# The Comparison of Typical Organic Solvents and Ionic Liquid for More Environmentally and User-Friendly Pyocyanin Extraction

Joanna Honselmann Genannt Humme, Paula Ossowicz-Rupniewska, Martyna Jurkiewicz, Marcin Gano, Kamila Dubrowska, Adrian Augustyniak, Ewa Janus, Ewa Mijowska, Lidia Favier, Rafal Rakoczy

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Joanna Honselmann Genannt Humme, Paula Ossowicz-Rupniewska, Martyna Jurkiewicz, Marcin Gano, Kamila Dubrowska, et al.. The Comparison of Typical Organic Solvents and Ionic Liquid for More Environmentally and User-Friendly Pyocyanin Extraction. Industrial and engineering chemistry research, 2024, 64 (1), pp.781-787. 10.1021/acs.iecr.4c04206. hal-04867350

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# The Comparison of Typical Organic Solvents and Ionic Liquid for More Environmentally and User-Friendly Pyocyanin Extraction

Joanna Honselmann genannt Humme,\* Paula Ossowicz-Rupniewska, Martyna Jurkiewicz, Marcin Gano, Kamila Dubrowska, Adrian Augustyniak, Ewa Janus, Ewa Mijowska, Lidia Favier, and Rafał Rakoczy

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Cite This: *Ind. Eng. Chem. Res.* 2025, 64, 781–787

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Supporting Information

**ABSTRACT:** Pyocyanin is a pigment that recently gained importance as a valuable chemical. Most often, the method for extraction is based on the use of chloroform. Therefore, an efficient and more environmentally friendly method is vital for further use. We compared organic solvents and ionic liquids as pyocyanin extractants. We assessed the extraction efficiency using a pyocyanin standard and pyocyanin extracted directly from the bacterial culture. The results showed that only chloroform, dichloromethane, and ethyl acetate were useful for pyocyanin extraction among the tested organic solvents. Extraction with chloroform was the most efficient method. Notably, the ionic liquid could also be favorable for the extraction process, exhibiting antibacterial properties and being separated from the pigment by adsorbing pyocyanin on clays and desorbing it with ethanol. The extraction of pyocyanin can be led by using alternative extractants, i.e., dichloromethane or ionic liquid, and therefore, it can be less hazardous for the environment and the user.

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## 1. INTRODUCTION

Phenazines constitute a diverse group of pigments synthesized in the natural environment by a myriad of microorganisms, including bacteria (e.g., *Pseudomonas*, *Streptomyces*, *Sorangium*, *Brevibacterium*, *Burkholderia*, *Bacillus*, *Erwinia*, *Pantoea*, *Vibrio*, *Pelagiobacter*, *Dermacoccus*) and archaea (*Methanosarcina*).¹ Among these pigments, pyocyanin (PYO), produced by *Pseudomonas aeruginosa* and *P. paraeruginosa*, is one of the most extensively studied. Over the past decades, PYO has garnered significant attention not only for its role as a virulence factor but also for its potential applications in agriculture, cancer, mitochondrial disease therapy, energy generation in microbial fuel cells, or as a dye for fabric or paper.²⁻⁵

PYO is produced in *P. aeruginosa* cultures and necessitates purification from the medium for its quantification and subsequent utilization. Among the methods used for pyocyanin quantification are spectrophotometric, chromatographic, and voltammetric analyses or surface-enhanced Raman spectroscopy.⁶ The most frequently reported technique for this purpose involves organic solvent extraction, predominantly chloroform (TCM), followed by sample acidification and spectrophotometric quantification. Upon acidification, PYO undergoes a distinct color change from blue (neutral or basic pH) to pink (acidic pH).

The conventional extraction process entails the utilization of organic solvents, which may pose environmental hazards. Consequently, in adherence to the principles of green chemistry, there is a growing impetus to explore more

environmentally benign alternatives. However, solvent selection is often constrained by considerations of chemical activity, toxicity, and other properties, rendering it a challenging endeavor.⁷ Thus, this study aims to compare PYO extraction using TCM, a well-established method, to extractions employing alternative organic solvents. Following guidelines established by companies and international consortia for green chemistry, we opted to evaluate organic solvents proposed as substitutes for TCM, including dichloromethane (DCM), as well as other less hazardous options such as ethyl acetate (EA), 2-methyltetrahydrofuran (Me-THF), and methyl *tert*-butyl ether (MTBE).

