## 3 Results
### Automated Ribosomal Intergenic Spacer Analysis (ARISA) Profiles  
The automated ribosomal intergenic spacer analysis showed that the FASTD kit generated the fluorograms with the highest number of peaks. Fewer numbers of peaks were provided by FAVOR and PUREL (Figures S1-S3). In the case of activated sludge samples, the number of peaks ranged from 4 to 23. The mean number of peaks was highest for FASTD (20.0 \(\pm\) 2.45 peaks), followed by FAVOR (17.6 \(\pm\) 1.99 peaks) and PUREL (12.6 \(\pm\) 4.79 peaks). For the biofilm, the number of peaks ranged from 6 to 26. The mean number of peaks was highest with FAVOR (16.5 \(\pm\) 3.42) peaks), followed by PUREL (13.0 \(\pm\) 5.75 peaks) and FASTD (12.1 \(\pm\) 5.11 peaks). In contrast, for the digestate, the number of peaks ranged from 17 to 30. The mean number of peaks was highest with FASTD (25.3 \(\pm\) 4.06 peaks), followed by FAVOR (20.6 \(\pm\) 1.50 peaks) and finally PUREL (19.5 \(\pm\) 4.81 peaks). In general, looking at all samples together, the number of peaks was highest for FASTD (19.2 \(\pm\) 6.74 peaks), then FAVOR (18.4 \(\pm\) 2.96 peaks), and the lowest for PUREL (15.0 \(\pm\) 5.95 peaks). The Mann-Whitney test found statistically significant differences in the number of peaks between FASTD and PUREL and between PUREL and FAVOR. Figure 1: Result of metagenomic DNA electrophoresis. DNA was extracted using three kits (FASTD, PUREL, and FAVOR). DNA was extracted from activated sludge (S1–S8), biofilm (S11–S18), and digestate from an anaerobic reactor (S21–S28). M—molecular marker M110–M1000 (Gdansk DNA). The genetic distance between samples shown in the form of dendrograms varied between the tested kits (Figures S4-S6). There were no statistically significant differences in genetic distance values obtained by particular pairs of DNA purification method and overall genetic distance for each type of sample. However, some pairs of DNA purification kits gave more similar results than others. Activated sludge samples analyzed by FASTD and PUREL were clustered together four times (samples S1, S5, S7, and S8), PUREL and FAVOR paired three times (samples S2-S4), whereas FASTD and FAVOR only once (sample S6). In the biofilm, PUREL and FAVOR were clustered together four times (samples S14 and S16-S18). FASTD and FAVOR were grouped three times (samples S12, S13 and S15), whereas FASTD and PUREL were only grouped once (sample S11). In digestate, the FASTD and PUREL methods were usually clustered together (samples S22-S26 and S28). Two times, PUREL and FAVOR were grouped together (samples S21 and S27). It was assumed that kits that are outgrouped more often are less reliable. For activated sludge, FAVOR was outgrouped 4 times; for biofilm, FASTD was out-grouped 4 times; and for digestate, FAVOR was outgrouped six times. In the case of activated sludge, the highest mean value of the Margalef index was noticed for FASTD (3.37 \(\pm\) 0.40), then for FAVOR (3.32 \(\pm\) 0.43), then PUREL (2.30 \(\pm\) 0.92) (Figure 2). The Mann-Whitney pairwise test showed a statistically significant difference between FASTD and PUREL (\(p\) = 0.01), as well as between PUREL and FAVOR (\(p\) = 0.013). In the biofilm, the highest mean value of the Margalef index was noticed in FAVOR (2.59 \(\pm\) 0.60), then in PUREL (2.03 \(\pm\) 1.04), and lowest in FASTD (1.91 \(\pm\) 0.83). The observed differences were not statistically significant. In digestate, FASTD showed the highest mean value of the Margalef index (3.96 \(\pm\) 0.61). The intermediate value was noticed in FAVOR (3.45 \(\pm\) 0.28), whereas the lowest was in PUREL (3.18 \(\pm\) 0.77). The observed differences were not statistically significant. A principal component analysis (PCA) was performed to construct a global picture of the relations between DNA yield, DNA purity, and biodiversity expressed in values of the Margalef index (Figure 3). DNA yield and DNA purity explained 67.32% of total variation. The obtained results confirm a stronger effect of the kit used for DNA extraction and purification.