4.1. Endophytes and compost synergistically improved B. curtipendula growth
Both compost and endophytes have established positive impacts on plant growth and re-vegetation of mine waste (Larney and Angers 2012; Santoyo et al. 2016; Rho et al. 2018). Organic amendments like compost are often required for successful plant growth in acidic, polymetallic tailings (Yang et al. 2016; Xie and van Zyl 2020). As observed in other studies, the municipal waste compost added during planting of B. curtipendula seedlings was an important source of nutrients and organics that facilitated plant germination and growth (Fig. 1). These improvements were realized regardless of whether the seeds were coated with microbial endophytes, and in some instances (e.g., for nitrogen) were independent of whether a seed was planted (Fig. 2). Our roughly 5% (w/w) compost additions are lower than those used for field scale re-vegetation of similar acidic metalliferous mine tailings in semi-arid environments (e.g., 15% w/w compost addition; Gil-Loaiza et al. 2016). We expected that the added diazotrophic endophytes coated on the seeds would have a similar function as compost, providing additional nutrients and soil microorganisms to improve plant growth (Wood et al. 2016) while reducing the bioavailability of phytotoxic elements (e.g., Cd, Pb) (Barzanti et al. 2007; Ma et al. 2016). However, per plant biomass yields for B. curtipendula grown with a combined compost and endophyte addition were only about 25% of those observed for buffalo grass grown in acidic polymetallic tailings (albeit in tailings with nearly 10 times less lead; Solís-Dominguez et al. 2012). This suggests further growth improvements in these tailings may be realized with higher compost addition rates. Lead toxicity may also be reduced with more compost. Lead (and other phytotoxic elements) sorbs onto compost (Geebelen et al. 2002; Smith 2009; Forján et al. 2016) and in our experiment Pb was mobilized into and stabilized in the compost layer.
Coating B. curtipendula seeds with endophytes improved plant growth and germination with compost, but there was no independent benefit of the endophyte-coated seeds on plant growth in the absence of compost (Fig. 1). In fact, we observed marginal decreases in root growth with the endophyte coating alone. Although endophytes can be mutualistic, there can be metabolic costs to the plant-endophyte relationship. The ultimate impact on plant growth is thus dependent on the biotic and abiotic environment (Hoeksema et al. 2010; Partida-Martinez and Heil 2011). For instance, under an environmental stress like nutrient limitation, endophytes would only improve plant growth when that environmental stress is alleviated (Cheplick et al. 1989). The high metal concentrations of the tailings may have killed endophytes during seed germination (McGrath et al., 1995) when they were planted directly into the tailings rather than between the compost-tailings interface. It is also possible that the endophytes—which are derived from poplar and willow species (Doty et al. 2005; Doty et al. 2009; Xin et al. 2009; Firrincieli et al. 2015)—were unable to colonize B. curtipendula since endophyte communities can be species-dependent (Wang et al. 2012; Papik et al. 2020). However, endophyte species specificity can be plastic (Cobian et al. 2019), and the endophytes we used can colonize and improve the growth of both herbaceous (e.g., rice, corn) and woody plants in non-contaminated environments (Khan et al. 2015; Kandel et al. 2017b). Therefore, we suspect that the harsh environmental conditions of the tailings without a compost topdressing (e.g., nutrient limitations or metal toxicity) may have inhibited the growth benefits of the added endophytes.
But even when compost was present, and where we observed improvements in growth with an endophyte-coated seed, the sequence of only one of the added endophytes (Rhizobium sp.) was uniquely identified. The finding of an additional six sequences of the ten added endophytes in uncoated seeds indicates B. curtipendula has indigenous endophytes with ≥ 99% sequence identity with the inoculants. Identifying endophytes in plants grown from field-harvested seeds is not unexpected. Endophytes are ubiquitous in plants and finding an endophyte-free plant would be the exception rather than the rule (Partida-Martinez and Heil 2011). Although the endophytes selected were functionally redundant, finding the sequences of endophytes in both the endophyte coated and uncoated seeds weaken the causal link between endophyte presence and improved growth. However, it is possible that Rhizobium sp. was responsible for the growth benefits observed through direct (e.g., N2 fixation) or indirect mechanisms (e.g., localized pH changes) (Kuiper et al. 2004; Valentín-Vargas et al. 2014).
