Bioresource Technology 154 (2014) 171–177

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Bioresource Technology

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Effect of light on the production of bioelectricity and added-valuemicroalgae biomass in a Photosynthetic Alga Microbial Fuel Cell

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.12.049

⇑ Corresponding author. Tel.: +351 217163636.E-mail address: cristina.matos@lneg.pt (C.T. Matos).

Luísa Gouveia a, Carole Neves a, Diogo Sebastião a, Beatriz P. Nobre a,b, Cristina T. Matos a,⇑a Laboratório Nacional de Energia e Geologia, I.P. Unidade de Bioenergia, Estrada do Paço do Lumiar, 1649-038 Lisboa, Portugalb Instituto Superior Técnico, Centro Química Estrutural, DEQ, Av. Rovisco Pais, 1, 1049-001 Lisboa, Portugal

h i g h l i g h t s

� Bioelectricity and value pigmentsproduction in a PAMFC.� A light intensity increase resulted in a

consequent increase of the PAMFCpower.� The maximum power produced was

62.7 mW/m2 with a light intensity of96 lE/(m2 s).� Light intensity and PAMFC operation

potentiated the carotenogenesis inthe cathode.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 September 2013Received in revised form 6 December 2013Accepted 11 December 2013Available online 18 December 2013

Keywords:Microbial fuel cellChlorella vulgarisPigmentsBioelectricity

a b s t r a c t

This study demonstrates the simultaneous production of bioelectricity and added-value pigments in aPhotosynthetic Alga Microbial Fuel Cell (PAMFC). A PAMFC was operated using Chlorella vulgaris in thecathode compartment and a bacterial consortium in the anode. The system was studied at two differentlight intensities and the maximum power produced was 62.7 mW/m2 with a light intensity of 96 lE/(m2 s). The results showed that increasing light intensity from 26 to 96 lE/(m2 s) leads to an increaseof about 6-folds in the power produced. Additionally, the pigments produced by the microalga wereanalysed and the results showed that the light intensity and PAMFC operation potentiated the caroteno-genesis in the cathode compartment.

The demonstrated possibility of producing added-value microalgae biomass in microbial fuel cellcathodes will increase the economic feasibility of these bioelectrochemical systems, allowing the devel-opment of energy efficient systems for wastewater treatment and carbon fixation.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The bioelectrochemical systems, such as microbial fuel cells(MFC) are able to convert the energy stored in any biodegradablesubstrate directly into electricity. The possibility of using light irra-diation to potentiate the production of electricity in MFCs hasreceived increased attention in the last decade, with the develop-ment of different systems and concepts capable of converting lightinto bioelectricity (Rosenbaum et al., 2010). These systems are

commonly known as photo microbial fuel cells (PhotoMFCs) andcould make use of the cost-free solar radiation to generate energy.Recently, several works proposed the use of photosynthetic micro-organisms as biocatalysts, for oxidation and reduction reactionsthat occur in the anode and cathode compartments of a MFC.Rosenbaum et al. (2010) describes several PhotoMFCs systems. Afew works report the use of photosynthetic bacteria for electronproduction in the anode compartment. Bacteria such as Rhodo-pseudomonas and other purple non-sulfur bacteria have been iden-tified in anode biofilms (Xing et al., 2008). The use of microalgae inMFCs has also been reported both in the cathode and anode com-partments. Raman and Lan (2012) described a system where green

