Biodegradation of phenol by Chlamydomonas reinhardtii

Article abstract of DOI:10.1007/s11120-020-00756-5

The data presented in this particular study demonstrate that the biodegradation of phenol by Chlamydomonas reinhardtii is a dynamic bioenergetic process mainly affected by the production of catechol and the presence of a growth-promoting substrate in the culture medium. The study focused on the regulation of the bioenergetic equilibrium resulting from production of catechol after phenol oxidation. Catechol was identified by HPLC-UV and HPLC-ESI-MS/MS. Growth measurements revealed that phenol is a growth-limiting substrate for microalgal cultures. The Chlamydomonas cells proceed to phenol biodegradation because they require carbon reserves for maintenance of homeostasis. In the presence of acetic acid (a growth-promoting carbon source), the amount of catechol detected in the culture medium was negligible; apparently, acetic acid provides microalgae with sufficient energy reserves to further biodegrade catechol. It has been shown that when microalgae do not have sufficient energy reserves, a significant amount of catechol is released into the culture medium. Chlamydomonas reinhardtii acts as a versatile bioenergetic machine by regulating its metabolism under each particular set of growth conditions, in order to achieve an optimal balance between growth, homeostasis maintenance and biodegradation of phenol. The novel findings of this study reveal a paradigm showing how microalgal metabolic versatility can be used in the bioremediation of the environment and in potential large-scale applications.

Full text of DOI:10.1007/s11120-020-00756-5

Photosynthesis Research
https://doi.org/10.1007/s11120-020-00756-5
ORIGINAL ARTICLE
Biodegradation of phenol by Chlamydomonas reinhardtii
1
1
1
1
Theocharis T. Nazos · Leonidas Mavroudakis · Spiros A. Pergantis · Demetrios F. Ghanotakis
Received: 22 January 2020 / Accepted: 22 April 2020
©
Springer Nature B.V. 2020
Abstract
The data presented in this particular study demonstrate that the biodegradation of phenol by Chlamydomonas reinhardtii
is a dynamic bioenergetic process mainly aꢀected by the production of catechol and the presence of a growth-promoting
substrate in the culture medium. The study focused on the regulation of the bioenergetic equilibrium resulting from produc-
tion of catechol after phenol oxidation. Catechol was identiꢁed by HPLC-UV and HPLC-ESI-MS/MS. Growth measure-
ments revealed that phenol is a growth-limiting substrate for microalgal cultures. The Chlamydomonas cells proceed to
phenol biodegradation because they require carbon reserves for maintenance of homeostasis. In the presence of acetic acid
(
a growth-promoting carbon source), the amount of catechol detected in the culture medium was negligible; apparently,
acetic acid provides microalgae with suꢂcient energy reserves to further biodegrade catechol. It has been shown that when
microalgae do not have suꢂcient energy reserves, a signiꢁcant amount of catechol is released into the culture medium.
Chlamydomonas reinhardtii acts as a versatile bioenergetic machine by regulating its metabolism under each particular set
of growth conditions, in order to achieve an optimal balance between growth, homeostasis maintenance and biodegradation
of phenol. The novel ꢁndings of this study reveal a paradigm showing how microalgal metabolic versatility can be used in
the bioremediation of the environment and in potential large-scale applications.
Keywords Phenol · Catechol · Biodegradation · Chlamydomonas reinhardtii · Bioenergetics · HPLC-ESI-MS/MS
Abbreviations
Introduction
HPLC-ESI-MS/MS High-performance liquid chroma-
tography-electrospray ionization
tandem mass spectrometry
Increased industrial activity has led to the release and accu-
mulation of signiꢁcant amounts of phenolic compounds in
the environment (Stoilova et al. 2006). Phenol and its deriva-
tives are common contaminants of terrestrial and aquatic
environments and as a result, these compounds have been
categorized as priority pollutants by the US Environmen-
tal Protection Agency (EPA) (Agarry et al. 2008; Du et al.
2009). An approach for the decrease of high concentrations
of phenolic compounds from ecosystems relies on their
biodegradation by microorganisms (Van Schie and Young
2000). Metabolic pathways, various bioreactor types, ener-
getic approaches and a signiꢁcant number of microorgan-
isms (mainly bacteria and fungi) have been identiꢁed (Al-
Khalid and El-Naas 2012).
