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High Abundance of Fluorescent Biological Aerosol Particles in Winter Beijing, China Siyao Yue, Hong Ren, Songyun Fan, Lianfang Wei, Jian Zhao, Mengying Bao, Shengjie Hou, Jianqiong Zhan, Wanyu Zhao, Lujie Ren, Mingjie Kang, Linjie Li, Yanlin Zhang, Yele Sun, Zifa Wang, and Pingqing Fu ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.7b00062 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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High Abundance of Fluorescent Biological Aerosol Particles in Winter Beijing, China Siyao Yue1,2, Hong Ren1,2, Songyun Fan1, Lianfang Wei1,2, Jian Zhao1,2, Mengying Bao3, Shengjie Hou1, Jianqiong Zhan1, Wanyu Zhao1, Lujie Ren4, Mingjie Kang1, Linjie Li1,2, Yanlin Zhang3, Yele Sun1,2, Zifa Wang1,2, and Pingqing Fu1,2,4* 1

State Key Laboratory of Atmospheric Boundary Layer Physics and Atmospheric Chemistry, Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing 100029, China

2

College of Earth Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

3

Yale-NUIST Center on Atmospheric Environment, Nanjing University of Information Science and Technology, Nanjing 210044, China 4

Institute of Surface-Earth System Science, Tianjin University, Tianjin 300072, China

*Corresponding author e-mail: [email protected] (P. Fu)

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TOC art:

ABSTRACT. Primary biological aerosol particles (PBAP) such as pollen, fungal spore, bacteria and virus represent a major subset of particulate matter for both coarse and fine aerosols. Although PBAP affect climate and human health, large uncertainty of their sources and characteristics still remains, especially in urban atmosphere. Here we combined online fluorescent biological aerosol particle measurements and offline fluorescent and biomarker analysis in winter Beijing, a city suffering from severe air pollution. Our results show that the abundance of fluorescent biological particles was higher than many other natural environments. Bioaerosol loadings were much enhanced during mealtime, indicating local emissions of bioaerosols from human activities. The contributions of fungal spore to total organic carbon (OC) from fluorescent bioaerosol analyses were estimated to be around 10% both in clean and polluted periods. This study highlights the importance of human activities to emit bioaerosols into the atmosphere and the potential uncertainties of modeling bioaerosol effects in climate systems.

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Keywords: bioaerosol, WIBS, PBAP, fungal spore, mannitol

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INTRODUCTION Primary biological aerosol particles (PBAP) including proteins, virus, bacteria, fungal spores, algae, cyanobacteria, pollens, plant and animal debris,1-5 are major sources of atmospheric aerosol matter.6, 7 PBAP can influence weather processes and climate dynamics by serving as ice nuclei,8-10 harm human health through transported viruses11 and allergens,12, 13 and can also affect ecosystem functions through hydrological cycles.5, 10, 14, 15 Although many studies of PBAP have been conducted, estimation of terrestrial PBAP emission is still less understood, ranging from 56 to 1000 Tg yr–1,16, 16, 17 which indicates that large uncertainties of modelling their physical and chemical influences in the climate system still exist.18-21 This is partly due to the omission of some emission sources. For example, current model studies of bacteria and fungal spores only consider the natural emission processes based on land-use types and meteorological parameters like humidity and temperature.22-26 However, anthropogenic sources of PBAP, such as indoor environments,27-29 waste treatments30 and human beings,6 were not included. It was frequently found that the concentrations of bacteria and fungal spores were higher in urban than rural environments, suggesting urban environments are source of bioaerosols.31-36 Characterization, quantification and intercomparison of PBAP are also challenged by the diverse characterization methods.37 Currently, analytical techniques for characterizing PBAP include biological, chemical and physical methods.6, 15, 37, 38 Cultivation, traditional and modern DNA sequencing have been applied to airborne fungi and bacteria quantification.4,

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Biomolecules such as proteins and metabolites (like endotoxin, mannitol) can be analyzed by mass spectrometry.2, 42-44 Auto-, stained or laser induced fluorescence for online or assay based enumeration of biomolecules are usually used as fast quantification of bioaerosols.45-49 In recent years, real-time UV based instruments such as Wideband Integrated Bioaerosol Sensor (WIBS) 4 ACS Paragon Plus Environment

