Microbe, enzyme or mineral? A riddle in the soil.

Standard
Soil is the most microbially diverse habitat on Earth, and contains twice as much carbon as living plants and the atmosphere combined. Source: National Geographic

Soil is the most microbially diverse habitat on Earth, and contains twice as much carbon as living plants and the atmosphere combined. Source: National Geographic

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.

Scanning electron microscope image of iron oxidizing bacteria Acidovorax sp. BoFeN1, encrusted in iron minerals. Microbes, enzymes and minerals are often intimately associated in soils, making it difficult to separate out their contributions to biochemical reactions. Source: Eye of Science, Reutlingen

Scanning electron microscope image of iron oxidizing bacteria Acidovorax sp. BoFeN1, encrusted in iron minerals. Microbes, enzymes and minerals are often intimately associated in soils, making it difficult to separate out their contributions to biochemical reactions. Source: Eye of Science, Reutlingen

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.

ResearchBlogging.org

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

Advertisements

When infection is a good thing: sulfur-eating bacteria enlist viruses to help acquire energy

Standard

“Black smokers”, deep sea vents where hot, sulfur-rich fluids bubble up from beneath the ocean floor, are considered hotspots of microbial activity. They may even be where life originated. Credit: Wikimedia Commons

Life is no cake walk at the ocean floor, where carbon is scarce and light nonexistent.  At least near deep ocean vents, mineral-rich water bubbles up from magma beneath the crust, providing both heat and a source of energy. In these alien environments, lithotrophs– bacteria that eat minerals instead of organic carbon- have staked out a niche by evolving some creative metabolic strategies.

But minerals are a poor source of energy compared to organic matter. Lithotrophs are slow-growing critters, easily outcompeted when carbon is abundant. They need all the help they can get. And it turns out, they might very well get help… from an unlikely source. A study published last week in Science Express reports how viruses may be helping deep marine bacteria eat sulfur.

Viruses are everywhere- in soils, skies, oceans, plants and animals, even deep beneath the ocean floor.  In spite of their ubiquity, it’s not well-known how viruses influence marine bacteria and the nutrient cycles they drive.

To study the impact of viruses on sulfur-eating bacteria, a team of scientists led by Dr. Karthik Anatharaman at the University of Michigan collected samples from five different hydrothermal vents : four in the Western Pacific Ocean and one in the Gulf of California. They used shotgun metagenomic sequencing to look at the genomes of the bacteria and viruses present in these environments.

The researchers found five different virus “types” that carried two genes involved in sulfur metabolism. Known as rdsrA and  rdsrC, these genes encode different pieces of dissimilatory sulfite reductase, an enzyme that breaks down elemental sulfur.

How and why did viruses come to acquire these genes? Not technically considered alive, viruses don’t really need enzymes because they don’t perform metabolism on their own. A virus’s sole purpose is to infect a host with its genetic code so that it can turn the host cell into a virus factory. But during viral replication, pieces of  DNA can be accidentally snipped out of the host and integrated into the viral genome. The scientists found bacteria in the same samples carrying both the rdsrA and rdsrC genes. It’s likely that accidental incorporation of host DNA is how viruses ended up with sulfur genes in the first place.

While acquiring sulfur genes may have been a complete accident, viruses appear to have been actively maintaining those genes for a long time.

When the scientists compared rdsrA and rdsrC from viral and bacterial genomes, they found something interesting: completely different DNA sequences surrounding the genes. If viruses had acquired sulfur genes during recent replication errors, some of the nearby DNA would probably have come along for the ride as well. That completely different DNA sequences surround the sulfur metabolism genes in bacteria and viruses suggests viruses have been passing along rdsr genes, generation to generation, for a very long time. In other words, sulfur metabolism genes are being maintained  in viruses by natural selection.

Why would deep sea viruses bother to maintain sulfur metabolism genes they don’t use?  It turns out these viruses may actually have a use for sulfur genes, albeit an indirect one: helping out their hosts. By carrying genes that bacteria can make use of, viruses may assist their hosts in acquiring energy. The ability of sulfur-eating bacteria to acquire energy is ultimately limited by how quickly they can decode their sulfur genes to build enzymes that can do the work. But if a bacteria were to contain viruses with extra copies of the genes it needs, that could help speed the process along. In essence, when a sulfur virus infects a sulfur bacteria, it donates genes that its host can use.

