Treatment of Olive Mill Wastewaters by Pseudomonas putida mt-2: Toxicity Assessment of Untreated and Treated Effluent

Abstract

The aim of this work was to examine the performance of Pseudomonas putida mt-2 in treating olive mill wastewater (OMW) effluent after dilution with sterilized water (33%, v/v) to reduce its bactericide effect. P. putida significantly reduced the color and phenolic compounds in OMW by 75% and 66%, respectively. Dissolved chemical oxygen demand and biochemical oxygen demand removals reached 85.3% and 92.5%, respectively. Genotoxicity of OMW, before and after biodegradation with P. putida mt-2, was evaluated in vitro, using SOS chromotest, and in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations. Results indicated that OMW showed a significant ability to induce DNA damage, evaluated by SOS and chromosome aberration assay systems. This toxicity was imputed to the presence of phenolic compounds of OMW. However, the toxicity of OMW was significantly reduced after 48 h of aerobic incubation with P. putida mt-2. The present study demonstrates that P. putida mt-2, incubated under aerobic conditions, has a metabolism that enables it to degrade OMW and, especially, to detoxify the effluent mixtures.

Introduction

The Mediterranean region represents the most important olive-growing area in the world. The extraction of olive oil yields great quantities of olive mill wastewater (OMW) commonly called vegetation water. Around 30 million m³ of OMW are produced annually in the Mediterranean area (Borja et al., 1993). Tunisia is one of the largest olive oil producers in the world, with an average annual production of 200,000 tons. This activity produces highly pollutant wastewater and/or solid residue, depending on the olive oil extraction process (600,000 m³ OMW). This effluent has a great negative impact on the environment (Justino et al., 2009). In fact, OMW is characterized by high concentration of several organic compounds including sugar, tannin, pectin, lipids, and phenolic substances (Hamdi, 1996; Ergüder et al., 2000), which are responsible for their high chemical oxygen demand (COD) and biochemical oxygen demand (BOD). These concentrations generally vary between 80 and 200 g/L for COD (Boari and Mancini, 1990; Scioli and Vollaro, 1997) and 12–63 g/L (Cossu, 1993) for BOD.

Recently, Mekki et al. (2006), Komilis et al. (2005), Iconomou et al. (2002), and Aliotta et al. (2002) reported the phytotoxicity of polyphenols from OMW on seed germination and plant growth. Yesilada and Sam (1998) reported their toxic effects on the soil bacterium Pseudomonas aeruginosa. Other authors (Fiorentino et al., 2003) reported the toxic potential of this matrix on the Pleurotus ostreatus of the fresh water food chain.

Because olive oil is a typical Mediterranean product, the treatment of OMW is of crucial importance and a common problem in several European and Mediterranean countries. Several physical methods such as adsorption (Aktas et al., 2001), electrocoagulation, and flocculation/coagulation (Panizza et al., 2006) and chemical methods such as hydrogen peroxide and ozone (Minh et al., 2008) treatment were investigated for OMW depollution. These treatments are generally expensive and produce large amounts of sludge (Robinson et al., 2001).

Biological treatments used for OMW are aerobic and anaerobic biodegradation. The aerobic pretreatment of OMW by fungi and bacteria was demonstrated to be a promising way to reduce pollutants for wastewaters. A plethora of aerobic biological processes, technologies, and microorganisms have been tested for the treatment of OMW, aimed at reducing the organic load, dark color, and toxicity of the effluents (Morillo et al., 2009). COD removing have been widely reported for anaerobic process (Morillo et al., 2009) and a reduction of more than 80% in COD was noticed in the presence of some anaerobic microorganisms. Recently, an innovative process for the treatment of olive mill wastewater has been upscaled from lab scale to pilot plant (Khoufi et al., 2006, 2009). This process combines the electro-Fenton reaction followed by anaerobic digestion and ultrafiltration as a posttreatment to completely detoxify the anaerobic effluent and remove its high-molecular-mass polyphenols. Working in a semicontinuous mode, removal efficiencies are 50% for COD and 95% for monophenolic compounds.

