Ice-nucleating particles (INPs) are atmospheric aerosol particles
that can strongly influence the radiative properties and precipitation onset
in mixed-phase clouds by triggering ice formation in supercooled cloud water
droplets. The ability to distinguish between INPs of mineral and biological
origin in samples collected from the environment is needed to better
understand their distribution and sources. A common method for assessing the
relative contributions of mineral and biogenic INPs in samples collected
from the environment (e.g. aerosol, rainwater, soil) is to determine the
ice-nucleating ability (INA) before and after heating, where heat is
expected to denature proteins associated with some biological ice nucleants.
The key assumption is that the ice nucleation sites of biological origin are
denatured by heat, while those associated with mineral surfaces remain
unaffected; we test this assumption here. We exposed atmospherically
relevant mineral samples to wet heat (INP suspensions warmed to above 90
In the absence of nucleation sites, cloud water droplets can supercool to
temperatures below around
To represent the impact of INPs on clouds in our models, we must improve our understanding of the global distribution and temporal variability of INPs. However, much uncertainty remains regarding the distribution, sources, and relative ice-nucleating ability (INA) of INPs throughout the Earth's atmosphere (Kanji et al., 2017; Huang et al., 2021; Murray et al., 2021). Two important general categories of INPs are mineral dust (Hoose et al., 2010; DeMott et al., 2003; Vergara-Temprado et al., 2017) and biogenic materials (Vergara-Temprado et al., 2017; Creamean et al., 2013).
Laboratory and field data indicate that mineral dusts often dominate the INP
population relevant for mixed-phase clouds below around
Biological INPs are capable of nucleating ice at much warmer temperatures
than all but the most active minerals and can include primary biological
particles (PBAPs) such as bacteria, fungal spores, pollen grains, fragments
of terrestrial organic material such as cellulose (Hiranuma et al., 2015b, 2019) and macromolecules of marine biogenic origin (Schnell
and Vali, 1976; Warren, 1987; Wilson et al., 2015; McCluskey et al., 2018a).
Atmospheric concentrations of ice-active bacteria, fungal spores and pollen
grains are much smaller than mineral dusts (Hoose et al., 2010). Estimates
of the mass of PBAPs emitted to the atmosphere annually range from low
hundreds to
Biological INPs tend to nucleate ice at temperatures where they may initiate secondary ice production processes (Morris et al., 2014; Field et al., 2017), thus amplifying their effect in clouds. Biogenic INPs could also play an important role in feedback processes in the rapidly warming Arctic climate, as increasing surface temperatures may expose new terrestrial sources in thawing permafrost (Creamean et al., 2020) or newly exposed glacial outwash sediments (Tobo et al., 2019) or reveal new marine reservoirs as the sea ice coverage is reduced (Hartmann et al., 2020). While the INA of mineral dust from various arid sources around the world is relatively similar (within around a factor of 10) (Niemand et al., 2012; Atkinson et al., 2013), the INA of biological material varies massively between the various sources, which makes predicting the INP population of biological material particularly challenging.
Much effort in the past decade has been put into not only collecting and identifying biogenic INPs or their markers in the environment, but also determining their relative contributions to the total measured INP population (Huang et al., 2021). While techniques such as genomic sequencing (Garcia et al., 2012; Huffman et al., 2013; Hill et al., 2014) and microscopy (Huffman et al., 2013; Sanchez-Marroquin et al., 2021) can reveal the presence of biological species in an aerosol sample that has been found to contain INPs, it remains difficult to characterise the ice-nucleating ability of these species over other constituents (e.g. mineral dusts) when a sample's INA is analysed by, for example, a droplet freezing assay alone. To date, no high-throughput technique has been established that can directly identify both the composition and nucleation temperatures of a specific INP type within a sample. However, a widely used methodology for performing an indirect assessment of the contribution of mineral vs. biogenic INPs involves treating a collected aerosol sample (or other INP-containing media) with heat and comparing its INA spectrum before and after heating. Changes in INA can then be related to the presence and domination of biogenic INPs over inorganic INPs based on several assumptions, as discussed below. This heat treatment procedure has the advantages of being suitable for high-throughput offline sample analysis and does not require specialised equipment or the addition of reagents to selectively degrade biological material such as hydrogen peroxide (Suski et al., 2018; O'Sullivan et al., 2014; Tobo et al., 2019), lysozyme (Joyce et al., 2019; Henderson-Begg et al., 2009) or guanidinium hydrochloride (Conen and Yakutin, 2018). We have compiled a list of past studies which have employed heat tests to detect biological INPs with the conditions and method of INP detection in Table 1.
