Further characterisation of isolates

2.2.3.1

Production of indole-3-acetic acid

A modified method for IAA detection from Sawar and Kermer (1995) was carried out. The IAA growth medium consisted of 5 g of glucose, 0.025 g yeast extract, and 0.204 g of L-tryptophan (Sigma- Aldrich, USA, CAS 73-22-3) in 1 L and the growth medium was autoclaved. The ten EPS isolates were propagated in 3 mL of tryptic soy broth (TSB) for 24 h followed by two washing steps with the IAA medium before inoculating 500 µL aliquots of the washed culture into 10 mL of the IAA growth medium in McCartney bottles. After incubating for 72 h (20°C, 250 rpm) in the dark, 1 mL of cell culture was centrifuged for 10 min (15,7000 × g). A 90 µL aliquot of the supernatant was then incubated with 60 µL of freshly prepared Salkowski reagent in 96 well plates (Costar®, flat bottom, Bio-Rad, California, U.S.) in the dark for 30 min. Salkowski reagent consisted of 1 part of 500 mM iron

chloride (FeCl3) and 49 mL of 35 % perchloric acid (HClO4). The concentration of IAA in each culture

medium was determined by comparison with a standard curve. The standard curve was made from a stock 0.1 % IAA solution at 6, 15, 30, 45, 60, 90 and 120 µg IAA mL-1 _{concentrations and incubated}

with Salkowski reagent as described for the treatment samples above. Colour changes from colourless to pink were measured at 530 nm using the FLUOstar Omega microplate reader (BGM Labtech, Ortenberg, Germany). The negative control (blank) comprised a culture filtrate that had not been inoculated with bacteria which was incubated under the same conditions as the treatments and the value from the negative control was subtracted before calculations. IAA produced by each isolate was measured in triplicate.

2.2.3.2

Siderophore production

The siderophore assay is based on competition for iron between the Fe(III) complex of an indicator dye, Chromazurol S (CAS, Merck KGaA, Darmstadt, Germany), where a strong chelator produced by siderophore-producing rhizobacteria removes the iron from the dye resulting a colour change from blue to orange. The colour change can be detected by a modified CAS plate assay (Schwyn & Neilands 1987) (Figure 2.3.1). To identify siderophore producing rhizobacteria, the CAS agar medium was prepared by mixing three solutions. Solution I consisted of 50 mL autoclaved distilled water, 60.5 mg Chromazurol S, 10 mL filter sterilised iron chloride (FeCl3) solution (1 mM FeCl3 in 10 mM HCl);

solution II consisted of 40 mL autoclaved distilled water, 72.9 mg cetrimonium bromide (hexadecyl- trimethyl-ammonium bromide); and Solution III consisted of 850 mL distilled water, 6.4 g Na2PO4, 0.3

g KH2PO4, 2.5 g NaCl, 2.5 g NH4Cl, 30.24 g PIPES (2-[4-(2-sulfoethyl)piperazin-1-yl]ethanesulfonic

acid), 12 g of 50% NaOH, 15 g agar and adjusted the pH to 6.8 with sterilised 50% NaOH and the mixture was autoclaved. Filter sterilised glucose with a pH of 6.8 (20 mL, 20 %), and filter sterilised casamino acid (30 mL, 10 %, pH 6.8) was also added to solution III and the pH was maintained at 6.8. Adjust Solution II to pH 6.8 using filter sterile 4 M NaOH before mixing with Solution I followed by adding the mixture to Solution III. The CAS agar was settled into sterile petri dishes before the green agar mixture turned into blue when setting. To standardise the assays, 1 µL of an overnight culture (with approximately ~105 _{cfu) of each isolate was aliquoted onto the CAS agar plates, which were}

incubated for 7 days at 25°C. The development of yellow halos indicated the production of siderophores (Figure 2.3.1).

2.2.3.3

Hydroxyapatite solubilisation

To quantify the amount of soluble P released during P solubilisation, as well as to obtain an organic acid profile released by each isolates using High Performance Liquid Chromatography (HPLC), liquid cultures were used. The HSU HydroxP liquid culture medium used in this study was modified from the HSU buffer as described in Giles et al., (2014), where additional iron (II) sulfate was included and calcium phytate precipitate was replaced with HydroxP that is known to be a less soluble form of calcium phosphate (-log Ksp = 116.8) (Dorozhkin & Epple, 2002) (Appendix A.2.4). The reason for using

the HSU medium is because this medium contains a low salt concentration, and is used for maintaining positive selection pressure on wildtype bacteria to produce essential amino acids and vitamins. Furthermore, to maintain a constant solute ion at the stationary phase for HPLC separation, low salt concentration as well as chloride ions was used as the major anion in the HSU solution to retain the ion effect on the HPLC column.

Quantification of soluble P released by ten EPS isolates were performed by growing each isolate in HSU liquid culture medium with the only P source from HydroxP (Ca5(PO4)3(OH)). This was carried out

in 250 mL Erlenmeyer flasks containing 50 mL of HSU liquid culture medium (55.5 mM glucose, 1.38 mM K2SO4, 2.63 mM MgCl2, 4.67 mM NH4Cl, 0.27 mM CaCl2, 24.98 mM NaCl, and 0.3 g of HydroxP,

Appendix A.2.4, Figure 2.2.2). The HSU HydroxP medium was adjusted to pH 6.5 and the flasks were covered with non-absorbent cotton before autoclaving. Before starting the HSU liquid culture assay, each isolate was grown in LB broth for 24 h at 25°C except isolate Ha185, which was grown for 48 h, and was shaking at 250 rpm until the OD600 reached ~4.0. At this OD600, each isolate was pelleted by

centrifugation for 3 min at 5900 × g at room temperature. The starter culture was prepared by resuspending each cell pellet independently to a final OD600 ~2.0 with filtered sterilised HSU liquid

culture medium that contained no HydroxP. Each flask was inoculated with 100 µL of the starter culture to give a bacterial suspension of ~106 _{cfu mL}-1_{, which was confirmed by serial dilution plating}

(Appendix B.2). The HSU liquid culture medium was incubated at 25°C and shaking at 250 rpm in a Raytek orbital incubator. One millilitre of each sample was pelleted after day 3 and day 7 post inoculation and centrifuged at 15,700 × g for 10 min. The supernatant was filtered by 0.22 um PVDF syringe filter (13 mm, Thermofisher scientific, Massachusetts, USA) before the amount of soluble P was measured by Murphy and Riley’s method (1962) as described in Section 2.2.1.1. Colony forming units (CFUs) was also enumerated for each HSU liquid culture at day 3 and day 7 by serial dilution plating (Appendix B.2). This experiment was performed at three independent times for statistical analysis.

Figure 2.2.2 Photographs of the liquid culture assay. Insoluble hydroxyapatite (HydroxP) is a white powder (A), 50 mL uninoculant HSU HydroxP medium in Erlenmeyer flasks and 1.5 mL aliquot in Eppendorf tube (B1 and C1 respectively), HSU HydroxP medium with isolate Ha185 inoculation for 7 days at 25°C in Erlenmeyer flasks and 1.5 mL aliquot in Eppendorf tube (B2 and C2 respectively). Note the clearing in C2 indicating solubilisation of HydroxP insoluble particles by Ha185 isolate after 7 days incubation.