FEMS Microbiology Letters, 362, 2015, fnv072

doi: 10.1093/femsle/fnv072
Advance Access Publication Date: 29 April 2015
Research Letter

R E S E A R C H L E T T E R – Physiology & Biochemistry

Laccase-like enzyme activities from chlorophycean

green algae with potential for bioconversion
of phenolic pollutants
Benjamin Otto1,2,∗ , Carl Beuchel3 , Christiane Liers4 , Werner Reisser1 ,
Hauke Harms2 and Dietmar Schlosser2
1
Institute of Biology, General and Applied Botany, University of Leipzig, 04103 Leipzig, Germany, 2 Department
of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, 04318 Leipzig, Germany,
3
Faculty of Natural Sciences III, Institute of Agricultural and Nutritional Science, Martin Luther University of
Halle-Wittenberg, 06099 Halle (Saale), Germany and 4 Department of Environmental Biotechnology, TU
Dresden, International Institute Zittau, Markt 23, 02763 Zittau, Germany
∗ Corresponding author: Department of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, 04318 Leipzig, Germany.
Tel: +49-341-235-1329; Fax: +49-341-235-2247; E-mail: benjamin.otto@t-online.de
One sentence summary: In chlorophycean algae, laccases are prevalent within the Moewusinia group and may contribute to the breakdown of
environmental contaminants like triclosan and 17α-ethinylestradiol.
Editor: Claire Remacle

ABSTRACT
In order to explore the abundance and potential environmental functions of green algal laccases, we screened various algae
for extracellular laccase-like activities, characterized basic features of these activities in selected species and exemplarily
studied the transformation of environmental pollutants and complex natural compounds by the laccase of Tetracystis aeria.
Oxidation of the classical laccase substrate ABTS was found to be widespread in chlorophycean algae. The oxidation
activity detected in members of the ‘Scenedesmus’ clade was caused by an unknown thermostable low-molecular-mass
compound. In contrast, species of the Moewusinia, including Chlamydomonas moewusii and T. aeria, excreted putative ‘true’
laccases. Phenolic substrates were oxidized by these enzymes optimally at neutral to alkaline pH. The Tetracystis laccase
efficiently transformed bisphenol A, 17α-ethinylestradiol, nonylphenol and triclosan in the presence of ABTS as redox
mediator, while anthracene, veratrylalcohol and adlerol were not attacked. Lignosulfonate and humic acid underwent
slight (de)polymerization reactions in the presence of the laccase and mediator(s), probably involving the oxidation of
phenolic constituents. Possible natural functions of the enzymes, such as the synthesis of complex polymers or
detoxification processes, may assist the survival of the algae in adverse environments. In contaminated surface waters,
laccase-producing green algae might contribute to the environmental breakdown of phenolic pollutants.

Keywords: phenol oxidases; microalgae; biotransformation; aromatics; xenobiotics; phycosphere

