Introduction

Global demand for metallic elements is surging because of the technological shift required for a net-zero carbon emission transition (European Commission 2020; IEA 2021; Mudd 2020; Xu et al. 2020). Base metal mining occurs around the world, with Australia being a major producer of lead (Pb), zinc (Zn), copper (Cu), silver (Ag) and nickel (Ni) (Mudd et al. 2017; Spitz and Trudinger 2019). There are > 70 major base metal mines/fields across Australia (Mudd 2007). One of the most significant production centres are the Mt Isa deposits in northwest Queensland, which have the largest reserves of Pb, Zn and Ag in the world (Williams 1998) and hosts some of the leading global producers of base metals, including the Mt Isa Mines and Dugald River Mine. The tailings material generated from base metal mining generally contains high concentrations of potentially toxic trace elements, including arsenic (As), cadmium (Cd) and thallium (Tl). Large volumes of this potentially hazardous material is generated as ore-grades continue to decline whilst metal demand and production surge (Jowitt et al. 2020; Mudd 2020). Mine tailings management is one of the major issues confronting the mining industry, governments, and the society at large, with deleterious repercussions on the environment and public health if inappropriately managed (Entwistle et al. 2019). Environmentally friendly and cost-effective approaches are required to holistically address the increasing risk of tailings contamination from active and abandoned mines. However, in many mine tailings, their inherent geochemical characteristics present strong challenges for effective rehabilitation (Huang et al. 2012).

Hyperaccumulator plants have the unique ability to tolerate and accumulate high concentrations of potentially toxic trace elements into their shoots (Reeves 2006). Notional threshold values in dry shoot matter for recognition of hyperaccumulation are set at 100 µg g−1 for Cd, selenium (Se) and Tl; 300 µg g−1 for cobalt (Co) and Cu; 1000 µg g−1 for As and nickel (Ni); 3000 µg g−1 for Zn; and 10,000 µg g−1 for manganese (Mn) (van der Ent et al. 2013). On this basis, there are currently 746 hyperaccumulator species known globally: 532 Ni, 53 Cu, 42 Co, 42 Mn, 41 Se, 20 Zn, seven Cd, five As, two Tl, and two rare earth elements (REEs) (Reeves et al. 2018a,b). Hyperaccumulation has both fundamental (e.g., understanding ion homeostasis in plants) and applied (e.g., development of phytoextraction and biofortification) importance (Chaney and Baklanov 2017; Clemens 2017; van der Ent et al. 2015, 2017).

Phytoextraction involves the in situ cultivation of selected hyperaccumulator plants on a metal-rich substrate followed by harvesting and subsequent processing of the metal-rich biomass to recover valuable metal(loid)s and/or to remove hazardous elements from their biomass for safe disposal (Anderson et al. 1999; Nkrumah et al. 2016, 2018, 2019). Phytoextraction can be cost-effective and environmentally friendly and potentially generate economic gains from valuable metals, such as Ni, Co, and Tl from unconventional resources such as mine tailings (Corzo Remigio et al. 2020; van der Ent et al. 2021). Production of high-purity metal salts from the harvested biomass may in the future partly meet the growing demand for critical metals required for the global net-zero carbon emission transition (Nkrumah et al. 2022). The main limitation of phytoextraction is the number of known hyperaccumulator plant species with the right combination of desirable traits, including high biomass production and a high accumulation capacity for the target element (Nkrumah et al. 2021). Furthermore, hyperaccumulator plant species typically tolerate and accumulate only one or two elements simultaneously (van der Ent et al. 2019). This presents a problem at many metal-contaminated sites, including base metal mine tailings, because they are intrinsically polymetallic. Considering the inherent polymetallic characteristics of most base metal mine tailings, it may not be useful to target single metal(loid) during phytoextraction. However, this drawback may be overcome by selecting a purposeful assemblage of hyperaccumulator plant species that in concert tolerate and accumulate multiple metal(loid)s thereby providing synergistic conditions for optimum growth and metal yield of all cultivated species.

