ISSN 0026-2617, Microbiology, 2018, Vol. 87, No. 3, pp. 308–316. © Pleiades Publishing, Ltd., 2018.

Original Russian Text © A.G. Zavarzina, T.A. Semenova, O.V. Belova, A.V. Lisov, A.A. Leontievskii, A.E. Ivanova, 2018, published in Mikrobiologiya, 2018, Vol. 87, No. 3.

EXPERIMENTAL ARTICLES

Laccase Production and Humic Acids Decomposition

by Microscopic Soil Fungi
A. G. Zavarzinaa, b, *, T. A. Semenovac, O. V. Belovad, A. V. Lisova, d, e,
A. A. Leontievskiid, e, and A. E. Ivanovaa
aDepartment
of Soil Sciences, Lomonosov Moscow State University, Moscow, Russia
b
Paleontological Institute, Russian Academy of Sciences, Moscow, Russia
c
Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia
d
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia
e
Pushchino State Institute of Natural Sciences, Pushchino, Russia
*e-mail: zavarzina@mail.ru
Received October 19, 2017

Abstract—Laccase (para-diphenol:oxygen oxidoreductase, EC 1.10.3.2) is a phenol oxidase widespread in

fungi and bacteria. In basidiomycetes, this enzyme is involved in the transformation of lignin and humic sub-
stances (HS) in soil. The role of laccases of soil ascomycetes and deuteromycetes in HS degradation is not
established, and conditions of the enzyme production have been poorly studied. In the present work soil
micromycetes, potential laccase producers, were isolated from typical soils of the forest, steppe, and forest-
steppe zones of European Russia by plating on agar media with ABTS (2,2'-azino-bis(3-ethylbenzothiazo-
line-6-sulphonic acid, sodium salt)) as the substrate. Their abundance, species composition, conditions of
laccase production, and its relation to humic acids (HA) degradation in liquid and solid media were studied.
Out of 68 strains isolated, 20 exhibited ABTS oxidation at initial plating on agar media. In pure cultures on
agar media, oxidation was less pronounced, but in the presence of HA laccase production by some strains was
higher than without HA. Significant and weak extracellular laccase production in liquid medium was
observed for Acremonium murorum (Corda) W. Gams Z1710 and Botritis cinerea Pers. ex Fries Z1711, respec-
tively. The level of laccase production by A. murorum was the same without inducers and in the presence of
HA, while B. cinerea produced laccase only without inducers. No direct correlation was found between the
presence of laccase and/or its activity and ability of the fungi to decolorize (degrade) HA. In liquid media
active laccase producer A. murorum caused lower HA decolorization (43%) than B. cinerea (62%) and the
fungi lacking extracellular laccase (54–81%). The role of micromycete oxidative systems in HA degradation
requires further investigation.

Keywords: micromycetes, laccase, humic substances, humification, carbon cycle

DOI: 10.1134/S0026261718030153

Dark-colored humic substances (HS) and their
organomineral compounds make up 60–80% of soil
organic matter (Orlov, 1990), which is the largest res-
ervoir of organic carbon in the biosphere. The pro-
cesses of HS formation and destruction largely deter-
mine CO2 flows between the soil and the atmosphere
and affect the global climate; however, the nature and
biochemical stability of these compounds and the role
of individual groups of soil microorganisms in their
transformation remain uncertain.
It is considered (Grinhut et al., 2007) that the main
agents for destruction of HS, as well as of lignin, are
white rot basidiomycetes and ligninolytic litter sapro-
trophs producing high redox potential peroxidases,
with manganese-dependent peroxidase (EC 1.11.1.13)
being the most widespread (Hofrichter, 2002). The
ligninolytic complex of basidiomycetes also includes a
phenol oxidase laccase (para-diphenol:oxygen
oxidoreductase, EC 1.10.3.2). The enzyme catalyzes
the oxidation of phenolic substrates and aromatic
amines by molecular oxygen with the formation of
phenoxy radicals and quinones, which initiate sponta-
neous reactions including the cleavage of C–C bonds
in aromatic substrates, their demethoxylation,
demethylation, and polymerization, as well as forma-
tion of reactive oxygen species (Thurston, 1994).
Being involved in depolymerization/polymerization
reactions, laccase can be considered a universal
enzyme for humification. In contrast to ligninolytic
peroxidases, which are peculiar solely to white rot
basidiomycetes and related litter fungi, laccase is also
produced by ascomycetes, deuteromycetes (Baldrian,
2006) and bacteria (Alexander and Zhulin, 2000),
being the most widespread oxidative enzyme in soils

