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Biotechnology Letters 24: 20472051, 2002.

2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Biodegradation of phenols by microalgae


Gabriele Pinto1, , Antonino Pollio1 , Lucio Previtera2 & Fabio Temussi2
1 Dipartimento

di Biologia Vegetale, Universit`a Federico II, Via Foria 223, I-80139 Napoli, Italy
di Chimica Organica e Biochimica, Universit`a Federico II, Via Cynthia 4, I-80126 Napoli, Italy
Author for correspondence (Fax: +39-081-450165; E-mail: gabriele.pinto@unina.it)
2 Dipartimento

Received 3 May 2002; Revisions requested 22 May 2002/11 July 2002; Revisions received 9 July 2002/7 October 2002; Accepted 8 October
2002

Key words: biodegradation, microalgae, phenols

Abstract
Two green microalgae, Ankistrodesmus braunii and Scenedesmus quadricauda, degraded phenols (each tested at
400 mg ml1 ) selected from olive-oil mill wastewaters, within 5 days, with a removal greater than 70%. Green
algae may, therefore, represent an alternative to other biological treatment used for the biodegradation of phenolcontaining wastewaters.

Introduction
Phenolic compounds are among the most frequently
occurring pollutants of surface waters as they are the
waste products of many industrial activities. Their
abatement in sewage treatment plants is often unsatisfactory because phenolics are highly toxic to anaerobic
and aerobic bacteria (Capasso et al. 1995). For this
reason the phenol detoxification potential of different
microrganisms, mainly bacteria and fungi, has been
extensively studied (Kahru et al. 1998). In the last
decade the possible use of the unicellular chrysophyte,
Ochromonas danica, has been investigated for the
degradation of phenols in the dark and in aerobic conditions. This alga is able to carry out the metacleavage
of exogenous phenol, utilizing the connected reactions
for its energetic requirements (Semple et al. 1999). On
the other hand, Ochromonas is not completely suitable
for the treatment of wastewaters as it is not suitable
for mass cultivation. As with other Chrysophytes, this
organism possesses an endogenous mechanism of regulation, limiting the number of vegetative cells within
a population (van den Hoek et al. 1995).
In general, the algae most frequently used in wastewater treatment are green unicellular or coenobic algae
(Hoffmann 1998). In addition, algal systems with filamentous cyanobacteria have been proposed for the

secondary and tertiary treatment of wastewaters (Talbot & de la Noe 1993) since the intensive algal
growth in open ponds causes increases in pH which
can be tolerated by cyanobacteria.
Both cyanobacteria and green algae are sensitive to
phenolics whose toxicity is related to the number and
to the polarity of the substituents on the aromatic ring
(Della Greca et al. 1992). However, some algal strains
can metabolise phenylpropanoids such as -asarone at
104 M (Pollio et al. 1993).
The objective of this study was to screen bluegreen and green algae in the search for phenolresistant strains. The strains so far isolated have been
tested for their ability to remove from the culture media simple phenols selected among those found in
wastewaters from olive oil mill wastewaters, whose
toxicity is due mainly to the presence of large amounts
of these compounds.
Materials and methods
The algae listed in Table 1 were obtained from the
Cambridge Collection of Algae and Protozoa, UK
(CCAP), from the Algal Collection at the University
of Texas at Austin, USA (UTEX), from Sammlung
of Algenkulturen, Gttingen, Germany (SAG) and
from Algal Collection at the University of Naples

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Fig. 1. Structure of selected phenols.

Fig. 2. Removal (%) of phenols by T 76 Scenedesmus quadricauda after 5 or 10 days of exposure. The initial concentration of each phenol was
400 mg ml1 . Operating conditions: pH 6.2, 25 C, total irradiance: 100 E m2 sec1 , provided by daylight fluorescent lamps. The tests
were carried out in 100 ml Erlenmeyer flasks containing 50 ml of algal suspension (mean SD, n = 2).

