8. Composition of biological ice nuclei in short: quite a bit of research already 50 years ago

1. 8. Composition of biological ice nuclei • in short: • quite a bit of research already 50 years ago • very selective knowledge in small sub-areas developed • much is still missing towards a complete understanding • recently, new interest emerged, triggered by the recognition that there is a large gap in our understanding and also by new measurement possibilities

2. Bio - Ice Nuclei (IN) Do we need to care? data base for distribution of bacteria from Burrows (2009): simulated global transport of bacteria in a general circulation model - … - In order to improve understanding of this topic, more measurements of the bacterial content of the air and of the rate of surface-atmosphere exchange of bacteria will be necessary. Burrows et al. (2013): model parameters contribute surprisingly much to uncertainty (as much as observations) Murray et al., Chem. Soc. Rev., (2012) talk by Ulrich Pöschl at the EGU “Life is in the air and it interacts with precipitation” We suggest that the following research activities should be pursued in future studies of atmospheric biological aerosol particles: (1) develop efficient and reliable analytical techniques for the identification and quantification of PBAP (primary biological aerosol particles); (2) apply advanced and standardized techniques to determine the abundance and diversity of PBAP and their seasonal variation at regional and global scales (atmospheric biogeography); (3) determine the emission rates, optical properties, IN and CCN activity of PBAP in field measurements and laboratory experiments; (4) use field and laboratory data to constrain numerical models of atmospheric transport, transformation and climate effects of PBAP.

3. Bio - Ice Nuclei (IN) Do we need to care? Several recent reviews highlighted a need for quantifying number and source of biological IN in the atmosphere (e.g. Szyrmer and Zawadzki, 1997; Möhler et al., 2007; Ariya et al., 2009; DeMott and Prenni, 2010; Martin et al., 2010; DeMott et al., 2011; Despres et al., 2013). Pratt et al. (2009): examined 46 atmospheric cloud ice-crystal residues with ATOFMS; 33% (15) were biological residues, 50% (23) mineral dust; 60 % (14) of the dust particles likely a mix of biological material with mineral dust Prenni et al. (2009): IN over Amazon rainforest are a combination of mineral dust and biological particles (bioparticles locally emitted, dust imported from Sahara); above approx. −25 C; biological IN appeared to dominate over mineral dust IN, and IN concentration and abundance can be almost entirely explained by these biological particles DeMott and Prenni (2010): the most important carbonaceous particles that may be acting as ice nuclei above −15 C may be biological particles Conen et al. (2011): organic material was found to enhance the ice nucleation of soil dust (IN ability lost by heat treatment) Prenni et al. (2013) & Huffman et al., ACPD (2013): rain at a forest in Colorado increased IN concentrations in atmosphere, some of these IN were biological (DNA sequencing and other methods)

4. Bio - Ice Nuclei (IN) • from bacteria • from pollen • from fungal spores (fungi and lichen) • - (leaf litter / biomass burning) • oceanic sources? (phytoplankton / bacteria / others?) • others? (e.g. terpenes and other tree oils; freeze-tolerant insects)

5. INA bacteria – what we know Vali (1968): natural soils were rich in organic material -> better sources of IN than basic clay constituents of these soils Schnell & Vali (1972): IN from decaying vegetation Maki et al. (1974), Lindow et al. (1978): related IN to bacterial species (Pseudomonas syringae and other related bacteria, mostly of the Pseudomonas-familiy (e.g. P. fluorescens, P. viridiflava, Pantoea agglomerans, Erwinia herbicola, Xanthomas campestris) found in soils, on vegetation, airborne, in precipitation and also lakes (e.g. Morris et al. (2008): “The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle”) Wolber et al. (1986): ice nucleating ability due to a protein anchored in the cell membrane Orser et al. (1985), Green & Warren (1985): the protein is encoded by a single gene which is highly conserved among ice nucleating bacteria after the mid-1980ies: much research particularly on P. syringae (reduction of frost damage to crops – ”ice minus bacteria”, “Frostban”, first release of a genetically modified organism into the environment) Pseudomonas syringae foto: G. Vrdoljak, U.C. Berkeley

