Rhamnolipids: Highly Compatible Surfactants for the Enzymatic

Research Article pubs.acs.org/journal/ascecg

Rhamnolipids: Highly Compatible Surfactants for the Enzymatic Hydrolysis of Waste Frying Oils in Microemulsion Systems Ignacio Moya-Ramírez,* Miguel García-Román, and Alejandro Fernández-Arteaga Chemical Engineering Department, Faculty of Sciences, University of Granada, Avenida Fuentenueva s/n, Granada 18071, Spain S Supporting Information *

ABSTRACT: In this work, we explore a novel application for rhamnolipid (RHL) biosurfactants as emulsifiers for water-in-oil microemulsions, focused on enhancing the state of the art of oil hydrolysis in emulsion systems. We show that RHL can form microemulsions with a stability comparable to that provided by the widely used synthetic surfactant bis(2-ethylhexil) sulfosuccinate (AOT). In addition, we test RHLbased microemulsions as reaction media for the enzymatic hydrolysis of waste frying oil (WFO) to produce added-value products. To this aim, we analyze the influence on the hydrolysis degree of several key parameters, such as the amount of cosurfactant, the water/surfactant molar ratio, and the oil volume fraction in the organic phase. Remarkably, under the same conditions RHL leads to a hydrolysis degree 35% higher than that of AOT. Furthermore, RHL also increases the average hydrolysis rate and shows an extraordinary enhancement of the enzyme stability in comparison to that of AOT. Our results demonstrate that RHL can be considered as a potential renewable substitute for synthetic AOT in microemulsions, and pioneer a more sustainable valorisation of WFO. In a wider perspective, they highlight new opportunities for the use of RHL in other enzyme-based processes. KEYWORDS: Biosurfactant, Rhamnolipid, Waste frying oil, Recycling, Reverse micelle, Enzyme/lipase

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INTRODUCTION In an increasingly populated world, global challenges such as the depletion of natural resources (food, water, and energy), environmental pollution, and climate change, have become major social concerns which will need to be addressed in the coming decades. A culture of sustainability is emerging in the 21st century, and the ideal of alternative and fully renewable generation of energy and chemicals as a way to shift to a postpetroleum society is becoming progressively widespread.1 This fact has a direct impact on fields such as biotechnology, which is involved in the production of renewable chemicals. A clear reflection of this is the considerable scientific and industrial interest in compounds such as biosurfactants in the last three decades. These surface active molecules are naturally synthesized by bacteria, filamentous fungi, yeasts and plants, and because of their natural origin. These molecules possess excellent biodegradability and biocompatibility as well.2 Indeed, biosurfactants bring the opportunity to create a new generation of surfactants, which are extensively used worldwide, through green and even renewable processes.3 Traditionally, research in the field of biosurfactants has been connected to two main objectives, i.e., (i) maximize the process yield by using different fermentation strategies or by genetic engineering and (ii) reduce the production costs by using waste and renewable feedstocks.4,5 These efforts have shown considerable progress and thus encouraged the rise of new research lines oriented toward applications of these bioproducts and looking for possible advantages of biosurfactants compared © 2017 American Chemical Society

to their synthetic homologues. Thanks to their structural diversity, they can be used in a wide range of applications, from soil remediation to antimicrobial agents or additives in food and cosmetic formulations.2,6−9 Another interesting use of biosurfactants is the formulation of emulsions since these systems require the presence of amphiphilic molecules to mediate between both phases for their stabilization. For example, the phase behavior of microemulsions containing rhamnolipids and sophorolipids has been previously evaluated.10,11 Rajabi and Luque reported a highly efficient esterification reaction in a micellar system prepared with a glucose-derived synthetic bioinspired surfactant (N-alkanoyl-N-methyl-1-glucamine polyol).12 Leng and coworkers used rhamnolipids to prepare glycerol-in-diesel microemulsions as fuel upgrading.13 Finally, Peng et al. employed an emulsion prepared with rhamnolipid to extract enzymes (laccase and cellulase).14,15 The same authors also reported a method to degrade polycyclic aromatic hydrocarbons by means of a reverse micellar medium entrapping laccase inside the micelles.16 However, with the exception of these few works, the use of biosurfactants as key components in reaction media is almost unexplored.17 This approach becomes of special interest when the reaction takes place in an emulsion including enzymes because biosurfactants could be more compatible with the Received: April 3, 2017 Revised: June 21, 2017 Published: July 13, 2017 6768

