First evaluation of the effect of microorganisms on steady state hydroxyl radical concentrations in atmospheric waters.

Clouds are complex multiphasic media where efficient chemical reactions take place and where microorganisms have been found to be metabolically active. Hydroxyl radical is the main oxidant in cloud water, and more generally in the atmosphere, during the day and drives the cloud oxidative capacity. However, only one measurement of the steady state hydroxyl radical concentrations in cloud water has been reported so far. Cloud chemistry models are used to estimate the hydroxyl radical concentrations with values ranging from 10-12 to 10-15 M that are surely overestimated due to a lack of knowledge about the speciation of the organic matter acting as a sink for hydroxyl radicals. The aim of this work is to quantify the concentration of hydroxyl radicals at steady state in rain and cloud waters and to measure the impact of native microflora on this concentration. First, the non-toxicity of terephthalic acid as probe is controlled before the analysis in real atmospheric water samples. Higher concentrations of hydroxyl radicals are found in cloud waters than in rain waters, with a mean value "1.6 ±â€¯1.5" × 10-16 M and "7.2 ±â€¯5.0" × 10-16 M for rain and cloud waters respectively and no real impact of microorganisms was observed. This method allows the measurement of steady state hydroxyl radical levels at very low concentrations (down to 10-17 M) and it is biocompatible, fast and easy to handle. It is a useful tool, complementary to other methods, to give a better overview of atmospheric water oxidant capacity.

Detection of semi-volatile compounds in cloud waters by GC×GC-TOF-MS. Evidence of phenols and phthalates as priority pollutants.

Although organic species are transported and efficiently transformed in clouds, more than 60% of this organic matter remains unspeciated. Using GCxGC-HRMS technique we were able to detect and identify over 100 semi-volatile compounds in 3 cloud samples collected at the PUY station (puy de Dôme mountain, France) while they were present at low concentrations in a very small sample volume (<25 mL of cloud water). The vast majority (∼90%) of the detected compounds was oxygenated, while the absence of halogenated organic compounds should be specially mentioned. This could reflect both the oxidation processes in the atmosphere (gas and water phase) but also the need of the compounds to be soluble enough to be transferred and dissolved in the cloud droplets. Furans, esters, ketones, amides and pyridines represent the major classes of compounds demonstrating a large variety of potential pollutants. Beside these compounds, priority pollutants from the US EPA list were identified and quantified. We found phenols (phenol, benzyl alcohol, p-cresole, 4-ethylphenol, 3,4-dimethylphenol, 4-nitrophenol) and dialkylphthalates (dimethylphthalate, diethylphthalate, di-n-butylphthalate, bis-(2-ethylhexyl)-phthalate, butylbenzylphthalate, di-n-octyl phthalate). In general, the concentrations of phthalates (from 0.09 to 52 µg L-1) were much higher than those of phenols (from 0.03 to 0.74 µg L-1). To our knowledge phthalates in clouds are described here for the first time. We investigated the variability of phenols and phthalates concentrations with cloud air mass origins (marine vs continental) and seasons (winter vs summer). Although both factors seem to have an influence, it is difficult to deduce general trends; further work should be conducted on large series of cloud samples collected in different geographic areas and at different seasons.

Environmental scenarii for the degradation of oxo-polymers.

The fate of oxo-polymers in nature is strongly dependent on environmental conditions, mainly on the intensity and duration of sunshine, which vary with the season and the climate. In this work, we report the effect of different scenarii on the production and the molecular composition of oligomers released from oxo-biodegradable HDPE films. Under our experimental conditions, the duration of accelerated weathering corresponded to a period of 3 months to 3 years of exposure to outside conditions under temperate climate. In addition, the oligomers were extracted in three different solvents: i) water to mimics the natural environment; ii) acetone and chloroform to identify oligomers trapped in the polymer matrix. The combination of high-resolution mass spectrometry and 1H NMR spectroscopy gives an extensive picture of the relative concentrations and the structural compositions of the extracted oligomers in the different tested conditions. In particular, the masses, the number of oxygen and carbon atoms could be determined for up to 2283 molecules. Globally the concentration and the size of oligomers increased with the duration of extraction, the level of aging of the polymer and the use of non-polar solvents. Surprisingly, the presence of highly oxidized molecules in acetone and chloroform extract, suggested an important swelling of HPDE films in these solvents and a better diffusion of these oligomers in the matrix. In nature, the biodegradability of oligomers could result from processes occurring both at the molecular (oxidation) and the macromolecular (diffusion and release) levels.

