Arthrobacter aurescens strain TC1 was isolated without enrichment by plating atrazine-contaminated soil directly onto atrazine-clearing plates. A. aurescens TC1 grew in liquid medium with atrazine as the sole source of nitrogen, carbon, and energy, consuming up to 3,000 mg of atrazine per liter. A. aurescens TC1 is metabolically diverse and grew on a wider range of s-triazine compounds than any bacterium previously characterized. The 23 s-triazine substrates serving as the sole nitrogen source included the herbicides ametryn, atratone, cyanazine, prometryn, and simazine. Moreover, atrazine substrate analogs containing fluorine, mercaptan, and cyano groups in place of the chlorine substituent were also growth substrates. Analogs containing hydrogen, azido, and amino functionalities in place of chlorine were not growth substrates. A. aurescens TC1 also metabolized compounds containing chlorine plus N-ethyl, N-propyl, N-butyl, N-s-butyl, N-isobutyl, or N-t-butyl substituents on the s-triazine ring. Atrazine was metabolized to alkylamines and cyanuric acid, the latter accumulating stoichiometrically. Ethylamine and isopropylamine each served as the source of carbon and nitrogen for growth. PCR experiments identified genes with high sequence identity to atzB and atzC, but not to atzA, from Pseudomonas sp. strain ADP.s-Triazine rings are common scaffolds for the synthesis of industrial chemicals; they are found in pesticides, plastic resins, dyes, and explosives (20). s-Triazine herbicides are widely used in modern agriculture, where they kill susceptible plants by coordinating to the quinone-binding protein in photosystem II, thereby inhibiting photosynthetic electron transfer (19). Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-s-triazine) is one of the most widely used herbicides in the United States for the control of broadleaf weeds in corn, sorghum, and sugarcane (1). Other s-triazine herbicides include ametryn, atratone, cyanazine, prometryn, and simazine.The fate of s-triazine compounds in the environment depends on the metabolic activities of soil microorganisms (7,14). Since 1994, a number of laboratories have independently isolated, by enrichment culture, several genera of gram-negative bacteria capable of atrazine dechlorination (4,16,22,31,33,35,36,38). Subsequent to dechlorination, metabolism of hydroxyatrazine liberates nitrogen to sustain bacterial growth. More recently, gram-positive bacteria from the genera Nocardioides (32) and Arthrobacter (22) have been shown to grow on atrazine, the former using atrazine as the sole source of carbon and nitrogen for growth. Streptomyces strain PS1/5 was also shown to metabolize several s-triazine herbicides in the presence of additional carbon and nitrogen in the growth medium (29).It is now established that bacteria metabolize melamine and the triazine herbicides such as atrazine via enzyme-catalyzed hydrolytic reactions (6,11,12,35). The enzymatic basis of atrazine mineralization has been most extensively studied in Pseudomonas sp. strain ADP (3,8,16,17...

Hydraulic fracturing is a method of oil and gas extraction from shale in which substantial volumes of water return to the surface containing chemicals and microorganisms. This paper begins to address the microbial composition and aqueous chemistry and the potential for intrinsic and enhanced bioremediation of these waters. The waters from a gas and oil shale in the Marcellus and Bakken regions, respectively, were analyzed for inorganic elements, organic chemicals, microbial taxonomic composition, and biodegradative capabilities. The waters were highly saline, reaching NaCl concentrations up to 3.5 N, but no significant levels of radioactive elements were detected. More than 1,000 organic compounds were separated and identified by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. The major classes of organic compounds, in order of decreasing abundance, were aliphatics, cycloaliphatics, single-ring aromatics, and polycyclic aromatic compounds. The bacterial genera found natively in the waters were identified by sequencing the 16S rRNA genes within the extracted DNA. The major genera identified included strains known to thrive under saline conditions (Halanaerobium, Marinobacter, Oceanimonas, Streptohalobacillus) and degrade petroleum hydrocarbons (Thauera, Pseudomonas, Marinobacterium, Williamsia, Colwellia). Microbial populations were extracted from the Bakken shale waters, encapsulated within silica gels, and then reintroduced into their waters of origin. Both intrinsic biodegradation from the free native microorganisms and enhanced biodegradation with the addition of encapsulated bacteria were observed. In total, this paper begins to better define the properties of waters derived from hydraulic fracturing and suggests a potential for the application of bioremediation to remove organic contaminants.

We performed the first field-scale atrazine remediation study in the United States using chemically killed, recombinant organisms. This field study compared biostimulation methods for enhancing atrazine degradation with a novel bioaugmentation protocol using a killed and stabilized whole-cell suspension of recombinant Escherichia coli engineered to overproduce atrazine chlorohyrolase, AtzA. AtzA dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine. Soil contaminated by an accidental spill of atrazine (up to 29,000 p.p.m.) supported significant populations of indigenous microorganisms capable of atrazine catabolism. Laboratory experiments indicated that supplementing soil with carbon inhibited atrazine biodegradation, but inorganic phosphate stimulated atrazine biodegradation. A subsequent field-scale study consisting of nine (0.75m3) treatment plots was designed to test four treatment protocols in triplicate. Control plots contained moistened soil; biostimulation plots received 300p.p.m. phosphate; bioaugmentation plots received 0.5% (w/w) killed, recombinant E. coli cells encapsulating AtzA; and combination plots received phosphate plus the enzyme-containing cells. After 8 weeks, atrazine levels declined 52% in plots containing killed recombinant E. coli cells, and 77% in combination plots. In contrast, atrazine levels in control and biostimulation plots did not decline significantly. These data indicate that genetically engineered bacteria overexpressing catabolic genes significantly increased degradation in this soil heavily contaminated with atrazine.

TrzN, the broad-specificity triazine hydrolase from Arthrobacter and Nocardioides spp., is reportedly in the amidohydrolase superfamily of metalloenzymes, but previous studies suggested that a metal was not required for activity. To help resolve that conundrum, a double chaperone expression system was used to produce