Bioremediation of PAHs the Co-Culture of White Rot Fungi and Bacteria a FRED in silico Emerge

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Int.J.Curr.Microbiol.App.Sci (2015) 4(8): 358-371

Original Research Article

Bioremediation of PAHs the Co-Culture of White Rot Fungi and

Bacteria a FRED in silico Emerge

Subash Nachimuthu* and Sasikumar Chinnagounder

PG & Research Department of Biotechnology, Nehru Memorial College (Autonomous), Puthanampatti 621 007, Tiruchirappalli, Tamil Nadu, India

*Corresponding author

A B S T R A C T

Introduction

PAHs are highly carcinogenic or mutagenic (Fang et al., 2006) and they have been classified by the Environmental Protection Agency (EPA) of the United States as priority pollutants. The EPA has listed 16 PAHs as hazardous because of their high carcinogenic or mutagenic potentials (Perera

et al., 2005; Taneja and Masih, 2006;

Sharma et al., 2006). Possible factors for PAHs increased release into the environment include volatilization, photo-oxidation, chemical oxidation, bioaccumulation and adsorption on soil

particles (Chang et al., 2000). They occur as colourless, white/ pale yellow solids with low solubility in water, high melting and boiling points and low vapour pressure. With an increase in molecular weight, their solubility in water decreases; melting and boiling point increases and vapour pressure decreases (Clar 1964; Patnaik, 1999). However, it is not known how rapidly or completely the human lungs absorb PAHs. Drinking water and swallowing food, soil or dust particles that contain PAHs become the routes for these chemicals to enter in to the ISSN: 2319-7706 Volume 4 Number 8 (2015) pp. 358-371

http://www.ijcmas.com

The present work focuses on collection and identification of white rot fungi and bacteria effective degradation of PAHs. Studies revealed Manganese peroxidase (MnP) and lignin peroxidase (Lip) as predominant enzymes in T. versicolor with good ligninolytic potential, while laccase reported maximum in P. chrysosporium. Investigation showed that PAHs degradation was higher in LSF bacterial and fungal co cultivation of T. versicolor with P. putida and effective organisms were noticeably declined in SSF. Anthracene was reported with high degradability by T.

versicolor + P. putida. It was also observed that co-cultivation of bacteria and

white rot fungi possessed high amount of biosurfactant production. Bio-informatic studies employing docking analysis to support the findings were studied using the ligninolytic enzymes and PAHs interactions. FRED analysis revealed that the maximum interactions occurred with anthracene as MnP>LiP>Laccase. The docking scores obtained between anthracene and co-cultivation of T. versicolor +

P. putida was significant when compared to control.

K e y w o r d s White rot fungi, Biosurfactant, Ligninolytic enzymes, PAHs

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body, but absorption is generally slow when PAHs are swallowed (USHHS, 1995). Bioaccumulation is an alternative strategy for microbial cells to cope with PAHs toxicity, since the latter are stored to microbial lipid fraction and might be transformed to more water-soluble molecules (Subashchandrabose et al., 2013). Taking the severity in the hazardous effects of PAH s into account the present study employed collection of PAHs from incomplete combustion of organic material such as oil, petroleum gas, coal, and wood and the study of the possible role of microorganisms in PAHs degradation revealed that two main groups are involved in the oxidation and subsequent mineralization of these compounds: soil bacteria and white rot fungi. The degradation of PAHs is limited by its solubility (Sims and Overcast, 1983) as soil bacteria are found to effectively degrade low molecular weight PAHs. White rot fungi can oxidize more condensed PAHs molecules with up to six aromatic rings, limiting water solubility (Bezalel et al., 1996; Wolter et al., 1997) and decrease their toxicity (Kotterman

et al., 1998).

Ligninolytic enzymes produced by basidiomycetes fungi such as lignin peroxidase (Lip), manganese peroxidase (Mnp) and laccase have been suggested to play a key role in Lignin degradation (Vares

et al., 1994). It was assumed that PAHs and

other organic pollutant degradation can be catalyzed by the extracellular ligninolytic enzyme system of basidiomycetes fungi (Bezalel et al., 1996; Pickard et al., 1996; Novotny et al., 2004). Hence the bioremediation process is employed with a view to protect the living organisms, most often microorganisms, plants, or both, or products produced from living organisms to degrade, detoxify, or sequester toxic chemicals present in natural waters and soils.

