Ligninolytic Activity of White Rot Fungi from GGV ...

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Cambria et al. (2011) studied that Laccase production, enhanced in white rot fungus Rigidoporus lignosusby the addition of some phenolic and aromatic.

Cryptogam Biodiversity and Assessment

Cryptogam Biodiversity and Assessment

Vol 1, No. 2 (2016), e-ISSN :2456-0251, 00-00

Ligninolytic Activity of White Rot Fungi from GGV campus as Bioremediation of Organic Pollutants Nikki Agrawal and Sushil Kumar Shahi* Department of Botany, Guru Ghasidas Vishwavidyalaya, Bilaspur- 495009, Chhattisgarh Publication Info Article history: Received : 15-04-2016 Accepted : 20-09-2016 DOI: http://dx.doi.org/ 10.21756/cab.v1i2.6894 Key words: Laccase, Lignin peroxidase, Manganese peroxidase, Organopollutants, White rot fungi.

Abstract White rot fungi constitute a diverse physiological group are capable of transforming and mineralizing a wide range of organopollutants. The Ligninolytic enzymes of white rot fungi are substrate specific, essential for Lignin degradation and organic pollutant remediation. In the present study white rot fungi were collected from the north region of Chhattisgarh of Guru Ghasidas Vishwavidyalaya (GGV) Campus. 40 species were collected and isolated then Qualitative and Quantitative screening for the Ligninolytic enzyme assaywere carried out. Out of 40 species, 5 species show potent Ligninolytic activity. In future we can utilize these fungi for the degradation of organic pollutants.

*Corresponding author: Sushil Kumar Shahi Email: [email protected]

1. INTRODUCTION

molecules (Lee et al. 2007). White rot fungi are able to secrete Ligninolytic enzymes, which are Laccases, Lignin peroxidase (LiP) and Manganese peroxidase (MnP), that are associated with Ligninolytic activities (Hadibarata et al. 2012). Mansur et al. (1997) identified Laccase Gene Family in the new Lignin Degrading Basidiomycete CECT 20197. Arora and Gill (2000) studied Laccase production by white rot fungi under different nutritional condition. Ligninolytic and Lignocellulosic enzymes of Ganoderma lucidum in liquid medium studied by Sasidhara et al. (2014). Eggert et al. (1996) investigated the Ligninolytic system of White rot fungus Pycnoporus cinnabarinusand also done the purification and characterization of Laccase. Cambria et al. (2011) studied that Laccase production, enhanced in white rot fungus Rigidoporus lignosusby the addition of some phenolic and aromatic compounds.Makela et al.(2013)studied the effect of nutrient source in the production of Ligninolytic enzymes by white rot fungi Phlebia radiata. The main objectives of in this workto collect white rot fungi from GGV campus,then investigate their Ligninolytic activity for further remediation of organic pollutants.

In the progress of science and technology, several organic pollutant releases into the environment from agricultural, chemical, oil, paper, textile and other industries. Organic pollutants are the product of incomplete combustion of fossil fuels are ubiquitous in nature. The continuous release of these compounds exhibit carcinogenic, mutagenic, teratogenic effect in the biosphere, they become a serious global concern in world wide. The Unites States Environmental Protection Agency (USEPA) has recognized 16 PAHs (Polycyclic aromatic hydrocarbons) as priority pollutants, including naphthalene, anthracene, phenanthrene, pyrene (Hadibarata et al. 2012). Last few decades, many researchers focused on the degradation of organic pollutants, with the help of fungi to develop bioremediation technologies for cleaning our environment ecofriendly. White rot fungi are the member of Basidiomycetes, play an important role in the degradation of organic pollutants. When simple carbon or nitrogen sources are not available for fungi, they will secrete an enzyme system which is able to degrade a complex polymer to simpler

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Agrawal and Shahi

Table 1: Qualitative assay for Ligninolytic enzyme production. S. No.

Fungal isolates

Mycelial growth and oxidation characteristics Laccase Activity

LiP Activity

MnP Activity

Fungal Colour Oxidation Fungal Colour Oxidation Fungal Colour Oxidation colony zone scale (a) colony zone scale (a) colony zone scale (a) diameter diameter diameter diameter diameter diameter (mm) (mm) (mm) (mm) (mm) (mm) 1.

MDG1

90

30

++

15

25

++

85

75

++++

2.

MDG2

93

43

+++

20

30

++

70

90

+++++

3.

FTG3

85

35

++

25

15

+

56

60

+++

4.

