Algae are amongst the most efficient photosynthetic organisms with fast growth rates, diverse products and tolerance to extreme environments. Diatoms, green algae and cyanobacteria (also referred to as the blue-green algae, Cyanophyta) are the major primary producers in the aquatic ecosystem, contributing to carbon dioxide removal, photo-oxygenation and also serving as sources of valuable biochemicals [1]. The biomass productivity of microalgae was estimated to be 50 times higher than switchgrass, which is the fastest growing terrestial plant [2]. With the increased interest in alternative energy sources, algae are being investigated as feedstock for biodiesel, bioethanol, biohydrogen and bioelectricity production [3], [4], [5]. Microalgae have oil content exceeding 80% DW, grow fast and can be mass cultured using open ponds or enclosed photobioreactors [6]. The filamentous cyanobacterium Anabaena was reportedly the first to be used for hydrogen generation [7], [8]. In 1997, electricity was generated using 2-hydroxy-1,4-naphthoquinone as an electron shuttle between Synechococcus sp. (UTEX 2380) and a carbon-cloth anode [9]. The current intensity increased with increasing cell concentration, reaching 320 µAcm⁻² [9]. A bioreactor (microbial fuel cell) with an air cathode and a graphite-felt anode coated by a biofilm of bacteria and algae, generated electricity when irradiated. On day 10, the voltage output and current density produced by the reactor were 0.32 V and 8.6 µAcm⁻² respectively [10].

Biological components have since then been introduced into fuel cells (FCs), giving rise to microbial fuel cells (MFCs). MFCs were designed to operate as a new generation of solar cells called biological photovoltaic devices (Biophotovoltaic, BPV) [11]. Both the MFCs and BPV share similar functions; where MFCs generate electricity from the metabolic process of living microbes, whereas BPV produce electricity from light energy via the light harvesting apparatus of photosynthetic organisms [12]. Previous BPV studies have utilized various exogenous soluble mediator compounds to facilitate electron transfer such as 5 mM ferricyanide as mediator between the biological materials and the anode [11]. In their paper, a new BPV device was fabricated with several advantages over the previous experiments such as multiple microchannels to facilitate multiple simultaneous experiments, removal of the need for external energy supply and a new design to cater for investigation with various types of biological materials instead of solely a single type. Results indicated a direct relationship between effects of cell density, electron mediator concentration and light intensity with the efficiency of the BPV device. The biological materials used in this study were intact Synechocystis cells and thylakoid membranes isolated from the cells, which generated total power output of 4.71 and 9.28 nWµmol Chl⁻¹ respectively. In another study, McCormick et al. [13] replaced the use of exogenous mediators with the development of biofilms. Several algae were grown directly on an Indium tin oxide- Polyethylene terephthalate (ITO-PET) anode on a sandwich type or an open air design. This work demonstrates the ability to produce simple, portable BPV devices without the need of an artificial electron mediator. The Synechococcus biofilm in this work produced a peak power output at 1.03×10⁻² Wm⁻² under 10 Wm⁻² of white light [13]. Biofilm development offers several advantages by increased power output due to direct contact between cell and electrode and reduced internal potential losses. In 1684, Antonie van Leeuwenhoek [14] reported the existence of animalcules on teeth, representing the first scientific report on biofilms. Microorganisms are able to attach to surfaces and form a hydrated polymeric matrix termed “extra-cellular polymeric substances (EPS)” that hold the biofilm together [15]. In an EPS matrix, there are interstitial water channels, which separate bacterial cells from each other, thereby allowing the transportation of nutrients, oxygen and genes [16]. Biofilms composed of microorganisms attached to surfaces, form a hydrated polymeric matrix consisting of polysaccharides, protein and nucleic acids [15]. There is a growing interest in the study of artificial phototrophic biofilms. In biofuel production, the cultivation of algae as biofilms reduces costs due to reduction of water volume and power required for pumping. This increases biomass and also avoids costly harvesting and dewatering technologies [17]. Traditional methods of monitoring changes in biomass of microphytobenthic biofilms are destructive, for example, pigment sampling by using syringe coring method. This method involves pulling the syringe plunger up while pushing the syringe to the sediment to create counter pressure [18]. Fluorescence techniques have been employed to measure photosynthetic performance of biofilms in sediments without interfering with the sediment surface [19]. Radiant energy absorbed by chlorophyll can undergo one of three fates: (i) used for photosynthesis (ii) dissipated as heat or (iii) re-emitted as chlorophyll fluorescence [20]. Hence, by measuring the yield of chlorophyll fluorescence, information about the efficiency of photochemistry and heat dissipation can be generated using a pulse amplitude modulation (PAM) fluorometer (Diving PAM, Walz, Germany) [21]. Photosynthesis parameters such as electron transport rate (ETR) can be calculated to measure the efficiency of photochemistry of Photosystem II (PSII) [22].

