Evaluation of the influences of solution path length and additives concentrations on the solar photo-Fenton degradation of 4-chlorophenol using multivariate analysis

This study reports the photodegradation of 4-chlorophenol (4-CP) in aqueous solution by the photo-Fenton process using solar irradiation. The influence of solution path length, and Fe(NO3)(3) and H2O2 concentrations on the degradation of 4-CP is evaluated by response surface methodology. The degradation process was monitored by the removal of total organic carbon (TOC) and the release of chloride ion. The results showed a very important role of iron concentration either for TOC removal or dechlorination. on the other hand, a negative effect of increasing solution path length on mineralization was observed, which can be compensated by increasing the iron concentration. This permits an adjustment of the iron concentration according to the irradiation exposure area and path length (depth of a tank reactor). Under optimum conditions of 1.5 mM Fe(NO3)(3), 20.0 mM H2O2 and 4.5 cm solution path length, 17 min irradiation under solar light were sufficient to reduce a 72 mg C L-1 solution of 4-CP by 91 (c) 2006 Elsevier B.V. All rights reserved.

4-chlorophenol biodegradation by Arthrobacter chlorophenolicus A6

A microorganism was isolated which could grow on unusually high concentrations of the toxic pollutant 4-chlorophenol. Taxonomic studies showed that the microorganism constituted a novel species within the genus Arthrobacter and it was named Arthrobacter chlorophenolicus A6. A. chlorophenolicus A6 was chromosomally tagged with either the gfp gene, encoding the green fluorescent protein (GFP), or the luc gene, encoding firefly luciferase. When the tagged cells were inoculated into 4-chlorophenol contaminated soil they could completely remove 175 µg/g 4-chlorophenol within 10 days, whereas no loss of 4-chlorophenol was observed in the uninoculated control microcosms. During these experiments the gfp and luc marker genes allowed monitoring of cell number and metabolic status. When A. chlorophenolicus A6 was grown on mixtures of phenolic compounds, the strain exhibited a preference for 4-nitrophenol over 4-chlorophenol, which in turn was preferred over phenol. Analysis of growth and degradation data indicated that the same enzyme system was used for removal of 4-chlorophenol and 4-nitrophenol. However, degradation of unbstituted phenol appeared to be mediated by another or an additional enzyme system. The luc-tagged A. chlorophenolicus A6 gave valuable information about growth, substrate depletion and toxicity of the phenolic compounds in substrate mixtures. The 4-chlorophenol degradation pathway in A. chlorophenolicus A6 was elucidated. The metabolic intermediate subject to ring cleavage was found to be hydroxyquinol and two different pathway branches led from 4-chlorophenol to hydroxyquinol. A gene cluster involved in 4-chlorophenol degradation was cloned from A. chlorophenolicus A6. The cluster contained two functional hydroxyquinol 1,2-dioxygenase genes and a number of other open reading frames presumed to encode enzymes involved in 4-chlorophenol catabolism. Analysis of the DNA sequence suggested that the gene cluster had partly been assembled by horizontal gene transfer. In summary, 4-chlorophenol degradation by A. chlorophenolicus A6 was studied from a number of angles. This organism has several interesting and useful traits such as the ability to degrade high concentrations of 4-chlorophenol and other phenols alone and in mixtures, an unusual and effective 4-chlorophenol degradation pathway and demonstrated ability to remove 4-chlorophenol from contaminated soil.

A comparative study of the photophysics and photochemistry of 4-chlorophenol adsorbed on silicalite and beta-cyclodextrin

The photochemistry and photophysics of 4-chlorophenol (4-CP) were studied onto two model solid supports, silicalite and beta-cyclodextrin (beta-Cl)), using time resolved diffuse reflectance techniques and product degradation analysis. The results have shown that the photochemistry and photophysics of 4-CP are different from solution and depend on the solid. Ground state diffuse reflectance and time resolved luminescence demonstrated the inclusion of the probe in both substrates. 4-CP exhibits room temperature luminescence in both hosts, being structured and much more intense in beta-CD. The emission was assigned to phosphorescence of the inclusion complex. Transient absorption demonstrated the formation of the unsubstituted phenoxyl radical and of 4-chlorophenoxyl radical in beta-CD. In silicalite only the later was detected. The studies of the photodegradation products indicate that phenol is the main photoproduct in beta-CD. In silicalite the chromatographic analysis indicates the presence of products that involve the ring cleavage. (C) 2002 Elsevier Science B.V. All rights reserved.