6. INA bacteria – what we know not each bacteria of a INA species has the protein on it (only formed under certain circumstances (Morris et al. (2004)) single INA protein estimated to be around 120-180 kDa and the ice active surface area of a single protein is about 4nm*32nm (Garnham et al. (2011)) when present, the proteins aggregate (Schmidt et al. (1997)) two or three distinct protein complex sizes seem to exist: on P. syringae, one consisting of at least two up to a few proteins, occurring in “1 of 300” up to “almost all” cells (Yankofsky et al. (1981) and inducing freezing at about -7°C to -10°C the larger ones being about 60 times as large, occurring in about 10-3 to 10-6 of the cells of P. syringae and inducing freezing already at –2°C testing for INA protein complexes: treatment with chemicals which destroy proteins (e.g. guanidinium chloride) or heat (approx. 90°C) or DNA sequencing INA protein complexes connected to mineral surfaces may be preserved (Kleber et al. (2007)) Garcia et al. (2012) found many INA gene carrying matter washed off from vegetation in two U.S. High Plains agricultural regions, but only little was found in the atmosphere Mortazavi et al. (2008) find that different bacteria isolated from snow samples induced freezing at ≈−15 ◦C Monteil et al. 2012 find that P. syringae is frequently found on decaying plant material / soils DeLeon-Rodrigues et al. (2013) examined airborne samples in the Carribean after two hurricanes and reported that 20% of the total particles in sub-mm-range were viable bacteria cells (fungal cells: one order of magnitude less present) Pseudomonas syringae sketch: hawashpharma. blogspot.de

7. INA bacteria – one of the open issues Kotzloff et al., J. Bacteriol., (1983) Murray et al., Chem. Soc. Rev., (2012) Hartmann et al., ACP, (2013) Frozen fraction of droplets with P. syringae (directly or from Snomax). Drop freezing assays often use VERY high concentrations of cells per droplet, which leads to higher freezing temperatures than would be observed for a single bacteria. Freezing temperature at which 50% of all droplets of P. syringae and E. herbicola froze, in dependance on pH.

8. INA bacteria – one of the open issues Hartmann et al., ACP, (2013) Kotzloff et al., J. Bacteriol., (1983) Murray et al., Chem. Soc. Rev., (2012) Frozen fraction of droplets with P. syringae (directly or from Snomax). Drop freezing assays often use VERY high concentrations of cells per droplet, which leads to higher freezing temperatures than would be observed for a single bacteria. Freezing temperature at which 50% of all droplets of P. syringae and E. herbicola froze, in dependance on pH.

9. pollen – what we know Diehl et al. (2001), (2002) found different pollen to induce freezing below -10°C von Blohn et al. (auch Diehl), 2005: 4 types of pollen particles in the immersion freezing mode ranged from −13.5 C to −21.5 C - … BUT: the fraction of pollen grains that are effective IN have not yet been determined Pummer et al. (2012) found different types of pollen and even the pollen washing waters to be ice active at -18°C IN ability due to INA macromolecules which are on the exine of the pollen grain (likely polysaccarides) INA macromolecules (about 10nm, mass 100 to 300 kDa) much smaller than whole pollen grain (25µm)  atmospheric relevance! multiplication of pollen fragments by rain bursting was already shown (Schäppi et al., Clin. Exp. Allergy, 1999) a rough estimate (Augustin et al., ACPD, 2013) yielded that each of the birch pollen carried several thousands of these macromolecules – other pollen types have less BUT: the polysaccarides are not identified, yet (they comprise only a small part of the large amount of material on/in the pollen (Augustin et al. (2013) -> roughly 10-6 of the mass that can be washed off the pollen)) www.infovisual.info Birch pollen grain, Pummer et al., (2012)

