Qualitative screening

The screening of cellulose-degrading bacteria is illustrated in Figure 1. The rotten dahlia sample was weighed and treated with sterile water. The mixture was fully shaken on a constant-temperature shaking table. The suspension was aspirated with a sterile pipette to obtain sample solutions with different dilutions and coat on Bengal red culture medium for 48 h. Single bacteria with yeast colony characteristics and large colonies were selected and repeatedly marked and separated on a yeast extract peptone dextrose culture medium until a single colony was obtained. Microscopic examination was used to establish pure bacterial cultures, which were preserved in Yeast extract peptone dextrose agar. The purified strains’ ability to secrete cellulase was assessed using the Congo red method (Danso et al., 2022). Pure isolates of single colonies were collected with sterile needles and inoculated separately on agar plates containing carboxymethylcellulose as the sole carbon source to screen the cellulolytic activity of the isolated yeasts. The inoculated plates were incubated at 37°C for 3 days. The yeast growth was observed until the strains that could grow stably on the medium were selected. The plates were stained with a 10 mL aliquot of Congo red dye solution (2.5 g/L) for 30 min, the dye solution was discarded, and the cultures were washed with 15 mL of 1 M NaCl for 10 min. The cellulose degradation activity of yeast was analyzed by measuring the CMC clearance zone around the yeast colony vs. the yeast colony diameter in millimeters. In addition, the hydrolytic capacity (HC) was estimated by the ratio of CMC clearance zone to yeast colony diameter (Danso et al., 2022). The potential yeast with the highest activity (highest HC value) among the isolated strains was selected based on plate assays, and further analyzed and characterized.

Qualitative and quantitative screening of cellulose-degrading bacteria.