In a recent paper, Luimstra et al. [23] produced a cost effective microbial fuel cell design that can be used on algae and cyanobacteria. Several strains of benthic cyanobacteria were screened and displayed electrogenic qualities suitable for microbial fuel cell purpose. The paper also describes a particularly good green alga, P. pseudovolvox that demonstrated greater electrogenic activity compared to the others. However, they also highlighted that the mechanisms used in the microorganisms to donate electrons have to be determined at this moment of time. Ng et al. [24] reported that Chlorella sp. (UMACC 313) and Spirulina platensis (UMACC 159) are able to form biofilms on ITO anode. Chlorella and Spirulina formed biofilms with coverage and maximum relative electron transport rate (rETRmax) of 99.46% and 140.796 µmol electrons m⁻² s⁻¹ and 80.70% and 153.507 µmol electrons m⁻² s⁻¹ respectively. Results indicate the potential for generating electrical energy from these microalgae using biophotovoltaic platforms.

The objectives of the present study are to (i) establish libraries of 16 strains of algae for selection of most suitable strains for application in biophotovoltaic platforms; (ii) investigate biofilm formation of selected algae on Indium tin oxide (ITO) and glass and determine the photosynthetic efficiency of the biofilms; (iii) Produce and test biofilms on Indium tin oxide (ITO) in a biophotovoltaic (BPV) device for electric power generation. Glass and ITO were chosen as substrates for biofilm formation to compare the adhesion of algae cells to these surfaces. Glass is hydrophilic with high surface energies and ITO is hydrophobic with low surface energy respectively [25]. ITO was an early favorite for hole injection cathode with good transparency and conductivity [26]. ITO was also successfully used to cultivate healthy biofilms for MFCs application [13].

The preliminary screening of 16 strains, comprising 14 local strains belonging to the blue-green and green algae, and two diatoms from the CCMP, allowed the selection of eight out of the 16 strains that were characterized in terms of growth rate, biochemical composition (protein, lipid and carbohydrate contents) and photosynthetic efficiency, for further studies on biofilm formation. The 16 strains were cultured till stationary phase to observe the batch culture characteristics. At stationary phase, the lipid contents are expected to be higher than at exponential phase of culture, as lipids are accumulated upon reaching stationary phase, as opposed to protein [30]. This was clearly observed with most of the strains except for the Cyanophyta (Synechoccus elongatus UMACC 105) and two Chlorophytes (Chlorella vulgaris UMACC 051 and Chlorococcum UMACC 207), where protein was highest at day 15. In general, the Chlorophytes contained higher lipid than the other strains, with Chlorella sp. (UMACC 256) having the highest lipid (63.64%DW) contents on day 15.

