The genetically modified bacterium turns greenhouse gas CO2 into ethylene - an energy dense fuel and major industrial feedstock - reversing the largest CO2-emitting process in the petrochemical industry and with an added bonus of O2 Dr. Mae-Wan Ho
Scientists at the US Department of Energy’s National Renewable Energy Laboratory in Golden, Colorado, have succeeded in turning carbon dioxide into ethylene, an energy-dense fuel and most widely produced feedstock for the petrochemical industry worldwide. The report was published online in the journal Energy & Environmental Science .
This is a major breakthrough in terms of a truly renewable and sustainable zero-carbon energy source; and perhaps especially in regenerating oxygen from carbon dioxide (although this aspect was not mentioned by the scientists themselves).
Ethylene (H2C=CH2) is made exclusively from fossil fuels in the largest CO2-emitting process in the industry. It is also the most widely produced feedstock for the chemical industry worldwide. Ethylene is used in the synthesis of diverse plastics and textiles such as polyester. It has been used to produce high-grade ethanol for the past 50 years, and can be polymerized into gasoline.
A total of 132.9 million tons of ethylene was produced in 2010, with a projected 5 % growth annually through to 2015. Its manufacture involves steam cracking long chain hydrocarbons from petroleum or ethane. Oil supplies are finite and steam cracking generates large amounts of greenhouse gases: 1.5 – 3.0 tons of CO2 per ton of ethylene. The team led by Justin Ungerer have taken the first steps in devising a sustainable, carbon-neutral alternative by coaxing a bacterium to convert CO2 into ethylene.
Using CO2 as feedstock is particularly appealing as it sequesters carbon and recycles CO2 instead of burying it in the ground (see  Carbon Capture and Storage A False Solution, SiS 39). Ethylene is a gas and can be collected directly from the space above the reaction mixture, saving cost and energy in harvesting and extracting compared to conventional lipids or bioethanol produced by algae. Furthermore, ethylene is not a food source for microbes, which eliminates feeding and contamination problems associated with sugar or fatty acid secretions. And to top it all, ethylene is not toxic to the bacterium producing it, unlike ethanol and butanol, and can be produced in larger amounts .
The cyanobacterium Synechocystis sp. PCC6803 has been enlisted for the job. The overall equation of the process is as follows:
2CO2 + 2H2O → C2H4 + 3O2 (1)
Thus photosynthetic production of one ton of ethylene could sequester 3.14 tons of CO2, not counting the savings of nearly as much again in CO2 emission compared with ethylene produced conventionally from fossil fuel.
The other important aspect the authors have not commented on is the release of O2, which I shall deal with later. As in normal photosynthesis, the bacterium uses sunlight to split water into 4H and O2; the 4H go to reduce 2CO2 into ethylene, releasing two extra molecules of oxygen from the carbon dioxide in addition to the one from water.
Ethylene is a natural plant hormone regulating germination, senescence and fruit ripening among other processes. In plants, it is synthesized from methionine in a three step reaction. Most bacteria make ethylene from methionine in a different two-step reaction, which is not very efficient. Many plant pathogens such as Pseudomonas syringae synthesize ethylene during infection to overcome the host defence. They do so directly from the tricarboxylic acid (TCA) cycle intermediate a-ketoglutarate in an efficient single-step reaction catalysed by the ethylene forming enzyme (EFE) that also produces succinate as a secondary product .
The enzyme from Pseudomonas syringae pv. Phaseolicola had been cloned and expressed in the yeast Saccharomyces cerevisiae, which released ethylene as a by-product of glucose fermentation as a result . EFE was also previously expressed in the cyanobacteria Synechococcus elongatus  and Synechocystis .
Ungerer and colleagues solved the problems of gene stability and poor productivity in Synechocystis by redesigning the efe gene . They also adjusted growth conditions to improve yield.
Modifying the efe gene
Previous attempts to genetically modify Synechococcus failed because the efe gene lost activity within three subcultures. The inactivation was due to duplications of the sequence CTATG, leading to truncation of the protein. The sequence occurs three times within the efe gene, and duplications were found at all three sites. Changing the sequence without changing the codon for amino acids solved the problem. This also allowed the codons to be optimized for improved expression in Synechocystis. Expressing one or two copies of the optimized gene had no effect on growth rate or morphology of the colonies.
The enzyme was stably expressed and produced high levels of ethylene over four successive subcultures. The pea plant promoter was found to be the best for expression.
Improving conditions of growth for production
In the usual growth medium, ethylene production peaks early and then steadily declines as the culture reaches the stationary phase. Numerous tests failed to identify a single limiting factor, suggesting that multiple components in the medium became limiting. Indeed, increasing the concentration of the medium (to 5x) resulted in increased peak level production, and remained higher for a longer period thereafter.
Increase copy number of efe gene
Expressing a second copy of efe gene driven by the same pea plant promoter increased production by twice as much, and at no apparent increase in metabolic burden to the strain compared with the wild type bacterium.
