Study of the biosorption of phenol by the cell walls of brewing yeast

. Sorption capabilities of cell walls of yeast Saccharomyces cerevisiae with respect to phenol have been investigated. Elemental analysis established the composition of yeast cell walls: 47.8 – 48.6% C; 5.7-6.1% N and 6.86% H. The surface of the sorbent determined by BET, was 118.6 m 2 /g, ash content – 18-22%. Using FTIR-spectroscopy, it is shown that the prevailing mechanism of phenol adsorption is physical sorption, and not complexation. The adsorption abilities of yeast cell walls with respect to phenol were studied relative to pH, initial phenol concentration, sorbent dose and activation time. It is shown that the sorption efficiency increases in protonated solutions with pH<3. Experimental data were analyzed using Freundlich and Langmuir models. The model of the adsorption isotherm is more precisely described by the Langmuir equation with R 2 0.9987. The calculated maximum capacity is 18.9 mg of phenol per 1 g of dry sorbent. The study showed that the cell walls of the yeast Saccharomyces cerevisiae can be used as a new adsorption material for eliminating phenol from aqueous solutions.


Introduction
Phenol and its derivatives belong to the main organic pollutants of the environment [1,2].Phenol itself belongs to the second group of ecotoxicants and has an irritating and damaging effect on skin and mucous surfaces.With prolonged contact with phenol, with its content in air and water media exceeding the concentration of 1 mg/L, poisoning of the body, damage to internal organs, manifestation of hepatocancerogenesis occurs.For this reason, the experts of the World Health Organization strictly set the maximum permissible concentration of phenol in waste and surface waters < 1 mg/L, and for fish farms 0.1 mg/L [3].
The main sources of pollution with phenol and its derivatives are coal, oil, wood processing enterprises, as well as the leather, pharmaceutical industries and agricultural enterprises that use insecticides and pesticides for processing their lands, in most cases being phenol derivatives.
To monitor the phenol content in the environment and food, hygienists use the so-called phenol index, which is the sum of phenol and all its derivatives in the analyzed object, which should not exceed 0.25 mg/kg.Such high requirements make it necessary to remediate wastewater and domestic discharges according to this indicator [4].
To remove phenol and its derivatives, various methods are currently used, including precipitation, ion exchange, activated carbon adsorption, ozonation, photolysis under ultraviolet irradiation [5][6][7][8][9].Each of these methods has its advantages and disadvantagesfirst of all, their high cost, the complexity of the regeneration of sorbent (activated carbon) and the inability to process large volumes of wastewater.The solution to this problem will be to find affordable and inexpensive adsorbents that can effectively replace natural and synthetic adsorbents and ion exchangers in the remediation of wastewater and surface water contaminated with phenols.
In recent decades, many researchers have considered the possibility of using various microorganisms that are "waste" of the pharmaceutical and food industries in the commercialization of biosorption technologies for remediation of industrial effluents [10][11][12][13][14][15].Laboratory experiments convincingly show that seaweed, mycelial fungi, yeast, agricultural waste can be used for these purposes.
The present work is devoted to the study of the biosorption of phenol by a biosorbent based on the cell walls of yeast Saccharomyces cerevisiae, a poorly utilized large-tonnage brewing waste.

Preparation of biosorbent
A biosorbent based on yeast cell walls was obtained from the sedimentary yeast Saccharomyces cerevisiae W-37, selected from a cylindrical-conical tank after filtration of the main product -beer.Sedimentary yeast was subjected to the following treatment: the biomass was centrifuged using a PC-6 centrifuge at 5000 rpm (~3600 g), the yeast precipitate was washed with distilled water until a clear solution was obtained, autoclaved at 130 0 C for 1.5 hours and dried in a vacuum camera at 65 °C.The dried biomass was crushed in an electric grinder and sifted through a sieve with a hole diameter of 0.3-0.5 mm.

