16S rRNA gene sequencing of MBRA bacterial communities

MBRA content samples (1 mL) were centrifuged at 14000 rpm for 1 min to obtain a pellet, which was stored at −80 °C until further use. Total DNA was extracted with QIAamp DNA Mini kit (QIAGEN) using a modified protocol. Pellets were re-suspended using 360 μL of buffer ATL and homogenized in MagnaLyser (Roche) at 7000 rpm for 70 s. Next, 40 μL of proteinase K was added followed by an incubation at 55 °C (1 h). In the next step 200 μL of buffer AL was added followed by an incubation at 70 °C (30 min). After the addition of 200 μL of 96–100% ethanol the content was transferred into column tubes and the subsequent steps followed the protocol provided in QIAamp DNA Mini kit. Extracted DNA was stored at −80 °C until further use.

Bacterial community structure was determined by sequencing the V3V4 variable region of the 16 S rRNA gene. Libraries were prepared according to the 16 S Metagenomic Sequencing Library Preparation (Illumina) protocol using the primer pair Bakt_341F (5′-CCTACGGGNGGCWGCAG-3′) – Bakt_805R (5′-GACTACHVGGGTATCTAATCC-3′) (approximately 460 bp fragment⁴⁷). Sequencing was performed on the Illumina MiSeq platform (paired-end sequencing, 2 × 300 bp).