Substantial unrealised opportunities exist in Australia for the discovery of hyperaccumulator plant species considering the high level of plant diversity and widespread metalliferous soils (Abubakari et al. 2021a,b; Lottermoser et al. 2008). However, the majority of the limited local hyperaccumulator plant species discovered so far either produce low biomass or are very slow growing and therefore, not suitable for economic phytoextraction (Batianoff et al. 1990; Bidwell et al. 2002; Fernando et al. 2009; Severne 1974). Recent field surveys have led to the discovery of Australian native species that may have greater potential. This includes the finding of one of the strongest Se hyperaccumulator plant species known in the world, Neptunia amplexicaulis (Fabaceae) endemic to Central Queensland, which may open new opportunities for a Se phytoextraction industry in regional Australia (Harvey et al. 2020). In addition, the discovery of the polymetallic (Zn-Cu-Cd) hyperaccumulator Crotalaria novae-hollandiae (Fabaceae) from the Dugald River Zn-Pb gossan in Central Queensland may find applications in phytoremediation and possibly phytoextraction in Australia (Tang et al. 2022).

Elucidating elemental distribution at the organ, tissue, cellular or subcellular levels can provide insights into the mechanisms underlying the high tolerance and accumulation physiologies in these species (van der Ent et al. 2018). Synchrotron and laboratory-based micro-X-ray fluorescence technology permits mapping of a wide range of elements in plant organs and tissues (van der Ent et al. 2021). Information on elemental distribution coupled with elemental concentrations and growth data could aid in developing improved hyperaccumulator cultivars and optimising the agronomic parameters required for practical phytoextraction (Broadhurst et al. 2009; van der Ent et al. 2017). For example, the co-localization of Ni and Mn in the leaf tissues of Odontarrhena (Alyssum) species (Brassicaceae) suggests that employing this species for Ni phytoextraction on Mn-rich soils may affect Ni yields (Broadhurst et al. 2009).

This study aims to investigate the responses of model polymetallic hyperaccumulator plants to typical polymetallic tailings and to provide a proof-of-principle for polymetallic phytoextraction on Zn-Pb base metal mine tailings from Australian mines. Some of the species used in this study will not be suitable for application in the semi-arid tailings in Central Queensland. However, it is hoped that the proof-of-concept may ultimately be adopted employing local native species if they are discovered in Australia.

Materials and methods

Tailings material

The tailings material used for this study included Zn-Pb base metal mine tailings from the Dugald River and Mt Isa Mines. The Mt Isa Mines commenced production in 1931 (Mudd 2007), whereas the Dugald River Mine became operational in 2017 (Creus et al. 2021). The chemical properties of the respective tailings materials are presented in Table 1). The Dugald River Mine tailings have high Mn, Zn, Cd, Tl and Pb total concentrations, whilst the Mt Isa Mine tailings also have high Cu, Co, As and Tl concentrations, but lower Zn and Mn concentrations (see Table 1; Forsyth et al. 2015; Huang et al. 2015; Tang et al. 2022). Notably, the tailings from the Dugald River Mine are not yet weathered because they have only recently been produced, and this may influence the phytoavailable metal concentrations (e.g., Zn, see Table 1).

Table 1 Initial chemical properties of the substrates used in this study. The elemental concentrations of the solutions extracted by total and diethylenetriaminepentaacetic acid (DTPA) methods are given in mg kg−1
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Plant species

