Thierry Lebeau

Enhanced phytoextraction of an agricultural Cr- and Pb-contaminated soil by bioaugmentation with siderophore-producing bacteria

Bioaugmentation-assisted phytoextraction may enhance the phytoextraction efficiency thanks to larger metal mobilization by microbial metabolites. Green fluorescent protein-tagged cells of Pseudomonas aeruginosa, Pseudomonas fluorescens or Ralstonia metallidurans, able to produce siderophores, were inoculated in an agricultural soil containing Cr (488 mg kg(-1)) and Pb (382 mg kg(-1)) and maize was cultivated. Bacteria were inoculated as free or immobilized cells in Ca-alginate beads, with skim milk in the aim at improving both the bacterial survival and the in situ siderophore production. Skim milk addition increased inoculated Pseudomonads concentration in soil. Soil inoculation with free cells of R. metallidurans supplied with skim milk increased Cr accumulation in maize shoots by a factor of 5.2 and inoculation with immobilized P. aeruginosa cells supplied with skim milk increased Cr and Pb uptake by maize shoots by a factor of 5.4 and 3.8, respectively. However total metal taken up by the whole plant decreases almost always with bioaugmentation. Translocation factor also increased with P. aeruginosa or R. metallidurans by a factor of 6 up to 7. Inoculated bacteria concentration in soil was correlated with metals in the exchangeable fraction. Cr and Pb concentrations in the exchangeable fraction were correlated with metal contents in shoots or roots. Our results suggest that bioaugmentation-assisted phytoextraction is a relevant method in the aim at increasing the phytoextraction rate which usually limits the use of phytoremediation technologies.

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

Abstract. 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.

Diatom cultivation and biotechnologically relevant products. Part I: Cultivation at various scales

Abstract. Biotechnological applications of diatoms are still in development. Further development at the industrial scale will depend on optimisation of the culture process with the aim of reducing costs. Because of the photoautotrophic status of the majority of diatoms, microalgal cultures suffer from the limitation of light diffusion, which requires the development of suitable photobioreactors. Thus, genetically engineered microalgae that may be cultivated in heterotrophic conditions present a new opportunity. Other limiting factors, such as nutrients (phosphate, nitrogen, silicon), pH, temperature, bioturbation and many more must be taken into account. Most of the time, metabolic stress conditions lead to an overproduction of the products of interest, with a decrease in biomass production as a consequence. Outdoor cultures in open ponds are usually devoted to aquaculture for the feeding of shrimps and bivalve molluscs (commercial production), while closed axenic indoor/outdoor photobioreactors are used for biotechnological compounds of homogeneous composition (still at the laboratory scale). In addition to the optimum culture conditions that have to be taken into account for photobioreactor design, the localisation of produced metabolites (intra- or extracellular) may also be taken into account when choosing the design. Microalgal cell immobilisation may be a suitable technique for application to benthic diatoms, which are usually sensitive to bioturbation and/or metabolites which may be overexpressed.

New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine–iron uptake pathway

Pyoverdine (PvdI) is the major siderophore secreted by Pseudomonas aeruginosa PAOI in order to get access to iron. After being loaded with iron in the extracellular medium, PvdI is transported across the bacterial outer membrane by the transporter, FpvAI. We used the spectral properties of PvdI to show that in addition to Fe(3+), this siderophore also chelates, but with lower efficiencies, all the 16 metals used in our screening. Afterwards, FpvAI at the cell surface binds Ag(+), Al(3+), Cd(2+), Co(2+), Cu(2+), Fe(3+), Ga(3+), Hg(2+), Mn(2+), Ni(2+) or Zn(2+) in complex with PvdI. We used Inductively Coupled Plasma-Atomic Emission Spectrometry to monitor metal uptake in P. aeruginosa: TonB-dependent uptake, in the presence of PvdI, was only efficient for Fe(3+). Cu(2+), Ga(3+), Mn(2+) and Ni(2+) were also transported into the cell but with lower uptake rates. The presence of Al(3+), Cu(2+), Ga(3+), Mn(2+), Ni(2+) and Zn(2+) in the extracellular medium induced PvdI production in P. aeruginosa. All these data allow a better understanding of the behaviour of the PvdI uptake pathway in the presence of metals other than iron: FpvAI at the cell surface has broad metal specificity at the binding stage and it is highly selective for Fe(3+) only during the uptake process.

