## 4 Results and Discussion
### Next-Generation Sequencing Results  
The microbial community of groundwater sampled in the C5 cluster was analysed to explore the possible influence of microbial activity (H\({}_{2}\)S and CH\({}_{4}\) production) on the unusual deuterium content detected in C5-waters. The 16S rRNA gene sequences of both bacteria and archaea identified in this study have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the accession number PRJNA631131. Based on analysis of the sequences obtained from the C5 cluster with NGS technologies, the broad diversity of the microbial community in terms of relative abundance of bacterial genera has been highlighted (Table 1). Regarding the main aim of the present study, the analyses showed a bacterial community characterised by aerobic and anaerobic/facultative anaerobic genera. Many of these genera are common in bacterial communities found in salty groundwater and marine environments. Halophilic bacteria can be considered moderate halophiles or extreme halophiles based on the concentration of salts present in water and soils (e.g., _Alcanivorax_[67] and _Marinomonas_[68] in Table 2); in contrast, halotolerant bacteria, despite growing better without NaCl, can tolerate high quantities of salt [69] (e.g., _Arcobacter_[70] and _Pseudomonas_[71] in Table 2). In addition, some of the bacterial genera can exploit sulfate reduction (_Desulfatiglans_[72], _Desulfovibrio_[73], and _Shewanella_[74] in Table 2) as a metabolic pathway. Sulfate reduction is a form of anaerobic respiration, typical of sulfate-reducing bacteria (SRB) or sulfur-reducing bacteria, and as such typically occurs in anoxic environments and leads to the production of hydrogen sulfide (H\({}_{2}\)S); however, some studies have found that this process can also occur in the presence of oxygen [71; 75]. Figure 11: Relation between \(10^{3}\)\(\delta^{2/1}\)H and T.U. in the investigated waters. Waters from the C5 wells exhibit a significant \(10^{3}\)\(\delta^{2/1}\)H vs. T.U. correlation. Regarding the archaeal component, the most abundant genus found in the analysed groundwater samples (C5a-c) was _Candidatus Nitrosopumilus_[76] (Table 3). This genus is typically found in marine environments where ammonia can be oxidised to nitrite. Moreover, halophilic genera were identified, such as _Halococcus_[77] (Table 3), a genus capable of living in the presence of very high concentrations of NaCl (up to 4.5 M NaCl). Methanogenic genera, such as _Methanobacterium_[78], _Methaninicococcus_[79], and _Methanolobus_[80] were also found (Table 3). Methanogenesis is a strictly anaerobic metabolic pathway that leads to the formation of methane, and s performed only by methanogenic archaea. Thus, the biomolecular investigations demonstrated that C5-groundwaters are characterised by bacterial (_Desulfitiglans_, _Desulfovibrio_, and _Shewanella_) and archaeal (_Methanobacterium_, _Methanomicococcus_, and _Methanolobus_) genera able to produce H2S and CH4, respectively, therefore suggesting that microbial activity is a factor of utmost importance to be taken into consideration when analysing the isotopic signature of local groundwater.