Soils rich in biological ice-nucleating particles abound in ice-nucleating macromolecules likely produced by fungi

Abstract

Soil organic matter carries ice-nucleating particles (INPs) the origin of which is hard to define and that are active at slight supercooling. The discovery and characterization of INPs produced by the widespread soil fungus Mortierella alpina permits a more targeted investigation of the likely origin of INPs in soils. We searched for INPs with characteristics similar to those reported for M. alpina in 20 soil samples from four areas in the northern midlatitudes and one area in the tropics. In the 15 samples where we could detect such INPs, they constituted between 1 and 94 % (median 11 %) of all INPs active at -10 ∘C or warmer (INP-10) associated with soil particles < 5 µm. Their concentration increased overproportionately with the concentration of INP-10 in soil and seems to be greater in colder climates. Large regional differences and prevalently high concentrations allow us to make inferences regarding their potential role in the atmosphere and the soil.

Full text

Biogeosciences, 15, 4381–4385, 2018
https://doi.org/10.5194/bg-15-4381-2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License.
Soils rich in biological ice-nucleating particles abound in
ice-nucleating macromolecules likely produced by fungi
Franz Conen1,* and Mikhail V. Yakutin2,*
1Department of Environmental Sciences, University of Basel, Bernoullistr. 30, 4056 Basel, Switzerland
2Institute of Soil Science and Agrochemistry, Siberian Branch of the Russian Academy of Sciences,
Academician Lavrentyev Avenue, 8/2, 630090 Novosibirsk, Russia
*Both authors contributed equally to this work.
Correspondence: Franz Conen (franz.conen@unibas.ch)
Received: 9 February 2018 – Discussion started: 20 March 2018
Revised: 20 June 2018 – Accepted: 9 July 2018 – Published: 18 July 2018
Abstract. Soil organic matter carries ice-nucleating particles
(INPs) the origin of which is hard to define and that are active
at slight supercooling. The discovery and characterization of
INPs produced by the widespread soil fungus Mortierella
alpina permits a more targeted investigation of the likely ori-
gin of INPs in soils. We searched for INPs with characteris-
tics similar to those reported for M. alpina in 20 soil samples
from four areas in the northern midlatitudes and one area in
the tropics. In the 15 samples where we could detect such
INPs, they constituted between 1 and 94 % (median 11 %)
of all INPs active at −10 ◦C or warmer (INP−10) associ-
ated with soil particles < 5µm. Their concentration increased
overproportionately with the concentration of INP−10 in soil
and seems to be greater in colder climates. Large regional
differences and prevalently high concentrations allow us to
make inferences regarding their potential role in the atmo-
sphere and the soil.
1 Introduction
Soils could be a relevant source of ice-nucleating particles
(INPs) found in the atmosphere, and INPs from soils are
also found in precipitation (Creamean et al., 2013, 2014)
and in rivers (Moffett, 2016; Larsen et al., 2017). Organic
matter, or biological residues, associated with soil particles
may contribute a major share to atmospheric INPs active at
temperatures warmer than −10 ◦C (Schnell and Vali, 1976;
Szyrmer and Zawadzki, 1997; Conen et al., 2011; O’Sullivan
et al., 2014; Creamean et al., 2013; Tobo et al., 2014; Hill
et al., 2016). Recent progress in this field of research has
been made by the detailed characterization of INPs produced
by the widespread soil fungus Mortierella alpina (Fröhlich-
Nowoisky et al., 2015). Plating and cultivation have allowed
(Fröhlich-Nowoisky et al., 2015) to identify M. alpina as
an INP-producing organism through DNA sequencing fol-
lowed by phylogenetic analysis. Together with the earlier
discovery of Fusarium avenaceum and Fusarium acumina-
tum as sources of INPs with similar characteristics (Pouleur
et al., 1992), this new INP source raises the question of the
more general relevance of cell-free INPs produced by fungi
in soils. Trying to identify and count or determine the mass of
these fungi in soil could be one approach. However, this ap-
proach would not account for the fact that INPs produced by
the organisms can be washed off, may be preserved, may ac-
cumulate in the soil, and may be exported from a watershed
during intense rainfall (Larsen et al., 2017). In a first attempt
to gauge the potential relevance of cell-free, ice-nucleating
macromolecules likely derived from fungi, we looked for
INPs in soils that match the challenge tests described for M.
alpina (Fröhlich-Nowoisky et al., 2015).
2 Material and methods
We collected grab samples (100 to 300 g of material per sam-
ple) from the surface of arable soils (Table 1) in Novosi-
birsk (Western Siberia), Saskatoon (Saskatchewan) and Col-
mar (France), from grasslands in La Brévine (Switzerland),
and from tropical mountain forests around Ranau (Borneo).
