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ISSN 0003 6838, Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 3, pp. 287­295. © Pleiades Publishing, Inc., 2013. Original Russian Text © O.I. Klein, N.A. Kulikova, A.I. Konstantinov, T.V. Fedorova, E.O. Landesman, O.V. Koroleva, 2013, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2013, Vol. 49, No. 3, pp. 292­300.

Transformation of Humic Substances of Highly Oxidized Brown Coal by Basidiomycetes Trametes hirsuta and Trametes maxima
O. I. Kleina, N. A. Kulikovaa, b, A. I. Konstantinovc, T. V. Fedorovaa, E. O. Landesmana, and O. V. Korolevaa
a

A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow, 117071 Russia b Department of Soil Science, Moscow State University, Moscow, 119991 Russia c Department of Chemistry, Moscow State University, Moscow, 119991 Russia e mail: klein_olga@list.ru
Received September 19, 2012

Abstract--The ability of white rot basidiomycetes Trametes hirsuta and Trametes maxima to transform coal humic substances (HS's) under the conditions of solid phase cultivation in the presence or absence of an eas ily available source of carbon (glucose) has been studied. It was shown that during the growth of the fungal strains used in media containing HS's, destructive and condensation processes of HS transformation pro ceeded simultaneously. Based on a comparative physicochemical analysis of the initial HS's and HS's trans formed by the fungi, it was established that, despite the introduction of glucose may favor a deeper trans formation of HS's by basidiomycetes, the general direction of their modification is dominant reduction or oxidation and is determined by the physiological biochemical peculiarities of the strain used. DOI: 10.1134/S0003683813030101

INTRODUCTION Since Coen and Gabriel for the first time showed in 1982 that fungi are able to grow directly on coal and are capable of metabolizing this substance [1], the processes of biological conversion of coal, first of all, brown coal, have become the subject of multiple inves tigations [2­7]. Recently, the interest towards the biological con version of coal has been increasing steadily which is determined by the negative consequences for the envi ronment emerging as a result of using coal as an energy source. This field is especially urgent for countries which are characterized by a high level of coal produc tion, including Russia, which takes second place after the Federal Republic of Germany by this parameter. The production of brown coal comprises about 90 mil lion tons per year in our country [8]. As compared to conversion by chemical or physical methods, microbi ological and enzymatic approaches possess a number of advantages such as the lack of necessary setting of high temperatures and pressure [9]. Despite promising uses for the biological approach to the conversion of coal, its development requires an accurate selection of a microorganism, on the basis of which the creation of a bioconversion technique is being planned. This is determined by the peculiarities of coal as a substrate which includes a number of lim itations for a suggested microorganism. One of the principal properties of coal is its toxicity, which com plicates its biodegradation, is attributed to the pres

ence of hard degradable compounds, for example, polycyclic aromatic hydrocarbons, as well as to the increased heterogeneity of the structure and solid phase state [8]. These indicated properties of coal, which are negative for its biotransformation, are responsible for the relatively small range of microor ganisms which are capable of degrading coal; first of all, such microorganisms include basidiomycetes fungi, which produce lignolytic enzymes. These microorganisms belong to a small group of microor ganisms which possess a unique system of extracellular ligninolytic enzymes and, due to this feature, are capable of degrading hard degradable substances with a complex phenol structure, such as lignin and coal. It was established that a leading role in this process belongs to the lignin degradation system, which includes enzymes of the ligninolytic complex: lignin peroxidase, Mn peroxidase, and laccase, as well as low molecular weight secondary metabolites [10­ 14]. The efficiency of the lignin degradation process under the action of this system considerably depends on the availability of carbon sources. It was shown that the presence of an easily available carbon source in a nutrient medium intensifies the process of lignin bio degradation [14]. Nevertheless, the relationship between the efficiency of coal degradation and the composition of a medium remains unexplored. The aim of the present study is to compare the bio logical degradation of brown coal under the action of producers of high active peroxidases and laccases--