Received: 17 February 2015; Accepted: 26 April 2015


C FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

1
 2 FEMS Microbiology Letters, 2015, Vol. 362, No. 11
INTRODUCTION
While being traditionally recognized as important primary pro-
ducers, the abilities of green algae to attack recalcitrant environ-
mental pollutants and complex natural compounds become in-
creasingly apparent (Semple, Cain and Schmidt 1999; Ghasemi,
Rasoul-Amini and Fotooh-Abadi 2011; Subashchandrabose et al.
2013). For instance, micropollutants like the xenoestrogen 17α-
ethinylestradiol (EE2) can be biotransformed by green microal-
gae (Della Greca et al. 2008).
Laccases (EC 1.10.3.2, benzenediol: O2 oxidoreductases) are
extracellular multicopper oxidases involved in the breakdown
of recalcitrant aromatic compounds, which combine the one-
electron oxidation of their substrates with the reduction of oxy-
gen to water (Giardina et al. 2010). Hitherto only one laccase
has been purified and characterized in a green alga (Otto and
Schlosser 2014), and genes encoding algal laccases were just re-
cently identified (Zhao et al. 2013). Possible environmental func-
tions and the abundance of green algal laccases are virtually
unknown. Like for fungal laccases, the decolorization of recal-
citrant synthetic dyes in the presence of redox-mediating com-
pounds was demonstrated for the laccase from the green soil
alga Tetracystis aeria (Otto and Schlosser 2014). Several other
green algae were shown to oxidize typical laccase substrates
(La Russa 2009; Chiaiese et al. 2011; Kılıç et al. 2011), but the na-
ture of the potentially laccase-catalyzed reactions was not fur-
ther studied.
In this study, we assessed the prevalence of laccase-like ac-
tivities by screening a range of chlorophycean algae from differ-
ent phylogenetic groups. Next, we characterized basic features
of these activities in selected species. To proceed toward under-
standing the possible functions and eco-physiological impacts
of laccases from green algae, we furthermore studied the abil-
ity of the T. aeria laccase to oxidize recalcitrant xenobiotic and
natural compounds, covering environmental pollutants, lignin
model compounds and complex macromolecules such as lignin
and humic acid.
MATERIALS AND METHODS
Screening of chlorophycean green algae
for ABTS-oxidizing activities
Algal strains given in Table S1 (‘Supplementary material’) were
obtained axenically from the ‘Culture Collection of Algae at
the University of Göttingen’ (SAG; Germany). Algae were plated
onto 1.6% agar plates with Bold’s Basal Medium containing
vitamins (Starr and Zeikus 1993), 20 μM CuSO4 as laccase in-
ducer (Otto, Schlosser and Reisser 2010) and 1 mM 2,2-azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; 98% purity;
Sigma; Deisenhofen, Germany). Agar media for Dunaliella parva
and Dunaliella salina additionally contained 1 M NaCl. Plates
were incubated for 8 weeks employing a light:dark cycle of
14:10 h under light intensities of 40 μmol photons m−2 s−1 at
20◦ C. The appearance of a blue-greenish coloration of the agar
around algal colonies indicated extracellular oxidation of ABTS.
Experiments were done in duplicates.
Phylogenetic tree reconstruction
A cladogram of the screened species was constructed based on
a comprehensive maximum-likelihood (ML) phylogenetic tree
(Fig. S1 in ‘Supplementary material’). The tree was inferred by
using a published alignment of partial 18S rRNA gene sequences
comprising 317 chlamydomonadal algae (Nakada, Misawa and
Nozaki 2008), which was complemented by further including
and manually aligning publicly available 18S rRNA sequences
(GenBank; Benson et al. 2013) of 45 chlorophycean algae (the
alignment is available upon request). ML inference was done us-
ing RAxML 8.0.24 (Stamatakis 2014) as available on the CIPRES
Portal (Miller, Pfeiffer and Schwartz 2010), employing 100 rapid
bootstrap inferences followed by a thorough ML search accord-
ing to the GTR +  substitution model. Sequence and tree editing
was done using MEGA 6.0 (Tamura et al. 2013). The algal strains
used for the ABTS-oxidizing activity screening were allocated to
their phylogenetic positions using the 18S rRNA gene sequences
of the respective strains where available, or of closely related
strains in some cases, as detailed in Table S1 (‘Supplementary
material’).
Characteristics of oxidative activities in selected algae
Chlamydomonas moewusii (SAG 11–11), T. aeria (SAG 89.80) and
Scenedesmus vacuolatus (SAG 211–8b) were grown in Bold’s Basal
Medium containing 20 μM CuSO4 (Otto, Schlosser and Reisser
2010). Cultures were filtered (Whatman No. 1 filter paper; GE
Healthcare; Little Chalfont, UK) to obtain cell-free culture super-
natants after 3, 6 and 1 week of cultivation, respectively. Proteins
in culture supernatants were concentrated and exchanged to 25
mM Tris–acetate (pH 9.0) by ultrafiltration on a 10 kDa cut-off
Omega polysulfone membrane using a 150 mL stirred cell (Pall
Life Sciences; Dreieich, Germany). ABTS-oxidizing activities and
pH activity profiles for the common laccase substrates ABTS, 2,6-
dimethoxyphenol (99% purity; Aldrich; St. Louis/MO, USA) and
syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine;
≥98% purity; Sigma), which is considered to be a unique lac-
case substrate (Harkin, Larsen and Obst 1974), were recorded
as previously described (Otto and Schlosser 2014). ABTS oxida-
tion was assayed before and after heat inactivation (100◦ C for
30 min) and in the presence of 0.2 mM H2 O2 , 1000 U mL−1 cata-
lase (from bovine liver; Fluka; Buchs, Switzerland) or 50 μM
NaN3 . Statistical comparisons were done by the two-sided Dun-
nett’s test (α = 0.05), following Levene’s test and a one-way
ANOVA using SigmaPlot 11 (Systat Software; San Jose/CA, USA);
data of C. moewusii were square-root transformed to establish
equality of variances.
Biotransformation of recalcitrant compounds
by T. aeria laccase
Experiments were carried out in 0.1 M citrate–0.2 M phosphate
buffer (pH 6.5). The compounds bisphenol A (BPA; 98.5% pu-
rity; Dr Ehrenstorfer GmbH; Augsburg, Germany), nonylphe-
nol (technical mixture; 85% purity; Fluka), EE2 (98% purity;
Sigma), triclosan (99.8% purity; Calbiochem; San Diego/CA,
USA), adlerol [1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-
1,3-propanediol; synthesized as described by Hatakka et al.
(1991)] [using 25 mM methanolic stock solutions containing 10%
(w/v) Tween 80] and veratrylalcohol (96% purity, Aldrich; using
a 25 mM aqueous stock solution) were added at 0.25 mM. An-
thracene (p.a.; Merck; Darmstadt, Germany) (using a 10 mM ace-
tonic stock solution) was added at 100 μM; the solubility was in-
creased by including 1% (w/v) Tween 80 in the reaction mixture
(Johannes, Majcherczyk and Hüttermann 1996). Concentrated
culture supernatants of T. aeria had been obtained as described
earlier (Otto and Schlosser 2014) and were added to give laccase
activities of 0.1 U mL−1 . Heat-inactivated samples (100◦ C for 15
min) were used as controls. Where indicated, the redox medi-
ators ABTS or syringaldehyde (SGD; 98% purity; Aldrich) were