172 L. Gouveia et al. / Bioresource Technology 154 (2014) 171–177

microalga Chlamydomonas reinhardtii transformation F5 was usedin the anode to produce electrons. Additionally, the use of microal-gae biomass has been reported as a substrate for bacteria in thePhotoMFCs anode compartments (Strik et al., 2008). However,the most reported concept is the use of microalgae in the cathodecompartment for oxygen production to the cathodic reaction, as itwas studied in the present work. Besides C. reinhardtii a few puremicroalgae strains have been used in PhotoMFCS, such as: Chlorellavulgaris (Wang et al., 2010; Zhang et al., 2011; Zhou et al., 2012),Spirulina platensis (Fu et al., 2009) and Pseudokircheneriella subcap-itata (Xiao et al., 2012). Cyanobacteria such as Anabaena have alsobeen reported as biocatalysts in the MFC cathodes (Pandit et al.,2012). Important results have also been achieved with microalgaeconsortium developed in the MFC cathodes (He et al., 2009; Striket al., 2010; Chandra et al., 2012; Jiang et al., 2012).

The concept of the PhotoMFCs studied in this work, herebydenominated by Photosynthetic Algae Microbial Fuel Cell (PAMFC)(represented in Fig. 1) is composed by an anode (inoculated with abacteria consortium) and a cathode (inoculated with C. vulgarismicroalga), separated by an ion exchange membrane. Typically,bacteria in the anode oxidise organic compounds and produceselectrons and protons. The electrons are transferred from the an-ode to the cathode electrode, through an external circuit producingelectricity. Protons are transferred to the cathode compartmentthought the ion exchange membrane. In the cathode, microalgaein the presence of light and carbon dioxide, produces oxygen (viaphotosynthesis), which together with protons and electrons (fromthe anode compartment), forms water completing the cathodicreaction. The advantage of these systems lies with the possibilityof treating biodegradable wastes (by bacteria in the anode), to-gether with CO2, nitrogen and phosphorous fixation (by microalgaein the cathode) concomitant with the production of bioelectricity.

Microalgae cultivation depends on several parameters, such aslight, temperature, nutrients and pH. Light (quality, intensity anddark/light regimes) is one of the most important parameter, con-tributing deeply for growth and composition of microalgae bio-mass (fatty acid and pigment profiles) (Khoeyi et al., 2012).Besides light, nutrients are also crucial for biomass growth andcomposition. Under nutrient stress limitation conditions (particu-larly nitrogen) microalgae increase their production of lipids, car-bohydrate and/or pigments (Gouveia et al., 2009, 1996; Mirandaet al., 2012).

Microalgae biomass and/or its extracts could be used for differ-ent applications, such as food and feed (e.g., pigments, polyunsat-urated fatty acids, antioxidants) and biofuels (e.g., oils for biodiesel,carbohydrate for bioethanol and biohydrogen). Biohydrogen andbiogas could be also produced from microalgae biomass leftoversthrough dark fermentation and anaerobic digestion, respectively(Nobre et al., 2013). Microalgae biomass is a recognised added-va-lue product and is sold for human and animal nutrition up to 50 €/

Fig. 1. Schematic representation of the PAMFC concept.

kg. Extracted microalgae pigments such as b-carotene can cost upto 2150 €/kg or astaxanthin 7 €/mg (Wiffels et al., 2010). The mic-roalgae biomass and all added-value compounds, such as pigmentscould be used even after microalgae performance in a cathode of aPAMFC (after production of bioelectricity).

The aim of this study was to assess the potential of a PAMFC forproducing bioeletricity and valuable microalga biomass (contain-ing high added-value compounds–pigments). The PAMFC wasoperated with an enriched bacteria consortium in the anode anda pure microalga culture C. vulgaris in the cathode. The power pro-duction of this combined system was evaluated under two differ-ent light intensity conditions and under nitrogen starvation inthe culture medium. After each PAMFC trial, the microalga biomasswas analysed to quantify and identify its pigment profile. The ef-fect of light intensity and nitrogen starvation on the pigment con-tent and profile of the microalga biomass were also evaluated. TheC. vulgaris strain used in this work was isolated by LNEG (Ex INETI)and was previously well studied in terms of carotenogenesis pro-cess (Gouveia et al., 1996). This alga contains a good pigment pro-file and it was studied for several applications, from animal feed tofood products (Marques et al., 2011). Therefore, it could be a goodcandidate for further biomass valorization after its use in the PAM-FC cathode.

