Life in the Clouds

In 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 ice-nucleating 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 putting things in cul-de-sacs and leaving them for a long time,” muses Sands. Indeed, his idea lay dormant for nearly 30 years. Hair ice in a Pacific Northwest forest near Vancouver, British Columbia. Photograph: Lesley Evans Ogden. 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. 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 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 ice-nucleating 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. 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. 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.” Virginia Walker, of Queen's University, beside some of her lab equipment, which is used to identify ice-nucleating organisms. Photograph: Lesley Evans Ogden. 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 protein, putting the water molecules in line so that ice can form at temperatures of about –2°C. Walker recently isolated the ice-nucleating 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 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. 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 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. 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. 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 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 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. Pseudomonas bacterial isolate glowing under ultraviolet light. Photograph: Tom Hill. 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 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 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. 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. 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 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, is an atmospheric scientist investigating biological ice He explains that much of the over the importance of biological ice-nucleating particles in the that they are probably important in some clouds but not “The impact on clouds the atmosphere and the climate is not at all he we know that ice formation is very but we have not what it is that ice to form. This is the temperature in which biological such as bacteria, may be the ice clouds of the of a at temperatures biological are probably much important than dust, he plants, crops, and high of ice-nucleating organisms, including P. syringae, but new research is that INA are not just in by at University and at the Pacific Northwest National has INA that are in the With this source of these organisms and that of the is in you to understand how they could be so widely says Christner. biological ice is the focus of State University and his colleagues and student has developed a test to one the INA bacteria, in the of and have set up an for sampling biological ice nucleators in They are with such as and to stimulate an the diversity, and of INA organisms as the and near where and her colleagues are particles for Photograph: Christner with a of microbes. at Morris Sands A cycle Earth and use through biological ice nucleators in the atmosphere. 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