Impact of ionic liquids on extreme microbial biotypes from soil

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Cite this: Green Chem., 2011, 13, 687

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Francisco J. Deive,a,b Ana Rodrıguez,a,b Adelia Varela,a,c Catia Rodrıgues,a Maria C. Leitao,a

Jos A. M. P. Houbraken,d Ana B. Pereiro,a,b Marıa A. Longo,b M. Angeles Sanroman,b Robert A. Samson,a,d

Luıs Paulo N. Rebeloa and Cristina Silva Pereira*a,e

Received 26th July 2010, Accepted 15th December 2010DOI: 10.1039/c0gc00369g

This work aims at identifying, amongst extreme soil biotypes at locations of high salinity and highhydrocarbon load, microbial strains able to survive short or long-term exposure to the presence ofselected ionic liquids. We have evaluated the impact of ionic liquids on the diversity of the soilmicrobiota to identify which microbial strains have higher survival rates towards ionic liquids, andconsequently those which might possibly play a major role in their biotic fate. To the best of ourknowledge, this is the first study of this kind. Soils, from a region in Portugal (Aveiro) weresampled and the bacterial and fungal strains able to survive after exposure to high concentrationsof selected ionic liquids were isolated and further characterised. We have mainly focused on twotypes of cations: imidazolium – the most commonly used; and cholinium – generally perceived asbenign. The surviving microbial strains were isolated and taxonomically identified, and the ionicliquid degradation was analysed during their cultivation. The continuing exposure of themicrobial strains to petroleum hydrocarbons is likely to be the basis for their acquired resistanceto some imidazolium salts; also, the higher capacity of fungi – compared to bacteria – to grow,even during their exposure to these liquid salts, became evident in this study.

Introduction

Soils and sediments, a fundamental part of any ecosystem,are being continuously affected by man-made chemicals. TheEuropean Environment Agency has recently stated that thereare about 250 000 contaminated locations urgently requiringremediation, and current estimates of the number of potentiallypolluting activities might increase that number to nearly threemillion.1 Not surprisingly, the European Community regulationof chemicals and their safe use, REACH, in force since 2007,became mandatory for any chemical produced above one tonneper annum, aiming to increase industry awareness of hazards andrisk management.2

Globally, there is an increasing demand for sustainablesolvents and processes. This fact naturally generates a high

aInstituto de Tecnologıa Quımica e Biologica – Universidade Nova deLisboa (ITQB-UNL), Apartado 127, 2780-901, Oeiras, Portugal.E-mail: www.itqb.unl.ptbDepartment of Chemical Engineering. University of Vigo, PO Box36310, Vigo, SpaincINRB/L-INIA (ex-EAN), Av. da Republica, Quinta do Marques,2784-505, Oeiras, PortugaldCBS-KNAW Fungal Biodiversity Centre, Utrecht, P.O.Box85167-3508AD, The NetherlandseInstituto de Biologia Experimental e Tecnologica (IBET), Apartado 12,2780-901, Oeiras, Portugal. E-mail: spereira@itqb.unl.pt

interest in ionic liquids. Ionic liquids have become recognisedas a major advance in the development of novel solventsdue to a unique combination of properties (their usual non-volatility and non-flammability, high thermal stability, andrecyclability) in combination with the capacity for fine-tuningtheir physical and chemical properties by choosing anion-cationcombinations.3 Their unique features mainly rely on their flexibledoubly dual nature (cation versus anion; low-charge versus high-charge density nano-domains).4 There are numerous examplesof ionic liquids applications, covering very distinct functions, e.g.catalysts,5 electrolytes,6 and solvents,7 either at an exploratoryphase or already employed in industrial processes.8 Within thelatter there are obviously some ionic liquids that the productionlevel per annum surpasses the tonne magnitude and that arecurrently undergoing REACH registration (e.g. [C2mim][X](X = Cl-, [C2SO4]-, [C1SO3]-, [O2CMe]-) and [C4mim]Cl),9

but, undoubtedly, this number will increase. One of the majoradvantages of (aprotic) ionic liquids relative to typically volatileorganic solvents is the low threat that they often pose tothe atmosphere under common use.10,11 However, some ionicliquids may seriously contaminate the environment in the eventof accidental leakage or wastewater disposal, representing aserious ecotoxicological concern to both terrestrial and aquatichabitats.12 There is increasing interest in the behaviour ofionic liquids in soil, due to its critical role in their transport,reactivity and bioavailability;13,14 whilst in soil some ionic liquids,

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Fig. 1 Chemical structures of all ionic liquids used.(a) 1-ethyl-3-methylimidazolium or 1-butyl-3-methylimidazolium chloride ([C2mim]Clor [C4mim]Cl); (b) 1-ethyl-3-methylimidazolium ethylsulfonate or propylsulfonate or butylsulfonate ([C2mim][C2SO3] or [C2mim][C3SO3]or [C2mim][C4SO3]); (c) 1-ethyl-3-methylimidazolium methylsulfate or ethylsulfate ([C2mim][C1SO4] or [C2mim][C2SO4]) and 1-butyl-3-methylimidazolium methylsulfate ([C4mim][C1SO4]); (d) 1-ethyl-3-methylimidazolium ethanoate, ([C2mim][O2CMe]; (e) 1-ethyl-3-methylimidazoliumDL-lactate, ([C2mim][lac]; (f) cholinium chloride ([N111(EtOH)]Cl); (g) cholinium DL-lactate ([N111(EtOH)][lac]).

e.g. imidazolium ionic liquids, have been reported to stronglyaffect the growth of plants,15 further demonstrating their highpersistence and eco-toxicological risk.

