Scientists brave ‘world’s worst water’ to watch wild bacteria evolving

By Ed Yong | April 26, 2012 4:00 pm

For almost a decade, Jillian Banfield has been travelling to a place that “pushes the limits of human endurance” – Richmond Mine in Northern California. Its abandoned caverns can reach 48 degrees Celsius and 100 per cent humidity. They are low in oxygen. They contain possibly the most acidic naturally occurring water on Earth, with a pH value of -3.6.

But even in these conditions, there is life. Bacteria grow within the cave, floating in thin films on top of its hot, acidic water. They are the lords of their extreme world, and they provide an unrivalled opportunity to study how wild microbes evolve.

The mine ecosystem is extremely simple. The dominant species is a bacterium called Leptospirillum that lives in sulphuric acid and eats iron. Only a handful of other microbes share the mine, and most migrants would simply die. This is an ideal community for keen scientists – it’s small, well-defined, not very diverse, and self-contained. “The fact that it’s a simple, closed community makes it feasible to observe the evolution of the main players, without worrying about genotypes coming in from outside,” says Richard Lenski from Michigan State University, who was not involved in the study.

The microbes are the reason why Banfield has repeatedly braved the toxic mine since 1995, recently joined by colleague Vincent Denef. To work in such harsh conditions, they need protective clothing. In the most oxygen-deprived regions, they can only work for a few minutes at a time. Even so, they have visited the mine several times a year and collected samples of Leptospirillum from several different sites. Now, they have analysed the DNA of their samples to chart the bacterium’s evolution.

On average, the bacteria accrued 1.4 mutations in every billion DNA letters, each generation. That’s near the top end of what people estimated based on lab experiments. “All current estimates stem from organisms reared in the laboratory and there is uncertainty whether change occurs at the same rate in the wild. This study shows that similar rates apply,” says Martin Polz from MIT.

The Leptospirillum strains that dominate the mine come in six different genetically distinct ‘genotypes’, numbered I to VI. Each type shares around 94 per cent of their DNA with the others – for comparison, we share 96 per cent of our DNA with chimps.

Types II to VI are all branches of the same dynasty, which has lived in the mine for at least 50,000 years. Type I is a more recent arrival. Since the 1960s, it has repeatedly swapped genes and fused with the early colonisers to create the other five types. The first of these fusions happened in the late 60s and produced the Type VI bacteria. Two more fusions in the 1980s produced types IV and V, and another one about ten years ago produced type III. This is the one that dominates the mine today.

Each of these events followed the same pattern. A late-colonising Type I microbe mingled with an early coloniser and transferred some of its genes across (which is typical for bacteria).  In the recipient cell, both sets of DNA merged to create a new type. In most cases, the descendants of that new hybrid then rose in numbers, until it dominated the mine.

It’s not clear why each emergent hybrid managed to establish itself. It could be due to dumb luck. The Richmond mine environment changes from season to season. For example, an influx of water during the rainy season might have washed away some groups at random, and given others a competitive advantage.

But Denef and Banfield found some telltale signs of natural selection at work in the bacterial genes. For example, the changes that accompanied the emergence of new types were unusually likely to affect control genes, which affect how other genes are used and activated. This suggests that the new types flourished because their hybrid genomes made them better-adapted to their environments.

It’s even possible that humans were involved. We were still using Richmond mine until the 1990s, and more recent decades have seen extensive efforts at cleaning up its toxic waters. We could have changed the mine’s environments in ways that favoured some Leptospirillum lineages over others. However, as Banfield says, “We cannot directly connect specific historical events to specific evolutionary steps.”

Denef and Banfield’s study shows just how quickly wild bacteria can evolve. By fusing their genomes together, they can diverge greatly in just a matter of years, and rapidly adapt to environmental changes. In a related editorial, Edward DeLong from MIT puts it beautifully: “It turns out that “genome” is a verb, not a noun—a process, not a product.”

Other scientists have done something similar, albeit without descending into a toxic cave to do so. Since 1988, Richard Lenski has been growing 12 populations of bacteria in his lab, in constantly a refreshed set of 12 flasks. They’re now past their 55,000th generation, and they’ve already told us much about the nature of evolution. And last year, Tami Lieberman and Jean-Baptiste Michel showed how a bacterium called Burkholderia has evolved while infecting the same sets of lungs over 20 years.

Lenski says that these studies show “the tremendous value of collecting and preserving time-series of microbial samples.” Rather than capturing a mere stills of bacterial diversity, these samples can show us the entire movie of life evolving under our noses.

Reference: Denef & Banfield. 2012. In Situ Evolutionary Rate Measurements Show Ecological Success of Recently Emerged Bacterial Hybrids. Science

Photo by Mitch Jones

More on bacterial evolution

CATEGORIZED UNDER: Bacteria, Evolution, Select

Comments (20)

  1. How is a pH of -3.6 possible? By my understanding that would require a solution of 4000M H+ (10^-3.6), which would mean 4 kg of protons in 1 kg of water. Where has my logic gone wrong? I guess temperature or the activity of the ions must be having some additional effect.