Photochemistry of 4-chlorophenol on cellulose and silica

The photochemistry of 4-chlorophenol (4-CP) was studied on silica and cellulose, using time-resolved diffuse reflectance techniques and product degradation analysis. The results have shown that the photochemistry of 4-CP depends on the support, on the concentration, and also on the sample preparation method. Transient absorption and photoproduct results can be understood by assuming the formation of the carbene 4-oxocyclohexa-2,5-dienylidene in both supports. On cellulose, at concentrations lower than 10 mumol g(-1), the carbene leads to the unsubstituted phenoxyl radical, and phenol is the main degradation product. At higher concentrations a new transient resulting from phenoxyl radicals coupling was also observed, and dihydroxybiphenyls are also formed. The reaction of the carbene with ground-state 4-CP was also detected through the formation of 5-chloro-2,4'-dihydroxybiphenyl. 4-Chlorophenoxyl radical and degradations products resulting from its coupling were also detected. Oxygen has little effect on the photochemistry of 4-CP on cellulose. On silica the transient benzoquinone O-oxide was formed in the presence of oxygen. Benzoquinone and hydroquinone are the main degradation products. In well-dried samples the formation of hydroquinone is reduced. At higher concentrations the same products as detected on cellulose were observed. 4-CP undergoes slow photochemical decomposition under solar radiation in both supports. The same main degradation products were observed in these conditions.

Determinação voltamétrica de 4-clorofenol sobre o eletrodo de diamante dopado com boro utilizando a voltametria de onda quadrada

The anodic voltammetric behavior of 4-chlorophenol (4-CF) in aqueous solution has been studied on a Boron-doped diamond electrode using square wave voltammetry (SWV). After optimization of the experimental conditions, 4-CF was analyzed in pure and natural waters using a Britton-Robinson buffer with pH = 6.0 as the supporting electrolyte. Oxidation occurs at 0.80 V vs Ag/AgCl in a two-electron process controlled by adsorption of the species. The detection limits obtained were 6.4 µg L-1 in pure water and 21.5 µg L-1 for polluted water taken from a local creek, respectively. The combination of square wave voltammetry and diamond electrodes is an interesting and desirable alternative for analytical determinations.

TiO2-Photocatalyzed degradation of phenol in saline media in an annular reactor: hydrodynamics, lumped kinetics, intermediates, and acute toxicity

The photocatalytic degradation of phenol in aqueous suspensions of TiO2 under different salt concentrations in an annular reactor has been investigated. In all cases, complete removal of phenol and mineralization degrees above 90% were achieved. The reactor operational parameters were optimized and its hydrodynamics characterized in order to couple mass balance equations with kinetic ones. The photodegradation of the organics followed a Langmuir-Hinshelwood-Hougen-Watson lumped kinetics. From GC/MS analyses, several intermediates formed during oxidation have been identified. The main ones were catechol, hydroquinone, and 3-phenyl-2-propenal, in this order. The formation of negligible concentrations of 4-chlorophenol was observed only in high salinity medium. Acute toxicity was determined by using Artemia sp. as the test organism, which indicated that intermediate products were all less toxic than phenol and a significant abatement of the overall toxicity was accomplished, regardless of the salt concentration.

Electrooxidation of glyphosate herbicide at different DSA (R) compositions: pH, concentration and supporting electrolyte effect

This paper has investigated the electrochemical oxidation of glyphosate herbicide (GH) on RuO(2) and IrO(2) dimensionally stable anode (DSA (R)) electrodes. Electrolysis was achieved under galvanostatic control as a function of pH, GH concentration, supporting electrolyte, and current density. The influence of the oxide composition on GH degradation seems to be significant in the absence of chloride; Ti/Ir(0.30)Sn(0.70)O(2) is the best electrode material to oxidize GH. GH oxidation is favored at low pH values. The use of chloride medium increases the oxidizing power and the influence of the oxide composition is meaningless. At 30 mA cm(-2) and 4 h of electrolysis, complete GH removal from the electrolyzed solution has been obtained. In chloride medium, application of 50 mA cm(-2) leads to virtually total mineralization ( release of phosphate ions = 91%) for all the evaluated oxide materials. (C) 2008 Elsevier Ltd. All rights reserved.

Transcriptional profiling of Gram-positive Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant phenolic compounds.

Arthrobacter chlorophenolicus A6 is a Gram-positive, 4-chlorophenol-degrading soil bacterium that was recently shown to be an effective colonizer of plant leaf surfaces. The genetic basis for this phyllosphere competency is unknown. In this paper, we describe the genome-wide expression profile of A.chlorophenolicus on leaves of common bean (Phaseolus vulgaris) compared with growth on agar surfaces. In phyllosphere-grown cells, we found elevated expression of several genes known to contribute to epiphytic fitness, for example those involved in nutrient acquisition, attachment, stress response and horizontal gene transfer. A surprising result was the leaf-induced expression of a subset of the so-called cph genes for the degradation of 4-chlorophenol. This subset encodes the conversion of the phenolic compound hydroquinone to 3-oxoadipate, and was shown to be induced not only by 4-chlorophenol but also hydroquinone, its glycosylated derivative arbutin, and phenol. Small amounts of hydroquinone, but not arbutin or phenol, were detected in leaf surface washes of P.vulgaris by gas chromatography-mass spectrometry. Our findings illustrate the utility of genomics approaches for exploration and improved understanding of a microbial habitat. Also, they highlight the potential for phyllosphere-based priming of bacteria to stimulate pollutant degradation, which holds promise for the application of phylloremediation.

