Nano Zero-Valent Iron Mediated Metal(loid) Uptake and Translocation

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Environmental Processes

Nano zero-valent iron mediated metal(loid) uptake and translocation by arbuscular mycorrhizal symbioses Songlin Wu, Miroslav Vosátka, Katarina Vogel-Mikus, Anja Kav#i#, Mitja Kelemen, Luka Šepec, Primož Pelicon, Roman Skála, Antonio Roberto Valero Powter, Manuel Teodoro, Zuzana Michálková, and Michael Komárek Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05516 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 17, 2018

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Environmental Science & Technology

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Nano zero-valent iron mediated metal(loid) uptake and

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translocation by arbuscular mycorrhizal symbioses

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Songlin Wu ,*, Miroslav Vosátka‡, Katarina Vogel-Mikus§,#, Anja Kavčič§, Mitja Kelemen#,

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Luka Šepec #, Primož Pelicon #, Roman Skála∥,⊥, Antonio Roberto Valero Powter†, Manuel

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Teodoro†, Zuzana Michálková†, Michael Komárek†

†

6 7 8 9 10 11 12 13 14 15 16

†

Department of Environmental Geosciences, Faculty of Environmental Sciences, Czech

University of Life Sciences Prague, Kamýcká 129, 165 00 Prague-Suchdol, Czech Republic ‡

Department of Mycorrhizal Symbioses, Institute of Botany, Czech Academy of Sciences,

272 53 Pruhonice, Czech Republic §

Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101,

SI-1000 Ljubljana, Slovenia #

Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia

∥

Institute of Geology of the Czech Academy of Sciences, Rozvojová 269, CZ-165 00 Prague

6, Czech Republic ⊥

Institute of Geochemistry, Mineralogy and Mineral Resources, Faculty of Science, Charles

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University in Prague, Albertov 6, CZ-128 43 Prague 2, Czech Republic

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*Corresponding author, E-mail address: [email protected] (Songlin Wu)

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ACS Paragon Plus Environment


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TOC art

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ABSTRACT: Nano zero-valent iron (nZVI) has great potential in the remediation of

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metal(loid)-contaminated soils, but its efficiency in metal(loid) stabilization in the

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plant-microbe continuum is unclear. This study investigated nZVI-mediated metal(loid)

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behavior in the arbuscular mycorrhizal (AM) fungal-maize (Zea mays L.) plant association.

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Plants with AM fungal inoculation were grown in metal(loid)- (mainly Zn and Pb)

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contaminated soils (Litavka River, Czech Republic) amended with/without 0.5% (w/w) nZVI.

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The results showed that nZVI decreased plant metal(loid) uptake but inhibited AM

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development and its function in metal(loid) stabilization in the rhizosphere. AM fungal

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inoculation alleviated the physiological stresses caused by nZVI and restrained nZVI

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efficiency in reducing plant metal(loid) uptake. Micro-proton-induced X-ray emission

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(µ-PIXE) analysis revealed the sequestration of Zn (possibly through binding to thiols) by

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fungal structures in the roots and the precipitation of Pb and Cu in the mycorrhizal root

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rhizodermis (possibly by Fe compounds originated from nZVI). XRD analyses further

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indicated that Pb/Fe mineral transformations in the rhizosphere were influenced by AM and

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nZVI treatments. The study revealed the counteractive effects of AM and nZVI on plant

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metal(loid) uptake and uncovered details of metal(loid) behavior in the AM fungal-root-nZVI

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system, calling into question nZVI implementation in mycorrhizospheric systems.

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INTRODUCTION

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Nano zero-valent iron (nZVI), which has been successfully introduced for groundwater

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remediation, also has great potential for the remediation of soils contaminated with

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metal(loid)s (e.g., Zn, Pb, Cr, As) due to its large specific surface area, high surface reactivity 3



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and strong capability for reducing oxidized metal(loid)s species (e.g. Cr(VI)).1-5 In the soil,

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nZVI can usually decrease metal(loid) bioavailability through adsorption, reduction etc2, 6,

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and promote subsequent plant growth and associated microbial development, leading to the

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restoration and revegetation of metal(loid) contaminated soils. However, nZVI is easily

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oxidized into Fe (oxy)hydroxides in the environment,7,8 and its functions of metal(loid)

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stabilization can be influenced by plant and soil microbe activities,9 which can transform

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nZVI through direct interactions with nZVI by their exudates (or metabolites) such as