List of past studies in which heat treatments were used to infer the presence of biological INPs in samples of various environmental media.
Continued.
CFDC: continuous flow diffusion chamber. DFA: droplet freezing assay.
The identification of the presence of biogenic INPs using a heat test is
based on the assumption that heat will inactivate biogenic (often but not
always explicitly proteinaceous) INPs, yielding a reduction in ice
nucleation temperatures following the treatment, while the INA of inorganic
INPs (likely to be dominated by mineral dust) will remain unaffected (Conen
et al., 2011). In addition to merely determining the presence of biogenic
INPs, this method has also been used by some researchers to quantify the
abundance of biogenic INPs in their samples by evaluating the magnitude of
the INA reduction (Christner et al., 2008a, b; Joly et al., 2014; Joyce et al., 2019). The assumption that protein-bearing biological INPs associated with
bacteria and fungi can lose at least some of their INA when sufficiently
heated (up to 100
Finally, several studies have demonstrated the apparent lability of mineral INPs kept in deionised water at room temperature over hours to days, wherein the immersion mode INA gradually decreased, which has been observed with samples of K-feldspar (Harrison et al., 2016; Peckhaus et al., 2016), quartz (Harrison et al., 2019; Kumar et al., 2019a) and ATD (Perkins et al., 2020). It is reasonable to predict that elevated temperatures could accelerate the “ageing” behaviour seen with these minerals, leading to an INA deactivation on the timescale of a biological INP heat test.
Overall, this highlights that the potential for the false positive “detection” of biogenic INPs through the loss of mineral INA when using heat treatments has yet to be ruled out. Here, we aim to validate the heat test in its current form by fully characterising how mineral INPs respond to heating both in air and in water compared to biogenic INPs. We achieve this via a laboratory study in which we tested the immersion mode INA of a set of atmospherically relevant mineral samples before and after two types of heat treatment. We also performed equivalent tests on a set of biogenic INP analogue samples for direct comparison to the mineral INP results and as a positive control to ensure that the heat treatments would reproduce the known heat sensitivity behaviour of biogenic INPs.
We employed two methods of heat treatment: (1) direct heating of the sample in aqueous suspension (wet heating) and (2) heating the sample while in dry powder form prior to immersion in water (dry heating). This enabled us to investigate whether the deactivation behaviour of a sample depends on the medium in which it is heated, as previous studies have involved heating samples in either the wet or dry mode but not both. Our rationale for this is that an INP sample's potential chemical or physical reactions to heating in water or air may differ as these are fundamentally different treatments. Where possible, we also characterise the heat sensitivity of the important subclasses of mineral INPs and then discuss how this could affect interpretations of biogenic INP heat test results and how this can inform us in the development of a more robust protocol. While our primary objective was to empirically evaluate commonly employed heat tests, we also discuss the physical reasons for the changes in INA found in our results, which may prove useful for future studies on the fundamental mechanisms of how mineral surfaces nucleate ice. This work may also be pertinent to emerging practical applications for mineral-based ice-nucleating agents in fields such as cell cryopreservation (Daily et al., 2020; Wragg et al., 2020; Morris and Lamb, 2018).
