Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Abstract

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.

Abbreviations

AN‐3

medium for fungal development

Lac

laccase

LiP

lignin peroxidases

ME

malt extract medium

MnP

Mn‐dependent peroxidases

PDA

Potato dextrose agar

PDB

potato dextrose broth

1. Introduction

Lignocellulose is one of the most abundant renewable carbon sources on Earth, thus representing a very important component in many biotechnological processes. It is composed of three polymers; cellulose, hemicellulose and lignin, together with small amounts of other components, like acetyl groups, minerals and phenolic substituents 1. Lignocellulose has evolved to resist degradation and this robustness or recalcitrance of lignocellulose stems from the crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin–hemicellulose matrix 2. Lignin is a highly cross‐linked polymer formed by polymerization of 4‐hydroxyphenylpropanoid monomers (monolignols) through various ether and carbon–carbon bonds. This polymer provides strength and rigidity to plants and is rather resilient towards degradation 3. The most effective lignin breakdown becomes possible mainly by fungi belonging to the Basidiomycota phylum. These fungi typically produce one or more of the three principal extracellular ligninolytic enzymes, phenol oxidase (laccase) (Lac, E.C. 1.10.3.2), lignin peroxidase (LiP, E.C. 1.11.1.14) and Mn‐dependent peroxidase (MnP, E.C. 1.11.1.13) 4, 5. Many white‐rot fungi simultaneously attack lignin, hemicellulose and cellulose, whereas some other white‐rot fungi preferentially work on lignin in a selective manner 6.

Fungal laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a variety of phenolic compounds, diamines and aromatic amines, coupled with a full, four‐electron reduction of O₂ to H₂O. Hence, they are capable of degrading lignin. It is one of a few enzymes that have been studied since the nineteenth century 7. MnP is a heme glycoprotein that catalyzes the oxidation of Mn²⁺ to Mn³⁺ in the presence of H₂O₂. Mn³⁺, complexed with a dicarboxylic acid chelator such as oxalate, acts as a diffusible redox mediator to oxidize lignin and phenolic lignin compounds. This enzyme belongs to the commonly occurring class II peroxidase group in basidiomycetous fungi and is capable of oxidizing and depolymerizing natural and synthetic lignins as well as entire lignocelluloses (milled straw or wood, pulp) in cell‐free systems (in vitro) 8. Clade of LiP also belongs to the class II of fungal secretory peroxidases (oxidoreductases) 9. These enzymes oxidize non‐phenolic lignin substructures by abstracting one electron and generating cation radicals, which are then decomposed chemically 4.

This group of enzymes is highly versatile in nature and possesses a great potential for application in several biotechnological processes including delignification of lignocellulosic biomass for biofuel production, food, laundry detergents, paper and pulp industries, bioremediation of chemical pollutants etc. 10, 11. The ligninolytic enzymes can successfully replace the conventional chemical processes of several industries. The biotechnological significance of these enzymes has led to a drastic increase in the demand for these enzymes in recent times.

Among all investigated fungi, white‐rot basidiomycetes are known as the most effective producers of Lac, MnP and LiP 7, 8, 11, 12, 13 that are synthesized during their secondary metabolism 14. Considerable attention is focused on Phanerochaete chrysosporium, Phlebia radiata, Coriolus versicolor, Cyathus stercoreus, Ceriporiopsis subvermispora, Trametes versicolor, T. trogii, Pleurotus ostreatus, P. pulmonarius, Schizophyllum commune, etc. 10, 12, 15, 16. Some of them contain all three classes of lignin‐modifying enzymes, while the others contain only one or two of these enzymes 4. Many producers of Lac, MnP and LiP secrete isoenzymes, which differ in stability and catalytic features 17. The tropical white‐rot strains Pycnoporus coccineus and Coriolus versicolor have been described as producers of all three enzymes, Lac, MnP and LiP 18. According to the study of Heinzkill et al. 17, Panaeolus papilionaceus produced only Lac, while P. sphinctrinus produced Lac and two peroxidases. Differential expression of MnP and Lac in white‐rot fungi has been reported by Scheel et al. 19. In most cases, the synthesis of these three ligninolytic enzymes is differently affected by the cultural culture conditions 11, 15, 18. Moreover, even among known producers, the enzyme levels are very low and various physicochemical parameters need to be optimized 13. MnP production by the white‐rot fungus P. chrysosporium BKM‐F‐1767 showed a significant dependence on some activators added to the nutrient medium (Mn²⁺, Tween 80, phenylmethylsulphonylfloride) 20. Elisashvili and Kachlishvili 21 reviewed that a specific feature of white‐rot basidiomycetes is their lack of universal conditions equally good for synthesis of different ligninolityc enzymes.