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and Ultraviolet aerodynamic particle sizer (UV-APS) have been applied to study fluorescent bioaerosols in forest, rural, urban and other environments.50-53 This study aims to understand the abundance of PBAP in a heavy polluted megacity (Beijing) in northern China by online and offline fluorescence techniques and offline measurements of organic molecular markers. During dry winter, since vegetation and agricultural activities are lowest throughout the year and no rain events occur which could substantially emit fungal spores into the air,52 anthropogenic activities would relatively contribute more bioaerosols than that in summer time. We report the abundance, size distribution and diurnal variations of FL-resolved fluorescent biological aerosol particles (FBAP). Estimated fungal spore concentrations and organic carbon (OC) contributions from fungal spores are compared between online fluorescence method and offline molecular marker technique, highlighting the possible discrepancy of determining fungal spores in current modelling studies.

MATERIALS AND METHODS Sampling. WIBS measurements (January 29 – February 15, 2015) and filter sampling of total suspended particles (TSP) were performed on the rooftop (8 m a.g.l.) of Hexi building, which is situated at the Tower campus of Institute of Atmospheric Physics (IAP), a typical urban site in northern China.52 Three-hour TSP samples (N=16) were collected during a highly polluted haze event to the following clean period. WIBS Analyses. Individual airborne particles were inhaled into the measuring chamber of WIBS through stainless and hoc tubes. Optical sizes of particles were determined from the scattered light signal (635 nm diode laser). Fluorescent emissions of particles were detected in two wavelength ranges (310 – 400 nm and 420 – 650 nm) excited by xenon wavelengths 5 ACS Paragon Plus Environment

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centered at 280 nm and 370 nm successively. The size cut for total particle analysis was set to 0.8 µm.52, 54 Fluorescence thresholds were determined as the average fluorescence intensities plus 3-fold standard deviations by using forced trigger mode data. Definitions of fluorescenceresolved (FL-resolved) particles were illustrated in Figure S1.50, 54-59 Offline Fluorescence and Factor Analyses of EEMs. A fluorometer (Fluoromax-4, Horiba) and a UV-Vis spectrophotomer (U3900H, Hitachi) were used to analyze the excitation-emission matrix of water-soluble contents of aerosols.52,

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PARAFAC model with non-negativity

constraints was applied to analyze the factors of EEMs (drEEM 0.2.0 MATLAB toolbox).61 Three components were resolved and designated as HULIS-2, HULIS-1 and PLOM (protein-like organic matter).46, 62 OC Measurement. OC was determined by an OC/EC Carbon Aerosol Analyzer (Sunset Laboratory Inc.) following a NIOSH protocol.63 Average OC concentration of blank samples was less than 9.3% of real samples. Organic Marker Analyses. For each sample, a filter aliquot was extracted for three times with dichloromethane/methanol (2:1, v/v) under ultra-sonication for 10 minutes. Solvent extracts were filtered through quartz wool and concentrated by a rotary evaporator and nitrogen-stripped to dryness. Derivatization was then performed by reaction with 30 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 10 µL of pyridine and 1% trimethylsilyl chloride at 70 °C for 3 hours. The derivatives were diluted with 30 µL of n-hexane containing 1.43 ng µL–1 of the internal standard (C13 n-alkane) prior to gas chromatography/mass spectroscopy (GC/MS) analysis. GC/MS measurements were performed on an Agilent model 7890 GC coupled to Agilent model 5975C MSD. The mass spectrometer was operated on Electron Ionization (EI) mode at 70 eV and scanned over the range of 50 – 650 Da. The response

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factors for target compounds were determined with authentic standards. Recoveries of sugars were better than 80%. Field blanks were subtracted from the real samples.

RESULTS AND DISCUSSION Abundance of FAP. The number concentrations of non-FAP and FAP during clean and polluted periods are shown in Figure 1 and Table S2. Separation of clean and polluted periods was based on the number concentration of total aerosol particles (sum of non-FAP and FAP, Figure S3, Table S3). During polluted periods, the abundance of total particles increased by more than one order of magnitude, with the largest enhancement of two orders (Table S3). Four clean and four polluted periods were discerned. During clean periods, the number concentrations of non-FAP and FAP were 4.40 × 103 ± 2.12 × 103 L–1 (average ± sdev) and 642 ± 297 L–1, respectively. And for polluted periods, the loadings surged to 61.8 × 103 ± 44.2 × 103 L–1 and 15.3 × 103 ± 16.8 × 103 L–1, which increased by 14.0 and 23.9 folds, respectively. The variations of both non-FAP and FAP were much higher during polluted periods than clean periods. The average fluorescence intensity of PLOM (Table S5) was around 5.2 times higher during the polluted period than the clean period, which was similar with the FL1 number concentration ratio (6.0). Fractions of FAP in total particles were 13.3% to 16.5% for clean and polluted periods. More than half of FAP were FL B aerosols representing the highest fraction of FAP (clean: 65.9%, polluted: 65.5%, Figure 1), which was similar to that in Nanjing.64 FL1 particles were relatively more abundant in clean periods (24.5%) than in polluted periods (10.1%), while FL C particles showed opposite features. The contribution of FL BC to FAP were relatively constant during clean and polluted periods.