308
 LACCASE PRODUCTION AND HUMIC ACIDS DECOMPOSITION
(Burns et al., 2013). At the same time, its role in
humus transformation is yet unclear, especially in the
case of the enzyme of micromycetes.
Micromycetes (deuteromycetes and ascomycetes)
are the key components of the cellulolytic microbial
consortium in soils. In contrast to basidiomycetes,
their with the hyphae concentrated mainly in the litter
(Osono, 2007), micromycetes are also widespread in
the humus horizon and underlying layers (Mirchink,
1988), which is indicative of their potential involve-
ment in the utilization of humus. Indeed, the ability of
micromycetes to partially oxidize and mineralize
humic substances has been shown in a number of
works (Gramss, 1999; Rezacova et al., 2006). How-
ever, the role of laccases in HS transformation and the
conditions of production of this enzyme in ascomyce-
tes have been insufficiently studied.
The goals of the present work were (1) to determine
the abundance and species composition of micromy-
cetes–potential laccase producers–in the typical soils
of the forest, forest steppe and steppe zones of the
European Russia; (2) to study the conditions of lac-
case production in isolated strains and their ability to
degrade humic substances.
MATERIALS AND METHODS
Soils. Micromycetes were isolated from the sam-
ples of sod-podzolic soil (SPS), Moscow
oblast, Abramtsevo, spruce forest with dead cover
(56.226819° N, 37.951602° E); gray forest soil (GF),
Moscow oblast, Pushchino, grassy herbal meadow
(54.835180° N; 37.575512° E) and grassy broadleaf
forest with dead cover (54.835061° N; 37.572558° E);
typical chernozem (TC), Kursk oblast, Bolshoye Sol-
datskoye settlement, gramineous-herb meadow and
grassy broadleaf forest with dead cover (51.330888° N,
35.51146° E); and leached chernozem (LC), Lipetsk
oblast, Polibino settlement, meadow-gramineous
motley grass. The carbon and nitrogen contents in the
soils were measured with a CNH analyzer (Karlo-
Erba, Italy); the soil pH value was determined in the
water extracts at a soil/solution ratio of 1 : 2.5.
Humic acids. The experiments on humic acid deg-
radation were performed with a HA preparation
obtained by extraction with 0.1 M NaOH from the A1
horizon of sod-podzol soil. The high-ash fractions
were removed by salting-out with 0.4 M NaCl, fol-
lowed by re-precipitation of the substances of the
supernatant with HСlconc. The ash content in the puri-
fied preparation was 9%.
Isolation of micromycetes, potential laccase pro-
ducers, was performed by inoculating the material of
water extracts from fresh soils (the soil/solution ratio
of 1 : 10) on an acidified Czapek agar medium
(pH 5.0) containing the following (g/L): sucrose, 20;
NaNO3, 2; K2HPO4 · 3H2O, 1; MgSO4 · 7H2O, 0.5;
KCl, 0.5; FeSO4 · 7H2O, 0.01; agar, 20; and 0.25 g/L
MICROBIOLOGY Vol. 87 No. 3 2018
309
of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid, sodium salt)) as a laccase substrate
(Steffen et al., 2002). Czapek medium was autoclaved
for 30 min at 1 atm (Zvyagintsev, 1991). ABTS was dis-
solved in 5–10 mL of sterile distilled water and added
to the medium before dispensing it into plates. Inocu-
lation was performed from serial dilutions: 1 : 100,
1 : 1000, and 1 : 10000. The inoculated plates were
incubated in a thermostat at 24°С for 1–2 weeks
(depending on colony growth pattern). Development
of green zones around the colonies due to ABTS oxi-
dation was monitored beginning from day 3 of growth.
The fungi were isolated into pure cultures on day 7–
14. The strains oxidizing ABTS during primary inocu-
lation were reinoculated on an ABTS-containing agar
medium to confirm the reaction.
Species identification. The pure fungal cultures
were identified according to the generally accepted
guides on the basis of cultural and morphological
characteristics. The modern taxonomic positions of
the species were given according to the MycoBank
Fungal (www.mycobank.org) and Index Fungorum
(www.indexfungorum.org) databases. The names of
teleomorphic stages are given for the species forming
