ACP Atmospheric Chemistry and Physics ACP Atmos. Chem. Phys. 1680-7324 Copernicus Publications Göttingen, Germany 10.5194/acp-13-5751-2013 Immersion freezing of ice nucleation active protein complexes Hartmann S. 1 Augustin S. 1 Clauss T. 1 Wex H. 1 Šantl-Temkiv T. 2 Voigtländer J. 1 Niedermeier D. 1 Stratmann F. 1 Leibniz Institute for Tropospheric Research, Permoserstr. 15, 04318 Leipzig, Germany Aarhus University, Stellar Astrophysics Centre, Department of Physics and Astronomy, 120 Ny Munkegade, 8000 Aarhus, Denmark 14 06 2013 13 11 5751 5766 Copyright: © 2013 S. Hartmann et al. 2013 This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/3.0/ This article is available from https://acp.copernicus.org/articles/13/5751/2013/acp-13-5751-2013.html The full text article is available as a PDF file from https://acp.copernicus.org/articles/13/5751/2013/acp-13-5751-2013.pdf

Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between −5 °C to −38 °C. Snomax™ is an industrial product applied for artificial snow production and contains <i>Pseudomonas</i> syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. <br><br> In our experiments, ice fractions increased steeply in the temperature range from about −6 °C to about −10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. <br><br> We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and <i>P. syringae</i> bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached to the outer membrane of intact bacteria or membrane fragments, (c) the temperature range in which heterogeneous droplet freezing occurs, and the fraction of droplets being able to freeze, both depend on the actual number of INA protein complexes present in the droplet ensemble, and (d) possible artifacts suspected to occur in connection with the drop freezing method, i.e., the method frequently used by biologist for quantifying ice nucleation behaviour, are of minor importance, at least for substances such as <i>P. syringae</i>, which induce freezing at comparably high temperatures. The last statement implies that for single ice nucleation entities such as INA protein complexes, it is the number of entities present in the droplet population, and the entities' nucleation rate, which control the freezing behaviour of the droplet population. Quantities such as ice active surface site density are not suitable in this context. <br><br> The results obtained in this study allow a different perspective on the quantification of the immersion freezing behaviour of bacterial ice nucleation.

 References Ahern, H. E., Walsh, K. A., Hill, T. C. J., and Moffett, B. F.: Fluorescent pseudomonads isolated from Hebridean cloud and rain water produce biosurfactants but do not cause ice nucleation, Biogeosciences, 4, 115–124, <a href="http://dx.doi.org/10.5194/bg-4-115-2007">https://doi.org/10.5194/bg-4-115-2007</a>, 2007. Amato,&nbsp;P., Parazols, M., Sancelme, M., Laj,&nbsp;P., Mailhot,&nbsp;G., and Delort,&nbsp;A.