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For intercomparison, considering the conversion factor of UV-APS concentration to WIBS-4 FL3 (2.7),65 FL3 abundance of WIBS-4 (5.02 × 103 L–1) was similar to that measured by UVAPS (~2300 L–1) in polluted periods in another study conducted in Beijing.41 It should be noted that currently the conversion factor has been only reported in one field comparison study, which pointed that UV-APS was less sensitive to smaller fluorescent aerosols ( 3 µm.

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Mealtime enhancements of the abundance of FL1 particles (FL A, AB, ABC) and FL B particles were found in the diurnal patterns of the FL-resolved FAP, whereas this phenomenon was absent for non-FAP in clean periods (Figure 3, Figure S7 and S8). The predominant enhancements were in the size range between 2 and 4 µm for FL1 particles, and around 1 µm for FL BC and C particles and in both ranges for FL B aerosols (Figure S9-S12 for size-resolved diurnal plots). In polluted periods, however, the highest number concentration of FL1 occurred at night. These enhancements were most possibly due to the relatively stable atmospheric conditions and the downward movement of the planetary boundary layer which trapped particles and enhanced adsorption of non-biological fluorophores such as PAHs, particularly low volatile PAHs containing more rings, onto existing particles.73 However, local mealtime enrichments of these FAP were also cognizable in polluted periods. To attribute these FAP as FBAP, exclusions of possible interferences, such as PAHs, secondary organic aerosol (SOA) and HULIS,45,

66, 74

are discussed as follows. First, as

extensively studied in winter Beijing, PAHs were mainly emitted from coal combustion for heating, followed by diesel and gasoline vehicle exhausts,73, 75-78 and showed a diurnal peak at night time.77 If PAHs contributed mainly to these enhancements, stronger enrichment of these FAP should appear at night.75, 77, 79 A study in Beijing reported that nearly half of particulate PAHs were less than 1.1 µm.73 However, even for sub-micron FL1 particles, their abundances were relatively low at night during clean periods (Figure S10). Second, the quantum yields of SOA are usually two orders of magnitude lower than that of typical biological fluorophores, like tryptophan, tyrosine and flavins.74 SOA reside largely in the accumulation mode, peaking at ~ 450 nm in winter Beijing.77 Also, the values of two fluorescent indices for water soluble contents of clean-day TSP samples (HIX: 1.17 – 2.02; BIX: 1.11 – 1.65) resided in different ranges from

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those of O3 and OH oxygenated limonese and α-pinene SOA (HIX: 4.2 – 6.1; BIX: 0.57 – 0.82),74 suggesting different sources of major fluorophores. Finally, HULIS fluorescence has been recognized in three main characteristic spectral positions (Ex/Em, nm): (i) 300-370/400-500, (ii)312/380-420 and (iii) 237-260/380-460, which do not coincide with that of FL1 channel (280/310-400). The fluorescence ability of dry humic and fulvic acids was also comparably much lower than typical biomolecules.45, 56, 66, 74 Since HULIS are not chemically identical, it is still of high uncertainty to determine their spectral characteristics from limit HULIS surrogates. Besides, most of the spectral properties of HULIS aerosols have been studied in dissolved states, which makes it difficult to compare with online measurement of aerosols in relatively dry state. In conclusion, although these interferences cannot be fully eliminated, combining the size distribution and diurnal features, it is the best estimate of this online fluorescence-based method to attribute these FL1 particles as FBAP. In other studies, diurnal variations of FBAP were found to have diverse modes. A diurnal peak at night in natural environments was frequently observed,47 which was found to be correlated with humidity, wind speed and temperature. However, no correlations were found for all FLresolved FAP in clean periods with meteorological parameters (relative humidity and temperature Figure S13, S14). Thus, considering the mealtime enhancements of these FBAP in Beijing, they most likely originated from anthropogenic activities. Cooking activities could be possible sources of these bioaerosols, originating from the ventilation system of kitchens or from the exhausted indoor kitchen air.80 Ventilation pipes of kitchen can provide nutrient matrix for microbial growth since organic materials and water vapor from cooking can be accumulated. Besides, food material, food-borne microorganisms and bacteria grown on the surfaces of kitchens are natively biological materials.81 It was also found that indoor-to-outdoor ratio of

