2.1. Isolation of bacteria

Bacteria were isolated from natural sources such as marine (M) (water column and mangrove sediment), Gastrointestinal region (GI) (fish gut, sheep and goat rumen), Herbivore (H) animal residue (Nilgai, Bison, Elephant, Spotted deer, Sambhar deer, Cattle). Serial dilution was performed from 10¹ to 10⁷ using 0.89% saline solu- tion. Tryptone Soya agar, TSA (Tryptone 15 g/l, Soya peptone 5 g/l, NaCl 5 g/l and Agar 15 g/l, p^H (at 25 C) 7.3 0.2) and Zobell Marine agar, ZMA (Peptone 5 g/l, Yeast extract 1 g/l, Ferric citrate 0.1 g/l, NaCl 19.45 g/l, MgCl₂ 8.8 g/l, Na2SO₄ 3.24 g/l, CaCl₂ 1.8 g/l, KCl 0.55 g/l, NaHCO₃ 0.16 g/l, KBr 0.08 g/l, Strontium Chloride 0.034 g/l, Boric acid 0.022 g/l, Sodium silicate 0.004 g/l, Sodium fluorate 0.0024 g/l, Ammonium nitrate 0.0016 g/l, Disodium phosphate 0.008 g/l, Agar 15 g/ l, p^H 7.6 0.02) for isolation and enumeration of heterotrophic marine bacteria and Carboxymethyl Cellulose agar (KH2PO₄ 0.5 g/l, MgSO₄ 0.25 g/l, Cellulose 2 g/l, Agar 15 g/l, Gelatin 2 g/l and p^H 6.8e7.2) for other samples.

2.2. Estimation of hydrolytic activity

• CMC medium: 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 incu- bated for 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, lu- gol’s iodine was added so as to cover the samples in the petriplates, which aids as an indicator for cellulose degradation (in an agar medium) and pro- vides the basis for a rapid and sensitive screening test for cellulolytic bacteria. Cellulose degrading potential of positive isolates were estimated by calcu- lating hydrolysis capacity as given in Eq. (1) (Gupta et al., 2012). The col- onies that degraded cellulose forms clear 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 (2e3.9) and high (above >4) (Dar et al., 2015)
  

  Hydrolysis capacity^(1/4)(diameter of zone/diameter of colony)

• Seaweed hydrolysate: Ulva intestinalis (UI) (previously known as Enteromor- pha intestinalis) and Ulva lactuca (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 determined.

2.3. Monitoring of bacterial growth

Bacterial strains were prioritized based on the hydrolytic capacity, and were cho- sen for further study. Bacterial growth was monitored through absorbance of 600 nm at every 24 h interval upto 72 h. Based on this, enzyme activity was calcu- lated with the plot of growth curve considering absorbance vs time. Protein con- centration 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) 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. Enzy- matic activity refers to the amount of enzyme that releases 1 mmol 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 3.5e14%).

2.4. Crude enzyme production, growth condition and biochemical characterization

The inocula with higher activity of cellulase was transferred to the production me- dium containing salts (0.5% Yeast extract, 3.5% artificial 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 p^H at 25 C 7.5 0.5) supplemented with 1.5% CMC as a sole source of carbon and p^H was adjusted to 7.5e8.0 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 U. intestinalis and U. lactuca were sub- jected to enzyme hydrolysis at 55 C p^H 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

Identification of bacterial strain with highest hydrolytic activity was done through using 16S rDNA sequencing. Genomic DNA was isolated and quantity was measured using Nano Drop Spectrophotometer and the quality was deter- mined using 2% agarose gel. A single band of high-molecular weight DNA was observed. 16S rRNA gene was amplified by 16S rRNAF and 16S rRNAR primers. A single discrete PCR amplicon band of 1500 bp was observed when resolved on Agarose gel. The PCR amplicon was purified to remove contami- nants. Forward and reverse DNA sequencing reaction of PCR amplicon was car- ried out with forward primer and reverse primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of 16S rRNA gene was generated from forward and reverse sequence. 16S rRNA gene sequence was used to carry out BLAST with the database of NCBI genbank database. Based on maximum identity score first ten sequences were selected and aligned using multiple alignment software program Clustal W. Distance matrix was generated and the phylogenetic tree was constructed by using MEGA 7 (Tamura et al., 2004; Kumar et al., 2016).

Fig. 1. Hydrolytic activities of all the Cellulose degrading bacteria.