&nbsp;M.: Microorganisms isolated from the water phase of tropospheric clouds at the Puy de Dôme: major groups and growth abilities at low temperatures, Fems Microbiol. Ecol., 59, 242–254, 2007. Ansmann,&nbsp;A., Tesche,&nbsp;M., Seifert,&nbsp;P., Althausen,&nbsp;D., Engelmann,&nbsp;R., Fruntke,&nbsp;J., Wandinger,&nbsp;U., Mattis,&nbsp;I., and Müller,&nbsp;D.: Evolution of the ice phase in tropical altocumulus: SAMUM lidar observations over Cape Verde, J. Geophys. Res.-Atmos., 114, D17208, <a href="http://dx.doi.org/10.1029/2008JD011659">https://doi.org/10.1029/2008JD011659</a>, 2009. Attard,&nbsp;E., Yang,&nbsp;H., Delort,&nbsp;A.-M., Amato,&nbsp;P., Pöschl,&nbsp;U., Glaux,&nbsp;C., Koop,&nbsp;T., and Morris,&nbsp;C.&nbsp;E.: Effects of atmospheric conditions on ice nucleation activity of Pseudomonas, Atmos. Chem. Phys., 12, 10667–10677, <a href="http://dx.doi.org/10.5194/acp-12-10667-2012">https://doi.org/10.5194/acp-12-10667-2012</a>, 2012. Augustin,&nbsp;S., Hartmann,&nbsp;S., Pummer,&nbsp;B., Grothe,&nbsp;H., Niedermeier,&nbsp; D., Clauss,&nbsp;T., Voigtländer,&nbsp;J., Tomsche,&nbsp;L, Wex,&nbsp;H. and Stratmann,&nbsp;F.: Immersion freezing of birch pollen washing water, Atmos. Chem. Phys. Discuss., 12, 32911–32943, <a href="http://dx.doi.org/10.5194/acpd-12-32911-2012">https://doi.org/10.5194/acpd-12-32911-2012</a>, 2012. Burrows, S. M., Elbert, W., Lawrence, M. G., and Pöschl, U.: Bacteria in the global atmosphere – Part 1: Review and synthesis of literature data for different ecosystems, Atmos. Chem. Phys., 9, 9263–9280, <a href="http://dx.doi.org/10.5194/acp-9-9263-2009">https://doi.org/10.5194/acp-9-9263-2009</a>, 2009. Christner,&nbsp;B.&nbsp;C., Morris,&nbsp;C.&nbsp;E., Foreman,&nbsp;C.&nbsp;M., Cai,&nbsp;R.&nbsp;M. and Sands,&nbsp;D.&nbsp;C.: Ubiquity of biological ice nucleators in snowfall, Science, 319, 1214–1214, 2008. Clauss,&nbsp;T., Kiselev,&nbsp;A., Hartmann,&nbsp;S., Augustin,&nbsp;S., Pfeifer,&nbsp;S., Niedermeier,&nbsp;D., Wex,&nbsp;H., and Stratmann,&nbsp;F.: Application of linear polarized light for the discrimination of frozen and liquid droplets in ice nucleation experiments, Atmos. Meas. Tech., 6, 1041–1052, <a href="http://dx.doi.org/10.5194/amt-6-1041-2013">https://doi.org/10.5194/amt-6-1041-2013</a>, 2013. Cochet,&nbsp;N. and Widehem,&nbsp;P.: Ice crystallization by <i>Pseudomonas syringae</i>, Appl. Microbiol. Biotech., 54, 153–161, <a href="http://dx.doi.org/10.1007/s002530000377">https://doi.org/10.1007/s002530000377</a>, 2000. Conen, F., Morris, C. E., Leifeld, J., Yakutin, M. V., and Alewell, C.: Biological residues define the ice nucleation properties of soil dust, Atmos. Chem. Phys., 11, 9643–9648, <a href="http://dx.doi.org/10.5194/acp-11-9643-2011">https://doi.org/10.5194/acp-11-9643-2011</a>, 2011. DeLeon-Rodriguez,&nbsp;N., Lathem,&nbsp;T.&nbsp;L., Rodriguez-Rojas,&nbsp;L.&nbsp;M., Barazesh,&nbsp;J.&nbsp;M., Anderson,&nbsp;B.&nbsp;E., Beyersdorf,&nbsp;A.&nbsp;J., Ziemba,&nbsp;L.&nbsp;D., Bergin,&nbsp;M., Nenes,&nbsp;A., and Konstantinidis,&nbsp;K.