In addition to organic solvents, ionic liquids (ILs) have emerged as promising solvents in the extraction processes, particularly in the context of extracting valuable substances from biological samples.⁸⁻¹⁴ The unique properties of ILs, such as low volatility, high thermal stability, and tunable chemical structures, make them ideal candidates for efficiently extracting bioactive compounds. Ghadge¹⁵ utilized the IL tetrabutylammonium hydroxide (TBAOH) for melanin (brown pigment) extraction from *Streptomyces hyderabadensis*

Received: November 4, 2024

Revised: December 10, 2024

Accepted: December 11, 2024

Published: December 24, 2024

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culture. ILs are also frequently mentioned as antimicrobial agents.<sup>16–19</sup> However, ILs remain rather costly reagents. Therefore, separation techniques are needed to recover the IL from extracted substances. Among them are distillation, extraction, adsorption, membrane separation, crystallization, force field, or combined methods.<sup>20</sup>

This study evaluates whether selected organic solvents can effectively replace TCM while exhibiting reduced adverse impacts on users and the environment, comparable PYO extraction efficiency, and lower costs. Moreover, we aimed to verify if ionic liquids can be utilized for pyocyanin extraction and replace the organic solvents. We also evaluated the possibility of pyocyanin separation from ionic liquid ([Chol]-[NTf₂]) using the adsorption/desorption approach on clays. Finally, we tested the toxicity of the IL to reference bacteria.

## EXPERIMENTAL SECTION

**Chemicals and Microbiological Model.** All solvents were of analytical grade and purchased from Chempur (Poland) (TCM, DCM, and EA) or Merck (Germany) (MeTHF, MTBE). Choline bis(trifluoromethylsulfonyl)imide ([Chol][NTf₂], 99%) was provided by IoLiTech (Germany). PYO standard in the form of blue powder and acetonitrile for HPLC (gradient grade, ≥ 99.9%) were purchased from Merck (Germany).

The PYO-producing strain used in the research was *Pseudomonas aeruginosa* ATCC 27853. The strain was kept at –20 °C in Trypticase Soya Broth medium (Biomaxima, Poland) supplemented with 10% v/v glycerol and revived on Trypticase Soya Agar (Biomaxima, Poland) medium at 37 °C. The strain was cultivated in King's A broth (20.00 g/L of K₂SO₄, 1.64 g/L MgCl₂, 1% v/v of glycerol, pH = 7.0).

**Extraction of PYO Standard.** Initially, the efficacy of organic solvents (TCM, DCM, EA, Me-THF, MTBE) and ionic liquid [Chol][NTf₂] was visually assessed using PYO standard dissolved in King's A broth and allowed to choose the most promising extractants. Next, the PYO standard (Merck, Germany) solutions at concentrations of 10, 20, 50, and 100 µg/mL were prepared to simulate extraction from the culture. The extraction was conducted according to the standard method with slight modifications.<sup>21,22</sup> Briefly, 5 mL of the PYO solution was transferred to a test tube, and 3 mL of the solvent was added. The mixture was incubated on a rotary shaker (60 rpm, room temperature) for 1 h, followed by centrifugation to separate the phases (5 500 rpm, 10 min). The organic phase (blue) was collected and transferred to another tube containing 1 mL of 0.2 M HCl. The mixtures were vortexed for 30 s and centrifuged (5500 rpm, 2 min). PYO's acidic phase (pink) extracted with organic solvents was collected for spectrophotometric quantification in a microplate reader (λ = 520 nm). PYO concentration was calculated from the standard curve and expressed in µg per mL of the culture. In the case of the IL, the acidified IL phase was collected for HPLC analysis (described below).

**Extraction of PYO from *Pseudomonas aeruginosa* Cultures.** *P. aeruginosa* ATCC 27853 was cultivated in 10 mL of King's A broth inoculated with an overnight culture (OD₆₀₀ = 0.5, 1:100) at 32 °C for 72 h without agitation. After incubation, the culture was collected and centrifuged to remove the cells (5500 rpm, 15 min). Extraction with each solvent and the ionic liquid was performed in triplicates according to the above-mentioned method. The samples were

subjected to HPLC quantification, which is described in detail below.