Fungi have been used for bioremediation especially white rot fungi (Bennett and Faison, 1997). These are wood degrading microorganisms that produce special oxidases that help to degrade the plant polymer lignin as well as great variety of chemicals, including many environmental pollutants. Other oxidative fungal enzymes like laccase appear to initiate degradation of many xenobiotic molecules (Paszczynski and Crawford, 1995). While bacteria s constitute as a class of microorganisms actively involved in the degradation of organic pollutants from contaminated sites. A number of bacterial species are known to degrade PAHs. Most of them, representing biodegradation efficiency, are isolated from contaminated soil or sediments. Long term petrochemical waste discharge, oil degrading bacteria are capable of degrading PAHs to a considerable extent (Walter et al., 1991; Schneider et al., 1996; Trzesicka and Ward, 1995). In addition, certain bacterial strains, which were related to the species

Alicycliphilus denitrificans, Microbacterium arabinogalactanolyticum and Shinellazo ogloeoides can be considered as effective

anthracene degraders, showing degradation (Spyridon et al., 2015).

Three oxidative enzymes are commonly found in extracellular ligninolytic cultures of White rot fungi. It is clear that different combinations of the known enzymes are produced by various lignin degrading fungi, suggesting that there is more than one successful strategy for lignin biodegradation (Hatakka, 1994). Laccase is produced by most white rot fungi (Hatakka, 1994), but normally not in Phanerochaete chrysosporium (Kirk and Farrell, 1987). It

has a high biotechnological interest as demonstrated by several studies including green processes, such as wood pulp delignification, dye decolorization in the textile industry, ethanol production, wine processing, treatment of polycyclic aromatic

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hydrocarbons, bioremediation and for the realization of biosensors with strong preferences given to Mn(II) as its reducing substrate (Salis et al., 2009; Ferraroni et

al.,2014). The redox potential of the Mn

peroxidase is lower than that of lignin peroxidase and it has only shown capacity for oxidize in vitro phenolic substrates (Vares, 1996).Lignin peroxidase (LiP) was first discovered in Phanerochaete chrysosporium(Glenn et al., 1983; Tien and

Kirk, 1983) and it produced considerable extent of white rot fungi. This enzyme is an extracellular hemeprotein, dependent of H2O2, with an unusually high redox potential and low optimum pH (Gold and Alic, 1993). It showed little substrate specificity, reacting with a wide variety of lignin model compounds and even unrelated molecules (Barr and Aust, 1994).

A major factor controlling the biodegradation of PAHs is their bioavailability to microbial degradation which may be limited because of low aqueous solubility of the contaminants and their absorption on to soil (Guiotet al., 2005). Microorganisms have a large surface to volume ratio, produce a variety of surfactants that adsorb to and alter the conditions prevailing at interfaces (Rosenberg, 2006).

The application of the practice in bioinformatics such as protein ligand docking tools offers a rapid means of identifying new potential targets for bioremediation (Drews, 2000). Subsequently have come up other studies related for docking using FRED Version-VIDA-2.1.2 in Open Eye (Arun et al., 2008). In the present investigation, PAHs degrading wild basidiomycetes isolates were collected from

Kolli hills of the Eastern Ghats, Namakkal

district, Tamil Nadu were compared to those of a bacterial isolate recovered from oil spilled soil from Manali (CPCL) Chennai

and to their corresponding co-cultures in liquid state fermentation (LSF), solid state fermentation (SSF) and in PAH. The production of biosurfactants seems to be a prerequisite so co-cultivation of the white rot fungi and the bacterial isolates have been investigated and reported. A simultaneous study was also carried out using in silico approach by employing docking tools, FRED version 2.2.5 Open Eye software to support the findings.