A set of atmospherically relevant ice-nucleating materials was assembled into two broad classes of “mineral” and “biogenic” for heat treatment experiments. The mineral class comprised samples of ground minerals (either purchased from vendors in a milled form or milled in-house from a bulk mineral using a planetary ball mill) and commercially available dust proxies. Details of the identity, provenance and purity of each of these are provided in Tables 2 and 3 background information on each class of mineral, and their significance as atmospheric INPs is provided in Sect. S1 of the Supplement. Most emphasis was placed on the feldspar and silica classes of minerals as these have previously been shown to be the most ice-active mineral classes in immersion mode freezing experiments (Atkinson et al., 2013; Harrison et al., 2019; Peckhaus et al., 2016) and therefore likely control the INA of a mineral dust assemblage of mixed mineralogy. Several of our samples have been analysed in the past using the same method and instrumentation as we employed here (Atkinson et al., 2013; Whale et al., 2017; Harrison et al., 2016, 2019), and of these only Atkinson quartz showed a deviation (slight loss in activity) in INA since they were last tested. This indicates that the INA of the mineral samples remains largely stable while in storage. The remaining mineral samples were clay-based samples, the dust surrogates NX illite and ATD, and, finally, calcite.
Sample information for mineral-based INP samples. Sources of data for purity and specific surface area (SSA) are detailed in the annotations.
References:
Sample information for biological-based INP samples.
We included five different samples of K-feldspar in our survey (see Fig. 2a) in order to represent the diversity of this group of minerals. These included BCS-376 microcline, which was studied previously (Atkinson et al., 2013) and is considered generally representative of the INA of standard K-feldspars (Harrison et al., 2016); amazonite and TUD#3 microcline, which are samples of microcline that show exceptionally high INA compared to typical variants of microcline and the other K-feldspar polymorphs for reasons that are still unclear (Harrison et al., 2016; Welti et al., 2019; Peckhaus et al., 2016); and Eifel sanidine, which exhibits much lower INA compared to the other samples due to a lack of features related to exsolution microtexture (Kiselev et al., 2021; Whale et al., 2017).
Three samples of plagioclase feldspar were included (see Fig. 3), two of which – BCS-375 albite (Atkinson et al., 2013; Harrison et al., 2016) and TUD#2 albite (Peckhaus et al., 2016) – are predominantly composed of the albite endmember, and labradorite – a plagioclase that features a Ca composition between 50 % and 70 % that of anorthite. BCS-375 albite contains quartz (4.0 %) and K-feldspar (16.7 %) impurities, while TUD#2 albite contains at least 90 % plagioclase feldspar, with the remaining 10 % of the content being unknown (based on X-ray diffraction (XRD) data; Atkinson et al., 2013; Peckhaus et al., 2016). The presence of K-feldspar in the former may mean that the observed activity is related to the presence of this component. No information is available on the mineral impurities present in the labradorite sample. However, as plagioclase feldspar of labradorite composition is typically only found in basalts and gabbros, it is unlikely to coexist with quartz or K-feldspar since these rarely occur in these types of igneous rocks.
We included three samples of silica (see Fig. 4a): two
To represent clays, we included samples that represent the main classes of clay minerals but also different samples of the same mineral to account for impurities which, due to the generally low INA of clays, may control the INA of the sample. Two samples of kaolinite (KGa-1b kaolinite and Fluka kaolinite), two samples of montmorillonite (SWy-2 montmorillonite and Sigma montmorillonite) and one sample of chlorite (Chlorite) are included. An illite-rich sample (NX illite) was included, but we classed this along with Arizona Test Dust (ATD) as a mineral dust analogue – these being commercially available dusts of composite mineralogy which have been used in the past as representative surrogates of atmospheric mineral dust. Finally, powder from a ground pure calcite crystal was used to represent the carbonates.
