ll OPEN ACCESS iScience Article Ecological engineering of iron ore tailings into useable soils for sustainable rehabilitation Songlin Wu, Yunjia Liu, Gordon Southam, ..., Narottam Saha, Yong Sik Ok, Longbin Huang l.huang@uq.edu.au Highlights Soil aggregate formation through eco-engineering of inhospitable Fe ore tailings Microbial and rhizosphere activities drove soil aggregate formation in tailings Mineral weathering was enhanced and colloidal Fe(III)-Si mineral gel was generated Conceptual model was proposed for aggregate development with mineral gel as backbone Wu et al., iScience 26, 107102 July 21, 2023 ª 2023 The Author(s). https://doi.org/10.1016/ j.isci.2023.107102 mailto:l.huang@uq.edu.au https://doi.org/10.1016/j.isci.2023.107102 https://doi.org/10.1016/j.isci.2023.107102 http://crossmark.crossref.org/dialog/?doi=10.1016/j.isci.2023.107102&domain=pdf iScience Article Ecological engineering of iron ore tailings into useable soils for sustainable rehabilitation Songlin Wu,1 Yunjia Liu,1 Gordon Southam,2 Tuan A.H. Nguyen,1 Kurt O. Konhauser,3 Fang You,1 Jeremy J. Bougoure,4 David Paterson,5 Ting-Shan Chan,6 Ying-Rui Lu,6 Shu-Chih Haw,6 Qing Yi,1 Zhen Li,1 Lachlan M. Robertson,1 Merinda Hall,1 Narottam Saha,1 Yong Sik Ok,1,7 and Longbin Huang1,8,* SUMMARY Ecological engineering of soil formation in tailings is an emerging technology to- ward sustainable rehabilitation of iron (Fe) ore tailings landscapes worldwide, which requires the formation of well-organized and stable soil aggregates in finely textured tailings. Here, we demonstrate an approach using microbial and rhizosphere processes to progressively drive aggregate formation and develop- ment in Fe ore tailings. The aggregates were initially formed through the agglomeration of mineral particles by organic cements derived from microbial decomposition of exogenous organic matter. The aggregate stability was consol- idated by colloidal nanosized Fe(III)-Si minerals formed during Fe-bearing primary mineral weathering driven by rhizosphere biogeochemical processes of pioneer plants. From these findings, we proposed a conceptual model for progressive aggregate structure development in the tailings with Fe(III)-Si rich cements as core nuclei. This renewable resource dependent eco-engineering approach opens a sustainable pathway to achieve resilient tailings rehabilitation without resort- ing to excavating natural soil resources. INTRODUCTION Billions of tons of mine tailings are generated annually from extracting and processing of metal andmineral ores,1–3 stockpiled in about 4,800 mine tailings storage facilities (TSFs),4 occupying >240,000 ha of land worldwide.4 Iron (Fe) ore tailings are one of the most challenging global tailings liability,5 with over 1.4 billion tons of tailings generated each year.6 These tailings are not only detrimental to the local envi- ronment, but they also have a significant CO2 footprint associated with rehabilitation and mine closure. Moreover, the traditional soil cover methods consume non-renewable natural soil resources and destroy the local soil ecosystem because of the reliance on excavating and transporting large volumes of soil from natural landscapes. Although Fe-ore tailings pose little pollution risks of heavy metals (unlike metal mine tailings, such as Pb-Zn tailings), they are not suitable for direct colonization of soil microbes and plants. This is because of their finely textured and highly compacted physical structure, and adverse chemical properties (i.e., alkaline pH, saline conditions, low organic matter and available nutrients).7 Past efforts to revegetate tailings have largely failed, because the effectiveness of remediation methods were short-lived, without acceler- ating pedological processes (e.g., mineral weathering and aggregation) for soil structure and hydro- geochemical stability development in the tailings.8 A sustainable approach is urgently required to carry out ecological rehabilitation of Fe ore tailings storage dams without secondary damages to natural land- scapes for soil excavation. Evidence so far has demonstrated that one way forward is to develop tailings into soil-like substrates (termed herein technosol) with soil structures capable of supporting sustainable vegetation cover.7,9 This approach adopts ecological engineering (eco-engineering) using abiotic and biotic inputs (i.e., organic matter, pioneer plants, soil microbes, and irrigation) within the context of soil pedogenesis.9 The critical step in this eco-engineering process is to initiate and accelerate formation of functional soil aggregates in the finely textured tailings, for diverse plant colonization.10 1Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane,QLD 4072, Australia 2School of Earth & Environmental Sciences, The University of Queensland, Brisbane,QLD 4072, Australia 3Department of Earth & Atmospheric Sciences, University of Alberta, Edmonton, AB T6G 2E3, Canada 4Centre for Microscopy, Characterisation and Analysis, University of Western Australia, 35 Stirling Hwy, Crawley, Perth, WA 6009, Australia 5Australian Synchrotron, Melbourne, VIC 3168, Australia 6National Synchrotron Radiation Research Centre, Hsinchu Science Park, Hsinchu 30092, Taiwan 7Korea Biochar Research Center, APRU Sustainable WasteManagement Program & Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Korea 8Lead contact *Correspondence: l.huang@uq.edu.au https://doi.org/10.1016/j.isci. 2023.107102 iScience 26, 107102, July 21, 2023 ª 2023 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 ll OPEN ACCESS mailto:l.huang@uq.edu.au https://doi.org/10.1016/j.isci.2023.107102 https://doi.org/10.1016/j.isci.2023.107102 http://crossmark.crossref.org/dialog/?doi=10.1016/j.isci.2023.107102&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ Stable and functional aggregates are key physical units underpinning the development of soil structure for regulating water retention, gaseous exchanges, soil organic matter and nutrient dynamics and biology capacity (e.g., root penetration and microbial colonization).11,12 The formation of soil aggregates involves assemblage of soil clay mineral particles with organic matter in natural soil.11 Previous studies with natural soil aggregates have emphasized the role of transient organic cements rather than mineral cements in the formation and stability of soil aggregates.13,14 In fact, ferric iron (Fe(III))-rich amorphous minerals have been considered as potentially important reagents contributing to aggregation in natural soil formed from Fe- rich parent lithologies.15,16 Microbes and plants play critical roles in stimulating bioweathering of primary minerals, especially ferrous iron (Fe(II))-bearing phyllosilicates and the generation of amorphous Fe(III) rich secondary minerals.17,18 Iron ore tailings can be treated as engineered parent materials rich in Fe bearing phyllosilicates, such as biotite and amphibole, without risks of heavy metal toxicity.7 These primary minerals can be weathered to secondary and amorphous Fe(III) rich mineral cements19,20 because of microbial and rhizosphere activities.19,21 These amorphous Fe(III) minerals act as core nuclei for cementing together finely textured mineral particles, adsorbing and sequestering organic carbon, and forming water-stable aggre- gates. As a result, it is critical to resolve how to initiate and accelerate the generation of amorphous Fe(III) mineral cements for aggregating fine mineral particles and sequestering organic matter into Fe ore tailings. In the present study, we demonstrate stable aggregate development in the Fe ore tailings through an eco-engineering procedure initiated with tolerant microbes and pioneer plants. It was found that the aggregate stability increased with more colloidal Fe(III)-silica (Si) rich amorphous minerals in the tailings. The amorphous Fe(III)-Si cement formation could be stimulated through