What’s a virus to gain from all of this? Remember, a virus’s sole purpose is replication. Staying in your host’s good graces has its perks. An “infected” bacteria that can acquire energy faster than its neighbors may be able to grow and reproduce faster- leading to more infected offspring, and more viruses. By supplementing host metabolism, viruses may help ensure their continued survival.

This study sheds light on a potentially widespread, mutually beneficial ecological interaction between bacteria and viruses. Lithotrophs in the deep ocean play an important role in the global sulfur cycle and have done so for billions of years. By giving an adaptive advantage to sulfur-eaters and helping them survive, it’s possible viruses have played an equally important role in the geochemical evolution of our planet.

ResearchBlogging.org

Anantharaman, K., Duhaime, M., Breier, J., Wendt, K., Toner, B., & Dick, G. (2014). Sulfur Oxidation Genes in Diverse Deep-Sea Viruses Science DOI: 10.1126/science.1252229

Who cheats and who eats? An evolutionary conundrum.

Standard

cheating_rect-460x307

Say what you will about our other vices, human beings did not invent cheating. Microbes have been doing it for billions of years. You see, for microbes, cheating can sometimes be an evolutionary advantage. And this can cause it to get out of hand really quickly.

Bacteria “cheat” by stealing each other’s lunch. They do it everywhere, all the time, and unwittingly. To understand why, we first need to consider how bacteria feed themselves.

Has it ever occurred to you what a convenience it is that most things we like eat are comparable to us in size? Probably not. But think about it for a second. Sure, some foods are quite a bit smaller than us (think nuts, berries and grains), while cows, bison and mastodons are certainly larger. But, in general, most things we eat are close enough to us in size that we can see, touch and handle them.

If this seems like a trivial observation, you may be surprised to learn it’s not always the case. Most of the things a bacteria would like to eat are thousands to millions of times too large for it to ingest. But bacteria have devised a clever way around this situation. They secrete enzymes, specialized proteins, out of their cells and into their environments. These so-called exoenzymes swim around chiseling bite sized fragments off anything they can: leaf scraps, insect remains, animal carcasses. Those fragments, sometimes a single molecule in size, can then be ingested by microbes and converted into energy.

Many bacteria rely entirely on exoenzymes for their food. However, enzymes are costly to build. All of the carbon and nutrients that go into making an enzyme could be spent elsewhere- on growth, reproduction, or cellular repair. Therefore, exoenzyme production is strictly regulated according to a simple rule anyone who has ever taken an introductory econ class will be familiar with: marginal revenues must exceed marginal costs. In other words, if you don’t get much benefit from that enzyme you just built, don’t build another one.

Here’s where cheating comes in. Exoenzymes are costly for the individual to produce, but they increase resource availability for the community. In econ-speak, exoenzymes are a public good. Once released into the “wild”, an enzyme is free to diffuse away from its producer. It can get stuck to a mineral, become inactivated by freezing or desiccation, or be eaten by another enzyme.  Given the risk associated with enzyme production, cheating can become an attractive alternative. Why produce enzymes yourself if you can enjoy free lunch thanks to your neighbors’ enzymes?

This type of microbial freeloading can become a major problem. The more cheaters are present in a population, the less return a producer will get on its’ exoenzymes. Too many cheaters, and suddenly it’s not worth anyone’s while to produce enzymes at all. Paradoxically, this leads to a situation where everyone’s starving, because no one can spare the resources needed to get food.

From a microbial perspective an exoenzyme is a tool for acquiring food. But on a global scale, exoenzymes serve a higher purpose. They are the engine that digests the dead, recycling carbon and nutrients back the living. They are the reason the surface of our planet is not piled high with the dead bodies of every living organism that ever was. Clearly, then, microbes have found ways of coping with the potentially debilitating effect of cheaters.

