## 3 Results  
### Digestate Samples and DNA Isolation  
We isolated DNA from digestates retrieved from 17 biogas plants, 2 wastewater treatment plants (WWTP), and 1 laboratory-scale reactor, all located in either Sweden (SW) or Germany (GER). These AD processes were chosen as they use conventional feedstocks/substrates such as organic household waste, slaughterhouse waste, manure, sewage sludge, and agricultural products (Table 1). Thirteen operate under mesophilic temperature (37-42 \({}^{\circ}\)C), three under hyper-mesophilic temperature (44 \({}^{\circ}\)C), and four under thermophilic temperature (48-52 \({}^{\circ}\)C) (Table 1). The digestate samples, hereafter referred to simply as samples, were taken directly from the methanogenic reactors without any further storage. All German and one Swedish sludge sample(s) were pre-frozen for transportation. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Sample ID**} & \multicolumn{6}{c}{**Substrate**} & \multirow{2}{*}{**Operation**} & \multirow{2}{*}{**Status**} & \multirow{2}{*}{**cidsDNA (ng/uL)**} \\ \cline{2-2} \cline{5-10}  & **Organic Household Waste** & & & & & & & & & \\ \hline \multirow{5}{*}{Sweden} & 01-SW* & o & & & & & & & & \\  & 02-SW & o & o & & & & & & & \\  & 03-SW & & & & o & & & & & \\  & 04-SW & & & o & & & & & & \\  & 05-SW & & & & & & & & & \\  & 06-SW & & & & & & & & \\  & 07-SW & o & o & & & & & & \\  & 08-SW & & & & o & & & & \\  & 09-SW & o & o & & & & & & \\  & 10-SW & & & o & & & & & \\ \hline \multirow{5}{*}{Germany} & 01-GER & & & & & o & & & \\  & 02-GER & & & o & & o & Hyper-mesophilic & Frozen & 282 \\  & 03-GER & & & o & & o & Hyper-mesophilic & Frozen & 410 \\  & 04-GER & & & o & & o & & & \\  & 05-GER & & & o & & & & & \\  & 06-GER & & & o & & & & & \\  & 07-GER * & & & o & & & & & \\  & 08-GER & o & & & & & & \\  & 09-GER * & & & o & & & & & \\  & 10-GER & & & o & & & & \\ \hline \hline \end{tabular}
* Laboratory-scale reactor.
* Mainly crop residues, grass, and/or slages.
* Digestate samples retrieved from reactors operated in parallel at the same biogas plant.  
\end{table} 
Table 1: Main substrate and temperature conditions in the 20 anaerobic digestion processes analysed in this work. The concentration of double-stranded DNA (dsDNA) after the DNA isolation and prior library preparation is also given as an average. The DNA isolation protocol described in this work was pre-tested and optimised on sample 01-SW before being applied to the other 19 reactor samples. Sample 01-SW was from a laboratory-scale reactor mimicking the operation of a large-scale biogas plant. The protocol was optimised for the largest possible DNA fragment length and high DNA purity for optimal nanopore activity and sequencing yield. The full protocol, with each step described in detail, is made available online at protocols.io [19]. The protocol includes all necessary and optimised steps to improve DNA yield and length, with brief explanations. The most significant improvements in overall DNA yield and length are shown in Figure 1. Each DNA isolation was performed in duplicate, and the average concentrations are summarised in Table 1. The concentration differences between the duplicates were less than 50 ng/\(\upmu\)L in all cases. An average of around 350 ng/\(\upmu\)L dsDNA could be retrieved via this protocol across all reactor samples. In brief, we first compared the reactor sample with controlled-growth culture samples using the DNA isolation kit from the FastDNA Spin Kit for Soil (MP Biomedicals) according to the manufacturer. As expected, we observed more digested DNA in the sludge samples, with significantly lower DNA length to yield ratio than in the cultures (Figure 1A). To improve overall DNA yield and fragment length, we modified the washing steps and the total amount of sludge sample used. The DNA yield could be slightly increased by increasing the amount of sludge used. Performing an additional humic acid (HA) washing step improved the yield and reduced the number of smaller DNA fragments.