Unveiling Microbial Communities: Methodological Biases in DNA Extraction and 16S rRNA Sequencing Skew Microbial Profiles in Anaerobic Fermentation

Modern molecular analyses have revolutionized the study of microbial communities, yet DNA extraction and sequencing remain critical sources of bias. This study investigated the impact of seven different DNA extraction protocols and two 16S rRNA hypervariable regions (V1–V3 and V3–V4) on the profiling of a complex anaerobic fermentative biomass selected for medium-chain fatty acids production. Microscopic analysis established a baseline community dominated by Actinobacteria (53% ± 2%) and Firmicutes (47% ± 3%). The results demonstrate that Kit1 and Kit5 provided the highest DNA yields (up to 603 ng/μL) and the most effective recovery of these hard-to-lyse phyla, although they introduced a slight taxonomic bias toward Actinobacteria. In contrast, protocols relying on intensive chemical lysis without robust mechanical disruption (Kit4) significantly underestimated total bacterial abundance and showed the lowest purity. 16S rRNA gene sequencing revealed that the V3–V4 region provided higher alpha-diversity and a more balanced representation of the community core compared to V1–V3, which was more susceptible to extraction-related variability and overrepresented the genus Olsenella. Our multi methodological approach reveals significant biases introduced by both extraction technique and 16S rRNA gene region. This evidence highlights that protocol optimization is mandatory for achieving an accurate and comprehensive characterization of microbial ecosystems.