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Life in the Clouds
LESLEY EVANS OGDEN
Bringing new life to the science of the water cycle.
I
n 1978, in northeastern Montana,
David Sands, of Montana State
University, was investigating the
pesky problem of crop damage by
Pseudomonas syringae, a bacteria that
can harm crops by means of its icenucleating abilities. At that time, the
ability of certain microbes to cause
ice nucleation and freezing injury in
plants was assumed to be strictly a
bad thing. After a copper treatment,
all signs suggested that Sands had successfully eliminated the disease-causing organism in 3.6 square kilometers
seeded with wheat. However, 3 weeks
later, the farmer called with bad news:
The disease was back.
Sands returned, got in a small airplane, and flew over the field, sticking
his hand out of a tiny round window to collect samples of air every
152 meters. “Sitting there with a pile
of dishes and hopefully not using the
vomit bag, we got in the cloud, which
is not fun in a small little Cessna,
and that’s where there were ice crystals,” says Sands. Where these ice crystals struck the Petri dish, they saw
Pseudomonas syringae, an organism
Sands has affectionately nicknamed
“Sue.” Finding Sue in the clouds, he
proposed an idea that he called bioprecipitation. Clearly, lightweight bacteria go up, but they must also come
down, “and they can do it best if they
can nucleate in a cloud giving them
enough weight to get down before
ultraviolet light kills them.” Sands and
his colleagues published their hypothesis in Időjárás, the Journal of the
Hungarian Meteorological Service,
in 1982. “Science has funny ways of
Hair ice in a Pacific Northwest forest near Vancouver, British Columbia.
Photograph: Lesley Evans Ogden.
putting things in cul-de-sacs and leaving them for a long time,” muses Sands.
Indeed, his idea lay dormant for nearly
30 years.
Now, a surging interest in climate
science and an advancement of technologies in the fields of genetics,
microbiology, geophysics, meteorology, plant pathology, and statistics—
plus an injection of funding—have
created the conditions for further
exploration of this idea. Whereas the
evapotranspiration of water from
plants is already a well-known link in
the water cycle, researchers are now
investigating how tiny life forms, too,
might be components of this critical
cycle, perhaps also influencing our
weather and climate in previously
unappreciated ways.
Cool ice
A vivid example of the abilities of
ice-nucleating organisms is hair ice. A
white filamentous substance resembling bleached cotton candy draped
over dry twigs, hair ice is formed by ice
crystallization helped along by microorganisms living on plants. Certain
types of bacteria or fungi can cause
the nucleation of ice crystals into these
bizarrely curvaceous, hairy formations.
Speeding up ice formation would
be a suicide mission for most living
BioScience 64: 861–867. © 2014 Ogden. All rights reserved.
doi:10.1093/biosci/biu144
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organisms, given the damaging effects
of freezing. But for some life forms, it is
a secret weapon. Some ice-nucleating
organisms are plant pathogens. Under
the right conditions—typically at temperatures higher than those under
which ice would normally form—
microbes, such as certain strains of
P. syringae, stimulate the freezing of
plant tissue. The physical effects of
ice crystals may lead to plant cells’
being damaged and leaky. And when
the ice melts, the plant is coated with
a layer of water. Both of these properties help any nearby bacteria enter and
find food. Historically, scientific interest has been focused on better understanding the biology of pathogenic
ice-nucleating organisms because of
their detrimental effect on crops. Frost
damage inflicts approximately $1 billion in crop loss per year in the United
States alone, and ice-nucleating organisms on plant surfaces help frost form.
However, not all ice-nucleating organisms are pathogens, which presents a
vexing problem. If it is not just a means
to invade, what other role does ice
nucleation play? As with all bacteria
on surfaces exposed to wind, tiny icenucleating microbes are swept up and
carried into the atmosphere, where
their ability to form ice and concentrate it into heavy snowflakes, hail, or
raindrops allows them to get down to
Earth again. So ice nucleation probably
plays an important role helping these
tiny organisms disperse.
