# Biomethane Potential Test: Influence of Inoculum and the Digestion System
## 2 Materials and Methods
### Substrates  
Five substrates with different nutrient compositions were chosen, where hay (S-HAY) and dried maize silage (S-DMS) represent widely used substrates and triglyceride fodder (S-TGF), concentrated fodder (S-CON) and microcrystalline cellulose (S-MCC) represent model feedstock with high content of fat, protein and fiber, respectively (Table 6).  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Code** & **DM \%** & **oDM \%** & **Ash** & **pH** & **Nitrogen** & **NH4+–N** & **VFA mg** & **Alkalinity** \\  & & **\%\%M** & **mg kg\({}^{-1}\)** & **mg kg\({}^{-1}\)** & **mg kg\({}^{-1}\)** & **kg\({}^{-1}\)** & **mg kg\({}^{-1}\)** \\ \hline I-LRS & 4.4 \(\pm\) 0.0 & 61.7 \(\pm\) 0.1 & 1.7 \(\pm\) 0.0 & 8.28 & 4280 \(\pm\) 30 & 3747 \(\pm\) 22 & 30 \(\pm\) 2 & 15.8 \\ I-BGP & 6.8 \(\pm\) 0.0 & 68.0 \(\pm\) 0.2 & 2.2 \(\pm\) 0.0 & 7.78 & 4620 \(\pm\) 0 & 2811 \(\pm\) 60 & 23 \(\pm\) 2 & 13.5 \\ I-LRD & 2.9 \(\pm\) 0.1 & 71.9 \(\pm\) 0.3 & 0.8 \(\pm\) 0.0 & 8.23 & 2115 \(\pm\) 5 & 1472 \(\pm\) 10 & 31 \(\pm\) 2 & 7.1 \\ I-WWP7 & 2.6 \(\pm\) 0.0 & 57.5 \(\pm\) 0.6 & 1.1 \(\pm\) 0.0 & 8.27 & 3040 \(\pm\) 40 & 1659 \(\pm\) 19 & 103 \(\pm\) 23 & 5.0 \\ I-WWP14 & 2.3 \(\pm\) 0.3 & 58.7 \(\pm\) 0.9 & 1.0 \(\pm\) 0.1 & 8.27 & 3155 \(\pm\) 65 & 1961 \(\pm\) 18 & 321 \(\pm\) 2 & 5.0 \\ \hline \hline \end{tabular}
\end{table}
Table 4: Dry matter (DM), organic dry matter (oDM), ash, pH, nitrogen, ammonium (NH4+–N), volatile fatty acid (VFA) and alkalinity with standard deviation for inoculum from a 400 L laboratory reactor (I-LRS), a 2.5 m\({}^{3}\) laboratory reactor (I-LRD), a biogas plant (I-BGP) and a wastewater treatment plant 7 d degassing and 14 d degassing (I-WWP7, I-WWP14).  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Code**} & \multicolumn{8}{c}{**Trace Element Concentration, mg kg\({}^{-1}\)dm**} \\ \cline{2-10}  & **Fe** & **Ni** & **Co** & **Mo** & **W** & **Mn** & **Cu** & **Se** & **Zn** \\ \hline I-LRS & 3244 & 15 & 3 & 7 & 2 & 316 & 91 & 1 & 378 \\ I-BGP & 2710 & 22 & 3 & 6 & 2 & 386 & 418 & 2 & 362 \\ I-LRD & 1131 & 5 & 1 & 10 & 1 & 177 & 98 & 1 & 284 \\ I-WWP & 92,096 & 18 & 5 & 6 & 20 & 220 & 797 & 0 & 643 \\ Reco-mended [28] & 750–5000 & 4–30 & 0.4–10 & 0.05–16 & 0.1–30 & 100–1500 & 10–80 & 0.05–4 & 30–400 \\ \hline \hline \end{tabular}
\end{table}
Table 5: Trace element (TE) concentration (dry matter base) of inoculum from a 400 L laboratory reactor (I-LRS), a 2.5 m\({}^{3}\) diluted from laboratory reactor (I-LRD), a biogas plant (I-BGP) and a wastewater treatment plant (I-WWP) together with the recommend range according to [28].  
Hay (S-HAY) (marstall Wiesen-Cobs, marstall GmbH, Oberstaufen, Germany) is a mixture of more than 50 different grass and herb species from the first cut from Allgau (South Germany) in the year 2012. S-HAY was dried by hot air, chopped to 16 mm, milled with a laboratory mill (Pulverisette 19, Fritsch GmbH, Markt Einersheim, Germany) and stored at -20 \({}^{\circ}\)C.  
Maize silage (S-DMS) was taken from Unterer Lindenhof (Eningen unter Achalm, Germany). S- S-DMS was dried and milled with the laboratory mill.  
Triyglyceride fodder (S-TGF) was provided as a homogenous standard material with well-known molecular structure (C\({}_{18}\)H\({}_{32}\)O\({}_{8}\)N) and a known theoretical biogas yield of 609 L kg\({}_{\text{oDM}}\)\({}^{-1}\)[8]. S-TGF was stored at 4 \({}^{\circ}\)C.  
Concentrated fodder (S-CON) (Raiffeisen Kraftftutterwerke Sud GmbH, Wurzburg, Germany) was milled with the laboratory mill and stored at -20 \({}^{\circ}\)C.  
Microcrystalline cellulose (S-MCC) (CAS: 9004-34-6, Acros Organics, Pittsburgh, PA, USA) was used as standard substrate with known biogas yield of 745 L kg\({}^{-1}\)\({}_{\text{oDM}}\) with a methane percentage of 50% [5]. S-MCC had an average particle size of 50 \(\upmu\)m and was stored at room temperature.