them during the cultivation; the names of the anamor-
phic stage are given for other species from the division
Ascomycota. For a number of strains with positive
reaction to ABTS, additional species identification
was performed by analysis of the nucleotide sequences
of the rDNA region ITS1-5.8S-ITS2. DNA was iso-
lated as described by Glushakova et al. (2011), but with
three cycles of heating to 65°С (1 h), shaking on a Vor-
tex (10 min), and freezing. PCR was performed using
the primers ITS1 and NL4 and ScreenMix for PCR
(Evrogen, Moscow) as described by Glushakova et al.
(2011). PCR products were purified and sequenced
according to Sanger’s method (Syntol, Moscow, Rus-
sia). Purification was performed with the BigDye X
Terminator Purification Kit (Applied Biosystems,
United States). Sequencing was performed with the
primer ITS4. The sequencing was carried out with a
BigDye Terminator v. 3.1 Cycle Sequencing Kit
(Applied Biosystems, United States) followed by anal-
ysis of the reaction products in an Applied Biosystems
3130xl Genetic Analyzer. Nucleotide sequences were
analyzed with the BLAST software packages; the
strains were identified using the NCBI database
(http://blast.ncbi.nlm.nih.gov/).
The data were statistically processed using Micro-
soft Office EXCEL 2010. Cluster analysis of the struc-
ture of fungal communities was performed by the rela-
tive abundance indices in STATISTICA 8.0. The clus-
ters were combined by Ward’s method using the
Euclidian distance.
Decolorization of HAs by pure micromycete cultures
on agar media; relationship with laccase production.
For studying the relationship between laccase produc-
tion and HA degradation by micromycetes, as well as
310 ZAVARZINA et al.
the effect of HAs on laccase production by the fungi,
the ABTS-oxidizing strains were inoculated on petri
dishes with Czapek medium with HAs at a concentra-
tion of 1 mg/mL or without HAs, using the pieces of
mycelium (0.5 cm in diameter) pre-grown for 4 days in
petri dishes with wort agar. The dishes were incubated
at 24○С for 9 days; then the HA-decolorizing activity
of the strain was assessed by the diameter of the decol-
oration zone (cm) around the mycelium. Laccase
activity in the mycelium was also tested. To this end,
on day 9 of growth (the variants of cultivation both
with and without HAs) the pieces of mycelium (10 mm
in diameter) were cut out of the central part of fungal
colonies and placed onto petri dishes with agarized
20 mM acetate buffer (pH 5.0) containing 1 mM
ABTS. The dishes with mycelium discs were incu-
bated for 24 h at 29○С (this temperature was used to
accelerate the oxidation reaction); laccase activity was
assessed by the diameter of a dark green zone around
the mycelium disc.
Laccase production and HA degradation by micro-
mycetes during cultivation in a liquid medium. The
strains producing laccase on agar media were culti-
vated in a liquid medium containing the following
(g/L): glucose, 20; peptone, 3; yeast extract, 1; MgSO4 ·
7H2O, 0.5; K2HPO4 · 3H2O, 0.3; pH 6.8. The cultiva-
tion was performed in 20-mL test tubes at 24○С for
14 days without stirring; the volume of the medium
was 3 mL. The 0.1 mM CuSO4, guaiacol, ferulic acid,
2,6-dimethoxyphenol, vanillic acid, or HA at a con-
centration of 1 mg/mL were used as laccase
inducers. At certain time intervals, the medium was
stirred, 100-μL samples were taken, and laccase and
peroxidase activities in these samples were deter-
mined. Laccase activity was determined by the rate of
oxidation of 1 mM ABTS in 20 mM acetate buffer
(pH 5.0) with spectrophotometric determination of
the oxidized product in a Shimadzu spectrophotome-
ter (Japan) at 420 nm, ε420 = 36000 M–1 cm–1 (Hein-
fling et al., 1998). Peroxidase activity was determined
by the difference in ABTS oxidation in the presence
and absence of 0.1 mM H2O2 in the reaction mixture.
The amount of the enzyme forming 1 μM of the prod-
uct per 1 min at 30°C was taken as a unit of activity.
HA decoloration in the liquid culture was determined
by the change in culture liquid absorption at 465 nm