Nevertheless, ionic liquids’ impact on soil function, namelyon the diversity of the soil microbiota, have yet to be adequatelyinvestigated. Moreover, while affecting the ecology of theexposed niche, the most tolerant species will possibly play amajor role in pollutant mitigation. The most commonly usedionic liquids, e.g. imidazolium,16 and pyridinium,17 are poorlybiodegradable, although side chain modifications may enhancetheir primary biodegradability potential.

It constitutes the aim of the present study to focus on twodistinct families of ionic liquids and diverse soil biotypes, toevaluate the existence, if any, of microbial species able totolerate ionic liquids up to a molar concentration magnitude,and of those which might play a major role in the chemicals’biodegradation (i.e. biotic decay).

Since ionic liquids are (low-temperature) molten salts carryingfunctionalised ions one could hypothesise that microbial com-munities isolated from locations of high salinity and/or highload of hydrocarbon pollutants might demonstrate higher sur-vival rates towards ionic liquids. From an anthropocentric pointof view, both aforementioned soil biotypes may be regarded asextreme, and, certainly, some of the microorganisms able to sur-vive under those conditions may be regarded as extremophiles,

which generally display high biocatalytic potential.18,19 Soils weresampled from an Aveiro region (500 km2), in NW Portugal,which is located at the mouth of the river Vouga, with anestuary characterised by great expanses of marshes and lagoons.This area was selected for soil sampling due to the diversity ofecosystems located nearby, namely extreme soil biotypes in saltmarsh and industrial locations, and also the normal soil biotypesinside a Natural Park (forest soil). The bacterial and fungalstrains able to survive after exposure to high concentrations ofselected ionic liquids were isolated and further characterised.

Materials and methods

Chemicals

The ionic liquids used in this study (see Fig. 1), in-cluding source and grade, were as follows: 1-ethyl-3-methylimidazolium chloride, [C2mim]Cl (Iolitec, purity ≥98%),1-ethyl-3-methylimidazolium ethylsulfate, [C2mim][C2SO4] (Sol-vent Innovation, purity ≥98%), 1-ethyl-3-methylimidazoliumethanoate, [C2mim][O2CMe] (Iolitec, purity ≥95%); 1-butyl-3-methylimidazolium methylsulfate, [C4mim][C1SO4] (Fluka,purity ≥95%), cholinium chloride, [N111(EtOH)]Cl (Sigma-Aldrich, purity ≥95%). 1-butyl-3-methylimidazolium chloride,[C4mim]Cl, 1-ethyl-3-methylimidazolium lactate, [C2mim][lac]

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and cholinium lactate, [N111(EtOH)][lac] were preparedby QUILL (Queen’s University Ionic Liquids Laboratory,Belfast, UK), and 1-ethyl-3-methylimidazolium ethylsulfonate,[C2mim][C2SO3], 1-ethyl-3-methylimidazolium propylsulfonate,[C2mim][C3SO3], and 1-ethyl-3-methylimidazolium butylsul-fonate, [C2mim][C4SO3] were prepared in our lab according toliterature procedure.20 These ionic liquids were characterisedand their purity confirmed by a combination of 1H NMR,13C NMR, electro-spray ionisation mass spectrometry, halidecontent, CHN elemental analysis and water content.

All ionic liquids were vacuum-cleaned of volatile impurities(10-1 Pa) under strong stirring at a moderate temperature ofcirca 320 K for several hours prior to their use. The absence ofthermal degradation was verified by 1H NMR. The collectedspectra were then used for a complete assessment of the ionicliquids’ biodegradability.

Sample collection

In each selected location in the region of Aveiro, Portugal, threedifferent spots were sampled, namely, (i) a saltwork marsh, (ii) anindustrial regional pole (Estarreja), and (iii) the natural reserveof Sao Jacinto (normal biotype – negative control). The soilsampling (April, 2009) consisted of collecting five individualsoil samples in each quadrant defined by 1 ¥ 1 m2, using a 3 cmdiameter gauge auger at a single depth: 0–20 cm. Then, the fivesamples of a given quadrant and depth were pooled together foranalysis. Each soil sample (pool of a given quadrant) was sieved(<2 mm) and conserved (dark, 4 ◦C) until analysis.

Soil characterisation

Chemical. Each soil sample was initially characterised usingstandard methods,21 namely humidity, pH (ISO 10390 : 1994);organic nitrogen, organic carbon (for wet and dry samplesby the Walkley-Black method,22 or by the European Standard12879 (ISO 12879 : 2000), respectively), and particle size analysis(PSA).

Total petroleum hydrocarbons. Soil samples (10 g) were sub-mitted to solvent-fast extractions by sequentially using solventsof increasing polarity, namely hexane, then dichloromethane,and finally, chloroform (1 : 10, w/v, with each solvent repeatedtwice). Solvents were evaporated to a constant weight, andthen Total Petroleum Hydrocarbon (TPH) content in soil wasdetermined gravimetrically accordingly to the method describedby Mishra et al.23

Microbial. The microbial community in soil was extractedby adding to each sample 0.1% (w/v) of peptone solution(1 : 10 w/v). Each suspension was homogenised for 1 h, at roomtemperature, with soft agitation (100 rpm). In order to define thetotal number of cultivable microbial strains in each soil sample,either fungal or bacterial, a small aliquot of the peptone extractswere used to inoculate specific solid media containing 10 mg L-1

of chloramphenicol or 75 mg L-1 of cycloheximide, selectivebactericidal and fungicidal agent, respectively. The numberof colony forming units (CFUs) were defined for bacteria inLysogeny broth (LB) agar media (containing per L of distilledwater: 10 g of trytone, 5 g of yeast extract, 10 g of NaCl and 15 gof agar), and for fungi also in Malt Extract Agar (MEA, Merck)

and dichloranglycerol 18% agar (DG18, Oxoid) media, after sixdays of incubation at 27 ◦C in the dark (CFUs were registereddaily). Each soil sample was analysed in triplicate.

