## 4 Discussion  
The extraction and purification of DNA is a crucial stage in metagenomic analysis; however, kits dedicated for the types of biomass analyzed in this study are still not available. Because the number of kits for metagenomic DNA extraction is still increasing, making the right decision is a hard task. In this paper, we evaluated three commercially available kits to identify which is suitable for sampling activated sludge, biofilm, and anaerobic digestate. The obtained results showed that for the activated sludge and digestate, the most promising is the FASTD method. This method allowed the highest DNA yield to be obtained and provided the highest biodiversity. For the biofilm with a high content of EPS, the FAVOR is recommended. The DNA yields measured with fluorometry were always highest when the FASTD kit was used, but other parameters should also be taken under consideration when metagenomic DNA is purified. Additionally, the DNA integrity analysis performed by gel electrophoresis showed that FASTD provided more disintegrated DNA than PUREL. It is not a disadvantage if short DNA fragments are going to be analyzed in further steps. However, if longer DNA fragments are needed (e.g., for third-generation sequencing techniques such as those developed by Oxford Nanopore Technologies and Pacific Biosciences), alternative kits should be considered. The most commonly used method for nucleic acid assessment is spectrophotometry, which relies on the absorbance of light by nucleic acids at different wavelengths. Alternatively, nucleic acids can be measured using fluorometry, which is shown to be a much more accurate and reproducible method than spectrophotometry, which is known to overestimate DNA or RNA quantity by manifolds [20; 21]. In our study, the values of DNA concentration were significantly higher when the spectrophotometric method was used for DNA concentration analysis. The differences were more significant when the values of the A\({}_{260}\)/A\({}_{280}\) ratio were higher than 2.0. The differences between the results obtained by spectrophotometry and fluorometry were not significant when DNA purity was higher, as in the case of the PUREL method. It confirms previous suggestions that the fluorometric method should be used for DNA concentration assessment when DNA extract is not ideally purified. Figure 3: PCA biplot (scores and loadings) presenting the correlations between DNA obtained from different types of biomass and the different kits used. Dots—activated sludge, diamonds—biofilm, and triangles—anaerobic digestate. Blue color—FASTD, red color—PUREL, and green color—FAVOR. The best kit should show the highest alpha diversity. Thus, such a kit should generate the highest biodiversity estimates and minimize the bias of not extracting DNA from the specific taxonomic group [4]. Our results showed that the best kits for describing microbial communities are the FASTD and FAVOR kits. In activated sludge and digestate, the highest values were obtained using FASTD, whereas in biofilm with a high content of EPS, the FAVOR kit provided higher values of biodiversity. The advantage of the FASTD kit, in light of its high efficiency in DNA extraction, is not surprising, but the superiority of the FAVOR kit over the PUREL kit could surprise. The index of biodiversity in the biofilm with a high content of EPS was lowest for the FASTD kit, although the DNA yield was almost three times higher than in the PUREL and FAVOR methods. This could suggest that presence of EPS may be an obstacle in obtaining DNA from some taxa of bacteria when the FASTD kit is used. In our work, we used microfluidic chip electrophoresis to obtain a picture of the biodiversity of analyzed samples. The study based on the analysis of the RIS showed the differences in biodiversity. The peak number, although limited by PCR bias, provided reliable information about biodiversity differences caused by DNA extraction using various kits. The main advantage of this method is its simplicity and fast execution time [15].