after deduction of the control (the media without
HAs).
RESULTS AND DISCUSSION
Abundance and species composition of micromycetes
in soils. The soils under study are typical of the south
taiga zone (sod-podzolic soil), forest steppe zone (gray
forest soil), and steppe zones (leached and typical
chernozems). The soils were characterized by acidic
reaction ; the pH values of water extracts were 3.8–
4.2 in sod-podzolic soil (in all layers except for that of
6–10 cm (pH 5.0)), 5.6–6.5 in gray forest soil
(meadow), 3.5–5.6 in gray forest soil (forest), and
4.9–5.6 in chernozems. The strains (potential laccase
producers) were isolated and counted on ABTS-con-
taining agar media. The search of producers of oxida-
tive enzymes by formation of colored zones of oxida-
tion of specific substrates, including ABTS, is exten-
sively used for both basidiomycetes and ascomycetes
(Steffen et al., 2002; Kiiskinen et al., 2004; Gochev
and Krastanov, 2007).
Altogether 68 micromycete strains were isolated
from the soils; 20 of them oxidized ABTS. In all soils
under study, the number of micromycetes was highest
in the layer of 0–10 cm and decreased along with
depth, correlating with the carbon content (Table 1).
In the litter (L) and humus horizons (A1) of the soils,
there was a wide variety of species compared to the
underlying layers, and the meadow biocenoses of gray
forest soil and typical chernozem were more diverse by
species composition compared to those of forest soils
(Table 1). The most frequent representatives of identi-
fied micromycetes in all soils belonged to the genera
Trichoderma and Penicillium. The species composition
and distribution of micromycetes in the soil profile
were in agreement with literature data (Mirchink,
1988). The decrease of fungal abundance with depth is
caused by decreased soil aeration, on the one hand,
and decreased availability of the substrates, on the
other hand. Cluster analysis revealed the soil fungal
communities to fall both compositionally and struc-
turally into two large clusters: the soils under forest
vegetation and the soils under meadow-steppe vegeta-
tion (Fig. 1). In the “forest” cluster, there was a well-
marked difference of fungal communities of sod-
podzolic soil from the communities of gray forest soil;
in the “steppe” cluster, there was a substantial differ-
ence of fungal communities between leached cherno-
zem and typical chernozem and gray forest soil
(meadow).
The fungi with the positive reaction of ABTS oxi-
dation in primary platings made up 15–30% of the
total number of identified species in each soil under
study. The abundance of ABTS-oxidizing micromy-
cetes was 10–35% of the total fungal number in the
upper layer (0–10 cm) in all soils under study and up
to 50% in the A1 horizon of the sod-podzolic soil
(Table 1). The maximum frequency of occurrence and
abundance were typical for the following ABTS-oxi-
dizing species: Acremonium murorum (Corda)
W. Gams identified in gray forest soil, leached and
typical chernozems; Oidiodendron griseum Robak
occurring in sod-podzolic soil, gray forest soil and
typical chernozem; and Isaria fumosorosea Wize iso-
lated from sod-podzolic soil, gray forest soil (forest)
and typical chernozem (meadow) (Table 1). The abil-
ity to oxidize ABTS varied in different strains of the
same species. For example, ABTS was oxidized by the
strain Umbelopsis isabellina (Oudem.) W. Gams from
sod-podzolic soil, but not by the strain from gray for-
MICROBIOLOGY Vol. 87 No. 3 2018
 Table 1. Abundance and species composition of micromycetes in soils
Total number,
Soil, horizon Corg, % Number of species** Species composition of micromycetes in soil profiles***
thousand CFU/g*
Sod-podzolic soil
L (0–1) 35.0 2471 (549) 8 (3) Umbelopsis isabellina,*** Oidiodendron griseum, Isaria fumosorosea, Acremonium sp.,
A1 (1–6) 2.85 599 (299) 7 (2) Aureobasidium pullulans, Cladosporium cladosporioides, Mortierella sp., Paecilomyces
A1E (6–10) 2.22 177 (0) 5 (0) carneus, Paecilomyces sp., Penicillium janczewskii, P. verruculosum, Scopulariopsis brevicaulis,