Materials and Methods Collection of white rot fungi

The basidiomycetes fungal sporocarps were collected from two different regions of Kolli hills (110 30' to 210 0' N Latitudes and 770 22' to 850 20' E Longitudes) a part of Eastern Ghats, located in Namakkal district, Tamil Nadu, India. The fungal sporocarps were carefully collected (Mueller et al., 2004). Out of two sets, one set of the samples were preserved for culturing the mycelia and another set of the samples was used for the study of morphological (color, shape and odour) characteristics, microscopical characteristics and biochemical reactions (Kaul, 1997). Individual basidiomes of the preserved samples of white rot fungi were cultured by the procedure suggested by Kaul (1997) on Potato Dextrose Agar (PDA) medium which was supplemented with streptomycin (50 mg L-1). The plates were incubated at 25°C for seven days. The samples were stored and cultured in a refrigerator and they were sub-cultured once in a month.

Molecular ITS identification of isolated white rot fungi

White rot fungal strain identification was carried out by amplifying partial ITS (Rochelle et al., 1995). The PCR product was sent to Rajiv Gandhi Centre for

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Biotechnology Kerala for sequencing and the identified sequences were compared with the ITS sequence available in the

public nucleotide databases

(http://www.boldsystems.org). Distances were calculated and phylogenetic trees were constructed using the neighbour-joining method (CLUSTAL W software) then the sequences were analyzed through MEGA3 package and submitted to Gen Bank (Drummond et al., 2010).

Isolation and molecular identification of Polycyclic Aromatic Hydrocarbon (PAH) biodegrading bacteria present in oil spilled soil

The oil spilled soil was collected from the oil refinery station nearby area at Chennai Petroleum Corporation Limited (CPCL), Manali, Chennai, India. The PAHs biodegrading bacterial isolates were collected from oil spilled soil and they were identified based on their microscopic, morphological and biochemical characteristics (Holt et al., 1994) and by partial sequencing of their 16S rRNA. The isolation of DNA was carried out according to Rochelle et al. (1995). Strain identification was studied by amplifying partial 16S rRNA by using the method of Weisburg et al. (1991).

The sequences of the partial 16S rRNA were compared with 16S rRNA sequence available in the public nucleotide databases at the National Center for Biotechnology Information (NCBI) by using their World Wide Web site (http://www.ncbi. nlm.nih.gov) and the BLAST (Basic Local Alignment Search Tool) algorithm using CLUSTAL W software. Distances were calculated and phylogenetic trees were constructed using the neighbor-joining method. The MEGA3 package was used for all the above fore said analysis (Drummond

et al., 2010).

Screening of white rot fungi for Ligninolytic enzyme producing potential

The white rot fungal cultures were inoculated in basal broth and incubated at room temperature. After incubation the triplicates of each flask were harvested by centrifuging the broth at 10,000 rpm for 20 minutes. The supernatants were extracted for the present study of ligninolytic enzymes such as Lip (Vijaya and Singaracharya, 2005), Mnp (MarioCarlous et al., 2002) and laccase (Ramakrishna et al., 2004).

Biosurfactant production

The biosurfactant activities of the selected organisms were tested by the following methods: dry weight, oil spreading technique, and emulsion test assay, hemolysis activity according to Mulligan et

al. (1984). The estimation of biosurfactant

activity was carried out by using oil spreading technique (Morikawa et al., 2000) and emulsion test (Cooper and Goldenberg, 1987). A sodium lauryl sulphate was used as control in the above said estimation process.

Biodegradation of PAHs

The biodegradation of the selected white rot fungi, the bacterial isolates recovered from oil spilled soil and co-cultures was studied in LSF and solid state following the modified method of Steffen et al. (2002). For PAHs biodegradation studies in solid state, paddy straw bits (2.5 g) containing 80% moisture were taken in flasks. The flasks were sterilized at 15 Lb for 30 minutes. For co-culture studies 100 mL of the log phase bacterial and white rot fungal isolates were inoculated simultaneously along with each of the selected white rot fungi (3 agar plugs). The organisms were grown as stationary cultures for 3 days at 26°C. The PAHs dissolved in dimethyl

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formamide were then added at a concentration of 1mg L-1 to each flask. The contents of the flasks were mixed and incubated in dark at 24°C. Controls were also treated in the same way but not inoculated with the organism. The flasks were harvested at 10 and 20 days after PAHs addition for the extraction (Arun et al., 2008).

