Bio-delignification ability of locally available edible mushrooms for the biological treatment of crop residues

Transcription

1 Indian Journal of Biotechnology Vol 11, April 2012, pp Bio-delignification ability of locally available edible mushrooms for the biological treatment of crop residues Ch Vijya 1 and R Malikarjuna Reddy 2 * 1 Departmen of Marine Biology, Vikrama Simhapuri University, Nellore , India 2 Department of Dairy Science, Jawahar Bharati Degree and PG College, Kavali , India Received 6 August 2010; revised 1 May 2011; accepted 21 August 2011 The crop residues, such as, paddy straw, have low digestibility and, therefore, efforts have been made in the present study for microbial and enzymatic delignification of crude fiber to improve its nutritive value before feeding to animals. Two types of edible mushrooms were collected from the market and screened qualitatively for their ability to produce ligninases through guaiacol assay. The oyster mushroom showed maximum lignolytic activity and, thus, it was used in the study for the treatment of paddy straw. After sequencing of the mushroom culture used, it was identified as Schizophyllum commune. The cellulolytic activity and lignolytic activity of S. commune was assayed. The organism showed high cellulolytic as well as lignolytic activity at C temperature and at neutral ph. As low cellulolytic and higher lignolytic activities are desirable for successful biodelignification of straw, different chemicals at various concentrations were used to suppress the cellulolytic activity. Of all the chemicals used, veratryl alcohol at 30 mm concentration was able to suppress the cellulolytic enzyme activity by 90%, while enhancing the lignolytic activity of the organism by 50-60%. Therefore, on the basis of present study, S. commune could be recommended for delignification of crop residues with the addition of appropriate quantity of veratryl alcohol. Keywords: Biodelignification, edible mushrooms, lignolytic enzymes, paddy straw, Schizophyllum commune Introduction Crop residues are lingo-cellulosic biomass used traditionally for cattle feed in India. Among various cereal straws available, paddy straw has long been considered as maintenance feed and important energy feed for ruminants 1. Paddy straw has three major components, viz., cellulose, hemicellulose and lignin, and among these components, cellulose and hemicellulose are easily broken down in the rumen of the cattle by extracellular enzymes of many bacteria and fungi 2. But lignin, being an aromatic heteropolymer of phenyl proponoid units, resists easy degradation by microorganisms of the rumen. Therefore, the digestibility of the paddy straw is only 40-45% in cattle and buffaloes, the proportion of total digestible nutrients (TDN) is about 45%, while digestible crude protein (DCP) is almost zero 3,4. Thus, efforts have been made to find the ways of microbial and enzymatic modifications, or delignification of paddy straw, to improve its bio-digestibility and upgrade its nutritive value before it is fed to the *Autho for correspondence: Tel: rmreddy58@gmail.com animals in order to improve the milk productivity of dairy animals. Further, feeding of nutritive feed to the animals reduces the emission of methane by 20-30%, whereas feeding of crop residues of low quality increases the methane emissions, which is one of the causes of global warming 5. Among various microorganisms, bacteria have less lignolytic activity and more cellulolytic activity and, hence, they are not considered for the delignification process 6. In this context, fungal mycelia could be preferred because the physiological requirement of their growth would be simpler. Soil fungi, such as, Aspergillus niger, Humicola fuscoatra and Penicillium notatum, are proven potential lignin degrading organisms but the straw treated with these organisms has low palatability and, thus, its acceptability by the cows and buffaloes is low 7. White-rot fungi have drawn special attention in this context because they selectively degrade lignin present in plant material and improve the digestibility and nutrient availability in rumen 8. Further, to depolymerise and mineralize lignin, white-rot fungi have developed a non-specific oxidative system including several extracellular enzymes, such as,

