Measurement of decolourisation efficiency

The decolourisation experiment was carried out in Erlenmeyer flasks (250 mL) containing 100 mL of previously prepared sterile melanoidins containing medium. The flasks were inoculated with overnight grown fresh culture of four identified bacterial strains, i.e., K. pneumoniae ({"type":"entrez-nucleotide","attrs":{"text":"KU726953","term_id":"1040467831","term_text":"KU726953"}}KU726953), S. enterica ({"type":"entrez-nucleotide","attrs":{"text":"KU726954","term_id":"1040467832","term_text":"KU726954"}}KU726954), E. aerogenes ({"type":"entrez-nucleotide","attrs":{"text":"KU726955","term_id":"1040467833","term_text":"KU726955"}}KU726955), and E. cloaceae ({"type":"entrez-nucleotide","attrs":{"text":"KU726957","term_id":"1040467835","term_text":"KU726957"}}KU726957). The each bacterial culture was inoculated for their exponential phase with highest optical density (O.D.) such as K. pneumoniae (OD₆₂₀ 0.24), S. enterica (OD₆₂₀ 0.24), E. aerogenes (OD₆₂₀ 0.24), and E. cloaceae (OD₆₂₀ 0.24). The different bacteria were inoculated in flask in equal ratio, i.e., 1:1:1:1 and incubated at 37 ± 1 °C under shaking flask condition (120 rpm) for 192 h. The sample were collected at every 24 h interval during incubation and centrifuged at 10,000×g for 10 min at 4 °C for measurement of decolourisation efficiency. Melanoidins decolourisation efficiency was monitored by measuring the change in absorbance maxima of the melanoidins at 295 nm after scanning of absorbance maxima using a UV–Vis spectrophotometer (Evolution-201, Thermo Scientific, USA). Simultaneously, the control flasks without any bacterial inoculation were also incubated in shaker with the same temperature and shaking speed. Finally, the bacterial cell in flask was found in ratio of 2:1:2:2. This indicated the optimum ratio of different bacteria in consortium for maximum decolourisation of molasses-melanoidins. Therefore, the ratio of different bacteria in consortium was selected in ratio of 2:1:2:2 for optimisation of decolourisation process at different nutritional and environmental parameters. The decolourisation efficiency was expressed as percent (%) of decolourisation:

where A_i is the initial absorbance and A_f is the final absorbance of medium after decolourisation at the λ_max 295 nm.