Optimal Intensity and Biomass Density for Biofuel Production in aThin-Light-Path PhotobioreactorAadhar Jain,†,⊥ Nina Voulis,‡,⊥ Erica E. Jung,† Devin F. R. Doud,‡ William B. Miller,§

Largus T. Angenent,‡ and David Erickson*,†

†Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York 14853, United States‡Biological and Environmental Engineering, §Department of Horticulture, Cornell University, Ithaca, New York 14853, United States

*S Supporting Information

ABSTRACT: Production of competitive microalgal biofuelsrequires development of high volumetric productivity photo-bioreactors (PBRs) capable of supporting high-densitycultures. Maximal biomass density supported by the currentPBRs is limited by nonuniform distribution of light as a resultof self-shading effects. We recently developed a thin-light-pathstacked photobioreactor with integrated slab waveguides thatdistributed light uniformly across the volume of the PBR.Here, we enhance the performance of the stacked waveguidephotobioreactor (SW-PBR) by determining the optimalwavelength and intensity regime of the incident light. Thisenabled the SW-PBR to support high-density cultures,achieving a carrying capacity of OD730 20. Using a geneticallymodified algal strain capable of secreting ethylene, weimproved ethylene production rates to 937 μg L−1 h−1. This represents a 4-fold improvement over a conventional flat-platePBR. These results demonstrate the advantages of the SW-PBR design and provide the optimal operational parameters tomaximize volumetric production.

■ INTRODUCTION

Driven by the need for sustainable alternatives for fossil fuels,energy research today is focused on a range of renewableresources, including biomass. Algae are of particular interest asa sustainable feedstock for transportation fuel because of theirsalient advantages over terrestrial crops, including higher areayields,1−3 smaller water footprint, ability of certain strains togrow in brackish or saline water, and applicability of wastestreams as a low-cost source of CO2, nitrogen, andphosphorus.4−10 In addition to biofuels, algae cultivation isalso used for production of food and feed supplements,11,12

showing the versatility of algal products and furtherencouraging the development of high efficiency photobior-eactors (PBRs).Current commercial algae cultivation relies on open ponds,

primarily because of low capital costs.6,7,13 This technology,however, has a number of disadvantages if used for large-scaleproduction of bulk commodities such as biofuel.13,14 Theseshortcomings include low biomass densities,5−7 requirement oflarge surface areas, contamination,4,14 incompatibility withcultivation under atmosphere with elevated CO2 levels,5 andhigh water losses due to surface evaporation.5

Production of high-volume commodities, therefore, requiresgrowth systems to overcome these limitations throughdeployment of alternative technologies, such as closedPBRs.13,15,16 Although conventional closed PBRs (e.g., flat-

plate and tubular reactors) enable algae cultivation in morecontrolled environments than open ponds, they still suffer fromsuboptimal light distribution.2,11,17 Under standard operationregimes, algae close to the illuminated surface are photo-inhibited, whereas algae in the interior of the reactor arephotolimited.13,18−23 These effects are mitigated by employingmixing strategies that circulate the algae cells across the lightgradient and thus ensure an adequate average light exposure.24

However, the energy requirements for mixing are high,amounting even in simple systems to 3 W m−2, close to theenergy eventually harvested from algal biomass5 and con-tributing to an overall energy efficiency ratio of less thanone.25,26 Mixing also accounts for 13−52% of total constructionand operation costs of conventional closed PBRs.27

In an earlier publication,28 we addressed the limitationsposed by simultaneous photoinhibition and photolimitation inconventional closed PBRs. We embraced a holistic PBR designby (1) delivering light through optical waveguides for optimallight distribution inside the reactor, (2) aiming for densecultures facilitating high volumetric product yields, and (3)envisaging utilization of product-secreting algae. The latter

Received: October 28, 2014Revised: April 14, 2015Accepted: April 24, 2015Published: April 24, 2015

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© 2015 American Chemical Society 6327 DOI: 10.1021/es5052777Environ. Sci. Technol. 2015, 49, 6327−6334

would simplify postprocessing steps and avoid costly andtechnically challenging harvesting and product extractionsteps,29 which can contribute up to 50% of the total productcost.6

