The use of hydroxyl-radical-generating systems for the treatment of olive mill wastewaters.
ABSTRACT Three hydroxyl-radical producing biomimetic systems, composed of CuII, hydrogen peroxide and pyridine, glucaric or succinic acid, were able to perform decolorization of olive mill wastewaters (OMW) >85 % within 3 d combined with a significant removal of total phenols (>75 %). The systems consisting of 50 mmol/L succinic acid, 5-10 mmol/L CuSO4 and 100 mmol/L H2O2 were the most effective at OMW treatment, and led to the reduction of phenol contents to <1 % along with high decolorization (>88 %) and acceptable values of chemical oxygen demand.
Folia Microbiol. 51 (4), 337–341 (2006)
The Use of Hydroxyl-Radical-Generating Systems
for the Treatment of Olive Mill Wastewaters
P. BALDRIANa, G.I. ZERVAKISb, V. MERHAUTOVÁa, S. NTOUGIASb, C. EHALIOTISc, F. NERUDa
aInstitute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czechia
fax +420 241 062 384
bInstitute of Kalamata, National Agricultural Research Foundation, 24100 Kalamata, Greece
cDepartment of Natural Resources and Agricultural Engineering, Agricultural University of Athens, 11855 Athens, Greece
Received 3 January 2006
Revised version 4 April 2006
ABSTRACT. Three hydroxyl-radical producing biomimetic systems, composed of CuII, hydrogen peroxide
and pyridine, glucaric or succinic acid, were able to perform decolorization of olive mill wastewaters (OMW)
>85 % within 3 d combined with a significant removal of total phenols (>75 %). The systems consisting of
50 mmol/L succinic acid, 5–10 mmol/L CuSO4 and 100 mmol/L H2O2 were the most effective at OMW
treatment, and led to the reduction of phenol contents to <1 % along with high decolorization (>88 %) and
acceptable values of chemical oxygen demand.
advanced oxidation processes
benzene, toluene, ethylbenzene, xylenes
chemical oxygen demand
olive mill wastewaters
The olive-oil manufacturing process yields huge amounts of liquid wastes (commonly termed olive
mill wastewaters), which have a dark brown color and possess a high organic load (Dias et al. 2004). Dispo-
sal of OMW in water and soil receptors causes biotoxic effects, mainly attributed to their high content in phe-
nolic compounds (Capasso et al. 1992; Bonari et al. 1993; Casa et al. 2003).
Research on OMW treatment focused on the removal of phenolics and decolorization mainly through
the application of biological processes. These can include the use of anaerobic bacterial consortia (Tsonis
and Grigoropoulos 1993; Borja and Gonzalez 1994; Erguder et al. 2000), aerobic bacteria isolated from OMW
(Di Gioia et al. 2002), and fungi (Sayadi and Ellouz 1993; Zervakis and Balis 1996; Jaouani et al. 2003;
D’Annibale et al. 2004; Koukol et al. 2004). However, the rather low biodegradation rate combined with
problems arising at the scaling-up of such processes (e.g., maintaining optimum conditions for the growth of
microorganisms, economic feasibility, etc.) have limited their practical application.
One of the promising alternatives to the treatment of wastes by microorganisms is the use of radi-
cal-generating systems. The participation of the activated oxygen species (like hydrogen peroxide, hydroxyl
radicals, superoxide anion, etc.) during the degradative processes has been discussed many times in connect-
ion with the catalysis of ligninolytic enzymes, laccases and peroxidases, and a hypothesis on the operation of
these radicals in fungi has been developed (Hammel et al. 2002). Most of the pertinent research has been
focused on the Fenton reaction (Wood 1994). Fungi produce a variety of chelating compounds, sideropho-
res, catecholate and hydroxamate derivatives, organic acids (e.g., oxalic, succinic, ascorbic) that in combina-
tion with other transition metals (copper, cobalt, manganese, etc.) also catalyze Fenton-type reactions, and
can lead to the oxidation of different substrates (Takao 1965; Watanabe et al. 1998). It can be anticipated
that similar reactions occur in vivo and perhaps also participate in the decolorization of OMW by white-rot
fungi (Kissi et al. 2001). From this analogy we have developed several biomimetic radical-producing sys-
tems that can effectively degrade different organic compounds including synthetic dyes, oligocyclic (‘poly-
cyclic’) aromatic hydrocarbons (PAHs) or BTEX (Verma et al. 2003; Nerud et al. 2001, Shah et al. 2003).
