Investigating the Impact of Planting Density of Juncus pauciflorus on the Phytoremediation of Arsenic-Contaminated Mine Waste with the Addition of Biochar and Bacillus subtilis

Phytoremediation is a cost-effective and sustainable method for the remediation of minewaste contaminated with heavy metals such as arsenic (As). However, mine waste soil is often nutrient-limited, especially in nitrogen (N), impairing plant growth and phytoremediation. This study aimed to assess how planting density together with soil amendments, biochar, and an isolated indigenous nitrogen-fixing bacterium (NFB) ( Bacillus subtilis ) affect the efficacy of phytoremediation by Juncus pauciflorus of an As-contaminated mine waste soil from Bendigo, Victoria. Three plant densities, including 9, 26, and 44 plants/m 2 , were grown in As-contaminated mine waste soil amended with biochar (10% w / w ) and B. subtilis (8.1 × 10 8 CFU/mL) and incubated for 100 days. Plant biomass, plant As uptake, soil As concentration, bacterial abundance (total and NFB using 16S and nifH gene copy numbers, respectively), and total soil N were assessed. Juncus pauciflorus at a higher density (44 plants/m 2 ) promoted the greatest biomass and total As uptake, 70.22 g/m 2 and 209.53 mg/m 2 , respectively. Plant density significantly influenced the root–shoot partitioning of As. Higher densities increased shoot uptake (BAF soil→shoot ), and TF root→shoot values remained >1 across all treatments, confirming the active translocation of As to the shoots, suggesting both phytostabilisation and phytoextraction potential by J. pauciflorus . Planting density significantly reduced soil As, ranging from 8000 mg/kg to 9500 mg/kg, compared to the initial concentration (13,032 mg/kg). The abundance of 16S and nifH genes was stable among treatments, ranging from 7 log 10 copies/g to 12 log 10 copies/g. TN content in soils amended with 44 plants/m 2 contained the highest TN content at day 33, approximately 7000 mg/kg. This study is the first to report that higher planting density of J. pauciflorus amended with biochar and NFB provides the strongest phytoremediation performance in highly As-contaminated mine soil by enhancing As uptake and accumulation in aboveground biomass. Most importantly, the results show that plant density also regulates the plant’s remediation strategy, shifting J. pauciflorus between phytostabilisation at dense planting and greater phytoextraction at lower density. These findings support the use of native plants in combination with biochar and microbial amendment as a sustainable strategy for remediating As-contaminated mine waste.