How, then, do microbial communities keep the cheaters in check? A group of microbial ecologists led by Dr. Steve Allison at the University of California, Irvine, devised a simple experiment using Pseudomonas fluorescens, a common soil bacteria, to investigate how and when cheating occurs. The study was published last week in the journal Frontiers in Microbiology.

The scientists first obtained two different strains of P. fluorescens. One strain had the genetic capacity to produce protease– a protein-decomposing enzyme. The other strain, a cheater, lacked the ability to produce the protease enzyme. The researchers created mixed cultures of these two strains and monitored the abundance of cheaters, producers, and protease over time.

When cheaters and producers were grown in 50/50 mixed cultures, protease activity declined to near zero. Rampant cheating apparently deterred everyone from making protease. More surprisingly, in cultures that contained only protease producers, enzyme activity still declined to near zero. Why? In an environment full of enzyme producers, cheaters have a selective advantage because they can get a free lunch. It’s possible that a genetic mutation resulting in an inability to produce protease- a cheater mutation- swept through the population.

So, given that cheaters cheat, and producers become cheaters who then cheat, how is it that microbial decomposition hasn’t ground to a halt? The answer, it turns out, may lie in the structure of the microbial environment. The experiments I just described were performed in liquid cultures. In liquid, enzymes move about freely via diffusion. The result is a cheater’s paradise, an environment where resources are abundant everywhere. At least for a short while.

But what about in a more structured environment, like soil? In soil, bacteria and enzymes adhere to solid particles. Depending on moisture levels, diffusion in soil can be rapid or very slow. If a producer’s exoenzymes stay close to home, they are more likely to provide their maker with benefits. In this case, the producers may actually have incentive to continue making enzymes. To test this idea, the researchers repeated their experiment, only this time, they grew P. fluorescens on solid agar in petri dishes. The results were quite different. Cheaters did not sweep the population. Instead, populations remained a patchwork of producers and cheaters.

In spite of its simplicity, this experiment has a profound implication for our understanding of microbial ecology. It demonstrates that environments with greater spatial structure favor a diversity of life strategies. In other words, increased environmental heterogeneity facilitates coexistence.

This finding is not unique to microbial ecology- we see a similar principle playing out across much larger scales. The introduction of invasive plants and animals to new ecosystems represents a breakdown of spatial boundaries; this results in the mixing of once-separate communities. We need only go to a kudzu-ridden forest in the Southern US, or read scifi-like stories of the cane toad devastation in Australia, to see for ourselves the link between spatial isolation and diversity. When populations are separate, they experience unique environmental challenges, leading to diverse adaptations and evolution. When systems become too mixed, diversity can lose out to a lower common denominator: who can hoard the most resources, grow and reproduce the fastest.

Ultimately, as systems become too well-mixed, too homogenous, they grow vulnerable to collapse. Remember, if everyone’s a cheater, no one eats.

ResearchBlogging.org
Allison, S., Lu, L., Kent, A., & Martiny, A. (2014). Extracellular enzyme production and cheating in Pseudomonas fluorescens depend on diffusion rates Frontiers in Microbiology, 5 DOI: 10.3389/fmicb.2014.00169

Amazon Mycorenewal Project launches campaign to clean up world’s largest oil spill with microbes

Standard

Deep in the heart of the Ecuadorian Amazon lies one of the worst environmental disasters in human history. Over the past several decades, oil companies have discharged more than 18 billion gallons of petroleum contaminated wastewater into the Sucumbíos region in northeastern Ecuador. The contamination, which spans a geographic region roughly the size of Rhode Island, is described by the media as “Amazonian Chernobyl.”

Oil drilling in Lago Agrio, one of the many oil-contaminated sites in Sucombios, Ecuador

Oil drilling in Lago Agrio, one of the many oil-contaminated sites in Sucombios, Ecuador

Three years ago, an Ecuadorian court ruled that Chevron should pay $18 billion US dollars of damages for the pollution in Sucumbíos. However, the oil giant has so far refused to pay remediation costs, and a bitter legal battle continues to rage.

