Scientists at the Department of Energy's Oak Ridge National Laboratory and the University of Cincinnati achieved a breakthrough in understanding the vulnerability of microbes to the butanol they produce during fermentation of plant biomass. The discovery could pave the way for more efficient production of domestic fuels, chemicals and materials.
The team used ORNL's neutron scattering capabilities and molecular dynamics simulations to analyze the fermentation process producing butanol, an energy-packed alcohol that can be used as a biofuel, solvent or chemical feedstock.
Methods developed so far to biologically produce the alcohol face a major hurdle: butanol is toxic to the very microorganisms that produce it. This toxicity limits the amount of butanol that can be generated during fermentation, presenting a challenge to biobased production.
Scientists focused their analysis on specialized regions within the microbes' cell membranes called membrane domains that play a critical role in organizing proteins and maintaining cell stability. Using tiny, bubble-like structures called liposomes that mimic cell membranes, researchers found that butanol tends to accumulate unevenly around the membrane, causing smaller domains to merge into larger ones and prompting the thinning of some regions of the membrane, as described in the journal Langmuir . Exposure to butanol ultimately triggered changes in the membrane's organization associated with cell stress and less-efficient fermentation.
By identifying for the first time the specific mechanisms of toxicity, scientists can, for instance, work toward developing microbial strains with stronger, more resistant membranes, identify microorganisms with greater tolerance to butanol, or develop other methods to reduce membrane thinning.
Neutrons, simulations expose toxicity effects
Researchers investigated the processes occurring during fermentation using the Biological Small-Angle Neutron Scattering instrument , or Bio-SANS, part of the Center for Structural Molecular Biology , or CSMB, located at the High-Flux Isotope Reactor, a DOE Office of Science user facility. Using neutrons allowed for nondestructive testing of the membrane mimic, letting scientists see the structures and arrangements of molecules. The instrument is supported by the DOE Biological and Environmental Research program's Biological Systems Science Division.
Neutrons generated by the reactor probed details of the sample, revealing that membrane domain size increased as the amount of butanol rose, and uncovering the membrane thinning effect.
Bio-SANS gave scientists the ability "to peer at what is happening at the nanometer-length scale to the structure of the membrane," said Jon Nickels, principal investigator for the project and associate professor of chemical and environmental engineering at the University of Cincinnati.
The instrument "was able to resolve where the butanol was going in the membrane," said Hugh O'Neill, project collaborator and CSMB director at ORNL. "That's much tougher to do with X-rays, which let you see overall thickness. Neutrons give you the ability to probe the interior of the membrane to help determine how the butanol is distributed."
The team then leveraged molecular dynamics simulations, a computer-based method calculating how atoms and molecules move and interact over time, to get a detailed, dynamic view of molecular behavior. The simulations supported experimental observations and revealed details on how butanol accumulated at the membrane domain interface.
The simulations were run on a supercomputer at the National Energy Research Scientific Computing Center, a DOE Office of Science user facility at Lawrence Berkeley National Laboratory. The results provided "a complete atomistic picture that can tell us a great deal about these systems and guide future experiments," Nickels said.
The study was supported by the Solvent Disruption of Biomass and Biomembranes Science Focus Area at ORNL, also known as the Biofuels SFA, funded by the DOE Biological and Environmental Research program's Biological Systems Science Division. The SFA aims to provide fundamental insights into how solvents like butanol alter the structure and arrangement of plant cell walls and microbial membranes.
"This could be a new fundamental mechanism for solvent toxicity, where the solvent does not have to disrupt the 'bulk' membrane but, rather, targets a 'weak' spot in the membrane - the domain interface," said Brian Davison, chief scientist for systems biology and biotechnology and lead for the Biofuels SFA at ORNL.
Leveraging biological expertise, big science tools
The butanol project was a "key step in testing a novel hypothesis about the way alcohols are interacting with cells in the fermentation process. We investigated the biophysical basis for this hypothesis, and now we've demonstrated that it physically checks out," Nickels said.
The findings "provide us with new targets to reduce the influence of these fermentation products," said Luoxi Tan, first author and a postdoctoral researcher at ORNL. "We now know to ask if more stable membrane domains could significantly reduce cell stress during fermentation, resulting in more efficient conversion and higher butanol titers."
"ORNL's neutron science capabilities and deep expertise in biology and computational science were key to this project," Davison said. "The SFA structure enabled the formation of a multidisciplinary 'A-Team' that led to a full analysis of the process. You could have 'just' had the neutron scattering that showed you that the domain sizes were increasing. But without the molecular dynamics simulations you wouldn't understand why."
The project represented a successful collaboration between academia and the DOE national labs, Nickels added. "ORNL has an ideal suite of capabilities and expertise for studying the structure of cell membranes," he said. "Once you have completed your analysis with neutrons, you can develop models to fit the data and extract things like the membrane partitioning of the alcohol for a highly accurate molecular structure."
UT-Battelle manages ORNL for DOE's Office of Science, the single largest supporter of basic research in the physical sciences in the United States. The Office of Science is working to address some of the most pressing challenges of our time. For more information, please visit energy.gov/science . - Stephanie Seay
