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

Ecotoxicology and Environmental Safety 184 (2019) 109634

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

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Srikanta Pal, Abhijit Sar, Bomba Dam∗ Microbiology Laboratory, Department of Botany (DST-FIST & UGC-DRS Funded), Institute of Science, Visva-Bharati (A Central University), Santiniketan, West Bengal, 731235, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Hypersaline lakes Moderate and extreme halophiles Bacteria and archaea Ionic liquid tolerance Active efflux Acidic proteome

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 1.7fold 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.

1. Introduction Ionic liquids (ILs), are salts with large organic cations, such as imidazolium and are preferred over volatile organic solvents due to their low vapor pressure, non-flammability, thermo-stability and recycling properties (Dadi et al., 2006). Hydrophilic imidazolium-based ILs with short-alkyl side chain, such as 1-ethyl-3-methylimidazolium chloride, [Emim]Cl or 1-butyl-3-methylimidazolium chloride, [Bmim]Cl can efficiently solubilize and deconstruct lignocellulosic biomass and thus are preferred solvents in biofuel production (Brandt et al., 2013; Dadi et al., 2006; Docherty and Kulpa, 2005; Klein-

∗

Marcuschamer et al., 2011). These IL-treated biomass becomes porous, amorphous and more accessible to cellulase for hydrolysis (Dadi et al., 2006). They have also been advocated as one of the most efficient solvent for catalysis and synthesis in other industries and bioreactor technologies (Moniruzzaman et al., 2010; Quijano et al., 2010; Welton, 1999). However, their use in industrial settings is constrained by their toxicity towards the presently used microorganisms or their enzymes (e.g., cellulases) (Bubalo et al., 2017; Docherty and Kulpa, 2005; Megaw et al., 2013; Santos et al., 2012). In fact, these two, and other similar ILs have been found to be toxic to most microorganisms, and are now characterized as “contaminants on the horizon” or “contaminants

Corresponding author. E-mail address: [email protected] (B. Dam).

https://doi.org/10.1016/j.ecoenv.2019.109634 Received 5 April 2019; Received in revised form 26 August 2019; Accepted 1 September 2019 Available online 11 September 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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spread on plates of heterotrophic media that were adjusted in terms of their pH, and salt concentration as in the original sample and incubated at 37 °C. For example, 13% salinity samples with pH 9.8 were spread on culture plates with pH 9.8 and 13% final NaCl concentration and so on. The heterotrophic media used contain (per liter) 5 g tryptone, 3 g yeast extract, 2 g KCl, and NaCl (as mentioned in respective experiment/ figure) with desired initial pH attained using 20% Na2CO3 solution, as commonly recommended for halophiles (Schneegurt, 2012). Natronomonas strains isolated from the lake brines were grown in heterotrophic medium having (per liter) 5 g tryptone, 3 g yeast extract, 4 g KCl, and NaCl (as mentioned in respective experiment/figure). Haloferax was isolated in course of this study from a saline pocket of Sunderban mangrove forest, West Bengal (21° 39′ N, 88° 20’ E). Haladaptatus sp. R4 was obtained from a solar evaporation saltern in Ramnagar, West Bengal, India (Sen et al., 2016). Both were grown in salt mix medium that contain (per liter): 660 mL of 30% modified salt water solution (described below), 3 g yeast extract, and 5 g tryptone, pH 7.4. Salt water solution contains (per liter) NaCl (as mentioned in respective experiment/figure), 30 g MgCl2.6H2O, 35 g MgSO4.7H2O, 7 g KCl, 5 mL 1 M CaCl2.2H2O, and 2 mL of 1 M Tris-Cl buffer (pH 7.5) (Savage et al., 2007; Schneegurt, 2012). Growth experiments of bacteria was also performed in minimal salt media that contain (per liter): NaCl (optimum for the isolate), 2 g KCl, 0.5 g NaHCO3, 3 g K2HPO4, 0.3 g KH2PO4, 0.2 g (NH4)2SO4, and 5 g glucose (Schneegurt, 2012). [Emim]Cl (98% purity), [Bmim]Cl (99% purity) and 1-ethyl-imidazole (95% purity) were purchased from Sigma, Germany. ILs of desired stocks was prepared in deionized water and filter through 0.22 μm filter paper before use. [Emim]Cl or [Bmim]Cl used for different growth experiments (concentrations mentioned in respective figures and tables) were added to the respective media that contain in addition the optimum amount of NaCl required by the particular bacteria/archaea. Growth was monitored from the increase in OD600 values with time. Aqueous solution of several imidazolium-based ILs, such as 1-butyl-3methylimidazolium tetrafluoroborate, 1-octyl-3-methylimidazolium bromide, 1-decyl-3-methylimidazolium bromide, 1-Dodecyl-3-methylimidazolium bromide, and 1-Butyl-3-methylimidazolium octyl sulphate are known to form micelle (Wang et al., 2007). But [Emim]Cl or [Bmim]Cl (at the used concentrations) does not impart any change in physical appearance of the culture media and looked similar to the nonIL control. Previous study with [Bmim]Br also reported no micelle formation (Wang et al., 2007). In fact, critical aggregation concentration of [Emim]Cl and [Bmim]Cl is very high (> 2 M) (Mao and Zhu, 2013), compared to that used (≤0.5 M) in the present study. Besides, bacteria may aggregate in medium with high IL concentrations, typically above critical micelle concentrations, but no change was observed in physical appearance either with naked eye or under microscope after addition of respective culture inoculums.