Pollution stress on aquatic microbial communities

Thousands of chemical compounds enter the natural environment but many have unknown effects and consequences, in particular at low concentrations. This thesis work contributes to our understanding of pollution effects by using bacteria as test organisms. Bacteria are important for this question because some of them degrade and transform pollutants into less harmful compounds, but secondly because they themselves can be inhibited in their reproduction by exposure to toxic compounds. When inhibitory effects occur this may change the composition of the microbial com¬munity in the long run, leading to altered or diminished ecosystem services by those communities. As a result chemicals of anthropogenic origin may accumulate and per¬sist in the environment, and finally, affect higher organisms as well. In addition to acquiring basic understanding of pollutant effects at low concentrations on bacterial communities an applied goal of this thesis work was to develop bacteria-based tests to screen new organic chemicals for toxicity and biodégradation. In the first part of this work we developed a flow cytometry-based assay on SYT09 plus ethidium-bromide or propidium-iodide stained cells of Pseudomonas ûuorescens exposed or not to a variety of pollutants under oligotrophic growth conditions. Flow cytometry (FC) allows fast and accurate counting of bacterial cells under simul¬taneous assessment of their physiological state, in particular in combination with different fluorescent dyes. Here we employed FC and fluorescent dyes to monitor the effect that pollutants may exert on Pseudomonas ûuorescens SV3. First we designed an oligotrophic growth test, which enabled us to follow population growth at low densities (104 - 10 7 cells per ml) using 0.1 mM sodium acetate as carbon source. Cells in the oligotrophic milieu were then exposed or not to a variety of common pollutants, such as 2-chlorobiphenyl (2CBP), naphthalene (NAH), 4-chlorophenol (4CP), tetradecane (TD), mercury chloride (HgCl2) or benzene, in different dosages. Exposed culture samples were stained with SYT09 (green fluorescent dye binding nucleic acids, generally staining all cells) in combination with propidium iodide (PI) or ethidium bromide (EB), both dyes being membrane integrity indicators. We ob- served that most of the tested compounds decreased population growth in a dosage- dependent manner. SYT09/PI or SYT09/EB staining then revealed that chemical exposure led to arisal of subpopulations of live and injured or dead cells. By modeling population growth on the total cell numbers in population or only the subpopulation of live cells we inferred that even in stressed populations live cells multiply at rates no different to unexposed controls. The net decrease in population growth would thus be a consequence of more and more cells being not able to multiply at all, rather than all cells multiplying at slower rates. In addition, the proportion of injured cells correlated to the compound dosage. We concluded that the oligotrophic test may be useful to asses toxicity of unknown chemicals on a variety of model bacteria. Mul¬tiple tests can be run in parallel and effects are rapidly measured within a period of 8 hours. Interestingly, in the same exposure tests with P. fluorescens SV3 we observed that some chemicals which did not lead to a reduction of net population growth rates did cause measurable effects on live cells. This was mainly observed in cells within the live subpopulation as an increase of the EB fluorescence signal. We showed that SYT09/EB is a more useful combination of dyes than SYT09/PI because PI fluorescence tend to increase only when cells are effectively dead, but not so much in live cells (less then twofold). In contrast, EB geometric mean fluorescence in live cells increased up to eightfold after exposure to toxic compounds. All compounds even at the lowest concentration caused a measurable increase in EB geometric mean fluorescence especially after 2 h incubation time. This effect was found to be transient for cells exposed to 2CBP and 4CP, but chronic for cells incubated with TD and NAH (ultimately leading to cell death). In order to understand the mechanism underlying the observed effects we used known membrane or energy uncouplers. The pattern of EB signal increase in chemical-exposed populations resembled mostly that of EDTA, although EB fluorescence in EDTA-treated or pasteurized cells was even higher than after exposure to the four test chemicals. We conclude that the ability of cells to efflux EB under equilibrium conditions is an appropriate measure for the potential of a chemical to exert toxicity. Since most bacterial species possess efflux systems for EB that all require cellular energy, our test should be more widely relevant to infer toxicity effects of chemical exposure on the physiological status of the bacterial cell. To better understand the effect of toxicant exposure on efflux defense systems, we studied 2-hydroxybiphenyl toxicity to Pseudomonas azeiaica HBP1. We showed that 2-HBP exerts toxicity even to P. azelaica HBP1, but only at concentrations higher than 0.5 mM. Above this concentration transient loss of membrane polarization and integrity occurred, which we conclude from staining of growing cells with fluorescent dyes. Cells finally recover and resume growth on 2HBP. The high resistance of P. azelaica HBP1 to 2-HBP was found to be the result of an efficient MexABOprM- type efflux pump system counteracting passive influx of this compound into the membrane and cellular interior. Mutants with disrupted mexA, mexB and oprM genes did no longer grow on 2-HBP at concentrations above 100 μ\4;, whereas below this concentration we found 2-HBP-concentration dependent decrease of growth rate. The MexAB-OprM system in P. azeiaica HBP1 is indeed an efflux pump for ethidium bromide as well. By introducing gfp reporter fusions responsive to intracellular 2- HBP concentrations into HBP1 wild-type or the mutants we demonstrated that 2HBP enters into the cells in a similar way. In contrast, the reporter system in the wild-type cells does not react to 2-HBP at an outside concentration of 2.4 μ\4;, whereas in mutant cells it does. This suggests that wild-type cells pump 2-HBP to the outside very effectively preventing accumulation of 2-HBP. 2HBP metabolism, therefore, is not efficient enough to lower the intracellular concentration and prevent toxicity. We conclude that P. azelaica HBP1 resistance to 2-HBP is mainly due to an efficient efflux system and that 2HBP in high concentrations exerts narcotic effects on the bacterial membrane. In the part of this thesis, we investigated the possibilities of bacteria to degrade pollutants at low concentrations (1 mg per L and below). As test components we used 2-hydroxybiphenyl, antibiotics and a variety of fragrances, many of which are