*5.1. Impact of Fe-Based Ions and NP Addition*

To quantitatively unveil the impact of the concentration of Fe-ion/Fe NPs and size effects upon the HY and HER in BioH2 generation, the collected values from the literature (Table 1) were statistically analyzed through our previously established ANN-RSM method and the results are shown in Figure 3.


**Table 1.** Comparison of BioH2 production with the addition of Fe-based nanoparticles.

In this table, MEG refers to mono ethylene glycol, SC refers to substrate concentration, MSJ denotes Marcroalgea Saccharina Japonica, NMBL refers to *R. sphaeroides* NMBL-02 and *E. coli* NMBL-04, MC refers to mixed consortia, BA refers to *Bacillus anthracis* PUNAJAN 1, CP refers to *C. pasteurianum*, EA refers to *E. aerogenes* ATCC13408, EC refers to *E. cloacae*, Cl refers to Clostridium, Ca refers to *C. acetobutylicum* NCIM 2337, SJ refers to sugarcane juice, CAS refers to cassava starch.

**Figure 3.** Statistical analysis of HY and HER. HY refers to H2 yield; HER refers to the H2 evolution rate. (**A**) Particle size and NP concentrations versus HY, (**B**) particle size and NP concentrations versus HER.

The effects of NPs size and NPs concentration together with the binary combined impact upon the HY and HER were extensively explored. Regarding HY, it was found that the size of the NPs together with the concentration of NPs were both statistically significant to the H2 yield amongst the surveyed literature's reports of experimental conditions. From Figure 3A, it is indicated that the HY tended to approach the highest value in the range of NP size (81–100 nm) and NP concentration (406–604 mg L<sup>−</sup>1). For HER, it was found that the size of NPs, the concentration of NPs, and Fe2+/Fe3+ were all significant to HER. For the combined effects (NP size and concentration), on the other hand, these effects were found to be statistically insignificant to HER. The 3D plot of HER versus NPs size and NPs concentration (Figure 3B) also tended to show the highest region of HER located at the size range of 81–100 nm. Among the collected literature reports, the HER seemed to be more appreciably and directly related to the relatively larger size of the particle, which might be quite contradictory to some findings. This indicates that the manipulation of NPs ideally in size range of 81–100 nm is favorable for both high HY and HER. Reducing the size of NPs could improve the quantum dot effect, thus improving the electron transport. In contrast, the electron transport phenomena in extracellular media during cultivation is quite complicated and some factors such as osmosis condition and the activity of the fermentation broth might be counter-effective to the nanoparticle size effect for enhancing BioH2 generation. Currently, very few works have been done to elucidate the mechanisms of this size impact upon selective enhancement of HY and HER. From our statistical analysis, a reasonable explanation for the ideal size effect is that the nanoparticle size of 81–100 nm is more thermodynamically stable than NPs with a smaller size during fermentation, since Fe-based NPs with smaller size are easier to agglomerate and form large Fe-based particles and deteriorate the electron transport performance in extracellular conditions. The fabrication of composites (e.g., Fe@graphene) is a promising strategy to enable the stable existence of small-sized nanoparticles; however, it has not been widely investigated.
