Characteristics of bacterial community in fog water at Mt. Tai: similarity and disparity under polluted and non-polluted fog episodes

: Bacteria, widely distributed in atmospheric bioaerosols, are indispensable component in fog water system and play an important role in atmospheric hydrological cycle. However, little is known about the bacterial community dynamics and ecological 15 function, especially under the increasing serious air pollution events in North China Plain. Here we have a comprehensive characterization of bacterial community structure, variation and environmental influence about fog water collected at Mt. Tai under polluted and non-polluted fog episodes from 24 Jul to 23 Aug 2014. Using the Miseq 16S rRNA gene sequencing, the facts that fog water harbored a highly diverse 20 bacterial community and the predominant phyla of Proteobacteria, Bacteroidetes, Cyanobacteria and Firmicutes were investigated. The abundant genera Acinetobacter , Stenotrophomonas , Pseudomonas , and Empedobacter originated from a wide range of habitat included opportunistic pathogenic and functional species, suggesting the bacterial ecological and healthy importance in fog water should be concerned. 25 2 Clustering analysis including hierarchical cluster (Hcluster) and principal coordinate analysis (PCoA) indicated a significant disparity between polluted and non-polluted samples. Potential pathogens were significant group in the polluted samples, whereas a more diverse ecological function group of bacteria were identified in the non-polluted samples using linear discriminant analysis effect size (LefSe). 5 Community structure discrepant performed by redundancy analysis (RDA) indicated PM 2.5 have negative impact on bacteria, playing vital role in shaping microbial community structure. PM 2.5 was possibly associated with different origins and pathways of air mass using source tracking by the backward trajectory and wind analysis, mainly related to the long-term transport combing with local regional 10 emission processes. This work furthered our understanding of bacterial ecological characteristics in the atmospheric aqueous phase, highlighted the potential influence of environmental variables on bacterial community over fog process, which will provide fundamental acquaintance of bacterial community response in fog water under increasing pollution stress.

Clustering analysis including hierarchical cluster (Hcluster) and principal coordinate analysis (PCoA) indicated a significant disparity between polluted and non-polluted samples. Potential pathogens were significant group in the polluted samples, whereas a more diverse ecological function group of bacteria were identified in the non-polluted samples using linear discriminant analysis effect size (LefSe). 5 Community structure discrepant performed by redundancy analysis (RDA) indicated PM 2. 5 have negative impact on bacteria, playing vital role in shaping microbial community structure. PM 2.5 was possibly associated with different origins and pathways of air mass using source tracking by the backward trajectory and wind analysis, mainly related to the long-term transport combing with local regional 10 emission processes. This work furthered our understanding of bacterial ecological characteristics in the atmospheric aqueous phase, highlighted the potential influence of environmental variables on bacterial community over fog process, which will provide fundamental acquaintance of bacterial community response in fog water under increasing pollution stress.

Introduction
Fog is the near-surface cloud and aerosol system composed of tiny droplets suspended in the atmosphere. In the atmosphere, numerous pollutants could be dissolved or suspended in fog, which may induce complex effects on environment security and human health. Over the past decades, studies on fog/cloud water are mainly focused 5 on the physicochemical properties (Aikawa et al., 2001;Boris et al., 2015;Ferná ndez-Gonzá lez et al., 2014). Recently, with the in-depth understanding of the characteristics of fog, bioaerosols in fog have been the upcoming focus.
Studies have showed that living microorganisms, including bacteria, fungi and yeasts, are present in fog or clouds (Burrows et al., 2009). As the first study on biological 10 particles in fog water, Fuzzi et al (1997) suggest the bacterial replication in foggy days. Afterwards, with the development of detection techniques, microorganisms in fog/cloud water are more systematically studied (Amato et al., 2007c;Delort et al., 2010;Vaï tilingom et al., 2012). Combined with the field investigations and lab experiments, diverse bacterial communities are identified, and the bacterial 15 metabolically active in fog/cloud water are also demonstrated. In atmospheric aqueous phase, microorganisms can act as cloud condensation nuclei and ice nucleation, which have potential impact on precipitation processes (Amato et al., 2015;Mortazavi et al., 2015). Moreover, microorganisms in fog/cloud water are available to metabolize organic carbon compounds and influence photochemical chemical reactions 20 (Vaï tilingom et al., 2013), involve in the nitrogen cycling (mineralization and nitrification) (Hill et al., 2007), degrade organic acids (formate, acetate, lactate, succinate) and associate with carbon recycling (Amato et al., 2007a;Vaï tilingom et al., 2010), and therefore participate in a series of complex and diverse biochemical metabolic activities.

