If you’re a science fiction fan, you’ve probably encountered the notion of alien rocks bringing life to Earth. But could Earth rocks bring microbial life to alien worlds? Check out my Motherboard piece on lithopanspermia to find out!
If you’re a science fiction fan, you’ve probably encountered the notion of alien rocks bringing life to Earth. But could Earth rocks bring microbial life to alien worlds? Check out my Motherboard piece on lithopanspermia to find out!
The Ecological Society of America (ESA) annual conference, once dominated by discussions of lions, tigers and bears (or so I’ll pretend), has become a convergence point for microbial ecologists. From amphibian skin microbiomes to biological soil crusts and more, the diversity of microbial talks this year was astounding.
As the conference winds down, I wanted to share just a few of the coolest microbial findings that stuck with me from the last couple days at ESA. What was your favorite microbial talk of the week? Post about it in the comments, or better yet, tweet about it! And don’t forget to tag me (@themadstone) as well as the official ESA microbial ecology twitter (@ESAmicrobe), if you do ;)
Can dormancy explain microbial biogeography?
Dr. Jay Lennon gave a great talk on dormancy; a subject that’s particularly fascinating to me in light of the vast (and probably rather dormant) microbial populations living in deep, subterranean environments.
Dormancy, otherwise known as metabolic inactivity, is an important strategy for surviving stressful environments. Conditions such as extreme drought, cold, or lack of food can cause microbes to enter a dormant state. Dormant microbes are fascinating not only for their ability to survive extreme conditions, but to do so for thousands to millions of years.
Lennon’s research group is particularly interested in applying principles of biogeography- how diversity scales across space- to microbial ecology. But understanding spatial patterns in microbial diversity is tricky because: 1) microorganisms are enormously diverse compared with macroorganisms and 2) unlike, say, mountain lions, which may be very genetically similar across kilometer scales but diverse over thousands of kilometers, genetically distinct populations of microbes can exist at the millimeter scale. Scientists find that microbial communities in soil samples taken 1 centimeter apart from each other can be up to 90% genetically distinct.
Here’s where dormancy comes in. Lennon’s group can separate the active from dormant members of microbial communities, by looking at the number of RNA transcript copies for a ubiquitous bacterial gene. RNA transcripts, the intermediate product between a DNA “blueprint” and an active protein, are well correlated with metabolic activity. Lennon finds differences in how diversity scales with space when examining the dormant versus active portions of a microbial community. Simply put, dormant microbial communities are more homogenous across space than their active counterparts. Furthermore, active microbial communities tend to become dominated by select taxa, probably those with some fitness advantage in the local environment. Dormant communities are less likely to be dominated by a few individual taxa, but rather represent a well-distributed “seed-bank” of genetic diversity.
The difference in how dormant versus active microbial communities scale with space may seem like a nuance, but this information could go a long way toward helping ecologists understand the distribution of microbial diversity across our planet. Lennon’s results suggest dormant microbes may represent up to 40% of a community. That’s no small portion of the total genetic diversity. Furthermore, dormant microbes, as a potentially long-term seed-bank, may help replenish the diversity of a community following extinction events.
Pine needle bacteria that soak up nitrogen
Did you know that nearly all leaves on Earth host their own microbiomes? Neither did I! But perhaps it’s not too surprising, given we’re now studying the microbiomes of cell phones and sewers. Anyway, it’s pretty cool. It’s extra cool in alpine ecosystems, where scientists are finding that these leaf-dwellers may play an important role in making nitrogen available to plants.
Dr. Carolin Frank’s lab studies nitrogen cycling in high, cold alpine ecosystems dominated by coniferous trees. Alpine ecosystems have long puzzled ecologists, because their soils and vegetation often contain more nitrogen than can be easily explained. Dr. Frank and colleagues hypothesized the “missing nitrogen” might come from nitrogen fixers- bacteria that carry a special enzyme which allows them to convert atmospheric N2 gas to ammonia- found in an unlikely place.
The team went out to a high-altitude forest in Colorado, collecting pine needles and bringing them back to the lab. In the lab, they placed the needles in sealed jars and added a chemical called acetylene. Acetylene, a simple carbon compound, can be chemically converted by nitrogenase, the same enzyme used to fix dinitrogen gas. This is a quick and cheap way of assessing whether a sample contains the nitrogenase enzyme, and hence nitrogen-fixing bacteria.
