Morphological and biochemical characterization of bacterial isolates

Morphological characterization of nine bacterial isolates was done using Gram staining and biochemical characterization was performed by IMViC test, starch hydrolysis and utilization of carbohydrates (lactose, fructose and dextrose).

Dye Decolourization Assay

Dye decolourization ability of nine bacterial isolates was carried out following the standardized method described earlier by our group (Ghatak & Das, 2018). Five different dyes (malachite green, crystal violet, congo red, nigrosin and safranin) were used for this purpose. Dye decolourization was assessed by the decrease in absorbance at respective wavelengths of the dyes. Inoculums (1 ml) of different bacterial isolates were separately given in 10 ml nutrient broth (pH 7.0) and were grown overnight at 37°C. The following day, dyes (1 ml in each tube) were added in each set at a concentration of 50 ppm and were again incubated at 37°C. Initial absorbance (‘0’ hr reading) was taken immediately after the addition of the dyes. Decolourization of the dyes was observed within 4 hrs. of incubation. Followed by incubation, 2 ml of culture was collected from each set and cells were removed by centrifugation and the supernatant was directly (without further dilution) used for taking absorbances at respective wavelengths. The percentage of dye decolourization was estimated using the following formula:

Percentage of Decolourization =

$\frac{\left[\right(Initial absorbance - Final absorbance\left)\right]}{Initial Absorbance}\times 100$

All the experiments were repeated thrice and the data in the graph is represented as Mean ± SE.

Assay of Amylase and Laccase

Amylase assay was carried out by conventional method (Bernfeld, 1955) and laccase assay was performed using the method described earlier by Das and Ghatak, 2019. All the assays were performed in triplicate and results are represented as Mean ± SE.

Sample Collection and Initial Screening

Total 15 different bacterial isolates were screened for their dye decolourization ability out of which only nine isolates showed dye decolourization and hence further studies were performed with those nine bacterial isolates. These isolates were designated as GW and numbered from 1 to 9.

Morphological and Biochemical Characterization of Bacterial Isolates

Samples were screened by using nutrient agar medium and discrete isolated colonies were taken for morphological study. Morphological analysis showed that all nine isolates were gram positive in nature and either rod shaped or cocci. Other than GW1 all the isolates were found to be indole negative. GW2, GW6 and GW7 showed positive methyl red test while others were found to be negative. All isolates were negative for VP test except GW8 and GW9. Positive citrate test was observed only for GW1, GW2, GW4 and GW5. Interestingly, GW1 and GW6 showed starch hydrolysis ability and hence these two were further used for in vivo amylase assay. However, the strains did not ferment lactose, fructose or dextrose. Biodegradability is considered as the most cost-effective methods hence substantial efforts have been made by different groups of researchers to employ microorganisms for this purpose (Rebekah & Sharphudhin, 2016) whose prerequisite is to characterize particular strains. Results of biochemical and morphological characterizations are represented in Table-1.

Dye Decolourization Assay

Dye decolourization ability of nine bacterial isolates was studied based on the decolourization of five different dyes which are commonly used in textile and other industries (Durve, Gupta, & Naphade, 2012; Kumar & Saravanan, 2015). According to our earlier study (Ghatak et al., 2018), the optimum concentration for the dyes were selected to be 50 ppm for each of the dyes and dye decolourization was represented in percent decolourization. Absorbances were recorded at 619 nm for malachite green, 586 nm for crystal violet, 495 nm for congo red, 570 nm for nigrosin and 515 nm for safranin. Among all the dyes, highest decolourization was observed for malachite green and crystal violet for all the isolates which could be correlated to an earlier study by (Das, 2019) . GW1, GW2, GW6, GW7, GW8 and GW9 all showed substantial decolourization of malachite green, however; the best result was documented for GW7 with a decolourization ability of 93.54%. Same result was also observed for crystal violet with GW7 showing the highest (89.4%) dye decolourization. In case of congo red and nigrosin, the isolated strains did not show marked decolourization effect; however, GW1 and GW8 showed 41.66% and 37.26% decolourization respectively for congo red and GW8 and GW9 showed 41.53% and 45.04% decolourization respectively for nigrosin. On the other hand, for safranin GW6 was found to be most effective (57.15%). Bacterial reduction of dyes often is associated with the activities of different enzymes but the difference in mechanism of metabolism among different bacteria could affect the efficiency of decolourization (Singh, Singh, & Singh, 2015). As GW1 and GW6 showed starch hydrolysis ability so they were screened for the presence of secretory amylase. The assay was performed at three different time points (24 hrs, 48 hrs and 72 hrs) and enzyme activity was highest at 72 hrs. GW6 gave better result (47.5 nmole/min/ml) than GW1 (16.1 nmole/min/ml) at 72 hrs (Figure 2A). Different scientific works established the role of laccase in biodecolourization of dyes (Singh et al., 2015). We observed that GW1, GW3, GW4, GW8 and GW9 all showed presence of secretory laccase with increase in activity on longer incubation. GW9 recorded highest activity with a value of 16.34 nmole/min/ml at 72 hrs (Figure 2B). It is of utmost importance to investigate the exact metabolic mechanism of these enzymes secreted by dye decolourizing bacteria to ensure the effectiveness of this method in a non-toxic manner.