Microbial community analyses of composts are influenced by particle size fraction and DNA extraction method

Composting plays a key role in sustainable agriculture by converting organic waste into a valuable soil conditioner. The process is driven by complex microbial communities, whose characterization is essential for optimizing the composting process and compost quality. Molecular techniques such as amplicon sequencing are commonly used for this purpose. However, sampling procedures and DNA extraction methods, key steps in the sequencing workflow, often vary across studies, challenging comparability. We investigated two aspects of sample preparation that may influence compost microbial analyses. For DNA extraction, often fine fractions (<2 mm) are used. However, compost has a heterogeneous structure, including coarse particles. To assess the effect of particle size, we separately sequenced bacterial and fungal communities of the fine (0-2 mm) and coarse (2-10 mm) fractions of three composts. In addition, DNA was extracted using a carboxyl-affinity-based magnetic method and a silanol-affinity-based filter method to evaluate the impact of the extraction technique. We found that the coarse fraction had higher bacterial richness and distinct bacterial and fungal community structures compared to the fine fraction. DNA extraction method also influenced bacterial community profiles, with the magnetic bead method improving coverage, particularly for Bacillota. Although the effects of particle size and extraction method were small compared to the overall diversity among composts, we recommend including coarse particles in sequencing analyses and using standardized DNA extraction protocols, especially for studies aiming at high-resolution community analyses.