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Oxidative enzymes activity and hydrogen peroxide production in white-rot fungi and soil-borne micromycetes co-cultures

Oxidative enzymes activity and hydrogen peroxide production in white-rot fungi and soil-borne... Fungal co-cultures appear to be advantageous for ligninolytic enzyme (LE) production compared to single fungal strains. The aims of this study were (1) to determine the type of fungal interactions in the co-cultures of two white-rot fungi (WRF, Pycnoporus sanguineus and Trametes maxima) and eight soil-borne micromycetes (SBM), (2) to determine the laccase and manganese peroxidase (MnP) activities and the hydrogen peroxide (H O ) production in two compatible fungal and 2 2 micromycetic co-cultures in submerged fermentation, and (3) to understand the effect of H O on LE production by WRF 2 2 through a dose-response bioassay. In the co-culture of SBM and Pycnoporus sanguineus, the main interaction was deadlock at a distance, whereas T. maxima showed competitive antagonism and replaced the SBM. In the agar plates, Purpureocillium lilacinum (27.8-fold increase) and Beauveria brongniartii (9.4-fold increase) enhanced the laccase and MnP activities of P. sanguineus,and Metarhizium anisopliae (Ma129) (0.83-fold increase) and Trichoderma sp. SP6 (22.6-fold increase) similarly enhanced these activities in T. maxima. In submerged fermentation, P. lilacinum also increased the laccase and MnP activities of P. sanguineus. The laccase activity of T. maxima only increased in the co-culture with B. brongniartii. The co-cultures achieved higher H O production compared to the WRF monoculture, which played a vital role in the increase of LE. The dose-response 2 2 assays revealed that low concentrations of H O (2.94 and 14.69 mM) enhance the laccase and MnP activities in WRF. 2 2 . . . Keywords Fungal co-culture Hydrogen peroxide production Laccase Manganese peroxidase Introduction cultures appears to be advantageous over the use of a single fungal strain during numerous biotechnological processes, In the last decade, the number of studies on the such as the production of LE (Pan et al. 2014), drug discovery mycoremediation of polluted water (Pan et al. 2014;Liet al. (Bertrand et al. 2014a), metabolite production (Rateb et al. 2016) and soil (Yanto and Tachibana 2014; Yanto et al. 2017) 2014), and lignin degradation (Song et al. 2011). Fungal co- using ligninolytic enzymes (LE) from fungal co-cultures, rath- cultures enable the synergistic utilization of the metabolic er than single fungal strains, has significantly increased. pathways of both species present in the co-culture (Bertrand According to Bader et al. (2010), a fungal co-culture is defined et al. 2014b). In nature, most transformations (i.e., lignin and as an anaerobic or aerobic incubation of different fungal spe- organic matter degradation) occur via the combined metabolic cies under aseptic conditions. The utilization of fungal co- pathways of different microorganisms. In this context, the application of fungal co-cultures is one potential means of producing LEs for industrial (Kumar et al. 2018), environ- * Chan-Cupul Wiberth mental (Kumar et al. 2017), and biotechnological processes wchan@ucol.mx (Rateb et al. 2014;Bawejaet al. 2016). Fungal co-culture has been reported as an effective strategy for increasing the LE activities of white-rot fungi (WRF) un- Biological Control and Applied Mycology Laboratory, Faculty of Biological and Agro-Livestock Sciences, University of Colima, der solid-state fermentation (Kuhar et al. 2015) and sub- Tecoman, Colima, Mexico merged fermentation (Díaz-Rodríguez et al. 2018). In partic- Department of Biological and Agricultural Engineering, University ular, many studies have focused on the interactions between of California Davis, Davis, CA, USA edible mushrooms (Basidiomycetes) and the micromycete ge- Micromycetes Laboratory, Institute of Ecology (INECOL A. C.), nus Trichoderma (Deuteromycetes). Initially, these co- Xalapa, Veracruz, Mexico 172 Ann Microbiol (2019) 69:171–181 cultures were studied because Trichoderma was found to be Materials and methods an antagonistic fungus of edible mushrooms (Savoie and Mata 2003; Velázquez-Cedeño et al. 2004;Mataet al. 2005; Zhang Fungal source et al. 2006;Flores et al. 2010). However, studies examining a wider range of WRF in co-cultures with soil-borne Two WRF and eight SBM were used. One of the WRF, micromycetes (SBM) and their effects of LE production are Trametes maxima, was isolated and identified by Chan- scarce (Bader et al. 2010). Cupul et al. (2016). The carpophore of another WRF, Additionally, the laccase (EC 1.10.3.2, p-diphenol oxygen Pycnoporus sanguineus, was collected in Villa de Alvarez, oxidoreductase) and manganese peroxidase (MnP, EC Colima, Mexico (location 19°30′32.70″ N, 103°63′058″ W), 1.11.1.13) produced by WRF in co-culture systems have several and isolated using the methodology of Chaparro et al. (2009). environmental, industrial, and biotechnological applications The utilized SBM were obtained as follows: Paecilomyces (Kumar et al. 2017; Kumar et al. 2018;Zhongetal. 2018). For carneus were donated, isolated, and identified by Heredia example, laccases are used to degrade endocrine disruptors and Arias (2008). Aspergillus sp., Beauveria brongniartii, (phytoestrogens, bisphenols, phthalates, antibiotics, Metarhizium anisopliae (strains Ma129 and Ma258), organohalogenated compounds, and parabens) through Penicillium hispanicum, Purpureocillium lilacinum,and mycoremediation (Barrios-Estrada et al. 2018) and are also wide- Trichoderma sp. (strain SP6) were isolated from agricultural ly used in the food (e.g., to remove phenols in wine), textile (e.g., soils in Colima, Mexico, and were reactivated in potato dex- to bleach denim fabrics), pulp and paper (e.g., to bleach pulp), trose agar (PDA) from the Fungal Culture Collection of the and biofuel (e.g., to pretreat lignocellulose materials) industries Faculty of Biological and Agricultural Sciences of Colima as well as for organic synthesis of compounds (e.g., to synthesize University. anticancer drugs and antibiotics) (Rodríguez-Couto 2018). The biotechnological applications of laccases have recently been ex- Co-cultures on agar plates plored in the field of nanobiotechnology (i.e., the creation of a laccase-based biosensor; Das et al. 2017) and biomedicine (i.e., Fungal interspecific interactions were evaluated in dual cul- the detection of insulin, morphine, and codeine; Rodríguez- ture experiments in Petri dishes (90 mm ø) containing 20 mL Couto 2018). Meanwhile, MnP has been successfully used for of modified Sivakumar (Sivakumar et al. 2010) culture medi- biopulping and bioleaching in the paper industry (Saleem et al. um containing the following (g/L): glucose (20), yeast extract 2018), for azo dye decolorization in the textile industry (Sen et al. (2.5), KH PO (1.0), (NH ) SO (0.05), MgSO (0.5), CaCl 2 4 4 2 4 4 2 2016; Zhang et al. 2018) and for the degradation of phenol com- (0.01), FeSO (0.01), MnSO (0.001), ZnSO (0.001), CuSO 4 4 4 4 pounds in bioethanol production (Rastogi and Shrivastava 2017). (0.002), and bacteriological agar (18). A 6-mm plug from the Both laccase and MnP from fungal co-cultures have been used margin of a 7-day-old culture of WRF was inoculated on the simultaneously to degrade xenobiotics in the mycoremediation border of a Petri dish to establish the co-culture; then, 5 μLof of petroleum hydrocarbons (Yanto and Tachibana 2014), indigo a SBM spore suspension (1 × 10 spores/mL) was inoculated dye (Pan et al. 2014), atrazine (Chan-Cupul et al. 2016), mala- on the other side of the Petri dish. Co-cultures were incubated chite green (Kuhar et al. 2015), sulfamethoxazole (Li et al. 2016), in the dark at 75% relative humidity (RH) and 25 °C for at and synthetic brilliant green industrial carpet dye (Kumari and least 7 days. Petri dishes inoculated with individual fungal Naraian 2016). species were used as controls. For each co-culture, five repli- Despite recent advances, a wider range of fungal spe- cates and their respective controls were used. A total of 18 co- cies needs to be studied to discover those that are capable cultures of the interactions between T. maxima or P. of enhancing LE activities in WRF. The fungal interaction sanguineus and eight SBM were studied. The methods for types between fungi in co-cultures also need to be deter- evaluating the response variables are described below. mined. In addition, the role of specific molecules such as hydrogen peroxide on LE production needs to be further Antagonism index The antagonism of each species was deter- explored (Gönen 2018). Therefore, the objectives of this mined using the rating scale proposed by Badalyan et al. study were (1) to examine the types of interspecific inter- (2002, 2004). The gross outcomes of combative interactions actions that occur between two WRF and eight SBM on can be either replacement, where one fungus gains territory agar plates in fungal co-cultures, (2) to determine the LE from the other, or deadlock, where neither fungus gains head- activities of WRF in co-cultures with SBM on agar plates, way (Boddy, 2000). Three main types of interactions (A, B, or (3) to evaluate the LE activities and H O production of C) and four subtypes (C ,C ,C ,orC ) were identified. 