continuous microbial and rhizo- sphere driven biogeochemical processes. By comparing the newly formed aggregate structure in tailings with those of native Fe rich soils, we then develop a conceptual model for stable aggregate development resulting from continuous generation and accumulation of Fe(III)-Si rich cement during mineral weathering of the Fe ore tailings. We propose that this process is the critical step toward the development of soil structure for ecological rehabilitation. RESULTS Physical and chemical changes in tailings subject to eco-engineering inputs In a glasshouse experiment, rapid pH neutralization was achieved and geochemical properties were improved within one month after amending alkaline Fe-ore tailings with 2% (w/w) Lucerne hay (shoot biomass of Alfalfa, Medicago sativa) and native soil microbes (Figure 1, Table S1A). The resultant tailings were sufficiently improved to have developed physical and chemical conditions to accommodate success- ful colonization by pioneer plant species for 3.5 months (Figure 1, Table S1A), including Atriplex amnicola, Maireana brevifolia, and Sorghum spp. Hybrid cv. Silk. By this stage the eco-engineered tailings are consid- ered as an ‘‘early technosol’’ supporting the proliferation of pioneer plants (Figure 1). The colonizing pioneer plants further advanced the development of ‘‘early technosol’’ toward ‘‘advanced technosol’’ through rhizosphere driven biogeochemical processes, leading to further improved physical and chemical properties in tailings (Figure 1, Table S1). The ‘‘advanced technosol’’ is expected to be structurally and functionally capable of supporting the colonization of diverse native keystone plant species and rhizo- sphere microbes such as mycorrhizal symbiosis. These accumulative impacts of microbial and rhizosphere processes in the technosol significantly (p < 0.05) shifted the development of key physical and chemical properties away from those in the initial tailings (Figures 2A and 2B; Table S1) and toward the onset of early and advanced technosol (ANOSIM test, p < 0.05; Figure 2B). Typically, the pH decreased from extreme alkaline (>9.5) in initial tailings to circumneutral (8.5–8.9) in ‘‘early technosol’’ and ‘‘advanced technosol’’ (p < 0.05). Total organic carbon (TOC) concentration increased from<0.5 g kg�1 to 2–4 g kg�1 in technosols (p < 0.05, Table S1). Plant colonization decreased electrical conductivity (EC) value significantly in ‘‘advanced technosol’’ (p < 0.05, Tables S1B and S1C). However, at this stage the resultant ‘‘advanced tech- nosol’’ have not yet reached the same property state of native Fe-rich soil at the Fe ore mine site according to principal component analysis (PCA) of physical and chemical variation (ANOSIM test, p < 0.05; Figure 2B). According to PCA analysis, macroaggregate percentage and MWD value were key contributors to PC1 (contributing 54.3% of the variation). Similarly, microaggregate percentage was the dominant contributor to PC2 (contributing 19.7% of the whole variation) (Figure 2A). These values indicate that aggregate stability (physical property) was the dominant factor differentiating properties of tailings, technosols, ll OPEN ACCESS 2 iScience 26, 107102, July 21, 2023 iScience Article and native soil. Furthermore, the percentage of macro- and micro-aggregates, as well as the value of mean weight diameter (MWD, a common indicator of soil aggregate stability11 and soil physical functionality22) increased progressively during the eco-engineering processes toward technosol formation (p < 0.05, Figures 2C–2E). Microstructure and cementation of aggregates formed in ‘‘early technosol’’ There was a heterogeneous distribution pattern of small-sized and irregular Fe-bearing minerals adhered onto quartz particles within macroaggregates (Figures 3A–3C). The Fe concentrations varied from 0 to 50% w/w (Figures 3B and 3C) across selected aggregate transections as revealed by synchro- tron-based X-ray fluorescence microscopy (XFM) analysis. The Fe-bearing minerals were closely associ- ated with biotite, amphibole and FeOx (representing Fe oxides and/or hydroxides), according to the mineral liberation analyzer (MLA) and BSE-EDS analyses (Figures 3D–3H), which were distributed randomly amongst the quartz grains. The dominant presence of biotite (a K rich mica) was further sup- ported by the strong Fe-K association (i.e., areas with Fe: K ratio of �9.98) in the tri Fe-K-Ca mapping (Figure S1) and correlation mapping (Figure S2). Little mineral cement was detected in adjoining spaces surrounding primary mineral particles in the tailings (Figures 3D and 3F). As characterized by synchro- tron-based C 1s NEXAFS analysis (Figure 3I) and high resolution C 1s XPS (Figure S3), the organic carbon (OC) in the aggregates formed in the ‘‘early technosol’’ were mainly composed of aromatic, carboxyl, phenolic, alkyl and O-alkyl rich compounds. The NanoSIMS analysis revealed that those OC in the aggre- gates was heterogeneously distributed at micron scale and mainly associated with Fe rich minerals (Figures 3J–3L). Microstructure and cementation of aggregates formed in ‘‘advanced technosol’’ During the ‘‘advanced technosol’’ formation, the percentage of microaggregates and MWD value increased significantly in response to colonization of various plant species (p < 0.05, Figure 2D), except for Sorghum spp. plants that could not survive in ‘‘early technosol’’ without 10% (w/w) native soil amend- ment (Figure S4). The aggregates in the ‘‘advanced technosol’’ contained mostly quartz and biotite, Figure 1. A schematic diagram illustrating the eco-engineering Fe ore tailings into soil-like substrates Process of ‘‘early technosol’’ formation – characteristic of organic matter and microbial inoculation inputs for pH neutralization, geochemical improvement, and alleviation of physical compaction (early aggregate formation). The ‘‘early technosol’’ can support colonization of pioneer tolerant plants. Process of ‘‘advanced technosol’’ formation – characteristic of the development of well buffered geochemical properties and improved physical structures (advanced aggregate formation). The ‘‘advanced technosol’’ is expected to be structurally and functionally capable of supporting the colonization of diverse native keystone plant species and associated microbes. The photos on the right show growth of Atriplex amnicola, Maireana brevifolia, and Sorghum spp. Hybrid cv. Silk. in tailings for 3.5 months after transplantation. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 3 iScience Article amphibole and FeOx, but more irregular Fe-Si rich minerals were found in the pore sites surrounding those primary mineral particles within microaggregates from pioneer plant colonized techno- sols (Figure 4). Figure 2. Overall variations of physical and chemical properties, as well as water stable aggregate distribution in the tailings, technosols and the native soil (A and B) Principal component analysis (PCA) of physical and chemical characteristics (pH, EC, TOC, Feox, Alox, Fed, Ald, Pox, Kox, Mgox and Caox, as well as aggregate fractions and stability, all data were standardized) in initial tailings, ‘‘early technosol’’, ‘‘advanced technosol’’ and native soil. ANOSIM test indicates the significant difference between tailings/technosols and native soils (R = 0.999, p = 0.001), initial tailings and early technosol (R = 1; p = 0.02), initial tailings and advanced technosol (R = 0.999, p = 0.001), early technosol and advanced technosol (R = 0.807, p = 0.001). (C–E) The distribution and mean weight diameter (MWD) of water stable aggregates in (c) early technosol, (d) advanced technosol, and (e) native Fe rich soil surrounding the tailing site. The water stable aggregate classification: macroaggregates (250–2000 mm), microaggregates (53–250 mm), discrete particles (<53 mm). Data are represented as mean G SEM. Different