Fo
Minimum ꢃuorescence that corre-
sponds to the time that all photosyn-
thetic reaction centers are open
Maximal ꢃuorescence that corre-
sponds to the time that all reaction
centers are closed
Fmax
F ,
v
Variable ꢃuorescence (F −Fo)
v
max
F /F
Photosynthetic eꢂciency
max
MRM
Multiple Reaction Monitoring
Tris-Acetate-Phosphate medium
used for the growth of microalgae
and in experimental cultures
Tris-Phosphate medium used in
experimental cultures
TAP
TP
The ꢁrst step of the aerobic biodegradation of phenol
includes the conversion of phenol to catechol by the enzyme
phenol hydroxylase that uses molecular oxygen. The cat-
echol aromatic ring can be cleaved either by ortho or meta
pathways. In the ortho pathway (β-ketoadipate pathway),
the enzyme 1,2-dioxygenase cleaves the bond between the
two carbons connected with the hydroxide groups, to form
*
Demetrios F. Ghanotakis
ghanotakis@uoc.gr
1
Department of Chemistry, University of Crete, Vasilika
Voutes, Heraklion, Crete 70013, Greece
Vol.:(0
1
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cis, cis- muconic acid. This intermediate is subsequently
metabolized through multiple steps to succinic acid and
acetyl-CoA. In the meta-cleavage pathway, ring cleavage
takes place in the adjacent carbon–carbon bond of the one
hydroxide group by 2,3-dioxygenase. As a result, 2-hydroxy-
muconic semialdehyde is formed, which is ꢁnally converted
to acetaldehyde and pyruvate (Fig. 1). It appears that the
strategy followed is initially to decrease the complexity of
the mother compound followed by its conversion to products
that can be utilized by microbial metabolism (Williams and
Sayers 1994; Van Schie and Young 2000; Zhou et al. 2016).
These pathways have been found to exhibit many similarities
both in bacteria and unicellular microalgae.
be used for a series of applications such as biodiesel produc-
tion (Delrue et al. 2016).
Phenol and its substituted derivatives inhibit growth of
green microalgae (Shigeoka et al. 1988). The presence of
phenol in cultures of marine microalgae upregulates genes
attributed to the expression of antioxidant enzymes and
biosynthesis of carotenoids (Zhou et al. 2017), production
of reactive oxygen species (ROS) as well as a decrease in
chlorophyll content (Duan et al. 2017). Despite these obser-
vations, several studies have proposed the eꢂcient use of
freshwater and marine microalgae for biodegradation of
phenolic compounds and have provided a few mechanistic
insights. Semple and Cain (1996) elucidated the mecha-
nism of phenol biodegradation in the brown chrysophyte
microalga Ochromonas danika. They showed that phenol
was initially converted to catechol, which was subsequently
catabolized through the meta-cleavage pathway. The cyano-
bacterium Synechococcus PCC 7002 was able to perform
extracellular biodegradation of phenol via the ortho cleavage
pathway. A signiꢁcant ꢁnding was that phenol was only bio-
degraded in the dark, while under photoautotropic and pho-
toheterotrophic conditions no biodegradation activity was
observed (Wurster et al. 2003). Otto and Schlosser (2014)
isolated and characterized an extracellular laccase, produced
by the green microalga Tetracystis aeria, that is related to
the oxidation of phenolic compounds. The freshwater green
alga Monoraphidium braunii was able to degrade signiꢁcant
amounts of bisphenol-A, while at the same time physiologi-
cal changes, such as a decrease of photosystem II activity
and decreases in cell size, were also observed (Gattullo et al.
Green microalgae are a class of well adapted microorgan-
isms that can grow in unfavorable environmental conditions
because they possess versatile metabolic networks (Mata
et al. 2010; Satyanarayana et al. 2011; Koller et al. 2014).
Microalgae have been successfully grown in municipal, agri-
cultural and industrial waste-waters in order to reduce their
phosphorous, nitrogen, micronutrient and organic content
(
Guldhe et al. 2017). The use of microalgae for wastewater
bioremediation applications has been proposed as a win–win
bioreꢁnery paradigm, because the resulting biomass could
2
012). The alga Cyclotella caspia was shown to biodegrade
nonylphenol. At increased concentrations of this phenol,
the chlorophyll a content and cell growth rate were signiꢁ-
cantly decreased. Biodegradation rates of nonylphenol were
high after an adaptation period of 48 h, while after 144 h
no further biodegradation of the substance was observed
(
Liu et al. 2013). The marine microalga Tetraselmis marina
was found to detoxify and degrade 2,4-dichlorophenol using
a glucose transfer followed by a malonyl transfer (Petrout-
sos et al. 2008). Recently, Das et al. (2015) discovered that
Chlorella pyrenoidosa biodegraded phenol both via ortho
and meta-cleavage pathways. The ortho pathway was found
to be signiꢁcantly more active than the meta pathway. A
remarkable observation was the accumulation of catechol,
cis-cis muconic acid and 2,3-hydroxy-muconic semial-
dehyde intermediates in the growth medium. Spirulina
maxima was found to biodegrade phenol up to a concen-
1
3
tration of 400 mg/L in 24 h and studies with C-labeled
carbon demonstrated that 30% of the phenol carbon was
assimilated in biomass along with its mineralization to CO
2
Fig. 1 Metabolic pathways for aerobic biodegradation of phenol.
