Indu Bhushan Prasher #1, Manju* 2 1,2 Mycology and Plant Pathology Laboratory, Department of Botany, Panjab University Chandigarh, India

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1 Optimization Of Physical And Nutritional Factors For Growth And Ligninolytic Activity Of Porostereum Spadiceum, A Noble White Rot Fungi From North-Western Himalayas (India). Indu Bhushan Prasher #1, Manju* 2 1,2 Mycology and Plant Pathology Laboratory, Department of Botany, Panjab University Chandigarh, India 2 Corresponding author: manjujaswal9@gmail.com Abstract Porostereum spadiceum (Pers.) Hjortstam & Ryvarden, Syn. Fung. (Oslo), a wood inhabiting fungi has been studied in vitro for physiological studies in relation to ligninolytic activity. The maximum mycelial growth in terms of average mycelial weight was obtained with Glucose Peptone at 28 C temperature with ph 4 after days of incubation. Lignin Peroxidases (LiP), Mangnese Peroxidases (MnP) and Laccase activity were also analysed in days of incubation. It was found that after 18 days of incubation LiP activity was the highest expressed enzymatic activity in comparison to MnP and Laccase. D (+) xylose was the best carbon source with which maximum mycelial gowth was obtained and least growth occurred in medium containing D (+) raffinose. Highest LiP activity was expressed in the medium supplemented with glucose as carbon source. The best inorganic nitrogen source for the growth of was Sodium nitrate, whereas least mycelia growth of the fungus was observed in Sodium nitrite and there was no growth in ammonium acetate. The best organic nitrogen source for the mycelial growth of fungus is DL-valine, whereas with L-methionine it has shown very little growth. The fungus expressed no LiP activity with any of the Inorganic and organic source of nitrogen. Keywords Wood decaying fungi, Agaricomycetes, Physiological studies, Lignin degradation I. INTRODUCTION Micro-organisms have an emerged as promising tools for bioremediation in present era of industrialization. Wood rotting fungi being magnificent organisms, have aroused an upsurge interest these days for the studies aimed at the better understanding of the unique enzymes they possess. The lignocellulolytic enzymes of Agaricomycetes have a key role in the effective bioconversion of plant residues. White rot fungi belonging to this particular group of fungi are able to degrade cellulose and lignin during the developmental process of fruiting bodies formation during their growth on dead trees ([1], [2]). It possess extracellular enzymes, mainly LiP, MnP and laccase which contribute to its ability to degrade lignocellulose [3]. Lignin is considered as the most recalcitrant component of lignocellulosic material to degrade ([4], [5]). Various chemicals are employed in paper and pulp, food, textile and dye industries, bioremediation, cosmetics, and many other industries for lignin degradation which are toxic and remain as effluents in physical environment. The potential applications of ligninolytic enzymes in industrial and environmental technologies require very large amount of such enzymes at low cost. Therefore, we need new organisms which can synthesise these enzymes in large quantity. Fungi being eco-friendly source of lignin and cellulose degrading enzymes can be used as an alternative to traditional methods employed in such industries. Therefore considering the LMEs producing potential of Porostereum spadicium, the present work was initiated for extracellular LiP production through optimization of different physical and nutritional factors, as nature and composition of culture medium regulate ligninolytic enzymes production by the fungus ([6], [7], [8], [9]). 411