known to be difficult to biodegrade. By using accurate counting of low numbers of bacterial cells we could demonstrate that specific growth on these compounds is possible. We demonstrated the accuracy of FC counting at low cell numbers (down to 103 bacterial cells per ml). Then we tested whether bacterial population growth could be specifically monitored at the expense of low substrate concentrations, us¬ing P. azelaica HBP1. A perfect relationship was found between growth rate, yield and 2-HBP concentrations in the range of 0.1 up to 5 mg per L. Mixing P. azelaica within sludge, however, suggested that growth yields in a mixed community can be much lower than in pure culture, perhaps because of loss of metabolic intermediates. We then isolated new strains from activated sludge using 2-HBP or antibiotics (Nal, AMP, SMX) at low concentrations (0.1-1 mg per L) as sole carbon and energy sub¬strate and PAO microdishes. The purified strains were then examined for growth on their respective substrate, which interestingly, showed that all strains can not with¬stand higher than 1 or 10 mg per L concentrations of target substrate. Thus, bacteria must exist that contribute to compound degradation at low pollutant concentrations but are inhibited at higher concentrations. Finally we tested whether specific biomass growth (in number of cells) at the expense of pollutants can also be detected with communities as starting material. Hereto, we focused on a number of fragrance chemicals and measured community biomass increase by flow cytometry cell counting on two distinct starter communities: (i) diluted Lake Geneva water, and dilute activated sludge from a wastewater treatment plant. We observed that most of the test compounds indeed resulted in significant biomass increase in the starter community compared to a no-carbon added control, but activated sludge and lake Geneva water strongly differed (almost mutually ex¬clusive) in their capacity to degrade the test chemicals. In two cases for activated sludge the same type of microbial community developed upon compound exposure, as concluded from transcription fragment length polymorphism analysis on community purified and PCR amplified 16S rRNA gene fragments. To properly test compound biodegradability it is thus important to use starter communities of different origin. We conclude that FC counting can be a valuable tool to screen chemicals for their biodegradability and toxicity. - Des milliers de produits chimiques sont libérés dans l'environnement mais beaucoup ont des effets inconnus, en particulier à basses concentrations. Ce travail de thèse contribue à notre comprehension des effets de la pollution en utilisant des bacteries comme des organismes-tests. Les bacteries sont importantes pour etudier cette ques¬tion car certaines d'entre elles peuvent degrader ou transformer les polluants, mais également parce qu'elles-mmes peuvent tre inhibees dans leur reproduction après avoit ete exposees à ces composes toxiques. Quand des effets inhibiteurs ont lieu, la composition de la communauté microbienne peut tre changee à long terme, ce qui mène à une reduction du service d'ecosystème offert par ces communautés. En consequence, après leur liberation dans l'environnement, les produits chimiques d'origine anthropogenique peuvent soit s'y accumuler et per¬sister, exerant ainsi des effets encore inconnus sur les organismes vivants. En plus d'acquérir des connaissances de base sur les effets des polluants à basses concentra¬tions sur les communautés microbiennes, un but applique de cette thèse était de développer des tests bases sur les bacteries afin d'identifier de nouveau composes pour leur toxicité ou leur biodégradation. Dans la première partie de ce travail, nous avons developpe un test base sur la cytometrie de flux (FC) sur des cellules de Pseudomonas fluorescens colorees par du bromure d'ethidium ou de l'iodure de propidium et exposees ou non à une palette de polluants sous des conditions de croissance oligotrophique. La cytometrie de flux est une technique qui connaît de nombreuses applications dans la microbiologie environ¬nementale. Cela est principalement du au fait qu'elle permet un comptage rapide et precis ainsi que l'évaluation de l'état physiologique, en particulier lorsqu'elle est combinée h des colorations fluorescentes. Ici, nous avons utilise la technique FC et des colorants fluorescents afin de mesurer l'effet que peuvent exercer certains pollu¬ants sur Pseudomonas ûuorescens SV3 . D'abord nous avons conu des tests oligo- trophiques qui nous permettent de suivre la croissance complète de cellules en culture h des densites faibles (104 -10 7 cellules par ml), sur de l'acetate de sodium à 0.1 mM, en presence ou absence de produits chimiques (2-chlorobiphenyl (2CBP), naphthalène (NAH), 4-chlorophenol (4CP), tetradecane (TD), chlorure de mercure(II) (HgCl2)) à différentes concentrations. Afin de montrer le devenir des bacteries tant au niveau de la cellule individuelle que celui de la population globale, après exposition à des series de composes chimiques, nous avons compte les cellules colorees avec du SYT09 (col¬orant fluorescent vert des acides nucléiques pour la discrimination des cellules par rapport au bruit de fond) en combinaison avec l'iodure de propidium (PI) ou le bromure d'ethidium (EB), indicateurs de l'intégrité de la membrane cellulaire avec FC. Nous avons observe que de nombreux composes testes avaient un effet sur la croissance bacterienne, resultant en une baisse du taux de reproduction de la pop¬ulation. En outre, la double coloration que nous avons utilisee dans cette etude SYT09/PI ou SYT09/EB a montre que les produits chimiques testes induisaient une reponse heterogène des cellules dans la population, divisant celle-ci en sous- populations "saine", "endommagee" ou "morte". Les nombres de cellules à partir du comptage et de la proportion de celles "saines" et "endommagees/mortes" ont ensuite ete utilises pour modeliser la croissance de P. ûuorescens SV3 exposee aux produits chimiques. La reduction nette dans la croissance de population est une consequence du fait que de plus en plus de cellules sont incapables de se reproduire, plutt que du fait d'une croissance plus lente de l'ensemble de la population. De plus, la proportion de cellules endommagees est correllee au dosage du compose chimique. Les résultats obtenus nous ont permis de conclure que le test oligotrophique que nous avons developpe peut tre utilise pour l'évaluation de la toxicité de produits chimiques sur différents modèles bacteriens. Des tests multiples peuvent tre lances en parallèle et les effets sont mesures en l'espace de huit heures. Par ailleurs, nous en déduisons que les produits chimiques exercént un effet sur la croissance des cellules de P. ûuorescens SV3, qui est heterogène parmi les cellules dans la population et depend du produit chimique. Il est intéressant de