Bond-line structures a phenol; b catechol; c cis, cis-muconic acid;
d succinic acid; e acetyl-CoA; f 2-hydroxymuconic semialdehyde; g
pyruvate and h acetaldehyde
(
Lee et al. 2015). In the marine microalga Lingulodinium
polyedrum phenol biotransformation was catalyzed by glu-
tathione S-transferase, phenol hydroxylase and catechol
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2
,3-dihydroxygenase metabolic pathways, while at the same
networks regarding the carbon uptake and utilization, and
moreover it can grow in autotrophic, heterotrophic and mixo-
trophic conditions (Hippler et al. 1998; Johnson and Alric
2013). Depending on growth conditions, Chlamydomonas
cells exhibit a diꢀerent response to exogenous conditions
that induce stress (Endo and Asada 1996). In a recent publi-
cation we showed that the green microalga Chlamydomonas
reinhardtii biodegraded significant amounts of phenol
(Nazos et al. 2017). This work was focused on the bioener-
getics of the biodegradation process under conditions aꢀect-
ing photosynthesis. It was shown that biodegradation of
phenol by Chlamydomonas reinhardtii is a photoregulated,
aerobic process. High concentrations of phenol were found
to induce higher levels of biodegradation as a response to
stress, while at the same time the presence of an alternative
organic carbon source (acetic acid) had a prominent role in
the alleviation of stress eꢀects.
time phenol was found to induce the activity of antioxidant
enzymes as a response to oxidative stress (Martins et al.
2
015).
Apart from the mechanistic studies presented above, sev-
eral published studies have focused on the role of bioener-
getic conditions (such as the presence of an alternative car-
bon source and light intensity that provide suꢂcient energy
to microalgal cells) in the biodegradation of phenolic com-
pounds. Priyadharshini and Bakthavatsalam (2016) modeled
and optimized phenol biodegradation by Chlorella pyrenoi-
dosa and found out that it was aꢀected by the interaction
of various factors, such as phenol concentration, initial
algal concentration and reaction time. It has been reported
in the literature that the presence of an alternative organic
carbon source plays an important role in the alleviation of
toxicity eꢀects of phenolic compounds on green microal-
gae (Megharaj et al. 1992). Theoretical studies suggest that
the presence of an alternative carbon source that promotes
growth could inhibit the process of biodegradation because
both substrates require suꢂcient amounts of oxygen in order
to be metabolized (Lika and Papadakis 2009). Papazi and
Kotzabasis (2007) showed that the biodegradability of dif-
ferent types of monosubstituted phenols by Scenedesmus
obliquus was based on the selection of appropriate culture
conditions as well as the type of phenolic compound tested.
Based on their data, it was proposed that monosubstituted
halogenated phenols were biodegraded initially by dehal-
ogenation followed by ꢁssion of the phenolic ring. When
The research presented here provides new results on
biodegradation of phenol by axenic cultures of the green
microalga Chlamydomonas reinhardtii, including characteri-
zation of a catechol molecule that is produced as metabo-
lite. In addition, the role of the presence or absence of an
alternative organic carbon source on the regulation of the
bioenergetic equilibrium of the biodegradation of phenol by
Chlamydomonas reinhardtii is characterized.
Materials and methods
cultures were supplemented with glucose or CO as carbon
Organism and growth conditions
2
sources, an increased biodegradability took place, while the
biodegradation process was found to be photosynthetically
dependent. On the other hand, low concentrations of phenol
and monosubstituted methylphenols exhibited an increased
biodegradability in the absence of an alternative carbon
source in the culture medium (Papazi and Kotzabasis 2007,
The CC-125 strain (wild type) of Chlamydomonas rein-
hardtii was used in all experiments. Mother cultures were
grown photoheterotrophically in Tris- Acetate- Phosphate
(TAP) medium (Harris 2009) at 25 °C, under a continuous
−
2 −1
light intensity of 70–80 μmol photons·m ·s for ꢁve days
−
1
2
008). Biodegradation of para-cresol by Scenedesmus cells
in Erlenmeyer ꢃasks shaken at a rate of 140 min . For the
preparation of experimental cultures, cells were ꢁrst col-
lected by centrifugation at 1000 g for 3 min., washed twice
by resuspension in fresh medium (depending on each par-
ticular experimental condition); 50 mL aliquots were trans-
ferred into 100 mL Erlenmeyer ꢃasks closed tightly with
septa. Initial cell concentration in the experimental cultures
was found to be a two-step process. Removal of the methyl
group from the phenolic ring led to the production of phe-
nol, which was further metabolized (Papazi et al. 2012).
Regarding the biodegradation of dichlorophenols, Scened-
esmus cells were found to follow a rational strategy, based
on a bioenergetic balance between the type of the compound,
growth, as well as the presence of glucose in the culture
medium (Papazi and Kotzabasis 2013). All these data prove
the metabolic versatility of photosynthetic microorganisms
and demonstrate that they can be eꢂcient systems for the
biodegradation of phenols.