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Trametes hirsuta and Trametes maxima basidio mycetes--in complete nutrient media and in the absence of an easily available source of carbon (poor medium). MATERIALS AND METHODS T. hirsuta (Wuff.:Fr.) Pil. and T. maxima (Mont.) David & Rajchenb strains were used for experiments which were obtained from the collection of cultures of the Komarov Botanical Institute, Russian Academy of Sciences. In order to study the degradation of coal by the selected basidiomycetes strains, they were solid phase cultivated using leonardite, i.e., coal of a high decomposition degree (Solntsevskii coal deposit, Sakhalin), according to [15]. During solid state cultivation, 20 g of crushed leonardite with a particle size of 1 mm and 5 mL of complete or poor nutrient medium were placed in flasks of 750 mL in volume. During growing fungi in the complete medium, a nutrient medium of the fol lowing composition was used (g/L): glucose (10.0), peptone (3.0), NaNO3 (3.0), KH2PO4 (0.6), ZnS4 22 (0.001), K2HPO4 (0.4), FeS4 72 (0.0005), MnS4 72 (0.05), MgS4 72 (0.5), CaCL2 (0.5), CuSO4 (0.25), pH 6.0. The poor medium without an easily available source of carbon had a similar composition, but it did not contain glu cose. Afterwards, mycelium of fungi was planted (the inoculate volume is 20 mL). During the experiment, humidity was maintained at a level of 80­90% in the flasks. Coal was sterilized by autoclaving (120°, 1 atm, 30 min). Cultivation was carried out for 30 days at 28°. The basic enzymes of the ligninolytic complex, i.e., lignin peroxidase, Mn peroxidase, and laccase, were monitored in order to estimate the functional state of the grown basidiomycetes. Determination of lignin peroxidase activity. The activity of the enzyme was determined by the rate of veratric alcohol oxidation to veratric aldehyde by mea suring the decrease in absorption at 310 nm in 0.1 M Na tartrate buffer, pH 3.0 [16]. Determination of the Mn peroxidase activity. The activity was assayed according to [17] using Mn2+ as a substrate. The oxidation of 1 moL of Mn(II) for 1 min was taken as a unit of activity. Assay of laccase activity. The activity of laccase was determined spectrophotometrically at a wavelength of 410 nm using a 10 mM chromogenic catechol sub strate in 0.1 M Na acetate buffer at pH 4.5 [18]. The increase in the optical density at one unit for 1 min was taken as a unit of activity. The rates of enzymatic reac tions were recorded using PerkinElmer spectropho tometers (United States). In order to convert the enzymatic activities into international units (IU),

standard units were normalized using the extinction coefficient of the substrate used. In order to control changes in leonardite during cultivation of basidial fungi, after the termination of the experiment, humic substances (HS's) were extracted from it and their physicochemical properties were described. For this purpose, 0.1 M NaOH was added to leonardite at a weight ratio of 1 : 2, the pasty mass was quantitatively transferred into a dialysis bag with a pore size of 12­14 kDa and were dialyzed against distilled water for 48 h with multiple water replacements at a room temperature for the depletion of excess alkaline. HS's were then centrifuged for 20 min at 3000 g, and the supernatant was collected. The obtained solution of HS's was dried in a drying oven at a temperature of 50°. HS's produced from leonardite, subjected to the same procedures (auto claving, nutrient media addition, and incubation) but without the introduction of fungi, were used as a con trol sample. The principal physicochemical properties of the HS's were characterized according to parame ters, such as the elemental composition, molecular mass (MM), and the group structure composition. Elemental composition. An analysis of C, H, and N was carried out using an 1106 elemental analyzer (Carlo Erba Strumentazione, Italy). The content of oxygen was measured according to the difference between the mass of the sample and the total content of ash and CHN. The ash was determined by combus tion of the preparation in a muffler at 850° for 40 min. Determination of the MM. The MM of HS's was assayed using exclusion chromatography according to the technique described in [19]. Fractionation of preparations was performed using a column filled with a Toyopearl HW 50(S) gel (Japan); polysterol sul fonates with MMs of 4.48, 14.0, 20.7, 45.1, and 80.8 kDa were used as calibration substances (Poly mer Standard Service, Germany). The concentration of HS's in the analyzed samples was found to be 40 mg/L. The mobile phase was made by phosphate buffer (0.028 M, pH 6.8); the elution rate was 1 mL/min. A UV detector was used in order to record HS's at the column outlet according to absorption at 254 nm. Average MMs were calculated using the Gel Treat program [20]. Group structure composition. The group structure composition was analyzed by 13C NMR spectroscopy. Specters were recorded using an Avance spectrometer (Bruker, Germany) at a retention time of 4 s. The indi cated relaxation delay allows for the complete relax ation of carbon nuclei of almost all types which ensures the quantitativeness of 13C NMR specters pro duced [21]. The concentration of HS's in the analyzed samples comprised about 30 g/L. The content of car bon in different structural fragments was determined by integrating the corresponding spectral regions
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0.25 mm 0.35 mm 0.44 mm 0.41 mm 0.32 mm 0.24 mm 0.34 mm 0.47 mm

