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Efficacy of Phanerochaete chrysosporium (MTCC 767) and Trichoderma viride (MTCC 167) on the biodegradation of sugarcane bagasse.

INTRODUCTION

The green manure revolution in India which encouraged the discriminate use of fertilizers to obtain two to three crop yields per year, led to an abdunce of crop residues. The quantity of agricultural residues and by products produced in India is 1396 million tonnes (MT). In India, sugarcane is the leading cash crop, the only source of sugar in the country that has been identified as the crop of immediate attention and is grown in an area of 3.93m.ha with annual production of 1.7 MT.

Bagasse is the dry dusty pulp that remains after the extraction of juice from the stems of sugarcane. The raw material that is left over remains as a pollutant, so when treated properly it solves the problem of disposal. It is the malted cellolosic fibre residue from sugarcane that has been processed in a sugar mill. It contains considerable quantities of cellulose, a beta linked glucose polymer, which is difficult to break down into glucose. In addition, it contains hemicellulose, which is a more complex polymer of several sugars including xylose and arabinose. Entwined around the two sugar polymers is lignin, a polymer that does not contain sugar. (Bohlmann, 2003). Its C: N ratio is always greater than 150:1. Under natural conditions, it takes 18 months to obtain mature bagasse compost. (Huang et al.,1996).

The fungi like Phanerochaete chrysosporium are robust organisms and they have a capacity to hasten the process of biodegradation (Lata et al, 2000).

Cellulose a major constituent of agricultural residues, can be degraded by a wide range of organisms. Trichoderma viride has been the most studied organism, because of its capacity to produce cellulase enzyme complex which converts cellulose into soluble sugar, glucose. (Lata et al., 2002).

Sundar (1998) reported that sugarcane produces huge quantities of foliage, about 40 percent of the total biomass (about 15-20 t ha-1). Usually, farmers dispose sugarcane trash by burning in the field, causing environmental pollution and damage to soil microflora.

Hence, the present investigation was carried out to convert bagasse into a nutrient enriched organic manure by inoculating Phanerochaete chrysosporium and Trichoderma viride.

MATERIALS AND METHODS

The present study was conducted to analyse the biodegradation rate of bagasse using Phanerochaete chrysosporium, Trichoderma viride and a mixed culture of Trichoderma viride and Phanerochaete chrysosporium as a fungal innoculant.

Collection of Materials

Phanerochaete chrysosporium and Trichoderma viride were bought from Institute of Microbial Technology, Chandigarh, India. Fresh bagasse samples were collected from the sugarcane industry in Trichy.

Growth Medium For Fungal Culture - Malt Extract For Phanerochaete chrysosporium (MTCC 767)

Malt extract medium with a composition of malt extract (20.0g), glucose (20.0g), peptone (1.0g), agar (20.0g) and distilled water (1000 ml) were taken in sterilized petridishes.Under aseptic conditions, Phanerochaete chrysosporium was inoculated. The bacterial growth was suppressed by the addition of 1ml of 10,000ppm Streptomycin Sulphate solution and growth pattern was noticed.

Potato Dextrose Agar Medium For Trichoderma viride (MTCC 167)

From peeled Potato (250 g) PDA medium was prepared by adding dextrose (20 g) and agar (15 g). The volume was made into 1000ml.

Prepration Of The Samples

Sterilized and dry bagasse (100 g) was taken in a perforated, labeled, thick gauge polythene bags and the fungal mass (about 20 g) was added to it, under aseptic conditions. The rate of degradation of bagasse was determined at 30 and 60 days.

Biochemical Analysis

Biochemical analysis was determined following the meth0d

* Cellulose (Updegroff, 1969)

* Estimation Of Lignin (Goering and Vansoest , 1975)

* Estimation of organic carbon (Walkley and Black, 1934)

* Estimation Of Total Nitrogen (Humphries, 1956)

* Estimation Of Phenol by Folin -Ciocalteau Method (Bray and Thrope, 1954)

* Total Soluble Sugars (Dubois et al, 1951)

* Reducing Sugars By Dinitroslicylic Acid Method (Miller, 1972)

* Estimation Of Non Reducing Sugars

Enzymology

Prepration Of Bagasse Sample For Enzymology

1g of the bagasse sample was macerated in 5ml of 0.1 M phosphate buffer (pH 6.5 for peroxidases and polyphenol oxidase and 6.8 for catalase) in a pre - chilled porcelain mortar and pestle. The homogenate was centrifuged in a refrigerated centrifuge (10,000 x g for 15 minutes). The supernatant served as enzyme source.