noter que dans les mmes tests d'exposition avec P. ûuorescens SV3, nous avons observe que certains composes qui n'ont pas conduit à une reduction du taux de la croissance nette de la population, ont cause des effets mesurables sur les cellule saines. Ceci a ete essentiellement observe dans la portion "saine" des cellules en tant qu'augmentation du signal de la fluorescence de 1Z4;[4;. D'abord nous avons montre que SYT09/EB était une com¬binaison de colorants plus utile que celle de SYT09/PI parce que la fluorescence du PI a tendance à augmenter uniquement lorsque les cellules sont effectivement mortes, et non pas dans les cellules saines (moins de deux fois plus). Par opposi¬tion, la fluorescence moyenne de l'EB dans les cellules saines augmente jusqu'à huit fois plus après exposition aux composes toxiques. Tous les composes, mme aux plus basses concentrations, induisent une augmentation mesurable de la fluorescence moy¬enne de 1Z4;[4;, plus particulièrement après deux heures d'incubation. Cet effet s'est revele tre transitoire pour les cellules exposees aux 2CNP et 4CP, mais est chro¬nique pour les cellules incubees avec le TD et le NAH (entranant la mort cellulaire). Afin de comprendre les mécanismes qui sous-tendent les effets observes, nous avons utilise des decoupleurs d'energie ou de membrane. L'augmentation du signal EB dans les populations causee par des produits chimiques ressemblait à celle exerce par le chelateur des ions divalents EDTA. Cependant, les intensités du signal EB des cellules exposees aux produits chimiques testees n'ont jamais atteint les valeurs des cellules traitees avec l'EDTA ou pasteurises. Nous en concluons que le test oli- gotrophique utilisant la coloration (SYT09/)EB des cellules exposees ou non à un produit chimique est utile afin d'evaluer l'effet toxique exerce par les polluants sur la physiologie bacterienne. Afin de mieux comprendre la reaction d'un système de defense par pompe à efflux après exposition à une toxine, nous avons étudié la toxicité du 2-hydroxybiphenyl (2-HBP) sur Pseudomonas azeiaica HBP1. Nous avons montre que le 2-HBP exerce une toxicité mme sur HBP1, mais uniquement à des concentrations supérieures à 0.5 mM. Au-dessus de cette concentration, des pertes transitoires d'intégrité et de polarization membranaire ont lieu, comme cela nous a ete montre par coloration des cellules en croissance. Les cellules sont finalement capables de se rétablir et de reprendre leur croissance sur 2-HBP. La forte resistance de P. azeiaica HBP1 h 2-HBP physiologie bacterienne s'est revele tre le résultat d'un système de pompe h efflux de type MexABOprM qui contre-balance l'influx passif de ce compose h travers la membrane. Nous avons montre, en construisant des mutants avec des insertions dans les gènes mexA, mexB and oprM et des fusions avec le gène rapporteur gfp, que l'altération de n'importe quelle partie du système d'efflux conduisait à accroître l'accumulation de 2-HBP dans la cellule, en comparaison avec la souche sauvage HBP1, provoquant une diminution de la resistance au 2-HBP ainsi qu'une baisse du taux de reproduction des cellules. Des systèmes d'efflux similaires sont répandus chez de nombreuses espèces bactériennes. Ils seraient responsables de la resistance aux produits chimiques tels que les colorants fluorescents (bromure d'ethidium) et des antibiotiques. Nous concluons que la resistance de P. azelaica HBP1 à 2-HBP est principalement due à un système d'efflux efficace et que 2-HBP, à des concentrations elevees, exerce un effet deletère sur la membrane bacterienne. En se basant sur le comptage des cellules avec la FC, nous avons developpe ensuite une methode pour evaluer la biodegradabilite de polluants tels que le 2-HBP ainsi que les antibiotiques (acide nalidixique (Nal), ampicilline (AMP) ou sulfamethoxazole (SMX)) à de faibles concentrations lmg par L et moins), par le suivi de la croissance spécifique sur le compose de cultures microbiennes pures et mixtes. En utilisant un comptage precis de faibles quantités de cellules nous avons pu demontrer que la croissance spécifique sur ces composes est possible. Nous avons pu illustrer la precision du comptage par cytometrie de flux à faible quantité de cellules (jusqu'à 10 3 cellules par ml). Ensuite, nous avons teste s'il était possible de suivre dynamiquement la croissance de la population de cellules sur faibles concentrations de substrats, en utilisant P. azelaica HBP1. Une relation parfaite a ete trouvee entre le taux de croissance, le rendement et les concentrations de 2-HBP (entre 0.1 et 5 mg par L). En mélangeant HBP1 à de la boue active, nous avons pu montrer que le rendement en communauté mixtes pouvait tre bien inférieur qu'en culture pure. Ceci étant peut tre le résultat d'une perte d'intermédiaires métaboliques. Nous avons ensuite isole de nouvelles souches à partir de la boue active en utilisant le 2-HBP ou des antibiotiques (Nal, AMP, SMX) h basses concentrations (0.1-1 mg par L) comme seules sources de carbone et d'energie. En combinaison avec ceci, nous avons également utilise des microplaques PAO. Les souches purifiees ont ensuite ete examinees pour leurs croissances sur leurs substrats respectifs. De faon intéressante, toutes ces souches ont montre qu'elles ne pouvaient pas survivre à des concentrations de substrats supérieures à 1 ou 10 mg par L. Ainsi, il existe des bacteries qui contribuent à la degradation de composes à basses concentrations de polluant mais sont inhibes lorsque ces concentrations deviennent plus hautes. Finalement, nous avons cherche à savoir s'il est possible de detecter une croissance spécifique à une biomasse au depend d'un polluant, en partant d'une communauté microbienne. Ainsi, nous nous sommes concentre sur certains composes et avons mesure l'augmentation de la biomasse d'une communauté grce à la cytometrie de flux. Nous avons compte deux communautés de depart distinctes: (i) une dilution d'eau du Lac Léman, et une dilution de boue active d'une station d'épuration. Nous avons observe que la plupart des composes testes ont entrane une augmentation de la biomasse de depart par rapport au control sans addition de source de carbone. Néanmoins, les échantillons du lac Léman et de la station d'épuration différaient largement (s'excluant mutuellement l'un l'autre) dans leur capacité à degrader les composes chimiques. Dans deux cas provenant de la station d'épuration, le mme type de communauté microbienne s'est developpe après exposition aux composes, comme l'a démontré l'analyse TRFLP sur les fragments d'ARN 16S purifie de la communauté et amplifie par PCR. Afin de tester correctement la biodegradabilite d'un compose, il est donc important d'utiliser des communautés de depart de différentes origines Nous en concluons que le comptage par cytometrie de flux peut tre un outil de grande utilité pour mettre en valeur la biodegradabillite et la toxicité des composes chimiques.