7
−1
was adjusted to be equal to 3.633 (± 0.528) ·10 cells·mL .
Two different experimental culture conditions were
tested; one with added acetic acid (1.048 g/L) as an alter-
native carbon source and another containing only phe-
nol. In the ꢁrst experimental condition, TAP was used as
growth medium while in the second cells were cultivated in
Tris–phosphate (TP) medium. The ꢁnal phenol concentra-
tion in the experimental cultures was 4.0 mM (376.4 mg/L).
Phenol was dissolved in double distilled water, and the solu-
tion was ꢁltered through a 0.2 μm syringe ꢁlter. The phenol
Chlamydomonas reinhardtii is a well-studied model
microorganism that has provided many insights in the elu-
cidation of the photosynthetic mechanism and genetics in
microalgae (Goodenough et al. 1995; Rochaix et al. 1998;
Harris 2009). This microalga possesses versatile metabolic
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stock solution’s concentration was 0.5 M. Parallel cultures
of Chlamydomonas cells in TAP and TP mediums were the
corresponding controls for the two experimental conditions
described.
methanol: water: acetic acid (50:49:1) at a ꢃow rate of
−1
0.5 mL· min . Technique was operated in the negative Mul-
tiple Reaction Monitoring (MRM) mode and identiꢁcation
of catechol in the sample was carried out by comparing the
retention time of the selected reaction of catechol fragmenta-
tion products in the culture sample with those obtained for
the catechol standard. All measurements were performed
using the following operation parameters: electrospray volt-
age−3.5 kV, sheath gas pressure 35 (arbitrary units), auxil-
iary gas pressure 10 (arbitrary units), ion transfer capillary
temperature 320 °C and source collision- induced dissocia-
tion (CID) 12 eV.
All experiments were repeated at least three times.
All nutrient media and glassware were autoclaved at
1
20 °C for 20 min, in order to avoid contamination. The
experiments were performed in a temperature-con-
trolled room (25 °C) under a continuous light intensity
−
2 −1
of 70–80 μmol photons·m ·s . Cultures were shaken at
−
1
1
40 min on a rotary shaker. Experiments were carried out
in a laminar ꢃow hood ꢁtted with a UV lamp and sterilized
with ethanol.
Determination of growth
High‑performance liquid chromatography (HPLC)
for the analysis of phenol and catechol
Cell growth was determined by cell counting using an
improved Neubauer cell counting chamber (Milliscience)
and a Nikon Eclipse E800 optical microscope. Cell density
For phenol and catechol analysis, culture samples were cen-
trifuged for 5 min at 1000 g. The supernatants were ꢁltered
through a 0.2 μm syringe ꢁlter and were diluted with the
mobile phase and then injected through a 20 μL loop into
the HPLC system. Analyses were performed using an HPLC
system equipped with a high precision pump (LC-10AD,
Shimadzu), a UV–Vis detector (SPD 10AV, Shimadzu) and a
C18 column (Grace Smart RP18, 250 mm l and 4.6 mm ID,
−
1
was expressed as number of cells · mL . Culture samples
were collected every 24 h with a sterile syringe. Sample
collection was carried out in a laminar ꢃow hood with a UV
lamp sterilized with ethanol.
Fluorescence induction measurements
5
μm particle size). Analyses were performed at 25 °C. An
isocratic HPLC method, using as a mobile phase methanol:
The fluorescence induction measurements were carried
out using the Handy Plant Eꢂciency Analyser (Hansatech
Instruments, Kings’ Lynn, Norfolk, UK). The parameter that
was measured was the maximal photosynthetic eꢂciency
−
1
water: acetic acid (50:49:1), and a ꢃow rate of 1.0 mL· min
according to the protocol of Lovell et al. (2002). Detection
was carried out by measuring absorbance at 279 nm and
quantiꢁcation by integration of known quantities of phenol
and catechol.
(
(
F /F ) according to the method of Strasser and Strasser
v max
1995). This method is based on the measurement of a fast
ꢃuorescence transient with a 10 μs resolution for 1 s after a
Catechol identiꢀcation with electrospray ionization
mass spectrometry coupled with high‑performance
liquid chromatography (HPLC‑ESI‑MS/MS) using
the multiple reaction monitoring (MRM) method
1
0 min dark sample adaptation and yields a dynamic meas-
urement of a photosynthetic sample at a given physiological
state. Fluorescence was measured at 12-bit resolution and
excited by three light-emitting diodes providing a saturating
−
2 −1
red (650 nm) light intensity of 3000 μmol photons·m ·s .
−
1
For the identiꢁcation of the catechol product, a 500 μg L
catechol standard and a culture sample collected after 5 days
of incubation, diluted 100 times in methanol: water: acetic
acid (50:49:1), were infused in a TSQ Quantum triple quad-
rupole mass spectrometer (Thermo Scientiꢁc, San Jose, CA,
Sample preparation and observation with optical
and ꢁuorescence microscopy
−
1
USA) through a syringe pump at a ꢃow rate of 20 μL·min .
