Yarrowia lipolytica Adhesion and Immobilization onto Residual Plastics.

Research in cell adhesion has important implications in various areas, such as food processing, medicine, environmental engineering, biotechnological processes. Cell surface characterization and immobilization of microorganisms on solid surfaces can be performed by promoting cell adhesion, in a relatively simple, inexpensive, and quick manner. The adhesion of Yarrowia lipolytica IMUFRJ 50682 to different surfaces, especially potential residual plastics (polystyrene, poly(ethylene terephthalate), and poly(tetrafluoroethylene)), and its use as an immobilized biocatalyst were tested. Y. lipolytica IMUFRJ 50682 presented high adhesion to different surfaces such as poly(tetrafluoroethylene) (Teflon), polystyrene, and glass, independent of pH, and low adhesion to poly(ethylene terephthalate) (PET). The adhesion of the cells to polystyrene was probably due to hydrophobic interactions involving proteins or protein complexes. The adhesion of the cells to Teflon might be the result not only of hydrophobic interactions but also of acid–basic forces. Additionally, the present work shows that Y. lipolytica cell extracts previously treated by ultrasound waves (cell debris) maintained their enzymatic activity (lipase) and could be attached to polystyrene and PET and used successfully as immobilized biocatalysts in hydrolysis reactions.


Introduction
The industrial application of polymeric materials, especially plastics, has grown intensively due to their suitable mechanical, chemical, and physical properties and low cost. Several industries, such as packaging, automotive, electronic, as well as biomedical industries, use these materials for several purposes. The massive dissemination of plastics is having a negative environmental impact, mainly on the sea [1]. Several researchers have been studying the biodepolymerization of plastics [2,3], but still the reuse/recycle of those materials seems to be a simpler and more practical alternative.
Immobilizing microorganisms on solid surfaces is a relatively simple, inexpensive, quick, and hence popular process [4]. Researchers have already achieved the adhesion of microbial cells to solid polymer surfaces [5][6][7][8]. Some have tested these immobilized catalysts in important industrial reactions, for example biodiesel production [6]. For this purpose, the cells must adhere strongly to a surface to avoid desorption during the reaction with consequent loss of the catalyst [7].
The investigation of the mechanisms of cell adhesion is essential for various fields such as food processing, medicine, environmental engineering, biotechnological process in biphasic medium,
Y. lipolytica W29 (ATCC20460; CLIB89) was donated by the Biological Engineering Center from Universidade do Minho (Braga, Portugal). This strain was used to compare the adhesion characteristics of two different strains of the same species.

Samples Preparation
For all methods used to characterize the cell surface, the collection and preparation of samples was performed as follows: cells grown for 48 h on YPD medium were harvested (3000 g, 10 min), washed twice with distilled water, and resuspended in different buffers. This procedure was performed in order to ensure the complete removal of any substance that was not covalently linked to the cell surface, in particular, a possible surfactant.

Adhesion Assays
The adhesion tests were based on the assays of cell adherence to polystyrene proposed by Rosenberg [20] and modified by Lehocky et al. [7] to measure hydrophobicity. For these tests, the cells were resuspended in phosphate buffer, at pH 3.0, 5.0, 7.0, or 9.0 and ionic strength varying from 10 −4 to 10 −1 M, until they reached an optical density at 570 nm (OD 570 ) of 0.70. One milliliter of the cell suspensions was poured onto the various adhesion surfaces and left to settle for 24 h. For each sample, the supernatant was then removed by inserting the dish (10 times) in a 2 L Becker containing 1.5 L of deionized water agitated at 1000 rpm. After 2 h, the dish was observed with an Olympus optical microscope BX 51 (Olympus Europa SE & Co., Hamburg, Germany), and the images obtained were treated by an image analysis procedure to determine cell surface coverage values.

Image Analysis
The obtained images were processed with a program developed in Matlab ® 6.1 (MathWorks, Natick, MA, USA) as reported by Freire et al. [21]. The image analysis performed followed a three-step sequence: image binarization, droplet quantification, and evaluation of statistical parameters. The binarization consisted in the conversion of the captured image to black and white and in noise removal. The second step quantified the cells in the image, yielding parameters such as the area occupied by the cells. During the last step, a statistical analysis of the data acquired from several images was performed in order to evaluate the average area occupied by the cells and its standard deviation.

Pronase Treatment
Yeast cells harvested by centrifugation and washed twice were suspended in 0.01 M Tris-HCl buffer to give an OD 570 of 10. Pronase was added to the cell suspension at a concentration of 0.1 mg mL −1 , and the cells were harvested after incubation for a determined period of time in a shaking water bath at 37 • C [22].