added at 0.5 mM. Reaction mixtures of 0.1 mL were placed into
HPLC vial–glass inserts (0.2 mL) for direct analysis at the indi-
cated incubation times. During incubation, samples were kept
sealed on a horizontal shaker (120 rpm) at room temperature.
Lignosulfonate (Na–salt; ≥93% purity; Carl Roth; Karlsruhe,
Germany) and humic acid (depur.; Carl Roth) (0.5 mg mL−1 ; from
25 mg mL−1 stock solutions in H2 Obidest and 0.1 M NaOH, respec-
tively) were treated as described above, but using reaction vol-
umes of 0.2 mL. Incubation over 3 days was carried out as de-
scribed above. All experiments were done in triplicate.
Chromatographic analysis of recalcitrant compounds
Enzymatic conversion of BPA, nonylphenol, EE2, triclosan,
adlerol, veratrylalcohol and anthracene was determined by ul-
tra performance liquid chromatography (UPLC) using an Acquity
UPLC system (Waters; Eschborn, Germany) equipped with a BEH
C18 column (2.1 × 50 mm, 1.7 μm particle size; Waters) as de-
scribed previously (Jahangiri et al. 2014). Samples of 3.3 μL were
directly injected from the incubation vials. As a modification, ve-
ratrylalcohol in the presence of SGD was measured by using an
isocratic flow of 19% (v/v) methanol, which had been adjusted
to pH 3 with phosphoric acid. Elution was monitored at 250 (an-
thracene) and 278 nm (all other compounds). Compound con-
centrations were calculated based on calibration curves estab-
lished with external standards.
Samples containing lignosulfonate and humic acid were an-
alyzed by size exclusion chromatography using a D-7000 HPLC
system (Merck/Hitachi; Darmstadt, Germany; Tokyo, Japan)
equipped with a HEMA MCX 1.000 Å column (PSS GmbH; Mainz,
Germany) as previously described (Singh, Harms and Schlosser
2014). Chromatograms were recorded at a wavelength of 250 nm
within a UV-Vis range of 220–600 nm. Calibration was done us-
ing polystyrene sulfonate sodium salt molecular mass standards
(PSS GmbH).
RESULTS AND DISCUSSION
Occurrence of ABTS-oxidizing activities
in chlorophycean green algae
A range of green algae of the class Chlorophyceae were screened
for extracellular oxidation of ABTS. Out of 50 tested species,
20 unambiguously oxidized ABTS (Fig. 1). Species were selected
with a certain emphasis on the group Moewusinia (sensu Nakada,
Misawa and Nozaki 2008) also comprising the laccase-producing
T. aeria. All of the 12 Moewusinia species oxidized ABTS, with
10 of them showing strong or very strong oxidation inten-
sities (Fig. 1). ABTS oxidation was also detected in all four
species of the ‘Scenedesmus’ clade (sensu Pröschold et al. 2001).
Other ABTS-oxidizing algae belonged to the Stephanosphaerinia,
Oogamochlamydinia (though ambiguous) and Chloromonadinia (all
groups sensu Nakada, Misawa and Nozaki 2008) (Fig. 1).
Oxidation of ABTS, 2,6-dimethoxyphenol and/or syringal-
dazine had previously been detected in Chlamydomonas
pitschmannii, S. vacuolatus and Scenedesmus ovalternus (La Russa
2009; Chiaiese et al. 2011) and is confirmed by our results (Fig. 1).
ABTS oxidation was also reported in Ankistrodesmus braunii
(Selenastraceae; Chiaiese et al. 2011) and Gonium sp. (Reinhardtinia;
Kılıç et al. 2011). Laccase-encoding genes were demonstrated for
Chlamydomonas reinhardtii and Volvox carteri (Reinhardtinia; Zhao
et al. 2013), while no extracellular enzyme activity was detected
in the latter species or in other members of the Selenastraceae
and Reinhardtinia in our study (Fig. 1). Possibly, laccase genes are
Otto et al. 3
not expressed or the enzymes are not released extracellularly
