Recently, we have investigated the use of known purple nonsulfur bacteria (PNSB) strain for the photoheterotrophic production of hydrogen using representative volatile fatty acids as substrate under two different headspace gases (Ventura et al., 2016). Based on our findings, it was identified that the Rhodobacter sphaeroides KCTC 1434 favored an argon gas-filled culture condition compared to a nitrogen as headspace gas. Under argon headspace, the H2 substrate conversion efficiency (SCE) and H2 yield is increased. Individual acid utilization shows that the strain had high SCE in both butyrate and propionate but not in acetate. In mixed-acid cultivation, however, resulted in a lower H2 generation and drastic increase of medium pH.

Based on these results, it could be inferred that there is a need to further study the metabolic activity of certain H2-producing microorganism such as PNSB. The diversity of their metabolism in the context of H2 production needs further understanding through physiological optimization, process improvement, or genetic manipulation. More importantly, an identification of the best strain for this process may need a lot of work especially in finding the best and robust strain to be used for H2 production.

        Integrated bio-H2 production pathway

    There are two ways to produce bio-H2 integrating the dark and photofermentation process. The first method is the two-stage bio-H2 production (Table 1). Based on the initial findings using some pure substrate, food carbon source, starch, whey and other organic wastes – the highest H2 yield is around 7 mol/mol hexose. This is roughly half of the theoretical yield of glucose to H2 (12 mol H2) fermentation using the integrated process. On the other hand, the combined process (meaning single reactor, two-phase fermentation) the maximum yield of H2 produced is around 7.2 mol/mol hexose using algal biomass as substrate (Table 2).

        Table 1. H2 production in sequential two-stage process (Rai and Singh, 2016)

        Table 2. H2 production in combined dark and photo fermentation process (Rai and Singh, 2016).

                  Based on Tables 1 and 2, the strains used in the dark fermentation stages are commonly a consortium of anaerobic fermenting bacteria. This is typical dark-fermentation inoculation setup especially if substrate is obtained from industrial or agricultural organic wastes. The photofermentation stage, however, uses pure PNSB strains such as Rhodobacter, Rhodobium, and Rhodopseudomonas species.

    The theoretical amount of H2 produced in 1 mole of hexose such as glucose is 12 mol. In the dark fermentation stage, 1 mol of glucose is typically limited to 4 mol H2 due to formation of other fermentation products (Reactions 1 and 2). On the other hand, photofermentation utilizing 1 mol of acetate or butyrate obtained in the preceding fermentation will produce 4 and 10 mol H2 (Reactions 3 and 4), respectively. Hence, the burden of this process could be mainly attributed to the photobiological-H2 performance of the strain used in the photofermentation stage.

C6H12O6 + 2H2O → 2CH3COOH + 4H2 + 2CO2                    (Reaction 1)

  C6H12O6 + 6H2O → CH3(CH2)2COOH + 2H2 + 2CO2       (Reaction 2)      

  CH3COOH + 2H2O → 4H2 + 2CO2                                         (Reaction 3)

  CH3(CH2)2COOH + 6H2O → 10H2 + 4CO2                             (Reaction 4)

    Several reports investigated the direct utilization of hexose using PNSB (Abo-Hashesh et al., 2011); however, PNSB prefer a more reduced substrate such as VFAs for bio-H2 production (Table 3). PNSB could utilize also hexoses but in the expense of lower H2 yield due to a thermodynamically not feasible H2 production pathway in an unmodified strain (McKinlay, 2014).

Substrate Conversion Efficiencies

     Substrate conversion efficiency (SCE) is defined as the amount of actual H2 (in moles) produced over the theoretical amount of H2 produced from the consumed substrate (in moles). Based on Table 3, it could be understood that there is a selectivity of substrate consumption among PNSB in converting the substrate in H2.

Table 3.  H2 Substrate Conversion Efficiencies (SCE) of different PNSB.