letters above the columns show significant difference between different treatments based on Tukey test at p < 0.05. Note: ‘‘TL’’, Tailings + Lucerne hay (2% w/w); ‘‘TLS’’, Tailings + Lucerne hay (2% w/w) + Native soil (10% w/w); ‘‘Aa’’, Atriplex amnicola; ‘‘Mb’’, Maireana brevifolia; ‘‘SHS’’, Silk sorghum grass (Sorghum spp. Hybrid cv. Silk). ll OPEN ACCESS 4 iScience 26, 107102, July 21, 2023 iScience Article Figure 3. The microstructure of aggregates from the ‘‘early technosol’’ as revealed by synchrotron based X-ray fluorescence microscopy (XFM), mineral liberation analysis (MLA), and backscattered-scanning electronmicroscope-energy dispersive X-ray spectroscopy (BSE-SEM-EDS) analysis (A) A thin section of tailing macroaggregates under light microscopy. (B) Fe mapping of the tailing macroaggregate thin sections as revealed by XFM analysis. (C) Fe concentrations across the selected area of the thin sections of aggregates in ‘‘b’’ (the distance in X axis is from the left to the right). (D–F) BSE-SEM images showing the microstructure of the aggregates and their mineral distribution. (G) XRD spectra of macroaggregates (250–2000 mm) and microaggregates (53–250 mm) in early technosol eco-engineered from tailings. Note: Qz = Quartz; Mag = magnetite; Bt = Biotite (K(Mg,Fe)3(AlSi3O10)(F,OH)2); Rct = Richterite ((Na,K)2(Mg,Mn,Ca)6Si8O22(OH)2), an amphibole; Mfr = magnesioferrite (Mg(Fe3+)2O4), FeOx = Fe oxides, and/or hydroxides. (H) k space Fe K-edge EXAFS spectra (weight 3) (line) and linear combination fitting (LCF, dashed) of early technosol aggregates. The right column shows the results of the fitting Fe K-edge EXAFS spectra. The fitting parameters: For Macro-aggregate: R factor = 0.069, Chi-square = 41.6, Reduced chi-square = 0.23; For Micro-aggregate: R factor = 0.092; Chi-square = 50.6; Reduced chi-square = 0.28. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 5 iScience Article The increasing accumulation of mineral cements in aggregates was further delineated through detailed examination of the mineralogy and OC forms in different fractions of microaggregates (i.e., colloidal and non-colloidal fraction) in comparison with that in bulk tailings (Figures 5 and 6). According to the linear combination fitting (LCF) of Fe K edge XAFS spectra, minimal changes in Fe phases were detected in the bulk tailings, which was mainly composed of biotite and magnetite-like minerals, regardless of tailings treatments (Figures 5A and S5). The proportion of biotite-like minerals decreased in colloidal and non- colloidal fraction of microaggregates, compared to that in the bulk tailings (Figure 5A). This was further confirmed by XRD analysis that revealed the reduction of crystalline primary minerals (i.e., biotite and amphibole minerals) and the emergence of mixed layered minerals (such as illite and smectic groups) in the colloidal and non-colloidal fractions of the microaggregates (Figures 5B and S6). By contrast, Fe(III)- Si-short range ordered (SRO) like minerals were found to be dominant in the colloidal fraction (Figure 5A); they appeared to be around 10 nm in size and were identified as Fe-Si rich poorly crystalline minerals (Figures 5C and S7). Plant colonization in the tailings consistently increased the percentage of the colloidal fraction in themicro- aggregates (Figure 5D) and the proportion of Fe(III)-Si-SRO like minerals within the colloidal fraction (Fig- ure 5A). Ferrous iron (Fe(II)) oxidation into Fe(III) (Figure S8), and coupled co-precipitation with silica, formed a highly amorphous structure with few double corner interactions around the Fe core (Figure S9). This presented a high capability for further interactions with organics and other minerals. These colloidal Fe(III)-Si-SRO minerals were readily associated with organic carbon as evidenced by elevated OC contents in colloidal fraction, compared to those in the non-colloidal fraction of microaggre- gates and bulk tailings (Tables S1 and S2). The functional groups of the organics within microaggregates weremainly composed of carboxyl, alkyl, O-alkyl, carbonyl, and aromatic groups in both colloidal and other fractions (Figures 6 and S10). Comparatively, plant colonization increased the percentage of aromatic, car- boxylic, and carbonyl groups, but decreased the percentage of O-alkyl groups (from polysaccharides) in the colloidal fraction, as revealed by synchrotron C 1s NEXAFS analysis (Figure 6). This was confirmed by ATR-FTIR analysis, which revealed that plant colonization enhanced peaks at around 1633 cm�1 and 1423-1436 cm-123 (Figure S11), indicating the possible association of aromatic and/or carboxyl groups with minerals (such as Fe(III)-Si rich SRO minerals) in the rhizosphere colloidal fraction. Geochemical changes during rhizosphere driven ‘‘advanced technosol’’ formation The plant colonization altered geochemical characteristics of the tailings porewater. Generally, the pore- water pH was circumneutral (7.2–8.2) but it was generally increased by plant colonization with time (Fig- ure S12, p < 0.05). By contrast, the electrical conductivity (EC) value was decreased by plant colonization with time (p < 0.05, Figure S12). Porewater TOC and TN concentrations increased with time, but they were generally reduced by plant colonization (Figure S12). Oxalic, formic, acetic, citric, lactic and tartaric acids were observed in tailings porewater (Figure S13). Plant colonization generally decreased oxalic acid concentration but increased acetic acid concentration in porewater (Figure S13, p < 0.05). The concen- trations of K and Mg in porewater were 10-1,000 times higher than that of Fe, Al and Si (Figure S14). Plant colonization generally increased porewater Fe, Al, K and Mg concentrations during the first month, but decreased (or had no effects on) those elemental concentration after 2 months’ cultivation (Figure S14, p < 0.05). Consistently, plant shoots and roots took up high quantities of K, Mg, Fe and Al from the tailing technosols (Tables S3 and S4). Rhizosphere microbial community development in ‘‘advanced technosol’’ As plant colonization drove the development of the ‘‘early technosol’’ toward the ‘‘advanced technosol’’, the prokaryotic microbial community diversity and composition shifted (Figure 7). According to Illumina sequencing, there were above 15,000 reads for all tailing samples, which were classified into 150–800 OTUs based on 97% sequence (Figure S15). The Rarefaction curves showed that the depth of the sequencing for tailings and technosol samples was adequate for characterizing prokaryotic microbial Figure 3. Continued (I) Synchrotron based C 1s NEXAFS spectra of aggregates from early technosol. Note: 1, 285.2 eV, aromatic C; 2, 286.5 eV, phenolic, pyrimidine or imidazole C; 3, 287.4 eV, alkyl C; 4, 288.3 eV, carboxyl C; 5, 289.3 eV, O-alkyl C. (J–L) Two-dimensional NanoSIMS images for the dispersed aggregates from early technosol. The red is 56Fe16O, the green is 12C, and the blue is 27Al16O. The range of counts for these images are: 56Fe16O, 0–300; 12C, 0–100; 27Al16O, 0–120. ll OPEN ACCESS 6 iScience 26, 107102, July 21, 2023 iScience Article communities (Figure S15). The OTU richness increased dramatically from round 200 in ‘‘early technosol’’ to above 400 in ‘‘advanced technosol’’ (p < 0.05, Figure S16). The Shannon index of microbial community increased from ca. 3.6 in ‘‘early technosol’’ to >5.5 in ‘‘advanced technosol’’ (p < 0.05, Figure S16). Neither soil amendment nor plant colonization influenced microbial diversity (Figure S16). Compared with ‘‘early technosol’’, the ‘‘advanced technosol’’ contained more Acidobacteria, Bacteroi- detes, Chloroflexi, Cyanobacteria, Planctomycetes and Verrucomicrobia, but less Proteobacteria and Fir- micutes (Figure 7A). Furthermore, plant colonization resulted in different microbial community patterns Figure 4. Backscattered-scanning electron microscope (BSE-SEM) showing the structure of the microaggregates from ‘‘advanced technosol’’ with various plant colonization treatments Note: the meanings of ‘‘TL’’, ‘‘TLS’’, ‘‘Aa’’, ‘‘Mb’’ and ‘‘SHS’’ are shown in the caption of Figure 2. For the minerals in BSE figures (verified by mineral liberation analysis, energy-dispersive spectroscopy, and X-ray diffraction analysis), ‘‘Bt’’ represents biotite, ‘‘FeOx’’ represents Fe oxides, and/or hydroxides; ‘‘Qz’’ represents quartz; ‘‘Amph’’ represents amphibole. Black areas are organic matter that has infilled pore spaces. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 7 iScience Article according to PCA analysis (ANOSIM test, p < 0.05, Figure 7B). Plant colonization increased percentage of microbes involved in mineral weathering, such as Verrucomicrobiaceae (belong to phylum Verrucomicro- bia),24 endolithic Ellin6075 (belong to phlum Acidobacteria),25 Xanthomonadaceae (belong to phylum Pro- teobacteria, reported to be OM degrader26) and Comamonadaceae (order-Burkholderiales)27 at family level (Figure S17). At genus level, some well-known mineral weathering bacteria such as Bacillus spp., Helothiobacillus spp., Solibacillus spp., Streptomyces spp., Geobacter spp., Thiobacillus spp., and Myco- bacterium spp.18 were induced by pioneer plant (such as M. brevifolia) colonization (Table S5). Figure 5. Mineral phases and morphology in different fractions of ‘‘advanced technosol’’ subject to different plant colonization treatments (A) Fe phases in bulk tailings, colloids and non-colloidal fraction (the mineral deposits left after colloid extraction) in microaggregates, as revealed by linear combination fitting (LCF) of k space Fe K-edge EXAFS spectra (k weight 3) (EXAFS spectra and LCF fitting parameters are in Figure S5). (B) XRD spectra (2 theta 6-15�) of whole bulk tailings, colloids and non-colloidal fractions in microaggregates from different treatments (Note: Bt = Biotite; ML = Mixed layered mineral such as illite and smectite group; Amp = Amphibole such as richterite; Kln = Kaolinite). (C) Transmission electron microscopy coupled with selected area electron diffraction and energy-dispersive X-ray spectroscopy (TEM-SAED-EDS) results showing nanophases in colloids of the ‘‘advanced technosol’’ with Maireana brevifolia colonization. The electronic diffraction is superimposed on 20 nm scale bright-field TEM images, indicating the presence of amorphous nanosized Fe(III)-Si mineral structure in colloids of microaggregates. (D) Percentage of colloids inmicroaggregates from ‘‘advanced technosol’’ with different plant colonization treatments. Data are represented asmeanGSEM. Note: themeanings of ‘‘TL’’, ‘‘TLS’’, ‘‘Aa’’, ‘‘Mb’’ and ‘‘SHS’’ are shown in the caption of Figure 2. Different letters show significant difference between different treatments based on Tukey test (p < 0.05). ll OPEN ACCESS 8 iScience 26, 107102, July 21, 2023 iScience Article Microstructure and cementation of aggregates in Fe rich native soil Mineral particles in native soil aggregates were consistently surrounded and cemented together by Fe-Si rich minerals within soil aggregates. The Fe concentrations across the section of soil aggregates accounted up to 10% w/w, with a high degree of spatial heterogeneity (Figures 8A–8C). BSE-SEM-EDS confirmed that the individual quartz grains were cemented by Fe(III)-Si rich cements (Figures 8E–8I). Further MLA, XRD and Fe K edge XAFS analyses indicated that the Fe(III)-Si rich cements were composed of amixture of Fe(III)-rich amorphous minerals (ferrihydrite-silica complexes) and Fe(III)-bearing clay minerals (i.e., Fe(III)-kaolinite and hematite) (Figures 8D–8K). This structural arrangement was a common feature observed in most of the soil aggregates examined (Figs, S18 and S19), suggesting its ubiquity in the Fe-rich soil aggregates. In addition, some large quartz grains were surface-coated by Fe(III)-Si rich cements (Figure S20), thus pro- moting chemical reactions with neighboring particles and forming stable and resilient aggregates under natural conditions. According to the three-color mapping of Fe-K-Ca across aggregate sections, the Fe(III)-Si cements were not associated with K or Ca (Figure S21). EDS analysis of the Fe(III)-Si rich cements indicated that the Si: Al ratio was around 1.15 (0.15), and the Si: (Al+Fe) ratio around 0.83 (0.18) (based on the EDS of 20 spots in the Fe(III)-Si rich cements). As indicated by C 1s NEXAFS analysis (Figure 8L) and C 1s XPS analysis (Figure S22), aromatic, phenolic, carboxyl, aliphatic, O-alkyl and carbonyl groups were found in the soil aggregates. Organic C was primarily located at the surfaces of Fe-rich mineral grains (e.g., Fe oxyhydroxides, Fe bearing kaolinite) in the soil aggregates according to NanoSIMS analysis (Figures 8M and 8N). The XRD and Fe K edge XAFS analysis of soil colloidal fraction and whole microaggregates revealed that those Fe(III)-Si rich minerals were mainly present in the colloidal fraction and composed of mainly Fe-Si-SRO, hematite and Fe(III)-kaolinite (Fig- ure S23). These colloidal particles were around 100 nm in diameter according to FE-SEM analysis (Figure S23). DISCUSSION Biological weathering and transformation of minerals are essential processes in natural soil formation.28 Here, we demonstrated the key drivers including tolerant microbes and pioneer plants in driving eco-en- gineered soil formation in Fe ore tailings through early and advanced technosol development. By adding a renewable resource, such as plant biomass, into the tailings, heterotrophic microbes can be stimulated to Figure 6. Organic carbon forms in colloidal fractions within microaggregates from ‘‘advanced technosol’’ subject to various plant colonization treatments (A and C) Synchrotron based C 1s NEXAFS spectra of colloidal fraction. Note: 285.2 eV, aromatic C; 286.4 eV, phenol C; 287.4 eV, alkyl C; 288.4 eV, carboxyl C; 289.3 eV, O-alkyl C; 290.4 eV, carbonyl C. (B and D) The column plots showed the proportion of different organic C forms as analyzed by peak fitting. Note: the meanings of ‘‘TL’’, ‘‘TLS’’, ‘‘Aa’’, ‘‘Mb’’ and ‘‘SHS’’ are shown in the caption of Figure 2. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 9 iScience Article decompose organic matter, leading to organic acid generation that not only neutralizes alkaline pH con- ditions in the tailings, but also facilitates the initial formation of aggregates.21,29 The functions of tolerance microbial decomposition of exogenous OM stimulated the early pedogenesis, toward forming ‘‘early tech- nosol’’ that is capable of survival of pioneer plants. The colonizing pioneer plants further advanced the development of ‘‘early technosol’’ toward ‘‘advanced technosol’’ (Figure 1) through rhizosphere biogeo- chemical processes,19,29–31 leading to improved physical and chemical conditions (Figure 2, Table S1) for various key stone native plant species colonization. Consistent with expected barriers of the compacted tailings, the aggregate stability was the dominant factor differentiating tailings, technosols, and native soil (Figure 2), highlighting the importance of the aggregate development in eco-engineered tailings-soil formation. Microbial generation of organic cements to initial aggregate formation Microstructural and microspectroscopic analysis of the tailings undergoing ‘‘early technosol’’ formation re- vealed that organic cementing agents played a key role in initiating organo-mineral interactions and aggre- gation of tailings particles, as the extensive mineral weathering of Fe-bearing minerals had not yet occurred to generate secondary mineral cements (Figure 3). In the tailings admixed with OM and the soil inoculum, various chemoheterotrophic microbes, that belong to the phyla of Proteobacteria and Fir- micutes, developed and attributed as key OM decomposers in the tailings32,33 (Figure 7). These tolerant microbes drove the