MICROBIOLOGY
Eh (10–20) 0.84 249 (14) 6 (1) Tolypocladium inflatum, Trichoderma sp., T. polysporum, Trichosporiella cerebriformis,
EB (20–30) 0.52 130 (13) 6 (2) Mycelia sterilia, unidentified
Gray forest soil, forest

Vol. 87
A1 (0–5) 4.33 1023 (341) 8 (2) Absidia spinosa, Acremonium butyri, Botrytis cinerea, Clonostachys rosea, Monocillium
(5–10) 2.94 546 (195) 7 (1) bulbillosum, U. isabellina, Mortierella sp., Paecilomyces carneus, Isaria fumosorosea,
AB (10–20) 1.29 427 (36) 9 (1) Penicillium janczewskii, Penicillium sp., Trichoderma sp., T. hamatum, T. koningii, T. virens,

No. 3
B (20–30) 0.58 132 (0) 4 (0) Trichosporiella cerebriformis, unidentified
Gray forest soil, meadow

2018
A1 (0–5) 2.44 646 (114) 9 (1) Acremonium murorum, Alternaria alternata, A. pullulans, B. cinerea, Exophiala sp.,
(5–10) 2.01 443 (74) 15 (2) Gilmaniella humicola, Sphaerostilbella aureonitens, Clonostachys rosea, Monodictys glauca,
(10–20) 1.53 209 (35) 9 (1) Mortierella macrocystis, Umbelopsis vinacea, P. carneus, Purpureocillium lilacinum,
AB (20–30) 0.89 131 (13) 6 (0) P. janczewskii, P. simplicissimum, P. aurantiogriseum, Talaromyces funiculosus,
B (30–40) 0.71 227 (15) 6 (1) Talaromyces verruculosus, Sporothrix sp., Talaromyces stipitatus, P. glaucoalbidum,
ВС (40–50) 0.57 100 (33) 8 (3) T. hamatum, T. koningii, Zygorhynchus moelleri, Mycelia sterilia, unidentified
Leached chernozem
A1 (0–5) 297 (59) 10 (1) Acremonium murorum, A. alternata, Aspergillus wenti, A. pullulans, Cylindrocarpon didymium,
A1 (5–10) 4.56 653 (115) 8 (1) Humicola fuscoatra, Penicillium sp., P. aurantiogriseum, Talaromyces funiculosus,
A1 (10–40) 76 (7.6) 8 (1) P. simplicissimum, P. miczynskii, P. aurantiogriseum, Talaromyces variabilis, T. hamatum,
A1 (40–60) 2.28 3.5 (1.4) 5 (2) T. harzianum, T. koningii, T. virens, T. viride, Mycelia sterilia
Typical chernozem, meadow
A1 (0–5) 4.99 640 (0) 12 (0) Absidia spinosa, Acremonium charticola, A. murorum, A. alternata, A. pullulans,
A1 (5–30) 3.56 333 (91) 10 (2) Cylindrocarpon didymium, Fusarium oxysporum, Clonostachys rosea, Myrothecium verrucaria,
AB (30–40) 2.69 30 (18) 7 (3) Mucor hiemalis, Isaria farinosa, Purpureocillium lilacinum, I. fumosorosea, Penicillium sp.,
AB (40–50) 2.02 4 (1.7) 7 (2) P. aurantiogriseum, P. janczewskii, P. thomii, P. waksmanii, Talaromyces rugulosus, Phoma sp.,
В1 (50–70) 1.86 3 (0.6) 6 (2) T. hamatum, T. koningii, T. viride, Myrmecridium schulzeri, Mycelia sterilia
Typical chernozem, forest
LACCASE PRODUCTION AND HUMIC ACIDS DECOMPOSITION