However, despite the growing importance of these enzymes, there is little information available as regards the production of extracellular Lac, MnP and LiP by autochthonous fungal strains. Furthermore, the optimization of the cultivation parameters is being considered as a prerequisite to a more effective process of enzyme production. Thus, there is a broad field of investigation on new effective producers of ligninolytic enzymes. The aim of the present work was (i) to isolate ligninolytic fungi from Bulgarian forest soil samples, (ii) to select a high producer strain and (iii) to compare the effect of a wide range of culture conditions on the production of Lac, MnP and LiP.

2. Materials and methods

2.1. Isolation and identification of ligninolytic fungi

Samples of forest soil were collected from Plovdiv region, Bulgaria, at a depth of 5–10 cm by random mixed sampling method by removing upper litter layer. Media and isolation techniques were used as described earlier 22. The plates were incubated at 28°C for up to 10 days. Fungi growing on the agar medium were transferred by subculturing from hyphal tips, colonies or spores to fresh beer agar. They were maintained at 4°C on beer agar, pH 6.3. All strains belong to the Mycological Collection of the Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences.

Using micromorphology and colony characteristics on various media, the isolated pure cultures were identified to genus 23, 24, 25. Molecular analyses were carried out as described by Savino et al. 26.

2.2. Culture media and cultivation

Colonized agar plugs on potato dextrose agar (PDA) plates for 10 days at 30°C were used as inoculum for subsequent experiments. For the purpose of temperature characteristic, both white‐rot fungi, Trametes trogii and T. hirsuta, were cultivated individually in plates of 9 cm in diameter with PDA medium. Each strain was inoculated centrally by employing material from one‐week‐old cultures. Triplicates of each plate were incubated in the dark at 4, 12, 25, 37 and 42°C for 21 days. The diameter of the colony was measured every 3 days.

For liquid cultivation, potato dextrose broth (PDB), malt extract medium (ME) and AN‐3 medium 27 were used. Each culture was conducted in 300 mL Erlenmeyer flasks containing 50 ml medium and three 10‐mm plugs of active fungus cultured on PDA, and incubated for 4, 7 10 or 14 days. The effects of different parameters on growth and enzyme activity were evaluated. Parameters considered were:

• growth temperature: cultivation was performed on PDB medium at 10, 25 and 35°С for 10 days;

• different media: cultivation was done in PDB, ME and AN‐3 for 4, 7 and 10 days at 25°C;

• type of cultivation: shaken (on a shaker at 150 rev min⁻1), and unshaken cultivation was carried out on PDB medium for 10 days at 25°C;

• volume ratio of medium/air ‐ 1:6, 1:10, 1:20 was achieved in 300, 500 or 1000 mL Erlenmeyer flasks containing 50 mL medium. The fungal strain was cultivated on PDB medium for 7 and 14 days at 25°C;

• inoculum size: 1, 3, 5 or 10 agar plugs (10‐mm diameter) were used for the fungal strain cultivation on PDB medium for 7 and 14 days at 25°C;

• three types of supplements, Tween 80 (0.1, 1.0, and 2.0%), glycerol (0.5, 1.0, 2.0, and 5.0%), veratryl alcohol (0.05, 0.5, and 1.0%) were added. A control was prepared by growing the fungus in the basal medium without any compounds.

All the assays were performed in triplicate.

