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Cite this:Phys.Chem.Chem.Phys.,

2014, 16, 17327

Thylakoid direct photobioelectrocatalysis: utilizingstroma thylakoids to improve bio-solar cellperformance†

Michelle Rasmussen and Shelley D. Minteer*

Thylakoid membranes from spinach were separated into grana and stroma thylakoid fractions which

were characterized by several methods (pigment content, protein gel electrophoresis, photosystem

activities, and electron microscopy analysis) to confirm that the intact thylakoids were differentiated into

the two domains. The results of photoelectrochemical experiments showed that stroma thylakoid

electrodes generate photocurrents more than four times larger than grana thylakoids (51 � 4 nA cm�2

compared to 11 � 1 nA cm�2). A similar trend was seen in a bio-solar cell configuration with stroma

thylakoids giving almost twice the current (19 � 3 mA cm�2) as grana thylakoids (11 � 2 mA cm�2) with

no change in open circuit voltage.

Introduction

The light-dependent reactions of photosynthesis in higherplants take place in the thylakoid membrane located insidethe chloroplast.2 This electron transport system consists of fourlarge protein complexes and several small redox species.3 Theinitial electron donor is water which is oxidized to O2 byphotosystem II (PS II). The electrons produced by this reactionare transferred to the cytochrome b6f complex by plastoquinonewhich is able to move through the membrane. Cytochrome b6facts as a proton pump because the reduced plastoquinone isoxidized and its protons are released into the thylakoid lumen.From the cytochrome complex the electrons are transferred tophotosystem I (PS I) by plastocyanin and then on to ferredoxin.At this point the electrons can be transferred by two differentpathways.1 Ferredoxin can be oxidized by ferredoxin–NADP+

reductase while producing NADPH. In the alternate pathway,the reduced ferredoxin transfers its electrons to plastoquinone,completing a loop for a cyclic pathway. The proton gradientgenerated during photosynthesis is used for production of ATPby ATP synthase.2

The thylakoid membrane is highly folded in order toincrease surface area, similarly to the cristae in mitochondria,and exists in two configurations.1 The majority of the systemconsists of stacked discs called grana thylakoids. These stacks

are connected by smaller membrane fragments called stromalamellae or stroma thylakoids.4 As seen in Fig. 1, the compo-nents of these structures are different. ATP synthase is locatedin the stroma thylakoids and the outside membrane of thegrana because a large portion of the complex must be exposedto the stroma for ATP production. Cytochrome b6f is distri-buted through the thylakoid membrane.3 Separation of the twophotosystems is required to prevent the transfer of slightlyhigher energy photons from PS II to PS I. Therefore, PS II islocated almost exclusively inside the grana stacks while PS I isconfined to the stroma thylakoids.1

Solar energy conversion with biological catalysts has beenstudied by many research groups using a number of speciesincluding individual photosystems or reaction centers,5–8 thyla-koids,9–11 and chloroplasts.12,13 Other groups have synthesizedmolecular species to mimic the reaction centers of photosyntheticcomplexes.14,15 However, no research groups have evaluated the

Fig. 1 Schematic of thylakoid membrane which illustrates the compo-nents contained in grana and stroma thylakoids.1

Departments of Chemistry and Materials Science and Engineering,

University of Utah, 315 S 1400 E Rm 2020, Salt Lake City, UT 84112, USA.

E-mail: minteer@chem.utah.edu; Tel: +1-801-587-8325

† Electronic supplementary information (ESI) available: Isolation and fractiona-tion of thylakoid membranes and protein gel electrophoresis results. See DOI:10.1039/c4cp02754j

Received 23rd June 2014,Accepted 1st July 2014

DOI: 10.1039/c4cp02754j

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photobioelectrocatalytic abilities of the two types of thylakoids andtheir potential for increasing the energy conversion in bio-solar celldevices which is crucial for their future application. In this work westudy the solar energy conversion of grana and stroma thylakoidelectrodes isolated from spinach. The photocurrent was measuredamperometrically with thylakoid modified electrodes which werethen connected with a laccase oxygen reduction biocathode forbio-solar cell tests.

Experimental methodsFractionation of thylakoid membranes

Organic spinach was purchased from a local supermarket.Thylakoid membranes were isolated and fractionated intograna and stroma thylakoid fractions following a procedurefrom literature.16 A more detailed description of the procedurecan be found in the ESI.† Isolated thylakoids were resuspendedin buffer containing 10 mM MgCl2, 10 mM NaCl, and 10 mMtricine, pH 7.8 and stored at �20 1C until further use.