A1 (0–10) 6.96 310 (39) 6 (1) Absidia spinosa, A. murorum, Aspergillus versicolor, A. pullulans, Pseudogymnoascus
A1 (10–30) 3.77 150 (48) 10 (4) pannorum, Clonostachys rosea, Mortierella sp., Mucor racemosus, O. griseum,
AB (30–40) 1.95 58 (5.8) 7 (1) P. janczewskii, P. aurantiogriseum, P. simplicissimum, Talaromyces funiculosus,
AB (40–50) 1.52 50 (30) 2 (1) T. verruculosus, Phoma sp., Myrmecridium schulzeri, Microascus brevicaulis, T. viride,
T. polysporum, Trichosporiella cerebriformis, unidentified, Mycelia sterilia
* The number of ABTS-oxidizing isolates is given in parentheses.
** The number of ABTS-oxidizing species is given in parentheses.
*** The species oxidizing ABTS during primary inoculation are in boldface.
311
312 ZAVARZINA et al.
est soil (forest); among the strains of the species Peni-
cillium janczewskii K.M. Zalessky, which occurred
nearly in all soils, ABTS oxidation was shown only for
the strain from typical chernozem (meadow) (Table 1)
and occurred in the zone of contact with Trichoderma
sp. or yeasts. Such reaction in the contact zone
between the mycelia of two micromycete species not
oxidizing ABTS separately has been revealed previ-
ously (our unpublished data) and demonstrates coop-
erative interactions during substrate oxidation by
micromycetes.
ABTS oxidation by pure micromycete cultures on
agar and in liquid media; the effects of inducers on lac-
case production. Eleven strains of ABTS-oxidizing
micromycetes were isolated in pure culture (Table 2).
Species identification of these strains was performed
by cultural, morphological as well as by molecular
techniques, especially in the cases when species iden-
tification was difficult (the strains of the genus Tricho-
derma) or impossible (the strains of sterile mycelium).
As a result of nucleotide sequence analysis of the
ITS1-5.8S-ITS2 region, strain Z1705 was identified as
T. composticola (99.8% homology); strains Z1701,
Z1701a and Z1707 were identified as representatives of
the species T. atroviride (100% identity with T. atrovir-
ide NG 13, GenBank sequence accession number
HQ115671); strain Z1706 was identified as Leucophell-
inus irpicoides (98.8% homology); strain Z1708 was
identified as a representative of the genus Cladospo-
rium (99.4% homology was obtained with the species
С. allicinum and С. bruhnei). Strain Z1703 was more
precisely affiliated to O. truncatum (96.1% homology).
When the pure cultures were reinoculated on
ABTS-containing Czapek agar medium, the ability of
most strains to oxidize this substrate was weaker (rep-
resentatives of the genus Trichoderma) or absent (e.g.,
in I. fumosorosea Z1704 and L. irpicoides (Table 2)).
ABTS was very poorly oxidized by O. truncatum in
pure culture. The conditions of laccase production by
ascomycetes are insufficiently understood. In fungi,
laccase is an inducible enzyme, especially in the case
of wood-degrading basidiomycetes showing only the
basal level of laccase production without an inducer
(Galhaup et al., 2002; Terrón et al., 2004). Laccase
inducers are low-molecular aromatic compounds
(phenol compounds, aromatic alcohols, acids, alde-
hydes), metal ions (copper, manganese, cadmium),
and polyphenol compounds (lignin, HA) (Piscitelli
et al., 2011). Ascomycetes are generally characterized
by laccase production without inducers (Palonen
et al., 2003). Nevertheless, an increase in laccase pro-
duction in response to the addition of inducers has
been shown for some ascomycetes (Gomaa and Mom-
taz, 2015), although to a far lesser extent than typically
observed in basidiomycetes. Laccase inducers in asco-
mycetes are first and foremost copper ions and low-
molecular aromatic compounds (Edens et al., 1999;
Saito et al., 2003). Laccase induction in the presence
of HA had not been studied and, therefore, we per-
1
2
3
4
5
6
7
25 30 35 40 45 50 55
Euclidean distance
Fig. 1. Cluster analysis of micromycete communities com-
pared by the indices of relative species abundance. Sym-
bols: 1—sod-podzolic soil; 2—gray forest, forest; point 1,
3—gray forest, forest; point 2, 4—gray forest, meadow; 5—
typical chernozem; point 1, 6—typical chernozem;
point 2, 7—leached chernozem.
formed relevant experiments both on solid and in liq-
uid media.
The HA effects on laccase production on agar
media were ambiguous (Table 3). Laccase production
in T. composticola Z1705, A. murorum Z1710, and
B. cinerea Z1711 was higher in the presence of HA,
while I. fumosorosea Z1704 showed low activity only in
the presence of HA. On the contrary, low activity of
I. fumosorosea Z1709 was demonstrated only in the
absence of HA. The fungus Cladosporium allicinum
(bruhnei) Z1708 did not oxidize ABTS in pure culture
with or without HA. The production of oxidative
enzymes in the presence and absence of HA and other
inducers in a liquid culture was investigated in the
fungi oxidizing ABTS on agar media (Table 3). The
production of laccase and peroxidase was measured,
both enzymes being capable of ABTS oxidation.
During the cultivation, no peroxidase activity was
found in any fungus. The fungi I. fumosorosea Z1704
and Z1709 and T. composticola did not produce laccase
in liquid media. Botritis cinerea showed a low laccase
activity only in the variant without inducers: it
appeared on day 2 of cultivation, reached 0.3 U/mL
on day 7, and remained at this level until the end of
cultivation (data not shown). The strain A. murorum
Z1710 demonstrated equally active laccase production
in the absence of inducers and in the presence of HA
(Fig. 2). In the presence of low-molecular phenol
inducers or copper ions in the medium, laccase pro-
duction by this strain decreased. Probably, it is due to
the toxic effects of phenol compounds and copper ions
as it was shown for the ascomycete Cladosporium clad-
osporioides (Halaburgi et al., 2011).
In the present study, a number of strains demon-
strating oxidative activity during the growth on solid
MICROBIOLOGY Vol. 87 No. 3 2018
 Table 2. Species identification of pure cultures, ABTS oxidation on Czapek agar medium