of emerging concern” (Bubalo et al., 2014; Koutinas et al., 2019; Richardson and Ternes, 2018). In addition, the highly soluble nature of [Emim]Cl and [Bmim]Cl increases the chance of environmental release either by accident or as industrial effluents if used as the preferred solvent in future. Thus, there is a need to understand the impact of these ILs on different group of microorganisms. In addition, attempts must be made to isolate and characterize microorganisms tolerant to them and reveal their molecular mechanism(s). The knowledge can be translated further into raising IL-tolerant members for industrial use. In fact, several attempts have been made to isolate bacteria those are tolerant/resistant to, or capable of degrading the imidazolium-based ILs from diverse environments. For instance, Enterobacter lignolyticus has been isolated from rain forests (DeAngelis et al., 2011; Khudyakov et al., 2012), Shewanella oneidensis from marine system (Alvarez et al., 2015), Rhodococcus erythropolis and many other bacterial genera from IL-enriched salt marsh, seawater, industrial soils, compost and activated sludge from municipal waste water treatment plants (Deive et al., 2011; Koutinas et al., 2019; Megaw et al., 2013; Santos et al., 2012; Simmons et al., 2014). A detailed list of such microorganisms, and the possible mechanism of their [Emim]-based IL tolerance is provided in Supplementary Table S1. Interestingly, almost all these bacteria are either themselves halotolerant and/or the genera to which they belong have known halotolerant representatives (Supplementary Table S1). Moreover, many of the environments chosen for isolating IL-tolerant microorganism have 1–5% salinity (Supplementary Table S1). But the response of moderate halophilic bacteria, and particularly extremophilic archaea towards [Emim]Cl or [Bmim]Cl is not known. In fact, no study till date has reported the possible effect of ILs on archaea in general. In addition, the direct relationship between salinity, IL-tolerance and microbial groups has never been assessed. This study therefore aimed to evaluate the relationship between salt and [Emim]Cl tolerance by microorganisms, using bacterial and archaeal strains isolated from different salinity samples within India's largest inland hypersaline water body, Sambhar Lake, Rajasthan. In addition we also tried to evaluate the mechanism for [Emim]Cl tolerance by moderate halophilic bacteria; and discuss the reason for archaeal sensitivity towards the IL. 2. Materials and methods 2.1. Sampling Sambhar Lake in Rajasthan, India is the largest inland hypersaline water body in India that remains dry almost throughout the year resulting in variations in physicochemical parameters in different segments of the lake (Upasani and Desai, 1990). Lake sediments have unique seasonal and diurnal fluctuations in temperature (10–49 °C), nutrient content, and salinity (2%–35%). The water activities in lake sediments/brines although not measured directly, are supposed to vary among samples and possibly reach values as low as 0.75 in the salt saturated brines (Grant, 2004). Sediments (top 10 cm) from four different regions of the lake with total salt concentrations ( ± 1%) of 2% (26° 53′ N, 75° 03′ E), 7% (26° 54′ N, 75° 04′ E), 13% (26° 55′ N, 75° 08′ E), and 19% (26° 56′ N, 75° 08′ E); and brine water (without lower sediment) from 35% salinity saltern (26° 54′ N, 75° 09’ E) were collected aseptically in sterile 50 mL falcon tubes and brought to the laboratory in ice buckets and stored at 4 °C (for culture-based work) or −20 °C (for DNA isolation) until further use. They are named respectively as S2, S7, S13, S19, and S35. Salinity and pH were measured at 25 °C using a portable multiparameter meter fitted with pH and electrical conductivity probes (Hana Instruments, USA).

2.3. Determination of minimum inhibitory concentrations (MIC) MIC of the isolates were determined in triplicate in heterotrophic media supplemented with IL concentrations of 50–500 mM of [Emim]Cl or 50–300 mM of [Bmim]Cl, with increments of 50 mM. Those with no growth in 50 mM were further checked with 1, 5, 10, 15, 20, 30, and 40 mM of the respective IL. Concentrations above 500 mM were not checked. Growth (OD600) was measured periodically up to one week for moderate and one month for extreme halophiles. MIC was determined from the lowest concentration at which no increase in OD600 value was recorded. 2.4. Preliminary screening for biodegradation Isolates were streaked onto minimal salts media containing [Emim]Cl (125 mM or 250 mM) or [Bmim]Cl (50 mM) as their sole carbon source and plates were incubated for two months and periodically examined for growth (Megaw et al., 2013). Similar incubations

2.2. Growth conditions of bacteria and archaea Total microbial count was made from appropriately diluted samples 2

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different concentrations of NaCl (0.7, 1.0, 1.7, 2.0, 3.0 M); or those with added [Emim]Cl (125 mM) or [Bmim]Cl (50 mM) were used. NaCl or [Emim]Cl induced efflux capability of the particular isolate has been reported by our group in a recent methods paper (Pal et al., 2019). Briefly, 2 mL culture (OD600 = 0.6) was harvested (5000×g, 10 min), washed and resuspended in equal volume of 50 mM sodium phosphate buffer (containing respective percentage of NaCl as in growth media) and 0.27 M glucose was added as energy source (Paixao et al., 2009). A time scan for 30 min was performed immediately after adding EtBr (final concentration 1 μg mL−1) in a fluorescence spectrophotometer (Hitachi F-7100, Japan). Fluorescence activity of EtBr is known to remain stable at concentrations of NaCl used in the assay (Timcheva et al., 1997).

were also done in liquid media. 2.5. Enrichment of sediment and brine samples 5 g sediments or 5 mL brines were mixed with 5 mL one-tenth diluted heterotrophic media for nutrient supply in petri-plates and supplemented with 100 mM [Emim]Cl. Plates were incubated at 37 °C for two weeks, except the brine set was kept for four weeks. Control sets were also incubated similarly without [Emim]Cl. 2.6. Adaptation experiment Halomonas sp. and B. agaradhaerens were grown in 250 mM [Emim]Cl containing heterotrophic medium, washed twice and referred as IL-adapted cells. These cells were then sub-cultured five times in ILfree medium and termed as de-stressed cells. Control sets contain washed cells grown in heterotrophic condition (without IL). IL-adapted, de-stressed, and control cells were used as starter culture to inoculate both heterotrophic, and minimal salt media supplemented with or without 250 mM [Emim]Cl. Growth was measured periodically from increase in OD600 value with time.

2.10. Estimation of cell membrane leakage after [Emim]Cl treatment Cell membrane leakage by [Emim]Cl-treatment was measured from increase in A260 and A280 values signifying release of nucleic acids and proteins in buffer. Heterotrophically grown cells (OD600 = 0.6) (without any IL) of bacteria and archaea were harvested (5000×g, 10 min), washed and resuspended in 50 mM sodium phosphate buffer containing NaCl (respective optimum concentrations). To the resting cells, 100 mM [Emim]Cl was added and incubated for 2 h. Sample was collected periodically, centrifuged (5000×g, 10 min) and A260 and A280 were measured in supernatant against a blank control set incubated without the IL. However, prior to measurement 100 mM [Emim]Cl was added to the blank set to neutralize any absorbance by the IL. Centrifuged supernatant after 2 h incubation were used as template (3 μl) in PCR reactions for 30 cycles with 16S rRNA gene specific primers for bacteria (5′-AGAGTTTGATCITGGCTCAG-3′ and 5′-CTGCTGC CTCCCGTAGG-3′) (Banerjee et al., 2018); or archaea (5′-CCGACGGTGAGRGRYGAA-3′and 5′-TTMGGGGCATRCNKACCT-3′) (Baker et al., 2003). 5 μl PCR products from each reaction was loaded in 1% agarose gel and electrophoresed to visualize bands on a gel documentation system (Bio-Rad, USA) and their intensities were measured by Image J software.

