Abstract
Manganese peroxidase (MnP), a crucial enzyme in biodegradation of lignin, is synthesized by most white rot fungi. To obtain novel enzymes with superior biodegradation potential, MnP-producing wild isolates were evaluated for their ability to degrade recalcitrant azo dyes, sulfonephthalein dyes, and kraft lignin. Of 30 wild isolates screened, 18 tested positive for lignin modifying enzymes (LMEs). Thirteen of these isolates were positive for both laccase and MnP, whereas four produced only laccase, and one produced lignin peroxidase alone. The isolates were identified as Clitopilus scyphoides MH172162 (AGUM004), Ganoderma rasinaceum MH172163 (AGUM007), and three Schizophyllum species: MH172164, MH172165, and MH172166 (KONA001, AGUM0011, and AGUM021). The Fourier-transform infrared spectroscopy (FTIR) analysis of dye degradation and kraft lignin samples with AGUM004 and AGUM007 revealed biotransformation. The former could not completely degrade Reactive Black 5 and bromocresol green, but it could completely (100%) decolorize bromophenyl blue, bromothymol blue, and Remazol brilliant blue R. The latter efficiently degraded almost all tested dyes. Both degraded kraft lignin. The screened hyper MnP-producing wild AGUM004 and AGUM007 were shown to be potential dye degraders in addition to having lignin degrading abilities.
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Screening of Wild Basidiomycetes and Evaluation of the Biodegradation Potential of Dyes and Lignin by Manganese Peroxidases
Ramya G. Rao,a,b Aarthi Ravichandran,a Giridhar Kandalam,c Samanta Ashish Kumar,d Senani Swaraj,d and Manpal Sridhar a,*
Manganese peroxidase (MnP), a crucial enzyme in biodegradation of lignin, is synthesized by most white rot fungi. To obtain novel enzymes with superior biodegradation potential, MnP-producing wild isolates were evaluated for their ability to degrade recalcitrant azo dyes, sulfonephthalein dyes, and kraft lignin. Of 30 wild isolates screened, 18 tested positive for lignin modifying enzymes (LMEs). Thirteen of these isolates were positive for both laccase and MnP, whereas four produced only laccase, and one produced lignin peroxidase alone. The isolates were identified as Clitopilus scyphoides MH172162 (AGUM004), Ganoderma rasinaceum MH172163 (AGUM007), and three Schizophyllum species: MH172164, MH172165, and MH172166 (KONA001, AGUM0011, and AGUM021). The Fourier-transform infrared spectroscopy (FTIR) analysis of dye degradation and kraft lignin samples with AGUM004 and AGUM007 revealed biotransformation. The former could not completely degrade Reactive Black 5 and bromocresol green, but it could completely (100%) decolorize bromophenyl blue, bromothymol blue, and Remazol brilliant blue R. The latter efficiently degraded almost all tested dyes. Both degraded kraft lignin. The screened hyper MnP-producing wild AGUM004 and AGUM007 were shown to be potential dye degraders in addition to having lignin degrading abilities.
Keywords: Manganese peroxidase; Clitopilus scyphoides; Ganoderma rasinaceum; Biodegradation; Dye decolorization; Lignin
Contact information: a: Bioenergetics and Environmental Sciences Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560 030, India; b: PhD Scholar, Department of Biochemistry, Jain University, Bengaluru, 560011, Karnataka, India; c: Knowledge management and Biostatistics Section, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, 560 030, Karnataka, India; d: Animal Nutrition Division, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, 560 030, Karnataka, India;
* Corresponding author: manpalsridhar@ yahoo.co.uk
INTRODUCTION
Fungi play important roles in biodegradation and bioremediation. White rot fungi (WRF), a group within the class Basidiomycetes, have the unique ability to degrade and mineralize recalcitrant hetero-polymer lignin, which is the most abundant organic material on Earth (Erden et al. 2009; Cesarino et al. 2012). White rot fungi are ubiquitous in nature and grow as saprophytes on dead and decaying trees, usually in the forest ecosystem, and are exceptional in their ability to degrade lignin selectively. They possess a set of nonspecific enzymes called lignin modifying enzymes (LME), which degrade lignin and other recalcitrant compounds such as munitions waste, pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, bleach plant effluent, synthetic dyes, synthetic polymers, and wood preservatives (Pointing 2001). These LME are a set of extracellular enzymes secreted by WRF, and are mainly comprised of laccase, manganese peroxidase (MnP), lignin peroxidase (LiP), and versatile peroxidase (VP), along with oxidases, which generate extracellular H2O2. Different species of white rot fungi may secrete one, two, or all of these enzymes (Wesenberg et al. 2003).
