Copper phytoavailability in vineyard topsoils as affected by pyoverdine supply.

Pyoverdine (Pvd) is a bacterial siderophore produced by some Pseudomonads species that can bind copper in addition to iron in soil. Pvd is expected to alter the dynamics and the ecotoxicity of Cu in vineyard soils. This study investigated the extent to which the mobility and the phytoavailability of Cu varied among vineyard soils with different pH and how they were affected by a supply of Pvd. Pvd was supplied (or not) to ten vineyard topsoils with pH ranging from 5.9 to 8.6 before metal was extracted with 0.005 M CaCl2. Cu mobility was assessed through its total concentration and Cu phytoavailability through its free ionic concentration measured in the CaCl2 extract. Cu mobility varied by a factor of six and Cu phytoavailability by a factor of 5000 among the soil samples. In the CaCl2 extract, the concentration of Cu2+ was not correlated with the concentration of total Cu but was correlated with pH. This revealed that Cu phytoavailability depends to a great extent on Cu complexation in soil pore water, the latter being highly sensitive to pH. Adding Pvd enhanced the mobility of Cu in the soils including in carbonate soils. The Pvd-mobilization factor for Cu varied from 1.4 to 8 among soils, linked to the availability of Fe and Al in the solid phase and to Pvd partitioning between the solid and the liquid phase. Adding Pvd reduced the concentration of Cu2+ in CaCl2 extract, which challenges the idea of using Pvd-producing bacteria to promote Cu phytoextraction.

How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunflower?

The aim of this study was to compare the efficiency of siderophore desferrioxamine B (DFOB) and EDTA in increasing the phytoextraction of metals in sunflower. A 28-day pot experiment was conducted in a metal-contaminated soil supplied with 200µmolkg-1 of DFOB or EDTA. Pore water was collected and pseudo-polarographic analyses were conducted to assess the impact of the two chelators on the mobility and speciation of metals in the liquid phase. Our results showed that DFOB is not an efficient mobilizing agent of divalent metals in soil. Adding DFOB selectively increased the mobility of trivalent metals while the supply of EDTA simultaneously increased the mobility of both trivalent and divalent metals. EDTA significantly reduced the labile fractions of Cd, Cu, (Pb) and Zn measured in the porewater. The labile concentration of Cd and Zn measured in presence of EDTA was even less than that measured in the control. As expected from the pore water analysis, the addition of DFOB did not affect the phytoextraction of any divalent metals. In contrast, the addition of EDTA enhanced Cu and Ni phytoextraction in sunflower 2.0 to 2.8 fold for Cu and 1.3 to 2.3 fold for Ni, depending on the cultivar. This result supports different hypotheses regarding the forms and the related pathways in which metals are taken up in presence of EDTA. Based on the results obtained for Ni, whose uptake is rate limited by its internalization across the cell membrane, the direct uptake of metal-EDTA complexes via the non-selective apoplastic pathway is hypothesized to contribute the most to the overall uptake of metals in presence of EDTA, even added at "low" concentrations.

A quick rhizobacterial selection tests for the remediation of copper contaminated soils.

AIMS: The main objective of the study is to develop and improve quick bacterial tests to select the best candidates for the bioaugmentation of metal-contaminated soil, coupled with phytoextraction. METHODS AND RESULTS: Bacteria isolates (181) were selected from a collection originated from a Cu-contaminated sediment, on the basis of several miniaturized biochemical tests adapted to the copper contamination. Amongst them, we used a growth soil based-medium to select metal-tolerant bacteria, and their ability to grow and mobilize metals by mean of metabolites (siderophores, organic acids) was also assessed. CONCLUSION: The result of the bacterial selection tests showed differences in presence or absence of copper, especially for phosphate-solubilizing strains which ability decreased by 53% in the presence of copper hydroxide phosphate as compared to the standard tricalcium phosphate test. A promising Pseudomonas putida was selected from the collection. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlined the importance of choosing significant selection tests regarding the nature of the metal occurring in the soil to be cleaned-up to assess the real potential of each bacterial strain for subsequent soil bioaugmentation purposes.

Remediation of sediment and water contaminated by copper in small-scaled constructed wetlands: effect of bioaugmentation and phytoextraction.