2 II. MATERIAL AND METHODS A. Microorganism and culture conditions Porostereum spadiceum (Pers.) Hjortstam & Ryvarden, Syn. Fung. (Oslo), a wood inhabiting fungi was isolated on Malt extract agar (Malt Extract g, Agar agar g, distilled water to make 1 ml) from dead culms of Bambusa sp. collected from Hamirpur, Himachal Pradesh, India. The stock cultures were maintained at ± 4 C. For all the experiments the fungus was sub cultured on same medium and grown on ± 24 C. B. Identification The fungus was morphologically identified macroscopically and microscopically according to the morphological details as provided in literature [1]. The cultural isolates was further confirmed by amplifying rrna genes using the universal primer set: ITS1 (5-TCCGTAGGTGAACCTGCGG-) and ITS4 (5- TCCTCCGCTTATTGATATGC-) [11]. The sequencing results were assembled and compared with public databases Genbank ( by using the BLAST N sequence match routines. Evolutionary analyses were conducted in MEGA7 [12]. C. Procedure The effects of various physical and nutritional factors on growth and reproduction of Porostereum spadiceum were observed in still cultures grown in 1 ml Erlenmeyer flask containing 25 ml of the basal medium as triplicates (autoclaved at 15 psi pressure for 15- mins). Each flask was inoculated with an agar plug of 1 mm diameter cut from the margin of a five days old culture with a mycelial inoculum equivalent to 2.8 mg dry weight. Three replicates were maintained for each parameter for statistical studies. At the end of each experiment, the mycelia were harvested through pre-weighed Whatman filter paper No. one and dried at 5 C to a constant weight in a hot air oven and their dry weights were measured using an electronic balance (Sartorius Analytical BL 21S). The final ph of the culture filtrate of the individual replicate was checked over Digital ph Meter 813. D. The experiments 1) Basal media: The fungus was grown in 12 different basal media viz. Raulin s, Richard s, Dox s, Coon s, Brown s-i, Brown s- II, Glucose-peptone, Glucose-nitrate, Czapek s-i, Czapek s-ii, Asthana & Hawker s and Elliot s medium for 1 days (selected tentatively). 2) Temperature: In the experiment on the effect of temperature, the fungus was incubated at 16,, 24, 28 and 32 C, in basal medium (Glucose-peptone) with a ph 5 (selected arbitrarily) for 1 days of incubation. 3) H-ion concentrations: In the experiment on the effect of ph, the ph levels of the medium were adjusted 3-9 with a difference of unit ph. The ph of each aliquot was adjusted to a separate unit value aseptically with sterile 1N-HCl and 1N-KOH and checked over Digital ph Meter 813. The flasks were incubated for 1 days at 28 C. 4) Days of incubation: The effect of days of incubation on growth and reproduction of the fungus was studied for days, at 28 C and at ph-4. (found optimum). 5) Carbon source: The effect of the carbon sources (fructose, glucose, lactose, maltose, pectin, raffinose, sorbose, starch, sucrose and xylose) on Porostereum spadiceum mycelial biomass production and enzymatic potential was evaluated at 28 C, ph 4. and after days of incubation. The glucose of the glucose-peptone medium was substituted singly by each of the carbon compounds so as to provide.333 g/l of carbon - a substituent of glucose (1 g/l) in the basal medium. Control without additional glucose source was run in parallel. 6) Inorganic and organic nitrogen source: The effect of different nitrogen sources was evaluated, replacing the peptone by different amino acids and inorganic nitrogen compounds (2. g/l) at optimum conditions i.e. at 28 C and ph 4. in Xylose supplemented basal medium for days of incubation. Control without additional nitrogen source was run in parallel. 7) Quantitative assays for lignin peroxidase, manganese peroxidase and laccase activity: The culture filtrate was assayed for enzymatic activity. All enzymes were assayed spectrophotometrically using THERMO SCIENTIFIC Evolution 1 UV spectrophotometer. Lignin peroxidase (LiP) activity and Manganese (II) peroxidase (MnP) activity was determined using the method as of [3] whereas Laccase activity was determined using guaiacol as a substrate as given in [13]. 412

3 8) Data analysis: All the experiments were carried out in triplicates. The means of three replicate values for all data in the experiments obtained were tested in one way ANOVA at P=.5 using PASW Statistics 16 software and Tukey s test was used to evaluate differences between treatments. Graphs were made by using the software Origin6. III. RESULTS A. Molecular identification: DNA from culture was successfully extracted and amplified using the ITS primer pair. After sequencing, blast results revealed the species identity of the unknown fungi to be Porostereum spadiceum. The optimal tree with the sum of branch length = is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The sequence obtained has been submitted at NCBI GenBank (accession no. MF ). (Fig. 1) KJ Uncultured Basidiomycota clone W-Bas16 KJ Uncultured Basidiomycota clone JQ Ganoderma carnosum strain KU Tyromyces galactinus KX Ceriporiopsis carnegieae voucher KJ Bjerkandera adusta cf. KR Fungal sp. CSE26 HQ Fungal sp. HJF51 Query Porostereum spadiceum JQ Polyporales sp. 1 SR-12 strain KU Bjerkandera atroalba JN Bjerkandera adusta isolate KT Ganoderma carnosum strain V2ERb JQ312.1 Polyporales sp. 1 SR-12 strain 646 KF Porostereum spadiceum isolate VPCI 7/P/12 KC Bjerkandera sp. F1 FJ Uncultured fungus clone S8 JX Porostereum spadiceum KUC KX Bjerkandera fumosa KC Polyporales sp. IIIM-2 HQ Fungal sp. HJF14 AY Rhizoctonia sp. Fig.1 Phylogenetic analysis of partial ITS rdna gene sequence of Porostereum spadiceum and related microorganisms. Built with the help of MEGA 7. software by the neighbor-joining method with Bootstrap values (1 replicate runs). B. Effect of basal medium: The fungus exhibited optimal growth on glucose peptone medium in comparison to the other media. Mycelial growth was incomplete, superficial and submerged in all the sets of media except for Glucose peptone in which growth was complete. It did not reproduce and developed thin walled hyphae. Prominent branching was observed microscopically in Richard s and Czapek s-ii media. There is no growth in Raulin s media but there was formation of chlamydospores. The ph of medium changed from 5. to 4.9. (Fig. 2) 413