On the other hand, aerobic bacteria appeared to be very effective against some low-molecular-mass phenolic compounds [Ramos-Cormenzana et al. (1996) evaluated the reduction of the phenolic contents of OMW by Bacillus pumilis, obtaining a biodegradation up to 50% of phenolic compounds] but are relatively ineffective against the more complex polyphenolics responsible for the dark coloration of OMW (Morillo et al., 2009). According to Morillo et al. (2009), available scientific information shows that fungi are more effective than bacteria at degrading both simple phenols and the more complex phenolic compounds present in olive mill wastes. The reason for this lies in the structure of the aromatic compounds present in OMWs; they are analogous to that of many lignin monomers. It should be highlighted that, in general, a close relationship has been found between the decrease of phenolic content and the decrease of phytotoxicity (Morillo et al., 2009).

However, comparing anaerobic to aerobic biodegradation, the former process requires generally higher capital investment, expert labor, and transport of waste from generation point to treatment point, resulting in higher fuel costs and higher emissions. Further, it has been a traditional practice to use composting of these wastes as a preferred aerobic biodegradation treatment, partially because of the reasons mentioned earlier and also because of the seasonal production of these wastes.

Pseudomonas putida mt-2 has been described in previous works to be able to decolorize and mineralize azo dyes (Ben Mansour et al., 2007, 2009a, 2009b, 2009c, 2009d, 2009e). In the present study, as far as azo dyes are aromatic and phenolic compounds (Ben Mansour et al., 2007), we have opted to assess the ability of P. putida mt-2 to decolorize and reduce phenolic contents in OMW and to detoxify it.

The aim of this work was to evaluate the ability of P. putida mt-2 to decolorize and reduce phenolic compounds in OMW under continuous shaking, allowing a better oxygenation of the culture, and then the removals of the dissolved COD and BOD of OMW before and after biodegradation are determined. The genotoxicity of P. putida-treated and untreated OMW was assessed in vitro and in vivo by using the SOS chromotest and by observing chromosome aberrations frequency.

Materials and Methods

Chemicals

Vinblastin (Gedeon Richter Ltd.). Giemsa was obtained from Fluka. Methanol and acetic acid were obtained from Prolabo. The ethylenediaminetetraacetic acid (EDTA), zearalenone o-nitrophenyl-d-galactopyranoside, and p-nitrophenylphosphate (PNPP) were purchased from Sigma–Aldrich. Aroclor 1254 was obtained from Supelco.

Animals

The mice used for the experiments were female white BALB/C, selected from mice of similar age and weight (20–25 g). Animals were kept for 1 week before the experiments for acclimatization and were maintained on food (conventional chow) and water ad libitum.

Bacterial strains

P. putida mt-2 (DSM 3931) used for the decolorization assays was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The strain harbors the plasmid pWWO encoding oxygenases for the degradation of toluene and related aromatic hydrocarbons (Franklin et al., 1981; Hugo et al., 1998). Cells from stock cultures were used for biodegradation studies after preculturing in nutrient broth.

Escherichia coli PQ 37 strain was kindly provided by Pr. Quillardet (Institut Pasteur of Paris). The complete genotype, as well as strain construction details, has been described by Quillardet and Hofnung (1985). Frozen permanent copies of the tester strain were prepared and stored at −80°C.

Characteristics of the OMW

The original OMW used in the present study was obtained, in 2008, from an olive oil production plant located in Ouled Jaballah, center of Tunisia. The OMW was derived from discontinuous process for extraction of olive oil (chemical characteristics: pH 5.1; COD: 93 g/L; N: 1,340 mg/L; P: 720 mg/L; K: 6,200 mg/L; phenols: 8,400 mg/L; glucose: 1,200 mg/L) (Mechri et al., 2008).

The OMW was stored at −20°C prior to the biodegradation and toxicity studies.

Microorganisms and culture procedures

P. putida mt-2 was grown at 30°C in 250-mL flasks containing 50 mL of medium under rotary shaking incubation at 200 rpm. The growth medium contained yeast extract (10 g/L), peptone (3 g/L), and glucose (5 g/L). Fresh OMW (5 mL), sterilized with a 0.45-μm filter (Nalgene™; Labware), used for inducing bacteria metabolization system, was then added to the culture medium.