in substantial amounts under the employed culture conditions.
Since the ABTS-oxidation reaction proceeds rather slowly at
neutral pH (Fig. 2) also applied in the agar plate growth medium,
low oxidation activity might possibly not have been detected in
some species. However, the very strong ABTS-oxidizing activity
detected for several species in the screening (Fig. 1) indicates
that substantial activities were clearly detectable with this
approach.
Since ABTS oxidation apparently is prevalent within the
Moewusinia and the ‘Scenedesmus’ clade, the oxidative activi-
ties were further characterized in the corresponding represen-
tatives C. moewusii and S. vacuolatus. Tetracystis aeria was in-
cluded for comparison. After 3, 6 and 1 week of liquid cultivation,
ABTS-oxidizing activities of 27, 18 and 16 U L−1 were obtained
in C. moewusii, T. aeria and S. vacuolatus culture supernatants,
respectively.
Putative laccases in the Moewusinia group
The ABTS-oxidizing activities of cell-free culture supernatants
from C. moewusii and T. aeria were successfully concentrated dur-
ing ultrafiltration (Table 1). The activity of these concentrates did
not significantly change in the presence of catalase and the ad-
dition of hydrogen peroxide caused no significant changes for
T. aeria and a slight inhibition for C. moewusii (Table 1), indi-
cating the absence of substantial peroxidase activities (Edens
et al. 1999). In the presence of sodium azide, a potent inhibitor
of metal-containing enzymes (Wood 1980), ABTS oxidation by
C. moewusii and T. aeria supernatants markedly decreased to
≤40%. Activity fully disappeared upon boiling, as expected for an
enzyme.
Culture supernatants of C. moewusii and T. aeria displayed
an increasing oxidation of ABTS with decreasing pH and bell-
shaped pH profiles toward the unique laccase substrate sy-
ringaldazine (Harkin, Larsen and Obst 1974) with optima at pH
7.0 (Fig. 2a and b). Neutral optima were also obtained for 2,6-
dimethoxyphenol oxidation by C. moewusii, while the T. aeria su-
pernatant displayed two optima at pH 7.0 and 9.0 (Fig. 2a and b),
which resembles previous findings (Otto, Schlosser and Reisser
2010; Otto and Schlosser 2014). Altogether, the features of the
oxidative activities of both algae are in favor of an enzymatic
reaction characteristic of laccase (Baldrian 2006).
The Moewusinia group comprises unicellular flagellates
(Chlamydomonas spp.) as well as single-celled and tetrad-forming
coccoids (Chlorococcum spp., Tetracystis spp.). Tetracystis aeria may
utilize exogenous carbon sources heterotrophically (Brown and
Bold 1964), while C. moewusii is an obligate autotroph (Lewin
1950). Both terrestrial and aquatic algae are among the ABTS-
oxidizing species (Fig. 1). A remarkable number of Moewusinia
species thrive in harsh environments. For instance, among the
species tested in this study, C. pitschmannii and Chlorococcum
elkhartiense are acidophilic (Pollio et al. 2005) and Tetracystis isobi-
lateralis is highly desiccation tolerant (Trainor and Gladych 1995).
Non-enzymatic oxidants in the ‘Scenedesmus’ clade
Upon ultrafiltration of S. vacuolatus culture supernatant, ABTS-
oxidizing activity was not concentrated and identical activi-
ties were detected in both the retentate and the flow-through
(Table 1), indicating a low-molecular-mass oxidizing agent. Ac-
cordingly, crude (i.e. non-concentrated) supernatants were used
to further characterize the oxidants. Catalase caused a cer-
tain decrease in activity, possibly indicating the involvement of
 4 FEMS Microbiology Letters, 2015, Vol. 362, No. 11