Draft Genome Sequence of Rhodococcus enclensis 23b-28, a Model Strain Isolated from Cloud Water.

The whole genome of Rhodococcus enclensis 23b-28, a bacterial strain isolated from cloud water, was sequenced. This microorganism is equipped with genes able to degrade aromatic compounds and could thus play a role in complex organic matter decomposition in cloud water.

Draft Genome Sequence of Pseudomonas graminis PDD-13b-3, a Model Strain Isolated from Cloud Water.

The whole genome of Pseudomonas graminis PDD-13b-3, a strain of bacteria isolated from cloud water, was sequenced. This showed that this microorganism is equipped with genes that could potentially be involved in its survival in the atmosphere and clouds: those for oxidative stress and carbon starvation responses, DNA repair, and iron uptake.

Draft Genome Sequence of Pseudomonas syringae PDD-32b-74, a Model Strain for Ice-Nucleation Studies in the Atmosphere.

We report here the whole genome sequence of Pseudomonas syringae PDD-32b-74, a gammaproteobacterium isolated from cloud water. This microorganism is equipped with ice-nucleation protein and biosurfactant genes that could potentially be involved in physicochemical processes in the atmosphere and clouds.

Characterization of oxidized oligomers from polyethylene films by mass spectrometry and NMR spectroscopy before and after biodegradation by a Rhodococcus rhodochrous strain.

The objective of this work was to develop a new approach to assess the specificity and the efficiency of biodegradation of oxidized oligomers extracted from aged HDPE polyethylene films and to bring insight on the mechanisms occurring during biodegradation. 1H NMR spectroscopy and LC Orbitrap™ mass spectrometry were combined together with data processing using Kendrick mass defect calculation and Van Krevelen Diagram. We showed that the molecular weight of extracted oligomers was lower than 850 Da with maximum chain length of 55 carbon atoms. The oligomers were divided into 11 classes of molecules with different oxidation state ranging from 0 to 10. All classes included series of chemically related compounds including up to 19 molecules. 95% of the soluble oligomers were assimilated by a strain of Rhodococcus rhodocchrous after 240 days of incubation. Large highly oxidized molecules completely disappeared while the other classes of molecules were still represented. Molecules containing 0-1 oxygen atom were less degraded. A strong shift to smaller molecules (<450 Da, 25 carbon atoms) was observed suggesting that longer molecules disappeared more rapidly than the smaller ones. It opens new perspectives on biodegradation processes as not only intracellular ß-oxidation must be considered but also extracellular mechanisms leading to chain cleavages.

In vivo 31P and 13C NMR investigations of Rhodococcus rhodochrous metabolism and behaviour during biotransformation processes.

AIMS: The strain Rhodococcus rhodochrous OBT18 was isolated from a water treatment plant used to decontaminate industrial effluents containing benzothiazole derivatives. Aims of the work are to study the central metabolism of this strain and more specifically its behaviour during biodegradation of 2-aminobenzothiazole. METHODS AND RESULTS: In vivo(13)C and (31)P NMR experiments showed that this strain contains storage compounds such as polyphosphates, glycogen and trehalose and produces biosurfactants containing trehalose as sugar unit. Trehalose can be synthesized after reversion of the glycolytic pathway. In vivo(31)P NMR experiments showed that energy metabolism markers such as the intracellular pH and the ATP concentration did not change during biotransformation processes when R. rhodochrous was exposed to potentially toxic compounds including iron complexes and (* )OH radicals. Also R. rhodochrous recovers the normal values of ATP and pH after anoxia/reoxygenation cycle very quickly. CONCLUSIONS: Rhodococcus rhodochrous carbon and energy metabolism is well adapted to different stresses and consequently to live in the environment where conditions are constantly changing. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to understand the behaviour of this bacterium in natural environments but also in water treatment plants where iron and UV light are present.

First isolation and characterization of a bacterial strain that biotransforms the herbicide mesotrione.

AIM: The aim of this study was to find and characterize a fungal or bacterial strain capable of metabolizing mesotrione, a new selective herbicide for control of broad-leaved weeds in maize. METHODS AND RESULTS: This strain was isolated from cloud water and showed close phylogenetic relationship with strains belonging to the Bacillus genus, based on 16S rRNA gene alignment. Kinetics of mesotrione degradation were monitored by high-performance liquid chromatography and in situ(1)H nuclear magnetic resonance spectroscopy at different concentrations. Mesotrione was completely biotransformed even at 5 mmol l(-1) concentration. 2-Amino-4-methylsulfonyl benzoic acid (AMBA) was identified as one of the metabolites, but was not the major one. CONCLUSIONS: This study reports the first rapid mesotrione biotransformation by a pure bacterial strain and the formation of several metabolites including AMBA. SIGNIFICANCE AND IMPACT OF THE STUDY: This bacterium isolated from cloud water is the first pure strain capable of rapidly degrading mesotrione.