To achieve this, we have demonstrated a short-light-pathstacked photobioreactor (SW-PBR) with internal lightdistribution through optical slab waveguides. Incident light iscoupled into the sides of each slab waveguide and is propagatedalong the length of the waveguide via total internal reflection.The surface of the optical waveguide is etched to createrandomly distributed scratches, thus changing the incident lightangle, thereby disrupting the propagating light wave andreleasing the light into the bioreactor. Combined with shortlight paths (∼1 mm), the waveguides enable the delivery ofoptimal light intensities throughout the entire volume of theSW-PBR, avoiding both photolimitation and photoinhibition aswell as reducing the need for energy-intensive and costlymixing. The resulting 3D architecture both reduces the amountof land area required and supports high-density cultures,leading to a high areal productivity. For the SW-PBR weshowed an 8-fold higher biomass accumulation than achieved ina control PBR without optical waveguides.28

Aiming to avoid costly cell-harvesting steps, we used aproduct-secreting Synechocystis sp. PCC 6803 2x EFE. Thisalgae strain had been genetically modified to secrete the biofuelprecursor ethylene.30 In an earlier work,28 we had demon-strated consistent production of ethylene for over 45 days usingthe SW-PBR. We had achieved a 2-fold increase in volumetricethylene production rates as compared with a flat-plate PBR.The earlier achieved results validate the design and perform-ance of the SW-PBR, indicating its promise as a viable

photobioreactor technology. In the present work, we exploredmethods to further improve yields and energy efficiencies. Weoptimized production over wavelength and intensity of thesupplied light.Photoautotrophic algae preferentially use certain wavelengths

in the photosynthetic active radiation (PAR) range, dependingon the collection of light harvesting pigments present in theirphotosynthetic machinery.22 Chlorophyll constitutes the mostimportant group of these pigments and absorbs in the blue(450−475 nm) and the red spectral range (630−675 nm).17,22

Excitation of chlorophyll molecules in the reaction centers ofphotosystem I and II requires energy equivalent to thatcontained in photons with a wavelength of 700 and 680 nm,respectively.22 Algae should, therefore, be supplied with light inthe red spectral range to minimize energy dissipation. Theefficacy of algal production under red light has been previouslyshown by multiple studies.1,31−34

In addition to wavelength, the intensity of the supplied lightis another important parameter determining algal growth. Theoptimum intensity for many algal species lies in the range of100−400 μE m−2 s−1.5,12,22 Photolimitation occurs at lowerlight intensities, when insufficient photons are available to carryout photosynthesis at the rate required for optimal cell growth.Photoinhibition, on the other hand, leads to cell damageinduced by high light intensities, adversely affecting growth.22

Although algal growth thus follows a concave function withrespect to intensity, photosynthetic efficiency (PE) decreaseswith increasing intensities as photosynthetic centers becomemore saturated.22,34,35 Therefore, design of algal productionsystems needs to account for both the optimal growth windowand sufficient PE.1,34,36−39

Figure 1. (a) Ten-stack 3D printed photobioreactor in operation. The reactor is illuminated from each side by red (630 nm) LED banks. The light iscoupled into the waveguides, which transport and evenly distribute the light inside the reactor. The photomask prevents uncoupled light fromentering the reactor. The side chimney connects the layers and allows collection of the produced gas. The influent port is used to inoculate thereactor before operation and collect displaced liquid during operation. The produced gas is extracted through the effluent port. (b) Schematic of theSW-PBR with 10 waveguides. (c) Schematic of the waveguide structure. A thin coverslip is attached on top of an etched glass slide.

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Here, we first present the effects of variation of wavelengthand intensity of the supplied light on ethylene production ratesof Synechocystis sp. PCC 6803 2xEFE. Second, we describe theeffect of high-density culture on ethylene production rates todetermine the carrying capacity of the SW-PBR. Third, usingthe collected data, we report the photosynthetic efficienciesachieved in the present system. The results provide us withoptimal illumination conditions and biomass densities tomaximize ethylene production in the SW-PBR.