BTG2

80

39

++

10

25

++

50

60

+++

5.

PNB2

75

30

++

16

20

+

63

80

++++

a - Oxidation scale measured on the 10th day of cultivation on medium: + diameter of the oxidized zone 0-20 mm, ++ zone diameter 21-40 mm, +++ zone diameter 41-60 mm, ++++ zone diameter 61-80 mm, +++++ zone diameter up to 81 mm.

2. MATERIAL AND METHODS

plate contains Sabouraud Dextrose Agar (SDA) media containing (in gram per litre): Dextrose (40), Peptone (10), and Agar (15). Antibiotic solution was added to inhibit bacterial growth. It was then sealed with parafilm and left horizontally overnight at 250C. If the basidia are mature, they will fall on agar surface. The next day, the agar plate surface was checked to see whether spores have been dropped. If spores are present on the agar surface, transfer a piece of agar containing each spore onto the SDA plate. The SDA plate was incubated in 250C until their mycelium grow about 1 to 2 cm. A small piece of mycelium with agar is then cut and transferred to another SDA plate and the culture was checked after few days, if there is no contamination, a pure culture was obtained (Choi et al. 1999).

2.1 Collection of white rot fungi White rot fungi which grown on wood surface were collected from the north region of Chhattisgarh of GGV Campus in the month of August – September 2014. Samples were collected in sterilized polythene bags for further isolation and investigation. 2.2 Isolation of white rot fungi using the spore drop method Sterilized the surface of fungal mycelium with the help of alcohol and antibiotic solution, a piece of the cap or gill tissue is cut from the fruiting body and was placed on the inside of the top of a Petri dish using Vaseline. Petri

Table 2: Quantitative assay for Ligninolytic enzyme production in MDG2 fungal isolate. Days

Laccase activity (U/mL)

LiP activity (U/mL)

MnP activity (U/mL)

Specific Laccase activity (U/mg)

Specific MnP activity (U/mg)

Biomass (mg)

5

0.3214 ± 0.02

*ND

5.6000± 0.28

0.8830

15.5556

0.2460± 0.05

10

1.8567± 0.03

ND

10.8956± 0.06

4.1667

24.1053

0.2300± 0.05

15

0.4319± 0.05

ND

8.6000 ± 0.29

1.2461

24.6701

0.2000± 0.02

20

0.4000± 0.02

ND

8.2480± 0.08

1.3333

27.4933

0.2363 ± 0.06

25

0.5213± 0.06

ND

24.1280± 0.17

2.7803

130.2105

0.2468± 0.04

30

0.6542± 0.05

ND

20.1020± 0.06

2.6615

80.8283

0.2410± 0.03

Values are mean of triplicates with standard deviation. * ND -not detected

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Cryptogam Biodiversity and Assessment

(a) Fig.1:

(b)

(c)

Screening of (a) laccase (b) lignin peroxidase (c) manganese peroxidase production in solid media of MDG2 cultured mycellium.

2.3 Maintenance of White rot fungi in culture

phenol red (w/v) was employed for the screening of Manganese peroxidase enzyme activities (Kuwahara et al. 1984, Ali et al. 2012). Plates containing screening medium were inoculated by 6 mm fungal inoculum. The diameter of the colored zone produced was measured.

Once a pure culture has been obtained, it can be maintained on an SDA slant at 40C and also stored in 30% glycerol at -200C to improve storage life and preserve microorganism for further investigation.

2.5 Quantitative Screening for Ligninolytic enzyme production

2.4 Qualitative Screening of collected fungi for Ligninolytic activity (Laccase, Lignin peroxidase and Manganese peroxidase)

Fungi which are positive for Ligninolytic enzyme activity, grow on basal medium containing (gm/L) Glucose - 2, Peptone - 5, Yeast extract - 5, K2HPO4 - 1, KH2PO4 - 3, CaCl2 – 1, MgSO4 – 1 (Ugoh and Ijigbade 2013) Ligninolytic enzyme production was screened in 5 day interval within 30 days.

2.4.1 Laccase assay The fungal isolates were screened for Laccase production by growing them on Potato dextrose agar (PDA g/L: Potato infusion- 200, Dextrose- 20, Agar- 20) plates containing 0.01% Guaiacol (D’Souza et al. 2006). Petri dishes (15 cm in diameter) each containing 25 mL of medium were used. 6 mm diameter fungal discs were taken from the periphery of the 7 day old cultures grown. Plates containing screening media were incubated at 30°C for 7 days in a static incubator. Guaiacol oxidised in the presence Laccase and produced colour zone. The diameter of the colored zone was analyzed.

