# Structure of Microbial Communities When Complementary
## 2 Materials and Methods
### Analytical and Chromatagraph Methods  
Performance of the process was monitored by analytical characterizations of all samples and by the volume and quality of the obtained biogas. Total and volatile solids (TS, VS), chemical oxygen demand (COD), total nitrogen (Kjeldahl, TN), ammonium (NH4\({}^{+}\)-N), and pH, were assayed according to standard methods [31]. The total content of phenols was determined by a colorimetric method [32], using caffe acid as the standard for the calibration curve. To measure the antioxidant capacity, a radical scavenging activity of samples against a stable DPPH radical (2,2-diphenyl-2-picrylhydrazyl hydrate) method was used [33]. A calibration curve was made using a Trolox 1.5 mM solution as the standard antioxidant (dissolved in ethanol), made in triplicate. The radical activity was calculated following antioxidant activity Equation (1):  
\[\%\text{DPPPH inhibition}=[(\text{Abs}_{b}-\text{Abs}_{b})/\text{Abs}_{b}] \times 100 \tag{1}\]  
where: Abs\({}_{b}\) is the absorption of blank (t = 0 min), Abs\({}_{s}\) is the absorption of tested sample (t = 30 min). The antioxidant activity was expressed in TEAC (Trolox Equivalent Antioxidant Capacity). The decreasing of the DPPH solution absorbance indicated an increase of the DPPH radical-scavenging activity [34].  
Analytical measures of described parameters were performed at the beginning (IN) and at the end (OUT) of the assay experimental time. The biogas production was monitored daily with a pressure transducer, expressed to standard conditions of temperature and pressure (STP: 0 \({}^{\circ}\)C, 1 bar) defined by IUPAC (International Union of Pure Applied Chemistry). The methane content of biogas, collected in each unit headspace, was measured by the injection of 0.5 mL gas sample weekly into a gas chromatograph (Varian CP 430-GC),  
\begin{table}
\begin{tabular}{l c c c} \hline \hline
**Effluents** & **Test Time** & **Mixture** & **Test Unit** \\  & **(d)** & & **Label** \\ \hline - Olive mill wastewater & & 100\% PE & P \\ (OMW) & & 70\% PE + 30\% OMW & 70P + 30O \\ - Piggery effluent & & 50\% PE + 50\% OMW & 50P + 50O \\ (PE) & & 20\% PE + 80\% OMW & 20P + 80O \\ \hline \hline \end{tabular}
\end{table}
Table 1: Anaerobic digestion in batch conditions.  
equipped with a thermal conductivity detector and a Porapack S column of 1/8\({}^{\prime\prime}\)x 3 m. Column, injector, and detector temperatures were 50, 80, and 100 \({}^{\circ}\)C, respectively. Nitrogen was utilized as the carrier gas (20 mL/mn). Quantification of each gas produced was performed by comparing the obtained graphical peak areas with patterns of an injected gas mix at the beginning of each analysis. Volatile fatty acids (VFA) were evaluated by using a gas chromatograph (Hewlett Packard 5890), furnished with a flame ionization detector and a 2 m \(\times\) 2 mm Carpopack B-DA/4% Carpowax 20 M (80-120 mesh) column. Nitrogen was the carrier gas (30 mL/mn). Temperature of the column, injector, and detector was 170, 175, and 250 \({}^{\circ}\)C, respectively. Total VFAs (acetate, propionate, butyrate, iso-butyrate, iso-valerate, and valerate) concentrations were expressed as acetic acid.  
The biogas production was followed for about 73 days. All values of methane yield (L CH\({}_{4}\)/kg SV\({}_{\text{in}}\)) are presented under STP conditions and divided by the mass of volatile solids of substrate fed in the beginning of the assay. The primary energy yield (kWh/kg VS\({}_{\text{in}}\)) of the tested mixtures was calculated using the lower methane heating value of 9.97 kWh/m\({}^{3}\) CH\({}_{4}\).