Species identification ABTS oxidation*

Strain Sampling site

by molecular methods (% homology),
by morphological characteristics in primary screening in pure culture
NCBI sequence accession number

MICROBIOLOGY
Z1701 Trichoderma spp. Trichoderma viridescens (93.8 %) GF, forest, AB 10–20 cm +++ +

Vol. 87
Z1701a GF, forest, A1 0–5 cm +++ −

No. 3
Z1707 GF, forest, A1 5–10 cm ++ −

2018
Z1705 Trichoderma composticola (99.8%) SPS, L 0–1 cm ++ ++
SUB3314097 Seq_ID2 MG646338

Z1711 Botritis cinerea Botritis cinerea GF, forest, A1 5–10 cm ++ ++

Z1704 Isaria fumosorosea I. fumosorosea (96.1%) GF, forest, A1 5–10 cm + −

Z1709 GF, forest, A1 5–10 cm ++ −

Z1706 Sterile mycelium Leucophellinus irpicoides (98.8%) GF, meadow, AB 30–40 cm ++ +

SUB3314097, Seq_ID6 MG646340

Z1710 Acremonium murorum A. murorum (98.5%) LC, A1 50–60 cm +++ +++

SUB3314097, Seq_ID10 MG 646342
LACCASE PRODUCTION AND HUMIC ACIDS DECOMPOSITION

Z1703 Oidiodendron griseum O. truncatum (96.1%) TC, forest, A1B 30–40 cm ++ +

SUB3314097 Seq_ID4 MG646339

Z1708 Sterile mycelium Cladosporium allicinum (bruhnei) TC, meadow, AB 40–50 cm ++ −

(99.4%) SUB3314097 Seq_ID8
MG646341

* Coloration: +++, intense; ++, medium; +, weak; −, absent.