2 2 A1 B1 A2 B2 two compatible fungal co-cultures (WRF-SBM) in sub- Type A and B are deadlock interactions consisting of mutual merged fermentation, and (4) to understand the effect of inhibition at mycelial contact (A) or at a distance (B) wherein H O on the LE production of WRF in submerged fer- neither organism is able to overgrow the other. Type C is 2 2 mentation through a dose-response bioassay. fungal replacement and overgrowth without initial deadlock. Ann Microbiol (2019) 69:171–181 173 The intermediate subtypes are C (partial), C (complete Co-culture experiments in liquid fermentation A1 A2 replacement after initial deadlock at mycelial contact), C B1 (partial), and C (complete replacement after initial deadlock Two fungal co-cultures from the Petri dish bioassays were B2 at a distance). A score was assigned to each type or subtype of selected to study in liquid fermentation using Sivakumar cul- interaction: A = 1.0, B = 2.0, C = 3.0, C = 3.5, C = 4.0, ture medium without agar: T. maxima-B. brongniartii and P. A1 B1 C =4.5, and C = 5.0. The antagonism index (AI) was then sanguineus-P. lilacinum. Co-cultures were established in A2 B2 calculated for each species according to Badalyan et al. (2004) Erlenmeyer flasks (250 mL) with 120 mL of Sivakumar cul- using Eq. 1,where n = number (frequency) of each type or ture medium. Flasks were inoculated with three plugs of T. subtype of interaction. maxima or P. sanguineus (5 mm Ø) and three plugs of B. brongniartii or P. lilacinum (5 mm Ø); the SBM was added AI ¼ AðÞ n  1:0þ BðÞ n  2:0þ CðÞ n  3:0 3 days after inoculating the WRF. Monocultures of WRF and SBM were used as controls. Co-cultures and monocultures þ CðÞ n  3:5þ CðÞ n  4:0þ CðÞ n  4:5 A1 B1 A2 were incubated on a rotating shaker (120 rpm) at 25 °C and þ CðÞ n  5:0 ð1Þ B2 75% RH for 4 days. Five replicates were established. Culture samples were collected daily. After being centrifuged (10,000×g, for 10 min), the supernatants were used to deter- Determination of enzyme activities mine the H O production and LE activities (laccase and 2 2 MnP). The fungal enzyme extracts (FEE) were prepared by taking seven mycelial discs (6 mm Ø) from the deadlock zone of Hydrogen peroxide determination The content of H O in the 2 2 each co-culture and placing them in a test tube containing FEEs was determined using the iodide/iodate method accord- 7 mL of sterile distilled water. The test tubes were stirred for ing to Klassen et al. (1994). The blank absorbance was deter- 2 h at 120 rpm in a horizontal shaker. Subsequently, the test mined by substituting the FEE with a sterile Sivakumar cul- tubes were centrifuged at 10,000×g for 10 min. Finally, the ture medium in the reaction mixture. The content of H O was 2 2 mycelium-free supernatant was collected from each sample to then calculated by substituting with H O reagent (30%, J. T. 2 2 determine laccase and MnP activities. Baker™) according to absorbance along the standard curve at known concentrations (y = 0.0303x + 0.0067, P < 0.05, r = Laccase The protocol from Sunil et al. (2011) was used to 0.992). measure laccase activity using ABTS (2, 2′-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich®, USA) Response of ligninolytic enzymes produced as the substrate. The assay mixture contained 0.5 mM ABTS, by white-rot fungi to H O concentration 2 2 0.1 M sodium acetate buffer (pH 4.5), and a sufficient amount of FEE. ABTS oxidation was monitored at 420 nm (ε420, A dose-response assay was used to study the effect of H O on 2 2 4 −1 −1 3.6 × 10 M cm ) during 5 min. One unit was defined as laccase and MnP activities in WRF. Erlenmeyer flasks with the amount of laccase that oxidized 1 μmol of ABTS substrate Sivakumar culture medium (120 mL) were used for the mono- per minute. Laccase activity was expressed as volumetric ac- cultures of T. maxima and P. sanguineus;flasks were inocu- tivity (U/L). lated with three agar-mycelial plugs and incubated as men- tioned previously. After 3 days, solutions of H O were added 2 2 Manganese peroxidase Manganese peroxidase activity was to the flasks using a micropipette to obtain concentrations of 3+ 2+ measured by the reduction of Mn to Mn in the 2.93, 14.69, 29.39, and 293.99 mM in the culture medium. presence of phenol red (Sigma-Aldrich®, USA) accord- Five replicates per sample were used to measure the H O 2 2 ing to Glenn and Gold (1985). The oxidation of phenol concentrations; a control without H O was also included. 2 2 −1 −1 red was measured at 610 nm (ε610, 22.0 mM cm ). Laccase and MnP activities were measured for 4 days. The reaction mixture contained 0.1 mM MnSO , 0.1 mM H O , 0.01% (wt./vol) phenol red, 25 mM lac- 2 2 tate, 0.1% (wt./vol) bovine serum albumin, and 20 mM Results and discussion sodium succinate buffer (pH 4.5). The mixture was in- cubated for 5 min at room temperature. After incuba- Antagonism index in interspecific interactions tion, the reaction was ended by the addition of NaOH (80 mM). One unit of MnP activity was the amount of Table 1 shows the AI for the fungal co-cultures of the WRF, P. enzyme needed to form 1 mmol of oxidized phenol red sanguineus (Fig. 1) and T. maxima (Fig. 2), and the eight (mL/min). Manganese peroxidase activity in the FEE SBM. In the P. sanguineus co-cultures, inhibition at a distance was expressed as volumetric activity (U/L). (B) was the main type of interaction. The SBM that showed 174 Ann Microbiol (2019) 69:171–181 Table 1 Antagonism index values and competitive reactions between The evaluation of fungal interactions on agar plates is use- the white-rot fungi: Pycnoporus sanguineus and Trametes maxima and ful for selecting two compatible fungal strains and for subse- soil-borne micromycetes in co-culture quently testing their effect on the enhancement of ligninolytic Soil-borne micromycetes White-rot fungi AI enzyme activities in liquid cultures. Two fungal species that show deadlock at mycelial contact (interaction type A) are P. sanguineus T. maxima preferable in comparison to fungal interactions characterized by partial or complete replacement, as occurred with Beauveria brongniartii BA 3 Trichoderma sp. and T. maxima (C ). In a previous study, B2 Purpureocillium lilacinum BA 3 Badalyan et al. (2004)reported that Trichoderma shows com- Penicillium hispanicum BC 7 B1 petitive antagonism against xylotrophic mushrooms, such as Trichoderma sp. (SP6) C*C*7 A1 B2 Coriolus versicolor, Ganoderma lucidum, Polyporus varius, Metarhizium anisopliae (Ma 129) B C 5.5 A1 and Pleurotus ostreatus, among others. In the present study, Aspergillus sp. A C 4.5 A1 the main interactions were partial (C ) or complete (C ) A1 A2 Paecilomyces carneus BB 4 replacement following deadlock. Metarhizium anisopliae (Ma 258) B A 3 Notably, P. sanguineus wasunabletoreplace theSBM. *Situation when the SBM was able to overgrowth and replaced the WRF Some strains of P. ostreatus can replace SBM such as Trichoderma viride and Trichoderma pseudokoningii this interaction type were B. brongniartii, P. lilacinum, P. (C interaction type, Badalyan et al. 2004). In contrast, A2 hispanicum, M. anisopliae (Ma 129 and Ma 258), and P. the studied strain of Trichoderma sp. SP6 was able to par- carneus. Aspergillus sp. achieved a deadlock with P. tially replace both P. sanguineus and T. maxima;these re- sanguineus (A). Trichoderma sp. (SP6) was able to overgrow placements led to an increase in laccase and MnP activities and completely replace P. sanguineus (C ,Fig. 1). in both WRF. Dwivedi et al. (2011) reported inhibition at a A1 Trametes maxima was competitive against P. hispanicum distance between Pycnoporus sp. (MTCC 137) and (C ), partially replacing this latter species after initial dead- Penicillium oxalicum (SAU -3.510) in agar plate interac- B1 E lock at a distance. In addition, T. maxima was slightly com- tions. Specifically, the mycelial growth of Pycnoporus sp. petitive against Metarhizium anisopliae (Ma 129) and (MTCC 137) reduced while that of P. oxalicum increased. Aspergillus sp. and was able to partially replace both More recently, Arfi et al. (2013) reported on the ability of micromycetes after exhibiting deadlock at mycelial contact Pycnoporus coccineus to overgrow the mold Botrytis (C ). On the other hand, T. maxima was completely replaced cinerea as well as the ability of Coniophora puteana to A1 by Trichoderma sp. (SP6, C ). Additionally, some SBM such overgrow P. sanguineus. These fungal co-cultures led to B2 as B. brongniartii, M. anisopliae (Ma 258), and P. lilacinum an increase in the gene transcript level of 1343 (B. cinerea) appeared to be compatible with T. maxima and exhibited dead- to 4253 (C. puteana) in comparison to the monoculture of lock at mycelial contact (A). Finally, P. carneus showed dead- P. coccineus; transcripts converge toward a limited set of lock at a distance against T. maxima (B, Fig. 2). roles, including detoxification of secondary metabolites. Fig. 1 Interspecific interactions types between Pycnoporus sanguineus deadlock at distance between M. anisopliae (Ma 129) and P. sanguineus, (Ps) and soil-borne micromycetes: (I) deadlock at distance between B. (VI) deadlock at contact between P. sanguineus and Aspergillus sp. (As), brongniartii (Bb) and P. sanguineus, (II) deadlock at distance between P. (VII) deadlock at distance between P. carneus (Pc) and P. sanguineus,and lilacinum (Pl) and P. sanguineus, (III) deadlock at distance between P. (VIII) deadlock at distance between M. anisopliae (Ma 258) and P. hispanicum (Ph) and P. sanguineus, (IV) partial replacement of P. sanguineus sanguineus after a deadlock at contact with Trichoderma sp. (SP6), (V) Ann Microbiol (2019) 69:171–181 175 Fig. 2 Interspecific interactions between T. maxima (Tm) and soil-borne deadlock at mycelial contact, (V) partial replacement of M. anisopliae micromycetes: (I) inhibition at mycelial contact between B. brongniartii (Ma 129) by T. maxima, after an initial deadlock at mycelial contact, (VI) (Bb) and T. maxima, (II) inhibition at mycelial contact between P. partial replacement of Aspergillus sp. (As) by T. maxima, after an initial lilacinum (Pl) and T. maxima, (III) partial replacement of P. hispanicum deadlock at mycelial contact, (VII) inhibition at distance between P. (Ph) by T. maxima, after an initial deadlock at distance, (IV) partial carneus (Pc) and T. maxima, and (VIII) inhibition at mycelial contact replacement of T. maxima by Trichoderma sp. (SP6), after an initial between M. anisopliae (Ma 258) and T. maxima In addition, barrages of brown pigments are commonly laccase activity when co-cultured with B. brongniartii (27.6 U/ found in mycelial contact zones; these could be associated L), P. lilacinum (47.2 U/L), Trichoderma sp. (30.1 U/L), M. with ligninolytic enzyme activities, principally those of anisopliae Ma129 (26.4 U/L), and Aspergillus sp. (14.1 U/L) laccase, as well as with the formation of melanin compounds on agar plates (Table 2). In particular, P. lilacinum achieved the that protect fungal hyphae (T. maxima) from SBM attack. highest fold increase in laccase activity (27.8) when co-cultured Also, mushrooms are known to release laccase as a defensive with P. sanguineus. Therefore, this fungal co-culture was select- response against mycelial invasion. In this respect, enzymes ed for the submerged fermentation experiment. In regard to help mushrooms to adapt to antagonistic environments or en- MnP activity, both B. brongniartii (9.4 U/L) and P. lilacinum vironmental stress (Divya and Sadasivan, 2016 (5.9 U/L) significantly enhanced (F = 3.61, P = 0.0056) the MnP activity of P. sanguineus (1.0 U/L) by 7.7- and 4.5-fold, respectively (Table 2). Ligninolytic enzyme activities in agar plates The ligninolytic enzyme activity of P. sanguineus on agar plates under co-culture conditions has not been previously Pycnoporus sanguineus interactions Pycnoporus sanguineus (1.6 U/L) significantly increased (F =8.41, P =0.00001) its reported. However, Van Heerden et al. (2011) did demonstrate Table 2 Laccase and MnP activities of Pycnoporus sanguineus in co-culture with soil-borne micromycetes in agar plates Interactions Laccase MnP Vol. activity (U/L) Increase fold Vol. activity (U/L) Increase fold P. sanguineus 1.6 ± 0.2 d – 1.0 ± 0.5 c – P. sanguineus-B. brongniartii 27.6 ± 2.9 b 15.9 ± 1.78 b 9.4 ± 3.7 a 7.7 ± 3.1 a P. sanguineus-P. lilacinum 47.2 ± 5.3 a 27.8 ± 3.24 a 5.9 ± 0.9 ab 4.5 ± 0.9 ab P. sanguineus-P. hispanicum 2.8 ± 0.4 d 0.7 ± 0.24 d 2.8 ± 0.2 bc 1.6 ± 0.2 bc P. sanguineus-Trichoderma sp. 30.1 ± 5.4 b 17.4 ± 3.28 b 1.5 ± 0.3 c 0.5 ± 0.1 c P. sanguineus-M. anisopliae (Ma129) 26.4 ± 2.5 b 15.5 ± 1.52 b 3.1 ± 0.5 bc 1.8 ± 0.4 bc P. sanguineus-Aspergillus sp. 14.1 ± 0.1 c 7.6 ± 0.57 c 1.9 ± 0.2 c 0.8 ± 0.2 c P. sanguineus-P. carneus 3.7 ± 0.7 d 1.3 ± 0.41d 1.7 ± 0.4 c 0.6 ± 0.3 c P. sanguineus-M. anisopliae (Ma258) 3.8 ± 0.2 d 1.3 ± 0.11 d 2.9 ± 0.7 bc 1.7 ± 0.7 bc F= 8.41 12.65 4.02 3.61 P= 0.00001* 0.00001* 0.0017* 0.0056* *Indicates significant values Means with the same letter in row are not significantly different (LSD, P = 0.05). Vol, volumetric 176 Ann Microbiol (2019) 69:171–181 Table 3 Laccase and MnP Interactions Laccase MnP activities of Trametes maxima in co-culture with soil-borne Vol. activity (U/L) Increase fold Vol. activity (U/L) Increase fold micromycetes in agar plates T. maxima (control) 67.5 ± 4.3 bc – 1.1 ± 0.1 c – T. maxima-B. brongniartii 115.9 ± 13.2 a 0.72 ± 0.19 a 9.2 ± 1.5 b 6.8 ± 1.2 bc T. maxima-P. lilacinus 69.9 ± 4.3 b 0.06 ± 0.01 b 11.1 ± 3.0 b 8.4 ± 2.5 b T. maxima-P. hispanicum 30.4 ± 6.6 de – 3.4 ± 0.9 c 1.9 ± 0.8 cd T. maxima-Trichoderma sp. SP6 85.4 ± 13.3 b 0.55 ± 0.09 28.0 ± 4.7 a 22.6 ± 3.9 a ab T. maxima-M. anisopliae 123.7 ± 21.6 a 0.83 ± 0.31 a 2.7 ± 0.5 c 1.3 ± 0.4 d (Ma129) T. maxima-Aspergillus sp. 61.3 ± 0.8 bc – 1.5 ± 0.1 c 0.3 ± 0.1 d T. maxima-P. carneus 10.1 ± 2.1 e – 3.0 ± 0.5 c 1.5 ± 0.4 d T. maxima-M. anisopliae 39.6 ± 5.3 cd – 6.2 ± 1.3 bc 4.8 ± 1.2 (Ma258) bcd F= 13.65 3.11 17.80 16.72 P= 0.00001* 0.0559* 0.00001* 0.00001* *Indicates significant values Means with the same letter in row are not significantly different (LSD, P = 0.05). Vol, volumetric that co-cultures of P. sanguineus with Aspergillus flavipes in weight), respectively. These increases were higher than those wood chips of Acacia mearnsii, Eucalyptus dunnii, produced by the SBM. The synthesis of enzymes by fungal Eucalyptus grandis,and Eucalyptus macarthurii altered the organisms is known to differ between strains and species. For chemical composition of each tree species in different ways. example, T. versicolor did not produce MnP activity in a study Also, the authors demonstrated that the co-culture of P. conducted by Hiscox et al. (2010). However, this enzyme was sanguineus-A. flavipes resulted in a higher cellulose content induced in co-cultures of T. versicolor and its fungal compet- and lower lignin content in degrading wood chips compared to itors; the highest production was found with S. gausapatum. the monoculture of P. sanguineus (Van Heerden et al. 2008). Meanwhile, in the present study, the highest fold increase Given this context, fungal co-cultures could have relevant (22.6) was found with Trichoderma sp. SP6. More recent- applications given the ecology of lignin decomposition, al- ly, Kuhar et al. (2015) studied the fungal co-culture of two though ligninolytic enzymes were not evaluated by these pre- basidiomycetes, T. versicolor and Ganoderma lucidum,on vious authors. agar plates. Laccase activity in the fungal co-culture was 1.1 U/g, representing a 1.75-fold increase in comparison to Trametes maxima interactions Trametes maxima (67.5 U/L) the monocultures of T. versicolor (0.4 U/g) and G. lucidum significantly increased (F =13.65, P = 0.00001) its laccase ac- (0.1 U/g). tivity when co-cultured with B. brongniartii (115.9 U/L) and M. anisopliae Ma129 (123.7 U/L) by 0.72- and 0.83-fold, Co-culture experiments in liquid fermentation respectively (Table 3). In addition, three species of SBM, B. brongniartii (9.2 U/L), P. lilacinum (11.1 U/L), and Co-culture of Pycnoporus sanguineus and Purpureocillium Trichoderma sp. SP6 (28.0 U/L) were able to significantly lilacinum Purpureocillium lilacinum significantly increased enhance (P < 0.05) the MnP activity of T. maxima (1.0 U/L). the laccase activity of P. sanguineus (48 h = 0.91 U/L and The highest fold increases in the MnP activity of T. maxima 96 h = 0.73 U/L) at 48 (t = − 4.7897, P = 0.0030) and 96 h were 8.4 and 22.6, which were caused by P. lilacinum and (t =2.1057, P = 0.0399) of fermentation by 2.09- (2.81 U/L) Trichoderma sp. SP6, respectively (Table 3). and 2.64-fold (2.69 U/L), respectively (Fig. 3A). Meanwhile, Studies of fungal co-cultures using Trametes sp. are scarce. MnP activity was strongly and significantly increased in the In a previous study, Hiscox et al. (2010) co-cultured Trametes co-culture with P. lilacinum at 48 h. Specifically, in the co- versicolor (216 mU/wet weight) with other basidiomycetes on culture, the laccase activity at 48 (t = − 8.4065, P =0.0001), −07 agar plates. The results indicated that Stereum gausapatum, 72 (t = − 28.4098, P =1.25 ), and 96 h (t = − 50.7384, P = −09 Daldinia concentrica, Bjerkandera adusta,and Hypholoma 3.93 ) was 24.19, 58.11, and 61.89 U/L, respectively; in the fasciculare enhanced the laccase activity of T. versicolor by monoculture, the laccase activity was 9.49 (48 h), 5.90 (72 h), 139- (3218.9 mU/wet weight), 3.06- (877.8 mU/wet weight), and 5.04 U/L (96 h, Fig. 3B). Thus, the laccase activity of the 3.03- (871.6 mU/wet weight), and 0.93-fold (417.1 mU/wet co-cultures increased by 1.55- (48 h), 8.83- (72 h), and 11.27- Ann Microbiol (2019) 69:171–181 177 Fig. 3 Laccase (A and D) and MnP (B and E) activities, and H O production (C and F) in two fungal co-cultures: Pycnoporus sanguineus- 2 2 Purpureocillium lilacinum (A, B, and C) and Trametes maxima-Beauveria brongniartii (D, E, and F) fold (96 h). The H O content of the fungal co-culture extract by 2.08- and 1.33-fold at 48 and 96 h of fermentation, 2 2 was higher during the evaluation period compared to the respectively. monoculture extracts (Fig. 3C). Specifically, the H O values 2 2 in the co-culture ranged from 4.90 (t = − 3.2046, P =0.0184) Co-culture of Trametes maxima and Beauveria brongniartii to 7.97 mg/L (t = − 3.5567, P = 0.0119), while those of the The laccase activity of T. maxima increased in the presence of monocultures ranged from 0.78 to 1.50 mg/L. Beauveria brongniartii (24 h = 100.17 U/L and 48 = 87.77 U/ However, in other studies, the laccase activities of P. L) at 24 (t = − 3.1653, P = 0.0194) and 48 h (t = − 7.9183, P = sanguineus in monocultures were variable. Eugenio-Eugenio 0.0002) of fermentation. The laccase activities of the co- et al. (2009) and Vikineswary et al. (2006)reported values of cultures were 113.87 and 129.66 U/L at 24 and 48 h, respec- 320 mU/L (0.32 U/L) in submerged fermentation and < 5.0 U/ tively, while the monoculture achieved 100.17 and 87.77 U/L g of the substrate in solid-state fermentation, respectively. in the same time frame (Fig. 4D). The MnP activity of T. These values are low (Eugenio-Eugenio et al., 2009) and high maxima during the co-culture was not enhanced by B. (Vikineswary et al. 2006) with respect to those found for the brongniartii (Fig. 2E). The co-culture achieved the highest co-culture of P. sanguineus and P. lilacinum (2.69 U/L), re- levels of H O at 48 (11.16 mg/L, t = − 7.1149, P =0.0003) 2 2 spectively. However, few data are available on P. sanguineus and 72 h (15.71 mg/L, t = − 4.7665, P = 0.0031) in compari- under co-culture conditions. Baldrian (2004) reported that son to those of the T. maxima monoculture (48 h = 6.85 mg/L Trichoderma harzianum was able to increase the laccase ac- and 72 h = 13.40 mg/L). tivity of P. sanguineus by 1.5-fold in liquid fermentation. Trametes (synonym of Cerrena) maxima was previously Meanwhile, in the present study, laccase activity increased studied in co-culture with the basidiomycete Coriolus 178 Ann Microbiol (2019) 69:171–181 Response of enzymes produced by white-rot fungi to H O concentration 2 2 At 24 h of exposure to 14.69 mM (531.7 U/L) of H O , T. 2 2 maxima (430.0 U/L) significantly (F = 59.78, P = 0.00001) enhanced its laccase activity by 0.23-fold. However, the highest concentration of H O (293.99 mM) strongly 2 2 inhibited laccase activity in T. maxima (Fig. 4A). At 48 (F = 24.99, P =0.00001) and 72 h (F =35.35, P =0.00001) of ex- posure, both 2.93 and 14.69 mM of H O led to significant 2 2 increases in the laccase activity of T. maxima; the correspond- ing laccase values were 1182.0 and 1122.5 U/L, respectively, representing 0.46- (at 2.93 mM) and 0.39-fold (at 14.69 mM) increases, respectively. However, the highest H O concentra- 2 2 tion caused strong inhibition of laccase activity at 48 and 72 h, corresponding with laccase activities of 11.3 and 28.1 U/L, respectively. At the end of the evaluation period, the laccase activity did not simply increase with increasing H O concen- 2 2 tration. The highest values of laccase activity ranged from 735.7 to 831.1 U/L, although the highest concentrations of H O reduced the laccase activity to 32.6 U/L (Fig. 4A). 