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Role of Pleurotus ostreatus and Gloeophyllum sepiarium in the Hydrolysis of some common agro-wastes.

Introduction

The current global energy crisis led credence to inevitable depletion of World's energy supply and there is now an increasing world-wide interest in finding alternative sources of energy (15). In order to meet the challenges of generating sufficient and sustainable energy, a potentially viable alternative is to use cellulosic biomass which is the most bioenergy feedstock in the world for biofuel and bioenergy production (14). The lignocellulosic biomass is mostly wasted in the form of preharvest and postharvest agricultural losses and wastes of food processing industries. Sadly, much of these lignocellulose wastes are often disposed of by biomass burning, which is not restricted to developing countries alone, but is considered a global phenomenon (8).

However, due to their abundance and renewability, there has been a great deal of interest in utilizing these lignocellulosic wastes for the production and recovery of many value-added products, among which is bioethanol (3). Therefore, agronomic residues such as corn stober (corn cobs and stalks), sugarcane waste, wheat or rice straw, forestry and paper mill discards, can be converted to bioethanol (15). The long-term benefits of using waste residues as lignocellulosic feedstocks will be to introduce a sustainable solid waste management strategy for a number of lignocellulosic waste materials, contribute to the mitigation in greenhouse gases through sustained carbon and nutrient recycling, reduce the potential for water, air, and soil contamination associated with the land application of organic waste materials, and to broaden the feedstock source of raw materials for the bioethanol production industry (9).

The brown rot basidiomycetes cause the most destructive type of wood decay and are important contributors to biomass recycling. These basidiomycetes are unusual in that they rapidly depolymerize the cellulose in wood without removing the surrounding lignin that normally prevents microbial attack (16). Baig et al. (1) reported the use of cellulolytic enzymes of Trichoderma lignorum in the saccharification of banana agrowaste. Also, in ethanol production using rice husk and bagasse, Patel et al. (11) reported the use of Trichoderma reesei, Phanerochaete chrysosporium and Pleurotus sajor-cajus among others in pretreatment of these lignocellulosic wastes. Similarly, in a related research, Chandel et al. (2) reported the use of Phanerochaete chrysosporium, Cyathus stercoreus, Cythus bulleri and Pycnoporous cinnabarinus in hydrolysing lignocellulosic wastes leaving cellulose and hemicellulose network in the residual portion. However, despite this, there is scanty information on the use of Pleurotus ostreatus and Gloeophyllum sepiarium in hydrolysing various lignocellulosic wastes. Therefore, the objective of this study was to compare the use of these two fungi in hydrolysing rice husks, millet husks, guinea corn stalks and groundnut shells which form postharvest wastes in this locality.

Materials and Methods

Collection and processing of samples

Sample collection and processing was carried out in accordance with the methods described by Oyeleke and Jibrin (10). The four agro-wastes (Groundnut shell, Rice husks, Millet husks, and Guinea corn stalks) were collected from agro-processing centers in Sokoto metropolis of Sokoto State. One kilogram (1kg) of each agro-waste sample was collected in a clean polythene bags and transported immediately to the laboratory. The agro-wastes were dried and ground to a powder form using a Waring blender (Binatone) and used for hydrolysis.

Stock pure cultures

Stock pure cultures of Pleurotus ostreatus and Gloeophyllum sepiarium were obtained from the Department of Microbiology of Federal University of Technology, Minna (Nigeria) and were maintained on Potato Dextrose Agar (PDA) incorporated with 1% streptomycin.

Spawn production

This was carried out in accordance with the method of Pathmashini et al. (12) using sorghum grains. The sorgum grains were cleaned manually to remove inert matter, stubble and debris. The cleaned grains were soaked in 0.5% CuS[O.sub.4] for 10 minutes and the soaked grains were thoroughly washed and soaked in tap water for 2 hours. The excess water were then drained followed by the addition of rice brain, CaC[O.sub.3] and MgSO4 at the rate of 10%, 2%, and 0.2% respectively on dry weight basis of the grains. The additives were thoroughly and evenly mixed with the grains. The glass jars were sealed using cotton wool and aluminium foil and autoclaved at 121[degrees]C for 30 minutes and allowed to cool for 24 hours. The pH of the medium was adjusted to 6.5 by the addition of 1M solution of NaOH. The medium was then inoculated with the mycelial culture of P. ostreatus and G. sepiarium and incubated at ambient laboratory temperature (35 [+ or -] 2[degrees]C) for 7 days.