Biochemical characterization

Protein concentrations of the thylakoid fractions were determinedusing a Pierce BCA assay kit. The amount of total carotenoid andchlorophylls a (Chl a) and b (Chl b) in each thylakoid fraction wasdetermined spectroscopically using a literature procedure.17 Absor-bances at 470 nm, 646 nm, and 663 nm were obtained for eachfraction diluted 1 : 100 in 80% (v/v) acetone. Pigment content(in mg mL�1) was calculated using the following equations:17

Chl a = 12.21A663 � 2.81A646

Chl b = 20.13A646 � 5.03A663

Carotenoids = (1000A470 � 3.27Chl a � 104Chl b)/229

Thylakoid fraction samples (20 mg for grana and stromathylakoids, 40 mg for full thylakoids) in SDS buffer were subjectedto electrophoresis in a 4–20% Tris-HEPES SDS-PAGE gel. The gelwas stained with Coomassie blue G-250. For TEM imaging,samples were prepared following a procedure adapted fromliterature.18 Samples were first fixed in hypotonic buffer with2% (v/v) glutaraldehyde and 3% (v/v) paraformaldehyde for 1 hand then suspended in 1.7% molten agarose at 37.8 1C for15 min. The samples were post-fixed with 1% (w/v) osmiumtetraoxide in a solution containing 0.1 M sodium cacodylate(pH 7.4), 5 mM CaCl2, and 0.5% potassium ferricyanide for 1 h.The samples were then rinsed with water and stained with 2%(w/v) uranyl acetate, dehydrated, embedded in Epon, and poly-merized overnight. Thin slices (70 nm) were cut and post-stainedwith uranyl acetate. Images were obtained with a JEM 1400transmission electron microscope (JEOL, Japan).

Photosystem activity assays

The activities of Photosystem I and II were determined for thethylakoid fractions using a procedure from literature.19 Photo-system I activity was measured by mixing 840 mL of 50 mM

HEPES (pH 8.0), 10 mM NaCl, 5 mM MgCl2, 100 mM sorbitol,1 mM NH4Cl, and 0.25 mM methyl viologen with 30 mL 1 mMascorbate (made fresh), 10 mL 3 mM 2,6-dichlorophenolindophenol(DCPIP, made fresh), and 10 mL 1 mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). A 100 mL aliquot of thylakoids containing10 mg Chl was added and the mixture was equilibrated in the darkfor 3 min. The mixture was then exposed to light and the increasein absorbance at 580 nm due to oxidation of DCPIP was observed.

Photosystem II activity was measured by mixing 870 mL of50 mM HEPES (pH 7.0), 10 mM NaCl, 5 mM MgCl2, 100 mMsorbitol, and 1 mM NH4Cl, with 30 mL 3 mM DCPIP. A 100 mLaliquot of thylakoids containing 10 mg Chl was added and themixture was exposed to light. The absorbance decrease at580 nm was recorded.

The following equation was used to calculate the activities ofPSI and PSII:

mol DCPIP (mg Chl)�1 s�1 = (DA580/min)(volume)/e(mg Chl)

where mmol DCPIP is the amount consumed during the reac-tion, volume is the reaction volume in the cuvette in mL, e is18 mL mmol�1, and Chl mg is the total mass of Chl in thesample. The specific activity is calculated by dividing theactivity by the protein concentration.

Photoelectrochemical analysis

All photoelectrochemical measurements were obtained with a CHInstruments CHI660 electrochemical workstation and the lightsource was a 250 W halogen lamp at 5200 lumens. Electrodes werefabricated according to a previously reported procedure.10 Briefly, a250 mL aliquot of the thylakoid sample was mixed with 7.5 mL ofcatalase from Aspergillus niger (Sigma, 6.69 kU mg�1, 31.4 mg mL�1)and 50 mL of this mixture was applied to a 1 cm2 Toray carbon paperelectrode. The electrodes were allowed to dry for approximately onehour, and then a thin layer of silica was vapor deposited usingtetramethyl orthosilicate. Modified electrodes were stored at 4 1Covernight. Laccase biocathodes were fabricated following apreviously reported procedure.11

Thylakoid electrodes were tested in two configurations.Amperometry was performed at 450 mV vs. Ag/AgCl in 0.1 MpH 5.5 citrate buffer in the absence and presence of light todetermine the photocurrent generated by each of the thylakoidfractions. The same experiment was also performed in thepresence of photocurrent inhibitors (DCMU and methyl violo-gen). The thylakoid electrodes were also tested in a bio-solarcell configuration by connecting with a laccase oxygenreduction biocathode. All experiments were performed intriplicate and reported uncertainties correspond to the standarddeviation of those triplicate measurements.