2 2 In regard to MnP activity (Fig. 4B), T. maxima significantly Fig. 4 Laccase (A) and MnP (B) activities of Trametes maxima under enhanced (F = 110.31, P = 0.00001) its MnP activity by 0.51- stress condition by hydrogen peroxide concentrations fold at 24 h of exposure to 14.69 mM of H O . Both 29.39 and 2 2 293.99 mM of H O reduced the MnP activity of T. maxima 2 2 from 3.35 U/L to 0.83 and 1.18 U/L, respectively (Fig. 2B). At hirsutus. In this case, both strains produced laccase at rates of 48 h, MnP activity was not increased by the H O concentra- 2 2 approximately 63.0 and 415.0 nkatal/L respectively, in mono- tions; instead, H O concentrations of 14.69, 29.39, and 2 2 cultures. However, these values were low compared to those 293.99 mM significantly inhibited MnP activity, leading to of the co-culture of both species (510 nkatal/L, Koroleva et al. decreases of 11.75 U/L to 5.40, 3.75, and 0.73 U/L, respec- 2002). The levels of laccase achieved in the co-culture of these tively. At 72 and 96 h, the H O concentrations of 2.93 and 2 2 two strains represented a 7.1- and 0.22-fold increase compared 14.69 mM were able to increase the MnP activities by 0.51- to the monocultures of Cucurbita maxima and C. hirsutus, and 0.93-fold (at 72 h) and by 0.28- and 0.44-fold (96 h), respectively. Baldrian (2004) reported that the SBM T. respectively. As previously occurred, both 29.39 and harzianum, Acremonium sphaerospermum, Fusarium 293.99 mM of H O inhibited the MnP activity of T. maxima 2 2 reticulatum, Humicola grisea,and Penicillium rigulosum (Fig. 2B). were able to increase the laccase activity of T. versicolor under Pycnoporus sanguineus exhibited less laccase activity liquid fermentation by 9-, 14-, 16.7-, 16-, and 55.6-fold, re- than T. maxima.However, P. sanguineus significantly in- spectively, after 3 days of interaction. creased its laccase activity under H O stress. At 24 h of 2 2 Contrary to Koroleva et al. (2002), Baldrian (2004), and the exposure, laccase activity in the P. sanguineus control was present study, Xiao et al. (2004)reported that Trametes sp. absent. However, the H O concentrations of 14.69 mM 2 2 AH28-2 reduced its laccase activity by 30% and 11% when and 29.39 mM activated laccase synthesis by P. co-cultured with Aspergillus oryzae and Gloeophyllum sanguineus, leading to a laccase production of 4.15 and trabeum, respectively. More recently, Díaz-Rodríguez et al. 5.94 U/L respectively (Fig. 5A).At48 h,theH O con- 2 2 (2018) reported that a non-laccase producing soil microfungi centrations of 14.69, 29.39, and 293.99 mM enhanced the Penicillium commune GHAIE86 could increase the laccase laccase activity of P. sanguineus (15.3U/L)by1.2- activity of Funalia floccosa LPSC232 (20 mU/mL) by 3- (34.9 U/L), 3.1- (63.1 U/L), and 1.5-fold (39.2 U/L). At fold (60 mU/mL) under submerged fermentation; in addition, 72 h, the H O concentrations of 29.39 and 293.99 mM 2 2 P. commune was able to induce two laccase isoenzymes in F. enhanced the laccase activity of P. sanguineus (17.32 U/ floccosa. Interestingly, one function of SBM species appears L) by 68.8- (1210.5 U/L) and 36.3-fold (645.4 U/L). By to be the enhancement of laccase activity in WRF. However, the end of the experiment (96 h), 2.93 mM of H O en- 2 2 the mechanisms behind and the reasons for this phenomenon hanced the laccase activity of P. sanguineus (56.12 U/L) are still under discussion. by 14.71-fold (882.0 U/L, Fig. 5A). Ann Microbiol (2019) 69:171–181 179 binding then modifies the absorbance of laccase and facilitates the removal of fluoride (a xenobiotic compound). Furthermore, the catalytic activity of MnP is well known to be dependent on H O ; however, this enzyme is also 2 2 inactivated by excess H O .According toBermeketal. 2 2 (2002), the oxidation of native MnP by an equivalent amount of H O forms active compound I, which subsequently oxi- 2 2 2+ 3+ dizes Mn to Mn . In this process, compound II is formed and reduced back to the native MnP, oxidizing an additional 2+ 3+ 2+ Mn to Mn .Mn is a mandatory substrate for compound II. When excess concentrations of H O are present, native 2 2 MnP is directly converted to compound III, which is an inac- tive form of the enzyme. In addition, Li et al. (1995)demon- strated that the white-rot fungus Phanerochaete chrysosporium produces MnP mRNA following the addition 2+ of a low amount of H O in the absence of Mn , the natural 2 2 substrate of MnP. These responses were observed in this study, as low amounts of H O (2.93 and 14.69 mM) stimu- 2 2 lated MnP activity, whereas high amounts (29.39 and 293.99 mM) inhibited MnP activity. The source of H O in co-culture systems is unclear and 2 2 will be the target of future studies. However, both WRF and Fig. 5 Laccase (A) and MnP (B) activities of Pycnoporus sanguineus SBM are able to produce different levels of H O via distinct under stress condition by hydrogen peroxide concentrations 2 2 metabolic pathways. Similarly, Urzúa et al. (1998)reported that Ceriporiopsis subvermispora is able to generate H O 2 2 In regard to MnP activity (Fig. 5B), at 24 h, the H O through the oxidation of the organic acids that it secretes. 2 2 concentrations of 2.4 (8.7 U/L), 14.69 (11.1 U/L), and Also, SBM are known to produce H O as a result of glucose 2 2 293.99 (8.8 U/L) mM significantly (F = 13.86, P = 0.0004) degradation by glucose oxidase (GOx), especially under the enhanced the MnP activity of P. sanguineus (1.5 U/L) by stress conditions caused by xenobiotics, as reported by 4.9-, 6.89-, and 5.0-fold, respectively. At 48 h, only Zúñiga-Silva et al. (2016). 14.39 mM of H O was required to significantly increase 2 2 MnP activity (F =4.64, P = 0.0224) by 1.8-fold. However, at 72 h, H O concentrations of 2.93, 14.69, and 29.39 mM were Conclusions 2 2 required to achieve 3.0-, 12.8-, and 1.0-fold increases, respec- tively, in MnP activity, corresponding with MnP activities of In this study, the interspecific interactions and enzyme activ- 11.29, 38.40, and 5.68 U/L, respectively. Meanwhile, the con- ities of the co-cultures of two WRF and eight SBM were trol only achieved a MnP activity of 2.8 U/L. Finally, at the evaluated in addition to the effects of H O on WRF. The main 2 2 end of evaluation period (96 h), both 2.93 (27.8 U/L) and type of interspecific interaction exhibited by the WRF 14.69 (44.9 U/L) mM of H O significantly increased (F = Pycnoporus sanguineus was deadlock at a distance, while T. 2 2 15.01, P = 0.0003) the MnP activity of P. sanguineus (5.9 U/ maxima partially replaced three of the studied SBM, L), corresponding with 3.7- and 8.5-fold increases, respective- confirming its dominance. Purpureocillium lilacinum en- ly (Fig. 5B). hanced to a large extent the laccase activity of P. sanguineus, Hydrogen peroxide differentially increased the laccase ac- and B. brongniartii was the principal enhancer of the MnP tivity of the WRF. This suggests that H O could be respon- activity of P. sanguineus.Both B. brongniartii and M. 2 2 sible for the increase in laccase and MnP activities in fungal anisopliae (Ma129) were the best enhancers of laccase activ- co-cultures, as the H O in fungal extracts from both the T. ity in T. maxima,while Trichoderma sp. (SP6) increased the 2 2 maxima-B. brongniartii and the P. sanguineus-P. lilacinum MnP activity of T. maxima to a large extent. In submerged co-cultures was high in comparison to the monocultures of fermentation, P. sanguineus increased its laccase and MnP both WRF. In fungal organisms, H O plays an important activity in co-culture with P. lilacinum. Meanwhile, T. maxima 2 2 role and influences enzymes activity. Branden et al. (1971) did not increase its MnP activity but did slightly increase its studied the interaction of a fungal laccase with H O and 2 2 laccase activity at the initial evaluation points. Finally, the co- found that H O molecules are able to bind to one specific cultures P. sanguineus-P. lilacinum and T. maxima-B. 2 2 2+ Cu of the four copper atoms of the laccase molecule. This brongniartii achieved higher H O production in comparison 2 2 180 Ann Microbiol (2019) 69:171–181 Biotechnol Adv 32:1180–1204. https://doi.org/10.1016/j. to the WRF monocultures, suggesting that H O plays an 2 2 biotechadv.2014.03.001 essential role in increasing ligninolytic enzyme activities. Bertrand S, Schumpp O, Bohni N, Monod M, Gindro K, Jean-Luc W This conclusion was also confirmed by the dose-response as- (2014b) De novo production of metabolites by fungal co-cultures of says, wherein low concentrations of H O (2.94 and Trichophyton rubrum and Bionectria ochroleuca. 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Oxidative enzymes activity and hydrogen peroxide production in white-rot fungi and soil-borne micromycetes co-cultures