■ MATERIALS AND METHODSStacked Waveguide Photobioreactor Design and

Assembly. The SW-PBR consisted of a 3D frame with slotsto stack slab waveguides vertically above each other (Figure 1).The dimensions of the SW-PBR were 7.5 cm × 2.5 cm × 3 cm(length × width × height), with a 2 mm gap separating twowaveguides and a total liquid volume of 16 mL. The SW-PBRhad two ports: (1) a bottom influent port for inoculation and(2) a top effluent port for gas product removal (Figure 1a).The frame was 3D printed from a photocurable resin

(VeroClear, Objet Geometries Inc.). It was coated by ParyleneC via a vapor deposition process to reduce gas permeability ofthe SW-PBR. The waveguides were placed into dedicated slotsin the frame and fixed to the frame by using polydimethylsilox-ane (PDMS) (Dow Chemicals, Midland, Michigan, USA) as aresin. This also ensured that any gaps (due to manufacturingerrors in the 3D printing process) between the frame and thewaveguides were sealed by the cured PDMS. Because PDMS isgas-permeable, the assembled reactor was subjected to a secondcoat of Parylene C to ensure gas impermeability. To preventuncoupled light from entering the SW-PBR, the gaps betweenthe slab waveguides were covered by a photomask (printedfrom the same photocurable resin as the frame). Aluminum foilwas attached to the photomask to improve reflectancecharacteristics.Waveguide Fabrication. The SW-PBR waveguides were

fabricated by affixing thin coverslips (VWR Micro CoverGlasses, Radnor, Pennsylvania, USA) to chemically etchedborosilicate microscope slides (VWR VistaVisio MicroscopeSlides, Radnor, Pennsylvania, USA). Glass etching paste(Armour Etch, Hawthorne, New Jersey, USA) was applied onthe glass slides for 7 h and subsequently washed away, creatingrandomly distributed surface defects for improved lightscattering from the waveguide surface. The thin coverslipswere attached on top of each glass slide using uncured PDMSas a resin and curing the assembly for 2 h at 80 °C. Attaching acoverslip on top of the glass slide changed the medium(cladding) adjacent to the etched glass surface from water(refractive index = 1.33) to air (refractive index = 1) (Figure1c). Because of the higher index contrast between the core andthe cladding of the waveguide now, the critical angle wasreduced, hence, increasing the amount of light scattered fromthe waveguide.28

Model Organism. The model organism in all experimentswas Synechocystis sp. PCC 6803 2x EFE. This geneticallymodified strain was selected for its ability to secrete ethylene asa gaseous product.30 Ethylene is both a biofuel precursor and animportant feedstock chemical in the chemical industry. Prior toinoculation, algal cultures were grown in semibatch in gastight 1L reactors with 20 mL of liquid phase at 30 °C and continuousbroad spectrum illumination (100 μE m−2 s−1). The culture wasgrown in 5-fold concentrated BG-11 buffered by TES (4.6 gL−1) and augmented with 25 mg L−1 spectinomycin and 200

mg L−1 kanamycin. Carbon was provided both via the gaseousphase (5% CO2 atmosphere) and via the liquid medium (20mM NaHCO3). These conditions allowed long-term steady-state maintenance of a dense algal culture at an OD730 of 60.Aliquots were taken from the semibatch culture and, afterdilution in growth medium to the required density, used asinoculum for the SW-PBR.

Experimental Setup and Procedure. The SW-PBR wasinoculated at a starting OD730 of 10 (for experiments run at470, 630, and 660 nm), 20 (for experiments at 630 nm), and 30(for experiments at 630 nm). The OD was determined using aspectrophotometer with a standard 1 cm cuvette at awavelength of 730 nm, using an appropriate dilution factor.The bioreactors were illuminated from both sides via an LEDbank (Figure 1c) of the specified wavelength (630 or 660 nm)and at five levels of intensity (35, 52, 69, 86, and 104 μE m−2

s−1). The incident light was coupled into the waveguides,propagated along the length of the waveguides, andsubsequently emitted to the algal culture by the scatteringsurface of the waveguides. The dissolved bicarbonate in thegrowth medium served as the sole source of carbon in the SW-PBR. During the course of each experiment, the bioreactorinfluent port was connected to a sterile bottle for collection ofthe displaced culture volume, and the effluent port on top ofthe side chimney was sealed by a septum to retain the secretedgaseous products in the SW-PBR.Upon illumination by the LED bank, the algal culture grew