After finding that the pine needles did indeed host nitrogen fixers, the group used genetic techniques to look more closely at who exactly is there. The most prominent bacteria in all pine needle samples was genetically similar to Gluconacetobacter -a nitrogen fixer found in sugarcane.
So, nitrogen fixers are living in pine needles and helping their host trees take up nitrogen. If replicated in other coniferous forests, these findings could transform our understanding of alpine and boreal ecosystems, helping explain how coniferous trees persist on very nitrogen-limited soils.
Ocean acidification and marine calcifiers: it’s more complicated than you thought.
Or at least, it’s much more complicated than an ignorant terrestrial scientist like myself thought. See, my understanding of ocean acidification went something like this: more CO2 in the atmosphere means more CO2 dissolving in the surface ocean. When this CO2 dissolves, it reacts with water to produce acid, lowering the pH of the ocean. This is bad news for organisms that build exoskeletons from calcium carbonate (CaCO3), because the protons that create acidity get in the way of calcium carbonate formation. Sound good?
Well, like most natural phenomena when examined closely, the answer is quite a bit more complicated. Dr. Deborah Iglesias-Rodruigez enlightened me today as to some of that complexity. Iglesias-Rodruigez studies Coccolithophores, a common group of marine phytoplankton (photosynthetic organisms) that precipitate a beautiful, calcium carbonate exoskeleton. But in order for these little guys to create their exoskeletons, they must first take up some form of carbon into their cells.
It turns out Coccolithophores have evolved to take up the predominant form of carbon found in seawater: bicarbonate, or HCO3-. And it also turns out this is the particular form of carbon that becomes more abundant during ocean acidification. So- does that mean acidification is good for calcium carbonate formation, after all?
Well, not quite. Although Coccolithophores seem to take up more bicarbonate under more acidic conditions, this isn’t exactly a good thing. Iglesias-Rodruigez finds that, in acidic conditions, Coccolithophores can start over-calcifying: precipitating more calcium carbonate than they need to, which is energetically wasteful. Moreover, once that calcium carbonate is precipitated, it gets slowly eaten away by the more-acidic seawater.
There was more detail to be found in this presentation, including some interesting stuff about differences in microbial cell volume mediating ocean acidification effects, but I’ll leave that for another day. One ocean chemistry lesson today was enough for my brain to juice on.
Imagine you were forced to live in perpetually subzero temperatures, with no oxygen, no light, and way more salt than your system could handle. How would you manage? One way might be to get extremely small. At least, that seems to be what’s happening in a frozen Antarctic lake that’s cut off from the rest of the world by 27 meters of perennial ice.
Lake Vida, Antarctica, has come under biological scrutiny recently. It’s an fascinating environment for a number of reasons. For one, it represents a unique combination of extreme conditions. Vida’s high salt concentrations keep the lake’s water liquid at -13.4ºC, or 7.9 ºF. And, even more intriguing, this super-chilled salt bath has been cut off from the outside world for nearly 3,000 years.
The microbial inhabitants of Lake Vida have had a unique opportunity to evolve in complete isolation. For microbial ecologists, this means a potential goldmine of novel adaptations and genetically unique organisms.
So far, Lake Vida’s microbes have lived up to expectations. In a study published recently in the journal Applied and Environmental Microbiology, Dr. Alison Murrary and colleagues find Lake Vida’s brine is teeming with some very tiny critters. These ultrasmall microbes, or ultramicrocells, are roughly 200 nanometers in diameter, just undercutting the theoretical “lower size limit” for a single-celled organism. In addition, these tiny critters display some fascinating adaptations for handling the stress of life in cold, salty brine.
Murray and colleagues used several techniques to characterize the ecology of Lake Vida brine samples collected in 2010, including scanning electron microscopy, spectroscopy, and x-ray diffraction.
In their recent study, the scientists observed two cell populations in Lake Vida’s brine. One population of rod-shaped bacteria ranged in size from ~0.4-1.5 µm, while a smaller class of spherical bacteria were approximately ~0.2 µm, or 200 nanometers, in diameter. This second class, designated the “ultrasmalls”, was 100 times more abundant than their larger counterparts. Even smaller particles that ranged in size from 20-140 nanometers were also abundant.