decomposition of added OM even under the extremely alkaline pH conditions in the tailings, producing various organic acids34 (rich in aromatic, carboxyl, phenolic groups, Figure 3I). Figure 7. Variations of prokaryotic microbial community in early technosol and advanced technosol subject to different plant colonization treatments (A) Prokaryotic microbial community composition at the phylum level, and (B) multivariate principal component analysis (PCA) of prokaryotic microbial community (Illumina data) at the OTU level in tailings of different treatments. Note: ‘‘ET’’, early technosol, the meanings of ‘‘TL’’, ‘‘TLS’’, ‘‘Aa’’, ‘‘Mb’’ and ‘‘SHS’’ are shown in the caption of Figure 2. ll OPEN ACCESS 10 iScience 26, 107102, July 21, 2023 iScience Article These acids then initiated pH neutralization and mineral weathering and acted as organic cements for or- gano-mineral association.35,36 These OC was distributed in a heterogeneous pattern, possibly because of their selective adsorption onto the Fe/Al bearingminerals, which were also randomly distributed (Figure 3). Figure 8. The microstructure of aggregates in natural Fe rich soils surrounding Fe-ore mine site, as revealed by synchrotron based X-ray fluorescence microscopy (XFM), mineral liberation analysis (MLA), and backscattered-scanning electron microscope-energy dispersive X-ray spectroscopy (BSE-SEM-EDS) analysis (A) Thin sections of aggregates under the light microscope. (B) Fe mapping as revealed by XFM analysis. (C) Fe concentrations across the selected area (white line) in ‘‘b’’, the distance in X axis representing top of thin section to bottom. (D) MLA mapping of different minerals in soil aggregates (yellow is quartz (Qz), blue is Fe bearing kaolinite-like minerals (Fe-Kln), red is ferric oxyhydroxide). (E–H) BSE-SEM micrographs revealing the microstructure of the soil aggregates (Note: Fe-Si rich cements include Fe bearing kaolinite like minerals and Fe (oxy)hydroxides, as well as Fh-Si (ferrihydrite-silica co-precipitates)). (I) EDS spectra showing the elemental composition of the selected area in ‘‘h’’. (J), XRD spectra of macroaggregates (250–2000 mm) and microaggregates (53–250 mm) in soil (Qz-Quartz; Kln-Kaolinite; Hem-Hematite; No-Nontronite). (K) k space Fe K-edge EXAFS spectra (weight 3) (line) and linear combination fitting (LCF, dashed) of soil aggregates. The right column shows the results of the fitting Fe K-edge EXAFS spectra. The fitting parameters: For Macro-aggregates: R factor = 0.017; Chi-square = 26.9; Reduced chi-square = 0.15; For Micro-aggregates: R factor = 0.047; Chi-square = 70.1; Reduced chi-square = 0.39. (L) Synchrotron based C1s NEXAFS spectra of soil aggregates. Note: 1, 285.2 eV, aromatic C; 2, 286.4 eV, phenolic C; 3, 287.4 eV, alkyl C; 4, 288.4 eV, carboxyl C; 5, 289.3 eV, O-alkyl C. (M and N), Two-dimensional NanoSIMS images for the native Fe rich soil aggregates. The red is 56Fe16O, the green is 12C, and the blue is 27Al16O. The range of counts for these images are: 56Fe16O, 0–300; 12C, 0–100; 27Al16O, 0–120. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 11 iScience Article The binding of OC compounds onto minerals under circumneutral pH conditions may have been driven by reactions between the OH groups of Fe/Al-rich minerals with carboxyl, aromatic and phenolic groups, forming polar covalent X-O-C bonds (X = Fe, Al, or Si)37 or via hydrophobic interactions between hydrophobic organic compounds (e.g., those aromatic, and/or aliphatic dominated organics) and min- erals.38 As illustrated by the layer-by-layer model,39 the initial association of organics with mineral surfaces functions as a nucleation site for the formation of stable aggregates.13 However, there was no mineral ce- ments in the adjoining spaces that connecting different mineral particles in the tailings (Figures 3D and 3F). Therefore, the aggregates were mainly supported by organic cements in the ‘‘early technosol’’, which we believe are transitional cements that may undergo rapid microbial decomposition, leading to aggregate dispersion. Generation of colloidal Fe(III)-Si rich cements to enhance aggregate stability After extensive physical, chemical and biological improvements, the resultant ‘‘early technosols’’ would have functions to support the growth of tolerant pioneer plants (Figure 1). All pioneer plants grew well in the ‘‘early technosol’’ (Figure 1), except Sorghum spp. that died because of the possible salt stress resulting from the extensive K dissolution; porewater K concentration increased to around 1000 mg L�1 (Figure S14) during initial weathering of primary minerals. The root activities of colonizing plants would sub- sequently enhance mineral weathering to form secondary mineral cements for the formation of a new class of more stable aggregates in emerging ‘‘advanced technosol’’ (Figures 2 and 4). Themicrostructure withinmicroaggregates showed that the secondary mineral phases such as irregular Fe- Si rich minerals, were generated. These, in turn, cemented quartz, biotite and FeOx particles in the ‘‘advanced technosols’’ after plant colonization (Figure 4), leading to the elevated stability of aggregates. These mineral cements were probably amorphous Fe(III)-Si short range ordered (SRO)minerals, which were found to be enriched in colloidal fractions within aggregates (Figure 5). It is reported that the colloidal frac- tion was small in size (below 1 mm) and high in chemical reactivity with other minerals and organics,40,41 acting as an important constituent of mineral cements for forming aggregates.42 The Fe(III)-Si rich SROminerals are considered to have resulted from the co-precipitation of dissolved Fe(III) and Si species (Figure 9A) generated from the weathering of primary minerals (such as biotite) in the tail- ings.19,20 Typically, the Fe(II) cations liberated from weathered minerals may be rapidly oxidized to Fe(III) (Figure S8), to form Fe(III) oxyhydroxide minerals (e.g., ferrihydrite) under circumneutral pH conditions through processes like hydrolysis and polymerization.43 The co-presence of amorphous/soluble silica hin- ders Fe(III) mineral polymerization, favoring the formation of Fe(III)-Si rich SROminerals.44 This may explain why Fe(III)-Si-SRO were so abundant in the colloidal fraction (Figure 5A). These nanosized Fe(III)-Si-SRO minerals have a high affinity for organic molecules,45 forming close association with organics as nuclei sites to agglomerate other mineral particles (i.e., primary mineral biotite and quartz), leading to the formation of stable aggregate structure. The mixed layered minerals (e.g., illite and smectite) in the non-colloidal frac- tions of microaggregates (Figure S6), possibly resulting from the solid-alteration of biotite minerals in the tailings,46 may also partially contribute to the aggregate formation. Rhizosphere processes enhancing formation of colloidal Fe(III)-Si-SRO like minerals Plant colonization can influence the mineralogical composition in colloidal fraction via alteration of pH, ionic strength, and/or organic groups in the tailing solutions (Figure 9). The circumneutral to slightly alka- line pH conditions (Figure S12) and the organic acid generation (Figure S13) by root activities facilitated the formation of ferrihydrite or Fe(III)-Si-SRO minerals (Figure 9A).20 It is noted that the initial weathering of biotite like minerals and the rapid release of K and Mg into porewater elevated salinity of the tailings (Fig- ure S14), which could hinder the Fe(III)-Si-SRO formation.47 However, plant uptake of these elements ulti- mately lowered porewater ionic strength (Tables S3 and S4, Figures S12 and S14), which, in turn, would have progressively favored Fe(III)-Si-SRO formation (Figure 9B)19,20 and diminished sylvite and aphthitalite min- erals in colloidal fraction (Figure S6). In addition, the changes of composition and abundance of individual organic