2.5.1 Laccase assay 3 mL of reaction mixture containing 1.5 mL acetate buffer (10 mM, pH 5.0), 1 mL guaiacol (2 mM) and 0.5 mL of the enzyme extract was incubated at 25°C for 2 h and absorbance read at 450 nm. The enzyme activity has been expressed in international units per mL of enzyme extract (U/mL) and the specific activity was expressed as units of total activity per mg of culture filtrate protein (Sandhu and Arora 1985).

2.4.2 Lignin peroxidase assay The fungal strains were screened for Lignin Peroxidase enzyme production were inoculated in Lignin Peroxidase screening medium (g/L: Glucose - 4.0, Glycerol - 0.7, L histidine - 0.05, CuSO4 - 0.01, NaNO3 - 0.18, NaCl 0.18, KCl - 0.05, CaCl2.H2O - 0.05, KH2PO4 - 0.1, FeSO4.H2O - 0.005, MgSO4.7H2O - 0.05, Guaiacol – 10 mm (v/v), H2O2 – 10 mm, Agar - 2.0). The plates were incubated by 6 mm fungal inoculum for 7 days and the reddish brown color change in the screening medium was analyzed (Sivakami et al. 2012; Atalla et al. 2010).

2.5.2 Lignin Peroxidase assay According to Archibald 1992, 3 mL of reaction mixture contained 1 mL of 125 mM sodium tartrate buffer (pH 3.0), 0.5 mL of 0.16 mM azureB, 0.5 mL of the culture filtrate and 0.5 mL of 2 mM hydrogen peroxide. After adding hydrogen peroxide reaction was initiated. One unit of enzyme activity was expressed as an O.D. decrease at 651 nm of 0.1 units per minute per mL of the culture filtrate. 2.5.3 Manganese Peroxidase assay

2.4.3 Manganese peroxidase assay

Manganese peroxidase activity was measured by the oxidation of DMP at 468 nm. 3 mL reaction mixture

Czapek-Dox agar medium containing 0.0025%

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Fig. 2:

Laccase enzyme production by MDG2 fungal strain.

Fig. 3:

MnP (Manganese peroxidase) enzyme production by MDG2 fungal strain.

contained 0.5 mL culture filtrate, 1 mL of sodium tartrate buffer (50 mM, pH 4.0), 1 mL of 2 mM DMP (2,6dimethoxy phenol). The reaction was started by the addition of 0.5 mL of 0.4 mM hydrogen peroxide (de Jong et al. 1992).

medium. Results existing in Table1 showed that fungal strain MDG2 isolated from GGV campus showed highest significant dark brick red colour zone (30.00 mm). Figure1b shows LiP production by MDG2 fungal strain in LiP screening media.

3. RESULTS AND DISCUSSION 3.1 Isolation of white rot fungi: Fourty fungal isolates (Choi et al. 1999) were isolated from the GGV campus. Different sample code given to each collected fungi. Isolated fungi are the wood rotted fungi basically the member of the family Basidiomycetes.

3.2.3 Manganese peroxidase assay–Primary screening for MnP was performed. Results existing in Table 1 showed that fungal strain MDG2 isolated from GGV campus showed oxidation of phenol red to form yellow zone (90.00 mm). Figure 1c shows Laccase production by MDG2 fungal strain in PDA media. Figure 1c shows MnP production by MDG2 cultured mycellium in Czapex-Doxmedia.

3.2 Qualitative assay for Ligninolytic enzyme production

3.3 Quantitative Screening for Ligninolytic enzyme production

3.2.1 Laccase assay-Guaiacol oxidation is one of the most appropriate qualitative assay for Laccase production among fungi. Isolated fourty fungal species were screened for guaiacol oxidation. Results showed that five fungal isolates MDG1, MDG2, FTG2, BTG2, PNB2 shown halo of intense brown colour zone under and around the colony and other remaining isolates did not exhibit an ability to oxidize Guaiacol, indicating the lack of Ligninolytic enzyme activity. Results existing in Table1 showed that MDG2 fungal isolate showed highest significant reddish brown colour zone (43.00 mm) and radial growth, is a positive correlation between the colour zone and radial growth. So MDG2 isolate selected as best strain for qualitative assay. Figure 1a shows Laccase production by MDG2 fungal strain in PDA media. Similarly Atalla et al. (2010) found Pleurotus ostreatus and Trematosphaeria mangrovei showed 32.00 mm, 26.00 mm reddish brown colour zone around the mycelium respectively.

