Comparison of amperage output of the three nutritional modes

Figure 5-4: The current output (A/g) as a function of electron flux.

The dark red line denotes the maximal current outputs and NADH_mfc production rate, while the area represents all

allowable current outputs and electron production rates. The round dotted arrow line indicates the maximal current output and corresponding electron production rate when the growth rate is set to 5% of the predicted maximum growth rate (0.007981h- 1_).

Table 5-8: Comparison of the predicted amperages and power outputs of the three modes.

Nutritional mode Conditions Biomass productio n rate (h-1₎ Electron (mmol gDW-1 _h-1₎ Amperage (A gDW-1₎ Coulombic efficiency (CE%) W gDW-1 Heterotrophic _Boyle's experimental data 0.035 39.71 1.064 49.64% 0.8835 Autotrophic 0.059 26.89 0.7206 40.87% 0.5988 Mixotrophic 0.066 82.31 2.206 56.46% 1.831 Heterotrophic 5% of optimal autotrophic growth rate 0.007981 42.56 1.141 53.20% 0.9467 Autotrophic 26.25 0.7035 39.90% 0.5839 Mixotrophic 88.36 2.368 60.61% 1.966 y = 0.0268x 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 Cu rr e n t o u tp u t (A g D W -1)

Electron flux (mmol gDW-1_h-1₎

2.368 0.7034 1.141 88.36 26.25 42.56 Autotrophic growth Heterotrophic growth Mixotrophic growth

Two groups of the metabolic states were chosen as the references for the inherent capability of C. reinahrdtii for current output and coulombic efficiency. The first group of reference metabolic states was simulated based on the growth rates experimentally obtained [315]. The second group of reference states were based on the assumption that 5% optimal growth would be the minimum viable growth rates in practise. Based on the computed current outputs for the 5% optimal growth, mixotrophic cultivation (2.368 A/gDW and 60.61% CE) is more suitable for the electricity generation than the phototrophic (0.7035 A/gDW and 39.90% CE) and heterotrophic growths (1.141 A/gDW and 53.20% CE). The photoautotrophic metabolism achieved relatively low coulombic efficiency values, because the photosynthesis is much less efficient in conversion of absorbed light into chemical energy than the acetate-dependent respiration. Although photoautotrophic metabolism produced the lowest current (0.7035 A/gDW), this cultivation condition is still competitive for use in MFCs, since the carbon source is no longer required reducing the operation costs.

It is difficult to compare the computed outputs with other MFCs results in the literature, as published values were calculated based on per electrode area and the electrodes have different designs such as mesh, plate or multi layers. Another output unit is based on amperage per litre of the culture, but, as exemplified in a 2005 study of C. reinhardtii based MFC [210], the cell density number was usually not provided. Nevertheless, here based on the dry mass of the C. reinhardtii of 48 pg/cell [187] and an observed concentration of 1g/L [341], the computed maximum output of C. reinhardtii under mixotrophic mode in the present study can be converted into 113.7 pA/cell and about 2.368 A/L respectively. This value is much higher than the previously measured maximum current output of 16.68 µA/(L culture) from an MFC based on in situ oxidation of hydrogen photosynthetically produced by C. reinhardtii [210].

Two recent studies have evaluated the electrogenic activity of mixed microalgae in anaerobic and oxygenic MFCs under the mixotrophic nutritional mode [264, 342]. The mixed

microalgae was dominated by the presence of Ankistrodesmus, Chlorella, Oscillatoria,

Scenedismus, Diatom and Cosmarium [343]. They suggested that it might be the Bc1 complex in the electron transfer chain that is the electron donor for the electricity generation. The results showed that under aerobic conditions, the mixed microalgae produced 8.571 µA/cm2 during daytime and 0.1429 µA/cm2 during the night [343]. Under the anoxygenic condition, the same mixed microalgae could output a stable current density of 45.71 µA/cm2 [342]. Since the cell density was not specified in these studies, these literature data could not be

directly compared with the presently computed amperage output for the pure culture of C. reinhardtii.

The present study showed that the current outputs were determined by the uptake of the substrates, i.e., acetate and photons. A maximum observed acetate uptake rate of 10 mmol/gDW/h was used as the upper limit for modelling the three nutritional modes. Nonetheless, a relatively conservative photo uptake rate, 145 mmol/gW/h, was used for modelling the mixotrpohic and photoautotrophic modes. This photon uptake rate is lower than the upper limit used in the original model file, i.e., 646.1 mmol/gDW/h, which indicates that the current outputs calculated for photoautotrophic and mixotrophic growths might be conservatively computed in the present study.

The present study did not regard anaerobic conditions as a prerequisite for modelling the electricity generation and allowed oxygen consumption by the organism. The aerobic metabolism possesses a higher efficiency in degradation of organic compounds in algal species, but the presence of oxygen may intercept the electrons to be diverted to the anode in MFCs in practice [343]. Nonetheless, an aerobic MFC that allows the existence of oxygen in an anodic chamber has also been recently developed [46, 278].