The selected model species included Noccaea caerulescens (Brassicaceae) targeting zinc, Biscutella laevigata (Brassicaceae) and Silene latifolia (Caryophyllaceae) targeting thallium, Phytolacca octandra (Phytolaccaceae) targeting manganese, and Pityrogramma calomelanos (Pteridaceae) targeting arsenic. Furthermore, we employed the well-known metal tolerant species capable of producing large amounts of carboxylate-rich root exudates, Lupinus albus (Fabaceae). The intensively studied hyperaccumulator N. caerulescens from Europe has the ability to hyperaccumulate Ni, Zn, Pb and Cd with accessions differing in their ability to tolerate and accumulate these metals (Assunção et al. 2003; Deng et al. 2014; Gonneau et al. 2014; Schwartz et al. 2003). The level of foliar accumulation in N. caerulescens is extreme as it can accumulate up to 52,000 µg g−1 foliar Zn (Zhao et al. 2003), whilst it can also attain 3410 µg g−1 foliar Cd (Reeves et al. 2001, 2018a,b). Biscutella laevigata can attain > 30,000 µg Tl g−1 when growing on soils with ~ 400 µg Tl g−1 (Pošćić et al. 2015) and S. latifolia up to 1500 µg Tl g−1 (Escarré et al. 2011). Pityrogramma calomelanos is an As hyperaccumulator that can reach up to 8350 μg As g−1 in its fronds (Francesconi et al. 2002). Phytolacca octandra and L. albus produce large amounts of root exudates rich in carboxylic acids, thereby increasing phytoavailable metal concentrations (Dinkelaker et al. 1989; Lambers et al. 2015). Moreover, Phytolacca species are Mn hyperaccumulators and may be beneficial in reducing Mn-toxicity to co-cultivated plants via the removal of available Mn from the rhizosphere (Pollard et al. 2009). Phytoavailability of the target metal(s) in the soil or substrate is a key consideration for the effectiveness of phytoextraction (Nkrumah et al. 2016, 2021) and we hypothesize that co-cultivating strong root exudates-producing plants with selected model hyperaccumulator plants could increase their metal accumulation, especially in relatively low phytoavailable metal substrates. Finally, as a legume, L. albus is a nitrogen-fixing species which may improve the fertility of the substrate for the co-cultivated hyperaccumulator plants.

Experimental design

The seed accession of N. caerulescens (Navacelles, France, non-metalliferous) was chosen purposely because it has a particularly high Zn bioconcentration factor (ratio of shoot Zn concentrations to soil Zn concentrations) (Escarré et al. 2000). The seed accessions of B. laevigata and S. latifolia originated from Saint-Laurent-le-Minier, Southern France (Zn-Pb-Cd-Tl metalliferous site). Seeds of P. octandra were collected near Brisbane, Australia, while seeds of L. albus were purchased from a commercial supplier in Brisbane and Pityrogramma calomelanos sporelings were collected from north Queensland. The experiments were conducted in plastic boxes (45 × 30 × 12 cm, L, W, H) in which a 1 cm layer of plastic granules (for irrigation) was covered by a plastic mesh and then filled with tailings mixes consisting of 90:5:5 and 75:10:15 (w/w/w) tailings/peat moss/perlite for Dugald River Mine and Mt Isa Mine tailings, respectively. Seeds of N. caerulescens, B. laevigata and S. latifolia were germinated separately on Gelzan gel in 2 mL Eppendorf tubes (made from 0.5-strength Hoagland’s solution). The seeds were then vernalized for one week at 3 °C and acclimatized for four days at 25 °C (van der Zee et al. 2021). Seeds of P. octandra were treated with sulphuric acid for 15 min and rinsed with water before sowing in a 1:1 mix of perlite and vermiculite. The seedlings (approx. 1 cm in size) were transplanted to the boxes. Four treatments with 16 biological replicates were undertaken on the Dugald River Mine substrate: B. laevigata only (hyperaccumulation of Tl) (D_B only), B. laevigata + N. caerulescens (hyperaccumulation of Zn and Tl) (D_B + N), B. laevigata + L. albus (hyperaccumulation of Tl) (D_B + L) and N. caerulescens only (hyperaccumulation of Zn) (D_N only). In addition, seven treatments with 16 biological replicates were undertaken on the Mt Isa Mine substrate: B. laevigata only (hyperaccumulation of Tl) (M_B only), B. laevigata + P. calomelanos (hyperaccumulation of As and Tl) (M_B + P), B. laevigata + L. albus (hyperaccumulation of Tl) (M_B + L), N. caerulescens only (hyperaccumulation of Zn) (M_N only), B. laevigata + P. octandra (hyperaccumulation of Mn and Tl) (M_B + Phy), S. latifolia only (hyperaccumulation of Tl) (M_S only) and S. latifolia + P. octandra (hyperaccumulation of Mn and Tl) (M_S + Phy). In the case of polyculture, eight replicates of each species were used. The plants were grown for a total period of 12 weeks with 12 hours light/day (provided by LEDs B200, Valoya, 350 µmol m-2 sec-1 photosynthetic photon flux density) at 25–20°C day/night temperature, harvested, and then separated into root and shoot fractions for analysis as described below.