2. Methods

2.1. Photosynthetic algae microbial fuel cell (PAMFC)

The PAMFC used in this study (see Fig. 2, for a schematic repre-sentation of the experimental apparatus) was a reactor similar tothe one used in Matos and Lopes da Silva (2013), composed of amodule with two identical cylindrical compartments (with a vol-ume of 50 mL, each) separated by a proton exchange membrane(Nafion 117). The two electrodes were of plain graphite (3 mmthick, 97% (metallic basis) Graphite foil from Alfa Aesar). Both theelectrodes and the membrane had a working area of 12.6 cm2.The cathode electrode was coated with 0.5 mg/cm2 of platinum(using 10% Pt/C from Aldrich). The produced voltage (E) was mea-sured online by a multimeter (UNI-T UT803) connected to a com-puter, where current (I) and power (P) production was calculatedby E = I R and P = I E. Pre-treatment of the Nafion membrane wasperformed by submerging the membrane in solutions of H2O2

Fig. 2. Experimental set-up: (1a) PAMFC schematic representation; (1b) PAMFCphotograph; (2) external resistance; (3) multimeter; (4) computer for on-lineacquisition.

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(3% (v/v) for 1 h at 80 �C) and H2SO4 (0.5 M for 1 h at 80 �C) andwashing with boiling water after each solution treatment.

All the tests were operated at closed circuit using a conductivetitanium wire and an external resistor (R) of 1 kX.

2.2. Anolyte and catholyte

The anolyte solution used in all experiments was a nutrientsolution with the same composition as described in Matos andLopes da Silva (2013), for 1 g/L basis (adapted from Oh et al.,2004): PBS 50 mM (pH 7), 10 mL of a saline solution (EDTA, 2.88;MgSO4�7H2O, 7.67; MnSO4�H2O, 0.63; NaCl, 1.25; FeSO4�7H2O,0.13; CaCl2�2H2O, 0.13; CoCl2�6H2O, 0.13; ZnCl2, 0.16; CuSO4�5H2O,0.013; AlK(SO4)2�12H2O, 0.013; H3BO3, 0.013; Na2MoO4�2H2O,0.036; NiCl2�6H2O, 0.03; Na2WO4�2H2O, 0.031) and 5 mL of a di-luted (100�) vitamin solution (biotin, 0.2; folic acid, 0.2; pyridox-ine, 1; riboflavin, 0.5; B12 vitamin, 0.01; p-aminobenzoic acid, 0.5;B5 vitamin, 0.5). The carbon source used was 20 mM of acetate.

The catholyte solution was a C. vulgaris culture medium adaptedfrom Gouveia et al. (1996) with a 50 mM PBS solution. This culturemedium, composed by (for 1 g/L basis) KNO3, 1.250; KH2PO4, 1.250;MgSO4�7H2O, 1.000; CaCl2, 0.084; H3BO3, 0.111; FeSO4�7H2O, 0.050;ZnSO4�7H2O, 0.088; MnCl2�4H2O, 0.014; MOO3, 0.007; CuSO4�5H2O,0.016; Co(NO3)2�6H2O, 0.005; Fe�EDTA, 0.5 was used for microalgainoculum preparation and PAMFC operation.

2.3. Microalga inoculum growth

C. vulgaris (INETI 58) strain from LNEG_UB was the microalgaused on PAMFC studies and was inoculated in the cathode com-partment in each batch test.

Prior to the inoculation in the PAMFC, the microalga was grownin 1.5 L bubble column plastic reactors, at controlled temperature(25 �C), under continuous cool-white fluorescent lamps (PhilipsTL-D 18 W/54-765) providing a light intensity of 59 lE/(m2 s)and agitated by bubbling filtered compressed air. When theOD540 nm of the algae culture reached 0.6, which correspondsapproximately to a 0.5 g/L of biomass dry weight, the biomasswas harvested by centrifugation (9000 rpm, 20 min, 8 �C). The col-lected biomass was inoculated in the PAMFC in order to obtain 2 g/L of microalga dry weight in the cathode compartment.

