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Pinpointing lipid accumulation in algae

Pinpointing lipid accumulation in algae

By Patti Koning

Algae has great promise as a potential source of biofuel — it grows easily and abundantly and can be rich in triacylglycerols, a lipid that can be extracted and converted into fuel. But after 30 years of research, little is known about the mechanism that produces triacylglycerols, the missing link that could take us from algae to “oilgae.”

CATCH AND RELEASE FOR ALGAE — Using laser trapping Raman spectroscopy, Huawen Wu and Seema Singh (both 8634) are able to analyze lipid accumulation in a single algae cell without changing or harming it, something not possible with conventional methods. This technique will greatly accelerate research into candidates for algal biofuel production. (Photo by Randy Wong)

“Certain algae are known to be really good candidates because of their lipid accumulation,” says Blake Simmons (8630). “One of the biggest problems is a lack of fundamental understanding about how these algae do what they do, what environments they do it in, and how we can manipulate them to make them more attractive from a biofuel production standpoint.”

Sandia researchers may have found the key to solving that problem with a new method of analyzing algae at the cellular level. Over the past two years, Seema Singh (8634) has led a Laboratory Directed Research and Development (LDRD) project to develop a real-time in situ method for lipid profiling using laser-trapping Raman spectroscopy (LTRS). While the LDRD project is studying microalgae, the method is universal and can be used for several applications in the field of lipidomics.

“We believe our method has the potential to transform the field of algal biofuel research,” says Seema. “Now we can analyze a single cell’s chemical fingerprint, not just once, but over and over and examine how different factors affect lipid production. The overarching goal is multifactorial correlation of biotic and abiotic factors to algal growth and lipid accumulation.”

The method combines two existing technologies — laser trapping and Raman spectroscopy — that have been widely used for many years. The novelty of Seema’s approach is bringing the two together to study algae. 

After months of work to select the best algae candidates, the team began with Raman spectroscopy as a way to develop chemical fingerprints of single-cell algae. “The ‘ah-ha’ moment came when we were saying it would be great if we could just observe these events on a single-cell basis,” Blake says. “Then we realized we could, that the technologies had been demonstrated separately, but that no one had combined these techniques for analyzing lipids in microalgal cells before.”

Seema brought postdoc Huawen Wu (8634) onto the project because of his experience using LTRS to study lipids in human cells. Algae, he says, are comparatively easier because their lipids are more abundant.

 The laser immobilizes a single cell in an optical trap; once immobilized, the cell is interrogated with a Raman spectroscopy system. “You can see what lipids are present, the degree of unsaturation, and even the relative quantitation of the absolute lipids present. Then you let the cell go,” Blake explains. “It’s basically a catch-and-release program we developed for algae.”

 That catch-and-release aspect is important because other methods can significantly perturb and even destroy the algae in the process of analysis. Two methods are commonly used to study algae today — extraction and fluorescence labeling.

In the extraction method, researchers grow and harvest algae, and then extract the lipids to analyze them. And then start over. The process is slow, involves tricky chemicals and laborious fractionation, and ultimately doesn’t yield much specific information unless you use very expensive mass spectrometry equipment.

“It’s an Edisonian, empirical approach that doesn’t easily get down into the important details of lipid composition in the algae,” says Blake.

The fluorescence labeling method doesn’t fare much better. “It’s a brute force method,” says Blake. “The dye doesn’t like being in water, so it partitions to the lipid within the algae. You can then use that signal as the dye is taken up by the cell to get a rough metric of how much lipid is there. You still don’t know what kind of lipid or how much, and it doesn’t work for all cells. And it’s toxic to the algae.”

To exacerbate the problem, algae themselves are rich in fluorescent pigments and create interference. Sometimes they are just too bright to be seen clearly under a fluorescent microscope.   

In contrast, the LTRS method is fast, yielding a complete analysis within a few seconds, and insensitive to culture conditions. A researcher can repeat the analysis over and over within the same sample while adjusting parameters.

“You can grow a culture, test it at one temperature and again at an increased temperature, or even as the culture is getting warmer,” says Seema. “We’re able to track these changes, get a handle on the fundamental biological process of lipid production, and establish a constitutive relationship between growth conditions and lipid yield and profile.”

This method also allows researchers to quickly amass hundreds of data points, which then enable the development of algorithms to conduct a ratiometric analysis of lipid, chain length, and degree of unsaturation.

“At the outset, we wanted to demonstrate the ability to grab a cell and understand its total profile. Then we got greedy and began wondering if we could use this method to predict melting temperature,” says Seema. “Now we can use this calibration plot for predictions. You bring me any algae, or any other cell for that matter, and we can probe it and make a prediction on its melting temperature within plus or minus 1 degree. This allows us to gain insight into a particular algae’s suitability for a biofuels application.”

Because of Sandia’s unique experience with Raman spectroscopy for microalgae, the corporation is now partnering with BaySpec to commercialize a pond-deployable algal analyzer.

The project received a phase one National Science Foundation Small Business Innovation Research grant and Seema is hopeful it will be funded for phase two.

This work was published in the March 2011 issue (vol. 108, 3809–3814) of the Proceedings of the National Academy of Sciences USA. The research also was recently highlighted by the Nature Publishing Group and the LIPID MAPS consortium in the March 23, 2011, article, “Techniques: Lipids probed by Raman spectroscopy in living cells.”

“One beauty of this method is it isn’t limited to algae — you can use it to study virtually anything,” Seema says. “If we can develop something that’s easy to use and get it into the hands of researchers in academia, national labs, and industry, there is so much that can be discovered. The potential, I think, is limitless.” – Patti Koning

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