The main advantage of these systems is that the produced radicals oxidize nonspecifically a broad spectrum
of organic molecules and are particularly beneficial against mixtures of complex composition. Furthermore,
the reaction is not pH-dependent and proceeds well at pH 3–11, while Fenton’s reagent can only work under
acidic conditions in the range of pH 2–4 (Gregor 1992).
338 P. BALDRIAN et al. Vol. 51
The aim of this study was to test the ability of copper-based hydroxyl-radical-producing biomimetic
systems to decrease phenol content and to decolorize OMW, and to design an optimized procedure for OMW
MATERIALS AND METHODS
Chemicals. Glucaric acid, succinic acid, pyridine, hydrogen peroxide and CuSO4 were obtained
from Sigma (USA). All other chemicals were of the highest grade available.
Catalytic systems and decolorization. OMW were obtained from the olive-oil mill of the Kalamata
Agricultural Cooperative (Kalamata, Greece) equipped with three-phase extraction decanters. In all experi-
ments, OMW concentrations of 25 or 50 % (V/V) were used. OMW (50 %, V/V) were treated with three
radical-generating systems (final concentrations): (i) CuSO4 (1 mmol/L), pyridine (2 %, V/V), and H2O2
(100 mmol/L); (ii) CuSO4 (10 mmol/L), glucaric acid (15 mmol/L), and hydrogen peroxide (200 mmol/L);
(iii) CuSO4 (10 mmol/L), succinic acid (200 mmol/L), and H2O2 (100 mmol/L). The concentrations of the
individual system components were tested for optimum performance using the decolorization of the synthe-
tic dye Remazol Brilliant Blue R (Nerud et al. 2001). Each reaction (final volume of 1 mL) was done at 25 °C
in the dark. Hydrogen peroxide was added fresh after 1 and 2 d, and measurements were done after 2 and 3 d,
respectively. OMW were also treated with a control system, where only hydrogen peroxide was repeatedly
added. The addition of FeCl2 (final concentration of 10 mmol/L) was used after treatment to remove residual
hydrogen peroxide. OMW were also treated with the Cu2+–pyridine–H2O2 and Cu2+–succinic acid–H2O2
systems described above and a Fenton oxidation system (Table I) in order to examine the effectiveness of
phenolic degradation and OMW decolorization at lower waste concentrations, and for comparing the two
systems with the traditional Fenton reaction. Controls (absence of H2O2) for all reactions were also included.
The decolorization was measured with Lambda 11 UV-vis spectrophotometer (Perkin-Elmer, USA)
and expressed as the average (in %) of decrease of absorbance at 500, 525 and 600 nm; all measurements
were done in triplicate.
Table I. Decolorization and decrease in phenolics content (both in %)a of olive mill wastewaters after a 1-d treatment with three bio-
5 – – 200 25 72 42
aPer cent of the initial value (phenolic content in OMW before treatment was 1.96 ± 0.09 µg/mL).
Determinations of total phenolics and hydrogen peroxide. The concentration of total phenolics was
determined by the Folin–Ciocalteau method (the ability of phenolics to reduce phosphomolybdic–phospho-
tungstic reagent; Waterman and Mole 1994), the formation of a blue complex was determined as absorbance
A760. The phenolic content of the samples was expressed as syringic acid equivalents (10 µg/mL corresponds
to A760 = 0.377). The concentration of H2O2 was estimated using xylenol orange and glucitol (Wolff 1994).
Chemical oxygen demand. COD was determined by standard methods for the examination of water
and wastewater (American Public Health Association 1995) with potassium hydrogen phthalate used as
a standard for the calculation of COD.
RESULTS AND DISCUSSION
The treatment of 50 % (V/V) OMW with biomimetic systems at concentrations optimized pre-
viously for the decolorization of synthetic dyes (Nerud et al. 2001, Shah et al. 2003, Verma et al. 2003), caused
a 65–75 % decolorization of the original sample within 1 d (Table I). Fenton reaction failed to efficiently
2006 HYDROXYL-RADICAL-GENERATING SYSTEMS IN TREATMENT OF WASTEWATERS 339
decolorize OMW (<30 % decolorization). Phenolic content was reduced to 20–42 %, depending on the treat-
ment. The treatment of OMW with Cu and H2O2 (omitting the addition of ligand molecules) did not change
the color of the sample, and caused only an 18 % decrease of total phenolics.