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A fog occurrence is a complex process, in contaminated area, fog typically contains numerous pollutants, e.g., sulfate and nitrate ions, organic carbon compounds, and bacteria (Badarinath et al., 2007;Despré s et al., 2012;Ferná ndez-Gonzá lez et al., 2014;Mohan & Payra, 2009). Emissions and resuspension of bacteria by wind erosion or splashing water from various terrestrial environments into the atmosphere recruit 30 diverse airborne bacteria, which possibly involve opportunistic and functional bacteria.
will be deposited back to the land via dry or wet deposition processes, which may induce human risks through microbial pathogens dispersion and potential effect on the diversity and function of aquatic and terrestrial ecosystems. Therefore, to evaluate the potential ecological functional bacteria in fog water is urgent, especially for the polluted fog episodes. 5 It is noteworthy that airborne bacterial communities are closely related to environmental characteristics (Gao et al., 2016), and meteorological factors are often correlated with the observed bacterial community structure (Dong et al., 2016). For instance, studies about the relationships between ambient inhalable airborne and environmental parameters suggest temperature, relative humidity, PM 10 , PM 2.5 and 10 particle size have significant impact on the composition and dynamic of microbial communities (Adhikari et al., 2006;Bowers et al., 2013). However, due to the paucity of detailed and comprehensive studies of atmospheric bacterial composition, the understanding of the dynamic of bacterial community remains incomplete, particularly in the North China Plain. The North China Plain is the most important 15 agricultural and economic region in China, which has been suffering serious air pollution events in recent years, e.g., the severe fog and haze pollution in Beijing during January 2013 (Huang et al., 2014). During a polluted fog process, how bacterial community varied and which environmental factor play decisive role in shaping bacterial community structure are still unclear.

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In the present work, typical fog episodes under polluted and non-polluted weather were collected in the summit of Mt. Tai in North China Plain. To understand the dynamic of bacterial community, the Miseq 16S rRNA gene sequencing was performed, and analysis of similarities (ANOSIM) and linear discriminant analysis effect size (LEfSe) were executed to clarify the discrepant bacterial taxa. Moreover, 25 RDA analysis was applied to identify the pivotal environmental factor influencing bacterial community. Air mass back trajectory and wind direction and speed analysis were selected to definitude the most likely source and transmission paths of pollutants and bacteria.
(CASCC2) with a droplet size cut of 3.5 μm at the summit of Mt. Tai (36 o 18′ N, 117 o 13′ E, and 1534 m a.s.l) (Guo et al., 2012). The flow rate was 24.5 m 3 min −1 and fog water was collected on the strings flows down to Teflon bottles. The collected samples were stored at 4 o C until analysis.
In fog water, the pH and conductivity was detected with a Multi pH/COND/TEMP 5 6350 hand held Meter immediately after sampling. Hourly data, e.g., meteorological parameters, and PM 2.5 were measured to evaluate the air quality during fog episodes.

Illumina high-throughput sequencing and analyzing
Raw sequences were processed and analyzed using the QIIME package (Kuczynski et 10 al., 2011). The PE reads were firstly merged with overlap greater than 10 bp. Then, the adapter, barcodes and primers were removed from the merged sequences.
Subsequently, the trimmed sequences with length shorter than 200 bp, quality score lower than 25, homologous longer than 8 bp, contained ambiguous characters were screened. Finally, chimeric sequences were identified using the Usearch61 algorithm 15 and removed from the dataset. The optimized sequences were clustered into OTUs at the threshold of 97% similarity with the usearch61 algorithm. Single OTUs were removed and taxonomy was assigned to each OTU using the Ribosomal Database Project (RDP) classifier in QIIME, with a minimum confidence cutoff of 0. fog episodes at genus or higher taxonomy levels (Segata et al., 2011).

Intercation between bacterial community structure and environmental variables
To determine the relationship between bacterial community structure and environmental variables, a detrended correspondence analysis (DCA) was first

Microbial community in fog water
Seven fog episodes from 24 July to 23 August 2014 were observed. Detail information was summarized in Table 1. Fog episodes can be classified as polluted and non-polluted according to the average PM 2.5 mass concentration.
Information on the bacterial community of fog water has been very scarce, our study 20 provided comprehensive investigation of bacterial community under both polluted and non-polluted fog episodes. From the 13 samples collected during 7 fog episodes, a total of 232148 high quality sequences were obtained after quality filtering and OTUs ranged from 975 to 1258 (Table 2). This value was similar with the previous sequence-based survey of atmospheric bacteria (OTUs, 1214) (Katra et al., 2014).
Rarefaction curves of observed OTUs continued to rise with increasing numbers of sequences ( Figure S1), suggesting further sequencing will yield more species.
However, the average Good's coverage of 13 samples was 97.2% (Table 2) Across all samples collected from the 7 fog episodes, Proteobacteria was the dominant has reported the ability to survive in low concentrations of nutrients, metabolize a series of carbon compounds, events toxic compounds (Xu et al., 2006). Similar to Sphingomonas, members of Brevundimonas are well known for their ability to withstand extreme harsh environment (Kopcakova et al., 2014). Massilia, isolated from air samples, could participate in the biodegradation and transport of Phenanthrene (Gu et al., 2016). Empedobacter from Bacteroidetes are widely distributed in water habitats, since the human clinical origins, Empedobacter are ranked as potential pathogens (Hugo et al., 2005). Bacillus included in the phylum Firmicutes commonly found in soil and water, are also found in air samples 5 (Suominen et al., 2001). Similar to Pseudomonads, some strains of Bacillus could produce biosurfactants that can act as cloud condensation nuclei (Delort et al., 2010).
Moreover, members of Deinococcus from Deinococcus-Thermus are well known for their ability to withstand extreme radiation conditions that could potentially adapt to the cloud environment (Mattimore & Battista, 1996). The identification of bacteria 10 adapt to specific environments in fog/cloud water (low temperature, harsh nutrition and high radiation environment) with the potential role in the nucleation, metabolism of organic pollutants, demonstrated the potential importance of participation and influence the atmospheric biochemistry cycle. 15 Bioaerosols have been complex assemblages of airborne and exogenic microorganisms, many of which likely emissions and resuspension from various terrestrial environments, e.g., soil, water, plants, animals or human beings. In the atmosphere, bacteria including pathogenic or beneficial species can be attached to particles or incorporated into water droplets of clouds/fog. During fog process, they 20 can be deposited back to land via deposition and possibly induce infections to human health and impose effect on the diversity and function of aquatic and terrestrial ecosystems ( Figure 3).