and secreted gaseous products (including ethylene) as a resultof its photosynthetic activity. The formed gas bubbles displacedequal volumes of culture through the effluent port into thesterile effluent bottle. The design of the side chimney ensuredthat the secreted gaseous products were separated from theliquid volume and could, therefore, be easily extracted.At the end of each experimental run, the gaseous products

were extracted from the SW-PBR and analyzed. Subsequently,the whole culture volume was removed from the bioreactor,centrifuged, and subsequently resuspended in fresh medium.After two experimental runs in which the algae acclimatized tothe new culture conditions, the procedure was repeatedmultiple times for each combination of density, incident lightwavelength, and intensity. A new inoculum, which was takenfrom the semibatch culture, was used to inoculate the SW-PBRfor each new combination of experimental conditions.

Gas Extraction and Analysis. Gaseous products wereextracted from the bioreactor upon completion of eachexperimental run. As the gas was removed through the septumat the effluent port, the liquid culture displaced earlier into theconnected bottle was simultaneously pulled back into thebioreactor through the influent port. This prevented anydilution of the gaseous sample by the ambient air. Theconcentration of ethylene in the samples was analyzed using agas chromatograph (GC). (See the analysis procedure in anearlier work.28)

Measurement of Light Intensity. Because of theinaccessibility of the waveguides (except the top one) withinthe SW-PBR, it was not possible to measure the light emanatingfrom each individual waveguide. However, our previous work28

had established that the light emitted by the waveguides withinthe SW-PBR was uniform across layers and contributed to themeasurement obtained from the surface of the top waveguide.This could therefore be used to estimate the light intensityemanating from each waveguide by a single measurement from

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the surface of the top waveguide. From previous work,28 it wasfound that the intensity at the top was given by

= × − −− −

I n arr

( )1 (1 )1 (1 )

n

(1)

Where I(n) is the light intensity at the top when n layers of thestack are exposed, a is the uniform light intensity emanatingfrom each layer, and r is the fraction of light that is lost as itpasses through one waveguide layer above. By fitting themeasured data to the expression above, the value of r wasestablished experimentally to be 0.205. For this value of r, eq 1can be inverted to find the light intensity emanating from eachwaveguide as

= ×a I0.227 (10) (2)

where I(10) is the intensity measured from the surface of thetop waveguide when all 10 layers are exposed. Thus, wemeasured the intensity from the surface of the top waveguide ofan empty reactor and used eq 2 above to estimate the lightintensity emanating from each individual waveguide.Statistical Data Analysis. Statistical analysis on the

collected data was performed using the software package R.40

Separate general linear regression models were fit for ethyleneproduction rates and the PE of ethylene production. In bothmodels, the illumination wavelength, intensity, and culture ODwere used as independent variables. The overall family wiseerror rate (FWER) was controlled at 5% using the Rmultivariate marginal models (mmm) package. The results ofthe regression analysis can be found in the SupportingInformation (SI). The optimal values of OD and intensity forethylene production were calculated assuming a multiplicativeinteraction term between OD and intensity. Standard errors(SE) for the optimal values were calculated using the simplifiedvariance formula.41

■ RESULTSEthylene Production Rate Dependence on Light

Wavelength and Intensity. Experiments were conductedto evaluate and quantify the effect of wavelength and intensityof the incident light on the ethylene production rates of thegenetically engineered strain of algae Synechocystis sp. PCC6803 2x EFE in the SW-PBR. The tested wavelengths of theincident light were chosen to target the absorption peaks of thepigment chlorophyll, in the red and blue spectrum of visiblelight, with experiments conducted at red (630 nm), deep red(660 nm), and blue (470 nm) wavelengths. Experiments couldnot be conducted at the peak situated around violet (∼430 nm)because of the unavailability of a suitable illumination source atthat wavelength. Experiments were carried out at five intensitylevels with a culture of OD730 10. Experiments at blue (470 nm)wavelength were discontinued after ethylene production wasfound to be negligible (results not shown).Ethylene production rates measured for each of the

intensities at red and deep red are shown in Figure 2. Ethyleneproduction rates were dependent on the light intensity, with amaximum production at 69 μE m−2 s−1 for both red and deepred illumination. The concave nature of the curve (p value forregression coefficient associated with quadratic term in intensitywas below 0.001; see the SI) suggests that at lower intensities,the algae were photolimited because of the dominant self-shading effects, whereas at high light intensities the ethyleneproduction decreased as a result of photoinhibition. In the

literature, microalgal flux tolerance has been measured to be∼200−400 μE m−2 s−1 for the full solar spectrum,22 whichagrees well with the intensity values found here for photo-inhibition at red wavelengths (which are used 5 times moreefficiently than the full solar spectrum34). However, there wasno statistically significant difference between the red and thedeep red illumination sources, indicating that the algae werecapable of utilizing either of the wavelengths equally efficiently(p value of 0.92 for the regression coefficient associated withwavelength, which was subsequently removed from the model).

