Phenolic compounds in olive oil milling effluents:
An environmental problem or a raw material?
O. Tzoraki1, A.Voutetaki1, P. Ignatiadis1, N. Passadakis2 and V. Gekas1
1 Environmental Engineering Department,
2 Mineral Resourses Engineering Department,
Technical University of Crete, Campus 731 00, CHANIA,
Tel. 0821-37486, Fax 0821-37474, e-mail: vgekas@enveng.tuc.gr
ABSTRACT
Various phenolic compounds are contained in olive oil and are also found in the effluents
of two-phase or three-phase olive-oil milling treatments. In olive oil they are known for
their anti-oxidative properties and quality retention. During olive oil processing they
rather possess a disposal /treatment problem when under uncontrolled conditions pass
into the surface or subsurface waters, converted by oxidation and/or polymerization into
annoying (phytotoxic and antibacterial) pollutants.
Our Laboratory of Transport Phenomena participates in a project for the enhanced
utilization of nutritious olive poly-phenols. Rather facing poly-phenols as a pollution
problem, the idea is to recover them and use them as raw material for the development of
added value products in pharmacy, cosmetics and also in the food industry as food
additives.
Increasing attention has been paid in discovering a recovery and purification method of
polyphenols from the wastewater . They can be reused as a raw material in the olive oils
that have been extracted , or enhance the shelf life of various plant oils , where the
concentration of natural antioxidants
is very limited . Pharmaceutical applications , focusing on their antibacterial properties
are now being developed .
Our laboratory specializes in membrane technology. Our particular task is to apply
membrane operations, after appropriate pretreatment (possibly sedimentation), in order to
obtain poly-phenol recovery from various effluents (from two phase or three phase olive
oil milling factories). Various types of membranes and membrane unit operations (Microultra- and nanofiltration) will be applied in order to recover the important compounds,
taking into account chemical compatibility, flux and retention factors.
1. INTRODUCTION
The extraction and manufacture of olive oil in Greece, as well as, in other Mediterranean
countries, are carried out in small plants which operate seasonally and as a byproduct
generate olive oil mill wastewater (OMW), red to black, high conductive and acidic
liquid effluent. Apart from its extremely high chemical oxygen demand (COD) value 50
to 150 g/L, OMW is known for its high toxicity due to the presence of polyphenols. The
origin of polyphenols is the phenolic compounds, which are initially contained in to the
olive fruits and preferentially pass in to the aqueous phase of the waste effluent. In their
initial form the phenolic compounds are not dangerous, but they became so when the
effluent is disposed of into evaporation ponds or is discharged into surface water
receivers. During this disposal procedure the initial phenolic compounds create a
pollutant potential by removal of the main toxic compounds.
Over the last decade we have seen a gradual move from three – phase decanters to two –
phase ones. The latter have been viewed as more environmentally friendly, since they use
less amount of water. When two – phase decanters are used no liquid waste effluents are
produced, The two streams, that exit the decanter are (i) one comprising of olive oil and
(ii) the other one is the high moisture olive cake which is comprised of husks, seed oil
and concentrated OMW. All of the liquid wastes typically encountered in the usual three
- phase decanters are now part of the crude olive cake and they are present in a more
concentrated form. The current practice is to ship the olive oil – mill cake to the closest
olive kernel – oil-processing plant. In a nutshell, the environmental disposal problem of
OMW is transferred from the mill to the olive kernel – oil-processing plant.
The polyphenols are responsible for the dark color, phytotoxic effects and antibacterial
activity. Increasing attention has been paid to discovering a use for OMW and a wide
range of technological treatments are available nowadays for reducing their pollutant
effects and for their transformations into valuable products. Technologies, which are able
to treat OMW, are too expensive for the mostly small and medium size olive mills.
Actually, only processes, based on unit operations and especially membrane processes
could be used, especially when high additive value products are to be produced.
2. PHENOLIC COMPOUNDS IN OMW
Olive oil is produced by mechanical pressure from olive paste, which is obtained by a
process of milling and continuous washing with water (malaxation). This procedure
yields a considerable amount of wastewater. Generally, OMW contains salts, associated
with high biochemical oxygen demand (BOD), chemical oxygen demand (COD), lipids,
pectins, polysaccharides and polyphenols. In this by-product there is the presence of
natural antioxidants. HPLC analysis of the wastewater extract allows the separation of
several components with antioxidant properties, identified as phenols and flavonoids.
Among these are hydroxytyrosol, oleuropein, vanillic acid, and verbascoside. The
wastewater contains notable levels of phenolic compounds, which could be utilized as
antioxidant sources for fats and oils.
