ISIS Report 19/09/12

Photosynthetic Bacterium Converts CO₂ into Petrochemical & O₂

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

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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 [1].

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

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 [2] 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 [1].

Production of ethylene in cyanobacterium

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 [3].

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 [4]. EFE was also previously expressed in the cyanobacteria Synechococcus elongatus [5] and Synechocystis [6].

Ungerer and colleagues solved the problems of gene stability and poor productivity in Synechocystis by redesigning the efe gene [1]. They also adjusted growth conditions to improve yield.

Optimizing for ethylene production

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.

Semi-continuous culture

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.

Production in seawater

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 [7] 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.

Ethylene is an alternative carbon sink for photosynthesis

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.

A major breakthrough especially for regenerating oxygen

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 ([8] O2 Dropping Faster than CO2 Rising, SiS 44). The precise causes are unknown, but deforestation - recently for the production of biofuels (see [9] 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 [13] 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 [14], 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 [15] Green Energies - 100% Renewable by 2050, ISIS report.)

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