Long read 16S amplicon analyses and improved cultivation techniques as joined approach for the identification of viable bacterial populations in silage

Lactic acid bacteria (LAB) are crucial for the quality and preservation of silage. Exploration and isolation of the key microbiome are important for improving fermentation processes. While cultivation-dependent methods enable the isolation of LAB, they often overlook organisms with challenging cultivation requirements. Next-generation sequencing provides cultivation-independent microbiome insights. In contrast to commonly used methods, long-read sequencing platforms like the Pacific Biosciences (PacBio) single-molecule real-time (SMRT) platform offer species-level resolution. This study aims to investigate the benefits of a combined cultivation-dependent and -independent approach for silage microbiome analyses. The common isolation medium for LAB was supplemented with sterile pressed juice from plant material improving the cultivation conditions. PacBio amplicon sequencing provided an almost complete and cultivation-independent picture of the bacterial community. The use of mock communities and a live/dead discriminating treatment of the samples revealed that the analysis can be misleading if appropriate controls are not performed. Growth of plant-associated microorganisms can be supported by a plant juice containing isolation medium, allowing isolation of the dominant LAB from silage. Microbial dynamics can be represented using long-read sequencing. However, the use of controls such as a live/dead discrimination and mock communities is essential for the reliability of the data.