4.2. Greatest improvements in soil fertility with a combined compost and endophyte addition
The establishment of a functional belowground ecosystem is critical to short- and long-term phytostabilization success (Marques et al. 2009). This includes not only restoration of the microbial community (Valentín-Vargas et al. 2014; Zhou et al. 2020) but also increases in soil organic matter and improvements in pH and soil structure (Huang et al. 2012). The rhizosphere is a hotspot of biological and geochemical activity relative to the surrounding bulk soil (Kuzyakov and Blagodatskaya 2015; Kuzyakov and Razavi 2019). Thus, developing an expansive root network is a target for areas undergoing remediation (Karthikeyan and Kulakow 2003; Saravanan et al. 2020) as it effectively increases the volume of soil affected by root processes (Ma et al. 2018). Plant roots cause local changes in pH and exude organic acids—both of which can cause the desorption and mobilization of metal(loid) contaminants (Yang et al. 2006; Schwab et al. 2008) as well as soil carbon (Keiluweit et al. 2015). Carbon formation is typically faster and more efficient in the rhizosphere (Sokol and Bradford 2018), and the influence of an expanding rhizosphere on soil carbon storage is an active area of research (Fontaine et al. 2007; Guenet et al. 2018; Zhao et al. 2022). In addition to its importance to plant growth and soil fertility (Tiessen et al. 1994; Oldfield et al. 2019), restoration of organic carbon formation processes during phytostabilization can provide an indirect monetary benefit if we consider the social cost of carbon emissions ($10–90 USD per metric ton of CO2; Stern et al. 2007; Nordhaus 2007).
In our study, compost was critical to forming organic matter (carbon and nitrogen) and allowing for microbial growth in the tailings. Although there was an additional increase in soil carbon with endophyte-coated seeds—likely due to the larger developing root network—it did not measurably increase nitrogen concentrations. Independent of the impacts on organic matter and microorganisms, a larger root network is beneficial to the physical and hydrologic properties of tailings (Huang et al., 2012). The poor hydraulic conductivity and compacted structure of mine tailings can be improved after growth of pioneer plants like grasses, allowing for other plant species to establish (Guittonny-Larchevêque et al. 2016). Reclamation is more successful long-term when multiple plants are revegetating the mine waste, either by diverse initial plantings or from ecological succession (Holl 2002; Juge et al. 2021). Thus, cost-effective amendments that provide the best improvements to soil structure, hydrology, organic matter, and microbial biomass for native plants—like our combined lime, endophyte, and municipal waste compost additions—should be expected to provide the best long-term outcomes for restoring belowground ecosystems (Cooke and Johnson 2002).
4.3. Optimal phytostabilization with a combined compost and endophyte addition
We quantified concentrations of potentially toxic major, minor, and trace elements in multiple pools (plants, compost, tailings, water) to provide a complete picture of contaminant solubilization and stabilization during the experiment. Overall, the concentrations of potentially toxic elements between the different pools were not strongly correlated, so that each pool provided unique information on the dynamics of elemental transformations during our experiment. A seed coating of endophytes had two primary effects: increasing B. curtipendula foliar yields of Cd, Co, Mn, and Zn and marginally lowering solid phase concentrations of Cu, S, Fe, Pb, and Zn (figures in Online Resource 1). The endophytes added in the seed coating produce siderophores and solubilizes calcium phosphate (Doty et al. 2009; Khan et al. 2015; Kandel et al. 2017a). Both these functions can enhance the mobilization of metal(loids) in mine waste by chelating iron (siderophores) or solubilizing minerals by phosphate solubilization (Ma et al. 2016). Copper, Pb, and Zn are held in Fe-bearing phases in these tailings (Creamer et al. 2022b). Potentially, the production of siderophores by endophytes in the seed coating may have mobilized these elements, resulting in their preferential loss from pots with added endophytes. Production of siderophores by endophytes has been associated with higher plant metal uptake (Ma et al. 2011b), although in our study only Zn had higher foliar yields (out of the Fe-associated elements Cu, Pb, and Zn). In the water extracts, Cd, Co, Mn, and Zn—which are all mobile elements in soils at pH 5—were moderately correlated with each other (Spearman’s r > 0.5) and were less extractable from tailings with endophyte coated seeds. These elements (Cd, Co, Mn, Zn) were also the elements that had higher foliar yields with endophytes, suggesting a potential higher mobility (and thus plant uptake) of these elements with endophytes. Most of the observed benefits of endophytes for phytostabilization come from their improvements to plant growth rather than metal(loid) solubilization (Ma et al. 2011a; Wood et al. 2016). In fact, higher solubilization of potentially phytotoxic elements (e.g., Pb, Cd) may have caused the marginally lower rhizosphere development observed for plants grown from endophyte-coated seeds in the absence of compost. Although our study indirectly suggests greater solubilization of Fe- and mineral-associated elements, the higher phytostabilization with the combined endophyte and compost addition predominantly resulted from the enhancement of foliar biomass rather than enhanced accumulation. For mine waste with appreciable amounts of Cd, Co, Mn, or Zn (e.g., Ni-Co laterites), applying endophytes with similar Fe-chelating and mineral solubilizing functions, or that allow for rhizosphere expansion, may improve foliar yields from native plants to aid with extraction focused remediation (i.e., phytoextraction) provided phytotoxic effects of mobilized elements are mitigated (e.g., through compost addition).