The biogenic class of samples tested here included examples of material with
expected INA heat sensitivity in which proteins are responsible for ice
nucleation (Snomax® as a non-viable form of
All aqueous suspensions were prepared in 0.1
Each INP sample was subjected to heat treatment using two distinct methods:
a “wet heating” treatment wherein the INP was heated while in suspension and a “dry heating” treatment wherein the INP sample was heated in dry powder form in air and subsequently mixed with water for analysis. The “standard” temperature and duration for the wet heat test was 95
The wet heating treatment comprised of a sealed vessel containing the INP
suspensions, described in Sect. 2.2, immersed in an open boiling
water bath (hence at the boiling point of water at 1 atm: 100
The temperature profile of the liquid inside 20 mL borosilicate glass vials
and 50 mL polypropylene centrifuge tubes (Corning Falcon 352090) throughout
the wet heat treatment procedure was measured (Fig. A2 in Appendix A) by
inserting a thermocouple (Type K) through a small hole punched in the caps
of the vessels and was recorded using a data logger (TC-08,
In addition to our standard 30 min heat treatment, we performed extended wet
heat treatments of up to 24 h for selected samples by immersing the vessels
in a silicone oil bath heated to 100
Dry heating of samples was achieved by placing a 20 mL borosilicate glass
vial containing a maximum of 0.2 g of dry sample in a standard laboratory
oven at 250
The INA of the mineral-based and biogenic sample suspensions both before and
after heat treatments was determined by performing immersion mode droplet
freezing assays (Vali, 1971) using the Microlitre Nucleation by Immersed
Particle Instrument (
Analysis of the droplet freezing events allowed the determination of the
fraction of droplets frozen as a function of temperature,
Fraction of frozen droplet (
Quantification of a nucleator's INA was achieved by determining the surface
density of ice-active sites,
In Fig. 1 we have shown several examples of fraction frozen curves for heated (wet and dry) and unheated samples to illustrate the heat sensitivity
of a range of ice-nucleating materials. Similar plots for all materials we
have tested here are shown in Figs. S1 and S2. In order to present this
information in a more concise manner, we have plotted the same data in the
form of boxplots of droplet freezing temperatures of mineral samples
throughout the results section. In addition, changes in INA resulting from
the heat treatments were evaluated by calculating the freezing temperature
shifts between them and determining whether the shifts were significantly
larger than the instrumental error. This was simply taken as the difference
between the median droplet freezing temperature (
In general, the INA of K-feldspar samples did not respond substantially to
wet heating for 30 min with no significant reductions of
To discuss the reasons behind the deactivation of K-feldspar when wet heated for longer than 30 min, the nature of the ice-nucleating sites on minerals must first be considered. Ice nucleation on mineral surfaces such as feldspars has been shown to occur at specific sites that become active at a specific temperature (Holden et al., 2019, 2021). Topographical features associated with exsolution microtexture (Whale et al., 2017; Kiselev et al., 2021) have been proposed as the locations of the highly active sites on K-feldspar. Moreover, Kiselev et al. (2017) observed that ice crystals growing from the vapour phase on the surface of microcline originated on steps and cracks and were preferentially orientated between the basal face of ice and the (100) cleavage plane. More recent work suggests that cracks caused by exsolution microtexture may expose the (100) face of feldspars (Kiselev et al., 2021). The chemical and physical nature of these sites is still unclear; however molecular dynamics studies such as those by Pedevilla et al. (2017) show that having a high density of functional groups like silanol groups (Si-OH), where water can hydrogen bond with the mineral surface and potentially order (such as those exposed at the (100) cleavage plane), may be important for nucleating ice (Harrison et al., 2019).
The most obvious physical cause of the INA deactivation of K-feldspar by wet
heating would be the alteration of the mineral surface by dissolution via
hydrolysis. This leaves an amorphous “leached” layer at the surface (Lee et
al., 2008; Chardon et al., 2006), destroying or at least disrupting the
ice-active sites. Several studies have shown experimentally that acid
treatment deactivates K-feldspar INPs (Augustin-Bauditz et al., 2014; Kulkarni et al., 2015; Kumar et al., 2018). In pure water and at
near-neutral pH, however, the supply of H
Amazonite microcline, one of our two highly ice-active microcline samples,
was an exception to other K-feldspar samples in that short-term wet heating
resulted in a significant but small deactivation (
Dry heating had a stronger deactivating effect on the K-feldspar samples
than wet heating (Fig. 2a). Amazonite microcline showed the largest
A potential alternative explanation for the apparent dry-heat sensitivity of
K-feldspar is that there is a biological component mixed with the K-feldspar
samples which nucleates ice and is deactivated on heating and, due to the
high temperatures required for deactivation, is unlikely to be bacteria- or
fungus-derived. Peckhaus et al. (2016) discussed the potential for
biological ice-nucleating material in TUD#3. They achieved a deactivation
in TUD#3 microcline by treatment with hot aqueous H
Recalcitrant organic coatings have previously been proposed as the source of INA in mineral dusts that is lost upon dry heating (Paramonov et al., 2018; Peckhaus et al., 2016; Perkins et al., 2020). However others have reported that organic coatings suppress the INA of mineral dusts rather than enhance it (Boose et al., 2019; China et al., 2017; Pach et al., 2021) by blocking access to underlying active sites. For example, Pach et al. (2021) treated slices of a K-feldspar crystal from the same locality as TUD#3 microcline with oxygen plasma and observed an enhancement in INA which they attributed to the oxidation and removal of organic material from the surface that originated from ambient air. They suggested that the plasma treatment “unblocked” the surface pores which contained the most active IN sites, allowing water to enter during their freezing experiments.