Bulk analysis of plant tissue samples

All of the plant material samples were thoroughly washed with demineralised water and then oven dried at 70 °C for 3 days. The samples were weighed and then ground to a fine powder in an impact mill at 15,000 rpm (IKA Tube Mill 100 control with disposable cups with titanium blades) and then 100 ± 5 mg of each sample was weighed into 6 mL polypropylene tubes. The samples were pre-digested using 2 mL HNO3 (70%) for 24 h before digestion in a block heater (Thermo Scientific™ digital dry bath) for a 2-h programme (1 h at 70 °C followed by 1 h at 125 °C). The digestates were then brought to volume (10 mL) with ultrapure water before analysis with Inductively coupled plasma atomic emission spectroscopy (ICP-AES) with a Thermo Scientific iCAP 7400 instrument for macro-elements (Mg, P, S, K, Ca) and trace-elements (Mn, Fe, Cu, Zn, As, Cd, Tl, Pb) in radial and axial modes depending on the element and expected analyte concentration. All elements were calibrated with a 4-point curve covering analyte ranges in the samples.

Collection and analysis of tailings samples

Samples of the respective substrates collected from 5–20 cm depth were air-dried and sieved through a 630 µm screen. The pH was measured in a 1:2.5 substrate to water slurry after 2 h mixing on an end-over-end shaker and a 1-h rest. Sub-samples were weighed (100 ± 5 mg) in quartz digestion vessels and 5 mL HNO3 (70%) and 2 mL HCL (37%) were added. The samples were then digested for 15 min at 80% power using a ColdBlock system (CB15S 15 channel system, ColdBlock Technologies Inc) which uses high-intensity infrared irradiation to aid rapid acid digestion (Wang et al. 2014). The digestates were quantitatively transferred to 50 mL tubes, brought to volume (40 mL) and filtered (Whatman® Grade 1 filter paper) before analysis with ICP-AES. As a means of estimating potentially phytoavailable trace elements, the DTPA-extractant was used according to Becquer et al. (1995), which was adapted from the original method by Lindsay and Norvell (1978), with the following modifications: excluding TEA, adjusted to pH 5.3, 5 g soil with 25 mL extractant, and extraction time of one hr. the extracts were then filtered (Whatman® Grade 1 filter paper) before analysis with ICP-AES.

micro-X-ray fluorescence elemental mapping

The UQ micro-XRF facility is a custom-built system manufactured by IXRF which consists of two 50 kV sources (1000 μA) fitted with polycapillary focussing optics: XOS microfocus Mo-target tube producing 17.4 keV X-rays (flux of 2.2 × 108 ph s−1) focussing to 25 μm and a Rh-target tube producing 20.2 keV (flux of 1.0 × 107 ph s−1) focussing to 5 μm. The system is fitted with two silicon drift detectors (SDD) of 150 mm2 coupled to a XIA Mercury X4 signal processing unit. The fresh foliar samples were mounted between two sheets of 4 μm Ultralene thin film in a tight sandwich to limit evaporation and analysed within 10 min after excision. The mounted samples between Ultralene thin film were stretched over a Perspex frame magnetically attached to the x–y motion stage at atmospheric temperature (~ 20 °C). The UQ microXRF facility acquired the XRF spectra in mapping mode using the instrument control package, Iridium (IXRF systems), from the sum of counts at the position of the principal peak for each element. They were then exported into ImageJ as greyscale 8-bit TIFF files, internally normalised such that each image covered the full dynamic range and displayed using ImageJ’s “Fire” lookup table.

Statistical analyses

Statistical analyses were performed using OriginPro 2021 (https://www.originlab.com/). The elemental concentrations of the plant fractions were presented in Tables and plotted in Figures as mean ± standard error or standard deviation. Significant differences were determined by ANOVA, separated by Tukey’s honestly significant difference (HSD) test (p < 0.05) and indicated by different letters.