As depletion of medium components limits ethylene production, the researchers decided to adopt a semi-continuous culture. The cells were spun down and replaced with fresh medium every week; the culture resumed ethylene production at high levels immediately afterwards. A maximum production of 3 100 mL of ethylene L-1 h-1 was achieved. This peak production rate was maintained for three weeks with daily medium replacement in a mature culture with optical density at 730nm of 15. It is clear that continuous culture (continuous medium replacement) is important for maintaining peak production levels.
Increasing light intensity
Increasing light intensity 12-fold - from 50 to 600 mmol photons m-2 s-1 increased production levels to 5 700 mL-1 h-1.
In order to make the production commercially viable and sustainable, the team investigated whether seawater could substitute for the growth medium, which is expensive, and requires a lot of freshwater, an increasingly scarce resource (see  World Water Supply in Jeopardy, SiS 56). They found that seawater by itself did not support high production levels. However, seawater supplemented with 4 mg L-1 of phosphate and 150 mg L-1 nitrate sufficed to support rates comparable to those using 5x concentrated medium. These main nutrients can be derived from sewage, providing additional waste recycling.
This is good news, as production could be located in brackish water, sea bays, or coastal areas - without competing for arable land or fresh water - with appropriate safeguards against discharge of transgenic wastes into the environment.
Analysis of carbon flux shows that although ethylene production is highest in the early log growth phase, the percentage of carbon fixed into ethylene increases as the culture matures, and is greatest at the stationary phase. It reached 5.5 %, which surpassed published carbon flux into the TCA cycle of core metabolism. This suggests that there are significant alterations to the carbon flux in the ethylene forming strains, and ethylene is providing an alternative sink for the fixed carbon.
The scientists have made a major breakthrough in a new source of renewable energy, which substitutes for a high CO2-emitting production process for a most widely used feedstock in the chemical industry that hitherto has been made only from fossil fuels. Moreover, the peak productivity of 7 125 mg L-1 h-1 is higher than has been reported for other algal/cyanobacterial petrochemicals including isobutanol (3 500 mg L-1 h-1), isobutyraldehyde (6 200 mg L-1 h-1) and biodiesel (3 420 mg L-1 h-1). Ethylene has a higher energy density (47 MJ kg-1) and carbon content (92 % wt) than other biofuels: isobutanol (33 MJ kg-1) (64 % wt), isobutyraldehyde (31 MJ kg-1) (62 % wt), ethanol (23 MJ kg-1) (52 % wt), biodiesel (37 MJ kg-1) (avg. 75 %). This makes ethylene a very attractive fuel, in addition to being a widely used chemical feedstock.
Perhaps the most important aspect of the process is that it reduces CO2 to ethylene, releasing extra oxygen (see above). Oxygen has been depleting faster than CO2 is increasing in the atmosphere from burning fossil fuels, and this trend has accelerated since around 2002-2003 ( O2 Dropping Faster than CO2 Rising, SiS 44). The precise causes are unknown, but deforestation - recently for the production of biofuels (see  Biofuels for Oil Addicts and other articles in the series, SiS 30) - and phytoplankton die-offs [10-12] (Shutting Down the Oceans. Act I: Acid Oceans, Shutting Down the Oceans. Act II: Abrupt Plankton Shifts, and Shutting Down the Oceans Act III: Global Warming and Plankton; Snuffing Out the Green Fuse, SiS 31) may be among the main factors. The resulting deficit of photosynthesis means that oxygen is simply not replenished, while the sequestered carbon takes much longer to become released.
It has become clear to some of us that reducing CO2 is not enough; oxygen has its own dynamic and the rapid decline in atmospheric O2 must also be addressed. Although there is much more O2 than CO2 in the atmosphere - 20.95 % or 209 460 ppm of O2 compared with around 380 ppm of CO2 – humans, all mammals, birds, frogs, butterfly, bees, and other air-breathing life-forms depend on this high level of oxygen for their well-being  Living with Oxygen (SiS 43). In humans, the failure of oxygen energy metabolism is the single most important risk factor for chronic diseases including cancer and death. ‘Oxygen deficiency’ is currently set at 19.5 % in enclosed spaces for health and safety , below that, fainting and death may result.
Land and ocean photosynthesis each accounts for roughly half of all oxygen generation. Ocean phytoplankton is particularly important as it works on the shortest timescales. It is literally the first life line of the biosphere. When phytoplankton goes, oxygen does not just deplete from the oceans but from the atmosphere, as the water solubility of oxygen is very low. The result is mass extinctions of air-breathing animals, as had happened at the end of the Permian 251 million years ago; only this time round, the human species will be included in the death toll.
We must halt deforestation now and replant forests to protect our oxygen source on land. At the same time we need to avoid further warming and acidification of the oceans by switching to truly renewable ‘zero-carbon’ energy options like the one described in this article, as well as many others already available  Green Energies - 100% Renewable by 2050, ISIS report.)
Article first published 19/09/12
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Terry Pollock Comment left 19th September 2012 22:10:13
Again, Dr. Mae-Wan Ho connects vital dots in the science behind renewing our energy resources in a supportive way to the planet and its inhabitants. She clearly contrasts an elegant use of a photosynthetic GE bacterium's safe "disposal" of CO2 with flawed oil addiction and its toll on planet Earth. A wonder of an article that plants seeds of hope.
