# Microbiota and Metabolite Profiling of Spoiled Spanish-Style Green Table Olives
## Results and Discussion
### Microbiota in Brine Samples  
The lactic acid bacteria and yeast populations obtained by plate counting are presented in Figure 2. The lactic acid bacteria grew rapidly and high numbers were present from the first few days, which explain the rapid acidity increase and pH decrease, as shown in Figure 1. The LAB populations in the Gordal and Manzanilla controls seemed to be lower than in the other treatments, probably as a logical consequence of the importance that salt concentration has on bacterial growth. With regard to yeasts, it is important to highlight that their number were below those of the LAB; and this is an indication that the fermentation process developed normally. Apart from the culture-dependent analysis carried out throughout the fermentation process, microbial DNA was extracted and analyzed at the post-fermentation stage once spoilage was detected. The high throughput sequencing of the 16S- and ITS-PCR products generated a mean of 71,189.33 and \begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multirow{2}{*}{**Sample a**} & \multirow{2}{*}{**pH**} & **Tittrable Acidity** & **Combined Acidity** & **Salt** & **Total Phenols** \\  & & **(g L\({}^{-1}\)** **Lactic Acid)** & **(eq L\({}^{-1}\))** & **(g L\({}^{-1}\) NaCl)** & **(g L\({}^{-1}\) Gallic Acid)** \\ \hline \multicolumn{6}{c}{**Manzanilla Cultivar**} \\ \hline M1 (control) & 4.27 & 4.5 & 0.081 & 87 & 2.00 \\ M2 (control) & 4.19 & 4.5 & 0.078 & 87 & 2.05 \\ MFC1 & 4.84 & 6.5 & 0.134 & 36 & 3.59 \\ MFC2 & 4.92 & 5.2 & 0.128 & 37 & 3.34 \\ MFL1 & 4.96 & 3.7 & 0.134 & 36 & 2.07 \\ MFL2 & 5.01 & 3.4 & 0.128 & 37 & 2.25 \\ MCC1 & 4.66 & 5.9 & 0.113 & 39 & 3.90 \\ MCC2 & 4.73 & 6.1 & 0.108 & 38 & 3.78 \\ MCC1 & 4.96 & 3.0 & 0.085 & 35 & 2.90 \\ MCL2 & 4.89 & 3.6 & 0.083 & 37 & 2.91 \\ \hline \hline \end{tabular}  
* See Table 5 for the meanings of samples abbreviations.  
\end{table} 
Table 1: _Cont._  
Figure 2: The microbial count of lactic acid bacteria and yeasts during fermentation. The points are the means of duplicate fermenters. Where the error bars (range of data) are not visible, the determinations were within the symbols on the graph. See Table 5 for the meanings of samples abbreviations. 46,906.61 high-quality sequences per sample with an average length of 459 and 495 bp, respectively (Tables S1 and S2). Across all taxa, a total of 3 families and 10 genera were identified as predominant bacterial Operational Taxonomic Units (OTU relative abundance >0.1%). Likewise, 1 family and 5 species were identified for ITS. The microbial community was also analyzed using a diversity estimator ([PERSON], Simpson, and Chao1) (Tables S1 and S2). Manzanilla samples showed a higher diversity than Gordal for ITS amplicons and no clear differences were found for 16S samples. The main taxa found at the end of the post-fementation stage are displayed in Figure 3 for bacteria and Figure 4 for yeasts. Figure 3: The relative abundance (%) of the bacterial genera or family obtained by the pyrosequencing analysis at the post-fementation stage. The results of the G and M controls correspond to a blend of both duplicates. See Table 5 for the meanings of the samples abbreviations. The most striking result in relation to bacteria is the very high relative abundance of sequences belonging to the _Cardiobacteriaceae_ family, initially and tentatively allocated to the genus _Suttonella_, which were predominant in all the vessels except in both the Gordal and Manzanilla controls. These Gram-negative _Gammaproteobacteria_ have been mainly related to the upper respiratory tract of human and animal samples, both mammals and birds, manifesting pathogenicity in some cases [16]. However, they seem to be normal microorganisms in dolphins [17; 18], and penguin stomach contents [19] and even in seawater [20]. More unexpectedly, _Suttonella_ sp. has been previously found in defective table olives [8] and in spoiled fermented cucumbers [21].