Aerosols play a crucial role in cloud formation. Biologically derived materials from bacteria, fungi, pollen, lichen, viruses, algae, and diatoms can serve as ice nucleating particles (INPs), some of which initiate glaciation in clouds at relatively warm freezing temperatures. However, determining the magnitude of the interactions between clouds and biologically derived INPs remains a significant challenge due to the diversity and complexity of bioaerosols and limited observations of such aerosols facilitating cloud ice formation. Additionally, microorganisms from the domain Archaea have, to date, not been evaluated as INPs. Here, we present the first results reporting the ice nucleation activity of four species in the class Haloarchaea. Intact cells of
Through their impact on the atmospheric energy budget and hydrological cycle, clouds play a prominent role in shaping Earth's climate at both global and regional scales (Baker and Peter, 2008; Boucher et al., 2013). However, due to the complexity of the microphysical processes associated with cloud formation and dynamics, clouds remain some of the most poorly constrained atmospheric features in climate models. In turn, while atmospheric aerosols strongly impact cloud formation, albedo, lifetime, and precipitation formation processes, disentangling the relationships and feedbacks among aerosols, clouds, and precipitation remains a significant challenge (Sato and Suzuki, 2019; Stevens and Feingold, 2009). In particular, the role of aerosols called ice nucleating particles (INPs), which induce glaciation in the mixed phase and ice clouds, is highly uncertain relative to other aerosol–cloud processes (Kanji et al., 2017). Improving our understanding of such ice formation processes is crucial given that most precipitation, globally, is initiated via the ice phase (Lohmann and Feichter, 2005).
In the Earth's troposphere, pure water remains in a supercooled liquid state until below
Even though many laboratory and field-based investigations have alluded to the importance of biologically derived INPs, the relatively limited available observational data have caused models to produce equivocal results regarding the global significance of biological ice nucleation in cloud and precipitation formation (Burrows et al., 2013; Hoose et al., 2010a; Hummel et al., 2018; Phillips et al., 2009; Sesartic et al., 2012; Twohy et al., 2016; Vergara-Temprado et al., 2017). This modeling issue is further complicated by a very limited understanding and representation of secondary ice formation processes and their links to biologically derived INPs in clouds. Climate models of all scales require information on INP sources to accurately represent ice nucleation and, thus, cloud microphysics, especially considering that (1) biologically derived INPs form ice at cloud temperatures as high as
While there have been ongoing efforts to identify and characterize INPs in the eukaryotic and bacterial domains (Dreischmeier et al., 2017; Failor et al., 2017; Fröhlich-Nowoisky et al., 2016; Hill et al., 2014; Kanji et al., 2017; Kunert et al., 2019; Ling et al., 2018; Morris et al., 2008; Pummer et al., 2012; Qiu et al., 2019), to date, there has been no study that has attempted to identify INPs within the domain Archaea. This is in no small part due the fact that, until recently, the Archaea were believed to be largely relegated to marginal existences in extreme environments on our planet. Though recent studies have begun to reveal the true ubiquity and abundance of Archaea in Earth's ecosystems – including seawater, ocean sediment, plankton, soil, marine and terrestrial biofilms, and sea ice, where they can comprise up to 40 % of the microbial taxa in an ecosystem (Cavicchioli, 2011; Flemming and Wuertz, 2019; Hoshino and Inagaki, 2019; Junge et al., 2004; Mondav et al., 2014; Munson et al., 1997; Ochsenreiter et al., 2002; Santoro et al., 2019) – many Archaea remain uncultured and unculturable in laboratory settings, further complicating investigations into their possible propensities to serve as INPs. At the same time, however, the archaeal domain contains both unique cell wall compositions and cell surface structures not present in the other domains (Albers and Meyer, 2011). While appearing highly similar to the bacterial domain in both size and appearance, the archaeal cell envelope is distinct in several ways. In contrast to the bacteria, most cultured Archaea maintain a proteinaceous surface layer, or S layer, that provides the cell with structural stability. In many Archaea, the S layers provide the entirety of the cell envelope. Comparatively few Archaea contain additional cell envelope polymers, and the ones that do, do not produce the near-ubiquitous bacterial polymer peptidoglycan. Some Archaea do produce a structurally similar polymer, pseudomurein. The archaeal S layers are often composed of a single protein or glycoprotein arranged in symmetrical patterns, with hexagonal symmetry being the most common (Albers and Meyer, 2011). As with the bacteria, many surface-exposed proteins are modified in a variety of ways. In addition to the S layer itself, the archaeal domain contains its own regimen of surface proteins and structures that interact with the external environment. Thus, an entire domain worth of cell surface structures remains to be assessed for potential INP activity.
Here we present a first attempt to assess the potential for members of the domain Archaea to serve as INPs. We initiated our investigation with the following four members of the obligate halophilic lineage, Haloarchaea:
We opted to initiate our investigation with members of the Haloarchaea for a variety of reasons, including the following: (1) the diversity of cell surface compositions, (2) that they are relatively easy to culture compared to other archaeal lineages, and (3) that the regional dominance of the Haloarchaea in relatively large hypersaline environments. While few large-scale geographic areas are dominated by members of the domain Archaea, one notable exception is hypersaline bodies of water such as the north basin of the Great Salt Lake and the Dead Sea. These waterbodies extend over hundreds of square kilometers, often contain upwards of 90 % Archaea, and can impact the local climate and weather patterns (Carpenter, 1993). Thus, when investigating the impact of potential archaeal INPs in an environment, halophiles offer attractive starting points.