PAHs biodegradation analysis

HPLC (SHIMADZU, SPD-10 a VP) with silicon C18 column was used to separate and analyze PAHs under isocratic condition (solvent-acetonitrile: water=80:20 v/v; detection wavelength=254 nm). One milliliter of the extracted samples was added to 5 ml of methanol, and from this, 20 l was injected to the HPLC analyzer for the analysis of PAHs (Concentration of each PAHs in 20 l of the sample is 50 g). The PAHs present in the culture liquid were identified by the comparison of retention time with authentic chemicals. Based on the remaining PAHs present in the sample, the percentage of degradation of PAHs by the organisms was calculated (Arun et al., 2008).

FRED Open Eye software studies

X-ray crystal structures of ligninolytic enzymes laccase (1 Gyc), manganese peroxidase (MNP), and lignin peroxidase (ILLP) were collected from the Brookhaven Protein Data Bank in Pdb format. The structures of various PAHs were drawn in chemdraw (Version-Ultra vision-12.0.2, Cambridge Soft Corporation) and saved as MDL MOL file format. The enzyme ligand docking was carried out by using FRED (Version-VIDA-2.1.2, Open Eye structure software (Arun et al., 2008). The present study employs the updated version FRED (Version-VIDA-2.2.5). The scoring function employed was Chem gauss 3.

Results and Discussion White rot fungi

The white rot fungal sporocarps were collected at two different sites of Kolli hills. The general climatic conditions of Eastern Ghats, Tamil Nadu, India, the maximum rainfall (209 mm) and humidity (97.7%) during September and November, 2012 and minimum rainfall and humidity recorded in during April and June 2013 was 5 mm and 21.7% respectively.

Identification of white rot fungi for ITS sequence studies

A native PAH degrading white rot fungi were ITS sequenced and was compared against those available in the public databases (bold systems). The ITS sequence of the isolated white rot fungi closely matched with T. versicolor, T. sanguine and

P. chrysosporium (99% homology)

sequence available in the database. Based on this study and on the morphological characteristics, the white rot fungi were identified as T. versicolor, T. sanguine and

P. chrysosporium. The ITS sequence has

been deposited in Gen Bank database under accession number T. versicolor

(KM596814), T. sanguine (KM596815) and

P. chrysosporium (KM596813).

Isolation and molecular identification of polycyclic aromatic hydrocarbon (PAHs) biodegrading bacteria present in oil spilled soil

In the present study, an indigenous PAH degrading bacterial species were isolated from oil-spilled soil using enrichment culture. The 16S rRNA sequences of the isolates were closely matched with those of

P. putida and B. subtilis (99% homology) in

the database. Based on this study and on the morphological, cultural characteristics and

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biochemical characteristics, the bacteria were identified as P. putida and B. subtilis. The nucleotide sequence has been deposited in Gen Bank database under accession number P. putida (KM873035) and B.

subtilis (KM873031).The study reported are

on par to the findings and postulations ofReddy et al., 2010.

Screening of white rot fungi for ligninolytic enzyme producing potential

Several studies have reported the ability of the basidiomycetes fungi in degrading high molecular weight PAH compounds having more than three rings (Cerniglia, 1992). Ligninolytic enzymes of the mushroom fungi are implicated in the PAH biodegradation. Three kinds of ligninolytic enzymes viz, laccases, manganese peroxidase and lignin peroxidase are widely distributed in basidiomycetes fungi. Based on the mineralization of Lignin, these are grouped into brown rot fungi which cannot mineralize lignin and white rot fungi which can mineralize lignin. Besides the grouping, basidiomycetes fungi are classified on the basis of ecological nieches viz. soil inhabiting fungi, wood rotting fungi and mycorrhizae. The wood rotting fungi reportedly elaborate all the three ligninolytic enzymes (Steffen, 2003) while lignin peroxidase was not produced by soil inhabiting fungi.