4 Raulin's Richard's Dox's Coon's Brown's-I Brown's-II Czapek's-I Basal media Czapek's-II Glucose Peptone Glucose Nitrate Asthana & Hawker's Elliot's Fig.2 Growth (average mycelial dry weight- mg/25 ml) of Porostereum spadiceum with different basal media at 24 C after 1 days of incubation (taken tentatively). C. Effect of temperature: The fungus exhibited maximum growth at 28 C temperature. Thin walled septate hyphae were observed in all the temperatures. Clamped hyphae with abundant branching were observed at 28 C and 32 C. The final ph of basal media significantly increased from 5. to 5.4. The fungus produced complete, submerged, superficial and arial mycelial growth at all temperatures except 16 C. (Fig. 3) Temperature ( o C) Fig.3 Growth (average mycelial dry weight- mg/25 ml) of Porostereum spadiceum with different Temperatures after 1 days of incubation (taken tentatively). D. Effect of ph: The fungus exhibited highest optimum growth at ph 4.. The final ph increased from 4. to 5.3. Thin walled, vacuolated hyphae with clamps developed at 3 to 5. The fungus produced complete, superficial, submerged and cottony mycelial mat in all the treatments except ph 3 in which growth was incomplete and a single mycelial patch was observed in submerged, superficial form. (Fig. 4) 414

5 E. Effect of days of incubation: Highest mycelial growth was observed after th day of incubation and final ph of the media increased upto 5.1. An increase in LiP production upto day 18, MnP upto day and laccase upto day 16 was observed which decreased gradually thereafter (Fig. 5). Out of three enzymes screened maximum activity was shown for LiP. Submerged as well as superficial, incomplete, white cottony mycelial mat was produced upto 8 days of incubation after which the growth was complete and arial mycelium was observed. Thin walled, clamped, septate and hyaline hyphae were observed upto 1 days of incubation. Thick walled hyphae were observed after 15 th day with sparse branching which continued to grow till th day of incubation ph 3. ph 4. ph 5. ph 6. ph 7. ph 8. ph 9. H-ion Concentration Fig.4 Growth (average mycelial dry weight- mg/25 ml) of Porostereum spadiceum with different ph at 28 C after 1 days of incubation (taken tentatively) Day 2 Day 4 Day 6 Day 8 Day 1 Day 12 Day 14 Day 16 Day 18 Day Day 22 Day 24 Day 26 Day 28 Day Days of Incubation LiP MnP Laccase Day 2 Day 4 Day 6 Day 8 Day 1 Day 12 Day 14 Day 16 Day 18 Day Day 22 Day 24 Day 26 Day 28 Day Fig.5 Growth (average mycelial dry weight- mg/25 ml) and ligninolytic enzyme production of Porostereum spadiceum in relation to days of incubation on Glucose Peptone medium at 28 C and ph Absorbance 415

