Metagenomics of clouds and atmosphere

Dr. Tina Santl Temkiv, Dr. Kai Finster and Dr. Ulrich Gosewinkel Karlson Cloud and Atmosphere Metagenomics SpringerReference Cloud and Atmosphere Metagenomics Synonyms Metagenomics of airborne and cloudborne bacteria Definition Previously only considered a dispersal route for microorganisms, the atmosphere has recently been added to a long list of environments on Earth that could serve as bacterial habitats. Diverse bacterial communities are present in the atmosphere up to high altitudes as well as in cloud and fog droplets. By entering the droplets, airborne bacteria gain access to a liquid environment and diverse organic compounds and potentially affect atmospheric chemistry and physics. Introduction The atmosphere is the most important conduit for bacterial dispersal. The mean global emissions from terrestrial surfaces amount to between 2.0*1016 and 5.6*1016 CFU per second (Burrows et al. 2009b). Emissions from marine surfaces are considered significantly lower. Marine bacteria get aerosolized through bubble bursting, whereas wind and temperature are proposed to be the main factors influencing uplift of bacteria from terrestrial surfaces (Burrows et al. 2009a). While bacteria remaining in the boundary layer of the atmosphere typically get transported only locally, those that enter the free atmosphere have long residence times, which allows them to overcome distances as long as several thousand kilometers (Burrows et al. 2009b). Despite the restricted knowledge on how microbial dispersal influences patterns of microbial distribution, bacteria are generally not considered to be dispersion limited. However, as airborne bacteria are confronted with a unique set of physical challenges, the atmosphere may act as a selective barrier distinguishing between more ubiquitous and more endemic groups of bacteria. In the atmosphere, bacteria are exposed to harsh environmental conditions such as desiccation, UV radiation, reactive oxygen species, and low temperature, which all affect bacterial survival and activity. Much of the atmosphere is highly unsaturated with water vapor, causing the free cytoplasmic water to instantaneously evaporate from bacterial cells, preventing bacterial activity. Only a limited number of bacteria can tolerate the removal of water from the cells, prolonged desiccation and multiple cycles of drying and rewetting. A highly efficient way of desiccation tolerance is the formation of resting stages, e.g., spores in some Gram-positive bacteria. High levels of UV radiation, characteristic for the atmosphere, can either directly damage bacterial DNA or may do so through the formation of free radicals and reactive oxygen species in the cytoplasm. A well-known mechanism of photoprotection in bacteria is synthesis of carotenoid pigments, which can react with free radicals and quench singlet molecular oxygen (1O2 *). A large proportion of cloud- and airborne bacteria are indeed able to synthesize pigments (Delort et al. 2010; Fahlgren et al. 2010). The temperature of the troposphere decreases with height, reaching on average -55 °C at the top of the troposphere. Low temperatures constrain bacteria by decreasing rates of biochemical reactions and increasing the viscosity of water. Formation of ice crystals, which may mechanically rip the cell membrane and prevent solution chemistry, poses an additional problem for airborne bacteria. Many cloudborne bacteria are closely related to strains adapted to other cold environments (Bowers et al. 2009, 2012) and are able to grow at low temperatures (Sattler et al. 2001; Delort et al. 2010). As passing through the atmosphere is an essential part of life for many bacterial species, these may have evolved different advantageous adaptations, acquired either due to aerosolization or in ground habitats exposed to similar types of stress. Despite these adaptations, it is not likely that aerosolized bacteria are active before entering the liquid phase of the atmosphere. Cloud Water as a Bacterial Habitat Airborne bacteria may get sucked into the clouds (e.g., by convective movement of air masses), scavenged by cloud droplets, and wet deposited to terrestrial and aquatic environments. This way clouds may play an