acids (e.g., the increase of acetic acid, Figure S13) in the rhizosphere would have enhanced mineral weathering and Fe(III)-Si-SRO formation.48 The organic functional groups such as aromatic and/or carboxyl groups (Figure 6) may complex Fe cations to form Fe-OM complexes, as has previously been demonstrated in numerous natural and man-made environments.49 These hindered Fe, Si and Al polymerization,50 and facilitated the formation of SRO mineral-OM complexes (Figure 9B).51 In sum, the association between SRO minerals and organics acts as nucleus underpinning the formation of water stable aggregates and ll OPEN ACCESS 12 iScience 26, 107102, July 21, 2023 iScience Article organic matter stabilization,14 both of which are fundamental and essential processes leading to soil for- mation in the tailings. Rhizosphere processes may have also accelerated mineral weathering and amorphous mineral formation through stimulating key microbial development in tailings. Particularly, plant colonization increased the percentage of functional microbes involved in mineral weathering, such as Fe phyllosilicate and/or rock associated bacteria Verrucomicrobiaceae (belong to phlum Verrucomicrobia)24 and endolithic Ellin6075 (belong to phlum Acidobacteria)25 at family level (Figure S17), as well as Bacillus spp., Helothiobacillus spp., Solibacillus spp., Streptomyces spp., Geobacter spp., and Thiobacillus spp.18(Table S5). These ongoing enrichment of diverse mineral associated microbes in rhizosphere further contributed to the pro- gressive weathering of Fe bearing minerals to form increasing amounts of secondary Fe(III)-Si minerals, advancing aggregate structure development in the technosols. Conceptual synthesis of mineral cements generation and aggregate development in tailing- soil formation The study has demonstrated the development of stable aggregate structure in Fe ore tailings through continuous mineral weathering and amorphous Fe(III)-Si mineral formation which was driven by microbes and pioneer plant root activities. The Fe(III)-Si rich mineral cements are critical to soil aggregate stability, which was also revealed through detailed characterization of mineral makeup, distribution and organo- mineral association in native Fe rich soil (Ferralsol) aggregates from the local Fe-ore mine site (Figure 8). The Fe(III)-Si rich mineral cements in soil aggregates mostly consisted of amorphous Fe(III) oxyhydroxides and Fe(III) bearing 1: 1 aluminosilicates (kaolinite, or halloysite) (Figures 8 and S18–S20), which most likely came from the weathering of primary minerals (i.e., biotite, amphibole) with K and/or Ca depletion.52 The isomorphic substitution of Al3+ by Fe3+ in the phyllosilicate structure could then have caused structural dis- order and increased surface area and reactivity of kaolinite.53,54 In addition, amorphous Fe(III) oxyhydrox- ides may have co-precipitated with secondary 1:1 phyllosilicates (e.g., via electrostatic interactions) to form ‘‘gel’’ like Fe(III)-rich mineral cements.36,55 These Fe(III)-Si rich cements in soil aggregates were found to be Figure 9. Diagram showing processes and possible mechanisms underlying rhizosphere driven mineral weathering and Fe(III)-Si mineral formation in Fe ore tailings (A) Eh-pH diagram showing the key geochemical evolution pathways during pioneer plant driven secondary Fe-Si mineral (especially Fe-Si rich short range ordered (SRO) minerals) formation coupled with primary mineral weathering in the advanced technosols. (B) Diagram summarizing plant root driven mineral weathering and secondary colloidal Fe(III)-Si-SRO-OM complex formation. The primary minerals (biotite, amphibole, magnetite) were expected to have undergone microdivision by root effects, forming micro- and nano-sized particles in colloids. These small-sized mineral particles are easily to be dissolved under functions of organic groups exuded by roots, causing the release of Fe2+, Fe3+, silica, and aluminum, as well as salt elements such as K+, Mg2+ and Ca2+. The salt elements were further taken up by halophyte plants, which stimulated further mineral weathering. The Fe2+ could be oxidized to Fe3+, which would co-precipitate together with silica in the presence of organics under rhizosphere modified geochemical conditions, leading to the formation of Fe(III)-Si-SRO-OM complexes. Note: Bt: Biotite; Amp: Amphibole; Mag: Magnetite; Fh: Ferrihydrite; OM: Organic matter; SRO: Short range ordered minerals. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 13 iScience Article mostly in the colloidal fraction (Figure S23), which not only confer strong physical stability, but also provide affinitive surfaces for interactions with carboxylic dominated organics (Figure 8L), underpinning organo- mineral association and nucleation sites for aggregate structure development.39 However, it is important to point out that the proportion of colloidal Fe(III)-Si rich cements in aggregates in the ‘‘advanced technosol’’ (0.4–0.8% w/w) remained much lower than those of native soil aggregates (3.5% w/w). This difference may explain the lower stability of the technosol aggregates than that of native soil aggregates. The aggregate structure and stability development in the tailings may be enhanced by increasing the generation of Fe(III)-Si cements during the eco-engineering processes and the technosol development over the time. From the perspective of natural pedogenetic processes, Fe(III)-Si rich mineral cements in soil aggregates should have formed via the long-term primary mineral weathering driven by continuous plant root and microbial activities.52 A conceptual model has thus been proposed to demon- strate procedures of aggregate development in the tailings (Figure 10). Briefly, the initial aggregates in ‘‘early technosol’’ can be formed through cementing effects of organic ma- terials derived frommicrobial OM decomposition, which intimately associate with tailing minerals. The ag- gregates are progressively consolidated by accumulating Fe(III)-Si rich mineral cements generated from the weathering of primary minerals (such as biotite) and the formation of colloidal Fe(III)-Si short range or- dered (SRO) minerals, which are stimulated by ongoing rhizosphere activities of tolerant plants in the developing technosol. Those colloidal Fe(III)-Si SRO minerals would be polymerized into ferrihydrite-Si, goethite and even hematite, during long-term aging. These more stable secondary minerals together with Fe(III)-Si SRO minerals would form new Fe(III)-Si rich mineral cements, serving as reactive sites for association with other mineral particles and organics to enhance aggregate stability under the semi-arid climatic conditions (Figure 10). The development of soil aggregate structure in ‘‘advanced technosol’’ sub- sequently facilitates the colonization of various plant/microbial communities, from pioneer plant species to diverse keystone plant species, in association with key soil microbes such as mycorrhizal fungi.56 These plant and rhizosphere microbial activities in the tailings would consistently stimulate the long-term soil aggregate development, through enhancing mineral weathering, Fe(III)-Si rich cements formation and Figure 10. A conceptual model for eco-engineering stable soil aggregate structure in Fe ore tailings The early aggregates (formed in ‘‘early technosol’’) could be initiated through microbial decomposition of exogenous organic matter and resultant organics cementation. The transition from early aggregates into advanced aggregates could be achieved through the accumulation of Fe(III)-Si rich cements during mineral weathering and geochemical reactions driven by intensive pioneer plant colonization. Mature aggregates can be progressively formed via long-term biotic and abiotic mediated processes of mineral weathering and Fe(III)-Si rich cements generation and accumulation, which collectively