Results

Growth performance on the mine tailings

Noccaea caerulescens grew well in all of the treatments (Fig. 1), but the biomass on the Mt Isa Mine substrate was significantly higher than that on the Dugald River Mine substrate (p < 0.05) (Fig. 2D). On the Dugald River Mine substrate, the biomass of the monoculture treatment was not significantly different from the treatment co-cultured with B. laevigata (p > 0.05). For B. laevigata, the growth was significantly reduced on the Dugald River Mine substrate, except when co-cultured with N. caerulescens (Fig. 1F, G, H, 3D). The plants in the Mt Isa Mine substrate grew well irrespective of the type of culture and the biomass production was similar to that co-cultured with N. caerulescens in the Dugald River Mine substrate (p > 0.05). The S. latifolia grew well in the Mt Isa substrate in both the monoculture treatment and co-culture with P. octandra and did not differ significantly from the biomass of B. laevigata growing on a similar substrate (p > 0.05) (Fig. 4D). Lupinus albus grew well in the Mt Isa Mine substrate but showed severe toxicity symptoms in the Dugald River Mine substrate (Fig. 1). Phytolacca octandra and P. calomelanos grew well in the Mt Isa Mine tailings.

Fig. 1
figure 1

The status of the cultivated plants at harvest, A-D: Mt Isa Mine substrate (A: Noccaea caerulescens only; B: Biscutella laevigata co-cultured with N. caerulescens; C: B. laevigata only; D: B. laevigata co-cultured with Lupinus albus) and E–H: Dugald River Mine substrate (E: N. caerulescens only; F: B. laevigata co-cultured with N. caerulescens G: B. laevigata only; H: B. laevigata co-cultured with L. albus). There were 16 plants per box and the plants were grown for 12 weeks

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Fig. 2
figure 2

Elemental concentrations A) Zn, B) Mn and C) Tl, and D) shoot biomass of Noccaea caerulescens grown on the Dugald River Mine and Mt Isa Mine substrates (Mono: monoculture and Poly: co-cultured with Biscutella laevigata). Key to symbols of boxplots: open squares are the ± mean, whiskers are ± standard deviation (SD), circles are outliers. Mean ± standard error followed by the same letter are not significantly different (p > 0.05) according to the Tukey test

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Fig. 3
figure 3

Elemental concentrations A) Tl, B) Zn and C) Mn, and D) shoot biomass of Biscutella laevigata grown on the Dugald River Mine and Mt Isa Mine substrates (Mono: monoculture, Poly BL: co-cultured with Lupinus albus, Poly BN: co-cultured with Noccaea caerulescens). Key to symbols of boxplots: open squares are the ± mean, whiskers are ± standard deviation (SD), circles are outliers. Mean ± standard error followed by the same letter are not significantly different (p > 0.05) according to the Tukey test

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Fig. 4
figure 4

Elemental concentrations A) Tl, B) Zn and C) Mn, and D) shoot biomass of Biscutella laevigata and Silene latifolia grown on the Mt Isa Mine substrate (Mono B: monoculture B. laevigata, B + P: B. laevigata co-cultured with Phytolacca octandra, P Mono S: monoculture S. latifolia, S + P: S. latifolia co-cultured with P. octandra). Key to symbols of boxplots: open squares are the ± mean, whiskers are ± standard deviation (SD), circles are outliers. Mean ± standard error followed by the same letter are not significantly different (p > 0.05) according to the Tukey test

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Elemental accumulation by the selected plant species

Noccaea caerulescens accumulated higher Mn and Zn concentrations in the Dugald River Mine substrate compared to the Mt Isa Mine substrate (> tenfold, p < 0.05) (Fig. 2, Tables 2 and 3). On the Dugald River Mine substrate, the Mn and Zn concentrations in the monoculture treatment were significantly higher than when co-cultured with B. laevigata (> threefold; p < 0.05). Biscutella laevigata accumulated higher Tl concentrations on the Mt Isa Mine substrate than that on the Dugald River Mine substrate (> threefold, p < 0.05) (Fig. 3A, Tables 2 and 3). However, an opposite pattern to foliar Tl was observed for foliar Mn and Zn concentrations in the respective substrates (Fig. 3). In the Dugald River Mine substrate, the Mn and Zn concentrations in the monoculture treatment were significantly higher than that co-cultured with N. caerulescens (Fig. 3B, C). Co-culture with P. octandra significantly increased Tl concentrations in S. latifolia (p < 0.05), whereas the effect on Mn and Zn concentrations was not significant (p > 0.05) (Fig. 4, Tables 2 and 3). Phytolacca octandra accumulated considerable concentrations of Mn, Fe, Cu, Zn and Cd, whereas Mn and As were the only trace elements accumulated in relatively higher concentrations by L. albus and P. calomelanos, respectively (Tables 2 and 3). The treatment effects on the substrate DTPA-extractable trace elemental concentrations were not apparent (Table 4). For the macro elements, B. laevigata and P. calomelanos accumulated relatively higher P compared to P. octandra (Table 3). Lupinus albus and P. octandra had relatively lower K concentrations compared to B. laevigata (~ twofold, p < 0.05). Biscutella laevigata accumulated significantly higher Ca than all the other species (p < 0.05).

