Moderate halophilic bacteria, but not extreme halophilic archaea can alleviate the toxicity of short-alkyl side chain imidazolium-based ionic liquids

Imidazolium-based ionic liquids (IL) with short-alkyl side chain such as 1-ethyl-3-methyl-imidazolium chloride ([Emim]Cl) and 1-butyl-3-methyl-imidazolium chloride ([Bmim]Cl) has immense application potential including in lignocellulosic bioenergy production. But they are toxic to most microorganisms, and those isolated from different environments as IL-tolerant have salt tolerance capabilities. This study evaluates the relationship between salt and [Emim]Cl tolerance of microorganisms using different salinity sediments (2-19%) and brines (35%) of India's largest inland hypersaline lake, Sambhar in Rajasthan as the model system. While samples with 2% and 35% salinities do not yield any [Emim]Cl (100 mM) tolerant colonies, others have 6-50% colonies tolerant to the IL. Similar trend was observed with 50 mM [Bmim]Cl. Moderate halophilic isolates of genera Halomonas and Bacillus (growth in 0.7-3.0 M NaCl) isolated from the sediments could grow in as high as 375 mM [Emim]Cl, or 125 mM [Bmim]Cl facilitated by higher synthesis, and uptake of organic osmolytes; and up to 1.7-fold increased activity of active efflux pumps. [Bmim]Cl was more toxic than [Emim]Cl in all performed experiments. [Emim]Cl-adapted cells could trounce IL-induced stress. Interestingly, enrichment with 100 mM [Emim]Cl resulted in increase of IL-tolerant colonies in all sediments including the one with 2% salinity. However, the salt saturated brines (35%) do not yield any such colony even after repeated incubations. Extreme halophilic archaea, Natronomonas (growth in 3.0-4.0 M NaCl) isolated from such brines, were exceedingly sensitive to even 5 mM [Emim]Cl, or 1 mM [Bmim]Cl. Two additional extremophilic archaea, namely Haloferax and Haladaptatus were also sensitive to the tested ILs. Archaeal sensitivity is possibly due to the competitive interaction of [Emim]⁺ with their acidic proteome (15.4-17.5% aspartic and glutamic acids, against 10.7-12.9% in bacteria) that they maintain to stabilize the high amount of K⁺ ion accumulated by salt-in strategy. Thus, general salt adaptation strategies of moderate halophilic bacteria help them to restrain toxicity of these ILs, but extremophilic archaea are highly sensitive and demands meticulous use of these solvents to prevent environmental contamination.