()

1 mm

(b)

1 mm

(c)

1 mm

(d)

10 µm

(e)

10 µm

(f)

10 µm

Fig. 1. SEM of leonardite samples after its interaction with T. hirsuta + T. maxima strains: (a, d) control, (b, e) medium without glucose, (c, f) medium with glucose, (a­c) â25, and (d­f) â300.

(ppm): 5­108 are atoms of C unsubstituted of O and N substituted aliphatic fragments (CAlk), 108­165 are atoms of C unsubstituted of O and N substituted aromatic fragments (CAr), 165­187 are C atoms of carboxyl and ether groups (CCOO), 187­220 are C atoms of ketone and chinone groups (CC=O). Scanning electron microscopy (SEM). The SEM technique was used in order to visualize the changes in leonardite. After the termination of the combined cul tivation of the studied strains, leonardite particles were dried to a constant mass at 50° and mounted with a conductive double sided adhesive sheet. In order to image the surface of coal, a high resolution scanning electron microscope equipped with an autoemissive JSM 7500F cathode was used (JEOL, Japan). Images were obtained in a regime of low energetic secondary electrons, since this regime provided the highest reso lution level. At the energy of a primary beam of 1 keV, the resolution was 1.5 nm; at 5 keV, 1 nm. Before micros copy, a metallic film was placed on the samples with a width of 5 nm using a magnetron spraying system. RESULTS AND DISCUSSION According to the SEM results, the analyzed strains of basidiomycetes actively interacted with leonardite that resulted in a marked loosening of its surface (Figs. 1a­1c), directly indicating the ability of fungi to degrade coal of a high decomposition degree using
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them as a source of nutrient substances. Previously, the ability of coal biosolubilization was demonstrated for basidiomycetes, such as Gymnopus erythropus [22], Clit ocybula dusenii and Nematoloma frowardii [23], Phaner ochaete chrysosporium [24], and Trametes versicolor and Lentinula edodes [25]. At a higher magnification (Figs 1d­1f), a system of hyphae is clearly seen which demonstrates the growth of the studied fungi with leonardite. In the medium without glucose, single hyphae are seen on the surface and hyphae grown inside leonardite particles, whereas in the complete medium, in which the growth of the fungus culture was more intense, mycelium formed a layer on the surface of coal. Intense surface growth of basidiomycetes did not allow for the visualization of their growth inside leonardite. An analysis of the elemental composition of initial and transformed HS's of leonardite showed that the changes observed are only determined by the strain used but little depend on the presence of an easily available carbon source in the medium. In the case of T. hirsuta cultivation, changes in the carbon and hydrogen contents were hardly observed, while a decreased content of hydrogen and carbon in HS's was observed for T. maxima (Table 1). Taking into account that the nitrogen content hardly changed, one may suppose that T. hirsuta cultivation is accompa nied by a partial reduction of HS's, cultivation of T. maxima, by their oxidation.
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Table 1. Elemental composition and MMs of initial HS's and those transformed by the white rots T. hirsuta and T. maxima Content, % Sample C Control T. hirsuta T. maxima T. hirsuta + T. maxima T. hirsuta T. maxima T. hirsuta + T. maxima 72.4 75.3 70.4 69.5 73.9 69.2 70.0 H 5.8 5.9 5.1 5.1 5.6 5.2 4.9 N 3.3 Medium without glucose 3.6 3.1 2.9 Medium with glucose 3.7 3.0 3.2 21.2 19.7 19.5 31.0 22.7 25.7 22.5 20.2 19.9 26.9 28.5 25.1 Ash 22.4 14.3 MM, kDa