maintain the hydrological and geochemical stability of the aggregates. ll OPEN ACCESS 14 iScience 26, 107102, July 21, 2023 iScience Article organicmatter stabilization (Figure 10). This would lead to a quasi-stable soil-like substrate, with strong and mature aggregate structure for supporting long-term resilient revegetation and ecosystem build-up in the tailing sites. It is important to point out that we have emphasized the key role of bioweathering and secondary Fe(III)-Si mineral cements in the formation of stable soil aggregate structure in Fe ore tailings undergoing soil for- mation. These secondary Fe(III)-Si minerals in themicroaggregates significantly increaseOC sequestration, because of their high reactivity and specific surface area.41 These organo-mineral complexes embedded in microaggregates contribute to the long-term retention of OC.14 The organo-mineral association formed through interactions of these secondary Fe(III)-Si minerals with functional organics are of critical impor- tance to not only soil structure development, but also the development of biogeochemical functions in the technosols for resilient ecological rehabilitation of tailings landscapes.57 We have demonstrated the approach to harness biological drivers of tolerant microbes and pioneer native plants as the key soil formation factors in accelerating critical processes of bio-generation of mineral cements (i.e., amorphous Fe(III)-Si minerals) and formation of microaggregates. This process is the key to unlock the barrier to soil formation in the mechanically compacted tailings without functional physical structure. Ecological rehabilitation of large areas of Fe-ore tailings landscapes worldwide requires large volumes of natural soils by excavating natural landscapes, which is not a sustainable option. Our findings have opened a cost-effective and sustainable pathway to achieve ecological rehabilitation of Fe-ore tailings without nat- ural soil, potentially solving the big environmental issues of thousands of hectares of mine tailing sites glob- ally. The merits of environmental and economic sustainability lie in the fact that our approach to eco-engi- neer tailings into functional soil for supporting revegetation does not require the supply of manymillions of cubic meters of natural soil at mine sites for rehabilitating hundreds to thousands of ha of tailings land- scape. Furthermore, the access to natural soil supply is beyond the reach of many mine sites, regardless of financial capability. Compared to conventional method by relying on excavated topsoil to construct soil cover, the rehabilitation of Fe ore tailings through the proposed eco-engineering pathway has been estimated to save up to 50–70% of the rehabilitation costs. For instance, in Australia, the current price of natural soil supply from offsite ranges is from $30 to 120/m3, including handling and transport expenses, which is translated to the cost of $300,000–1,200,000 per ha if using natural soil to create 1 m soil cover across a tailings landscape. As a result, the translation of this eco-engineering approach in field operations would lead to rapid rehabilitation progress and potential savings worth many billions of dollars, if adopted across the thousands ha of Fe ore mine tailings landscapes worldwide. Our work has proved the concept of eco-engineering of Fe ore tailings into soil-like technosol through a pot experiment under glasshouse conditions. Soil aggregate structure were formed and progressively developed in tailings to support soil functionality during eco-engineering processes driven by tolerant mi- crobial community and pioneer plants. The initial aggregates in ‘‘early technosol’’ contained little Fe(III)-Si cements and were mainly held together by organic cements. Later, rhizosphere biogeochemical processes of pioneer plants induced the generation of colloidal Fe(III)-Si SRO minerals, which sequestrated organic carbon and consolidated the aggregate structure and stability. The importance of Fe(III)-Si rich cements in underpinning aggregate structure was confirmed in native Fe rich soil surrounding the investigated tailing site. Based on these findings, we propose a conceptual model for progressive aggregate structure devel- opment in the tailings with Fe(III)-Si rich cements as core nuclei. The eco-engineering process proposed here can be tailored for local resource availability and climatic conditions in future scale-up field trials before adoption at remote mine sites. Limitations of the study In this study, we have demonstrated the approach to employ tolerant microbes and pioneer plants as the key drivers in accelerating in situ mineral weathering and secondary mineral cement formation for aggre- gate development toward eco-engineered soil formation in Fe ore tailings. The concept has been proved under the short-term glasshouse conditions. However, it is essential to scale up the proof of concept into the operational methodology by conducting long-term broadacre field trials at Fe ore tailings landscape, with consideration of climate and parent material variability across spatial distance and different field op- erators. Furthermore, because this study was only carried out using themagnetite alkaline Fe ore tailings as an example, it is necessary to investigate the suitability of this approach in eco-engineering soil formation in other tailings, such as hematite Fe ore tailings and Cu tailings. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 15 iScience Article STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d METHOD DETAILS B Experimental procedures B Micro-spectroscopic analysis d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.107102. ACKNOWLEDGMENTS The work is financially supported by Australian Research Council Linkage Project (LP160100598), Kar- ara Mining limited, and The Botanic Gardens and Parks Authority (BGPA). S. Wu also acknowledges the UQECR funding (613767). XFM mapping was undertaken on the XFM beamline at the Australian Synchrotron, part of ANSTO (AS182/XFM/13331). The XAS analysis was undertaken on the XAS beam- line at the Australian Synchrotron, part of ANSTO (Project Reference No: AS191/XAS/14392), as well as 01C1, 17C1 and 20A1 beamline in National Synchrotron Radiation Research Centre (NSRRC), Taiwan. The authors also thank Dr Jin-Ming Chen in Beamline 20A1 and Dr Jyh-Fu Lee in beamline 17C1, NSRRC, Taiwan for technical support in XAS analysis. Dr Jeremy Wykes at XAS beamline of Australian synchrotron is also acknowledged for technical support in XAS analysis. NanoSIMS analysis was done at Center for Microscopy, Characterization and Analysis at University of Western Australia. The authors acknowledge staffs in Australian Microscopy & Microanalysis Research Facility at the Cen- ter for Microscopy and Microanalysis, The University of Queensland for assistance in XRD, XPS and BSE-SEM-EDS analysis. The Australian Center for Ecogenomics, the University of Queensland, Australia was acknowledged for Illumina sequencing analysis. Dr Elaine Wrightman in JKMRC, SMI, UQ has been acknowledged for support in MLA analysis. We also thank Dr Shuncai Wang and Jing- fang Xue for help on the plant cultivation, maintenance, and harvest, as well as lab analysis. AUTHOR CONTRIBUTIONS S.W.: Conceptualization, Experimental design and conduction, Methodology, Data analysis, Writing-orig- inal draft, and review & editing; Y.L.: Methodology (Plant harvest, XFM analysis); Z.L., L.R., and Q.Y.: Meth- odology (Plant harvest, wet sieving); F.Y.: Methodology (Microbial community analysis); J.J.B.: Methodol- ogy (NanoSIMS analysis); D.P.: Methodology (XFM analysis); S.C.H.: Methodology (C 1s NEXAFS analysis); T.S.C. and Y.R.L.: Methodology (Fe K edge XAFS analysis); M.H. andN. S.: Methodology (ICP-OES analysis); G.S., K.K., T.N., and Y.S.O.: Writing-review & editing; L.H.: Conceptualization, Experimental design, Project administration, Funding acquisition, Writing-review & editing. DECLARATION OF INTERESTS The authors declare no competing interests. INCLUSION AND DIVERSITY We support inclusive, diverse, and equitable conduct of research. 