&nbsp;T.: Microbiome of the upper troposphere: Species composition and prevalence, effects of tropical storms, and atmospheric implications, Proc. Natl. Acad. Sci., 110, 2575–2580, <a href="http://dx.doi.org/10.1073/pnas.1212089110">https://doi.org/10.1073/pnas.1212089110</a>, 2013. DeMott,&nbsp;P.&nbsp;J.: Quantitative descriptions of ice formation mechanisms of silver iodide-type aerosols. Atmos. Res., 38, 63–99, 1995. DeMott,&nbsp;P.&nbsp;J., Sassen,&nbsp;K., Poellot,&nbsp; M.&nbsp;R., Baumgardner,&nbsp;D., Rogers,&nbsp;D.&nbsp;C., Brooks,&nbsp;S.&nbsp;D., Prenni,&nbsp;A.&nbsp;J. and Kreidenweis,&nbsp;S.&nbsp;M.: African dust aerosols as atmospheric ice nuclei. Geophys. Res. Lett., 30, 1732, <a href="http://dx.doi.org/10.1029/2003GL017410">https://doi.org/10.1029/2003GL017410</a>, 2003. Després,&nbsp;V.&nbsp;R., Huffman,&nbsp;J.&nbsp;A., Burrows,&nbsp;S.&nbsp;M., Hoose,&nbsp;C., Safatov,&nbsp;A.&nbsp;S., Buryak,&nbsp;G., Fröhlich-Nowoisky,&nbsp;J., Elbert,&nbsp;W., Andreae,&nbsp;M.&nbsp;O., Pöschl,&nbsp;U. and Jaenicke,&nbsp;R.: Primary biological aerosol particles in the atmosphere: a review, Tellus B, 64, 15598, <a href="http://dx.doi.org/10.3402/tellusb.v64i0.15598">https://doi.org/10.3402/tellusb.v64i0.15598</a>, 2012. Fuzzi,&nbsp;S., Mandrioli,&nbsp;P., and Perfetto,&nbsp;A.: Fog droplets – An atmospheric source of secondary biological aerosol particles, Atmos. Environ., 31, 287–290, 1997. Garnham,&nbsp;C.&nbsp;P., Campbell,&nbsp;R.&nbsp;L., Walker,&nbsp;V.&nbsp;K. and Davies,&nbsp;P.&nbsp;L.: Novel dimeric beta-helical model of an ice nucleation protein with bridged active sites, BMC Struct. Biol., 11, <a href="http://dx.doi.org/10.1186/1472-6807-11-36">https://doi.org/10.1186/1472-6807-11-36</a>, 2011. Green,&nbsp;R.&nbsp;L. and Warren,&nbsp;G.&nbsp;J.: Physical and functional repetition in an bacterial ice nucleation gene, Nature, 317, 645–648, 1985. Govindarajan,&nbsp;A.&nbsp;G. and Lindow,&nbsp;S.&nbsp;E.: Size of bacterial ice-nucleation sites measured in-situ by radiation inactivation analysis, Proc. Nat. Acad. Sci. USA, 85, 1334–1338, 1988. Hartmann,&nbsp;S., Niedermeier,&nbsp;D., Voigtländer,&nbsp;J., Clauss,&nbsp;T., Shaw,&nbsp;R.&nbsp;A., Wex,&nbsp;H., Kiselev,&nbsp;A., and Stratmann,&nbsp;F.: Homogeneous and heterogeneous ice nucleation at {LACIS}: operating principle and theoretical studies, Atmos. Chem. Phys., 11, 1753–1767, <a href="http://dx.doi.org/10.5194/acp-11-1753-2011">https://doi.org/10.5194/acp-11-1753-2011</a>, 2011. Hoose,&nbsp;C., Kristjánsson,&nbsp;J.&nbsp;E. and Burrows,&nbsp;S.&nbsp;M.: How important is biological ice nucleation in clouds on a global scale?, Environ. Res. Lett., 5, 024009, <a href="http://dx.doi.org/10.1088/1748-9326/5/2/024009">https://doi.org/10.1088/1748-9326/5/2/024009</a>, 2010. Hoose,&nbsp;C. and Möhler,&nbsp;O.: Heterogeneous ice nucleation on atmospheric aerosols: A review of results from laboratory experiments, Atmos. Chem. Phys., 12, 9817–9854, <a href="http://dx.doi.org/10.5194/acp-12-9817-2012">https://doi.org/10.5194/acp-12-9817-2012</a>, 2012. Joly,&nbsp;M., Attard,&nbsp;E., Sancelme,&nbsp;M., Deguillaume,&nbsp;L., Guilbaud,&nbsp;C., Morris,&nbsp;C.