Enzymatic hydrolysis of the agro-wastes

This was carried out according to the methods described by Gupta et al. (6) by setting two sets of 500ml capacity conical flasks. The conical flasks were labeled A, B, C and D for the samples and A1, B1, C1 and D1 for the controls respectively.

In each of the flask 50g of the designated agrowastes was placed and 500ml of distilled water added. The flasks were plugged with cotton wool and aluminium foil and then sterilized at 121[degrees]C for 30 minutes. One set of flask was inoculated with 5ml suspension of Pleurotus ostreatus and the other set with Gloeophyllum sepiarium. The flasks were incubated at ambient laboratory temperature (35 [+ or -] 2[degrees]C) for 5 days on an orbital shaker. After the 5 days period, the samples were filtered through whattman filter paper No1 and the glucose yield of the filterates were determined. For the control, also 50g of the agro-wastes was put in another set of conical flasks and 500ml of distilled water added. The flasks were incubated at ambient laboratory temperature (35 [+ or -] 2[degrees]C) for 5 days on an orbital shaker. Each set of flasks was replicated three times.

Reducing sugar determination

The reducing sugar yield of both the untreated and treated agrowastes was determined in accordance with the methods of Thiammaih (13). The glucose yield was determined using Benedict quantitative test with glucose as standard. A standard glucose concentrations of 0mmol/L, 2.50mmol/L, 5.00mmol/L, 7.50mmol/L and 100mmol/L were prepared. Then a blank (5.00mmol/L) was put in 2ml of Benedict solution and was used to zero the spectrophotometer. Then 5ml of each concentration was put in a test tube and 2ml of Benedicts reagent added. The mixture was boiled in a water bath for 5minutes. Using the spectrophotometer, the absorption of each concentration was taken at 477nm and the values were used to plot graph of absorbance against concentration. Then 5ml of each filterate and 2ml of Benedicts reagent were added and boiled for 5minutes in a water bath. Absorption of each filterate was determined using spectrophotometer (6100 model, Jenway, U.K.) and the reducing sugar concentration were extrapolated from the standard glucose curve.

Statistical analysis

Data generated were subjected to statistical analysis using one-way analysis of variance (ANOVA) and paired sample t-test using SPSS (version 14.0) statistical package to establish significant differences at 95% confidence limit. Data were expresses as mean [+ or -] standard error means (S. E). Results with significant difference (p[]0.05) were alphabetically ranked

Results and Discussion

A comparative study was carried out on the utilization of Pleorotus oestreatus and Gloeophyllum sepiarium in hydrolyzing some common agrowastes namely rice husk, millet husk, guineacorn stalk and groundnut shell. The results of the reducing sugar yield of the four agrowastes before hydrolysis revealed that groundnut shells had the highest yield of reducing sugar of 46.33 [+ or -] 0.02mmol/L while the least reducing sugar yield of 16.65 [+ or -] 0.01mmol/L was obtained with guineacorn stalks (Table 1).

However, after subjecting the agrowastes to enzymatic hydrolysis, high yield of reducing sugar (41.23 [+ or -] 0.67mmol/L) was obtained with guineacorn stalks using Gloeophyllum sepiarum while the lowest reducing sugar yield of 13.62 [+ or -] 0.60mmol/L was obtained using the same organism. Pleorotus ostreatus produced a high reducing sugar yield of 33.64 [+ or -] 0.20mmol/L with rice husks and a lower yield of 23.37 [+ or -] 0.11mmol/L with groundnut shells (Table 2).

