*2.4. Phylogeny*

To assess the phylogenetic position, 16S rRNA gene sequences were either acquired from previously published sources, or newly sequenced in this study. A list of accession numbers to partial 16S rRNA gene sequences attained from public repositories was provided in Tables A1 and A2. From unsequenced strains, DNA was extracted via the phenol chloroform method [32], and sequenced using Sanger technique [33]. Primer set 27F and 1492R, 5-AGAGTTTGATCCTGGCTCAG-3 and 5-GGTTACCTTGTTACGACTT-3 , respectively, was used to achieve a total contiguous 16S rRNA gene sequence length of >1400 bp per strain. New sequences of 16S rRNA genes from previously unidentified AAP strains were deposited to GenBank under accession numbers (MW970346–MW970408) as listed in Tables A1 and A2. Genetic relation of 16S rRNA gene sequences acquired from each AAP isolate were compared to the archived sequences of type species using the web-based software Basic Local Alignment Search Tool, BLAST [34]. Phylogenetic trees were constructed via MEGA X software [35] with 1000 bootstraps [36], using Maximum Likelihood method to align all AAP 16S rRNA gene sequences to one another based on the General Time Reversible model [37]. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.

#### *2.5. Siderophore Isolation and Concentration from C. halotolerans*

Strain EG19 was grown at 28 ◦C on MA agarized (2%) plates in the dark for 5 days. Pink-purple colonies developed with brown hue dispersed in agarized medium (Figure 1A), which corresponded to zone of clearing on Fe-Chromeazurol S Assay (Figure 1B). To obtain liquid cultures, EG19 was inoculated at 5% in Fe-free MA and grown for 5 days at 28 ◦C shaking at 200 rpm in the dark. Fe-free MA was prepared as described in Chromeazurol S Assay, excluding Fe from the trace element solution. In addition, after all components were added, 5 g/L of Chelex resin enclosed in dializing tubing was placed in the medium for 1 h prior to autoclaving. This step allowed the Chelex resin to remove traces of Fe introduced through the complex nutrients such as yeas<sup>t</sup> extract, bactopeptone and casamino acids [38].

After the cultivation of *C. halotolerans*, Fe-free medium became dark brown. The pinkpurple cells were pelleted in ~450 mL bottles at 10,000 rpm for 30 min using a Beckman J2HS centrifuge and a JA-10 rotor (Figure 1C). Siderophore containing supernatant was collected and 1.25 L was transferred into 2 L Erlenmeyer flasks (Figure 1D) to freeze overnight at −20 ◦C. Highly concentrated dark brown-pigmented high-salt slurry was formed predominantly on top of ice, while the lower part of ice was close to colourless. The flask with frozen material was removed from freezer and allowed to defrost at room temperature inserted upside down in a new collection beaker. Thawed supernatant was fractioned into 250 mL batches, with concentrated siderophore thawed and collected first, leaving frozen medium behind (Figure 1D), both observable visually (Figure 1E), and from the absorbance spectrum (Figure 1F).

A batch-type method of siderophore purification was used in this study [39]. Here, the combined freeze-concentrated samples were adjusted to pH 6.0 and XAD 7-HP resin was added (20 g resin per L of supernatant). This slurry was shaken at 200 rpm for 1 h on a rotary shaker and then filtered through a glass Millipore filter funnel that would collect resin, but allow supernatant to easily flow through. Concentrated siderophore required several extractions with resin to remove all pigment from supernatant. Siderophore-boundresin (now brown in colour) was thoroughly washed with ddH2O to remove all residual salts and other soluble contaminants. Vacuum assisted drying using Millipore system was performed, ddH2O discarded, and the cleaned siderophore-bound-resin was soaked

in methanol for 30 min to release the siderophore. Pigmented methanol was collected, and resin was soaked once more using the same batch of solvent for additional extraction. Methanol extracts were combined and then evaporated to dryness. The resulting dark brown powder was the presumed highly concentrated siderophore, and kept at −20 ◦C until further testing. Unbound-resin was washed with methanol, then soaked with ddH2O prior to reuse.

**Figure 1.** Extraction and concentration of brown pigment excreted by *C. halotolerans*. (**A**) Strain EG19 released a brown pigment into MA medium. (**B**) Fe-CAS plate with siderophore production by EG19. (**C**) Pelleted cells under supernatant. (**D**) Pigment concentrated with freezing-out technique. (**E**) Collected fractions during thawing with concentrated pigment released first. (**F**) Absorbance spectrum of each fraction.

#### *2.6. Purification and Fe-Chelation of Siderophore from C. halotolerans*

Concentrated siderophore powder from resin purification was resuspended in ddH2O (10 mg in 50 μL), and decimally diluted up to 10−3. In addition, a solution containing 5 mg of concentrate in 100 μL ddH2O was filtered through an Amicon® Ultra 0.5 mL 3000 Da spin column manufactured by Millipore Ireland, which removed any contaminating proteins over that size. This filtered concentrate was also decimally diluted up to 10−2. Once the dilutions were made, 10 μL of each sample, and an aliquot of freeze–thawed concentrate, were individually mixed with 10 μL of loading buffer, prior to filling into the wells of a Mini-Protean tris-tricine gel, 16.5% from Bio-Rad, Hercules, CA, USA. Loading buffer contained 200 mM tris-HCl, pH 6.8, 2% SDS, 40% glycerol and 0.04% Coomassie Brilliant Blue G-250 (CBB) from Bio-Rad, USA. Running buffer was made up of 1 M tris, 1 M tricine and 1% SDS at pH 8.3. Wells were 30 μL, filled with 20 μL of sample (10 μL of sample, 10 μL of loading buffer). Protein ladder was a C6210—color marker ultra-low range (M.W. 1060–26,600) from Sigma-Aldritch, St. Louis, MO, USA. Gel electrophoresis was run at 100 volts for 1 h prior to being stained in a CBB solution (1 g of CBB dissolved in 1 L of [50% MeOH, 10% glacial acetic acid, 40% H2O]). The gel was then destained with a mixture containing 5% acetic acid and 20% MeOH overnight.

Fe chelation was tested with the concentrated siderophore powder from the resin concentration step, the brown pigment filter-purified below 3 kDa, as well as with a sample from the remaining proteins that were >3 kDa from the spin filtration procedure. Each fraction was solubilized, where 10 mg of dried pigment was dissolved in 500 μL of 60% MeOH, prior to applying each solution into a blank diffusion disk, manufactured by Oxoid in the UK, allowing it to become dry and concentrated within the disk. These were then placed on a Fe-CAS plate and left to react with chromeazurol S overnight.