The plant phenols are aromatic secondary metabolites that embrace a considerable range
of substances possessing an aromatic ring bearing one or more hydroxy substituents.
Phelolic compounds present in olives are conventionally characterized as ‘polyphenols”
an unfortunate term since not all are polyhydroxy derivatives. In particular a number of
compounds, namely cinnamic acid, elenolic acid, shikimic acid and quinic acid, are
treated in the present discussion as phenolics because of metabolic considerations
although they lack a phenolic group or even an aromatic ring. Plant polyphenols have
been classified into 15 major groupings distinguished by the number of constitutive
carbon atoms in conjuction with the structure of the basic phenolic skeleton (Table 1).
The range of known phenolics is thus vast but of the various groups only the benzoic
acids, cinnamic acids, flavonoids and iridoids are of major significance in olives.
Additional structural complexity is introduced by the common occurrence of certain
phenolics as the O – glycosides in which one or more of the phenolic hydroxy groups is
bound to a sugar or sugars by an acid – labile hemiacetal bond. Glucose is the most
commonly encountered sugar with rhamnose and the disaccharide, rutinose (6 – O-a-Lrhamnosyl-D-glucose) also encountered. Acylation of the glycosides in which one or
more of the sugar hydroxys is derivatised with an acid, such as acetic or ferulic acid, is
occasionally observed.
Table 1: Major classes of fruit phenolic compounds in olives
Number
C atoms
Basic skeleton
Class
Example
7
C6-C1
Benzoic acid
Vanillic acid
Protocatechuic acid
9
C6-C3
Hydroxycinnamic acids
Caffeic acid
15
C6-C3-C6
Flavonoids
Anthocyanins
Flavonoids glycosides
Cyanidin
N
of
Iridoids
Lignins
Tannins
Rutin
Oleuropein
All of the phenolic compounds possess several common biological and chemical
properties; namely, antioxidant activity, the ability to scavenge both active oxygen
species and electrophiles, the ability to inhibit nitrosation and to chelate metal ions, the
potential for autoxisation, and the capability to modulate certain cellular enzyme
activities. The phenolics in olives are now recognized for their antimicrobial activity,
molluscicidal properties, their preventative role in dacus oleae infestations and resistance
to ather parasite invasions. Phenolics in olives have attracted attention as antioxidants.
More specifically, antioxidant activity in refined olive oil decreased in the series
hydroxytyrosol, caffeic acid > burylated hydroxytoluene (BHT) > protocatechuic acid,
syringic acid.
The ecological problem is mainly due to the compounds of phenolic nature of OMW,
which are responsible for the dark color, phytotoxic effects and antibacterial activity. In
order to know which group of phenolics is involved in the resistance to and/or inhibition
of biological treatments was investigated the separation by ultrafiltration technique, using
membranes with a cutoff 8 and 60 kDa, three groups of aromatics. The F1 fraction is low
molecular-mass (F1<8 kDa), F2 fraction is medium molecular-mass (8 kDa < F2 <60
kDa)and the F3 fraction is the high molecular-mass fraction (F3 > 60 kDa).
Untreated F1 fraction contains three families of aromatic compounds which could
correspond to simple phenolics, o- diphenols and monomeric flavoids in a first group,
hydrolysable tannins in a second group and some condensed tannins and anthocyanins in
the higher hydrodynamic volumes. F2 and F3 contain complex and darkly colored
polyphenols such as ‘humic acid like’ compounds, that are made up by phenolic
monomers as p-coumaric, benzoic acid derivatives and substituted phenoxyethanol.
F1 fraction is well degraded by aerobic bacteria. Bioadegradation of high concentrations
of simple and low molecular-mass phenolic compounds could be achieved by new
isolated or genetically – engineered bacteria under optimized aerobic process technology.
By contrast, high molecular – mass polyphenols are degraded particularly by white rot
fungi, processes requiring longer treatment periods. Furthermore, the comparison of the
biodegradation of the isolated fractions in continuous anaerobic digestion showed that the
toxicity of F1appeared rapidly at HRT = 14.5 days as the methane yield decreased
drastically. In this case, only low molecular – mass phelolics (smaller than about 800 D)
can penetrate the cell membrane. This toxicity may possibly be eliminated by longer
treatments and by increasing biomass concentration in the reactor. However, the
mechanism by which high molecular – mass polyphenolics inhibit methanization is not
known.
Most of the published research on OMW treatment has dealt with the anaerobic treatment
of OMW but failed due to the high phenolic content of OMW and their potential
inhibition of methanogenic bacteria. This toxicity was attributed to low molecular – mass
compounds such as phenolics and lipids without any experimental arguments. By contrast
it was shown that colored compounds inhibited the anaerobic digestion of OMW in
UASB reactors.
