EPS accumulated on the microbially colonised, PEL-HS mineral-coated glass beads packed into the glass column reactors when operated in the same way as described in Section 3.5.3. Two columns were taken down at the end of the experimental run and EPS harvesting experiments were conducted on each column as described in Section 3.10.1, alongside the other assays described above. Capsular (firmly bound) EPS recovered was analysed in terms of composition as described in Section 3.10.2. The loosely bound EPS was not of sufficient concentration to analysis.
3.10.1 EPS harvesting from the unsaturated flow-through ore bed
The colonised beads were suspended in 40 ml of 0K medium (pH 2.5) followed by settling out of the beads and centrifugation of the suspension (7,500 rpm for 20 min at 4 °C; Beckman Avanti Centrifuge). The supernatant, containing the loosely bound EPS fraction, was retained and stored at 4 °C for further processing and analysis.
The bound or capsular EPS fraction was recovered from the cell pellet using 10 ml of 30 mM dicyclohexyl-18-crown-6-ether (CE) in Tris buffer as described by Aguilera et al. (2008), followed by gentle agitation (30 rpm) for 2 hours at 4 °C to solubilise the bound EPS fraction. This was subsequently harvested (supernatant fraction) by centrifugation (7,500 rpm for 20 min at 4 °C; Beckman Avanti Centrifuge) as previously described and stored at 4 °C for further processing. A second extraction was then performed on the same sample by adding a further 10 ml of 30 mM CE, and the samples were agitated for a further 2 hours at 4 °C. As before, the supernatant fraction was harvested by centrifugation and retained for further processing. All the samples were filtered twice through a 0.22 μm filter (Merck Millipore), before being dialysed (Pur-A-lyzer Mega 20 ml dialysis kit, Sigma Aldrich, 3.5 kDa cut-off) against sterile MilliQ water (water volume was always greater than 25 times the total sample volume to be dialysed) for 12 hours with water exchanged twice at 6 hour intervals. Samples were subsequently stored at 4 °C until quantitative EPS chemical compositional characterisation was carried out.
3.10.2 EPS biochemical characterisation
Microbial EPS is generally made up of a combination of components, including (but not limited to) polysaccharides, DNA, and lipids. Standard biochemical colorimetric assays, analytical and molecular techniques were used to measure these EPS components.
3.10.2.1 EPS sugar content analysis
The total carbohydrate content of the EPS was determined using a combination of the Dubois phenol-sulfuric assay (Dubois et al., 1956), using glucose as a standard, and an assay modified by Michel et al. (2009), which incorporated Fe3+ into the glucose standard to correct
for any interference caused by the presence of iron in bioleaching systems. The complex carbohydrates are digested into sugar monomers, which are then detected by the creation of an orange-yellow colour. Briefly, a 200 μL sample and 200 μL of 5 % phenol were added into a test tube and mixed by vortexing. Concentrated H2SO4 (1 ml) was added onto the sample
and immediately vortexed. Samples were allowed to stand at room temperature for 10 minutes 20 minutes. Aliquots of 250 μl were
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This assay provides a combined quantitative indication of sugar content. In order to gain more detailed information on the composition of the extracted polysaccharide, the sugar monomers present in the polysaccharides were analysed using gas chromatography mass spectroscopy (GC-MS) at the Central Analytical Facility (CAF; Stellenbosch University). Each sample was prepared by adding 1 ml of 70 % (v/v) methanol to the samples and vortexing. Extraction was done in an oven at 60 °C for 3-4 hours and 250 µl of each of the extracted sample was transferred into a 2 ml tube and completely dried under a gentle stream of nitrogen. Subsequent to that, the samples were derivatised by adding 100 µl of 2 % methoxyamine in pyridine at 40 °C and incubated for 2 hours, followed by the addition of 50 µl N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) and derivatised again at 60 °C for 30 minutes. The samples were vortexed and transferred to a vial before injecting them into a GC-MS instrument for separation. Separation was performed using a gas chromatograph (6890N, Agilent technologies network) coupled to an Agilent technologies inert XL EI/CI Mass selective detector (MSD) (5975, Agilent technologies Inc., Palo Alto, CA). The GC-MS system was coupled to a CTC Analytics PAL autosampler. Separation of sugars was performed on a 30 m DB-5MS capillary column of 0.25 mm ID and 0.25 µm film thickness in which helium was used as the carrier gas at a flow rate of 1 ml min-1. The injector temperature was maintained at 250
°C and 1 µl of the sample was injected in 10:1 split ratio. The oven temperature was programmed at 80 °C for 5 min; and then ramped up to 250 °C at a rate of 8 °C min-1 for 1
minute; followed by a ramping rate of 20 °C min-1 for 5 min until 320 °C and eventually to a
maximum temperature of 325 °C and held for 0.25 min. The MSD was operated in a full scan mode and the source and quad temperatures were maintained at 240 °C and 150 °C, respectively. The transfer line temperature was maintained at 250 °C. The mass spectrometer was operated under electron impact mode at ionization energy of 70 eV, scanning from 40 to 650 m/z
3.10.2.2 EPS DNA content analysis
The DNA content was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific). An aliquot of 2 μL of EPS sample was placed on the Nanodrop and the concentration measured and recorded.
3.10.2.3 EPS lipid content analysis
The lipid content of the sample was measured based on Smedes and Askland (1999) method. Briefly, the samples were freeze dried overnight and, in order to solubilise complex lipids that are not soluble in methanol, 500 μl of hexane was added to freeze-dried pellet. A further 100 μl C17-TAG internal standard in hexane (1 mg ml-1) and 1 ml of basic catalyst (0.5 N NaOH in
constant agitation at 300 rpm. The samples were allowed to cool for 5 minutes at room temperature, before 1 ml of acid catalyst (5 % (v/v) HCl in methanol) was added before repeating the vortex and incubation step. The samples were allowed to cool for another 5 minutes before the addition of 400 μl deionised H2O to stop the reaction. Hexane (300 μl) and
100 μl C19-methyl ester internal standard in hexane (1 mg ml-1) were added and vortexed well
in order to extract the lipids. The samples were analysed using GC.
The GC run was conducted based on a method adapted from Griffiths et al. (2010). Fatty acid methyl esters (FAME) extracts (1 µl), containing internal standards that included glyceryl triheptadecanoate (C17-triacylglyceride) and methyl nonadecanoate (C19-methyl ester) that were added before and after transesterification respectively. Each sample was injected into a Varian 3900 GC equipped with a flame ionisation detector and SupelcoWax 10 column (30 m × 320 mm × 1.0 μm film thickness) (Supelco, USA). Standard split/splitless injection was used with a split of 100 and an injector temperature of 270 ºC. The column temperature was increased from 180 ºC to 260 ºC at 2 ºC min-1. Nitrogen (2 ml min-1) was used as the carrier
gas and the detector temperature was 260 °C. Peaks were identified by retention time using Supelco 37 Component FAME and C14:0 to C22:0 FAME mixtures. Peak areas were used to quantify each FAME relative to the internal standards. Differences in the response factor of the detector to the range of FAMEs in the samples were negligible. The total fatty acid content was calculated by adding all the individual FAME peak areas.
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