Tuesday Tool caseous microtome edition

I think the tool today is...

The cheese plane.

Six months ago, after milking the goaties, I took the twelve liters of milk into the kitchen and got it to about 30 degrees C.  I inoculated it with a mixture of mesophilic bacteria--some specialized in fermenting lactose to short-chain fatty acids (SCFAs), some specialized in fermenting lactose into SCFAs but also some interesting ketones and aldehydes and whatnot.  After letting them do their thing for an hour, I added some coagulant--a protease from a fungus that has the same specificity as the major component of rennet.  Over the course of about an hour, the combination of acid and coagulant curdled the milk.

I cut the curd up into chunks about a centimeter on a side, and slowly warmed the curds and whey up to 39C.  After about an hour, I removed half of the whey and replaced it with cool water, bringing the temperature down to the mid-20's and removing much of the lactose from the mix.  A half hour later, I removed all the whey and put the curds into a form, and pressed them with ever-increasing pressure for about ten hours.  The curds mostly stuck together, but preserved some of their individuality.

I removed the nascent cheese from form the next day, and immersed it in a saturated brine solution, and let it sit there for twelve hours.  As far as salt is concerned, cheese is a liquid, and it dissolved into the cheese.

Then, I let the cheese dry out for a bit, and then put it into a wine 'fridge in my basement, set to about 12 C.  And there it sat; I'd turn it every so often, and brush off the mold.  The exterior turned a golden yellow color, and turned hard and tough and horny.  The interior lost some moisture, becoming quite firm.  The curds, having been "washed," did not stick together as completely as they might in other cheese processes, leaving a few gaps or "mechanical spaces" in the cheesemaker's parlance. As it aged, organic compounds in the cheese became further oxidized and recombined; though the bacteria were mostly dead, their enzymes lived after them and continued their work.

Today, I dug the cheese out of the basement, brushed off a bit of mould, halved the 2 kg wheel with a cleaver, and then cut one of the halves in half.  I shaved off a bit with the cleaver and tasted it--it was very, very good.  The taste had elements of a tangy cheddar, elements of Parmesan, a little carmelized sweetness, a pleasant salty buttery-ness, and a firm texture.  What one should do with such a cheese is serve it after a nice dinner with some fino sherry, some membrillo or raisins and almonds...and cut it with a cheese plane.

Tomorrow I will go out and thank the goaties for their miraculous ability to eat spring grass and alfalfa hay and water and turn it into such amazing milk.

Use and misuse

National conventions are terrific ways to spread diseases.  People jetting in from all over, immune systems weakened by the stress of travel, confined in small rooms, mixing and mingling in shared facilities, then jetting back to wherever.  It would be hard to design a better system to distribute pathogens.

Goats have their own conventions, and the same rules obtain.  Mostly, goat shows are small affairs, a couple of hundred goats from a dozen counties, and the risks are slight.  However, the Real Doctor recently took five of our animals to the national show of the American Dairy Goat Association.  The goats spent four hours in a truck, then five days in six-by-four pens in a barn with hundreds of other goats, then another four hours to get back home.  While at the show, they were in the competition, which meant walking around in an arena used by zillions of other goats from all over the U.S.A. 

Our beasties did better than we had expected--nobody won their classes, but they all made the first cut and finished between 15th and 10th in their classes, and got some nice notices.  So nobody got ribbons, but they did (despite a lot of effort and sanitizing) get some ickies. 

By the last day, Opera had developed a fever and was looking pretty listless.  Some banamine (works in goats like acetomeniphen in us) perked her up, and a course of ceftiofur (a cephalosporin antibiotic) has left her right as rain.  Helen had a slightly elevated temperature too, which was also treated.  But, in the two weeks since the show, a cough has spread through our kids, one at a time, and it may be correlated with a couple of the young kids getting the poops.  Right now, Helen is back on ceftiofur after developing another fever, and Cavatina is finally coming off of a fever that spiked at 107.4 and has had her miserable for four days.  So, hurrah! for ceftiofur and banamine and their judicious use.

So far, ceftiofur works.  It works against Gram-positives and -negatives, and penecillin-resistant bugs.  But for how much longer?  Every dose of antibiotic, especially if I am not careful with it, is an additional bit of selection pressure that will favor the spread of antibiotic resistance genes in the environment.   (Genes, not bacteria.  First, genes are much easier to detect, since they don't need to be cultured.  Second, the bacterial predisposition to promiscuous lateral gene transfer means that once a gene is out there, it will get around.)

So I read in Science a charming article about the hog farming and spread of antibiotic resistance genes in waterways in China.  Antibiotics are a wonderful tool for rescuing sick animals, but it wasn't long after they were introduced that somebody noticed that you can just give your young animals lots of antibiotics all the time and they will grow faster.  Never mind that they will excrete most of the antibiotic in their urine, and it will go out into the environment and give that selective nudge to the genes that will make antibiotics useless, profit is to be had!

Needless to say, hog farming in China is epic in its scale.  So is the abuse of antibiotics.  About half of the antibiotics produced in China are used to fatten up the little piggies.  How much is that?  About 100,000 tons. 

Since I am human, I have a hard time visualizing numbers over 100 or 1000.  So, a ton of water is a cubic meter.  We'll assume that the density of dry antibiotic is the same as water, which is true to an order of magnitude.  So, I am picturing a mound of antibiotic, as long and as wide as a football field, and as tall as the tips of the field goal posts.  Or maybe a cube, half a football field on each side.  Now that is some selection pressure.

(Question for those interested--how big a mound of antibiotics is used in American meat production?  Answer here, if you highlight it:  Ten thousand tons.   Picture a cube about twenty meters on a side.  Still some selection pressure.) 

Sometimes, I feel like scientists giving humanity antibiotics was like giving a hormone-raddled teenager the keys to a Ferrari.  Are we really mature enough to not wreck ourselves?

Friday Flora Fuming Fewments Edition

This week's flora are an anonymous consortium of thermophilic anaerobic microbes.  They are not much to look at, so no picture, and until such time as the internet can transmit odor, I can't really convey their impact.

In the African nation of Gabon, there is a town called Oklo that stands atop some rich deposits of uranium.  Time and the geology of the area created a circumstance that has been observed only once.  Some billions of years ago, the concentration of fissionable uranium in these deposits was high enough that they could form a natural nuclear reactor.  By itself, though, the uranium was not sufficient.  The spontaneous fission of a U235 nucleus releases a neutron too energetic to trigger the fission of another U235 nucleus.  As in any human-made reactor, a "moderator" is required to slow the neutrons, and as in many human-made reactors, water served this role.  At Oklo--and as far as we know, only at Oklo--groundwater flowed through the uranium deposits, slowing the neutrons, allowing a chain reaction, which then released energy.