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fungi was especially higher in winter (58.2) than summer (3.17),36 suggesting a much stronger indoor source of bioaerosols in bio-inactive season.28 As anthropogenic activities are common and nearly consistent throughout a year, it is proposed that these human activities should be considered as an important source of PBAP. This study motivates the need for further laboratory, field and model studies of the release of PBAP of anthropogenic sources. FAP of Mixed Origins. The normalized size distributions of FL C and BC particles were similar to non-FAP, showing a dominant fine mode (< 2 µm). This feature was also observed in sub-urban Nanjing in Autumn.64 However, it was different from the aircraft observations over south border of the US (peaked at 6 µm) in autumn.56 The predominant fine size distribution of FL3 was also similar to UV-APS observations in winter Beijing82 and urban Helsinki.67 This predominant fine mode was slightly enhanced in polluted periods, which was frequently suggested as interferences from PAHs.45, 54, 64, 83 However, diurnal peaks for both fine and coarse FL C and BC particles did not appear at night as for PAHs, but at around 8:00. Coarse FL BC particles peaked at around 19:00, which was same as FL B. FL-size-resolved correlation coefficients between non-FAP and FL C (R2 = 0.02 – 0.31)/BC (R2 = 0.04 – 0.34) were much lower for clean periods than that in polluted episodes (R2 = 0.38 – 0.68 and R2 = 0.18 – 0.68, respectively; in Supplement Excel Table). The correlation coefficients (R2) between FL C (FL BC) and FL1 particles were no more than 0.13 (0.25) (Table S7). These features suggested that comparing with FL1 biological particles, FL C, BC particles have mixed origins, including biological aerosols different from FL1 FBAP, non-biological aerosols and possible particles internally mixed by bio- and non-biological matter.

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Estimated Contribution of Fungal-Spores to OC. Average mass concentrations of arabitol (2.5 ng m–3) and mannitol (3.9 ng m–3) were of the same level as those measured by Liang et al.31 for PM10 in winter Beijing. Estimation of the number concentration of fungal spores was very similar from arabitol and mannitol (Table 1). The UV-LIF method for detecting fungal spores was investigated in many previous field and laboratory studies.56, 83-85 The FAP in the size mode of 2 – 5 µm were frequently attributed to fungal spores, with evidence from Sporewatch and SEM.65 And it was found that WIBS FL1 particles (> 3 µm) were positively correlated with Ascospores optically counted from Sporewatch sampling.70 Cluster analysis of WIBS-4 FAP measured at a mid-latitude forest site assigned FAP with strong FL1_280 fluorescence intensities but with minor FL2_280 and FL2_370 signals as fungal spores, which were supported by the correlations with relative humidity.86 Although in another study conducted in Killarney, a coastal site, the correlation of FL1 particles with fungal spores were less significant, this was attributed to the large abundance of smaller bacteria.65 However, the author noted that Cladosporium could not be effectively detected by the FL1 channel, and Ascospores’s fluorescence in FL2 and FL3 channels were very weak. In previous laboratory tests of fluorescent properties of some fungal spores, like Tritirachium, Aspergillus versicolor, all of them can be classified as FL A, FL AB, and FL ABC, while Sychophalastrum spores also appeared as FL B particles56. Another recent laboratory investigation of the fluorescent properties of aerosolized bacteria, fungi and pollen grains reported that more than 90% of bacteria (< 1 µm) were FL A aerosols, more than 90% of fungal spores (2 – 9 µm) were FL A, FL AB or FL ABC particles.85 Although intercomparison of different WIBS instruments revealed shift of relative abundance of fungal spores among different FL types, most of them still remained in the FL1 category. Among those studied fungal spores,