Results and discussion

Fractions of thylakoid membranes isolated from spinach werecharacterized to confirm separation of grana and stromathylakoids. Pigment content of the fractions was determinedspectroscopically. The ratio of Chl a to Chl b is lower for grana

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thylakoids (2.0 � 0.2) than stroma thylakoids (3.9 � 0.2) whichis consistent with results reported by other researchgroups.16,20 Most of the Chl b is contained in the light harvest-ing proteins of PSII which leads to a low Chl a : b ratio in thegrana fraction where the PSII is located.21,22 The ratio of totalChl to carotenoids is similar for the two fractions (4.0 � 0.2 forthe grana fraction and 4.2 � 0.4 for the stroma) which is alsoexpected. The total Chl and carotenoids in thylakoids isdistributed approximately equally between PS I and II whichleads to similar ratios for the grana and stroma fractions.16,23

The thylakoids fractions were also evaluated using proteingel electrophoresis. A number of bands are observed in all threesamples (full, grana, and stroma thylakoids, see Fig. S1 in theESI†). However, the band at approximately 60 kDa, which is thetwo core subunits of PS I (PsaA and PsaB)20 is much darker inthe stroma fraction (S) compared to the grana (G) and fullthylakoid (T) fractions. The band near 25 kDa, which is thelight-harvesting center (LHC) of PS II,20 is darker in the granafraction. These results indicate that the grana fraction is PSII-enriched while the stroma fraction contains most of the PS I.

The activity of the two photosystems was determined foreach of the thylakoid fractions. As expected, the grana fractionshows more PS II activity (0.07 � 0.01 U mg�1 Chl) than thestroma fraction (0.03 � 0.005 U mg�1 Chl). The stroma fractionshows much higher PS I activity (0.38 � 0.03 U mg�1 Chl) thanthe grana fraction (0.13 � 0.02 U mg�1 Chl). These resultsconfirm that most of the PS I is contained in the stromafraction, while more of the PS II is found in the grana fraction.

Finally, the thylakoid fractions were observed using transmissionelectron microscopy (TEM). As seen in Fig. 2a, when the chloroplastsare lysed and removed, the thylakoid membrane loses much of itsthree-dimensional structure, but the membrane stacking remainsintact. The grana thylakoids (Fig. 2b) show a significant decrease inthe amount of stacked regions. However, the membrane itself isintact and the fragments are large (1.1 � 0.2 mm) compared to thestroma thylakoids (Fig. 2c). The stroma fraction shows smallmembrane fragments (0.25 � 0.07 mm) and no stacked regions.Although a mild detergent was used to solubilize the stroma

fraction, it is likely that this process broke the membrane sectionsup into the small fragments that are observed.

The characterization results confirm that grana and stromathylakoids were successfully separated into two fractions. Thegrana fraction contains much larger membrane fragmentswhich show almost twice the PS II activity and low PS I activity.The stroma fraction contains small membrane fragments withlarge PS I activity and little to no PS II activity.

Isolated thylakoid membrane fractions were immobilizedonto carbon paper electrodes for photoelectrochemical testing.Amperometry was performed at 450 mV vs. Ag/AgCl to measurethe photocurrent generated by the thylakoid fractions. Fig. 3shows representative data for a stroma thylakoid electrode. Thecurrent is allowed to stabilize in the dark before illuminating.The current stabilizes again and the light is turn off. This isrepeated several times. The photocurrent is calculated by sub-tracting the current in the dark from the current in the light.Stroma thylakoid electrodes generate much larger photocurrent(51 � 4 nA) compared to grana thylakoid electrodes (11 � 1 nA).However, different amounts of thylakoid were immobilizedon the electrodes. For more accurate comparison, the photo-current was normalized by dividing by either the protein or Chlconcentration. In both cases the stroma thylakoids show muchlarger normalized currents, 2 � 0.2 mA mg�1 protein and 11.5 �0.9 mA mg�1 Chl, than grana electrodes, 0.66 � 0.09 mA mg�1

protein and 2.1 � 0.3 mA mg�1 Chl.Thylakoid modified electrodes were also inhibited with two

herbicides: DCMU which inhibits PS II and methyl viologenwhich inhibits PS I photocurrent. It should be clarified herethat while DCMU is an inhibitor of PS II, methyl viologen doesnot physically inhibit PS I. Instead it acts as an electronacceptor for PS I and prevents the transfer of electrons fromPS I to the electrode. As expected, when grana thylakoidelectrodes are exposed to DCMU there is a greater than 25%decrease in photocurrent (see Fig. 3, right), because most of thePS II is contained in that fraction. Alternatively, there is littleto no decrease (o10%) when exposed to methyl viologen.However, when stroma thylakoid electrodes are inhibited with