Screening the impact of ionic liquids on the soil microbiota

Short-term exposure: cultivation in solid media. In order todefine the total number of microbial strains able to survive inmedia containing selected ionic liquids, the peptone extractsof the soil samples were used to inoculate standard media(LB, without any antimicrobial agent) containing 1 M ofeach ionic liquid, namely [([N111(EtOH)]Cl, [C2mim]Cl and[C2mim][C2SO4] (added before media jellification). The cultures(duplicates) were incubated at 27 ◦C in the dark and the numberof CFUs registered daily, during six days. Replicates of thecultures were incubated, under identical conditions, for twomonths in order to evaluate degradation, if any, of the ionicliquids during solid state cultivation.

The water activity (aw) of each solid media (with or withoutionic liquids) was determined with a portable water activityindicator (HydroPalm AW1) following the manufacturer’s in-structions at 25 ◦C with resting periods of 5 min (triplicates).

Long-term exposure: cultivation in liquid media. In order tocircumvent the limited bioavailability of the ionic liquids whichis likely to occur in the solid media and test longer exposureperiods, submerged cultures in 0.1% peptone (w/v) mediacontaining 0.5 or 1 M of each ionic liquid (Fig. 1) were inoculatedwith the soil microbial extracts (duplicates). After 2 months’incubation, at 27 ◦C in the dark with agitation (100 rpm), analiquot of the cultures was used to inoculate standard solidmedia (MEA, DG18 and LB, without any antimicrobial agent).The number of microbial CFUs in each medium was defineddaily, during 6 days (same conditions reported above).

Ionic liquid’s biodegradation analysis. After two months ofincubation, the microbial culture extracts from solid and liquidmedia were analysed by 1H NMR, and the latter also by LiquidChromatography (LC), in order to evaluate the extension of theionic liquids’ degradation, if any. The solid media were collected,crushed and then extracted with water during 18 h underagitation. Solids in suspension were removed by centrifugation(15 700 g during 20 min), and the clean aqueous extracts wereconserved at -20 ◦C until further analysis. The liquid mediawere cleaned by centrifugation (15 700 g during 20 min), andimmediately frozen at -20 ◦C until analysis. An aliquot of theculture extracts obtained from solid and liquid media was dilutedin deuterated water and analysed by 1H NMR spectroscopy(BRUKER Avance 400).

The residual levels of ionic liquid (quantifying separately thecation and the anion biodegradability) in the culture extracts(liquid media) were analysed by ultra-performance liquid chro-matography (UPLC) using a Waters (Waters Corporation, Mil-ford, USA) Acquity chromatographer with Photodiode Array(PDA) detector, cooling auto-sampler, and column oven. Dataacquisition was accomplished with the Empower 2 software,2006 (Waters Corporation). All solvents were of the highestanalytical grade, and water was obtained from a Milli-Q system(Millipore). Injections of the culture extracts (3 and 10 mL) weremade using a 10 mL loop operated in partial-loop with needle

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Table 1 Chromatographic conditions used for quantifying the residual levels of ionic liquid (quantifying separately the cation and the anionbiodegradability) in the culture extracts.

Ionic liquid ionDetection/nm Column Mobile phase

Flow rate/mL min-1

Retentiontime/min

[C2mim]+

[C4mim]+218218

Synergi Polar-RP column (150 ¥ 4.6 mm)packed with polar endcapped particles(4 mm, pore size 80 A) (Phenomenex,Torrance, USA), set at 26 ◦C

2% (v/v) acetonitrile and 98% (v/v)of an aqueous solution of 5 mMphosphate buffer, adjusted to pH 3

0.81.2

3.657.30

([NMe3(CH2CH2OH)]+ 215 2% (v/v) methanol and 98% (v/v)of an aqueous solution of 0.1%(v/v) heptafluorobutyric acidadjusted to pH 6

1.0 4.37

[lac]-

[O2CMe]-210210

Acquity UPLC HSS C18 (2.1 ¥ 150 mm),1.8 mm particle size column (WatersCorporation), set at 25 ◦C

2% (v/v) acetonitrile and 98% (v/v)0.1% H3PO4

0.30.3

1.831.99

[C2SO4]-

[C2SO3]-310310

PRP-x100 (4.1 ¥ 150 mm), 10 mm particlesize column (Hamilton Company, Reno,USA), set at 25 ◦C

3% (v/v) methanol and 97% (v/v)6 mM hydroxybenzoic acid (pH 10)

2.02.0

5.842.46

overfill and in full-loop mode, respectively. The chromatographicseparations were operated isocratically and the external standardmethod was used for the quantification of each ion. For eachionic liquid’s cation and anion, (i) the detection wavelength, (ii)the type of column (including temperature), (iii) the compositionof the mobile phase, (iv) the flow rate, and (v) the retention time,are depicted in Table 1.