The concentrations of metals and ligand molecules were varied to achieve the best performance
with the possibly lowest concentrations of the individual components (Tables II and III). Repeated addition
of hydrogen peroxide resulted in further significant decrease in phenolics and decolorization. In 50 % (V/V)
OMW experiments, the tested systems did not differ significantly as regards their decolorization efficiency;
decolorization of >85 % was achieved only after the second addition of hydrogen peroxide. The highest
OMW decolorization was achieved for Cu2+–succinic acid–H2O2 system at 25 % (V/V) OMW (>90 % of the
initial value). The highest decrease of phenolic content – >99 % – was recorded in OMW treated with the
succinic acid system containing 50–200 mmol/L succinic acid and 10 mmol/L CuSO4. The systems contain-
ing 50 mmol/L succinic and 5–10 mmol/L CuSO4 exhibited the lowest COD along and high decrease of
phenolics which is desirable for OMW treatment.
Table II. Decrease of phenolic content (%)a of olive mill wastewaters after a 1-, 2-, and 3-d treatment
with Cu–pyridine or Cu–glucaric acidb
Composition Decrease of phenolic content
Pyridine, % CuSO4, mmol/L 1 2 3
0.0 0.0 0 6 9
0.0 0.0 0 6 9
aExpressed as per cent of the initial value.
bH2O2 (100 mmol/L) was added after 0, 1 and 2 d.
Several AOP have already been tested for their ability to remove OMW phenolics. These include the
combined treatment with O3–UV and H2O2–UV (Benitez et al. 1996), UV and TiO2–UV (de Heredia et
al. 2001), TiO2 photodegradation (Poulios and Kyriacou 2002), photo-Fenton (Gernjak et al. 2003), and
above all the classical Fenton reaction (Rivas et al. 2001, 2003). Although the photocatalytic processes were
successful in the removal of phenolic compounds like vanillin, protocatechuic acid, syringic acid, 4-couma-
ric acid, 4-hydroxybenzoic acid, gallic acid or tyrosine in model systems under laboratory conditions, the
dark color and high contents of organic compounds contained in OMW would probably interfere with the
photoactivation. Fenton treatment with FeII and H2O2 exhibited the optimum performance in only a narrow
range of acidic pH values 2.0–3.5 (Rivas et al. 2003).
Copper(II) complex systems have recently been used for the degradation of a wide range of organic
molecules including lignin (Watanabe et al. 1998), oligocyclic aromatic hydrocarbons and BTEX (Gabriel et
al. 2000, 2004; Baldrian et al. 2005), and synthetic dyes (Salem 2001; Nerud et al. 2001; Shah et al. 2003).
The main advantage of these systems is their efficiency in a wide range of pH, which is especially important
for OMW treatment (OMW pH is 4.0–5.5). Another advantage is the high speed of reaction, which is com-
340 P. BALDRIAN et al. Vol. 51
pleted within several hours. Compared with biodegradation experiments (Kissi et al. 2001; Aggelis et al.
2002) the main disadvantage of the chemical system is the increase of COD.
Table III. Decrease of phenolic content (%)a and chemical oxygen demand (COD; %)b of olive mill wastewaters after treatment
with Cu–succinic acidc
Composition Decrease of phenolic content after
Succinic acid, mmol/L CuSO4, mmol/L 1 d 2 d 3 d
after 3 d
0 0 0 6 9 100
aExpressed as per cent of the initial value.
bMeasured after a 3-d incubation with subsequent addition of FeCl2 (final concentration of 10 mmol/L).
cH2O2 (100 mmol/L) was added after 0, 1 and 2 d; for further details see Materials and Methods.
Despite the fact that many AOP systems were effective only when low OMW concentrations were
used (Benitez et al. 1999, 2001), the systems examined in our study performed well at both low (25 %) and
high (50 %) concentrations of OMW. In conclusion, OMW treatment by the biomimetic system composed
of 50 mmol/L succinic acid, 5–10 mmol/L CuSO4 and 100 mmol/L H2O2 exhibited high phenol-removal
efficiency combined by high decolorization rate and acceptable COD values.
This work was performed in the frame of the Joint Research and Technology Programme between the Czech Republic and
Greece: Integrated treatment and remediation of recalcitrant agricultural and industrial wastes with high content in polyphenolics and
dyes (Ministry of Education, Youth and Sports of the Czech Republic–Greek General Secretariat of Research and Technology, ME686)
and by the Institutional Research Concept no. AV 0Z 5020 0510 of the Institute of Microbiology, Acad. Sci. Czech Rep.
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