# Screening of Plants and Indigenous Bacteria to Improve Arsenic Phytoextraction
## 2 Materials and Methods
### Preparation of Microcosms  
To set up the microcosm tests, four types of soil were chosen from those obtained from the sampling, representing both the soils of the external area (S2, S6) and the internal one (PZ, SW): S2 (1-1.6 m), S6 (0.3-0.6 m), PZ (0.3-1 m), and SW (1-2 m). Figure 1: List of arsenic-tolerant bacteria isolated from the As-contaminated soil and results of the tests performed on them. GenBank Accession number, metal tolerance, and in vitro PGP properties are shown. On these soils, two sets of microcosms were set up in succession, one for each mobilizing agent, testing first the phosphate and then the oxalate. Each batch included replicates to test the effect of adding PGPR bacteria from the first set of isolates. As for the plant species, in addition to _Cannabis sativa_, _Brassica juncea_ and _Zea mays_ have also been used, to evaluate which was the most suitable for the specific contamination of the site. The first set of tests was carried out according to the following scheme:  
* All 4 selected soil types were investigated, using about 300 g of soil for each microcosm. * Sowing involved all 3 plant species, in particular 0.4 g of _B. juncea_ seeds, 12 seeds of _C. sativa_, and 5 seeds of _Z. mays_, by germinating the seeds on moistened cotton and then transplanting them at the appropriate time. * Three different treatments were prepared for each soil and each plant, namely a control (CT), a treatment with 0.05 M KH\({}_{2}\)PO\({}_{4}\) (P), and a treatment with the further addition of growth-promoting bacteria (P+). All tests were performed in triplicate, for a total of 108 microcosms prepared for this first set. The duration of the microcosm test was on average around 30 days. The mobilizing agent was administered about 15 days after transplanting for both corn and hemp, and 20 days after sowing for Brassica, as this plant species developed somewhat slower than the others. A total dose of 10 mL of mobilizing agent was added to the microcosms, divided over 5 days to dilute the effect of a possible too-rapid absorption of the As. Each 2 mL daily dose was diluted with water to a volume of 10 mL. The plants were watered according to their need. The addition of phosphate was planned as, both from previous tests and from the literature, it has always been the best additive to solubilize arsenic and favor its absorption by plants [34]. At the end of the test, the plants were harvested by separating the aerial part from the roots. The plant samples were thoroughly washed and prepared for analysis. A second set of microcosms was then set up with the same plant species, using 0.2 M potassium hydrogen oxalate instead of KH\({}_{2}\)PO\({}_{4}\) as the mobilizing agent. This second set of tests was carried out according to the following scheme:  
* Two soils, S2 and SW, representative of the external and internal area, respectively, were investigated, and the amount of soil used for each microcosm was about 300 g.
* Three plant species, replicating the quantities and methods used for the first series of tests. * For each soil and each plant, four different treatments were investigated, namely a control (CT), a treatment with 0.2 M KHC\({}_{2}\)O\({}_{4}\) (O), and the same treatments with further addition of growth-promoting bacteria (CT+ and O+). Figure 2: Second set of arsenic-tolerant bacteria isolated from a different soil contaminated with As. GenBank Accession number, metal tolerance, and in vitro PGP properties are shown. All tests were carried out in triplicate, for a total of 72 microcosms prepared for this second set. The trial lasted approximately 30 days and treatment with PGPR (first set of isolates) began approximately 15 days after the transplant. A total dose of 25 mL of 0.2 M KHC2O4 was added to the microcosms, divided over 5 days. Each 5 mL daily dose was diluted with water to a volume of 10 mL. The plants were watered according to their need. Once again, the aerial part and roots of the plants were separated and washed thoroughly before being analyzed.