2 192 INDIAN J BIOTECHNOL, APRIL 2012 several kinds of laccases, peroxidases and oxidases, producing H 2 O However, the enzymatic composition of the lignolytic system depends on the fungal species, with laccase being the common component 12,13. White-rot fungi, such as, Pleurotus eryngii, P. ostreatus, Bjerkandra adusta, Phanerochaete chrysosporium, etc, have efficient lignin peroxidase, laccase and manganese peroxidase enzymes under wide range of culture conditions 8,11,14. Use of P. ostreatus and other organisms for delignification of paddy straw under solid state fermentation technique proved to be successful in improving its nutritive value 15,16. However, the availability of this mushroom culture to ordinary farmers in India is not an easy task. In these circumstances, edible mushrooms grown commercially using paddy straw as substrate are selected as culture for the purpose. This is because the production of mushrooms is now a house hold activity in many parts of the country and thus the availability of seed or culture would not be a problem for the production of delignified paddy straw or crop residues by the dairy farmers. Thus, an attempt is made to analyze the use of edible mushrooms to improve the nutritive value of paddy straw. Materials and Methods Screening of Ligninase Positive Fungi Two types of mushrooms, viz., oyster mushroom and milky mushroom, collected from the market were screened qualitatively for their ability to produce ligninases through guaiacol assay method. The mushroom which showed maximum lignolytic activity was used in the study for the treatment of paddy straw and its 16S rrna sequencing was done by RAS Life Sciences Ltd, Hyderabad for proper identification of the species. A small piece of each mushroom was cut, surface sterilized with mercuric chloride solution (0.2% in SDW), inoculated into malt extract broth and incubated at 37 o C for 7-10 d. Later, it was used for delignification of the straw repeatedly. The malt extract broth, after incubation for certain periods, was used for the enzyme activity studies. Estimation of Enzyme Activity During the process of incubation, edible fungi produce many lignolytic and cellulolytic enzymes to convert the less digestible lignin and cellulose into easily digestible carbohydrates and other nutrients. The relative activity of cellulolytic enzyme, endogluconase (C x ) was assayed by viscosity measurements as suggested by Akin et al 17, while the activity of exogluconase ( ), another cellulolytic enzyme, was estimated according to the procedure of Plummer 18 and expressed as mg/ml of reducing sugars liberated in 6 h. The activity of two lignolytic enzymes, lignin peroxidase () and laccase (), was estimated using the method suggested by Tien and Kirk 19. Influence of Temperature and ph In order to optimize the growth conditions of the mushroom, the effect of incubation temperature and ph on the enzyme activity was estimated. The malt extract broth inoculated with mushroom culture was incubated at different temperatures for 7 d and the same was used to estimate the residual activity of cellulolytic and lignolytic enzymes. The influence of ph on cellulolytic and lignolytic enzymes of edible fungi was also studied by using citrate buffer ranging from ph in malt extract medium. The influence of various chemicals and metal ions on the enzymatic activity was also observed by taking the chemicals at different concentrations in malt extract broth. Results and Discussion Screening of Edible Mushrooms for Lignolytic Activity Malt extract agar plates supplemented with 0.02% guaiacol and inoculated with mushroom culture were examined for the lignolytic ability of mushrooms after 7 d of incubation. The mushroom having the lignolytic enzyme activity developed circular zones of reddish brown colour due to the oxidation of guaiacol and noted as ligninase positive 20. Of the two varieties of mushrooms used, the diameter of reddish brown colour zone was higher (52 mm) in case of oyster mushroom as compared to milky white mushroom (24 mm) (Fig. 1). Thus, the oyster mushroom showed Fig:1 Ligninase activity of mushrooms in guaiacol medium: A. Milky white mushroom; & B. Oyster mushroom.