Eight out of the 16 strains, namely two Cyanophytes, five Chlorophytes and one diatom, were selected for the biofilm studies. The Cyanophytes were selected because blue-green algae produce abundant mucilage and are expected to form biofilms easily. The blue-green algae has higher potential to form a hydrated polymeric matrix termed extracellular polymeric substances (EPS) which encourages the biofilm formation [15]. Also from the preliminary screening, the Synechococcus elongatus (UMACC 105) and Spirulina platensis (UMACC 159) had high rETRmax values, indicating efficient photosynthesis. Chlorella vulgaris (UMACC 001) has been used for many studies in our laboratory and has shown ability to grow in wastewaters [43], [44], [45] and has potential for biofuel production [30]. In 2007, Wong and co-workers [46] compared the tolerance of Antarctic, tropical and temperate microalgae to ultraviolet radiation (UVR) stress. When the Chlorella vulgaris (UMACC 001) was exposed to ambient light, the specific growth rate was 0.16 d⁻¹ but decreased to 0.04 d⁻¹ when exposed to ultraviolet radiation (UVR) treatment. Vejesri et al. [30] reported that Chlorella vulgaris (UMACC 001) was a potential feedstock for biodiesel production due to high specific growth rate (μ = 0.42 d⁻¹) and high saturated fatty acid (SFA) content (68.2%DW). Chu and co-workers [43] reported that immobilized cultures of Chlorella vulgaris (UMACC 001) in alginate removed 48.9% of color from textile wastewater. Chlorella vulgaris (UMACC 001) showed high NPQ and strong photoprotection with higher capacity to cope with high irradiance. Chlorococcum (UMACC 207) had high rETRm as well as high protein (30.29%DW) content. Chlorella sp. (UMACC 313) and Chlorella vulgaris (UMACC 051) isolated from the aerobic pond for palm oil mill effluent (POME) treatment were selected because they formed biofilms on the base and sides of the culture flasks, and had very high lipid content. One marine alga Chorella sp. (UMACC 256) was included because it had the highest rETRmax value among the three marine strains. It was also shown that this strain had high specific growth rate (0.75 d⁻¹) and high saturated fatty acid (SFA) content (53.8%DW) in our previous study [30]. One strain from the CCMP culture collection was also selected for comparison with the UMACC cultures. The strain, Coscinodiscus wailesii (CCMP 2513) was a centric diatom with high rETRmax (166.32 µmol electrons m⁻² s⁻¹) and higher lipid content of the two diatoms. In addition to the photosynthetic parameter of rETRmax, consideration was also given to the biofuel potential of the strains, in terms of lipid content.

In the biofilm studies, strains like Synechococcus elongatus (UMACC105) and Chlorella (UMACC 313) were observed to have formed appreciable biofilms after only three days. This may be based on the high production of EPS by Synechococcus elongatus (UMACC 105) and the high growth rate of Chlorella (UMACC 313). Application of PAM fluorometry in the study of algae biofilms has provided valuable information on the operation of algal BPV platforms. One of the most important information is the non-destructive generation of light-response curves of photosynthetic activity [47]. The distance between the optical fiber-optics and the sample surface was set at 2 mm [48]. Eight selected strains starting with the same concentration of pure cultures at OD_{620 nm} at 0.5. All cultures were transferred into the same incubator at 24°C illuminated by cool white fluorescent lamps (30 µmol m⁻² s⁻¹) on a 12∶12 hour light-dark cycle to ensure that the only variable was the strain of algae in the biofilm development. E_k values were all relatively high and ranged from 582.724 to 761.731 µmol photons m⁻² s⁻¹ indicating that all biofilms were able to adapt to the high light intensities (307–1829 µmol photons m⁻² s⁻¹). Under high irradiances, an inverse relationship between NPQ and rETRmax was observed in all strains; higher NPQ values being matched by lower rETRmax values. The Chl-a content of the eight strains increased with the thickness of biofilms. The Chlorella species (UMACC 313 and UMACC 051) isolated from the aerobic pond used in POME treatment formed significant biofilms on day 3 on both substrate materials. However, the % SAC of the biofilm from the diatom Coscinodiscus wailesii (CCMP 2513) was low compared to other strains probably due to the need for longer time to form hydrated polymeric matrix. Even in the suspension culture during the preliminary screening, this diatom took 15 days to reach stationary phase (Table 2). The substrate effect was minimal as there was no significant difference of % SAC of all strains on both glass and ITO slides, except for the filamentous blue-green alga Spirulina platensis (UMACC 159) which grew better on ITO slides compared to glass slides with higher % SAC, Chl-a content and biofilm thickness. From this study, four strains, the Chlorophytes Chlorella (UMACC 051, UMACC 313) and the Cyanophytes Synechococcus elongatus (UMACC 105) and Spirulina platensis (UMACC 159) were identified to produce the most stable biofilms on ITO. The growth and viability of these microorganisms in a working BPV device would have a significant impact on the photocurrent that would be generated [13].

