Supplementary MaterialsAnnexure mmc1

Supplementary MaterialsAnnexure mmc1. and hydrolytic activity of 2.46 on hydrolysate, Buspirone HCl 3.06 on hydrolysate at 72 h of incubation. Total activity of enzyme of 2.11 U/ml and particular activity of 6.05 U/mg were recorded at 24 h. Enzyme hydrolysis of macroalgal biomass; and produced 135.9 mg/g and 107.6 mg/g of reducing sugar respectively. The study discloses that this enzyme extracted from salt tolerant bacteria is suitable for optimal saccharification of seaweed polysaccharides towards biofuel production. Cellulose degrading ability of bacterial strains were confirmed by streaking on CMC medium with composition KH2PO4 0.5 g/L, MgSO4 0.25 g/L, Gelatin 2 g/L, Agar 15 g/L, CMC-Na salt 2 g/L, which were incubated for Buspirone HCl 24 h, 48 h and 72 h at 35 C. Endoglucanase activity of the enzyme was ascertained by using CMC (Juturu and Wu, 2014). After incubation, lugol’s iodine was added so as to cover the samples in the petriplates, which aids as an indication for cellulose degradation (in an agar medium) and provides the basis for a rapid and sensitive screening test for cellulolytic bacteria. Cellulose degrading potential of positive isolates were estimated by calculating hydrolysis capacity as given in Eq. (1) (Gupta et?al., 2012). The colonies that degraded cellulose forms obvious zones, which were considered for further studies. Average values of diameters were considered for the colonies with uneven zones. Cellulolytic activity was identified as low (if the diameter of the zone was between 0.5-1.9), medium (2C3.9) and high (above 4) (Dar et?al., 2015) (UI) (previously known as (UL) were treated with 0.7 N and 0.5 N dilute acid and the hydrolysate obtained was supplemented with Agar 20 g/L, which was autoclaved for 121 C for 15 min. Later the seaweed medium was incubated for 24, 48 and 72 h and hydrolytic capacity of each strain decided. 2.3. Monitoring of bacterial growth Bacterial strains were prioritized based on the hydrolytic capacity, and were chosen for further study. Bacterial growth was monitored through LAT absorbance of 600 nm at every 24 h interval upto 72 h. Based on this, enzyme activity was calculated with the plot of growth curve considering absorbance vs time. Protein concentration of the crude enzyme was measured by Bradford method and standard plot was prepared taking bovine serum albumin (BSA) as standard (Bradford, 1976). The cellulase activity was quantified by spectrometric determination of reducing sugars by 3, 5-dinitrosalicylic acid (DNS) Buspirone HCl method (Miller, 1959) at different incubation time of 30 min, 24, 48 and 72 h. The release of reducing sugar was measured through the measurement of absorbance at 546 nm. Enzymatic activity refers to the amount of enzyme that releases 1 mol of reducing sugar per minute. Salt tolerance for the selected bacteria was determined by monitoring the growth (recorded the absorbance at 600 nm) in a broth medium at different NaCl concentrations (of 3.5C14%). 2.4. Crude enzyme production, growth condition and biochemical characterization The inocula with higher activity of cellulase was transferred to the production medium made up of salts (0.5% Yeast extract, 3.5% artificial Buspirone HCl sea water medium (NaCl 24.6 g/l, KCl 0.67 g/l, CaCl2.2H20 1.36 g/l MgSO4.7H2O 6.29 g/l MgCl2.6H2O 4.66 g/l, NaHCO3 0.18 g/l Final pH at 25 C 7.5 0.5) supplemented with 1.5% CMC as a sole source of carbon and pH was adjusted to 7.5C8.0 before sterilization at 121 C for 15 min. The culture was incubated at 35 2 C on rotary shaker at 150 rpm. After 24 h of incubation, the production medium was centrifuged at 12,000 rpm for 30 min at 4 C and supernatant was treated as crude enzyme (Trivedi et?al., 2011). Biochemical and morphological analysis were done according to Bergey’s Manual of Systematic Bacteriology. 2.5. Enzyme saccharification of macroalgal polysaccharide Dilute acid pretreated macroalgal biomass and were subjected to enzyme hydrolysis at 55 C pH 6.8 for 36 h and. The reducing sugar released was estimated every 6 h using DNS method (Miller, 1959). 2.6. 16S rDNA sequencing for strain identification.