Figure 1. Extracellular ABTS-oxidizing activities in chlorophycean green algae. The general habitats of the algae (according to the information provided by the SAG) and
the maximum intensities of ABTS oxidation (as inferred from the appearance of a blue-greenish coloration on agar plates) are shown. Species where ABTS oxidation
was present or absent are shown in blue and red, respectively. Species are assigned to a cladogram showing the major groups of chlorophycean algae (sensu Pröschold
et al. 2001; Lewis and McCourt 2004; Nakada, Misawa and Nozaki 2008; Krienitz et al. 2011). The presence of putative ‘true’ laccases and thermostable oxidants within
the Moewusinia (highlighted in green) and the ‘Scenedesmus’ clade (highlighted in yellow), respectively, was inferred from further studies on representative species
(C. moewusii, T. aeria and S. vacuolatus; shown in bold). Details on the algal strains used for the screening and an ML tree used for construction of the cladogram are
given in the ‘Supplementary material’ (Table S1; Fig. S1). The tree is rooted with Chlorella vulgaris and Tetraselmis convolutae as outgroup (not shown; see Fig. S1). 1 –
Caudivolvoxa; 2 – Xenovolvoxa; 3 – Chlamydomonadales; 4 – Sphaeropleales. ∗ Poor growth. a Habitats: S – Soil, F – Freshwater, M – Marine, E – Epiphyte (bark). b ABTS-oxidation
intensities were visually assigned to a scale of 0 (no oxidation) to 5 (very strong oxidation); two values are given in the case of differing intensities among duplicates.
c
ABTS oxidation was ambiguous, as only observed in one of the duplicate plates.