Within the LME, MnP is synthesized by almost all the lignin-degrading WRF and is the most crucial in lignin biodegradation. The MnPs are peroxide-dependent extracellular glycoproteins with one molecule of heme (Dashtban et al. 2010). In the presence of hydrogen peroxide, they oxidize Mn (II) to highly reactive Mn (III). Mn (III) further chelates with organic acids, which attack and oxidize lignin and other recalcitrant compounds (Van Aken and Agathos 2002). Since the discovery of MnP in P. chrysosporium (Glenn and Gold 1985), MnPs have been found in other basidiomycetes, such as Bjerkandera sp. strain BOS55 (Mester and Field 1997), Bjerkandera sp. (Palma et al. 2000), Agaricus bisporus (Lankinen et al.2001), Lenzites betulinus (Hoshino et al. 2002), Phanerochaete flavido-alba (de la Rubia et al. 2002), Phanerochaete sordida (Harazonoet al. 2003), Panus tigrinus (Lisov et al. 2003), Ganoderma lucidium (Novotnýet al. 2004), Dichomitus squalens (Eichlerová et al. 2005), Lentinula edodes (Boer et al. 2004), Nematoloma frowardii b19 (Hildén et al. 2008), Pleurotus pulmonarius (dos Santos Bazanella et al. 2013), Trametes versicolor, Pleurotus ostreatus, Irpex lacteus (Zhao et al. 2015), and Trametes hirsuta (Vasina et al. 2017).
The potential applications of MnPs rival those of laccases. They degrade both phenolic and non-phenolic lignin model compounds (Wang et al. 2018). They are efficient degraders of many recalcitrant compounds including different synthetic dyes (Qin et al. 2014), herbicides, polycyclic aromatic hydrocarbons, mycotoxins, estrogens, explosives, and antifouling compounds (Wang et al. 2018). The presence of MnP increases the degree of dye decolorization (Chagas and Durrant 2001; Christian et al. 2003).
Considering the wide application of MnPs, the current study was conducted to screen for wild WRF exhibiting higher MnP activities and identify their phylogenetic relationships. The wild isolates producing greater quantities of MnP were further evaluated for their ability to degrade recalcitrant azo dyes and sulfonephthalein dyes. They were tested for the biodegradation of kraft lignin. The extent of biodegradation of different dyes and kraft lignin was measured using spectrophotometry, and patterns of degradation were studied using Fourier-transform infrared spectroscopy (FTIR) techniques. The identified isolates producing MnP have not been explored previously.
EXPERIMENTAL
Material and Methods
Chemicals
Chemicals used in the study were of analytical grade unless otherwise stated. Reactive Black 5 (RB5), 2,2’-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) (ABTS), and Remazol brilliant blue R (RBBR) used for assay were procured from Sigma Aldrich (St. Louis, MO, USA).
Collection, isolation, and maintenance of white rot fungi
Fruiting bodies of 30 different WRF were collected in clean dry self-sealing polythene bags from forests in Agumbe, Madikere and in and around Shimoga (Karnataka State), India. In all cases, the substrate was represented by wood found in various stages of decay.
The cultures were marked with information such as number and isolation location. Pure cultures from collected samples were obtained by tissue culture technique (Pradeep et al. 2013). All pure cultures were maintained on PDA slants and stored at 4 °C for further use.
Screening for LME production
Initial screening for lignolytic enzyme production was carried out on 0.02% guaiacol-supplemented PDA. All cultures were inoculated on pre-solidified PDA medium supplemented with guaiacol and incubated for 7 days at room temperature to check the phenolic oxidation. The positive cultures from primary screening were then grown in basal medium which contained 0.1% KH2PO4, 0.05% C4H12N2O6, 0.05% MgSO4.7H2O, 0.001% of CaCl2 and yeast extract, and 0.0001% of CuSO4.5H2O, Fe2(SO4)3, and MnSO4.H2O. Fungal cultures pre-grown in PDB were used as inoculum. The cells were homogenized prior to inoculation, and 2% of culture suspension was added to the production medium. Flasks were maintained under continuous shaking at 80 rpm at 30 C for 7 days. Culture filtrates obtained were assessed for the production of laccase and other peroxidases. All experiments were performed in triplicate.