**Quantitative and Qualitative Determination of the Extracted PYO.** The phase containing an acidic form of PYO extracted with the chosen solvent was analyzed by using high-performance liquid chromatography. Samples were analyzed without additional purification before HPLC measurement. The analyses were conducted using a SHIMADZU Nexera-i LC-2040C 3D High Plus liquid chromatograph with a DAD/ FLD detector (Kyoto, Japan). The mobile phase consisted of solvent A (water-trifluoroacetic acid, 100:0.04, v/v) and solvent B (acetonitrile-trifluoroacetic acid, 100:0.04, v/v). The separation was achieved using Kinetex 3.5 µm XB-C18 100 Å LC column 150 × 4.6 mm i.d. (Phenomenex, Torrance, CA, USA) and a gradient elution at the temperature of 30 °C. A programmed change in mobile-phase composition during the analysis was as follows: 100%A maintained for 3 min, then a linear gradient to 30% A and 70% B in 27 min, a composition maintained for the next 10 min, then a linear gradient to 100% A in 10 min, and maintained until the end. The flow rate of eluent was 0.5 mL/min for 30 min, then 1 mL/min for the next 10 min, and finally 0.5 mL/min until the end (60 min from the starting time). The detection wavelength was 520 nm. The sample was injected thrice by a dosing loop with a volume of 50 µL. In the case of typical solvents, after acidification, pyocyanin in acidic form passed into an aqueous solution, while in the case of extraction using an ionic liquid, after acidification, the pink pyocyanin remained in the ionic liquid layer, and in this case, the analysis was performed in the ionic liquid layer. The rest of the procedure remained unchanged. The calibration curve for HPLC measurements is presented in Figure S1 (Supporting Information).

**Adsorption and Desorption of PYO on Clays.** The clays, i.e., montmorillonite and sepiolite, were characterized by XRD and BET methods (Supporting Information, Figure S2, Table S1). X-ray diffraction (Aeris, Malvern Panalytical, UK) was conducted in the range 10–90° with the copper lamp CuKα as a radiation source. The adsorption/desorption isotherms of N₂ were used to determine the specific surface area, total pore volume, and median pore width by using the BET method (BET-Micromeritrics ASAP 2460, Nocross, GA, USA).

To investigate the possibility of recovering PYO from the IL, we attempted to use a separation method based on the adsorption of PYO on clays, which was earlier described for PYO dissolved in aqueous solution.<sup>23</sup> We tested four concentrations of PYO (50, 20, 10, and 5 µg/mL) standard dissolved in King's A medium that was extracted using IL. Later, each concentration of PYO in IL was divided into two groups: pH 7 (blue) and pH 3 (pink, acidified with 0.2 M HCl). The adsorption was conducted using 100 mg of montmorillonite or sepiolite and 400 µL of the standard in IL (24 h, 20 °C, 200 rpm on an orbital shaker). Later, the samples were centrifuged (14 500 rpm, 10 min), and the supernatants were passed to HPLC to quantify the residual PYO in the IL. The pellets were washed with 400 µL of ethanol (24 h, 20 °C, 200 rpm on orbital shaker) to desorb PYO and centrifuged (14 500 rpm, 10 min), and the supernatants were passed to HPLC for PYO quantification in ethanol.

**Antibacterial Properties of IL on Model Microorganisms.** To assess whether the tested IL exhibits bacteriostatic or bactericidal properties against *Escherichia coli* (ATCC 25922),
<table><caption>Table 1. Results of Preliminary Testing of the Solvents<sup>a</sup></caption><thead><tr><th>Solvent</th><th>EA</th><th>DCM</th><th>TCM</th><th>Me-THF</th><th>MTBE</th><th>[Chol][NTf<sub>2</sub>]</th></tr></thead><tbody><tr><td>Evaluation basis</td><td>(slight blueish color in the upper phase)</td><td>(pronounced blue color of the lower phase)</td><td>(pronounced blue color of the lower phase)</td><td>(transparent upper phase)</td><td>(transparent upper phase)</td><td>(pronounced blue color of the lower phase)</td></tr></tbody></table>

<sup>a</sup>“+” - low visible PYO content, “++” - moderate visible PYO content, “+++” - high visible PYO content, “-” - no visible PYO.