&nbsp;E., Amato,&nbsp;P., and Delort,&nbsp;A.-M.: Ice nucleation activity of bacteria isolated from cloud water, Atmos. Environ., 70, 392–400, <a href="http://dx.doi.org/10.1016/j.atmosenv.2013.01.027">https://doi.org/10.1016/j.atmosenv.2013.01.027</a>, 2013. Kamphus,&nbsp;M., Ettner-Mahl,&nbsp;M., Klimach,&nbsp;T., Drewnick,&nbsp;F., Keller,&nbsp;L., Cziczo,&nbsp;D.&nbsp;J., Mertes,&nbsp;S., Borrmann,&nbsp;S., and Curtius,&nbsp;J.: Chemical composition of ambient aerosol, ice residues and cloud droplet residues in mixed-phase clouds: single particle analysis during the Cloud and Aerosol Characterization Experiment ({CLACE 6}), Atmos. Chem. Phys., 10, 8077–8095, <a href="http://dx.doi.org/10.5194/acp-10-8077-2010">https://doi.org/10.5194/acp-10-8077-2010</a>, 2010. Kanitz,&nbsp;T., Seifert,&nbsp;P., Ansmann,&nbsp;A., Engelmann,&nbsp;R., Althausen,&nbsp;D., Casiccia,&nbsp;C., and Rohwer,&nbsp;E.&nbsp;G.: Contrasting the impact of aerosols at northern and southern midlatitudes on heterogeneous ice formation, Geophys. Res. Lett., 38, L17802, <a href="http://dx.doi.org/10.1029/2011GL048532">https://doi.org/10.1029/2011GL048532</a>, 2011. Kawahara,&nbsp;H.: The structures and functions of ice crystal-controlling proteins from bacteria, J. Biosci. Bioeng., 94, 492–496, 2002. Kleber,&nbsp;M., Sollins,&nbsp;P. and Sutton,&nbsp;R.: A conceptual model of organo-mineral interactions in soils: self-assembly of organic molecular fragments into zonal structures on mineral surfaces, Biogeochemistry, 85, 9–24, 2007. Knutson,&nbsp;E.&nbsp;O. and Whitby,&nbsp;K.&nbsp;T.: Aerosol classification by electric mobility: Apparatus, theory and applications, J. Aerosol Sci., 6, 443–451, 1975. Levin,&nbsp;Z. and Yankofsky,&nbsp;S.&nbsp;A.: Contact Versus Immersion Freezing Of Freely Suspended Droplets By Bacterial Ice Nuclei, J. Climate Appl. Meteorol., 22, 1964–1966, 1983. Lindow,&nbsp;S.&nbsp;E., Arny,&nbsp;D.&nbsp;C., and Upper,&nbsp;C.&nbsp;D.: Bacterial ice nucleation – a factor in frost injury to plants, Plant Physiol., 70, 1084–1089, 1982. Lindow,&nbsp;S.&nbsp;E., Lahue,&nbsp;E., Govindarajan,&nbsp;A.&nbsp;G., Panopoulos,&nbsp;N.&nbsp;J., and Gies,&nbsp;D.: Localization of ice nuleation activity and the iceC gene product in <i>Pseudomonas syringae</i> and Escherichia-coli, Mol. Plant Microbe In., 2, 262–272, 1989. Lohmann,&nbsp;U.: Aerosol effects on clouds and climate, Space Sci. Rev., 125, 129–137, 2006. Maki,&nbsp;L.&nbsp;R. and Willoughby,&nbsp;K.&nbsp;J.: Bacteria as biogenic sources of freezing nuclei, J. Appl. Meteorol., 17, 1049–1053, 1978. Maki,&nbsp;L.&nbsp;R., Galyan,&nbsp;E.&nbsp;L., Chang-Chien,&nbsp;M.-M., and Caldwell,&nbsp;D.&nbsp;R.: Ice nucleation induced by <i>Pseudomonas syringae</i>, Appl. Microbiol., 28, 456–459, 1974. Möhler, O., Georgakopoulos, D. G., Morris, C. E., Benz, S., Ebert, V., Hunsmann, S., Saathoff, H., Schnaiter, M., and Wagner, R.: Heterogeneous ice nucleation activity of bacteria: new laboratory experiments at simulated cloud conditions, Biogeosciences, 5, 1425–1435, <a href="http://dx.doi.org/10.5194/bg-5-1425-2008">https://doi.org/10.5194/bg-5-1425-2008</a>, 2008. Monteil,&nbsp;C.