## 4 Discussion  
Keeping possible biases inherent to molecular technique and next-generation sequencing (NGS) in mind [9] (i.e., PCR, short-read sequences, pan-genome concept of bacterial evolution), many works demonstrated that NGS can be successfully used to improve our understanding of bacterial taxonomic structure and functional profile across aquatic [9; 17; 18; 19; 20; 21] and terrestrial ecosystems [22; 23; 24; 25]. One of the most commonly used NGS techniques (Illumina Miseq) offers paired-end reads, which increase almost two times of the non-merging read length (i.e., 580 bp for MiSeq reads of 300 bp with a 20 bp minimal overlap) [52]. In this study, we demonstrated that FAPROTAX was able to assign functions to prokaryotic taxa derived from both aquatic and terrestrial sources, especially soils. Even though aquatic samples tend to gain higher assignment than those of terrestrial samples, no significant difference was found on assignment percentage between different sample sources based on a limited number of publications (Table S3). Several previous studies used this tool to predict the functions of bacterial/archaeI communities residing in not only aquatic habitats but also in soil samples. For example, FAPROTAX was used to assess the impact of microbial inoculation and fertilizer application on soil bacterial functions involved in for C and N cycles [53; 54; 55]. Specifically, [PERSON] et al. [53] showed stimulation of denitrification in soil after _Sparnitam alteriflora_ invasion, as well as [PERSON] et al. [54] revealed a significant effect of straw incorporation and nitrogen fertilization on hydrocarbon degradation and nitrogen fixation. Similarly, [PERSON] et al. [55] showed an increase of aerobic nitrite oxidation in soil inoculated with multi-species inoculants. Moreover, FAPROTAX was utilized in several studies that aimed to compare the effect between different treatments or a response of site managements [56; 57; 58; 59; 15]. For instance, it was used to investigate the effects of bacterial functions after grazing prohibition and in plantation and natural forest [56; 15; 28]. The increase in denitrification and nitrification functions was found after soil tillage and forest-to-agriculture conversion [57; 58]. In addition to soil samples, FAPROTAX has also been helpful in predicting bacterial functions of animals and human microbiome. For examples, FAPROTAX was used to compare the functional richness of bacteria in gallbladder and gut between young and adult rabbits [60], \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Eosystems**} & **No. of N-Fixation** & **No. of Additional OTU** & \\  & **(FAPROTAX Assignment)** & **Manually** & **Additional Taxa Capable of N-Fixation** \\  & **Assignment)** & **Assigned to N-Fixation** & \\ \hline \hline \multirow{4}{*}{Agricultural Bulk soil} & \multirow{4}{*}{17} & \multirow{4}{*}{53} & _Bacillus megaterium_, _Bacillus cereus_, _Ensifer_, _Bradyrhizobium_, _Microvirga_, _Pullobacterium_, _Mesorhizobium_, _Allorhizobium-Nerhizobium-Paranthizobium-Rrizobium_, _Burkholderia-Cahalleroonia-Paranburkholderia_, _Devosia_, _Methylobacterium_ \\  & & & _Bacillus megaterium_, _Ensifer_, _Bradyrhizobium_, _Mesorhizobium_, _Phylobacterium_, _Microvirga_, _Devosia_, _Allorhizobium-Nerhizobium-Paranthizobium-Rrizobium_, _Burkholderia-Cahalleroonia-Paranburkholderia_, _Methylobacterium_ \\  & & & _Bacillus megaterium_, _Ensifer_, _Bradyrhizobium_, _Mesorhizobium_, _Dryolbacterium_, _Microvirga_, _Devosia_, _Allorhizobium-Nerhizobium-Paranthizobium-Rrizobium_, _Burkholderia-Cahalleroonia-Paranburkholderia_, _Methylobacterium_ \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Number of OTUs capable of N-fixation before and after adding data from previous studies and the names of additional taxa. infer biogeochemical processes, especially in nitrogen and manganese cycling occurring on human and mammalian skin [32] and show the impact of environmental factors on the functional diversity of bacteria in frog skin [14].