DOI: 10.1021/acssuschemeng.7b01008 ACS Sustainable Chem. Eng. 2017, 5, 6768−6775

Research Article

ACS Sustainable Chemistry & Engineering

phase samples of 1.25 mL were placed in glass test tubes. Subsequently, the aqueous phase containing the enzyme was added (to obtain the desired W0 value) and the hydrolysis reaction started. Samples were maintained at 37 °C under stirring at 120 rpm. After the desired time, the reaction was stopped by placing the tubes in a thermoblock at 100 °C for 2 min. Afterward, the free fatty acids (FFA) released during the hydrolysis were colorimetrically quantified by the method proposed by Kwon and Rhee.22 The lipase concentration was 0.2 g L−1 for all the experiments, referred to the bulk phase. The hydrolysis extent or reaction conversion was calculated as the percentage of hydrolyzed ester bonds with respect to their initial number. Lipase Stability Study. Lipase Stability in Aqueous Solution. Lipases, at a concentration of 16.66 g L−1, were incubated at 37 °C in aqueous solution containing either RHL or AOT at 50 mM in Tris buffer. Their lipolytic activity was measured at regular time intervals by the pH-stat method using tributyrin as the substrate.23 A blank was prepared containing only the buffer. One lipase unit (LU) was considered as the amount of enzyme which releases 1 μmol of butyric acid per minute. Additionally, other surfactant-enzyme compatibility tests were conducted, measuring the activity of the lipases in tributyrin emulsions obtained with RHL, AOT, or gum arabic (as control), and the effect of each surfactant on the desorption of the enzymes when these were previously adsorbed on the tributyrin/water interface, as described by Jurado et al.24 Briefly, for these desorption experiments, twice the amount of surfactant needed for the complete coverage of the interface was added (this amount being obtained from the works of Nave et al. and Ö zdemir et al. for AOT and RHL, respectively).25,26 The parameter desorption capacity (DC) was defined to describe the ability of the surfactant to desorb or interact with the enzyme at the interface:

enzyme than synthetic surfactants. Thus, we hypothesize that enzymes would show a synergy with biosurfactants due to their common biological origin when they are used instead of synthetic surfactants as emulsifiers to prepare emulsions. In this work we confirm this premise for the first time. Microemulsions have already been used as reaction media for the enzymatic hydrolysis of vegetable oils.18 A surfactant is essential for the formation of these microemulsions, such as sodium bis(2-ethylhexil) sulfosuccinate (AOT), the most extendedly used in previous studies in the field.19 In this work we aim to substitute this synthetic anionic surfactant with a biosurfactant (rhamnolipids) in an effort to develop a greener process for the valorisation of waste frying oils (WFOs). For this purpose, we prepare a reverse micellar system using rhamnolipids and compare it with a similar system stabilized by the synthetic surfactant AOT, in terms of extent and rate of the enzymatic hydrolysis of WFO and enzyme stability. We show that rhamnolipid-based microemulsions perform excellently, allowing us to improve our previously reported production process of free fatty acids, mono- and diglycerides from WFO,20,21 in an attempt to design a fully renewable process to recycle and give added value to this waste.

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EXPERIMENTAL SECTION

Chemicals and Reactants. Surfactants sodium bis(2-ethylhexil) sulfosuccinate (AOT, 98% purity) and rhamnolipids (RHL, 90% purity, MW= 567.46 g mol−1); solvents of ACS-grade isooctane (IO), hexane, tert-butyl alcohol (TB), 1-pentanol, isopropanol, and lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) from Thermomyces lanuginosus (TLL) and Pseudomonas f luorescens (PFL) were provided by Sigma-Aldrich (Saint Louis, MO, USA). Tributyrin, cupric acetate monohydrate, gum arabic, and tris(hydroxymethyl) aminomethane were provided by Panreac-Applichem (Barcelona, Spain). WFO, employed as the substrate for hydrolysis experiments, was kindly donated by the municipal collection system of the city of Granada, Spain. Its fatty acid profile, free fatty acid concentration, and other physicochemical parameters were detailed in a previous work.21 Emulsion Preparation and Characterization. Reverse micelles were used as the medium for the enzymatic hydrolysis of WFO. They consisted of an organic continuous phase and an aqueous dispersed one. The organic phase consisted of a solvent (IO), a cosurfactant (an alcohol), the substrate (WFO), and either RHL or AOT as the emulsifiers at a concentration of 50 mM. The aqueous phase was composed of the lipase solution at the desired concentration in 0.02 M Tris buffer at pH 8. The water/surfactant molar ratio (W0) ranged between 8.89 and 30.0, as determined in our previous work on the oil hydrolysis in reverse micelles using synthetic surfactants.20 For the preparation of the emulsion, a desired amount of the aqueous phase was added to the organic phase and the mixture was vortexed during 10 s. The obtained emulsions were characterized as follows. Phase Behavior. The phase behavior of the aforementioned system was studied by the titration method at 37 °C. Initially, the organic phase was prepared with the desired amount of IO, cosurfactant, and WFO. Then, aliquots of the aqueous phase (without the lipase) were added. The turbidity of the samples was assessed visually. We assumed that the reverse micellar region matched with fluid, transparent, and macroscopically isotropic samples which remained stable for several weeks. Dynamic Light Scattering. The droplet diameter distribution was measured in a Z-sizer Nano (Malvern Instruments, Malvern, UK) using a detection angle of 173°. Previously, the viscosity of the samples was measured with a rheometer (Haake VT 500, Thermo Scientific, Massachusetts, USA) using concentric cylinders of 4.1 and 3.5 cm internal and external diameters and 12.3 and 6.7 cm heights, respectively. All of them were found to behave as Newtonian fluids. WFO Hydrolysis. WFO hydrolysis was accomplished by lipases entrapped inside the dispersed micelles. For this purpose, organic