Table 2 Foliar elemental concentrations in mg kg−1 (trace elements) in the respective treatments. There were 16 plants per box. Mean ± standard error for each element followed by the same letter are not significantly different (p > 0.05) according to the Tukey test. ** deceased plants
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Table 3 Foliar elemental concentrations in g kg−1 (macro elements) in the respective treatments. There were 16 plants per box. Mean ± standard error for each element followed by the same letter are not significantly different (p > 0.05) according to the Tukey test. ** deceased plants
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Table 4 Final pH and diethylenetriaminepentaacetic acid (DTPA)-extractable trace elements concentrations in the soil of the respective treatments at harvest. The DTPA-extractable elemental concentrations are given in means ± standard error in mg kg−1. The extracts were analyzed by inductively-coupled plasma atomic emission spectroscopy (ICP-AES). Mean ± standard error followed by the same letter are not significantly different (p < 0.05) according to the Duncan-Waller K-ratio t-test
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Metal yield per treatment

In the Dugald River Mine tailings substrates, the N. caerulescens only treatment had the highest Mn and Zn yields, followed by B. laevigata and N. caerulescens co-culture treatment, whereas in the Mt Isa Mine tailings substrates, B. laevigata and P. octandra co-culture treatment had the highest Mn and Zn yields, followed by B. laevigata only treatment (Fig. 5). Moreover, B. laevigata and N. caerulescens co-culture treatment had the highest Tl yield in the Dugald River Mine tailings, whereas B. laevigata and P. calomelanos co-culture treatment had the highest Tl yield in the Mt Isa Mine tailings substrates.

Fig. 5
figure 5

Manganese, zinc and thallium yields (kg ha−1) of the respective treatments. The treatments included: Dugald River Mine substrate: B. laevigata only (D_B only), B. laevigata + N. caerulescens (D_B + N), B. laevigata + L. albus (D_B + L) and N. caerulescens only (D_N only); Mt Isa Mine substrate: B. laevigata only (M_B only), B. laevigata + P. calomelanos (M_B + P), B. laevigata + L. albus (M_B + L), N. caerulescens only (M_N only), B. laevigata + P. octandra (M_B + Phy), S. latifolia only (M_S only) and S. latifolia + P. octandra (M_S + Phy)

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Micro-XRF analysis of foliar elemental distribution

In N. caerulescens Zn was strongly enriched in the margin, with excess distributed in the lamina, whereas the midrib and the veins were particularly depleted (Fig. 6). Similarly, Mn was enriched in margin, but depleted in midrib, veins, and lamina. In contrast, K was enriched in the midrib, veins and lamina but depleted in the margin. Calcium was enriched in the lamina but depleted in the midrib, veins, and margin. For B. laevigata, Zn was primarily in the midrib and veins, with excess stored in the base of the trichomes (Figs. 7, 8). However, the lamina and the margin were much lower in Zn. This pattern of Zn distribution is opposite to that of N. caerulescens. Manganese was mainly stored in the trichomes, with excess distributed to the margin and tips. However, the midrib and the veins were depleted in Mn. Thallium was distributed primarily in the midrib and margin, with excess in the veins. However, the lamina and the trichomes were depleted in Tl. The trichome rays were enriched in Ca compared to the trichome base, lamina, and veins. Potassium was enriched in the midrib and lamina but depleted in the tips and trichome rays (Fig. 7).

