Urmila Gupta Phutela

Production of cellulases from Humicola fuscoatra MTCC 1409: Role of enzymes in paddy straw digestion

Cellulases were produced from fungus Humicola fuscoatra MTCC 1409 by solid state fermentation under different cultural conditions viz. pH, incubation temperature, inoculum size and days of incubation in order to optimize the conditions for maximum enzyme production. The potential of cellulase pretreatment to increase the digestibility of paddy straw was also ascertained. Maximum enzyme production was achieved at pH 6.0 of Mandel media and at temperature 45°C. Inoculum size of 1×107 spores/ml was found to be optimum for maximum enzyme production. Enzyme production increased with the increase in days of incubation from 2 to 6 days and then declined thereafter. Cellulase units at the concentration of 1, 1.5 and 2 µmole/g were exogenously added to paddy straw and change in chemical composition of paddy straw was determined after 18, 24, 30 and 36 h of treatment. With increase in enzyme concentration and incubation period, the content of neutral detergent fibre (NDF), acid detergent fibre (ADF), cellulose and hemicellulose reduced gradually with simultaneous increase in lignin and silica content. The concentrations of NDF, ADF, cellulose and hemicellulose decreased by 9.2, 5.9, 10.8 and 23.4% respectively, however lignin and silica content increased by 9.7 and 6.4% respectively as compared to control (with no cellulase added) at 2 µmole enzyme concentration after 36 h of pretreatment. These results show that the enzyme produced from cellulolytic fungus H. fuscoatra is capable of increasing paddy straw digestibility and thus enhancing the utilization of paddy straw for different purposes. Key words: Cellulase production, Humicola fuscoatra, paddy straw, paddy straw digestibility.

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:

POULTRY DROPPING BASED BIODIGESTED SLURRY FOR PROTEASE PRODUCTION BY Humicola fuscoatra MTCC 1409: OPTIMIZATION AND KINETIC STUDY

Protease is one of the most important groups of commercially produced enzymes. This study was aimed at the optimization and kinetics of protease production from poultry dropping based biodigested slurry by Humicola fuscoatra MTCC 1409. Four significant variables (pH, temperature, slurry concentration and inoculum concentration) were considered for optimization both by one variable at a time approach and response surface methodology. The maximum protease production in the poultry dropping based biodigested slurry was (531±1.37 U g -1 ) under the optimum conditions of pH (5), temperature (40°C), slurry concentration (25%) and inoculum concentration (10%). The protease production was found to be 3.38 fold higher under optimized conditions as compared to the non-optimized ones. The thermal inactivation of protease produced from biodigested slurry was investigated kinetically within temperature range of 30-70°C. The irreversible inactivation was well described by first order kinetics with k values increasing between 0.0028 to 0.0071 min -1 and t1/2 decreasing from247.70 to 98.10 mins. At higher temperature, there was significant decrease in residual activity. The activation energy, enthalpy, Gibbs free energy and entropy range calculated on the basis of residual activity experiments conducted at temperature range of 30-70°C was found to be 21.29, 18.44 to 18.78 kJ mol -1 , 89.01 to 98.47 kJ mol -1 and -0.23334 to -0.23181 kJ mol -1 respectively, suggesting the thermostability of enzyme. This is first report on optimization, kinetics and determination of thermodynamic parameters of protease production by Humicola fuscoatra MTCC 1409 from poultry dropping based biodigested slurry. * Corresponding author KEYWORDS

MORPHOLOGICAL AND STRUCTURAL CHANGES IN PADDY STRAW INFLUENCED BY ALKALI AND MICROBIAL PRETREATMENT

Paddy straw is a lignocellulosic waste rich in holocellulose (cellulose+hemicellulose) content. It can be used as a good substrate for biogas, bioethanol and biodiesel production. However, the recalcitrant cell wall components i.e. lignin and silica are the main deterrent to efficient utilization of paddy straw. This stringent sheath of lignin and silica does not allow fermenting microbes to access holocellulose. Pretreatment of paddy straw is, therefore, crucial to get rid of lignin and silica. In this context, paddy straw was pretreated with various alkalis viz. NH 3 , Na 2 SO 3 , Na 2 CO 3 and NaOH in the current study. All the alkali pretreatments were supplemented with microwave irradiations (720 W, 18°C) for 30 minutes. Paddy straw was also pretreated microbially with Pleurotus florida for 15 days via spawning. Morphological and structural changes in the pretreated paddy straw were visualized via Scanning Electron Microscopy (SEM). The straw turned remarkably fragile with enhanced bulk density and surface wettability after 4% NaOH-30 min microwave pretreatment. The pretreated straw was also found to lack silicified cuticle layer and lignin sheath which exposes straw sugars (cellulose and hemicellulose) to fermenting microbes.