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Springer Journals
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Copyright © 2018 by Università degli studi di Milano
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-018-1413-4
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Abstract

Fungal co-cultures appear to be advantageous for ligninolytic enzyme (LE) production compared to single fungal strains. The aims of this study were (1) to determine the type of fungal interactions in the co-cultures of two white-rot fungi (WRF, Pycnoporus sanguineus and Trametes maxima) and eight soil-borne micromycetes (SBM), (2) to determine the laccase and manganese peroxidase (MnP) activities and the hydrogen peroxide (H O ) production in two compatible fungal and 2 2 micromycetic co-cultures in submerged fermentation, and (3) to understand the effect of H O on LE production by WRF 2 2 through a dose-response bioassay. In the co-culture of SBM and Pycnoporus sanguineus, the main interaction was deadlock at a distance, whereas T. maxima showed competitive antagonism and replaced the SBM. In the agar plates, Purpureocillium lilacinum (27.8-fold increase) and Beauveria brongniartii (9.4-fold increase) enhanced the laccase and MnP activities of P. sanguineus,and Metarhizium anisopliae (Ma129) (0.83-fold increase) and Trichoderma sp. SP6 (22.6-fold increase) similarly enhanced these activities in T. maxima. In submerged fermentation, P. lilacinum also increased the laccase and MnP activities of P. sanguineus. The laccase activity of T. maxima only increased in the co-culture with B. brongniartii. The co-cultures achieved higher H O production compared to the WRF monoculture, which played a vital role in the increase of LE. The dose-response 2 2 assays revealed that low concentrations of H O (2.94 and 14.69 mM) enhance the laccase and MnP activities in WRF. 2 2 . . . Keywords Fungal co-culture Hydrogen peroxide production Laccase Manganese peroxidase Introduction cultures appears to be advantageous over the use of a single fungal strain during numerous biotechnological processes, In the last decade, the number of studies on the such as the production of LE (Pan et al. 2014), drug discovery mycoremediation of polluted water (Pan et al. 2014;Liet al. (Bertrand et al. 2014a), metabolite production (Rateb et al. 2016) and soil (Yanto and Tachibana 2014; Yanto et al. 2017) 2014), and lignin degradation (Song et al. 2011). Fungal co- using ligninolytic enzymes (LE) from fungal co-cultures, rath- cultures enable the synergistic utilization of the metabolic er than single fungal strains, has significantly increased. pathways of both species present in the co-culture (Bertrand According to Bader et al. (2010), a fungal co-culture is defined et al. 2014b). In nature, most transformations (i.e., lignin and as an anaerobic or aerobic incubation of different fungal spe- organic matter degradation) occur via the combined metabolic cies under aseptic conditions. The utilization of fungal co- pathways of different microorganisms. In this context, the application of fungal co-cultures is one potential means of producing LEs for industrial (Kumar et al. 2018), environ- * Chan-Cupul Wiberth mental (Kumar et al. 2017), and biotechnological processes wchan@ucol.mx (Rateb et al. 2014;Bawejaet al. 2016). Fungal co-culture has been reported as an effective strategy for increasing the LE activities of white-rot fungi (WRF) un- Biological Control and Applied Mycology Laboratory, Faculty of Biological and Agro-Livestock Sciences, University of Colima, der solid-state fermentation (Kuhar et al. 2015) and sub- Tecoman, Colima, Mexico merged fermentation (Díaz-Rodríguez et al. 2018). In partic- Department of Biological and Agricultural Engineering, University ular, many studies have focused on the interactions between of California Davis, Davis, CA, USA edible mushrooms (Basidiomycetes) and the micromycete ge- Micromycetes Laboratory, Institute of Ecology (INECOL A. C.), nus Trichoderma (Deuteromycetes). Initially, these co- Xalapa, Veracruz, Mexico 172 Ann Microbiol (2019) 69:171–181 cultures were studied because Trichoderma was found to be Materials and methods an antagonistic fungus of edible mushrooms (Savoie and Mata 2003; Velázquez-Cedeño et al. 2004;Mataet al. 2005; Zhang Fungal source et al. 2006;Flores et al. 2010). However, studies examining a wider range of WRF in co-cultures with soil-borne Two WRF and eight SBM were used. One of the WRF, micromycetes (SBM) and their effects of LE production are Trametes maxima, was isolated and identified by Chan- scarce (Bader et al. 2010). Cupul et al. (2016). The carpophore of another WRF, Additionally, the laccase (EC 1.10.3.2, p-diphenol oxygen Pycnoporus sanguineus, was collected in Villa de Alvarez, oxidoreductase) and manganese peroxidase (MnP, EC Colima, Mexico (location 19°30′32.70″ N, 103°63′058″ W), 1.11.1.13) produced by WRF in co-culture systems have several and isolated using the methodology of Chaparro et al. (2009). environmental, industrial, and biotechnological applications The utilized SBM were obtained as follows: Paecilomyces (Kumar et al. 2017; Kumar et al. 2018;Zhongetal. 2018). For carneus were donated, isolated, and identified by Heredia example, laccases are used to degrade endocrine disruptors and Arias (2008). Aspergillus sp., Beauveria brongniartii, (phytoestrogens, bisphenols, phthalates, antibiotics, Metarhizium anisopliae (strains Ma129 and Ma258), organohalogenated compounds, and parabens) through Penicillium hispanicum, Purpureocillium lilacinum,and mycoremediation (Barrios-Estrada et al. 2018) and are also wide- Trichoderma sp. (strain SP6) were isolated from agricultural ly used in the food (e.g., to remove phenols in wine), textile (e.g., soils in Colima, Mexico, and were reactivated in potato dex- to bleach denim fabrics), pulp and paper (e.g., to bleach pulp), trose agar (PDA) from the Fungal Culture Collection of the and biofuel (e.g., to pretreat lignocellulose materials) industries Faculty of Biological and Agricultural Sciences of Colima as well as for organic synthesis of compounds (e.g., to synthesize University. anticancer drugs and antibiotics) (Rodríguez-Couto 2018). The biotechnological applications of laccases have recently been ex- Co-cultures on agar plates plored in the field of nanobiotechnology (i.e., the creation of a laccase-based biosensor; Das et al. 2017) and biomedicine (i.e., Fungal interspecific interactions were evaluated in dual cul- the detection of insulin, morphine, and codeine; Rodríguez- ture experiments in Petri dishes (90 mm ø) containing 20 mL Couto 2018). Meanwhile, MnP has been successfully used for of modified Sivakumar (Sivakumar et al. 2010) culture medi- biopulping and bioleaching in the paper industry (Saleem et al. um containing the following (g/L): glucose (20), yeast extract 2018), for azo dye decolorization in the textile industry (Sen et al. (2.5), KH PO (1.0), (NH ) SO (0.05), MgSO (0.5), CaCl 2 4 4 2 4 4 2 2016; Zhang et al. 2018) and for the degradation of phenol com- (0.01), FeSO (0.01), MnSO (0.001), ZnSO (0.001), CuSO 4 4 4 4 pounds in bioethanol production (Rastogi and Shrivastava 2017). (0.002), and bacteriological agar (18). A 6-mm plug from the Both laccase and MnP from fungal co-cultures have been used margin of a 7-day-old culture of WRF was inoculated on the simultaneously to degrade xenobiotics in the mycoremediation border of a Petri dish to establish the co-culture; then, 5 μLof of petroleum hydrocarbons (Yanto and Tachibana 2014), indigo a SBM spore suspension (1 × 10 spores/mL) was inoculated dye (Pan et al. 2014), atrazine (Chan-Cupul et al. 2016), mala- on the other side of the Petri dish. Co-cultures were incubated chite green (Kuhar et al. 2015), sulfamethoxazole (Li et al. 2016), in the dark at 75% relative humidity (RH) and 25 °C for at and synthetic brilliant green industrial carpet dye (Kumari and least 7 days. Petri dishes inoculated with individual fungal Naraian 2016). species were used as controls. For each co-culture, five repli- Despite recent advances, a wider range of fungal spe- cates and their respective controls were used. A total of 18 co- cies needs to be studied to discover those that are capable cultures of the interactions between T. maxima or P. of enhancing LE activities in WRF. The fungal interaction sanguineus and eight SBM were studied. The methods for types between fungi in co-cultures also need to be deter- evaluating the response variables are described below. mined. In addition, the role of specific molecules such as hydrogen peroxide on LE production needs to be further Antagonism index The antagonism of each species was deter- explored (Gönen 2018). Therefore, the objectives of this mined using the rating scale proposed by Badalyan et al. study were (1) to examine the types of interspecific inter- (2002, 2004). The gross outcomes of combative interactions actions that occur between two WRF and eight SBM on can be either replacement, where one fungus gains territory agar plates in fungal co-cultures, (2) to determine the LE from the other, or deadlock, where neither fungus gains head- activities of WRF in co-cultures with SBM on agar plates, way (Boddy, 2000). Three main types of interactions (A, B, or (3) to evaluate the LE activities and H O production of C) and four subtypes (C ,C ,C ,orC ) were identified. 