The use of plants and microorganisms to mitigate sediment contaminated by copper was studied in microcosms that mimic the functioning of a stormwater basin (SWB) connected to vineyard watershed. The impact of phytoremediation and bioaugmentation with siderophore-producing bacteria on the fate of Cu was studied in two contrasted (batch vs. semi-continuous) hydraulic regimes. The fate of copper was characterised following its discharge at the outlet of the microcosms, its pore water concentration in the sediment, the assessment of its bioaccessible fraction in the rhizosphere and the measurement of its content in plant tissues. Physico-chemical (pH, redox potential) and biological parameters (total heterotrophic bacteria) were also monitored. As expected, the results showed a clear impact of the hydraulic regime on the redox potential and thus on the pore water concentration of Cu. Copper in pore water was also dependent on the frequency of Cu-polluted water discharges. Repeated bioaugmentation increased the total heterotrophic microflora as well as the Cu bioaccessibility in the rhizosphere and increased the amount of Cu extracted by Phragmites australis by a factor of ~2. Sugar beet pulp, used as a filter to avoid copper flushing, retained 20% of outcoming Cu and led to an overall retention of Cu higher than 94% when arranged at the outlet of microcosms. Bioaugmentation clearly improved the phytoextraction rate of Cu in a small-scaled SWB designed to mimic the functioning of a full-size SWB connected to vineyard watershed. Highlights: Cu phytoextraction in constructed wetlands much depends on the hydraulic regime and on the frequency of Cu-polluted water discharges. Cu phytoextraction increases with time and plant density. Cu bioaccessibility can be increased by bioaugmentation with siderophore-producing bacteria.

Effect of pyoverdine supply on cadmium and nickel complexation and phytoavailability in hydroponics.

Siderophores are chelators with a high selectivity for Fe(III) and a good affinity for divalent metals, including Cd(II) and Ni(II). Inoculation with siderophore-producing bacteria (SPB) has thus been proposed as an alternative to chelator supply in phytoremediation. Accurate assessments of the potential of this association require a dissection of the interaction of siderophores with metals at the soil-root interface. This study focuses on pyoverdine (Pvd), the main siderophore produced by Pseudomonas aeruginosa. We first assessed the ability of Pvd to coordinate Ni(II). The stability constant of Pvd-Ni(II) (log K (L'Ni) = 10.9) was found to be higher than that of Pvd-Cd(II) (log K (L'Cd) = 8.2). We then investigated the effect of a direct supply of Pvd on the mobilization, speciation, and phytoavailability of Cd and Ni in hydroponics. When supplied at a concentration of 50 µM, Pvd selectively promoted Ni mobilization from smectite. It decreased plant Ni and Cd contents and the free ionic fractions of these two metals, consistent with the free ion activity model. Pvd had a more pronounced effect for Ni than for Cd, as predicted from its coordination properties. Inoculation with P. aeruginosa had a similar effect on Ni phytoavailability to the direct supply of Pvd.

Contrasting effects of pyoverdine on the phytoextraction of Cu and Cd in a calcareous soil.

Enhanced metal phytoextraction by the use of siderophore-producing bacteria (SPB) has received a lot of attention in the past decade. Bacterial siderophores are able to bind a wide range of metals other than iron and thus should enhance their phytoavailability in contaminated matrices. However, the impact of bacterial siderophores in the soil-plant transfer of metals is not yet fully elucidated, as underlined by the opposing results reported in the literature regarding the efficiency of coupling phytoextraction with bioaugmentation by SPB. The present study focuses on one bacterial siderophore, the pyoverdine (Pvd), produced by Pseudomonas aeruginosa. The coordination properties of Pvd towards Cd(II) and Cu(II) were determined and the effect of Pvd supply was assessed on (i) the mobility (CaCl2 extractions), (ii) the phytoavailability (DGT measurements) and (iii) the phytoextraction of Cd and Cu, in a calcareous soil. The stability constant of Pvd-Cu (KL'Cu=10(20.1)) was found much higher than that of Pvd-Cd (KL'Cd=10(8.2)). The major finding was the agreement observed between Pvd coordination properties and Pvd impact on metals phytoextraction. Pyoverdine, supplied at 250 µmol kg(-1) soil, enhanced the mobility, the phytoavailability and the phytoextraction of Cu while the fate of Cd was not affected. All these results were compared to those reported for chelate-assisted phytoextraction. Their relevance in using SPB for phytoremediation is discussed.

Quantification of the 16S-23S rRNA internal transcribed spacers of Burkholderia xenovorans strain LB400 using real-time PCR in soil samples.