Taxonomic characterisation of the microbial strains

Fungi. The fungal colonies were isolated by transfer tofresh standard media (without ionic liquids). The isolates werethen cultivated for eight days on MEA (conditions describedabove), and their preliminary taxonomic evaluation was carriedout based on the colony morphology, either by macroscopicand/or microscopic analysis. DNA extraction was performedusing the Ultra Clean Microbial DNA Isolation Kit (MoBioLaboratories) and amplifications of a part of the b-tubulingene and the ITS regions (including 5.8S rDNA) were donein a GeneAmp PCR system 2720 (Applied Biosystems) thermo-cycler using the primers Bt2a and Bt2b, and V9G and LS266,respectively. Primer sequences are as follows: Bt2a, 5¢-GGTAAC CAA ATC GGT GCT GCT TTC-3¢; Bt2b, 5¢-ACC CTCAGT GTA GTG ACC CTT GGC-3¢;V9G, 5¢-TTA CGT CCCTGC CCT TTG TA-3¢; LS266, 5¢-GCA TTC CCA AAC AACTCG ACT-3¢. The PCR products were sequenced directly inboth directions with primers Bt2a and Bt2b, and V9G andLS266. All sequencing reactions were purified by gel filtrationthrough Sephadex G-50 (Amersham Pharmacia Biotech, Piscat-away, NJ), equilibrated in double distilled water and analysed onthe ABI PRISM 310 Genetic Analyzer (Applied Biosystems).Contigs were assembled using the forward and reverse se-quences with the program SeqMan from the LaserGene package(DNAStar Inc.). Sequence similarity searches were performed inpublic databases of GenBank (http://www.ncbi.nlm.nih.gov/)with BLAST (version 2.2.6) and in internal databasesat the CBS-KNAW Fungal Biodiversity Centre (theNetherlands).

Bacteria. The streak plate method, consisting of a mechani-cal dilution of the microbial cultures in liquid media containingthe selected ionic liquids (see section Long-term exposure:cultivation in liquid media) on the surface of agar plates served toisolate the bacterial strains. These, in turn, were grown in freshLB media and their preliminary taxonomic evaluation was donebased on their microscopic morphology and Gram stain test.

DNA was extracted from pure cultures and amplificationof the 16S rDNA sequences was performed in a GeneAmpPCR system 2400 (Applied Biosystems) thermo-cycler usingdegenerated primer based on conserved sequence of 16S rDNA.Primer sequences are as follows: 5¢-AGA-GTT TGA TC/TA/CTGG CT-3¢ and 5¢-TAC GGC/T TAC CTT GTT ACG ACT-3¢.PCR amplified fragments were purified on Microspin columns(Amersham Pharmacia Biotech), and cycle sequencing in a Ge-neAmp PCR system 2400 (Applied Biosystems) thermo-cycler.Multiple alignments of sequences were created by ClustalX,version 1.81,24 and subsequently compared with sequences inpublic databases of GeneBank (http://www.ncbi.nlm.nih.gov/)with BLAST, version 2.2.6.

Results and Discussion

Soil characterization

The physicochemical characteristics of the sampled soils (forest,salt marsh and industrial locations in the Aveiro region), namelyhumidity, pH, and C/N ratio (Table 2) allowed us to initiallydefine the heterogeneity of the soil samples collected at thesame location and their main characteristics, which may greatlyinfluence the soil microbiota. The humidity in the soil samplescollected in the natural park were higher, by ~2 and >9 times,when compared with the salt marsh and the industrial ones,respectively. In addition, soil pH values (mean 7, varyingbetween 4.6 to 7.6) and their classification (PSA method) assandy, loam and sandy-loam, were in good agreement with thosepreviously observed by Rodrigues et al. in soils collected inthe same Portuguese region,21 which was considered to be an

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Table 2 Physicochemical characterisation and total petroleum hydrocarbons (TPH) content of the soil samples. Reported data correspond to themaximum and minimal values (triplicates) determined in independent soil samples collected at a given location.

Soil locations Humidity (%) pH O.C. (%) O.N. (%) PSA classification TPH/mg kg-1

Forest 62.2 ± 0.2 4.6 ± 0.01 18.6 ± 0.5 0.52 ± 0.009 Sandy/Sandy-loam n.d.66.4 ± 4.3 6.1 ± 0.01 50.1 ± 1.6 1.89 ± 0.096

Salt marsh 25.5 ± 1.0 5.6 ± 0.01 0.7 ± 0.1 0.07 ± 0.003 Loam 128 ± 2734.0 ± 1.0 6.8 ± 0.03 2.2 ± 0.2 0.13 ± 0.003 620 ± 84

Industrial 2.5 ± 0.04 6.2 ± 0.03 0.6 ± 0.01 0.05 ± 0.000 Sandy 3312 ± 3927.0 ± 0.17 7.6 ± 0.01 1.0 ± 0.21 0.06 ± 0.003 34806 ± 4287

O.C., organic carbon; O.N., organic nitrogen; PSA, particle size analysis; n.d. not detected.

accurate characterisation of climate conditions and the parentmaterials of the Aveiro region. Soils from the Natural Park weresurrounded by dense vegetation, and presented, as expected,the highest carbon and nitrogen contents with, for example,organic carbon values (~20 and 50%) reasonably similar to thoseobserved in other forest soils, which were shown to vary between29 to 50%.25 Likewise, carbon and nitrogen levels in salt marshand industrial soils were comparable to those reported for othersimilar soils, even when geographically detached, e.g. salt marshsoils in Georgia,26 and South Carolina,27 and industrial soils inSouth Korea.28

In order to estimate the possible load of organic pollutantsin these soils, the amount of Total Petroleum Hydrocarbons(TPH) present in each location was determined (Table 2). Withthe exception of the forest soil samples, which have reportedundetectable levels of TPH (<0.01 mg per kg soil), both extremebiotypes were contaminated with petroleum hydrocarbons, dis-playing high heterogeneity in the samples collected at the samelocation. Nevertheless, as typically found in other industrialsoils,29 TPH values determined in the industrial soils wereexceptionally elevated, higher than those in the salt marsh byone or two orders of magnitude.