3 VIJYA & REDDY: DELIGNIFICATION ABILITY OF EDIBLE MUSHROOM, S. COMMUNE 193 maximum lignolytic activity and it could be used for the treatment of paddy straw. After sequencing 16S rrna of the oyster mushroom culture used, it is identified as Schizophyllum commune (Acc.No. X , S. commune small subunit 16S rrna sequencing: Maximum score=1977; Total score obtained=1977; Query coverage=96%; E. value=0.0; Max. ident=99%). Estimation of Lignolytic and Cellulolytic Enzymes The ability S. commune for the bio-delignification of paddy straw was further studied under in vitro conditions using malt extract broth and malt extract broth amended with 20% paddy straw. Initiatly the activity of lignolytic enzymes in malt extract broth was low (70-84 U/mL) up to 7 th d of incubation and then rose to U/mL at 21 st d of incubation with increase in the period of incubation (, 125 U/mL and, 138 U/mL; Table 1). On the other hand, the production of cellulolytic enzymes was maximum ( -100 REA and -220 mg/ml) at 7 th d of incubation and declined with the further increase in incubation period. The correlation coefficients of period of incubation and enzyme activity indicate a strong negative correlation ( for Cx & for ) between cellulolytic activity and period of incubation and strong positive correlation ( for & for ) between lignolytic activity and period of incubation. The delignification studies carried out in malt extract broth amended with 20% paddy straw also indicated the same trend in the production of extracellular lignolytic and cellulolytic enzymes by S. commune (Table 1). However, the presence of paddy straw enhanced the lignolytic enzyme activity by about 10-20% compared to the enzymatic activity in malt extract broth alone. This is because the lignolytic system of white rot fungi is activated in the presence of lignin compounds, such as, paddy straw, during secondary metabolism of fungal growth and is regulated by the availability of nutrients, oxygen, trace metals and ph 21. The rise in the lignolytic enzyme activity of S. commune in the presence of straw or lignin is a desirable feature required for any organism that could be used for the bio-delignification of crop residues. By juxtaposing both cellulolytic and lignolytic enzyme activities expressed by S. commune, it is striking to note that the activity of cellulolytic enzymes is higher compared to lignolytic enzymes (Table 1). However, in the biological treatment of crop residues, higher lignolytic activity and lower cellulolytic activity of the organism is to be needed. This is due to the fact that the rumen microflora of cows and buffaloes possess cellulolytic activity but they are devoid of lignolytic activity 2. If the organism exhibits more cellulolytic activity during the treatment there will be loss in the dry matter or quantity of the crop residues used for the treatment. Thus, it is necessary to suppress the cellulolytic activity of the organism intended for the microbiological treatment of straw. Therefore, an attempt is made to explore the influence of physicochemical parameters such as ph and temperature in order to suppress the activity of extracellular cellulolytic enzymes released by S. commune. Influence of Temperature on Enzyme Activity The optimum temperature recorded for the Cx and activity of S. commune was 45 and 55 o C, respectively (Table 2). Contrary to this, and enzymes exhibited maximum activity (115 U/mL & 92 U/mL, respectively) at 30 and 25 o C, respectively, indicating that C is the optimum temperature for bio-delignification by S. commune 22 (Table 2). Thereafter, the activity of these enzymes declined with the rise in incubation temperature. Thus, it is evident from the above results that low temperatures favour the activity of and enzymes, while comparatively higher temperatures favour the activity of C x and. This clearly indicates that the Table 1 Activity of cellulolytic ( & ) and lignolytic ( & ) enzymes produced by S. commune after 7, 14 and 21 d of incubation at 37 C and at ph 5.5 Incubation period (d) Malt extract broth only Malt extract broth amended with 20% paddy straw Cellulolytic enzymes Lignolytic enzymes Cellulolytic enzymes Lignolytic enzymes Cx: Endogluconase, : Exogluconase, : Lignin peroxidase, : case