Catalase Activity (Luck, 1974)

Procedure

5ml of (0.067 M) phosphate buffer (pH 6.8) was pipetted out into two conical flasks. 3ml of 0.3N hydrogen peroxide was added to each flask. To one of the flasks, 0.1 ml enzyme extract was added and mixed well and incubated for 15 min at room temperature. After 15 min, 0.1 ml enzyme extract was added to the second flask (control). Immediately, 10 ml of 2N sulphuric acid was added to both the flask to arrest the reaction. The contents were titrated against 0.01 N potassium permanganate and the titre values were noted. End point was the appearance of light pink colour. Difference between the titre values (control and test) was the volume of permanganate consumed equivalent to enzyme activity. Catalase activity was determined from the volume of 0.01 N Potassium permanganate consumed by the enzyme.

Peroxidase Activity (Putter, 1974)

The spectrophotometer was adjusted to read zero at 430 nm with a cuvette containing 3ml of 0.5 M pyrogallol solution and 0.1ml enzyme extract. Immediately, 5 ml of one per cent hydrogen peroxide was added to the test cuvette and mixed well. The change in absorbance was recorded for every thirty seconds up to three minutes. Protein content of the enzyme source was determined following the method of Lowry et al. (1951).

Polyphenol oxidase activity (Mayer and Harel, 1979) E.C.1.10.3.1 Prepration Of The Sample

About 1 g of bagasse sample was macerating in a pre - chilled mortar and pestle with 20 ml medium containing 50 mM tris - Hcl (pH 7.2), 0.4 M sorbitol and 10mM sodium chloride. The homogenete was centrifuged (20,000 g for 10 minutes) and the supernatant was used as an enzyme source.

Procedure

2.5 ml of 0.1M phosphate buffer (pH 6.5), 0.3 ml of catechol solution (0.01M) were added into a cuvette and the spectrophotometer was adjusted to read zero at 495 nm. Swiftly, 0.1 ml of the enzyme extract was added and mixed well. The change in the absorbance was recorded for every thirty seconds up to three minutes. Protein content of the enzyme source was determined following the method of Lowry et al., (1951).

RESULTS AND DISCUSSION

Cellulose And Lignin Content

The treatment [T.sub.2] (mixed culture inoculated bagasse) showed maximum decrease in cellulose and lignin content of 16.22 per cent and 8.64 per cent followed by [T.sub.3] Phanerochaete chrysosporium inoculated bagasse with 9.03 per cent lignin content and 12.01 per cent cellulose over the raw bagasse with 31.83 per cent cellulose content and 12.47 per cent lignin content.

Similar result was obtained by Ortega et al. (1992) who also found reduction in cellulose and lignin content of sugarcane bagasse to 47 per cent and 55 per cent respectively.

Organic Carbon content

A drastic reduction in organic carbon content was recorded in mixed culture inoculated bagasse [T.sub.2] treatment (21.13 per cent) followed by [T.sub.3], Phanerochaete chrysosporium inoculated bagasse (22.10 per cent) compared to [T.sub.1] raw bagasse (41.13 per cent).

According to Hussein et al. (1988) the gradual decrease in organic carbon content was due to biological oxidation by the fungi.

Nitrogen Content

The sample [T.sub.2] (Phanerochaete chrysosporium and Trichoderma viride inoculated sample) recorded maximum nitrogen content of 1.03 per cent from 0.33 per cent followed by [T.sub.4] (Trichoderma viride inoculated samples) with 0.82 per cent.

The result is in accordance with the findings of Nallathambi and Marimuthu (1993). They found that among the different Pleurotus sp tested, P. platypus could increase the total nitrogen content of sugarcane bagasse to 0.97 per cent from 0.58 per cent.

C: N Ratio

The C: N ratio was narrowed down in [T.sub.2] (mixed culture inoculated samples) from 149:1 to 21:1 followed by [T.sub.4] (Trichoderma viride inoculated sample) from 149:1 to 26:1.

Total Phenolic Content and Non-Reducing Sugars

A significant decrease in total phenolic content and non - reducing sugars was registered, which varied from 0.96 mg [g.sup.-1] to 0.51 mg [g.sup.-1] and 0.42 mg [g.sup.-1] to 0. 29 mg [g.sup.-1] in mixed culture inoculated [T.sub.2] samples with in 60 days of decomposition.

The result is in accordance with the result of Saroja et al. (1999) who found that the total phenolic content of sugarcane bagasse was decreased from 58.40 mg [g-.sup.1] to 45.28 mg [g.sup.-1] during 30 days of spawning with Pleurotus spp.

