Abstract. Within the last years pollen grains have gained increasing attention due to their cloud-forming potential. Especially the discovery that ice nucleating macromolecules (INMs) or subpollen particles (SPPs) obtained from pollen grains are able to initiate freezing has stirred up interest in pollen. INMs and SPPs are much smaller and potentially more numerous than pollen grains and could significantly affect cloud formation in the atmosphere. However, INMs and SPPs are not clearly distinguished. This has motivated the present study, which focuses on birch pollen and investigates the relationship between pollen grains, INMs, and SPPs. According to the usage of the term SPP in the medical fields, we define SPPs as the starch granules contained in pollen grains. We show that these insoluble SPPs are only obtained when fresh pollen grains are used to generate aqueous extracts from pollen. Due to the limited seasonal availability of fresh pollen grains, almost all studies have been conducted with commercial pollen grains. To enable the investigation of the SPPs we develop an alternative extraction method to generate large quantities of SPPs from commercial pollen grains. We show that INMs are not bonded to SPPs (i.e. can be washed off with water). Further, we find that purified SPPs are not ice nucleation active: after several times of washing SPPs with ultrapure water the ice nucleation activity completely disappears. To our knowledge, this is the first study to investigate the ice nucleation activity of isolated SPPs. To study the chemical nature of the INMs, we use fluorescence spectroscopy. Fluorescence excitation–emission maps indicate a strong signal in the protein range (maximum around λex = 280 nm and λem = 330 nm) with all ice nucleation active samples. In contrast, with purified SPPs the protein signal is lost. We also quantify the protein concentration with the Bradford assay. The protein concentration ranges from 77.4 µg mL−1 (highly concentrated INMs) to below 2.5 µg mL−1 (purified SPPs). Moreover, we investigate the connection between proteins and ice nucleation activity by treating the ice nucleation active samples with subtilisin A and urea to unfold and digest the proteins. After this treatment the ice nucleation activity clearly diminished. The results indicate a linkage between ice nucleation activity and protein concentration. The missing piece of the puzzle could be a glycoprotein which exhibits carboxylate functionalities, can bind water in tertiary structures, and displays degeneration and unfolding of its secondary structure due to heat treatment or reaction with enzymes. Even though purified SPPs are not ice nucleation active they could act as carriers of INMs and distribute those in the atmosphere.

Terrestrial ecosystems can influence atmospheric processes by contributing a huge variety of biological aerosols (bioaerosols) to the environment. Several types of biological particles, such as pollen grains, fungal spores, and bacteria cells, trigger freezing processes in super-cooled cloud droplets, and as such can contribute to the hydrological cycle. Even though biogenic particles are known as the most active form of ice nucleation particles (INPs), the transport to high tropospheric altitudes, as well as the occurrence in clouds, remains understudied. Thus, transport processes from the land surface into the atmosphere need to be investigated to estimate weather phenomena and climate trends. To help fill this knowledge gap, we developed a drone-based aerosol particles sampling impinger/impactor (DAPSI) system for field studies to investigate sources and near surface transport of biological INPs. DAPSI was designed to attach to commercial rotary-wing drones to collect biological particles within about 100 m of the Earth’s surface. DAPSI provides information on particulate matter concentrations (PM10 & PM2.5), temperature, relative humidity, and air pressure at about 0.5 Hz, by controlling electrical sensors with an onboard computer (Raspberry Pi 3). Two remote-operated sampling systems (impinging and impacting) were integrated into DAPSI. Laboratory tests of the impinging system showed a 96% sampling efficiency for standardized aerosol particles (2 µm polystyrene latex spheres) and 84% for an aerosol containing biological INPs (Betula pendula). A series of sampling missions (12 flights) were performed using two Phantom 4 quadcopters with DAPSI onboard at a remote sampling site near Gosau, Austria. Fluorescence microscopy of impactor foils showed a significant number of auto-fluorescent particles < 0.5 µm at an excitation of 465–495 nm and an emission of 515–555 nm. A slight increase in ice nucleation activity (onset temperature between −27 °C and −31 °C) of sampled aerosol was measured by applying freezing experiments with a microscopic cooling technique. There are a number of unique opportunities for DAPSI to be used to study the transport of bioaerosols, particularly for investigations of biological INP emissions from natural sources such as birch or pine forests.

Abstract. Silver birch (Betula pendula) is known to contain ice-nucleating macromolecules (INMs) to survive in harsh environments. However, little is known about the release and transport of INMs from birch trees into the atmosphere. In this study, we conducted in situ and in vivo investigations on INMs from nine birches growing in an alpine valley (Ötztal, Austria). A detailed analysis of drill cores showed that INM concentration increases towards outer layers, reaching its maximum near the surface. Aqueous extracts from the surfaces of leaves, bark, primary wood and secondary wood contained INMs (34∕36) with concentrations ranging from 9.9×105 to 1.8×109 INMs cm−2. In a field study, we analysed the effect of precipitation on the release of these INMs attached to the surface of the trees. These experiments showed that INMs are splashed and aerosolized into the environment during rainfall events, at concentrations and freezing temperatures similar to in vivo samples. Our work sheds new light on the release and transport of INMs from birch surfaces into the troposphere. Birches growing in boreal and alpine forests should be considered an important terrestrial source of INMs.

