Nidhi Sahni

Effect of thermophilic fungus Humicola fuscoatra MTCC 1409 on paddy straw digestibility and biogas production

The ability of Humicola fuscoatra MTCC 1409 to pretreat paddy straw for enhancing its digestibility and biogas production was investigated in this study. The potential of pretreatment of paddy straw was studied at regular intervals of 0, 5, 10, 15 and 20 days by determining the change in chemical composition of paddy straw like NDF, ADF, cellulose, hemicellulose, lignin and silica. Results indicated that the pretreatment of paddy straw with culture for 10 days was appropriate for increasing paddy straw digestibility and biogas production. The pretreatment significantly reduced the concentrations of NDF, ADF, cellulose and hemicellulose in the paddy straw by 16.9, 5.5, 56.1 and 42.9 % respectively. Reducing sugars and hydrolysis rate also increased significantly with pretreatment. These results showed that Humicola fuscoatra is an efficient cellulolytic fungus which is capable of increasing paddy straw digestibility and hence biogas production increased by 27.7% within 10 days pretreatment. The microscopic structural changes were examined by scanning electron microscopy (SEM) under reasonable conditions. INTRODUCTION In the recent years, there has been an increased interest in the development of technologies for exploiting renewable energy sources such as biomass (especially energy crops) for energy/ power generation either directly or indirectly through various conversion routes (Kashyap et al. 2003). Anaerobic digestion is a biological process in which biodegradable biomass is decomposed in the absence of oxygen by the sequential action of hydrolytic, acetogenic and methanogenic bacteria to produce biogas. Biogas is a mixture of CH4 (50-65%), CO2 (30-45%), H2 (0-3%), N2 (1-5%), CO (0-0.3%), H2S (0.1-0.5%), O2 and water vapors (traces) (Pauss et al. 1987) Paddy straw consists of cellulose (35-40%), hemi-cellulose (2024%), lignin (8-12%), ash (14-16%) and extractives (10-12%) which are associated with each other (Saha, 2003). Although, paddy straw has high cellulose content but the lignin complex and silica incrustation shields the microbial action for biogas production. Therefore, the paddy straw needs to be pretreated in order to enable cellulose to be more accessible to the microbial/enzymatic attack. Microbial pretreatment employs the use of micro-organisms especially fungi such as Pleurotus ostreatus, P chrysosporium NRRL 6361, Ceriporiopsis subvermispora and Cyathus stercoreus (Gammal et al. 1998; Taniguchi et al. 2005). Thermophiles are a good source of novel catalysts that are of great industrial interest. The thermophiles have more stable enzymes as compared to mesophiles (Li et al. 2005). Thermophilic enzymes are also active at low temperature. Thermophiles developed more rapidly to higher peaks as compared to mesophiles and stability of obligate thermophiles increased with process temperature. Enzymes synthesized by thermophiles and hyper-thermophiles are known as thermozymes. These enzymes are typically thermostable or resistant to irreversible inactivation at high temperature. Thermozymes can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. The main advantages of performing process at higher temperature are reduced risk of microbial contamination, lower viscosity, improved transfer rates and improved solubility of substrates. Due to these multifarious potentialities, they appear to be natureborne biotechnologists. No doubt, reports are available for biological pretreatment of paddy straw using mesophilic fungi; however there is negligible work done on pretreatement using thermophilic fungi. Therefore, the present study was undertaken to optimize the conditions for thermophilic fungal pretreatment of paddy straw by Humicola fuscoatra MTCC 1409 and to study the implications of enhanced paddy straw digestibility on biogas production. MATERIALS AND METHODS Procurement of the materials Paddy straw was procured from the research field of Punjab Agricultural University, Ludhiana after harvesting of the crop. The paddy straw was chopped to 3-4 cm with a chopping machine and was stored in polythene bags at room temperature. Microbial culture of H. fuscoatra MTCC 1409, was procured from Institute of Microbial Technology, Chandigarh and was maintained on yeast peptone soluble starch (YPSS; yeast extract = 0.4%, soluble starch = 1.5%, K2HPO4 = 0.1%, MgSO4 = 0.1% & agar = 2.0%) agar slants. The culture was stored in refrigerator after sub-culturing at monthly intervals. Digested cattle dung slurry was procured from a working biogas plant of School of Energy Studies for Agriculture, PAU, Ludhiana. Biological pretreatment of paddy straw For the preparation of inoculum of Humicola fuscoatra MTCC 1409, wheat grains were washed and boiled for 20-30 minutes. The excess water was drained off. The grains were then mixed with 2% gypsum (CaSO4) and 4% CaCO3 and dispensed into empty glucose bottles (250 g/bottle). The bottles were plugged and autoclaved for 90 minutes. After cooling, the bottles were inoculated with 5mm bits of 7-8 days old culture of H. fuscoatra MTCC 1409 and incubated at 50±2°C. The mycelium impregnated grains were used to inoculate paddy straw. Chopped paddy straw was soaked in water overnight. The excess water was drained off, so as to have approximately 65-70% moisture content. It was then mixed with inoculum at 10% w/w ratio (i.e. 25 g inoculum /250 g PS). After proper mixing, paddy straw was filled in polythene bags and incubated at 50±2°C for different times i.e. 0, 5, 10, 15 and 20 days, respectively. After the completion of required incubation, each set of treated paddy straw was removed and used to determining the change in chemical composition, dry matter loss, reducing sugars and hydrolysis rate of paddy straw. Chemical analysis Dry matter (DM) loss was determined by difference between dry weight of sample before and after pretreatment and described as percentage of initial weight of sample. Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined by the standard method of AOAC (2000). Hemicellulose content was estimated as the difference between NDF and ADF while cellulose, lignin and silica were estimated by acid detergent lignin (ADL) as per standard procedure (AOAC, 2000). Estimation of reducing sugars and hydrolysis rate The total amount of reducing sugars was determined by 3, 5-dinitrosalicilic acid colorimetry (DNS method) (Ghose, 1987). Sample preparation: Total sugars were extracted from the sample as follows:

Role of Coriolus versicolor MTCC 138 in ligno-silica complex removal of paddy straw and its implication on biogas production

Paddy straw pretreated with Coriolus versicolor MTCC 138, is a ligno-cellulolytic fungus, used to enhance its biodegradability. The potential of microbial pretreatment on paddy straw digestibility was investigated at regular intervals of 5, 10, 15, 20 and 25 days by determining the change in proximate (TS% & VS%) chemical composition (cellulose, hemicellulose, lignin and silica content). Results suggest that, 5 days of pretreatment was appropriate. The pretreatment significantly (P ≤ 0.05) reduced the concentrations of cellulose, lignin and silica in the paddy straw by 19.3, 19.1 and 32.5%, respectively. The present data showed that C. versicolor is capable of producing high quantity of lignocellulolytic enzymes for the reduction of lignocellulose biomass in less incubation time. However, biogas production was found to increase by 26.2% in 5 days pretreated sample.

Isolation and Preliminary Screening of Paddy Straw Degrading Thermphilic Fungi

The present study was aimed at isolation, purification and screening of paddy straw degrading (lignocel- lulolytic) thermophilic fungi for enhancing digestibility. A total of 80 cultures were isolated from paddy straw, farm yard manure and soil. These isolated cultures then purified and screened qualitatively and quantitatively on agar plates. Remazol brilliant blue (RBB) dye and guaiacol used as substrate to evaluate lignolytic activity in eighty isolated thermophilic fungi. A total of 68 fungi decolorized the RBB during the growth and only 19 fungi showed redness zone on guaiacol. Three isolates namely T10, T14 and T17 are the potential paddy straw degraders which can used for enhancing biogas production. Paddy straw, being a lignocellulose, predominantly contains cellulose (35-40%), hemi-cellulose (20-24%), lignin (8-12%), ash (14-16%) and extractives (10-12%) which are associated with each other (Maiorella, 1985). Hemi-cellulose serves as a connection between lignin and cellulose fibres and provides more rigidity to the whole cellulose-hemicellulose-lignin net- work (Laureano-Perez et al. 2005). Lignin provides structural support to the plant, impermeability and resistance to deg- radation. Rice plant, a typical silicon accumulating organism, accumulates about 10% silicon in the paddy straw obtained from rice plant (Van Soest, 2006). This silicon forms complex with lignin as lignin-silica complex which further restricts the accessibility to the cellulose. MATERIALS AND METHODS Isolation and purification of lignocellulolytic fungi The fungal cultures were isolated from different samples like soil, compost, digested slurry and plant debris. One gram of sample was vortexed with 99 ml of sterilized distilled water to make uniform suspension. Heavy particles were allowed to settle and clear supernatant was used for serial dilution. One ml of serially diluted sample was pour plated on paddy straw agar medium (PSA), each containing chloramphenicol (50 mg/l) and incubated at 50±2°C. The isolated colonies were transferred thrice on fresh agar plates to purify the cultures. Screening of lignocellulolytic fungi The isolated and purified cultures were qualitatively screened for its lignocellulose degradation potential by agar plate as- say method (Okino et al. 2000). Remazol brilliant blue (RBB) and guaiacol were used as indicator dyes for lignin degra- dation and potency index was calculated by the following formula: Potency index = size of clearance zone (cm 2 )/size of colony