Further analysis using x-ray spectroscopy indicated that both ultrasmalls and nanoparticles had granular, iron-rich surface coatings. Interestingly, these coatings resemble iron oxide minerals found in old, weathered soils. It was also common for ultrasmalls to possess exopolysaccharides- long, filamentous proteins- connecting them to the nanoparticles.
Exopolysaccharides can serve many functions for microorganisms. In this case, the scientists speculate exopolysaccharides act as a nucleation site for iron particles- that is, a surface to which iron particles can precipitate in solid form. The resultant “iron exoskeleton” may be a unique adaptation for protection against extreme cold.
The nanoparticles remain something of a mystery, but the scientists hypothesize these may also be a part of an elaborate ultrasmall survival strategy. The size and morphology of the nanoparticles suggests they may, in fact, be extracellular membrane vesicles- pieces of cells that have popped off their parent and become self-contained storage units . Other scientists have found that microbes produce such vesicles in response to temperature stress. Like a storage unit, vesicles allow microbes to sweep their house clean, removing unnecessary clutter. One sort of unwanted baggage for the Lake Vida ultrasmalls may be misfolded proteins. Protein misfolding is a common problem in subzero environments. Harboring useless misfolded proteins represents a drain on valuable cellular resources.
Lots of open questions remain regarding the ecology of Lake Vida’s ultrasmalls. Perhaps the biggest question is why exactly these microbes are so tiny. There are a number of possibilities to be explored. Smallness is a response to stressful environments across all domains of life. Hyperosmotic stress- the result of being bathed in a super salty liquid- may result in water loss and cell shrinkage. Or ultrasmalls may be expending so much energy dealing with the cold that they don’t have the extra resources required to grow bigger.
Answering these questions will help scientists understand how microbes may cope with extreme environments not only on Earth, but on icy extraterrestrial worlds as well.
Kuhn, E., Ichimura, A., Peng, V., Fritsen, C., Trubl, G., Doran, P., & Murray, A. (2014). Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida, Antarctica Applied and Environmental Microbiology, 80 (12), 3687-3698 DOI: 10.1128/AEM.00276-14
The cutting edge of robotics may not be a smarter Siri or a less-creepy humanoid Japanese robot. It might be a swarm of bacteria, compelled to do our bidding through a remotely controlled magnetic field.
Some of the biggest technological advances of the past two decades have involved scaling things down. The development and continual improvement of microprocessors has revolutionized home computing. And the emerging field of nanomedicine promises to transform biomedical science in many ways: from targeted drug delivery to tissue repair at the cellular level.
However, working at the microscale (thousandths of a millimeter) or nanoscale (millionths of a millimeter) poses major challenges. To create a circuit out of microscopic components, we need very small tools that can do the work for us. We need microscopic robots, or microbots.
But building useful microbots is no easy task. Power supply has always been one of the toughest challenges. Some bots contain a very lightweight battery. Others possess a coin cell that scavenges vibrational or light energy from its surroundings. Telling our microbots where to go is another hurdle. Microbots typically don’t work alone- we usually need a swarm of them to perform a task. And we’d like to be able to direct that swarm with the utmost precision.
Some scientists think the solution to our microbot woes can be found in nature. Bacteria are essentially organic “machines” that use light or chemical energy to move about and do work. They fit the size criteria, and they’re everywhere. Rather than re-invent the wheel, what if we could train bacteria to do work for us?
To answer this question, microroboticists are now looking to a group of bacteria that possesses an astounding property: magnetism.
So-called magnetotactic bacteria are promising because their motion can be guided by an externally applied magnetic field. Given the right magnetic field, we might be able to coordinate the motion of thousands to millions of magnetotactic bacteria at once.
Magnetotactic bacteria are a genetically diverse group of organisms thought to have evolved during the early Proterozoic, some 2.5 billion years ago, when rising atmospheric oxygen concentrations were reducing the amount of dissolved iron in Earth’s oceans. It’s believed this caused some bacteria to start stockpiling iron. Eventually, these iron stores were adapted to form magnetosomes, crystalline structures found in the cell membranes of modern magnetotactic bacteria. Magnetosomes align in a chain, allowing the bacterium to orient itself like a compass needle to the local magnetic field.