After cultivation of P. putida mt-2 on nutrient broth supplemented with 10% of OMW (which constitute the enzymatic induction medium), the exponential phase culture (∼2 g/L of dry cells concentration) was centrifuged (1,700 g for 10 min) to harvest cells that were transferred into a second flask (50 mL in a 250-mL flask) containing only OMW at 33% (v/v in sterilized water). At this time, OMW was used with the aim of following its dissolved COD and BOD removals. Biodegradation test was conducted in Erlenmeyer flasks at 30°C under oxygenated conditions assured by agitation (200 rpm) and continuous air injection.

After 48 h of incubation, culture medium was centrifuged (1,700 g for 10 min) and filtered on a 0.45-μm filter (Nalgene; Labware) to perform the toxicological tests. Decolorization was assayed by the measurement of absorbance at 390 nm using Pharmacia Biotech Novaspec II spectrophotometer.

The COD was determined using the method described by Knechtel (1987). The BOD was measured by the manometric method with a respirometer BSB-Controller, model 620 T.WTW.

Percentage of decolorization was calculated as follows: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes {10} {9} {7} {6} \begin{document} \begin{align*} {\rm Decolorization} \, (\%) = ({\rm DO}_{\rm i} - {\rm DO}_{\rm f}/{\rm DO}_{\rm i}) \times 100 \end{align*} \end{document}

where DO_i is the absorbance before incubation with P. putida and DO_f is the absorbance after incubation with P. putida.

The cell dry concentration was estimated by recording absorbance at 660 nm (A660) and according to the following relation: Cell dry concentration (g/L)=0.61×A660 (Ben Mansour et al., 2007).

All decolorization, COD, and BOD experiments were performed in three sets. Abiotic (without microorganism) controls were always included.

Total phenol determination

Phenolic compounds were prepared as follows: samples (10 mL of the supernatant culture decolorization medium after 48 h of incubation) were acidified with HCl to pH 2 and extracted three times with ethyl acetate (v/v) at ambient temperature. The three organic fractions were combined and dried with anhydrous Na₂SO₄ for 30 min. The extract was concentrated to dryness in a rotary evaporator and redissolved with 10 mL of mixture methanol/water (60/40) (Ayedi and Hamdi, 2003). Total phenol content was measured using the Folin–Ciocalteu phenol reagent (Merck), which involved the successive addition of 5 mL sodium carbonate (200 g/L) and 2.5 mL Folin–Ciacalteu's phenol reagent to 50 mL sample. After 60 min at 20°C, the absorbance was measured at 720 nm. The blank was composed of the same reagents except that the 10 mL of tested compound was replaced by 10 mL of distilled water.

Phenolic content was expressed as mg of gallic acid equivalents per mg of dry weight through the calibration curve of gallic acid.

Toxicity assessment

In vitro genotoxicity

Activation mixture: The S9 microsome fraction was prepared from the liver of rats treated with Aroclor 1254 (Maron and Ames, 1983). The composition of the activation mixture is the following per 10 mL of S9 mix: salt solution (1.65 M KCl+0.4 M MgCl₂·6H₂O) 0.2 mL; G6P (1 M) 0.05 mL; NADP (0.1 M) 0.15 mL; Tris buffer (0.4 M, pH 7.4) 2.5 mL; Luria broth medium 6.1 mL; S9 fraction 1 mL (Quillardet and Hofnung, 1985).