6 F. Effect of carbon source: The maximum average growth in terms of mycelial dry weight was observed with D (+) Xylose followed by sorbose, starch, fructose, glucose, lactose, maltose, pectin, sucrose and raffinose. The ph of all the medium with different carbon sources shifted towards increase with maximum 6.6 in the media containing pectin as carbon source. Mycelial growth was incomplete, submerged as well as superficial in control and lactose whereas in rest of the carbon sources, a complete, submerged, superficial, with white mycelial mat and aerial growth was observed. Maximum LiP activity was observed with glucose as carbon source whereas least activity was shown with lactose. Maximum MnP activity was observed with D- Xylose as carbon source whereas least activity was shown with sorbose. As far as laccase activity is concerned it is highest with pectin and least with sorbose as carbon source. Among three of the enzymes laccase activity was highly expressed. (Fig. 6) Control D (-) Fructose D (+) Glucose D (+) Lactose Maltose Pectin D (+) Raffinose L (-) Sorbose Starch Sucrose D (+) Xylose Carbon Source LiP MnP Laccase Control D (-) Fructose D (+) Glucose D (+) Lactose Maltose Pectin D (+) Raffinose L (-) Sorbose Starch Sucrose D (+) Xylose Fig.6 Growth (average mycelial dry wt.-mg/25ml) and ligninolytic enzymes activity of Porostereum spadiceum in relation to different carbon sources at 28 C and ph 4. after days of incubation F. Effect of inorganic nitrogen: Average Mycelial growth was maximum in Sodium nitrate followed by Potassium nitrate, Ammonium nitrate, Ammonium oxalate, Ammonium phosphate, Ammonium sulphate and Ammonium chloride. There is no growth in Ammonium acetate and a little growth was shown with Sodium nitrite. Mycelial growth was incomplete, submerged, and superficial in control, ammonium chloride, ammonium oxalate, and ammonium phosphate and ammonium sulphate. Mycelial patches are formed in case of Ammonium chloride and Ammonium nitrate. There was complete superficial submerged growth in Ammonium nitrate, Potassium nitrate and Sodium nitrate. The ph level increased from initial ph 4 in ammonium acetate, ammonium oxalate, ammonium nitrate, potassium nitrate, sodium nitrate and sodium nitrite whereas it decreased in ammonium chloride, ammonium phosphate and ammonium sulphate. No LiP activity was shown by the fungus with any of the inorganic nitrogen source. Maximum MnP and laccase activity was observed with ammonium oxalate and sodium nitrite respectively. However no enzymatic activity was observed with the inorganic nitrogenous sources such as ammonium acetate, ammonium chloride, ammonium nitrate, and ammonium phosphate and ammonium sulphate. (Fig. 7) Absorbance 416

7 Control Ammonium acetate Ammonium chloride Ammonium nitrate Ammonium oxalate Ammonium phosphate Ammonium sulphate Potassium nitrate Sodium nitrate Sodium nitrite Control Ammonium acetate Ammonium chloride Inorganic Nitrogen source MnP Laccase Ammonium nitrate Ammonium oxalate Ammonium phosphate Ammonium sulphate Potassium nitrate Sodium nitrate Sodium nitrite Absorbance Fig.7 Growth (average mycelial dry wt.-mg/25ml) and ligninolytic enzymes activity of Porostereum spadiceum in relation to different inorganic nitrogen sources at 28 C and ph 4. after days of incubation G. Effect of organic nitrogen source: Maximum average growth of mycelium was observed in DL-Valine. The fungus produced incomplete, submerged mycelial mat as well as superficial hyphae with light gelatinous texture in basal media containing L-cysteine, L-cystine, glutamic acid, DL-methionine, serine and tryptophan. Growth was complete, superficial, submerged and aerial in rest of the inorganic nitrogen containing basal media. Final ph of all the media were shifted towards an increase, except the media containing L- cysteine, L-cystine and DL- methionine. (Fig. 8). As far as the enzymatic activity is concerned the fungus did not show LiP activity with any of the organic nitrogen source. It showed maximum MnP and laccase activity with the medium containing L-tyrosine as organic nitrogen source. It did not show any of the enzymatic activity with the organic nitrogen sources such as L-α amino-n butyric acid, DL-alanine, L- arginine HCL, L-cysteine HCL and L-leucine. 417