2 2 A1 B1 A2 B2 two compatible fungal co-cultures (WRF-SBM) in sub- Type A and B are deadlock interactions consisting of mutual merged fermentation, and (4) to understand the effect of inhibition at mycelial contact (A) or at a distance (B) wherein H O on the LE production of WRF in submerged fer- neither organism is able to overgrow the other. Type C is 2 2 mentation through a dose-response bioassay. fungal replacement and overgrowth without initial deadlock. Ann Microbiol (2019) 69:171–181 173 The intermediate subtypes are C (partial), C (complete Co-culture experiments in liquid fermentation A1 A2 replacement after initial deadlock at mycelial contact), C B1 (partial), and C (complete replacement after initial deadlock Two fungal co-cultures from the Petri dish bioassays were B2 at a distance). A score was assigned to each type or subtype of selected to study in liquid fermentation using Sivakumar cul- interaction: A = 1.0, B = 2.0, C = 3.0, C = 3.5, C = 4.0, ture medium without agar: T. maxima-B. brongniartii and P. A1 B1 C =4.5, and C = 5.0. The antagonism index (AI) was then sanguineus-P. lilacinum. Co-cultures were established in A2 B2 calculated for each species according to Badalyan et al. (2004) Erlenmeyer flasks (250 mL) with 120 mL of Sivakumar cul- using Eq. 1,where n = number (frequency) of each type or ture medium. Flasks were inoculated with three plugs of T. subtype of interaction. maxima or P. sanguineus (5 mm Ø) and three plugs of B. brongniartii or P. lilacinum (5 mm Ø); the SBM was added AI ¼ AðÞ n  1:0þ BðÞ n  2:0þ CðÞ n  3:0 3 days after inoculating the WRF. Monocultures of WRF and SBM were used as controls. Co-cultures and monocultures þ CðÞ n  3:5þ CðÞ n  4:0þ CðÞ n  4:5 A1 B1 A2 were incubated on a rotating shaker (120 rpm) at 25 °C and þ CðÞ n  5:0 ð1Þ B2 75% RH for 4 days. Five replicates were established. Culture samples were collected daily. After being centrifuged (10,000×g, for 10 min), the supernatants were used to deter- Determination of enzyme activities mine the H O production and LE activities (laccase and 2 2 MnP). The fungal enzyme extracts (FEE) were prepared by taking seven mycelial discs (6 mm Ø) from the deadlock zone of Hydrogen peroxide determination The content of H O in the 2 2 each co-culture and placing them in a test tube containing FEEs was determined using the iodide/iodate method accord- 7 mL of sterile distilled water. The test tubes were stirred for ing to Klassen et al. (1994). The blank absorbance was deter- 2 h at 120 rpm in a horizontal shaker. Subsequently, the test mined by substituting the FEE with a sterile Sivakumar cul- tubes were centrifuged at 10,000×g for 10 min. Finally, the ture medium in the reaction mixture. The content of H O was 2 2 mycelium-free supernatant was collected from each sample to then calculated by substituting with H O reagent (30%, J. T. 2 2 determine laccase and MnP activities. Baker™) according to absorbance along the standard curve at known concentrations (y = 0.0303x + 0.0067, P < 0.05, r = Laccase The protocol from Sunil et al. (2011) was used to 0.992). measure laccase activity using ABTS (2, 2′-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich®, USA) Response of ligninolytic enzymes produced as the substrate. The assay mixture contained 0.5 mM ABTS, by white-rot fungi to H O concentration 2 2 0.1 M sodium acetate buffer (pH 4.5), and a sufficient amount of FEE. ABTS oxidation was monitored at 420 nm (ε420, A dose-response assay was used to study the effect of H O on 2 2 4 −1 −1 3.6 × 10 M cm ) during 5 min. One unit was defined as laccase and MnP activities in WRF. Erlenmeyer flasks with the amount of laccase that oxidized 1 μmol of ABTS substrate Sivakumar culture medium (120 mL) were used for the mono- per minute. Laccase activity was expressed as volumetric ac- cultures of T. maxima and P. sanguineus;flasks were inocu- tivity (U/L). lated with three agar-mycelial plugs and incubated as men- tioned previously. After 3 days, solutions of H O were added 2 2 Manganese peroxidase Manganese peroxidase activity was to the flasks using a micropipette to obtain concentrations of 3+ 2+ measured by the reduction of Mn to Mn in the 2.93, 14.69, 29.39, and 293.99 mM in the culture medium. presence of phenol red (Sigma-Aldrich®, USA) accord- Five replicates per sample were used to measure the H O 2 2 ing to Glenn and Gold (1985). The oxidation of phenol concentrations; a control without H O was also included. 2 2 −1 −1 red was measured at 610 nm (ε610, 22.0 mM cm ). Laccase and MnP activities were measured for 4 days. The reaction mixture contained 0.1 mM MnSO , 0.1 mM H O , 0.01% (wt./vol) phenol red, 25 mM lac- 2 2 tate, 0.1% (wt./vol) bovine serum albumin, and 20 mM Results and discussion sodium succinate buffer (pH 4.5). The mixture was in- cubated for 5 min at room temperature. After incuba- Antagonism index in interspecific interactions tion, the reaction was ended by the addition of NaOH (80 mM). One unit of MnP activity was the amount of Table 1 shows the AI for the fungal co-cultures of the WRF, P. enzyme needed to form 1 mmol of oxidized phenol red sanguineus (Fig. 1) and T. maxima (Fig. 2), and the eight (mL/min). Manganese peroxidase activity in the FEE SBM. In the P. sanguineus co-cultures, inhibition at a distance was expressed as volumetric activity (U/L). (B) was the main type of interaction. The SBM that showed 174 Ann Microbiol (2019) 69:171–181 Table 1 Antagonism index values and competitive reactions between The evaluation of fungal interactions on agar plates is use- the white-rot fungi: Pycnoporus sanguineus and Trametes maxima and ful for selecting two compatible fungal strains and for subse- soil-borne micromycetes in co-culture quently testing their effect on the enhancement of ligninolytic Soil-borne micromycetes White-rot fungi AI enzyme activities in liquid cultures. Two fungal species that show deadlock at mycelial contact (interaction type A) are P. sanguineus T. maxima preferable in comparison to fungal interactions characterized by partial or complete replacement, as occurred with Beauveria brongniartii BA 3 Trichoderma sp. and T. maxima (C ). In a previous study, B2 Purpureocillium lilacinum BA 3 Badalyan et al. (2004)reported that Trichoderma shows com- Penicillium hispanicum BC 7 B1 petitive antagonism against xylotrophic mushrooms, such as Trichoderma sp. (SP6) C*C*7 A1 B2 Coriolus versicolor, Ganoderma lucidum, Polyporus varius, Metarhizium anisopliae (Ma 129) B C 5.5 A1 and Pleurotus ostreatus, among others. In the present study, Aspergillus sp. A C 4.5 A1 the main interactions were partial (C ) or complete (C ) A1 A2 Paecilomyces carneus BB 4 replacement following deadlock. Metarhizium anisopliae (Ma 258) B A 3 Notably, P. sanguineus wasunabletoreplace theSBM. *Situation when the SBM was able to overgrowth and replaced the WRF Some strains of P. ostreatus can replace SBM such as Trichoderma viride and Trichoderma pseudokoningii this interaction type were B. brongniartii, P. lilacinum, P. (C interaction type, Badalyan et al. 2004). In contrast, A2 hispanicum, M. anisopliae (Ma 129 and Ma 258), and P. the studied strain of Trichoderma sp. SP6 was able to par- carneus. Aspergillus sp. achieved a deadlock with P. tially replace both P. sanguineus and T. maxima;these re- sanguineus (A). Trichoderma sp. (SP6) was able to overgrow placements led to an increase in laccase and MnP activities and completely replace P. sanguineus (C ,Fig. 1). in both WRF. Dwivedi et al. (2011) reported inhibition at a A1 Trametes maxima was competitive against P. hispanicum distance between Pycnoporus sp. (MTCC 137) and (C ), partially replacing this latter species after initial dead- Penicillium oxalicum (SAU -3.510) in agar plate interac- B1 E lock at a distance. In addition, T. maxima was slightly com- tions. Specifically, the mycelial growth of Pycnoporus sp. petitive against Metarhizium anisopliae (Ma 129) and (MTCC 137) reduced while that of P. oxalicum increased. Aspergillus sp. and was able to partially replace both More recently, Arfi et al. (2013) reported on the ability of micromycetes after exhibiting deadlock at mycelial contact Pycnoporus coccineus to overgrow the mold Botrytis (C ). On the other hand, T. maxima was completely replaced cinerea as well as the ability of Coniophora puteana to A1 by Trichoderma sp. (SP6, C ). Additionally, some SBM such overgrow P. sanguineus. These fungal co-cultures led to B2 as B. brongniartii, M. anisopliae (Ma 258), and P. lilacinum an increase in the gene transcript level of 1343 (B. cinerea) appeared to be compatible with T. maxima and exhibited dead- to 4253 (C. puteana) in comparison to the monoculture of lock at mycelial contact (A). Finally, P. carneus showed dead- P. coccineus; transcripts converge toward a limited set of lock at a distance against T. maxima (B, Fig. 2). roles, including detoxification of secondary metabolites. Fig. 1 Interspecific interactions types between Pycnoporus sanguineus deadlock at distance between M. anisopliae (Ma 129) and P. sanguineus, (Ps) and soil-borne micromycetes: (I) deadlock at distance between B. (VI) deadlock at contact between P. sanguineus and Aspergillus sp. (As), brongniartii (Bb) and P. sanguineus, (II) deadlock at distance between P. (VII) deadlock at distance between P. carneus (Pc) and P. sanguineus,and lilacinum (Pl) and P. sanguineus, (III) deadlock at distance between P. (VIII) deadlock at distance between M. anisopliae (Ma 258) and P. hispanicum (Ph) and P. sanguineus, (IV) partial replacement of P. sanguineus sanguineus after a deadlock at contact with Trichoderma sp. (SP6), (V) Ann Microbiol (2019) 69:171–181 175 Fig. 2 Interspecific interactions between T. maxima (Tm) and soil-borne deadlock at mycelial contact, (V) partial replacement of M. anisopliae micromycetes: (I) inhibition at mycelial contact between B. brongniartii (Ma 129) by T. maxima, after an initial deadlock at mycelial contact, (VI) (Bb) and T. maxima, (II) inhibition at mycelial contact between P. partial replacement of Aspergillus sp. (As) by T. maxima, after an initial lilacinum (Pl) and T. maxima, (III) partial replacement of P. hispanicum deadlock at mycelial contact, (VII) inhibition at distance between P. (Ph) by T. maxima, after an initial deadlock at distance, (IV) partial