Contact Angle Measurement
Contact angles were measured by the sessile drop technique on the samples prepared previously, using an OCA 15 PLUS apparatus, Dataphysics (Filderstadt, Germany). The measurements were performed at room temperature using three different liquids with known surface tensions: water, formamide, and diiodomethane. Ten separated contact angle readings for each testing liquid were averaged to obtain representative contact angle values which were used for further evaluation according to the Young-Good-Girifalco-Fowkes equation [23]. The total surface energy (γ tot ) and their components Lifshitz-van der Waals (γ LW ) and Lewis acid-base (γ AB )-which in has a positive (γ + ) and a negative (γ − ) component-were calculated.
2.5. Testing the Immobilization of Cell Debris in Solid Surfaces 2.5.1. Production of Cells with Enzymatic Activity (Cell Debris) Y. lipolytica cells obtained after the fermentation process (4 L bench New Brunswick MF-114, Sci. Inc., USA, bioreator, containing 3 L of YPRFO medium w/v: yeast extract 1%; peptone, 2%; residual frying oil 2.5% v/v) were washed with distilled water and 200 mM MOPS (3-morpholinopropane-1-sulfonic acid) buffer pH 7.0 (Merk, São Paulo, Brazil) and then centrifuged at 4 • C, 4600× g, for 5 min. the cells were resuspended in MOPS buffer and sonicated in a 20 kHz horn-type sonicator (ultrasonic mixing sonicator, DES500, Unique Group, São Paulo, Brazil) in an ice water bath, in two stages of constant acoustic power of 150 W and frequency of 20 kHz, for 9 min. After centrifugation (4 • C, 4600× g, for 5 min), the sonicated biomass (cell debris with lipase) was resuspended in 200 mM MOPS buffer pH 7.0 and frozen for subsequent measurement of enzyme activity. This sonicated biomass with high lipase activity [16,17] was used as a catalyst. It was resuspended in MOPS buffer, let adhere to solid surfaces (polystyrene and PET), as described in Section 2.4.2, and used as an immobilized biocatalyst.

Determination of Enzymatic Activity
The determination of the enzymatic activity of lipase in cell debris and cell debris immobilized on solid surfaces (polystyrene and PET) was performed by measuring the hydrolysis of p-nitrophenyl laurate (pNP-laurate) [16]. In this method, 25 mL of 560 µM pNP-laurate dissolved in 50 mM potassium phosphate buffer (pH 7.0) containing 1% (v/v) dimethyl sulfoxide (DMSO) was mixed, at 37 • C, either with 0.1 mL of cell debris resuspended in phosphate buffer or with the cell debris immobilized on the solid surfaces. The production of p-nitrophenol (product of the enzymatic reaction) was followed during 100 s in a HACH spectrophotometer, DR/4000U, (Loveland, CO, USA) at λ = 410 nm (the extinction coefficient under these conditions was 10.052 × 1/mol/cm). One lipase unit (U) is defined as the amount of enzyme which releases 1 µmol of p-nitrophenol per minute at pH 7.0 and 37 • C.

Solid Surface Characterization
The characterization of the surfaces used in the adhesion tests was carried out by contact angle measurement using the sessile drop technique. The acid-base theory was used to calculate the surface energy of the samples. The total surface energy of a surface i, γ i tot , consists of an apolar, or Lifshitz-van der Waals, component, (γ i LW , which comprises the dispersion as well as the induction and orientation contributions to the van der Waals interactions) and a polar, or Lewis acid-base component (γ i AB ) [24]: According to Lewis, the acid-base interaction can be determined by Equation (2): where γ i + is the electron donor, and γ i − is the electron acceptor of the acid-base part of the surface energy.
The surface components, γ i LW , γ i + , and γ i − , can be determined by contact angle measurements, with at least three different liquids (of which two must be polar), using Young's equation in the following form: where j refers to the studied material, i to the testing liquid, and θ to the measured contact angle. The liquids used in our experiments and their characteristic parameters are listed in Table 1. The surface free energy and the corresponding contributions calculated using Equation (3) are presented, in Table 2. According to the definition of Rijnaarts et al. [26] a surface is classified as hydrophilic for 0 • < θ w (contact angle between water and the surface) < 20 • , intermediately hydrophobic for 20 • < θ w < 50 • , and hydrophobic for 50 • > θ w . According to this classification, glass is hydrophilic (θ w = 16.6 • ), and polystyrene (θ w = 90.5 • ), Teflon (θ w = 97.3 • ), and PET (θ w = 77.8 • ) are hydrophobic. Although polystyrene, PET, and teflon are all classified as hydrophobic, these surfaces present different characteristics. Completely apolar compounds have no electron donor (or electron acceptor sites and thus undergo maximum hydrophobic interactions. PET, being the material with the lowest electron donor/electron acceptor characteristics, was studied in the present work (Table 2). Polystyrene presents high electron donor and acceptor surface tension components, like glass. The difference between these two surfaces is their degree of hydrophobicity.

