Optimal intensity and biomass density for biofuel production in a thin-light-path photobioreactor.

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Optimal Intensity and Biomass Density for Biofuel Production in a Thin Light-path Photobioreactor Aadhar Jain, Nina Voulis, Erica E Jung, Devin F. R. Doud, William B Miller, Largus T. Angenent, and David Erickson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5052777 • Publication Date (Web): 24 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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Environmental Science & Technology

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Optimal Intensity and Biomass Density for

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Biofuel Production in a Thin Light-path

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Photobioreactor

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Aadhar Jain1ǂ, Nina Voulis2ǂ, Erica E. Jung1, Devin F.R. Doud2, William B. Miller3, Largus T.

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Angenent2, and David Erickson1*

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1 - Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY,

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14853, USA

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2 - Biological and Environmental Engineering, Cornell University, Ithaca, NY, 14853, USA

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3 – Department of Horticulture, Cornell University, Ithaca, NY, 14853, USA

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*

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ǂ

To whom correspondence may be addressed: [email protected] ; Ph: 607-255-4861 Both authors contributed equally to this work.

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Production of competitive microalgal biofuels requires development of high volumetric

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productivity photobioreactors (PBRs) capable of supporting high density cultures.

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Maximal biomass density supported by the current PBRs is limited by non-uniform

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distribution of light due to self-shading effects. We recently developed a thin light-path

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stacked photobioreactor with integrated slab waveguides, which distributed light

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uniformly across the volume of the PBR. Here, we enhance the performance of the stacked

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waveguide photobioreactor (SW-PBR) by determining the optimal wavelength and

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intensity regime of the incident light. This enabled the SW-PBR to support high density 1 ACS Paragon Plus Environment


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cultures, achieving a carrying capacity of OD730 20. Using a genetically modified algal

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strain capable of secreting ethylene, we improved ethylene production rates to 937 µg L-1 h-

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1

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results demonstrate the advantages of the SW-PBR design and provide the optimal

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operational parameters to maximize volumetric production.

. This represents a fourfold improvement over a conventional flat plate PBR. These

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INTRODUCTION

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Driven by the need for sustainable alternatives for fossil fuels, energy research today is focused

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on a range of renewable resources, including biomass. Algae are of particular interest as a

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sustainable feedstock for transportation fuel due to their salient advantages over terrestrial crops,

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including higher area yields1–3, smaller water footprint, ability of certain strains to grow in

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brackish or saline water, and applicability of waste streams as a low-cost source of CO2, nitrogen

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and phosphorus4–10. Besides biofuels, algae cultivation is also used for production of food and

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feed supplements11,12, showing the versatility of algal products and further encouraging the

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development of high efficiency photobioreactors (PBRs).

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Current commercial algae cultivation relies on open ponds, primarily due to low capital costs6,7,13.

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This technology, however, has a number of disadvantages if used for large-scale production of

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bulk commodities such as biofuel13,14. These shortcomings include low biomass densities5–7,

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requirement of large surface areas, contamination4,14, incompatibility with cultivation under

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atmosphere with elevated CO2 levels5, and high water losses due to surface evaporation5.

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Production of high-volume commodities, therefore, requires growth systems to overcome these

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limitations through deployment of alternative technologies such as closed PBRs13,15,16. Although

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conventional closed PBRs (e.g., flat-plate and tubular reactors) enable algae cultivation in more

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controlled environments than open ponds, they still suffer from suboptimal light distribution2,11,17.

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Under standard operation regimes, algae close to the illuminated surface are photoinhibited,

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while algae in the interior of the reactor are photolimited13,18–23. These effects are mitigated by

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employing mixing strategies, which circulate the algae cells across the light gradient, and thus

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ensure an adequate average light exposure24. However, the energy requirements for mixing are

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high, amounting even in simple systems to 3 W m-2, close to the energy eventually harvested

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from algal biomass5 and contributing to an overall energy efficiency ratio of less than one25,26.

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Mixing also accounts for 13% to 52% of total construction and operation costs of conventional

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closed PBRs27.