Reversible and irreversible pollutant-induced bacterial cellular stress effects measured by ethidium bromide uptake and efflux.

Chemical pollution is known to affect microbial community composition but it is poorly understood how toxic compounds influence physiology of single cells that may lay at the basis of loss of reproductive fitness. Here we analyze physiological disturbances of a variety of chemical pollutants at single cell level using the bacterium Pseudomonas fluorescens in an oligotrophic growth assay. As a proxy for physiological disturbance we measured changes in geometric mean ethidium bromide (EB) fluorescence intensities in subpopulations of live and dividing cells exposed or not exposed to different dosages of tetradecane, 4-chlorophenol, 2-chlorobiphenyl, naphthalene, benzene, mercury chloride, or water-dissolved oil fractions. Because ethidium bromide efflux is an energy-dependent process any disturbance in cellular energy generation is visible as an increased cytoplasmic fluorescence. Interestingly, all pollutants even at the lowest dosage of 1 nmol/mL culture produced significantly increased ethidium bromide fluorescence compared to nonexposed controls. Ethidium bromide fluorescence intensities increased upon pollutant exposure dosage up to a saturation level, and were weakly (r(2) = 0.3905) inversely correlated to the proportion of live cells at that time point in culture. Temporal increase in EB fluorescence of growing cells is indicative for toxic but reversible effects. Cells displaying high continued EB fluorescence levels experience constant and permanent damage, and no longer contribute to population growth. The procedure developed here using bacterial ethidium bromide efflux pump activity may be a useful complement to screen sublethal toxicity effects of chemicals.