2.7. Stress enzyme activity Halomonas sp. and Bacillus agaradhaerens were grown in heterotrophic media (OD600 = 0.8) either with or without 250 mM [Emim]Cl, harvested, washed, and resuspended in sodium phosphate buffer with 1 M NaCl (final OD600 = 1.0). 2 mL culture was sonicated (30 s, 5 times) in ice at cycle-1, amplitude-100% (Heilscher, Germany), centrifuged in a microfuge (Hitachi, Japan) (10,000×g, 10 min, 4 °C) and supernatant was used as cell free extract (CFE) for assays of catalase, peroxidase and superoxide dismutase (SOD) (Banerjee et al., 2019). 2.8. Quantification of osmolytes Organic osmolytes were estimated from 20 mg (dry weight) heterotrophically grown cells of Halomonas sp. and B. agaradhaerens either without or with 250 mM [Emim]Cl. For proline estimation (Bates et al., 1973), cells were suspended in 1 mL aqueous sulfosalicylic acid (3%), sonicated (as mentioned in section 2.7), centrifuged, and to the supernatant 1 mL each of acid-ninhydrin and glacial acetic acid were added and incubated for 1 h at 100 °C. After cooling in ice, 2 mL toluene was added and vortexed at high speed for 15–20 s. Toluene present in upper layer was pipetted, warmed to room temperature, and A520 was measured against a blank set of toluene. For glycine betaine and choline (Grieve and Grattan, 1983) cell pellets were resuspended in deionized water, sonicated, centrifuged and supernatant was diluted (1:1) with 1 M H2SO4 and cooled in ice for 1 h. To 0.5 mL solution, cold KI-I2 (0.2 mL) was added, gently vortexed and incubated at 4 °C. After 16 h, the solution was centrifuged (10,000×g for 15 min at 4 °C) and pellet was dissolved in 9 mL of 1, 2 dichloroethane by vigorous vortexing. After incubating for ∼2 h, A365 was measured that correspond to total quaternary amines including choline and glycine betaine. Choline was estimated similarly, except the post-sonication supernatant was diluted (1:1) with potassium phosphate buffer (0.2 M, pH 6.8). Glycine betaine was estimated by subtracting amount of choline from total quaternary amines.

Pure cultures were molecularly identified from their 16S rRNA gene sequence homologies. Briefly, PCR was performed with genomic DNA (100 ng) used as template, and universal bacterial 16S rRNA gene specific primers 8F (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492R (5′ GGTTACCTTGTTACGACTT 3′) (Sar et al., 2018). DNA which did not yield PCR product with bacterial primer pair were amplified using archaea specific primers A571F (5′ GCYTAAAGSRNCCGTAGC 3′) and A1204R (5′ TTMGGGGCATRCNKACCT 3’) (Baker et al., 2003). PCR products were gel eluted, sequenced on Sanger platform, and BLASTN analysis was performed to find the closest relative. GenBank accession numbers are provided in Table 1. Osmolyte synthesis/uptake gene(s) in genomes of representative bacteria and archaea were identified using RAST platform (Aziz et al., 2008). For this, complete genome sequences of one or more representative genera were downloaded from NCBI database, annotated (with preserved gene calls) in RAST server, categorized into SEED subsystems and searched for the desired gene(s). Percentage of acidic amino acids was calculated from translated protein sequences.

2.9. Efflux assay

3. Results

Ethidium bromide (EtBr) fluorescence was used to monitor involvement of active efflux pump(s) (Martins et al., 2013; Pal et al., 2019) in NaCl or [Emim]Cl tolerance. The assay relies on the fact that EtBr (at sub-lethal dose) can enter cell passively by diffusion through general pores, but would be effluxed out only by active pumps (Paixao et al., 2009). Halomonas sp. grown in heterotrophic media with

3.1. [Emim]Cl and [Bmim]Cl-tolerant microorganisms in lake samples

2.11. 16S rRNA gene sequencing and bioinformatic analysis

The total salinity of lake sediments, S2, S7, S13, and S19; and brine, S35 used for the study were 2%, 7%, 13%, 19%, and 35% respectively. The pH was also very high and ranged from 9.1 to 10.5 (Supplementary Table S2). Total heterotrophic microbial load in the sediments were 3

4

MH593030 (99) Moderate Sambhar 8 6–10 1.0 0.7–3.0 Positive (375) 84 > 500 200

MH593029 (99) Moderate Sambhar 8 6–10 1.0 0.7–3.0 Positive (375) 71 > 500 250

Moderate Sambhar 9 7–11 1.0 0.7–3.0 Positive (375) 76 > 500 200

MH593031 (99) Moderate Sambhar 8 6–10 1.0 0.7–2.0 Positive (250) 70 450 100

MH620452 (100)

Salisedimini-bacterium halotolerans

Halotolerant Sambhar 7.5 5–9 0.4 0.1–0.7 Slow (75) 47 200 30

MH620451 (99)

Cellulomonas sp.

Genome sequence (Sen et al., 2016) Extreme Solar saltern 6 5–7 3.0 2.0–4.0 Slow (75) 20 150 15

Haladaptatus sp. R4

Bacillus locisalis

Halomonas sp.

Bacillus agaradhaerens

Archaea

Bacteria

h

Extreme Sunderban 7 5–8 3.0 2.0–4.0 Slow (50) 10 150 15

MH593039 (99)

Haloferax sp.

MH593032 to MH 593038 (99) Extreme Sambhar 9 8–10 4.0 3.0–5.0 No (5) 0 <5 <1

Natronomonas pharaonis (7 strains) i

b

Sambhar: Sambhar Lake sediments/brines; Solar saltern: A saltern in West Bengal (Sen et al., 2016); Sunderban: Saline pocket in Sunderban mangrove forest. Positive growth for optimizing pH and NaCl concentration in heterotrophic media refers to at least 50% growth as in the optimum set determined from OD600 value. c NaCl concentrations tested, 0.1, 0.4, 0.7, 1.0, 2.0, 3.0, 4.0, and 5.0 M. Concentrations > 5 M was not used as salt crystals are formed after incubation. d Heterotrophic media with optimum NaCl and pH was used. Positive refers to growth with up to a maximum 10-times longer lag-phase. Archaeal members have very slow or no growth with [Emim]Cl. Numbers within bracket is the highest concentration of [Emim]Cl that supports the growth. e Growth response was similar with 1-ethyl-imidazole, used at these concentrations. f Heterotrophic CFU count after 12 h incubation in suitable buffer with [Emim]Cl (concentrations used is the same as mentioned in row above), expressed as percentage as compared to the control set (buffer only). g MIC was determined using IL concentrations of 50–500 mM with increment of 50. Those with no growth in 50 mM were further checked with 1, 5, 10, 15, 20, 30, and 40 mM of the IL. h Three additional Haloferax strains (generous gift from a colleague) also showed similar IL-sensitivity. i All seven strains showed similar properties.