*Staphylococcus aureus* (ATCC 33591), and *P. aeruginosa* (used in this study as a pyocyanin producer — ATCC 27853), we subjected the cultures to 4-h-long incubation with IL. Shortly, the overnight bacterial cultures were diluted 1:100 in fresh TSB medium and incubated with IL (in the volumetric ratio of 5:3) for 4 h on an orbital shaker at 20 °C. After that, the cultures were shortly centrifuged to allow good phase separation, and the broth phase was aliquoted to the wells of the polystyrene 96-well microplate. The viability of the cells was tested using a resazurin assay (10% v/v added to the culture).²⁴ The cultures subjected to the same conditions but with no addition of IL served as the controls.

**Statistical Analysis.** Descriptive statistics (mean, standard deviation) were calculated for each group of replicates. Means were compared by one-way ANOVA with Tukey's posthoc test. Cases with *p* ≤ 0.05 were considered significantly different.

## ■ RESULTS AND DISCUSSION

**Extraction of PYO Standard.** Initial testing allowed us to notice that only the two tested organic solvents, TCM and DCM, effectively extracted PYO (Table 1). Moreover, [Chol][NTf₂] also effectively extracted pyocyanin from the medium. The use of EA led to partial extraction of PYO. However, the product was barely visible, and its hue was relatively light compared to that of TCM, DCM, or [Chol][NTf₂]. We also initially tested other ILs (Supporting Information, Table S2.). However, they were characterized by lower extraction efficiencies, higher density, viscosity, or both, impeding the extraction process. Therefore, further research was continued only with TCM, DCM, and EA, and two other solvents (Me-THF and MTBE) were excluded.

Further testing of TCM, DCM and EA used for PYO standard extraction from the King's A medium proved that TCM was the most effective among the organic solvents for the extraction of the desired compound (Figure 1.). Nevertheless, the differences between the extraction efficiency of TCM and DCM did not differ significantly in the case of lower PYO concentrations, i.e., 10 and 20 µg/mL (one-way ANOVA with Tukey's test, at *p* ≤ 0.05). The extraction with EA resulted in a very low concentration of extracted PYO in all tested concentrations, and therefore, EA can be considered the least efficient solvent among all tested. Interestingly, IL [Chol][NTf₂] was the most effective extractant in the PYO standard extraction in the 100 µg/mL concentration. Nevertheless, the IL was as effective or less effective than TCM in the case of lower PYO concentrations. Re-extraction was a vital step in separating almost all PYO from the medium in the case of TCM and DCM (Supporting Information, Table S3). The amount of PYO extracted in the second extraction was significantly higher in the case of DCM than that of TCM and EA at all tested concentrations.

**Extraction of PYO from Pseudomonas aeruginosa Cultures.** TCM was identified as the most effective PYO extractant for *P. aeruginosa* cultures (Table 2), reaching 158.81

<figure><img src="image_12.png" /><figcaption>**Figure 1.** Concentration of PYO standard in samples after extraction from King's A medium; superscripts with different letters indicate statistically significant differences with *p* ≤ 0.05, and error bars represent standard deviation.</figcaption></figure>

<table><caption>Table 2. Results of Extraction of PYO from the Bacterial Culture in King's A Medium<sup>a</sup></caption><thead><tr><th>Solvent</th><th>PYO extraction [µg/mL] average value ± SD</th></tr></thead><tbody><tr><td>TCM</td><td>158.81 ± 14.65<sup>a</sup></td></tr><tr><td>DCM</td><td>102.49 ± 5.17<sup>b</sup></td></tr><tr><td>EA</td><td>21.47 ± 0.62<sup>d</sup></td></tr><tr><td>[Chol][NTf<sub>2</sub>]</td><td>62.59 ± 11.94<sup>c</sup></td></tr></tbody></table>

<sup>a</sup>Superscripts with different letters indicate statistically significant differences with *p* ≤ 0.05, and errors represent standard deviation.