However, this release of energy, as heat, caused the groundwater to boil.  The reactor effectively killed itself--until it could cool down, and water flowed into it, bringing it back to life.  Some modeling and experimentation suggests that the reactor would be "on" for about a half hour, and "off" for two and a half, and that this cycle lasted for thousands of years.

...........................................................

Today, tucked away in a corner of our main pasture, there stands a majestic compost pile.  I'm kind of embarrassed by it; it is in a poor location, on soil rather than concrete, and it is not covered.  It reminds me of the Oklo reactor because of its relationship to water.  It has been accumulating raw material all summer, and with each new dose--which carried with it a deal of moisture--it would ferment, steam, fume and stink for a few days.  It would cook off all the freshly added water, and the reactions would slow down and stop.  After a light rain earlier this summer, it again seethed for a bit, and died.

The reactions, of course, are not nuclear.  There's a lot of fermentation, to be sure--the sheep's manure and used bedding contains lots of reduced carbon.  Where there's fermentation and anaerobiosis, there's going to be methanogenesis and acetogenesis, forms of respiration using carbon dioxide in the place of oxygen.  There's also respiration going on using sulfate in place of oxygen; the stink of hydrogen sulfide attests to that fact.  All these reactions produce energy for the microbes carrying them out.  The second law of thermodynamics tells us that the universe can't allow any reaction to happen without a tax being paid to entropy, so a sizeable chunk of the energy from these reactions is lost as heat--heat that makes the compost warm to the touch, and that has dried the pile out every time it has been wetted.

Now we've just had a good rainfall, thoroughly soaking the pile.  Hoooo-weee!  It is reacting like mad, smelling and steaming.  And so the cycle repeats, a natural reactor getting drenched with water and generating heat to turn itself off, then cooling down and starting over--though probably not for thousands of years, and while the stink might be annoying, it's not producing anything as bad as plutonium. 

About that paper on organic food...

A good biology paper will make you think about life; a few of them make you think about your own life.  A research paper came out this week that made a bit of a splash, and prompted a lot of people—a lot of non-scientists, who may not be used to thinking scientifically—about what they are doing and why.  The paper was a meta-review: essentially, an attempt to compare lots of different studies, with different methodologies, different foci, and different motivations, and reach a coherent conclusion.  The question that the authors were trying to answer is in the title of their paper: “Are Organic Foods Safer or Healthier Than Conventional Alternatives?”

The answer is very, very close to “no,” followed by some asterisks.  Some of the asterisks are whether the government approved levels of pesticides are actually safe, and that while the levels of bacterial contamination were comparable, the contaminants in conventional foods were more likely to be antibiotic resistant.  But on the whole, no difference. 

Looking at NPR and the NY Times blogs and their commenters, a few have their faith shaken by facts, but most have their faith reinforced by being liberated from facts.  They turn to attacks on the paper and its authors.  There’s also a fair amount of triumphal crowing from folks who (like my dad, a biochemist) refuse to buy organic on principle. 

So, as a person who tends to buy organic and pay attention to science, what do I think?  I think I’m going to have a heart attack and die of not surprise.  Whether produce is good and good for you or not—as long as it falls within guidelines for pesticides and other contaminants—is a matter of whether the farmer was competent and the food has gotten from farm to you in a timely and careful fashion.  So, why do I tend organic?

I’ll set aside human reasons, although conventional ag inevitably causes worker exposure to nasty chemicals, which is a good reason to favor organic.  I’ll set aside whether there is any safe level of pesticides in food, because there isn’t yet any scientific agreement on the subject.  I’ll also set aside (for now) the effects of using huge amounts of antibiotics that can leak into the environment.  Instead, I’ll (predictably) focus on microbes, since they make the world go ‘round and are more important than people in the long run. 

I love long-term studies (I mean, what is our planet but a long term environmental study?).  The Swiss government started just such a study in 1978, comparing three variations on farming for typical Swiss crops:  conventional agriculture with pesticides, herbicides, and chemical fertilizer supplemented with animal manure, organic farming with no herbicides or pesticides and only animal or plant manures, and “biodynamic” farming.   They did not address which produce is better for you, or which tastes better; they’re Swiss, so the conventional stuff was within pretty tight government regulations, and they’re scientists, so all the crops were equally fussed-over.  What they did compare was the performance of the farms as if they were factories, and the health of the farms over the course of decades.  (Just to simplify things, I’m lumping together the results of biodynamic and organic practices, since they were essentially the same.)

I’ll start with the result my dad would point out:  yields in the organic fields just weren’t as high as in the conventional fields.  Potatoes, beets, barley, wheat, it didn’t matter, yields from the organic fields rarely equaled those from the conventional fields, and were generally about 80% of the conventional yields.  This is not a trivial point in a world that’s trying to feed 7 billion people with limited amounts of cropland and ever-more-difficult access to water. 

However, there are other factors that are limiting, and energy is right up there.  Comparison of the energy inputs to get those crop outputs is illuminating.  The researchers figured out how much energy was needed for farming activities such as tilling, and added the substantial energy for making mineral fertilizers, nitrogen, pesticides, and herbicides.  Over a six-year period, they discovered that organic farming took slightly more than half as much energy per hectare, so even though yields per acre were slightly reduced, organic farming was still vastly more efficient.  (I’ll also note that about when this study was published, my dad abandoned his SUV for a Prius.)

Soil is the factory that produces food.  Clearly, organically farmed soil is a different kind of factory from conventionally farmed soil—more efficient with energy, though less efficient with space.  It’s the architecture and the workers in the factory, and how they interact and affect each other, that make the difference. 

In this study, there is a visible difference between organic and conventional soils. 

In this picture of winter wheat seedlings, the biodynamically farmed soil shows more weeds, but the soil looks friable and there are plenty of worm casts.  These differences are quantifiable; water drainage is improved, as well as the ability of the soil to cohere. 

The workers in the factory of soil are microbes and small invertebrates.   It’s not too surprising that organically and biodynamically farmed soil has a lot more life in it—the Swiss study found twice as many earthworms, spiders, and beetles, and much more root-associated fungi.  The sheer mass of microbes was higher, as was both their genetic diversity and (as has been found in similar studies) their enzymatic and metabolic diversity.  We are constantly told that a more diverse workplace is better, and at least in the work done in the soil, this seems to be the case.  To really understand this, though, we need to see what these workers do.