DC = (V0 − V1)/V0 × 100 where V0 and V1 are the hydrolysis reaction rates before and after the addition of the surfactant, respectively. Lipase Stability in Microemulsions. Water-in-oil microemulsions were prepared as described previously, but without adding WFO to the organic phase. These microemulsions were incubated in the darkness at 37 °C. At specific time intervals, 1 mL of microemulsion was added to 0.25 mL of WFO, starting the hydrolysis reaction with W0 = 15.6 and Φ = 0.2. The FFA released after 1 h of hydrolysis were measured as detailed above. Anaclitic Techniques. Gas chromatography analysis was used to detect the presence of esters in the reaction mixture. It was carried out in an Agilent 7890A chromatograph (Agilent Technologies, Santa Clara, USA) equipped with a capillary column of fused silica Omegawax (0.25 mm × 30 m, 0.25 μm standard film; Supelco, Sigma-Aldrich) and a flame ionization detector as described by Camacho Paez et al.27 The distribution of the different isomers of the rhamnolipid used was measured by UPLC-MS as described by Rudden et al.28 It was found that the relative abundance of mono- and dirhamnolipids is 65.5 and 34.5%, respectively, the monorhamnolipid−C10−C10 being the major component among the different isomers with a relative abundance higher than 50% (Table S1). All the results presented where error bars are shown, are the mean plus/minus the standard deviation of at least three experiments.

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RESULTS AND DISCUSSION Phase Diagrams: Microemulsion Formation with RHL. Water-in-oil (W/O) microemulsions are dispersions of nanometer-sized water droplets (reverse micelles) in a hydrophobic continuous phase, stabilized by an amphiphile at the interface between them. These systems show isotropicity and thermodynamic stability.29,30 For the preparation of our microemulsions, we selected a mixture of isooctane and WFO as the continuous phase.19,31 In addition, we added an alcohol (1-pentanol, 6769


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Figure 1. Phases observed at different oil volume fraction (Φ, top lines) and isooctane/tert-butanol volumetric ratio (IO/TB, bottom lines) for microemulsions prepared with RHL (a) and AOT (b).

isopropanol, or tert-butanol (TB)) as cosurfactant to modify the polarity of the organic phase and to enable solubilization of the highly hydrophilic RHL.32 The three alcohols gave rise to positive results in terms of RHL solution and WFO hydrolysis (data not shown). However, the use of an alcohol together with a lipase may lead to the esterification of the alcohol with the fatty acids released during the hydrolysis, an undesirable secondary reaction. To check if this reaction took place, the concentration of esters was measured by gas chromatography for the three alcohols. Esters were not detected when using TB, while 1-pentanol yielded the highest concentrations (see Figure S1). This can be explained by the steric hindrance for the enzymatic esterification of the tertiary alcohol TB, which is higher than that for secondary alcohols (such as 2-propanol) or primary alcohols (1-pentanol).33 Consequently, we selected tert-butanol as the cosurfactant for the hydrolysis experiments. Subsequently, we tested the ability of RHL to form microemulsions and compared it with that of AOT under the same conditions at a constant concentration of 50 mM for both surfactants. For this purpose, we analyzed the phase behavior as a function of the solvent/cosurfactant volumetric ratio, IO/TB, and the WFO volume fraction, Φ (Figure 1). Two regions were differentiated: (i) an isotropic phase showing long time stability and corresponding to the aforementioned reverse micellar system and (ii) a multiphase region when it was not possible to emulsify all the water. In addition, a tight transition phase with a turbid color was observed. We detected the reverse micelle region for RHL under all the assayed conditions with the exception of IO/TB = 90/10 (Figure 1). Even though AOT emulsions were able to incorporate higher amounts of water, RHL gave rise to stable microemulsions. An increase in both the oil volume fraction and the IO/TB ratio restricted the microemulsion region for both surfactants, with less water being emulsified. The amount of water that can be incorporated into a microemulsion depends on several parameters, such as the rigidity of the interface or the nature of the bulk oil phase, among others.34 When alcohols are used as cosurfactants, they tend to move to the interface, interacting with the surfactant by increasing its tail volume and reducing the effective surface area. Thus, alcohols reduce the interfacial tension and facilitate the formation of a microemulsion, which results in an increase of the water emulsification of the system.35,36 As for the effect of the oil volume fraction, Φ, we observed a reduction of the amount of water emulsified when Φ increased, in agreement with previous works.36,37