± 14.65 µg/mL in comparison to 102.49 ± 5.17 µg/mL with DCM. Interestingly, promising results were obtained using ionic liquid for extraction - [Chol][NTf₂], for which 62.59 ± 11.94 µg/mL was obtained. Once again, EA was the least efficient extractant, providing only 21.47 ± 0.62 µg/mL. The results obtained for extracting PYO with IL from the culture are significantly lower than those obtained from the pure medium. It may result from a plethora of organic moieties in the culture or the limited extraction capacity of IL.²⁵ Nevertheless, the extraction was still selective toward PYO and should be further investigated by tuning the extraction conditions to exceed our current study's goal.

Replacement of TCM was proposed a few times in the literature. Cequier-Sanchez²⁶ compared the extraction of lipid and fatty acids from animal and plant samples using two setups, one with TCM/methanol and the second with DCM/methanol. The authors concluded that DCM could replace TCM and underlined the lower costs and toxicity of DCM when compared to TCM. Wang²⁷ summarized the organic solvents used for the extraction of homoserine lactones, signaling molecules responsible for quorum sensing in bacteria. Among the most popular extractants, TCM, DCM, EA, ethyl
<figure><img src="image_13.png" /><figcaption>**Figure 2.** Adsorption and desorption of PYO on clays a) montmorillonite at pH 3, b) montmorillonite at pH 7, c) sepiolite at pH 3, and d) sepiolite at pH = 7.</figcaption></figure>

ether, and hexane are considered. However, only the first two are effective and provide similar yields. Regarding PYO extraction, some researchers decided to replace TCM with DCM.²⁸,²⁹ However, only El-Fouly³⁰ tested several solvents (TCM, toluene, *n*-heptane, petroleum ether, hexane, benzene, diethyl ether, and DCM) to extract PYO. They concluded that TCM, DCM, and benzene extracted PYO, with the first two being more efficient than benzene. Nevertheless, the authors did not provide quantitative data to support these findings. Therefore, to the best of our knowledge, our work is the first to quantitatively assess the extraction of PYO using different solvents.

**Adsorption and Desorption of PYO on Clays.** The investigation of the adsorption of PYO from IL showed that both examined clays adsorbed PYO, irrespective of the pH (Figure 2). However, sepiolite was proven to adsorb almost all of the pyocyanin from IL at all tested concentrations. In contrast, montmorillonite adsorbed from 56.5 to 83.0% of PYO at pH 3 and from 50.8 to 97.7% at pH = 7. The higher the concentration of PYO, the lower the amount of PYO adsorbed on montmorillonite. No such tendency was observed on sepiolite. However, in the case of desorption, it was noted that the concentration of the desorbed PYO from montmorillonite was growing with the ascending concentrations of the pigment and reached around 40% of the initial PYO in the IL (for 50 µg/mL of PYO at all tested pH values). Nevertheless, in the case of sepiolite, the pH was a deciding factor for desorption. At pH 3, the concentration of the desorbed PYO

was higher the higher the initial standard concentration and reached the maximum of ca. 52% for the 50 µg/mL of PYO standard. Nonetheless, at pH 7, practically no PYO was desorbed to ethanol. We attempted desorbing PYO from sepiolite at pH 7 using acidified ethanol to verify if the pH was the deciding factor. Nevertheless, still no or very little pyocyanin was desorbed. It may suggest that PYO and sepiolite are firmly bound together at pH = 7. We also tested whether adding another volume of ethanol after the first desorption results in further PYO release from the clays. In the case of montmorillonite and sepiolite at pH 3, we found that some residual PYO was desorbed. Again, the higher the PYO standard concentration, the higher the amount of desorbed PYO (up to 10% of the initial value of PYO).

Adsorption of PYO was previously investigated using sepiolite, palygorskite, montmorillonite, and microporous magnesium oxide-templated carbon to reduce the cytotoxicity exerted by PYO.²³,³¹ Fashina and Deng²³ showed that PYO was adsorbed in the interlayer of montmorillonite, in the tunnels of sepiolite, and on the external surface of palygorskite. It was proven by molecular dynamics simulations that PYO is adsorbed in the interlayer spaces of montmorillonite and is oriented differently based on its protonation.³²

Based on the results, we propose sepiolite as an effective clay for PYO adsorption, as it separates PYO and the IL. However, additional experiments should be conducted to assess the possibility of the IL reuse in another extraction. Moreover, the
desorption process should be subjected to thorough optimization to increase the amount of desorbed PYO.