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low-molecular-weight organic acids (LMWOAs).10-12 The transformation of nZVI may further

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influence its adsorption capacity1, 2. Moreover, despite the fact that metal(loid)s are adsorbed

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by nZVI, they may be re-dissolved due to plant and soil microbe activities,9, 13 which can

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influence metal(loid) transformation through altering redox and pH value14, or complexing

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metal(loid)s with their exudates.15 Additionally, nZVI can also cause toxicity to plants and

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microbes through the generation of reactive oxygen species (ROS), leading to cell membrane

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disruption and oxidative stress.10, 16 Therefore, it is essential to address the effects of plants

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and microbes on nZVI efficiency in metal(loid) stabilization, as well as the potential influence

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of nZVI on plant and microbial activities.

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Arbuscular mycorrhizal (AM) fungi are ubiquitous soil fungi that can form symbiosis with

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more than 80% terrestrial plants. The fungi obtain carbohydrates from plant partners and, in

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return, provide plants with mineral nutrients (e.g., phosphorus and nitrogen).17 AM fungi can

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also help plants to adapt to various stresses, e.g., drought, salt.18, 19 Additionally, it has been

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well-established that AM symbiosis can enhance plant resistance to metal(loid)s, mostly

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through the stabilization of metal(loid)s in the roots or rhizosphere, implying their promising 4


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potential in the phytoremediation and revegetation of metal(loid) contaminated soils.20-23 In

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fact, a recent field trial study indicated that AM fungi, together with Fe bearing phyllosilicate

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amendments could help plant survive in metal-contaminated soils.24 AM fungi can usually

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interact with metal(loid)s through the direct complexation of metal(loid)s by their exudates

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and cell wall or by indirectly influencing metal(loid) speciation through the alteration of the

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micro-environment in the mycorrhizosphere.22, 23 Similarly, AM fungi may also interact with

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nZVI and its aging products (Fe oxides) or directly interact with metal(loid)s adsorbed on the

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nZVI surface, thus influencing the nZVI efficiency in metal(loid) stabilization. In addition,

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nZVI may also influence AM formation and development and thus affect its functions

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towards metal(loid) stabilization. However, little information is available about the

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metal(loid)-nZVI-AM fungi interactions in the plant-soil system, which would be of critical

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importance in the implementation of nZVI and AM fungi in the phytoremediation and

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revegetation of metal(loid) contaminated soils. Therefore, it is urgent to investigate whether

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and how AM fungal inoculation influence nZVI functions towards metal(loid) stabilization in

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plant-soil system.

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The present study investigated the influence of AM symbiosis on the efficiency of nZVI in

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metal(loid) stabilization in alluvial metal(loid)-contaminated soil (originating from mining

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and smelting) in the Příbram District of the Czech Republic, which has been heavily

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contaminated with Zn and Pb (as well as Cd, As and Cu).25 The study was based on a natural

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metal(loid)-contaminated soil, which is different from many previous studies that used

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artificial metal(loid)-contaminated soils26-28 and the results would be thus more

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environmentally relevant. The AM fungus Rhizophagus irregularis and maize (Zea mays L.) 5



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were selected for the study, as they have been proven to be resistant to heavy metal stress in

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previous studies.29, 30 The plants inoculated with/without AM fungus were cultivated in soils

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amended with/without nZVI. Plant physiological characteristics and metal(loid)s uptake,

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translocation and transformation were investigated. In particular, cellular distributions of

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metal(loid)s and mineral nutrients in plant roots were investigated by employing scanning

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electron microscopy equipped with electron dispersive X-ray spectroscopy (SEM-EDS) and

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micro-proton-induced X-ray emission (µ-PIXE) analysis, which proved to be a powerful tool

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for the analysis of elemental distribution in plant tissues.31 In addition, solid phases of

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metal(loid)s (including Fe) in the mycorrhizosphere soil were analyzed by X-ray diffraction

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(XRD) to track their transformation. We hypothesized the following: (1) nZVI amendment

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can decrease metal(loid) uptake by plants but negatively affect AM development and

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functions in metal(loid) immobilization; (2) AM fungal inoculation can restrain nZVI

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efficiency towards reducing plant metal(loid) uptake and regulate metal(loid) translocation in

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roots; and (3) AM symbiosis can influence nZVI functions through altering metal(loid)

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bioavailability and Fe mineral transformation in the rhizosphere.