This study establishes a new real-time PCR assay (using SYBR Green™ detection) for the identification and the direct quantification of specific individual Burkholderia xenovorans strain LB400 from DNA samples of soil and sediment. Specific primers were designed to amplify a 190-bp fragment of the 16S-23S rRNA internal transcribed spacers (ITS) from LB400. The specificity of primers was evaluated using 21 strains. The detection limit of the real-time PCR was analysed on soil samples inoculated with LB400 and was of 6 copies (10(5)  CFU g(-1) of dry sample). The 16S-23S rRNA ITS primers developed in this work for rapid quantification of LB400 were validated. The assay allowed the quantification of LB400 as pure strain and among the indigenous microbial community in samples of soil and sediment (105-day experiment).

Decontamination of a polychlorinated biphenyls-contaminated soil by phytoremediation-assisted bioaugmentation.

A 70 day pot experiment was conducted for the cleaning-up of a PCBs-contaminated soil (104 mg kg(-1) soil DW) using bioaugmentation with Burkholderia xenovorans LB400 (LB400) assisted or not by the use of tall fescue (Festuca arundinacea). The total cultivable bacteria of the soil were higher with the presence of plants. Real-time PCR showed that LB400 (targeting 16S-23S rRNA ITS) survived with abundance related to total bacteria (targeting 16S rRNA) being higher with fescue (up to a factor of three). Bioaugmentation had a positive effect on fescue biomass and more specifically on roots (by a factor of three). PCB dissipation (sum of congeners 28, 52, 101, 118, 153, 180) averaged 13 % (bioaugmented-planted) up to 32 % (non bioaugmented-planted), without any significant difference between treatments. Basically our results demonstrated that indigenous bacteria were able to dissipate PCBs (26.2 % dissipation). PCB dissipation was not related to the abundance of LB400 or to the total bacterial counts. Bioaugmentation or fescue altered the structure of the bacterial community of the soil, not the combination of both. Principal component analysis showed that bioaugmentation tended to improve the control of the process (lower variability in PCB dissipation). Opposite to that bioaugmentation increased the variability of the structure of the bacterial community.

Herbicide mitigation in microcosms simulating stormwater basins subject to polluted water inputs.

Non-point source pollution as a result of wine-growing activity is of high concern. Stormwater basins (SWB) found downstream of vineyard watersheds could show a potential for the mitigation of runoff water containing herbicides. In this study, mitigation of vinery-used herbicides was studied in microcosms with a very similar functioning to that recorded in SWB. Mitigation efficiency of glyphosate, diuron and 3,4-dichloroaniline (3,4-DCA) was investigated by taking into account hydraulic flow rate, mitigation duration, bioaugmentation and plant addition. Mitigation efficiency measured in water ranged from 63.0% for diuron to 84.2% for 3,4-DCA and to 99.8% for glyphosate. Water-storage duration in the SWB and time between water supplies were shown to be the most influential factors on the mitigation efficiency. Six hours water-storage duration allowed an efficient sorption of herbicides and their degradation by indigenous microorganisms in 5 weeks. Neither bioaugmentation nor plant addition had a significant effect on herbicide mitigation. Our results show that this type of SWB are potentially relevant for the mitigation of these herbicides stemming from wine-growing activity, providing a long enough hydraulic retention time.

Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples".

Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring.

[Anastomotic stricture after radical prostatectomy for prostate cancer]. / Sclérose de l'anastomose urétrovésicale après prostatectomie totale pour cancer.

The present paper intends to review diagnosis and treatment issues of bladder neck anastomosis stricture after radical prostatectomy for localised prostate cancer. Even though cancer control is not necessarily a concern, quality of life may be greatly altered. Patients may suffer from dysuria, urgency and the feeling of incomplete bladder emptying. Flowmetry, cystoscopy and cystography contribute to its diagnosis. Treatment should be graded according to the severity of the disease and the quality of life of the patient. Cold-Knife incisions and pneumatic dilatation are the first line treatments. Holmium laser shows good results on the stricture in a second line treatment. A two-stage strategy with an endoluminal stent followed by artificial urinary sphincter implant is the ultimate option to manage severe strictures, while maintaining acceptable quality of life. Continence sparing is the challenge of the treatment of this type of stricture.

[Technical aspects of laparoscopic robot-assisted pyeloplasty]. / Aspects techniques de la pyéloplastie laparoscopique robot-assistée.