The concentration of the high molecular – mass polyphenols depends on olive variety,
cultivation system, maturity of the fruit, extraction processes used and particularly the
time and mode of conservation of this waste before treatment. These high molecular –
mass compounds should be removed completely or partially from OMW prior to
treatment by aerobic activated sludge or anaerobic digestion. A pretreatment step is
required using physico – chemical methods or fungi capable of decreasing the organic
load and degrading or modifying the high molecular – mass phenolic compounds in
OMW biotreatments. Physicochemical treatments such as decolorization through resin or
coagulation – flocculation with Ca(OH)2 resulted in reducing the concentration of high
molecular – mass colored compounds.
The treatment of the phenolic compounds of OMW with membrane processes is shown to
be a very successful method. With hydrophilic Microfiltration and Ultrafiltration
membranes is it possible to separate the polyphenols. The challenge in the application of
a membrane process in OMW is concentrated on the additional difficulty due to the
presence of olive oil and the resulting problem of compatibility of the membrane material
with the particular application.
3. ANALYTICAL METHODS
Vegetation waters are stored at -25 0C. About 20 ml of them are let to defrost and fitered
using a Buchner funnel apparatus. 5 ml of vegetation waters are mixed well with 5 ml
sodium diethyldithiocarbamate(DIECA) 20 mg/l in methanol, to inhibit
polyphenoloxidase and lipoxygenase activities.
Solid Phase Extraction(SPE) is used to separate the phenolic compounds present in the
water extract. In oder to activate the cartridge, 5ml of ethyl ether is let to pass and then
5ml of distilled water. Two milliliters of this mixture is added to a 5g/20ml C18 cartridge.
To recover the phenolic compounds 600 ml of Ethyl ether as extraction solvent is used to
pass through the cartridge under vacuum pressure. The eluate is collected and the organic
solvent is evaporated in rotary evaporator at 350C. The residue is dissolved in 5ml of
Methanol and stored at -200C until it is used.
The concentration of total polyphenols in the methanolic extract is estimated with FolinCiocalteau reagent. The procedure consisted of addition of some drops of water, dilution
of 0.1 ml or a suitable aliquot of the extract (up to 0.4 ml ) in a 10 mL volumetric flask,
and addition of 0.25 ml of Folin - Ciocalteau. After 3 minutes, 1 ml of saturated (35 %)
Na2CO3 solution was added. The content was mixed and diluted to volume with water.
The extinction was measured after 1 hr at 725 nm against a reagent blank. Caffeic acid
served as a standard for preparing the calibration curve ranging 0-100 μg/10 ml assay
solution. The determination is done by spectrophotometer(UV-Visible) at 725 nm
according to Gutfinger,T; Polyphenols in Olive Oils JAOCS.
4. MEMBRANE OPERATIONS
The phenolic compounds contained initially in the vegetation waters are of a low
molecular mass in a range which typically belongs to the Nanofiltration (NF) separation
range. Polyphenols, as macromolecular substances, could be removed by Ultrafiltration
(UF), phenols could be recovered by Nanofiltration.






In our laboratory, we intend to use both membrane unit operations.
Applying Ultrafiltration to a model system being an aqueous solution 1% (w/v)
consisting of
Water, 99%
Caffeic acid SIGMA: C 0625
180 Da
Vanillic acid, SIGMA: V 2250
168 Da
Gallic acid SIGMA: G 7384
170 Da
Tyrosol , EXTRASYNTHESE S.A.
138 Da
Oleuropein: EXTRASYNTHESE S.A.
540 Da
The model system will be exposed to ambient conditions in order to study the stability of
the initial phenolic compounds with time. As the phenols will polymerize their molecular
mass will increase and their retention by UF membranes
The aim will be achieved by measuring the retention coefficient of the Ultrafiltration
membrane as a function of time
We have installed stirred cell modules (model 8200) made by Amicon and we are going
to test plyethersulphone as well as PVDF membranes of a cutoff of 10 000 Da.
Nanofiltration experiments aim at the direct recovery of low molecular mass phenols.
A crossflow module will be useed in this case, since higher transmembrane pressures are
required which cant be obtained with the stirred cell kind of modules.
1.
2.
3.
The criteria of successful application are
Permeate Flux
Retention with the respect to various phenols
Compatibility
The last point is a critical point, especially for Nanofiltration membranes. Usually,
commercial NF membranes are made of materials such as cellulosics or crosslinked
nylon said to give compatibility problems with phenolic compounds.
