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Bacterial photosynthesis can be divided into two types depending on the presence or absence of oxygen for the metabolism of bacteriochlorophyll, a bacterial photosynthetic pigment. Oxygenic photosynthesis is carried out by cyanobacteria and prochlorophytes, whereas anoxygenic photosynthesis can be generally mediated by purple bacteria, green sulfur bacteria, heliobacteria and others [24]. Photosynthetic anoxygenic bacteria are a very diverse groups of bacteria which carry out bacteriochlorophyll dependent photosynthesis as a metabolic process [25]. The anoxygenic phototrophic bacteria can be broadly grouped into different classes (Figure 1), based on their photosynthetic pigments and electron donors [24, 25]. Depending on the electron donors used, purple bacteria can be further divided into purple sulfur bacteria (use sulfur compounds as electron donors) and non-sulfur bacteria (use organic substances as electron donor).

Some drawbacks of this photofermentative system as pointed by Hellenbeck and Benemann [26] include inherent high energy demand associated with the nitrogenase enzyme, lower photo conversion efficiencies and economic issues of anaerobic photobioreactors covering large areas. These drawbacks can be overcome by effective design and operation of the photobioreactors (PBRs) and selecting proper strains or enrichment of PNSB for an efficient conversion to photo- H2.

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Figure 1. Classification of anoxygenic photosynthetic bacteria.

6.2.2. Purple non-sulfur bacteria (PNSB)

Among the anoxygenic bacteria, the PNSB exhibit very diverse morphological, biochemical and metabolic properties [27]. PNSB are gram-negative photo-heterotrophs, which normally carry out photosynthesis under anaerobic conditions. Although PNSB are facultative anaerobes, they can also grow chemotropically under oxygenic conditions using oxygen as electron acceptor [25]. Different from purple sulfur bacteria, which use elemental sulfur as the electron donor, PNSB typically use organic electron donors such as organic acids, however, they can also use hydrogen gas as electron donor [24].

PNSB can utilize various types of carbon sources such as short-chain organic acids and glucose. The theoretical photofermentative conversion of different organic acids, typically present in DFE, to H2 can be expressed by the following reactions (Equations 4-7) [12, 28, 29]:

Lactate: 𝐶3𝐻6𝑂3+ 3 𝐻2𝑂 → 6 𝐻2+ 3 𝐶𝑂2 (4) Acetate: 𝐶𝐻3𝐶𝑂𝑂𝐻 + 2 𝐻2𝑂 → 4 𝐻2+ 2 𝐶𝑂2 (5) Propionate: 𝐶3𝐻6𝑂2+ 4 𝐻2𝑂 → 7 𝐻2+ 3 𝐶𝑂2 (6) Butyrate: 𝐶₄𝐻₈𝑂₂+ 6 𝐻₂𝑂 → 10 𝐻₂+ 4𝐶𝑂₂ (7)

However, the conversion ability of different PNSB for different substrates varies [29, 30]. Some species prefer a certain sole carbon source, while H2 yields seem to be higher with mixed sources

112 by differences in their reduction states and the associated metabolism for the assimilation of different carbon sources [28, 31, 32]. Similarly, when the carbon source is acetate, most of the reducing power of the PNSB is utilized for the synthesis of PHB rather than H2 [31, 33].

6.2.3. Photosystem of PNSB

The photosynthetic apparatus of PNSB is simple as it contains only one photosystem (PS), unlike the two PS in algae and cyanobacteria. PNS bacterial cells contain bacteriochlorophyll α or β located on cytoplasmic membrane. The PS of PNSB contains the light harvesting complexes that absorb photons initiating a charge (electron-hole) separation through excitation (Figure 2). Electrons that are liberated from organic acids are transported around through a number of electron carriers, i.e. the cytochrome C2 complex, cytochrome bc1 complex (Cyt bc1) and

quinone Q (Figure 2). The transfer of electrons across the membranes creates a large proton gradient which drives the synthesis of ATP from ADP by ATP synthase (Figure 2) [34, 35]. The extra energy in the form of ATP will be used to reduce ferredoxin-fd. Then, the ATP and reduced ferredoxin drives the proton reduction to hydrogen by nitrogenase [6]. Thus, as a result of anoxygenic photosynthesis, conversion of organic substances into H2 takes place.

Figure 2. Schematic representation of mechanisms of photofermentative H2 and

PHB production in PNSB (adapted and modified from [15, 31, 35]). PHB Synthesis ATP Synthase Photo system Light Energy Substrate CO2+ H+_{+ e}- H+ CO2reduction to

fixed carbon source Nitrogenase

H+ ADP +Pi ATP H2 Hydrogenase 2H+_{+ 2e}- e- e- NH4+ H+ H+ Membrane Periplasm Cytoplasm TCA Cycle - e- C2 Cyt bC1 Q Fd

113 Nitrogenase and hydrogenase are the two enzymes that strongly influence hydrogen production: nitrogenase promotes its production, whereas hydrogenase consumes hydrogen (Figure 2). Besides the light conditions, the PF culture medium should be under nitrogen limitation and oxygen should be absent, as their presence inhibits the nitrogenase activity [8, 31, 36]. The activity of the nitrogenase enzyme is of fundamental importance for efficient photo-H2

production [26]. Equations 8 and 9 explain the effect of N2 on the metabolism of PNSB [5]: With dinitrogen:

𝑁2+ 8 𝐻++ 8 𝑒−+ 16 𝐴𝑇𝑃 → 2 𝑁𝐻3+ 𝐻2+ 16 𝐴𝐷𝑃 + 16 𝑃𝑖 (8)

Without dinitrogen:

8 𝐻++ 8𝑒−+ 16 𝐴𝑇𝑃 → 4 𝐻2+ 16 𝐴𝐷𝑃 + 16 𝑃𝑖 (9)

The presence of nitrogen, either in gaseous form or in the culture medium, can thus inhibit the activity of the nitrogenase enzyme that synthesizes molecular H2. Therefore, substrates with a

high C/N ratio are more suitable for H2 conversion in these systems. Thus, the nitrogen content

in the substrate (e.g. DFE) should be considered when coupling PF to dark fermentation.

6.2.4. PHB accumulation by PNSB

PNSB accumulate poly-β-hydroxybutyrate (PHB), an intracellular storage of carbon and energy formed under physiological stress, particularly, at high carbon to nitrogen (C/N) ratio, higher ammonia concentration or sulphur deprived conditions [11, 37, 38]. The production of PHB and H2 functions as the way to dissipating the excess reducing power and the PHB synthesis

competes with the H2 production (Figure 2). Thus, depending on the aim of the process, the PF

can be directed towards H2 production by suppressing the PHB synthesis through genetic

engineering of the PNSB [39]. Kars and Gündüz [31] reviewed the different genetic manipulation strategies to improve photofermentative biohydrogen production. They proposed to modify the acetate assimilation pathways that share the common biosynthetic route of PHB.

After the deletion of the PHB producing gene from R. sphaeroides KD131, the H2 production

rate was increased from 36.1 mL H2 L-1 h-1 to 43.8 mL H2 L-1 h-1 [39], in accordance with the

study of Hustede et al. [33] who observed an increase in cell growth and H2 production when

eliminating the gene for PHB synthesis in Rhodobacter sphaeroides.

In addition, PNSB produce light harvesting bacterial pigments (bacteriochlorophylls and carotenoids) that can be of commercial interests [40]. This ability of PNSB has been highlighted in a few older studies and need to be explored again [41, 42].

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