All these results showed that it is possible to prepare W/O microemulsions using RHL, with a performance close to that of the synthetic AOT. This would widen its applicability, for example, for the realization of enzymatic reactions. Concerning the effect of the relative proportion of mono- and dirhamnolipids on the enzymatic behavior, it is known that the two isomers show different physicochemical properties38 and thus may influence the stereospecificity of the reaction or the affinity for specific substances. Indeed, some previous works have taken advantage of the different self-assembly behavior of mono- and dirhamnolipids in applications such as disruption of microbial films39,40 or drug delivery.7 However, separation of the two isomers to produce either pure isomers or mixtures of specific isomer proportions is still very expensive.41 Thus, these refined rhamnolipid formulations can only be used in highadded value applications at the moment. Enzymatic Oil Hydrolysis in W/O Microemulsions Prepared with Rhamnolipids. Once we confirmed the viability of preparing microemulsions with RHL, we investigated the potential use of these microemulsions as reaction media for the hydrolysis of WFO. Three key parameters were considered: the IO/TB volumetric ratio, the water/surfactant molar ratio (W0), and the oil volume fraction (Φ). Effect of the IO/TB Volumetric Ratio. The IO/TB volumetric ratio effect was studied using PFL and TLL lipases. In general terms, microemulsions prepared with RHL led to higher hydrolysis degrees when compared to those obtained with AOT (Figure 2). Actually, PFL and TLL showed a hydrolysis degree 35% higher when using RHL as emulsifier at an IO/TB ratio of 75/25. Such a better performance suggests a higher compatibility of both enzymes with this biosurfactant, a feature that could be highly advantageous for the improvement of enzymatic reactions in W/O microemulsions, compared to traditional surfactants. Both enzymes showed their best performance, i.e., highest hydrolysis degree, when the ratio IO/TB was 75/25 (Figure 2). As mentioned above, the use of an alcohol as a cosolvent is necessary to allow RHL solubilization. However, because it partitions between both phases, TB could interfere with the enzymatic hydrolysis reaction. The reduction in performance at IO/TB = 50/50 could be explained by the partial solubilization of TB in the water cores of the microemulsion, with a subsequent denaturation of the enzyme.42 The increase on the hydrolysis degree of RHL microemulsion between IO/TB 90/ 10 and 75/25 can be clarified considering that the alcohol also acts as cosurfactant and thus directly interacts with the main 6770


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Figure 3. Effect of water/surfactant molar ratio (W0) on the hydrolysis degree with both PFL and TLL lipases and with RHL or AOT as surfactant. For experimental details see Figure 2.

Figure 2. Effect of isooctane/tert-butanol volumetric ratio (IO/TB) on the WFO hydrolysis degree for microemulsions prepared with RHL or AOT as emulsifiers (50 mM in the organic phase) and with PFL or TLL lipases (0.2 g L−1 referred to the bulk phase). Hydrolysis were carried out during 48 h at 37 °C and under stirring at 120 rpm. W0 = 13.3 and WFO volume fraction (Φ) of 0.2. The hydrolysis degree was calculated considering the FFA released and the initial amount of ester bonds in the WFO.

similar variation with W0, in this case with a clear optimum at W0 = 15.0. PFL exhibited a considerable better performance than TLL, particularly in the microemulsions containing RHL. However, with this enzyme we did not observe a clear bellshaped profile in the hydrolysis degree with the W0 variation. Instead, PFL showed a constant and slight increase in the hydrolysis degree with increasing W0. This lower dependence on the amount of water as compared to TLL can be attributed to the higher affinity of PFL for the interface,46 which would make it less sensitive to the microenvironment conditions of the micelle and thus, to the W0 value. Additionally, it should be noted that the marked decrease in the reaction extent observed at W 0 > 30 when using RHL occurred outside the microemulsion domain, that is, when the biosurfactant was not able to allocate any more water inside the micelles (see Figure 1). In order to provide complementary information, we conducted DLS measurement at different W0 values for emulsions prepared with both surfactants. In the case of RHL and in the absence of water, micelles with an average diameter of 0.1 μm were detected (see Figure S2). When water is added to the system, micelles swell increasing their diameter up to values around 1 μm. Interestingly, the micelle diameter did not increase with W0 as expected.44,47 If micelles had been swollen only with water, then the increase of W0 from 8.89 to 20 would have implied approximately a 1.4-fold increase of the diameter. However, the almost constant diameter suggests that TB was present at a considerable level inside the micelles; thus, an increase in the amount of water just had a slight effect on the micelle diameter. These observations are in good agreement with the trend observed for the hydrolysis degree of PFL at different W0 values, whereas TLL seemed to be more sensitive to the presence of water in the micelle. Unfortunately, we were not able to measure the diameter of micelles formed with AOT, either with or without water because of limitations in the instrument sensitivity. The different behaviors observed for the two lipases mark the importance of selecting a highly compatible enzyme−surfactant pair, which allows taking full advantage of using a biosurfactant as emulsifier. However, the most interesting result here was the confirmation of a better compatibility between the lipases and the biosurfactant compared to the synthetic AOT, widely used for microemulsion stabilization. Study of the Reaction Kinetics and Φ Effect. We studied the kinetics of the enzymatic WFO hydrolysis in microemulsions prepared with RHL and AOT as a function of the WFO volume fraction (Φ). Only the enzyme PFL was used in

