Microbial adaptation to ionic liquids

Abstract

One out of 10 microorganisms from extreme locations was adapted to the presence of common families of ionic liquids, which have lately emerged as “contaminants on the horizon”. A 10-fold higher tolerance was concluded for the ionic liquid-resistant strain. A biopolymer was secreted as an adaptation response.

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Over the last years, environmental concerns have highlighted the extensive and increasing importance of implementing industrial processes using greener solvents. In particular, the replacement of volatile organic compounds with ionic liquids has set the pace for the achievement of truly revolutionary processes. Although the low vapour pressure¹ of ionic liquids may reduce the air pollution with respect to the typical volatile organic compounds, some of them show a high solubility in water, thus becoming persistent pollutants in both the aquatic and the soil environment.² These neoteric solvents can be considered as new emerging contaminants since they are already used with some extant processes at an industrial scale in companies such as BASF (BASIL, aluminium plating, cellulose dissolution), and the annual production of some of them surpasses the ton magnitude. Accordingly, ionic liquids are assumed to gain environmental relevance and they have recently been reported as “contaminants on the horizon”.³

Therefore, the proposal of efficient methods for their removal falls into one of the priorities established in the current global environmental water policies. Among the existing alternatives for pollutants remediation, biological methods stand out as more environmentally sustainable ones and they bear a rather positive social image, instead of their chemical and physical counterparts. Up to date, most biological assays for ionic liquids as pollutants have been defined under static laboratory conditions and with the same type of microorganisms that despite their importance are usually unrealistic, failing to reproduce the numerous abiotic and biotic processes occurring in the environment. In general, the studies of environmental fate and toxicity of ionic liquids have shown that the most common families present a considerable toxicity, which varies across organisms and trophic levels.⁴ Generally speaking, different ionic liquids have been reported to be highly toxic to microorganisms due to different mechanisms: be it through increase in osmotic pressure, a modification of membrane fluidity and structure, or an alteration of enzymatic activity.⁵ Since ionic liquids pose a breakthrough in the chemical industry, the hunt of novel bacterial strains and/or engineered existing strains for ionic liquid tolerance is critical. One solution to this problem could be placed in extreme microorganisms, which would play a role as “ionic liquids-metabolizers”. Our preliminary data⁶ allowed us to conclude that the environmental pressure caused by high petroleum hydrocarbon load and, to a lesser extent, by high-salinity in soil, augmented the microbial capacity to actively grow or to survive short or long periods of exposure to ionic liquids. Starting from this premise, we have bet in this kind of microorganisms as viable candidates for ionic liquids bioremediation. With this aim, several commercial families of ionic liquids have been proposed as chemical pressure in the culture medium to select the most promising microbial strain in terms of ionic liquid endurance. Their structure is shown in Fig. 1.

Fig. 1 Structure of ionic liquids used.

Considering the basic definition of ionic liquids as molten salts it makes sense to test the response of marine bacteria like Shewanella oneidensis and Halobacterium salinarum as representative halotolerant microorganisms. In relation to the ionic liquids role as organic compounds, Staphylococcus warneri, Pseudomonas stutzeri, and Consortium C26b are also interesting since they are bacteria commonly found in industrial polluted areas.^7,8 Moreover, thermophilic microorganisms are getting increasing attention in biotechnology due to the fact that their enzymes are better suited to operate under harsh industrial processes. For this reason, Anoxybacillus flavithermus and Thermus thermophilus HB27 were chosen as representative thermophiles to analyse their tolerance to the presence of ionic liquids. Finally, two white-rot fungi with demonstrated capacity to degrade persistent contaminants were also included in this initial screening: Phanerochaete chrysosporium and Trametes versicolor. Their growth curves in the absence of ionic liquids are shown in ESI (Fig. S2 and S3†).

The ionic liquids toxicity was evaluated by means of their minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC), through microorganisms cultivation in 96-well plates in mineral medium supplemented with glucose as carbon source (10 g L⁻¹), ionic liquids concentrations 0.005, 0.010, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, and 1.5 M, and the growth was monitored by UV spectrometry at 600 nm. Although no differences were observed for the MIC values, the analysis of the MLC data (listed in Table 1) confirmed that the microbial agents obtained from polluted locations (P. stutzeri, St. warneri and Consortium C26b) and the marine bacteria (S. oneidensis) show a higher resistance to thrive under the pressure of these neoteric solvents. The hypothesis that both hydrocarbon load and salinity could improve the possibilities of survival is thus confirmed, in agreement with our previous findings.⁶ The analysis of the selected cations in terms of toxicity reveals that phosphonium is the one leading to a greater lethal effect. The information coming from the literature about the hazards of this family is still scarce and not conclusive, although the initial data provided by Coutinho and coworkers allow confirming our results.⁹ In relation to the anion, a slightly higher toxicity of the [C₁SO₄] is observed. This seems to contradict the statement that a longer alkyl chain leads to higher toxicity.¹⁰ Nonetheless, it should be noted that the first member of a family is usually an outlier (not following an extrapolation of the trend presented by the others), so that could explain this behaviour.

