## 2 Materials and Methods
### Experimental Design
This approach constitutes the standard methodology utilized in bench-scale laboratory biomethane volumetric determinations [32; 33; 34]. Agit \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**(VS\({}_{\text{WHDS}}\)/VS\({}_{\text{SY}}\))** & **1:0** & **4:1** & **3:2** & **2:3** & **1:4** & **0:1** \\ \cline{2-7}
**Bioreactor Designation** & **WHDS Only** & **Mix-1** & **Mix-2** & **Mix-3** & **Mix-4** & **SY Only** \\
**Parameter** & & & & & & \\ \hline WHDS (g wet basis) & 54.728 & 43.783 & 32.837 & 21.891 & 10.946 & 0.000 \\ SY (g wet basis) & 0.000 & 2.816 & 5.632 & 8.448 & 11.263 & 14.081 \\ Total carbon (g dry basis) & 1.725 & 1.537 & 1.349 & 1.161 & 0.974 & 0.786 \\ Total nitrogen (g dry basis) & 0.100 & 0.091 & 0.083 & 0.074 & 0.065 & 0.056 \\ carbon to nitrogen (C/N) ratio & 17.250 & 16.890 & 16.253 & 15.689 & 14.985 & 14.036 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The mass characteristics of the different co-digestion substrate mixtures investigated. Figure 2: Labeled schematic representation of the experimental set-up for assessing the co-digestion of substrate mixtures of VS\({}_{\text{WHDS}}\) to VS\({}_{\text{SY}}\) mass ratios of 1:0, 4:1, 3:2, 2:3, 1:4 and 0:1.  
was undertaken twice daily to enable uniform availability of useful substrates to microbes and thus provide further growth impetus for aceticlastic methanogens present in the substrates. The pressure of 760 mmHg was maintained by ensuring that the connecting reservoir tank was open to the atmosphere with daily measured biomethane gas volumes normalized to the standard temperature condition of 273.15 K using the ideal gas law. Pressure compensation (to \(\approx\)760 mmHg) was achieved by ensuring that barrier liquid level in the audiometer was returned to the liquid level in the reservoir after daily biomethane potentials were recorded. Although negligible contributions to the biomethane potentials were anticipated from the degradation of residual biomass present with the inoculum (after the degassing operation), a control bioreactor containing only the inoculum was also subjected to a similar anaerobic degradation process. The reported biomethane potentials from the different bioreactors were subsequently corrected using the biomethane potentials obtained from the control bioreactor containing only the inoculum. Finally, to facilitate a low-cost and efficient estimation of the percentage biomethane content (volumetric basis) of the total volume of biogas generated from the co-digestion substrates, each bioreactor was equipped with a similar gas measurement setup as described above, with the only modification being the barrier solution utilized. The NaOH liquid barrier was replaced with an acidified (pH of 2)-saturated NaCl solution as the barrier solution to enable the determination of the total biogas volume as utilized in a previous study [35]. This is because the acidified-saturated NaCl solution will prevent the dissolution of the major components of the biogas of CO\({}_{2}\) and CH\({}_{4}\) such that the volume of biogas, in terms of its major components, can be measured [35; 36]. Experiments for determining biomethane potential and biogas potential for each substrate mixture were undertaken simultaneously with biogas volumes recorded on days with associated biomethane yields. The percentage biomethane content (volumetric content basis) of biogas was subsequently calculated. Possible synergizing and antagonistic effects that may arise from the application of the ACD pathway were investigated by assessing the characteristic co-digestion performance index (CPI) of each substrate mixture [37]. Generally, if the CPI is greater than 1, the presence of synergizing effects is demonstrated; if the CPI is less than 1, the presence of antagonizing effects is established. However, if a CPI value is equal to 1, it suggests that the ACD process occurs independently of Synergizing and antagonistic effects [38].