lnrrrnarionai Biodeterioraiion & Riodegradrrrion(1996) 277-282

ELSEVIER

PII:

SO964-8305(96)00062-5

1997 Published by Elsevier Science Limited

Printed in Great Britain. All rights reserved
0964-8305196 $15.00 + 0.00

Problems of Identifying Phenolic Compounds

During the Microbial Degradation of Olive Mill
Wastewater
G. Knupp, G. Riicker,b A. Ramos-Cormenzanat
S. Garrido Ho~os,~ M. Neugebaue#
T. Ossenkopb

&

Fachbereich fir Bauingenieurwesen, Labor fir Abfallwirtschaft, Siedlungswasserwirtschaft, Umweltchemie (LASU) , Fachhochschule
Mtinster, D-48149 Mtinster, Germany
Pharmazeutisches lnstitut Poppelsdorf, Rheinische Friedrich- Wilhelms- Universittit Bonn, Kreuzbergweg 26, D-531 15 Bonn, Germany
Departamento de Microbiologia, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain
dDepartamento de Ingenieria Quimica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain

The main objectives of the presented Spanish-German

collaboration are the
purification of alpechin by biodegrading phenolic compounds and the investigation of metabolites during fermentation prior to its safe disposal. In addition to
12 well-known compounds, 3,4-dihydroxyphenylglycol
was also identified in
untreated Spanish and Italian alpechin samples using a GC/MS method. The
qualitative composition of the Italian and Spanish samples differ. First results of
degradation tests of reference substances are reported: Arthrobacter is capable of
completely
transforming
added tyrosol to the corresponding
4-hydroxyphenylacetic acid and after 139h of fermentation no traces of tyrosol can be
identified. In contrast, only traces of phenylacetic acid are produced by Bacillus
pumilus after 139h of fermentation of tyrosol. 0 1997 Published by Elsevier
Science Limited. All rights reserved

chemical and biological methods have been

tested to purify the wastewater.
A
possible
method
of
biotechnological
purification through microbiological degradation of
the phenolic compounds is being tested among
others at the Departamento
de Microbiologia,
Granada in collaboration with the Fachhochschule
Miinster and the Universitat Bonn. The main
objective of this collaboration is the identification of
might
metabolites
that
occur
during
the
fermentation to optimize the system and to make
sure that no toxic intermediates are created. The
efforts towards the identification of phenolic
compounds in olive mill wastewater and first results
of their possible biodegradation are reported here.
For the purpose mentioned above the following
investigations are planned and on-going. First,
defined reference substances+affeic
acid, tyrosol
and 4-hydroxybenzoic acid-as
selected by the
Spanish
partners,
will
be
subjected
to
biodegradation in appropriate culture media, one
by one, in order to get familiar with the
degradation products to be expected. With more
information about the fate of an individual type

Olive mill wastewater

is produced
in large
quantities during the processing of olive oil. The
Spanish generic term for this wastewater is
alpechin. Alpechin causes severe problems in the
environment
due to its high chemical and
and
its
biochemical
oxygen
demand
antimicrobial and phytotoxic effects. These wellknown
ecologically
harmful
properties
are
assigned
to phenolic
compounds
such as
aromatic acids, alcohols and aldehydes present
in alpechin (Moreno et al., 1987; Capasso et al.,
1992). An overview of the phenolic compounds
as described in the literature by several authors
(Fedeli, 1977; Balice & Cera, 1984; Martinez
Nieto et al., 1992) and are given in Fig. 1. Many
of them e.g. caffeic acid, tyrosol and 4hydroxybenzoic acid are well known precursors
and products in the pharmaceutical,
chemical
and food industries. The undesirable aspects of
the alpechin make the direct waste disposal or
the reutilization
of the rich organic
and
inorganic content difficult. Different physico*New address: Fachochschule Rhein-sieg, FB Chemie und
Werkstofftechnik, D-53754, Sankt, Augustin.
277

G. Knupp et al.

278
CCQH

-i-)