Mass spectra were obtained with an electrospray source in
the negative ionization mode through tandem mass spec-
trometry (MS/MS) in order to obtain the fragmentation pat-
tern of catechol. Having acquired the fragmentation pattern
of catechol, the method was coupled with a High-perfor-
mance liquid chromatography system (HPLC–ESI–MS/
MS). For the reversed phase HPLC–ESI–MS/MS method, a
C18 column (Grace Smart RP18, 250 mm l and 4.6 mm ID,
Culture samples were collected after a cultivation period
of ꢁve days, using a sterile pipette tip. The observation of
the cells was performed using a Nikon Eclipse E800 opti-
cal microscope and an optical oil-immersion Plan Apo
60×lens and a CCD ProgRes CF camera. For the ꢃuores-
cence microscopy, excitation was produced by a UV lamp;
ꢃuorescence detection utilized a B-2A ꢁlter that allows only
wavelengths in the region of 450–490 nm to pass. The chlo-
roplast photosynthetic units exhibit red color ꢃuorescence
emission.
5
μm particle size) was used. The mobile phase was
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Photosynthesis Research
Results and discussion
Identiꢀcation of the production of catechol
by cultures of Chlamydomonas reinhardtii grown
in the absence of acetic acid
Samples from cultures of Chlamydomonas reinhardtii cul-
tivated in TP medium were collected after an incubation
period of 10 days. After centrifugation the supernatant from
cultures that had been incubated with 4.0 mM initial phe-
nol concentration had a brownish color, a fact that could be
attributed to the production of one or more metabolites by
algal cells after phenol biotransformation (Fig. 2). No such
color was observed for the samples of the corresponding
cultures in TP medium where phenol was not present. This
macroscopic observation was accompanied by changes in the
chromatographic proꢁle of the samples collected from the
cultures (Fig. 3). More speciꢁcally, chromatographic proꢁles
of culture samples collected at the beginning of the experi-
ments and after a 10-day growth period exhibited signiꢁcant
diꢀerences. The peak that corresponds to phenol (retention
time of 4.73 min) was found to decrease after 10 days of
incubation, while at the same time appearance of three new
peaks with retention time of 2.02, 2.32 and 3.15 min were
observed. These peaks could be attributed to the production
of possible metabolites. A comparison with the chromato-
grams of control cultures and the TAP medium indicated
that the peak with a retention time 1.78 min corresponds
Fig. 3 HPLC–UV proꢁles of a standard phenol sample, b standard
catechol sample and c culture supernatant from the initial (gray line)
and from the 10th (black line) day of the experiment, using methanol:
water: acetic acid (50:49:1) mobile phase with a ꢃow rate of 1.0 mL/
min. The retention time of each peak is shown with the corresponding
number. Peak intensity was measured using the detector’s arbitrary
units
to one or more components of the TAP medium (data not
shown). The peak with a retention time of 3.15 min probably
corresponds to catechol, as shown by a comparison with the
elution proꢁles of a catechol standard under the same HPLC
operating conditions. To clarify these observations, further
analysis with tandem mass spectrometry was carried out.
Figure 4 shows the comparison of the MS/MS spectra of
the peak with m/z 109 of the standard catechol sample, which
−
corresponds to the deprotonated ion of catechol, [M–H] ,
and the ꢁltered supernatant of the sample collected from the
algal cultures after ꢁve days of incubation. The fragmenta-
tion patterns (Fig. 4a, b, respectively) of the two MS/MS
spectra, exhibit similar fragment ions with m/z values of
108, 91, 81, 65, and 53. These fragments are in agreement
Fig. 2 Supernatant and pellet of ten-day old culture samples after
centrifugation at 1000 g for 5 min. The left tube, control culture; right
tube, culture incubated with 4.0 mM initial phenol concentration in
the absence of an alternative carbon source (TP medium)
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Photosynthesis Research
Fig. 4 ESI–MS/MS spectra of the peak with m/z=109 observed
ture sample collected from the after an incubation period of 5 days
in TP medium. Cells were grown in TP medium with an initial phe-
nol concentration of 4.0 mM under continuous light intensity of
−1
in the negative ion mode. a 500 μg L standard catechol sample,
b culture sample collected after a ꢁve-day incubation period in TP
medium with an initial phenol concentration equal to 4.0 mM, c cul-
70–80 μmol photons·m− ·s
2
−1
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Photosynthesis Research
with those of the fragmentation patterns of catechol previ-
ously described in the literature (Binkley et al. 1994). A
diꢀerence between the two MS/MS spectra is that the spec-
trum of the standard catechol sample shows a low intensity
peak with m/z 41, which is not present in the culture sample.