Abstract. Within the last years pollen grains have gained increasing attention due to their cloud forming potential. Especially the discovery that ice nucleating macromolecules (INM) or subpollen particles (SPP) obtained from pollen grains are able to initiate freezing has stirred up interest in pollen. INM or SPP are much smaller and potentially more numerous than pollen grains and could significantly affect cloud formation in the atmosphere. However, INM and SPP are not clearly distinguished and explanations on how these materials could distribute in the atmosphere are missing. In this study we focus on birch pollen and investigate the relationship between pollen grains, INM and SPP. According to the usage of the term SPP in the medical fields we define SPP as the starch granules contained in pollen grains. We develop an extraction method to generate large quantities of SPP and show that INM are loosley attached to SPP. Further, we find that purified SPP are not ice nucleation active: after several times of washing SPP with ultrapure water the ice nucleation activity completely disappears. To our knowledge this is the first study to investigate the ice nucleation activity of isolated SPP. To study the chemical nature of the INM we use fluorescence spectroscopy. Fluorescence excitation-emission maps indicate a strong signal in the protein range (maximum around λex = 280 nm and λem = 330 nm) that correlates with the ice nucleation activity. In contrast, with purified SPP this signal is lost. We also quantify the protein concentration with the Bradford assay. The protein concentration ranges from 77.4 μg mL−1 (Highly concentrated INM) to below 2.5 μg mL−1 (purified SPP). The results indicate a linkage between ice nucleation activity and protein concentration. Even though purified SPP are not ice nucleation active they could act as carriers of INM and distribute those in the atmosphere.

&lt;p&gt;Within the last years pollen grains have gained increasing attention due to their cloud-forming potential. Especially the discovery that ice nucleating macromolecules (INMs) or subpollen particles (SPPs) obtained from pollen grains are able to initiate freezing has stirred up interest in pollen. INMs or SPPs are much smaller and potentially more numerous than pollen grains and could significantly affect cloud formation in the atmosphere.&lt;/p&gt;&lt;p&gt;However, INMs and SPPs are not clearly distinguished. This has motivated the present study, which focuses on birch pollen and investigates the relationship between pollen grains, INMs and SPPs. According to the usage of the term SPPs in the medical fields, we define SPPs as the starch granules contained in pollen grains. We show that these insoluble SPPs are only obtained when fresh pollen grains are used to generate aqueous extracts from pollen. Due to the limited seasonal availability of fresh pollen grains almost all studies have been conducted with commercial pollen grains. To enable the investigation of the SPPs we develop an alternative extraction method to generate large quantities of SPPs from commercial pollen grains. We show that INM are not bonded to SPPs (i.e. can be washed off with water). Further, we find that purified SPP are not ice nucleation active: after several times of washing SPPs with ultrapure water the ice nucleation activity completely disappears. To our knowledge, this is the first study to investigate the ice nucleation activity of isolated SPPs.&lt;/p&gt;&lt;p&gt;To study the chemical nature of the INMs, we use fluorescence spectroscopy. Fluorescence excitation-emission maps indicate a strong signal in the protein range (maximum around &amp;#955;&lt;sub&gt;ex&lt;/sub&gt; = 280 nm and &amp;#955;&lt;sub&gt;em&lt;/sub&gt; = 330 nm) with all ice nucleation active samples. In contrast, with purified SPP the protein signal is lost. We also quantify the protein concentration with the Bradford assay. The protein concentration ranges from 77.4 &amp;#956;g mL&lt;sup&gt;-1&lt;/sup&gt; (Highly concentrated INM) to below 2.5 &amp;#956;g mL&lt;sup&gt;-1&lt;/sup&gt; (purified SPP). Moreover, we investigate the connection between proteins and ice nucleation activity by treating the ice nucleation active samples with subtilisin A and urea to unfold and digest the proteins. &amp;#160;After this treatment the ice nucleation activity clearly diminished. The results indicate a linkage between ice nucleation activity and protein concentration. The missing piece of the puzzle could be a glycoprotein, which exhibits carboxylate functionalities, can bind water in tertiary structures and displays degeneration and unfolding of its secondary structure due to heat treatment or reaction with enzymes. Even though purified SPPs are not ice nucleation active they could act as carriers of INMs and distribute those in the atmosphere.&lt;/p&gt;&lt;p&gt;Reference of the study: Burkart, J., Gratzl, J., Seifried, T. M., Bieber, P., and Grothe, H.: Isolation of subpollen particles (SPPs) of birch: SPPs are potential carriers of ice nucleating macromolecules, Biogeosciences, 18, 5751&amp;#8211;5765, https://doi.org/10.5194/bg-18-5751-2021, 2021.&amp;#160;&lt;/p&gt;