5. POTENTIAL USES OF RECOVERED PHENOLIC COMPOUNDS
The well known effect of the Mediterranean diet in lowering the incidence of several
pathogenic pathologies , including heart diseases and cancer is greatly contributed to the
use of extra virgin olive oil and the presence of a large amount of dietary antioxidants ,
known with the generic name phenolic compounds. Phenolic compounds , apart from
product stability and extended shelf life , exert a variety of biochemical and
pharmacological roles including anti-inflammatory and anti-neoplastic activities , making
olive oil a unique fruit .
.
It is clear that the recovery of poly-phenols from OMW ,would make these compounds
valuable precursors in the pharmaceutical ,chemical and food industries (Recently , the
biggest olive producer in Greece , has launched a new type of olive oil highly enriched in
poly-phenols) . At the same time , classical biological processes designed for reducing
COD , nutrients and microbiological load will be accelerated .
References
Danielle Ryan and Kevin Robards (1998) “Phenolics compounds in olives”
Analyst, May 1998, Vol.123 (31R –44R)
2.
I Angelikaki, B.K. Ahring (1997) “Codigestion of olive oil mill wastewaters
with manure, household waste or sewage sludge” Biodegradation 8:221-226, 1997,
Kluwet academic publishers, printed in the Netherlands.
3.
J.Κ. Scott, J.O. Oloidi, I.F. McConvey “Emulsion membrane filtration applied
to phenol recovery” Process technology group, ICI FCMO, Hexagon house, Blackley
1.
Κουτσαυτάκης Α. Στεφανουδάκη Ε. (1994) “Ελαχιστοποίηση αποβλήτων με
ελαιουργεία δυο φάσεων” Πρακτικά διεθνούς διημερίδας Σητεία 16-17 Ιουνίου 1994,
Γεωτεχνικό επιμελητήριο Ελλάδος, Οργανισμός ανάπτυξης Σητείας
5.
Οικονόμου Δ., Κουτσαυτάκης Α., Στεφανουδάκη Ε.(1994) “Αξιοποίηση
αποβλήτων ελαιουργείων με τεχνολογία μεμβρανών” Πρακτικά διεθνούς διημερίδας
Σητεία 16-17 Ιουνίου 1994, Γεωτεχνικό επιμελητήριο Ελλάδος, Οργανισμός ανάπτυξης
Σητείας
6.
N. Ινιωτάκης, Σ. Μαράκης, Π. Μιχαηλίδης(1994) “Μια συμφέρουσα
εγκατάσταση διαχείρισης του ελαιολάδου” Πρακτικά διεθνούς διημερίδας Σητεία 1617 Ιουνίου 1994, Γεωτεχνικό επιμελητήριο Ελλάδος, Οργανισμός ανάπτυξης Σητείας
7.
Ν. Καλογεράκης, Ε. Διαμαντόπουλος, Α. Γυπάκης (2000) ”Ολοκληρωμένη
διαχείριση υγρών αποβλήτων ελαιοτριβείων στην Κρήτη” Workshop on Energy from
Wastes: Enviromental and Agricultural Advantages, 17/04/00))
8.
Maurizio servili, Maura Baldioli, Roberto Selvaggini, Enrico miniati, Alceo
Macchioni and Gianfrancesco Montedoro (1999) “High perjormance liquid
chromatography evaluation of phenols in olive fruit, virgin olive oil, vegetation
waters, and pomace
and 1D- and 2D- Nuclear magnetic resonance
characterization” JAOCS, Vol. 76. no.7
9.
Sami Sayadi, Noureddine Allouche, Mohamed Jaoua, Fathi Aloui, (1999)
“Detrimental effects of high molecular mass polyphenols on olive mill wastewater
biotreatment” Process Biochemistry 35 (2000) 725-735
10.
Στέλλιος Τσόνις, Βασιλική Τσόλα, Σωτήριος Γρηγορόπουλος (1989) “Systematic
characterization and chemical treatment of olive oil mill wastewater” Toxicological
and environmental chemistry, Vols.20-21, pp.437-457, Gordon and Breach, Science
publishers, Inc. Printed in Great Britain.
11.
Στεφανουδάκη-Κατζουράκη Ε., Α. Κουτσαυτάκης (1994) “Χαρακτηριστικά
αποβλήτων από ελαιουργεία δυο και τριών φάσεων” Πρακτικά διεθνούς διημερίδας
Σητεία 16-17 Ιουνίου 1994, Γεωτεχνικό επιμελητήριο Ελλάδος, Οργανισμός ανάπτυξης
Σητείας
4.