We might think of plants as rugged individualists, gamely taking sunlight and water and CO₂ and pulling themselves up by their own bootstraps.  In reality, they depend upon soil microbes, both bacteria and fungi, for making many (or most) of their nutrients available and delivering them to their roots.  These microbes break down the components of wood so that the elements therein can be absorbed by plants; they convert chemically inert atmospheric nitrogen into a form that the plant can absorb; symbiotic fungi called mycorrhizae, which grow both in soil and extend their threads into the cells of plant roots, capture these liberated nutrients and inject them directly into the plants.  All the players in this system, plants, fungi, and bacteria, have evolved to work with each other, and all fail to thrive in the absence of the others.  A plant is a visible expression of the health of the soil community.

The Swiss study, as well as studies on Italian rice, Dutch onions, California strawberries, and other combinations of crop and soil, have found that the diversity of the soils microbes and mycorrhizae are higher in organic soils.  (Indeed, as soils go, the champions are wild, uncultivated soils, with many different types of plants growing in them—but that’s not agriculture.)   These soils show increased ability to break down manure, an increased ability to mobilize nutrients such as nitrogen and phosphorus, and increased interaction between mycorrhizae and plant roots.     

When the Swiss researchers examined conventionally farmed soil, they found limited microbial diversity, and reduced metabolic diversity (that is, the number of different types of biological reactions occurring).  However, they found increased metabolic activity (that is, the amount of microbial nutrient consumption aimed at just making energy to live, as measured by the amount of CO₂ the microbes exhaled).   In the simplified environment of conventional soil, the microbes had to work harder to do less.  This is not a fluke; a similar observation was made in comparing organic and conventional strawberry fields in California.

This illuminates the gross productivity and efficiency differences seen between the conventional and organic systems in the Swiss study.  The conventional soils had greater yield, but (because they are less efficient factories) they required much higher inputs of material and energy.  Organically farmed soils are healthier.  Arguing for conventional farming because arable land is a scarce resource ignores the fact that, unless there is a large input of energy and skill, conventional farming can result in the degradation and loss of that same scarce resource

Of course, factories have more than one product; even the most efficient factory will produce some waste.  Even here, organic farming has some benefits, and these benefits also are a result of the more diverse and efficient microbial community in organically-farmed soils. 

The job of any factory is to convert raw materials into a mix of useful products and waste, hopefully with an emphasis on the former.  Farmers, whether organic or conventional, add raw materials to their soil factory, and they are particularly mindful of the nitrogen they add.  Organic farmers add various forms of manure for their nitrogen content—chicken or cow wastes, or composted legumes.  Conventional farmers will supplement or replace these nitrogen sources with calcium nitrate or anhydrous ammonia (as an aside—production of this fertilizer consumes upwards of 1% of the global human energy budget).  This is the raw material that enters the factory; some of the nitrogen gets incorporated into the plants, but a lot of it will disappear as waste.  And here is where there is a significant difference between conventional and organic soils, again due to their microbial composition. 

Nitrogen compounds are neat.  Most of the earth’s nitrogen is in the form of nitrogen gas (N₂) in our atmosphere; this is inert, so chemically unreactive that it is used to protect precious documents and Guiness beer.  A few microbes have learned how to “fix” this atmospheric nitrogen, to make ammonia (NH₃), which is like rocket fuel for plant growth.  Lots of soil microbes love to eat ammonia too, but rather than using it for growth, they oxidize it for energy; in the process called nitrification, they take ammonia and make it into nitrate (NO₃-).  Nitrate is a mixed blessing; plants can use it, though not nearly as well as ammonia.  Mostly it leaches out of the soil and pollutes waterways, leading to algal blooms and their resultant die-offs and dead zones.  Microbes can also take nitrate in the soil and use it for respiration the same way we use oxygen, in a process called denitrification.  Some denitrifiers convert the nitrate into nitrous oxide (N₂O), which disappears from the soil as a gas; it’s not a good thing, given that it can degrade ozone and is also, gram for gram, about 300 times more effective as a “greenhouse gas” than carbon dioxide.  Other denitrifiers use the nitrate more effectively, and convert it back to nitrogen gas. 

Either way, as a result of this nitrogen cycle, a farmer can add nitrogen to the soil and watch some of it disappear as waste; it’s just a matter of whether the added nitrogen disappears by leaching (and polluting the water) as nitrate, by going into the atmosphere as pollution in the form of nitrous oxide, or by going into the atmosphere as benign nitrogen.   Since the nitrogen cycle is largely driven by microbes, and since organic and conventional farming techniques result in different soil microbiota, it seems like a reasonable hypothesis the way nitrogen leaves the soil would differ in organic and conventional situations. 

No matter what form of agriculture, human activity dominates the addition of nitrogen to the soil.  Conventional farmers add over 80 million metric tons of ammonia to the soil every year, and organic farmers add manure.  This, combined with using legumes in crop rotation, determines the start of the nitrogen cycle.  However, according to a study comparing organic and conventional apple orchards in Washington state, the fate of the nitrogen differs significantly.

In the organically fertilized orchard, nitrogen was added in the form of manure; the soil microbiota broke down the manure, so nitrogen entered the soil more slowly, making it easier to be assimilated.  Of the nitrogen that was not used by the trees and left the soil, only 10% leached out as nitrate.  Because of the denitrifying microbes in the soil, 10% was denitrified to N₂O, and 80% was denitrified to harmless nitrogen gas. 

In the conventional orchard, nitrogen was added in the form of calcium nitrate, a common agricultural fertilizer.  The same amount of nitrogen was added, and the trees grew as well, with the same amount of nitrogen in their leaves and a comparable amount of nitrogen leaving the orchard as waste.  Here, only 20% of the nitrogen left by microbial denitrification, half as N₂O and half as nitrogen gas.  The remaining 80% of the added nitrogen left by leaching out of the soil as harmful nitrate.  There is a striking correlation between the richer microbiota of the organic orchard and the increased ability of the soil to process nitrogen into environmentally benign forms—with, as the authors of this study note, no effect on the yield of fruit. 

Which brings us back to the whole question of whether or not to go organic, and thanks to the news-making review, we can ignore questions of nutrition.  Those who argue against organics point to increased cost, and less efficient use of land.  I think that some of the costs of conventional agriculture are distributed or hidden—increased energy inputs per acre, and the costs of dealing with increased pollution.  Land use may be less efficient in the short term, but unless there is active and conscientious management of conventional soils (another hidden cost), organic soils are healthier and more sustainable. 