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In an earlier publication28, we addressed the limitations posed by simultaneous photoinhibition

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and photolimitation in conventional closed PBRs. We embraced a holistic PBR design by: (1)

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delivering light through optical waveguides for optimal light distribution inside the reactor, (2)

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aiming for dense cultures facilitating high volumetric product yields, and (3) envisaging

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utilization of product-secreting algae. The latter would simplify post-processing steps and avoid

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costly and technically challenging harvesting and product extraction steps29, which can

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contribute up to 50% of the total product cost6.

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To achieve this, we have demonstrated a short light-path stacked photobioreactor (SW-PBR)

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with internal light distribution through optical slab waveguides. Incident light is coupled into the

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sides of each slab waveguide and is propagated along the length of the waveguide via total

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internal reflection. The surface of the optical waveguide is etched to create randomly distributed

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scratches, thus changing the incident light angle, thereby disrupting the propagating light-wave

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and releasing the light into the bioreactor. Combined with short light-paths (~1 mm), the

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waveguides enable the delivery of optimal light intensities throughout the entire volume of the

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SW-PBR, avoiding both photolimitation and photoinhibition as well as reducing the need for

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energy intensive and costly mixing. The resulting 3D architecture both reduces the amount of

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land area required and supports high density cultures, leading to a high areal productivity. For

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the SW-PBR we showed an eightfold higher biomass accumulation than achieved in a control

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PBR without optical waveguides28.

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Aiming to avoid costly cell-harvesting steps, we used a product-secreting Synechocystis sp. PCC

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6803 2x EFE. This algae strain had been genetically modified to secrete the biofuel precursor

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ethylene30. In an earlier work28, we had demonstrated consistent production of ethylene for over

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45 days using the SW-PBR. We had achieved a two-fold increase in volumetric ethylene

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production rates as compared to a flat-plate PBR. The earlier achieved results validate the design

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and performance of the SW-PBR, indicating its promise as a viable photobioreactor technology.

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In the present work we explored methods to further improve yields and energy efficiencies. We

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optimized production over wavelength and intensity of the supplied light.

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Photoautotrophic algae preferentially use certain wavelengths in the photosynthetic active

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radiation (PAR) range, depending on the collection of light harvesting pigments present in their

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photosynthetic machinery22. Chlorophyll constitutes the most important group of these pigments

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and absorbs in the blue (450-475 nm) and the red spectral range (630-675 nm)17,22. Excitation of

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chlorophyll molecules in the reaction centers of photosystem I and II requires energy equivalent

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to that contained in photons with a wavelength of 700 and 680 nm, respectively22. Algae should,

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therefore, be supplied with light in the red spectral range to minimize energy dissipation. The

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efficacy of algal production under red light has been previously shown by multiple studies1,31–34.

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Besides wavelength, intensity of the supplied light is another important parameter determining

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algal growth. The optimum intensity for many algal species lies in the range of 100 to 400 µE m-

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available to carry out photosynthesis at the rate required for optimal cell growth. Photoinhibition

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on the other hand, leads to cell damage induced by high light intensities, adversely affecting

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growth22. While algal growth thus follows a concave function with respect to intensity,

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photosynthetic efficiency (PE) decreases with increasing intensities as photosynthetic centers

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become more saturated22,34,35. Therefore, design of algal production systems needs to account for

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both the optimal growth window and sufficient PE1,34,36–39.

s-1

5,12,22

. Photolimitation occurs at lower light intensities, when insufficient photons are

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Here, we first present the effects of variation of wavelength and intensity of the supplied light on

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ethylene production rates of Synechocystis sp. PCC 6803 2xEFE. Second, we describe the effect

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of high density culture on ethylene production rates to determine the carrying capacity of the 5 ACS Paragon Plus Environment


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SW-PBR. Third, using the collected data, we report the photosynthetic efficiencies achieved in

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the present system. The results provide us with optimal illumination conditions and biomass

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densities to maximize ethylene production in the SW-PBR.

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MATERIALS AND METHODS

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Stacked Waveguide Photobioreactor Design and Assembly

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The SW-PBR consisted of a 3D frame with slots to stack slab waveguides vertically above each

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other (Figure 1). The dimensions of the SW-PBR were 7.5 cm x 2.5 cm x 3 cm (length x width x

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height) with a 2-mm gap separating two waveguides, and a total liquid volume of 16 mL. The

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SW-PBR had two ports: (1) bottom influent port for inoculation, and (2) top effluent port for gas

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product removal (Figure 1(a)).