important role in bacterial dispersal. Bacteria could in turn influence the microphysics of clouds, as they may be involved in the nucleation http://www.springerreference.com/index/chapterdbid/303413 © Springer-Verlag Berlin Heidelberg 2014 28 Apr 2014 04:10 1 Dr. Tina Santl Temkiv, Dr. Kai Finster and Dr. Ulrich Gosewinkel Karlson Cloud and Atmosphere Metagenomics SpringerReference of cloud droplets and ice crystals, with possible implications for local or global patterns of precipitation. In fact, cloud and fog droplets are the most hospitable part of the atmosphere, giving access to nutrients and water as well as shading against damaging UV radiation. Many airborne bacterial strains remain viable; thus, it is conceivable that cloud- and fogborne bacteria may be involved in chemical transformations of organic compounds. If a part of airborne bacteria that are omnipresent in the troposphere retain active metabolism, the atmosphere, and cloud water in particular, may be considered an overlooked bacterial habitat. Just as the atmosphere, cloud water is a stressful environment, which is often characterized by low pH and temperature, presence of toxic compounds, reactive oxygen species, and multiple cycles of drying and wetting or freezing and thawing. Due to extreme conditions and very short residence times of bacteria in clouds, it is unlikely that extensive bacterial biomass would form in clouds (Sattler et al. 2001). The atmosphere contains a diverse assembly of organic compounds that are present as particles, gases, or dissolved in cloud or fog droplets. The dominant class of airborne organics are biogenic volatile organic compounds (VOCs), among which terpenoids are considered most important. Many low-molecular-weight biogenic VOCs are also present, including methanol, ethylene, formaldehyde, ethanol, acetone, and acetaldehyde. Highly reactive chemical species, present in the atmosphere, oxidize these so-called primary organic aerosols, which results in the formation of secondary organic aerosols. These are less volatile, more prone to condensation, and thus more likely to enter cloud droplets. During their formation, clouds accumulate large amounts of different chemicals in a small volume of liquid, becoming important in transformation of atmospheric organic compounds. The concentration of dissolved organic carbon (DOC) in cloud droplets indicates that clouds are eutrophic environments, containing on average 3.6 mg DOC per liter of cloud water (Marinoni et al. 2004). Bacterial cell numbers reported for cloud water range between 1,500 (Sattler et al. 2001) and 430,000 (Hill et al. 2007) per ml. Assuming an initial cloud droplet diameter of 10 μm, this implies that cloud droplets are sparsely populated, although nutrient rich environments. The most dominant dissolved organics in cloud water are aldehydes and carboxylic acids, primarily originating from the gas phase (Marinoni et al. 2004). Carboxylic acids accounted for 18-71 % of total DOC, with monocarboxylic (formic, acetic, lactic, glycolic, glyoxylic, propionic) acids dominating (71 %) over dicarboxylic (oxalic, glutaric, succinic, maleic, malonic, and tartaric) acids (Marinoni et al. 2004). Significance of Cloudborne Bacteria for Atmospheric Processes Biotransformation of Organic Compounds in the Atmosphere It was long believed that the chemistry of organic compounds in the atmosphere is controlled exclusively by free radicals and oxidants. However, recent studies identified active cloudborne bacteria as an alternative route for the transformation of organic compounds in the atmosphere. In situ growth of the indigenous bacterial community on the bulk of compounds naturally present in cloud water and snow samples at 0 °C was studied by Sattler et al. ( 2001). By measuring the uptake rates of 3H-thymidine, serving as a measure of bacterial growth, and 14C-leucine, used as a measure of bacterial production, they found that bacteria could grow at atmospherically relevant temperatures. The generation times in cloud water were between 3.6 and 19.5 days, allowing the bacterial biomass to increase for up to 20 %, assuming a 1-day lifetime of the cloud. Ariya et al. (2002) showed that airborne microbes were efficient in metabolizing