Where present, aboveground vegetation and litter were re-
Published by Copernicus Publications on behalf of the European Geosciences Union.

4382 F. Conen and M. V. Yakutin: Soils rich in biological ice-nucleating particles
Table 1. Details of sample origin, including mean annual temperature (MAT) and precipitation (MAP). In each area, between three and
six samples (N) were collected. A sample consisted of 100 to 300 g of soil collected from the surface within a radius of a few metres. The
maximum distance between samples (Dmax) ranged from 4 to 106 km.
Location, Novosibirsk, Saskatoon, La Brévine, Colmar, Ranau,
region south-western Northern Jura Upper Rhine Borneo
Siberia Great Plains Mountains Valley
Coordinates latitude 54◦380to 55◦180N 52◦040to 52◦080N 46◦590N 48◦000to 48◦050N 05◦590to 06◦030N
longitude 82◦440to 84◦230E 106◦290to 106◦370W 06◦360E 07◦190to 07◦230E 116◦420E
Altitude (m a.s.l.) 120–150 500–520 1050 200 450–690
Land use arable crops arable crops grassland arable crops mountain forest
MAT (◦C) 1.7 2.6 4.9 10.9 27
MAP (mm) 448 354 1597 607 2880
Sampling time May, Jun 2013 Oct 2014 Sep 2014 Oct, Nov 2014 Mar 2014
N6 3 4 3 4
Dmax (km) 106 12 4 10 8
moved before sampling. Samples were air-dried and dry-
sieved (< 63 µm). From each sample, 1 g of dry particles
(< 63µm) was weighed into a 50mL centrifuge tube contain-
ing 20 mL of 0.1% NaCl, was shaken for 2 min by hand, and
was allowed to settle for another 10 min. About 10 mL sus-
pension was withdrawn from the top of the suspension and
passed through a syringe filter with 5 µm pore size (sterile
cellulose acetate filter; Sterlitech Corporation, Kent, USA);
9 mL of it was put into a pre-weighed aluminium tray; 1.0
to 1.5 mL was put into another tube together with the proper
amount of 0.1 % NaCl to create a 1 :20 dilution of the sus-
pension. The tray and its content were dried at 80 ◦C and
reweighed, and the mass of particles < 5 µm was determined
from the difference to a control tray prepared with only an
NaCl solution. The tube containing the 1 :20 dilution of the
suspension was analysed for INPs on a freezing nucleation
apparatus (Stopelli et al., 2014) in 52 aliquots of 100 µL in
0.5 mL tubes and, if necessary, further diluted to a concen-
tration at which most, but not all, of the 52 tubes were frozen
at −10 ◦C. Final concentrations of particles < 5 µm ranged
from 0.02 to 15.5 µg mL−1, with a median of 1.0 µg mL−1.
The remainder of the suspension with the final concentra-
tion was then passed through a 0.22 µm syringe filter (same
material and supplier as above) and partitioned into three
portions. One portion was analysed for INPs without further
treatment; the other two portions were either heated to 60 or
95 ◦C for 15 min in a water bath before being analysed in the
same way. From the original suspension and a 6 M solution
of guanidinium chloride (> 99.5 %; Roth GmbH +Co. KG,
Karlsruhe, Germany) we prepared a similarly diluted suspen-
sion of particles < 0.22µm and analysed it for INPs after 1 to
2 h of storage at room temperature. Guanidinium chloride de-
activates bacterial and fungal INPs but not pollen (Pummer
et al., 2012, 2015). Blank samples of 0.1 % NaCl solution
did not freeze at −10 ◦C. Our criteria for what we presume
are cell-free fungal INPs were an activation temperature of
−6.5◦C or warmer (INP−6.5) that is retained after heating
to 60 ◦C but which is deactivated by heating to 95◦C and
by 6 M guanidinium chloride. For practical reasons (small-
est mesh filter size available), we relaxed the size criterion
(< 300kDa) in Fröhlich-Nowoisky et al. (2015) to < 0.22 µm.
This may seem generous, but it still excludes other potential
INP−6.5that are associated with cells and are not detached
macromolecules. However, bacterial INPs have been found
to not withstand heating to 60 ◦C, with the exception of ice-
nucleating entities resistant to boiling produced by Lysini-
bacillus sp. (Failor et al., 2017). Pollen-derived INP macro-
molecules are not sensitive to guanidinium chloride or boil-
ing (Pummer et al., 2012, 2015). Thus, the only INPs that are
currently known to match the applied test criteria are from
fungal sources.