The most obvious advantage to a magnetotactic microbot is that its motion can be guided by a user-generated magnetic field. But bacteria are also advantageous because they possess flagella. These rotating, tail-like structures act as a propeller, allowing bacteria to swim about quickly and change direction with ease. And the energy required to turn a flagellum is generated by the bacterium’s own metabolism. Power source, check. Motor, check.
So, what’s it going to take for magnetotactic bacteria to become our microbots of choice? One challenge scientists are now focusing on is 3-D aggregation. Using a single electromagnet, it’s simple business to line a bunch of magnetic bacteria up in a 2-D sheet. Forming a 3-D swarm is more challenging. But if doable, 3-D configurations would have major advantages. A 3-D swarm would be much easier to guide through the intricacies of the human vascular system. It would also be able to build 3-D objects more efficiently.
In a paper recently published in the Journal of International Robotics Research, scientists used Magnetococcus marinus, a spherical, magnetotactic bacterium possessing 2 bundles of flagellar “propellors”, to explore the possibility of creating 3-D bacterial clusters with magnets.
Using remotely powered pairs of electromagnetic coils, the researchers applied three different time-varying magnetic field sequences to a liquid culture of M. marinus. They were able to generate both 2-D and 3-D bacterial configurations. Then they managed to steer these swarms through a complex network of glass tubes intended to mimic capillaries.
These basic steps are laying the foundation of being able to one-day guide armies of magnetotactic bacteria to carry out many tasks. In the future, swarms of magnetic “biobots” might be used for targeted cancer treatments, transporting microscale objects, assembling microcircuits, and even microscale magnetic resonance imaging.
de Lanauze, D., Felfoul, O., Turcot, J., Mohammadi, M., & Martel, S. (2013). Three-dimensional remote aggregation and steering of magnetotactic bacteria microrobots for drug delivery applications The International Journal of Robotics Research, 33 (3), 359-374 DOI: 10.1177/0278364913500543
Miles beneath our feet, Earth’s rocky crust may seem a cold, dead place. On closer inspection it’s anything but.
Microbes have been making a home on and in rocks since…well, since the beginnings of life, some 3.5 billion years ago. The traditional view of rock-dwelling microbes is one of sparse, energy-starved survivalists eking out an existence on a hostile frontier. These critters are at the mercy of their environments, their numbers dictated by physical factors – things like temperature and pH.
A study published in Geomicrobiology Journal, led by graduate student Aaron Jones at the University of Texas at Austin, challenges the assumption that subsurface microbes are passive survivalists. In a series of lab experiments, Jones demonstrates that mineral-dwelling microbial communities are uniquely adapted to their environments on the scale of individual mineral grains. Moreover, his research suggests microbes and minerals share a partnership that stretches back through geologic time.
Jones was interested in how mineralogy- the composition of the minerals that form rocks- influences the abundance and diversity of microbes. In particular, he was interested in sulfur oxidizers- bacteria that strip electrons from sulfur to acquire energy. Sulfur oxidizers are common in other subsurface environments including deep ocean vents. Their metabolism generates strong acids that eat away at certain minerals, forming cracks, pores, even caves.
In the lab, Jones set up a series of “bioreactors”- tanks in which liquid of a specific chemical composition flows over thin sheets of rock. He used a range of rocks that differ in their mineralogy. These included several rocks composed of calcium carbonate (limestone, dolostone and calcite) and several made of silica-rich minerals (albite, microcline and quartz). Jones inoculated his bioreactors with samples of a sulfur-metabolizing biofilm collected from a Wyoming cave spring.
After three weeks of incubation, he measured bacterial growth, extracted DNA and used pyrosequencing to examine the composition and diversity of the microbial communities formed on different rock types.
Different rocks, different microbes
Growth patterns differed substantially across the carbonate and silicate rocks. On carbonates, bacteria grew in thick, filamentous biofilms. By contrast, growth was much sparser on silicates, with bacteria either accumulating in small clumps or spreading out individually.
Community composition also differed among different rocks. The bacterial phyla Alphaproteobacteria dominated on most carbonates. Sulfur oxidizers that thrive at near-neutral pH were common, representing 17-45 % of the community.
A different picture emerged on the silicate rocks. Members of the phyla Firmicutes and Actinobacteria- also known as Gram-positive bacteria- were found here. While community composition was relatively similar among the carbonates, communities that grew on quartz were very different than those found on albite or microcline. Jones observed fewer sulfur oxidizers on silicates, and those he found tended to be more acid-tolerant.