Enzyme Assay
Culture filtrate was used as the enzyme source to determine the ligninolytic enzymes activity. A sample of 500 µL was taken to check different enzyme production. Laccase production was assessed by a measurement of the enzymatic oxidation of ABTS at 420 nm (Kinnunen et al. 2016).
The reaction mixture contained 0.8 mM ABTS, 0.4 M sodium acetate buffer (pH5.2), and 500 µL of 0.5 mg/mL catalase. The MnP activity was assayed by the oxidation of 4 mM MnSO4in 50 mM sodium malonate buffer (pH 4.5), in the presence of 0.4 mM H2O2. Oxidation of MnSO4 was measured by increase in OD at 270 nm (Wariishi et al. 1992).
Lignin peroxidase was determined by Azure B assay (Archibald 1992). Reaction mixture for assay contained 0.04 mM Azure B, 100 mM sodium tartarate (pH 4.5), and 0.4 mM H2O2. Oxidation of Azure B was determined by the decrease in OD at 651 nm. Production of versatile peroxidase was assessed by RB5 assay (Jarosz-Wilkołazka et al. 2009). Oxidation of RB5 was determined in 100 mM sodium tartrate buffer at pH 3 with 10 µM of RB5. The reaction was initiated by addition of 0.1mM H2O2 and assessed through decrease in absorbance at 598 nm.
Identification and Phylogenetic Analysis of WRF
The proficient strains unveiling hyper MnP activity were identified in a separate study carried out by Rao et al. (2018). Genomic DNA of selected WRF were isolated by the microwave method, which was then amplified using PCR with ITS 1 (5′-TCCGTAGGTGAACCTGCG G- 3′) and ITS 4 (5′-TCCTCCGCTTATTGATAT G-3′) as forward and reverse primers, respectively. The amplified product comprised of 18s rDNA, 28s rDNA, and5.8s rDNA, along with ITS1 and ITS4 regions, was sequenced. The ITS sequence obtained was then subjected to sequence comparison through BLAST. Culture names were assigned based on more than 99% sequence similarity.
To study the morphology of identified isolates, culture stained with lacto-phenol blue dye, was examined under microscope. The morphology of the fungus and details of hyphae structure and spores were observed at 60x magnification (Pradeep et al. 2013).
The identified isolates were studied for their evolutionary relationship with their phylogeny using MEGAX software (Kumar et al. 2018). A total of 44 related species’ DNA sequences comprised of 18s rDNA, 28s rDNA, and5.8s rDNA, along with ITS1 and ITS4 regions, were selected; the data were retrieved from NCBI. Maximum likelihood statistical method was selected to compute the evolutionary distance (Tamura et al. 2004). Bootstrap method with 1000 replicates was adopted to find the phylogenetic evaluation (Felsenstein1985). To construct the phylogenetic tree, Nearest-Neighbor-Interchange (NNI) was considered (Saitou and Nei 1987). All positions containing gaps and missing data were eliminated.
MnP Production
The biosynthesis trend of MnP from AGUM004 and AGUM007 was studied using modified production media (Takano et al. 2004). Production media was composed of 0.4g K2HPO4, 0.6 g KH2PO4, 0.5 g MgSO4, 0.03 g MnSO4, 0.01 g CaCO3, 1 g NH4H2PO4, 0.25 g yeast extract, 10 g glucose, 0.5 g Tween 80, and 8.3 g sodium malonate in 1000 mL of distilled water. Then 50 mL of production media in 250-mL shaken flasks were inoculated with 2% well grown culture in LME basal medium and incubated for 7 days at 30 ºC with constant shaking at 120 rpm. Activities of MnP and laccase were monitored from 0 h to 10 days at intervals of 24 h. Experiment was carried out in 5 replicates.