2.3. Enzyme activities assay

Plate assay of laccase activity was determined as described by Srinivasan et al. 28. As a positive control, laccase from T. versicolor (Sigma‐Aldrich GmbH) was used. The development of an intense bluish green color around the wells was considered a positive test for laccase activity.

Lac activity was determined using ABTS (2,2'‐azinodi‐3‐ethyl‐benzothiazoline‐6‐sulphuric acid) (Sigma‐Aldrich GmbH) as a substrate 29. LiP was assayed by the veratryl alcohol oxidation method 30 and MnP through catalyses the oxidation of Mn⁺² to Mn⁺³ by H₂0₂ 30. All enzymes were expressed in enzyme units per litre i.e.U/L, where one enzyme unit is expressed as the μmol of the product formed per minute.

2.4. Measurement of biomass content

Dry weight determination was performed on samples of mycelia harvested throughout the culture period. The culture fluid was filtered through a Whatman No. 4 filter. The separated mycelia were washed twice with distilled water and dried to a constant weight at 105°C.

2.5. Statistical evaluation of the results

All experiments were triplicated and all data were analyzed using one‐way analysis of variance (ANOVA) and theTukey's test. The bars indicate standard deviation (SD) (n = 3).

3. Results

3.1. Isolation and identification of lignolytic fungal strains

The isolation procedure on Cooke agar medium gave rise to 20 fungal cultures from the forest soil samples. The isolates included Aspergillus sp., Penicillium sp., Paecilomyces sp., and two fungal strains with general macrocharacters of the Phylum Basidiomycota.

Among all fungal strains, only 4₆ and 7₁ showed Lac activity values with ABTS substrate, forming a green zone around the colonies (Fig. 1). These fungal strains were selected for further studies.

Figure 1.

Screening of fungal isolates for Lac production on PDA medium. Isolates: 1 – 4₆, 2 ‐ 7₁, 3 – 3₁₃, 4 ‐ 2₁, 5 – 3₁₂, 6 ‐ 3_18.

Using classical taxonomy based on morphology, both fungal strains with general macrocharacters of the Phylum Basidiomycota were identified as Trametes spp. Identification of species of the genus Trametes is still unsettled mainly due to the problem of separation of the species based on morphological characteristics 31. Molecular methods have been widely applied to identify a large number of them. The study of the 18S rDNA sequences can provide important complementary information for the definition of species and their appropriate identification. The molecular taxonomic affiliation of the two investigated fungal strains was performed on the basis of the comparison with 18S rDNA sequences of reference organisms published in the Gene sequence database of NCBI Data Bank. The validation of the genotypic versus the phenotypic analyses indicated that the investigated strain is closely related to the species Trametes trogii and T. hirsta.

3.2. Growth and ligninolytic enzyme production at different temperatures

Typical colony growth for T. trogii was detected at temperature ranging from 4 to 42°C, but T. hirsuta could grow within the temperature range of 12–42°C (Fig. 2A). The optimal temperature range for both strains was 35–37°C. Their mycelium expanded to the margin of 9 cm Petri dishes within 21 days at 37°C. Mycelium even could grow at 42°C, which is the lethal temperature for most filamentous fungi.

Figure 2.

(A) Colony growth of the fungal strains under different temperatures after 7 or 21 days of cultivation on agar medium: (A) T. trogii. (B) T. hirsuta. (B) Positive solid‐plate ABTS oxidation of the T. trogii and T. hirsuta incubated for 10 days at different temperatures on PDA: front view (1) and reverse view (2). The bars indicate standard deviation (SD) (n = 3).

T. trogii and T. hirsuta displayed temperature‐dependent Lac production (Fig. 2B). Both strains showed positive reactions on the indicator plates tested and showed similar results. After cultivation at 4 and 15°C, a clear green zone with an average diameter of 20 mm was observed around the inoculum block on the plate of PDA. At enhanced temperatures the green zone expanded to the margin of the dish and simultaneously turned into strong violet.