carneus (Pc) and T. maxima, and (VIII) inhibition at mycelial contact replacement of T. maxima by Trichoderma sp. (SP6), after an initial between M. anisopliae (Ma 258) and T. maxima In addition, barrages of brown pigments are commonly laccase activity when co-cultured with B. brongniartii (27.6 U/ found in mycelial contact zones; these could be associated L), P. lilacinum (47.2 U/L), Trichoderma sp. (30.1 U/L), M. with ligninolytic enzyme activities, principally those of anisopliae Ma129 (26.4 U/L), and Aspergillus sp. (14.1 U/L) laccase, as well as with the formation of melanin compounds on agar plates (Table 2). In particular, P. lilacinum achieved the that protect fungal hyphae (T. maxima) from SBM attack. highest fold increase in laccase activity (27.8) when co-cultured Also, mushrooms are known to release laccase as a defensive with P. sanguineus. Therefore, this fungal co-culture was select- response against mycelial invasion. In this respect, enzymes ed for the submerged fermentation experiment. In regard to help mushrooms to adapt to antagonistic environments or en- MnP activity, both B. brongniartii (9.4 U/L) and P. lilacinum vironmental stress (Divya and Sadasivan, 2016 (5.9 U/L) significantly enhanced (F = 3.61, P = 0.0056) the MnP activity of P. sanguineus (1.0 U/L) by 7.7- and 4.5-fold, respectively (Table 2). Ligninolytic enzyme activities in agar plates The ligninolytic enzyme activity of P. sanguineus on agar plates under co-culture conditions has not been previously Pycnoporus sanguineus interactions Pycnoporus sanguineus (1.6 U/L) significantly increased (F =8.41, P =0.00001) its reported. However, Van Heerden et al. (2011) did demonstrate Table 2 Laccase and MnP activities of Pycnoporus sanguineus in co-culture with soil-borne micromycetes in agar plates Interactions Laccase MnP Vol. activity (U/L) Increase fold Vol. activity (U/L) Increase fold P. sanguineus 1.6 ± 0.2 d – 1.0 ± 0.5 c – P. sanguineus-B. brongniartii 27.6 ± 2.9 b 15.9 ± 1.78 b 9.4 ± 3.7 a 7.7 ± 3.1 a P. sanguineus-P. lilacinum 47.2 ± 5.3 a 27.8 ± 3.24 a 5.9 ± 0.9 ab 4.5 ± 0.9 ab P. sanguineus-P. hispanicum 2.8 ± 0.4 d 0.7 ± 0.24 d 2.8 ± 0.2 bc 1.6 ± 0.2 bc P. sanguineus-Trichoderma sp. 30.1 ± 5.4 b 17.4 ± 3.28 b 1.5 ± 0.3 c 0.5 ± 0.1 c P. sanguineus-M. anisopliae (Ma129) 26.4 ± 2.5 b 15.5 ± 1.52 b 3.1 ± 0.5 bc 1.8 ± 0.4 bc P. sanguineus-Aspergillus sp. 14.1 ± 0.1 c 7.6 ± 0.57 c 1.9 ± 0.2 c 0.8 ± 0.2 c P. sanguineus-P. carneus 3.7 ± 0.7 d 1.3 ± 0.41d 1.7 ± 0.4 c 0.6 ± 0.3 c P. sanguineus-M. anisopliae (Ma258) 3.8 ± 0.2 d 1.3 ± 0.11 d 2.9 ± 0.7 bc 1.7 ± 0.7 bc F= 8.41 12.65 4.02 3.61 P= 0.00001* 0.00001* 0.0017* 0.0056* *Indicates significant values Means with the same letter in row are not significantly different (LSD, P = 0.05). Vol, volumetric 176 Ann Microbiol (2019) 69:171–181 Table 3 Laccase and MnP Interactions Laccase MnP activities of Trametes maxima in co-culture with soil-borne Vol. activity (U/L) Increase fold Vol. activity (U/L) Increase fold micromycetes in agar plates T. maxima (control) 67.5 ± 4.3 bc – 1.1 ± 0.1 c – T. maxima-B. brongniartii 115.9 ± 13.2 a 0.72 ± 0.19 a 9.2 ± 1.5 b 6.8 ± 1.2 bc T. maxima-P. lilacinus 69.9 ± 4.3 b 0.06 ± 0.01 b 11.1 ± 3.0 b 8.4 ± 2.5 b T. maxima-P. hispanicum 30.4 ± 6.6 de – 3.4 ± 0.9 c 1.9 ± 0.8 cd T. maxima-Trichoderma sp. SP6 85.4 ± 13.3 b 0.55 ± 0.09 28.0 ± 4.7 a 22.6 ± 3.9 a ab T. maxima-M. anisopliae 123.7 ± 21.6 a 0.83 ± 0.31 a 2.7 ± 0.5 c 1.3 ± 0.4 d (Ma129) T. maxima-Aspergillus sp. 61.3 ± 0.8 bc – 1.5 ± 0.1 c 0.3 ± 0.1 d T. maxima-P. carneus 10.1 ± 2.1 e – 3.0 ± 0.5 c 1.5 ± 0.4 d T. maxima-M. anisopliae 39.6 ± 5.3 cd – 6.2 ± 1.3 bc 4.8 ± 1.2 (Ma258) bcd F= 13.65 3.11 17.80 16.72 P= 0.00001* 0.0559* 0.00001* 0.00001* *Indicates significant values Means with the same letter in row are not significantly different (LSD, P = 0.05). Vol, volumetric that co-cultures of P. sanguineus with Aspergillus flavipes in weight), respectively. These increases were higher than those wood chips of Acacia mearnsii, Eucalyptus dunnii, produced by the SBM. The synthesis of enzymes by fungal Eucalyptus grandis,and Eucalyptus macarthurii altered the organisms is known to differ between strains and species. For chemical composition of each tree species in different ways. example, T. versicolor did not produce MnP activity in a study Also, the authors demonstrated that the co-culture of P. conducted by Hiscox et al. (2010). However, this enzyme was sanguineus-A. flavipes resulted in a higher cellulose content induced in co-cultures of T. versicolor and its fungal compet- and lower lignin content in degrading wood chips compared to itors; the highest production was found with S. gausapatum. the monoculture of P. sanguineus (Van Heerden et al. 2008). Meanwhile, in the present study, the highest fold increase Given this context, fungal co-cultures could have relevant (22.6) was found with Trichoderma sp. SP6. More recent- applications given the ecology of lignin decomposition, al- ly, Kuhar et al. (2015) studied the fungal co-culture of two though ligninolytic enzymes were not evaluated by these pre- basidiomycetes, T. versicolor and Ganoderma lucidum,on vious authors. agar plates. Laccase activity in the fungal co-culture was 1.1 U/g, representing a 1.75-fold increase in comparison to Trametes maxima interactions Trametes maxima (67.5 U/L) the monocultures of T. versicolor (0.4 U/g) and G. lucidum significantly increased (F =13.65, P = 0.00001) its laccase ac- (0.1 U/g). tivity when co-cultured with B. brongniartii (115.9 U/L) and M. anisopliae Ma129 (123.7 U/L) by 0.72- and 0.83-fold, Co-culture experiments in liquid fermentation respectively (Table 3). In addition, three species of SBM, B. brongniartii (9.2 U/L), P. lilacinum (11.1 U/L), and Co-culture of Pycnoporus sanguineus and Purpureocillium Trichoderma sp. SP6 (28.0 U/L) were able to significantly lilacinum Purpureocillium lilacinum significantly increased enhance (P < 0.05) the MnP activity of T. maxima (1.0 U/L). the laccase activity of P. sanguineus (48 h = 0.91 U/L and The highest fold increases in the MnP activity of T. maxima 96 h = 0.73 U/L) at 48 (t = − 4.7897, P = 0.0030) and 96 h were 8.4 and 22.6, which were caused by P. lilacinum and (t =2.1057, P = 0.0399) of fermentation by 2.09- (2.81 U/L) Trichoderma sp. SP6, respectively (Table 3). and 2.64-fold (2.69 U/L), respectively (Fig. 3A). Meanwhile, Studies of fungal co-cultures using Trametes sp. are scarce. MnP activity was strongly and significantly increased in the In a previous study, Hiscox et al. (2010) co-cultured Trametes co-culture with P. lilacinum at 48 h. Specifically, in the co- versicolor (216 mU/wet weight) with other basidiomycetes on culture, the laccase activity at 48 (t = − 8.4065, P =0.0001), −07 agar plates. The results indicated that Stereum gausapatum, 72 (t = − 28.4098, P =1.25 ), and 96 h (t = − 50.7384, P = −09 Daldinia concentrica, Bjerkandera adusta,and Hypholoma 3.93 ) was 24.19, 58.11, and 61.89 U/L, respectively; in the fasciculare enhanced the laccase activity of T. versicolor by monoculture, the laccase activity was 9.49 (48 h), 5.90 (72 h), 139- (3218.9 mU/wet weight), 3.06- (877.8 mU/wet weight), and 5.04 U/L (96 h, Fig. 3B). Thus, the laccase activity of the 3.03- (871.6 mU/wet weight), and 0.93-fold (417.1 mU/wet co-cultures increased by 1.55- (48 h), 8.83- (72 h), and 11.27- Ann Microbiol (2019) 69:171–181 177 Fig. 3 Laccase (A and D) and MnP (B and E) activities, and H O production (C and F) in two fungal co-cultures: Pycnoporus sanguineus- 2 2 Purpureocillium lilacinum (A, B, and C) and Trametes maxima-Beauveria brongniartii (D, E, and F) fold (96 h). The H O content of the fungal co-culture extract by 2.08- and 1.33-fold at 48 and 96 h of fermentation, 2 2 was higher during the evaluation period compared to the respectively. monoculture extracts (Fig. 3C). Specifically, the H O values 2 2 in the co-culture ranged from 4.90 (t = − 3.2046, P =0.0184) Co-culture of Trametes maxima and Beauveria brongniartii to 7.97 mg/L (t = − 3.5567, P = 0.0119), while those of the The laccase activity of T. maxima increased in the presence of monocultures ranged from 0.78 to 1.50 mg/L. Beauveria brongniartii (24 h = 100.17 U/L and 48 = 87.77 U/ However, in other studies, the laccase activities of P. L) at 24 (t = − 3.1653, P = 0.0194) and 48 h (t = − 7.9183, P = sanguineus in monocultures were variable. Eugenio-Eugenio 0.0002) of fermentation. The laccase activities of the co- et al. (2009) and Vikineswary et al. (2006)reported values of cultures were 113.87 and 129.66 U/L at 24 and 48 h, respec- 320 mU/L (0.32 U/L) in submerged fermentation and < 5.0 U/ tively, while the monoculture achieved 100.17 and 87.77 U/L g of the substrate in solid-state fermentation, respectively. in the same time frame (Fig. 4D). The MnP activity of T. These values are low (Eugenio-Eugenio et al., 2009) and high maxima during the co-culture was not enhanced by B. (Vikineswary et al. 2006) with respect to those found for the brongniartii (Fig. 2E). The co-culture achieved the highest co-culture of P. sanguineus and P. lilacinum (2.69 U/L), re- levels of H O at 48 (11.16 mg/L, t = − 7.1149, P =0.0003) 2 2 spectively. However, few data are available on P. sanguineus and 72 h (15.71 mg/L, t = − 4.7665, P = 0.0031) in compari- under co-culture conditions. Baldrian (2004) reported that son to those of the T. maxima monoculture (48 h = 6.85 mg/L Trichoderma harzianum was able to increase the laccase ac- and 72 h = 13.40 mg/L). tivity of P. sanguineus by 1.5-fold in liquid fermentation. Trametes (synonym of Cerrena) maxima was previously Meanwhile, in the present study, laccase activity increased studied in co-culture with the basidiomycete Coriolus 178 Ann Microbiol (2019) 69:171–181 Response of enzymes produced by white-rot fungi to H O concentration 2 2 At 24 h of exposure to 14.69 mM (531.7 U/L) of H O , T. 2 2 maxima (430.0 U/L) significantly (F = 59.78, P = 0.00001) enhanced its laccase activity by 0.23-fold. However, the highest concentration of H O (293.99 mM) strongly 2 2 inhibited laccase activity in T. maxima (Fig. 4A). At 48 (F = 24.99, P =0.00001) and 72 h (F =35.35, P =0.00001) of ex- posure, both 2.93 and 14.69 mM of H O led to significant 2 2 increases in the laccase activity of T. maxima; the correspond- ing laccase values were 1182.0 and 1122.5 U/L, respectively, representing 0.46- (at 2.93 mM) and 0.39-fold (at 14.69 mM) increases, respectively. However, the highest H O concentra- 2 2 tion caused strong inhibition of laccase activity at 48 and 72 h, corresponding with laccase activities of 11.3 and 28.1 U/L, respectively. At the end of the evaluation period, the laccase activity did not simply increase with increasing H O concen- 2 2 tration. The highest values of laccase activity ranged from 735.7 to 831.1 U/L, although the highest concentrations of H O reduced the laccase activity to 32.6 U/L (Fig. 4A). 