The goal is aspirational; right now, organic stuff is more expensive, and that’s a hardship for some.  Many farmers (not to mention some pretty enormous agribusinesses) are pretty set against organic growing.  There’s also situations that are really difficult to address with anything but conventional means.  I am an example; I am using Crossbow to clean up blackberries and poison oak and vinca that have accumulated after several years of neglect.  But, the goal here is a transition to organic, and it is doable and right.

So, imagine I offered you a couple of MP3 players for sale; they are functionally identical, and both will fill your ears and satisfy your musical desires.  However, one costs 20% more than the other.  What’s the difference?  One is made in a coal-powered factory that produces a large amount of toxic wastes and causes damage to its local environment, while the more expensive one is from a renewably-powered factory that actually collects and recycles waste, cleaning its environment.  Which would you choose?

Galván, Guillermo A.,  István Parádi, Karin Burger, Jacqueline Baar,  Thomas W. Kuyper, Olga E. Scholten, and Chris Kik (2009).  Molecular diversity of arbuscular mycorrhizal fungi in onion roots from organic and conventional farming systems in the Netherlands.  Mycorrhiza 19(5): 317-328.  Onions, with their weak roots, are quite dependent upon mycorrhizae; since the farms were in polders, the soils were very new to agriculture, but even so, mycorrhizae were present. 

Kramer, Sasha B., John P. Reganold, Jerry D. Glover, Brendan J. M. Bohannan, Harold A. Mooney (2006).  Reduced nitrate leaching and enhanced denitrifier activity in organically fertilized soils.  Proceedings Natl. Acad. Sci. USA 103: 4522-4527.  A neat paper about denitrification, free access. 

Lumini, E., M. Vallino, M. M. Alguacil, M. Romani, and V. Bianciotto (2011).  Different farming and water regimes in Italian rice fields affect arbuscular mycorrhizal fungal soil communities.  Ecological Applications 21 (5): 1696-1707.

Maeder, Paul, Andreas Fliessbach, David Dubois, Lucie Gunst, Padruot Fried, Urs Niggli (2002).  Soil Fertility and Biodiversity in Organic Farming.  Science 296: 1694-1697. This paper documents the Swiss long-term experiment; since this was published, many more details have come out. 

Orr, Caroline H., Angela James, Carlo Leifert, Julia Cooper, and Stephen P. Cummings (2011).  Diversity and Activity of Free-Living Nitrogen-Fixing Bacteria and Total Bacteria in Organic and Conventionally Managed Soils.  Appl. Env. Micro. 77(3): 911-919. 

Reeve JR, Schadt CW, Carpenter-Boggs L, Kang S, Zhou J, Reganold JP (2010).  Effects of soil type and farm management on soil ecological functional genes and microbial activities. International Soc. Microbial Ecol. Journal 4(9): 1099-1107.  Good paper, underlines the microbial difference between organic and conventional soils.  Also, for brother M:  Watsonville strawberries. 

Smith-Spangler, Crystal, and, Margaret L. Brandeau, Grace E. Hunter, J. Clay Bavinger, Maren Pearson, Paul J. Eschbach; Vandana Sundaram, Hau Liu, Patricia Schirmer, Christopher Stave, Ingram Olkin, and Dena M. Bravata (2012).  Are Organic Foods Safer or Healthier Than Conventional Alternatives?: A Systematic Review.  Annals of Internal Medicine 157(5): 348-366.

;-) ;-) (wink, wink)

No living thing is in equilibrium with its environment. I mean this in a physical-chemical sense; ecologists will discuss how ecosystems are at equilibrium, meaning that populations stay constant. However, to a biochemist, equilibrium is the same thing as death. A system at equilibrium has zero energy, and since life by any definition requires energy, equilibrium is death. It follows that if you can measure how far out of equilibrium a living thing is, you can get a clue as to how much energy it has at its disposal.

The way almost all living things get energy is sort of like the way energy is stored in a battery:

In a battery, there’s too many electrons over at one end, and not enough at the other. This potential difference is a form of energy. We can let the battery get to equilibrium by letting the charges become uniformly distributed, and we can use that movement to do some work for us:

If it achieves equilibrium—an equal concentration of charges on either end—no more work can be done.

A living cell functions almost exactly the same way. The only changes are that the charge difference is between inside and outside, and the charged particles are (positively-charged) protons (H+) instead of (negatively charged) electrons.

And, just like a battery, this potential difference stores energy. If we let the cell get to equilibrium by letting the charges become uniformly distributed, the cell can use that movement to do some work. Instead of powering light bulbs, cells have enzymes in their membranes that use that energy to make a chemical form of energy, ATP, or wiggle their flagella around so they can move, or pack food into the cell and expel wastes.

Also like a battery, equilibrium means no more work can be done.

Establishing the disequilibrium in a battery takes work, and making the proton gradient across a cell membrane also takes work. Respiring cells use the oxidation of food to move protons out of the cell. Cells that can use light for energy have evolved a couple of mechanisms to move protons out of the cell. The simplest of these is called proteorhodopsin. It’s an enzyme that straddles the cell membrane, like so:

When it’s hit by light...

it can use that energy to change its chape, grab a proton from inside the cell and force it out of the cell (and away from equilibrium). Like so:

To understand the physiology of a cell, it’s nice to know the charge difference, or membrane potential, of the cell. Keeping track of membrane potential is pretty straightforward in cells like ours. Our cells are large enough that we can make tools that can investigate to our cell membranes the way that a stethoscope can check in on a guinea pig. However, bacterial cells are much too small for this approach, and their membranes are covered up by cell walls. So until recently, we haven’t been able to probe the membrane potential of individual bacterial cells, despite knowing that membrane potential is essential for the well-being of the cell.

A bunch of researchers at Harvard, led by Adam Cohen, have devised (and are in the process of patenting) and exceedingly clever way to look at membrane potential in single bacterial cells. Remember proteorhodopsin, which absorbs light energy and uses it to pull a proton out of the cell? The reason it’s able to absorb light energy is that it has a color. Grabbing onto a proton changes the energy state of the molecule, so it changes its color a little bit. So, Cohen and his gang figured out a way to modify proteorhodopsin, essentially reversing its normal behavior: if a proton were forced onto this modified enzyme, it would not only change color, it would fluoresce. How can a proton be forced onto this modified proteorhodopsin? Simply have a higher-than-normal concentration of protons inside the cell. So, here's a normal cell, and the engineered proteorhodpsin is dark:

And here's a depolarized cell. The relatively high concentration of protons inside the cell forces one onto the engineered bacteriorhodopsin, and it fluoresces:

Bear in mind that what triggers the light is not the concentration of protons, just the difference in concentration across the cell membrane.