Enzymology Catalase

Catalase activity was expressed maximally to 40.43 [Ul.sup.-1] in [T.sub.2] (Mixed culture inoculated bagasse) with in 60 days of biodegradation than the raw bagasse [T.sub.1] (11.8 [Ul.sup.-1]). Catalase activity was expressed minimally (24.83 [Ul.sup.-1]) in [T.sub.3] (P. chrysosporium inoculated bagasse sample).

The result concided with the result of Morpeth (1987) who reported that the activity of the enzyme catalase in P. chrysosporium increased gradually and reached a maximum after 4 to 6 days of growth.

Peroxidase

A significantly highest peroxidase activity of 63.93 [Ul.sup.-1] was recorded in [T.sub.2] (Mixed culture Sample) and the least activity of 21.87 [Ul.sup.-1] was recorded in [T.sub.4] (T. viride inoculated samples).

Similar result was obtained by Vijaya and Singaracharya (2005). During their investigation on delignification of paddy straw by white - rot fungus, Pleurotus ostreatus, they found that the fungus produced highest amount of lignin peroxidase of 80 U[ml.sup.-1].

Polyphenol Oxidase

A significantly higher polyphenol oxidase activity of 34.33 to 41.47 [Ul.sup.-1] was recorded in the sample [T.sub.2] (bagasse degraded by mixed culture), compared to the sample [T.sub.3] (P.chrysosporium inoculated samples) and [T.sub.4] (T.viride inoculated bagasse).

Saroja et al. (1999) during their study on the utilization of Pleurotus sp AM-I for the biodegradation of the lignocellulosic waste, observed maximum polyphenol oxidase activity of 0.0051 enzyme units for sugarcane bagasse on the 10th day of spawning.

CONCLUSION

Due to the activity of lignolytic fungus Phanerochaete chrysosporium and cellulolytic fungus Trichoderma viride, carbon minerilization and oxidation occurs in sugarcane bagasse during biodegradation (with in 60 days ). This resulted in a narrowing down of C: N ratio (21:1) and increased nitrogen content (1.03 per cent) making it suitable for use, as organic manure for agricultural crops.

Thus, the efficacy of lignolytic activity of Phanerochaete chrysosporium and cellulolytic activity of Trichoderma viride, can be exploited for the acceleration of decomposition process. This efficacy can be utilized for converting waste into value added product, organic manure. It offers a practical and environmentally safer technique for sustainable agriculture.

REFERENCES

(1.) Bohlmann.G..2003. Ethanol from bagasse. Business research and private client consulting for world wide chemical industry. PEP Review.

(2.) Dubois. 1951. Cited by Mahadevan, A. and Sridhar, R. 1986. Methods in physiological plant pathology. Sivakami publications, Madras. pp. 146-147.

(3.) Goering, H.D. and Van Soert, P.J. 1975. Forage fibre analysis US, Department of Agriculture. Agric. Res. Service, Washington, DC.

(4.) Huang, C.M., Chen, W.C., Sheen, H.K., Li, S.W. and Wang, L.H. 1996. Recycling and utilization of bagasse. Report of the Taiwan Sugar Research Institute, 151: 41-53.

(5.) Humphries, E.C. 1956. Mineral composition and ash analysis. K. paech and M.V. Tracey (eds). In : Modern methods of plant analysis, 1, Springer verlag, Berlin. pp. 468-502.

(6.) Hussein, J. M., El. Sadik, M.K., Adbel, R.S., and Badran, N.M. 1988. Biological degradation of different organic manures in Egyptian soils and their effects on soil microorganisms. Egyptian. J. Microbial., 23: 209-221.

(7.) Lata, R.N, Sangeetha, P. and Verma. 2002. Solid state fermentation of sorghum straw with cellolytic Trichoderma viride strains and its effect on wheat in conjunction with Azotobacter chroococum strain W5. Indian. J. Microbiol. 40: 57-60.

(8.) Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall,R.H. 1951. Protein measurement with folin-phenol reagent. J. Biol. Chem., 193: 265-275.

(9.) Luck, H. 1974. Methods in enzymatic analysis-2 (H.U. Bergmeyer ed.). Academic press, New York. p. 885.

(10.) Mayer, A.M. and Harel, E. 1979. Polyphenol oxidase in plants. Phytochemistry, 18:193-215.

(11.) Miller, G.L. 1972. Anal. Chem., 31426.

(12.) Morepth, F.F. 1987. Intracellular oxygen - metabolizing enzymes of Phanerochaete chrysosporium. J. Gen. Microbiol., 133 : 3521-3525.