Fig. 6
figure 6

Laboratory micro-X-ray fluorescence (µXRF) maps of Zn, Mn, K and Ca of hydrated leaves of Noccaea caerulescens (monoculture) grown on the a) Dugald River Mine substrate and b) Mt Isa Mine substrate

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Fig. 7
figure 7

Laboratory micro-X-ray fluorescence (µXRF) maps of Zn, Mn, Tl and Ca of hydrated leaves of Biscutella laevigata (monoculture) grown on the a) Mt Isa Mine substrate and b) Dugald River Mine substrate

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Fig. 8
figure 8

Laboratory micro-X-ray fluorescence (µXRF) maps of Zn, Mn, K and Ca of hydrated leaves of Biscutella laevigata co-cultured with Noccaea caerulescens on the Dugald River Mine substrate

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For P. octandra, Zn was enriched in the lamina, but depleted in the midrib, veins, and tip, opposite to the pattern observed for K (Fig. 9). Manganese was enriched in the margin compared to the lamina. The midrib and the veins were particularly depleted in Mn. Calcium was enriched in the lamina and leaf margins but depleted in the midrib and veins. For L. albus, Zn was depleted in the lamina but relatively enriched in the midrib, veins, and margin (Fig. 10). Manganese was enriched in the margin but depleted in the midrib, veins, and lamina. A similar distribution pattern was observed for Ca but that of K was opposite. For P. calomelanos, Zn and K were enriched in the rachis and pinna but depleted in the pinnules; an opposite distribution pattern was observed for Ca (Fig. 10).

Fig. 9
figure 9

Laboratory micro-X-ray fluorescence (µXRF) maps of Zn, Mn, K and Ca of hydrated leaves of Phytolacca octandra grown on the Mt Isa Mine substrate

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Fig. 10
figure 10

Laboratory micro-X-ray fluorescence (µXRF) maps of Zn, Mn, K and Ca of hydrated leaves of 1) Noccaea caerulescens only on Mt Isa Mine substrate; 2) Noccaea caerulescens only on Dugald River Mine substrate; 3) Noccaea caerulescens co-cultured with Biscutella laevigata on Dugald River Mine substrate; 4, 11, 12) Pityrogramma calomelanos co-cultured with B. laevigata on Mt Isa Mine substrate; 5) Lupinus albus co-cultured with B. laevigata on Mt Isa Mine substrate; 6) Biscutella laevigata co-cultured with P. calomelanos on Mt Isa Mine substrate; 7) Biscutella laevigata co-cultured with N. caerulescens on Mt Isa Mine substrate; 8) Biscutella laevigata only on Mt Isa Mine substrate; 9) Biscutella laevigata only on Dugald River Mine substrate; 10) Biscutella laevigata (flower) on Mt Isa Mine substrate

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Discussion

Noccaea caerulescens performed well in all of the treatments in the Dugald River Mine and Mt Isa Mine substrates, including substrates with relatively higher Mn and Zn concentrations, and accumulated these metals at high concentrations in its leaves (reaching ~ 1 wt% Mn and ~ 1.4 wt% Zn) (Figs. 1, 2, Tables 2 and 3). This finding shows that N. caerulescens is highly tolerant to elevated Mn and Zn and is a prime candidate for phytoextraction on these substrates. The elemental maps reveal co-localization of Mn and Zn (together with Ca) in the leaf tips and margins (Fig. 5), suggesting similar storage mechanisms (Callahan et al. 2016). However, limited research exists on Zn distribution in N. caerulescens at the whole leaf level (Callahan et al. 2016; do Nascimento et al. 2021) as opposed to that at the cellular and sub-cellular levels (Küpper et al. 1999). Recent evidence suggests that the Zn distribution in whole leaves of N. caerulescens may be accession dependent (van der Zee et al. 2021), with some accessions accumulating Zn in the cells surrounding the central and secondary veins (do Nascimento et al. 2021), whereas other accessions preferentially store Zn at the leaf tip (Callahan et al. 2016). The Mn localization in the margins of N. caerulescens is consistent with that of the Mn hyperaccumulator P. octandra (Fig. 9), Mn hypertolerant L. albus (Fig. 10; Blamey et al. 2018) and several other Mn hyperaccumulator plants, including Gossia bidwillii, G. fragrantissima, Denhamia bilocularis, D. silvestris and P. americana (Abubakari et al. 2021a,b, 2022; Xu et al. 2006). The Mn localization in the margins of these species is attributed to increasing transpiration rates closer to leaf margins which result in greater Mn accumulation in leaf marginal areas (Abubakari et al. 2021a,b).