The biofilm samples were dark adapted for at least 15 minutes before PAM measurement. When cultures were placed in the dark, minimum (F_o) and maximum (F_m) fluorescence both increased, as did the maximum PSII quantum efficiency (F_v/F_m). After dark adaption, the reaction centres were in a relaxed stage, and ready to receive light for photosynthesis [19]. The maximum quantum yield F_v/F_m was obtained when all reaction centres were opened and was proportional to the fraction of reaction centres capable of converting absorbed light to photochemical energy [49]. F_v/F_m is often used as an indicator of photosynthetic capacity. Various studies have reported values of F_v/F_m ranging between 0.1 to 0.65 for natural populations of microalgae [50]. Results in the present study indicated that most samples were in a healthy condition, as the F_v/F_m values were all above 0.800 except for Synechococcus elongatus UMACC 105 with a F_v/F_m value of 0.799. Serôdio et al. [51] reported rapid light-response curves of chlorophyll fluorescence in benthic microalgae from a mesotidal estuary located in the west coast of Portugal. The values of photosynthetic parameters were as follows: alpha (α): 0.249–0.473, rETRmax (µmol electrons m⁻² s⁻¹): 118.9 to 424.0, E_k (µmol photons m⁻² s⁻¹): 474.1 to 821.4. A similar study carried out by Cartaxana et al. [52] on the same area reported PAM fluorometry as a tool to assess photophysiology of intertidal microphytobenthic biofilms. The microphytobenthos communities were dominated by diatoms, resulting in the following photosynthetic parameters; alpha (α): 0.657, rETRmax (µmol electrons m⁻² s⁻¹): 331, E_k (µmol photons m⁻² s⁻¹): 582.72–761.73. In our study, the range of photosynthetic parameters for suspension cultures of the 16 strains were: alpha (α): 0.31–0.91, rETRmax (µmol electrons m⁻² s⁻¹): 64.68–166.32, E_k (µmol photons m⁻² s⁻¹): 72.73–473.85 and F_v/F_m: 0.43–0.80. For biofilms of the eight strains, the range of photosynthetic parameters were: alpha (α): 0.48–0.75, rETRmax (µmol electrons m⁻² s⁻¹): 369.19–508.40, E_k (µmol photons m⁻² s⁻¹): 582.72–760.45 and F_v/F_m: 0.80–0.85. Our results showed that algal biofilms have better photosynthetic performance than suspension cultures. Results showed that the algal strains used in this study have high photosynthetic efficiency indicating their potential for use in BPV platforms. In addition, high rETRmax and E_k values obtained from the present study reflected an adequate adaptation to high irradiance [53], and was observed in the two Cyanophytes (Synechococcus elongatus UMACC 105) and the diatom (Coscinodiscus wailesii CCMP 2513). The NPQ values for each strain were compared and an increase in NPQ with changing light intensities was observed in all cases of the biofilms (Figure 3). The change in NPQ is expected due to photoprotection of photosystem II to avoid light induced damage [54]. In the case of the Cyanobacteria (Cyanophyta), the resulting changes in NPQ have been reported to be triggered by the light activation of orange carotenoid protein found in phycobilisomes [55]. In comparison, the NPQ in diatoms is mediated by the light-harvesting complex stress-related (LHCSR) protein and the conversion of diadinoxanthin (Ddx) to diatoxanthin (Dtx) [56]. In green algae (Chlorophyta), the controlled change in pH and its effect on the xanthophyll cycle has a predominated effect on NPQ [54]. In the present study, the Chlorophyte strains were observed to have higher ability (higher NPQ values) to photoprotect themselves than the Cyanophytes and diatoms. The Cyanophytes (blue-green algae) were observed to have the lowest value or in some cases, no NPQ, indicating that they have the lowest capacity for photoprotection. There was no clear correlation between the F_v/F_m values and the NPQ values of the 16 strains in the screening experiment as well as the eight strains used for the biofilm study, except where the lowest F_v/F_m values of the two Cyanophytes also corresponded to the lowest NPQ values (Tables 2 & 4 and Figures 2 & 3). Interestingly the low NPQ values of the two Cyanophytes and two diatoms corresponded to higher E_k values, and may indicate that these strains may in fact be tolerant of higher irradiance. This however, has to be confirmed by detailed studies on the relationship between the various parameters. In this study, the four strains, Cyanophytes Synechococcus elongatus UMACC 105, Spirulina platensis UMACC 159 and Chlorophytes Chlorella UMACC 051 and UMACC 313, were selected for the BPV study based on strong ability to form biofilms; however their NPQ ranged from low to high values, to allow the correlation of NPQ to photo current production in a working BPV device. The diatoms had high rETRmax but very low NPQ, and were not selected due to poor ability to form biofilms. On analysis of data from the BPV studies, it was apparent that the Cyanophyte Synechococcus elongatus (UMACC 105), was able to give highest performance in the BPV device but it had the lowest NPQ value. The maximum power density was similar for the other Cyanophyte Spirulina sp. (UMACC159) and two Chlorophytes Chlorella (UMACC 051 and 313), which had higher NPQ. Further studies would need to be carried out to correlate the NPQ of the strains with the ability to extract charge from biofilms in a working BPV device.

