Colony forming unit (CFU) tests were used to determine the basic structure and numbers of microorganisms in the biofilm. The following selective solid agar media (Oxoid Ltd., UK) were used to determine 1) the total cell numbers, 2) the number of eukaryotic cells, 3) that of Pseudomonas, and 4) that of the primary n-butyl acetate degraders, respectively: 1) Standard plate count agar CM 0463, 2) Rose Bengal chloramphenicol agar base CM 549 with selective supplement SR 78, 3) Pseudomonas agar base CM 559 with C-N supplement SR 102, and 4) BSM agar. The last of them was based on the BSM medium solidified with 10 g L -1 of a pure agar, Agar bacteriological LP0011; the dishes were placed in a desiccator with n-butyl acetate vapors. All agars were purchased from Oxoid Ltd. UK.
Samples of packing materials covered with the biofilm were taken at a depth of 20 cm from the top of each biofilter. A standard sample procedure was applied previously published for CFM analyses. [16] (link) To observe the complex bioreactor ecology, the entire volume, 50 mL of BSM medium, was applied at once to each biofilter to wash out any possible protozoa, metazoa and mite. The leachate was collected and examined by microscopy using an Olympus BX40 microscope equipped with a Canon EOS 700D camera. Samples were taken on 119th and 300th days of the biofilter operation.
Samples of packing materials covered with the biofilm were taken at a depth of 20 cm from the top of each biofilter. A standard sample procedure was applied previously published for CFM analyses. [16] (link) To observe the complex bioreactor ecology, the entire volume, 50 mL of BSM medium, was applied at once to each biofilter to wash out any possible protozoa, metazoa and mite. The leachate was collected and examined by microscopy using an Olympus BX40 microscope equipped with a Canon EOS 700D camera. Samples were taken on 119th and 300th days of the biofilter operation.