## 3 Results and Discussion
### Indirect Roxarsone Reduction Experiment at Different Time Intervals  
The roxarsone concentration was found to decrease when HFO was included in the incubations of both strains of MR-1 and Cellu-2a (Figure 2A,B). However, the decrease patterns were different from each other. In strain MR-1, there was a lag phase of roxarsone decrease to 10 days, with a relatively rapid decrease of roxarsone from 10 days to 15 days (Figure 2A), while roxarsone decreased rather rapidly to 5 days with strain Cellu-2a (Figure 2B). It seems that there was a rather direct reduction of roxarsone in Cellu-2a incubations even with HFO, as indicated by the similar pattern of roxarsone decrease in the direct reduction experiment (Figure 1B).  
Figure 1: Optical density for bacterial cell growth and concentration of roxarsone in the direct roxarsone reduction experiments with strain MR-1 (**A**) and strain Cellu-2a (**B**).  
In the indirect experiment with strain MR-1, ferrous iron was found to increase up to 10 days and started to decrease gradually from 10 days to 30 days (Figure 3A). The decrease of Fe(II) may be caused by heterogeneous reduction of rovarsone on the solid Fe(II) phases of the partially reduced HFO, which is, in turn, supported by the rapid decrease of rovarsone from 10 days in Figure 2A. This suggests that the reduced Fe(II) on HFO might have acted as a reductant for rovarsone. In the previous publication by [PERSON] et al. [15], it was reported that strain MR-1 reduced 1 mM rovarsone almost completely within 40 h in the presence of soluble phase of ferric-citrate, which was much faster than the incubation without ferric-citrate. However, in this study, with solid phase Fe of HFO, the rovarsone decrease was delayed (Figure 2A), compared to the direct reduction without HFO (Figure 1A). Once reduction of HFO generated Fe(II) on solid surface of HFO, rovarsone decreased relatively rapidly (Figure 2A). From this observation, it could be speculated that the reduction of the rovarsone decreases with increasing temperature.  
Figure 2: Roxarsone concentration in the indirect rovarsone reduction experiments with strain MR-1 (**A**) and strain Cellu-2a (**B**).  
that roxarsone would react both with soluble Fe(III) and solid phase Fe(II). In addition, the differences of incubation conditions such as concentrations of electron donor, electron acceptor, and media composition might have affected the microbial metabolic activities on roxarsone reduction in both studies. In the case of strain Cellu-2a, there was no observation of decrease of the generated Fe(II) (Figure 3B), and ferrous iron concentration increased up to 1.91 \(\pm\) 0.06 mM, which was a near complete reduction of the initial HFO (1.96 \(\pm\) 0.03 mM) (Figure 3B). Looking at the roxarsone decrease and Fe(II) production patterns together in the Cellu-2a incubations (Figures 2B and 3B), it seemed that there was no indirect reduction of roxarsone by the generated Fe(II). Instead, the strain Cellu-2a might have reduced roxarsone directly even with added HFO in fermentation conditions.  
Figure 3: Concentrations of 0.5 N HCl-extractable Fe(II) and Fe(III) from HFO in the indirect roxarsone reduction experiments with strain MR-1 (**A**) and strain Cellu-2a (**B**).