The results revealed that G. sepiarum produced the highest reducing sugar yield of 41.23 [+ or -] 0.67mmol/L from guineacorn stalks while P. oestreatus produced a high yield of 33.64 [+ or -] 0.20mmol/L from rice husks. The lowest yield was obtained from groundnut shells with a yield of 13.62 [+ or -] 0.60mmol/L and 23.37 [+ or -] 0.11mmol/L for G. sepiarum and P. oestreatus respectively. This was not surprising because guineacorn stalk contained more carbohydrate in its structural components than the other agrowastes used. Also, the high lignin content of the other agrowastes may inhibits enzymatic hydrolysis of the cellulose in the lignocellulose biomass. These findings were in agreement with the study of Epstein et al. (5) who reported lower yield of reducing sugars from apple and grape juices. Also, the variations in the reducing sugar yield of the isolates from these agrowastes may be as a result of the difference in N and C/N ration of the agrowastes. Dundar et al. (4) reported that different N and C/N ratio of substrates affect the yield performance of the fungi and for high performance yield the ratio must be 50 or higher than 50. The results indicated that there was significant difference (p[]0.05) between the yield of reducing sugar when substrates were hydrolyzed by the test organisms to that of the controls. This collaborated well with the work of Hatakka (7) who reported that Pleorotus sp produced sugar concentrations three times higher than that obtained from controls without fungal pretreatment. Similarly, Baig et al. (1) reported glucose concentration of 20mg/ml from banana wastes when treated with Trichoderma lignorum. The implication of these findings are that the ability of the Pleorotus ostreatus and Gloeophyllum sepiarium to produced high yields of reducing sugar from these agrowastes indicated their potentiality in hydrolyzing these wastes. Also, these agro-wastes can be exploited as cheap carbon source for industrial production of bioethanol thereby tackling or reducing their disposal problems.

References

[1] Baig, M. M. V.; Baig, M. L. B.; Baig, M. I. A.; Yasmeen, M. (2004). Saccharification of banana agro-wastes by cellulolytic enzymes. Afr. J. Biotechnol, 3 (9): 447-450

[2] Chandel, A. K.. ; Chan, E. S.; Rudravaran, R.; Narasu, L. M.,; Rao, L. V.; Ravindra, P. (2007). Economics and environmental impact of bioethanol production technologies: An appraisal. Biotechnol. Mol. Biol. Rev, 2 (1): 014-032

[3] Demirbas, A. (2008). Products from lignocellulosic materials via degradation processes. Energy Sources Part A: Recovery, Utilization and Environmental Effects, 30 (1): 27-37

[4] Dundar, A.; Acey, H.; Yildiz, A. (2009). Effect of using different lignocellulosic wastes for cultivation of Pleurotus ostreatus (Jacq) P. Kumm. on mushroom yield, chemical composition and nutritional value. Afr. J. Biotechnol, 8 (4): 662-666

[5] Epstein, J. L.; Vieira, M.; Aryal, B.; Vera, N.; Solis, M. (2010). Developing Biofuel in the Teaching Laboratory: Ethanol from Various Sources. J. Chem. Educ, 87 (7): 708-710

[6] Gupta, R.; Sharma, K. K.; Kuhad, R. C. (2009). Simultaneous saccharification and fermentation of Prosopis jutiflora, a woody substrate for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis- NCIM 3498. Biores. Technol, 100: 1214-1220

[7] Hatakka, A. I. (1983). Pretreatment of wheat straw by white rot fungi for enzymatic saccharification of cellulose. Eur. J. Appl. Microbiol. Biotechnol, 18: 350-357

[8] Howard, R. L.; Abotsi, E.; Jansen van Rensburg, E. L.; Howard, S. (2003). Lignocellulosic Biotechnology: Issues of Bioconversion and Enzyme Production. Afr. J. Biotechnol, 2 (12): 602-619

[9] Mtui, G. (2009). Recent advances in Pretreatment of Lignocellulosic wastes and Production of Value-added Products. Afr. J. Biotechnol, 8 (8): 1398-1415

