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The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic ␣ subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant ␣ subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase ␣ subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an ␣ subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.Aromatic hydrocarbons such as benzene, toluene, and ethylbenzene are common contaminants of soil and groundwater (41) and are listed as priority pollutants by the U.S. Environmental Protection Agency (http://www.epa.gov/waterscience /criteria/wqcriteria.html). One of the most attractive means to remove these compounds from polluted environments is through bioremediation, which can be achieved by natural attenuation, by stimulation of the local microbial activity through the addition of nutrients and/or electron acceptors, or by bioaugmentation (65).The aerobic degradation of aromatic compounds is frequently initiated by Rieske nonheme iron oxygenases, which catalyze the incorporation of two oxygen atoms into the aromatic ring to form arene cis-diols (30), followed by a dehydrogenation reaction catalyzed by a cis-dihydrodiol dehydrogenase to give catechol or substituted catechols which serve as substrates for oxygenolytic aromatic ring cleavage. Rieske nonheme iron oxygenases are multicomponent enzyme complexes composed of a terminal oxygenase component (iron-sulfur protein [ISP]) and different electron transport proteins (a ferredoxin and a reductase or a combined ferredoxin-NADH-reductase) (13). The cataly...

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Lab-scale and pilot-scale activated sludge bioreactors with integrated microfiltration membranes were operated over a period of up to three years. During the entire operation period no excess sludge was removed from the bioreactors apart from sampling, resulting in highly concentrated biomass in the reactors. The dry weight of the sludge ranged from 15 to 23 g MLSS l–1 for a plant fed with municipal wastewater and up to 60 g ll–1 for a lab-scale plant fed with high strength molasses. Stable biomass concentrations were reached at F/M ratios as low as approximately 0.07 kg COD (kg MLSS)–1 d–1. The degradation performance of the analyzed reactors was high and stable. Direct microscopical studies revealed high amounts of free suspended cells and at various times also high numbers of filamentous bacteria. Surprisingly only low numbers of protozoa were observed during most of the time. By use of fluorescent in situ hybridization (FISH) only about 40% to 50% of all bacteria emitted probe conferred fluorescence signals sufficient for detection, compared to around 80% cells detectable in conventional activated sludge. Studies on oxygen consumption rates indicated that the biomass in the bioreactor was substrate limited. These data suggest that substrate is mainly oxidized and not used for growth purposes which offers the possibility to operate membrane bioreactors with significantly reduced secondary sludge production.

Resin acids are tricyclic diterpenoids naturally synthesized by trees that are released from wood during pulping processes. Using a newly designed primer set, genes similar to that encoding the DitA1 catalytic alpha-subunit of the diterpenoid dioxygenase, a key enzyme in abietane resin acid degradation by Pseudomonas abietaniphila BKME-9, could be amplified from different Pseudomonas strains, whereas ditA1 gene sequence types representing distinct branches in the evolutionary tree were amplified from Burkholderia and Cupriavidus isolates. All isolates harbouring a ditA1-homologue were capable of growth on dehydroabietic acid as the sole source of carbon and energy and reverse transcription polymerase chain reaction analysis in three strains confirmed that ditA1 was expressed constitutively or in response to DhA, demonstrating its involvement in DhA-degradation. Evolutionary analyses indicate that gyrB (as a phylogenetic marker) and ditA1 genes have coevolved under purifying selection from their ancestral variants present in the most recent common ancestor of the genera Pseudomonas, Cupriavidus and Burkholderia. A polymerase chain reaction-single-strand conformation poylmorphism fingerprinting method was established to monitor the diversity of ditA1 genes in environmental samples. The molecular fingerprints indicated the presence ofa broad, previously unrecognized diversity of diterpenoid dioxygenase genes in soils, and suggest that other bacterial phyla may also harbour the genetic potential for DhA-degradation.