8 IV. DISCUSSION Cultivation of this particular fungus has given significant results in terms of relationship of physical and nutritional factors with ligninolytic activity. The optimal medium in terms of highest average mycelial growth is Glucose-Peptone at temperature 28 C and ph 4 after days of incubation. This is in accordance with the studies conducted as in [14] and [15], who reported that two strains of Fusarium coeruleum could tolerate a ph range of 3-11 and F. solanii isolate grew well at 28 C respectively. It is reported that fast growing fungi usually have high temperature optima [16] and various reports suggested fungi being able to tolerate temperature range of C- C ([17], [18], [19], []). LiP activity being highest expressed as compared to MnP and Laccase, attains maximum activity at 18 th day of incubation and decreases gradually afterwards. MnP and Laccase activity was maximum on 18 th and 16 th days of incubation respectively and decrease afterwards. Cultivating P. spadiceum in optimum medium with different carbon sources revealed D Xylose as the best carbon source for mycelial growth. It was reported that Hexagonia apiaria and Lenzites sterioides, exhibited better mycelial growth with D-Xylose whereas Trematus pini have shown lesser growth with this particular carbon source [21]. In comparison to MnP and Laccase, LiP activity was best expressed and among all the carbon sources used, it is highest with the medium containing glucose as carbon source followed by fructose, which is in accordance with the results of some previous studies [22]. The reason for this is the easily oxidisable nature of glucose in comparison to other substrates making it more favourable for growth and LiP production [23]. Observations regarding effect of inorganic nitrogenous source revealed sodium nitrate to be the best in terms of average mycelial growth followed by potassium nitrate. In utilizing sodium nitrate, it resembles Dictyoarthrinium synnmaticum [24]. However, insignificant growth was observed with ammonium acetate and sodium nitrite respectively. This is in contrast to the findings of ([25], [26], [27], [28]), according to which ammonium nitrogens are assimilated in general and nitrates were moderately poor for fungal growth. Similar results were shown with sodium nitrite for Agaricus bisporus and Lentinus edodes as a non-available source of nitrogen ([29], []). However Beauveria bassiana and some of the other fungi have shown better growth when sodium nitrite was used as the sole nitrogen source ([31], [21]). In case of organic nitrogenous compound maximum mycelial growth was shown with DL-valine. It has been reported previously that 99% mycelial growth was shown by Polyporous serialis and Polyporous rubescen with DL-Valine as in [32]. 418

9 There was a decrease in final ph level of the basal medium with some the organic and Inorganic nitrogen sources which can be connected to de novo organic acids production during secondary metabolism [33]. The fungus doesn t show LiP activity with both inorganic and organic source of nitrogen. Reference [34] have reported earlier that LiP production in Bjerkandera sp. was highly stimulated by high nitrogen, depending on the combination of initial ph and the type of N-source. The presence of MnP and laccase activities are consistent with the previous findings that MnPlaccase combination is the most common group of enzymes in the white rot fungi ([35], [36]). It is also proposed that the activity of MnP and/or laccase may be sufficient for lignin degradation in some of the fungi whereas LiP production in some white rot fungi is highly dependent on the culture conditions and is strain related [36]. V. CONCLUSION These findings indicates that growth substrate may play an influential role in production of ligninolytic enzymes. Optimization of various physical and nutrional factors which enhance the activity of such enzyme can be very helpful for the exploitation of such organisms in biotechnological applications. These results may provide sustainable means for cultivation of fungi for further applications. ACKNOWLEGDEMENT The authors are thankful to UGC (SAP DRS-III) for financial support and the Chairperson, Department of Botany, Panjab University, Chandigarh for infrastructural facilities. REFERENCES 1. Sun, Y. and Cheng, J., Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresource Technol. 2, 83: Mtui, G. Y. S., Recent advances in pretreatment of lignocellulosic wastes and production of value added products. Afr. J. Biot. 9, 8: ISSN Atalla, M. M., Zeinab, H. K., Eman, R. H, Amani, A. Y. and Abeer, A. A. E. A., Screening of some marine-derived fungal isolates for lignin degrading enzymes (LDEs) production. Agri. Biol. J. Nor. Amer. 1, 1(4): ISSN : Himmel Michael E., Shi-You Ding, David K. Johnson, William S. Adney, Mark R. Nimlos, John W. Brady, Thomas D. F., Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production Science 7, 315: Sánchez C., Lignocellulosic residues: Biodegradation and bioconversion by fungi. Biotechnology Advances 9, 27: Zakariashvili, N. G. and Elisashvili, V. I., Regulation of Cerrena unicolor lignocellulolytic activity by a nitrogen source in culture medium. Microbiol (Moscow) 1992, 62: Galhaup, C., Wagner, H., Hinterstoisser, B. and Haltrich, D., Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb. Technol. 2, : DOI: 1.116/S (1) Songulashvili, G., Elisashvili, V., Wasser, S. P., Nevo, E. and Hadar, Y. Basidiomycetes laccase and manganese peroxidase activity in submerged fermentation of food industry wastes. Enzyme Microb. Technol. 7, 41: Abdel-Azeem, A. M. and Salem, F. M., Biodiversity of laccase producing fungi in Egypt. Mycosphere 12, 3(5): 9-9. Doi /mycosphere/3/6/4 1. Prasher, I. B., Wood-rotting non-gilled Agaricomycetes of Himalayas. Fungal Divers. Research Series (Springer Netherlands), White, T. J., Bruns, T., Lee, S. and Taylor, J., Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M. A., Gelfand, D. H., Sninsky, J. H., White, T. J. PCR Protocols, : San Diego, USA: Academic Press, 199, pp

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