Alternatively the loss of a (non-organic) volatile component during dry
heating may alter K-feldspar in a way that reduces its INA. As described
above, amazonite microcline is a green- or turquoise-coloured variant of
microcline and was observed here to lose its green colouration upon dry
heating. This phenomenon has previously been observed (Hofmeister and
Rossman, 1985) and was correlated to the loss of water molecules that were
structurally bound within the feldspar crystal lattice. Although amazonite
is a relatively rare variety of microcline, all feldspars contain a minor
water component either as lattice-bound H
BCS-375 albite and TUD#2 albite showed no significant changes to their
Boxplot showing freezing temperatures before (black) and after heat treatments (red for wet heat, blue for dry heat) for all plagioclase feldspar samples along with clean water blank and handling blank runs.
Atkinson quartz, Fluka quartz and fused quartz all exhibited similar
reactions to both wet and dry heat treatments (see Fig. 4a). In each case,
the INA experienced significant deactivation upon wet heating (
Being sensitive to wet heat, yet virtually resistant to dry heat treatment, is an indirect but strong indication that the heat labile ice-nucleating sites on Atkinson quartz, Fluka quartz and fused quartz are not biological in nature. This is because our dry heat treatment would be expected to reduce the activity of all biological INPs (as our results with biogenic materials show in Sect. 3.2). In addition, it is interesting that the glassy fused quartz sample had very similar responses to both dry and wet heat. This indicates that the active sites on these three silica samples are not dependent on crystallinity. Given that Bombay chalcedony was the exception in this mineral class in that it was insensitive to heat, it seems that the active sites on this material were distinct to the other silica samples we studied. The high INA and stability to heat of Bombay chalcedony are comparable to several of the K-feldspar samples. Bombay chalcedony is also a microcrystalline material possessing micropores, much like K-feldspar, and this may give rise to stable active sites (Harrison et al., 2019).
Given the INA deactivation in silica samples upon heating appears to be
abiotic and only occurs in water, but not dry heat, the most obvious
explanation is that it is due to the accelerated dissolution of surface
features associated with the active sites. Active sites are thought to be
most abundant where defects and fractures occur, as milling has consistently
been found to increase the INA of quartz (Zolles et al., 2015; Kumar et al., 2019a; Harrison et al., 2019). They may also be the most unstable sites as Harrison et al. (2019) observed measurable ageing in quartz samples
(including Atkinson quartz and Fluka quartz) that were immersed in room-temperature water for only 1 h. Our wet heat treatment of Atkinson quartz
resulted in an INA deactivation of similar magnitude (
A similar apparent phenomenon of room-temperature ageing being accelerated
by heating has also been observed for BCS-376 microcline K-feldspar
(Harrison et al., 2016), except that the process appears to be much slower.
At room temperature, INA deactivations of similar magnitude (up to 2
While neither kaolinite sample was significantly sensitive to dry heat (the Fluka sample was only marginally sensitive), the Fluka kaolinite showed clear sensitivity to wet heating while KGa-1b kaolinite did not (see Fig. 5). We can perhaps attribute this to the comparatively purer state of the latter (96 % kaolinite) compared to the former (83 %) that includes a 6 % component of quartz, which was shown to be sensitive to wet heating in Sect. 3.1.3.