surfactant molecules.43,44 This phenomenon implies several effects which in conjunction could explain the observed maximum. First, short-chain alcohols reduce the rigidity of the W/O interface which could enhance the behavior of the enzyme entrapped in the aqueous microenvironment.44 Furthermore, as stated previously, TB increases the solubility of rhamnolipids and thus promotes the formation of the microemulsion. Additionally, it allows a deeper penetration of the oil in the interface and thus enhances contact with the lipase.36 As expected, the influence of the TB concentration was more pronounced for RHL than for AOT. This was probably due to the fact that AOT is soluble enough in IO itself while RHL needs the presence of TB to dissolve in the organic medium. Finally, when comparing the behavior of the two enzymes, the hydrolysis extent was higher for PFL than for TLL for all the IO/TB ratios (Figure 2). Furthermore, PFL showed a lower reduction of its hydrolytic ability with increasing TB concentrations, while TLL seemed to be more sensitive to deactivation through the interaction with the alcohol. Further details about the enzyme deactivation caused by TB will be discussed in the section “Enzyme Stability in W/O Microemulsions”. Influence of W0 on Hydrolysis Extent and Micelle Size. The water/surfactant molar ratio (W0) is a key factor influencing enzymatic reactions carried out in microemulsions, and having a direct impact on the micelle size, enzyme location, and microenvironment conditions.45 For this reason we have assessed the effect of W0 on the hydrolysis extent of WFO of RHL-stabilized microemulsions and compared it with AOT. The enhancement in the hydrolysis degree for RHL in comparison to AOT was observed at different water/surfactant molar ratio (W0), except for TLL at W0 ≥ 20 (Figure 3). Thus, these results confirm that both lipases have a better performance with the assayed biosurfactant than with the synthetic and extensively used AOT. The improvement was higher when using PFL. Indeed, the behavior against W0 variation was different for both lipases. In the case of TLL in microemulsions prepared with AOT, an optimum was obtained between W0 = 15.0 and 20.0, showing a slight shift to higher values when compared to the system without TB.20 When using RHL and the TLL lipase, the hydrolysis degree showed a 6771


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Figure 4. Hydrolysis time course for the lipase PFL, at W0 = 15.0, IO/TB = 75/25, and at different WFO volume fractions (Φ = 0.1, 0.2, and 0.4) in microemulsions prepared with RHL (left) and AOT (right) as surfactant. The rest of the conditions are the same than those on Figure 2.

Figure 5. Evolution of the enzyme residual activity of lipases from (a) PFL and (b) TLL incubated in aqueous solution of RHL or AOT 50 mM in Tris 0.02 M pH 8 buffer. The blue line corresponds to a control experiment for which no surfactant was added to the buffer. Activity was measured through butyric acid release during the hydrolysis of tributyrin in O/W emulsion at 37 °C (in lipase units per gram of enzyme, UL/g).

hydrolysis of the FFA esters). Taking into account that the hydrolytic activity of PFL did not change significantly within this range of W0, it seems probable that the observed reduction of the hydrolysis rate was caused by the saturation of the interface by the released FFA, which would have hindered contact between the enzyme and the substrate.48,49 However, if these results are analyzed in terms of the FFA released (see Figure S3), then it can be appreciated how an increase in Φ prompted a higher FFA release. The highest amount of FFA was released with AOT at Φ = 0.4 and RHL at Φ = 0.2 and 0.4. In any case, at high WFO fractions the reaction extent is so low that the recovery of unreacted triglycerides would be unavoidable. For this reason, lower Φ values are preferred for the valorisation of WFO. Enzyme Stability. Enzymatic lipolysis reactions in surfactant-stabilized systems are strongly dependent on a number of parameters such as the interaction between the enzymes, surfactants and other compounds of the system, the ionic nature of the surfactant and its concentration, pH, or affinity of enzyme and substrate at the interface.50 Therefore, these systems are highly variable and depend on the pair enzyme/surfactant. Consequently, and with the aim of gaining further knowledge on the interaction of the enzyme with the amphiphile and the presence of TB, we studied the lipase activity and stability under different experimental conditions. Influence of the Surfactant on the Lipase Activity and Stability in Aqueous Environments. Rhamnolipids, as well as AOT, are anionic surfactants. 10 Since the electrostatic interaction of anionic surfactants with enzymes could lead to partial or complete unfolding of the protein tertiary structure,24,51 we analyzed the effect of the two surfactants on the enzyme stability over time.

