PADDY STRAW COMPOSTING USING PHANEROCHAETE CHRYSOSPORIUM WITH VARIED TREATMENTS

Navneet Kaur Gill 1* , Dr Sandeep Sharma 2 , Dr (Mrs) Maninder Arora 3 and Dr (mrs) Surekha Bhatia 4 . 1. Department of Microbiology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, Punjab, India. 2. Deparment of soil science, college of agriculture, Punjab,Agricultural University, Ludhiana-141004, Punjab, India. 3. Department of processing and food engineering, College of Agricultural Engineering and Technology, Punjab,Agricultural University, Ludhiana-141004, Punjab, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Lignocellulosic materials are the most abundant agricultural residues in the world, primarily composed of cellulose (36.2%), hemicelluloses (19.0%) and lignin (9.9%). Hydrolysis of these materials under natural condition is slow. Because lignin surrounds the cellulose and forms a physical barrier, which restricts microbial enzyme attack. Microorganisms including fungi as well as actinomycetes and other bacteria, have an important role in increasing digestibility of lignocellulolytic material. Biological treatment of agricultural residues is a new method for the improvement of their digestibility (Jalk et al., 1998). White-rot fungi, belonging to the wood-decaying basidiomycetes, as lignocellulolytic microorganisms are able to decompose and metabolize all plant cell constituents (cellulose, hemicellulose and lignin) by their enzymes (Eriksson et al., 1990). Most of the white-rot fungi degrade lignin and cellulose simultaneously. A selective white-rot fungus, Ceriporiopsis subvermispora is known to selectively degrade lignin in softwood and hardwood (Okano et al., 2005) and Phanerochaete chrysosporium, Pleurotus ostreatus, Ceriporiopsis subvermispora, Cyathus stercoreus can be used to increase the digestibility of paddy straw (Taniguchi et al., 2005).
Although cellulolytic fungi occur in all major fungal taxa (Coughlan, 1985), but there are relatively few groups of microorganisms that can produce the ligninolytic enzymes. The white-rot fungi enzyme complexe increases the accessibility of cell wall structure. Lignin is oxidised and degraded by a ligninase system (Rodrigues et al., 2008) composed by lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. In addition, cellulases, hemicellulases and esterases are also considered to be extremely important in the degradation process of lignocellulosic biomass (Panagiotou et al., 2007). These enzymes should act in synergy to facilitate the complete degradation of cell walls. Solid state fermentation is an advantageous method to degrade ligninocellulosic compounds and improve the digestibility. Fungi grown under these conditions not only bring better ligninolysis but also improve's its digestibility by enhancing the accessibility of holocellulose. Further, growth of fungal mycelium contributes in increasing the total protein content of the feed (Fazaeli, 2007).
Microbial composting is an effective environmentally sound alternative for degradation of paddy straw. All sorts of cereal straw may not be available for composting because of their fodder value. Paddy straw has limited use as animal feed because of its high oxalic acid and silica content. Composting of paddy straw enhance sustainable agriculture and environment protection by improving the physical, chemical and biological properties of soil (Mylavarapu and Zinati, 2009), which ultimately results in better plant growth and yield. The aim of composting is sanitation, eliminating pathogenic microorganisms and reducing the volume of the wastes (Zibiliske et al., 1998). Chemical and physicochemical pretreatment methods such as strong acid and steam explosion, respectively, break the lignin structure for enzymatic contact with the cellulose. However, such methods also bring about higher operational costs and hazardous waste (Sun and Cheng, 2002). The physical, chemical and physico-chemical treatments are still restricted in terms of safety concerns, costs and potential negative environmental consequences (Phutela et al., 2011). Hence biodegradation, serves as an attractive option that is both energy-saving and environmentally friendly (Scott et al., 1998). In this study, keeping in mind the poor nutritive quality of paddy straw, the experiments were designed to degrade lignin and cellulose by using lignocellulolytic fungi with different treatments, study the biochemical changes of straw constituents and improvement of its degradability.

Material and methods:-Enzyme Assay:-
The activity of cellulases (FPU, CMCase and Xylanase) was estimated in the culture fifi ltrates after 15 and 30 days solid state fermentation of paddy straw method described by Sandhu and Kalra (1982). Laccase activity was assayed in the culture filtrates by the method of Dhaliwal et al (1991).

Composting under field conditions:-Preparation of inoculums:-
Two kg of sorghum (Sorghum bicolor) grains were soaked in water for three hours. After soaking, water was decanted out. The grains were boiled in 0.2 per cent dextrose solution for 15 minutes. Sorghum grains were transferred into conical flasks (250 ml) at the rate of 70 g each. Addition of CaSO 4 and CaCO 3 was done at the rate of 2% and 4%, respectively and autoclaved at 120˚C at 15 lbs psi for 15 minutes. Flasks containing grains were inoculated with 5 mm discs of 5 days old fungal cultures and mixed by hand shaking. Incubate flasks at 28±2˚C for 7 days.