hydrogen peroxide in the reaction. Contrariwise, hydrogen per-
oxide (0.2 mM) addition caused a certain inhibition of ABTS ox-
idation. Activity was only slightly affected in the presence of
sodium azide, possibly due to chemical reactions of azide with
electrophilic compounds (Sugumaran 1995). ABTS oxidation re-
mained completely unchanged during extensive boiling of the
S. vacuolatus culture supernatant (Table 1), strongly suggesting a
non-enzymatic oxidant.
The pH activity profiles of S. vacuolatus culture supernatant
(Fig. 2c) differed remarkably from the supernatants of the other
algae tested (Fig. 2a and b). ABTS and 2,6-dimethoxyphenol oxi-
dation sharply increased with decreasing pH within a strongly
acidic range below pH 4.0 (Fig. 2c), again clearly indicating a
non-enzymatic oxidant. Syringaldazine oxidation was negligible
(Fig. 2c).
Thus, the oxidative activity of S. vacuolatus is obviously not
(directly) caused by an enzyme, but rather by a thermostable
low-molecular-mass compound. Non-enzymatic oxidation reac-
tions may, e.g., result from interactions of stabilized redox-active
transition metals with reactive oxygen species (Euler and Bolin
1909; Gómez-Toribio et al. 2009; Jeon et al. 2012).
Biotransformation of recalcitrant compounds
by T. aeria laccase
When released into the environment, laccases might poten-
tially attack a broad range of natural, but also recalcitrant xeno-
biotic compounds. The substrate spectrum of green algal lac-
cases was studied in order to further explore their possible nat-
ural functions and their potential relevance for bioremediation.
 Otto et al. 5