4 194 INDIAN J BIOTECHNOL, APRIL 2012 Table 2 Influence of temperature on cellulolytic ( & ) and lignolytic enzymes ( & ) produced by S. commune Temperature ( o C) Cellulolytic enzymes Lignolytic enzymes Cx: Endogluconase, : Exogluconase, : Lignin peroxidase, : case cellulolytic activity of the organism could be reduced by reducing the incubation temperature to o C, while the activity of lignolytic enzymes would remain at optimum at this temperature. The data is subjected to the statistical analysis (Single factor ANOVA) and the results showed that the temperature of incubation has significant bearing on the activity of both cellulolytic and lignolytic enzymes of S. commune. Influence of ph on Enzyme Activity The ph dependence of the cellulolytic and lignolytic enzyme activity of S. commune was examined at the temperature of 37 C in the ph range of with a ph interval of 0.5 using acetate buffer and the results are presented in Table 3. The maximum Cx activity (145 REA) was found in the acidic ph 6.0, while the highest activity (285 mg/ml) was recorded at lower ph 5.0. On the other hand, the optimum ph for the lignolytic enzymes of S. commune fell between ph 5.5 and 7.0 ( at ph 6.5 & at ph 5.5). 23,24 Thus, the optimum phs for both cellulolytic and lignolytic enzymes of S. commune lie in the close proximity and, therefore, by changing the ph of the medium the activity of cellulolytic enzymes could not be suppressed substantially, while keeping the activity of lignolytic enzymes at the optimum. Statistical analysis (Single factor ANOVA) of the data indicates that variation in the ph of the medium influences the activity of both cellulolytic and lignolytic enzymes of S. commune. As the change in incubation temperature and ph of the medium was unable to suppress the activity of cellulase enzymes substantially, some Table 3 Effect of ph on cellulolytic and lignolytic activity of S. commune incubated at 37 C. ph Cellulolytic enzymes Lignolytic enzymes other mean is necessary for the successful delignification of paddy straw without causing loss in dry matter of the straw. Influence of Metal Ions and Chemicals on Enzyme Activity An attempt was made to study the effect of chemicals, such as, Cu, Mg, Zn, EDTA, TCA, NaN 3 and veratryl alcohol on the activity of cellulolytic and lignolytic activity of S. commune (Table 4). Except veratryl alcohol, all the chemicals used did not exhibit the desired effect on both cellulolytic and lignolytic enzymes 21,25. Among the three metal ions, Mg and Zn enhanced the activity of cellulolytic enzymes (Cx), while lowered the lignolytic activity to a lesser extent. The presence of Cu moderately reduced the activity of cellulolytic enzymes, while enhanced the lignolytic activity to a extent of 5-10%. The addition of EDTA and NaN 3 in all concentrations completely suppressed the activity of both cellulolytic and lignolytic enzymes. Further, TCA enhanced the cellulolytic activity (Cx) and suppressed the lignolytic activity and, thus, its amendment could not be considered. On the other hand, the desirable feature, i.e., the suppression of cellulolytic activity and simultaneous rise of lignolytic activity for successful delignification, was observed in three concentrations of veratryl alcohol and, hence, recommended for its amendment in the treatment of straw. The addition of veratryl alcohol at 10 mm concentration suppressed the Cx and activity by about 80 and 75%, respectively. On the other hand, the same concentration of veratryl

5 VIJYA & REDDY: DELIGNIFICATION ABILITY OF EDIBLE MUSHROOM, S. COMMUNE 195 Table 4 Influence of different chemicals on cellulolytic and lignolytic enzymes by S. commune after 10 d of incubation at 37 0 C Chemical agent Conc. (mm) Cx Lip Control Cu Mg Zn EDTA TCA NaN Veratryl alcohol Cx: Endogluconase, : Exogluconase, : Lignin peroxidase, : case alcohol enhanced the activity of from 110 to 145 U/mL, recording an increase of about 30% enzyme activity (Table 4). In the case of enzyme, the same trend was recorded. The increase in the concentration of veratryl alcohol from 10 to 30 mm enhanced the lignolytic enzyme activity, while cellulolytic enzyme activity was further inhibiting 43,44. It has been demonstrated that veratryl alcohol functions via an induction type of mechanism affecting only certain ligninase positive species 26,27. Thus, the present study indicates that S. commune could be used for successful bio-delignification of crop residues by the addition of veratryl alcohol at appropriate concentration. The idea of exploiting crop residues treated with white-rot fungi is a novel approach where the delignified straw could be efficiently used as animal feed. 8,16 The study indicates that the paddy straw mushroom (S. commune) expressed significantly higher lignin degrading enzyme system at normal atmospheric temperature of o C and at neutral ph. The higher cellulolytic enzyme activity of the organism could be regulated by the addition of veratryl alcohol at mm concentration to the straw. However, the regulation of production of individual ligninolytic enzymes is a complex phenomenon. In these circumstances, solid state fermentation