Now a grassroots effort known as the Amazon Mycorenewal Project (AMP) hopes to take remediation into their own hands- and to the people of Sucumbíos. The scientists, international volunteers and Ecuadorians driving the AMP believe the key to remediation lies in microorganisms thriving in petroleum-contaminated soils. Today, the AMP launched an indiegogo campaign to raise money for research that will determine whether naturally occurring bacteria, fungi and plants can be used to degrade the toxic petro-waste that has plagued the region.

Bioremediation, or using microbes to clean up our environmental messes, is not a new concept. Many microorganisms have been deployed across the world to degrade a range of environmental pollutants, including PCBs, gasoline, radioactive waste and mercury. Several years ago, a group of undergraduate researchers from Yale University visited the Amazon and discovered fungi that eat polyurethane plastic, a synthetic, petroleum-derived material. The enzymes microbes use to degrade synthetics are probably used in nature to decompose lignocellulose, the stuff that makes wood “woody”.

What makes the AMP unique is their ecological approach to bioremediation. By culturing bacteria and fungi that already thrive in petroleum-contaminated environments, the AMP hopes to develop communities of bioremediators that are “naturally suited” to their habitat. Preliminary studies conducted over the last several years show that local fungi, grown in the lab on petroleum-enriched substrates, develop increased resistance to petroleum toxicity.

Cultures of "petrophiles", wild fungi isolated from petroleum contaminated soils in the Ecuadorian Amazon

Cultures of “petrophiles”, wild fungi isolated from petroleum contaminated soils in the Ecuadorian Amazon

The other unique aspect of AMP’s approach is the organization’s integration with local communities. Mia Maltz, a fungal ecologist and PhD student at the University of California, Irvine, has been working with the AMP since 2007. Over the last seven years Mia and other members of the AMP have taught mushroom cultivation and mycoremediation (bioremediation using fungi) techniques to locals. By designing and teaching low-cost bioremediation strategies, AMP is empowering locals to do bioremediation on their own.

 “A lot of what we’re doing is very low-tech, easy for local people to do” says Mia. “That’s really the goal here. We want things to be inexpensive and cost-effective. The science here is very simple-  encouraging the natural process of lignocellulose decomposition. We want to show people the simplicity of what’s happening, help them internalize it, so they can tap into a process that’s a huge part of the web of life.”

This summer, a team of scientists and volunteers will work in Sucumbíos to identify and cultivate microorganisms capable of degrading different hydrocarbons present in petroleum. In collaboration with scientists in the United States, the AMP hopes to use metagenomic analyses to profile entire microbial communities and document key microbial taxa and genes involved in petroleum degradation.

AMP volunteer searching for petro-tolerant fungi in contaminated sites

AMP volunteer searching for petro-tolerant fungi in contaminated sites

Ultimately, AMP aims to develop inexpensive biofiltration systems. Biofiltration is the process of filtering contaminated water through living organisms (microbes and plants) which detoxify and remove pollutants. The AMP envisions a biofiltration system consisting of several chambers containing different assemblages of petro-degrading bacteria and fungi. After microbial filtration, the wastewater will be fed to plants that can tolerate high concentrations of heavy metals and other residual contaminants.

AMP volunteers are currently developing prototype biofiltration systems. “We want these to be modular and flexible” says Mia. “We plan on licensing everything in the creative commons to make our technology accessible to local communities around the world.”

The Amazon Mycorenewal Project brings together a broad range of expertise to tackle this enormous pollution challenge. Co-founder Bob Rawson is president of two bioremediation companies (International Wastewater Solutions and the Pseudonym Corporation) and has over 36 years of experience cleaning up contaminated soils. Joanna Zlotnik, program director for AMP, has worked as an environmental geologist for ten years, conducting site assessments and doing remediation work. Collaborator Tradd Cotter is the founder and director of Mushroom Mountain LLC. Tradd brings over 22 years of experience in commercial and experimental mushroom cultivation and mycoremediation research.

Check out the Amazon Mycorenewal Project indiegogo campaign and make a contribution here.

Powering our future with electrigenic bacteria

Standard

Trillions of microbes live on us, in us, and around us, quietly sharing our bodies and our cities. But in the future, some microbes may have to work for their real estate. In fact, they may quite literally become the generators that power our lives. How? By putting them to work inside electricity-generating fuel cells.