2 2 In regard to MnP activity (Fig. 4B), T. maxima significantly Fig. 4 Laccase (A) and MnP (B) activities of Trametes maxima under enhanced (F = 110.31, P = 0.00001) its MnP activity by 0.51- stress condition by hydrogen peroxide concentrations fold at 24 h of exposure to 14.69 mM of H O . Both 29.39 and 2 2 293.99 mM of H O reduced the MnP activity of T. maxima 2 2 from 3.35 U/L to 0.83 and 1.18 U/L, respectively (Fig. 2B). At hirsutus. In this case, both strains produced laccase at rates of 48 h, MnP activity was not increased by the H O concentra- 2 2 approximately 63.0 and 415.0 nkatal/L respectively, in mono- tions; instead, H O concentrations of 14.69, 29.39, and 2 2 cultures. However, these values were low compared to those 293.99 mM significantly inhibited MnP activity, leading to of the co-culture of both species (510 nkatal/L, Koroleva et al. decreases of 11.75 U/L to 5.40, 3.75, and 0.73 U/L, respec- 2002). The levels of laccase achieved in the co-culture of these tively. At 72 and 96 h, the H O concentrations of 2.93 and 2 2 two strains represented a 7.1- and 0.22-fold increase compared 14.69 mM were able to increase the MnP activities by 0.51- to the monocultures of Cucurbita maxima and C. hirsutus, and 0.93-fold (at 72 h) and by 0.28- and 0.44-fold (96 h), respectively. Baldrian (2004) reported that the SBM T. respectively. As previously occurred, both 29.39 and harzianum, Acremonium sphaerospermum, Fusarium 293.99 mM of H O inhibited the MnP activity of T. maxima 2 2 reticulatum, Humicola grisea,and Penicillium rigulosum (Fig. 2B). were able to increase the laccase activity of T. versicolor under Pycnoporus sanguineus exhibited less laccase activity liquid fermentation by 9-, 14-, 16.7-, 16-, and 55.6-fold, re- than T. maxima.However, P. sanguineus significantly in- spectively, after 3 days of interaction. creased its laccase activity under H O stress. At 24 h of 2 2 Contrary to Koroleva et al. (2002), Baldrian (2004), and the exposure, laccase activity in the P. sanguineus control was present study, Xiao et al. (2004)reported that Trametes sp. absent. However, the H O concentrations of 14.69 mM 2 2 AH28-2 reduced its laccase activity by 30% and 11% when and 29.39 mM activated laccase synthesis by P. co-cultured with Aspergillus oryzae and Gloeophyllum sanguineus, leading to a laccase production of 4.15 and trabeum, respectively. More recently, Díaz-Rodríguez et al. 5.94 U/L respectively (Fig. 5A).At48 h,theH O con- 2 2 (2018) reported that a non-laccase producing soil microfungi centrations of 14.69, 29.39, and 293.99 mM enhanced the Penicillium commune GHAIE86 could increase the laccase laccase activity of P. sanguineus (15.3U/L)by1.2- activity of Funalia floccosa LPSC232 (20 mU/mL) by 3- (34.9 U/L), 3.1- (63.1 U/L), and 1.5-fold (39.2 U/L). At fold (60 mU/mL) under submerged fermentation; in addition, 72 h, the H O concentrations of 29.39 and 293.99 mM 2 2 P. commune was able to induce two laccase isoenzymes in F. enhanced the laccase activity of P. sanguineus (17.32 U/ floccosa. Interestingly, one function of SBM species appears L) by 68.8- (1210.5 U/L) and 36.3-fold (645.4 U/L). By to be the enhancement of laccase activity in WRF. However, the end of the experiment (96 h), 2.93 mM of H O en- 2 2 the mechanisms behind and the reasons for this phenomenon hanced the laccase activity of P. sanguineus (56.12 U/L) are still under discussion. by 14.71-fold (882.0 U/L, Fig. 5A). Ann Microbiol (2019) 69:171–181 179 binding then modifies the absorbance of laccase and facilitates the removal of fluoride (a xenobiotic compound). Furthermore, the catalytic activity of MnP is well known to be dependent on H O ; however, this enzyme is also 2 2 inactivated by excess H O .According toBermeketal. 2 2 (2002), the oxidation of native MnP by an equivalent amount of H O forms active compound I, which subsequently oxi- 2 2 2+ 3+ dizes Mn to Mn . In this process, compound II is formed and reduced back to the native MnP, oxidizing an additional 2+ 3+ 2+ Mn to Mn .Mn is a mandatory substrate for compound II. When excess concentrations of H O are present, native 2 2 MnP is directly converted to compound III, which is an inac- tive form of the enzyme. In addition, Li et al. (1995)demon- strated that the white-rot fungus Phanerochaete chrysosporium produces MnP mRNA following the addition 2+ of a low amount of H O in the absence of Mn , the natural 2 2 substrate of MnP. These responses were observed in this study, as low amounts of H O (2.93 and 14.69 mM) stimu- 2 2 lated MnP activity, whereas high amounts (29.39 and 293.99 mM) inhibited MnP activity. The source of H O in co-culture systems is unclear and 2 2 will be the target of future studies. However, both WRF and Fig. 5 Laccase (A) and MnP (B) activities of Pycnoporus sanguineus SBM are able to produce different levels of H O via distinct under stress condition by hydrogen peroxide concentrations 2 2 metabolic pathways. Similarly, Urzúa et al. (1998)reported that Ceriporiopsis subvermispora is able to generate H O 2 2 In regard to MnP activity (Fig. 5B), at 24 h, the H O through the oxidation of the organic acids that it secretes. 2 2 concentrations of 2.4 (8.7 U/L), 14.69 (11.1 U/L), and Also, SBM are known to produce H O as a result of glucose 2 2 293.99 (8.8 U/L) mM significantly (F = 13.86, P = 0.0004) degradation by glucose oxidase (GOx), especially under the enhanced the MnP activity of P. sanguineus (1.5 U/L) by stress conditions caused by xenobiotics, as reported by 4.9-, 6.89-, and 5.0-fold, respectively. At 48 h, only Zúñiga-Silva et al. (2016). 14.39 mM of H O was required to significantly increase 2 2 MnP activity (F =4.64, P = 0.0224) by 1.8-fold. However, at 72 h, H O concentrations of 2.93, 14.69, and 29.39 mM were Conclusions 2 2 required to achieve 3.0-, 12.8-, and 1.0-fold increases, respec- tively, in MnP activity, corresponding with MnP activities of In this study, the interspecific interactions and enzyme activ- 11.29, 38.40, and 5.68 U/L, respectively. Meanwhile, the con- ities of the co-cultures of two WRF and eight SBM were trol only achieved a MnP activity of 2.8 U/L. Finally, at the evaluated in addition to the effects of H O on WRF. The main 2 2 end of evaluation period (96 h), both 2.93 (27.8 U/L) and type of interspecific interaction exhibited by the WRF 14.69 (44.9 U/L) mM of H O significantly increased (F = Pycnoporus sanguineus was deadlock at a distance, while T. 2 2 15.01, P = 0.0003) the MnP activity of P. sanguineus (5.9 U/ maxima partially replaced three of the studied SBM, L), corresponding with 3.7- and 8.5-fold increases, respective- confirming its dominance. Purpureocillium lilacinum en- ly (Fig. 5B). hanced to a large extent the laccase activity of P. sanguineus, Hydrogen peroxide differentially increased the laccase ac- and B. brongniartii was the principal enhancer of the MnP tivity of the WRF. This suggests that H O could be respon- activity of P. sanguineus.Both B. brongniartii and M. 2 2 sible for the increase in laccase and MnP activities in fungal anisopliae (Ma129) were the best enhancers of laccase activ- co-cultures, as the H O in fungal extracts from both the T. ity in T. maxima,while Trichoderma sp. (SP6) increased the 2 2 maxima-B. brongniartii and the P. sanguineus-P. lilacinum MnP activity of T. maxima to a large extent. In submerged co-cultures was high in comparison to the monocultures of fermentation, P. sanguineus increased its laccase and MnP both WRF. In fungal organisms, H O plays an important activity in co-culture with P. lilacinum. Meanwhile, T. maxima 2 2 role and influences enzymes activity. Branden et al. (1971) did not increase its MnP activity but did slightly increase its studied the interaction of a fungal laccase with H O and 2 2 laccase activity at the initial evaluation points. Finally, the co- found that H O molecules are able to bind to one specific cultures P. sanguineus-P. lilacinum and T. maxima-B. 2 2 2+ Cu of the four copper atoms of the laccase molecule. This brongniartii achieved higher H O production in comparison 2 2 180 Ann Microbiol (2019) 69:171–181 Biotechnol Adv 32:1180–1204. https://doi.org/10.1016/j. to the WRF monocultures, suggesting that H O plays an 2 2 biotechadv.2014.03.001 essential role in increasing ligninolytic enzyme activities. Bertrand S, Schumpp O, Bohni N, Monod M, Gindro K, Jean-Luc W This conclusion was also confirmed by the dose-response as- (2014b) De novo production of metabolites by fungal co-cultures of says, wherein low concentrations of H O (2.94 and Trichophyton rubrum and Bionectria ochroleuca. 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Annals of Microbiology – Springer Journals

Published: Dec 15, 2018

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