Degradation of wheat straw by Fibrobacter succinogenes S85: a liquid- and solid-state nuclear magnetic resonance study.

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with 13C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of 13C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than alpha-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.

Identification of new derivatives of sinigrin and glucotropaeolin produced by the human digestive microflora using 1H NMR spectroscopy analysis of in vitro incubations.

One- and two-dimensional (1)H NMR spectroscopy were used to study the biotransformation of two dietary glucosinolates, sinigrin (SIN), and glucotropaeolin (GTL) by the human digestive microflora in vitro. The molecular structures of the new metabolites issued from the aglycone moiety of the glucosinolate were identified, and the modulation of carbon metabolism was studied by quantifying bacterial metabolites issued from the xenobiotic incubation in the presence or absence of a source of free glucose. Unambiguously and for the first time, it was shown that SIN and GTL were transformed quantitatively into allylamine and benzylamine, respectively. The comparison of the kinetics of transformation of SIN and GTL with and without glucose clearly showed that the presence of glucose did not modify either the nature of the metabolites or the rate of transformation of the glucosinolates (complete degradation within 30 h). The main end products of the glucose moiety of glucosinolates were characteristic of anaerobic carbon metabolism in the digestive tract (acetate, lactate, ethanol, propionate, formate, and butyrate) and similar to those released from free glucose. This work represents the first application of (1)H NMR spectroscopy to the study of xenobiotic metabolism by the human digestive microflora, demonstrating allyl- and benzylamine production from glucosinolates. Whether these amines are produced in vivo from dietary glucosinolates remains to be established. This would reduce the availability of other glucosinolate metabolites, notably cancer-protective isothiocyanates.

In situ 1H NMR study of the biodegradation of xenobiotics: application to heterocyclic compounds.

In vivo or in situ nuclear magnetic resonance (NMR) offers a powerful tool to study the degradation of xenobiotics by microorganisms. Most studies reported are based on the use of heteronuclei, and experiments with xenobiotics have been limited because specifically labeled xenobiotics are not commercially available, with the exception of 19F and 31P. wn>1H NMR is, thus, of great interest in this area. To avoid problems caused by the presence of water and intrinsic metabolite signals, some studies were performed using a deuterated medium or specific detection of protons linked to the 13C-15N enriched pattern. We report here the application of in situ 1H NMR, performed directly on culture media, to study the metabolism of heterocyclic compounds. In this review, we show that a common pathway is involved in the biodegradation of morpholine, piperidine, and thiomorpholine by Mycobacterium aurum MO1 and Mycobacterium sp. RP1. In all cases, the first step is the cleavage of the C-N bond, which results in an amino acid. Thiomorpholine is first oxidized to sulfoxide before the opening of the ring. The second step is the deamination of the intermediate amino acid, which leads to the formation of a diacid. We have shown that the cleavage of the C-N bond and the oxidation of thiomorpholine are initiated by reactions involving a cytochrome P450.

In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85.

Sodium gradients (DeltapNa) were measured in resting cells of Fibrobacter succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5- [thulium(III) 1,4,7,10-tetraazacyclododecane-N',N",N"'-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a DeltapNa of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).

Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy.

1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1-(13)C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was < or = 4. Both labelled (1-(13)C and 6-(13)C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.

In situ (1)H NMR study of the biodegradation of xenobiotics: application to heterocyclic compounds.

In vivo or in situ nuclear magnetic resonance (NMR) offers a powerful tool to study the degradation of xenobiotics by microorganisms. Most studies reported are based on the use of heteronuclei, and experiments with xenobiotics have been limited because specifically labeled xenobiotics are not commercially available, with the exception of (19)F and (31)P. (1)H NMR is, thus, of great interest in this area. To avoid problems caused by the presence of water and intrinsic metabolite signals, some studies were performed using a deuterated medium or specific detection of protons linked to the (13)C-(15)N enriched pattern. We report here the application of in situ (1)H NMR, performed directly on culture media, to study the metabolism of heterocyclic compounds. In this review, we show that a common pathway is involved in the biodegradation of morpholine, piperidine, and thiomorpholine by Mycobacterium aurum MO1 and Mycobacterium sp. RP1. In all cases, the first step is the cleavage of the C-N bond, which results in an amino acid. Thiomorpholine is first oxidized to sulfoxide before the opening of the ring. The second step is the deamination of the intermediate amino acid, which leads to the formation of a diacid. We have shown that the cleavage of the C-N bond and the oxidation of thiomorpholine are initiated by reactions involving a cytochrome P450.

