Sandia LabNews

Small-scale technologies for large-scale biofuels production

Small-scale technologies for large-scale biofuels production

By Patti Koning

Small but speedy Ñ Rajiv Bharadwaj holds up the chip that is the basis of a high-throughput method to evaluate the effectiveness of digestion methods. The method, featured on the cover of Analytical Chemistry this month, provides quantitative information about all sugars in a sample, not just the monosaccharides.

You could call November “Microfluidics meets Bioenergy Research” month. Work led by Rajiv Bharadwaj and Aarthi Chandrasekaran (both 8621), and performed at the Joint Bio Energy Institute (JBEI), this month appears in three prominent scientific journals, including on the cover of Analytical Chemistry.

Small but speedy — Rajiv Bharadwaj holds up the chip that is the basis of a high-throughput method to evaluate the effectiveness of digestion methods. The method, featured on the cover of Analytical Chemistry this month, provides quantitative information about all sugars in a sample, not just the monosaccharides. (Photo by Randy Wong)

“We’re coming back from a 35-year hiatus on bioenergy research. There was a lot of effort in the 1960s and 1970s, but when gas prices tanked, the lack of funding and political will brought the work to a halt,” says Anup Singh (8621). “With the crises in oil-producing nations and the sudden spike in oil prices a few years ago, research has started up again with a major influx of funds from government and industry.” Anup is the manager of the biosystems and bioengineering group at Sandia and director for high-throughput chemical analysis at JBEI.

Because of that long pause, the tools to conduct basic research are sorely out of date. Advances in other areas of biology often don’t easily translate to bioenergy.

“We need tools that allow researchers to screen processes and molecules in a high-throughput manner and do assays faster and cheaper with smaller amounts,” Anup says. “The microfluidics core at JBEI has been examining the state of the art to see how the lack of technology is slowing down researchers in terms of getting to their biological end goals. We now have some great successes, with a common theme of increasing speed and efficiency, to share with the rest of the bioenergy community.”

Rajiv is the lead author of a paper titled “Microfluidic glycosyl hydrolase screening for biomass-to-biofuel conversion,” the cover story for the Nov. 15 issue of Analytical Chemistry. The other authors (all part of JBEI) are Zhiwei Chen (8634), Supratim Datta (8634), Bradley Holmes (8634), Rajat Sapra (8634), Blake Simmons (8630), Paul Adams (Lawrence Berkeley National Laboratory), and Anup.

This paper describes a new method for screening the effectiveness of digestion, the process by which treated biomass is converted into fermentable sugars. To evaluate a particular digestion method, researchers need to know the profile of the various sugars it produces. The characterization of various oligosaccharides produced during biomass digestion is critical for the design of suitable reactors, enzyme cocktail compositions, and biomass pretreatment schemes.

The state-of-the-art screening technique is high-performance liquid chromatography (HPLC), which takes several hours to complete. Rajiv’s research moved this screening process to a chip, drastically reducing the amount of sample needed and slashing the time from hours to just minutes.

“Our goal was to create something you can keep running all day and process hundreds of samples in the time it used to take to do one. This method is much more sophisticated because it provides quantitative information about everything in the sample, not just the monosaccharides,” he says.

This chip-based screening method also has potential for industrial settings, Rajiv adds. “Industrial production will be greatly scaled up from what we are doing in the lab, so the ability to troubleshoot quickly is essential,” he explains. “You can also use this method to prescreen each batch of biomass digestion so you don’t waste fermenter time.”

Another project of Rajiv’s, described in the paper, “High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration,” appears in Bioresource Technology. The other authors are April Wong (8621), Bernard Knierim (JBEI), Seema Singh (8934), Bradley, Manfred Auer (JBEI), Blake, Paul, and Anup.

This project tackles the logistical problem of accurately metering small amounts of insoluble biomass for enzymatic digestion. The metering of ionic liquid (IL) pretreated biomass is typically performed either by weighing biomass or dispensing a biomass slurry solution. Both of these approaches are cumbersome and prone to variability, especially for high-throughput screening.

Rajiv hit upon a novel solution by observing that when IL-solublized biomass is placed in a microwell containing water, the biomass precipitates to the bottom. In this volumetric metering method, a researcher washes the biomass with water and adds the enzyme, all within the same microwell. No need for weighing biomass or metering slurry.

This method is also an ideal way to prepare a pure sample of biomass for imaging. “The imaging people see this as an imaging platform,” Rajiv says. “That wasn’t our original intent, but it’s another application of this method.”        

Speed and efficiency also are the focus of the third JBEI paper. “A microscale platform for integrated cell-free expression and activity screening of cellulases” appears in the November issue of the Journal of Proteome Research. Aarthi is the lead author with contributions from Rajiv, Joshua Park (8634), Rajat, Paul, and Anup. 

This work addresses the problem of screening hundreds of thousands of variants of cellulases, which is currently done by expressing into E. coli or yeast, a process that can take weeks.

“The question is, ‘can we bypass the entire process by doing the first level of screening in something that is higher throughput, much faster, and simpler?’ And expressing cell-free, with no need for living E. coli?” Aarthi asks.

The research team developed a first-pass screening device for quantitative large-scale screening of cellulase variants. They adapted commercial off-the-shelf cell-free expression kits to express a large number of celullases. Using a microfluidics platform, the scientists integrated an assay for evaluation and connected it to a fluorescent readout, enabling what Aarthi terms “a one-stop shop.”

“It’s a quick, simple solution to a very costly problem,” she says. “The microfluidics platform achieves the entire process of transcription, translation, and activity screening within two or three hours, compared with the days necessary for conventional cell-based cellulase expression, purification, and activity screening.”

By performing expression and screening within the same reaction volume, researchers can express many variants of cellulases and know almost immediately if they are active or not. Scaling down the dimension and volume to a microfluidics platform accelerates the process and reduces the amount of reagent needed, a tremendous cost savings.

“The methods described in these papers will enable scientists at JBEI and beyond to push the boundaries of bioenergy research,” Anup says. “The faster we can screen and evaluate components of the biomass to biofuel process, the closer we get to moving biofuels from a concept to a reality.”

JBEI is now one of three new DOE Bioenergy Research Centers. This San Francisco Bay Area scientific partnership is led by Lawrence Berkeley National Laboratory and includes Sandia, the University of California (UC) campuses at Berkeley and Davis, the Carnegie Institution for Science, and Lawrence Livermore National Laboratory.