Maryann Dominguez Comment left 20th September 2012 19:07:28
I love Dr. Mae-Wan Ho's articles. I just have to comment here that while this discovery on the one hand presents good results and what may seem as hope, on the other hand, as much as we are faced with all the dangers created by the pursuit and exploitation of petrochemicals, we are again creating dangers - this time UNKNOWN dangers – in the pursuit of seeking to replace all that with genetic manipulation of life. We are again exchanging one danger for a greater one! What is being done is the same as in medicine. Yes, the DOMO thingy that Dr. Ho talks about. The need remains to focus on resolving our ills from the CAUSES up! There was a time we did not depend on synthetics (at least not so life-threateningly). Man has succeeded in satisfying man's basic needs, what more aggrandizements does he seek? But now, in modern society, a demand for something is created WHEN that something IS created - but creation is not synonymous with need nor with man's advancement. The “demand” of my high school’s Economics days are not the same “demand” of today. Today we create for whimsy...for the pursuit of individual wealth and not man's - human - advancement. And so how do we call ourselves an "advanced society" by destroying our Earth and biology? ...by being so proud of our "intellectuality"? ... so egocentric with our technological brilliance? Can we not now see its dullness?? ...we actually see, smell, hear, touch, and taste it now everyday - yet we are not conscious of it...but yet we are sick from it. And THAT is how literally senseless experimental man has become. We must build our future constructs from new and heart-ier foundations. THAT is where our hope may grow from... THAT is where our creativity...our intellectuality and brilliance is needed...is screaming at us to correct. NOT in the creation of more Faux Frankensteins to replace Failed Frankensteins. That was our error to begin with! We seem to have been the Barbarians we were seeking to transform. WHO are we and WHAT do we want to become? Already there is talk of “moving” to a new planet once we have “exhausted” our resources – what mentality! WE ARE our resources. Let’s become resourceful with our brilliance and build WITH LIFE… NOT against LIFE. …Let’s build FROM LIFE… NOT transform LIFE. …In seeking to transform LIFE we mutilate and desecrate all LIFE, to our own life’s degeneration. We need NOT mount a scientific study to confirm this… the evidence is before all your senses. USE what you’ve been given, as it has been given to you. For LIFE CREATED YOU. LIFE has needed no scientific instruction, no philosophic theory, no laureate discourse, no engineering savvy, not even conscious mind, for its cells’ mathematically and harmonically-precise self-creation into complex systems and bodily organs with definite functions and localities, needing of only those Earth provided essences vibrating with them in a synergistic dance – momentum and rhythm self-perpetuating, self-creating, self-healing. Not to recognize, nor to acknowledge, when synthetics or any institution, construct, structure, procedure, protocol interfere with the magnificence of this natural living dance within infinity's music, is not a simple breach of etiquette… it is irrationally and simply doing so to the tune of human genocide. I hope, feverishly, my daughters live to see… smell, hear, touch, and taste things as LIFE originally created them… for look around your virtual man-made-life… they are, sadly, almost all gone.
Rory Short Comment left 20th September 2012 19:07:58
Wonderful news, GE being used in a way which would seem to be beneficial long term for us all rather than in providing short term profits to agri-businesses.
Joel Hinrichs Comment left 21st September 2012 13:01:44
Doing the math (exercise left to reader) .0057 cc per liter per hour comes out to ~65 g per liter per year. To replace 132.9 million tons would require roughly 10**7 square meters one meter deep, or in english 2 miles on a side one yard deep. Ramping up laboratory micro liters per liter per hour to megatons per square mile per year is another exercise left to the reader. 132.9 million tons C2H4 sequesters 415.7 million tons C2O. That's minuscule, but a good start. Question: Don't use ambient flowing water; pipe in [for example] that 10E8 cubic meters of water from the ocean; now can we make it into a four square mile biodome alongside every fossil fuel burning plant and capture all its CO2? At 360 parts per million in the atmosphere, to find 415 million tons of CO2 you must move 1.15 TRILLION tons of air. Better to capture it at a reasonable source; and feed the release O2 back into the plant to make its burn more efficient. By the time we will truly run out of carbon based fuels, at least a generation of scientists will have obsoleted all of the above.
Frank Rowson Comment left 21st September 2012 20:08:43
Maryanne stated it well with her comments on science;it is constantly being used to cure a problem it caused in the first place.The best Carbon sequestration will come from the return to sustainable agriculture,restoring the soil, especially by nurturing mycorrhiza and increasing the amount of glomalin. Unless this is done no amount of GE will help, but it will keep some scientists in work, scientists who have yet to make the paradigm shift to research the causes of the problems, not the symptoms.
Phil Kortis Comment left 10th October 2012 07:07:20
What are appropriate safeguards against discharge & what is the likelihood they would followed, enforced, and effective? esp. if large quantities of growing-medium water are needed?