The present flux model was built based on the mass balance and stoichiometric conversion rules with respect to the objective function that maximises the growth rate. Nevertheless, in eukaryotic cells, other objectives such as maximising ATP or entropy could become more dominant in influencing the NADH production than maximization of the growth rate. For example, in a multicellular system, the unlimited growth rate leads to the formation of a tumor and the primary objectives of some pancreas cells could be maximizing production of insulin. Also, regulatory mechanisms could take effect to prevent the NADH flux re-directed outside for MFCs. All these unidentified factors may impose burdens for the cell in achieving the optimal metabolic state for NADH extraction for MFCs.

This study investigated cytosolic NADH as an electron source for electricity generation, since the cytoplasm is where the main cellular metabolism takes place and the putative mediator that passes through the cell membrane should reach the cytoplasm first. It is also possible for the mediator to travel through the cytoplasm to arrive in the next barrier, the membrane of the

mitochondria and chloroplast, and subsequently form a redox cycle with the reducing electron shuttles within these two organelles. Nonetheless, this putative MET mode is expected to be less practical than cycling of a cytosolic NADH/NAD+ pair to supply electrons to the anode. Future study could extend the present findings to further investigate the organelles such as mitochondria and chloroplast as the electron supplier with the methods provided here. This could be done in a similar way as the electron transfer from c-type cytochrome that was modelled in the study of G. sulfurreducens [280].

Although the use of NADH obviates the electrical potential loss during the redox chemical molecules conversion [22, 41] and has been suggested as the optimal intracellular electron source for mediated electron generation process [36], the electricity generation may also target other electron carriers as the election suppliers, including a range of metabolites involved in primary (e.g, NADPH [20], ferredoxin [344] and quinone pool [99]) and secondary (e.g., serotonin [345]) metabolisms. The primary metabolites are the compounds that are essentially connected to the microbial metabolism, whereas secondary metabolites are not directly connected to the main metabolism and consist of a variety of different compounds [51]. Besides, the primary metabolites are similar in all groups of living organisms, while the secondary metabolites can be different for various organisms [51]. It is generally accepted that the availability of NADH determines the production of secondary metabolites and the higher energy organic molecules that are building blocks for the biomass.

The present modelling could not differentiate the regeneration capability of NADH and NADPH. In other words, NADH and NADPH are exchangeable for achieving the same maximum reducing power for current production. This may be attributed to the incomplete set of constraints in the model (e.g. enzyme capacity, regulatory, thermodynamic, or other

constraints). For example, the network model used did not consider material fluxes and signalling interactions between compartments. In the future, these fluxes and interaction information can be obtained through isotopic tracers such as 13C, or measurement of compartment concentrations such as using fluorescence resonance energy transfer (FRET) techniques [346].

Generally, NADH is commonly used in reactions related to energy metabolism [21, 31],

biosynthesis of cellular components and defence systems against oxidative stresses [32]. In addition, synthesis of NADH is more energetically efficient than NADPH in the heterotrophic nutritional mode [347]. Since the previous experimental MFC studies could not explain the roles of NADH and NADPH in the electricity generation in practice [61] and maintaining a proper NADH/NAD+ balance is much more important to biomass production [348], here we presume the metabolic perturbation caused by the current output would have a direct impact on the energy metabolism, instead of NADPH-dependent anabolic reactions.

5.4

Conclusions

Analysis of the metabolic flux models predicted that C. reinhardtii has a potential to output current at up to 2.368 A/gDW in the mixotrophic nutritional mode, 0.7035 A/gDW in the photoautotrophic mode, and 1.141 A/gDW in the heterotrophic mode. These computed high current values will serve as the theoretical maximum limits of the current output for C. reinhardtii and are expected to be achieved through the metabolic engineering strategies such as adaptive evolution and gene knockout strategies. Nevertheless, the practical current

production reliant on the metabolic activity elucidated here, necessitates development of proper mediators able to pass through the eukaryotic cell wall and efficiently extract the NADH from the identified enzymes.

This study has also identified a set of essential reactions that can regenerate NADH efficiently to sustain the high current production in each case of the three nutritional modes. It is shown that the three modes shared two common reactions, catalysed by alcohol dehydrogenase (glycerol, NAD) (EC: 1.1.1.2) and 3-Hydroxybutanoyl-CoA: NAD+ oxidoreductase (EC: 4.2.1.17; 5.3.3.8), for supplying NADH at a high rate. Unlike the mixotrophic and

photoautotrophic modes, the elevated current production in the heterotrophic mode involved combined mechanisms of two other organelles, i.e., glyoxysome and chloroplast, which produced NADH and transported them out to the cytoplasm for electron extraction. These results could represent a promising starting point for future studies to tune engineering solutions to harvest optimal current yields with minimal energy costs.