2.4. PAMFC operation

An anode enriched in a previous work (Matos and Lopes da Sil-va, 2013), with a bacterial consortium was used in this study in theanode compartment. The PAMFC was operated in batch cycles withaddition of fresh anolyte and catholyte solutions. For each cycle,50% of anode compartment volume was replaced with fresh ano-lyte solution (with 20 mM of acetate) and the culture of the micro-alga C. vulgaris was also added in cathode compartment at initialconcentration of 2 g/L. Both compartments are initially flushedwith nitrogen gas to remove oxygen. The PAMFC was operated insuccessive batch cycles at room temperature, with the two com-partments magnetically stirred at 200 rpm. When the output volt-age reached a minimum value, each cycle was consideredcompleted. Samples from the beginning and the end of each cyclewere collected for analyses of the medium and biomasscompositions.

2.5. Studies of light intensity during PAMFC operation

To study the effect of light intensity on the performance of thePAMFC and on the biomass pigment profile, the PAMFC was oper-ated under a constant fluorescent light intensity using one (26 lE/(m2 s)) or two (83 or 96 lE/(m2 s)) fluorescent lamps (a Philips TL-

D 14W/54 and a City Bright TL 4018 11W). Two batch cycles wereperformed for each light intensity.

2.6. Studies of nitrogen starvation in the PAMFC cathode

To study the effect of nitrogen starvation on C. vulgaris culturemedium and its implication on the performance of the PAMFCand on the biomass pigment profiles, the PAMFC cathode wasoperated without the addition of nitrate to the microalga culturemedium. The PAMFC was operated during two anode batch cycles(two acetate pulses of 20 mM) without changing the microalgaesuspension in the cathode.

2.7. Chemical measurements

At the beginning and end of each cycle, samples were taken inorder to measure the pH, acetate and inorganic carbonconcentration.

The acetate concentration of the samples was determined byHPLC using a differential refractometer detector, Merck LaChrom7, and an Aminex HPX-87H column (BioRad, USA), connected toa Merck Hitachi autosampler L-7200 and a pump L-7100. The mo-bile phase was 0.01 N of H2SO4 (flow rate 0.4 mL/min).

The inorganic carbon concentration present in the samples wasdetermined by a total carbon analyser SHIMADZU TOC-5050A.

2.8. Pigment extraction, quantification and identification

The quantification of the pigments was carried out using themicroalgal freeze-dried biomass and acetone as a solvent, accord-ing to Nobre et al. (2013). Approximately 20 mg of biomass weremixed with small glass beads (760 ± 10 mg) and 3 mL of acetone.The mixture was placed alternately in an ice bath and in a vortex.The extracts were centrifuged at 3900 rpm for 8 min and collected.The extraction procedure was repeated until both precipitate resi-due and supernatant became colourless. Initial screening for pig-ment identification was carried out by visible absorption spectra(380–700 nm) of the obtained extracts using a spectrophotometer(UV-2401 PC SHIMADZU). The total pigments concentration wascalculated by the Beer–Lambert law at maximum absorbance mea-sured, 430 nm. The optical extinction coefficient (E1%

1 cm) value usedwas 2109 (Mendes et al., 1995).

The pigments present in the extracts were identified by thinlayer chromatography (TLC) and their profile was determined byhigh-performance liquid chromatography (HPLC) (Hewlett PackardHPLC, 1100 series equipped) with a UV/VIS detector (k = 430 nm).A l-Boundapak C18 column and a Vydac 201 TP column were used.The mobile phase, set at 1 mL/min was, respectively, (methanoland 0.2% H2O)/acetonitrile (75:25 v/v) and methanol/acetonitrile(90:10). Carotenoid standards of astaxanthin (98% Sigma), cantha-xanthin (10% roche), lutein (90% Sigma), echinenone (98% Sigma)and beta-carotene (97% Sigma) were used to identify and quantifythe carotenoids in the extracts by TLC and HPLC.