313
314 ZAVARZINA et al.
Table 3. ABTS oxidation by fungi in the presence and absence of HAs. HA decoloration by fungi
Diameter of ABTS oxidation zone, cm
Fungal strain
medium with HA
A. murorum Z1710 4.5
B. cinerea Z1711 5.5
I. fumosorosea Z1704 0.7
I. fumosorosea Z1709 − 0.8
T. composticola Z1705 5 0.8
C. allicinum (bruhnei) Z1708 − −
media (Table 3) did not show it when grown in liquid
media. This phenomenon is known (Kiiskinen et al.,
2004), although its mechanism has not yet been inves-
tigated. Ascomycetes were shown to produce constitu-
tive laccases, which do not require the presence of
inducers, are spore- or cell wall-associated and are
probably involved in the production of such pigments
as melanins (Law and Timberlake, 1980; Holker et al.,
2002).
HA decolorization by micromycetes on agar media
and in liquid culture; relationship with laccase produc-
tion. The soil and wood-decomposing fungi are capa-
ble of HA degradation only in the course of cometab-
olism, i.e., in the presence of easily assimilated sub-
strates, such as glucose. At the same time, it is believed
that HA transformation by micromycetes is associated
with the activity of oxidative enzymes, including lac-
case (see the review by Zavarzina et al., 2011). We have
performed experiments on decolorization of the HA
preparation by the fungi on Czapek agar medium, with
the subsequent testing of the mycelium for its ability to
oxidize ABTS. Unambiguous correlations between
ABTS oxidation and HA decolorization on agar media
have not been established. HA decolorization cor-
related with intense ABTS oxidation only in A. muro-
rum and T. composticola. The strain Botritis cinerea,
which oxidized ABTS most intensely, did not decolor-
ize HA. On the contrary, the fungi intensively decolor-
izing HA (Isaria fumosorosea Z1704 and C. allicinum
(bruhnei)) either oxidized ABTS very poorly or did not
oxidize it at all (Table 3).
The same absence of correlation between the oxi-
dative ability of the strains and their ability to decolor-
ize HA was demonstrated by the fungi cultivated in liq-
uid media (Fig. 3). During 14 days of cultivation, the
fungi C. allicinum (bruhnei) and Isaria fumosorosea
Z1709 showed a strong HA-decolorizing activity: 81
and 67%, respectively, although laccase and peroxi-
dase production was not found in these fungi. The
fungus Isaria fumosorosea Z1704 not producing these
enzymes decolorized HA to a large extent (54%). The
fungus T. composticola not producing laccase in liquid
media but producing it on agar media (Table 3) decol-
orized HA insignificantly (7%). The fungus A. muro-
rum, in spite of the highest laccase production (Figs. 1,
Diameter of HA
medium without HA decolorization zone, cm
3.5 1.1
2.5 −
− 8.2
−
−
7.1
2), decolorized HA to a lesser extent (43%) than
B. cinerea (62%), which showed insignificant laccase
production in liquid media.
Thus, there was no direct dependence between lac-
case production and/or activity and the HA-decolor-
izing ability of the fungi, with the exception of A. mur-
orum. While the high HA-decolorizing ability in the
absence of oxidative exoenzymes was described for
ascomycetes earlier (Gramss et al., 1999), the causes
of this phenomenon remain uncertain. The presence
of HA transformation mechanisms in white rot basid-
iomycetes related to the potential function of cyto-
chromes P450 has been recently proposed by Zahmat-
kesh et al. (2016); however, cytochromes P450 are
intracellular enzymes, while soil HAs are mainly mac-
romolecular compounds not penetrating into the cells.
HA oxidative destruction probably involves nonenzy-
matic oxidative systems, e.g., OH radicals, which can
be produced extracellularly due to hydroquinone-
mediated redox cycling of Fe2+ ions (Fenton reaction).
The process is known in brown rot basidiomycetes
(Arantes and Goodell, 2014) and has been recently
described in some ascomycetes (Krueger et al., 2016;
Rojas-Jimenez et al., 2017). The causes of intensive
HA degradation by the fungi not producing laccase
20
Laccase activity, U/mL
16 1
2
12
8 3
4
4 5
6
7
0 2 4 6 8 10 12 14
Days
Fig. 2. Laccase production in A. murorum Z1710 in the
media: without inducers (1); with HA (2); with vanillic
acid (3); with ferulic acid (4); with guaiacol (5); with cop-
per ions (6); and with 2,6-dimethoxyphenol (7).
MICROBIOLOGY Vol. 87 No. 3 2018
 LACCASE PRODUCTION AND HUMIC ACIDS DECOMPOSITION
HA decoloration, % Laccase activity, U/mL
100 16
80
12
60
8
40
4 sis by metal ions, Microbiology (UK), 2002, vol. 148,
20
0 0
Z1704 Z1709 Z1708 Z1705 Z1710 Z1711
Fig. 3. Decolorization of humic acids (color loss, % of the
initial optical density at 465 nm) by micromycetes (black
columns) and laccase activity (gray columns) in culture
liquid on day 14 of cultivation: Z1704, Z1709—Isaria
fumosorosea, Z1708—Cladosporium allicinum (brunei),
Z1705—Trichoderma composticola, Z1710—A. murorum,
Z1711—B. cinerea.
and the role of ascomycete laccases in the oxidative
degradation of humus need further investigation.
Thus, it was shown that micromycetes demonstrat-
ing oxidative ability when growing on ABTS-contain-
ing agar media constituted up to 30% of the number of
species and the total number of micromycetes in sod-
podzolic soil, gray forest soil, and chernozems. The
ABTS oxidation ability decreased in pure cultures of
most species. In liquid media, laccase production was
found only in A. murorum and B. cinerea, suggesting
the possibility of constitutive synthesis of laccases in
the strains oxidizing ABTS on agar media. The most
intensive HA-decolorizing activity in liquid media was
demonstrated by the fungi not producing laccase. The
mechanisms of oxidative action of ascomycetes
towards humic substances will be a subject of our fur-
ther studies.
ACKNOWLEDGMENTS
The work was supported by the Russian Science
Foundation, project no. 17-14-01207.
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Translated by E. Makeeva
MICROBIOLOGY Vol. 87 No. 3 2018