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Cladosporium, Fusarium, Pennicillium and Aspergillus were four of the six most abundant fungi genera in Beijing.87 To give an estimation of fungal spores from WIBS measurement, for particles in the size range of 1.8 – 5.0 µm, FL A + FL AB were regarded as a lower estimate of fungal spores (number concentration, NwibsL), and FL A + FL AB + FL ABC were set as a higher estimate (NwibsH). In the latter clean period, the estimated NwibsL and NwibsH were similar to each other, which were 81 and 87 times higher than that estimated from the fungal spore tracers (arabitol and mannitol), respectively (Table 1)88. For the contribution of OC from fungal spores, the molecular marker based method only resulted in a very small OC fraction (~ 0.11%), whereas fungal spores accounted for 10% in average from the WIBS estimation. In another study of fungal spores in Beijing89, 90, the conversion factors were 0.49 and 0.38 pg per spore for mannitol and arabitol, respectively. Compared with the factors from the study in Vienna88, it will cause increase of fungal spore estimation by 3.47 (mannitol) and 3.16 (arabitol) folds. As an example, based on mannitol, the number concentration of fungal spore would be estimated to be 3.82 L-1 and 13.3 L-1 for clean and polluted periods, respectively, which are still much lower than those estimated from WIBS (lower estimate NwibsL, 89.2 L-1and 428 L-1). According to Healy et al. (2014)65, dismissing the smaller particle influence, taking a moderate conversion factor as 8 between WIBS and Sporewatch count, WIBS estimate of fungal spores would be still nearly one order of magnitude higher than the molecular marker method. Modelling studies of PBAP or specific kinds of biological aerosols, such as fungal spores or bacteria are still sparse. The modeled annual mean fungal spore number concentration in Beijing was reported to be < 25 L–1.24 Considering that fungal spore loading was the smallest in winter Beijing,91 the modeled abundance should be even lower. And this value was one order of

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magnitude lower than the estimated NwibsL/NwibsH (225/256 L-1) from WIBS for the whole measuring period. In another study, the number concentrations of measured fluorescent biological particles (UV-APS, WIBS3 and WIBS4) were also higher than simulated fungal spores, especially for urban Kalsruhe in October.23 For a semi-arid temperate forest without strong influence of human activities,92 the fungal spore number concentration estimated from arabitol and mannitol were found to be consistent with the model results, which was driven by natural processes with input of leaf area index and atmospheric water vapor.24, 84 This may be due to that current model studies of bacteria and fungal spores only consider the natural emission processes based on land-use types and meteorological parameters like humidity and temperature, neglecting their anthropogenic release.22-26 These contrasting results highlight the gaps between traditional molecular marker based fungal spore characterization and the relatively new online fluorescence measurement. Several reasons can result in this large discrepancy. First, it has been reported that airborne bacteria usually cluster together to form into particles in the size range of around 3 µm.85 Second, fragments of plant and animal debris that contain proteins can also be interferences.6 And third, it may also be possible that fungi or fungal fragments growing in ventilation pipes for cooking usage are forcedly blown into the atmosphere, in which process fungi do not fully produce enough arabitol or mannitol that are usually contained in fungal spores. Thus, using the same conversion factor should cause an underestimation for fungal spores. This large discrepancy should be further investigated to better constrain the contribution of fungal spore and primary biological materials to atmospheric particulate matter. The possible new PBAP emission source of human activities may exert a large driven force to organic aerosol transformation and the possible health stress may be much more severe than current knowledge.

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The outflows of PBAP to other regions from urban environments by atmospheric circulation should be readdressed especially during haze events in East and South Asia.

ASSOCIATED CONTENT Supporting Information The following supporting information are available free of charge. Supporting description of analyzing methods, figures and tables (PDF) Supporting table (XLSX)

AUTHOR INFORMATION Corresponding Author * Phone : +86-10-8201-3200; e-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors are grateful for the financial support from the National Natural Science Foundation of China (Grant Nos. 41475117, 41625014 and 41571130024). REFERENCES 1. Lindemann, J.; Constantinidou, H. A.; Barchet, W. R.; Upper, C. D., Plants as sources of airborne bacteria, including ice nucleation-active bacteria. Appl. Environ. Microbiol. 1982, 44, (5), 1059-1063. 2. Fu, P.; Kawamura, K.; Okuzawa, K.; Aggarwal, S.; Wang, G.; Kanaya, Y.; Wang, Z., Organic molecular compositions and temporal variations of summertime mountain aerosols over Mt. Tai, North China Plain. J. Geophys. Res. Atmos. 2008, 113, (D19), D19107.

