microorganisms in snow.
Distinct microbial communities formed by MP interference may exhibit various microbial functions. For instance, the functions related to phototrophy, nitrogen fixation, and nitrate reduction were significantly decreased by exposure to submicrometer plastics [
56]. Here, we observed that alterations in microbial communities were consistent with the prediction of metabolic pathways. Studies have shown that MPs significantly increase the abundance of the microbial genes involved in soil nitrogen cycling [
23]. Amino acid and vitamin metabolism pathways are increased in biofilms colonizing MP substrates [
54]. In addition, MPs provide a new microbial niche and serve as vectors for the transport of other pollutants (pathogens, heavy metals, and organic pollutants) [
29], [
57]. The future climate is predicted to increase the network complexity and modularity of the plastisphere microbiota and change the function of keystone taxa in plastisphere networks [
58]. Thus, plastic-related microbes can adapt to environmental changes by altering their community composition and functions.
5. Conclusions
MPs may act as vectors for chemicals and pathogens and represent a potential threat to the ecosystem and human health. Our detection of MPs in snow samples together with previously published measurements of MPs on snowy mountains and glaciers demonstrate global-scale atmospheric pollution with these emerging contaminants. Fibers were the most common type of plastic found in the snow samples, and the presence of MPs was found to alter the diversity and function of microorganisms in snow. Press perturbations of microplastics can affect the microbial population density, dominant species, and functional characteristics in snow. Although MPs can accumulate in snow over time, as global warming causes large-scale melting of snow and ice, large amounts of potential MPs or their associated complex pollutants are inevitably released back into other ecosystems.
Acknowledgments
This work was supported by the funds for the National Natural Science Foundation of China (52070183), the International Cooperation and Exchange of the National Natural Science Foundation of China (51820105011), and the Program of the Youth Innovation Promotion Association of Chinese Academy of Sciences (2019044).
Compliance with ethics guidelines
Hongwei Yu, Junrong Shao, Huawei Jia, Diga Gang, Baiwen Ma, and Chengzhi Hu declare that they have no conflict of interest or financial conflicts to disclose.
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