All the pigment extractions were carried out in duplicate.

3. Results and discussion

3.1. Effect of light intensity on PAMFC performance

In order to study the effect of light intensity on the performanceof the PAMFC, four batch cycles were performed using one or twodifferent florescent lights, directed to the cathode compartmentand inoculated with C. vulgaris culture (2 g/L of initial concentra-tion). Fig. 3 shows the results achieved for a light intensity of

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Fig. 3. Voltage and power density generation in acetate-fed PAMFC, under continuous fluorescent light intensity with 26 lE/(m2 s) (a) and 96 lE/(m2 s) (b). Initial microalgaconcentration was 2 g/L in the cathode compartment.

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26 lE/(m2 s) (first two batch cycles) (Fig. 3a) and of 96 lE/(m2 s)(third and fourth batch cycles) (Fig. 3b).

The results clearly show an increase in voltage (and power) out-put with higher light intensity. As shown for the first two batch cy-cles presented in Fig. 3a, after a start-up period, the voltage outputof the PAMFC illuminated with 26 lE/(m2 s) reach a steady statevalue of 110 mV (10 mW/m2) during 4 days. After this period, thevoltage output drop due to the total acetate depletion.

In opposition, the third and fourth batch cycles presented inFig. 3b showed that with high light intensity (96 lE/(m2 s)), highervoltage outputs up to 280 mV (maximum power output 62.7 mW/m2) were maintained for periods of 2 days, after which acetate wascompletely consumed. A valley in the voltage output is observed,which corresponds to the night period, suggesting that naturalday light in the laboratory contributes cumulatively for electricityproduction. The cycles of voltage output were shorter at high lightintensity, indicating a faster acetate oxidation by the bacterial pop-ulations. This result shows that the photosynthetic activity in thecathode increases: the electron flow from one compartment tothe other and the reduction (in the anode) of higher oxygen con-centrations (produced by microalgae) being transported throughthe ion exchange membrane (separating the anode and cathodecompartment).

The analyses of the inorganic carbon (IC) source revealed a finalconcentration of 16 mg/L IC (for the higher light intensity trial),which demonstrated that the microalga consumed the inorganiccarbon initially present in the medium (70 mg/L of IC added inthe form of bicarbonate), proving that this system is able to fix car-bon in the microalgae biomass. This result is in accordance withthe observed consumption of IC in a PAMFC reported by Caoet al. (2009). Additionally, the bacteria respiration emits CO2 (finalinorganic carbon in the anode was 42 mg/L of IC, for the higherlight intensity trial), which can permeate the membrane and bealso consumed by the microalga.

The achieved results demonstrated that the power outputs arereproducible for each light intensity tested. The maximum poweroutput achieved of 62.7 mW/m2 (1.6 W/m3) is in the same orderof magnitude of several PhotoMFCs reported in the literature(41 mW/m2, Strik et al., 2010), (2.2 W/m3, Xiao et al., 2012),(68 mW/m2, Zhang et al., 2011). Pandit et al. (2012) reported amaximum power output of 29.7 mW/m2 for a system were thephoto biocathode was sparged with air, and this value increasedto 57.8 mW/m2 when the same biocathode was sparged with airenriched with CO2 (in addition to the carbonate added in thegrowth medium). Therefore, for the system presented in this studyit could also be possible to optimise the power output by spargingCO2 (which could be obtained from an industry gaseous waste

stream) and not only using the carbonate initially present in themedium.