Ice 101
Grade-schoolers learn in science class
that the freezing point of water is 0
degrees Celsius (°C). However, in very
pure water, unless ice is already present, water does not actually form ice
crystals until it reaches much lower
temperatures. “If you have a tiny drop
of very pure water, even at –40°C or
so, that water may not freeze,” explains
Virginia Walker, biology professor at
Queen’s University, in Ontario. In liquid water, molecules move quickly.
As the temperature goes down, so too
does the speed of their movement,
such that they eventually line up in
hexagonal shapes to form a crystal.
Virginia Walker, of Queen’s University, beside some of her lab equipment, which
is used to identify ice-nucleating organisms. Photograph: Lesley Evans Ogden.
Walker’s analogy: “If they have a template of ice, they’ve essentially got a
line of soldiers showing them how to
line up, and they can just add on, holding hands, one by one.”
In nature, the best nucleator is
ice itself. “The second best,” explains
Walker, “is bacteria.” Ice-nucleating
bacteria have proteins that imitate a
template of ice. They allow the water
molecules to associate with that
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protein, putting the water molecules
in line so that ice can form at temperatures of about –2°C.
Walker recently isolated the icenucleating protein of Pseudomonas
borealis, a nonpathogenic bacterium. It
is a beneficial soil bacterium thought
to help plants fix nitrogen. In soil
samples collected at a research station
300 kilometers north of Yellowknife, in
the Canadian Arctic, Walker expected
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to find lots of freeze-resistant bacteria
from which she could isolate antifreeze proteins. But using a method
that separates dirt from ice-nucleating
organisms through their adherence to
a popsicle formed using a cold metal
rod, she isolated one organism that
sped up—rather than slowed down—
freezing. “We couldn’t explain why it
had this ability,” says Walker. So in a
paper in Cryobiology (doi:10.1016/j.
cryobiol.2014.06.001), she postulated
that the formation of ice provides a
means of dispersal, bringing the bacteria back down from their aerial journeys in rain, snow, or hail.
The recognition that microbes can
be widely dispersed in the air is not new.
In 1921, scientists from the University
of Minnesota collected spore samples
from a US Army plane. They were
monitoring the movement of an epidemic of stem rust, a wheat pathogen
caused by aerially transported fungal spores of Puccinia graminis. In
over 50 sampling flights from April to
July at altitudes of up to 3300 meters,
rust spores and other plant pathogenic fungi were detected during all
of the flights, at all of the sampled
altitudes. It was the first demonstration
that microorganisms are present in
the atmosphere at the cloud level and
beyond and marked the foundation of
interest in the field of aerobiology, a
discipline that has largely focused on
how the atmosphere affects the microorganisms it transports. That focus is
beginning to shift. Scientists are eager
to understand whether microbes also
affect the atmosphere, in turn affecting
weather and climate.
A microbe named Sue
One of the most intensely studied biological ice nucleators is the bacterium
P. syringae. Not all strains of it have
ice-nucleating abilities, but those that
do can use the damaging effects of
ice as their lunch ticket. The study of
P. syringae has an interesting history.
In the 1960s and 1970s, there was a
strong focus on its molecular biology. After identifying its ice-nucleating
gene and snipping it out, researchers in
the 1980s requested permission from
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The popsicle machine in Virginia Walker’s lab at Queen’s University is used
to isolate ice-nucleating active microbes. The ice-nucleating organisms in
the murky liquid water (soil mixed with water) adhere to the cold rod as the
popsicle of clear ice forms. Photograph: Lesley Evans Ogden.
the US government to release and test
this modified strain for controlling
frost damage on crops. This sparked
the beginning of activism surrounding
genetically modified organisms, and
the initial trial was sabotaged.
Cindy Morris, senior research scientist of the Plant Pathology Research
Unit at the French National Institute
for Agricultural Research, in Avignon,
has long studied P. syringae. She is one
of several scientists following up on the
foundational work on this first-known
ice-nucleating organism as members
of two independent groups—a group
of meteorologists and physicists led
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by Gabor Vali, at the University of
Wyoming, in Laramie, and a group led
by Chris Upper and Dean Arny, which
Morris joined while she was a graduate student in plant pathology at the
University of Wisconsin.