In order to validate the above stated hypothesis, the O/C and H/C atomic ratios were calculated which allow for estimating the degree of oxidation and unsat uration of HS's. The results of the calculations pre sented in the Van Crevelen diagram (Fig. 2) show a bidirectional change in the HS properties. In the case of T. hirsuta, both studied atomic ratios decreased simultaneously; i.e., HS's were modified via the reduction and dehydration route in this basidiomycete strain. During the transformation of HS's under the action of T. maxima or in the case of co cultivation of T. hirsuta and T. maxima, a decreased H/C ratio was also observed; at the same time, the O/C parameter was shown to increase; i.e., one may suppose that oxi dative processes, such as carboxylation, proceed.
/ 0.98 2 5 0.90 4 0.86 3 7 0.82 0.10 0.15 0.20 0.25 0.30 / 6

1

0.94

Fig. 2. Van Crevelen diagram for (1) initial leonardite HSs and those transformed by T. hirsuta and T. maxima fungi: 2­4, a medium without glucose; 5­7, a medium with glu cose; 2 and 5, T. hirsuta; 3 and 6, T. maxima; 4 and 7, T. hirsuta + T. maxima.

The relationship between HS transformation and the reduction capacity of fungi was previously demon strated for the majority of basidiomycetes, including representatives of the genera Polystictus, Hypholoma, Trametes, and Polyporus [10]. Based on the screening results of more than 20 strains of fungi, only those strains were established to possess an ability to deco lour HS solutions, in the presence of which the reduc tion of oxybenzoic and methoxybenzoic acids to pri mary alcohols is observed. Thus, reduction should be considered as a necessary stage in the degradation of HS's by basidiomycetes. It is supposed that cellobiose dehydrogenase may be responsible for the reduction processes, i.e., flavocytochrome capable of one elec tron reduction of aromatic radicals or two electron reductions of chinones with simultaneous oxidation of semiacetal sugar hydroxyl to produce carbonyl [26]. Therefore, the reduction of aromatic acids to alcohols may be regarded as a primary stage in the transforma tion of HS's by basidiomycetes. A decreased H/C ratio was recorded for all studied samples which indicates a decreased percentage of ali phatic chains in the structure of HS's. It is of interest to note that the changes observed during HS modifica tion in the presence of T. hirsuta are similar to the changes characteristic of conversion of fulvic to humic acids of soils, whereas the HS transformation under the action of T. maxima is similar to the processes which take place during the formation of fulvic acids from plant remains [27]. Increase in the MMs of HSs in all studied samples (Table 1) indicate that polymerization processes occur during HS transformation which are likely related to indirect enzymatic degradation based on the forma tion of radicals as main and by products of enzymatic reactions with a further initiation of radical processes [28]. Another explanation for the recorded polymer
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TRANSFORMATION OF HUMIC SUBSTANCES OF HIGHLY OXIDIZED BROWN COAL Table 2. Group structure composition of initial HS's and HS's transformed by the white rots T. hirsuta and T. maxima Content, % Sample CAlk Control 31.7 CAr 46.0 CCOO 18.3 Medium without glucose T. hirsuta T. maxima T. hirsuta + T. maxima 27.7 28.3 19.9 52.8 45.3 56.1 15.3 18.1 18.6 Medium with glucose T. hirsuta T. maxima T. hirsuta + T. maxima 26.5 21.2 28.1 47.6 53.8 42.8 18.3 18.9 16.7 7.5 6.1 12.5 1.8 2.5 1.5 4.2 8.3 5.4 1.9 1.6 2.8 CC=O 4.5 1.5