From 2000, the robot-assisted laparoscopic approach has been developed for the management of ureteropelvic junction obstruction (UJO) with equivalent outcomes to conventional laparoscopic access regarding functional results. This system has simplified the suturing and has improved the precision of operative technique. The main surgical steps of the transperitoneal laparoscopic robot-assisted pyeloplasty are as follows: four or five port arrangement; initial dissection and early identification of the ureteropelvic junction; renal pelvis section; transection of the ureter and preparation of a spatula; continuous posterior suture; confection of a handle racket suture; placement of a double J stent; ending of the anastomosis. Outcomes after robotic and pure laparoscopic pyeloplasties are equivalent nowadays. Despite the financial cost, it seems easier and technically feasible and accessible for surgeons accustomed to the laparoscopic techniques and even beginners to learn the robotic technique if the system is available in their institution with success rate (radiologic and clinical) almost similar with those obtain with open techniques.

Simultaneous mineralization of glyphosate and diuron by a consortium of three bacteria as free- and/or immobilized-cells formulations.

A bacterial consortium able to mineralize two herbicides, glyphosate (Pseudomonas 4ASW) and diuron (Arthrobacter sp. N4 and Delftia acidovorans), was cultivated in both a synthetic culture medium without phosphate and a sediment extract medium. In the aim at optimizing glyphosate and diuron mineralization, all the combinations, i.e., free and/or immobilized cells in Ca-alginate beads were tested. With the synthetic medium, the simultaneous mineralization of glyphosate and diuron required at least the immobilization of Pseudomonas 4ASW. Conversely, with the sediment extract medium, only the mineralization of diuron was observed, most probably, because of both nutrient deficiency and phosphate in the sediment extract medium.

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized Arthrobacter sp. and Delftia acidovorans.

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l(-1)) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l(-1)) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l(-1 )h(-1) vs. 0.06 mg l(-1 )h(-1) with free cells in co-culture).

Long-term incomplete xylose fermentation, after glucose exhaustion, with Candida shehatae co-immobilized with Saccharomyces cerevisiae.

Saccharomyces cerevisiae and Candida shehatae were co-immobilized in an agar sheet which was introduced in an original two-chambered bioreactor asymmetrically fed in a batch mode with a mixture of glucose and xylose in a ratio of 35:15. The two sugars were consumed simultaneously. All glucose was fermented but only 20% of xylose. After incubation, yeast cells recovered from different areas of the agar sheet (close to, called Hi, and distant from, called Ho, the substrate chamber) were cultured as suspended cells in fresh culture medium provided with xylose or the sugar mixture. Xylose utilization by gel released Hi yeasts was significantly delayed compared to the Ho culture. Ethanol consumption by Hi yeasts in the two-substrate medium occurred after glucose exhaustion despite the presence of xylose. The waste medium resulting from incubation of the immobilized-cell structure inhibited xylose utilization by C. shehatae. Our results suggested that batch fermentation most probably favoured this incomplete xylose fermentation.

Diatom cultivation and biotechnologically relevant products. Part II: current and putative products.

While diatoms are widely present in terms of diversity and abundance in nature, few species are currently used for biotechnologically applications. Most studies have focussed on intracellularly synthesised eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA) used for pharmaceutical applications. Applications for other intracellular molecules, such as total lipids for biodiesel, amino acids for cosmetic, antibiotics and antiproliferative agents, are at the early stage of development. In addition, the active principle component must be identified amongst the many compounds of biotechnological interest. Biomass from diatom culture may be applied to: (1). aquaculture diets, due to the lipid- and amino-acid-rich cell contents of these microorganisms, and (2). the treatment of water contaminated by phosphorus and nitrogen in aquaculture effluent, or heavy metal (bioremediation). The most original application of microalgal biomass, and specifically diatoms, is the use of silicon derived from frustules in nanotechnology. The competitiveness of biotechnologically relevant products from diatoms will depend on their cost of production. Apart from EPA, which is less expensive when obtained from Phaeodactylum tricornutum than from cod liver, comparative economic studies of other diatom-derived products as well as optimisation of culture conditions are needed. Extraction of intracellular metabolites should be also optimised to reduce production costs, as has already been shown for EPA. Using cell immobilisation techniques, benthic diatoms can be cultivated more efficiently allowing new, biotechnologically relevant products to be investigated.

Cadmium biosorption by free and immobilised microorganisms cultivated in a liquid soil extract medium: effects of Cd, pH and techniques of culture.