Soil ecosystems are surely one of the most extremely rich bio-types one can find, and usually present high chemical, structuraland biological heterogeneity – only in a few grams of soil awealth of micro-niches may be found.30 In order to investigatethe natural abundance and functional dominance of eitherfungal or bacterial strains in the sampled soils, the number ofCFUs (i.e. cultivable microorganisms) growing in standard solidmedia, despite its inherent limitations, was investigated (Table 3).The number of bacterial CFUs, relative to fungal CFUs, inforest soils and industrial soils were higher by approximatelyone or two/three orders of magnitude, respectively. A cleardominance of the prokaryotic organisms, relative to fungi, wasobserved in the salt marsh soil samples (higher by four/fiveorders of magnitude). This is not surprising since many hyper-saline environments around the globe, despite active fungi, aregenerally dominated by prokaryotic organisms.31

Ionic liquid effects on soil microbiota

While analysing ionic liquids’ eco-toxicity, studies have generallyfocused on either the use of single model species or standardbioassays, which, despite their functionality and significance,may lack a true ecological meaning.12 As a result, the ecologyof the exposed niche is not truly enlightened (e.g. speciesinteractions). Moreover, the environmental mitigation of any

Table 3 Number of microbial colony forming units (CFUs) per g ofsoil, in soils collected at distinct locations. CFUs were counted onto thesurface of standard solid media. Media water activity (aw) values arealso presented.

Fungal CFUs Bacterial CFUs

Soil location MEAa DG18a LBa LBb

Salt marsh 9.2 ¥ 103 1.5 ¥ 103 4 ¥ 104 1.6 ¥ 108

Industrial >104 4.7 ¥ 103 8 ¥ 104 1.4 ¥ 106

Forest >104 >104 105 6 ¥ 105

aw 1 ± 0.001 0.96 ± 0.001 1 ± 0.001 1 ± 0.001

n.d., not detected.a Media supplemented with chloranphenicol. b Mediasupplemented with cycloheximide.

chemical will certainly involve the most tolerant species. Thisconstitutes the aim of the present study, while focusing onsoil biotypes, to evaluate the existence, if any, of microbialspecies able to tolerate ionic liquids up to molar concentrationmagnitude, and putatively investigate which microbial strainsmay play a major role in the chemicals’ biodegradation (i.e.biotic decay). The experimental approach used is depicted inFig. 2.

Fig. 2 Schematic view of the experimental approach used.

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Table 4 Number of soil microbial strains (colony forming units, CFUs) able to actively grow in the presence of 1 M [N111(EtOH)]Cl and [C2mim]X,with X = Cl- and [C2SO4]-. Hyper-saline control media (3.4 M NaCl) was also included. CFUs were counted onto the surface of LB solid media.Media water activity (aw) values are also presented.

CFUs per gram soil

[N111(EtOH)]Cl [C2mim]Cl [C2mim][C2SO4] NaCl

Soil biotype F B F B F B F B

Salt marsh 102 / 8 ¥ 102 0 3 ¥ 102 /4 ¥ 102 0 2 ¥ 103 / 3 ¥ 103 0 0 >106

Industrial 4 ¥ 103 / 7 ¥ 103 0 8 ¥ 102 / 103 0 4 ¥ 104 / 6 ¥ 104 0 0 0Forest 0 0 0 0 0 0 0 0

aw 0.980 ± 0.003 0.966 ± 0.000 0.961 ± 0.001 0.882 ± 0.001

F, fungi; B, bacteria.

The effect of short-term exposure (six days) on microbialabundance and the diversity of the soil microbial communities,has initially been screened for two very distinct families of ionicliquids. Preference was initially given to cultivation in a solidmedia (LB media containing 1 M of [C2mim]Cl, [C2mim][C2SO4]and [N111(EtOH)]Cl) since it may constitute a better simulationof the soil environment than cultivation in liquid media.The selection of [C2mim][X] (with X = Cl- and [C2SO4]-)and [N111(EtOH)]Cl, was clearly justified because these ionicliquids display high/moderate antimicrobial activity and verylow biodegradability potential,16,32 and high biocompatibilityand readily biodegradability potential,33,34 respectively. In thepresence of these ionic liquids the number of bacterial CFUs wasnull (Table 4). The solid media MEA/LB and DG18 reported,as expected, water activity values (aw) of 1 and 0.96, respectively.LB media supplementation with [N111(EtOH)]Cl, [C2mim]Cl and[C2mim][C2SO4] reduced aw to 0.98, 0.97 and 0.96, respectively,which might have contributed to reduce the number of microbialCFUs. However, they did not play a major role in the numberof bacterial CFUs since the aw of the hyper-saline control media(0.88) was sufficient to support growth of some bacterial strainsfrom the salt marsh soil. The hyper-saline control media (3.4 MNaCl) is generally used to select halotolerant microorganisms(aw ~ 0.85).31 Herein, the use of this media allowed us to evaluatethat apparently none of the halotolerant bacterial strains wereable to form colonies in media supplemented with the testedionic liquids.