The increase in the light intensity resulted on a power densityincrease of about 6-folds. This result may be due to the fact thathigh light intensity (96 lE/(m2 s)) promotes higher photosyntheticactivity and the production of oxygen, which will be available forthe PAMFC cathodic reaction, resulting in a maximization of thevoltage output.

The microalgae biomass concentration was also positively af-fected by the high light intensity, reaching a concentration of2.8 g/L, against 2.2 g/L for 96 and 26 lE/(m2 s), respectively (theinitial microalgae biomass concentration was the same in bothcases, 2.0 g/L). This increase was possible even after only 2 daysof operation, against the 5 days of operation under the lowestlight intensity. Therefore, under the tested conditions the high lightintensity allowed for an increase of the microalgae growth rate upto 10 times higher than the rate achieved for the lower lightintensity.

Several authors report the influence of light and dark cycles inPAMFCs systems (Strik et al., 2010; Wang et al., 2010; Zhanget al., 2011; Xiao et al., 2012; Chandra et al., 2012) showing thatduring dark lower or no power is produced (when microalgae isused in the cathode for oxygen production).

3.2. Effect of light intensity on microalga biomass pigment compositionduring PAMFC operation

The total pigment content and pigment profile of C. vulgaris bio-mass extracts were determined in order to evaluate the productionof these added-value compounds and the differences in their pro-file, after PAMFC operation under different light intensities. The to-tal pigment content of C. vulgaris biomass extracts obtained, after5 days of PAMFC operation, at 26 lE/(m2 s) was 0.6% w/w (gpigment/100 gdry microalgal biomass). Using higher light intensity (96 lE/(m2 s)),the total pigment content in the extracts of C. vulgaris increased to0.8% (w/w) (gpigment/100 gdry microalgal biomass) after only 2 days ofoperation. This proves that by using higher light intensity it is pos-sible to increase the microalga pigment productivity. The TLC ofthe microalga extracts showed the presence of lutein, canthaxan-thin and b-carotene, which was also confirmed by HPLC by com-parison of the retention times with the standards. Astaxanthinand echinenone were not found in all the extracts analysed.

Fig. 4 represents differences between the initial inoculum com-position (Cinoculum) (as % of total pigments) and the compositionafter PAMFC operation (CPAMFC), with the two light intensities(Cinoculum (%) � CPAMFC (%)). This Figure depicts the increases ordecreases in the percentages of each pigment content for both

Fig. 4. The positive and negative increment of each pigment, extracted frommicroalgae biomass collected from PAMFC trials, under different light intensities 26and 96 lE/(m2 s). The increment was the difference between the percentagepigment content in the microalgae inoculum (initial content) and the percentagecontent in the biomass collected after the PAMFC trials (final content): Cinoculum

(%) � CPAMFC (%).

Fig. 5. The positive and negative increment of each pigment, extracted frommicroalgae biomass collected from a PAMFC trial, under nitrogen stress in theculture medium. The increment was the difference between the percentagepigment content in the microalgae inoculum (initial content) and the percentagecontent in the biomass collected after the PAMFC trial (final content): Cinoculum

(%) � CPAMFC (%).

