To understand the phylogeny and taxonomy of eight new toluene-degrading denitrifying isolates, we performed a 16s rRNA sequence analysis and a gas chromatographic analysis of their cellular fatty acids and examined some of their biochemical and physiological features. These isolates had 165 rRNA sequence signatures identical to those of members of the beta subclass of the Proteobacteria. The levels of similarity were as follows: 97.9 to 99.9% among the new isolates; 91.2 to 92.4% between the new isolates and Azoarcus sp. strain S5b2; 95.3 to 96.2% between the new isolates and Azoarcus sp. strain BH72; and 94.8 to 95.3% between the new isolates and Azoarcus indigens VB32T (T = type strain). Phylogenetic trees constructed by using the distance matrix, maximum-parsimony, and maximum-likelihood methods showed that our eight denitrifying isolates form a phylogenetically coherent cluster which represents a sister lineage of the previously described Azoarcus species. Furthermore, the fatty acid profiles, the cell morphology, and several physiological and nutritional characteristics of the eight isolates and the previously described members of the genus Azoarcus were also similar. In contrast to the previously described members of the genus Azoarcus, the eight new isolates were capable of degrading toluene under denitrifying conditions. We concluded that these toluene-degrading denitrifiers are members of a new species of the novel nitrogen-fixing genus Azoarcus. We propose the name Azoarcus toZuZyticus for these toluene-degrading denitrifying isolates and designate strain To14 the type strain.

Avaliou-se a produção de matéria seca (MS), bem como as concentrações de nitrogênio, fósforo, potássio e carbono do tecido vegetal. O cultivo consorciado de plantas de cobertura proporcionou produção de matéria seca estatisticamente semelhante àquela da aveia e do nabo em culturas isoladas e superior à da ervilhaca. O acúmulo de N na fitomassa dos tratamentos envolvendo consórcio de aveia + ervilhaca não diferiu daquele da ervilhaca isolada e, na média dos três anos, foi superior ao da aveia isolada em 32 kg ha -1 de N. Consorciando aveia + ervilhaca, houve um aumento médio de 67 % na relação C/N da fitomassa, em relação à ervilhaca. As plantas de cobertura proporcionaram maior produção de MS e foram mais eficientes no acúmulo de N, P e K do que a vegetação espontânea do pousio invernal. Os resultados indicaram que o consórcio de aveia + ervilhaca e de aveia + nabo no outono/inverno proporcionou maior

Enrichments capable of toluene degradation under 02-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from different geographic regions of the world. Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm. From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different. Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene. meta-Xylene was the only other substrate used anaerobically and was used by only one isolate. All isolates were motile gram-negative rods, produced N2 from denitrification, and did not hydrolyze starch. All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes. All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe. Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp. Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus. The activity and isolation of at least one toluene-degrading denitrifier from the majority of the habitat types studied suggest that microbes with the capacity to grow anaerobically on toluene are common in nature.

A copper-containing nitrite reductase gene (nirU) from Pseudomonas sp. strain G-179 was found in a 1.9-kb EcoRI-BamHI DNA fragment. The coding region contained information for a polypeptide of 379 amino acids. The encoded protein had 78% identity in amino acid sequence to the nitrite reductase purified from Achromobacter cycloclastes. The ligands for type 1 copper-and type 2 copper-binding sites found in A. cycloclastes were also found in Pseudomonas sp. strain G-179, suggesting that these binding sites are conserved. Upstream from the promoter, two putativeJfr boxes were found, suggesting that an FNR-like protein may be involved in regulation of the nitrite reductase gene under anaerobic conditions. When the 1.9-kb clone was used to probe Southern blots for similar sequences in DNAs from different denitrifiers, hybridization bands were seen for 15 of 16 denitrifiers known to have nitrite reductases containing copper. Except for Pseudomonas stutzeri JM300, all denitrifiers tested that have nitrite reductases containing heme c,d, showed no or weak hybridization to this probe. Thus, this structural gene may be useful as a probe to detect denitrifiers with copper-containing nitrite reductases.

We characterized the bacterial populations that grew in a Moffett Field, Calif., aquifer following three sequential field tests of phenol-or toluene-driven cometabolism of trichloroethene (TCE). Reducing the toluene and phenol concentrations in most-probable-number (MPN) tubes from 50 to 5 ppm increased the population density measured for these degraders by 1.5 and 1 log units, respectively, suggesting that natural populations might be quite sensitive to these substrates. Phenol and toluene degraders were isolated from the terminal MPN dilution tubes; 63 genetically distinct strains were identified among the 273 phenol-and toluenedegrading isolates obtained. TCE was cometabolized by 60% of the genetically distinct strains. Most strains (57%) grew on both phenol and toluene, and 78% of these strains hybridized to the toluene ortho-monooxygenase (TOM) probe. None of the strains hybridized to probes from the four other toluene oxygenase pathways. Gram-positive strains comprised 30% of the collection; all of these grew on phenol, and 47% of them also grew on toluene, but none hybridized to the TOM probe. Among the gram-negative strains, 86% of those that grew on both toluene and phenol hybridized to the TOM probe, while only 5% of those that were TOM-positive grew on toluene alone. A larger proportion of TCE degraders was found among gram-negative than gram-positive strains and among organisms that grew on phenol than those that grew on toluene. Hybridization of strains to the TOM probe was somewhat predictive of their TCE-cometabolizing ability, especially for strains isolated on toluene, but there was also a significant number (20%) of strains that hybridized to the TOM probe but were poor TCE cooxidizers. No Moffett Field isolates were as effective as Burkholderia cepacia G4 in cooxidizing TCE. Most of the aquifer strains ranged from moderately effective to ineffective in TCE cooxidation. Such populations, however, apparently accounted for the successful phenol-and toluene-stimulated TCE removal that occurred during the field assessment of this remediation process. This suggests that naturally occurring communities of only moderate TCE-cooxidizing ability may support successful TCE bioremediation as long as the phenol or toluene present is not limiting. This activity, however, may not be sustainable for the long term, because TCE-inactive populations that consumed toluene at rates equal to that of the best TCE degraders were present and hence would be expected to eventually dominate the community.