One hundred microliters of overnight culture of E. coli PQ37 was added to 5 mL of fresh Luria broth medium supplemented with ampicillin at 10 μg/mL and then the medium was incubated for 2 h at 37°C. One milliliter of this culture was diluted with 9 mL of fresh Luria broth or 9 mL of S9 mix. Fractions (0.6 mL) were distributed into glass test tubes containing 20 μL/assay of OMW before or after biodegradation. β-Galactosidase (β-gal) and alkaline phosphatase (AP) activities were assayed on the E. coli PQ37 cultures after 2 h of incubation at 37°C with shaking. To determine the constitutive AP activity (toxicity assay), 2.7 mL P-buffer (Tris 1 M and sodium dodecyl sulfate 3.5 mM and adjusted to pH 8.8) was added to 0.3 mL of E. coli PQ37 culture (8×10⁸ bacteria/mL). The tubes were maintained at 37°C. The reaction was started by addition of 0.6 mL of PNPP solution (4 mg/mL in P-buffer) and stopped by addition of 1 mL of 2 M HCl. After 5 min, 1 mL of 2 M Tris buffer was added to restore the color measured spectrophotometrically at 405 nm. Enzyme units were calculated as for β-gal. The protocol used to determine the β-gal activity induced by DNA-damaging compounds was the same as for AP except that B-buffer (Na₂HPO₄ 113 mM, NaH₂PO₄·7H₂O 45.8 mM, KCl 10 mM, MgSO₄·7H₂O 0.1 mM, sodium dodecyl sulfate 3.5 mM, and 2.7 mL/L β-mercaptoethanol and adjusted to pH 7) replaces P-buffer, and o-nitrophenyl-d-galactopyranoside (4 mg/mL) replaces PNPP. The reaction was stopped with 2 mL of 1 M Na₂CO₃. The induction factor (IF) was calculated as the ratio of R_c/R₀, where R_c is equal to β-gal activity/AP activity determined at concentration c and R₀ is equal to β-gal activity/AP activity in the absence of the tested compound. Aflatoxin and nifuroxazide were used as positive controls, respectively, in the presence and absence of S9 mix.

In vivo toxicity

Animal treatments: Animals were randomly divided into three groups:

1. Animals given a single dose of culture medium run in the presence of P. putida mt-2 and without OMW as negative control group.

2. Animals given a single dose of zearalenone (4 mg/kg) were used as positive control group.

3. Animals given a single dose of, respectively, OMW before and after biodegradation.

Untreated or treated OMW was administrated intraperitoneally (5 mL/kg bw). Twenty-four hours before sacrifice, animals were given a suspension of yeast powder (100 mg/500 μL) to accelerate mitosis of bone marrow cells. Vinblastin at a final concentration 250 μg/mL (200 μL; 4 mg/kg bw) was injected into the animals at 45 min before sacrifice, to block dividing cells in metaphase. Finally, animals were sacrificed by cervical dislocation.

In vivo chromosome aberration assay

Bone marrow preparation: Bone marrow cells were obtained according to the technique of Yosida and Amano (1965). Briefly, femur and tibia were removed immediately after animal sacrifice and bone marrow was flushed out with KCl solution (0.075 M, 37°C) using a syringe. The bone marrow cell suspension was incubated for 20 min at 37°C and centrifuged at 3,500 rpm for 10 min. The supernatant was discarded, the pellet was resuspended in 5 mL of a fixative solution (acetic acid/methanol, 1:3, v/v) and centrifuged (3,500 rpm for 10 min), and the supernatant was discarded again. This step was repeated three times to clean the pellet. Finally, the pellet was resuspended in 1 mL of the aforementioned fixative solution and used for chromosome preparation.

Chromosome preparation: Chromosomes were prepared according to the technique of Evans et al. (1960). The cell suspensions were dropped on glass slides, giving smears that were blazed on a flame for 5 s and then air-dried for conservation at room temperature and/or directly stained with Giemsa. Giemsa working solution was freshly prepared (4 mL in 100 mL phosphate buffer [0.15 M, pH 7.2]). Slides were left for 15 min in this staining solution, then rinsed with water, and allowed to dry at room temperature.

Slide analysis: The slides were examined under 100× magnification objectives using an optical microscope (Carl Zeiss). Three hundred well-spread metaphases were analyzed per group for abnormalities. Metaphases with chromosome break, gap, ring, and centric fusion (robertsonian translocation) were recorded and expressed as percentage of total metaphases per group.

Statistical analysis

Data are expressed as mean±standard deviation from three replicates. The statistical analyses were performed with STATISTICA edition 99 France. Duncan test was used to compare tested compounds versus control. Difference was considered significant when p<0.05.

