Increased membrane fluidity, which causes cofactor leakage and loss of membrane potential, has long been documented as a cause for decreased cell growth during exposure to ethanol, butanol, and other alcohols. Reinforcement of the membrane with more complex lipid components is thus thought to be beneficial for the generation of more tolerant organisms. In this study, organisms with more complex membranes, namely, archaea, did not maintain high growth rates upon exposure to alcohols, indicating that more complex lipids do not necessarily fortify the membrane against the fluidizing effects of alcohols. In the presence of alcohols, shifts in lipid composition to more saturated and unbranched lipids were observed in most of the organisms tested, including archaea, yeasts, and bacteria. However, these shifts did not always result in a decrease in membrane fluidity or in greater tolerance of the organism to alcohol exposure. In general, organisms tolerating the highest concentrations of alcohols maintained membrane fluidity after alcohol exposure, whereas organisms that increased membrane rigidity were less tolerant. Altered lipid composition was a common response to alcohol exposure, with the most tolerant organisms maintaining a modestly fluid membrane. Our results demonstrate that increased membrane fluidity is not the sole cause of growth inhibition and that alcohols may also denature proteins within the membrane and cytosol, adversely affecting metabolism and decreasing cell growth.

Despite extensive studies on microbial and enzymatic lignocellulose degradation, relatively few Archaea are known to deconstruct crystalline cellulose. Here we describe a consortium of three hyperthermophilic archaea enriched from a continental geothermal source by growth at 90 °C on crystalline cellulose, representing the first instance of Archaea able to deconstruct lignocellulose optimally above 90 °C. Following metagenomic studies on the consortium, a 90 kDa, multidomain cellulase, annotated as a member of the TIm barrel glycosyl hydrolase superfamily, was characterized. The multidomain architecture of this protein is uncommon for hyperthermophilic endoglucanases, and two of the four domains of the enzyme have no characterized homologues. The recombinant enzyme has optimal activity at 109 °C, a halflife of 5 h at 100 °C, and resists denaturation in strong detergents, high-salt concentrations, and ionic liquids. Cellulases active above 100 °C may assist in biofuel production from lignocellulosic feedstocks by hydrolysing cellulose under conditions typically employed in biomass pretreatment.

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