This designed membrane protein doesn’t help the cell out any—it can’t do what normal proteorhodopsin does—but it does serve as a sensitive monitor of the membrane potential of a single bacterial cell. It is, in effect, a very sensitive tool. Cohen’s group embedded single bacterial cells in a gel matrix, hooked one end of the gel matrix up to the positive end of a battery and the other end up to the negative end. The electrical field pushed all of the protons in the matrix towards one end of the cell, depolarizing the other end. Sure enough, that end of the cell lit up like a Christmas tree. Here it is in cartoon form...

and the actual microscope view, as they flipped the polarity from one end of the cell to the other:

So far, this is just a cute tool. Unlike our cells, which are so large that small patches of the membrane can depolarize, bacterial cells are so large that they essentially can’t have any regionalized depolarization under normal conditions. Also, this modified enzyme only lights up when there’s essentially no difference in charge across the cell’s membrane—which condition, if sustained, is the same thing as death. So, what’s the point?

Here’s where this gets interesting, and actually tells us something new and unexpected about how bacterial cells live. When Cohen looked at a bunch of these cells under the microscope, he saw them “blink” at him at irregular intervals. Whatever the reason, something about the cells’ membrane potential would periodically just give out. (This movie is sped up four times actual speed.)

The modified bacteriorhodopsin essentially senses the difference in proton concentration across the cell membrane. So, there’s a couple ways that a blink could happen—either too many protons inside the cell, or not enough protons outside the cell. “Concentration of protons” is simply a long way to say “pH,” so to test this possibility, Cohen loaded his cells with a pH indicator (like Litmus paper, only in convenient liquid form!). When a cell “blinked,” its internal pH stayed the same.

The cell uses this proton concentration difference to do stuff—make ATP, and rotate its flagella, among other things. It’s kind of a rude trick, but if you glue a cell’s flagellum down, the whole cell rotates…

…as long as there is a proton gradient. (By the way, that movie is actually slowed down three times!) When Cohen’s group examined a cell that was “blinking,” they found that the blink correlated with the cell’s rotation slowing or stopping. Since the concentration of protons inside the cell is remaining constant, the concentration of protons outside the cell must drop during a blink. (I’m not going to include a movie of a cell…not moving.)

So why would a cell blink? Why would it periodically deprive itself of energy? There’s one thing that it’s much easier for a cell to do if it no longer has a membrane potential, and that is push positively charged substances out of the cell. There are some toxins and metals that are harmful to the cell that are positively charged, and cells are known to spit these out of the cell under stressful conditions. So, perhaps this is happening during a blink: the cell deliberately depolarizes, and purges itself of toxins. Cohen tried feeding his cells a toxic dye, and sure enough, it could be observed flowing out of the cell only during a blink.

So, this Cohen’s cleverly modified protein ends up being more than just an example of clever engineering; it tells us something new about how cells work. Until very recently, the only way we could study the physiology of bacterial cells was by studying the average properties of billions of cells all at once. Very useful information can be gained that way (just ask the US Census Bureau), but vital information about how individuals work is lost. This modified proteorhodpsin is one of several neat tools that have emerged in the last decade that allow us to study single cells or even single proteins. Just as doing interviews with individuals would change your views of census data, these new tools are changing how we view the lives of bacteria.

Joel M. Kralj, Daniel R. Hochbaum, Adam D. Douglass, and Adam E. Cohen (2011). Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein. Science 333: 345-348.

When 3.4 billion years old you reach, look as good you will not

I finally got a chance to read the latest microbial news that is creating a buzz, the discovery of some tantalizing maybe-fossils of cells over 3.35 billion years old. (News reports here and here; a couple of blogs’ opinions here and here.) I’m not a geologist, so I can’t adequately judge everything about the paper, but as a microbiologist I can offer my two cents.

I’ll confess that I rolled my eyes a bit when I first heard the news, for several reasons. First, I’m not a fan of science by press release. Fortunately this is actually in the legit literature*. Second, the topic of ancient microfossils is one that leads to a lot of squinting at grainy pictures and seeing what one wishes to see. Third (and partly because of the very subjective nature of interpretation) this has been a field of heated, occasionally quite personal debate, and I am conflict-averse. Finally, I’m not sure it matters.

Here’s the problem. It would be nice to find a beautifully preserved, clearly cellular structure over 3 billion years old with unambiguous evidence of biological functions. It would also be nice if a unicorn came by and pooped golden bricks for me, and about as likely. First, it’s really hard to find nice sedimentary, fossil-bearing rocks that are that old—plate tectonics being what it is, any really old rocks are likely to have been recycled, squeezed and cooked so much that fossils wouldn’t exist. Second, any life present over 3 billion years ago would have been unicellular and very small. It’s hard to find small things. Finally, it’s hard to find something that old that is unambiguously biological. Non-biological processes can generate mineral patterns that look cell-ish, and even mimic some of the chemical processes of life. Remember the martian meteorite that was supposed to have evidence of life on Mars?

So, here’s this report from Martin Brasier and his associates at the University of Western Australia. It’s actually pretty good.

To solve the first problem, they found one of the few places on earth with really old, relatively un-metamorphosed rock—the boonies of Western Australia. These are sandstones, sedimentary rock that was laid down on top of some volcanic rock that’s about 3.5 billion years old, then buried by more volcanic rock 3.35 billion years ago. That this rock is sandwiched between these layers is nice—the volcanic stuff is relatively easy to date, and it helps to protect it from modern organisms (there are some bacteria that live in surprisingly deep hot basalts; this geological setting minimizes the chances of contamination by these organisms, but does not eliminate it).

To solve the problem of finding fossils, they looked in likely places—places where carbonaceous traces were present. Here’s some pictures of what they found:

These are, of course, not convincing on their own to the layman or the specialist. Which brings up the third problem, and the meat of the report. As the authors say with modest understatement, “determining the biogenicity of putative [3 billion year old] microfossils is notoriously difficult.”

The authors provide some pretty good structural arguments. The candidate microfossils are cell-shaped, cell-sized (unlike the supposed martian fossils), and (like modern cells, but unlike some geological artifacts) quite uniform in size. They are located in the sediments like normal cells would be in such an environment. Some pretty amazing work with spectrometry shows that the things that look like cell walls are actually where most of the carbon is found. So, the geology is good and the taphonomy (I love that word—it means the conditions of fossil formation) is pretty good.