**Antibacterial Properties of IL.** The test showed that the IL exhibited bactericidal properties against all tested micro-organisms. It was previously reported that various ionic liquids exert such properties, and they were even proposed as antibacterial agents for air purification and combating nosocomial infections.16,17 Hassan 202218 investigated the toxicity of the [NTf₂] anion and concluded it to be strongly antibacterial in combination with imidazolium and pyridinium cations. In our research, we identified that 4-h-long exposure of *E. coli*, *S. aureus*, and *P. aeruginosa* to [Chol][NTf₂] resulted in complete inhibition of aerobic respiration in resazurin reduction test (Figure 3) when compared to the control

<figure><img src="image_14.png" /><figcaption>**Figure 3.** Antibacterial properties of IL tested on model micro-organisms (EC – *E. coli*, EC+IL – *E. coli* + [Chol][NTf₂], SA – *S. aureus*, SA+IL – *S. aureus* + [Chol][NTf₂], PA – *P. aeruginosa*, PA+IL – *P. aeruginosa* + [Chol][NTf₂].</figcaption></figure>

cultures. The control *E. coli* culture immediately reduced resazurin (highest read at time point 0), while *S. aureus* exhibited the highest fluorescence after 45 min and *P. aeruginosa* after 240 min.

The toxicity of ionic liquids is a crucial matter defining the safety of their use and was a subject of numerous studies.33–36 So far, it has been concluded that cation [Chol] is characterized by relatively low toxicity.34 However, the [Chol][NTf₂] was reported to cause a fall in respiration of the activated sludge while applied in the concentration of 50 mg/L, whereas the concentration of 10 mg/L did not cause such effects.37 Nevertheless, this raises concerns regarding the release of [Chol][NTf₂] to wastewater plants. Couling38 studied the toxicity of [Chol][NTf₂] on *Aliivibrio fischeri* (formerly *Vibrio fischeri*, a well-known bacterial ecotoxicity indicator) and concluded that [Chol][NTf₂] can be considered relatively nontoxic in comparison to other tested ILs and stated that choline-based ILs might be more environmentally friendly than aromatic ILs. On the other hand, Mena et al.37 underlined higher toxicity of [Chol][NTf₂] to *Aliivibrio fischeri* when compared to [Chol][Cl] and [Chol][Ac]. It was reported that the activated sludge37 did not degrade [NTf₂] anion. However, Concepcion et al.39 identified bacteria *Pantoea agglomerans* able to biodegrade [Chol][NTf₂]. It suggests that removal of IL from the environment is possible.

However, there is a shortage of research verifying the toxicity of [Chol][NTf₂] to mammalian cells, which is important to verify user toxicity. The only available article reported that the presence of [NTf₂] anion decreased the toxicity of ILs to CaCo-2 and HT-29 cell lines compared to other anions and independently of the cation.40

Laird et al.41 reported that the Editorial Advisory Board of Organic Process Research and Development (OPRD) advised encouraging scientists and engineers to minimize using certain solvents, e.g., TCM. Macmillan et al.42 concluded that pharmaceutical companies are increasing the sustainability of their processes by adopting green chemistry rules. One of the proposed is to use more environmentally and user-friendly solvents. They deemed chlorinated solvents, i.e., TCM and DCM, as significantly hazardous to humans and the natural environment and concluded that if possible, they should be replaced with other solvents. Byrne et al.43 underlined that TCM and DCM are likely carcinogenic. Nevertheless, in Pfizer's color-coded solvent selection guidelines, DCM is suggested to replace other chlorinated solvents, e.g., TCM, when nonchlorinated ones cannot be used. Welton44 summarized the process of choosing green solvents and highlighted that several companies have released guidelines to help users choose solvents. In the combined green solvent selection guide, EA and Me-THF are proposed as replacements for DCM, while DCM is the suggested replacement for TCM. The authors underlined that a low environmental impact of the solvent is crucial. However, a typical trade-off of changing the solvent to a greener alternative is a significant decrease in extraction efficiency. Our work shows that TCM can only be replaced with DCM, but the total elimination of chlorinated solvents and maintaining the same extraction efficiency are problematic in the case of PYO in the tested setup. Even though EA did extract PYO, which could be described as a desirable and “green” solvent, the amount of extracted PYO was relatively small. Therefore, regardless of its recommendation in green chemistry, EA cannot be considered to be a good extractant for PYO extraction.

