The Importance of Biological Particles to the Ice Nucleating Particle Concentration in a Coastal Tropical Site

Atmospheric aerosol particles that can nucleate ice are referred to as ice nucleating particles (INP). Recent studies have confirmed that aerosol particles emitted by midand high-latitude oceans can act as INPs. This very relevant information can be included in climate and weather models to predict the formation of ice in clouds, given that most of them do not consider the oceans as 5 a source of INPs. Very few studies to sample INPs have been carried out in tropical latitudes, and there is a need to evaluate their availability to understand the potential role that marine aerosol may play in the hydrological cycle of tropical regions. This study presents results from the first measurements obtained during a field campaign conducted in the topical village of Sisal, located on the coast of the Gulf of Mexico of the Yucatan 10 peninsula in Mexico in January-February 2017, and one of the few data sets currently available at similar latitudes. Aerosol particles sampled in Sisal are shown to be very efficient INPs, with onset freezing temperatures as high as -3 ◦C (in some cases), similar to the onset temperature for Pseudomonas syringae. The results show that the INP concentration in Sisal is higher than at other locations sampled with the same type of INP counter. Air masses arriving in Sisal during the passage 15 of cold fronts have, surprisingly, higher INP concentrations than the campaign-average, despite their lower total aerosol concentration. Biological particles were likely found to be very important in ice cloud formation at this tropical 1 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1215 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 6 December 2018 c © Author(s) 2018. CC BY 4.0 License.

most important oceanic sources of INPs are the southern oceans, north Atlantic, and north Pacific (Burrows et al., 2013;Yun and Penner, 2013;Wilson et al., 2015;Vergara-Temprado et al., 2017); 55 however, those conclusions were drawn with little or no data from tropical latitudes.

Ice nucleating particles
To determine the INP concentrations in the ambient air, aerosol particles were collected on hydrophobic glass cover slips (HR3-215; Hampton Research) with the help of a Micro-Orifice Uniform Deposit Impactor (MOUDI 110R, MSP). Identical substrate holders as those described in Mason et al. (2015a) were used to keep the glass coverslips at a location on the impaction plate 125 where particle concentrations varied by a relatively small amount. The MOUDI has eight stages at which particles are separated and collected as a function of their aerodynamic diameter (cut-sizes are 0.18 µm, 0.32 µm, 0.56 µm, 1.0 µm, 1.8 µm, 3.2 µm, 5.6 µm, and 10.0 µm). The flow through the MOUDI is 30 L min −1 and the typical sampling time was 6 h. It has been recognized that when sampling with a MOUDI under dry conditions (i.e, RH below approximately 60 %), aerosol parti-130 cles can bounce from the impaction plates moving to lower stages (Winkler, 1974;Chen et al., 2011;Bateman et al., 2014). Although this is a known artifact when using this technique, this may not have been an issue in the current study given that the ambient RH was typically above 67 %. The glass substrates containing the ambient aerosol particles were stored in petri dishes at 4 • C prior to their analysis.

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The INP concentrations were measured with a cold cell coupled to an optical microscope with an EC Plan-Neofluar 5 X objective (Axiolab, Zeiss) following the MOUDI-DFT method described by Mason et al. (2015a). Activation scans were conducted between 0 • C and -40 • C at a cooling rate of -10 • C per minute for particles collected on stages two to seven. Stage one was not taken into account given that the aerosol concentration on the glass substrates was typically very low, whereas 140 in stage eight the number concentration of particles deposited on the glass substrates was too high which inhibited the proper formation of water drops.

Chemical composition
A second MOUDI (100NR, MSP) was operated simultaneously to collect aerosol particles for chemical composition analysis with particle sizes ranging from 0.18 µm to 10.0 µm. Particles were col-145 lected on 47 mm Teflon filters (Pall Science) for 48 h at a flow rate of 30 L min −1 . The filters were weighed prior and after the sampling and stored in petri dishes at 4 • C until they were analyzed.
The filters were first analyzed for elemental composition and followed by ion-cation concentration analysis, performing two different analyses on each filter.
Cycle conditions were as follows: initial denaturation at 94 • C for 1 min; followed by 35 cycles at 94 • C for 1 min, 56 • C for 30 s, 72 • C for 1.5 min; and a final extension at 72 • C for 5 min. The

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PCR products were examined for size and yield using 1.0 % (w/v) agarose gels in TAE buffer. After successful amplification, the obtained products were sequenced using a PRISM 3730 automated sequencer (Applied Biosystem Inc.). DNA sequences were edited and assembled using the Seq-Man and Edit Seq software (Chromas Lite, Technely Slom Pty Ltd. USA). Sequence similarity analysis was performed using the BLAST software (http://www.ncbi.nlm.nih.gov/BLAST).