Some more convincing evidence comes from the chemistry of the supposed fossils. The researchers determined not just that carbon and nitrogen and sulfur were present, but which isotopes of carbon and sulfur were present. (Isotopes are versions of atoms with different numbers of neutrons—the number of protons and electrons remains the same, but an atom may have more or fewer neutrons. So, most carbon has 6 neutrons per atom, but a very small amount of carbon has 7 neutrons per atom.)

Isotope fractionation is one of the more reliable fingerprints of life. When living things such as plants and microbes take carbon dioxide from the atmosphere and make it into part of a living thing, they preferentially take the certain isotopes of carbon. To “fix” carbon dioxide, a biological enzyme first has to catch a molecule of the gas. The enzyme is not all that great at this, so it tends to catch relatively more of the heavier, slower-moving molecules that have heavier carbon isotopes. So, it’s possible to examine a carbon sample’s isotope ratios and determine whether it’s of biological origin. (Not only that, but different carbon-fixing enzymes are better or worse at snatching carbon dioxide from the atmosphere, so with modern carbon samples it’s possible to determine what sort of organism actually did the job—we can even determine how much of you is made from corn-based carbon vs. carbon fixed by wheat and other crops!)

The carbon isotopes reported with these microfossils check out as biological, which is reassuring. There are some arcane ways to make slight isotope imbalances without using biological processes, but the carbon compounds that are made are not consistent with what was found.

The researchers also presented evidence of isotope fractionation with sulfur found in the samples. This is particularly suggestive about what may have occurred so long ago. One place you may think of sulfur in your own biology (especially after the posts from this last week!) is in flatulence—and in a very real way, what is being looked at here are 3.4 billion year old bacteria farts.

The way we get most of our energy is through what are called oxidation-reduction, or “redox” reactions. In these reactions, an electron goes from a high-energy situation to a low-energy situation. Electrons don’t really like to be attached to carbon, so that’s a high-energy situation; electrons love to be attached to oxygen, so that’s a low energy situation. So, we get energy by letting electrons move (accompanied by a proton, to make a hydrogen atom) from carbohydrates to oxygen. To a very rough degree, you can think of these reactions moving hydrogen atoms from carbon to oxygen:

C₆H₁₂O₆ + O₂ --> CO₂ + H₂O **

There’s plenty of evidence that 3 billion years ago, there was no oxygen in the atmosphere. However, something like this process probably went on, and goes on today in your intestines, where there is no oxygen available. Although electrons love to be on oxygen, sulfur is a pretty good second choice. So, the microbes in your gut can do this reaction:

C₆H₁₂O₆ + S --> CO₂ + H₂S

The cells won’t get as much energy, but they’ll get enough to live—and your farts will smell like rotten eggs.

Surrounding the supposed fossils, the researchers found grains of pyrite. If this metabolism (or one very like it, using hydrogen instead of carbohydrate and sulfate instead of sulfur) occurred, the resulting H₂S would react with iron in the environment and form pyrite. This reaction can happen without biological intervention, but if it’s done by life as we know it, you would again expect to see an enrichment of heavier isotopes of sulfur—and this is just what was found. The spatial relationship of the supposed fossils and the pyrite grains is also the same as what one sees with modern cells performing this type of metabolism.

So, Brasier and his colleagues present a very good (but I hesitate to say iron-clad) case for having discovered 3.4 billion year old evidence for life. Their geology and taphonomy are sound, and they provide a couple of lines of evidence consistent with life, especially life under the conditions of the young earth. How young was the earth when these maybe-cells maybe lived? There’s geological evidence that the young earth was pummeled by a massive barrage of meteorites. This “late heavy bombardment,” which would have made life impossible by vaporizing the oceans, ended about 3.8 billion years ago. So, as one news report has it, life may have emerged “very soon” after the late heavy bombardment—although I think of 400 million years as being a pretty long time.

This is some impressive work, and it may well be the “oldest evidence for cellular life.” However, as I mentioned, I’m not sure it matters beyond who gets bragging rights. Although it’s old, it doesn’t change anyone’s estimate about when life started. It doesn’t change our views of ancient biochemistry, as it is consistent with what has been predicted. It would be nice to know if the purported cells were Bacteria or Archaea, but that information can’t be recovered. Finally, unambiguous proof of cells that old (or for that matter, if we every find anything on Mars or Europa) is just about impossible.

On the bright side, I suppose it is nice not only to know that you had a great-great grandmother, but also to have a picture of her. It makes for a better lecture to show a slide of these pictures rather than a slide of isotope data.

*David Wacey, Matt R. Kilburn, Martin Saunders, John Cliff and Martin D. Brasier (2011). Microfossils of sulphur-metabolizing cells in 3.4-billion-year-old rocks of Western Australia. Nature Geoscience (Published online 21 August 2011 | DOI: 10.1038.

**Yes, I'm not balancing the equation; I'm just interested in the chemicals, not their amounts. If you're concerned, I encourage you to do the stoichiometry.

Nature vs. Nurture in the gut microbiota III: what's in a name?

(Part I, Part II)

The population of microbes in our gut are a personal trait, like height or weight. The precise make-up of that population, its balance of different types of microbes, is significant as it can have major beneficial or harmful effects on our health. Like many other traits, the composition of that population is partly due to nature—our genes create an environment welcoming to certain types of microbes—and nurture, the food we eat.

Given the importance of the composition of the microbial community to our health, it’s not surprising that there is a lot of interest in trying to figure out just how significant are the contributions of nature and nurture. Comparing genetically identical mice with a drastically simplified community of microbes in their gut, researchers found that changes in diet led to predictable changes in community structure. However, comparing genetically distinct strains of laboratory mice, a different group of researchers found that the subtle genetic differences between two inbred lines of mice led to measurable differences in the gut microbial community (see part I).

The roles of nature and nurture can be probed further by comparing the microbial community of different, but related, types of animals. What if two animals are closely related, but have different diets? What if they are only distant relatives, but have similar diets? An extreme example of the latter case is a comparison of the intestinal microbes of the tammar wallaby and the cow (see part II). While the comparison is informative, these two mammals are about as distantly related as possible, so a closer comparison is useful.

In a recent study from Washington University at Saint Louis, a group led by Jeffrey Gordon attempted to compare a family tree of mammals, a family tree of the microbes that lived in their guts, the diets of those mammals, and the genetic abilities of their microbes. Any four-way comparison gets complicated pretty quickly, but there are some clear facts that emerge from the fog of statistics. These facts emphasize some basic biology of microbes, as well as telling us something about how mammals and their microbes co-evolve.