dicarboxylic acids with turnover times of 1.5-10 days, which is comparable to the turnover times of liquid-phase oxidation by atmospheric oxidants. The authors suggest that dicarboxylic acids might serve as energy sources for airborne bacteria, which could in turn have an important role in the atmospheric chemistry of dicarboxylic acids. In a few studies, evidence of microbial activity in clouds was found. Hill et al. ( 2007) examined the activity of cloudborne bacteria in natural cloud water by employing tetrazolium dye, affirming that a large majority (76 %) of bacteria were metabolically active. In addition, ATP concentrations measured in cloud water samples collected over Puy de Dôme indicated potential microbial activity in the cloud droplets (Delort et al. 2010). This let Amato et al. (2007) to investigate the change in ATP concentration over time in a fresh cloud water sample. They found that after an initial lag phase, lasting for the first 45 h, a subsequent increase in ATP concentrations implied that bacteria were growing exponentially on nutrients naturally present in cloud water. Ice Nucleation-Active Bacteria and Their Influence on Rain Formation A major part of global precipitation involves the formation of ice crystals (Möhler et al. 2007). Freezing of pure water (homogenous freezing) is less common in the atmosphere compared to heterogeneous freezing, as the former requires http://www.springerreference.com/index/chapterdbid/303413 © Springer-Verlag Berlin Heidelberg 2014 28 Apr 2014 04:10 2 Dr. Tina Santl Temkiv, Dr. Kai Finster and Dr. Ulrich Gosewinkel Karlson Cloud and Atmosphere Metagenomics SpringerReference cloud droplets to supercool down to about -40 °C. In mixed-phase clouds, where temperatures usually do not fall so low, often only the presence of ice nucleators (INs), which facilitate freezing at higher temperatures, enables ice crystals to form. Many inorganic particles have been identified to act as important IN in clouds, but among the most active IN, which can cause ice nucleation at temperatures close to zero, are some species of Gram-negative epiphytic bacteria, e.g., Pseudomonas syringae (Morris et al. 2008). These bacteria carry genes for an ice nucleation-active (INA) protein, which forms oligomers anchored in the outer membrane of the cell. These INA protein oligomers promote the nucleation of water presumably by mimicking the surface of an ice crystal. By nucleating ice the epiphytic INA bacteria can damage plant tissues and thus gain access to nutrients. In addition, the INA protein could increase their probability to get wet deposited from the atmosphere. Recently, in a time-of-flight study of ice crystals in a cloud, one third of ice crystals were found to contain biological particles (Delort et al. 2010). Direct studies of biological ice nucleation in precipitation and in the atmosphere have shown that proteinaceous nucleators, likely bacterial IN, are common in precipitation, ranging from 4 to 490 IN per 1 l of precipitation (Christner et al. 2008). These numbers still imply that only a very low proportion of initial cloud droplets contain INA bacteria. Bowers et al. (2009) compared the densities of biological IN in clear and cloudy air and found that IN density significantly increased with increasing humidity, ranging between 0 and 91 biological IN per m 3 of air. It has been shown that 0.1 % of INA cells of Pseudomonas syringae could act as IN at simulated cloud conditions (Delort et al. 2010). Very little remains known about the abundance of cloudborne INA bacteria and their actual involvement in precipitation processes. Bacterial Diversity in the Atmosphere and Clouds Diverse bacterial communities have been described in the atmosphere (Radosevich et al. 2002; Maron et al. 2005; Bowers et al. 2009) and cloud water (Delort et al. 2010; Kourtev et al. 2011; Šantl-Temkiv et al. 2012). Large temporal and spatial variations are characteristic for the boundary layer bacterial community composition (Bowers et al. 2011, 2012 ), which are likely due to variable emission rates from different local sources together with the contribution of bacteria being transported across long distances. A few metagenomic studies, placing emphasis on bacterial diversity, were