Environmental activity of bacteria degrading pollutants

Une des meilleures techniques pour décontaminer l'environnement d'éléments toxiques (comme par exemple le dibenzofuan, DBF et le 4-chlorophenol, 4CP) déposés par l'homme, à bas coûts et sans le perturber considérablement, est sans doute la biorémédiation, et particulièrement la bioaugmentation. Malheureusement, si plusieurs microorganismes ont démontré leur efficacité à dégrader les composés toxiques en conditions de laboratoire, plusieurs tentatives afin de les utiliser dans l'environnement n'ont pas abouti. Ces échecs sont probablement le résultat des pauvres connaissances des réactions de ces mêmes microorganismes dans l'environnement. L'objectif de mon travail a été de mieux comprendre les réponses de ces bactéries au niveau de leurs gènes lorsqu'elles sont introduites ou prospèrent dans des conditions plus proches de la réalité, mais encore suffisamment contrôlées pour pouvoir élucider leur comportement. Le fait de résister à des conditions de sécheresse a été considéré en tant que facteur clé dans la survie des bactéries amenées à être utilisées pour la biorémédiation; cela implique une série de mécanismes utilisés par la cellule pour faire face au stress hydrique. Le chapitre II, par une approche métagénomique, compare les réactions de trois souches prometteuses pour la biorémédiation (Arthrobacter chlorophenolicus A6, Sphingomonas wittichii RW1 and Pseudomonas veronii 1YdBTEX2) vis-à-vis du stress hydrique simulé en conditions de laboratoire. L'objectif ici est de découvrir et de décrire les stratégies de résistance au stress, communes ou spécifiques, employées par les bactéries. Mes résultats montrent que les trois souches ont des sensibilités différentes au stress hydrique. Entre les traits communs trouvés, il y a une diminution de l'expression des gènes flagellaires ainsi qu'une augmentation de l'expression de solutes compatibles, mais qui sont souche-spécifiques. J'ai étudié plus en détail la réponse génomique de RW1 par rapport aux inoculations ainsi que sa croissance dans le sable contaminé et non-stérile (chapitre III), et je les ai comparé à des cultures en milieu liquide. Mes résultats indiquent que RW1 peut résister efficacement et peut croître dans des conditions presque sèches et peut également dégrader le contaminant (DBF, dans le cas présent) si les pré-cultures sont réalisées dans le même type de contaminant. Par contre, notre hypothèse du chapitre II se révèle fausse car le comportement de RW1 est très diffèrent de celui observé dans des conditions avec stress hydrique induit par l'addition de sel ou de PEG. Plus intéressant, les réponses de RW1 en milieu liquide sont très différentes de celles observées dans le sable, révélant ainsi que cette souche peut reconnaître le milieu dans lequel elle se trouve. Les mêmes expériences en sable contaminé, cette fois-ci avec 4CP, ont été réalisées pour A6 (chapitre IV) dans l'espoir de compléter la comparaison entre le stress hydrique et l'adaptation dans le sol. Malheureusement, il n'a pas été possible d'obtenir d'échantillons de bonne qualité pour les hybridations des microarrays afin d'étudier la réponse transcriptionnelle dans les différentes phases de croissance dans le sable (contaminé ou non). Toutefois, j'ai appris qu'Arthrobacter ne peut pas croitre dans les sols hautement contaminés si les conditions du sol sont très sèches, elles ont en effet besoin de suffisamment d'eau pour dégrader des quantités importantes de 4CP. Ces observations dirigent l'attention sur le fait que les études sur l'efficacité de l'inoculation de bactéries doivent être testées dans des conditions le plus proche possible de l'environnement ciblé, tout comme les concentrations optimales pour l'inoculum. Finalement, nous avons étudié le comportement de A6 dans la phytosphère avec deux dégrés d'humidité (chapitre V). A6 ne montre pas de réaction particulière face aux changements d'humidité, et à nouveau, ces réponses ne peuvent être liées aux changements d'expression des gènes observées dans les conditions de stress hydrique simulées. Cette étude a permis d'identifier la présence de composés phénoliques dans les feuilles qui peuvent potentiellement améliorer les propriétés de dégradation ou qui permettent d'effectuer de façon plus rapide la réaction de dégradation des contaminants dans un processus de phytoremédiation par A. chlorophenolicus.