HO-j=&

H~cooH

Brenzcatechol
4-Hydroxyphenylacetic
acid
3.4-Dihydroxyphenylacetic
acid

R=H
R = OH

R=H
R=OH
R = OCHs

Cumaric acid
Caffeic acid
Ferulic acid

OH
H

HO

COOH

R
RI =H
Rj=OH
[ RI = OH

R2 = H

Tyrosol

R2=H
R2 = OH

Hydroxytyrosol
3,4-Dihydroxyphenylglycol ]

Fig. 1. Phenolic

compounds

of alpechin

of phenol, it is planned to design the system in a

more complex manner to investigate interactions
in particular, using synthetic alpechin as a culture
medium. In a further step, phenol-free alpechin,
mixed with three reference substances will be
The
long-range
subjected
to fermentation.
objective will then be the identification
of
degradation products in natural alpechin after
fermentation. For this purpose, at each step of the
investigations, samples will be taken from the
fermentation
broth
and analysed.
A basic
requirement for all these investigations is the
development of a suitable analytical method for
the rapid, sensitive and unequivocal identification
of the phenolic compounds.
In the literature,
several methods have been described, e.g. TLC
(Vazquez Roncero et al., 1974; Capasso et al.,

Oily residue:
phenolic

R=H
R=OH

4-Hydroxybenzoic
acid
Protocatechuic acid

R = OCH~ Vanillic acid

described

in the literature.

1994), HPLC (Martinez Nieto et al., 1992), and

GC (Hamdi et al., 1992; Balice & Cera, 1984) or
GC/MS (Lopez Aparicio et al., 1977). In the
following, the GC/MS method used at Bonn and
the difficulties that might occur when analysing
untreated alpechin will be reported. Figure 2
shows a flowchart of this method.
alpechin-adjusted
to
The
pH4.5
with
hydrochloric acid and centrifuged if necessary-is
immediately extracted with ethyl acetate. After
evaporating the organic fraction to dryness an oily
residue is obtained. As the phenolic compounds
themselves are too polar to be separated by gaschromatography,
a suitable derivatization
is
necessary to enhance volatility and thermal stability.
This is carried out using N-methyl-N-trimethylsilyltrifluoracetamide
(MSTFA),
a reagent which

Water residue

t
Derivatization:
MSTFA
85C I5 min.

IrnD

k
Compound

GCiMS

Fig. 2. Flowchart

of the analytical

method.

ident!fying phenolic compounds during alpechin microbial degradaation

transforms
both
hydroxylic
and
carboxylic
functional groups into the corresponding TMSethers and -esters. It has been proven with the help
of reference substances that no intermediate
products due to non-quantitative
reactions are
produced using this reagent. The derivatized residue
is then separated by capillary-CC
and the
corresponding mass spectra are recorded.
When analysing alpechin in this way, a first idea
of the compound identity can be obtained by
comparing
times
in
the
retention
gas
chromatogram with those of reference substances.
Then, the identities of the phenolic compounds
are confirmed by comparing the mass spectra with
those of authentic reference substances. This
procedure is necessary because there are some
cases described in the literature where retention
times of compounds
from alpechin correlate
perfectly with those of reference substances
although they are not identical (Lopez Aparicio et
1977). By using
combined
GC/MS,
al.,
misinterpretations can be avoided.
With this method, extracts of untreated alpechin
from Spain were analysed. The Spanish alpechin
sample was obtained from the Granada area. In
addition, a sample of Italian alpechin from
Borgomaro in Liguria was analysed. Both samples
were kept frozen at -30C
until further
some
examination.
Table
1 gives
more
information about these two samples. It can be
seen that the Spanish alpechin has a chemical
oxygen demand, a biochemical oxygen demand
all of which are
and dry matter content
approximately double that of the Italian alpechin.
So far, 12 phenolic compounds in alpechin from
both origins have been identified using the GC/
MS method.
Fig. 3 shows two typical gasof ethyl acetate extracts of
chromatograms
untreated alpechin, prepared in the same manner.
Peak no. 1 in the chromatograms of Italian and
Spanish alpechin was identified as the TMSTable 1. Spanish
Sample-Important

COD (gl-I)
BODs (gl-)d
Dry matter (gl-)
pH (24C)

Alpechin
Sample
Parameters

vs

Italian

Alpechin

Spanish alpechin

Italian alpechin

49.0
4.2
35.1
4.9

80.4
11.5
73.0
5.2

Origin: Area of Granada.

bOrigin: Borgomaro, Liguria.
Chemical oxygen demand.
Biochemical oxygen demand.