Both strains could qualify as a high extracellular ABTS oxidizing activity producer as the dark green coloration appeared in the first week of incubation and the ratio diameter of the halo/diameter of the colony was greater than 1 12.

The mycelia biomass content of liquid cultures of T. trogii and T. hirsuta varied with growth temperature (Fig. 3A). The results showed that both lignolytic strains could grow at 10°C, but very slowly. Increasing the temperature to 25 and 35°C significantly enhanced the biomass content. The maximum biomass was observed at 35°C, which was about 1.60 and 1.25 g/100 mL for T. trogii and T. hirsuta, respectively. Thus, the best biomass producer was the T. trogii strain.

Figure 3.

(A) Biomass content of liquid cultures of T. trogii and T. hirsuta at temperatures 10, 25 and 35°С. (B) Effect of temperature on ligninolytic enzyme activity of T. trogii and T. hirsuta. The bars indicate standard deviation (SD) (n = 3).

Temperature dependence of Lac, MnP and LiP production after 10 days of cultivation is shown in Fig. 3B. The results demonstrated that both strains produced all three enzymes, i.e. Lac, MnP and LiP, but there were significant differences in the ligninolytic enzyme activity. The production of Lac and MnP by T. trogii was detected at all temperature levels. The maximum of Lac activity (2 500 U/L) was produced in the flasks incubated at 25°C and dropped twofold at 35°C. In contrast, MnP and LiP showed their maximum activities at 35°C (45.6 and 18.2 U/L, respectively). The maximum ligninolytic activity detected in the culture medium of T. hirsuta was many times lower than that of T. trogii. Furthermore, optimum Lac (790 U/L), MnP (1.50 U/L) and LiP (0.65 U/L) was reached at 25°C.

3.3. Selection of medium for biomass and enzyme production

Biomass mycelial production by T. trogii and T. hirsuta on different liquid media is reported in Fig. 4. A comparative analysis of the data showed that T. trogii (Fig. 4A) formed a larger amount of biomass than T. hirsuta (Fig. 4B). In addition, development dynamics outlined an upward curve where T. trogii increased by 25–33% compared to T. hirsuta. On the other hand, the data showed the best growth on PDB, followed by ME. The medium AN‐3 turned out to be unsuitable for development of this fungus as the biomass content obtained was 3‐4‐fold lower than that on PDB. Maximum biomass production was attained on the 10^th day after inoculation of the culture in the medium.

Figure 4.

Biomass production by T. trogii and T. hirsuta in different nutrient media for 4, 7 and 10 days. The bars indicate standard deviation (SD) (n = 3).

The extracellular ligninolytic enzyme activities were found to depend on the culture medium (Table 1). The production of all three enzymes was consistently higher for cultures grown on PDB than on MEA and AN‐3. PDB proved to be the best medium for Lac production by T. trogii. The Lac activity rose dramatically during the cultivation period – from 157 to 3062 and 8776 U/L on days 4, 7 and 10, respectively. The maximum was achieved after 10 days of cultivation. LiP and MnP reached maximum levels after 7 and 4 days, respectively, followed by an abrupt threefold decrease in activity.

Table 1.

Ligninolytic enzyme production by T. trogii and T. hirsuta cultivated on different nutrient media for 4, 7 and 10 days

Enzyme/activity [U/L]	T. trogii									T. hirsuta
	PDB			МЕ			AN‐3			PDB			МЕ			AN‐3
	4 d	7 d	10 d	4 d	7 d	10 d	4 d	7 d	10 d	4 d	7 d	10 d	4 d	7 d	10 d	4 d	7 d	10 d
Lac	173	3 062	8 776	71	801	1 545	49.7	336	587	57	406	771	41	201	245	30	35	49
LiP	0.60	0.72	0.32	0.55	0.21	‐	0,22	‐	‐	1.2	2.8	12.4	0.72	0.81	0.23	‐	‐	‐
MnP	7.69	6.15	2.05	6.15	2.12	1.09	3.10	2.54	0.78	3.1	2.8	1.4	2.7	1.5	0.7	0.9	0.3	‐

The same effect of growth media on the production of Lac and MnP was established for the strain T. hirsuta, but the maximum enzyme activities were significantly lower than those in the cultures of T. trogii. In contrast, T. hirsuta demonstrated higher LiP activity compared to T. trogii (12.40 versus 0.72 U/L).