Biosorbent characteristics
The physicochemical properties of the biosorbent were characterized using elemental analysis, estimation of the sorption surface area by the BET method using CO2 adsorption, as well as potentiometric and IR spectroscopic studies.The IR study of the biosorbent before and after phenol adsorption was carried out using a Shimadzu FT-IR 8400S Fourier transform IR spectrometer in the range of 400-4000 cm -1 by measuring the spectrum of yeast cell wall samples compressed into a tablet with KBr (1:50).Potentiometric titration of biomass was carried out in order to determine the functional groups available for biosorption, for which 1 g of biomass was treated with 50 ml of 0.1 n HCl for 3 hours at room temperature and shaking 150 rpm.Then potentiometric titration of the contents of the 0.1 n NaOH flask was performed using a glass pH electrode ESL-63-07.The signal was recorded using a universal ionomer I-130.Based on the obtained data, a titration curve was constructed and the pKa of the found functional groups was calculated.

Adsorbate
Model solutions with different phenol content in the concentration range 0-150 mg/l were prepared from a reagent qualified as "pure for analysis" by dissolving a phenol sample in bidistilled water.The phenol calibration scale was built by dilution of the initial solution.The determination of the equilibrium concentrations of phenol in solutions was determined by inversion voltammetry using a mercury-graphite film electrode as an indicator.Phenol concentration was monitored on a SF-46 spectrophotometer at a wavelength of 270 nm in 1-centimeter quartz cuvettes.

Sorption experiments
The study of the biosorption capacity of yeast cell wall biomass in relation to phenol was carried out by the method of equilibrium concentrations, for which 1 g of dry sorbent was introduced into a conical Erlenmeyer flask with 100 ml of a solution with a known concentration of phenol and shaken on a horizontal shaker AV-6S with a frequency of 150 rpm for 3 hours.After the activation time, the contents of the flasks were centrifuged at 4000 rpm (2850 g) and the concentration of phenol in the filler fluid was determined.The sorption capacity of biomass was calculated by the difference in the concentrations of the initial and final solutions according to the formula: where q is the capacity of the sorbent mg/g; C0 and Ce -the initial and final concentrations of phenol in solution, mg/L; V is the volume of the solution, l; m is the mass of the sorbent, g.
The effect of pH, sorbent dose, initial phenol concentration in the model solution, activation time on the sorption capacity of the biosorbent was studied.According to the obtained data, adsorption isotherms were constructed and its parameters were calculated in Freundlich and Langmuir coordinates.

pH effect
The effect of the pH of the solution on the sorption of phenol from model solutions was determined by the value of the sorption capacity of the biomass of yeast cell walls.To do this, 300 mg of sorbent was added to 100 ml of a solution with a phenol concentration of 100 mg/L and a certain pH value and shaken at room temperature on a shaker with a frequency of 150 rpm for 3 hours.Studies were carried out in the pH range of 1-12.The required pH value was determined by adding decimolar concentrations of HCl and NaOH to the analyzed solution.In all cases, it was ensured that the ionic strength of the solution did not exceed 0.05 M. Based on the results obtained, a graphical dependence of the sorbent capacity on the pH value was constructed.

The effect of the sorbent dose on the efficiency of phenol sorption
To establish the optimal dose of the sorbent, a different amount of yeast cell wall biomass was added to 100 ml of phenol solution with a concentration of 100 mg/L in the range of 0.1-3.0g.Sorption was carried out at room temperature for 3 hours with shaking at 150 rpm.After the activation and separation of the filler fluid was completed, the concentration of phenol in it was determined by centrifugation.

The effect of the initial concentration of phenol on the sorption efficiency
To establish the dependence of the sorption efficiency on the initial concentration of phenol in model solutions, 0.3 g of sorbent was added to 100 ml of a solution with different concentrations of phenol in distilled water and activation was carried out for 3 hours with shaking at 150 rpm.Upon completion of activation, the residual concentration of phenol in the solution was determined.

