Article pubs.acs.org/est
Extracellular Polymeric Substances from Bacillus subtilis Associated with Minerals Modify the Extent and Rate of Heavy Metal Sorption Robert Mikutta,†,* Anja Baumgar̈ tner,† Axel Schippers,‡ Ludwig Haumaier,§ and Georg Guggenberger† †
Institut für Bodenkunde, Leibniz Universität Hannover, Germany Federal Institute for Geosciences and Natural Resources (BGR), Hannover, Germany § Department of Soil Ecology, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, Germany ‡
S Supporting Information *
ABSTRACT: Extracellular polymeric substances (EPS) are an important source of organic matter in soil. Once released by microorganisms, a portion may be sorbed to mineral surfaces, thereby altering the minerals̀ ability to immobilize heavy metals. EPS from Bacillus subtilis were reacted with Ca-saturated bentonite and ferrihydrite in 0.01 M KCl at pH 5.0 to follow the preferential uptake of EPS-C, -N, and -P. The sorption kinetics of Pb2+, Cu2+, and Zn2+ to the resulting EPS-mineral composites was studied in single and binary metal batch experiments ([metal]total = 50 μM, pH 5.0). Bentonite sorbed much more EPS-C (18.5 mg g−1) than ferrihydrite (7.9 mg g−1). During sorption, EPS were chemically and size fractionated with bentonite favoring the uptake of low-molecular weight components and EPS-N, and ferrihydrite selectively retaining high-molecular weight and P-rich components. Surface area and pore size measurements by N2 gas adsorption at 77 K indicated that EPS altered the structure of mineral-EPS associations by inducing partial disaggregation of bentonite and aggregation of ferrihydrite. Whereas mineral-bound EPS increased the extent and rate of Pb2+, Cu2+, and Zn2+ sorption for bentonite, either no effect or a decrease in metal uptake was observed for ferrihydrite. The extent of sorption always followed the order Pb2+ > Cu2+ > Zn2+, which also prevailed in binary Pb2+/Cu2+ systems. In consequence, sorption of EPS to different minerals may have contrasting consequences for the immobilization of heavy metals in natural environments by inducing mineral-specific alterations of the pore size distribution and, thus, of available sorption sites.
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INTRODUCTION Prokaryotic and eukaryotic soil microorganisms exudate highmolecular weight extracellular polymeric substances (EPS) used for cell attachment to mineral surfaces, protection from dehydration and pollutants, or nutrient capture.1,2 Soil and sediment EPS are easily bioavailable C sources, thus, they are considered to play an important role in the bioaccumulation of metals in living organisms.3 As the majority of soil bacteria live attached to or in proximity of mineral particles, a portion of produced EPS react with mineral surfaces.2 Such reactions are fostered by the richness of EPS in functional groups such as phosphoryl, carboxyl, amide, amino, and hydroxyl groups.1,4 A number of studies have provided direct or indirect evidence that EPS from various sources are able to complex heavy metals.5−10 Because of their multiple functional groups, EPS modify the surface charge of minerals, thus influencing the minerals’ affinity toward heavy metals. Omoike and Chorover4 showed that EPS become chemically fractionated with respect to their major components upon interaction with minerals. Variable-charge surfaces of goethite (α-FeOOH) and amorphous Al(OH)3 predominantly retained P-containing structures, for example, nucleic and teichoic acids, and phospholipids.4,11 Sorption of EPS components may thus trigger a differential retention of heavy metals. Similar to other organic © 2012 American Chemical Society
substances, mineral-attached EPS might increase metal uptake to minerals by providing additional complexation sites,12 or reduce metal immobilization by blockage of nanometer-sized pores, that is, by impairing inter- or intraparticle diffusion,13,14 or by influencing the aggregation status of the respective EPS− mineral association.15 At present, only few studies have addressed the interaction of heavy metals with EPS−mineral associations. In a recent study, Fang et al.12 have shown by using equilibrium sorption isotherms, potentiometric and microcalorimetric titration that EPS either bound to goethite or montmorillonite increased the retention of Cu(II). They concluded that the type of dominating EPS−mineral interaction, that is, innersphere complexation in the case of goethite and hydrophobic/van der Waals forces for montmorillonite, control the quantity of available surface sites in the respective composite and subsequently the sorption of copper. So far, it remains unclear how mineral-bound EPS influence the aggregation status of the respective composite and how this alters the sorption of heavy metals. Received: Revised: Accepted: Published: 3866
December 13, 2011 February 27, 2012 March 14, 2012 March 23, 2012 dx.doi.org/10.1021/es204471x | Environ. Sci. Technol. 2012, 46, 3866−3873
Environmental Science & Technology
Article
sorption sites; the pH was readjusted by using 0.1 M NaOH or HCl. EPS stock solutions containing 0, 5, 10, 20, 40, 60, 80, 100, 200 mg l−1 EPS-C were adjusted to pH 5.0 and left 30 min for equilibration. Five milliliters of mineral suspension and 50 mL of EPS solution were mixed in polyethylene Nalgene centrifuge tubes corresponding to a maximal EPS-C dose of 100 mg g−1 mineral and shaken horizontally for 18 h at 25 °C. For the EPS−mineral associations used in the metal sorption experiments, a higher EPS-C dose of 391 mg g−1 mineral was chosen. Separation of the solution and solid phase was done by filtration using 0.1 μm polyethersulfone membranes (Supor100, Pall Life Sciences). The EPS−mineral associations were washed with 5 mL of 0.01 M KCl, frozen in liquid N2, and freeze-dried. The mass of adsorbed EPS-C, -N, and -P was calculated as the difference between initial and final concentrations in the filtrate. Organic N was calculated as total N minus [NO3− + NH4+]−N; and organic P as total P minus [PO43−]T−P. Inorganic anions and NH4+ were measured by ion chromatography (ICS-90; Dionex, Sunnyvale, USA). Sorption of EPS components was best described by the Freundlich equation with
This study focuses on the interaction of EPS derived from the Gram-positive soil bacterium Bacillus subtilis with ferrihydrite and bentonite and the subsequent effects on heavy metal sorption (Pb2+, Cu2+, Zn2+) to the respective EPS− mineral associations. Despite EPS from pure bacteria cultures do not directly resemble soil biofilm EPS, they provide an easyto-study model system devoid of trace metals frequently coextracted from natural EPS sources.16 Sorption envelopes were constructed at a rhizosphere-relevant pH of 5.0 to determine the capacity of the minerals to accumulate EPS and to follow the selective uptake of individual EPS structures. Sorption relevant parameters such as surface area, porosities, and charge properties of minerals and EPS−mineral associations were characterized by N2 adsorption at 77 K and electrophoretic mobility measurements. The sorption kinetics of Pb2+, Cu2+, and Zn2+ to the minerals and EPS−mineral associations was monitored for 24 h in single and binary metal systems.
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EXPERIMENTAL METHODS Preparation of mineral sorbents. Two-line ferrihydrite was hydrolyzed from a 0.1 M Fe(NO3)3 solution,17 frozen in liquid N2, and freeze-dried. Bentonite of a size-fraction