279

derivative of benzoic acid. The intense peak no. 2 in

the lower chromatogram belongs to brenzcatechol.
Although the chromatogram of Spanish alpechin
presents an intensive signal at the same scan, the
corresponding
mass-spectra revealed that this
compound
is not identical to brenzcatechol.
Brenzcatechol was identified in low quantities in the
peaks tailing (signal no. 2). By comparing the massspectra of the TMS-derivatives, peak no. 3 in both
chromatograms
was identified as tyrosol. This
substance is described in the literature as one of the
major compounds (Capasso et al., 1994). Peaks Nos 4
and 5 in the lower chromatogram are the signals of
derivatives of 4-hydroxybenzoic
acid and 4hydroxyphenylacetic acid, respectively. These two
compounds, clearly identified in the extracts of
Italian alpechin could only be found in trace amounts
in the Spanish alpechin. According to the retention
time from a mixture of derivatized authentic reference
substances, peak no. 6 of both chromatograms
correlates with the derivative of veratric acid.
However, comparison of the corresponding mass
spectra revealed that the substance in these two
chromatograms is not identical to veratric acid.
Although veratric acid is described in the literature as
a typical alpechin compound (Balice & Cera, 1984) it
has not yet been identified in the analysed samples. In
the leading edge of the dominant peak around scan
no. 450 two further well-known compounds were
identified in both extracts by their mass spectra:
vanillic acid (peak no. 7) and hydroxytyrosol (peak
no. 8). Peak no. 9 in the lower chromatogram belongs
to the TMS-derivative of protocatechuic acid.
Although the retention time of the corresponding
intense peak in the upper chromatogram correlates
with protocatechuic acid, the mass spectra are not
identical. In contrast to being present in great
amounts in extracts of Italian alpechin, only traces of
protocatechuic acid were found in Spanish alpechin
9. The
presence
of 3,4at
peak
no.
dihydroxyphenylglycol-a
well known compound in
olive fruits (Bianchi & Pozzi, 1994j-has not yet been
described in alpechin.
The TMS-derivative of this labile substance could
be identified as peak no. 10. There are three other
signals in the chromatograms worth mentioning:
signal no. 11 in the lower chromatogram of Italian
extract represents the TMS derivative of 3,4dihydroxyphenylacetic
acid. No corresponding
signal can be found in the upper chromatogram.
Conversely phenylpropanoic compounds have been
identified only in the Spanish alpechin so far. In the
upper chromatogram the corresponding signals of

G. Knupp

280

et al.

100
1

100
1:45

Spanish sample

300
5:15

200
3:30

400
7:oo

500
6:45

600
10:30

500
6~45

6dO
10:30

700
12:15

600 Scans
14:00 Time [min]

Italian sample

8
\

I .
100
1:45

200
3:30

300
515

400
7:oo

Fig. 3. Chromatograms

the TMS-derivatives of cumaric acid (no. 12) and of

caffeic acid (no. 13) can be found. A possible
explanation for their absence in Italian alpechin
might be a partial natural biodegradation of these
compounds
occurring prior to sampling. /IOxidation and/or m-hydroxylation
could create
protocatechuic acid. The strong intensity of the
protocatechuic acids peak, as shown before in the
would agree with this
lower chromatogram,
observation.
In the following discussion, two mass spectra of
tyrosol-TMS
are given as an example of
unequivocal identification. On the left side of
Fig. 4 the TMS-derivative of a tyrosol reference