291

CAr/CAlk

ization of products of partial HS degradation may be such an ability of the ligninolytic enzymatic complex of selected strains as the presence of high redox potential laccases, which as a rule possess condensing activity in relation to a wide range of phenolic sub strates [28]. In any case, these data indicate the simul taneous proceeding of destructive and condensation processes in HS's during their growing with T. hirsuta and T. maxima. Similar differently directed processes were previously shown when studying the interaction between white rot fungi and lignin [26]. It was estab lished that lignins of aspen and larch wood, trans formed under the action of Phanerochaete sanguine, could have MMs that exceed the MMs of lingins in initial wood by 1.5 times. An analysis showed that con densation processes mainly go through C5 atom of the aromatic ring. An analysis of the group structure composition of HS's by 13C NMR spectroscopy (Fig. 3, Table 2) con firmed the previously established regularities and allowed for establishing more precise changes which occur in HS's during their interaction with the ana lyzed basidiomycete stains. It was revealed that the decreased content of carbon in HS's is mainly deter mined by a decrease in the relative content of aliphatic structure fragments for practically all samples. This is evidenced by both a decreased CAlk parameter and growth in the degree of CAr/CAlk aromaticity. T. hirsuta + T. maxima sample in a medium with glu cose was an exception, for which the decreased per centage of carbon was attributed to a simultaneous decrease in the carbon content of both aliphatic and
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aromatic fragments that did not result in any increase in the CAr/CAlk aromaticity parameter. Therefore, one may suppose that when HS's are used as a source of nutrient substances by basidiomycetes, first of all, aliphatic chains are to be removed but not aromatic structures. A more marked decrease in the content of aliphatic groups in HS's during transformation by T. hirsuta and T. maxima strains was observed in the samples with a compete nutrient medium. This may be determined by both accelerated biomass growth and the resulting increased production of ligninolytic enzymes, and the use of glucose as a substrate for rad ical reactions. During the cultivation of T. hirsuta in a medium without glucose, a decreased content of CCOO carboxyl group carbon in HS's was observed which agrees with the data on the elemental composition analysis show ing a decrease in the oxygen content in HS's under these conditions. This confirms the hypothesis regard ing the reduction of carboxyl groups to alcohol ones during the first stage of HS transformation. In the presence of glucose, the carboxyl group carbon con tent did not change, whereas that of ketone and chi none increased. This possibly indicates the presence of deeper HS degradation processes, which also include oxidation of alcohols, formed during the first stage of transformation, to ketones. The decreased HS oxida tion level established by analyzing the elemental com position (Fig. 2) is likely determined by the loss of oxy gen substituted aliphatic fragments by HS's. Thus, the presence of easily available carbon source favors a deeper transformation of HSs by this fungal strain.
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ppm 250 I 200 150 () 100 50 0 II

ppm 250 200 150 100 50

ppm 0 250 (b) 200 150 100 50 0

ppm 250 200 150 100 50

ppm 0 250 (c) 200 150 100 50 0

ppm 250 200 150 100 50

ppm 0 250 200 150 100 50 0

Fig. 3. 13C NMR specters of initial HS's (C) and those transformed by (a) T. hirsuta, (b) T. maxima, and (c) T. hirsuta + T. max ima; I is a medium without glucose, and II is a medium with glucose.