Effect of activation duration on sorption efficiency
The effect of the duration of contact of the sorbent biomass with the adsorbate solution on the completeness and efficiency of phenol extraction was studied in the range of 0.5-36 hours.In each case, the solution (100 ml with an initial concentration of 20 mg/l of phenol in distilled water) was in contact for a certain time with 0.3 g of sorbent with particle sizes of ~ 200 mesh (~0.15 mm) under the conditions described in previous experiments.Similarly, the residual concentration of phenol in the solution was determined.

Results and discussion
It is known that the biosorption of pollutants from aqueous solutions by microorganisms can be carried out in various ways and be active or passive.It is obvious that active sorption is characterized by bioaccumulation and directly depends on the metabolic activity of microorganisms.Passive sorption does not depend on metabolic processes and can be carried out on the surface of both living and dead cells of microorganisms.At the same time, it is possible to implement various biosorption mechanisms, including both physical adsorption and chemisorption, accompanied by ion-exchange, chelating and complexing effects [16].The specific functional groups of the peptide-glucan complex of yeast cell walls are responsible for the chelating and complex-forming effects.

Determination of physicochemical parameters of biosorbent
Microscopy using staining yeast cells with methylene blue according to Fink [17] found that as a result of pre-preparation of yeast biomass, including harsh physical effects (washing, autoclaving, centrifugation, drying and mechanical grinding), there were no living cells left and only biosorption processes occurring on the surface of yeast cell walls can be considered.

Potentiometric titration of yeast cell wall biomass
Potentiometric titration of biomass saturated with H + ions, 0.1 M NaOH solution identified some functional groups of biopolymers that make up the yeast cell wall.Integral (a) and differential (b) curves of potentiometric titration are shown in Fig. 1.Based on the results obtained, the total static capacity of yeast cell walls in relation to H + ions was estimated, which was 2.96 mmol.The RCAs of each of the found functional groups were calculated using the Hendersen-Hasselbach equation.The results of the interpretation are presented in Table .1.All these functional groups are able to participate in biosorption processes.

FTIR spectroscopy of yeast cell walls
IR spectroscopy allows identification of functional groups of biopolymers of yeast cell walls.Figure 2 shows the IR spectra of yeast cell walls before (a) and after (b) saturation with phenol.Visualization of the IR spectrum and its assignment have shown that the IR spectrum of the cell membrane of S. cerevisiae yeast is very close to the IR spectrum of cellulose, with the presence of absorption bands of different intensity corresponding to hydroxyl, carboxyl, carbonyl (aldehyde-ketone), amino and amido groups, as well as in the nonspecific range of the spectrum -sulfohydryl and phosphoryl groups [19].
Comparison of IR spectra of yeast cell walls before and after their saturation with phenol shows that there are no significant changes in them.Some absorption bands shift to the low frequency region characteristic of -OH and -NH groups in the region of 3414 cm -1 ; 2925 cm -1 for CH-and 1645 cm -1 for NH-groups for amide I and in the region of 1541 cm -1 for amide II.This indicates that in the case of phenol adsorption, the prevailing mechanism may be physical sorption, and not complexation.

Effect of pH on phenol adsorption
It is known that the pH value affects the sorption characteristics of the sorbent, since both the degree of ionization and the ionization form of the adsorbed substance and the electrokinetic properties of the adsorbent surface, determined by the magnitude and sign of the ζ-potential, depend on it.We have previously shown that the ζ-potential of yeast cell walls at neutral pH has a value of -15 ÷ -18 mv and increases until the polarity sign changes to positive in acidic media.[20].
Figure 3 shows the dependence of the sorption capacity of the sorbent with respect to phenol on pH.The maximum efficiency of biosorption is observed at pH <3.The following explanation can be given for this effect.
Acidic properties of phenol are determined by its dissociation For phenol, pKa = 10.The degree of dissociation of the phenol molecule depending on pH can be calculated by the formula [21]:


This shows that with an increase in the pH value of the solution, the proportion of negatively charged phenolate ions increases.At the same time, competition for sorption sites between OH -and C6H5O -is possible and, as a consequence, a decrease in the sorption capacity of the sorbent [22].In an acidic environment, the ionized fraction of phenol molecules is small, but the surface of the sorbent acquires a positive ζ-potential, as a result of which favorable conditions are created for physical sorption due to electrostatic attraction.