Ethylene Production Rate Dependence on CultureDensity (OD730). We hypothesized that the short light path (1mm) in the SW-PBR should be capable of supportingconsiderably higher densities of algae culture, thus furtherincreasing the volumetric production rates. Experiments weretherefore conducted with higher density algal cultures at OD73020 and OD730 30. Because the earlier experiments indicated nosignificant difference between red and deep red illuminationsources, experiments with high culture densities were carriedout only with the red (630 nm) light source. Three levels oflight intensity were tested to determine the optimal operationpoint at high culture densities (Figure 3).The maximal ethylene production rate for the OD730 20 was

nearly 30% higher, 937 μg L−1 h−1 as compared with 715 μgL−1 h−1 observed for the OD730 10 culture, demonstrating thatthe SW-PBR was capable of supporting high culture densities.The maximal production rate for OD730 20 was found at anintensity of 86 μE m−2 s−1. This is higher than that observed forOD730 10 (69 μE m−2 s−1), which is indicative of a greatershading effects in the denser culture.A similar functional dependence on light intensity was

observed for OD730 30, although the production rates werelower than those for OD730 20, but greater than those for OD73010 (p value < 0.001) (Figure 3). Moreover, at OD730 30, theculture density decreased (data not shown) over the course ofthe experiment, suggesting that light attentuation due toabsorption by the algae was too substantial to penetrate eventhrough the small 1 mm light path. This indicates that themaximum carrying capacity of the SW-PBR, with a 2 mm

Figure 2. Volumetric ethylene production rates in the SW-PBRachieved by Synechocystis sp. PCC 6803 2x EFE algal strain (OD730 of10). The figure shows results obtained at two different wavelengths(630 and 660 nm) and five light intensity levels (35−104 μE m−2 s−1).The flags indicate standard errors.

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spacing between the stacks, lies between an OD730 20 andOD730 30 (equivalent to cell dry weight (CDW) of 9−14 gL−1).Photosynthetic Efficiency. In addition to volumetric

productivity, another important consideration is the PEexhibited by the algae inside the SW-PBR. PE is defined asthe ratio of the amount of energy stored by the algae to theamount of photon energy absorbed. PE can be defined in termsof (1) total biomass produced or (2) product, that is, ethylene,secreted. In both cases, as a conservative assumption, the entirelight incident on the algae (i.e., the light emanating from thesurface of an individual waveguide) was assumed to beabsorbed completely. The PEs for the above-describedexperiments were calculated in terms of lower heating valueof the ethylene produced. There was no significant differenceobserved in the PEs between red and deep red illuminationsources at all five intensities of the incident light (p value of0.57 for the regression coefficient associated with wavelength,which was subsequently removed from the model) (Figure 4).Although the production rates peaked at intermediateintensities at both wavelengths, the PE monotonicallydecreased with increasing intensity (p value < 0.001). Thisimplies that even though a rise in number of incident photonsled to more photons being utilized by the algae, thus improvingvolumetric production rates, they were overall utilized lessefficiently. A similar monotonically decreasing trend with anincrease in light intensity was also observed for higher ODcultures (Figure 5).Further, PE was found to be dependent on the culture

density, with the regression model indicating that PE is concavein OD (the p value for the regression coefficient associated withquadratic term in OD was below 0.01). The initial increase inPE can be attributed to a larger portion of the providedphotons being utilized by the greater number of algae in adenser culture. However, on further increase in the culturedensity, the light distribution within a stack becomessuboptimal with the algae near the waveguide incapable ofutilizing all provided photons and the algae in the interior ofthe stack being limited because of strong self-shading.