a

16S rRNA gene sequence accession no. (% identity to the nearest homolog) Halophilic nature Isolation source a Optimum pH (tested 4–12) pH supporting positive growth b Optimum NaCl (M) c NaCl (M) supporting positive growth b, c [Emim]Cl (mM) supporting growth d, e Cell viability (%) f MIC of [Emim]Cl (mM) g MIC of [Bmim]Cl (mM) g

Isolate

Table 1 Bacteria and archaea used in this study, their isolation source, and influence of pH, NaCl, [Emim]Cl, and [Bmim]Cl on their growth.

S. Pal, et al.

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(≤0.7 M, optimum 0.4 M). They could not tolerate even 100 mM [Emim]Cl and have poor growth in 75 mM of the IL (Table 1). Besides, seven additional isolates randomly picked from IL-free heterotrophic plates of saturated brines (S35) were extreme halophilic and could grow only in ≥3.0 M NaCl (optimum 4.0 M) (Table 1, Supplementary Fig. S2). Genomic DNA of all seven isolates does not yield any PCR product with primers specific for bacterial 16S rRNA gene, but were positive for the archaeal primers. Sequences of all seven isolates showed 99% homology with Natronomonas pharaonis. Interestingly, none could grow in as low as 5 mM [Emim]Cl even after 30 days prolonged incubation. The MIC for [Bmim]Cl was even lower (< 1 mM). 3.3. Physiological response in moderate halophiles to [Emim]Cl/[Bmim]Cl treatment

Fig. 1. Heterotrophic total count, and [Emim]Cl or [Bmim]Cl tolerant microbial load in Sambhar Lake sediments/brines. Total CFU count (control) of 2, 7, 13 and 19% salinity sediments, or 35% brines were determined on heterotrophic media supplemented with NaCl equivalent to respective salinity in samples. IL tolerant count was determined on similar media supplemented with 100 mM or 200 mM [Emim]Cl or 50 mM [Bmim]Cl. For clarity, data of 35% brine is also shown in inset. Values are mean of three independent enumerations ± standard deviation and are also provided in Supplementary Table S2.

To understand the mechanism of [Emim]Cl tolerance in moderate halophiles, Halomonas sp. and B. agaradhaerens, two of the best IL-tolerant bacteria isolated in course of this study and representatives of Gram negative and Gram positive members respectively were used in subsequent experiments. Both isolates can grow in nutrient limited minimal salt media supplemented with 250 mM [Emim]Cl (Fig. 3a and b). However, none could grow when [Emim]Cl (250 mM) or [Bmim]Cl (50 mM) was supplied as the sole carbon source (data not shown), suggesting their inability to degrade the ILs. Stress response was prominent in [Emim]Cl-treated sets as compared to IL-free controls with 1.2–2.3-fold, and 1.6–2.0-fold higher activities of catalase, peroxidase and superoxide dismutase for Halomonas sp. and B. agaradhaerens respectively (Fig. 3c and d).

sufficiently high (1.8 × 106 to 5.7 × 106 CFU g−1), as compared to the saturated brines (S35) (1.0 × 103 CFU mL−1) (Fig. 1 and Supplementary Table S2). However, the proportion of microorganisms tolerant to [Emim]Cl or [Bmim]Cl varied greatly. While S2 yield no colonies on plates supplemented with 100 mM [Emim]Cl, the trend changed in higher salinity samples (Fig. 1). Of the total heterotrophic load, 6% and 35% were tolerant to 100 mM [Emim]Cl in S7 and S13 respectively, that increased up to 50% in S19 (Fig. 1). Interestingly, saturated brines do not yield any IL-tolerant colony. Number of such colonies in each sample dropped when higher concentrations of [Emim]Cl (200 mM), or another IL, [Bmim]Cl was used at a much lower concentration (50 mM) (Fig. 1).

3.3.1. Compatible solute production and accumulation [Emim]Cl-stressed (250 mM) cells of Halomonas sp. and B. agaradhaerens compared to control produced 1.6, 11.6 and 7.3-fold; and 1.4, 11.0 and 4.9-fold higher proline, glycine betaine, and choline respectively (Fig. 3e and f). Even external addition of organic osmolytes (2 mM glutamate, proline or glycine betaine) in minimal salt media with 250 mM [Emim]Cl could substantially reduce the stress-induced lag-phase of both tested bacteria (Fig. 3a and b). Similar stress alleviation response was observed with 50 mM [Bmim]Cl (Supplementary Fig. S3).

3.2. Relation between microbial salt and [Emim]Cl/[Bmim]Cl tolerance To comprehend the rationale behind the observed variations in microbial IL-tolerance patterns in the samples, morphologically distinct colonies from 200 mM [Emim]Cl-containing plates were randomly picked, purified, and characterized in detail for their salt- and [Emim]Cl-tolerance capabilities. Three such bacteria, based on their 16S rRNA gene sequence homology, were molecularly identified as Halomonas sp. (99%), Bacillus agaradhaerens (99%), and Salisediminibacterium halotolerans (100%). The first two isolates showed positive growth in heterotrophic media with a wide range of salt (0.7–3.0 M NaCl, and 1.0 M optimum), as compared to S. halotolerans that can bear only 0.7–2.0 M NaCl (Table 1). They also have different [Emim]Cl tolerance capabilities with higher values (375 mM) recorded for the first two, but only 250 mM for the third (Table 1, Fig. 2). In fact, Halomonas sp., and B. agaradhaerens can grow (albeit slowly) even in 500 mM [Emim]Cl (Table 1). Tolerance pattern to [Bmim]Cl was similar with positive growth by the first two isolates in 125 mM of the IL, while only 50 mM for S. halotolerans (Supplementary Fig. S1). Thus, based on their NaCl and [Emim]Cl tolerance patterns bacteria of the lake were divided into two distinct groups, one with growth in wide range of NaCl and high concentrations of [Emim]Cl; and the other with growth in narrow range of NaCl and low concentrations of the IL. In fact, most of the bacteria isolated from high salinity (S7, S13, S19) sediments belong to either of these two groups and at least five and two additional members from both were characterized in course of this study (data not shown). Interestingly, a third group also exists that include isolates from IL-free heterotrophic plates of S2, with one representative member identified as Cellulomonas sp. (99% homology). They are halotolerant and grow only in very low NaCl concentrations

