3.8.1 Determination of SCFA (short chain fatty acids)
In order to analyse the SFCA’s produced by fermentation, a modified method of Pylkas et al., (2005) was used. This method has been used in previous work related to current study to anlyse the SCFA concentration (Stewart & Slavin, 2009). Samples (2 mL) that had been taken at the specific intervals and stored frozen were thawed and centrifuged for 30 minutes at 5000 x g at room temperature (In original method-3000 x g) . The supernatant (0.75 mL) was transferred to a sterile vial and vortexed with meta- phosphoric acid (20 %, 0.3 mL). Vials were incubated with meta-phosphoric acid at room temperature (25qC) for 30 min, after which samples were centrifuged for 20 min at 20,000 g for 10 min (In original method-5000 x g for 15 min) . The supernatant was analysed for SCFA by GLC. Samples were stored at -20 qC until required. Centrifugation conditions differed from the original method as these centrifugation comditions were found to produce more SCFA than the original centrifugation conditions.
3.8.2 Preparation of samples and standards for Gas Chromatography
Samples were thawed and the pH was adjusted to 6.5 with 4M KOH and 0.01 mL of 0.3M oxalic acid was added (Pylkas et al., 2005).
3.8.2.1 Chemicals and reagents used to prepare standards
Acetic acid (100%) was obtained from Merck (Darmstadt, Germany). Propionic acid (100%)), n-butyric acid (99%), were purchased from Sigma (St Louis, MO, USA). The chemicals were used as standards for GLC. 2-Ethylbutyric acid purchased from
Sigma-Aldrich (Chemie GmbH, Steinheim, Germany) was used as the internal standard. The water used in the experiment was purified using a Milli-Q® reagent water system (Millipore, Molsheim, France).
3.8.2.2 Experimental conditions for gas chromatography
Analyses were performed using an Agilent 6890N GC (Hewlett Packard, Palo Alto, CA, USA) with a flame ionization detector (FID). A fused-silica capillary column with a free fatty acid phase (DB-FFAP 125-3237, J&W Scientific, Agilent Technologies Inc., USA) of 30 m × 0.53 mm and with 0.50 μm film thickness was used.
Helium was the carrier gas at a flow rate of 14.4 mL/min. The initial oven temperature was 60 °C, maintained for 0.5 min, raised to 180 °C at 8 °C/min and held for 1 min, then increased to 250 °C at 20 °C/min, and finally held at 250 °C for 5 min. Glass wool (Supelco) was placed in the glass liner of the splitless injection port. These glass wool was prepared by immersing in H3PO4 (100 g/L) for 1 h and rinsed to remove the excess
acid before placing at 100 °C for 1 h. The temperature of the flame ionization detector and the injection port was 200 and 240 °C, respectively. Flow rates of nitrogen, hydrogen and air were 20, 30 and 275 mL/min, respectively. The injected sample volume was 1 μL, and the run time for each analysis was 16.5 min. Data handling was
carried out with HP ChemStation Plus software (A.09.xx, Agilent). Unidentofied peaks did not interfere with SCFA peaks and the appearance of ghost peaks was not
significant. One μL of water was injected before starting the analysis and after 10 runs to clean the column.
3.8.2.3 Preparation of stock standard solution
An aqueous stock standard solution was prepared for each acid with a concentration of 500 mM for acetic acid and 200mM for propionic acid and n-butyric acid. 2-Ethylbutyric acid solution (4 mM) in methanol (99.9%) was prepared as an
72 internal standard stock solution. All the stock standard solutions were stored at −20 °C. 3.8.2.4 Preparation of standard mix solution
A standard mix solution of acetic, propionic and butyric acids was prepared in water using stock solutions at 50:10:10, 35:7.5:7.5, 20:10:10, 25:5:5 and 15:2.5:2.5 mM (Appendix D shows the calculations used to prepare the solutions). Five standard mix solutions were used to accurately identify the retention time of the SCFA.
3.8.2.5 Standard curve
Individual calibration curves were obtained for each compound (acetate, propionate and butyrate) using the standard SCFA mixtures. Each standard mixed solution was diluted to have following, concentrations,
Mix solution (mL) Water (MiliQ, mL)
0 0.75 0.05 0.70 0.1 0.65 0.15 0.60 0.2 0.55 0.25 0.50
The different concentrations of each standard mix solution were treated as described in sample preparation for GLC (3.8.2). Forty micro liters (40 µL, 25mM) of internal standard was added to 960 µL of the prepared standard solutions to a final concentration of 1mM and to samples before injecting into the GLC. The linearity of response for each standard acid was tested five times by using the six concentrations described earlier in millimoles (mM). From the chromatograms, the peak area was plotted (response) against the concentration of each SCFA to find the calibration graph for each
SCFA. Calibration graphs were subjected to Excel regression analysis. Results of the graph were obtained as the slope a, intercept b and square correlation coefficients r2 for each acid analysed. Correlation coefficients were approximately 0.9998 for all three acids.
To examine the precision of the method and to calculate the concentration of the SCFAs, one standard mixture of the SCFAs was used after determining the retention time for analysed SCFAs. This was 20:10:10 mM. For experiments, a standard mix solution (20: 10:10 mM) was prepared to plot the calibration graph using the described dilutions and also standards were checked after 10 runs of the GLC each day.
3.8.2.6 Retention time of standards
The acetic, propionic and butyric acid samples were identified on
chromatograms by their specific retention times (min) under the above conditions (3.8.2.2) of GLC.
Acetic acid - 1.979± 0.003 Propionic acid - 2.471 ± 0.004 Butyric acid - 3.101 ± 0.003 2-ethyl butyric acid - 4.104 ± 0.003
3.8.2.7 Quantification
Quantification of the SCFA in the samples was done on the basis of peak area and relative response factor (Jouany, 1982; Zhao et al., 2006). Quantification was not based on the peak height, since peak height is less reliable. Peaks can be broadened, when the column is contaminated with compounds (non volatile) from previous injections. This broadening will cause less accuracy for peak heights but have no effect on the peak area.
74 RRF= Peak area of 1 mM of SCFA / Peak area of 1 mM internal standard
Concentration of SCFA mM in sample
= (Peak area of particular SCFA *Concentration of internal standard)/(RRF of particular SCFA * Peak area of internal standard)