Instead of soil clean-up, a process not very technically and economically suited to agricultural soil contaminated by heavy metals (with a low concentration of heavy metals but highly or potentially highly contaminated surfaces), the control of the transfer of cadmium from the soil to the crops may well be a convenient method. We tested the bacterium ZAN-044, the actinomycete R27 and a basidiomycete Fomitopsis pinicola isolated for their ability to biosorb Cd, in order to inoculate agricultural soils afterwards. We then compared the cadmium biosorption by viable microbial cells which were free or immobilised in alginate beads and incubated in a soil extract liquid medium at various pH values (5, 6 and 7) and cadmium concentrations (1 and 10 mg/l). The Cd concentration in the medium had the most important effect on the percentage of Cd biosorbed by the microorganisms, but the culture mode (free or immobilised cells) was not a side effect. In the case of F. pinicola and the actinomycete R27, the percentage of Cd biosorbed by free cells did not decrease when the Cd concentration in the medium increased (6-42% at the lowest Cd concentration to 11-48% at 10 mg Cd/l). On the other hand, with a low Cd concentration (1 mg Cd/l), the percentage of Cd biosorbed by the bacterium ZAN-044 was maximum (69%) at pH 7, while this bacterium did not grow at 10 mg Cd/l and it did not accumulate Cd. For the three micro-organisms tested, relatively low specific biosorptions of Cd were observed, when the microorganisms were cultivated with a soil extract medium ('poor' medium), comparatively to those with a 'rich' medium. Finally, the choice of microorganism for the inoculation of contaminated soils depends on the cadmium level in the medium and on the distribution of the metal between the biomass and the medium.

A new photobioreactor for continuous marennin production with a marine diatom: influence of the light intensity and the immobilised-cell matrix (alginate beads or agar layer).

In oyster ponds, the marine diatom Haslea ostrearia synthesises and excretes a hydrosoluble pigment of commercial interest called marennin. During the benthic stage, when algal cells are naturally immobilised in their own polysaccharides, marennin production is higher. To optimise this production, axenic cultures of H. ostrearia were immobilised in a polysaccharidic matrix (alginate or agar) and introduced into a new photobioreactor device for continuous marennin production. Solute diffusion was improved using an alginate beads monolayer, leading to higher levels of cell growth (a 2-fold higher cell concentration) and marennin productivity (7.57-8.80 mg day(-1) l(-1)). An increase in the light intensity (from 3.0 to 8.5x10(16) quanta cm(-2) s(-1)) led to an earlier and 1.3-fold higher production of marennin. However, the higher light intensity led to a higher rate of cell death [0.29 instead of 0.40 ng chlorophyll a (10(6) cells)(-1)]. Due to the secondary nature of marennin metabolism, it would be necessary to alternate between culture conditions favouring cell growth (moderate light intensity and no limiting nitrate supply) and those promoting marennin production (high light intensity and limiting nitrate supply).

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination.

The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27-43 days) and the volumetric productivity was maximum (18.7 mg day(-1) l(-1) gel) at the highest dilution rate (0.25 day(-1)) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer.

Continuous alcoholic fermentation of glucose/xylose mixtures by co-immobilized Saccharomyces cerevisiae and Candida shehatae.

Viable Saccharomyces cerevisiae and Candida shehatae cells were co-immobilized in a composite agar layer/microporous membrane structure. This immobilized-cell structure was placed in a vertical position between the two halves of a double-chambered, stainless-steel bioreactor of original design and applied to the continuous alcoholic fermentation of a mixture of glucose (35 g dm-3) and xylose (15 g dm-3). Various dilution rates and initial cell loadings of the gel layer were tested. Simultaneous consumption of the two sugars was always observed. The best fermentation performance was obtained at low dilution rate (0.02 h-1) with an excess of C. shehatae over S. cerevisiae in the initial cell loading of the gel (5.0 mg dry weight and 0.65 mg dry weight cm-3 gel respectively): 100% of glucose and 73% of xylose were consumed with an ethanol yield coefficient of 0.48 g g total sugars-1. In these conditions, however, the ethanol production rate per unit volume of gel remained low (0.37 g h-1 dm-3). Viable cell counts in gel samples after incubation highlighted significant heterogeneities in the spatial distribution of the two yeast species in both the vertical and the transverse directions. In particular, the overall cell number decreased from the bottom to the top of the agar sheet, which may explain the low ethanol productivity relative to the total gel volume.