The concept of using microbes to generate electricity, otherwise known as a microbial fuel cell, has been around for decades. In essence, a microbial fuel cell is a system that converts chemical energy into electrical energy by taking advantage of the natural oxidation (i.e., electron-release) that occurs as microbes digest organic matter. In this fuel cell, electrons released from microbial digestion are transferred to an electrode. As electrons travel along a charge gradient, they pass through an external electrical connection that harvests some of their energy in a battery or resistor.

Schematic of a microbial fuel cell, in which bacteria connected to an anode digest organic matter, and release electrons that travel to a cathode, generating electric current.

Schematic of a microbial fuel cell, in which bacteria connected to an anode digest organic matter, and release electrons that travel to a cathode, generating electric current. Credit: The Biodesign Institute, Arizona State University.

Scientists have envisioned many ways in which microbial electricity may one day power our lives, from small household electronics to automobiles to self feeding robots. The reality of the matter, however, is that the technology is not yet developed enough to produce substantial quantities of power in a cost-effective way. Most fuel cells today are so large that they can’t fit inside the electronics they are intended to power.

In working to improve microbial fuel cell technology, a lot of effort has been focused on electrochemical engineering. Many scientists are working to improve the efficiency of the electrode: making it better at grabbing and transferring electrons. However, equally important to the development of effective microbial fuel cells is understanding the microorganisms that power them.

In this aspect, one discovery in particular has given scientists hope: electrigens. These are organisms that can harvest energy by directly growing on electrodes. (This is in contrast to the many other organisms that have been scouted for fuel cell application, most of which are simply going about their business digesting organic matter, unaware that some of their precious electrons are being siphoned away to a battery). The most well-studied of the electrigens is Geobacter, an iron-breathing organism that lives in oxygen-free environments. Several species of Geobacter use electrically conductive pili (small antenna-like appendages) to transfer electrons from organic matter directly to iron oxides in the environment. In essence, the process by which Geobacter acquires energy can be co-opted for electricity generation by replacing iron oxides, which occur naturally in soils, with an electrode.

Geobacter coating iron oxide minerals. Credit: Wikipedia

Geobacter coating iron oxide minerals. Credit: Wikipedia

Electrigens represent a promising step towards the development of a sustainable fuel cell, one that can generate a high enough power outputs to be useful. That these bugs naturally thrive on electrodes cuts out one of the largest hurdles associated with microbial fuel cell development. One of the earliest examples of an electrigen-powered fuel cell is the Benthic Unattended Generator (BUG). BUGs live at the bottom of the ocean, producing electric current from organic matter. Their design includes a piece of graphite buried in oxygen-free sediments which serves as an anode. Electrons are collected on this anode when microbes break down sedimentary organic matter. Electrons are transferred to another piece of graphite (the cathode) sitting in the overlying water.

A sediment microbial fuel cell. Credit: Lovely 2006, Nature.

A sediment microbial fuel cell. Credit: Lovely 2006, Nature.

BUGs may one day be used to power monitoring devices and other electronic equipment at the bottom of the ocean. Using a similar concept, scientists have speculated on the possibility of generating electricity from oil spill remediation. Microbes munching away on oil liberate oodles of electrons. Perhaps some of these electrons could be used to power, say, the equipment needed for oil spill remediation.

My personal favorite future use of a fuel cell? Compost generators. Imagine, if you will, an in-home compost bucket that doubles as a generator, allowing you to literally extract power from your trash. That’s a future I’m ready for.

ResearchBlogging.org

Lovley, D. (2006). Bug juice: harvesting electricity with microorganisms Nature Reviews Microbiology, 4 (7), 497-508 DOI: 10.1038/nrmicro1442

Resurrecting ancient microbes to understand evolution

Standard
The ever-growing popularity of the zombie apocalypse is testament to our fascination with the idea of resurrection, or the reanimation of the dead. Credit: Robert D. Brown Inc.

The ever-growing popularity of the zombie apocalypse is testament to our fascination with the idea of resurrection, or the reanimation of the dead. Credit: Robert D. Brown Inc.