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MATERIALS AND METHODS

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Materials. The soil was collected in the alluvium of the Litavka River (Příbram District,

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Czech Republic), which has been contaminated with Zn, Pb, Cd, As and Cu. The soil

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physico-chemical characteristics are shown in Table 1. The methodologies of soil

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characterization are summarized in the Supporting Information (SI). The soil was sterilized by

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gamma radiation (20 kGrey) one week before the experiments. To increase soil porosity,

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clean quartz sand (with 1-2 mm diameter) was mixed with the soils (soil: sand = 5:1 w/w)

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before use.

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Maize (Zea mays) seeds were obtained from REIN SAAT (Austria). The seeds were surface

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sterilized in 10% H2O2 for 15 min, washed with Milli-Q water, and pre-germinated on moist

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ﬁlter paper until the emergence of radicles. The AM fungus Rhizophagus irregularis was

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isolated from metal-contaminated soils (pH 5) in the same area (Příbram, Czech Republic).30

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AM fungal inoculum included mycorrhizal roots, mycelium, spores (80 spores g-1) and quartz

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sand. Nano zero-valent iron was provided by Nano Iron (Czech Republic), with a size of

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50-100 nm (Figure S1), and the particles had a smooth sphere shape coated with a thin layer

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of Fe oxides that protected nZVI from further oxidation (further characteristics can be found

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in SI).

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Experimental Design. The soil was amended with/without 0.5% (w/w) nZVI (the level was

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based on a previous study32), and the plants were inoculated with/without AM fungi. The

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amendment of nZVI was carried out through homogeneously mixing dry powder air-stable

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nZVI particles with soils. As nZVI becomes very reactive in aqueous environments, the

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nZVI-amended soil was watered daily with distilled water (keeping the soil moisture at 55%

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of the water holding capacity (WHC)) to activate the nZVI and facilitate its functions on

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metal(loid)s stabilization for one month before plant and AM fungal cultivation. AM fungal

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inoculation was performed as follows: 700 g of soils amended with (or without) nZVI were

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first put into a pot, and 300 g of soil containing 30 g AM inoculum were then added. For

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non-inoculated controls, 30 g gamma radiation (20 kGrey) sterilized AM inoculum was added

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along with 10 mL of AM inoculum ﬁltrate to reintroduce microbes other than AM fungi in the 7



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AM inoculum. Additionally, soil filtrate (without AM fungi but with other indigenous

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microbes) was also reintroduced into the soil for all treatments to reintroduce the native soil

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microbial communities. Five germinated seeds were transplanted into each pot and 100 g soil

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was amended to support the seedlings. Seedlings were thinned to 2 per pot 1 week after

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emergence of cotyledon. To investigate the influence of plant growth on the efficiency of

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nZVI in soil metal(loid) stabilization, two bare soils with/without nZVI amendment were also

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employed. The experimental design is shown in Figure S2. There were 6 treatments, each

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with 6 replicates, totaling 36 pots. The experiment was conducted in a controlled greenhouse

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environment at 14/10h (light/dark) and 25/20°C (light/dark). The light intensity was

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500-1,100 µmol m-2 s-1, which was provided by natural light and supplementary lights from

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high-pressure sodium lamps. Each pot was watered daily to maintain a water content of 55%

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WHC. Basal nutrients were added to the soil twice per month to give a final level of 60 mg

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kg-1 N (in the form of NH4NO3), 60 mg kg-1 K (KNO3), and 15 mg kg-1 P (KH2PO4) in the soil

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to support plant growth.

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Harvest and Chemical Analysis. Plants grew for 65 days before harvest. One day before

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harvest, leaf water potential (Ψw, MPa) was determined to evaluate the plant water balance.

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The third leaf from the top of the plant shoot was collected for Ψw determination using the

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Scholander pressure chamber (model 600; PMS instruments Co., Corvallis, Oregon, USA).33

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At harvest, plant shoots and roots were washed carefully with distilled water and weighed

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(total fresh weight, Mft). Several fresh root segments were immediately prepared for µ-PIXE

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analysis, and a small amount of fresh roots and shoots were kept for malondialdehyde (MDA),

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chlorophyll determination and AM colonization assessment (the fresh samples were kept at 8


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-80°C until analysis). The remaining fresh plant samples were weighed (Mfr), freeze-dried at

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-50°C for 48 h, and weighed again (Mdr). The dry weight of the whole tissues (roots or shoots)

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(Mdt) were calculated as follows:

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Mdt = Mft × (Mdr/Mfr)

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assuming that the water content of the extracted tissues (for µ-PIXE, MDA, Chl and AM

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colonization assessment analysis) were the same with that of the remaining fresh tissues. The

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remaining dry tissues were then ground for macro-nutrients and metal(loid) determination.