Partial Purification and Characterization of Cellulolytic Enzymes Extracted from Trichoderma reesei Inoculated Digested Biogas Slurry

Alternative sources of energy are the demand of the modern world. For generating different alternate fuels, utilization of lignocellulolytic biomass is on top of the priority list, for which cellulase enzymes are required specifically. In this context, the aim of the presented paper was the partial purification and characterization of cellulolytic enzymes from Trichoderma reesei inoculated digested biogas slurry. The enzyme was produced at previously standardized conditions (Incubation period: 15 day, Spore concentration: 108 spores/ml, Slurry concentration: 25%). The cellulolytic enzymes viz. CMCase, Cellobiase and FilterPaperase produced were then partially purified by ammonium sulphate precipitation (0-30 and 30-80%) and dialysis followed by ion exchange chromatography using DEAE-cellulose column. Twelve fold purification was achieved for cellobiase. Specific activity of 20.18 U/mg was measured. Two isoforms of Cellobaise (C-I and C-II) were found with 21 and 32 fold purification, respectively. Upon characterization, the optimal pH and optimal temperature values for cellobiase came out to be 7.5 and 55°C for C-I and 25°C for C-II. Carboxymethyl cellulase was partially purified up to 10.4 fold with specific activity of 1.87 U/mg of protein and Fpase was purified to 11.3 fold with 1.47 U/mg of protein specific activity. Partially purified enzyme activities were compared with that of commercial enzymes. This is a novel work where cellulases were extracted and partially purified from digested slurry from biogas plant, which is very significant with reference to not only disposal of digested biogas slurry but also its value addition for industrial applications.

Statistical optimization of cultural conditions for manganese peroxidase production by Coriolus versicolor MTCC 138 from digested cattle dung slurry

The present study reports the production of manganese peroxidase enzyme using digested cattle dung slurry as a substrate by Coriolus versicolor MTCC 138. Digested cattle dung slurry was analyzed for proximate (total solids, volatile solids and pH), chemical (cellulose, hemicellulose, lignin and silica) and biochemical (total proteins and manganese peroxidase) composition. Manganese peroxidase production by C. versicolor MTCC 138, was optimized by a conventional ‘one variable at a time approach’ and then by Placket-Burman design. Optimized fermentation conditions for the maximum manganese peroxidase (78.0 U mL−1) enzyme production were 25% slurry concentration, 5 days of incubation, temperature 25°C and inoculum size of 106 spores mL−1. This enzyme titre obtained by statistical optimization was 1.86 fold than that achieved by ‘one variable at a time approach’. It revealed that the effect of different cultural conditions (slurry concentration, spore concentration and incubation period) have statistically significant effect on enzyme production.

Selection of Low Cost Substrate for Inoculum Preparation of Pleurotus Ostreatus MTCC 142 for Pretreatment of Paddy Straw

The present study was conducted to assess the selection of low-cost substrate for inoculum preparation of Pleurotus ostreatus Microbial Type Culture Collection (MTCC) 142 for pretreatment of paddy straw. P. ostreatus MTCC 142, a white-rot fungus, known for its cellulolytic and ligninolytic properties can be used to degrade lignin present in the cell wall of paddy straw. Wheat grains are the most commonly used substrate for growing P. ostreatus, but it is not cost-effective. P. ostreatus MTCC 142 was grown on different low-cost substrates, namely, wheat straw, paddy straw (grinded), paddy straw (chopped), wood shavings, cow dung, cow dung slurry, charcoal and wheat straw combinations, paddy straw and charcoal combinations. Observations were made alternatively at different time durations (2, 4, 6, 8 and 10 days) for the mycelia impregnation. Wheat straw and wheat straw combination with charcoal showed maximum mycelium impregnation by day 8. These substrates were further used to inoculate paddy straw for its degradation. Paddy straw degradation was observed through chemical and proximate analysis [total solids (TS), volatile solids (VS), cellulose, hemicellulose, lignin and silica] and by measuring its cellulolytic activity at different time durations (5, 10, 15 and 20 days). This study showed that the wheat straw and wheat straw combination with charcoal are promising substrates for inoculum preparation of P. ostreatus MTCC 142 and showed maximum cellulolytic activity, for wheat straw: Carboxymethyl cellulose activity (103.3 U ml−1) (control 95.54 Uml−1), cellobiase activity (320.86 Uml-1) (control 317.61 U ml−1) and total cellulase activity (52.39 Uml-1) (control 49.14 Uml-1) and for wheat straw and charcoal combination: CMC activity 103.30 U ml−1, cellobiase activity 319.84 Uml−1 and total cellulase activity 52.42 Uml−1, as on day 20. This further needs to be examined for ligninolytic enzyme studies, to exploit them as the potential substrates for inoculum development of paddy straw degradation.

Optimisation of Thermostable Laccase Production by Thermoascus aurantiacus MTCC 375 Using Paddy Straw as Substrate and Digested Biogas Slurry (DBS) as Medium in Solid State Fermentation

Optimisation of laccase production by Thermoascus aurantiacus microbial type culture collection (MTCC) 375 using paddy straw as substrate and digested biogas slurry as medium was carried out. Laccase production by a thermophilic mould, Thermoascus aurantiacus MTCC 375, was optimised in solid state fermentation (SSF) by a conventional ‘one variable at a time approach ‘andfurther by response surface methodology. Enzyme activity was strongly affected by three independent variables namely incubation temperature, incubation period and inoculum level. The optimised conditions for maximum laccase (715.0 Ug−1 of paddy straw) production were 45°C temperature, 107 spores ml−1 and 4 days incubation period. The enzyme titre (715.0 Ug−1 of paddy straw) attained in the validation experiment of this study is higher than those reported in the literature. An overall (2.65 fold) increase in laccase production was achieved in SSF due to statistical optimisation compared to that of a conventional ‘one variable at a time approach ’. All these cultural conditions were found to have statistically significant (P < 0.05) effect on enzyme production.

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