## LSU Historical Dissertations and Theses

1992

Dissertation

#### Degree Name

Doctor of Philosophy (PhD)

Previous researchers have reported the isolation of a diphenylether cleaving organism, Erwinia sp., using an enrichment medium containing lignin. A copper and dinitrophenol resistant mutant of this organism, Erwinia sp. Cu3614, has also been reported. To assess the effect of copper on the growth and biochemistry of this organism, continuous cultivation was used employing an apparently optimized medium containing ethanol as carbon source. Upon increasing the concentration of copper sulfate in the medium from 5 $\mu$g/ml to 10 $\mu$g/ml increases in maximum specific growth rate and growth yield were seen. Also decrease in the values for doubling time and the coefficient for maintenance energy were seen. At higher levels of copper sulfate a "non competitive" inhibition of growth was seen as indicated by the values calculated for substrate affinity constant, and inhibition constant. To assess this organism's ligninolytic ability, an assay for residual lignin was developed. The assay measured a reaction between diazotized sulfanilic acid and lignin in alkaline solution by spectrophotometric monitoring of the resulting adduct. Use of this technique indicated that Erwinia sp. Cu3614 could degrade up to 80% of lignin in batch cultures. Further evidence about the ligninolytic ability of this organism was provided by examination of electron micrographs of lignocellulosic substrates incubated with cell suspensions. An assay for monitoring diphenylether cleaving abilities was also developed using resazurin, a redox dye. In vivo assays with cells obtained from continuous culture studies indicated a linear relationship between the rates of reaction with resazurin and the amount of copper associated with cells. In vitro assays, employing cell free extracts and resazurin, were used to obtain a fraction enriched in diphenylether cleaving activity by a heat treatment procedure. This fraction could cleave methoxy groups from an arylether containing molecule, vanillin, to produce vanillyl alcohol and triphenylphosphate. Degradation of lignin in a continuous culture was also demonstrated.