Implications in human health and ecosystem
In the present study, the presence of potential pathogen sequences indicated occasional distribution and dispersion of pathogens in fog water. The levels of 25 opportunistic pathogens found in polluted fog episodes are comparable to non-polluted samples (  et al., 2006). Similarly, the Brevundimonas vesicularis and Brevundimonas diminuta can induce virulent infections, often associated with nervous system or bacteraemia (Gilad et al., 2009;Han & Andrade, 2005). Besides that, the Acinetobacter schindleri and Moraxella osloensis are associated with skin and wound infections, bacteremia and pneumonia (Banks et al., 2007;Nemec et al., 2001). Previous studies has showed  (Table 3). Excessive studies have illustrated their capability of metabolism of hydrocarbon compounds, even toxic pollutants, e.g., 15 aromatic compounds (Bock et al., 1996;Busse et al., 2003;Geng et al., 2009;Goyal & Zylstra, 1996). Stenotrophomonas rhizophila and Phyllobacterium myrsinacearum are two typical rhizospheric microorganisms. As plant-associated strains, S. rhizophila fulfill plant-protective roles and have been safely applied in biotechnology (Alavi et al., 2013). Phyllobacterium myrsinacearum, which is a predominant rhizospheric 20 bacterium, its capability of azotification has made the utilization in plant growth promotion and biological control of soil-borne diseases (Gonzalezbashan et al., 2000).
In addition to the potential impact on human health and ecosystem, there are extremophiles, e.g., Deinococcus aquatili, which is radiation-resistant and well Overall, fog water seems to harbor highly diverse bacterial communities in ecosystem, which may be the atmospheric mixing of diverse point sources origins in the rhizosphere, soil and water and possibly participate in the biodegradation of organic compounds in fog water. To find specialized bacterial groups within the polluted and non-polluted fog episodes, LEfSe is performed, which showed statistically significant differences. A total of 70 10 bacterial groups were distinct using the default logarithmic (LDA) value of 2.
Cladograms show taxa with LDA values higher than 3.5 for clarity ( Figure 5).

Consequently, 8 and 19 differentially represent bacterial taxa in polluted and
non-polluted fog episodes were detected.
Like Proteobacteria, the Gram-negative Gammaproteobacteria contains a series of ecologically and medically important bacteria, e.g., the pathogenic Enterobacteriaceae, Vibrionaceae and Pseudomonadaceae. The Xanthomonadales from this phylum has 20 been reported to cause disease in plants (Saddler & Bradbury, 2005

Environmental factors shaping bacterial community structure
To clarity the vital environmental variable in shaping bacterial community structure, RDA was performed to discern the genus-level structure with the selected 25 environmental factors ( Figure 6).  (Table S2). As aforementioned in section 3.3, the above bacteria were metabolically diverse groups found in various habitats.
Of the atmospheric environmental characteristics measured, PM 2.5 was the best predictor of variability in diversity levels within the dominant genera and strongly  Jones & Harrison, 2004). In the present study, air mass from the contaminated area through long-term transport or local regional emission combined with lower wind speeds, largely reduce the diffusion rate of pollutants and thus lead to the sustained high PM 2.5 during polluted fog episodes. Whereas in the non-polluted fog episodes, 15 higher wind speed was beneficial to the diffusion of pollutants and resulted in the lower PM 2.5 mass concentration, which finally led to the significant discrepancy of bacterial community structure. However, further research still needed to address the detailed interaction between bacterial community and environmental factors, and understanding the mechanism that how chemical composition influence microbial 20 community.

Figure 3
Schematic representation of the life cycle and potential influence on the ecosystem of bioaerosols in the atmosphere, modified from Poeschl (Poeschl, 2006).
The predominant identified bacteria species with potential ecological functions are indicated in the figure.    Abbreviates are as followed: AP, Alphaproteobacteria; BP, Betaproteobacteria; GP, Gammaproteobacteria; AC, Actinobacteria; BA, Bacteroidetes; CY, Cyanobacteria; DT, Deinococcus-Thermus; FR, Firmicutes. Biodegradation refers to the bacteria associated with the biodegradation of organic compounds, even toxic pollutants, e.g., aromatic compounds.