3.3.2. Active efflux pumps Halomonas sp. could tolerate up to 9 μg mL−1 of EtBr, but B. agaradhaerens or any other bacterial or archaeal strain used in the study were sensitive even to 0.5 μg mL−1 of the fluorescent substrate. As 0.5 μg mL−1 EtBr is the recommended minimum concentration for monitoring efflux activity (Pal et al., 2019), the assay was performed only with Halomonas sp. Washed bacterial cells incubated with the dye for 30 min had almost two-fold higher fluorescence (i.e., less efficient efflux activity) in sets with 0.7 or 1.0 M NaCl as compared to those with 1.7, 2.0 or 3.0 M of the salt (Fig. 4a). Addition of 125 mM [Emim]Cl in representatives of either groups (1.0 and 1.7 M NaCl), further decreased the respective fluorescence (or increased efflux activity) by 1.3 and 1.7fold (Fig. 4a). Even these IL-stressed cells of Halomonas sp. showed better growth performance in heterotrophic media supplemented with higher NaCl (1.7, 2.0, and 3.0 M) sets, as compared to the one with optimum NaCl requirement (1.0 M) (Fig. 4b). Efflux activity and growth response of Halomonas sp. in 1.0 and 1.7 M NaCl supplemented heterotrophic media with 50 mM [Bmim]Cl (Supplementary Fig. S4) was comparable to [Emim]Cl sets. 3.3.3. Adaptation and [Emim]Cl tolerance Halomonas sp. and B. agaradhaerens had similar growth in [Emim]Cl-adapted and non-IL control sets under heterotrophic condition (Fig. 5a, c). However, de-stressed and non-adapted cells of both bacteria had a prolonged lag phase. Similar trend was observed when 5

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Fig. 2. Growth response (OD600) of (a, b) Halomonas sp., (c, d) B. agaradhaerens, and (e, f) S. halotolerans under heterotrophic condition with different concentrations of (a, c, e) NaCl (0.7, 1.0, 2.0, 3.0, 4.0, and 5.0 M); or (b, d, f) [Emim]Cl (0, 125, 250, 375, and 500 mM). 1 M NaCl (optimum requirement for all tested bacteria) was added in sets with [Emim]Cl. Values are mean of three independent experiments ± standard deviation.

the cells were inoculated in nutrient deprived minimal salt media (Fig. 5b, d), suggesting [Emim]Cl-adapted cells to be more tolerant to the IL. Enrichment of Sambhar Lake sediments with 100 mM [Emim]Cl and a one-tenth diluted heterotrophic media for nutrient supply resulted in a considerable increase in [Emim]Cl tolerant colonies including in S2 (Supplementary Table S4). Interestingly, S2 had no [Emim]Cl tolerant colony initially (Fig. 1). In fact, one such IL tolerant isolate from the enrichment set of S2 sample have similar growth patterns in NaCl and [Emim]Cl as Halomonas sp., and B. agaradhaerens (Table 1, and Supplementary Fig. S5). The bacterium is a moderate halophile and identified as Bacillus locisalis (99% 16S rRNA gene homology, Table 1). But the saturated brines still lack any such tolerant colony even after several enrichment trials.

[Bmim]Cl were 150 mM and 15 mM respectively (Table 1). In fact, toxicity of the IL towards archaeal members was prominent from the 80–100% drop in CFU count of resting cells incubated with [Emim]Cl in suitable buffer for 12 h, while the same for the moderate halophilic bacteria was only 16–30% (Table 1). Even, the halotolerant Cellulomonas sp. performed better with only 47% drop in CFU count. In addition, archaeal cells incubated with 100 mM [Emim]Cl also showed sign of leakage with sharp increase in nucleic acid (A260 values) and protein (A280 values) contents in buffer supernatants (Fig. 6a and b). In this test, Haladaptatus again performed slightly better as compared to Natronomonas and Haloferax, while Halomonas sp. and B. agaradhaerens were stable. The buffer supernatants when used as template in PCR reactions with bacterial/archaeal 16S rRNA gene specific primers yield positive PCR product in archaeal sets but not in bacterial ones (Fig. 6c).

3.4. Extreme halophilic archaea and [Emim]Cl/[Bmim]Cl tolerance

4. Discussion

To verify whether [Emim]Cl-sensitivity as observed for Natronomonas isolates of Sambhar Lake was universal to extreme halophilic archaea, two additional genera, Haladaptatus sp. strain R4 and Haloferax sp. isolated from different sources were used. Both have an optimum NaCl requirement of 3.0 M. Interestingly, members of these two genera were also sensitive to [Emim]Cl, with very slow growth by Haladaptatus and Haloferax in 75 mM and 50 mM [Emim]Cl respectively (Table 1). The MIC of these two cells towards [Emim]Cl and

Halophilic bacteria of Sambhar Lake can tolerate toxicity of the two short-alkyl chain imidazolium-based ILs, with [Bmim]Cl being more toxic than [Emim]Cl (Table 1). The MIC of [Bmim]Cl for the bacterial isolates was half or less than that of [Emim]Cl, while for archaeal isolates tested, it was five to ten-fold lower (Table 1). Imidazolium-based ILs with long-alkyl side chain were reported to be more toxic than those with smaller ones in previous studies as well (Docherty and Kulpa, 2005; Sharma and Mukhopadhyay, 2018; Zhang et al., 2017). The MIC 6

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Fig. 3. Role of compatible solutes and stress enzymes in [Emim]Cl tolerance (250 mM) by (a, c, e) Halomonas sp. and (b, d, f) B. agaradhaerens. (a, b) Effect of external addition of 2 mM glutamate, proline, or glycine betaine on growth (OD600) in nutrient deprived minimal salt media without organic protein source. Production of (c, d) stress enzymes (in cell free extract); and (e, f) organic osmolyte (in washed cells). Values are mean of three independent experiments ± standard deviation. Result of unpaired t-test is depicted as *, P < 0.05; **, P < 0.001 and ns, not significant.