3 Results and discussion
We found what we presume are cell-free fungal INPs in all
samples with more than one INP µg−1particles < 5 µm ac-
tive at −10 ◦C (INP−10) (Figs. 1, 2). There might also have
been a contribution of cell-free fungal INPs in samples with
less than 1 INP−10 µg−1, but it was too small to be de-
tected. The latter applies to all four samples from tropical
Ranau (INP−10 < 0.1 µg−1) and one (of three) from the wine-
growing area around Colmar (INP−10 =0.3 µg−1). Higher
concentrations of INPs in cold compared to warm regions
were previously reported by Schnell and Vali (1976) and Au-
gustin et al. (2013). Guanidinium chloride reduced the num-
ber of INP−10 in all suspensions of particles < 0.22 µm to
below the detection limit (to 2 % or less of what we found
in suspensions prepared with 0.1 % NaCl), as did heating
to 95 ◦C. What we presume are cell-free fungal INPs were
therefore not derived from pollen (Pummer et al., 2012,
2015) or Lysinibacillus sp. (Failor et al., 2017), otherwise
they would still have been active after heating to 95 ◦C. Av-
eraged over all samples, 97 % (±9 %) of INP−6.5associ-
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F. Conen and M. V. Yakutin: Soils rich in biological ice-nucleating particles 4383
Figure 1. Ice-nucleating particles with characteristics of macro-
molecules released by certain fungi as a function of the total num-
ber of INPs active at −10 ◦C in soil particles < 5 µm. The trend line
was fitted to all data in the plot. Not plotted are four samples from
tropical Ranau (INP−10 < 0.1 µg−1) and one (of three) from the
wine-growing area around Colmar (INP−10 =0.3 µg−1) in which
we could not detect any INPs resembling macromolecules released
by fungi.
ated with particles < 5 µm passed through the 0.22 µm filter
and 86 % (±10 %) of those remained active after heating to
60 ◦C. Consequently, about five-sixths (0.97 ×0.86 =0.83)
of all INP−6.5found in soil particles < 5 µm matched char-
acteristics of cell-free fungal INPs. There might have been a
small contribution by Isaria farinosa (Huffman et al., 2013)
to the number of INP−6.5determined before heat treatment.
However, these INPs would have been deactivated after heat-
ing to 60 ◦C (Pummer et al., 2015) and would not have con-
tributed to the number of cell-free fungal INPs considered
in the present study. Smaller fractions of INP−10 passed the
challenge tests. On average 81 % (±6 %) passed through
0.22 µm and only half (51 %, ±9 %) of all INP−10 were also
active after heating to 60 ◦C.
Cell-free fungal INPs made up only 1/20th of INP−10
around Colmar but 2/3 of INP−10 around Novosibirsk. Re-
gression analysis of the ensemble of 15 samples with de-
tectable cell-free fungal INPs from all four areas on three
continents (Fig. 1) suggests that a doubling of INP−10 may
be associated with a tripling of the number of cell-free
fungal INPs (21.6=3). This trend not only applies across
the different areas investigated but also within certain ar-
eas (Saskatoon, La Brévine). We speculate that the great
plasticity in the contribution of cell-free fungal INPs results
from their property of being macromolecules that can be
Figure 2. Cumulative freezing spectra of the (untreated) samples
shown in Fig. 1 and a third sample from Colmar in which we could
not detect INPs resembling macromolecules released by fungi (low-
ermost curve).
washed off from the mycelium. In principle, the production
of a macromolecule requires less resources than the produc-
tion of a complete cell carrying an ice-nucleation-active en-
tity. Hence, an organism capable of releasing ice-nucleation-
active macromolecules has a greater range over which it can
potentially modify its surrounding in terms of ice nucleation.
4 Inferences from a wider context
4.1 Atmosphere
The frequency of clouds containing ice particles at moder-
ate supercooling is greater above fertile land than downwind
of a desert (Kanitz et al., 2011). One cause of this differ-
ence could be higher concentrations of INPs in fertile soils
compared to desert soils (Conen et al., 2011). Above fertile
land, however, atmospheric INP concentrations do not seem
to vary with INP concentration in soils. The 1 to 2 orders of
magnitude larger concentrations of INPs in soils at Novosi-
birsk, compared to soils in La Brévine and Colmar (Fig. 1)
leave little or no trace in the atmosphere. At Chaumont, a
site 30 km to the east of La Brévine and 120km to the south-
west of Colmar, we observed similar concentrations of atmo-
spheric INPs active at −8◦C or warmer to those we observed
in Novosibirsk (Conen et al., 2017). During April and May,
when arable soils are prepared for seeding and wind erosion
is most prevalent (Selegey et al., 2011), median values were
four INPs and seven INPs per cubic metre at Chaumont and
Novosibirsk, respectively. Thus, soils are unlikely the domi-
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4384 F. Conen and M. V. Yakutin: Soils rich in biological ice-nucleating particles
nant source of biological INPs in the atmosphere above the
cropland belt stretching from western Europe eastward all the
way to Novosibirsk. Still, the influence of soils as a source of
atmospheric INPs might appear unduly small in this com-
parison because of efficient atmospheric mixing within the
latitudinal band. Nevertheless, it is likely that vegetation and
leaves decaying at the soil surface make a larger contribu-
tion to atmospheric INP−10 (Schnell and Vali, 1976; Conen
et al., 2017). We conclude that cell-free fungal INPs asso-
ciated with soil dust probably have a minor influence on ice
formation in supercooled clouds, and regional differences be-
tween soils are masked by atmospheric mixing and relatively
larger contributions of INPs from vegetation and decaying
leaves.
