Transformation of atrazine to hydroxyatrazine in the environment may be underestimated by current assessment schemes since immobilization and further transformation of the metabolite can render parent-to-daughter compound ratios unreliable. This study reports significant C and N isotope fractionation of atrazine in transformation to hydroxyatrazine by Chelatobacter heintzii, Pseudomonas sp. ADP, and Arthrobacter aurescens TC1 highlighting an alternative approach to detecting this natural transformation pathway. Indistinguishable dual isotope slopes big up tri, open (= delta(15)N/delta(13)C approximately epsilon(N)/epsilon(C)) for Chelatobacter heintzii (-0.65 +/- 0.08) and Arthrobacter aurescens TC1 (-0.61 +/- 0.02) suggest the same biochemical transformation mechanism despite different hydrolyzing enzymes (AtzA versus TrzN). With Pseudomonas sp. ADP (also AtzA) significantly smaller fractionation indicates masking effects by steps prior to enzyme catalysis, while a distinguishable big up tri, open = -0.32 +/- 0.06 suggests that some of these steps showed slight isotope fractionation. Abiotic reference experiments reproduced the pattern of biotic transformation at pH 3 (enrichment of (13)C, depletion of (15)N in atrazine), but showed enrichment of both (13)C and (15)N at pH 12. This indicates that the organisms activated atrazine by a similar Lewis acid complexation (e.g., with H(+)) prior to nucleophilic aromatic substitution, giving the first detailed mechanistic insight into this important enzymatic reaction.

Methane is a major product of anaerobic degradation of organic matter and an important greenhouse gas. Its stable carbon isotope composition can be used to reveal active methanogenic pathways, if associated isotope fractionation factors are known. To clarify the causes that lead to the wide variation of fractionation factors of methanogenesis from H 2 plus CO 2 (a CO2ÀCH4 ), pure cultures and various cocultures were grown under different thermodynamic conditions. In syntrophic and obligate syntrophic cocultures thriving on different carbohydrate substrates, fermentative bacteria were coupled to three different species of hydrogenotrophic methanogens of the families Methanobacteriaceae and Methanomicrobiaceae. We found that C-isotope fractionation was correlated to the Gibbs free energy change (DG) of CH 4 formation from H 2 plus CO 2 and that the relation can be described by a semi-Gauss curve. The derived relationship was used to quantify the average DG that is available to hydrogenotrophic methanogenic archaea in their habitat, thus avoiding the problems encountered with measurement of low H 2 concentrations on a microscale. Boreal peat, rice field soil, and rumen fluid, which represent major sources of atmospheric CH 4 , exhibited increasingly smaller a CO2ÀCH4 , indicating that thermodynamic conditions for hydrogenotrophic methanogens became increasingly more favourable. Vice versa, we hypothesize that environments with similar energetic conditions will also exhibit similar isotope fractionation. Our results, thus, provide a mechanistic constraint for modelling the 13 C flux from microbial sources of atmospheric CH 4 .

Compound-specific stable isotope analysis by gas chromatography-isotope ratio mass spectrometry (GC-IRMS) is increasingly used to assess origin and fate of organic substances in the environment. Although analysis without isotopic discrimination is essential, it cannot be taken for granted for new target compounds. We developed and validated carbon isotope analysis of atrazine, a herbicide widely used in agriculture. Combustion was tested with reactors containing (i) CuO/NiO/Pt operating at 940 degrees C; (ii) CuO operating at 800 degrees C; (iii) Ni/NiO operating at 1150 degrees C and being reoxidized for 2 min during each gas chromatographic run. Accurate and precise carbon isotope measurements were only obtained with Ni/NiO reactors giving a mean deviation delta delta(13)C from dual inlet measurements of -0.1-0.2% per hundred and a standard deviation (SD) of +/- 0.4% per hundred. CuO at 800 degrees C gave precise, but inaccurate values (delta delta(13)C = -1.3% per hundred, SD +/- 0.4% per hundred), whereas CuO/NiO/Pt reactors at 940 degrees C gave inaccurate and imprecise data. Accurate (delta delta(15)N = 0.2% per hundred) and precise (SD +/- 0.3% per hundred) nitrogen isotope analysis was accomplished with a Ni/NiO-reactor previously used for carbon isotope analysis. The applicability of the method was demonstrated for alkaline hydrolysis of atrazine at 20 degrees C and pH 12 (nucleophilic aromatic substitution) giving epsilon(carbon) = -5.6% per hundred +/- 0.1% per hundred (SD) and epsilon(nitrogen) = -1.2% per hundred +/- 0.1% per hundred (SD).