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Global Change Biol. 24, 1762–1770. ll OPEN ACCESS iScience 26, 107102, July 21, 2023 19 iScience Article http://refhub.elsevier.com/S2589-0042(23)01179-3/sref71 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref71 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref72 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref72 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref72 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref72 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref72 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref72 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref72 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref73 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref73 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref73 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref73 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref73 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref73 https://doi.org/10.1021/acs.est.7b03715 https://doi.org/10.1021/acs.est.7b03715 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref75 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref76 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref77 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref78 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref78 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref78 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref78 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 http://refhub.elsevier.com/S2589-0042(23)01179-3/sref79 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Atriplex amnicola Nindethana Ltd., Australia N/A Maireana brevifolia Nindethana Ltd., Australia N/A Sorghum spp. Hybrid cv. Silk Pukalus’s farm in Injune origin, Queensland, Australia N/A Chemicals, peptides, and recombinant proteins Magnetite iron ore tailings Karara Mining Ltd N/A Hematite Sigma Aldrich CAS ID: 1317-60-8 Goethite Sigma Aldrich CAS ID: 20344-49-4 Magnetite Sigma Aldrich CAS ID: 7439-89-6 Siderite Sigma Aldrich CAS ID: 14476-16-5 Pyrite Sigma Aldrich CAS ID: 1309-36-0 Fe(III)-citrate Sigma Aldrich CAS ID: 3522-50-7 Fe(II)-gluconate hydrate Sigma Aldrich CAS ID: 699014-53-4 Fe(II)-oxide Sigma Aldrich CAS ID: 1345-25-1 Jarosite Sigma Aldrich CAS ID: 12207-14-6 Fe(III)-sulfate Sigma Aldrich CAS ID: 10028-22-5 Iron(III)-nitrate Sigma Aldrich CAS ID: 7782-61-8 Sodium silicate Sigma Aldrich CAS ID.: 6834-92-0 Oxalic acid Sigma Aldrich CAS ID: 144-62-7 Illite Ward’s science https://www.wardsci.com Vermiculite Ward’s science https://www.wardsci.com Epidote Ward’s science https://www.wardsci.com Biotite Ward’s science https://www.wardsci.com Olivine Ward’s science https://www.wardsci.com Critical commercial assays DNeasy PowerSoil Kit QIAGEN https://www.qiagen.com/au/ Oligonucleotides 926F (50-AAACTYAAAKGAATTGACGG-30) Australian Centre for Ecogenomics, the University of Queensland N/A 1392wR (50-ACGGGCGGTGWGTRC-30 ) Australian Centre for Ecogenomics, the University of Queensland N/A Software and algorithms QIIME2 (Quantitative Insights Into Microbial Ecology 2, version 2019.1) QIIME 2 development team https://qiime2.org/ SPSS statistics 18 IBM https://www.ibm.com/ Origin 2021 OriginLab https://www.originlab.com/ Excel Microsoft https://www.microsoft.com/ R (v3.4.1) R Project https://cran.r-project.org/ Diffracplus Evaluation Package V4.2 Bruker AXS, Germany https://www.bruker.com/ DEMETER software package CARS, University of Chicago https://bruceravel.github.io/demeter/ GeoPIXE CSIRO, Australia http://nmp.csiro.au/geopixe.html (Continued on next page) ll OPEN ACCESS 20 iScience 26, 107102, July 21, 2023 iScience Article https://www.wardsci.com https://www.wardsci.com https://www.wardsci.com https://www.wardsci.com https://www.wardsci.com https://www.qiagen.com/au/ https://qiime2.org/ https://www.ibm.com/ https://www.originlab.com/ https://www.microsoft.com/ https://cran.r-project.org/ https://www.bruker.com/ https://bruceravel.github.io/demeter/ http://nmp.csiro.au/geopixe.html RESOURCE AVAILABILITY Lead contact Further information and requests for resources should be directed to the lead contact, Longbin Huang (Email: l.huang@uq.edu.au) Materials availability All materials used in this study are available from the lead contact upon reasonable request. Data and code availability d All data that support the findings in this study are available in the article and its supplemental information. d This study did not generate original code. d Any additional information required to reanalyze the data reported in this work is available from the lead contact upon reasonable request. METHOD DETAILS Experimental procedures Tailings and soil collection The Fe-ore tailings was collected from the tailings dam (GPS: 29.19842�S 116.75972�E) within a magnetite Fe-ore mine site in Western Australia, which was rich in biotite mica, quartz, Fe(III) oxyhydroxides (described previously7,29) (Figure S24). The tailings were strongly alkaline (pH 9.5), with low level of organic carbon (0.1% w/w) and nitrogen (0.1% w/w), but high amount of quartz and primary phyllosilicate (biotite- like) in the form of fine particles (below 53 mm, with little water stable aggregate structure).7 Native Fe-rich soils were collected from a natural undisturbed site adjacent to the investigated Fe oremine site inWestern Australia (GPS: 29.19717o S, 116.76222o E) (Figure S24). The soil had a neutral pH, with 0.6% w/w organic carbon and 0.6% w/w nitrogen, with abundant kaolinite, quartz and hematite and Fe hydroxides (e.g. goethite, ferrihydrite) and well water stable aggregates.7 The details of the physical and chemical proper- ties of initial tailings and native Fe rich soils are in Table S1. As the soils were collected from the sorrounding area of the tailing site, the comparation is reasonable. Eco-engineering of the tailings In ‘‘early technosol’’ formation process, Fe ore tailings were throughly amended with 2% (w/w) Lucerne hay (ground to below 1 mm, 43.2% C and 4.1% N, with a C/N ratio of 10.6) (Petbarn Ltd., Australia), and Continued REAGENT or RESOURCE SOURCE IDENTIFIER OpenMIMS data analysis software National Resource for Imaging Mass Spectrometry http://nrims.harvard.edu CasaXPS software package Casa Software Ltd http://www.casaxps.com/ ImageJ National Institutes of Health, Bethesda, MD, USA https://imagej.nih.gov/ij/ CANOCO 5.0 Microcomputer Power, Ithaca, NY, USA http://www.canoco5.com/ Other FRITSCH Analysette 3-laboratory model of vibration sifting device Fritsch, Germany https://www.fritsch-international.com/ Cary 630 FTIR with Diamond ATR module Agilent Technologies, Palo Alto, CA, USA https://www.agilent.com/ Hitachi SU3500 SEM Hitachi, Japan https://www.hitachi-hightech.com/ Copper grid (200 mesh) ProSciTech Pty. Ltd., Queensland, Australia https://proscitech.com.au/ NanoSIMS 50L Cameca, Gennevilliers, France https://www.cameca.com/products/sims/ nanosims Hitachi HF5000 S/TEM Hitachi, Japan https://www.hitachi-hightech.com/ ll OPEN ACCESS iScience 26, 107102, July 21, 2023 21 iScience Article mailto:l.huang@uq.edu.au http://nrims.harvard.edu http://www.casaxps.com/ https://imagej.nih.gov/ij/ http://www.canoco5.com/ https://www.fritsch-international.com/ https://www.agilent.com/ https://www.hitachi-hightech.com/ https://proscitech.com.au/ https://www.cameca.com/products/sims/nanosims https://www.cameca.com/products/sims/nanosims https://www.hitachi-hightech.com/ inoculated with a native soil microbial inoculum (rich in Actinobacteria (62.3%), Chloroflexi (11.46%) and Proteobacteria (8.45%) at phylum level) in s