(DBV). All the strains were cultivated on Bold Basal


Medium (BBM), and the growth of each strain was
followed with a colorimeter. For paper disk bioassays,
an aliquot of each culture (2 ml) in the active phase of
growth was inoculated on a Petri dish containing BBM
(30 ml) solidified with 15% (w/v) agar. Filter paper
disks (sterile blanks, Difto Bacto Concentration Disks,
6 mm) were impregnated with catechol solution and
placed on each previously inoculated plate. The plates
were incubated upside down at 25 C with a total irradiance of 100 E m2 sec1 provided by daylight
fluorescent lamps located below the plates. Observations for the inhibition of growth of the plated algae
around the filter disk were made as soon as growth
became visible. The inhibition was measured as the
diameter of the no-growth zone including the paper
disk.
For the experiments of phenol removal by microalgae (Figure 1), besides catechol (1) the following

compounds were selected: tyrosol (2), hydroxytyrosol


(3), p-hydroxybenzoic acid (4), ferulic acid (5), pcoumaric acid (6), synapic acid (7), caffeic acid (8)
and vanillic acid (9). Each compound was added to
the algal suspension (density 2 g l1 ) to give 400 mg
l1 . Control flasks containing the phenols dissolved in
BBM medium without algae were prepared for each
experiment. All the tests were conducted under the
same conditions of temperature and light indicated
for paper disk bioassays. At the end of each test the
algae were centrifuged (3000 g15 min) and the supernatant was filtered on Millex filters and injected
in a HPLC-UV system using an RP-18 column. The
column was equilibrated with a mixture of A (H2 O
containing 1% acetic acid)-B (methanol containing
1% acetic acid) 9:1 (v/v) and using the following program: isocratic run for 25 min, followed by an increase
of B up to 60% in 30 min and a decrease down to 10%
in 5 min. The detector wavelength was set at 260 nm.

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Chlorophyta

Cyanopyta

Table 1. Inhibition of algal growth by catechol. Catechol was dissolved in acetone


(5 mg/200 ml); then each filter paper disk was impregnated with an appropriate
volume (2 l, 4 l, 6 l, 8 l) of the solution to bring the final amount of catechol
on the disk to the required value, namely 0.05,0.1,0.15 and 0.2 g. After evaporation of the solvent, the filter papers disks were placed on each previously inoculated
plate. + + + no inhibition; ++ diameters of inhibition zone from 6 to 14 mm; +
diameters from 15 to 22 mm; diameters from 23 to 30 mm.
Species

0.05 g

0.1 g

0.15 g

0.2 g

Anabaena flos-aquae
Aulosira terrestre
Fremyella diplosiphon
Fischerella ambigua

+++
+++
+++
+++

+++
+++

+++
+++

Nostoc commune
Nodularia sphaerocarpa
Phormidium autumnale
Scytonema hofmanni
Synechococcus leopolensis
Ankistrodesmus braunii
Chlamydomonas applanata
Chlorella emersonii
Chlorella saccharophila
Chlorella zofingiensis
Coccomyxa elongata
Coelastrum microporum

+++

+
+++
++
+++
+++
+++
+++

++

+++

+++
+
+++
++

+++

+++

+++

+++

+++
+

+++

+++

+++

+++

+++
+

+++

+++

Friedmania israeliensis
Mesotaenium caldariorum
Pseudococcomyxa adherens
Scenedesmus quadricauda
Selenastrum capricornutum
Stichococcus bacillaris

++

+++
+++
+
+++

+
+++
+
+++

+++

++

+++

For the determination of catechol, caffeic acid and


p-hydroxybenzoic acid uptake in algal cells the algae were separated from the medium by centrifugation
at 4000 g for 15 min. The cell pellet collected was
washed twice with BBM to solubilize the selected phenol from the external surface of the cells. Each time the
supernatant was collected and extracted as described
above. The algal pellet obtained after the second centrifugation was collected and extracted with methanol.
The extract was injected in the HPLC system (in the
conditions previously described) to check the presence
of the phenol.
The tests were carried out for five and ten days.
Each experiment, conducted in triplicate, was made
twice and each value represents the mean of the two
experiments. The standard error was never higher than
5%.