carried out on cloud water and air. Maron et al. (2005) used A-RISA patterns to study bacterial diversity of two airborne communities and found a high diversity, with bacterial species richness, evenness and rarefaction curves falling in the range of values typical for soil communities. Using pyrosequencing of the SSU rRNA genes, Bowers et al. (2009) found that atmospheric samples contained approximately 170 OTUs at a species level without exhausting the total diversity. Kourtev et al. ( 2011) employed aircraft-based sampling to investigate microbial communities of two clouds using DGGE and sequencing of SSU rRNA genes. They demonstrated that not only the atmosphere but also cloud water harbors a large diversity of bacteria. The number of DGGE bands was 17-21, showing that the diversity of the most abundant bacteria was comparable to metabolically diverse communities, e.g. bacterioplankton in lakes. Another study on bacterial diversity focused on investigating the diversity of a storm cloud, by the use of large individual hailstones functioning as replicate samples (Šantl-Temkiv et al. 2012). Sequencing the SSU rRNA genes, Šantl-Temkiv et al. (2012) detected 231 OTUs at the species level in nine replicates, and estimated that total bacterial species richness of the storm cloud was on the range of 1,800 OTUs, making it an environment with high diversity roughly comparable to that of soil and marine environments. Furthermore, a medium species evenness characteristic of the same community indicated that the community was balanced and had an ability to resist environmental stress. Employing metaanalysis of high-throughput pyrosequencing data obtained from numerous near-surface atmospheric samples, together with previously published sequences from terrestrial environments, Bowers et al. ( 2011, 2012) studied the spatial and temporal variability of airborne bacteria and its dependence on different sources. They found that bacterial composition was significantly affected by season and land-use type (forests, agricultural, and suburban areas), which was a result of changes in relative inputs from different local source communities. Dominant source communities were soils, plant surfaces, snow, and feces, all having different relative contributions depending on season and location. A compelling result of Bowers et al. (2011) was the overall similarity in bacterial community structure of atmospheric samples when compared to their source environments, which indicates the existence of a bacterial community that is characteristic for the atmosphere. Bowers et al. (2011) speculated that the atmospheric communities over terrestrial surfaces are a mixture of bacteria emitted from their source environments, with different groups having differential ability of survival in the atmosphere. http://www.springerreference.com/index/chapterdbid/303413 © Springer-Verlag Berlin Heidelberg 2014 28 Apr 2014 04:10 3 Dr. Tina Santl Temkiv, Dr. Kai Finster and Dr. Ulrich Gosewinkel Karlson Cloud and Atmosphere Metagenomics SpringerReference Although representatives from diverse bacterial phyla have been detected in atmospheric samples, generally Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes are considered to dominate the atmosphere. In addition, Plantomycetes (Šantl-Temkiv et al. 2012) and Acidobacteria (Bowers et al. 2009, 2012) were found as important members of some airborne communities, indicating a higher contribution of aquatic or soil bacterial sources. Members of Proteobacteria are generally the most abundant phylum both in dry air (Maron et al. 2005; Bowers et al. 2009 ; Zweifel et al. 2012) as well as in cloud water samples (Šantl-Temkiv et al. 2012). Some epiphytic species within the γProteobacteria represent an atmospherically important group, as they carry INA proteins. It is striking that members of the genus Pseudomonas were detected almost universally in different aerosol and cloud samples (Maron et al. 2005; Bowers et al. 2009; Delort et al. 2010; Fahlgren et al. 2010; Zweifel et al. 2012). Ahern et al. (2007), who retrieved a clone library of 256 sequences from two clouds and two simultaneously collected rain samples, found that the largest operational taxonomic units (OTUs) consisted