Effect of the adsorbent dose on the efficiency of phenol sorption
This parameter determines the equilibrium state of the adsorbent for a given phenol concentration.Figure 4 shows the dependence of the sorption capacity of the sorbent from the cell walls of yeast S. cerevisiae on its dose.As can be seen, the efficiency of phenol sorption decreases with an increase in the dose of the sorbent in a phenol-containing solution of more than 3 g/L.This can be explained by the fact that competition arises between labile active sites on the surface of the adsorbent for binding to phenol molecules, as a result of which the effective sorption capacity of the sorbent decreases.From the above dependence it can be seen that the biosorbent from the yeast cell walls is most effective at initial concentrations of phenol in water not exceeding 100 mg/l.At the same time, the maximum capacity of the sorbent is 18.9 mg of phenol per 1 g of dry sorbent.

Phenol adsorption isotherms
The adsorption isotherms of equilibrium phenol concentrations were described by Freundlich and Langmuir monomolecular adsorption models.These models are the most frequently used in the study of biosorption processes, allowing to establish quantitative characteristics of adsorption.
Figures 7 and 8 show linearized isotherms of phenol adsorption in Freundlich and Langmuir coordinates.The parameters of the adsorption isotherms and correlation coefficients are presented in Table 2.As can be seen from the values given, the process of phenol biosorption is described more accurately by the Langmuir model, which assumes the formation of a monolayer of sorbate (phenol) on the surface of the sorbent.
A comparison of the sorption capacity of the cell walls of S. cerevisiae yeast and other known phenol sorbents is presented in Table 3.

Conclusion
In the course of studies of the biosorption capacity of the cell walls of yeast Saccharomyces cerevisiae, it was found that the main mechanism of phenol sorption is physical sorption due to the recharge of the electrokinetic potential of the sorbent surface in an acidic medium and a change in the ionization form of phenol.Factors affecting the effectiveness of phenol sorption were studied: the dose of the sorbent, the concentration of which should be within 3 g/L; the optimal pH value <3; activation time 1-3 h; the initial concentration of phenol in solution is not more than 100 mg/L.By constructing models of phenol adsorption isotherms by yeast cell walls, it was shown that the sorption process is more accurately described by the Langmuir equation, which assumes the formation of a monolayer of sorbate (phenol) on the surface of the sorbent.
Given the availability of yeast biomass and the simplicity of its pre-preparation procedure, it can be concluded that the cell walls of yeast Saccharomyces cerevisiae can become a promising material for the manufacture of inexpensive sorbents that allow the concentration of phenolic pollutants from wastewater and surface waters for environmental purposes.

Fig. 2 .
Fig. 2. FTIR spectrum of a sample of S.cerevisiae yeast cell walls before (A) and after (B) saturation with phenol.

Figure 5
Figure 5 shows the phenol adsorption isotherm, showing the dependence of the sorbent capacity on the initial concentration of phenol.

Fig. 5 .
Fig.5.Effect of the initial concentration of phenol in solution on the sorption efficiency.

E3S
Web of Conferences 462, 02031 (2023) AFE-2023 https://doi.org/10.1051/e3sconf/2023462020313.7 Effect of activation time on the efficiency of phenol adsorptionKinetic characteristics of sorption processes are determined by the time of reaching equilibrium.The experimental results for determining the equilibrium time are shown in Fig.6, from which it follows that the adsorption capacity of yeast cell walls increases with increasing activation time and dynamic equilibrium occurs within 3 hours.At the same time, a significant part of the phenol from the solution (80%) is sorbed within the first hour.

Fig. 6 .
Fig.6.Influence of the sorbent contact time on the completeness of phenol extraction.

Table 1 .
Functional groups of cell walls of yeast S. cerevisiae revealed by potentiometric titration.

Table 2 .
Parameters of phenol adsorption isotherms

Table 3 .
Comparative characteristics of some phenol sorbents