Research by Morris and others has
demonstrated the broad global distribution of P. syringae. The researchers
have found that P. syringae strains vary
in the proportion of individual cells
with ice-nucleating abilities. Between
60 and 100 percent of the strains that
they have examined in rainwater are
ice-nucleation active (INA). In snow,
however—a type of precipitation initiated exclusively by ice formation in
clouds—they have found that 100 percent of the strains are INA.
At Louisiana State University, Brent
Christner heads up the National
Science Foundation (NSF)–funded
project Research on Airborne Ice
Nucleating Species (RAINS). Long
interested in INA organisms, he initially embarked on the research with
zero support. “All the research was
either bootlegged on other projects
or personally funded,” says Christner.
Working in Montana, Christner, a keen
skier, had the ski patrol at four local
resorts bag snow samples whenever
there was a fresh snowfall. He used
heat to separate living from nonliving
ice-nucleating particles in his samples, because heat denatures the INA
proteins, just as proteins in eggs are
denatured by cooking. Assuming that
plants, especially crops, were the main
source of these “ice bugs,” Christner
was surprised to find INA organisms
in all of the samples, regardless of
the season. “We’ve never analyzed a
precipitation sample that we didn’t
find biological ice nucleators in,” says
Christner. He is now examining patterns in the presence of INA organisms
in samples of glacial ice, recognizing this multilayer source as a useful
record of atmospheric conditions dating back centuries and a way of looking at the presence of INA organisms
over time.
The life cycle of P. syringae includes
eating, moving around, and multiplying on the ground. But because the
Pseudomonas bacterial isolate glowing under ultraviolet light.
Photograph: Tom Hill.
bacterium is so tiny and lightweight, it
gets picked up easily and dispersed by
the wind;coming down again is more
difficult. “There is no way that a particle that size will come out of the atmosphere without some active method to
bring it down, because it’s too light,”
explains Morris. Most net movement
of air is upward, because the Earth is
warm. So, to get down, the bacterium
has to get inside a bigger particle, such
as a raindrop or an ice crystal. When it
goes up, suggests Morris, it is strictly
about survival. “If you ever travel on
public transportation, you know there
is a big diversity and there are tons of
people and its crowded. But it’s not a
place to live.” The atmosphere, thinks
Morris, is like the Metro for microbes.
Getting back down to Earth is a
matter of survival for INA bacteria,
explains Christner, because, when they
are swept up into an atmospheric conveyor belt, “time is ticking, and due to
the stresses involved, they are in the
process of dying.” Up high, microbes are
desiccated and exposed to high doses
of ultraviolet radiation, so by removing
themselves from the atmosphere, they
have a shot at reproduction and continued survival. Intriguingly, though,
ice-nucleating microbes do not need
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to be alive to maintain their ability
to seed ice crystallization. Proteins
in their outer membranes retain the
physical shape that facilities ice crystallization even after death.
Much of Earth’s life in the clouds—
the abundance, diversity, flux, and distribution of biological ice-nucleating
organisms—remains to be investigated. This is no easy task. Studying
microbes in the atmosphere from normal aircraft has risks, and deliberately
flying into icy clouds is tricky. One of
the promising new research methods
is the use of unmanned aerial vehicles.
David Schmale, an associate professor
in the Department of Plant Pathology,
Physiology, and Weed Science at
Virginia Tech, is piloting this research.
He has developed drones kitted out
with Petri dishes for sampling airborne microorganisms from tens to
hundreds of meters above the ground,
launched from a special research facility in Blacksburg.
“The sampling devices operate like a
little clamshell” mounted on the leading edge of the plane’s wing or its
fuselage, explains Schmale. The Petri
dishes are closed during takeoff and
landing, but flipping a switch during
flight opens the sampler, which allows
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the collection and verification of various ice-nucleating organisms in the
atmosphere. Larger drones provide the
possibility of tracking microorganisms
during flight using a technology called
surface plasmon resonance, which
allows the viewing of the data in real
time through a ground control station.