Perhaps the most obvious conclusion here is that microbes do not select rocks at random. Different growth patterns among rocks tell us this. Better microbial growth on carbonates probably reflects a higher density of essential nutrients, particularly phosphorus. Moreover, two of the silicate rocks, albite and microcline, contain high concentrations of aluminum, a toxic element that restricts bacterial growth.
More subtly, however, there appears to be “selective relationship” between mineralogy and microbial ecology. In other words, the microbes making a living on any given rock are uniquely adapted to taking advantage of, or protecting themselves from, the specific chemical properties of that rock.
This relationship bears itself out in many ways. For one Jones found more acid-tolerant microbial communities on silicate rocks. Remember, acid is a byproduct of sulfur metabolism. Carbonate rocks have a high “buffering capacity”, meaning their minerals react with and neutralize acid. By contrast, silicate rocks, especially quartz, are pretty unreactive. Rather than eating away at minerals, acid accumulates at silicate surfaces. The presence of acid-loving bacteria on quartz suggests a feedback loop between sulfur metabolism, mineralogy and microbial community structure.
The presence of Gram-positive bacteria on silicates also underscores the relationship between microbial community composition and the mineral environment. The cell walls of Gram-positives possess unique chemical properties that promote bonding with silicate surfaces. This microbe-mineral binding, known as adhesion, is a key element of survival for most rock-dwelling bacteria. By adhering to a solid surface, bacteria can mine nutrients and form protective biofilms.
Even microbes living on the undesirable aluminum-rich rocks may possess unique adaptations. While few bacteria grew on albite or microcline, these sparse populations were highly diverse. The unique challenges presented by a toxic environment may serve to maintain competition, preventing any single group of organisms from gaining dominance.
Our planet’s rocky subsurface is the largest microbial habitat, but we still know very little about its inhabitants. Most microbial ecologists prefer to study environments they can see, feel and intimately relate to. Jones’s research points to complex ecological interactions between microbes and minerals in the deep, dark depths of the world. Rocks shape microbial communities, but microbes may also shape rocks in profound ways. This study lends evidence to a deep synergy between the geological and biological evolution of our planet.
Jones, A., & Bennett, P. (2014). Mineral Microniches Control the Diversity of Subsurface Microbial Populations Geomicrobiology Journal, 31 (3), 246-261 DOI: 10.1080/01490451.2013.809174
The love affair between industrial agriculture and the antibiotic industry has come into an uncomfortable spotlight of late. In 2011, 7.7 million pounds of antibiotics were sold to treat sick people in the United States. This compares with a whopping 29.9 million pounds of antibiotics fed to cattle, pigs and poultry.1 Regular antibiotics doses keep perpetually overcrowded animals from falling ill and dying en masse, but antibiotics are also widely used to hasten growth, shortening an animal’s time to slaughter and increasing profit.
What’s the consequence of all this unfettered antibiotic use? Multi-drug resistant strains, or “superbugs” are on the rise. Our ability to keep pace with resistance by producing new antibiotics is diminishing. It’s even been suggested that we’re now entering a post-antibiotic era.
In 2010, representatives of the FDA, U.S. Department of Agriculture and Center for Disease Control and Prevention testified before Congress that a definitive link exists between the overuse of antibiotics in animal agriculture and antibiotic resistant diseases in humans.
But in spite of mounting evidence, the meat industry has largely succeeded in lobbying against any antibiotic restrictions. A major thrust of the industry’s argument is the lack of direct evidence linking antibiotic resistant bacteria bred on animal farms to human disease.
Now, proponents of antibiotic regulation may have some powerful new evidence to fuel their case. Microbial ecologist Ludek Zuerkand colleagues at Kansas State University are finding that insects- particularly houseflies and cockroaches- may represent the missing link between animal farms and human population centers.
Their review paper on insects and antibiotic resistance is currently in press in the journal Applied and Environmental Microbiology.