3
1, 15

600 Scans
14:OO Time [min]

of alpechin extracts.

substance and on the right side the analysed

peak at scan no. 328 of Spanish alpechin are
shown. After EI ionization the molecular ion
occurs at m/z 282. The next peak at m/z 267
results from the loss of a methyl group. This
typical M- 15 fragment ion is prominent in all
mass spectra of trimethylsilylated
alcohols and
acids and can be used to detect the molecular
ion. a-Cleavage creates the base peak at m/z 179
which belongs to a cyclic tropylium-TMS
ether
cation. The signals at m/z 73 and 103 are created
by the TMS group. On the right, the mass
spectrum of tyrosol recorded at scan no. 328
from the Spanish alpechin extract can be found.

281

Identifying phenolic compounds during alpechin microbial degradation

Reference

Compound

substance

at peak # 3

179

179

73

262
267

1 I

IL L

SO

lb0

260

150

50

300

250

100

150

Fig. 4. EI/MS

of silylated

Ir

532

I300

lmouritv

48

24 h

HO~jCDOH

372

600
10:30

600
14:00

72

Scan
Time [mini

10:30

I.;.bosca
400
7:oo

200
3:30

10:30

14:o0

Time [min]

139 h

ho

i7:oo

250

tyrosol.

330
0~

3:30

200

fermented under usual conditions. Four samples

were taken after 24,48, 72 and 139h of fermentation
and extracted immediately. Figure 5 shows four
chromatograms monitoring this fermentation over
139h. With the GC/MS method described above it
can be shown that Arthrobacter
is capable of
oxidizing the added tyrosol to the corresponding 4hydroxyphenylacetic acid. Although no quantitative

As the two spectra show good correlation within

their fragmentation
patterns,
the compound
identity could be confirmed.
In the next step of the Spanish-German
collaboration, tyrosol was subjected to fermentation
by Arthrobucter for 139h. For this purpose tyrosol
was added to a typical culture medium in a
concentration
of lgl-
and then aerobically

400
7:oo

,I

m/z

m/z

.L

14:00

Fig. 5. Biodegradation

400
7:oo

200
3:30

Time [min]

of tyrosol

over

139h.

600
10:30

scan
14:00

Time [min

282

G. Knupp et al.

measurements have been carried out to date it can be

seen in the chromatograms that the peak at scan no.
330 belonging to tyrosol decreases while the
corresponding signal of 4-hydroxyphenylacetic acid
increases over the monitored time. In the fourth
the
chromatogram
tyrosol
has
completely
disappeared after 139h of fermentation. Now the
only prominent peak at scan no. 374 belongs to 4hydroxyphenylacetic acid. Both, retention times and
fragmentation patterns of the mass spectra match
those of our authentic reference substances. In
contrast, Bacillus pumilus was unable to completely
biodegrade tyrosol over the monitored time. So far,
next to the remaining tyrosol only traces of
phenylacetic acid could be identified. Extracts of the
fermentation broths of 4-hydroxybenzoic acid are
currently being analysed.

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Balice, V. and Cera, 0. (1984) Acidic phenolic fraction of the
olive vegetation water determined by a GC method.
Grasas y Aceites, 35, 178-180.
Bianchi, G. and Pozzi, N. (1994) 3,4-Dihydroxyphenylglycol,

major

C&z

phenolic

in

Olea

europaea

fruits.

Phytochemistry, 35, 1335-1337.

Capasso, R., Cristinzino. G., Evidente, A. and Scognamiglio,

F. (1992) Isolation, spectroscopy and selective phytotoxic
effects of polyphenols
from vegetable waste waters.
Phytochemistry, 31, 4125-4128.

Capasso, R., Evidente, A. and Vista, C. (1994) Production of

hydroxytyrosol
from
olive oil vegetation
waters.
Agrochimica, 38, 165-17 1.
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Hamdi, M., Garcia, J. L. and Ellouz, R. (1992) Integrated

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Lopez Aparicio, F. J., Garcia-Granados

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Roncero,
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