The fungi’s higher tolerance to the ionic liquids, relative tobacteria, was apparent, notwithstanding the fact that fungi inforest soils were unable to grow in the solid media containingthe tested ionic liquids. The fungal strains in both extremesoil biotypes demonstrated high resistance to [N111(EtOH)]Cl,[C2mim]Cl and [C2mim][C2SO4], with CFUs counts for indus-trial and salt marsh soils generally of a lower magnitude thanin the LB standard media (Table 4). One exception was noticed,since the number of fungal CFUs from the industrial soils wereof similar magnitude in both standard and [C2mim][C2SO4] LBmedia. The apparently high antimicrobial activity reported forboth chlorides at molar magnitude concentration was probablydue to the saline stress imposed, especially when comparedto the hyper-saline control media (i.e. media with NaCl)which completely inhibited fungal growth. Overall, these results

suggest that, at high concentrations, even the most benign ionicliquids may significantly reduce the microbial diversity in thesoil. It became apparent that the environmental pressure causedby high petroleum hydrocarbon load and, although secondarily,by the high salinity in soil, augmented the microbial capacityto actively grow in a media containing these liquid salts. Theeffect of hyper-salinity was not completely clarified since the saltmarsh soils also contained hydrocarbon petroleum pollutants(see Table 3).

In order to further comprehend the response of the soilmicrobial communities (extreme soil biotypes), their capacityto survive and to recover from long periods of exposure toionic liquids was analysed (Table 5). Preference was given tocultivation in liquid media so as to screen more rapidly and moreeconomically a larger number of ionic liquids. We have focusedon several [Cnmim]+ (n = 2 and 4) and [N111(EtOH)]+, combinedwith different anions: halide (Cl-), alkylsulfonate ([CnSO3]-, withn = 2, 3 and 4), alkylsulfates ([CnSO4]-, with n = 1 and 2) andcarboxylates (ethanoate and lactate) (Fig. 1). After two monthsof exposure, the microbial cultures were used to inoculate solidmedia free of any antimicrobial agent. This, in turn, enabledthe strains kept at a dormant stage in the presence of thechemical to recover in its absence. The trend of higher survivalof microbial strains from industrial soils relative to that of thesalt marsh was clear (Table 5). Moreover, the survival rates forfungal and bacterial strains were linked to the soil location, i.e.supremacy of fungi, relative to bacteria, was observed in theindustrial soil biotype and vice versa. Within the salt marsh soil,viable CFUs were annotated only in the less toxic ionic liquids,namely [N111(EtOH)]Cl and [C2mim][X], with X = Cl-, [O2CMe]-

and [C2SO4]-. The low contribution of these anions to overalltoxicity has been previously reported.33,35 The most benign ionicliquid (higher survival rates for both bacteria and fungi) wasundoubtedly [N111(EtOH)]Cl, agreeing with previous studieswhich have reported its high biocompatibility.36 In addition,regarding the toxicity of ionic liquids carrying chloride orlactate anions, it became obvious that [C2mim]+ displayed highertoxicity, in similarity to that previously reported.36,37 It is widelyaccepted that imidazolium ionic liquids display moderate/highantimicrobial activity, which is correlated with the length ofthe alkyl chain in the imidazolium ring.38 Our data (Table 5)demonstrated typical toxicity trends, since, at the level of the

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Table 5 Number of soil microbial CFUs able to survive for two months in the presence of several imidazolium and cholinium based ionic liquids(0.5 and 1 M), and to recover, actively growing onto the surface of standard solid-media depleted of antimicrobial chemicals.

CFUs per g soil

Salt marsh soil Industrial soil

Ionic liquid [IL]/M DG18 MEA LB DG18 MEA LB

[C2mim]Cl 0.5 — — >104 F/B >104 F >104 F/B >104 B/F1.0 — — >104 F/B >104 F >104 F/B >104 F/B

[C2mim][Lac] 0.5 — — — >104 F >104 F >104 F1.0 — — — — — —a

[C2mim][C2SO3] 0.5 — — — 103 F 2 ¥ 103 F 2 ¥ 103 F1.0 — — —a — — —

[C2mim][CnSO3], n = 3 and 4 0.5 / 1.0 — — — — — —[C2mim][C2SO4] 0.5 — — 2 ¥ 104 F / 2 ¥ 103 B — — 2 ¥ 104 F

1.0 — — 103 B — — 2 ¥ 103 F[C2mim] [O2CMe] 0.5 — — 5 ¥ 103 B — — —

1.0 — — 4 ¥ 103 B — — —[C4mim]Cl 0.5 — — — — — 2 ¥ 104 B

1.0 — — — — — —[C4mim]C1SO4 0.5 / 1.0 — — — — — —[N111(EtOH)]Cl 0.5 — >104 B >104 B 2 ¥ 103 F / >104 B >104 B/F >104 B/F

1.0 —a >104 B >104 B 3 ¥ 103 F / >104 B >104 B/F >104 B/F[N111(EtOH)][lac] 0.5 — — — 104 F >104 F >104 F

1.0 — — — — — —

F, fungal; B, bacterial. Blank entries correspond to absence of microbial growth.a Contamination detected.

cation, [C2mim]+ reported lower antimicrobial activity than[C4mim]+ and, at the level of the alkylsulfonate anions, [C2SO3]-

was less toxic than [CnSO3]- with n = 3 or 4. This findingfurther supports the possibility that the mode of toxicity of theseionic liquids may be associated with membrane disruption,39 aswas also indirectly demonstrated by their ability to augmentthe solubilisation of membrane proteins.40 The slightly highertoxicity of [C4mim]+ when carrying [C1SO4]- anion relative tothe halide [Cl]- is thought to be due to the higher contribution ofthe former anion to the overall ionic liquid toxicity.41 Finally, thehigher antifungal activity of [C2mim][O2CMe] when comparedwith the lactate one, has also been observed before,36 and isprobably due to the presence of an hydroxyl group in thelatter.