and even 3.0 M NaCl supplemented media (Fig. 2a, c). The cells however cannot biodegrade or biotransform the ILs as confirmed from absence of any growth of Halomonas sp. and B. agaradhaerens in minimal media with [Emim]Cl or [Bmim]Cl used as the sole carbon source. Earlier observations have also highlighted that imidazolium-based ILs with less than six alkyl side chains are poorly or even not biodegradable (Docherty et al., 2007; Jordan and Gathergood, 2015; LiwarskaBizukojc and Gendaszewska, 2013; Liwarska-Bizukojc et al., 2015). and strains with reported biodegradation capability are not so efficient, for example, Rhodococcus erythropolis and Brevibacterium sanguinis can degrade 1-ethyl-3-methylimidazolium chloride by up to 59% after prolonged incubation of two months (Megaw et al., 2013). Moderate halophiles employ two common adaptive strategies to alleviate the additional salt stress. The first one being uptake and synthesis of compatible solutes to maintain cellular osmotic balance (Grant, 2004; Oren, 2006). Ionic liquids being salts are also expected to change the cellular osmotic pressure and thus, similar adaptive feature might help these bacteria to tolerate them. In fact, in both Halomonas sp. and B. agaradhaerens organic osmolytes, such as proline, glutamate, glycine betaine or choline were uptaken (Fig. 3a and b) as well as synthesized (Fig. 3e and f) at higher concentrations in [Emim]Cl-

of Halomonas, B. agaradhaerens and B. locisalis towards [Emim]Cl are > 500 mM, which is on the higher end among those characterized for the property (Table 1). The [Emim]Cl tolerance attribute of these moderate halophiles are comparable in terms of MIC (562.5 mM) and mechanism to the best characterized strain for the property till date, i.e., Enterobacter lignolyticus SCF1 (Khudyakov et al., 2012; Ruegg et al., 2014). 4.1. Possible mechanism of [Emim]Cl/[Bmim]Cl tolerance in moderate halophilic bacteria Moderate halophilic bacteria could adapt to the toxic effect of [Emim]Cl or [Bmim]Cl, with those enduring broad range of salt concentrations tolerating higher concentrations of the ILs. However, stress response due to IL was prominent with higher activity of oxidative stress enzymes (Fig. 3c and d). Even, a delayed growth response or prolonged lag phase was also prominent in both heterotrophic rich as well as nutrient-deprived minimal salt media. The stress response in growth experiments with [Emim]Cl or [Bmim]Cl was not due to increase in total salinity of the media. This is evident from almost similar growth response of Halomonas sp. and B. agaradhaerens in 1.0 M, 2.0 M 7

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[Emim]Cl tolerance. Another possibility for better growth performance with increase in NaCl concentration could be hormesis. However, such possibility is ruled out from the fact that the growth was never triggered (at least in the tested amounts) beyond that observed in the corresponding control sets without [Emim]Cl (Fig. 4b) or [Bmim]Cl (Supplementary Fig. S4b). Even our experiments with different tested concentration of [Emim]Cl (Fig. 2b, d, f; Supplementary Fig. S5b) or [Bmim]Cl (Supplementary Figs. S1 and S5c) does not show any hormetic effect as observed for [Emim]Br or [Bmim]Br on the luminescence of Vibrio qinghaiensis sp.Q67 in a time-dependent manner (Zhang et al., 2013); and [Emim]Acetate on the growth of anaerobic Clostridium sp. and aerobic Pseudomonas putida (Nancharaiah and Francis, 2015). The observed stress response or delayed growth in sets with [Emim]Cl or [Bmim]Cl can also be argued to be due to the increased osmotic pressure by the additional amount of salt in the form of IL. However, such argument can be ruled out from the results of Fig. 4b and Supplementary Fig. S4b where the sets with 1.7 M (or higher) NaCl supplemented with the IL responded with better growth performance as compared to the 1.0 M NaCl set. If osmotic stress due to increased salt concentration would have been the sole reason for the delayed growth response, a reverse response was expected in the two sets. Rather, shortalkyl chain imidazolium-based IL tolerance by moderate halophilic bacteria is a general adaptive feature of these microorganisms and not a selection trait as confirmed from the adaptation experiment (Fig. 5). Even previous studies have also used enrichment of natural samples such as salt marsh, forest soil and industrial soil to study servility of microorganisms to short and long-term exposure of ILs (Deive et al., 2011). Based on these facts it can be argued that microbes present in the low (S2) and high (S35) salinity sediments/brines are possibly less adapted to fluctuations in stressed conditions and thus no IL-tolerant colonies were obtained. and enrichment of sediments of Sambhar Lake with 100 mM [Emim]Cl confirmed the same with an increased proportion of [Emim]Cl-tolerant CFU count in all samples including S2 (Supplementary Table S4) that lack any such colony initially. Thus, moderate halophilic bacteria, either present as a dominant member in natural environments as in S7, S13 and S19, or as a minor population as in S2 (that gets enriched after [Emim]Cl treatment), could in general tolerate very high concentrations of the IL possibly mediated by general halophilic adaptive strategies.

Fig. 4. [Emim]Cl tolerance and active efflux pumps. (a) Efflux assay to measure fluorescence intensity (excitation, 520 nm and emission, 590 nm for 30 min) of EtBr (1 μg mL−1) from washed cells (OD600 = 0.6) of Halomonas sp. pre-grown with NaCl (0.7, 1.0, 1.7, 2.0, 3.0 M) or 125 mM [Emim]Cl supplemented to sets with 1.0 or 1.7 M NaCl. AU refers to arbitrary unit. Graphs are representative of three independent time scan measurements. The assay was not possible with B. agaradhaerens or any other strain studied, as none could tolerate even 0.5 μg mL−1 EtBr. (b) Growth of Halomonas sp. with either 1.0, 1.7, 2.0 or 3.0 M NaCl, or the same media supplemented with 250 mM [Emim]Cl. Values are mean of three independent experiments ± standard deviation.

treated cells compared to control. Proline being a standard amino acid was produced in highest and almost equal amounts in the two sets. Furthermore, genome sequences of the representative bacterial genera that show prominent [Emim]Cl tolerance pattern (e.g., Halomonas sp. and B. agaradhaerens) do encode genes for uptake and synthesis of organic osmolytes, but similar genes were absent completely or partially in those which could not tolerate the IL, such as the three archaeal members and even in that of the halotolerant Cellulomonas sp (Supplementary Tables S3 and S5). The second adaptive feature used by moderate halophilic bacteria to combat salt stress is triggering of active efflux pumps (Oren, 2006; Tokunaga et al., 2004), and the same was found to be involved in [Emim]Cl and [Bmim]Cl tolerance in Halomonas sp. Higher efflux activity in [Emim]Cl and [Bmim]Cl treated sets compared to control suggests trigger of specific efflux pumps that are not induced by NaCl. On the other hand, better growth performance by IL-stressed cells of Halomonas sp. in higher NaCl supplemented media, as compared to sets with its optimum NaCl requirement (Fig. 4b), suggests involvement of some active pumps induced by higher NaCl concentrations that could also efflux out [Emim]Cl or [Bmim]Cl, thereby facilitating its growth. In fact, in a recent global transcriptome study of E. lignolyticus grown in [Emim]Cl and NaCl, both these type of transporters were identified, namely, specialized ones that are up-regulated in [Emim]Cl treated set alone, and those which are expressed equally in both sets (Khudyakov et al., 2012). Even, functional metagenomic cloning of E. lignolyticus genomic DNA in fosmids could identify a major facilitator superfamily protein to impart [Emim]Cl-tolerance in E. coli (Ruegg et al., 2014) suggesting transporters to be actively involved in