When you hear the word “resurrection”, what’s the first thing that comes to mind? Religious miracles? Zombie viruses? The end of the world?

Whatever your mental association, I’m willing to bet it’s not “an emerging scientific discipline.” Well, it just so happens a growing community of microbial ecologists are developing a new use for the traditionally mysticism-affiliated word. These scientists don’t just study modern microbes. Nor do they fashion themselves paleobiologists. No, these people actually straddle the boundary between life and death. They resurrect. And in doing so, they hope to gain insight into how evolution occurs: from single genes to entire communities.

I’ll admit Carl Zimmer beat me to this one , but the topic of resurrection ecology is too interesting and relevant for me to ignore. Zimmer’s article provides an excellent summary of many recent discoveries in the field- including the revival of a 1,500 year old Antarctic moss and the resurrection of a 30,000 year old virus frozen in Siberian permafrost. In previous posts, I’ve discussed both microbial resuscitation from ancient ice  as well as mechanisms some bacteria use to stay alive at subzero temperatures.

Today I’d like to dive a little deeper and really dissect what it is these resurrection ecologists are trying to do. Why resurrect an 8 million year old bacteria, or hatch 700 year old insect larvae? Other than the “cool” factor, is there any profound ecological or evolutionary insight to be gained from this research?

I’ll give you the punchline now: yes, there most certainly is. You see, resurrection of organisms from the past, a feat that was until quite recently considered impossible, offers scientists the opportunity to directly test hypotheses about why evolution occurs.

As an example, imagine a community of bacteria, happily swimming about in a shallow pool of water sitting on top of Siberian permafrost couple thousand years ago. One particularly cold winter, this water freezes and some of these bacteria get trapped in the ice. Perhaps it remains cold enough that the ice doesn’t melt again come spring. Over time, this ice, along with some hapless bacteria, get buried and compacted, frozen in space. But also frozen in time. While some of these guys may  have escaped cryosleep, continuing to grow, reproduce, and die, frozen bacteria don’t change. And because they remain static, evolution also freezes. No reproduction means no gene swapping, no adapting to new environmental pressures, no natural selection. Beyond their ice fortress, genetic cousins of the frozen bacteria are spreading their genes, mutating their DNA. Populations are growing, shrinking and migrating in response to their environment. They are evolving.

Fast forward to the present. Suppose you’re an ecologist studying the bacterial communities of a Siberian lake. You discover that a rare gene known as “cocA” is widespread. What sorts of selective pressures may have caused cocA to become so common? By collecting samples from the surrounding permafrost, you find traces of bacteria frozen in the ice. You extract and sequence their DNA, and determine, to your delight, these frozen organisms are a close genetic match to some of your lake bacteria. Similar, but different enough to infer that some evolution has occurred. Moreover, in all of your frozen samples, a different flavor of the cocA gene- let’s call it “colA” – is dominant.

Here’s where the resurrection part comes in. You’d really like to know how your bacterial communities shifted from colA-dominated to cocA-dominated. To do so, you try growing some of your frozen bacteria in the lab. You give them heat, carbon, and nutrients. Soon, you’ve got fossil bacteria happily multiplying on your lab bench. You have resurrected life.

 More importantly, you’ve got yourself a situation most paleontologists would kill for. You now have living replica of ancient organisms, and their  modern descendants. You set out to conduct a series of lab experiments in which you vary different environmental conditions- pH, nutrient concentrations, temperature- and monitor how your fossil bacteria change. Specifically, you track whether and how your gene of interest (colA) changes over time. What you are really doing is experimentally simulating evolution on an ancient organism. Ultimately, you might be able to determine what sorts of environmental changes must have occurred to lead to the modern, cocA-dominated descendants.

The concept of resurrection ecology is a powerful one. In no other subfield of biology can scientists directly study the ecology or evolution of an organism from a different time. Normally, scientists have to rely on fossils and other “proxies” to make inferences about past species, their ecology, and ultimately, the environment. Imagine what we might learn if we could resurrect a family of dinosaurs and tinker in the lab until we figured out what sorts of environmental conditions lead to the evolution of feathers.