  2. Can you clarify what pH -3.6 means? My understanding is that pH is the negative log of the H+ concentration. IANAC (I am not a chemist) but I don’t see how that can be a negative number.

  3. Terry

    I second that comment. What is a “minus pH?”

  4. Jon

    Rosie–all it would take is for the concentration to be > 1. That would make the -log(concentation) negative. Wikipedia claims that the concentration used for pH calculations is molarity ( So if there is more than 1 mole of H+ per liter of water, the molarity will be greater than 1. The log of something greater than 1 is gonna be positive, so the negative log of something greater than 1 is negative. Negative pH.

  5. Do notice, however, that none of these bacteria evolved into…I don’t know…some random higher crown-group amniote. Humans. I can’t make this joke anymore. It’s just so depressing.

  6. Jeramia Ory

    You are correct, Rosie. A pH of -3.6 is mathematically possible, but not chemically possible. It would mean a H+ concentration of 3900 molar. Pure water is 55 molar water. I’m guessing what they mean is if you try to measure the pH with a pH meter, it says -3.6.

  7. Marmauke

    It just means the concentration of H ions is greater than 1. In this case (ignoring the subtleties of pH), over 10^3 mol

  8. Marmauke

    … 10^3 mol / L.

  9. Brian

    pH 0 = 1M [H+]
    pH -1 = 10M [H+]
    pH -2 = 100M [H+]
    pH -3 = 1000M [H+]
    pH -3.6 = 4000M [H+]
    … and so on.

    There are obviously not 4000 moles of H+ in a liter of water, given that there’s only 55 moles of water in a liter of water. I suspect what they mean here is that the activity, not the analytic concentration, is a pH of -3.6. That means that the effect of H+ in this solution is /as if/ there were an analytic concentration of 4000M H+.

  10. Okay, sorry, I got the pH value from a different report, and really should have checked it myself. I’ve taken it off until I can do that.

  11. MadGenius

    TIL that pH can have a negative value. This might answer Rosie Redfield’s question

    (I’m not a chemist either)

  12. Dan Milton

    The bacteria may have been an isolated underground community for 50,000 years, but they can only have been in the mine since it was opened in the 1860’s.

  13. Jeramia Ory

    I’m guessing they used the methods in this paper to come up with -3.6. Brian’s right, we routinely use [H+] for pH calculation, but it is actually dependent on the activity, not the concentration. At low concentrations and low ionic strength, is is an acceptable approximation. At high ionic strength, it isn’t. This is why I call myself a biochemist, and not a chemist.

  14. Okay, the pH value is back. The source is this paper:

  15. Jeramia Ory

    From the PNAS article:

    “The reporting of negative pH values has been controversial, and for several good reasons.”


    “There is no generally accepted procedure for defining individual ion activity coefficients without some arbitrary assumptions.”

    I would add that it causes a great deal of confusion with the general public who have any recollection of their grade school chemistry. It seems sensationalist (the original usage, not yours) with the potential to distance the public from chemistry further.

  16. I never doubted you Ed. Thanks for the link to the PNAS paper. I knew negative pH was possible, just not so negative. It seems pH is more complex than I realised.

  17. I’m wondering (a bit superficially, admittedly) if pH meters actually *work* with such contaminated water (200 g/L metals and 760g/L sulfate!?!). I don’t dispute the idea of negative pH (I get the math) but given the chemical constraints (pH -3.6 = 4000 M H+), as pointed out above, I can’t get my head round what it means.

    I ‘m guessing there may pH meters that may be specifically designed for very extreme pH values (a bog-standard lab instrument is probably designed to operate from pH 1-10). Does anyone know?

  18. Chris

    As a chemist I must clear up some misconceptions about pH. The normal way it is presented in gen chem is pH = -log[H+]. This is a good approximation for normal lab acid concentrations. However at very high concentrations the activity of the H+, not the concentration of [H+] becomes important.

    pH = -log (activity of H+)

    (activity of H+) = (activity coefficient)*[H+]

    Think of working in the lab. If you are a dilute solution, you work very efficiently so you have an activity coefficient near one. Now in a concentrated solution you are constantly bumping into other people, waiting for chemicals or to use the scale, so your activity coefficient drops below one. You could also divvy up the work and make you more efficient so your activity coefficient could be greater than one.

    Additionally there is a temperature dependence with pH, so at these higher temps, the water acts more acidic. Neutral goes from pH=7 to pH=6.6 at 50 C. Not huge compared to the actual pH but still important.

    For very concentrated acids, they tend to have activity coefficients greater than one, giving pH much lower than would be expected. So yes while seeing negative pH values is not common and always worth a double check, it is not impossible.

  19. Chris

    @18 Chris (addendum)

    I should also add that the activity of the H+ is not just dependent on the [H+], but also the concentration of other ions in solution. And with concentrations of several molar, this is a big effect. At low concentrations the Debye–Hückel equation is used, but at these high concentrations, higher order approximations are needed.

  20. Mike McD

    Pity the comments has devolved in to what pH is or is not possible instead of discussing other more prevalent (IMO) thoughts such as: density dependant controls on population size; resource partitioning and competition by the different species of bacteria.

    I can fully understand the reasons for the pH debate, just think that the above I mention are far more interesting topics.


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