(13.) Nallathambi, P. and Marimuthu, T. 1993. Pleurotus platypus : A potent oyster mushroom for organic recycling of agricultural wastes. Mushroom Res. 2 : 75-77.

(14.) Ortega, G.M., Martinez, E.O., Betan Court, D., Gonzalez, A.E. and Otero, M.A. 1992. Bioconversion of sugarcane crop residues with white - rot fungi Pleurotus sp. World. J. Microbiol . Biotech., 8: 402-405.

(15.) Putter, J. 1994. Methods of enzymatic analysis. 2 (Ed. HU. Bergmeyer). Academic press, New York., P. 685.

(16.) Saroja, S., Anitha Das, Annapurani, S. and Srilatha, R. 1999. The utilization of Pleurotus species AM - I for the biodegradation of lignin cellulosic wastes. Int. J. Env. Educ and Inf. 18(2): 131- 36.

(17.) Sundar, B. 1998. Composting sugarcane trash and pressmud. In: Sugarcane cultivation. Vikas Publishing house Pvt. Ltd., Sugarcane Breeding Institute, Coimbatore .pp- 270-277

(18.) Updegraff, D.M. 1969. Semi-micro determination of cellulose in biological materials. Ann. Biochem., 32: 420-444.

(19.) Vijaya, C.H. and Singaracharya, M.A. 2005. Delignification of paddy straw by a whiterot fungus, Pleurotus ostreatus. Asian. J. Microbiol. Biotech. Env. Sc., 1: 77-83.

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D. Adlene Sangeeth * and C.K. Padmaja **

Department of Botany, Avinashilingam Deemed University,Coimbatore - 613043, India.
Table--1: Biochemical Parameters Of Bagasse During Biodegradation

 Organic Carbon Total Nitrogen C: N Ratio
Treatments (%) (%)

Days 30 60 30 60 30 60

T1 47.17 41.13 0.33 0.44 149:1 93:1
[T.sub.2] 26.80 22.10 0.79 1.03 34:1 21:1
[T.sub.3] 33.73 26.73 0.48 0.64 70:1 42:1
[T.sub.4] 23.67 21.13 0.60 0.82 39:1 26:1

 SE--0.62 SE--0.01
 CD--1.81 (P<0.01) CD--0.04 (P<0.01)

Table--2: Biochemical Parameters Of Bagasse During Biodegradation

Treatments Phenols Cellulose Lignin
 (mg [g.sup.-1]) (%) (%)

Days 30 60 30 60 30 60

[T.sub.1] 5.83 4.63 34.71 31.83 13.63 12.47
[T.sub.2] 0.96 0.51 25.10 16.22 11.87 8.64
[T.sub.3] 1.40 0.61 16.93 12.01 10.93 9.03
[T.sub.4] 0.92 0.73 12.27 10.25 12.27 10.25

 SE--0.10 SE--0.33 SE--0.10
 CD--0.30 CD--0.03 CD--

 (P<0.01) (P<0.01) (P<0.01)

Treatments Non-reducing
 sugars
 (mg [g.sup.-1])

Days 30 60

[T.sub.1] 0.74 0.61
[T.sub.2] 0.42 0.29
[T.sub.3] 0.47 0.33
[T.sub.4] 0.32 0.28

 SE--0.01
 CD--0.03

 (P<0.01)

Table--3: Enzyme Activity Of Of Bagasse By The Fungi During
Biodegradation

Treatments Catalase Peroxidase Polyphenol
 oxidase

Days 30 60 30 60 30 60

[T.sub.1] 6.83 11.80 12.23 14.40 9.20 12.37
[T.sub.2] 32.27 40.43 44.47 63.93 34.33 41.47
[T.sub.3] 21.07 24.83 32.00 55.90 28.67 40.30
[T.sub.4] 22.33 29.27 17.37 21.87 19.20 33.57

 SED--0.19 SED--0.18 SED--0.17
 CD--0.56 CD--0.54 CD--0.50
 (P<0.01) (P<0.01) (P<0.01)

[T.sub.1]--Raw bagasse sample

[T.sub.2]--Bagasse + Trichoderma viride + Phanerochaete
 chrysosporium in equal amounts.

[T.sub.3]--Bagasse + Phanerochaete chrysosporium.

[T.sub.4]--Bagasse + Trichoderma viride.
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Author:Sangeeth, D. Adlene; Padmaja, C.K.
Publication:Bulletin of Pure & Applied Sciences-Botany
Date:Jan 1, 2006
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