In general, the photosynthetic efficiencies associated withethylene production were low, with maximum values around0.125% (Figure 4). The low efficiencies are primarily due to thecurrent state of genetic modification of the organism, whichutilizes the majority of the fixed carbon for biomass productionwhile only a limited portion is channeled toward ethylenesynthesis.30 Consequently, only a small fraction of the energy isstored in ethylene molecules. With this consideration, we alsocalculated PE of biomass production under maximal ethyleneproduction conditions (i.e., for OD730 20 at 630 nm and 86 μEm−2 s−1). The biomass accumulation rate was ∼0.2 g L−1 h−1

(corresponding to an OD730 change of 1.39 units over 2.5 h),nearly 200 times more than the ethylene production rate,leading to a PE of light to biomass conversion of ∼15% (heat ofcombustion for Synechocystis sp. PCC 6803 is 0.026 J μg−1).

Figure 3. Volumetric ethylene production rates in the SW-PBRachieved by Synechocystis sp. PCC 6803 2x EFE algal strain at 630 nm.The figure shows results obtained at three different culture densities(OD730 of 10, 20, and 30) and five light intensity levels (35−104 μEm−2 s−1). The flags indicate standard errors.

Figure 4. Conversion efficiency from light incident on the algal cultureto energy contained in ethylene secreted by Synechocystis sp. PCC6803 2x EFE (OD730 10). The figure shows results obtained at twodifferent wavelengths (630 and 660 nm) and five light intensity levels(35−104 μE m−2 s−1). The flags indicate standard errors.

Figure 5. Conversion efficiency from light incident on the algal cultureto energy contained in ethylene secreted by Synechocystis sp. PCC6803 2x EFE at 630 nm. The figure shows results obtained at threeculture densities (OD730 of 10, 20, and 30) and five light intensitylevels (35−104 μE m−2 s−1). The flags indicate standard errors.

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This value is 2- to 3-fold higher than the PE published forconventional closed PBR.42 The improvement can be mainlyattributed to the use of red light, which minimizes energy lossesin photosystems I and II.

■ DISCUSSIONOptimization of Short-Light-Path Stacked PBR Vary-

ing Wavelength and Intensity. The results described aboveallow performance optimization of the SW-PBR. Twoillumination variables, namely, wavelength and intensity ofthe supplied light, were investigated. The experiments showedthat incident light of both red and deep red wavelengths isreadily utilized by Synechocystis sp. PCC 6803 2x EFE forgrowth and ethylene production. The obtained results areconsistent with earlier studies showing algal growth under redlight.1,31,33,34 From an overall efficiency point of view, the use ofred light is preferable because of lower energy dissipation perphoton captured in photosystem I or II. It is estimated thatphotosynthesis driven by red light is 5 times more energy-efficient than under broad-spectrum solar light.34 Because nosignificant differences were found in growth and ethyleneproduction under red and deep red light, the latter should bepreferred to improve photosynthetic efficiencies, albeit theachievable difference is only ∼5%.Light energy required per unit volume increases with the

density of the culture. In conventional PBRs, this is achieved bysupplying high light intensities at the reactor surface, withphotoinhibition as an unwanted side effect. Because of fast lightattenuation along the light path, intensities in the interior of thereactor rapidly become too low to support growth (i.e., result inphotolimitation13,20−23). A high surface-to-volume ratio is,therefore, desirable to uniformly distribute light across theculture volume. This is realized in the SW-PBR by using thestacked waveguide architecture combined with thin light paths.The highest production rate of 937 μg L−1 h−1 was attained atOD730 of 20 and an intensity of 86 μE m−2 s−1. This is a nearlytwo times improvement over the production rates of 510 μgL−1 h−1 reported earlier28 and a 4-fold improvement over themaximal production rate of 244 μg L−1 h−1 achieved in aconventional flat-plate PBR (3 cm light path; illuminated fromone side at 200 μE m−2 s−1).28

Even though the volumetric production rate was concave innature, as confirmed by the regression model, the productionlevels around the maximal production rate varied little withintensity. This was further borne out in the regression model bythe low coefficient values of the quadratic term in intensity,which is indicative of small differentials around the optimum(see the SI). Therefore, economic considerations could dictateoperation at a lower intensity to achieve higher PE because thelatter decreases with increasing intensity.22 This would reducethe area required for solar collectors, lowering the capitalinvestment costs.Demonstration of High Culture Density and Areal

