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

Abstract

<strong>Aims:</strong> 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.<br /> <strong>Methods and results: </strong>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.<br /> <strong>Conclusion:</strong> 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.