Dye Decolorization and Lignin Degradation Studies
To study the dye decolorization and delignification proficiency, hyper MnP-producing strains AGUM004 and AGUM007 were considered. For this, 20 mL of MnP production medium supplemented with 0.02% of RBBR, RB5, bromocresol green, bromothymol blue, and kraft lignin in 100 mL shaken flasks were inoculated with 2% well grown culture in LME basal medium and incubated for 7 days at 30 °C with constant shaking at 120 rpm. Uninoculated flasks with dyes/lignin were considered as control. Culture filtrates were examined on a spectrophotometer at the absorbance maxima for different dyes and lignin. Degradation efficacy of the culture was determined in terms of percentage of color removal and was calculated according to the formula given below,
(1)
where C is the current concentration of dye in a control sample (mg/L) and S is the current residual concentration of dye in the samples. Results were expressed in % degradation.
All the samples were also analyzed for dye and lignin breakdown pattern by using the FTIR technique. The FTIR analysis was carried out in the mid IR region of 400 cm-1 to 4000 cm-1. Graphs were reformed using Originpro software for comparison and interpretation. Absorption spectra in the region between 3100 cm-1 and 3600 cm-1 indicated the presence of exchangeable protons, typically from alcohol, amine, amide, or carboxylic acid groups, which was of less importance in interpreting the data (Coates 2000). Therefore, the region above 3000 cm-1 was omitted in the reformed graphs. The FTIR analysis was outsourced from SITC, University of Science and Technology, Cochin, India.
RESULTS AND DISCUSSION
Screening for LME Production
The 30 WRF were collected together to check the production of ligninolytic enzymes. Out of 30 cultures, 18 cultures were found to be positive for guaiacol oxidation in the primary screening, which showed an intense brown halo in and around the culture and indicated the production of ligninolytic enzymes, while 40% of the culture specimens failed to develop brown color around the mycelial growth (Fig. 1). Further investigation on lignolytic enzymes production in liquid medium, revealed that 13 WRF were both MnP and laccase producers, 4 cultures were only laccase producers, and only 1 culture was a lignin peroxidase producer. However, VP was not detected in any of the culture filtrates. Out of 13 MnP positive cultures, AGUM004 and AGUM007 were observed to secrete higher quantities of both MnP and laccase enzymes. Table 1 specifies the morphological appearance of positive cultures on PDA along with ability of guaiacol oxidation and lignolytic enzyme quantification.
Fig. 1. Guaiacol oxidation: control (uninoculated plate) (A); positive for guaiacol oxidation (B); positive culture on PDA without guaiacol (C); negative for guaiacol oxidation (D)
Identification and Phylogenetic Analysis of WRF
As AGUM004, AGUM007, KONA001, AGUM011, and AGUM021 were showing greater MnP activity, they were considered further for culture identification as well as phylogenetic studies. Isolates were identified as Clitopilus scyphoides (AGUM004), Ganoderma rasinaceum (AGUM007), and theother three as Schizophyllum species (KONA001, AGUM0011, and AGUM021).
Table 1. Morphological Appearance of WRF along with Primary Screening Result (Guaiacol reaction) and Quantification of their Lignolytic Enzymes
Fig. 2. Microscopic observation of (A) AGUM004; (B) AGUM007; (C) KONA001; (D) AGUM011; and (E) AGUM021
The PCR amplified DNA sequence through ITS1 and ITS4 primers were of 700 bp length comprised of ITS1, ITS2, and rDNA regions, which were deposited to Genbank with accession numbers MH172162 (Clitopilus scyphoides isolate AGUM004), MH172163 (Ganoderma rasinaceum isolate AGUM007), MH172164 (Schizophyllum sp. isolate KONA001), MH172165 (Schizophyllum sp. isolate AGUM011), and MH172166 (Schizophyllum commune isolate AGUM021). Figure 2 reveals microscopic observation of the type of hyphae, septa, clamp connections, and the features of the spores, which further confirmed these isolates belong to basidiomycetes. Morphological details revealed that Clitopilus scyphoides had septate hyphae and elliptical spores (Fig. 2A), Ganoderma rasinaceum formed typical almond-shaped basidiospores unique to the genus with septate hyphae (Fig. 2B), and the other three isolates of Schizophyllum had simple septate hyphae forming clamps with absence of basidiospores (Figs. 2C, 2D, and 2E).