The Federal Aviation Administration
regulates the operation of drones in
public airspace, and flying drones into
clouds is not permitted at the research
site. There are many questions remaining in the emerging field of aeroecology. “It’s what we like to call job
security,” jokes Schmale.
New takes on an old idea
About 12 years ago, after the bioprecipitation hypothesis had lain dormant
for decades, Morris and Sands brought
the idea back to the table. “I won’t
say it was dead, it just wasn’t being
worked on,” says Morris. Pseudomonas
syringae was being used commercially
for snowmaking, seeding clouds, cryopreservation, and frozen food preparation, but people were not thinking
more holistically about the cycle. In
2006, Morris and Sands succeeded in
securing funding to further explore the
role of microbes in the atmosphere and
water cycle. They set up the first interdisciplinary workshop on this subject, funded by the European Science
Foundation. Held in Avignon, the
meeting brought together a core group
of 25 people, who continue to work
together. That meeting, says Morris,
“was really fundamental in bringing
us together and teaching us how to
talk to each other,” no small feat for
participants from disciplines including
agriculture, microbiology, climatology,
atmospheric science, and geochemistry.
That scientific conversation is
continuing. Morris collaborates with
Christner on his RAINS research program, which includes funding for an
international early-career workshop
entitled “Microbes at the interface of
land-atmosphere feedbacks,” to be held
this month in Sainte-Maxime, France.
With Sands, Morris is facilitating the
training of a new generation of scientists who will explore this subject.
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This Petri dish, in one of David Schmale’s unmanned aerial vehicles, is used for
aboveground sampling of ice-nucleating organisms. The lid of the dish can be
opened and closed by the drone’s operator on the ground.
Photograph: David Schmale.
An unmanned aircraft (drone) loaded up with Petri dishes for sampling
organisms in the air. Photograph: David Schmale.
Biological ice nucleators have begun
to be included in climate models. Dust
and many other tiny nonliving airborne particles are well known for the
role they play in weather and climate
by scattering or absorbing solar radiation and serving as condensation and
ice nuclei. New global models consider some biological ice nucleators,
including bacteria, fungal spores, and
pollen. Modeling by Corinna Hoose,
a theoretical meteorologist at the
Karlsruhe Institute of Technology, in
Germany, suggests that, on a global
scale, ice nucleation in clouds is predominately performed by mineral
dust, not biological particles, implying that the microbes’ relative impact
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on global precipitation and climate is
small. Current models are necessarily
simplified, with coarse resolution, and
“cannot resolve single clouds, convective updrafts, and local sources of biological particles.” Nevertheless, using
higher-resolution regional models,
recent simulations for Europe found
that biological particles did not contribute significantly to atmospheric
ice-nuclei concentrations and ice formation in clouds. So debate over their
potential importance continues.
At the University of Leeds,
Benjamin Murray is an atmospheric
scientist investigating biological ice
nucleators. He explains that much of
the uncertainty over the importance
of biological ice-nucleating particles
(INPs) lies in the fact that they are
probably important in some clouds
but not others. “The overall impact
on clouds throughout the atmosphere
and the planet’s climate is not at all
clear,” he says. “Above –15°C, we know
that ice formation is very important,
but we have not identified what it
is that causes ice to form. This is
the temperature range in which biological INP, such as bacteria, may be
important.” Inside the thin, wispy ice
clouds of the upper troposphere, (the
cruising altitude of a transatlantic
jet), at temperatures below –40°C,
biological INPs are probably much
less important than desert dust, he
explains.
Land and sea
Terrestrial plants, particularly cereal
crops, grasses, and fruit trees, host high
numbers of ice-nucleating organisms,
including P. syringae, but new research
is revealing that INA are not just terrestrial in origin. Work by Daniel
Knopf at Stony Brook University
and Susannah Burrows at the Pacific
Northwest National Laboratory has
implicated INA diatoms that are
extremely abundant in the ocean. With
this newly identified marine source of
these organisms and “considering that
three-quarters of the planet is covered
in ocean, you begin to understand how
they could be so widely distributed,”
says Christner.
Wave-tank apparatus near La Jolla, California, where Christina McCluskey and
her colleagues are capturing ocean spray particles for analysis.