Selection of low cost materials for the sorption of copper and herbicides as single or mixed compounds in increasing complexity matrices.

Low cost materials (sugar beet pulp, corncob, corncob char, perlite, vermiculite, sand, sediment) have been tested for their ability to quickly sorb copper, glyphosate, diuron and 3,4-dichloroaniline (3,4-DCA) as single or mixed compounds. Tests have been performed in increasingly complex liquid matrices: ultra pure water (UPW), runoff water (RW) and sediment extract medium (SEM). Highest sorption levels in UPW are achieved with corncob char for Cu (93%), glyphosate (74%), diuron (98%) and 3,4-DCA (99%). Other ready-to-use adequate sorbents are sugar beet pulp for Cu and sand for glyphosate, diuron and 3,4-DCA. Sorption levels obtained in UPW are significantly altered in SEM as a result of its higher dissolved organic carbon concentration, tenuous changes being obtained with RW. Interactions between herbicides and Cu are pointed out: higher sorption level is observed for glyphosate in mixture with Cu, as it is observed with diuron and 3,4-DCA when mixed with all other pollutants. Langmuir model has been found to better fit the data for copper, whereas Freundlich one has been found more relevant for diuron and 3,4-DCA. Our results stress the need for studying adsorption in different matrices when searching for sorbents to be used in field conditions.

Effect of immobilization of a bacterial consortium on diuron dissipation and community dynamics

This work intended to study the relationship between diuron herbicide dissipation and the population dynamics of co-cultivated Delftia acidovorans WDL34 (WDL34) and Arthrobacter sp. N4 (N4) for different cell formulations: free cells or immobilization in Ca-alginate beads of one or both strains. GFP-tagged WDL34 and N4 Gram staining allowed analyzing the cell growth and distribution of each strain in both beads and culture medium in the course of the time. Compared to the free cell co-culture of WDL34 and N4, immobilization of WDL34 in Ca-alginate beads co-cultivated with free N4 increased the dissipation rate of diuron by 53% (0.141 mg ml(-1) h(-1)). In that case, immobilization strongly modified the final equilibrium among both strains (highest total N4 to WDL34 ratio). Our results demonstrated that the inoculant formulation played a major role in the cell growth of each cultivated strain possibly increasing diuron dissipation. This optimized cell formulation may allow improving water and soil treatment.

Alginate-entrapped Haslea ostrearia as inoculum for the greening of oysters

Entrapment in calcium alginate beads of the marine diatom, Haslea ostrearia, was successfully used for stock-culture managment and afterwards the sowing of ponds for the greening of oysters. After storage during almost 2 months, viable and cultivable cells were recovered from beads by dissolving alginate matrix but an original way lies in directly introducing beads in ponds and promoting natural cell leakage.

Bioaugmentation for In Situ Soil Remediation: How to Ensure the Success of Such a Process

Among bioremediation techniques, bioaugmentation is certainly the less easy to control, but at the same time the one exhibiting the greatest potential for the soil cleaning-up. The main challenge for the success of in situ soil bioaugmentation is based on the ability to manage the process in environments subject to variable conditions. Microorganisms are thus suggested to be selected not only for their ability to degrade organic compounds or to modify the chemical form of metals, but also by systematically considering some ecological traits. Bioaugmentation-assisted plants could help improve the stability of the microbial activity in the rhizosphere by supplying specific nutrients and ecological niche for inoculated microorganisms provided that both microorganisms and plants are diligently chosen. Tools at disposal for monitoring and controlling bioaugmentation are described in this chapter. These tools should be helpful to the better understanding of mechanisms involved in bioaugmentation as well as environmental impacts of such a technique.

Artificial cell-immobilization: a model simulating immobilization in natural environments?

A study of the marine diatom Haslea ostrearia was performed in controlled culture conditions. Biofilm formation by cells grown in liquid medium was studied through macroscopic events and extracellular polysaccharides (EPS) production. Alcian blue staining and image analysis methods were used to quantify EPS production in regard to the usual pattern (lag, exponential and stationary phases) of microalgal growth. Simultaneously, an artificial immobilization of H. ostrearia cells was carried out using an agar layer. Samples from liquid medium and artificially immobilized cultures were studied in scanning electron microscopy. They revealed the structure of the entrapping material and progressive changes of the natural biofilm along culture ageing. The use of artificially immobilized cultures for solute diffusivity investigations is discussed.

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

Abstract 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.