Table 1. ABTS-oxidizing activities of culture supernatants from se-

lected green algae after different treatments. Culture supernatants
were subjected to ultrafiltration to test for the retention of the oxi-
dizing agents on a 10 kDa cut-off membrane. Thereafter, the ABTS
oxidation of concentrated (C. moewusii and T. aeria) and crude super-
natants (S. vacuolatus) was measured in 0.1 M citrate–0.2 M phosphate
buffer (pH 4.0) under varied conditions.

Treatment C. moewusii T. aeria S. vacuolatus

Retention (10K) Yes Yes No

ABTS oxidation (%)a
Control 100 ±3 100 ±6 100 ±4
+ H2 O2 83b ±1 102 ± 10 90b ±4
+ Catalase 92 ±9 101 ±1 71b ±2
+ NaN3 40b ±1 28b ±3 75b ±1
Boiling 3b ±1 0b ±1 98 ±4

a
Data represent means ± SD from triplicate determinations. 100% (controls) refer
to absolute values of 170 (C. moewusii), 130 (T. aeria) and 10 U L−1 (S. vacuolatus).
b
Significantly different from the corresponding control values according to two-
sided Dunnett’s test (P < 0.05).

complete conversion of EE2, nonylphenol and triclosan within

Figure 2. pH activity profiles of culture supernatants from selected algae toward
selected substrates. Culture supernatants of C. moewusii (a) and T. aeria (b) were
concentrated by ultrafiltration. Culture supernatant of S. vacuolatus (c) was ap-
plied non-concentrated. 100% of ABTS, 2,6-dimethoxyphenol (2,6-DMP) and sy-
ringaldazine (SGZ) oxidation activities refer to absolute values of 380, 12 and
5.0 U L−1 (C. moewusii), 2000, 85 and 29 U L−1 (T. aeria) and 510, 280 and 1.0 (set
arbitrarily) U L−1 (S. vacuolatus), respectively. Data represent means ± SD from
triplicate determinations.
Tetracystis aeria was taken as representative for laccase-
producing green algae of the Moewusinia group. Since no other
phenol oxidase or peroxidase is detectable in the culture super-
natants of this alga (Otto, Schlosser and Reisser 2010; Otto and
Schlosser 2014), the observed catalytic activities can essentially
be attributed to the laccase.
BPA was readily attacked by the enzyme with and without
ABTS as redox mediator (100 and 44% transformation within
3 days, respectively; Fig. 3a). The presence of ABTS also led to
at most one day (Fig. 3a–d). These observations demonstrate the
potential of green algal laccases to contribute to the breakdown
of endocrine-disrupting chemicals.
The lignin-related phenolic SGD was less effective than ABTS
as a redox mediator toward BPA and EE2 (Fig. 3a and b) and not
(or only marginally) effective toward the other substrates tested
(Fig. 3c–g). This is consistent with previous findings, where ABTS
was clearly more efficient than SGD in mediating the decoloriza-
tion of two out of three synthetic dyes by the algal laccase (Otto
and Schlosser 2014).
A formation of highly colored products within the first hour
of incubation was observed in reactions containing ABTS in
addition to BPA, EE2 (purple coloration) or nonylphenol (gray-
ish coloration), probably due to the formation of oligo- or poly-
mers involving both oxidized substrates and redox mediators
(Mikolasch and Schauer 2009). When analyzed by liquid chro-
matography (see ‘Supplementary material’), UV-Vis spectra of
the products in all these cases showed three distinct absorbance
maxima, which obviously indicate aromatics (around 300 nm),
ABTS oxidation (420–430 nm) and coupling adducts (540–580 nm)
(Osman, Wong and Fernyhough 2006; Farnet et al. 2011).
No transformation of anthracene or the lignin model com-
pounds veratrylalcohol and adlerol was detected, not even in the
presence of ABTS (Fig. 3e–g). Generally, these non-phenolic aro-
matics can only be attacked by high redox potential enzymes,
mainly laccases or peroxidases of wood-rotting fungi (Li, Xu and
Eriksson 1999; Liers et al. 2010). The nature of the mediator, the
redox potential and the stability of the laccase are crucial for
the efficiency of such reactions (Call and Mücke 1997; Li, Xu and
Eriksson 1999).
The treatment of lignosulfonate and humic acid by the al-
gal laccases was followed by size exclusion chromatography
(Fig. 4a–e). For lignosulfonate, a chromatographic peak of high
molecular mass of ∼33 kDa slightly increased to ∼35 kDa, and
a peak of lower molecular mass of ∼5.1 kDa decreased to ∼4.7–
4.5 kDa in the presence of the redox mediators ABTS and SGD
(Fig. 4a), indicating a certain degree of concomitant polymeriza-
tion and depolymerization. Humic acid mainly displayed a low
molecular mass fraction of ∼1.1 kDa, shifting to ∼1.5 kDa in the
presence of ABTS (Fig. 4b). SGD showed no effect (Fig. 4b), indi-
cating polymerization reactions only in the presence of ABTS.
Concurrently, spectral changes of the eluted fractions appeared
 6 FEMS Microbiology Letters, 2015, Vol. 362, No. 11

Figure 3. Transformation of recalcitrant compounds by T. aeria laccase. BPA (a), EE2 (b), nonylphenol (c), triclosan (d), anthracene (e), veratrylalcohol (f) and adlerol
(g) were incubated with concentrated culture supernatant (0.1 U mL−1 laccase activity) in 0.1 M citrate–0.2 M phosphate buffer (pH 6.5). The redox mediators ABTS
and SGD were included at 0.5 mM. Heat-inactivated samples served as controls. Compound concentrations were determined by UPLC. Data show means ± SD from
triplicate determinations.

Figure 4. Transformation of lignosulfonate (a) and humic acid (b) by T. aeria laccase, as analyzed by size exclusion chromatography. The compounds (0.5 mg mL−1 ) were
treated with concentrated culture supernatant (0.1 U mL−1 laccase activity) in 0.1 M citrate–0.2 M phosphate buffer (pH 6.5) for 3 days. The redox mediators ABTS and
SGD were included at 0.5 mM. Heat-inactivated samples served as controls. Absorbances (250 nm) are averaged from triplicate determinations. The corresponding
apparent molecular masses are indicated. Representative UV-Vis spectra are derived from the 33–35 kDa (c) and 4.5–5.1 kDa (d) peaks of lignosulfonate and 1.1–1.5 kDa
peaks of humic acid (e); absorbances are normalized.

for the reactions with humic acid and ABTS (Fig. 4b) and with
lignosulfonate and both mediators (Fig. 4a). Shifts of distinct
absorbance maxima toward more unspecific spectra and addi-
tional peaks or shoulders around 550 nm (Fig. 4c–e) indicate a
polymeric character of the formed products (Liers et al. 2011).
Apparently, lignin-related compounds and humic sub-
stances can be modified to some extent by algal laccase in the
presence of redox mediators. In accordance with the inability
to oxidize non-phenolic lignin model compounds, oxidations
of phenolic structures of lignin or other aromatic compounds
 Otto et al. 7

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