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AM Colonization Assessment. AM colonization was determined following the methods of

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Phillips and Hayman (1970).34 In brief, fresh root sections (1-cm) were cleared by 10% (w/v)

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KOH, rinsed in 2% (v/v) HCl, and stained with 0.05% (w/v) Trypan blue. The intensity of the

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mycorrhizal colonization (M%) and the arbuscule abundance (A%) in the root system was

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assessed

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(www2.dijon.inra.fr/mychintec/Mycocalc-prg/download).35

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Plant Physiological and Chemical Analysis. Malondialdehyde (MDA) contents usually

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represent the level of lipid peroxidation and were measured using a modified thiobarbituric

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acid (TBA) method.36 The chlorophyll content in plant leaves was determined according to

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Wellburn and Lichtenthaler (1984).37 Details on MDA and chlorophyll determination are in

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the SI. The lyophilized and homogenized plant samples were digested in HNO3/H2O2 at

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210°C on an electric heating plate (HE1 TH1, Harry Gestigkeit GmbH, Germany) for 12h,

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diluted to 50-mL with Milli-Q water and filtered through a 0.22-µm membrane.

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Macro-nutrients (P, K, Ca, Mg) and metal(loid) concentrations (e.g., Zn, Pb, Fe, Cu, As) were

(1)

using

MYCOCALC

9


software


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determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, 720ES,

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Varian Inc., Palo Alto, California, USA).

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Micro-PIXE Analysis. The sample preparation for µ-PIXE was performed according to

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Vogel-Mikus et al. (2014)31 (see details in the SI). The µ-PIXE analysis was performed using

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a nuclear microprobe (voltage 3 MeV, beam size 0.8×0.8 µm, current 200 pA) at the Jožef

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Stefan Institute.38,

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initial raw data using the GEOPIXE II software package (http://nmp.csiro.au/Geopixe.html).40

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Element concentrations in different part of the roots (rhizodermis, cortex (including AM

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fungal structures), endodermis and vascular bundle) were calculated from the numerical

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matrices after the quantification of PIXE spectra by GEOPIXE II and extracted from the

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selected regions using ImageJ (https://imagej.nih.gov/ij/index.html).

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SEM-EDS Analysis. The thin sections of freeze-dried plant roots were analyzed by scanning

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electron microscopy (SEM, TESCAN Brno, Czech Republic), which was equipped for

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electron dispersive X-ray spectroscopy (EDS, Bruker-nano, Berlin, Germany) to detect the

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elemental distribution across the roots of different treatments.

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Soil Analysis. One week before harvest, soil solution samplers (mean pore volume size 0.15

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µm, Rhizosphere Research Products, Netherlands) were carefully inserted into the soils, and

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the soil solution was collected at harvest. Phosphorus and metal(loid) concentrations in soil

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solutions were analyzed by ICP-OES.

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At harvest, soil samples (50g each, stones were removed by sieving) were lyophilized and

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ground into a fine powder in an agate mortar in liquid nitrogen for X-ray powder diffraction

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(XRD) analysis. XRD analysis was conducted using a Bruker D8 Discover diffractometer

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The quantitative element-distribution maps were generated from the

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coupled with Johansson-type focusing Ge primary monochromator and a LynxEye linear

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silicon strip detector (see details in the SI). Soil pH and redox potential (Eh) were analyzed as

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described in the SI.

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Statistical Analysis. The mycorrhizal metal(loid) response (%MMeR) of roots was calculated

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using the individual metal(loid) content (determined as a product of metal concentration and

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plant dry weight) of AM roots and mean metal(loid) content of nonAM roots (Equation 2)

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at different nZVI treatments.

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% =

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Since there was a significant difference in A% between the control and nZVI-treated AM

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plants, we also took into account the A% weights, namely the “arbuscule-weighed %MMeR”

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(%AMMeR) (Equation 3).

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% = % %

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The mycorrhizal metal(loid) response data were analyzed using an independent t-test (P

Nano Zero-Valent Iron Mediated Metal(loid) Uptake and Translocation

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