10. lichen* and fungi – what we know Hyland et al. (1953): fungal spores in air and rain Marshall et al. (1996), Tormo et al. (2001): lichen found in airborne samples Jayaweera and Flanagan (1982): spores from Penicilliumdigitatumare ice active at -10°C Kieft (1988), Ashworth & Kieft (1992): IN from lichen (Rhizoplaca, Xanthoparmelia, andXanthoria), icenucleationbetween -1.9°C and -8°C (dropfreezingassay) Kieft & Ruscetti (1990), (1992): lichen contain proteinaceous IN, the active site is equal to those of bacteria Pouleur et al. (1992): ice activity in two fungi species, Fusariumacuminatum and Fusariumavenaceum Iannone et al. (2011): spores of Cladosporiumspores (2-4 mm) acted as IN around -30°C Huffman et al., ACPD, 2013: two new species of IN-active fungi (Isariafarinosaand Acremoniumimplicatum) (greates increase at (2-6 mm, i.e. the range for fungal spores and bacteria) – they also found a large increase in airborne bioparticles during rain (also described by Prenni et al. (2013)) *lichen (Flechten) are a symbiosis of a fungus and a photosynthetic partner (e.g. an algae)

11. phytoplankton – what we know Bigg (1973): STCZ characterized by mixing, overturning and upwelling water  prolific zone of marine plankton Schnell & Vali (1975), Schnell (1977, 1982): high IN conc. associated with living marine bacteria, associated with plankton, or with high conc. of biogenic materials in the same samples Rosinski et al. (1986, 1987, 1988): sulfate ions of marine origin enhance atmospheric IN activity Bigg & Leck (2001): bacteria and probable submicron fragments of marine organisms were suggested to be the source of IN in the Arctic ocean Knopf et al. (2011): marine diatoms (a kind of algae and among the most abundant phytoplankton) act as IN in a lab-study Sullivan at AGU 2012: “Ice nuclei emissions from sea spray produced by realistically simulated breaking waves“ (coupledocean-atmospherefacility, seePrather et al., PNAS, 2013) transfer of marine bacteria to the atmosphere  bubble bursting Burrows et al. (2013): marine biogenic IN (occurring in highly variable amounts) may play a dominant role for oceanic IN concentrations near the surface (model study based on data by Schnell & Vali, 1975) DeLeon-Rodrigues et al. (2013): examined airborne samples in the Carribean after two hurricanes and reported that 20% of the total particles in sub-mm-range were viable bacteria cells (fungal cells: one order of magnitude less) Source: http://earthobservatory.nasa.gov/images/.../rosssea_amo_2011022_lrg.jpg

12. open issues in general • amounts and distribution of biological IN in the atmosphere • not all species contributing bio-IN are known (might be impossible to ever identify them all) • for some species, it is not yet clear what it is that induces freezing (phytoplankton / leaf litter) • viability as the BIG FILTER!!! • even for those where the INA entity is know, the freezing mechanism is not clear (protein complex / polysaccarides) • abundance is an open issue – can/do these polysaccarides exist separately from the pollen / how can they become airborne / how to detect them (non viable and small!) – same for protein complexes (might stay ice nucleation active even when dead / fragmented; vesicles) • temperature where they are active / how should the freezing behaviour be reported / parameterized? (nucleation rates, n_s, …)

13. new methods • DNA-testing for the INA-gene (e.g. Garcia et al. (2012)) • new detection devices which use fluorescence induced by bioparticles (e.g. Huffman et al. (2013), • optical microscope coupled with a temperature-controlled flow cell and a CCD (e.g. Iannone et al. (2011)) • advanced microscopy (SEM) • IN counters become more numerous (many more than only Paul DeMott with his CFDC, e.g. LACIS, ZINC/PINC, FINCH, SPIN, …)

14. advises for future actions • bio-IN: • determine the different species • abundance (when, where, how many) • temperature range where they are active (similar for similar species?) • do measurements of ice activity on samples which contain a well defined number of INA entities (realistic for the atmosphere)