&nbsp;L., Guilbaud,&nbsp;C., Glaux,&nbsp;C., Lafolie,&nbsp;F., Soubeyrand,&nbsp;S., and Morris,&nbsp;C.&nbsp;E.: Emigration of the plant pathogen <i>Pseudomonas syringae</i> from leaf litter contributes to its population dynamics in alpine snowpack, Environ. Microbiol., 14, 2099-2112, 2012. Morris,&nbsp;C.&nbsp;E. and Sands,&nbsp;D.&nbsp;C.: From Grains to Rain: the link between landscape, airborne microorganisms and climate processes, e-book, accessed 26 April 2012, <a href="http://bioice.wordpress.com/">http://bioice.wordpress.com/</a>, 2012. Morris,&nbsp;C.&nbsp;E., Georgakopoulos,&nbsp;D.&nbsp;G., and Sands,&nbsp;D.&nbsp;C.: Ice nucleation active bacteria and their potential role in precipitation. J. Phys. IV, 121, 87–103, 2004. Morris,&nbsp;C.&nbsp;E., Sands,&nbsp;D.&nbsp;C., Vinatzer,&nbsp;B.&nbsp;A., Glaux,&nbsp;C., Guilbaud,&nbsp;C., Buffiere,&nbsp;A., Yan,&nbsp;S.&nbsp;C., Dominguez,&nbsp;H., and Thompson,&nbsp;B.&nbsp;M.: The life history of the plant pathogen <i>Pseudomonas syringae</i> is linked to the water cycle, ISME J., 2, 321–334, 2008. Mueller,&nbsp;G.&nbsp;M., Wolber,&nbsp;P.&nbsp;K., and Warren,&nbsp;G.&nbsp;J.: Clustering of ice nucleation protein correlates with ice nucleation activity, Cryobiology, 27, 416–422, 1990. Murray,&nbsp;B.&nbsp;J., O'Sullivan,&nbsp;D., Atkinson,&nbsp;J.&nbsp;D., and Webb,&nbsp;M.&nbsp;E.: Ice nucleation by particles immersed in supercooled cloud droplets. Chem. Soc. Rev., 41, 6519-6554, 2012. Niedermeier,&nbsp;D., Hartmann,&nbsp;S., Shaw,&nbsp;R.&nbsp;A., Covert,&nbsp;D., Mentel,&nbsp;T.&nbsp;F., Schneider,&nbsp;J., Poulain,&nbsp;L., Reitz,&nbsp;P., Spindler,&nbsp;C., Clauss,&nbsp;T., Kiselev,&nbsp;A., Hallbauer,&nbsp;E., Wex,&nbsp;H., Mildenberger,&nbsp;K., and Stratmann,&nbsp;F.: Heterogeneous freezing of droplets with immersed mineral dust particles – measurements and parameterization, Atmos. Chem. Phys., 10, 3601–3614, <a href="http://dx.doi.org/10.5194/acp-10-3601-2010">https://doi.org/10.5194/acp-10-3601-2010</a>, 2010. Niedermeier,&nbsp;D., Shaw,&nbsp;R.&nbsp;A., Hartmann,&nbsp;S., Wex,&nbsp;H., Clauss,&nbsp;T., Voigtländer,&nbsp;J., and Stratmann,&nbsp;F.: Heterogeneous ice nucleation: exploring the transition from stochastic to singular freezing behavior, Atmos. Chem. and Phys., 11, 8767–8775, <a href="http://dx.doi.org/10.5194/acp-11-8767-2011">https://doi.org/10.5194/acp-11-8767-2011</a>, 2011a. Niedermeier,&nbsp;D., Hartmann,&nbsp;S., Clauss,&nbsp;T., Wex,&nbsp;H., Kiselev,&nbsp;A., Sullivan,&nbsp;R.&nbsp;C., DeMott,&nbsp;P.&nbsp;J., Petters,&nbsp;M.&nbsp;D., Reitz,&nbsp;P., Schneider,&nbsp;J., Mikhailov,&nbsp;E., Sierau,&nbsp;B., Stetzer,&nbsp;O., Reimann,&nbsp;B., Bundke,&nbsp;U., Shaw,&nbsp;R.&nbsp;A., Buchholz,&nbsp;A., Mentel,&nbsp;T.