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Preparation of compost:-Paddy straw for composting was collected from the experimental area of Department of Soil Science, PAU, Ludhiana.
Method:-Unchopped paddy straw was weighed and filled in the pits of 29.5×10cm 2 in length and breath. Pits were inoculated with the selected fungal culture at the rate of 5.0% (w/w) and mixed throughly. Then the paddy straw was amended with FYM (3:1), urea (1%), Rock phosphate (1%) individually. The moisture content was maintained at 60% by adding water at different intervals of composting. The material was allowed to decompose for three months and turnings were given at 15  Scanning electron microscopy of paddy straw:-Paddy straw was immersed in 2.5% cacodylate buffered glutaraldehyde and kept at 4 0 C for 24 hours. The cells were washed with 0.1 M cacodylate buffer three times for 15 minutes each at 4 0 C. Secondary fixation was done with 1% osmium tetraoxide for 2 hours. Again three washings were done with 0.1 M cacodylate buffer each for 15 minutes at 4 0 C. After washing, dehydration was done three times with 30%, 50%, 70%, 80% and 90% ethanol each for 15 minutes and the dehydration step was completed by immersing samples in 100% ethanol (three times) each for 15 minutes. Finally, ethanol was decanted and the sample was placed in a desiccator for drying. Then the sample was placed on stub and sputter coated with gold ion sputter coater. The samples were imaged in Hitachi S-3400N SEM at 15 kV acceleration voltage.
Statistical analysis:-Data was analysed statistically using Analysis of Variance (ANOVA) appropriate for factorial experiment in completely randomized design, further mean separation of treatment effects was accomplished by using Fisher's protected least significant difference test. All data analysis was carried out by using SAS-software.
Lignin loss by T1 (Paddy straw + FYM) was only 6.5%, within 60days of composting. In comparison to control, maximum degradation in silica was observed in T5 (35.5%) followed by T4 (29.5%) and T3 (Paddy straw + FYM + Fungal culture) (26%) respectively after 60 days (Table.2). Beary et al (2002) have reported that a fungal bacterial consortium of Ceriporiopsis subvermispora and Cellulomonas sp enhances the sugarcane crop residue decomposition, when supplemented with 0.3% molasses. During 60 days of composting, all treatments showed decrease in total solids and volatile solids, but an increase in ash content was observed (Table. 3). Maximum change in total solids, ash content and volatile solids were brought by T5 (Paddy straw + FYM + Fungal culture + Urea + Rock phosphate) followed by T4 (Paddy straw + FYM+ Fungal culture + Urea) and T3 (Paddy straw + FYM + Fungal culture). Total solid contents gradually decreased from 29.6% (control) to 12.2%, indicating a loss of 54% by T5 in 60 days. Similarly 45% and 42% of total solids were degraded by T4 and T3 respectively after same days of composting. Totally different trend was followed in case of ash content. In T5, ash content gradually increased from 16.2% to 27.1% after 60 days. Maximum degradation in Volatile solids was by T5 (13.3%) on 60 th day of composting.

Scanning electrom microscopy (SEM) of paddy straw:-
Large fraction of holo-cellulose content was removed by composting, therefore, some physical changes were there in the straw. For this reason, SEM pictures of untreated paddy straw and paddy straw treated with T5 (Paddy straw + FYM + Fungal culture + Urea + Rock phosphate) were produced (Plate. 2). The distinct changes in surface structure were visible in the basic tissue of paddy straw. The untreated paddy straw exhibited a rigid and highly compact structure, whereas pretreated sample showed opening of the holo-cellulose fibrils due to creation of pores of different sizes. These structural analyses proved that composting of rice straw degraded the lignin and reduced the crystallinity of cellulose micofibrils. Micro-fibrils were separated from initial connected structure and are fully exposed, thus increasing the external surface area and porosity of paddy straw. Similar results were also reported by Yu et al (2009) reported that both morphological and structural characteristics were changed due to organic polar substances and inorganic silica partly dissolved, which leaves higher surface area with more pores of different sizes.

Control T5
537 (a) Longitudinal section of paddy straw before treatment (b) Longitudinal section of paddy straw after treatment with T5 (Paddy straw + FYM + Fungal culture + Urea + Rock phosphate Plate 2:-Scanning electron micrographs (SEM) of paddy straw before and after biological pretreatment, (a) longitudinal section of paddy straw before treatment, (b) longitudinal section of paddy straw after treatment with T5 (Paddy straw + FYM + Fungal culture + Urea + Rock phosphate.