Yields. We demonstrated that the carrying capacity of thecurrent SW-PBR lies between OD730 of 20 and 30,corresponding to a CDW of 9−14 g L−1. In addition, becauseof the stackable design, high areal yields between 115 and 173 gm−2 have been achieved. This represents a 7- to 10-foldimprovement in biomass density and a 4-fold improvement inareal density compared with the control conventional flat-platePBR.28 High volumetric productivities are advantageous both ifalgae are used as a biocatalyst secreting biofuel and if usedbiofuel precursors (as supported in the SW-PBR) or directly as

high-energy biomass. The downstream processing of thebiomass requires energy intensive dewatering (up to 1 kWhm−343), for which the cost per unit product can be reduced byharvesting dense cultures. Thus, production of high densitycultures, such as achievable in SW-PBR, will be keydeterminants for economic viability.2

Integrated System Design. The current SW-PBR is anintermediate step in the development of a full-growntechnology. Here, we have first optimized the illuminationparameters. A second bottleneck in PBR development isappropriate gas exchange. Sufficient CO2 needs to be providedas a carbon source for cell growth, and O2 must be removed toavoid toxic concentrations.6,44 High gas exchange rates are ofparticular importance in high-density cultures because of thehigh volumetric reaction rates catalyzed by these cultures.Integration of SW-PBR with membrane-based gas transfer, suchas that achieved through hollow fibers,6,44−47 is a promising wayto improve productivity by alleviating inhibitions possiblycaused by oxygen toxicity and carbon depletion. Alleviatingcarbon limitation by providing a continuous CO2 supply wouldarguably further increase the ethylene production rates,especially at higher light intensities and higher cell densities.Hollow fibers would further increase yields by preventingculture displacement due to gas production. In addition, thepresent reactor design supports secreted gas products, furthernecessitating adequate gas exchange to enable extraction of saidproducts. The ability to extract ethylene through hollow fibershas already been successfully tested in preliminary experiments(results not shown).In conventional systems, the most mixing energy is required

to circulate cells across the light gradient and guaranteeadequate gas transfer. The SW-PBR as described here (i.e.,using a combination of slab waveguides and short light path foroptimal light delivery, and integrated with hollow fibers for gasexchange) could reduce the amount of mixing required.Because mixing contributes 13−52% of the total constructionand operation costs of conventional closed PBR,27 a decrease inthe mixing requirements is key to making large-scaleproduction of biofuels cost-effective.7 Further research anddevelopment of scaled-up SW-PBR systems would be necessaryto quantify the energy and cost savings attainable.Similar to using visible light for operation, the ultraviolet

regime of the light spectrum can be used for disinfection of theSW-PBR when required. The efficacy of such treatment viaetched slab waveguides has also been recently demonstrated.48

Further improvements in efficiency can be obtained by usingflashing light of frequencies >1 Hz. This approach has beenproven to increase photosynthetic rates and energy efficien-cies.49,50

The presented work forms a first step in the optimization ofthe earlier developed stacked photobioreactor with internalillumination through slab waveguides. After optimizing onlyillumination, we observed 4-fold ethylene production rates andup to 10-fold higher biomass densities compared with aconventional flat-plate PBR. We suggested further improve-ments, including incorporation of hollow fibers for efficient gastransfer and integration of the stacked PBR with solar collectorsand spectral splitting technology to achieve integrated, holisticsystems for future algal biofuel production.

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■ ASSOCIATED CONTENT*S Supporting InformationResults of the regression analysis for ethylene production ratesand PE. The Supporting Information is available free of chargeon the ACS Publications website at DOI: 10.1021/es5052777.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 607-255-4861. E-mail: de54@cornell.edu.Author Contributions⊥A.J. and N.V. contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the Advanced Research ProjectAgency, Energy (DE-AR0000312). This work was performed inpart at the Cornell NanoScale Facility, a member of theNational Nanotechnology Infrastructure Network, which issupported by the National Science Foundation (Grant ECCS-0335765). We thank Jianping Yu, Ph.D. (NREL, Golden, CO)for providing the Synechocystis sp. PCC 6803 2x EFE algalstrain. We thank Rose Harmon for assistance with gas analyses.

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