This could be due to the lower concentration of catechol
in the culture sample, compared to the standard catechol
solution; in such a case, ions that are not abundant enough
cannot reach the detection limits. In addition, another dif-
ference between the two mass spectra is the presence of a
fragment with m/z 35 in the culture sample. This peak is
attributed to the fragmentation of a background ion with
m/z value of 109, as shown in the MS/MS spectrum of the
control culture sample from the same experiment (Fig. 4c).
HPLC-ESI-MS/MS analysis was used in order to identify
the presence of catechol in the culture medium. The multiple
reaction monitoring (MRM) method was used for the frag-
mentation reactions of 109 → 108 and 109 → 91, because
of the high abundance of the corresponding ions in the MS/
MS spectra (Fig. 4a, b). Figure 5 shows the chromatographic
elution proꢁles acquired for both the catechol standard and
the culture sample. In all cases only one peak with a reten-
tion time of 6.1 min was observed, which clearly proves the
presence of catechol in the culture medium. Thus, it can be
concluded that catechol is produced by the metabolism of
phenol by Chlamydomonas reinhardtii cells. At this point,
it is important to note that in experiments carried out under
the same conditions in the absence of the cells, neither a
decrease of phenol concentration nor of catechol production
were recorded (data not shown).
Corresponding HPLC-UV proꢁles received at the begin-
ning of the experiment and on the ꢁfth and tenth day of incu-
bation (Fig. 6) clearly show the accumulation of catechol in
the culture medium with a respective decrease of the phenol
peak. The same behavior was also observed for the other two
uncharacterized peaks.
Fig. 6 HPLC–UV proꢁle of the culture supernatant at the beginning
of the experiment (black line), after an incubation period of ꢁve (light
gray line) and ten (dark gray line) days, using the methanol: water:
acetic acid (50:49:1) mobile phase with a ꢃow rate of 1.0 mL/min.
Peak intensity is given in the detector’s arbitrary units. Cells were
grown in TP medium with an initial phenol concentration of 4.0 mM
under continuous light intensity of 70–80 μmol photons·m− ·s
2
−1
Fig. 5 HPLC–ESI–MS/MS chromatographic proꢁles, a mobile phase
c 109→91 and d 109→108 of samples collected from the algal cul-
ture after an incubation period of ꢁve days. Cells were grown in TP
medium with an initial phenol concentration of 4.0 mM under con-
methanol: water: acetic acid (50:49:1) mobile phase at a ꢃow rate of
0
.5 mL/min. Monitoring of the reaction a 109→91 and b 109→108
−1
−2 −1
in a 500 μg L standard catechol sample. Monitoring of the reaction
tinuous light intensity of 70–80 μmol photons·m ·s
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Photosynthesis Research
Impact of the presence or absence of an alternative
carbon source, acetic acid, in the culture medium
on the bioenergetic equilibrium of catechol
production by cultures of Chlamydomonas
reinhardtii
(Fig. 8) emphasizes the importance of an alternative carbon
source in the culture medium. In the absence of acetic acid,
an increased uptake of phenol by Chlamydomonas cells is
observed in the ꢁrst 48 h of incubation. This could be attrib-
uted to the fact that phenol is the only carbon source in the
medium; cells consume phenol in order to synthesize carbon
reserves. Compared to phenol removal rates in the absence
of acetic acid (Fig. 9a), the maximum rate was recorded 48 h
after the initiation of the experiment. Phenol removal was
found to take place in cycles, with the maxima recorded on
days 2, 5, and 8. For every maximum the value of the rate
appears to be lower than the previous one, which indicates
that after each cycle the ability of Chlamydomonas to take
up phenol decreases. An interesting fact is that at the same
time the catechol production rate starts to increase on the
third incubation day and reaches its maximum value on the
sixth experimental day, where phenol removal rate is almost
equal to zero; catechol is produced with an almost steady
rate until the end of the experiment. Phenol consumption
after the sixth day is signiꢁcantly lower compared to the
consumption observed at the beginning of the experiment.
It is interesting to note that the amount of catechol produced
in the medium was approximately 20% of the amount of total
phenol removed. These results show that catechol ring cleav-
age is the most energetically demanding step during phenol
biodegradation by Chlamydomonas cells. This is supported
by the fact that no growth of the cultures was observed
(Fig. 7a) despite the consumption of phenol when an alter-
native carbon source was not present in the medium. Tak-
ing into account previous observations (Nazos et al. 2017),
we conclude that phenol drives microalgal metabolism to
“maintenance”. The observed increase in the peak inten-
sity of the other metabolites during the experiment (Fig. 6)
As shown above, catechol is produced in the culture
medium during phenol biodegradation by Chlamydomonas
reinhardtii. The origin of this result was investigated. We
have already shown that biodegradation of phenol by Chla-
mydomonas reinhardtii is a dynamic bioenergetic process
aꢀected by a set of factors including light intensity, oxygen
availability, presence or absence of an alternative growth-
promoting organic substrate as well as phenol concentration
(
Nazos et al. 2017). Two parallel sets of experiments were
carried out, one in the presence of acetic acid as an extra car-
bon source, apart from phenol, in the culture medium (TAP
medium) and another where phenol was the only carbon
source for Chlamydomonas cells (TP medium). Figure 7a
presents the growth of Chlamydomonas cultures in these
conditions. In the absence of acetic acid, zero to negligible
biomass increase was observed. During experiment, ꢃasks
were sealed with septum and as a result no CO from the
2
atmosphere was getting inside. Cells consumed the CO that
2
existed in the air and this resulted to negligible growth. On
the other hand, in the presence of acetic acid phenol was
found to cause inhibition of microalgal growth when com-
pared to the corresponding cultures grown in TAP medium
without phenol. This result is in agreement with our previous
ꢁ
ndings, which demonstrated that high concentrations of
phenol inhibited algal growth (Nazos et al. 2017).