Zurek’s research team focuses on Enterococci, a group of bacteria responsible for illnesses ranging from urinary-tract infections to meningitis. Enterococci are also rather infamous for developing multi-drug antibiotic resistance. In one study, researchers measured the abundance of Enterococci in two swine production facilities in Kansas and North Carolina. The scientists examined houseflies, roaches and pig feces collected at both sites, finding Enterococci in 89% of all samples. Multi-drug resistant strains were found everywhere. Moreover, the drug-resistant strains found in flies and roaches were genetically identical to the strains found in swine feces, indicating insects acquired their pathogens from pigs.
In another study, the researchers screened houseflies collected from five fast food restaurants in a town in northeastern Kansas. Ninety seven percent of flies harbored Enterococci. The most abundant strain, Enterococcus faecalis, showed resistance to broad-spectrum antibiotics including tetracycline, erythromycin, ciprofloxacin and kanamycin. The scientists also identified transposons- snippets of DNA bacteria can swap during conjugation, their version of sex- that are associated with antibiotic resistant traits.
Ready-to-eat food from the same restaurants was also contaminated with antibiotic-resistant bacteria. Contamination was higher in summer than winter, corresponding with increased numbers of houseflies in restaurants.
From these investigations, the researchers concluded that “food served in restaurants is commonly contaminated with antibiotic-resistant Enterococci and that houseflies may play a role in this contamination.”
Not wishing to lose points for a lack of thoroughness, the scientists decided to test directly whether insects from animal farms can contaminate food. In another study, they collected flies from a cattle feedlot and brought them back to the lab. Within thirty minutes, the flies deposited roughly 1,000 antibiotic-resistant Enterococci on a hapless beef patty. This experiment was carried out using as few as five flies.
Houseflies give bacteria more than just a free ride from farm to food. They may also serve as an incubator. Several studies have shown that pathogenic strains of E.coli proliferate in the gut of common houseflies and can be transferred during feeding.
Using a fluorescent protein to tag and track bacteria, Zurek’s research team found Enteroccoccus density peaks in the fly’s crop, or foregut, roughly 48 hours after ingestion. Significantly, houseflies regurgitate the contents of their crop while feeding. In doing so, they can disseminate bacteria into their food and water. Zurek suggests houseflies serve as a “bioenhanced vector for bacteria” because of their dual role as incubator and locomotion.
The work of Zurek and his fellow scientists has profound public health implications. Through many lines of evidence, this body of research demonstrates a direct link between the antibiotic resistant bacteria on factory farms and antibiotic resistant bacteria in our food.
Of course, none of this is terribly surprising, is it? We’ve known since biblical times that flies are harbingers of disease. Included in the ten Biblical Plagues in the Book of Exodus is the Plague of Flies, which “came [as a] grievous swarm of flies into the house of Pharaoh, and into his servants’ houses, and into all the land of Egypt: the land was corrupted by reason of the swarm of flies.”
However, when it comes to an issue as personal (and political) as food, we sometimes tend to forget unpleasant truths. In his book in Eating Animals, an acclaimed work of investigative journalism on the modern meat industry, Jonathan Safran Foer writes, “Food choices are determined by many factors, but reason (even consciousness) is generally not high on the list.” As hard scientific evidence accumulates on the link between antibiotic resistance on animal farms and public health, one can only hope growing consumer consciousness will force the meat industry to take a hard look at its practices.
Zurek, L., & Ghosh, A. (2014). Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits Applied and Environmental Microbiology, 80 (12), 3562-3567 DOI: 10.1128/AEM.00600-14
When you look at soil, you probably see dirt. When I look at soil, I see billions of microorganisms, crawling atop one another, consuming the dead in a feasting frenzy that stops for nothing save a deep freeze. I see microbes and their enzymes, the digestive juices that break down, transform and release all the energy tied up in our planet’s terrestrial ecosystems.
Through their remarkable ability to decompose nearly anything that comes their way, soil microbes collectively represent a planetary recycling factory, one that takes carbon- the structural unit behind all living matter- from dead organic matter back to the atmosphere as carbon dioxide (CO2). Soils “exhale” CO2 for the same reason you and I exhale CO2. We’ve eaten something, broken down the carbon bonds that hold it together, and extracted all the energy that we could. CO2 is the generic waste product of cellular metabolism, the last bit of carbon that our own metabolic inefficiency precludes us from using. Ecologists have coined the term soil respiration to describe the collective exhale of microbial carbon decomposition.