Based on the results obtained so far, it was evident that the strain T. trogii is a better producer of ligninolytic enzymes, especially Lac, than T. hirsuta.

3.4. Influence of type of cultivation

Ligninolytic enzyme production was strongly affected by the type of cultivation, shaken or unshaken (Fig. 5). Aeration provided to T. trogii for 10 days of cultivation was important for the enzyme production as well as for fungal growth. Biomass content was significantly greater in the static than in the agitated cultures. As shown in Fig. 5A., it is apparent that the static conditions led to a 1.6 – 1.8‐fold increase in biomass production compared to the shaken cultures. The growth rates were significantly greater in the unshaken cultures (0.15 g/100 mL per day) than in the shaken cultures (0.092 g/100 mL per day).

Figure 5.

Effect of type of cultivation of T. trogii on: (A) biomass content and (B) ligninolytic enzyme production. The bars indicate standard deviation (SD) (n = 3).

The type of cultivation played a major role in ligninolyic enzyme expression (Fig. 5B). Significantly higher laccase activity was observed under static cultivation than under shaking. Static cultures of T. trogii produced levels of laccase activity that were more than threefold higher than those produced by shaken cultures (6 091 versus 1 847 U/L), even though the biomass was about 1.5‐fold higher on day 10, when peak laccase activities were observed. The same dependence on aeration was established about LiP and MnP activity, with an exception of MnP activity after 10 days of cultivation. Moreover, maximal MnP activity was seen after 7 days of cultivation.

3.5. Effect of volume ratio of medium/air

The volume ratio of medium/air not only affected fungal growth of unshaken cultures but also regulated the production of ligninolytic enzymes. In all variants, T. trogii showed good biomass yield after 7 and 14 days (Fig. 6A). Although the maximum biomass content was achieved after 14 days of cultivation, aeration had a more pronounced effect on growth in the early stages of cultivation (7^th day). Variant 2 presented the optimal conditions for biomass production. However, its highest enzyme activities showed different trends (Fig. 6B). Maximum Lac activity was observed for variant 1, although no significant ratio effect was detected after 14 days of cultivation. In contrast, variant 2 provided better conditions for production of LiP and MnP. In addition, MnP activity was only detected from the cultures grown at volume ratio 1:20.

Figure 6.

Changes in biomass content (A) and ligninolytic enzyme activities (B) depending on volume ratio of medium/air. The bars indicate standard deviation (SD) (n = 3).

3.6. Influence of inoculum size

As can be seen in Fig. 7A, the growth of T. trogii did not show a significant dependence of inoculum content. Although maximum biomass production was achieved using 5 agar plugs, all tested variants showed similar biomass content.

Figure 7.

Growth (A) and ligninolytic enzyme production (B) by T. trogii depending on inoculum size. The bars indicate standard deviation (SD) (n = 3).

The inoculum size plays an important role in the production of Lac, MnP and LiP (Fig. 7B). T. trogii demonstrated high Lac and LiP activity, which increased from the 7th to 14th day. While the strain utilized 3 agar plugs for maximum activity of both enzymes (11 085 and 2.5 U/L, respectively), the highest value for the MnP (7.8 U/L) was observed with 10 agar plugs. In addition, more active MnP production was observed in the earlier period of cultivation (7th day).

3.7. Effect of inducers on ligninolytic enzyme production

The used compounds had diverse effect on enzyme production (Fig. 8). They exerted maximum inductive effect on the production of Lac and MnP, while LiP showed an apparent decrease in activity. Cultures treated with Tween 80 at 0.1 and 1.0% showed about 2.8‐ and 2.3‐fold higher Lac and MnP activity, respectively, compared with the control. At the same time, while 0.05% veratryl alcohol caused only 38% increase in Lac production, the treatment with higher concentrations (up to 1%) resulted in a remarkably higher MnP activity (2.7‐fold higher than that in the variant without treatment). Among the tested inducers, the addition of glycerol at all used concentrations in the medium proved optimal with regard to laccase enzyme production. Maximum Lac activity was detected in the medium supplemented with 5% inducer, about 3.8‐fold higher as compared to the control. However, glycerol demonstrated concentration‐dependent inhibition of LiP production.