of sequences closely related to different Pseudomonas spp., including known INA bacteria P. syringae and P. fluorescens. Different strains of INA bacteria have been isolated from cloud water and precipitation (Morris et al. 2008). Despite the apparent omnipresence of Pseudomonas strains in the atmosphere, Ahern et al. (2007) did not find any INA strain, when trying to detect the presence of INA gene and phenotype among 80 Pseudomonas isolates from cloud water. More recently, Pseudomonas spp. closely related to known INA bacteria were commonly found in air above the atmospheric boundary layer (Zweifel et al. 2012). Some γ-Proteobacteria genera (e.g., Pseudomonas) and epiphytic members of α-Proteobacteria (e.g., Methylobacterium) that were detected in clouds may be able to grow in clouds (Kourtev et al. 2011; Šantl-Temkiv et al 2012) as they can utilize a variety of carbon sources present in clouds and have fast growth responses and high growth rates (Šantl-Temkiv et al. 2012). Bacterial strains belonging to Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, which were isolated from cloud water, were shown to grow on organic compounds found in clouds (formate, acetate, lactate, succinate, formaldehyde, and methanol) (Amato et al. 2007). Vaïtilingom et al. (2011) investigated the biodegradation of the same carboxylic acids in artificial cloud water medium at 5-17 °C. They used 17 cloudborne bacterial strains belonging to the genera Arthrobacter, Bacillus, Clavibacter, Frigoribacterium, Pseudomonas, Sphingomonas, and Rhodococcus. Overall, Pseudomonas and Rhodococcus strains were best in degrading tested compounds, whereas Arthrobacter, Bacillus, Clavibacter, and Frigoribacterium did not degrade any. As Gram-positive bacteria are less resistant to stress when in the vegetative state, they are likely present in the atmosphere in the form of resting stages. Resting stages increase the chances of these bacterial groups for long distance dispersal in a viable state, but strains forming resting stages are unlikely to play a role in atmospheric chemistry. It has recently become clear that, although being subject to large temporal and spatial variations, the airborne bacterial communities are distinct from their sources and can thus be considered characteristic of the atmosphere. Further carefully designed metagenomic studies of the atmosphere, such as done by Bowers et al. (2011), are needed to elucidate the selective role of the atmosphere during bacterial emission, residence, and deposition and its influence on the patterns of bacterial distribution. In addition, the actual bacterial involvement in the processes of atmospheric physics and chemistry also still needs to be confirmed. Summary Serving as a dominant route for bacterial dispersal, the atmosphere may act as a selective barrier influencing patterns of microbial distribution. Cloud droplets, offering a liquid environment and concentrated nutrients, may even support bacterial growth. Recent metagenomic studies revealed diverse bacterial communities, whose compositions are influenced mostly by season and location. Of the common bacterial phyla (Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes) generally described in atmospheric samples, groups of α- and γ-Proteobacteria appear to be particularly important. These groups have been in focus, as they may have impacts on formation of precipitation and chemical transformations in the atmosphere. Further studies are needed for clarification of the selective role of the atmosphere in bacterial dispersal as well as of bacterial involvement in atmospheric processes. References Ahern HE, Walsh KA, Hill TCJ, et al. Fluorescent pseudomonads isolated from Hebridean cloud and rain water produce biosurfactants but do not cause ice nucleation. Biogeosci. 2007;4(1):115-24. Amato P, Demeer F, Melaouhi A, et al. A fate for organic acids, formaldehyde and methanol in cloud water: their http://www.springerreference.com/index/chapterdbid/303413 © Springer-Verlag Berlin Heidelberg 2014 28 Apr 2014 04:10 4 Dr. Tina Santl Temkiv, Dr. Kai Finster and Dr. Ulrich Gosewinkel Karlson Cloud and Atmosphere Metagenomics SpringerReference biotransformation by micro-organisms. Atmos Chem Phys. 2007;7(15):4159-69. Ariya PA, Nepotchatykh O, Ignatovaand O, et al. Microbiological degradation of atmospheric organic compounds. Geophys Res Lett. 2002;29(22):2077-81. Bowers RM, Lauber CL, Wiedinmyer C, et al. Characterization of airborne microbial communities at a high-elevation site and their potential to act as atmospheric ice nuclei. Appl Environ Microbiol. 