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88. Bauer, H.; Claeys, M.; Vermeylen, R.; Schueller, E.; Weinke, G.; Berger, A.; Puxbaum, H., Arabitol and mannitol as tracers for the quantification of airborne fungal spores. Atmos. Environ. 2008, 42, (3), 588-593. 89. Liang, L.; Engling, G.; Cheng, Y.; Duan, F.; Du, Z.; He, K., Rapid detection and quantification of fungal spores in the urban atmosphere by flow cytometry. J. Aerosol Sci. 2013, 66, 179-186. 90. Liang, L.; Engling, G.; Du, Z.; Duan, F.; Cheng, Y.; Liu, X.; He, K., Contribution of fungal spores to organic carbon in ambient aerosols in Beijing, China. Atmos. Pollut. Res. 2017, 8, (2), 351-358. 91. Liang, L.; Engling, G.; Du, Z.; Cheng, Y.; Duan, F.; Liu, X.; He, K., Seasonal variations and source estimation of saccharides in atmospheric particulate matter in Beijing, China. Chemosphere 2016, 150, 365-377. 92. Kim, S.; Karl, T.; Guenther, A.; Tyndall, G.; Orlando, J.; Harley, P.; Rasmussen, R.; Apel, E., Emissions and ambient distributions of Biogenic Volatile Organic Compounds (BVOC) in a ponderosa pine ecosystem: interpretation of PTR-MS mass spectra. Atmos. Chem. Phys. 2010, 10, (4), 1759-1771.

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Figure 1. (a) Box plots of the number concentrations of fluorescent aerosol particles (FAP) and non-fluorescent aerosol particles (non-FAP) in clean (c1 – c4) and polluted periods (p1 – p4). The pie charts represent the fractions of FAP and non-FAP in total particles (>0.8 µm). The areas of the pie charts represent the relative number concentration of total particles. (b) Relative contributions of fluorescence-resolved (FL-resolved) particles in clean and polluted periods.

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Number Concentration

Number Concentration -1 (L(L-1))

(a) 250x10

FAP

84%

(b)

87%

84%

non-FAP

76%

200

16%

13%

16%

150

24% 87%

86%

88%

88%

100

13%

14%

12%

12%

50 0 c1

Fraction to FAP (%)

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p1

c2

p2

c3

p3

c4

p4

100

C BC B AC ABC AB A

80 60 40 20 0 c1

p1

c2

p2

c3

p3

c4

p4

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Figure 2. Normalized size distributions of FL-resolved FAP during clean (c1 – c4) and polluted periods (p1 – p4). Normalization was performed by dividing the highest value. The reported fluorescent aerosol particle categories are (a) FL A, (b) FL AB, (c) FL ABC, (d) FL B, (e) FL BC and (f) FL C.

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Figure 3. Diurnal variations of the number concentrations of FL-resolved FAP. Solid lines with shadows of standard deviations represent clean periods. Lines with circles are data for polluted periods. The reported fluorescent aerosol particle categories are (a) FL A, (b) FL AB, (c) FL ABC, (d) FL B, (e) FL BC and (f) FL C.

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Table 1. Number concentrations and OC fractions of fungal spore origins, estimated from mass concentrations of arabitol, mannitol and WIBS particle number concentrations (lower estimation for integration of FL A and FL AB, higher estimation for integration of FL A, FL AB and FL ABC) for clean and polluted periods. Clean period range

Polluted period

mean std

range

mean std

Number Concentrations (L–1) NAra

0.3 – 3.6

1.1

1.0

1.2 – 7.5

3.6

2.4

NManni

0.2 – 3.7

1.1

1.1

1.5 – 7.8

4.2

2.5

NwibsL

38.8 – 154

89.2

41.8

218 – 704

428

171

NwibsH

44.0 – 165

95.7

43.5

242 – 869

496

219

OC Fractions from Fungal Spores (%) OCFAra

0.03 – 0.35

0.11

0.09

0.03 – 0.28

0.11

0.10

OCFManni

0.02 – 0.37

0.11

0.10

0.03 – 0.30

0.12

0.10

OCFwibsL

3.3 – 15.1

9.5

4.5

8.3 – 11.8

10.2

1.6

OCFwibsH

3.6 – 16.0

10.2

4.6

9.2 – 14.5

11.7

2.2

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