Boxplot showing freezing temperatures before (black) and after heat treatments (red for wet heat, blue for dry heat) for clay-based mineral samples.
The results for the montmorillonite samples were harder to interpret because
both possessed quite low purities and showed responses to heat treatments
that are not easily explained by their feldspar and quartz components. A
notable result was that the INA of the Sigma montmorillonite sample
increased after dry heat treatment. An increase in INA in the deposition
mode after dry heating has previously been observed in a smectite-rich
Saharan dust sample that had been dry heated at 300
The results for chlorite, with its high purity (99.6 %), indicated heat lability in both the wet and dry heat modes. However, chlorite likely has only limited atmospheric importance as an INP due to both its relatively low INA and typically low (around 5 %) proportional make-up of airborne mineral dusts (Murray et al., 2012; Kandler et al., 2009; Glaccum and Prospero, 1980).
The boxplots with droplet freezing temperatures for NX illite and ATD are
shown in Fig. 6a. NX illite was unresponsive to wet heating, as was
previously demonstrated by O'Sullivan et al. (2015), but deactivated
after dry heating with a
As described above, K-feldspar is mostly only sensitive to dry heating while quartz is only sensitive to wet heating, which implies that the observed changes in INA for NX illite may be controlled by the K-feldspar component while the INA of ATD may be controlled by milled quartz particles. Alternative explanations to the deactivations include biological contamination. However, similar to the results obtained for the silica samples, the greater deactivation seen in ATD from wet heating compared with that from dry heating suggests that the heat labile component is not biological.
The calcite sample displayed a reduction in INA after wet heating (
Four biological INP analogue samples were subjected to the same wet and dry
heat treatments (95
Boxplot showing freezing temperatures before (black) and after heat treatments (red for wet heat, blue for dry heat) for biological INP samples along with handling blank data for the filtration procedure described in Sect. 2.2.
After the standard heat tests Snomax® (0.05 %
Birch pollen washing water (0.5 %
Overall, the results showed that the bacterially and fungally derived samples
(Snomax® and lichen) clearly suffered substantial deactivation by the standard wet and complete deactivation to standard dry heat treatments, while BPWW and MCC showed no or very little sensitivity to wet heating but did to dry heating at 250
We performed both wet and dry heat tests on a range of mineral and biological ice-nucleating materials and directly compared their characteristic INA responses to both modes of heat treatment. Our findings, summarised in Table 4, show that the general assumption that the INA of
minerals is insensitive to heat is too simplistic, and we identified
sensitivities characteristic to important mineral classes. For example,
quartz and plagioclase feldspar INPs were found to be sensitive to wet
heating in a comparable way to proteinaceous INPs (bacteria and lichen) but
were insensitive to dry heating at considerably higher temperature (250
Summary of the characteristic responses of classes of INPs to wet and dry heat treatments.
Notes: (Stable) denotes assumed stability as heating to higher temperatures resulted in no deactivation.
The biogenic INP samples showed the clear heat sensitivity of bacterially and
fungally derived INPs – and heat resistance of pollen and cellulose INPs in wet
mode while dry heating at 250
An implication of this work is that reduced INA of INP samples subject to a heat test may be incorrectly attributed to biological INPs when heated, particularly in wet mode. But crucially, since the INA of K-feldspar is not reduced by short-term wet heating, the standard wet heat test (30 min immersed in boiling water) remains a valid method for distinguishing sensitive proteinaceous INPs from mineral dusts, so long as the INA of the mineral dust component is controlled by K-feldspar. Nevertheless, the INA heat lability of some commonly occurring minerals raises the possibility that a false positive detection of biological INPs could be made following a wet heat test; i.e. a loss of INA of quartz or plagioclase feldspar may be misconstrued as a loss of biological INA. This could occur during a scenario in which a wet heat test is performed on a sample whose mineral component INA is dominated by its silica or plagioclase feldspar content rather than K-feldspar and in which sensitive proteinaceous biological INPs are absent. The importance of quartz and plagioclase feldspars as ice-nucleating components of mineral aerosols is second only to that of K-feldspar; hence the possibility of this scenario occurring should not be dismissed. However, feldspars and quartzes tend to be found together in desert dust assemblages; thus K-feldspar will likely control the INA of desert dust on most occasions.