Photograph: Christina McCluskey.
Additional resources.
Christner BC. 2012. Cloudy with a chance of microbes. Microbe Magazine. Available
at http://io.aibs.org/microbe.
Morris CE, Conen F, Huffman A, Phillips V, Pöschl U, Sands DC. 2012. Bioprecipitation:
A feedback cycle linking Earth history, ecosystem dynamics and land use through
biological ice nucleators in the atmosphere. Global Change Biology 20: 341–351.
doi: 10.1111/gcb.12447
Morris CE Sands DC. 2012. From Grains to Rain: The Link between Landscape,
Airborne Microorganisms and Climate Processes. Available at http://bioice.files.wordpress.com/2012/05/grainsrain_v26apr2012d.pdf.
Sands DC. 2012. TEDx Talk. The Rainmaker Named Sue. Available at www.youtube.
com/watch?v=_9ZeYxoWsuk.
Identifying oceanic biological ice
nuclei is the focus of Colorado State
University environmental microbiologist Thomas Hill and his colleagues Paul
DeMott and doctoral student Christina
McCluskey. Hill has developed a quantitative polymerase chain reaction test
to count one possible group, the INA
bacteria, in environmental samples. Off
the coast of La Jolla, California, Hill and
McCluskey have set up an apparatus
for sampling biological ice nucleators
in sea spray. They are experimentally
spiking seawater with nutrients such as
nitrogen and phosphorus to stimulate
an algal bloom, then assessing the composition, diversity, size, and abundance
of INA organisms as the algal bloom
progresses, peaks, and senesces.
“With crashing waves, you get sea
spray aerosol,” explains McCluskey. So
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as the waves crash, the apparatus collects sea spray particles in an enclosed
wave tank. A continuous flow diffusion chamber measures the number of
particles with ice-nucleating or cloudnucleating abilities. Other instruments,
such as an aerosol mass spectrometer,
allow them to look at the chemical
properties of single particles. They
are also examining the sea surface
microlayer. Sea spray or bubbles can
fling particles from this layer into the
atmosphere, where they are aerosolized and can play a role in cloud formation and ice nucleation. It’s a small
component of a larger project called
the NSF Center for Aerosol Impacts
on Climate and the Environment, led
by Kimberly Prather, distinguished
chair in atmospheric chemistry at the
University of California, San Diego.
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Many unknowns remain. DeMott and
his colleagues also plan to sample from
different environments—land, air, and
sea—including examining any INA
particles released in biomass burning.
Where do these waves of emerging research leave the bioprecipitation
hypothesis? “It’s credible; we just don’t
know the details,” says Morris, arguing that the idea of another previously
unappreciated role for microbes in the
biosphere is not so radical. “Everybody
knows that major shift in oxygen concentration on the planet was due to
microorganisms. We have 20 percent
oxygen in our atmosphere because
of planktonic unicellular green algae,”
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and there are major biogeochemical
cycles that involve bacteria, she adds. “I
don’t think anyone’s going to argue that
[the bioprecipitation feedback cycle] is
impossible.” The argument, she says,
“is how significant it is, and where [it
is] significant.” What’s controversial is
the idea that organisms are somehow
controlling their own weather. DeMott
suggests they are just one part of the
story of precipitation.
Whereas humans have synthesized
chemicals, such as silver iodide, that are
highly efficient at initiating ice crystallization, microbes with ice-nucleating
proteins are the Iceman superheroes
of the natural world. “I think there’s
a whole orchestra of things out there
[that can perform ice nucleation],” says
Hill, but “the biological components are
a large string section of that orchestra.”
How the musical score is written, who
is playing, and how loud remain to be
revealed. But to the scientists studying
these fascinating living icemakers, they
make beautifully mysterious music.
Lesley Evans Ogden is a science writer–producer
based in Vancouver, British Columbia. A
field ornithologist turned journalist, Lesley
had long suffered from the impression that
microorganisms were dull. Writing this Feature
cured her of this affliction. Find her on the Web
at lesleyevansogden.com and on
Twitter @ljevanso.
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