Long-range (1)H-(15)N heteronuclear shift correlation at natural abundance: a tool to study benzothiazole biodegradation by two rhodococcus strains.

The biodegradation of benzothiazole and 2-hydroxybenzothiazole by two strains of Rhodococcus was monitored by reversed phase high-pressure liquid chromatography and by (1)H nuclear magnetic resonance (NMR). Both xenobiotics were biotransformed into a hydroxylated derivative of 2-hydroxybenzothiazole by these two strains. The chemical structure of this metabolite was determined by a new NMR methodology: long-range (1)H-(15)N heteronuclear shift correlation without any previous (15)N enrichment of the compound. This powerful NMR tool allowed us to assign the metabolite structure to 2,6-dihydroxybenzothiazole.

Differentiation of mobile and immobile pesticides on anionic clays by 1H HR MAS NMR spectroscopy.

Adsorbed vs. intercalated MCPA (4-chloro-2-methylphenoxyacetic acid) in highly hydrated clays taken as a soil model were clearly distinguished by 1H HR MAS NMR; adsorbed herbicide gave sharp signals indicating high mobility while intercalated herbicide gave very wide unresolved spectra due to its strong interaction with the solid matrix.

Common degradative pathways of morpholine, thiomorpholine, and piperidine by Mycobacterium aurum MO1: evidence from (1)H-nuclear magnetic resonance and ionspray mass spectrometry performed directly on the incubation medium.

In order to see if the biodegradative pathways for morpholine and thiomorpholine during degradation by Mycobacterium aurum MO1 could be generalized to other heterocyclic compounds, the degradation of piperidine by this strain was investigated by performing (1)H-nuclear magnetic resonance directly with the incubation medium. Ionspray mass spectrometry, performed without purification of the samples, was also used to confirm the structure of some metabolites during morpholine and thiomorpholine degradation. The results obtained with these two techniques suggested a general pathway for degradation of nitrogen heterocyclic compounds by M. aurum MO1. The first step of the degradative pathway is cleavage of the C---N bond; this leads formation of an intermediary amino acid, which is followed by deamination and oxidation of this amino acid into a diacid. Except in the case of thiodiglycolate obtained from thiomorpholine degradation, the dicarboxylates are completely mineralized by the bacterial cells. A comparison with previously published data showed that this pathway could be a general pathway for degradation by other strains of members of the genus Mycobacterium.

13C and 1H NMR study of cellulose metabolism by Fibrobacter succinogenes S85.

Fibrobacter succinogenes S85, a cellulolytic rumen bacterium, is very efficient in degrading lignocellulosic substrates and could be used to develop a biotechnological process for the treatment of wastes. In this work, the metabolism of cellulose by F. succinogenes S85 was investigated using in vivo 13C NMR and 13C-filtered spin-echo difference 1H NMR spectroscopy. The degradation of unlabelled cellulose synthesised by Acetobacter xylinum was studied indirectly, in the presence of [1-13C]glucose, by estimating the isotopic dilution of the final bacterial fermentation products (glycogen, succinate, acetate). During the pre-incubation period of F. succinogenes cells with cellulose fibres, some cells ('non-adherent') did not attach to the solid material. Results for 'adherent' cells showed that about one fourth of the glucose units entering F. succinogenes metabolism originated from cellulose degradation. A huge reversal of succinate metabolism pathway and production of large amounts of unlabelled acetate which was observed during incubation with glucose only, was found to be much decreased in the presence of solid substrate. The synthesis of glucose 6-phophate was slightly increased in the presence of cellulose. Results clearly showed that 'non-adherent' cells were able to metabolise glucose very efficiently; consequently the metabolic state of these cells was not responsible for their 'non-adherence' to cellulose fibre.

Interactions between carbon and nitrogen metabolism in Fibrobacter succinogenes S85: a 1H and 13C nuclear magnetic resonance and enzymatic study.

The effect of the presence of ammonia on [1-13C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by 13C and 1H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The 13C enrichment of succinate and acetate was precisely quantified by 13C-filtered spin-echo difference 1H-NMR spectroscopy. The presence of ammonia did not modify the 13C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the 13C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by 13C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and 1H NMR. When cells were incubated in vivo with [1-13C]glucose, only 13C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.