Removal of phenol and production of catechol in the
absence or presence of acetic acid in the culture medium
Fig. 7 Graphs presenting the growth curves (a) and maximal photo-
synthetic eꢂciency (b) of Chlamydomonas reinhardtii cultures, dur-
ing experiments, grown in TAP and TP medium in the absence and
in the presence of 4.0 mM initial phenol concentration under con-
−2 −1
tinuous light intensity of 70–80 μmol photons·m ·s . Biomass was
expressed in number of cells·(mL culture)−
1
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Photosynthesis Research
Fig. 8 Graphs presenting phenol removal and catechol produc-
tion in the culture medium by Chlamydomonas reinhardtii cultures
a in the absence and b in the presence of acetic acid as an alterna-
tive carbon source, with initial phenol concentration of 4.0 mM.
Experiments were carried out under a continuous light intensity of
−2 −1
70–80 μmol photons·m ·s . Phenol removal and catechol produc-
tion were expressed in μmol·L−
1
Fig. 9 Change in the phenol consumption rate (black squares) and in
the catechol production rate (gray circles) by Chlamydomonas rein-
hardtii cultures incubated with an initial phenol concentration of
4.0 mM, under a light intensity of 70–80 μmol photons·m−2·s−1 in the
absence (a) and in the presence (b) of acetic acid
shows that Chlamydomonas reinhardtii follows a strategy
that has as a main target decreasing the concentration of
phenolic compounds in the culture medium rather than com-
plete oxidation of the other metabolites. Cells perform the
oxidation of phenol to catechol and then release catechol
that they cannot further degrade. As complete metabolism of
phenol is an energetically demanding process, under condi-
tions where phenol is the only carbon source present in the
culture medium, cells biodegrade only the amount of phenol
that is necessary for the maintenance of cellular components.
This hypothesis is supported by the comparative light and
ꢃuorescence microscopy studies (Fig. 10) of control cultures
incubated for ꢁve days in the absence of a carbon source
to exhibit empty spaces that include small vesicles. These
empty spaces are located in chloroplasts, as shown clearly by
ꢃuorescence microscopy images, and could be attributed to
the physiological phenomenon of autophagy. Autophagy is
an intracellular catabolic process that allows cells to recycle
useless or destroyed cellular material for maintenance of
homeostasis (Couso et al. 2018). These mechanisms have
been extensively studied in the literature (He and Klion-
sky 2009; Mizushima et al. 2011; Li and Vierstra 2012;
Liu and Bassham 2012). The process includes formation of
autophagosomes that transfer the “useless” cellular mate-
rial into speciꢁc vacuoles. In the Chlamydomonas genus it
has been observed that this process takes place under dep-
rivation of nutrients such as carbon and nitrogen (Goodson
et al. 2011; Perez-Perez et al. 2012; Goodenough et al. 2014;
Davey et al. 2014). In our case this result is expected if we
(
Fig. 10a, b) and cultures where phenol was the only carbon
source present in the medium (Fig. 10c, d). Compared to the
cells treated with phenol, the control cell cultures were found
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Photosynthesis Research
Fig. 10 Optical (a, c) and ꢃuorescence (b, d) microscopy images of ▸
Chlamydomonas reinhardtii cells. Cells were grown in TP medium
in the absence (a, b) and in the presence (c, d) of 4.0 mM initial
phenol concentration. Samples were collected after an incubation
period of ꢁve days. Cells were grown in TP medium with an initial
phenol concentration of 4.0 mM under continuous light intensity of
0–80 μmol photons·m− ·s
2
−1
7
take into consideration the fact that cells cultivated in TP
medium have remained for many days in the sealed ꢃask
without a supply of a carbon source. On the other hand,
in cultures where phenol was present, the autophagy phe-
nomenon was not observed. If we also take into account
the fact that removal of phenol takes place in the ꢁrst hours
of incubation, we could conclude that the Chlamydomonas
reinhardtii cells biodegrade phenol in order to cover their
carbon requirements for the preservation of their homeosta-
sis and cellular structure. In order to compare cell viability,
cells from all the examined experimental conditions (TAP
and TP in the presence and absence of phenol) were col-
lected 10 days after the end of the experiments, washed
twice with fresh TAP medium and diluted into fresh TAP
medium in order to obtain initial cell concentration equal to
5
−1
4
.5 (± 0.4)·10 cells·mL . Cells were left to grow under a
−
2 −1
light intensity of 70–80 μmol photons·m ·s for 3 days.