technology using white-rot fungi as bio-conversion agents is appropriate for up-gradation of lignocellulosic wastes with a view to enhance their feed value for developing countries like India 16. Depending upon the kind and extent of treatment, the wastes get upgraded in protein and energy. The product also gets enriched in fats, soluble sugars, vitamins, amino acids and, thus, could be used as animal feed. Acknowledgement The authors are thankful to University Grants Commission, New Delhi for the financial assistance provided for this research project. References 1 Chaudhry A S, Chemical and biological procedures to upgrade cereal straws for ruminants, Nutr Abstr Rev (Ser B), 68 (1998) Teeri T, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends Biotechnol, 15 (1997) Arora M, Sehgal V K, Thaper V K & Wadhwa M, Nutritional improvement of rice straw by higher fungi, in Fungi in biotechnology, edited by A Prakash (CBS Publishers, New Delhi, India) 1998, Vijaya C, Singaracharya M A, Narasu M L, Lakshmi M V & Reddy R M, Use of lignocellulolytic mutants of Pleurotus ostreatus in ruminant feed formulations, BioResources, 4 (2009) ( 5 Singh J, Verma H K & Sikka S S, Green cow, clean environment, Indian Dairyman, 62 (2010) Tripathi M K, Mishra A S, Misra A K, Vaithiyanathan S, Prasad R et al, Selection of white-rot basidiomycetes for bioconversion of mustard (Brassica compestris) straw under solid-state fermentation into energy substrate for rumen micro-organism, Lett Appl Microbiol, 46 (2008) Reddy R M, Vijaya C & Singaracharya M A, Acceptability of paddy straw treated through solid state fermentation technique using soil fungi, Asian J Microbiol Biotechnol Environ Sci, 10 (2008) Shrivastava B, Thakur S, Khasa Y P, Gupte A, Puniya A K et al, White-rot fungal conversion of wheat straw to energy rich cattle feed, Biodegradation, 22 (2011) Schoemaker H E, On the chemistry of lignin degradation, Recl Trav Chim Pays-Bas, 109 (1990)

6 196 INDIAN J BIOTECHNOL, APRIL Thurston C F, The structure and function of fungal laccases, Microbiology, 140 (1994) Camerero S, Sarkar S, Ruiz-Duenas F J, Martinez M J & Martinez A T, Description of a versatile peroxidase involved in natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate binding sites, J Biol Chem, 274 (1999) Hatakka A, Lignin-modifying enzymes from selected whiterot fungi: Production and role in lignin degradation, FEMS Microbiol Rev, 13 (1994) Pelaez F, Martinez M T & Martinez A T, Screening of 68 species of basidiomyteces for enzymes involved in lignin degradation, Mycol Res, 99 (1995) Cohen R, Hader Y & Ordan Y, Transcript and activity levels of different Pleurotus ostreatus peroxidases are differentially affected by Mn 2+, Environ Microbiol, 3 (2001) Chalamcherla V L, Singaracharya M A & Lakshmi M V, Amino acids profile of the lignocellulosic feed treated with cellulase-free lignolytic mutants of Pleurotus ostreatus, BioResources, 5 (2010) Puniya A K & Singh K, Solid state fermentation of lignocellulosics, in Fungi in biotechnology, edited by A Prakash (CBS Publishers, New Delhi, India) 1998, Akin D E, Rigsby L L, Sethuraman A, Morrison III W H, Gamble G R et al, Alteration in structure, chemistry and bioavialability of grass lignocellulose treated with the white-rot fungi, Ceriporiopsis subvermispora and Cyathus stercoreus, Appl Environ Microbiol, 61 (1995) Plummer D T, An introduction to practical biochemistry (Tata Mc Graw-Hill Publishing, New Delhi) 1993, Tein M & Kirk T K, Lignin peroxidase of Phanerochaete chrysosporium, Methods Enzymol, 161 (1988) Djarwanto & Tachibana S, Screening of fungi capable of degrading lignocellulose from plantation forests, Pak J Biol Sci, 12 (2009) Faison B D & Kirk T K, Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium, Appl Environ Microbiol, 49 (1985) Lang E, Gonser A & Zadrazil F, Influence of incubation temperature on activity of lignolytic enzymes in sterile soil by Pleurotus sp. and Dichomitus squalens, J Basic Microbiol, 40 (2000) Baldrian P, Fungal laccasess Occurrence and properties, FEMS Microbiol Rev, 20 (2006) Kumar A G, Sekaran G & Krishnamoorthy S, Solid state fermentation of Archras zapota lignocellulose by Phanerochaete chrysosporium, Bioresour Technol, 97 (2006) Wariishi H, Haung J, Duford H B & Gold M H, Reactions of lignin peroxidase compounds I and II with veratryl alcohol. Transient-state kinetic characterization, J Biol Chem, 266 (1991) Leisola M S A, Ulmer D C, Waldner R & Fiechter A, Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium, J Biotechnol, 1 (1984) Faison B D, Kirk T K & Farrell R L, Role of veratryl alcohol in regulating ligninase activity in Phanerochaete chrysosporium, Appl Environ Microbiol, 52 (1986)