Obviously this is a preposterous idea. Safety considerations aside (we all know how Jurassic Park turned out), dinosaurs reproduce too slowly to observe evolutionary change on useful timescales. Moreover their ranges are too large, and their species interactions too complex, to accurately simulate their habitats.

Microbes are a different story. They require only a petri dish to grow. They reproduce in minutes to hours. Changes in the genetic make-up of microbial communities can be observed within days or weeks. Antibiotic and pesticide resistance are powerful testimonial to the swift pace of microbial evolution.

Resurrection ecology thus represents a compelling new tool for understanding how genes, populations and communities evolve. But its significance may not be limited to the past. By informing population models with “biological archives”, scientists may be able to forecast adaption to future environmental changes. In other words, by helping scientists develop a deeper understanding of how evolution works, resurrection ecology may inform our expectations for the future, and allow us to prepare for it.

*******

A quick update: National Geographic’s cover story last week was on reviving extinct species! Check it out here.

ResearchBlogging.org

Orsini, L., Schwenk, K., De Meester, L., Colbourne, J., Pfrender, M., & Weider, L. (2013). The evolutionary time machine: using dormant propagules to forecast how populations can adapt to changing environments Trends in Ecology & Evolution, 28 (5), 274-282 DOI: 10.1016/j.tree.2013.01.009

Fossil viruses preserved in hot spring bacteria

Standard
Viruses, such as the Avian flu virus depicted in this scanning electron microscope image, are neither alive nor quite dead, but may have a lot to tell us about the evolution of life on Earth. Credit: 3DScience.com

Viruses, such as the Avian flu virus depicted in this scanning electron microscope image, are neither alive nor quite dead, but may have a lot to tell us about the evolution of life on Earth. Credit: 3DScience.com

Fossilized microbes have provided scientists many clues about origins of life. By comparison, little attention is given to viruses in the fossil record. Although technically non-living, there is no question these tiny packets of protein-sheathed DNA have shaped the evolution of most life on earth, including humans. But can viral particles, a fractional size of even the smallest bacteria, actually become fossils? A study recently published in the journal Geobiology argues they can. Here, scientists present the first experimental evidence of silicification– the encasement in silicate minerals- of viruses living in hot spring biofilms. Moreover, these viruses can become fossils while still inside their host cells.

Before we get into viral fossilization, let’s back up for a minute and briefly review what a virus is. Viruses are simple beings that strip the notion of life down to a single, fundamental purpose: propagation of genetic code. While sharing this feature with all life on Earth, viruses are not considered living because they lack the means to carry out genetic reproduction on their own. Instead, viruses propagate by injecting their genetic material into a host cell. One inside a host, a virus uses one of two strategies, or “life cycles”, to reproduce. In the lysogenic cycle, viral DNA integrates with host DNA, lying low and allowing itself to be replicated along with the oblivious host cell. In our own chromosomes, many introns, or non-coding pieces of “junk DNA”, are thought to be ancient viral particles that “went to sleep” inside our cells and never woke up. In the more active (and deadly) lytic cycle, the virus co-opts the host cell’s replication machinery, creating a virus factory that eventually spews forth thousands of baby viruses which proceed to infect other cells.

Conceptual model of the two general modes of viral biology: lytic and lysogenic cycles. Credit: allbiologytutors.blogspot.com

Conceptual model of the two general modes of viral biology: lytic and lysogenic cycles. Credit: allbiologytutors.blogspot.com

Which of these cycles a virus chooses depends a lot on the external environment. If conditions are good in the host cell, it may be advantageous to hunker down and stay put. If, on the other hand, the host cell is already sick or dying, the virus may prefer to replicate as much as possible before abandoning ship.

Hot springs host communities of highly specialized extremeophiles. These bugs are not only hyperthermophilic, or super heat-tolerating, but often cope with extremely acid or alkali conditions. One way they do so is by modifying the pH within their cells, making it closer to neutral. Perhaps unsurprisingly, most hot spring viruses follow a lysogenic cycle, enjoying the less-extreme environment offered by their host.