The monocultured B. laevigata in the high Mn and Zn substrate (Dugald River Mine) exhibited severe toxicity symptoms (Fig. 1G). The elemental maps show preferential accumulation of elevated Zn in the base of the trichomes, similar to that of Arabidopsis halleri and sunflower (Küpper et al. 2000; Li et al. 2019, 2021; Sarret et al. 2009). The movement of excess Zn into the base of trichomes, likely into cell wall, lowers the Zn concentrations in the remainder of the leaf, which is an important Zn detoxification mechanism (Li et al. 2019, 2021). It is likely that continued Zn loading, in excess of trichome base storage capacity, of the monocultured B. laevigata on the Dugald River Mine substrate increased the Zn concentrations in the remainder of the leaf, thereby partly contributing to its toxicity (Fig. 6). Future dosing trials could test the Zn concentration threshold at which toxicity starts in B. laevigata. In addition, time-resolved µ-XRF techniques could clarify the mechanism of Zn toxicity in B. laevigata, as has been demonstrated for Mn toxicity in soybean and sunflower (van der Ent et al. 2020). The storage of Mn in trichomes is an important detoxification mechanism for several Mn hypertolerant plants (Blamey et al. 1986, 2015; Broadhurst et al. 2009; McNear and Küpper 2014). Excess Mn in the trichomes due to continued Mn loading starts to accumulate in the apoplastic space or walls of the surrounding cells, as observed in Odontarrhena chalcidica (Alyssum murale) and sunflower (Blamey et al. 1986, 2015; McNear and Küpper 2014). This mechanism may partly explain the toxicity symptoms in the monocultured B. laevigata in the Dugald River Mine substrate. However, the B. laevigata plants co-cultured with N. caerulescens on the high Mn and Zn substrate did not show any toxicity symptom. It is possible that N. caerulescens reduced Mn and Zn availability to B. laevigata (Fig. 8; Table 4). Moreover, the monoculture B. laevigata grown in the Mt Isa Mine substrate did not experience any toxicity symptom partly because all the Mn accumulated in the trichomes, whilst Zn accumulated in the midrib and veins (Fig. 7).

Conclusions

A major drawback with phytoextracting a targeted metal from polymetallic substrate is that there may be other non-targeted metals that may be toxic to the selected hyperaccumulator plants, in a process described as ‘co-metallic effect’ (Anderson et al. 2013). The discovery of a communalistic relationship, at least in terms of growth, for these model hyperaccumulator plants overcomes this drawback, and suggests that, in principle, it may be possible to tailor-make a purposeful assemblage of hyperaccumulators for any polymetallic substrate. The facilitation of metal accumulation in selected hyperaccumulators by root-exudates producing species is also worth investigating further. We suggest that this nature-based metal phytoavailability enhancement may provide an alternative to the use of cost prohibitive and environmentally hazardous synthetic chelates, such as ethylene diamine tetraacetic acid (EDTA) (Grčman et al. 2001; Meers et al. 2005; Nowack et al. 2006). The relatively low foliar concentrations of Fe, Cu, As, Pb show the ability of the model species to biopurify the target metals, which is potentially beneficial in the eventual metal recovery process. The production of high purity metal salts could partly meet the growing demand for battery metals (Nkrumah et al. 2022). The findings in this study provide baseline information for developing in situ polymetallic phytoextraction of base metal mine tailings. However, long-term laboratory and field trials using a purposeful assemblage of hyperaccumulators for polymetallic mine tailings substrates under various climatic conditions are required to provide real-life polymetallic phytoextraction demonstration. Moreover, these long-term studies are needed to build economic viability of this approach while ascertaining the number of cropping years required to reduce the metals to acceptable concentrations. Apart from base metal mine tailings, other potential phytoextraction substrates are polymetallic in nature (e.g., ultramafic soils are usually enriched in Mn, Co and Ni) and could benefit from the same approach.