All four strains, the Cyanophytes Synechoccus elongatus (UMACC 105) and Spirulina platensis (UMACC 159), and the Chlorophytes Chlorella vulgaris (UMACC 051) and Chlorella sp. (UMACC 313), showed ability to produce electrical power outputs (Figure 4 a–d). Synechococcus elongatus (UMACC 105) showed higher power outputs compared to other strains. This may be due to the fact that Synechococcus elongatus (UMACC 105) registered a high rETRmax values (147.50 µmol electrons m⁻² s⁻¹), high E_k value and readily produced biofilms (84.68% SAC on ITO) which were able to accumulate a high level of charge. The Synechococcus produces unicells of small dimensions and with production of EPS, would form compact biofilms on the anode, compared to the filamentous Spirulina which like the Arthrospira used in the study by Inglesby et al. [39], formed porous biofilms. The maximum power density obtained in this study ranged from 1.12×10⁻⁴ to 3.13×10⁻⁴ Wm⁻². Inglesby et al. [39] reported very low power densities from 6.7 to a maximum of 24.8 µW m⁻² with a similar BPV device using biofilm of the Cyanophyte Arthrospira maxima on ITO as well, considering that the theoretical maximum power density achievable when using microalgae in microbial fuel cells is 2.8 Wm⁻². The low power density obtained in the present study reflects an unoptimised system with the results aimed only for comparison between the four strains. Optimisation of the process in terms of biofilm thickness and irradiance properties may improve the power output. Optimisation of temperature and light intensity in the BPV device may enhance power output, as was shown by Inglesby et al. [39], where an increase of temperature from 25 to 35°C increased power output from 9.9 to 24.8 µW m⁻² while there may be a limitation to how high the light intensity can be, due to photoinhibition. In the present study. Based on power output per Chl-a content, the four strains can be ranked as follows: Synechococcus elongatus (UMACC 105) (6.38×10⁻⁵ Wm⁻²/µgChl-a)>Chlorella vulgaris (UMACC 051) (2.24×10⁻⁵ Wm⁻²/µgChl-a)>Chlorella sp. (UMACC 313) (1.43×10⁻⁵ Wm⁻²/µgChl-a)>Spirulina platensis (UMACC 159) (4.90×10⁻⁶ Wm⁻²/µgChl-a). These values were obtained by dividing the maximum power density by the total Chl-a extracted from the whole algal biofilm on the ITO slides. However no correlation (P<0.05) was observed between power output and Chl-a content. Of the four strains, Chlorella vulgaris (UMACC 051) had highest lipid (31.09% DW) and crude protein (46.70% DW) contents, indicating additional advantages over the other strains. Although it is premature to speculate on the long-term usage of algal cultures in BPV devices, on a large scale, biomass may be generated continuously in the BPV. The biomass may be harvested on a regular basis to prevent over-buildup of the biofilm, which results in reduced bioactivity at the surface of the biofilm furthest from the anode. Photosynthetically efficient strains with high lipid and protein productivities, may have an added advantage over other strains, providing a valuable biomass that may enter an alternative energy-producing system like biodiesel production. This may overcome the lower energy output from algal BPV systems compared to commercially available photovoltaics [39]. However the additional cost of continuous nutrient supply to the biofilms on the anodes and cost of removal of surplus biomass from the biofilms may necessitate innovative design and operation of the BPV devices.

The present study indicated that local algal strains were good candidates for utilization in BPV platforms in future. According to our screening results, all eight strains were able to form biofilms on ITO anode surfaces with good photosynthetic performance. From this, four strains were selected for the BPV studies, based on their high photosynthetic performance and ability to produce biofilms. The Chlorophytes Chlorella species (UMACC 051, UMACC 313) and the Cyanophytes, Spirulina platensis (UMACC 159) and Synechococcus elongatus (UMACC 105), demonstrated exoelectrogenic activity and showed their capacity to produce significant electrical power outputs without the requirement of additional organic fuel. More work is required to further understand the mechanisms of harnessing light energy and converting them to electricity as well as to investigate the correlation between PAM data and the BPV power output.