After this period cell counts were made. In all cases similar
growth was observed; diꢀerence was less than 5%.
The behavior is diꢀerent in the presence of acetic acid
in the culture medium. In the beginning of the experi-
ment cells take up a small amount of phenol, and no phe-
nol degradation is observed until the ꢁfth day (Fig. 8b).
Signiꢁcant metabolic activity is recorded after that day,
when phenol removal increases continuously until the last
day of the experiment. It is worth mentioning that in that
day, cultures treated with phenol and acetic acid enter the
stationary phase of growth and as a result all available
reserves of acetic acid have been exhausted from the cul-
ture medium. With a double substrate in the medium, cells
choose to biodegrade the growth-promoting substrate (ace-
tic acid) ꢁrst, consistent with the growth of the cultures,
and then degrade the growth-limiting substrate (phenol).
This strategy is followed by microalgae in order to gain
energy reserves. An important observation is that catechol
production in the culture medium was found to be negli-
gible (less than 2% of the phenol removed). The proꢁle of
the rate of phenol removal during the experiment (Fig. 9b)
is signiꢁcantly diꢀerent, when compared to that observed
in the absence of acetic acid (Fig. 9a). After the cells reach
the stationary phase, a maximum rate is observed on the
ꢁ
fth experimental day, while after the sixth experimental
day cells exhibit continuous increase in the rate of phenol
removal. This fact underscores the importance of the pres-
ence of an organic substrate in the culture medium that
provides cells with suꢂcient energy reserves. In addition
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Photosynthesis Research
to these data, measurements of the maximum photosyn-
thetic eꢂciency revealed that the presence of acetic acid
assists microalgae in overcoming stress eꢀects caused by
the presence of phenol in the culture medium. In the pres-
ence of acetic acid, the values of the F /F
parameter
max
v
exhibit a decrease during the ꢁrst 24 h (acute stress caused
by the presence of phenol), while maximal photosynthetic
eꢂciency returns to high levels throughout the incubation
period. In contrast, in the absence of an alternative organic
carbon source, maximal photosynthetic eꢂciency gradu-
ally decreases during the experiment (Fig. 7b).
The results presented here and in a previous publica-
tion of our research group (Nazos et al. 2017), show that
Chlamydomonas reinhardtii cells adapt their metabolism
in order to achieve an optimal bioenergetic equilibrium
between growth, stress and phenol biodegradation. This
equilibrium is aꢀected signiꢁcantly by the production of
catechol and the presence or absence of a substrate for
energy production in the culture medium.
Conclusion
The results of the experiments reported here provide fur-
ther evidence for the hypothesis that biodegradation of
phenol by Chlamydomonas reinhardtii is a dynamic bio-
energetic process by focusing on regulation of the bio-
energetic equilibrium by the production of catechol by
the microalgae. Figure 11 summarizes schematically the
proposed bioenergetic model of the present study’s ꢁnd-
ings. When microalgae lack suꢂcient energy reserves,
phenol oxidation to catechol was found to take place; the
amount of catechol that could not be further biodegraded
was released into the culture medium. In the presence of
acetic acid, the amount of catechol found in the medium,
compared to the amount of phenol consumed, was negli-
gible. Phenol is a growth-limiting substrate that requires
signiꢁcant amounts of energy to be biodegraded. It was
biodegraded only when Chlamydomonas cells required
carbon reserves for the maintenance of their homeostasis.
Chlamydomonas reinhardtii was proven to act as a versa-
tile bioenergetic machine by regulating its metabolism to
achieve the best balance between growth and biodegrada-
tion of phenol. These data extend previous ꢁndings and
provide new unique metabolic and bioenergetic insights
in the biodegradation of phenol by microalgae.
Fig. 11 Proposed bioenergetic models of phenol biodegradation by
Chlamydomonas reinhardtii. a In the absence of acetic acid in the
growth medium, phenol uptake leads to pronounced stress in Chla-
mydomonas cultures that lasts throughout the experimental period.
No growth of the cultures is observed and approximately 20% of the
consumed phenol is released as catechol into the culture medium. b
In the presence of acetic acid growth of Chlamydomonas cultures
is observed and the acute stress caused by the presence of phenol
is decreased throughout the experiment. The amounts of catechol
released in the culture medium are less than 2% of the total phenol
amount consumed by Chlamydomonas cells
Compliance with ethical standards
Conflict of interest The authors declare that they have no conꢃict of
interest.
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