Adhesion of Cells on Solid Surfaces
Images of the adhesion assays are presented in Figure 1. The image obtained from the microscope was converted to grey scale ( Figure 1a) and subjected to image process analysis in Matlab ® 6.1 (Figure 1b). From this image some information could be obtained, such as the area (in pixels) covered by the cells. Therefore, it was possible to determine the ratio between cell-occupied area and total area, which is the parameter herein used to evaluate the adhesion of cells. Figure 1 also shows examples of the different surfaces used in this manuscript (polystyrene in Figure 1c, glass in Figure 1d, teflon in Figure 1e, and PET in Figure 1f) with adherent cells, after image process analysis. where j refers to the studied material, i to the testing liquid, and  to the measured contact angle.
The liquids used in our experiments and their characteristic parameters are listed in Table 1.  Table 2. According to the definition of Rijnaarts et al. [26] a surface is classified as hydrophilic for 0° < w (contact angle between water and the surface) < 20°, intermediately hydrophobic for 20° < w < 50°, and hydrophobic for 50° > w. According to this classification, glass is hydrophilic (w = 16.6°), and polystyrene (w = 90.5°), Teflon (w = 97.3°), and PET (w = 77.8°) are hydrophobic. Although polystyrene, PET, and teflon are all classified as hydrophobic, these surfaces present different characteristics. Completely apolar compounds have no electron donor (or electron acceptor sites and thus undergo maximum hydrophobic interactions. PET, being the material with the lowest electron donor/electron acceptor characteristics, was studied in the present work ( Table  2). Polystyrene presents high electron donor and acceptor surface tension components, like glass. The difference between these two surfaces is their degree of hydrophobicity.