4.2. Acidic proteome of archaea and [Emim]Cl tolerance The saturated brines (S35) of the lake did not yield any [Emim]Cl or [Bmim]Cl tolerant colonies either with native samples, or post enrichment. These are dominated by extreme halophilic archaeal member with all seven pure cultures isolated from S35 identified as Natronomonas sp. Other well known extreme halophilic archaeal genera, such as Haladaptatus and Haloferax were not retrieved from Sambhar Lake possibly because of high pH and low Mg2+ content there in. The media that are generally used for growth of these two extreme halophiles (as mentioned in Section 2.1) also have very high content of Mg2+ ions and a much lower pH (Savage et al., 2007; Schneegurt, 2012). None of the three extreme halophilic archaea could tolerate either of the tested IL (Table 1). Haladaptatus is well known for its adaptation and survivability in diverse salinity conditions, thus the name (Sen et al., 2016; Youssef et al., 2014) and was expected to perform better against [Emim]Cl. Even external addition of compatible solutes (2 mM of proline, glycine betaine choline, or even a combination of all three) to the respective [Emim]Cl-containing growth media could not help them to grow in IL containing media (data not shown). In fact, their representative genome sequences also lack genes for their uptake or synthesis, except for a few in Haladaptatus (Supplementary Table S3) as also reported earlier (Youssef et al., 2014). This might have possibly helped the later to grow albeit very slowly in 75 mM [Emim]Cl. Instead our data supports the fact that [Emim]Cl causes 8

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Fig. 5. Growth of IL-adapted, non-adapted, or de-stressed cells of (a, b) Halomonas sp. and (c, d) B. agaradhaerens in (a, c) heterotrophic, and (b, d) nutrient deficient chemically defined media (both with 1.0 M NaCl) in presence of 250 mM [Emim]Cl along with a non-IL control set. Values are mean of three independent experiments ± standard deviation.

high percentages (15.4–17.5%) in representative genome sequences of the former members as compared to the moderate halophilic bacteria (10.7–12.9%) except Salinibacter ruber (15.6%) (Supplementary Table S3). This bacterium is a well known extreme halophilic member with several properties similar to archaea (Oren, 2013; Oren et al., 2002). The maintenance of acidic proteome in extreme halophilic archaea and S. ruber is an adaptation strategy to counterbalance the net charge in cytoplasm that gets disturbed by accumulation of K+ ions by salt-instrategy to neutralize the osmotic balance (Oren et al., 2002). It might be a possibility that [Emim]+, also known to be a chaotropic agent (Zhao, 2006), interact with acidic moieties of proteins, and disrupt their stability. To check whether the [Emim]Cl-sensitivity of archaeal strains can be trounced by higher concentrations of K+ ion in the environment, growth experiments with all three archaea was repeated with 10-times higher KCl (40 g m L−1). However, this also gave negative results (data not shown), suggesting a strong and irreversible interaction of [Emim]+ to the acidic proteome.

membrane leakage in these extremophilic archaea leading to release of cytoplasmic biomolecules in the buffer (Fig. 6) and possibly have a general cidal effect on them. There are reports that showed interaction of these short-alkyl chain ILs with bacterial cell membrane leading to leakage and release of cellular components to the environment (Lim et al., 2014; Yoo et al., 2016). Unfortunately, visualization of cell structure after [Emim]Cl-treatment under scanning electron microscope was not possible as cells could not be clearly demarcated from NaCl crystals that were added in buffer to avoid osmotic rupture of archaeal cells. All these results suggest that extreme halophilic archaea are in general, very sensitive to [Emim]Cl. As opposite to the moderate halophilic or halotolerant bacterial members, the IL sensitivity pattern did not change after enrichment. 1-ethyl-imidazole, another structural homolog of [Emim]Cl that lacks the 3’ methyl group, and thus expected to be less toxic was also used. This homolog when used at similar concentrations of [Emim]Cl was also toxic to the archaeal members, while the bacteria could grow contentedly (Table 1). Thus, the imidazole ring structure with attached short-alkyl chain is in general toxic to archaeal members. In fact, imidazolium-based ILs particularly those with short-alkyl groups (e.g., [Bmim]Cl) were shown to specifically interact with negatively charged surface moieties of purified serum albumins of bovine (Shu et al., 2011) and human (Silva et al., 2014). And these interactions were shown to destabilize hydrogen bonds, nonpolar, and electrostatic interactions among amino acids that maintain protein three dimensional structures, conformation and eventually activity. Moreover, the compact and small size of [Emim]+ makes them more susceptible to electrostatic interactions with the negatively charged surface (Silva et al., 2014). Thus, it can be argued that similar destabilization of proteins might be the possible reason behind archaeal IL sensitivity. And to substantiate the argument, abundance of negatively charged amino acids (aspartate and glutamate) in genome encoded proteins of representative archaeal and bacterial members were searched for. Interestingly, these two amino acids are present in quite

4.3. Conclusion Thus, higher stress enzyme activities, uptake and synthesis of organic osmolytes, and active efflux systems in moderately halophilic bacteria, helps them overcome the toxic effect of imidazolium-based ILs with short-alkyl side chain such as [Emim]Cl or [Bmim]Cl. Even, among bacteria, strains that can tolerate higher range of salt stress perform better due to their general adaptive features. On the contrary, extreme halophilic archaea are highly sensitive to [Emim]+ that possibly destabilize their acidic proteome by competing with function of K+ ion accumulated by salt-in strategy to maintain osmotic pressure. Thus, this study highlights two major aspects. First, saline environments that supports growth of moderately halophilic bacteria are excellent target sites for isolation of efficient [Emim]Cl-tolerant bacteria. Even the IL-tolerant strains isolated in this study can also be carried forward for future bioprospecting in different industrial settings. In fact, in an 9