During the transformation of HSs by T. maxima strain in a poor medium, the content of ketone and chinone group carbon was observed to increase at a simultaneous insignificantly decreased content of ali phatic, aromatic, and carboxyl fragments. Taking into account the total increase in the degree of oxidation according to the data of the elemental composition anal ysis (Fig. 2), one may suppose that an HS transformation occurred via a mechanism of alcohol group oxidation to

produce ketones in this case. Since the content of car boxyl groups, which may serve as a source of alcohol groups, did not decrease in this case, aliphatic fragments became oxidized. In the presence of glucose, the CC=O content also increased; nevertheless, at the same time, the carboxyl group content also increased simulta neously. This indicates that, as in the case with T. hirsuta, the introduction of an easily available source of carbon leads to a more deep transformation of HS's.
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Table 3. Comparative assessment of the lignin peroxidase, Mn peroxidase, and laccase activities of the white rot fungi T. hir suta and T. maxima during their cultivation with leonardite Activity, IU Sample Lignin peroxidase, â10 5 6 3 15 4 10
3

Mn peroxidase, â10 Medium without glucose 18 8 7 Medium with glucose 10 8 14

3

Laccase 0.2 0.3 0.1 0.2 0.2 1.9

T. hirsuta T. maxima T. hirsuta + T. maxima T. hirsuta T. maxima T. hirsuta + T. maxima

Thus, the analysis of changes in the basic physico chemical properties of HS's during their transforma tion by the studied basidiomycete strains allows one to
Aromatic acids
reduction

propose the following sequence of basic processes of HS degradation under the action of enzymes of the ligninolytic complex of basidiomycetes:
Ketones Aldehydes
oxidation

Alcohols

oxidation

Alyphatic acids

Monitoring of the enzyme activity in culture fluids showed that the dynamics in the activity of all three studied enzymes displayed a complex character and depended on the phase of culture development. Therefore, for comparison, the mean values of activi ties for the studied period of HS transformation were used (Table 3). The data presented in Table 3 show that maximum laccase activity was observed in the ligninolytic com plex of T. hirsuta and T. maxima which is typical of the strains used [15]. In the medium without glucose, the investigated strains possessed similar activities of lig nin peroxidase and laccase but differed in the activity of Mn peroxidase which was significantly higher than in T. hirsuta. It was previously shown that, under these conditions under the action of T. hirsuta, the content of carboxyl groups in HS's was shown to decrease (Table 2). Transformation under these conditions under the action of T. maxima did not lead to a decreased content of carboxyl groups but was favor able for growth in the content of ketone and chinone fragments in HS's. Therefore, one may conclude that, under the action of Mn peroxidase, HS's became decarboxylated. In fact, correlation analysis showed a reverse linear dependence (r 2 = ­0.86) between the activity of Mn peroxidase which is found in the medium and the content of carboxyl groups in the HS's transformed (Fig. 4). The dependence obtained agrees with the data of other researchers which men tioned the decarboxylation of HS's under the action of fungal Mn peroxidase which catalyzes the formation
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of a low molecular weight mediator with a high redox potential, i.e., chelated Mn(III) ion [23]. When grown in a complete nutrient medium, the activities of Mn and lignin peroxidases were found to be higher in T. hirsuta than in T. maxima. The change in the enzyme profile resulted in that, during HS transformation, a more marked increase in the con tent of ketone and chinone groups was observed in T. maxima. Hence, one may suppose that the lignin peroxidase synthesized by T. hirsuta is responsible for the oxidation of alcohol groups in HS's to produce
CCOO, % 20 18 16 14 12 5 10 15 20 IU â 103

Fig. 4. Correlation field of variables: the Mn peroxidase activity and the content of carboxyl group carbon CCOO of HS's transformed by T. hirsuta and T. maxima. No. 3 2013

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30

25

HS's by the studied basidiomycete strains; however, the total direction of this modification: a predominant reduction or oxidation, is determined by the physio logical biochemical peculiarities of the strain. One may suppose that the domination of reduction pro cesses during HS transformation was determined by the presence of extracellular cellobiose dehydrogena ses in the composition of the complex. Nevertheless, in order to verify this hypothesis, additional research is needed.
5 10 15 20 IU â 103