Adhesion of Cells on Solid Surfaces
Images of the adhesion assays are presented in Figure 1. The image obtained from the microscope was converted to grey scale (Figure 1, a) and subjected to image process analysis in Matlab ® 6.1 (Figure 1, b). From this image some information could be obtained, such as the area (in pixels) covered by the cells. Therefore, it was possible to determine the ratio between cell-occupied area and total area, which is the parameter herein used to evaluate the adhesion of cells. Figure 1 also shows examples of the different surfaces used in this manuscript (polystyrene in Figure 1, c, glass in Figure 1, d, teflon in Figure 1, e, and PET in Figure 1, f) with adherent cells, after image process analysis.  Figure 2 shows the adhesion of Y. lipolytica IMUFRJ on polystyrene. It is possible to observe that the adhesion of cells to this material is relatively high at every pH and ionic strength studied, with more than 50% of surface area covered by the cells in most cases. At ionic strength of 0.1 M, the adhesion tended to decrease. Only at pH 9, the adhesion of the cells to polystyrene did not significantly vary with the ionic strength. Rijnaarts et al. [26] have shown that the isoelectric point (IEP) of a microorganism is an important parameter to predict the steric properties of cell surface polymers and their consequences for cell adhesion. The IEP of a cell surface is determined by the balance between the charges of anionic and cationic acid/base groups on the cell surface. In a previous work [14], we determined that the IEP of Y. lipolytica IMUFRJ 50682 is about 2.4, which indicates the presence of cell wall glucuronic acids or other polysaccharide-associated carboxyl groups. These polymers might inhibit cell adhesion at high ionic strength (0.1 M) because of steric interactions.   Figure 2 shows the adhesion of Y. lipolytica IMUFRJ on polystyrene. It is possible to observe that the adhesion of cells to this material is relatively high at every pH and ionic strength studied, with more than 50% of surface area covered by the cells in most cases. At ionic strength of 0.1 M, the adhesion tended to decrease. Only at pH 9, the adhesion of the cells to polystyrene did not significantly vary with the ionic strength. Rijnaarts et al. [26] have shown that the isoelectric point (IEP) of a microorganism is an important parameter to predict the steric properties of cell surface polymers and their consequences for cell adhesion. The IEP of a cell surface is determined by the balance between the charges of anionic and cationic acid/base groups on the cell surface. In a previous work [14], we determined that the IEP of Y. lipolytica IMUFRJ 50682 is about 2.4, which indicates the presence of cell wall glucuronic acids or other polysaccharide-associated carboxyl groups. These polymers might inhibit cell adhesion at high ionic strength (0.1 M) because of steric interactions.  Figure 2 shows the adhesion of Y. lipolytica IMUFRJ on polystyrene. It is possible to observe that the adhesion of cells to this material is relatively high at every pH and ionic strength studied, with more than 50% of surface area covered by the cells in most cases. At ionic strength of 0.1 M, the adhesion tended to decrease. Only at pH 9, the adhesion of the cells to polystyrene did not significantly vary with the ionic strength. Rijnaarts et al. [26] have shown that the isoelectric point (IEP) of a microorganism is an important parameter to predict the steric properties of cell surface polymers and their consequences for cell adhesion. The IEP of a cell surface is determined by the balance between the charges of anionic and cationic acid/base groups on the cell surface. In a previous work [14], we determined that the IEP of Y. lipolytica IMUFRJ 50682 is about 2.4, which indicates the presence of cell wall glucuronic acids or other polysaccharide-associated carboxyl groups. These polymers might inhibit cell adhesion at high ionic strength (0.1 M) because of steric interactions.  When glass was used as the adhesion material, the specific area occupied by the cells was smaller in comparison to the that measured when using polystyrene, though still large (around 45%), as Figure 3 depicts. It can also be observed that cell adhesion at a higher ionic strength (0.1 M) decreased. This adhesion behavior is similar to adhesion on polystyrene, which showed that at high ionic strength, adhesion was also inhibited by steric interactions. The difference between adhesion to polystyrene and adhesion to glass showed that the greater adhesion to polystyrene might be due to hydrophobic interactions between cells and this surface, as polystyrene (θ w = 90.5 • ) is much more hydrophobic than glass (θ w = 16.6 • ).
Polymers 2020, 12, x FOR PEER REVIEW 7 of 10 When glass was used as the adhesion material, the specific area occupied by the cells was smaller in comparison to the that measured when using polystyrene, though still large (around 45 %), as Figure 3 depicts. It can also be observed that cell adhesion at a higher ionic strength (0.1 M) decreased. This adhesion behavior is similar to adhesion on polystyrene, which showed that at high ionic strength, adhesion was also inhibited by steric interactions. The difference between adhesion to polystyrene and adhesion to glass showed that the greater adhesion to polystyrene might be due to hydrophobic interactions between cells and this surface, as polystyrene (w = 90.5°) is much more hydrophobic than glass (w = 16.6°).  Table 3 shows the results for the adhesion of Y. lipolytica IMUFRJ and W29 strains to polystyrene, PET, and glass at pH 3.0, 5.0, 7.0, and 9.0. It is possible to observe that the adhesion of Y. lipolytica W29 was weak, independent of the material or pH, in relation to that of Y. lipolytica IMUFRJ. This result shows that IEP and w cannot always predict the adhesion behavior and suggests that Y. lipolytica IMUFRJ has some surface components that induce adhesion.
The results showed that cell adhesion of Y. lipolytica IMUFRJ to polystyrene was relatively high, with more than 50% of surface area covered by the cells. This characteristic seems to be unique for this strain, since reduced adhesion was observed for Y. lipolytica W29. In our previous study [14], we showed, by other assays, differences between the surface properties of these strains.   Table 3 also shows that adhesion to PET was inferior to adhesion to both polyethylene and glass, in most cases. This might be related to the inferior electron donor and acceptor surface tension components of PET (Table 2), which indicates that adhesion also involves polar interactions.
Y. lipolytica IMUFRJ was treated with pronase in order to denature proteins in the cell wall and modify its characteristics, since proteins and protein complexes (for example, mannoproteins) are   Table 3 shows the results for the adhesion of Y. lipolytica IMUFRJ and W29 strains to polystyrene, PET, and glass at pH 3.0, 5.0, 7.0, and 9.0. It is possible to observe that the adhesion of Y. lipolytica W29 was weak, independent of the material or pH, in relation to that of Y. lipolytica IMUFRJ. This result shows that IEP and θ w cannot always predict the adhesion behavior and suggests that Y. lipolytica IMUFRJ has some surface components that induce adhesion. The results showed that cell adhesion of Y. lipolytica IMUFRJ to polystyrene was relatively high, with more than 50% of surface area covered by the cells. This characteristic seems to be unique for this strain, since reduced adhesion was observed for Y. lipolytica W29. In our previous study [14], we showed, by other assays, differences between the surface properties of these strains. Table 3 also shows that adhesion to PET was inferior to adhesion to both polyethylene and glass, in most cases. This might be related to the inferior electron donor and acceptor surface tension components of PET (Table 2), which indicates that adhesion also involves polar interactions.
Y. lipolytica IMUFRJ was treated with pronase in order to denature proteins in the cell wall and modify its characteristics, since proteins and protein complexes (for example, mannoproteins) are usually responsible for cell interactions with organic compounds, as reported by Amaral et al. [14]. In this test, teflon was also used as an adhesion surface since it was reported that Y. lipolytica present high adhesion to Teflon-like films [7]. Indeed, it can be noticed in Table 4 that adhesion of Y. lipolytica cells to Teflon was similar to adhesion to polystyrene (for 10 −1 M), despite the different surface characteristics of these two materials ( Table 2). It is possible to observe in Table 4 that pronase treatment significantly affected the adhesion of Y. lipolytica IMUFRJ to polystyrene and PET and, to a lesser extent, the adhesion to Teflon. Y. lipolytica IMUFRJ adhesion to glass was not modified by the denaturation of surface proteins. Therefore, the yeast components responsible for adhesion to polystyrene and glass must be completely different. The adhesion of cells to polystyrene was higher than that to other surfaces (glass and PET) and similar to that to Teflon at higher ionic strength. Polystyrene and Teflon have higher hydrophobicity (θ w > 90), suggesting that the adhesion of Y. lipolytica cells is probably related to hydrophobic interactions. Despite the lower hydrophobicity of glass, the total surface energy and that of its components are high. The Lifshitz-Van der Waals/acid-base (LW/AB) theory was used to obtain the total surface energy γ tot and that of its components, i.e., an apolar, or Lifshitz-an der Waals component, γ i LW (dispersion), and polar γ i AB acid-base component [18]. Adhesion of cells to glass was inferior than to adhesion to polystyrene, but still high (34% of coverage), which might be related to the acidic component (γ + ) of this material. It has been shown that Y. lipolytica IMUFRJ surface shows a more basic character (higher γ − ) and, therefore, acid-base forces might be related to the interaction between glass and cells. The treatment of cells with pronase did not influence adhesion to glass, as it did to polystyrene. However, for Teflon, a significant interaction with cells was also observed after pronase treatment, which indicates that acid-base forces might be related to the interaction between Teflon and cells, besides hydrophobic interaction forces. Dufrêne [9] has reviewed the studies related to forces in microbial cell adhesion and reported that cell adhesion is mediated by a multitude of molecular interactions that are specific (molecular recognition between receptors and ligands) or non-specific (hydrogen bonding, hydrophobic, van der Waals, electrostatic, and macromolecular forces).