To fully understand the interaction of Earth's climate with the microbial world, it is imperative to include the impact of the archaeal domain since, even when less prevalent, the possession of ice nucleation activity will enable a species to exert an outsized effect on its environs. And to fully understand the potential impact of the archaeal domain on Earth's climate, it is important to assess the potential for members of the Archaea to serve as INPs and contribute to cloud formation.
Cell cultures for all four haloarchaeal species tested were purchased from the DSMZ (German Collection of Microorganisms and Cell Cultures;
Samples produced from all four haloarchaeal species for ice nucleation testing. Initial sample cell concentrations and salinities are provided after culturing. Diluted cell concentrations and salinities were calculated based on the volume of culture mixed with media and dilution factor. The cell state is also provided after dilution.
Due to the high salinities of the media which would have caused significant freezing point depression – for reference, seawater is typically 30–35 ppt (Bodnar, 1993) and can depress the freezing point of water by
INP concentrations were measured using the Colorado State University (CSU) Ice Spectrometer (IS; Hiranuma et al., 2015; Mccluskey et al., 2018; Suski et al., 2018), which is an immersion freezing measurement device suited to testing aliquots of liquid culture. The IS is constructed using two 96-well aluminum incubation blocks, designed for incubating polymerase chain reaction (PCR) plates placed end-to-end and encased on their sides and base by cold plates. Immersion freezing temperature spectra were obtained by dispensing 50
Heat and peroxide treatments were conducted to isolate heat labile (e.g., proteinaceous) and organic INPs in the diluted samples (Barry et al., 2021; Creamean et al., 2020; Hill et al., 2016; Mccluskey et al., 2018; Perkins et al., 2020a; Suski et al., 2018; Tobo et al., 2014). Each sample (i.e., all dilutions listed in Table 1) was subjected to heat and peroxide treatments to obtain the heat labile (proteinaceous) and organic frozen fractions in addition to the total (unamended) frozen fractions. The stability (or lack thereof) of INPs to these treatments provides an indication of the composition. To assess the contribution of heat labile entities, a 1.5 mL aliquot of suspension was tested after heating to 95
Figure 1 shows the results of the
Freezing spectra for each of the haloarchaeal species diluted
As expected, ice nucleation activity did not occur in all tested haloarchaeal species, just as with bacteria (Karimi et al., 2020; Szyrmer and Zawadzki, 1997). Lysed cells of both
Both
Unamended, heat labile (heat), and organic (peroxide) frozen fractions for each of the three
Unamended, heat labile (heat), and organic (peroxide) frozen fractions for each of the
For the other three haloarchaeal species, only
Here, we present the first reported results on the ice nucleation activity of the domain Archaea. Specifically, we focus on four species within the Haloarchaea due to their diversity of cell surface compositions, ease of culturability, and regional presence within relatively large hypersaline environments. The freezing temperature ranges measured for the Haloarchaea species involved in this study are put into a broader context by comparing them to reported ranges for other known biologically derived INPs (Fig. 4). While not the most proficient of biologically derived INPs such as lichen, bacteria, fungi, viruses, algae, pollen, or pollen wash water, which nucleate ice at warmer temperatures, Haloarchaea fall within moderate freezing temperature ranges above phytoplankton exudates, diatoms, and fungal spores. However, this work is based on a limited subset of species, and future work should focus on other members of the Archaea.
Summary of approximate freezing temperature ranges reported for known biologically derived INPs. Haloarchaea data are those from
All Haloarchaea species were introduced to hyposaline conditions to reduce freezing point depression. While intact cells of
While halophilic Archaea are prominent in hypersaline environments throughout the globe, such as the Great Salt Lake and the Dead Sea, other members of the domain Archaea, such as methanogens and thermophiles, are prevalent in anoxic systems in seawater, sea ice, marine sediments, glacial ice, permafrost, hot springs, submarine hydrothermal vents, and hot, dry deserts (Amend and Shock, 2001; Collins et al., 2010; Oremland and Taylor, 1978; Price, 2007; Thauer et al., 2008; Thummes et al., 2007; Van Der Maarel et al., 1999). However, some studies allude to the fact that these extremophiles are not confined to extreme living conditions, which qualifies them as one of the most abundant prokaryotes on Earth (Delong, 1998). Thus, these microorganisms are more ubiquitous than one might think, are present in the atmosphere (Fröhlich-Nowoisky et al., 2014), and may affect cloud formation (Amato et al., 2017). Indeed, the order Halobacteriales, which contains
Data used in this paper can be accessed in the Supplement.
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JMC and MER were responsible for overseeing these experiments. JMC lead the writing of this paper. JEC, LN, and ADP prepared the cultures and ran the immersion freezing tests. TCJH and PJD helped with interpretation of the results. All authors contributed to the writing of this paper.
The authors declare that they have no conflict of interest.
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The authors would like to acknowledge Stephen Schmidt and Pacifica Sommers of the University of Colorado, Boulder, for assisting with the preservation and culturing of the Haloarchaea after its arrival in Colorado.
Julio E. Ceniceros was supported by the US Department of Commerce, National Oceanic and Atmospheric Administration, Educational Partnership Program (grant no. NA16SEC4810006). This work was funded by a NASA EPSCoR Research Infrastructure Development for the state of South Carolina.
This paper was edited by Denise Akob and reviewed by Brent Christner and three anonymous referees.