Table 1 MLC values of the selected microorganisms under the pressure of different ionic liquids. White colour indicates no growth in the tested range, and grey colour shows growth

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The comparison of the MLC values obtained with relevant literature data reveals that both the microbial agents obtained from polluted and marine locations are highly resistant to the studied ionic liquids, since concentration levels up to 1 M are tolerated. These values are higher to those reported in literature¹¹ for model bacteria and yeasts. Additionally, these microorganisms were able to survive at concentrations almost similar to those reported for the most biocompatible ionic liquids based on cholinium cations.¹² It is necessary to highlight that P. stutzeri was the bacterium leading to the highest values of biomass under the pressure of ionic liquids. Therefore, this bacterium was selected as a viable candidate for an acclimation process. After two months in a lab-scale bioreactor in the presence of [C₂C₁im][C₂SO₄] (200 μM), under controlled agitation, aeration and temperature, the microbial biomass was collected to further investigate the existence of some kind of acclimation. The analysis of this strain revealed MLC levels one order of magnitude higher for imidazolium and pyridinium cations, and 2 times higher for phosphonium-based ionic liquid. Additionally, cell concentration data (shown in Table 2, and graphically represented in ESI in Fig. S4 to S11†) allow concluding very high values for the adapted P. stutzeri, no matter the culture medium used (both rich and mineral). This is advantageous because the use of a mineral medium is preferred to approach future studies of bioremediation.

Table 2 Microbial growth of the selected microorganisms at maximum ionic liquid concentration in mineral (MM) and rich medium (RM). (−) no growth; (+) A₆₀₀ = 0.1–0.4; (++) A₆₀₀ = 0.4–0.6; (+++) A₆₀₀ > 0.6

	[C2Py][C2SO4]		[C2C1im][C1SO4]		[C2C1im][C2SO4]		[P4441][C1SO4]
	MM	RM	MM	RM	MM	RM	MM	RM
P. s.	+++	++	++	++	++	++	+	+
P. s. a	+++	+++	+++	+++	+++	+++	++	++
S. o.	+++	+++	++	+++	++	+++	+	++
St. w.	+++	++	++	+++	++	+++	++	+++
C26b	++	++	++	++	++	+++	+	++
H. s.	−	+	−	++	−	+	−	−
P. c.	−	+	−	++	−	+++	−	++
T. v.	−	+	−	++	−	+++	−	+
T. t.	−	+	−	+	−	+	−	+
A. f.	−	++	−	+	−	+	−	++

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Hence, the results obtained suggest that acclimation is taking place, which can be due to a phenotypic and/or genetic change. Up to date, no information has appeared in the literature indicating the viability of ionic liquid adaptation of microorganisms. It is also interesting to notice that the adaptation of P. stutzeri to imidazolium-based ionic liquids involved and acquired resistance to the stress of other commercially available ionic liquid families. Notwithstanding the fact that the specific mechanisms of toxicity are currently not well-understood, there are several research lines that point to different strategies to unravel the microbial response to the presence of ionic liquids, such as the modification of membrane permeability, enzyme detoxification, or the synthesis of metabolites allowing the entrapment of the contaminant, both extracellular- and intracellularly.¹³ In this sense, the ionic effect related to the presence of the ionic liquid in aqueous solutions should also be taken into account, since it could promote the observed microbial toxicity.² In this particular case, it becomes patent that the adaptation entails a clear visual change in the culture broth, as illustrated in Fig. 2. The formation of a biopolymer after 24 h of cultivation of the adapted P. stutzeri is evident. This response has been found to be one of the ways to protect the microbial communities from environmental stresses.¹⁴ In this particular case, the obtained biopolymer turned out to be a polysaccharide mainly composed by glucose, as elucidated from HPLC analysis (see experimental details and chromatogram Fig. S4 in ESI†).

Fig. 2 Visual aspect of P. stutzeri wild (left) and adapted (right) in the presence of ionic liquids.

Thus, the flexible nature of prokaryotic gene expression conferred a greater acclimation to the presence of different families of ionic liquids, by means of exopolysaccharide synthesis. The analysis of the wild strain of P. stutzeri and that adapted to the presence of ionic liquids by means of SEM microscopy (Fig. 3) makes it evident the presence of this polymer entrapping bacterial cells.

Fig. 3 SEM images of wild (left) and ionic liquids-adapted (right) P. stutzeri.

It should be noted that the polysaccharide expression is maintained even though the ionic liquid is removed from the media, which points to an alteration at the gene level. Therefore, further investigation of a global bacterial response at the transcriptome level could shed light on the understanding of the adaptation strategies followed by microorganisms to the presence of these emerging neoteric contaminants, and must be unavoidably tackled in future works. The synthesis of biopolysaccharides also confers special advantages for the formation of biofilms, which allow a higher withstanding to nutrient deprivation, pH changes, or contaminants charge swings.^15,16 Thus, the presence of these biopolymers could be beneficial for biosorption, bioaccumulation or biomineralization strategies.¹⁷

Conclusions

In this work, a preliminary screening among microorganisms from extreme biotopes (high temperature, hydrocarbon load and salinity) allowed confirming the suitability of extreme microorganism from locations with both high salt and hydrocarbon charge to thrive under the presence of common families of ionic liquids. The existence of microbial acclimation to the presence of ionic liquids was demonstrated for the first time by a two-month cultivation of P. stutzeri in a stirred tank bioreactor. Finally, the production of a biopolysaccharide was the permanent response obtained to the continuing exposure to the presence of ionic liquids.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra10283e

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