Results and discussion


Catechol, the main anti-algal phenol occurring in olive
oil mill waste-waters (Della Greca et al. 2001) was
used to select algal strains resistant to phenols. Paper
disk bioassays were carried out to assess the effects of
the compound at four different amounts, from 0.05 g
to 0.2 g, on selected strains of blue-green and green
algae (Table 1).
Catechol affected the growth of the majority of algae, and only five strains were found resistant to it,
namely SAG 1453.5a Nostoc commune, UTEX 2349
Scytonema hofmanni, CCAP 202.7a Ankistrodesmus
braunii, CCAP 211/9a Chlorella saccharophila, and
UTEX 76 Scendesmus quadricauda. Further tests
were carried out by preparing liquid cultures of each
resistant strain at 104 M catechol. Analyses of the incubation medium of each alga after 5 days of catechol
exposure showed that strains CCAP 202.7a and UTEX

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Fig. 3. Removal (%) of phenols by 202-7a Ankistrodesmus braunii after 5 or 10 days of exposure. The initial concentration of each phenol was
400 mg ml1 . Operating conditions: pH 6.2, 25 C, total irradiance: 100 E m2 sec1 , provided by daylight fluorescent lamps. The tests
were carried out in 100 ml Erlenmeyer flasks containing 50 ml of algal suspension (mean SD, n = 2).

76 removed more than 50% of the initial amount of


catechol from the medium, whereas strains CCAP
211/9a and UTEX 2349 respectively gave 45% and
30% catechol removal. Finally, strain SAG 1453.5a
did not significantly decrease the initial amount of catechol in the medium. The following experiments of
phenol removal used strain CCAP 202.7a or UTEX 76,
which were exposed for 5 and 10 days to each of the
most abundant phenols isolated from olive oil wastewater. In all the experiments a phenol concentration
was used which corresponded to catechol concentration in olive oil wastewaters (Della Greca et al.
2001).
The presence of a high phenol concentration in
the medium did not affect algal morphology for the
first five days but, in the following days, some algal
cells became larger, many oil droplets were observed
in the cytoplasm and some cells showed anomalous
shapes. The concentration of each phenol in the control flasks remained constant during the course of the
experiments. Both strains degraded phenols within
5 days, achieving, in some cases, a removal greater
than 70%, even though better results were obtained
with T76 (Figures 2 and 3). The only exception was
tyrosol, whose concentration in the medium was significantly lowered only by UTEX 76 S. quadricauda
after 10 days. The results obtained with microalgal
strains are comparable with those obtained with fungi
and bacteria. The basidiomycetes Coprinus sp., re-

moved 2538% catechol and 68100% ferulic acid


from the culture medium after 5 days (Guiraud et al.
1999), whereas Arthrobacter completely transformed
tyrosol to 4 hydroxyphenyl acetic acid within 136 h
(Knupp et al. 1996). Bacteria and fungi can metabolise
phenolic compounds to yield various biotransformation products, whereas no biotransformation was observed in the experiments carried out with algae, although these same strains, particularly UTEX 76, can
biotransform simple steroids (Della Greca et al. 1996)
The possible biosorption and accumulation of phenols into the algal cells were monitored on catecholtreated cells of CCAP 202.7a and UTEX 76 after
5 days, evidencing that neither in extracellular space,
nor in the cells themselves was any accumulation of
catechol observed. The same results were obtained
in experiments carried out with caffeic acid or 4hydroxybenzoic acid, selected from among the phenolics most efficiently removed by algae (Figures 2
and 3). These preliminary data suggest that phenolic compounds could be metabolised by green algal
cells. However, investigation of the enzymes involved
in phenol biodegradation of these strains is necessary
to understand whether the mechanisms of intracellular degradation are the same as those described for
Ochromonas danica (Semple et al. 1999).
These results indicate that green algae may represent an alternative to other biological treatment used
for the biodegradation of phenol-containing wastewa-

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ters. Further studies are in progress to establish if both
strains are able to carry out the degradation of phenolic
mixtures in the dark, and by using cell immobilization
technologies to improve the removed yields.

Acknowledgement
This work was performed in the framework of project
Ambiente Terrestre: Chimica per lAmbiente (Cluster 11-A) del Consorzio Interuniversitario La Chimica
per lAmbiente, financed by M.I.U.R. (Legge 488/92).

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