title: 16S rRNA from 7 hours of  industrial fermentation of 2G ethanol - Experiment 1
design: Taxonomic  analysis to identify bacterias was performed only for the industrial  fermentation (IN). For this, DNA extraction was performed at the four  sampling points as described previously. The primer pair used, targeting the  variable regions 3 and 4 of the 16S rRNA gene, was Bakt_341F  (CCTACGGGNGGCWGCAG) and Bakt_805R (GACTACHVGGGTATCTAATCC) (Herlemann et al.,  2011). Primer sequences within the 16S paired read sequences were trimmed out  with Cutadapt software (version 1.16) (Martin, 2011). Only the where both the  forward primer and the reverse primer could be identified in the R1 and R2  reads, respectively, were retained. Insertions, deletions or error rates  greater than 20% were not allowed for primer identification. Amplicon  sequence variants (ASVs) were obtained using DADA2 (version 1.8.0) (Callahan  et al., 2016). First, quality profiles were inspected to truncate the R1 and  R2 reads to 228bp and 215bp, respectively. Reads with more than two  expected errors or containing ambiguous bases were filtered out. Finally, R1  and R2 reads were independently denoised using the pseudo-pooling approach  and the pairs were merged to obtain ASV sequences. Taxonomic assignment of  the V3-V4 16S ASVs was performed with IDTAXA (Murali et al., 2018) using a  classifier trained with the GTDB (release 83) (Parks et al., 2018). The  confidence threshold was set to 50 and only ASVs with relative abundance  greater than 0.01% in at least two samples were used for subsequent analysis.  Correspondence analysis with the count data was carried out with the phyloseq  package (McMurdie and Holmes, 2013).