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of other imidazolium-based herbicidal ILs on the microbial population as they are recommended for direct environmental applications. Conflicts of interest The authors declare no conflict of interest. Acknowledgements This work was financially supported by Council of Scientific & Industrial Research (CSIR), India [No. (38(1410)/15/EMR-II)]. Srikanta Pal and Abhijit Sar are thankful to CSIR and University Grants Commission-Basic Scientific Research (UGC-BSR), India for their fellowships respectively. Haladaptatus sp. R4 and three stains of Haloferax were generous gifts from Dr. Subhrakanti Mukhopadhyay and Dr. Abhrajyoti Ghosh respectively. We thank Dr. Alakananda Haza for discussions regarding basic chemistry of ionic liquids. We would like to thank the editor and nine anonymous reviewers for their suggestions that greatly improved quality of the paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109634. References Klein-Marcuschamer, D., Simmons, B.A., Blanch, H.W., 2011. Techno‐economic analysis of a lignocellulosic ethanol biorefinery with ionic liquid pre‐treatment. Biofuels Bioprod. Biorefin 5, 562–569. Alvarez, M., Rodríguez, A., Sanroman, M., Deive, F., 2015. Microbial adaptation to ionic liquids. RSC Adv. 5, 17379–17382. Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., Formsma, K., Gerdes, S., Glass, E.M., Kubal, M., 2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9, 75. Baker, G., Smith, J.J., Cowan, D.A., 2003. Review and re-analysis of domain-specific 16S primers. J. Microbiol. Methods 55, 541–555. Banerjee, S., Sar, A., Misra, A., Pal, S., Chakraborty, A., Dam, B., 2018. Increased productivity in poultry birds by sub-lethal dose of antibiotics is arbitrated by selective enrichment of gut microbiota, particularly short-chain fatty acid producers. Microbiology 164, 142–153. Banerjee, S., Misra, A., Chaudhury, S., Dam, B., 2019. A Bacillus strain TCL isolated from Jharia coalmine with remarkable stress responses, chromium reduction capability and bioremediation potential. J. Hazard Mater. 367, 215–223. Bates, L., Waldren, R., Teare, I., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Brandt, A., Grasvik, J., Hallett, J.P., Welton, T., 2013. Deconstruction of lignocellulosic biomass with ionic liquids. Green Chem. 15, 550–583. Bubalo, M.C., Radosevic, K., Redovnikovic, I.R., Halambek, J., Srcek, V.G., 2014. A brief overview of the potential environmental hazards of ionic liquids. Ecotoxicol. Environ. Saf. 99, 1–12. Bubalo, M.C., Radošević, K., Redovniković, I.R., Slivac, I., Srček, V.G., 2017. Toxicity mechanisms of ionic liquids. Arh. Hig. Rada. Toksikol. 68, 171–179. Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904–910. DeAngelis, K.M., D'Haeseleer, P., Chivian, D., Fortney, J.L., Khudyakov, J., Simmons, B., Woo, H., Arkin, A.P., Davenport, K.W., Goodwin, L., 2011. Complete genome sequence of “Enterobacter lignolyticus” SCF1. Stand. Genom. Sci. 5, 69. Deive, F.J., Rodríguez, A., Varela, A., Rodrígues, C., Leitao, M.C., Houbraken, J.A., Pereiro, A.B., Longo, M.A., Sanromán, M.Á., Samson, R.A., 2011. Impact of ionic liquids on extreme microbial biotypes from soil. Green Chem. 13, 687–696. Docherty, K.M., Kulpa, C.F.J., 2005. Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids. Green Chem. 7, 185–189. Docherty, K.M., Dixon, J.K., Kulpa Jr., C.F., 2007. Biodegradability of imidazolium and pyridinium ionic liquids by an activated sludge microbial community. Biodegradation 18, 481–493. Grant, W., 2004. Life at low water activity. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 1249–1267. Grieve, C., Grattan, S., 1983. Rapid assay for determination of water soluble quaternary ammonium compounds. Plant Soil 70, 303–307. Jordan, A., Gathergood, N., 2015. Biodegradation of ionic liquids–a critical review. Chem. Soc. Rev. 44, 8200–8237. Khudyakov, J.I., D'Haeseleer, P., Borglin, S.E., Deangelis, K.M., Woo, H., Lindquist, E.A., Hazen, T.C., Simmons, B.A., Thelen, M.P., 2012. Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium, Enterobacter lignolyticus. Proc. Natl. Acad. Sci. U.S.A. 109, E2173–E2182. Koutinas, M., Vasquez, M.I., Nicolaou, E., Pashali, P., Kyriakou, E., Loizou, E., Papadaki,

Fig. 6. (a) Nucleic acid and (b) protein release from resting cells of bacteria and archaea incubated for 2 h in respective optimum salinity-maintained buffer with 100 mM [Emim]Cl. Values are mean of three independent incubations ± standard deviation. (c) The supernatant (3 μl) were also used as template in PCR reactions for 30 cycles with 16S rRNA gene specific primers (product size of 354 bp for bacteria and 454 bp for archaea). Lanes: M, 1 kb DNA ladder; 1, Halomonas sp.; 2, B. agaradhaerens; 3, Haladaptatus; 4, Haloferax; 5, Natronomonas; 6, 7, positive control, and 8, 9, negative (no template) control, for bacteria and archaea respectively. The bars above the respective PCR product bands represent the relative abundance of band intensities compared against that of the 16S rRNA gene amplified product of control bacteria (lane 6), and archaea (lane 7) respectively. Band pattern was recorded on a gel documentation system (Bio-Rad, USA); its intensity was measured by Image J software and expressed as arbitrary units (AUs) (Supplementary Table S6). The y-axis label is placed at the left, outside the gel image.

ongoing project we have successfully isolated several IL-stable cellulases, both from bacterial pure cultures as well as by functional metagenomic screening from Sambhar Lake sediments. These enzymes could effectively be used for in situ saccharification of alkali or IL pre-treated lignocellulosic biomass such as rice straw and thus could be used in biomass industry (manuscript communicated elsewhere). Secondly, the almost universal toxicity of extreme halophilic archaea towards [Emim]Cl or possibly to any similar ionic compounds poses a serious threat to their overall diversity due to any such contamination in future, particularly in hypersaline lakes or other extreme environments. Thus, meticulous use of these compounds has to be done to preserve the ecology of archaeal population in different natural sites. Moreover, considering the present limitations of [Emim]Cl and [Bmim]Cl being used on a massive scale, it would be noteworthy to evaluate the impact 10

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