Discovering Low Toxicity Ionic Liquids for Saccharomyces cerevisiae by Using the Agar Well Di ﬀ usion Test

: Ionic liquids (ILs) are new solvents widely used in many technologies due to their unique and advantageous physicochemical properties. In biotechnological applications, ILs can be used along with microorganisms such as Saccharomyces cerevisiae . Due to the enormous number of ILs that can be synthesized through the combination of di ﬀ erent anions and cations, it is necessary to have an easy and quick tool for the preliminary screening of their biocompatibility for being used in biotechnological applications. In this work, the agar well di ﬀ usion test was successfully applied as a rapid method to identify toxic / nontoxic ILs toward S. cerevisiae . Sixty-three ILs containing a diverse set of cations and anions were used. Through this methodology, nine fully biocompatible ILs toward S. cerevisiae were identiﬁed, including: [Bmim + ] [NO 3 − ], [HOPmim + ] [NO 3 − ], [Bmim + ] [NTf2 − ], [N 8,8,8,1 + ] [NTf2 − ], [S 2,2,2 + ] [NTf2 − ], [EMPyr + ] [NTf2 − ], [BMPi + ] [NTf2 − ], [Moxa + ] [MeSO 4 − ] and [Chol + ] [H 2 PO 4 − ]. The analysis of the results also provides preliminary rules to enable the design of biocompatible ILs with S. cerevisiae . In this context, the toxicity was mainly determined by the cation nature although some anions can also display a strong inﬂuence on the IL biocompatibility as the bistriﬂimide anion. Besides, it was observed that an increase in the alkyl chain length of cations, such as imidazolium or pyridinium, involves an increase in the IL toxicity.

At the same time, ionic liquids (ILs) are low-melting-point salts that have become increasingly attractive as green solvents for industrial applications. From an environmental point of view, their most important property is their practically zero vapor pressure. Furthermore, IL properties can be tailored for a specific application by accurately selecting the cation and the anion. Taking into account all these features, ILs are considered good candidates to be tested as extracting agents or solvents in

Results and Discussion
The biocompatibility of 63 ILs toward S. cerevisiae was assessed by using the agar well diffusion test previously described. The names of these compounds and the results obtained are shown in Tables 1-6. The importance of evaluating the compatibility of ILs with S. cerevisiae lies in the development of new biotechnological processes that combine ILs and S. cerevisiae as it has been recently reported by de los Ríos et al. [19]. In this previous work, they evaluated the toxicity of nine water-insoluble ILs toward S. cerevisiae using the agar diffusion test (in pure form and 3%v/v IL solution), specific growth rates (µ, h −1 ) in liquid media at 3% (v/v) IL and the final dry-weight concentration of yeast at 48 h [19]. The toxicity results found by the authors with the agar diffusion test were similar to those achieved in the rest of the toxicity assays evaluated (growth in liquid media and final dry weight concentration), which confirms the suitability of the well diffusion test as a simple method to estimate the toxicity of ILs toward S. cerevisiae [19]. In this context, the growth rate S. cerevisiae in the presence of ILs which exhibited zero inhibition radius (agar diffusion test, pure ILs) was similar to the growth rate of the control without IL. These preliminary results allow us to classify the toxicity of ILs as 'very toxic' when the inhibition radius is higher than 1 cm, as 'toxic' when de inhibition radius is between 1 and 0.5 cm, as 'low toxic' when it is between 0.5 and 0.0 cm and as 'biocompatible' when the inhibition radius is equal to zero. The IL [Omim + ] [dca − ] is very toxic and, on the contrary, [HOPmim + ] [NO 3 − ] and [Bmim + ] [NTf2 − ] can be considered biocompatible with S. cerevisiae (see Table 1). On the other hand, it was found that water solubility was not directly related to the toxicity of the ILs toward S. cerevisiae as can be observed in Table 1. For instance, some ILs that are water-insoluble are very toxic (e.g., [P 6,6,6,14 + ] [C 9 COO − ], see Table 2) while other water-soluble ILs are low toxic or even biocompatible with the yeast (e.g., [Omim + ] [dca − ], see Table 1). This implies that it is necessary to analyze the relationship between toxicity and IL structure. In the following sections, we systematically analyze the qualitative relationship between IL structure and toxicity. cerevisiae (see Table 1). On the other hand, it was found that water solubility was not directly related to the toxicity of the ILs toward S. cerevisiae as can be observed in Table 1. For instance, some ILs that are water-insoluble are very toxic (e.g., [P6,6,6,14 + ] [C9COO − ], see Table 2) while other water-soluble ILs are low toxic or even biocompatible with the yeast (e.g., [Omim + ] [dca − ], see Table 1). This implies that it is necessary to analyze the relationship between toxicity and IL structure. In the following sections, we systematically analyze the qualitative relationship between IL structure and toxicity.

