Discovering microbes naturally tolerant to salty liquids and understanding their mechanisms of tolerance should significantly enhance biofuel production.

—Michael Thelen

The team describes an in-depth characterization of native resistance of Enterobacter lignolyticus strain SCF1, a lignocellulolytic bacterium from tropical rain forest soil, to the toxic effects of 0.5 M 1-ethyl-3-methylimidazolium chloride, an ionic liquid commonly used for lignocellulose pretreatment, in an open access paper published in the Proceedings of the Natural Academy of Sciences (PNAS).

We looked into how SCF1 tolerates this ionic liquid by using high-throughput growth assays, composition analysis of the bacterial cell membrane, and by determining all of the genes that are differentially expressed when SCF1 cells are exposed to an ionic liquid.

—Michael Thelen

By mapping these analyses onto all of the known metabolic reactions identified in the genome sequence of SCF1, the team found that the bacteria can resist the toxic effects of an ionic liquid by adjusting the composition of membranes to reduce cell permeability, and by increasing a type of transport protein that can pump the toxic chemical out of the cell before damage occurs.

Different bacterial responses to ionic liquid. In (A), a fermentative bacterium is exposed to sugars for biofuel synthesis along with an ionic liquid (IL) used during the production process. The ionic liquid exerts a toxic effect soon after it enters the cell (B). In contrast, the rainforest isolate E. lignolyticus in (C) can tolerate relatively high levels of the ionic liquid, even as it secretes enzymes (D) that degrade plant cellulosic material to glucose and other sugars. A mechanistic model for bacterial resistance to ionic liquid includes: reducing membrane permeability to ionic liquid by remodeling the phopholipid bilayer (introduction of kinked fatty acids in lower left inset); removing intracellular ionic liquid through membrane transport proteins (efflux pumps shown as purple cones in lower middle inset); and, slowing the influx of ionic liquid by decreasing the number of porins (transmembrane cylinders in lower right inset). Green objects: glucose (rings) and cellulosic materials (sheets); red objects: ionic liquid; purple objects: enzymes. Click to enlarge.

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.

Other Livermore researchers included: Jane Khudyakov and Patrik D’haeseleer. Other institutions include: Lawrence Berkeley National Laboratory, Joint Genome Institute and Sandia National Laboratories. JBEI is one of three Bioenergy Research Centers established by the DOE Office of Science in 2007. It is a scientific partnership led by Lawrence Berkeley Lab and includes the Sandia National Laboratories, the University of California campuses of Berkeley and Davis, the Carnegie Institution for Science, and Lawrence Livermore National Laboratory.

Resources

• Jane I. Khudyakov, Patrik D’haeseleer, Sharon E. Borglin, Kristen M. DeAngelis, Hannah Woo, Erika A. Lindquist, Terry C. Hazen, Blake A. Simmons, and Michael P. Thelen (2012) Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium, Enterobacter lignolyticus PNAS doi: 10.1073/pnas.1112750109