Targeting enzymes

One of the things that ideally needs to happen for cellulosic ethanol to become commercially viable is a reduction in the amount of enzymes required to break down cellulose into glucose – from 10–15mg of enzymes per gram of cellulose at the moment to just 0.5–1.0mg. Although this seems like quite a tall order, there is actually an obvious route to doing this, and that’s to increase the proportion of amorphous cellulose released from plant biomass by pre-treatment processes.

In plants, cellulose exists as a highly ordered, crystalline polymer, comprising aligned cellulose nanofibers held together by hydrogen bonds. But if crystalline cellulose is exposed to a solvent at high temperatures and pressures, the aligned nanofibers fall apart to form a disorganized, amorphous structure. Unsurprisingly, cellulose-degrading enzymes find it much easier to break down this disorganized, amorphous cellulose into its component glucose molecules than crystalline cellulose. But current acid-based pre-treatment processes tend to the release the cellulose in its crystalline form rather than converting it into the amorphous form.

So what is needed is a pre-treatment process that can both release cellulose from biomass and convert it to the amorphous form, and that is what a team of US researchers, led by Shishir Chundawat at Rutgers University-New Brunswick, recently came up with. Their work builds on previous research showing that anhydrous liquid ammonia can convert crystalline cellulose to the amorphous form, by disrupting the hydrogen bonding network that holds the cellulose nanofibers together. But this still requires comparatively high temperatures and pressures, which prevents it being a cost-effective option for commercial cellulosic ethanol production.

As they report in a paper in Green Chemistry, Chundawat and his team managed to develop a version of this process that works at ambient temperatures and pressures. To do this, they simply treated the crystalline cellulose twice, albeit with slightly different ammonia-based substances. 

First off, they treated the crystalline cellulose with anhydrous liquid ammonia, which at ambient temperatures and pressures causes the crystalline structure to partially collapse, forming channels through the nanofibers. Next, they treat the cellulose with an ammonium thiocyanate salt dissolved in liquid ammonia, which can access deep into the cellulose through the channels, allowing it to fully disrupt the hydrogen bonding network and convert the cellulose to the amorphous form. Breaking down this amorphous form into glucose required just half the amount of enzymes required for breaking down crystalline cellulose.

Going one step better, a team of Japanese researchers led by Takayasu Kawasaki at Tokyo University of Science has managed to do away with enzymes entirely, by replacing them with a laser that can blast the cellulose into its component sugar molecules. Specifically, they used a near- and mid-infrared free electron laser to produce beams at various infrared wavelengths.

They did this because the bonds in many molecules, including cellulose, absorb infrared wavelengths. At low intensities, this absorption can be used to detect and identify these molecules by infrared spectroscopy, because different bonds absorb different wavelengths. At higher intensities, however, this absorption can eventually cause the bonds to break.

There are three main bonds in cellulose, between the carbon, oxygen and hydrogen atoms, and each absorbs different infrared wavelengths. Initially, Kawasaki and his team tried using a single wavelength to break all the bonds in powdered cellulose, but that didn’t work. So they then tried irradiating the cellulose with laser beams at the three wavelengths most efficiently absorbed by the three bonds, which worked much better. As they report in a paper in Energy & Fuels, this blasted the cellulose apart, producing glucose and cellobiose (a disaccharide that can easily be converted to glucose) at high yields.

Both teams worked initially with pure cellulose, but are confident that their processes should work with raw biomass as well. Chundawat and his team have already shown that their ammonia process can produce amorphous cellulose from corn stover, while Kawasaki and his team are now turning their laser on the other components of biomass.

The views represented here are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd. or of the SCI.