Chemostat approach for the directed evolution of biodesulfurization gain-of-function mutants.

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources.

Microbial desulfurization of alkylated dibenzothiophenes from a hydrodesulfurized middle distillate by Rhodococcus erythropolis I-19.

Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.

Development of a differential volume reactor system for soil biodegradation studies.

A bench scale experimental system was developed for the analysis of polycyclic aromatic hydrocarbon (PAH) degradation by mixed microbial cultures in PAH contaminated Manufactured Gas Plant (MGP) soils and on sand. The reactor system was chosen in order to provide a fundamental protocol capable for evaluating the performance of specific mixed microbial cultures on specific soil systems by elucidating the important system variables and their interactions. The reactor design and peripherals are described.

Dynamic response of naphthalene biodegradation in a continuous flow soil slurry reactor.

Periodic perturbations were used to evaluate the system stability and robustness of naphthalene biodegradation in a continuous flow stirred tank reactor (CSTR) containing a soil slurry. The experimental design involved perturbing the test system using a sinusoidal input either of naphthalene or non-naphthalene organic carbon at different frequencies during steady state operation of the reactors. The response of the test system was determined by using time series off-gas analysis for naphthalene liquid phase concentration and degradation, total viable cell counts, and gene probe analysis of naphthalene degradative genotype, and by batch mineralization assays.

Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation.

A bioluminescent reporter plasmid for naphthalene catabolism (pUTK21) was developed by transposon (Tn4431) insertion of the lux gene cassette from Vibrio fischeri into a naphthalene catabolic plasmid in Pseudomonas fluorescens. The insertion site of the lux transposon was the nahG gene encoding for salicylate hydroxylase. Luciferasemediated light production from P.