1-hexyl-3methylimidazolium dicyanamide
Liquid [19] 0.5 ± 0.2                             0.1 ± 0.0 Table 6. Biocompatibility data of piperidinium-, morpholinium-, oxazolinium-and sulfonium-based ionic liquids toward S.cerevisiae using agar well diffusion test. 0.1 ± 0.0 Table 6. Biocompatibility data of piperidinium-, morpholinium-, oxazolinium-and sulfonium-based ionic liquids toward S.cerevisiae using agar well diffusion test. 0.1 ± 0.0 Table 6. Biocompatibility data of piperidinium-, morpholinium-, oxazolinium-and sulfonium-based ionic liquids toward S.cerevisiae using agar well diffusion test. 0.1 ± 0.0 Table 6. Biocompatibility data of piperidinium-, morpholinium-, oxazolinium-and sulfonium-based ionic liquids toward S.cerevisiae using agar well diffusion test.   [31,72,73] 0.1 ± 0.0 Table 6. Biocompatibility data of piperidinium-, morpholinium-, oxazolinium-and sulfonium-based ionic liquids toward S.cerevisiae using agar well diffusion test.  According to the results obtained, an increase of the alkyl chain length in the IL cation (considering the same anion) involves an increase in the toxicity of the ILs to S. cerevisiae. This behaviour is found for the imidazolium cation (see Table 1  According to the results obtained, an increase of the alkyl chain length in the IL cation (considering the same anion) involves an increase in the toxicity of the ILs to S. cerevisiae. This behaviour is found for the imidazolium cation (see Table 1

Influence of the Alkyl Substituent of the Cation of Ionic Liquid on the Toxicity toward S. cerevisiae
According to the results obtained, an increase of the alkyl chain length in the IL cation (considering the same anion) involves an increase in the toxicity of the ILs to S. cerevisiae. This behaviour is found for the imidazolium cation (see Table 1

Influence of the Alkyl Substituent of the Cation of Ionic Liquid on the Toxicity toward S. cerevisiae
According to the results obtained, an increase of the alkyl chain length in the IL cation (considering the same anion) involves an increase in the toxicity of the ILs to S. cerevisiae. This behaviour is found for the imidazolium cation (see Table 1 According to the results obtained, an increase of the alkyl chain length in the IL cation (considering the same anion) involves an increase in the toxicity of the ILs to S. cerevisiae. This behaviour is found for the imidazolium cation (see Table 1 [11] studied the toxicity of nine immiscible ILs toward S. cerevisiae, used in biphasic systems in the synthesis of 2-phenylethanol catalyzed by this yeast. To this aim, S. cerevisiae was grown in the presence of the selected ILs. The results found are in line with those obtained in the present work since the longer the alkyl side chain on the imidazolium ring, the lower biocompatibility of the ILs. In other microorganisms, it was also found that the toxicity was directly related to the chain length of the alkyl substituent on the cation [19,[77][78][79][80]. The influence of an increasing chain length of the imidazolium cation moiety on the cytotoxicity in marine bacteria and two types of mammalian cell cultures were also evident in HeLa cells [81,82]. This effect is currently known as the 'side-chain effect' [83]. For highly lipophilic cations, cytotoxicity does not significantly increase with lipophilicity anymore. It is well known that lipophilicity relationships with biological activity are only linear over a restricted range [84]. The 'side-chain effect', in which carbon atoms are added, involves a high hydrophobic character in ILs. It would increase the possibility of their interaction with phospholipid bilayers of the cell membranes and the hydrophobic domains of the membrane proteins, leading to the disruption of the membrane physiological functions and, consequently, to cell death [81,82,85,86]. On the contrary, as can be observed in Table 1, the inclusion of an oxygen atom in the alkyl substituent of the imidazolium ring can significantly decrease the toxicity of imidazolium-based ILs. It has been observed that [HOPmim + ] [Cl − ] (RI = 0.1 cm) displayed much lower toxic effects than [Bmim + ] [Cl − ] (RI = 0.6 cm), so the substitution of the methyl by a hydroxy group converts a toxic IL into a slightly toxic IL. This behavior is in agreement with the work of Álvarez-Guerra and Irabien [87], who reported that the presence of oxygenated groups in the structure of cations can lead to a decrease in the ecotoxicity of the IL. In line with these results, Tether et al. [88] reported the decrease in the ionic liquid toxicity toward Escherichia coli and Staphylococcus epidermidis through the side chain oxygenation by using the agar diffusion test. In this context, the presence of the oxygen makes the IL more hydrophilic, and therefore, less toxic. It is also important to remark the high toxicity observed for [ The inclusion of the methyl substituent in the R2 position of the imidazolium ring could reduce the acidic proton in 2, so lowering the toxicity of the IL. This effect was also recently observed for Shewanella [89].

