Lb broth media

We used E. cloacae SBP-8 (Accession No. NAIMCC-B-02025), an environmental isolate obtained from rhizospheric soil (Singh et al., 2017 (link)). The culture was grown in Luria Bertani (LB) broth media (HiMedia) at 37°C with agitation (150 rpm) as and when required. The glycerol stocks made with 20% glycerol (SRL) were stored for further use at −70°C.

Three microliters of bacterial and actinomycetes cultures grown overnight in LB broth media (Himedia) were spot plated on CMC agar (0.2% NaNO₃, 0.1% K₂HPO₄, 0.05% MgSO₄, 0.05% KCl, 0.2% carboxy methyl cellulose (CMC) sodium salt, 0.02% peptone, and 1.7% agar) (HiMedia, India). A mycelial disc (0.3 cm dia each) of fungal colony was placed on the centre of the modified Cezapak Dox Agar (CDA) plate supplemented with CMC instead of cellulose powder and pH adjusted to 5.3 for screening cellulolytic activity. The culture plates were incubated at 20°C and 30°C. After 4 days of incubation, the culture plates were flooded with Gram’s Iodine for 2-3 min (Kasana et al.
2008 (link)). Positive colonies were determined by the relative cellulolytic activity index (RCAI) which compares the diameter of the clearing zone around the colony with the diameter of microbial colony.

The DNA was extracted from the E. coli isolates as described by Moore et al. [24 ] with a minor modification. Briefly, a single colony was subcultured in Luria Bertani (LB) broth media (HiMEDIA, Mumbai, India). A 1.5 mL bacterial culture was pelleted via centrifugation and subsequently resuspended and washed in 100 µL sterile deionized water. The 150 µL bacterial suspensions were lysed at 100 °C and then frozen at −20 °C. Finally, the suspensions were centrifuged at 10,000 rpm for 10 min, and 100 µL of supernatant containing DNA from each preparation was transferred into fresh Eppendorf tubes. The DNA preparations were quantified using a Nano-drop spectrophotometer and a ratio of 260 nm/280 nm was used to estimate the quality of the DNA. Moreover, gel electrophoresis was used to check the intactness of the isolated DNA.