Application of Cells Immobilized on Polymer Surfaces
Although it has been reported that Y. lipolytica cells present lipase enzymatic activity [15], a higher lipase activity is detected when the cells are treated with ultrasound, which results in cell debris associated with lipase [17]. Y. lipolytica cell debris were tested for lipase activity (hydrolysis of p-nitrophenyl laurate into p-nitrophenol), and a positive result was obtained, as depicted in Table 5 (before adhesion). This hydrolysis activity was determined after maintaining the cell debris resuspended in MOPS buffer (pH 7) for 24 h at 25 • C, simulating the same conditions that cells are exposed to when adhesion was performed (Section 2.4.2). Lipase in Y. lipolytica cell debris had previously been tested for its ability to hydrolyze lipolyzed milk fat and showed good thermal stability and best reaction conditions at 37 • C and pH 7.0 [17]. After adhesion of cell debris to polystyrene or PET, these surfaces were also tested for lipase activity, with the same amount of cell debris used in suspension. Table 5 shows that hydrolytic activity was still detected when these cell debris were adherent to polystyrene or PET. The reduction in lipase activity in relation to cell debris before adhesion might be related to the fact that not all cell debris were adherent to the surfaces, as already seen in this work for cell adhesion tests. This hypothesis is supported by the fact that inferior activity was detected for cell debris adherent to PET in relation to cell debris adherent to polystyrene (Table 5), in agreement with the fact that PET showed inferior cell adhesion. The possibility of attaching cell debris containing lipase activity to polystyrene and PET and of using this system as an immobilized biocatalyst suggest promising applications.

Conclusions
Y. lipolytica IMUFRJ 50682 presents high adhesion to different surfaces such as Teflon, polystyrene, and glass, independent of pH. Cell wall glucuronic acids or other polysaccharide-associated carboxyl groups might inhibit adhesion at high ionic strength (0.1 M) because of steric interactions. The adhesion to polystyrene may be due to hydrophobic interactions between cells and this surface involving proteins or proteins complexes. Cell extracts prepared using ultrasound waves (cell debris) maintained enzymatic activity (lipase), also adhered to polystyrene and PET, and were used successfully as immobilized biocatalysts in hydrolysis reactions.