Effect of the Ionic Liquid Cation on Toxicity toward S. cerevisiae
In order to study the effect of the cation structure, ILs with the same anion and different cation were compared. In general, the cation toxicity for other microorganisms has been higher for ILs containing aromatic cations, such as imidazolium and pyridinium cations, in comparison to nonaromatic cations, e.g., pyrrolidinium. A higher hydrophobic character of aromatic cations could increase the possibility of interaction with the cell membrane [81,82,85]. Furthermore, the planarity of the cation ring in imidazolium and pyridinium appeared to be also a relevant parameter for increasing IL toxicity, as reported in [90]. This fact could be due to the lower steric hindrance of the aromatic cation, which might favor the interactions with the lipidic membrane to a greater extent. For S. cerevisiae, a high toxicity was found for pyridinium and imidazolium cations compared with pyrrolidinium cations for the series [ [92] determined the maximum nontoxic concentration of choline-based ILs to S. cerevisiae finding significant biocompatibility when the ILs were water-soluble. The biocompatibility with choline-based hydrophilic IL was lower. All these results are in good agreement with those reported in the present study.
In the case of the phosphonium family, it was found that long alkyl chains promote higher toxic effects toward the bacterium Vibrio fisheri [83], following the 'side-chain effect' mentioned above. The toxicity values for phosphonium-based IL toward S. cerevisiae ranged from RI = 1.5 cm ([P 6 was obtained by determining the maximum nontoxic concentration of the IL for S. cerevisiae, resulting in toxicity toward S. cerevisiae [92]. The same result was obtained in this work by using the agar well diffusion test.
On the other hand, the morpholinum-based IL was found to be biocompatible with S. cerevisiae, which might be due to the inclusion of a heteroatom in the imidazolium ring and also to the low alkyl chain constituent of the aromatic ring. Finally, only a sulfonium-based IL was assessed, [S 2,2,2 + ] [NTf2 − ], which proved to be biocompatible with S. cerevisiae. These results might be related to the nature of its counteranion.

Effect of the Ionic Liquid Anion on Toxicity toward S. cerevisiae
The effect of the anion composition on the IL toxicity was analyzed by comparing ILs with different anions and the same cation. Several authors have reported that the toxicity of ILs on several microorganisms is directly related to the cation nature, while the anion seems to modulate the toxicity to some extent and in specific cases [77][78][79][80]93]. This behavior was observed in several ILs in which the toxicity values were similar when sharing the same cation but contain different anions. For example,  1 cm). This could be explained by the contribution of other factors to the toxicity values, such as anion nature, IL solubility, or the synergy effect between anion and cation nature. Synergy effects between the anion and the cation can occur, which make the complete isolation of individual anion and cation contributions difficult; and, as mentioned above, IL toxicity has been mainly correlated to the cation than to the anion. Another important consideration of the anion is that those with high lipophilicity or susceptible to hydrolysis could offer partially drastic effects. [PF 6 − ] could be included in this group, since it is well established the instability of ionic liquids containing this anion toward hydrolysis in contact with moisture-forming volatiles, e.g., HF, POF 3 , etc., which might pose potentially hazardous effects [94][95][96]; [NTf2 − ] could also be included due to its hydrophobicity, which is even higher than [PF 6 − ] [33,96].
For a better understanding of the influence of the anion nature on IL toxicity, an anion sequence for different cation families is presented in Table 7

Mechanisms of IL toxicity
Relatively few mechanisms have been suggested to explain the toxicity of ILs towards microorganisms, but membrane disruption is considered the most common [98,99]. The ability of the ILs to disrupt the cell wall of different microorganisms seems to be due to their hydrophobicity, caused by the length of the alkyl side chain of the cation or the presence of aromatic cations, which is in line with the results obtained in the present work.
Furthermore, the inclusion of a heteroatom such as oxygen leads to reduce the hydrophobicity of the IL and thus toxicity. Weuster-Botz [100] 3 COO − ]. They found that these ILs likely target mitochondria. High-throughput chemical proteomics showed the effects of ILs on mitochondrial protein levels. ILs induced abnormal mitochondrial morphology, as well as, altered the polarization of mitochondrial membrane potential, similar to valinomycin.

Ionic Liquids Biocompatible with S. cerevisiae
The agar well diffusion test can serve as an easy and quick decision-making tool when it comes to choosing sustainable ILs for biotechnological applications involving S. cerevisiae. In this case, ILs are deemed as biocompatible with S. cerevisiae when the radius of inhibition is equal to zero. In this sense, those classified as biocompatible are [Bmim + ] [NO 3 − ] in which the low toxicity could be explained by biocompatible with S. cerevisiae. It is also important to note that the agar well diffusion method for the assessment of IL toxicity toward S. cerevisiae has been validated not only by comparison with other methods in previous works of our research group ( [19]) but also with the results reported by other authors in this field, as commented throughout this work.

Conclusions
This work assesses the toxicity of a high number of ionic liquids toward S. cerevisiae using the agar well diffusion test. This method enables the easy and quick analysis of their biocompatibility toward S. The results obtained also allowed us to establish several toxicity-structure relationships which could even help to make important predictions about IL toxicity, without further experimentation. Thus, it was observed that an increase in the alkyl chain length in cations such as imidazolium or pyridinium involves an increase in the hydrophobicity of the ILs and therefore, an increase of their toxicity. Furthermore, the toxicity is mainly determined by the cation but, certain anions have a strong influence on IL toxicity as in the case of the [NTf2 − ] anion, which dramatically reduces the toxicity of the IL towards S. cerevisiae.