The ligninolytic enzyme production potential of the basidiomycetes fungal isolates were compared with that of the basidiomycetes fungal pure cultures. The ability of the various white rot fungal species to transform PAHs and other pollutants is similarly quite variable. This variability is due to differences in both the enzymology of the various white rot species and differences in growth and enzyme production responses of the fungi to different culture media (Arun et al., 2008;

Arun and Einey, 2011). The ligninolytic enzyme production potential is understood from the table 1. All the three isolated white rot fungal strain produced Laccase, Mnp and Lip which produced relatively higher amount of ligninolytic enzymes were selected for the present study and they were 14x10-6, 30.5x10-6 and 12.3x10-6 IU/ L-1 respectively.

Biosurfactant production

The biosurfactant activities of the selected organisms were tested by the following methods: dry weight, oil spreading technique, and emulsion test assay, hemolysis activity. Among the methods T.

versicolor was found to be the major

proficient biosurfactant producing strain. Willumsen and Karlson (1997) have suggested that the surfactant producing bacterial strains and basidiomycetes fungi were able to solubilize PAHs more efficiently than the non surfactant producing strains. Similar observations were made in the present study, where the efficient ligninolytic and PAH degrading mushroom

T. versicolor was found to be capable of

producing higher amount of biosurfactant. The efficiency of biosurfactant production was measured by the extent of hemolysis by

T. versicolor (1.6 cm) matching that of T. sanguine (1.4cm) and P. crysosporium

(1.2cm). The bacterial isolate showed minimum biosurfactant production P. putida (0.7cm) and B. subtilis (0.4cm) (Table 2). Biosurfactant produced by T. versicolor and

P. putida was extracted and characterized by

the oil spreading technique and emulsion test (Table 2), hemolytic activity. All the three fungal strains were utilized for the biosurfactant production. The surfactant producing bacterial strains and basidiomycetes fungi were able to solubilize

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PAHs more efficiently than the non-surfactant producing strains.The present study was showed the similar findings inefficiency of ligninolytic and PAH degrading white rot fungi and biosurfactant in T. versicolor and T. sanguine as reported by Reddy et al. (2010); Arun et al. (2011).

Co-culture studies on PAHs biodegradation in LSF and SSF

Several white rot fungal genera are reported to degrade PAHs in liquid state fermentation (Arun et al., 2008; Tony et al., 2009; Arun and Einey, 2011). The extent of PAH biodegradation by the selected fungal and bacterial co-culture with 10 days and 20days of incubation in LSF is presented in figures 1 and 2.

PAHs biodegradation by the mushroom fungal and bacterial co-culture ranged between 72.7% and 92.7% on 10 to 20days of incubation respectively with maximum degradation rate in LSF.

The amount of PAH biodegradation by the preferred fungal and bacterial co-culture with 10 days and 20 days of incubation in SSF is given in figure 3 and table 3. The white rot fungal and bacterial isolates degradation ranged between 9.7% and 15.7% with 20 days of incubation that slightly differed in contrast to the 10 days of SSF.

Among the co-culture, fungal and bacterial isolates in LSF and SSF on 10 days of incubation compared to SSF were found significantly augmentable in the rate of biodegradation by co-cultures of white rot fungi in LSF. Comparison of the present investigation with the findings of Arun and Einey (2011) reported better biodegradation with anthracene degradation by co-culture of

T. versicolor + P. putida more significant.

While Spyridon et al. (2015) has reported

using the bacterial monoculture 60 -70% when compared to the co-culture T.

versicolor + P. putida as an efficient

degrader.