Fig. 2 TEM images of thylakoid membrane fractions: (a) full thylakoids, (b) grana thylakoids, and (c) stroma thylakoids.

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methyl viologen, there is a large decrease (30%) in photocurrentbecause that fraction contains most of the PS I. There is nodecrease for stroma electrodes in DCMU. Previously we hadshown that when intact thylakoids are inhibited, there is morecurrent loss with a PS I inhibitor compared to a PS II inhibitor.24

This indicates that PS I contributed more to the photocurrentwhich is consistent with the results for the fractionated thylakoidswhere the stroma (containing PS I) shows larger photocurrents.This is also consistent with previous reports which showdecreased electrogenic effect with PS II compared to PS I.25,26

Finally, thylakoid modified electrodes were connected with alaccase oxygen reduction biocathode to test as a bio-solar cell.I–V curves were obtained by scanning the voltage and representa-tive curves can be seen in Fig. 4. For these experiments, the

electrodes were each modified with the same amount of protein.The open circuit voltages were the same for all three types ofthylakoid bio-solar cells in the absence and presence of light(see Table 1). However, the current responses were quite differ-ent. The stroma thylakoid electrodes showed much largercurrents over the potential range of 0 to 0.4 V compared to thegrana and full thylakoid electrodes. Above 0.4 V all three showonly small currents. The short circuit currents in the dark (ISC,D)and light (ISC,L) for the stroma electrodes were 13 � 3 mA and19 � 3 mA, respectively, which are significantly larger than thosefor the grana and full thylakoids. Additionally, the currentdifference between the light and dark for the stroma electrodesis more than twice as large which indicates that the stromafraction performs more photobioelectrocatalysis.

Conclusions

Stroma thylakoids immobilized onto carbon paper electrodesgenerated much larger photocurrents than grana thylakoids.The small size of the stroma thylakoid fragments, as seen withtransmission electron microscopy, allows more of the PS I closeto the carbon surface for direct electron transfer (DET) whichleads to more current. In the grana thylakoids, most of the PS IIis located inside the stacks which prevents it from doing facileDET. Only the PS II located towards the outside of the stacks

Fig. 3 (left) Representative amperometric results for a stroma thylakoid modified electrode in 0.1 M pH 5.5 citrate buffer at 450 mV vs. Ag/AgCl in a 3electrode configuration in the absence and presence of light (black line). The electrode was also inhibited with DCMU (blue line) and methyl viologen (redline). (right) Photocurrent measured at grana (black) and stroma (red) thylakoid modified electrodes in 0.1 M pH 5.5 citrate buffer at 450 mV vs. Ag/AgCl.

Fig. 4 Representative I–V curves for bio-solar cells (2-electrode cell)incorporating full thylakoids (black line), grana thylakoids (red line), andstroma thylakoids (blue line) in 0.1 M pH 5.5 citrate buffer in the presenceof light. The cathode was a laccase oxygen reduction electrode and thescan rate was 5 mV s�1.

Table 1 Results from I–V curves for bio-solar cells incorporating incor-porating full, grana thylakoids and stroma thylakoids in 0.1 M pH 5.5 citratebuffer in the absence and presence of light. The cathode was a laccaseoxygen reduction electrode and the scan rate was 5 mV s�1

OCVD (V) OCVL (V) ISC,D (mA) ISC,L (mA) DI (mA)

Grana 0.68 � 0.01 0.69 � 0.02 8 � 1 11 � 2 2.6 � 0.4Stroma 0.65 � 0.01 0.65 � 0.01 13 � 3 19 � 3 5.6 � 0.4Full 0.66 � 0.03 0.66 � 0.03 7 � 2 7 � 3 2 � 1

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would be close enough to the carbon surface which leads tosmaller currents.

Acknowledgements

Generous funding was provided by the National Science Foun-dation MRSEC Grant (#DMR 11-21252). The authors would alsolike to thank Dr Linda Nikolova for obtaining TEM images.

Notes and references

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