A family tree of the mammals comes out pretty much the same, regardless of whether you use genetic data or anatomical differences:

Looking at this family tree, there is little convergence between relatedness and diet. For instance, elephants, capybaras, gorillas, horses, and rabbits are all “hindgut-fermenting herbivores,” with a specific method of digesting cellulose—and yet, are found on different branches of the tree. Sheep, hyraxes, kangaroos, colobus monkeys, and pigs are all “foregut-fermenting herbivores,” with an anatomically distinct way of digesting plant matter—but again, they are only distant relatives. One can find groups, such as the primates, comprising foregut-fermenters, hindgut-fermenters, carnivores, and omnivores. So, genetics on a deep level does not determine diet.

If genetic heritage doesn’t determine diet, does it at least determine something about a mammal’s intestinal microbial community? To approach this question, you can imagine a goodly branch of that mammalian family tree. A diverse bunch of modern mammals evolved from a common ancestor:

We obviously don’t know anything of the DNA sequence of their common ancestor, but we can see the genetic similarities of the modern animals, and work backwards to predict what their ancestor was like, as well as which modern animals are most closely related. If the ancestor had a certain genetic composition—symbolized by the color blue—then its descendants would have evolved variations on that genetic composition—symboized by variations in the hue in the modern beasts.

Now, if genetic heritage determines an animal’s gut microbes, then the genetic character of that community should show a pattern of evolution sort of like the mammalian family tree:

This is a trickier tree than the previous one. Rather than looking at a lineage of organisms, we are looking at the composition of a community of thousands of species. We can make an analogy with the white pages of a phone book: in this tree, we are looking at how the content of a community’s phone book changes over time. (Also, it’s important to remind ourselves that we don’t know what the original community was like—we only have the present one, and we try to work backwards.)

So, what did Gordon’s group find? If mammals and their intestinal microbes co-evolved, then we could examine a diversity of modern mammals and the microbial communities in their poop—which is just what they did. We’d expect related mammals to have related communities. Lining up the tips of the family trees (that is, just currently living animals and the microbes in their poop), we’d see something like this:

There was good reason to expect this result, as similar patterns of co-evolution are seen with insects and their symbiotic bacteria. Co-evolution is common. But here’s what they found:

What to make of this? Obviously, genetic heritage has very little to do with the community structure of an animal’s intestinal microbes. However, Gordon’s group looked at the same data again. This time, instead of looking at mammals by their ancestry, they considered their diet:

Aaaaah, much better! So, it seems like diet—nurture, in our nature/nurture dichotomy—is a much better predictor of a mammal’s microbial community. (This is a much simplified version of the data presented; my apologies to Gordon et al, who studied 33 mammal species and hundreds of microbial species. It’s also useful to note that in several instances they looked at the communities of different individuals of the same species, and found little variation between them.)

Interestingly, despite the differences in community structure between mammals, all mammals have pretty much the same “core” community of microbes. To return to the metaphor of a phone book, any big city in this country will have roughly the same collection of names, it’s just that one city will have a lot more Schmidts and Webers and Mullers and another will have many more Vasquezes and Diazes and Gonzaleses.

Although there was a decent correlation between mammalian diet and community composition in Gordon’s data, his group found an even more significant relationship. The best correlation was not between mammalian diet and community composition. It was between mammalian diet and what genes were present in the microbial community.

Let’s consider that phone book analogy again. If I wanted to find somebody to turn some wheat into flour, I probably wouldn’t turn to the white pages and call up somebody named Muller or Miller. (Heck, I wouldn’t say I’m a good horticulturalist because I’m named Appleman.) Trades are not inherited with family names, and this is true in the microbial world as well. So, if you’re a vegetarian mammal looking for a microbe to turn your b-glucosides into monosaccharides, you can’t assume that you’re looking for Brevibacter—all you care about is whether the organism has the genes for doing the job.

Microbial genomes—the collection of genes a given species has—are incredibly fluid. Genes can disappear from one genome in the space of a few generations, and can establish themselves in a new genome in an equally short time. Genes can travel with relative ease between bacterial species that are barely related to each other. (There are many mechanisms for doing this, and it makes firmly describing a microbial species next to impossible.) So, when a hyrax feeds its intestinal microbes a blend of grass and insects, it is (unwittingly) selecting a specific assemblage of genes, not a specific community of microbes.

These results raise some interesting questions about evolution. The community of microbes that lives in a mammal’s gut—its microbiome—can easily be viewed as an organ like the lungs or kidneys. However, its inheritance and evolution are quite distinct from that of the rest of the animal. As Gordon’s group suggests, it would be fun to compare a lot of different carnivores, and try to extrapolate just when differences in their microbiomes evolved. Do such evolutionary jumps correlate with events such as continental drift or other significant milestones? Are the changes gradual or rapid? Can one community be dislocated by another? Just how fluid are the communities in the gut?

These questions bring us back to the tammar wallaby and its oddly non-methanogenic digestion. Interestingly, in this study, the microbiome of a kangaroo clearly grouped with other foregut-fermenting mammals, such as sheep and gazelles. It was an outlier in that group, but still clearly part of it and not some other group. Well, the tammar wallaby was found to have a small number of methanogens and acetogens in its gut, giving it at least some similarity to the sheep. The big difference in digestion (and flatus) may be traceable to the very small community difference of one organism with one interesting metabolic pathway, a factor that simply can’t be revealed by this sort of analysis. The situation is reminiscent of human societies, which can be hugely affected by a single individual—and knowing the name of that individual won’t tell you anything about how they affect society.

Brian D. Muegge, Justin Kucyznski, Dan Knigts, Jose C. Clemente, Antonio Gonzalez, Luigi Fontana, Bernard Henrissat, Rob Knight, and Jeffrey Gordon (2011). Diet Drives Convergence in Gut Microbiome Functions Across Mammalian Phylogeny and Within Humans. Science 332, 970-974.

Nature vs Nurture in the gut microbiota, part II: the music of wallaby farts

I recently wrote about a study showing the impact of host genetics on gut microbes. This was done by comparing different strains of mice, eating about the same diet. These animals are closely related, having had common ancestors only a hundred years ago. What if we looked at animals eating about the same diet, but whose most recent common ancestor lived a hundred million years ago?

Consider the tammar wallaby: This cute little kangaroo-let has recently found interest as a laboratory animal. As a marsupial, the last ancestor it shares with a cow lived over a hundred million years ago. It eats essentially the same diet as a cow. However, while cow farts are rich in methane, wallaby farts are not. This suggests that, despite identical diets, these animals have very different gut microbes. Until very recently, the nature of these differences was quite puzzling.