4.2 Soil
Postulated potential advantages to an organism capable of
catalysing ice formation at slight supercooling include the
cleavage of structures by ice formation to access otherwise
occluded resources (Paul and Ayres, 1991) and the accu-
mulation of water through growing ice from vapour in the
surrounding air (Kiefft, 1988). However, there is little ex-
perimental evidence to support these ideas in the context of
soil. To our knowledge, the most convincing evidence for
an accumulation of water in the form of ice was described
by Hofmann et al. (2015). Fascinating sculptures of hair ice
can form on the surface of dead wood infected by the fun-
gus Exidiopsis effusa through the mechanism of ice segrega-
tion. This mechanism transports slightly supercooled water
from inside the wood to a body of ice growing on the outside
of it. The heat released by the phase transition stabilizes the
front between liquid and ice, as long as water is supplied to
the growing ice at a sufficient rate. Although fungal activity
is responsible for shaping hair ice, ice segregation proceeds
under the same conditions without the fungus but then re-
sults in an ice crust. Temperatures recorded by Hofmann et
al. (2015) inside and outside of wood samples showed that
hair ice formation started when temperatures had decreased
to about −0.5 ◦C in one experiment and to −2.5 ◦C in an-
other experiment. In both cases, temperature inside the wood
increased sharply after the onset of ice formation and stabi-
lized near −0.2 ◦C through the heat released by ice forma-
tion, while temperature outside the wood continued to de-
crease. In one of the experiments, ice growth stopped when
outside temperature had decreased to −4◦C.
The same process of ice segregation as described by Hof-
mann et al. (2015) may also take place at the surface or within
the porous structure of soil, where larger pores are typically
air-filled and water is held in finer capillaries, similar to those
supplying water to the hair ice growing on wood. Visible phe-
nomena of water accumulating through ice segregation at or
near the soil surface include ice needles and ice lenses (Dash
et al., 2006). For a soil fungus to benefit from ice segregation,
it has to produce INPs active as close as possible to 0 ◦C.
We think that INP−6.5do not provide much of an advantage
in this context. Even in a very small volume of soil, pore
water is unlikely to supercool to that temperature. Further,
the volume of water that might be harvested through ice seg-
regation would be irrelevantly small if there are other INPs
active at the same temperature nearby, which is definitively
the case for all samples shown in Fig. 1. It can only be the
much rarer INPs active closer to 0 ◦C that potentially provide
the advantage of ice segregation to an INP-producing fungus
in soil. Pouleur et al. (1992) found about 1 in 104INP−6.5
was already active at −2.5 ◦C. The large numbers of cell-
free fungal INPs found in our samples may just be a proxy
for the soil-ecologically relevant INPs active closer to 0 ◦C.
The detection of the latter would require larger volumes of
soil (e.g. millimetre-size aggregates) tested under conditions
where temperature can be controlled with great stability and
high precision (e.g. within a dry-block temperature calibra-
tor).
Data availability. All data is is available upon request from the cor-
rensonding author.
Competing interests. The authors declare that they have no conflict
of interest.
Acknowledgements. The collaboration between the authors on the
issue of ice nuclei in soils was supported by the Swiss National
Science Foundation through grant number IZK0Z2-142484/1 to
Mikhail V. Yakutin for a short visit to Basel in summer 2012, during
which much of the method applied in this study was developed. We
thank Kirk Blomquist, Vitali Petrovich Baranov, and Simon Tresch
for providing samples from Saskatoon, Ranau, and La Brévine,
respectively. This manuscript has benefitted a lot from comments
and suggestions made by Cindy Morris and a second, anonymous
referee.
Edited by: Kees Jan van Groenigen
Reviewed by: Cindy Morris and one anonymous referee
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