Performing heat tests on minerals in parallel with biological samples
allowed the magnitude of mineral wet heat sensitivity to be put into
context. For example, Yadav et al. (2019), performed wet heat tests on
rainwater and dust samples collected from northern India, with a heat test
of ATD performed as a control. The results showed a resultant deactivation
of INA that was consistent with our results. The authors attributed this to
the presence of organic matter in their ATD sample. However, the magnitude
of deactivation (
We also consider the issue of whether the heat-sensitive active sites we found in our mineral samples are an artefact of the milling process and therefore not representative of particles present in the environment. The INA of quartz (Kumar et al., 2019a; Zolles et al., 2015; Harrison et al., 2019), hematite (Hiranuma et al., 2014) and also natural desert dusts (Boose et al., 2016) is increased by milling. This might imply that heat labile mineral INPs do not occur naturally. Conversely, it has been argued that quartz particles in desert dusts are naturally “milled” by collisions during the process of saltation prior to being lofted into the air (Harrison et al., 2019). If this is correct, then it would mean that only quartz INPs originating from desert dust, with their active surfaces exposed following saltation, would be wet heat labile, whereas quartz particles that have been in contact with water, for example in soil or sediments, would have already been aged and so may be less susceptible to further wet heat treatment.
Here, we provide some further caveats and considerations for the use of heat
tests to identify biological, specifically proteinaceous, INPs in
environmental and atmospheric samples.
In this study, we have tested and characterised the changes in
ice-nucleating ability of the principal mineral components of desert dust in
response to heat treatments in both wet and dry modes and in parallel with
biological INP analogues (bacterial, fungal, pollen and cellulose). The main
purpose of this was to assess the efficacy of heat treatments for the
“detection” of biological INPs in environmental sample media such as ambient aerosol, surface waters, soils and desert dusts. Understanding how the sources and distribution of biological INPs and mineral dust INPs differ in the environment may be crucial for understanding their current and future
impact on the climatic impacts of clouds. It has been previously assumed
that mineral INPs are inert to moderate heat treatments that are sufficient
to denature proteins. However, we found that while the INA of (most)
K-feldspars was unchanged on wet heating for 30 min, as expected, quartz and
plagioclase-rich feldspars were heat labile. The INA of quartz and
plagioclase-rich feldspar samples was unchanged when exposed to dry heat
(250
We suggest that the loss of INA on wet heating of quartz and plagioclase feldspars is related to aqueous dissolution of features acting as active sites on the mineral surface. This is supported by the observation that the relative dissolution rates of the different mineral types correlate with their relative heat sensitivities. Moreover, several studies have previously reported aqueous room-temperature ageing of mineral INP samples, and our results are consistent with the same process being accelerated by heating. As quartz and plagioclase feldspars are ubiquitous components of mineral dusts, this raises the possibility of false positives being produced by minerals in wet heat tests, which are more commonly used compared to dry heat tests. However, if the mineral-based INA of an environmental sample being tested for INPs is controlled by K-feldspar, then wet heat tests are valid.
Dry heating produced stronger deactivations compared to wet heating in the biological INP analogues, while overall being less likely to deactivate minerals. This could mean that dry heating has less potential to produce false-positive detection of biological INPs, so it could be a more appropriate method for INP heat tests since wet heating is the method usually employed in these investigations. However, this may be precluded by the finding that most of our K-feldspar samples exhibited dry heat deactivations. Due to its practical simplicity and potential for high throughput of samples, heat treatments will likely continue to be the primary method used in future studies where biological INPs need to be differentiated from other types present in a collected sample. Interpretation of results may by aided by identification of the mineral phases present in a sample using techniques such as XRD or SEM. Overall, we have highlighted potential limitations where INP heat tests are applied, and the need for deeper interpretation of results and has outlined possible improvements to INP heat treatment methods. Further studies should focus on finding the optimum physical conditions that would result in the most selective deactivations of biological INPs.
