<p style="text-align:center"><b>Figure 4C</b> Fusion of the plasmid in step one(4A) and plasmid 3.</p>

The first hydrogen production data using our system is the pink curve (curve 1) in Figure 1. It shows that lighting can induce hydrogen production in a closed system with nano rods (NR), mediator Methyl Viologen, and IPTG-induced bacteria transformed with fused plasmid. To prove that every element of the system is necessary and that it is our hydrogenase that produced the hydrogen rather than NR, we conducted a series of experiments.<p></p>

To see whether NR is necessary and whether the hydrogen is produced by the reaction between NR and water under lighting rather than our hydrogenase, we conducted the experiment where the system does not contain nano rods or contain only nano rods. The data is summarized in Figure 1A. The red curve (curve 2) represents the system with the transformed bacterial suspension but without nano rods (NR). The flat curve shows that the system without NR could not produce hydrogen with light; NR is necessary for the system. The black curve (curve 3) represents a system in which only NR and mediators are present, with no bacteria. The flat curve shows that it could not produce hydrogen, which proves that the elements of the bacteria is necessary in the synthesis of hydrogen.<p></p>

<p style="text-align:center"><b>Figure 2</b></p>

Hydrogen production evolution curve (Sensor Data/ Hydrogen amount vs Time) with different components. The pink curve (curve 1) in all pictures is the hydrogen production with all the components, nano rods (NR), IPTG induction, and the bacteria transformed with our hydrogenase plasmid. The rest are data with one or two components missing. In particular, data in the integrated picture are categorized into Figure 2A and 2B. Figure 2A shows the system with or without nano rods or with nano rods alone, and Figure 2B represents the system with or without induction. The curve 3 in each of the specific figure is the blank control with not transformed <em>E. coli</em> BL21. This series of experiments show that only when both nano rods (NR) and IPTG-induced transformed bacteria are present can the system produce hydrogen in a stable way.<p></p>

Another step in proving that it is that the hydrogenase is indeed responsible for hydrogen production is to contrast the production level between the induced and un-induced bacteria suspension. The experiment we conducted are summarized in Figure 6B In this set of experiment, the blue line (curve 4) acts as our blank control. In this group, we use the wild type BL21 cells without plasmid. Although we can see a positive oscillation during a short time in the curve, the production was not at high rate and is likely due to the native hydrogenase in <em>E. coli</em>. The green curve (curve 5) represents the transformed bacterial with no induction of IPTG after 12h cultivation. The flat curve shows that it could not produce hydrogen, which proves that the induction of the hydrogenase expression is necessary. To further confirm, we did another experiment using bacteria that have grown 36 hours with no induction. The purple curve (curve 6) clearly contrasts the induced BL21 and the non-induced one. With curve 4 to 6, we have demonstrated that, with the help of NR, it was our hydrogenase in the system that produced the hydrogen we detected.<p></p>

As mentioned earlier, hydrogenase catalyzes the reversible oxidation of molecular hydrogen (H2). Thus, when we “turn off” the production mode, we should be able to see the consumption of hydrogen by hydrogenase. In testing this bidirectional catalytic property, conducted an experiment where we turned on and turned off the light alternately. The data is shown below in Figure 7. During lighting period, the hydrogen production increases, until we shut off the light at points that correspond to the tips. The curve then goes downward, showing that the hydrogen concentration is lowered, an evidence of the consumption of hydrogen. It is noteworthy that the hydrogenase shows the greatest production rate at the beginning of lighting: a transient sharp rise can be observed at the valleys. It is also obvious that each period of “light-on light-off” gives similar curves, which implies that our hydrogenase is stable.

         <p style="text-align:center"><b>Figure 2</b> Verifying the bidirectional catalytic property of [FeFe] hydrogenase.</p>