Enrichment of Thermophilic Microoganisms for the Degradation of Permethrin and 3-Chlorobenzoate
Richard I. SHARP*, Trevor S. MARKS, and Sarah E MALONEY
Research Division, Centre for Applied Microbiology and Research, Porton Down, Salisbury, SP4 OJG, United Kingdom
Bubble columns have been developed to operate anaerobically at 75-80℃ under continuous culture to enrich for thermophilic microorganisms able to degrade organic pollutants. These systems encourage biofilm development and enable variation in gas blending and offer a more dynamic microbial environment enhancing liquid:gas and liquid: solid interfaces in order to more closely resemble the natural environments of the organisms. A variety of glass and porous borosilicate beads have been used in the bubble columns to simulate packed-bed reactors as used for waste water treatment. Permethrin and 3-chlorobenzoate degradation have been demonstrated in these systems.
Samples from geothermal sites in New Zealand were used to inoculate these systems. After 1 week at 80℃, phase-contrast microscopy showed fine rods and a few cocci. Gram stains showed fine Gram-negative rods only. Cell counts were approximately 105- 106 cells / ml. Attempts to subculture these enrichments onto gelrite plates were not successful.
These bubble column systems were run continuously for 9 months with cell counts in the liquid phase maintained at approximately 107-108 cells/ml. During this time microbial dehalogenation of 3-chlorobenzoate 0.5 mM was achieved at 75-80℃. The major metabolite, benzoic acid was detected within 1 week of incubation. Glass beads (5 mm diameter) were added to one bubble column to help encourage biofilm development using an inoculum from an existing 3-chlorobenzoate degrading culture. After 2 weeks operation, the beads appear blackened, and low levels of benzoic acid detected. Scanning electron micrographs of the glass beads show the development of a biofilm. A mixed culture growing anaerobically at 75-80℃, has also been established in a bubble column using Tween 80 as the main carbon source. Transformation of permethrin was demonstrated in this system. Microbially-mediated esterase cleavage of permethrin (100μM) was also achieved in bubble column systems (75-80℃), packed with porous borosilicate beads (5 mm diameter), using Tween 80 as the primary carbon source. Permethrin was hydrolysed to non-insecticidal products within one week of incubation. Scanning electron micrographs of the biofilms developed on these glass beads have been obtained.