L. Gouveia et al. / Bioresource Technology 154 (2014) 171–177 175

conditions. In what concerns the Fig. 4, the identified chlorophyllsinclude chlorophyll a and b, as well as its degradation products(pheophythin and pheophorbide). In this Figure the ‘‘other pig-ments’’ include more polar compounds, such as violaxanthin/neo-xanthin and other carotenes such as alpha-carotene. Comparingthe inoculum pigment content and the total content after PAMFCoperation with 26 lE/(m2 s), only slight changes were observedin the pigment profile. It can be seen from Fig. 4 that, when PAMFCwas operating under a 5 day cycle with 26 lE/(m2 s) of light inten-sity, there was an increase in the chlorophyll, b-carotene and lu-tein/zeaxanthin percentage content of 2–3%, while for ‘‘otherpigments’’ the percentage content decreased 4%. No canthaxanthinor other secondary carotenoids were found in these extracts. Onthe other hand, the changes on the pigment composition weremore significant during PAMFC operation under 96 lE/(m2 s) lightintensity (even after only 2 days). Chlorophyll content decreasedand ‘‘other pigments’’ increased considerably, together with asmall increase of b-carotene content. Additionally, this PAMFCoperation cycle showed the presence of canthaxanthin in the ex-tracts as well as a significant increase in more polar carotenoids,such as violaxanthin/neoxanthin (‘‘other pigments’’). These results,obtained after only 2 days of operation, clearly indicate that higherlight intensity stimulates the production of carotenoids. Theseoutcomes are in accordance with previous studies, which demon-strated that nutrient and light stresses induces the carotenogenesisprocess, characterized by the production of b-carotene and second-ary carotenoids, and decreased the content of chlorophylls in mic-roalgae biomass (Gouveia et al., 1996; Campenni’ et al., 2013).Secondary carotenoids act as protective agents of photosyntheticreaction centres, since part of light absorbed by accessory pig-ments is diverted from these systems. The increase of the amountof secondary carotenoids (‘‘other pigments’’) demonstrated themicroalgae photoprotection response to high light stress, where lu-tein/zeaxanthin was oxidised to canthaxanthin (Fig. 4) (Gouveiaet al., 1996; Campenni’ et al., 2013), which may increasing the eco-nomic potential of the PAMFC. It is important to underling that theidentified carotenoids were obtained after only 2 days of operation,meaning that other secondary carotenoids, as well as higheramount of total carotenoids, could be obtained for longer operationperiods, or by the induction of other stresses, such as nitrogen lim-itation and/or salt addition (Gouveia et al., 1996; Campenni’ et al.,2013).

The identified carotenoids are high added-value compoundsand powerful antioxidants with important applications in food,

feed, nutraceutical, pharmaceutical, and medicine. The obtainedresults demonstrate that the light intensity can be adjusted in or-der to stimulate the carotenoid production, during a PAMFC oper-ation. Additionally to the light stress studied in this work, differentmicroalgae culture stress conditions can be applied to the systemin order to potentiate the carotenogenesis in the cathode compart-ment. These stress conditions include nutrient limitation andosmotic stress. Operational conditions such as duration of dark/light regimes, microalgae and bacterial biomass concentrationmay also influence the carotenogenesis process and powerproduction.

3.3. Effect of nitrogen starvation on microalga biomass pigmentcomposition during PAMFC operation

In addition to light stress the PAMFC was also operated underculture medium nutrient stress. In this trial the PAMFC cathodewas operated without nitrate for 8 days (corresponding to two ace-tate pulses of 20 mM in the anode compartment) with a high lightintensity (83 lE/(m2 s)). During the trial C. vulgaris grew from theinitial concentration of 2 g/L to the final value of 3.3 g/L. The max-imum power output achieved was 61.7 mW/m2, similar to the oneachieved for the trial performed without nitrogen culture mediumstress under a light intensity of 96 lE/(m2 s) (see Section 3.1).Therefore, under the tested conditions of nitrogen limitation stresslow effect on the PAMFC power output was observed. Nevertheless,the total pigment content of C. vulgaris biomass extracts obtained,after 8 days of PAMFC operation under nitrogen starvation was1.2–1.3% w/w (gpigment/100 gdry microalgal biomass), which representsan increase of 117% from the initial inoculum pigment content of0.6% w/w (gpigment/100 gdry microalgal biomass). Fig. 5 shows (in simi-larity with Fig. 4) the evolution of the pigment profile of C. vulgaris,depicting increases or decreases in the relative percentages of eachpigment, after PAMFC operation. Similar to the previous presentedtrial (under 96 lE/(m2 s) light intensity and no nutrient stress) theresults show a decrease in the chlorophyll and lutein content witha significant increase of more polar carotenoids (‘‘other pigments’’).Additionally, pigments such as lutein esters were also identifiedshowing that there is an advance in the carotenogenesis process.

