Assessment of different screening methods for selecting biosurfactant producing marine bacteria

Different screening methods namely, hemolytic assay (HA), modified drop collapse (MDC), tilted glass slide (TGST), oil spread method (OSM), blue agar plate (BAP), hydrocarbon overlaid agar (HOA) plate, emulsification index (EI), emulsification assay (EA) were assessed for their efficiency to detect biosurfactant producing marine bacteria. Forty-five strains of bacteria, comprising 18 Acinetobacter and 27 other bacteria along with positive MTCC reference strains were examined. HA, MDC, TGST efficiently detected 15, 17 and 14 biosurfactant producers respectively. Five hemolytic cultures did not show any biosurfactant production in MDC, TGST, and/or OSM. The emulsification of kerosene was also poorer. These results suggest that HA is not totally reliable. Six bacterial isolates produced biosurfactant in OSM, and MDC as well as TGST. MDC and TGS tests demonstrated good activity for nine isolates and proved to be the essential methods. None of the bacteria produced glycolipid on BAP. Cultures showing >30% of emulsification with kerosene were found to be positive in at least one of the above mentioned screening methods. The reference strains, Gram negative bacterium MM73b produced 68% the highest emulsification and demonstrated biosurfactant production in modified drop collapse, tilted glass slide test with highest emulsification units of 213.8 (EU/ml) for petrol. In case of xylene, Acinetobacter spp. MM74 demonstrated 187.5, Acinetobacter spp. WB42 demonstrated 170.4 emulsification units. HOA plate identified 31 and 22 bacteria for diesel and crude oil degradation respectively. Thus, this method proved to be significant one. We suggest that single method is not suitable to identify all type of biosurfactants, and recommend that drop collapse, tilted glass slide test, oil spread method followed by emulsification assay are more suitable for primary screening.