A carbazole-utilizing bacterium was isolated by enrichment from petroleum-contaminated soil. The isolate,designated Sphingomonas sp. strain XLDN2-5, could utilize carbazole (CA) as the sole source of carbon,nitrogen, and energy. Washed cells of strain XLDN2-5 were shown to be capable of degrading dibenzofuran(DBF) and dibenzothiophene (DBT). Examination of metabolites suggested that XLDN2-5 degraded DBF to2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienic acid and subsequently to salicylic acid through the angulardioxygenation pathway. In contrast to DBF, strain XLDN2-5 could transform DBT through the ringcleavage and sulfoxidation pathways. Sphingomonas sp. strain XLDN2-5 could cometabolically degrade DBFand DBT in the growing system using CA as a substrate. After 40 h of incubation, 90% of DBT was transformed,and CA and DBF were completely removed. These results suggested that strain XLDN2-5 might be useful inthe bioremediation of environments contaminated by these compounds.

Previous research suggested that Pseudomonas spp. may attack the pyrrolidine ring of nicotine in a waysimilar to mammalian metabolism, resulting in the formation of pseudooxynicotine, the direct precursor of apotent tobacco-specific lung carcinogen. In addition, the subsequent intermediates, 6-hydroxy-3-succinoylpyridine(HSP) and 2,5-dihydroxypyridine (DHP) in the Pseudomonas nicotine degradation pathway are twoimportant precursors for drug syntheses. However, there is little information on the molecular mechanism fornicotine degradation via the pyrrolidine pathway until now. In this study we cloned and sequenced a 4,879-bpgene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine,HSP, and DHP were identified from resting cell reactions of the transformant containing the genecluster and shown to be identical to those of the pyrrolidine pathway reported in wild-type strain Pseudomonasputida S16. The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directlyto DHP was cloned, sequenced, and expressed in Escherichia coli, and the purified HSP hydroxylase (38 kDa)is NADH dependent. DNA sequence analysis of this 936-bp fragment reveals that the deduced amino acidshows no similarity with any protein of known function.

A new technology for 6-hydroxy-3-succinoyl-pyridine(HSP) production from (S)-nicotine in tobacco waste bywhole cells of a Pseudomonas sp. has been developed.Whendeionized water was used in the transformation reactionas a medium and the initial pH value of reaction mixturewas adjusted to 7.0, 1.45 g/L HSP was produced from 3 g/Lof nicotine in 5 h with 3.4 g/L of cells in a 5-L flask at 30°C. HSP could be easily purified from the reaction withoutperplexing separation steps. A quantity of 1.3 g of HSPwas recovered without impurity, and the overall yield of HSPwas 43.8% (w/w), based on an initial concentration of3.0 g/L of nicotine in reaction. This biotransformation madeit possible to convert nicotine in tobacco wastes withhigh nicotine content into valuable compounds.

Ethanol concentration and fermentation productivity using Saccharomyces cerevisiae were substantially increased in shake flask cultures with a normal inoculum by combining 3 methods: (a) by making nutrient additions to the standard medium for ethanol production, (b) by immobilizing the cells in alginate beads and (c) by using a glucose step-feeding batch process. Ethanol concentration by free yeast was improved from 5.9% (w/w) to 9.6% (w/w) when a further 0.8% yeast extract and 1% animal peptone were added to the standard 30% (w/v) glucose nutrient medium. This was further increased to 12.8% (w/w) by using alginate immobilized yeast. The ethanol concentration was increased again, to 15.0% (w/w) by using the glucose step-feeding batch process.