20

0

ACKNOWLEDGMENTS This study was supported by the Ministry of Educa tion and Science of the Russian Federation in the scope of the federal target program "Human Capital for Science and Education in Innovative Russia in 2009­2013," contract no. 8111. REFERENCES
1. Cohen, M.J. and Gabriele, P.D., Appl. Environ. Micro biol., 1982, vol. 44, pp. 23­27. 2. Scott, C.D., Scott, T.C., and Woodward, C.A., Fuel, 1993, vol. 72, pp. 1695­1700. 3. Ralph, J.P., Graham, L.A., and Catcheside, D.E.A., Appl. Microbiol. Biotechnol., 1996, vol. 46, pp. 226­ 232. 4. HÆlker, U., Fakoussa, R.M., and HÆfer, M., Appl. Microbiol. Biotechnol., 1995, vol. 44, pp. 351­355. 5. HÆlker, U. and HÆfer, M., Biotechnol. Lett., 2002, vol. 24, pp. 1643­1645. 6. Yuan, H.L., Yang, J.S., Wang, F.Q., and Chen, W.X., Appl. Biochem. Microbiol., 2006, vol. 42, pp. 52­55. 7. Gupta, A. and Birenda, K., Fuel, 2000, vol. 79, pp. 103­105. 8. Fakoussa, R.M. and Hofrichter, M., Appl. Microbiol. Biotechnol., 1999, vol. 52, pp. 25­40. 9. Faison, B.D., in Microbial Transformations of Lowrank Coals, Crawford, D.L., Ed., Boca Raton, FL: CRC Press, 1993, pp. 1­26. 10. Hurst, H.M., Burges, A., and Latter, P., Phytochemis try, 1962, vol. 1, pp. 227­231. 11. Solarska, S., May, T., Roddick, F.A., and Lawrie, A.C., Chemosphere, 2009, vol. 75, pp. 751­758. 12. Gramss, G., Ziegenhagen, D., and Sorge, S., Microbial Ecol., 1999, vol. 37, pp. 140­151. 13. Rezacova, V., Hrselova, H., Gryndlerova, H., Miksik, I., and Gryndler, M., Soil Biol. Biochem., 2006, vol. 38, pp. 2293­2299. 14. Moilanen, A.M., Lundell, T., Vares, T., and Hatakka, A., Appl. Microbiol. Biotechnol., 1996, vol. 45, pp. 792­799. 15. Klein, O.I., Kulikova, N.A., Stepanova, E.V., Sof'in, A.V., Filippova, O.I., Landesman, E.O., and Koroleva, O.V., Probl. Agrokhim. Ekol., 2011, no. 3, pp. 36­39.
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Fig. 5. Correlation field of variables: the MMs of HS's transformed by T. hirsuta and T. maxima and the activity of lignin peroxidase.

ketones that indicates the substrate specificity of lignin peroxidases [28]. It is interesting to note that a positive linear depen dence (r 2 = 0.73) was observed between the activities of lignin peroxidase and the MM of HS's in the two media used (Fig. 5). It is likely that the established effect of lignin per oxidase on the MMs of HS's indicates the involvement of this enzyme in the polymerization of products of partial HS degradation. This study showed that in the case of T. hirsuta the presence of an easily available source of carbon mainly influences the ratio of lignin peroxidase and Mn per oxidase in the produced multienzyme complex. In the absence of glucose, a higher activity of Mn peroxidase was recorded; in the presence of this substance, that of lignin peroxidase. A change in the ratio of these two enzymes resulted in that the process of reduced decar boxylation of HS's, which dominates under the condi tions of a poor medium and is likely the first stage in the transformation of HS's, changed into deeper pro cesses of oxidative destruction leading to the forma tion of ketone fragments and aliphatic carboxylic acids. Despite the occurrence of oxidative processes during the transformation of HS's by the studied T. hirsuta strain in the medium with glucose and with out, reduction processes dominated. During growing T. maxima, the influence of an easily available source of carbon on the production of multienzyme complex components was less marked: the activity of Mn peroxidase did not change, and the activity of lignin peroxidase decreased slightly upon the introduction of glucose into the nutrient medium. This resulted in fewer formed ketone groups in HS's and increased the content of carboxyl elements. Thus, these results indicate that the presence of glucose in a medium favors a deeper transformation of

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