Brookhaven biochemists engineered duckweed, an aquatic plant, to produce large quantities of oil. If scaled up, the approach could produce sustainable bio-based fuel without competing for high-value croplands while also potentially cleaning up agricultural wastewater. (Image: Brookhaven National Laboratory)

Scientists at the U.S. Department of Energy’s Brookhaven National Laboratory, with collaborators from Cold Spring Harbor Laboratory (CSHL), have engineered duckweed — one of nature’s fastest-growing aquatic plants — to produce high yields of oil. They did so by adding genes to the plant to push the synthesis of fatty acids, pull those fatty acids into oils, and protect the oil from degradation.

The scientists say that such oil-rich duckweed could easily be harvested to produce biofuels or other bioproducts.

The team engineered Lemna japonica, a strain of duckweed, to accumulate oil at close to 10 percent of its dry weight biomass. That’s a 100-fold increase over such plants growing in the wild — including yields more than seven times higher than soybeans, the current largest biodiesel source.

“Duckweed grows fast,” said John Shanklin, Ph.D., Biochemist and Chair of Biology, Brookhaven National Laboratory, who led the team. “It has only tiny stems and roots — so most of its biomass is in leaf-like fronds that grow on the surface of ponds worldwide. Our engineering creates high oil content in all that biomass.

“Growing and harvesting this engineered duckweed in batches and extracting its oil could be an efficient pathway to renewable and sustainable oil production.”

To bolster its case, since it’s an aquatic plant, oil-producing duckweed wouldn’t compete with food crops for coveted agricultural land, and it can even grow on runoff from pig and poultry farms — meaning it could potentially clean up agricultural waste streams as it produces oil, according to Shanklin.

Preliminary work showed that increased fatty acid levels triggered by the push gene can have detrimental effects on plant growth. To avoid such effects, Brookhaven Lab postdoctoral researcher Yuanxue Liang paired that gene with a promoter, which can be turned on via the addition of a small amount of a specific chemical inducer.

“Adding this promoter keeps the push gene turned off until we add the inducer, which allows the plants to grow normally before we turn on fatty acid/oil production,” Shanklin said.

Liang then created a series of gene combinations to express the improved push (W), pull (D), and protect (O) factors singularly, in pairs, and all together. The key findings show that the overexpression of each gene modification alone did not significantly increase fatty acid levels in Lemna japonica fronds. However, plants engineered with all three modifications accumulated up to 16 percent of their dry weight as fatty acids and 8.7 percent as oil when results were averaged across several different transgenic lines. The top plants accumulated up to 10 percent triacylglycerols (TAG) — better than 100 times the level of oil that accumulates in unmodified wild-type plants.

Some two-modification combos (WD, DO) increased fatty-acid content and TAG accumulation dramatically compared to their individual effects — also known as synergistic results.

“Future work will focus on testing push, pull, and protect factors from a variety of different sources, optimizing the levels of expression of the three oil-inducing genes, and refining the timing of their expression,” Shanklin said. “Beyond that we are working on how to scale up production from laboratory to industrial levels.”

The scale-up work includes designing the types of large-scale culture vessels for growing the modified plants, optimizing large-scale growth conditions, and developing methods to efficiently extract oil at high levels.

Here is an exclusive Tech Briefs interview (edited for length and clarity) with Shanklin.

Tech Briefs: What inspired the research?

Shanklin: The idea of harnessing duckweed to make oil from sunlight and CO2 from the atmosphere got me excited about this project. The fact that it can use nutrients (fertilizer) from agricultural runoff (e.g., poultry and hog farms) to make oil while cleaning up pollution was also appealing. Lastly, it won’t compete with regular crops for prime agricultural land.

Tech Briefs: What were the biggest technical challenges?

Shanklin: We had to figure out how to get genes into duckweed and how to knock out genes out that we want to silence. We did that in partnership with colleagues at Cold Spring Harbor. We were then able to introduce genes to make oil, but we found those made the duckweed grow very slowly and have misshapen fronds (duckweed equivalent of leaves). We figured out which gene was causing the growth problem and put that on what’s known as an inducible promoter, which is like a light switch we can turn on at any given time by adding a tiny bit of a small chemical molecule to the water on which the duckweed is growing.

Tech Briefs: Can you explain in simple terms how it works?

Shanklin: We have three oil-producing genes. Two are on all the time, and the third key gene is turned on by adding a small molecule inducer. We grow the duckweed normally until it’s at high density, then add the inducer, and in about four days the duckweed fills up with little oil droplets.

Tech Briefs: What’s the next step with regards to your research/testing?

Shanklin: We currently get 10 percent of dry weight as oil. We think if we test genes from additional sources, we could boost that to 20 percent.

Then we need to figure out the optimal growth method. It might be sealed containers from the size of a car to the size of a shipping container. Or it could be open ponds. For growth in the open we would need to deploy what is known as kill gene technology. This means that if it gets into the wild, it will die after a couple of generations and not become invasive. Making oil takes a lot of energy though, so it will be much weaker than its non-oil-making relatives and at a competitive disadvantage anyhow.

We are also working with chemical engineers to optimize the process of oil recovery and conversion to biodiesel.

Tech Briefs: How far away are we from this becoming completely ubiquitous?

Shanklin: The first commercial use might be five years away. It could produce transportation fuel on-site for companies that transport goods to markets. Being made from atmospheric CO2, it represents a step toward net zero carbon emissions.

Tech Briefs: Do you have any advice for scientists aiming to bring their ideas to market?

Shanklin: It’s a significant challenge to move from the bench to commercial applications. Seek out partners, don’t expect them to find you. Try and scale up to pilot scale from the laboratory bench scale to give potential partners confidence that it will work when they scale it up for commercial production. This is called “crossing the valley of death” in the trade, otherwise known as de-risking for industry.

Tech Briefs: Anything else you’d like to add?

Shanklin: Working in a team with BNL’s expertise in lipid biochemistry and the regulation of plant metabolism along with Cold Spring Harbor’s expertise in genomics and genetics has been very productive and fun.