It was in one such strange habitat, the Gumingquan hot spring in the Yunnan province of southern China, that Dr. Brian Jones and colleagues decided to investigate viral silicification. Why study fossilization in a geothermal hot spring? Geothermal waters, heated by subsurface magma from the Earth’s mantle, are often rich in silica and other minerals, such as calcium and iron, that promote fossilization. Moreover, thick, diverse biofilms hosting “virus-like” particles are known to inhabit the Gumingquan hot spring.

For their study, the researchers collected samples of these biofilms and brought them back to the lab. From biofilms containing many different Bacteria and Archaea, the scientists isolated Geobacillus lituanicus, a thermophilic, aerobic (oxygen-breathing) bacteria belonging to the phylum Firmicutes. They also isolated viral particles by liquifying samples of biofilm, separating out and removing all cells, and filtering the remaining liquid through a 0.22 µm filter- large enough to include viruses but small enough to remove most other microbial debris. Spherical, “virus-like” particles were further isolated and purified by infecting G.lituanicus cells with “viral extract”, and growing the infected cells on agar.

Once the researchers had purified both a host and virus-like particles, they set up a rather clever experiment to test for viral fossilization. They concocted a growth media for G. lituanicus containing sodium metasilicate, a silica-rich mineral. After adding G.lituanicus cells and virus-like particles to this media, they incubated the mixture for 22 days. At regular intervals, the scientists took samples and used transmission electron microscopy (TEM) to search for silicified viruses.

Using TEM, the scientists found numerous silica nanoparticles both inside and outside G. lituanicus cells. These circular particles consist of a core roughly 35-60 nanometers (nm) in diameter, surrounded by an outer shell approximately 30 nm thick. While these particles may just be small, silicified fragments of cellular detritus, there is good reason to think many of them are fossil viruses. For one, the fossil “cores” are similar in size and shape to the spherical virus-like particles used in the experiment. The silica nanoparticles had a “near normal”, or bell-curve, size distribution, another hallmark of biology. Finally, some of the silica nanoparticles had tails.

TEM images showing silica nanoparticles inside and outside cells. Note the light colored "virus-like cores" clearly visible in panels c and d, surrounded by a silicious shell. Credit: Peng et al. 2013

TEM images showing silica nanoparticles inside and outside cells. Note the light colored “virus-like cores” clearly visible in panels c and d, surrounded by a silicious shell. Credit: Peng et al. 2013

A few of you might be scratching your heads at this point, thinking something seems amiss. Remember, I told you most hot spring viruses are lysogenic (integrated into host DNA) most of the time. But in order for a virus to fossilize, it must have its own, independent structure. In other words, it must be lytic.

Does the discovery of fossil viruses within host cells negate the idea that hot spring viruses are typically dormant? Not necessarily. Actually, it may offer an explanation for when and why viral dormancy occurs. You see, all cells eventually die due to accumulated environmental stress. When a hot spring bacteria like G. lituanicus dies, its cell wall breaks open, causing hot, mineral-rich geothermal fluids to rush in. Suddenly, any viruses inside are exposed to all sorts of elements- heat, acid, etc.- they can’t necessarily tolerate. By precipitating a silica shell, these viruses may be able to survive long enough to find a new host.

Some of the proteins on a virus’s outer surface contain chemically-reactive molecules, such as carboxyl groups, that may aid in silica encrustation. The host cell itself may inadventently promote viral fossilization. Many alkaliphiles, like those in the alkaline Gumingquan waters, maintain their cellular pH at two or more units lower than the external environment. Geothermal waters that are undersaturated with silica at pH 12 may become supersaturated at pH 9 or 10, causing silicates to precipitate out as solids.

The strong evidence for viral silicification presented in this experiment suggests viruses may be preserved in the fossil record, particularly in rocks associated with alkaline geothermal systems. Some of the features of virus-like fossils noted in this study- distinct cores and shells, fossil “tails”, a concentration of fossil-like particles inside a host cell- may be useful for identifying the footprints of ancient viruses in the wild.

ResearchBlogging.org

Peng, X., Xu, H., Jones, B., Chen, S., & Zhou, H. (2013). Silicified virus-like nanoparticles in an extreme thermal environment: implications for the preservation of viruses in the geological record Geobiology DOI: 10.1111/gbi.12052