Evaluation Studies of Thermophilic Bacteria isolated from Rice Field Soil for Cellulose Digestion

In the present study cellulolytic bacteria were isolated from soil of rice field in aim to degrade various recalcitrant lignocellulosic biomaterials. Soil was collected from rice field at Lovely Professional University Campus & spreading was done upto serial 10-7 dilutions on CMC Agar Media. Out of 108 colonies from 10-1 and 78 colonies from 10-2 dilution of the soil sample, two bacteria RFS1 (Rice Field Soil 1) and RFS2 (Rice Field Soil 2) were screened on basis of zone of hydrolysis on Agar Medium supplemented with 2 % CMC. Gram Staining was done and both bacteria were found to be Gram +ve and Bacilli. For bacterial identification, the bacterial cultures were sent to Gentech Pvt. Ltd., Lucknow for 16S rRNA Sequencing and identified RFS1 as Bacillus coagulans and RFS2 as Geobacillus stearothermophilus. Enzyme activity optimization was done for media parameters-pH, Temperature and CMC concentration. Enzyme activity was found to be maximum at pH 5.0, temp. 40°C and CMC conc. 3% for Bacillus coagulans while for Geobacillus stearothermophilus, it was maximum at pH 6.0 , temp. 50°C and CMC conc. 2%. Pretreatment studies like Physical Pretreatment like Stream Explosion and Chemical Pretreatment with 0.25 N HCl, 0.25 N NaOH, 0.25 N Acetic Acid and 0.25 N Lactic Acid was done. Order of chemicals in delignification was Hydrochloric Acid > Lactic Acid >Acetic acid > Sodium Hydroxide .Steam Explosion was least efficient process in term of glucose yield.for Groundnut shell in case of both strains .Different substrates as Leaves of Mangifera indica ,Lab Cotton, Lab Filter Ppaer,Wheat Bran and Groundnut Shell were taken to confirm the effectiveness of lignin removal by HCl(0.25 N).Order of different materials giving glucose yield was Wheat Bran > Leaves of Mangifera indica > Lab Filter Paper > Groundnut Shell > Lab Cotton for both the strains.Our target was the hydrolysis of groundnut shell by isolated strains from the soil of rice field .So biochemical analysis of Groundnut shell was done showing Cellulose 35 % , Lignin 28.6 % ,Pectic Substances 3% , Organic Carbon Content 27.7 % and Nitrogen content 23.4 %.After biochemical analysis study of glucose yield versus cellulose hydrolysis was done.After 14th day of saccharification process Glucose Yield was 48.71 % and 58.07 % and Cellulose hydrolysis was 79.29 % and 90.67 % for Bacillus coagulans and Geobacillus stearothermophilus respectively.Partial Enzyme Purification was done with Ammonium Sulphate followed by Dialysis and Enzyme Activity was 0.0392 U, 0.0617 U while and Specific activity was 2.091 U/mg and 3.47 U/mg for Bacillus coagulans and Geobacillus stearothermophilus respectively.After dialysis,partially purified protein samples were run on SDS-PAGE and protein bands of 200 kDa were observed corresponding to the cellulase (116-205 kDa).For degradation studies of cellulose in groundnut shell Fourier Transform Infrared Spectroscopy was done and cellulose degradation was seen in wavenumber region ranges 1100-1400 cm -1 .