FRED Open eye software studies

Selected ligninolytic enzymes (Lip, Mnp, laccase) and PAH ligands naphthalene, acenaphthene, fluorene and anthracene were docked using FRED in Open Eye. In the present study, FRED- open eye software revealed that the anthracene ligand docked with all the three ligninolytic enzymes (Figure 4). The interaction of various PAHs with different enzymes decreased in the order of anthracene, fluorine, acenaphthene and naphthalene. Choi (2005) used FRED as the fastest algorithm requiring average 18 seconds, followed by average 46 seconds requiring DOCK programme. Arun et al., 2008 used PAH ligands-naphthalene, acenaphthene, fluorene, anthracene, and pyrene-were docked using FRED Version-VIDA-2.1.2 in Open Eye. Apart from the important factors such as concentration of enzymes, pollutants and their bioavailability, it is important to recognize that with all its margins in docking, it would be exceptionally constructive to recognize binders and non-binders from a pool of ligands and this would be a large amount than the conservative approach in the assortment of unambiguous enzymes for PAH biodegradation. Even though Arun et

al. (2008) used FRED version-VIDA-2.1.2

and the present study version-VIDA-2.2.5 was used and it is concluded that only not significant (0.2%) variations found between FRED revealed studies and in vivo studies. The present study has documented co-cultivation of T. versicolor + P. putida as an efficient strain in MnP production in LSF with maximum lignin degradation potential. The MnP was the predominant enzyme of

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ligninolytic activity studied in both bacterial and fungal isolates. The applicable importance of ligninolytic enzymes lies in the fact that they can contribute in breaking down recalcitrant pollutants that may help in reduction of some environmental pollution

problem. The wild basidiomycetes fungi T.

versicolor with oil degrading bacteria P. putida used in the present investigation

exhibited a remarkable activity in decreasing anthracene percentage in polluted soil.

Table.1 Ligninolytic activity of the isolated white rot fungi

Ligninolytic Activity (U mL-1) S.No. Isolated white rot fungi

Laccase MnP LiP

1 P. chrysosporium 14.00 17.12 7.61

2 T. versicolor 7.79 30.50 12.30

3 T. sanguine 8.07 19.40 12.00

Triplicate with a standard deviation of <3%.

Table.2 Quantitative assay of biosurfactant produced by bacterial and white rot fungi

S.No. Isolated white rot fungi Emulsion Index (%) Oil spreading technique Zone of hemolysis (cm) 1 P. chrysosporium 109 17 1.2 2 T. versicolor 127 57 1.6 3 T. sanguine 111 22 1.4 4 P. putida 119 27 0.7 5 B. subtilis 112 24 0.4 6 1% SDS 225 - -

Table.3 Biodegradation using bacterial, fungal Co-cultures at 20 days incubation in SSF

Biodegradation rate of various PAHs in %a S.No. Co-culture of bacteria and

fungi

Naphthalene Acenaphthene Fluorene Anthracene

1 T. versicolor + P. putida 6.5 7.8 12.5 15.7 2 T. sanguine + P. putida 7.5 4.2 6.2 9.4 3 P. chrysosporium + P. putida 8.2 5.3 3.3 3.2 4 T. versicolor + B. subtilis 6.4 2.4 4.1 12.2 5 T. sanguine + B. subtilis 10.2 7.4 8.2 10.2 6 P. chrysosporium + B. subtilis 4.4 4.1 6.3 6.2

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Figure.1 Biodegradation using bacterial, fungal Co-cultures at 10 days incubation in LSF

Figure.2 Biodegradation using bacterial, fungal co-cultures at 20 days incubation in LSF

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Figure.3 Biodegradation using bacterial, fungal Co-cultures at 10 days incubation in SSF

Figure.4 Docked poses interaction between enzymes of white rot fungi and PAHs

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Acknowledgement

The authors thank the University Grants Commission, New Delhi for financial support to carry out this work and we thank Mr. U. Suresh Kumar, DNA Examiner, Regional Facility for DNA Finger printing Rajiv Gandhi Centre for biotechnology, Thiruvanandapuram Kerala and Dr. K. Sathiskumar, Research Associate, Mother Theresa University, Kodaikannal for valuable help during the ITS sequencing. The Management and Principal of Nehru Memorial College (Autonomous), Puthanampatti and our unit research team members Mrs. J. Viji, Miss. R. Shalini and Miss. M. Nirmala Research Scholar, Department of Chemistry Periyar University Salem and Mrs. Dr. V. Meena for valuable help during the study period.

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