Our research has also shown that ionic liquids such as [Chol][NTf₂] can extract compounds, including PYO. So far, this IL was successfully used to extract phenolic compounds, o-cresol, o-xylene, and acetonitrile from aqueous solutions.45,46 Our experimental results show that [Chol][NTf₂] shows moderate efficiency in PYO extraction compared to traditional organic solvents such as TCM and DCM. Although its extraction capacity may be lower than that of TCM or even DCM, it is worth emphasizing that ionic liquids are characterized by different physicochemical properties, which may affect their use in specific extraction applications. Additionally, [Chol][NTf₂] may be the preferred solvent for extractions where sustainability aspects, such as green chemistry and minimizing toxicity, are a priority. However, it should be emphasized that our study focused mainly on the comparison of traditional organic solvents, and the analysis of the use of ionic liquids in PYO extraction would require further research.

### 3. CONCLUSIONS

Our study yielded valuable insights into the effectiveness of various solvents for extracting PYO. We confirmed that TCM remains the most effective extractant, followed by DCM, which offers a safer and more eco-friendly alternative. Additionally, our investigation sheds light on the effectiveness of ionic
liquids, exemplified by [Chol][NTf₂], suggesting their potential utility in specialized extraction applications. We also proposed the method for separating IL and pyocyanin using adsorption on clays and desorption with ethanol. Moving forward, research efforts should prioritize the development of safer and more sustainable extractants, such as ionic liquids, to minimize environmental impact and ensure worker's safety. Our study emphasizes the immediate applicability of [Chol]-[NTf₂] as a viable alternative. By acknowledging these findings, we hope to encourage further exploration and innovation in the field of extraction chemistry of fermentation products.

## ASSOCIATED CONTENT

### Data Availability Statement

The data generated in this research is available in Bridge of Knowledge data repository: https://mostwiedzy.pl/pl/open-research-data/pyocyanin-extraction-the-comparison-of-typical-organic-solvents-and-ionic-liquids,930103548948572-0?_share=8ff9f7631e374cb1.

### Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.iecr.4c04206.

Calibration curve for pyocyanin quantification; XRD and BET analysis of the clays; extraction efficiencies and properties of other tested ILs; data from the first and second extraction of pyocyanin (PDF)

## AUTHOR INFORMATION

### Corresponding Author

Joanna Honselmann genannt Humme - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland;

● orcid.org/0000-0001-6457-5127;

Email: joanna_jablonska@zut.edu.pl

### Authors

Paula Ossowicz-Rupniewska - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland

Martyna Jurkiewicz - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland; ● orcid.org/0000-0002-8807-6151

Marcin Gano - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland

Kamila Dubrowska - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland

Adrian Augustyniak - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland; Center for Advanced Materials and Manufacturing Process Engineering (CAMMPE), 71-065 Szczecin, Poland

Ewa Janus - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland

Ewa Mijowska - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland

Lidia Favier - Ecole Nationale Supérieure de Chimie de Rennes, University Rennes, CNRS, ISCR-UMR6226, F-35000 Rennes, France

Rafal Rakoczy - Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, 71-065 Szczecin, Poland; Center for Advanced Materials and Manufacturing Process Engineering (CAMMPE), 71-065 Szczecin, Poland

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.iecr.4c04206

### Notes

The authors declare no competing financial interest.

## ACKNOWLEDGMENTS

This work was supported by the National Science Centre, Poland within project no. 2021/41/N/ST8/01094 (PREL-UDIUM 20) granted to Joanna Jabłońska (Honselmann genannt Humme) and by the National Centre for Research Development (Poland) under LIDER Programme (LIDER14/0169/2023) granted to Adrian Augustyniak. Joanna Jabłońska (Honselmann genannt Humme) was supported by the Foundation for Polish Science (FNP).

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