&nbsp;F., and Stratmann,&nbsp;F.: Experimental study of the role of physicochemical surface processing on the IN ability of mineral dust particles, Atmos. Chem. Phys., 11, 11131–11144, <a href="http://dx.doi.org/10.5194/acp-11-11131-2011">https://doi.org/10.5194/acp-11-11131-2011</a>, 2011b. Orser,&nbsp;C., Staskawicz,&nbsp;B.&nbsp;J., Panopoulos,&nbsp;N.&nbsp;J., Dahlbeck,&nbsp;D., and Lindow,&nbsp;S.&nbsp;E.: Cloning and expression of bacterial ice nucleation genes in Escherichia-coli, J. Bacteriol., 164, 359–366, 1985. Pratt,&nbsp;K.&nbsp;A., DeMott,&nbsp;P.&nbsp;J., French,&nbsp;J.&nbsp;R., Wang,&nbsp;Z., Westphal,&nbsp;D.&nbsp;L., Heymsfield,&nbsp;A.&nbsp;J., Twohy,&nbsp;C.&nbsp;H., Prenni,&nbsp;A.&nbsp;J., and Prather,&nbsp;K.&nbsp;A.: In situ detection of biological particles in cloud ice-crystals, Nat. Geosci., 2, 397–400, <a href="http://dx.doi.org/10.1038/ngeo521">https://doi.org/10.1038/ngeo521</a>, 2009. Pummer,&nbsp;B.&nbsp;G., Bauer,&nbsp;H., Bernardi, J., Bleicher, S., and Grothe, H.: Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen. Atmos Chem Phys., 12, 2541–2550, <a href="http://dx.doi.org/10.5194/acp-12-2541-2012">https://doi.org/10.5194/acp-12-2541-2012</a>, 2012. Šantl-Temkiv,&nbsp;T., Finster,&nbsp;K., Sahyoun,&nbsp;M., Hartmann,&nbsp;S., Augustin,&nbsp;S., Stratmann,&nbsp;F., Wex,&nbsp;H., Clauss,&nbsp;T., Korsholm,&nbsp;U.&nbsp;S., Nielsen,&nbsp;N.&nbsp;W., Sorensen,&nbsp;J.&nbsp;H., and Karlson,&nbsp;U.&nbsp;G.: Airborne <i>Pseudomonas sp.</i> may increase the number of biological ice nuclei in the atmosphere by excreting ice nucleation active proteins, in preparation for Environ. Microbiol., 2013. Schnell,&nbsp;R.&nbsp;C. and Vali,&nbsp;G.: Atmospheric ice nuclei from decomposing vegetation, Nature, 236, 163–165, 1972. Schmid, D., Pridmore,&nbsp;D., Capitani,&nbsp;G., Battistutta,&nbsp;R., Neeser,&nbsp;J. R., and Jann,&nbsp;A.: Molecular organisation of the ice nucleation protein InaV from <i>Pseudomonas syringae</i>, Febs Lett., 414, 590–594, 1997. Seifert,&nbsp;P., Ansmann,&nbsp;A., Mattis,&nbsp;I., Wandinger,&nbsp;U., Tesche,&nbsp;M., Engelmann,&nbsp;R., Müller,&nbsp;D., Pérez,&nbsp;C., and Haustein,&nbsp;K.: Saharan dust and heterogeneous ice formation: eleven years of cloud observations at a&nbsp;Central European EARLINET site, J.&nbsp;Geophys. Res.-Atmos., 115, D20201, <a href="http://dx.doi.org/10.1029/2009JD013222">https://doi.org/10.1029/2009JD013222</a>, 2010. Skirvin, R. M., Kohler, E., Steiner, H., Ayers, D., Laughnan, A., Norton, M. A., and Warmund, M.: The use of genetically engineered bacteria to control frost on strawberries and potatoes. Whatever happened to all of that research?, Sci. Hortic., 84, 179–189, 2000. Southworth,&nbsp;M.&nbsp;W., Wolber,&nbsp;P.&nbsp;K., and Warren,&nbsp;G.&nbsp;J.: Nonlinear relationship between concentration and activity of a bacterial ice nucleation protein, J. Biol. Chem., 263, 15211–15216, 1988. Stephanie, D. and Waturangi,&nbsp;E.: Distribution of ice nucleation-active (INA) bacteria from rain-water and rain, HAYATI Journal of Biosciences, 18, 108–112, 2011. Stratmann,&nbsp;F., Kiselev,&nbsp;A., Wurzler,&nbsp;S., Wendisch,&nbsp;M., Heintzenberg,&nbsp;J., Charlson,&nbsp;R.