Results

Biodegradation

In experiments preceding the biodegradation assay, the diluted OMW exhibited no toxicity against P. putida mt-2 when using the agar disk diffusion method. In fact, P. putida reached a cell concentration of 3.71 g/L after 48 h of shaking incubation, which corresponds to a specific growth of 35.6×10⁻³ g/L/h and is similar to that obtained in other medium (mineral medium supplemented with 10 g/L of glucose) without OMW addition (32.3 g/L/h) (Ben Mansour et al., 2009b). In contrast, P. putida mt-2 growth was reduced in the presence of undiluted OMW. On the other hand, P. putida mt-2 was able to decolorize OMW and reduce the total phenolic content to an extent of 75% and 66%, respectively, after 48 h of oxygenated incubation. In the same way, the concentration of dissolved COD and BOD in diluted OMW (COD=31.5 g/L and BOD=28 g/L) significantly decreased after incubation for 48 h in the presence of P. putida mt-2 (85.3% and 92.5%, respectively).

Toxicity

In vitro genotoxicity

No genotoxicity was shown by the OMW before and after biodegradation without S9 metabolization system. In fact, the induction factor (IF) determined by the SOS chromotest, in the presence of various concentrations of the compounds, ranged between 1.03 and 1.23. According to Kevekords et al. (1999), a compound is classified as “not genotoxic” if the IF is less than 1.5, as “marginally genotoxic” if it is between 1.5 and 2, and as “genotoxic” when it is more than 2.

When extracts resulting from untreated and treated OMW were tested in the presence of the S9 mix (Table 1), IF increased and exceeded 2 for intact diluted OMW (IF=2.62).

Table 1.

Genotoxic Activity of Untreated and Treated Olive Mill Wastewater by P. putida mt-2

		Induction factor
Test compound	Dose (μL/assay)	−S9	+S9
Nifuroxazide^a (5 μg/assay)	10	5.43	—
Aflatoxin B1^a (1 μg/assay)	10	—	7.19
Untreated OMW	20	1.23	2.62
Treated OMW by P. putida mt-2	20	1.03	1.21

Genotoxic activity was evaluated by the SOS chromotest with Escherichia coli PQ37 in the presence or absence of the metabolic activation system (S9).

Nifuroxazide and aflatoxin B1 are the positive controls used in the absence or presence of the metabolic activation system S9, respectively.

OMW, olive mill wastewater.

In vivo toxicity

Chromosome aberrations: It is of note that our results clearly showed that the selected dose of OMW and its biodegradation derivatives administrated alone to animals did not have any toxic effect (mortality, body weight, feed intake, and size and shape of liver and kidney).

The volumes of the OMW before and after biodegradation injected to animals were chosen deliberately to obtain a compromise between chromosome damages and a sufficient number of scorable metaphases with, however, a limited lethality. Only structural aberrations induced by the different treatments were enumerated in the present study, with special emphasis on gaps, rings, breaks, and centric fusions. All of these types of structural abnormalities and their frequencies for both control and treated groups are presented in Table 2. Numerical chromosome abnormalities such as polyploidy and aneuploidy were not evaluated.

Table 2.

Different Types of Chromosome Aberration and Their Percentage in Bone Marrow Cells Treated by Olive Mill Wastewater Before and After Biodegradation with P. putida

	Structural aberration
Tested compound	Rings	Centric fusion	Chromosomal breaks	Gaps	Total aberration
Negative control	0	2.5±0.5	2.2±0.1	2.1±0.5	6±1
Zearalenone (20 mg/kg bw)	2.3±1.5	21.3±2.5^**	8.3±3.5^**	6±0.5^*	33.0±1^**
Untreated OMW (5 mL/kg bw)	6.50±1^**	11.5±1.5^**	06.5±1.5^*	4.5±2.5	28±2.5^**
Treated OMW (5 mL/kg bw)	0	5.5±1	2.5±0.5	2.5±0.5	10.5±0.5

Data are expressed as mean±standard deviation.

p<0.01; ^**p<0.001.

Chromosome aberrations, whatever the type, significantly increased in mice treated with OMW (28%) when compared with the nontreated control group (6%). Centric fusions represent the majority of chromosome abnormalities (about 11.5%), and gaps are the least frequent lesions, representing only 6.5%.

However, the ability to induce DNA damage decreased (10.5% of chromosome aberrations) when OMW obtained after 48 h of aerobic incubation with P. putida mt-2 was tested (Table 2).