2009;75(15):5121-30. Bowers RM, McLetchie S, Knight R, et al. Spatial variability in airborne bacterial communities across land-use types and their relationship to the bacterial communities of potential source environments. ISME J. 2011;5:601-12. Bowers RM, McCubbin IB, Hallar AG, et al. Seasonal variability in airborne bacterial communities at a high-elevation site. Atmos Environ. 2012;50:41-9. Burrows SM, Elbert W, Lawrence MG, et al. Bacteria in the global atmosphere - part 1: review and synthesis of literature data for different ecosystems. Atmos Chem Phys Discuss. 2009a;9:10777-827. Burrows SM, Butler T, Jöckel P, et al. Bacteria in the global atmosphere - part 2: modeling of emissions and transport between different ecosystems. Atmos Chem Phys. 2009b;9:9281-97. Christner BC, Morris CE, Foreman CM. Ubiquity of biological ice nucleators in snowfall. Science. 2008;319(5867):1214. Delort AM, Vaïtilingom M, Amato P, et al. A short overview of the microbial population in clouds: potential roles in atmospheric chemistry and nucleation processes. Atmos Res. 2010;98(2-4):249-60. Fahlgren C, Hagström Å, Nilsson D, et al. Annual variations in the diversity, viability, and origin of airborne bacteria. Appl Environ Microbiol. 2010;76(9):3015-25. Hill KA, Shepson PB, Galbavy ES, et al. Processing of atmospheric nitrogen by clouds above a forest environment. J Geophys Res. 2007;112(D11):1-16. Kourtev PS, Hill KA, Shepson PB, et al. Atmospheric cloud water contains a diverse bacterial community. Atmos Environ. 2011;45:5399-405. Marinoni A, Laj P, Sellegri K, et al. Cloud chemistry at the Puy de Dome: variability and relationships with environmental factors. Atmos Chem Phys. 2004;4:715-28. Maron P-A, Lejon DPH, Carvalho E, et al. Assessing genetic structure and diversity of airborne bacterial communities by DNA fingerprinting and 16S rDNA clone library. Atmos Environ. 2005;39:3687-95. Möhler O, DeMott PJ, Vali G, et al. Microbiology and atmospheric processes: the role of biological particles in cloud physics. Biogeosci. 2007;4:1059-71. Morris CE, Sands DC, Vinatzer BA, et al. The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. ISME J. 2008;2:1-14. Radosevich JL, Wilson WJ, Shinn JH, et al. Development of a high-volume aerosol collection system for the identification of air-borne micro-organisms. Lett Appl Microbiol. 2002;34:162-7. Šantl-Temkiv T, Finster K, Hansen BM, et al. The microbial diversity of a storm cloud as assessed by hailstones. FEMS Microbiol Ecol. 2012;81(3):684-695. Sattler B, Puxbaum H, Psenner R. Bacterial growth in supercooled cloud droplets. Geophys Res Lett. 2001;28(2):239-42. Vaïtilingom M, Charbouillot T, Deguillaume L, et al. Atmospheric chemistry of carboxylic acids: microbial implication versus photochemistry. Atmos Chem Phys Discuss. 2011;11:4881-911. Zweifel UL, Hagström Å, Holmfeldt K, et al. High bacterial 16S rRNA gene diversity above the atmospheric boundary layer. Aerobiologia. 2012;28(4):481-498. http://www.springerreference.com/index/chapterdbid/303413 © Springer-Verlag Berlin Heidelberg 2014 28 Apr 2014 04:10 5 Dr. Tina Santl Temkiv, Dr. Kai Finster and Dr. Ulrich Gosewinkel Karlson Cloud and Atmosphere Metagenomics SpringerReference Cloud and Atmosphere Metagenomics Dr. Tina Santl Temkiv Department of Physics and Astronomy, Aarhus University, Aarhus C, Denmark Dr. Kai Finster Department of Bioscience, Microbiology Section, Aarhus University, Aarhus, Denmark Dr. Ulrich Gosewinkel Karlson Department of Environmental Science,, Aarhus University, Roskilde, Denmark DOI: 10.1007/SpringerReference_303413 URL: http://www.springerreference.com/index/chapterdbid/303413 Part of: Encyclopedia of Metagenomics Editor: Dr. Karen E. Nelson PDF created on: April, 28, 2014 04:10 © Springer-Verlag Berlin Heidelberg 2014 http://www.springerreference.com/index/chapterdbid/303413 © Springer-Verlag Berlin Heidelberg 2014 28 Apr 2014 04:10 6