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Although specific growing media for actinobacteria were not used in this study, some actinobacteria colonies were able to grow on the TSA petri dishes; therefore, in some cases they were isolated and identified as follows. Genomic DNA was extracted using standard protocols reported previously for actinobacteria (Maldonado et al., 2009). The DNA preparations were then used as template for 16S rRNA gene amplification using the universal set of bacterial primers 27f and 1525r (Lane, The PCR amplification was achieved using a Techne 512 gradient machine using the protocol de-210 scribed in Maldonado et al. (2008). The expected product (size aprox 1,500 bp) was checked by horizontal electrophoresis (70 V, 40 min) and then purified using the QIAquick PCR purification kit (QI-AGEN, Germay) following the manufacturers instructions. Purified 16S rRNA gene PCR products were sent for sequencing to Macrogen (Korea) for the DyeDeoxy Terminator Cycle Sequencing kit (Applied Byosystems). Assembly of each 16S rRNA gene sequence was performed using Chromas 215 (www.technelysium.com.au) and checked manually with the SeaView software (Galtier et al., 1996).
Each assembled sequence was compared against two databases, namely, (a) the GenBank database (www.ncbi.nlm.nih.gov) by using the BLAST option and (b) the EZCloud (www.ezbiocloud.net) under its EZTaxon option. Both databases generated a list of the closest phylogenetic neighbors to each sequence and the EZTaxon, specifically provided the list of the closest described (type) species.

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At least 650 bp were employed for the analyzes. entire sampling period of 896±570 cm −3 and 0.026±0.022 cm −3 , respectively. The data reported by the CPC and the LasAir indicate that most of the aerosol particles were smaller than 300 nm.

Aerosol Concentration and Meteorology
A similar result was found by Rosinski et al. (1988) in the Gulf of Mexico (GoM) who found that the aerosol concentration for particles ranging between 0.5 µm and 1.0 µm was three to four orders 230 of magnitude smaller than particles ranging between 0.003 µm and 0.1 µm. A decrease in aerosol particle concentration was observed at the arrival and during the passage of two cold fronts (indicated by the vertical grey areas in Fig Hysplit back-trajectories from the measurement site were run for 72 h for each of the days of the campaign. In the absence of cold fronts A and B, the air masses arriving in Sisal had a very likely 240 large continental influence, associated with southerly winds ( Figure S1). However, when the cold fronts A and B reached the Yucatan Peninsula, northerly and northwesterly winds prevail, associated with more marine influence. The arrival of the cold fronts was also confirmed with the National Oceanic and Atmospheric Administration (NOAA) surface maps for January 22 and 29 ( Figure S2).
Air masses behind both cold fronts, flowing over the GoM, were characterized by lower aerosol 245 particle concentrations than air masses coming from the south to the site. This result agrees well with a large body of evidence indicating that marine air masses have lower aerosol particle concentration than continentally-influenced air masses (Patterson et al., 1980;Fitzgerald, 1991). Note, however that the number size distributions of the aerosol particles larger than 300 nm did not significantly change before, during or after the passage of the cold fronts ( Figure S3).

Ice Nucleating Particle Concentration ([INP])
A total of 41 samples (8 stages each) were collected during the Sisal field campaign to calculate the [INP] as a function of temperature and particle size. Some of these samples showed a high ice nucleating activity with high [INP] at temperatures close to 0 • C. In some cases the onset freezing temperatures were found to occur at temperatures as high as -3 • C.  Recalling that the air masses behind cold front B contained a lower aerosol particle concentration, this suggests that the marine particles in that air mass are more efficient INPs than in the air masses with more continental influence. Additionally, the chemical composition of the aerosol particles collected by Rosinski et al. (1988) indicate that the air masses in the GoM in July-August were largely influenced by mineral dust particles. This is further discussed below. were different for the temperatures considered, in contrast with the results obtained by Mason et al. (2015b) at the Pacific coast of Canada. At -15 • C the peak concentration was found to be in particles 300 ranging between 1.0 µm and 1.8 µm. This has been reported as the typical size for airborne bacteria (Burrows et al., 2009). Similar size distributions were obtained at -20 • C and -25 • C with a peak concentration for particles ranging in size between 3.2 µm and 5.6 µm. Finally, at -30 • C the peak was observed at smaller sizes (i.e., between 1.8 µm and 3.2 µm).