this study, based on the observations described in the previous sections. When comparing the behavior of both surfactants, three main phenomena could be observed (Figure 4). First, and most noticeable, the hydrolysis rate was considerably higher for RHL-stabilized microemulsions: After 1 h of reaction, 43.5 and 29.0% of the oil was hydrolyzed at Φ = 0.1 and 0.2 respectively, for the RHL system. In contrast, with AOT under the same conditions, the hydrolysis extents were only 29.7 and 20.7%. Second, as stated previously, RHL gave rise to significantly higher hydrolysis degrees. For example, at Φ = 0.1 and 0.2, the hydrolysis extents attained after 48 h of reaction were about 30 and 40% higher, respectively, with RHL than those achieved with AOT. Notably, with an hydrolysis degree higher than 57% after 48 h, the RHL system almost doubled the yield reported in our previous work using an AOT/isooctane/water system with a maximum hydrolysis degree of around 33% at Φ = 0.2.20 Finally, at Φ = 0.4, and as seen in the section “Phase Diagrams: Microemulsion Formation with RHL”, RHL were not able to form stable microemulsions. Therefore, under these conditions the system stabilized with RHL could not be strictly compared to that stabilized with AOT, despite their showing similar performance. All these results confirm that PFL entrapped on RHL-stabilized microemulsions showed not only a higher hydrolysis degree but also a faster hydrolysis rate which is also a key factor for a scalable and economically viable process. For both emulsifiers, a marked decrease in the hydrolysis rate was observed after the first 4−6 h of reaction (Figure 4). This could be due to both interface saturation and consumption of water, which is also involved in the hydrolysis reaction. In fact, after 48 h of reaction W0 theoretically decreased from 15 to 8.7 at Φ = 0.4 (estimated by considering the water needed for the 6772


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ACS Sustainable Chemistry & Engineering This set of experiments consisted of an incubation of the lipases in an aqueous phase with either AOT or RHL. At specific time intervals, the activity of both enzymes was measured. Figure 5 depicts the evolution of the lipolytic activity of PFL and TLL over time. A marked difference is observed between both surfactants. Remarkably, PFL and TLL incubated in RHL solutions maintained or even increased, respectively, their activity, compared to the control test. On the contrary, AOT caused a pronounced decrease of PFL and TLL activities (of 92 and 68%, respectively) after 144 h of incubation. Lipase deactivation caused by the interaction with AOT has been previously reported, provided its strong interaction with enzymes.52,53 In contrast, RHL did not have any negative effect on the lipase activity and even improved it in the case of TLL. The lipases showed different behaviors in tributyrin emulsions obtained with each surfactant. As showed in Table S2, in the case of PFL a higher activity was observed in emulsions prepared with RHL when compared with those prepared with AOT and gum arabic (control). However, this effect was not observed for TLL, which exhibited the highest activity with the control and the same activity when emulsions were prepared with either RHL or AOT (no statistically significant difference). In addition, we have studied the effect of each surfactant on the desorption of the enzymes when these were previously adsorbed on the tributyrin/water interface. As showed in Table S2, none of the surfactants displaced the enzymes from the O/ W interface. Indeed, the addition of any of them to the medium where tributyrin hydrolysis was already taking place improved the rate of the process, which could be due to a better adsorption of the enzyme on the interface or to the formation of a more active enzyme−surfactant complex Jurado et al.24 In the case of PFL, AOT showed a slightly higher improvement compared to RHL, whereas for TLL, RHL improved the rate of hydrolysis almost two times more than AOT. These observations are in alignment with those of Wang et al., who reported an increase of cellulose and xylanase activity in the presence of RHL.54 Although these results were obtained for enzymes suspended in an aqueous environment and thus not encapsulated in microemulsions, they go in accordance with the substantially better performance of RHL-stabilized microemulsions for WFO hydrolysis compared to those stabilized by AOT. Enzyme Stability in W/O Microemulsions. In an attempt to analyze the stability of PFL and TLL in the system under study, these enzymes were entrapped inside reverse micelles and incubated in the absence of substrate (WFO). The lipase hydrolytic activity was measured at specific time intervals in order to gain insight into their deactivation process. The effects of the presence of TB in the system and of the type of surfactant were studied. Both lipases retained more activity if RHL was used as emulsifier instead of AOT (Figure 6a). Remarkably, PFL and TLL showed a residual activity after 96 h of incubation of 73 and 99% compared to the observed activity at the beginning of the experiment in the RHL-stabilized emulsion. In contrast, when AOT was used, the residual activities were of 48 and 74% with PFL and TLL, respectively, at the end of the experiment. These results evidence that the use of RHL is doubly advantageous. First, it gives rise to a faster and deeper WFO hydrolysis than AOT. Second, it results in higher compatibility with the lipases by maintaining their activity for longer times.