In our wet heating experiments, the mineral INP suspensions were heated
while inside 20 mL borosilicate glass vials containing 10 mL of suspension.
Kumar et al. (2019a) observed that ageing of quartz INP suspensions over
several days occurred at room temperature in glass vials but not in
polypropylene centrifuge tubes. For this they proposed an alternative
explanation to the active sites on the quartz INP being irreversibly
degraded by ageing in water, in which silicic acid leaches out from the
glass vial walls and re-precipitates onto the active sites of the mineral,
effectively blocking them. When polypropylene is the suspension container,
however, the Si concentration remains too low for this to occur, so the INA
does not reduce. Therefore, to rule out that quartz wet heat deactivations
are only an artefact of heating in glass containers, we repeated our wet
heat test for Fluka quartz in an alternative glass vessel type (20 mL
non-borosilicate vial with 10 mL of INP suspension) and plastic vessels (50 mL propylene centrifuge tube and 1.5 mL polypropylene microcentrifuge tube
(Sarstedt Micro Tube 72.690) containing 10 and 1 mL of INP suspension
respectively) and compared the deactivations with those seen for our
standard wet heat treatment in borosilicate glass. The results in
Plot showing the fraction of droplets frozen (
Similar or larger wet heat deactivations occurred for the 1.5 mL
microcentrifuge tube (
Thermocouple measurements of suspension temperature inside both glass and plastic vessels during the wet heat treatment procedure.
Several mineral samples' INA was significantly deactivated by heating in this study, and we hypothesise that their ice-active sites are degraded by elevated temperature but also dependent on whether the mineral samples were heated while immersed in water or dry in air. Here we explore the dependence of additional variables on heat treatments using some of the mineral INP samples and the biogenic INP samples included in this study.
We used relatively concentrated suspensions of minerals and biogenic INPs for
the experiments shown in Sect. 3 in order to ensure their droplet freezing
temperatures were well above the instrumental background. However, there are
potential mechanisms for the concentration of the suspension itself to
affect the INA independently from the heat treatments. For example, species
dissolved from the mineral powders may potentially interact with the
nucleation to reduce the INA (Koop and Zobrist, 2009; Kumar et al., 2018;
Whale et al., 2018). Agglomeration of particles causing loss of INP surface
area has been proposed to cause lower-than-expected INA with increasing INP
concentrations (Emersic et al., 2015; Hiranuma et al., 2019). Also, more
concentrated suspensions are more likely to contain rarer, warmer
temperature IN sites which may be of a different nature and thus different
heat sensitivity to lower temperature IN sites. We therefore repeated both
wet and dry heat tests for BCS-376 microcline, Fluka quartz, NX illite and
ATD at both higher and lower concentrations than the standard 1 %
The resultant droplet freezing data are plotted in the
Plots of
In Sect. 3 all samples were subject to standard heat treatment conditions of 95
Boxplots of freezing temperatures for droplet freezing assays of suspensions of
The dataset for this paper, including raw droplet assay freezing data, is publicly available at the University of Leeds Data Repository –
The supplement related to this article is available online at:
The study was conceptualised by MID and BJM. MID designed and performed the experiments with scientific input from BJM and TFW. MID prepared the manuscript with contributions from all co-authors.
The contact author has declared that neither they nor their co-authors have any competing interests.
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The authors are grateful to Alex Harrison and Jim Atkinson, who originally sourced many of the mineral samples used in this study, and to Mark Holden, who provided the amazonite and Pakistan orthoclase samples. Ulrike Proske collected and preserved the lichen sample and performed initial measurements of its INA. Andrew Hobson and Andrew Connelly provided valuable laboratory support. Thomas F. Whale thanks the Leverhulme Trust and the University of Warwick for supporting an Early Career Fellowship.
This research has been supported by the Natural Environment Research Council (grant no. NE/L002574/1), the European Research Council (ERC, MarineIce (grant no. 648661)), along with Cytiva (formerly Asymptote Ltd), Cambridge, UK, the Leverhulme Trust and the University of Warwick for supporting an Early Career Fellowship (grant no. ECF2018-127).
This paper was edited by Mingjin Tang and reviewed by three anonymous referees.