Identification of the Potential INP Sources
The chemical analyzes of the sampled aerosol particles indicate that a large fraction of the particle mass (for sizes between 0.18 µm to 10.0 µm) are likely of marine origin (Figure 7(A-B)). Both 310 techniques, i.e., XRF and HPLC found that the main elements/cations/ions are sodium and chlorine.
The low concentrations of Ti, Cu, and Zn shows the very low probability of anthropogenic influence at the sampling site. However, although sulfate and ammonium can be emitted by natural sources, their presence, in addition to nitrates, indicate that the influence of anthropogenic activities to the aerosol population is not completely negligible. Finally, the low concentration of Al, Fe, and Si 315 suggest that mineral dust is not a major contributor of aerosol particle mass during the sampling period. The long-range transport of mineral dust particles from Africa to the Yucatan Peninsula and the GoM is very rare between January and February, compared with the more frequent transport probability during July-August (Rosinski et al., 1988;Kishcha et al., 2014). Rosinski et al. (1988) reported that the concentration of Al, Fe, and Si in July-August 1986 in the GoM were higher than 320 the sodium and chlorine concentrations, although the aerosol was supposed to be of marine origin.   Table 2 summarizes the identified bacteria before the arrival of cold front A and after the passage of cold fronts A and B. Additionally, Table 3 shows the fungi identification for the whole campaign.
To our knowledge this is the first time that airborne viable bacteria, and fungi are identified at this coastal location. Although biological microorganism characterization has been previously conducted in Mexico, those studies focused on health effects mainly (Santos-Burgoa et al., 1994;Guzman, 365 1998;Maldonado et al., 2009;Frías-De León et al., 2016;Ríos et al., 2016). Note that 76 % of the detected bacteria were Gram positive with Micrococcus, Staphylococcus, and Bacillus as the main identified genus ( Figure S6). As shown in Regarding fungi, different genus were also identified as shown in Table 3 with Cladosporium and 375 Penicillium as the most frequent ones (51 % and 11 %, respectively) as shown in Figure S6. This is in good agreement with the data reported by Després et al. (2012).
Several studies haven shown the good correlation between the concentration of fluorescent biological particles and the [INP]; however, from those studies it is highly uncertain if the good ice nucleating abilities can be attributed to a single microorganism specie (Mason et al., 2015b;Twohy 380 et al., 2016). Off-line methods as the one used here have been able to identify from rain water and cloud water specific microorganisms such as Pseudomonas syringae, Micrococcus, Staphylococcus, Cladosporium, Penicillium, Aspergillus among others, with some showing good ice nucleating ability (Amato et al., 2007(Amato et al., , 2017Delort et al., 2010;Failor et al., 2017;Stopelli et al., 2017;Akila et al., 2018).

Conclusions
Aerosol particles around Sisal (on the northwest coast of the Yucatan peninsula) were found to be efficient INPs with onset freezing temperatures as high as -3 • C, similar to the onset freezing temperature of the well known efficient INP Pseudomonas syringae (Wex et al., 2015) and Arctic sea surface microlayer organic-enriched waters (Wilson et al., 2015). The results show that the 390 INPs concentration in Sisal are comparable (in specific cases even higher) than at other locations studied using the same INP counter type, especially under the influence of cold fronts. This is an intriguing result given that the air masses behind the cold front contained lower aerosol particles concentrations. This deserves further analysis given than the Yucatan peninsula and the Caribbean region are impacted by this meteorological phenomenon during the winter and early spring months.

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The chemical analyzes performed on the sampled aerosol particles did not indicate the presence of mineral dust particles in significant concentrations (the combined mass concentrations of Al, Si, and Fe correspond to 5.1 % of the total particle mass measured by the XRF), not surprisingly since African dust is mainly transported to South America during January (Prospero et al., 2014). The The quantitative understanding of the importance of biological particles in ice particle formation is a challenging task for the cloud physics community. As shown here, even when combining biology with chemistry, physics, and meteorology, the results obtained are not as quantitative as would be desired. Efficient INPs such as those measured in Sisal could be very important for cloud glaciation. Schnell, R. and Vali, G.: Freezing nuclei in marine waters, Tellus, 27, 321-323, doi:10.1111Tellus, 27, 321-323, doi:10. /j.2153Tellus, 27, 321-323, doi:10. -3490.1975Tellus, 27, 321-323, doi:10. . tb01682.x, 1975