These results demonstrate that it is possible to stimulate themicroalga biomass growth and increase the pigment content by oper-ating the PAMFC for longer periods under culture medium nutrientstarvation, without significant effect on power production. Thus,allowing to obtain bioelectricity and valuable microalga biomass.

Fig. 6. Pigment profile composition of the microalgae biomass collected from a halfcell control and PAMFC trials, performed under the same conditions of lightintensity and agitation and with the same initial microalgae inoculums.

176 L. Gouveia et al. / Bioresource Technology 154 (2014) 171–177

3.4. Effect of PAMFC operation on microalga biomass pigmentcomposition

In order to determine if the PAMFC operation had any negativeor positive effect on microalga pigment production under lightstress conditions, a control cell (half PAMFC, identical to the PAM-FC cathode compartment, volume 50 mL) and a PAMFC, were oper-ated under the same light (83 lE/(m2 s) and agitation conditionsfor a period of 4 days. Afterwards, biomass of both control andPAMFC was submitted to extraction in order to evaluate the totalpigment content, as well as the pigment profile. The control celland the PAMFC were both inoculated with microalga C. vulgarisas inoculum at the same initial biomass concentration (2 g/L).The total pigment content of C. vulgaris biomass extracts obtainedwas 1.6% and 2.0% w/w (gpigment/100 gdry microalgal biomass) for thecontrol and PAMFC, respectively. This result demonstrates thatunder the tested conditions the PAMFC operation had a positiveimpact on pigment production. This observation is favourable forthe economic efficiency of the PAMFC that will allow the produc-tion of bioelectricity and simultaneously production of added-va-lue pigments.

Fig. 6 shows the pigment profile for both the control and PAMFCtrials. The results showed a similar pigment profile for both trials.The content on lutein was superior for the control and the PAMFCtrial produced slightly more pigments identified as ‘‘other pig-ments’’ in Fig. 6. Therefore, both trials showed evolution of thecarotenogenesis process, in accordance with microalgae growthunder light stress conditions. Overall, the PAMFC operation al-lowed for the production of added-value pigments without anynegative effect, under the tested conditions. Additionally, the bio-mass of the PAMFC trial showed the production of lutein esters(0.08%). The results indicate that the carotenogenesis was slightlyfaster in the PAMFC trial, since its extracts presented lutein estersand a higher percentage of ‘‘other carotenoids’’ such as, more polarcarotenoids: violaxanthin/neoxanthin. Longer operation periodswill confirm this observation. This result was also confirmed inthe trial performed under nutrient starvation were the lutein esterswere detected (0.62%) in the PAMFC samples and not in the halfcell control.

4. Conclusion

The simultaneous production of bioelectrical power and added-value pigments was demonstrated in a Photosynthetic Alga Micro-bial Fuel Cell. The results demonstrated that the performance of

the studied PAMFC was controlled by the photosynthetic activity,since power output and pigment production were directly affectedby light intensity conditions. The development and scale-up ofsuch PAMFC combined system at real sunlight conditions, could al-low for an economic and energy efficient wastewater treatmenttogether with carbon, nitrogen and phosphate fixation in themicroalgae biomass and obtaining valuable microalgae biomass(containing high added-value compounds) produced in the PAMFCcathode.

Acknowledgements

This work was part of a research project ‘‘Microalgae as a sus-tainable raw material for biofuels production (Biodiesel, Bioetha-nol, Bio-H2 and Biogas)’’ (PTDC/AAC-AMB/100354/2008)sponsored by the Portuguese Foundation for the Science and Tech-nology (‘‘Fundação para a Ciência e a Tecnologia’’ – FCT). Beatriz P.Nobre acknowledges FCT for the research Grant (SFRH/BPD/42004/2007).

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