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.

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.

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.

Temporal evolution of copper distribution in soil fractions, influence of soil pH and organic carbon level on copper distribution.

The present paper deals with the temporal evolution, over 6 years, of copper distribution in soil fractions using a sequential extraction procedure isolating five soil fractions which are operationally defined but commonly identified as : F1 = exchangeable metals, F2 = metals bound to organic matter, F3 = metals bound to manganese oxides, = metals bound to iron oxides and F5 = the residual fraction. The soil studied was a silty agricultural soil whose four plots were deliberately enriched with copper sulphate in October 1992 and one of them was also enriched with organic matter, another one with lime and the last one with both organic matter and lime. The great advantage of such an experimental procedure was that the five plots represent the same soil and thus had the same soil texture. It was then possible to fully describe anthropogenic copper behaviour, and to describe clearly the influence of pH and organic carbon level on copper distribution. Furthermore, the plots were subjected to natural climatic conditions allowing a temporal study of copper distribution under natural conditions. The total Cu level as well as Cu-F3 and Cu-F4 were clearly decreasing with time. Copper concentration in artificially defined soil fractions were described with multiple regression equations with the variable total copper content (TCu) and with the variables soil pH and soil organic carbon level when required. Cu-F1 and Cu-F2 depended on soil pH and soil organic carbon level, while Cu-F3 depended only on TCu. Copper level bound to iron oxides depended on soil pH.

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.