Zobell marine agar

A marine bacterium Bacillus megaterium was used for production of Lipopeptide29 . This bacterial strain was obtained from sea water of the Andaman and Nicobar Islands, India. For preparation of the primary inoculums, bacterial strain was maintained in Zobell marine agar (Himedia, Mumbai, India) and grown in Zobell marine broth following incubation for 12 h at 32 °C. Primary inoculums were inoculated in 250 ml Erlenmeyer flask containing glucose mineral salts medium (GMSM containing: glucose−25 g/L, NH₄NO₃–6 g/L, KH₂PO₄–0.028 g/L, K₂HPO₄–1.6 g/L, MgSO₄•7H₂O–0.3 g/L and CaCl₂•2H₂O–0.2 g/L) and the flask was kept in a shaking incubator at 180 rpm and 32 °C till mid-exponential growth of cells. The inoculum was further inoculated in 1 L Erlenmeyer flasks containing 200 ml GMSM and kept in incubator at 180 rpm and 32 °C. Fermentation broth samples were collected at different time for lipopeptide collection.

The marine bacteria were isolated from a marine sponge D. nigra as described by Selvin et al. [17 (link)]. Briefly, 1 cm³ of surface-sterilized sponge tissue was excised and the excised portion was thoroughly washed three times in sterile aged seawater to remove any bacteria within current canals. The tissue was homogenized with phosphate buffered saline using a tissue homogenizer. The resultant homogenate was serially diluted with sterilized aged seawater and preincubated at 40°C for 1 h for the activation of dormant cells. The aliquot was placed on various isolation media including marine sponge agar [33 (link)]. The inoculated plates were incubated at 30°C for 7 days in dark. The morphologically distinct colonies were reisolated and maintained on Zobell marine agar (HiMedia) at 4°C. The isolates were designated as marine sponge isolates (MSI). The isolated bacteria were screened for PHA production using viable colony staining method. Briefly, Zobell marine agar supplemented with solution of 0.25 mg Nile blue A (w/v in dimethyl sulfoxide) to give final volume of 0.5 μg dye ml⁻¹ medium. The colonies were directly examined for fluorescence under UV light to detect the accumulation of PHA. The efficient PHA producer was identified by Sudan black B staining method.

One ml of sea water sample was suspended in 9 ml of sterile distilled water and serially diluted. For isolating the pure culture of Bacillus, the dilutions were incubated at 30°C for 4 hours and heat shocked at 80°C for 3 to 5 minutes in a water bath to destroy all vegetative microbial cells. 100μl of sample from each dilution was taken and spread on Zobell marine agar (HiMedia, India). The plates were inverted and incubated at 30°C for 24 hrs.