Figure 6. Residual lipase activity (of PFL and TLL) incubated in microemulsions at W0 = 15 and 37 °C. Enzyme concentration was 0.2 g L−1, referred to the bulk medium. The organic phase composition was (a) IO/TB 75/25 in volume with either AOT or RHL 50 mM and (b) IO with AOT 50 mM. Relative enzyme activities are referred to the observed at the initial time of incubation.

tert-Butanol can also have an impact on the enzyme stability; therefore, we analyzed its effect by comparing the residual activity of microemulsions prepared with and without TB for the AOT system. Different deactivation patterns were observed between AOT microemulsions containing IO and TB in the organic phase (Figure 6a) and those obtained only with isooctane and AOT (Figure 6b). While TLL showed a better stability when TB is added to the organic phase, the presence of this alcohol boosted the deactivation of PFL. Regardless of this detrimental effect over PFL stability, if considering hydrolysis degree values instead of residual activity,then PFL performance improved when adding TB to AOT microemulsions increasing it from 3.31 to 6.78% (after 2 h reaction with enzymes incubated during 96 h).

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CONCLUSIONS In this work we have reported for the first time the use of a biosurfactant (rhamnolipids) as the stabilizer of a water-in-oil microemulsion, which is used as the medium for an enzymatic reaction. More specifically, the revalorisation of waste frying oils (WFO) resulting from lipase hydrolysis has been studied. The experimental results have been compared to those obtained with the synthetic and widely used surfactant AOT. Phase studies revealed a satisfactory behavior of RHL as emulsifier. Even though this biosurfactant showed lower emulsifying capacity than AOT under the same conditions, RHL gave rise to stable microemulsions, providing an appropriate system for the enzymatic hydrolysis of oil. In most of the assayed conditions, i.e., different solvent/ cosurfactant and water/surfactant ratios, microemulsions prepared with RHL led to higher WFO hydrolysis degrees when compared to those obtained with AOT with both PFL and TLL lipases. Therefore, in this case, the stability and interfacial compatibility between the enzyme and the surfactant must have had a greater effect than the emulsifying properties of the surfactant. Likewise, we observed a considerable increase in the hydrolysis rate for the RHL-stabilized systems compared to those of AOT within the first hours of reaction. Both achievements could be crucial for the development of a scalable and economically viable industrial process. Finally, we found that both lipases remarkably retained more activity if RHL was used instead of AOT, either in aqueous systems or in microemulsions. In this way, our results confirm that RHL shows an excellent compatibility with lipases when used in microemulsions and could allow a considerable improvement of the enzymatic hydrolysis of WFO in reverse micelle systems. 6773