The ionic liquid resistant microbial strains were isolatedand taxonomically identified so as to better understand thosewhich may play a major role in the ionic liquids’ bioticdecay in soils (Table 6). Amongst the fungal strains from theindustrial soil biotype able to grow in the presence of thetested ionic liquids, three of them were entomopathogenic fungi,identified as Paecilomyces lilacinus, Metarhizium anisopliae andBeauveria bassiana. Their potential for biotransformation ofhydrocarbons has been reported to be generally very high(thought to be associated with their higher ability to degradearomatic compounds in insect cell walls),42 e.g. P. lilacinus wasobserved to form a variety of biphenyl oxidation compoundsincluding ring cleavage products.43 The ubiquitous soil fungus:Penicillium brevicompactum, was unable to grow in the presenceof the tested ionic liquids, but survived a molar magnitudeconcentration of [C2mim][C2SO4]. This was not surprising sincethis fungus has been previously observed to tolerate extremelyhigh concentrations of some ionic liquids,33,36 and aromaticpollutants.44 Finally, the two strains isolated from salt marsh

soils belong both to the genus Penicillium, namely P. svalbardenseand P. roseopurpureum, and have previously been isolated inhyper-saline environments.45,46

Despite the fact that no bacterial strains were able to activelygrow in the presence of molar magnitude concentrations of theselected ionic liquids, twelve bacterial strains were able to surviveafter long periods of exposure (Table 6). Half of these strainswere Gram positive; nevertheless, they usually demonstrate,relative to Gram negative bacteria, higher susceptibility to ionicliquids,38 which is thought to be related to differences in their cellwall composition. Based on the phylogenetic homology analysis,the bacterial strains from the industrial soil that survived afterlong periods of exposure to [Cnmim]Cl (n = 2 and 4) and[N111(EtOH)]Cl, were {Bacillus cereus IMAU80004, unculturedsoil bacterium clone B5-4 and Micrococcus sp. BD-15}; and{Bacillus cereus MUJ and Serratia proteamaculans 568}, re-spectively. All strains, except the latter, of a genus ubiquitouslyfound in nature,47 were reported to have previously been isolatedfrom hydrocarbon polluted sites.48 In addition, those isolatedfrom the salt marsh soil biotype: Rhodococcuss erythropolis,Bacillus aquimaris, Sporosarcina luteola, Virgibacillus sp. jx15and three Pseudomonas spp. have often been reported to beinhabitants in hyper-saline environments,49–52 and generally alsoshow high capacity to degrade hydrocarbon.53 Based on the data,it seems likely that continuing exposure of the soil microbiota tohydrocarbons is the basis for their higher resistance (and survivalrates) to the tested liquid salts.

When both data sources, short and long periods of exposure toionic liquids, i.e. actively growing microorganisms and microbialsurvival rates, respectively, were simultaneously analysed it be-came evident that some microbial strains (mostly bacteria) werekept dormant in the presence of the ionic liquids. Nevertheless,during incubation the ionic liquid concentration in the liquid

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Table 6 Taxonomic characterisation of the microbial strains able to actively grow in solid media containing 1 M of selected imidazolium andcholinium based ionic liquids (fungi); or to survive prolonged exposure periods (0.5 or 1 M) recovering afterwards onto the surface of standardsolid-media depleted of antimicrobial chemicals (fungi and bacteria).

Microbial strains taxonomic classification (Ref. Gene Bank; similarity level)

Fungal species Bacterial species

Ionic liquid Salt marsh soil Industrial soil Salt marsh soil Industrial soil

[C2mim]Cl Penicillium roseopurpureumAF033415; 97%

Metarhizium anisopliaeFJ177505; 100%

Rhodococcus erythropolisAM921647; 100% (G+, R)

Bacillus cereus IMAU80004GU125426; 99% (G-, R/C)

— — — Soil bacterium cloneb B5_4FJ184330; 100% (G-, R)

[C2mim][Lac] — Paecilomyces lilacinusAY213665; 100%

— —

[C2mim][C2SO3] — Paecilomyces lilacinusAY213665; 100%

— —

— Penicillium brevicompactuma

AY373897; 100%— —

[C2mim][C2SO4] Penicillium svalbardenseCBS; 100%

Metarhizium anisopliaeFJ177505; 100%

Sporosarcina luteolaAB473560; 100% (G-, R)

Penicillium roseopurpureumAF033415; 97%

— Virgibacillus sp. jx15FJ539116; 99% (G+, R)

[C2mim][O2CMe] — — Bacillus aquimaris AB376670;99% (G+, R)

[C4mim]Cl — — — Micrococcus sp.BD-15GU085223; 100%(G+, C)

[N111(EtOH)]Cl — Paecilomyces lilacinusAY213665; 100%

Pseudomonas reactans PSR2GQ354529; 100% (G-, R)

Serratia proteamaculans568CP000826; 99% (G+, R)

— Beauveria bassianaAF291872; 100%

Pseudomonas sp.StFLB049AB506040; 99% (G-, R)

Bacillus cereus MUJGU125426; 99% (G+, R)

