Ionic liquids are environmentally benign organic salts often used as green chemistry substitutes for volatile organic solvents. At JBEI, ionic liquids are being used to pretreat cellulosic biomass to make it more digestible for microbes. Image: Roy Kaltschmidt, Berkeley Lab
In the search for technology by which economically
competitive biofuels can be produced from cellulosic biomass, the combination
of sugar-fermenting microbes and ionic liquid solvents looks to be a winner
save for one major problem: The ionic liquids used to make cellulosic biomass
more digestible for microbes can also be toxic to them. A solution to this
conundrum, however, may be in the offing.
Researchers with the U.S. Department of Energy
(DOE)’s Joint BioEnergy Institute (JBEI), a multi-institutional partnership led
by Lawrence Berkeley National Laboratory (Berkeley Lab), have identified a
tropical rainforest microbe that can endure relatively high concentrations of
an ionic liquid used to dissolve cellulosic biomass. The researchers have also
determined how the microbe is able to do this, a discovery that holds broad
implications beyond the production of advanced biofuels.
“Our findings represent an important first step in
understanding the mechanisms of ionic liquid resistance in bacteria and provide
a basis for engineering ionic liquid tolerance into strains of fuel-producing
microbes for a more efficient biofuel production process,” says Blake Simmons,
a chemical engineer who heads JBEI’s Deconstruction Division and one of the
senior investigators for this research.
Adds Michael Thelen, the principal investigator and
a member of JBEI’s Deconstruction Division, “Our study also demonstrates that
vigorous efforts to discover and analyze the unique properties of
microorganisms can provide an important basis for understanding microbial
stress and adaptation responses to anthropogenic chemicals used in industry.”
Thelen is the corresponding author and Simmons a coauthor
of a paper reporting the results of this research in the Proceedings of the National Academy of Sciences (PNAS).
The burning of fossil fuels releases nearly 9
billion metric tons of excess carbon into the atmosphere each year. Meanwhile
the global demand for gasoline and other petroleum-based fuels continues to
rise. Clean, green, and renewable fuels that won’t add excess carbon to the
atmosphere are sorely needed. Among the best candidates are advanced biofuels
synthesized from the cellulosic biomass in non-food plants. Such fuels could
displace petroleum-based fuels on a gallon-for-gallon basis and be incorporated
into today’s vehicles and infrastructures with no impact on performance.
To this end, researchers at JBEI have already
engineered a strain of E. coli bacteria to digest the cellulosic
biomass of switchgrass, a perennial grass that thrives on land not suitable for
food crops, and convert its sugars into biofuel replacements for gasoline,
diesel and jet fuels. A key to this success was the pretreatment of the
switchgrass with an ionic liquid to dissolve it.
“Unlike the starch sugars in grains, the complex
polysaccharides in cellulosic biomass are semicrystalline and deeply embedded
within a tough woody material called lignin,” Simmons says. “Lignin can be
removed and cellulose crystallinity can be reduced if the biomass is pretreated
with ionic liquids, environmentally benign organic salts often used as green
chemistry substitutes for volatile organic solvents.”
Current strategies for processing cellulosic
biomass into biofuels involve multiple production steps in which the bulk of
ionic liquids used to pretreat biomass can be washed out before the microbes
are added. However, to cut production costs, a “one pot” strategy in which
processing steps take place in a single vat would be highly desirable. This
strategy requires microbes that can tolerate and grow in ionic liquids used to
pretreat cellulosic biomass.
In search of such microbes, a team of JBEI
researchers led by microbiologist and PNAS paper co-author Kristen
DeAngelis ventured into the El Yunque National Forest in Puerto
Rico, a tropical rain forest where microbial communities have
demonstrated exceptionally high rates of biomass decomposition, and a tolerance
to high osmotic pressures of the sort generated by exposure to ionic liquids.
They returned with a prime candidate in the SCF1 strain of Enterobacter
“We first determined that the SCF1 strain of Enterobacter
lignolyticus grows in the presence of the ionic liquid [C2mim]Cl at
concentrations comparable to the concentrations that remain in the cellulose
after pretreatment and recovery,” Thelen says. “Next, through a combination of
phenotypic growth assays, phospholipid fatty acid analysis, and RNA sequencing
technologies, we investigated the mechanisms by which SCF1 tolerates this ionic
Working in collaboration with researchers at DOE’s
Joint Genome Institute, another multi-institutional partnership led by Berkeley
Lab, Thelen and Simmons and their JBEI colleagues developed a preliminary model
of ionic liquid tolerance for SCF1.
“Our model suggests that SCF1 bacteria resist the
toxic effect of the [C2mim]Cl ionic liquid by altering the permeability of
their cell membrane and pumping the toxic chemical out of the cell before
damage occurs,” Thelen says. “These detoxifying mechanisms are known to be
involved in bacterial responses to stress, but not in a coordinated manner as
we have shown for the response of SCF1 to ionic liquid.”
Thelen says the information gained from this study
will be used at JBEI to help engineer new fuel-producing microbes that can
tolerate ionic liquid pretreatments. Beyond biofuels, the techniques developed
in this study should also be applicable to the screening of microbial responses
to other chemical compounds, such as antibiotics.