Figure 8.

Response of ligninolytic enzyme production in the presence of: (A, B, C) ‐ Tween 80; (D, E, F) – glycerol; (H, I, J) –veratryl alcohol. A, D, H – Lac; B, E, I – LiP; C, F, J – MnP. The bars indicate standard deviation (SD) (n = 3).

4. Discussion

Literature about the potential of autochthonous fungi for ligninolytic enzyme production is relatively rare. Mycological data refer to fungal producers of these enzymes isolated from different substrates from Zimbabwe 32, Argentina 33, 34, Turkey 35 and Cuba 36, 37. There are also reports of ligninolytic enzyme production by fungi from diverse biotopes of Tunisia, Brazilian, Spain, and South Africa [see 12]. Information on the production of these enzymes from fungi isolated from Bulgarian soils is very scarce. Gochev and Krastanov 38 reported on Trichoderma spp., producer of laccase of Bulgarian origin.

In the present study, we isolated from Bulgarian forest soils two basidiomycetes belonging to the genus Trametes that are capable of producing ligninolytic enzymes. They were described as T. trogii and T. hirsuta based on their morphological and genetic characteristics. White‐rot fungi, such as species belonging to genera Phanerochaete, Phlebia, Dichomitus, Rigidoporus, Trametes, Bjerkandere, Pleurotus, Lentinus, etc., contain the best known sources of ligninolytic enzymes. 4, 39. Among the species of genus Trametes, T. hirsuta and T. trogii are considered as good producers. Although the large number of reports on effective producers, a selection of new strains capable of synthesizing significant amounts of ligninolytic enzymes evokes growing interest in research 40. Furthermore, the efficiency of degrading potential depends on the strain's ability to produce various ligninolytic enzymes. The selected strains T. hirsuta and T. trogii demonstrated lignin‐degrading activity (Lac, MnP and LiP) that is typically found in white‐rot fungi. This activity depends on growth temperature and medium. In all experiments, Lac in fungal cultures was significantly higher than that of peroxidases (MnP and LiP), which is compatible with the results of other authors [see 13]. According to Galliano et al. 41, Lac degrades lignins without synergistic cooperation with other ligninases, which has not been observed for peroxidase activity. Maybe this is the reason why good producers of Lac synthesize peroxidases to a lesser extent.

In secondary metabolic pathways such as Lac production, enzyme synthesis was not dependent on high biomass yields 42. However, in the present study laccase production was found to be highly related to mycelial growth. The used fungal strains grew well on PDB liquid medium under non‐shaking and non‐inducing conditions as can be seen by the large biomass of both cultures. The same conditions also contributed to the highest enzyme activity.

T. trogii was selected as a better producer of ligninolytic enzymes, especially Lac. This strain produced 8 776 U/L Lac after 10 days of cultivation in PDB medium at 25°C, which is compatible with the results about T. trogii CTM 10154 and CTM 10156 reported by Dhouib et al. 12. Similar data on initial Lac levels have been published about different T. trogii strains 43. Kocyigit et al. 44 selected T. trogii TEM H2 as the best Lac producer that reached a maximum level of 989.6 U/L on the 8th day of cultivation before experiments on culture condition optimization.