[10] Oyeleke, S. B.; Jibrin, N. M. (2009). Production of bioethanol from guinea corn husk and millet husk. Afr. J. Microbiol. Res, 3 (4): 147-152

[11] Patel, S. J.; Onkarappa, R.; Shobha, K. S. (2007). Fungal pretreatment studies on rice husks and bagasse for ethanol production. Electr. J. Environ. Agri. Food Chem, 6 (4): 1921-1926

[12] Pathmashini, L.; Arulnandhy, V.; Wilson, R. S. W. (2008). Cultivation of oyster mushroom (Pleurotus ostreatus) on saw dust. Cey J. Sci. (Biol. Sci.), 37 (2): 177-182

[13] Thimmaiah, S. K. (1999). Standard methods of biochemical analysis. Kaliani publishers, New Delhi, pp 1032-1040.

[14] Tushar, J.; Gerpen, J. V.; McDonald, A. (2010). Production of Fuel Ethanol from Woody Biomass. Proceedings of the 1st International Conference on 'New Frontiers in Biofuels, DTU', January 18-19, 2010, New Delhi

[15] Yan, L.; Tanaka, S. (2006). Ethanol fermentation from Biomass Resource: Current state and Prospects. Appl. Microbiol. Biotechnol, 69: 627-642

[16] Yoon, J. J.; Kim, Y. K. (2005). Degradation of crystalline cellulose by the brown-rot Basidiomycete Fomitopsispalustris. J. Microbiol. 487-492

* (1) Rabah A.B., (2) Oyeleke S.B., (1) Manga S.B., (3) Hassan L.G., Ibrahim A.D. and (4) Shehu K.

(1) Department of Microbiology, Usmanu Danfodiyo University, Sokoto

(2) Department of Microbiology, Federal University of Technology, Minna

(3) Department of Chemistry, Usmanu Danfodiyo University, Sokoto

(4) Department of Biological Science, Usmanu Danfodiyo University, Sokoto

Corresponding Author E-mail: abrabah2009@gmail.com
Table 1: Glucose yield of the four agro-wastes before hydrolysis.

Agro-wastes Glucose yield * (mmol/L)

Rice husks 20.51 [+ or -] 0.63b
Millet husks 20.36 [+ or -] 0.05b
Guineacorn stalks 16.65 [+ or -] 0.01c
Groundnut shells 46.33 [+ or -] 0.02a

* Results are means of triplicate experiments

Table 2: Glucose yield of the agrowastes hydrolysed with
the fungal isolates.

Agro-wastes Glucose yield * (mmol/L)

 G sepiarum P ostreatus

Rice husks 15.55 [+ or -] 0.40 (c) 33.64 [+ or -] 0.20 (a)

Millet husks 35.25 [+ or -] 0.16 (b) 32.31 [+ or -] 0.52 (b)

G/corn stalks 41.23 [+ or -] 0.67 (a) 31.54 [+ or -] 0.17 (c)

G/nut shells 13.62 [+ or -] 0.60 (d) 23.37 [+ or -] 0.11 (d)

Agro-wastes

 Control (1) Control (2)

Rice husks 13.25 [+ or -] 21.55 [+ or -]
 0.02 ([dagger]) 0.06 ([dagger])
Millet husks 20.39 [+ or -] 27.40 [+ or -]
 0.29 ([dagger]) 0.20 ([dagger])
G/corn stalks 33.36 [+ or -] 22.60 [+ or -]
 0.38 ([dagger]) 0.10 ([dagger])
G/nut shells 12.62 [+ or -] 0.02 20.40 [+ or -]
 0.20 ([dagger])

* Results are means of triplicate experiments

([dagger]) Indicated significant difference between sample and
control (p>0.05)
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Author:Rabah, A.B.; Oyeleke, S.B.; Manga, S.B.; Hassan, L.G.; Ibrahim, A.D.; Shehu, K.
Publication:International Journal of Biotechnology & Biochemistry
Date:Sep 1, 2011
Words:2377
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