## 3 Results and Discussion
### Microbial Analysis  
To identify the microorganisms involved in the DF process, a comparison of relative abundance across different process conditions was conducted (Figure 4). The analysis at the genus level revealed that _Bacteroides_ consistently dominated the microbial community, with relative abundance varying slightly depending on the nanoparticle conditions, ranging from 55% to 65%. _Bacteroides_, the phylum to which _Bacteroides_ belong, are known as proteolytic bacteria and are likely involved in the degradation of various proteins. These proteolytic microorganisms have the capability to metabolize amino acids, leading to the production of volatile fatty acids (VFAs) such as acetate, propionate, succinate, and ammonia (NH\({}_{3}\)) [41].  
Following _Bacteroides_, the subdominant genera identified include _Clostridium_, _Lachnoclstidium_, _Veillonella_, and _Robinsonella_. Initially, the addition of 50 ppm FeNPs resulted in a decrease in the relative abundance of _Clostridium_ from 10% to 6%. However, as the NPs concentration was increased to 100 ppm and 200 ppm, the relative abundance of _Clostridium_ rose to 19% under both conditions. _Clostridium_ is well-known as a classic H\({}_{2}\)-producing genus in DF [42]. While _Clostridium_ can utilize simple sugars for H\({}_{2}\) production, previous studies have demonstrated its ability to produce H\({}_{2}\) from more complex substrates as well [43]. The molecular analysis used in this study did not allow for a correlation between H\({}_{2}\) production and the relative abundance of Clostridium, as it does not reflect bacterial activity. When the NPs concentration was increased to 100 ppm and 200 ppm, the relative abundance of _Clostridium_ increased; however, this did not correspond to an enhancement in H\({}_{2}\) productivity. In fact, H\({}_{2}\) productivity decreased at FeNPs 200 ppm condition. Despite the overall increase in _Clostridium_ species with higher nanoparticle concentrations, there was a notable decline in species such as _Clostridium butylicum_, which are strongly associated with H\({}_{2}\) production.  
The subdominant genera identified in the study are not strictly associated with H\({}_{2}\) production. For example, _Lachnoclstidium_, which belongs to the family _Lachnospiraceae_,  
Figure 4: Structure of the microbial community at the genus level in the acidogenic broth under different conditions. The category “others” includes genera with a relative abundance of less than 1%.  
is primarily linked to the production of VFAs, including acetic, propionic, and butyric acids [44]. Many species within the _Lachoclostidium_ genus are strict anaerobes that ferment carbohydrates, leading to the synthesis of these organic acids. Notably, certain strains of _Lachoclostidium_ are particularly known for their ability to produce butyric acid. Similarly, _Robinsoniella_ has been shown to grow on a variety of polysaccharides and monosaccharides, including amygdala, arabinose, cellobiose, fructose, glucose, maltose, lactose, raffinose, starch, trehalose, xylan, and xylose [45]. Finally, _Veillonella_ is associated with the production of acetic and propionic acids as major end products [46]. Although these genera are not directly associated with H\({}_{2}\) production, their significant reduction under the highest NPs concentration condition (200 ppm) could be related to the observed decrease in H\({}_{2}\) production. The synergistic action and mutualistic relationships among mixed bacterial communities play a crucial role in the fermentation process for H\({}_{2}\) production.