Characterization and Control Strategies of an Integrated Chemical-Biological System for the Remediation of Toxic Pollutants in Wastewater: A case of study

In a previous work, a hybrid system consisting of an advanced oxidation process (AOP) named Photo-Fenton (Ph-F) and a fixed bed biological treatment operating as a sequencing batch biofilm reactor (SBBR) was started-up and optimized to treat 200 mg·L-1 of 4-chlorophenol (4-CP) as a model compound. In this work, studies of reactor stability and control as well as microbial population determination by molecular biology techniques were carried out to further characterize and control the biological reactor. Results revealed that the integrated system was flexible and even able to overcome toxic shock loads. Oxygen uptake rate (OUR) in situ was shown to be a valid tool to control the SBBR operation, to detect toxic conditions to the biomass, and to assess the recovery of performance. A microbial characterization by 16S rDNA sequence analysis reveals that the biological population was varied, although about 30% of the bacteria belonged to the Wautersia genus.

TiO(2)-PHOTOCATALYZED DEGRADATION OF PHENOL IN SALINE MEDIA IN AN ANNULAR REACTOR: HYDRODYNAMICS, LUMPED KINETICS, INTERMEDIATES, AND ACUTE TOXICITY

The photocatalytic degradation of phenol in aqueous suspensions of TiO(2) under different salt concentrations in an annular reactor has been investigated. In all cases, complete removal of phenol and mineralization degrees above 90% were achieved. The reactor operational parameters were optimized and its hydrodynamics characterized in order to couple mass balance equations with kinetic ones. The photodegradation of the organics followed a Langmuir-Hinshelwood-Hougen- Watson lumped kinetics. From GC/MS analyses, several intermediates formed during oxidation have been identified. The main ones were catechol, hydroquinone, and 3-phenyl-2-propenal, in this order. The formation of negligible concentrations of 4-chlorophenol was observed only in high salinity medium. Acute toxicity was determined by using Artemia sp. as the test organism, which indicated that intermediate products were all less toxic than phenol and a significant abatement of the overall toxicity was accomplished, regardless of the salt concentration.

Treatment of 1,10-phenanthroline laboratory wastewater using the solar photo-Fenton process

The red Fe2+-phenanthroline complex is the basis of a classical spectrophotometric method for determination of iron. Due to the toxicity of this complexing agent, direct disposal of the wastewaters generated in analytical laboratories is not environmentally safe. This work evaluates the use of the solar photo-Fenton process for the treatment of laboratory wastewaters containing phenanthroline. Firstly, the degradation of phenanthroline in water was evaluated at two concentration levels (0.1 and 0.01%, w/v) and the efficiencies of degradation using ferrioxalate (FeOx) and ferric nitrate were compared. The 0.01% w/v solution presented much higher mineralization, achieving 82% after 30 min of solar irradiation with both iron sources. The solar photo-Fenton treatment of laboratory wastewater containing, in addition to phenanthroline, other organic compounds such as herbicides and 4-chlorophenol, equivalent to 4500 mg L-1 total organic carbon (TOC) resulted in total degradation of phenanthroline and 25% TOC removal after 150 min, in the presence of either FeOx or ferric nitrate. A ratio of 1: 10 dilution of the residue increased mineralization in the presence of ferrioxalate, achieving 38% TOC removal after 120 min, while use of ferric nitrate resulted in only 6% mineralization over the same period. (c) 2007 Elsevier B.V. All rights reserved.

Influence of the iron source on the solar photo-Fenton degradation of different classes of organic compounds

In this work the influence of two different iron sources, Fe(NO3)(3) and complexed ferrioxalate (FeOx), on the degradation efficiency of 4-chlorophenol (4CP), malachite green, formaldehyde, dichloroacetic acid (DCA) and the commercial products of the herbicides diuron and tebuthiuron was studied. The oxidation of 4CP, DCA, diuron and tebuthiuron shows a strong dependence on the iron source. While the 4CP degradation is favored by the use of Fe(NO3)(3), the degradation of DCA and the herbicides diuron and tebuthiuron is most efficient when ferrioxalate is used. on the other hand, the degradation of malachite green and formaldehyde is not very influenced by the iron source showing only a slight improvement when ferrioxalate is used. In the case of formaldehyde, DCA, diuron and tebuthiuron, despite of the additional carbon introduced by the use of ferrioxalate, higher mineralization percentages were observed, confirming the beneficial effect of ferrioxalate on the degradation of these compounds. The degradation of tebuthiuron was studied in detail using a shallow pond type solar flow reactor of 4.5 L capacity and 4.5 cm solution depth. Solar irradiation of tebuthiuron at a flow rate of 9 L h(-1), in the presence of 10.0 mmol L-1 H2O2 and 1.0 mmol L-1 ferrioxalate resulted in complete conversion of this herbicide and 70% total organic carbon removal. (c) 2005 Elsevier Ltd. All rights reserved.