Discussion

In this work, P. putida mt-2 exhibited a decolorization activity and reduced significantly the total phenols in the OMW obtained from an olive oil production industry located in the center of Tunisia. The kinetic of P. putida mt-2 on OMW was very remarkable (75% of decolorization, 66% of phenolic content, 85.3% of COD, and 92.5% of BOD decrease after only 2 days of aerobic incubation with P. putida) when compared with the action of other biodegradation microorganisms. In fact, according to Tziotzios et al. (2007), the maximum phenolic and dissolved COD removal reached up to 82%–90% for the dilutions of 20%, 50%, and 100% in 11, 23, and 30 days, respectively, by olive fruit bacteria. In the same way, many tested fungi exhibited high ability to degrade OMW; however, it takes enormous time, which generally exceeds 5 days (Sayadi et al., 2000; Minh et al., 2008; Martinez-Garcia et al., 2009; Morillo et al., 2009). This result is very important as far as phenolic compounds are considered to be persistent, recalcitrant in the environment, toxic to most bacteria and fungi, and are used as a slimicide and disinfectant (Yesilada and Sam, 1998; Sayadi et al., 2000; Marques, 2001; Martinez-Garcia et al., 2009); however, they seem to not affect the growth of P. putida mt-2.

In this work, we evaluated the genotoxic potential of OMW before and after biodegradation in the presence of P. putida mt-2. The SOS chromotest with prokaryotic cells indicated that OMW (with or without shacked cultures of P. putida) did not show any genotoxic response in the absence of S9 microsomal mixture. It clearly indicated that these compounds were not directly genotoxic.

However, OMW metabolites obtained in aerobic incubation exhibited a strong genotoxicity when tested in the presence of S9 microsomal preparation. This genotoxicity was confirmed by in vivo toxicity test. In fact, we conducted experiments to evaluate the in vivo genotoxicity in mouse bone marrow. To this end, we evaluated the effect of a single injection of OMW before or after biodegradation, on mouse bone marrow, by monitoring its effects on chromosome aberrations. Chromosome aberrations, whatever the type, significantly increased in mice treated with diluted OMW; however, no genotoxic effect was observed with treated OMW. Results obtained with SOS chromotest were in accordance with those obtained with the chromosome aberrations assay system. In fact, based on the statistical analysis, significant results were found for intact OMW regarding the induction of several types of chromosome aberrations. It has been established that centric fusions (robertsonian translocations) are detected relatively at high frequencies compared with other types of chromosome abnormalities; this may be due to the fact that almost all mouse chromosomes are acrocentric. These types of chromosomes have the ability to merge with each other. So far, very less number of polyploidy or aneuploidy cases have been detected; only the structural chromosome aberrations were investigated.

In this study, the chromosome aberration test, recommended by regulatory authorities for the assessment of genotoxicity and mutagenicity of many chemicals and natural compounds, has provided positive data. These indirect genotoxic effects related to some of tested compounds can be explained by several mechanisms: generally, intact molecules undergo metabolization by hepatic cytochrome P450, leading to the formation of epoxides and/or reactive oxygen species (ROS) responsible of DNA damages (Ben Mansour et al., 2007).

However, toxicity of OMW decreased significantly after aerobic incubation in the presence of P. putida mt-2. In fact, several studies have shown that some phenolic compounds are responsible for the OMW toxicity (Capasso et al., 1991; Bonari et al., 1993; Quaratino et al., 2007). Thus, we believe that the observed decrease of toxicity should be ascribed at least partially to the reduction of phenolic content, when OMW was incubated with P. putida under aerobic conditions (66% decrease of phenolic content after 48 h of incubation with P. putida) and/or the composition changes of the phenolic contents after bioremediation. In a previous work, P. putida mt-2, incubated under good oxygenated conditions, was found to be able to oxidize aromatic amines and some phenol compounds, which undergo metabolization pathways involving, amongst other activities, oxygenases (Ben Mansour et al., 2009b).

Conclusion

Biological degradation through P. putida mt-2 is a very effective method in the removal of many organic pollutants (aromatic compounds) from wastewaters such as OMW. The advantages of this process are numerous, including high efficiency, simplicity in destroying the contaminants, and the nonnecessity of special equipment. The coloring phenolic content of OMW decreases significantly after 48 h of incubation in the presence of P. putida. In vivo and in vitro toxicity observed with untreated OMW extract seems therefore to be the results of the presence of phenolic compounds in the OMW.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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