&nbsp;J., Diehl,&nbsp;K., Wex,&nbsp;H., and Schmidt,&nbsp;S.: Laboratory studies and numerical simulations of cloud droplet formation under realistic supersaturation conditions, J.&nbsp;Atmos. Ocean. Tech., 21, 876–887, 2004. Turner,&nbsp;M.&nbsp;A., Arellano,&nbsp;F., and Kozloff,&nbsp;L.&nbsp;M.: Three separate classes of bacterial ice nucleation structures, J. Bacteriol., 172, 2521–2526, 1990. Turner,&nbsp;M.&nbsp;A., Arellano,&nbsp;F., and Kozloff,&nbsp;L.&nbsp;M.: Components of ice nucleation structures of bacteria, J. Bacteriol., 173, 6515–6527, 1991. Twohy,&nbsp;C.&nbsp;H. and Poellot,&nbsp;M.&nbsp;R.: Chemical characteristics of ice residual nuclei in anvil cirrus clouds: evidence for homogeneous and heterogeneous ice formation, Atmos. Chem. Phys., 5, 2289–2297, <a href="http://dx.doi.org/10.5194/acp-5-2289-2005">https://doi.org/10.5194/acp-5-2289-2005</a>, 2005. Vali, G.: Quantitative Evaluation Of Experimental Results On Heterogeneous Freezing Nucleation Of Supercooled Liquids, J. Atmos. Sci., 28, 402–409, 1971. Vali,&nbsp;G., Christensen,&nbsp;M., Fresh,&nbsp;R.&nbsp;W., Galyan,&nbsp;E.&nbsp;L., Maki,&nbsp;L.&nbsp;R., and Schnell,&nbsp;R.&nbsp;C.: Biogenic ice nuclei. Part II: Bacterial sources, J. Atmos. Sci., 33, 1565–1570, 1976. van Overbeek,&nbsp;L.&nbsp;S., Nijhuis,&nbsp;E.&nbsp;H.&nbsp;M., Koenraadt,&nbsp;H., Visser,&nbsp;J., and van Kruistum,&nbsp;G.: The role of crop waste and soil in <i>Pseudomonas syringae</i> pathovar porri infection of leek (Allium porrum), Appl. Soil Ecol., 46, 457–463, 2010. Ward,&nbsp;P.&nbsp;J. and DeMott,&nbsp;P.&nbsp;J.: Preliminary Experimental Evaluation of Snomax$^{\text{TM}}$ Snow Inducer, <i>Pseudomonas syringae</i>, as an Artificial Ice Nucleus, J. Weather Mod., 21, 9–13, 1989. Warren,&nbsp;G. and Wolber,&nbsp;P.: Molecular aspects of microbial ice nucleation, Mol. Microbiol., 5, 239–243, 1991. Warren,&nbsp;G.&nbsp;J. and Wolber,&nbsp;P.&nbsp;K.: Heterogeneous ice nucleation by bacteria, Cryo. Lett., 8, 204–217, 1987. Wilck, M., Stratmann, F., and Whitby, E.: A fine particle model for fluent: Description and application, in: Proc. Sixth Int. Aerosol Conf., 1269–1270, Chinese Association for Aerosol Research in Taiwan/International Aerosol Research Assembly, Taipei, Taiwan, 2002. Wolber,&nbsp;P.&nbsp;K., Deininger,&nbsp;C.&nbsp;A., Southworth,&nbsp;M.&nbsp;W., Vandekerckhove,&nbsp;J., Vanmontagu,&nbsp;M., and Warren,&nbsp;G.&nbsp;J.: Identification and purification of a bacterial ice-nucleation protein, P. Natl. A. Sci. USA, 83, 7256–7260, 1986. Wood,&nbsp;S.&nbsp;E., Baker,&nbsp;M.&nbsp;B., and Swanson,&nbsp;B.&nbsp;D.: Instrument for studies of homogeneous and heterogeneous ice nucleation in free-falling supercooled water droplets, Rev. Sci. Inst., 73, 3988–3996, 2002. Yankofsky,&nbsp;S.&nbsp;A., Levin,&nbsp;Z., Bertold,&nbsp;T., and Sandlerman,&nbsp;N.: Some basic characteristics of bacterial freezing nuclei, J. Appl. Meteorol., 20, 1013–1019, 1981.