ScienceDaily (May 14, 2012) — 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
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 co-author of a paper reporting the results of this research in the Proceedings of the National Academy of Sciences (PNAS).
Other co-authors are Jane Khudyakov, Patrik D'haeseleer, Sharon
Borglin, Kristen DeAngelis, Hannah Wooa, Erika Lindquist and Terry
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
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
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 lignolyticus.
"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 liquid."
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.
This work was supported by the DOE Office of Science.
Blake Simmons, in addition to his JBEI appointment, is also a
scientist with the Sandia National Laboratories. Michael Thelen, in
addition to his JBEI appointment, is also a scientist with the Lawrence
Livermore National Laboratory.
Note: Materials may be edited for content and length. For further information, please contact the source cited above.
- Jane I. Khudyakov,
Sharon E. Borglin,
Kristen M. DeAngelis,
Erika A. Lindquist,
Terry C. Hazen,
Blake A. Simmons,
and Michael P. Thelen. Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium, Enterobacter lignolyticus. PNAS Plus, 2012 DOI: 10.1073/pnas.1112750109