It’s useful to have a little background on what goes on in the gut of a cow eating grass. Most of what the animal eats is cellulose and other polysaccharides. These are compounds that are made by stringing together hundreds of sugar molecules, and they are tough—the table that I’m writing on and the cotton I’m wearing are made of polysaccharides. Breaking these polysaccharides down to release their constituent sugars is chemically very difficult, and breaking down the cellulose found in forage is almost impossible. Surprisingly, cows can not do this. It’s a job done by microbes living in the guts of these animals. Of course, the microbes that release the sugars from cellulose don’t do it out of charity to their hosts—as soon as the sugar is freed from the cellulose, they eat it. How does this help the cow?

You probably know fermentation as what yeasts do, taking sugar and making ethanol and CO₂ as wastes. However, there are other ways that microbes get energy by fermenting, and most of the microbes in a cow’s gut take sugar and make acetic acid, CO₂, and hydrogen gas as wastes. So, after the microbes have broken down the cellulose and fermented the sugars, the cow thanks the microbes nicely, and absorbs the acetic acid as a nutrient. But the CO₂ and hydrogen pose a real problem.

Picture a worker on an assembly line: he takes two widgets, screws them together, and passes them on to the next guy. He can keep working as long as the widgets keep coming down the assembly line and the next guy takes his finished product away. If the next guy falls asleep, the product will build up, and our worker will be unable to do his job. This is what could happen to the fermentation in a cow: CO₂ and hydrogen are the products of fermentation, and if they build up, the fermenting microbes will stop (and the cow will starve). Fortunately for the cow and the microbes, there are other microbes in its gut, called methanogens, that get energy by “burning” the hydrogen—they oxidize it, using CO₂ instead of oxygen, and produce methane. So, the production of methane in cow farts is absolutely necessary if the cow is to continue breaking down cellulose.

However, as was noted, wallaby farts don’t have so much methane, even though they eat the same amount of cellulose. How can wallabies and their microbes digest cellulose?

One possibility that seemed likely was that there were some other microbes in the wallaby gut that were able to eat CO₂ and hydrogen. It was already known that termites (who also eat cellulose) house bacteria called “acetogens” that combine hydrogen and CO₂ to make acetic acid rather than methane. So, it was thought that if an animal ate cellulose, it had to have either methanogens or acetogens in its gut to help the fermentation reactions along (and in fact, we have some of each in our guts—as evidenced by the flammability of farts—just not as many, since we don’t eat as many polysaccharides). When I first heard that wallaby farts were low in methane, I assumed that wallabies must be full of acetogens.

However, it’s always good to check your assumptions. An analysis of the wallaby gut community showed few methanogens, and somewhat more acetogens—but not enough to make fermentation (as it was understood) work. This was a real puzzle—without microbes to consume the CO₂ and hydrogen from fermentation, how could the wallaby eat cellulose? The answer, according to an international group of researchers, may be that methanogens and acetogens are unnecessary for the fermentation that happens in a wallaby gut—their microbes use a unique fermentation reaction that doesn’t produce hydrogen, and consumes CO₂.

The overall theme of fermentation by the cow’s microbes is to break sugars into smaller and smaller pieces—start with a molecule of six carbons, then go to two molecules of three carbons, then two molecules of two carbons and two molecules of CO₂ and some hydrogen atoms.

The researchers observed that a significant component of the microbial population of a wallaby gut was similar to a previously identified bacterium called Succinovibrio, named after its unusual metabolism. Sugar fermentation in Succinovibrio starts the same way as in the cow; however, in the final stages, the fragments of sugar molecules are partially reassembled to make a four-carbon compound, succinate. This process (which also consumes CO₂) provides the bacterium energy to live—and what’s more, the succinate gives the wallaby more energy than a similar amount of acetate gives to a cow.

The researchers showed very nicely that this sort of fermentation can go on in the wallaby’s gut, and that this could explain the way that wallabies are able to eat the same diet as a cow yet not produce so much methane. So, why do wallabies play host to Succinovibrio while cows play host to methanogens? It’s obviously not diet—the original studies showing differences in methane production were done with wallabies and cows on the same feed, and wallabies do host a few methanogens. There may be structural issues; cow anatomy is quite different from that of the macropodes. But most likely, as we have learned from studies on mice and humans, is that the genetic background of the host sets up a molecular environment that is particularly hospitable to specific microbes. The genomes of both the cow and the wallaby have been sequenced, so the information is there. We just have to do the hard work of understanding it.

There is a practical aspect to this question, beyond the flammability of wallaby farts. Methane is an extremely powerful greenhouse gas; it is over twenty times more efficient at retaining solar heat than CO₂. The human fondness for cattle grazing has increased methane production, contributing to global climate change. There are researchers who entertain the pipedream of converting cattle to a wallaby metabolism—producing more meat per munch of hay, and producing less pollution to boot. A noble cause, but I’ll probably only be able to eat a burger from such an animal when I pull up to the drive-through in my flying car.

Paul N. Evans, Lyn A. Hinds, Lindsay I. Sly, Christopher S. McSweeney, Mark Morrison, and Andr ́e-Denis G. Wright (2010). Community Composition and Density of Methanogens in the Foregut of the Tammar Wallaby (Macropus eugenii). Applied and Environmental Microbiology 75: 2598-2602.

Emma J. Gagen, Stuart E. Denman, Jagadish Padmanabha, Someshwar Zadbuke, Rafat Al Jassim, Mark Morrison, and Christopher S. McSweeney (2010). Functional Gene Analysis Suggests Different Acetogen Populations in the Bovine Rumen and Tammar Wallaby Forestomach. Applied and Environmental Microbiology 76: 7785–7795.

P. B. Pope, S. E. Denman, M. Jones, S. G. Tringe, K. Barry, S. A. Malfatti, A. C. McHardy, J.-F. Cheng, P. Hugenholtz, C. S. McSweeney, and M. Morrison (2010). Adaptation to herbivory by the Tammar wallaby includes bacterial and glycoside hydrolase profiles different from other herbivores. Proceedings Natl Acad Sci USA 107:14793-14798.

P. B. Pope, W. Smith, S. E. Denman, S. G. Tringe, K. Barry, P. Hugenholtz, C. S. McSweeney, A. C. McHardy, M. Morrison (2011). Isolation of Succinivibrionaceae Implicated in Low Methane Emissions from Tammar Wallabies. Science 333: 646-648.