For successful phytostabilization, potentially toxic trace elements should not be translocated into foliar plant tissues (reducing potential grazing animal exposure) and should instead be immobilized on plant roots or in the soil (Mendez and Maier 2008a; Xie and van Zyl 2020). Bouteloua curtipendula is thus suitable for phytostabilization because foliar translocation was low ( < < 1) under our experimental conditions, but bioconcentration of potential contaminants in the roots was high, particularly for Cd, Co, and Mn (root: soil ratios > > 1). The root concentrations of potentially toxic elements (i.e., the elements we would target for phytostabilization) were decreased in tailings with a compost topdressing. Lowered bioavailability and sorption of potentially toxic elements like Cd, Cu, Pb, Mn, and Zn after compost addition has been observed elsewhere (Geebelen et al. 2002; Smith 2009; Forján et al. 2016). Based on the observed mobilization of Cd, Mn, and Pb into the compost layer during our experiment and the lower water extractability of Pb, our results are consistent with lower bioavailability of Cd, Mn, and Pb with compost addition. Although compost lowered the concentrations of potentially toxic trace elements in both B. curtipendula roots and shoots, this translated to lower yields only in the shoots. Lowered foliar yields is beneficial for phytostabilization of tailings with high concentrations of potentially toxic trace elements (Mendez and Maier 2008a). In our study, this translated to a lower environmental risk to grazing animals.
4.4. Implications for mine site reclamation
The primary elements of concern in these tailings were As, Cd, Mn, Pb, and Zn. During the experiment only Mn and Pb (and to a lesser extent Zn) showed decreasing solid phase concentrations. If we consider the compost, water, and plants to be potential sinks for these elements, we recovered about 50% of the Zn and about 30% of the Mn, split equally between the water extract and the compost. However, less than 5% of the decrease in solid phase Pb was found in the plants, water, or compost, indicating that a substantial portion of the Pb was mobilized out of the bottom of the pot during the experiment. Despite high concentrations of these elements in B. curtipendula roots, because of their small size, less than 1% of these elements were recovered in B. curtipendula roots or shoots, suggesting higher stabilization potential for bigger plants (Sessitsch et al. 2013; Wood et al. 2016). Although suitable for enhanced Mn and Zn stabilization in B. curtipendula roots or shoots, simply growing larger plants would likely stabilize only a portion of this mobilized Pb as the bioconcentration factor (root: soil ratio) of Pb for our experiment ranged roughly between 0.2 and 1. Instead, this Pb could be immobilized by greater compost additions, by selecting organic amendments targeted for high Pb sorption (e.g., biochar; Park et al. 2013), by phosphate amendments to form lead phosphate (Miretzky and Fernandez-Cirelli 2008; Zeng et al. 2017), or by placing sorbents in areas of water outflow (Delkash et al. 2015).
Remediation of sulfidic polymetallic mine tailings is challenging, particularly when concentrations of phytotoxic metals and metalloids are exceedingly high. Here, we establish that applying municipal waste compost and lime, along with a seed coat of diazotrophic endophytes, improves the growth of a widespread perennial grass, Bouteloua curtipendula, and provides the greatest increases in soil organic matter and trace element stabilization. A similar approach employed in the field, focused on increasing pH and providing a source of microorganisms and nutrients, would likely improve remediation resultd (e.g., Gil-Loaiza et al. 2016). This combination is particularly suitable for improved phytostabilization of Cd, Mn, or Zn into B. curtipendula roots while lowering foliar concentrations, or for targeted foliar concentration of Co. Compared to other studies, the exceeding high Pb concentration in tailings—roughly 16,000 mg/kg compared to a typical range of 10–4500 mg/kg for sulfidic tailings and established plant toxicity levels of 100–500 mg/kg (Mendez and Maier 2008a; Xie and van Zyl 2020)—are a barrier to effective re-vegetation of these barren tailings piles. A combination of compost and endophytes apparently reduced the toxicity of Pb in the tailings by enhancing stabilization (compost) and mobilization (endophytes). Direct planting of B. curtipendula seedlings into tailings with similarly elevated Pb concentrations may require additional amendments to immobilize Pb both to prevent offsite transport and improve plant growth, while our presented approach is appropriate for sulfidic tailings with substantial Cd, Mn, and Zn contamination.