To improve ligninolytic enzyme production by selected strain T. trogii, further optimization of the culture conditions was carried out. Generally, agitation of the culture medium ensured uniform oxygen concentration in the whole culture volume, which plays a significant role in the growth and enzyme production by fungi. Most published research findings have revealed that agitated cultivation leads to the attainment of higher ligninoytic enzyme activities 45, 46. With the exception of the MnP activity after 10 days of cultivation, our results were negatively impacted by agitation. In addition, the increase in the volume ratio of medium/air did not affect enzyme production. This becomes especially evident for Lac production. Górska et al. 13 also suggested that the average biosynthesis of Lac by fungal strains belonging to genera Pleurotus, Lentinus, Trametes and Phanerochaete in unshaken cultures was higher than in shaken cultures. A negative effect of agitation of fungal cultures on Lac production has been reported also by other authors 47, 48, 49. This effect could be attributed to mechanical damage to the mycelium during agitation and its harmful effect on fungal growth and synthesis of enzymes as well as to changes in CO₂ concentration in the medium 13, 50.

Ligninolytic enzyme production can be controlled through the manipulation of the inoculum size. In this study, it was found that the number of used plugs from T. trogii had a slight influence on the biomass formation, but caused a more significant effect on LiP and MnP production. On the other hand, Lac activity was affected partially by the fungal inoculum size up to 5 plugs and to a lesser extent in the experiments with large inoculum (10 plugs). Presumably, after a certain limit, enzyme production decreases because of depletion of nutrients, which would result in a decrease in metabolic activity 51.

One of the most effective approaches to increase the yield is the supplementation of the nutrient medium with an appropriate inducer 45, 52. Different natural or synthetic agents have been used to induce the synthesis of ligninases 11, 13, 15, 43. This study confirms published data showing that supplemental Tween 80, veratryl alcohol and glycerol cause an increase in Lac and MnP activity in extracellular fluid of T. trogii. Among the tested substances, glycerol and Tween 80 were the best Lac inducers. A similar increase in Lac production in response to the addition of glycerol has been reported about different white‐rot fungi 22, 53. Furthermore, Tween 80 and veratryl alcohol significantly improve MnP production by T. trogii in every concentration used in the study. In agreement with these findings, both substances were described as good inducers for the production of MnP using Daedaleopsis confragosa, Fomes fomentarius, T. gibbosa, T. suaveolens and T. versicolor 52, Stereum ostrea 11, Phanerochaete chrysosporium 21. Unlike most published reports 11, 54, our results showed that veratryl alcohol slightly affected LiP activity of T. trogii, while glycerol and Tween 80 strongly reduced this activity. Similar data have been published on LiP activity of Coriolopsis gallica cultivated in the presence of Tween 80 55. Tonon and Odier 56 and Valli et al. 57 suggested that veratryl alcohol does not stimulate higher enzyme activity by inducing de novo protein synthesis, but protects the enzyme from inactivation by excess H₂O₂ produced by the fungus in the culture. Obviously, the effect of the widely used inducers on ligninolytic enzyme production depends on several factors and each strain responds in a particular way to each of these factors.

5. Concluding remarks

The present work has resulted in the identification of two autochthonous white‐rot fungal strains isolated from Bulgarian soil T. trogii and T. hirsuta, which possess the ability to synthesize different ligninolytic enzymes. T. trogii was the better producer of Lac, LiP and MnP. The production of these three enzymes was influenced by the growth temperature and medium, type of cultivation, inoculum size and presence of inducers. Maximum enzyme activities were found under static cultivation at 25°C in PDB medium supplemented with glycerol. The evaluated Lac activity was comparable to that reported in the literature for good producers of ligninolytic enzymes.

Practical application

Fungal strains, producers of ligninolytic enzymes, possess a great potential for application in several biotechnological processes including degradation of lignocellulosic biomass for food, laundry detergents, paper and pulp industries, bioremediation of chemical pollutants, etc. These fungi also play a very important role in the production of biofuels as a new alternative to pretreatment processes. Their ability to synthesize ligninolytic enzymes facilitates lignin degradation. In the present study, a more effective ligninolytic enzyme producer, Trametes trogii, was discovered, and cultivation conditions for enhanced production were optimized. The results were comparable to those of good producers published. Our findings could improve the operation of delignification. In addition, T. trogii could be a potent candidate for biotechnological production of laccase.

The authors have declared no conflict of interest.