Five fungi showed positive reaction for Ligninolytic enzyme activity. Out of five fungal strain MDG2 strain show maximum enzyme activity, were grown in liquid culture medium (Basal medium) for enzymeactivities. Where Laccase, Lignin peroxidase, Manganese peroxidase, Biomass and Protein of the culture filtrate were measured. 3.3.1 Laccase assay-MDG2 fungal strain produced maximum 1.8567 U/mL (Table 2, Figure 2) Laccaseenzyme and shows 4.1667U/mg Specific Laccase activity within 10 days of incubation. Eggert et al. (1996) reported Pycniporus cinnabarinus produced 1.2 U/mL Laccase on day 7 on basal liquid medium. Laccaseenzyme is most important enzyme in the degradation of Organic pollutants. Han et al. (2004) reported degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its Laccase, was isolated in Korea. 3.3.2 Lignin peroxidase (LiP) assay-LiP enzyme production was monitored by oxidation of Azure B in the presence of hydrogen peroxidase. The MDG2 fungal strain was not shown LiP activity.

3.2.2 Lignin peroxidase assay–Isolated fungi were screened for LiP enzyme production in LiP screening

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Cryptogam Biodiversity and Assessment

degradation of creosote polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 61:2631–2635

3.3.3 Manganese peroxidase (MnP) assay- DMP oxidize in the presence of hydrogen peroxidase is a suitable method for MnP enzyme production. MDG2 fungal strain produced 24.1280 U/mL (Table 2, Figure 3) MnP enzyme and shown 130.2105 U/mg Specific MnP activity.

Cambria MT, Ragusa S, Calabrese V, Cmbria A (2011) Enhanced Laccase production in white rot fungus Rigidoporus lignosus by the addition of selected Phenolic and Aromatic compounds. Appl. Biochem. Biotechnol. 163:415-422

4. CONCLUSION In this study, the fourty fungal species were isolated from the GGV campus of Chhattisgarh. These fungal isolates were screened for Ligninolytic enzyme activity. Out of the fourty cultured fungal strains five strains (MDG1, MDG2, FTG3, BTG2, PNB2) shown positive for Ligninolytic enzyme production. In these five strains, MDG2 fungal strain produced maximum 1.8567U/mL Laccase enzyme and 24.1280 U/mLMnP enzyme. Many researchers (Boganand Lamar 1995; Kim et al. 1998; Hadibarata and Tachibana 2010; Dehghanifard et al. 2013) reported Ligninolytic enzymes play a significant role in the organic pollutant degradation and it depends on the production and secretion of Ligninolytic enzymes. Thus we concluded that isolated white rot fungi can be used for the degradation of organic pollutants after detail investigation against harmfull pollutants.

Choi YW, Hyde KD, Ho WH (1999) Single spore isolation of fungi. Fungal Divers 3:29-38 D’Souza DT, Tiwari R, Sah AK, Raghukumar C (2006) Enhanced production of Laccase by a marine fungus during treatment of colored effluent and synthetic dyes. Enzyme Microb. Technol. 38:504-511. doi:10.1016/ j.enzmictec.2005.07.005 de Jong Ed, Field JA, de Bont JA (1992) Evidence for a new extracellular peroxidase Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55. FEBS Lett. 299(1):107-110. doi:10.1016/0014-5793(92) 80111-S Dehghanifard E, Jafari AJ, Kalantary RR, Mahvi AH, Faramarzi MA, Esrafili A (2013) Biodegradation of 2, 4-dinitrophenol with Laccase immobilized on nano-porous silica beads. Iran J Environ Health SciEng 10:25 Hadibarata T and Tachidana S (2010) Characterization of phenanthrene degradation by strain Polyporus sp. S133. J. Environ. Sci. 22(1):142–149.

5. ACKNOWLEDGEMENTS My sincere thanks to Head, Department of Botany, Guru Ghasidas Vishwavidyalaya, providing infrastructural facilities and thank to Prof. S. K. Chaturvedi (Department of Botany) Nagaland University and Mr. Anand Barapatre, SRF (Department of Occupational Health) JNARDDC Campus, Nagpur for their kind support and guidance to improve my research work. Also,thank to Guru Ghasidas Vishwavidyalaya, Bilaspur (C.G.) for providing the fellowship.

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