— Beauveria bassianaAF291871, 99%

Pseudomonas rhodesiae NO5FJ462694; 99% (G-, R)

[N111(EtOH)][lac] — Paecilomyces lilacinusAY213665; 100%

— —

R, rods; C, cocci. Blank entries correspond to absence of microbial growth.a Only fungus that despite its survival was unable to grow in mediacontaining 1 M of ionic liquid. b Uncultivated.

media may decrease due to readily and/or partially bioticdegradation and/or cellular sorption (i.e. adsorption to themembrane surface and uptake into the cell).54 Therefore, if theionic liquid concentration is significantly decreased, some strainsmay initiate active growth before its removal from the media,however, the method used here does not distinguish growth fromsurvival. In order to complete this study, the ionic liquids’ degra-dation by the microbial cultures (both solid and liquid) after 2months of incubation was monitored by NMR spectroscopy.The peaks attributed to the cholinium or the imidazoliumcations were conserved in the spectral analyses (data not shown),which may indicate partial mineralisation of both the anion andthe cation. In order to further investigate the biodegradabilityof the ionic liquids, the cultures where microbial survival hadbeen detected after two months of incubation were analysedby liquid chromatography. The biodegradability rate (%) ofeach cation ([Cnmim]+ (n = 2 and 4) and [N111(EtOH)]+) and ofeach anion ([lac]-, [C2SO3]-, [C2SO4]-,[O2CMe]-), as detected byliquid chromatography, are depicted in Table 6. Under aerobicconditions, cholinium has been previously reported to undergoalmost complete biodegradation (93%).34 However, after twomonths, the tested soil microbial cultures had only partiallydegraded it. In addition, it seems likely that amongst the micro-

bial strains able to survive a molar magnitude concentration of[N111(EtOH)]Cl there were both bacteria and fungi which were,either alone or within a mixed community of bacteria and fungi,able to degrade it. Previous studies showed, for example, that1-butyl-3-methylimidazolium bromide was not biodegradableand that, despite the side chain primary biodegradability po-tential, the biodegradability of the imidazolium ring cannot beaddressed.16,55 In the present study, even though some microbialstrains survived a molar magnitude concentration of [Cnmim]+

(n = 2 and 4), in all cases the degradation of the imidazoliumcation was observed to be null (Table 7), and no accumulationof 1-methylimidazole (which had previously been suggested asa putative breakdown product56) was observed. On the otherhand, the short chain anions (ethanoate, lactate, ethylsulfonateand ethylsulfate) were degraded, even if only partially, in allcases. It becomes apparent that, amongst the isolated microbialstrains, there are both fungi and bacteria which were able todegrade, up to a certain extent, these anions. It is not surprisingthat the biodegradability rates were only ~20%, since their degra-dation has been previously observed to be highly concentrationdependent, e.g. the efficiency of ethanoate degradation by fungidecreased 40% when concentration increased from 0.125 to0.25 M.33

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Table 7 Aerobic degradation by soil microbial strains of the cations [Cnmim]+ (n = 2 and 4) and [N111(EtOH)]+, and of the anions lactate, ethylsulfate,ethylsulfonate, ethanoate after two months of incubation in liquid media (ionic liquid initial concentration equal to 0.5 and 1 M).

Degradation rate (%)

Cation Anion

Ionic liquid [IL]/M Salt marsh soil Industrial soil Salt marsh soil Industrial soil

[C2mim]Cl 0.5/1.0 n.d. n.d. * *[C2mim][Lac] 0.5 — n.d. — 27.1[C2mim][C2SO3] 0.5 — n.d. — 8.9[C2mim][C2SO4] 0.5 n.d. n.d. 22.4 25.4

1.0 n.d. n.d. 2.3 20.8[C2mim][O2CMe] 0.5 n.d. — 18.5 —

1.0 n.d. — 11.3 —[C4mim]Cl 0.5 — n.d. — *[N111(EtOH)]Cl 0.5 20.3 10.1 * *

1.0 9.3 10.5 * *[N111(EtOH)][lac] 0.5 — 5.2 — 6.2

Average values of duplicates; standard error between duplicates is < 0.05%. n.d., not detected. Blank entries correspond to absence of microbialdegradation. *, not quantified.

Conclusions

It became apparent that the environmental pressure caused byhigh petroleum hydrocarbon load and, secondarily, by highsalinity in soil, augmented the microbial capacity to activelygrow or to survive short or long periods of exposure to ionicliquids. The conditions studied, despite the fact they do notintend to mimic any realistic pollution scenario, focused onionic liquids currently produced at remarkable quantities. Eventhe most benign chemicals provoke an immediate impact (asobserved for the cholinium chloride); however, environmentalrisk should be mainly defined by the chemical environmentalpersistence (and biodegradability), which ultimately determinesthe recovering capacity of the biotypes from the contaminatedecosystem. The microbial strains taxonomically characterisedhere are good candidates for advanced studies of remediationsolutions for possible spills of these liquids salts.

Acknowledgements

The work was partially supported by a grant from Iceland,Liechtenstein and Norway through the EEA financial mech-anism (PT015) and by FCT (Project QUIM/71331/2006). F.J. Deive wants to thank Fundacion Juana de Vega for a post-doctoral grant. The authors would like to thank M. L. Cravo,M. C. Pegado and A. M. Neves, specialist technicians fromUARN for their valuable assistance in sample characterisation.The authors would like to thank Dr P. Moran from UVigo forher valuable assistance in bacterial characterisation, and LuısMorgado (ITQB) for the graphical abstract design.

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