Breathing matters, but in soil, it’s complicated
This earthly exhale is an event of profound significance to the biosphere. It dictates how much carbon remains in soil, and how much returns to the atmosphere, where acts as a heat-trapping greenhouse gas that helps regulate Earth’s climate. Biogeochemists- scientists who study the planetary cycling of elements, like carbon- are working to understand how microbes contribute to the global exhale of Earth’s soils. What makes them breathe slower? Faster? How does this influence the distribution of carbon across a forest? Across the planet? Over a year? A century? Understanding what makes soil microbes breathe will allow scientists to make better predictions about our planet’s future. How will human disturbances, like climate change, urbanization, or fertilization affect this important carbon pathway?
In order to predict why soils exhale, we need to first understand who or what’s breaking down carbon. And it turns out, when you really get down into the dirt, things get a bit more complicated. You see, microbes don’t actually accomplish all that carbon digestion by themselves. Enzymes- proteins that act as catalysts by facilitating specific reactions- mediate nearly every step in soil carbon decomposition. Some of these enzymes reside inside living microbes, but many are released outside the cell, into the environment. These “extracellular enzymes”, or exoenzymes, act as independent entities from their microbial producers. This means exoenzymes can-and often do- persist in soil long after their microbial parent dies. By chemically associating with soil minerals, exoenzymes can form stable complexes that resist drought, pH changes, even attack and degradation by other enzymes.
And finally, to make matters even more complicated, some carbon decomposition occurs by entirely abiotic (non-living) processes. Certain soil minerals, such as iron oxides, strip electrons form organic matter in a process known as oxidation. Oxidation is important for the breakdown of large, complex molecules such as lignin, the primary building block of wood.
Exoenzymes and soil microbes represent two different pieces of the decomposition puzzle. Abiotic carbon oxidation represents a third. Since these different aspects of decomposition may respond differently to environmental change, we’d like a way of separately measuring their contributions to the soil carbon cycle. But separating exoenzymes, microbes and minerals, all of which not only coexist within nanometers of each other, but are often chemically bonded, is no easy task. Soil microbial ecologists have long recognized, sometimes acknowledged, but largely ignored our inability to separate the living from the non-living in soil carbon decomposition.
Separating the living and the dead
Finally, we may have a roadmap for solving a problem that has eluded microbial ecologists for years. Last month, a group of researchers led by Dr. Joshua Schimel at the University of California, Santa Barbara, published the first study that rigorously attempts to separate cellular carbon metabolism from exoenzyme activity in soils. The study is currently in press in the journal Soil Biology & Biochemistry.
To separate cellular from extracellular metabolism in soil, the researchers recognized the need for a method that could disable living cells while leaving exoenzymes intact. This is no trivial problem. Heat can be used to sterilize soil, but high heat can also disrupt exoenzyme activity and accelerate the break-down of other organic compounds. Toxic chemicals like sodium azide (NaN3) can inhibit most microbial activity, but a small fraction of microbes develop resistance.
Two potential methods the researchers decided to test are chloroform fumigation and gamma irradiation. Chloroform, a nonpolar and hydrophobic (water-avoiding) molecule, is chemically inert but nonetheless deadly. Chloroform buries itself in the lipid bilayer of cell membranes, causing membranes to become disorganized, and eventually rupture. While some exoenzymes are clearly not affected by chloroform, others may be. Gamma radiation is a powerful, ionizing radiation that punctures cell membranes and causes them to burst. Gamma radiation is frequently used to sterilize food and medical equipment. Very few studies have measured the effect of gamma radiation on exoenzymes. The scientists compared the efficacy of these to techniques to autoclaving, a more traditional sterilization method in which a sample is subjected to very high heat.
Having chosen several sterilization methods, the researchers set up an experiment to test how cellular and extracellular soil carbon metabolism would respond. They collected soils from a California grassland, and sterilized samples using each method. From each sterilized soil, they attempted to culture viable cells. They also added a vital stain (one that differentiates living and dead cells), and used fluorescent microscopy to evaluate the number of living and dead cells.
To evaluate extracellular metabolism, they measured the activity of eight exoenzymes produced by a wide range of soil microorganisms. The eight chosen enzymes degrade common components of soil organic matter (lignin, cellulose, chitin and proteins). They included six hydrolases, enzymes that break specific chemical bonds, and two oxidases, which indiscriminately strip electrons from organic molecules. Hydrolases like beta-glucosidase are important for degrading molecules with predictable, repeating structures, such as cellulose. Oxidases play a central role in breaking down larger, more complex structures, similarly to abiotic oxidation.