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(3) Marchant, R.; Banat, I. M. Biosurfactants: a sustainable replacement for chemical surfactants? Biotechnol. Lett. 2012, 34, 1597−1605. (4) Geys, R.; Soetaert, W.; Van Bogaert, I. Biotechnological opportunities in biosurfactant production. Curr. Opin. Biotechnol. 2014, 30, 66−72. (5) Dobler, L.; Vilela, L. F.; Almeida, R. V.; Neves, B. C. Rhamnolipids in perspective: Gene regulatory pathways, metabolic engineering, production and technological forecasting. New Biotechnol. 2016, 33 (1), 123−135. (6) Mulligan, C. N. Recent advances in the environmental applications of biosurfactants. Curr. Opin. Colloid Interface Sci. 2009, 14 (5), 372−378. (7) Gudiña, E. J.; Rangarajan, V.; Sen, R.; Rodrigues, L. R. Potential therapeutic applications of biosurfactants. Trends Pharmacol. Sci. 2013, 34 (12), 667−675. (8) Kiran, G. S.; Ninawe, A. S.; Lipton, A. N.; Pandian, V.; Selvin, J. Rhamnolipid biosurfactants: evolutionary implications, applications and future prospects from untapped marine resource. Crit. Rev. Biotechnol. 2016, 36 (3), 399−415. (9) Viisimaa, M.; Karpenko, O.; Novikov, V.; Trapido, M.; Goi, A. Influence of biosurfactant on combined chemical-biological treatment of PCB-contaminated soil. Chem. Eng. J. 2013, 220, 352−359. (10) Nguyen, T. T. L.; Edelen, A.; Neighbors, B.; Sabatini, D. A. Biocompatible lecithin-based microemulsions with rhamnolipid and sophorolipid biosurfactants: Formulation and potential applications. J. Colloid Interface Sci. 2010, 348, 498−504. (11) Nguyen, T. T.; Sabatini, D. A. Characterization and Emulsification Properties of Rhamnolipid and Sophorolipid Biosurfactants and Their Applications. Int. J. Mol. Sci. 2011, 12, 1232− 1244. (12) Rajabi, F.; Luque, R. An efficient renewable-derived surfactant for aqueous esterification reactions. RSC Adv. 2014, 4 (10), 5152− 5155. (13) Leng, L.; Yuan, X.; Zeng, G.; Chen, X.; Wang, H.; Li, H.; Fu, L.; Xiao, Z.; Jiang, L.; Lai, C. Rhamnolipid based glycerol-in-diesel microemulsion fuel: Formation and characterization. Fuel 2015, 147, 76−81. (14) Peng, X.; Xu, H.; Yuan, X.; Leng, L.; Meng, Y. Mixed reverse micellar extraction and effect of surfactant chain length on extraction efficiency. Sep. Purif. Technol. 2016, 160, 117−122. (15) Peng, X.; Yuan, X. Z.; Zeng, G. M.; Huang, H. J.; Zhong, H.; Liu, Z. F.; Cui, K. L.; Liang, Y. S.; Peng, Z. Y.; Guo, L. Z.; et al. Extraction and purification of laccase by employing a novel rhamnolipid reversed micellar system. Process Biochem. 2012, 47 (5), 742−748. (16) Peng, X.; Yuan, X.-Z.; Liu, H.; Zeng, G.-M.; Chen, X.-H. Degradation of Polycyclic Aromatic Hydrocarbons (PAHs) by Laccase in Rhamnolipid Reversed Micellar System. Appl. Biochem. Biotechnol. 2015, 176, 45−55. (17) La Sorella, G.; Strukul, G.; Scarso, A. Recent advances in catalysis in micellar media. Green Chem. 2015, 17, 644−683. (18) Biasutti, M. A.; Abuin, E. B.; Silber, J. J.; Correa, N. M.; Lissi, E. A. Kinetics of reactions catalyzed by enzymes in solutions of surfactants. Adv. Colloid Interface Sci. 2008, 136, 1−24. (19) Park, K. M.; Kwon, C. W.; Choi, S. J.; Son, Y.-H.; Lim, S.; Yoo, Y.; Chang, P.-S. Thermal deactivation kinetics of Pseudomonas fluorescens lipase entrapped in AOT/isooctane reverse micelles. J. Agric. Food Chem. 2013, 61, 9421−9427. (20) Moya-Ramírez, I.; García-Román, M.; Fernández-Arteaga, A. Waste Frying Oil Hydrolysis in a Reverse Micellar System. ACS Sustainable Chem. Eng. 2016, 4 (3), 1025−1031. (21) Moya-Ramírez, I.; Fernández-Arteaga, A.; Jurado-Alameda, E.; García-Román, M. Waste Frying Oils as Substrate for Enzymatic Lipolysis: Optimization of Reaction Conditions in O/W Emulsion. J. Am. Oil Chem. Soc. 2016, 93 (11), 1487−1497. (22) Kwon, D. Y.; Rhee, J. S. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Am. Oil Chem. Soc. 1986, 63 (1), 89−92.

Microemulsions can be considered as a promising organic media to perform biocatalysis,42 and our observations suggest that biosurfactants are quite promising components for these systems. As noted in this work, they could constitute the basis of a fully renewable process for the valorisation of WFO through its hydrolysis. This could offer a viable and versatile recycling strategy for WFO given the recent concerns about the use of diesel and biodiesel,55 which is the main end use for this waste nowadays.56 However, some challenges need to be addressed before achieving this. First, biosurfactant production should reach competitive prices in the market. This is expected to happen in the upcoming years thanks, for example, to the use of waste and/or renewable feedstocks.57,58 Second, although our results seem very promising, they suggest the need of further process optimization, including the use of a cosurfactant with a softer interaction with the enzyme or an enzyme stabilizer additive as well as a protocol for enzyme reutilization. Therefore, this work pioneers a new potential application for the recently emergent biosurfactants. Our results constitute a first step toward the replacement of a synthetic and potentially hazardous surfactant (AOT) with RHL, which has a microbial origin and a great biocompatibility and biodegradability. From a wider perspective, our findings on the excellent enzyme compatibility of RHL could be useful for the development of more environmentally sustainable processes in other enzymebased applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01008. Distribution rhamnolipids isomers, surfactant-enzyme compatibility tests, FFA-alcohol esters detected with different alcohols, droplet size of W/O microemulsion prepared with RHL, FFA concentration during WFO hydrolysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +34 958244075. ORCID

Ignacio Moya-Ramírez: 0000-0001-9584-7922 Alejandro Fernández-Arteaga: 0000-0002-5407-7877 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the University of Granada (project PP2015-08). We also acknowledge Dr. Morales-Medina ́ (University of Granada), Dr. Rodriguez-Arco (University of Bristol), and Teresa Quiroga Puertas for their kind and valuable help in this work.

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REFERENCES

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