To evaluate cellular metabolism, the researchers used a technique called “substrate induced respiration” (SIR). SIR involves adding a small molecule (typically an amino acid or glucose) that cells can metabolize completely, and measuring CO2 loss. The CO2 generated from an SIR experiment indicates the metabolic potential of live cells in a sample.
They found chloroform and gamma irradiation both led to a 1,000 fold reduction in the number of living cells. Autoclaving was a more powerful sterilization technique; reducing the number of live cells approximately 100,000 fold. Most exoenzyme activities were only modestly reduced by chloroform or gamma irradiation. However, sterilization did reduce the activity of two enzymes, alpha-glucosidase and beta- xylosidase by approximately 75%. It’s possible that consistent reductions in activity across all enzymes may have had a proportionally greater effect on these two, which had the lowest activity in non-sterilized soils.
From these initial findings, what can we conclude? Both chloroform and gamma radiation effectively sterilize soils (if we can accept the fact that a few hardy spores might resist these treatments). Both treatments also keep exoenzyme activity intact (keeping in mid low-activity soils will probably experience proportionally larger reductions). A few caveats here and there, but by and large both techniques appear promising.
But what about cellular metabolism? Did sterilization successfully eliminate this, as well? Your gut reaction might be that this is a silly question. If we’re killing all living cells, we must also be killing cellular activity, right? That’s at least what the researchers initially thought.
The SIR results tell a different story. The scientists added a number of different simple carbon substrates, all of which can are broken down during basic cellular metabolic processes like glycolysis and the citric acid cycle, or TCA cycle.Following sterilization, respiration from glucose and amino acid substrates was halted. However, respiration from TCA-cycle substrates, including pyruvate, citrate and ketoglutarate, was reduced but not eliminated. How could dead cells continue respiring? The authors speculate that certain dehydrogenases, the enzymes that break down TCA cycle-substrates, are able continue functioning outside the controlled environment of the cell. This finding was completely unexpected. It calls into question many assumptions about one of the most fundamental energy-harvesting processes in cellular biology, while further blurring the line between microbial metabolism and environmental metabolism.
Another surprise came from the oxidase enzymes. While all hydrolytic enzymes were killed by high heat in the autoclaving treatment, oxidative reactions persisted. The authors speculate the oxidative activity they measured in autoclaved soil is the result of abiotic processes, supporting the notion that a large amount of oxidative decomposition in soils can be caused by mineral catalysis.
Taken as a whole, this study finds both chloroform fumigation and gamma irradiation to be promising methods for separating cellular and extracellular metabolism. Some refinements are certainly needed- finding a way to separate living from non-living oxidation, for instance. But both methods already promise to unravel new mechanisms for microbial carbon metabolism, as highlighted by the finding that some TCA- cycle enzymes can persist after cell death. Now scientists can start using these sterilization treatments to measure the lifetime of exoenzymes in the environment. Right now we can only guess at how long exoenzymes stick around once they’re produced. Understanding the long-term persistence of these enzymes in the environment will help us to better gauge the capacity of soils- even in the absence of living cells- to continue digesting the planet’s carbon.
Blankinship, J., Becerra, C., Schaeffer, S., & Schimel, J. (2014). Separating cellular metabolism from exoenzyme activity in soil organic matter decomposition Soil Biology and Biochemistry, 71, 68-75 DOI: 10.1016/j.soilbio.2014.01.010
YOU KNOW YOU WANT TO...
Life from a Wellcome Trust perspective
the curious lives of microbes on the edge
Engaging and transformative reporting on the environment, energy, and sustainability
A Window into my Research Notebook
The latest oceanography literature, explained
the curious lives of microbes on the edge
300 words or more
this nerd's life
Musings on Matings and the Evolutionary Biology of Sex
Science interest, advocacy, and explanation
An Ocean Science Blog by Grad Students in Marine Science at UNC- Chapel Hill
the curious lives of microbes on the edge
How to write papers that get cited and proposals that get funded
IxD Lessons from Sci-Fi
What's Orange and Green?
The Blog of Astrobiologist Kuhan Chandru, PhD
Mushroom Cultivation Research and Education