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The frame was 3D printed from a photocurable resin (VeroClear, Objet Geometries Inc.). It was

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coated by Parylene C via a vapor deposition process to reduce gas permeability of the SW-PBR.

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The waveguides were placed into dedicated slots in the frame and fixed to the frame by using

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Polydimethylsiloxane (PDMS) (Dow Chemicals, Midland, Michigan, USA) as a resin. This also

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ensured that any gaps (due to manufacturing errors in the 3D printing process) between the frame

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and the waveguides were sealed by the cured PDMS. Since PDMS is gas permeable, the

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assembled reactor was subjected to a second coat of Parylene C to ensure gas impermeability. To

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prevent uncoupled light from entering the SW-PBR, the gaps between the slab waveguides were


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covered by a photomask (printed from the same photocurable resin as the frame). Aluminium

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foil was attached to the photomask to improve reflectance characteristics.

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Waveguide Fabrication

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The SW-PBR waveguides were fabricated by affixing thin cover slips (VWR Micro Cover

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Glasses, Radnor, Pennsylvania, USA) to chemically etched borosilicate microscope slides (VWR

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VistaVisio Microscope Slides, Radnor, Pennsylvania, USA). Glass etching paste (Armour Etch,

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Hawthorne, New Jersey, USA) was applied on the glass slides for seven hours, and subsequently

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washed away, creating randomly distributed surface defects for improved light scattering from

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the waveguide surface. The thin coverslips were attached on top of each glass slide using

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uncured PDMS as a resin and curing the assembly for two hours at 80°C. Attaching a coverslip

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on top of the glass slide changed the medium (cladding) adjacent to the etched glass surface from

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water (refractive index = 1.33) to air (refractive index = 1) (Figure 1(c)). Due to the higher index

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contrast between the core and the cladding of the waveguide now, the critical angle was reduced,

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hence increasing the amount of light scattered from the waveguide28.

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Model Organism

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The model organism in all experiments was Synechocystis sp. PCC 6803 2x EFE. This

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genetically modified strain was selected due to its ability to secrete ethylene as a gaseous

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product30. Ethylene is both a biofuel precursor and an important feedstock chemical in the

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chemical industry. Prior to inoculation, algal cultures were grown in semi-batch in gastight 1 L

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reactors with 20 mL liquid phase at 30°C and continuous broad spectrum illumination (100 µE 7 ACS Paragon Plus Environment


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m-2 s-1). The culture was grown in fivefold concentrated BG-11 buffered by TES (4.6 g L-1) and

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augmented with 25 mg L-1 spectinomycin and 200 mg L-1 kanamycin. Carbon was provided both

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via the gaseous phase (5% CO2 atmosphere) and via the liquid medium (20 mM NaHCO3).

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These conditions allowed long term steady-state maintenance of a dense algal culture at an OD730

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of 60. Aliquots were taken from the semi-batch culture and, after dilution in growth medium to

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the required density, used as inoculum for the SW-PBR.

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Experimental Setup and Procedure

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The SW-PBR was inoculated at a starting OD730 of 10 (for experiments run at 470, 630, and 660

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nm), 20 (for experiments at 630 nm), and 30 (for experiments at 630 nm). The OD was

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determined using a spectrophotometer with a standard 1 cm cuvette at a wavelength of 730 nm,

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using an appropriate dilution factor. The bioreactors were illuminated from both sides via an

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LED bank (Figure 1(c)) of the specified wavelength (630 nm or 660 nm) and at five levels of

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intensity (35, 52, 69, 86, and 104 µE m-2 s-1). The incident light was coupled into the waveguides,

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propagated along the length of the waveguides, and subsequently emitted to the algal culture by

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the scattering surface of the waveguides. The dissolved bicarbonate in the growth medium served

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as the sole source of carbon in the SW-PBR. During the course of each experiment the bioreactor

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influent port was connected to a sterile bottle for collection of the displaced culture volume,

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while the effluent port on top of the side chimney was sealed by a septum to retain the secreted

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gaseous products in the SW-PBR.

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Upon illumination by the LED bank, the algal culture grew and secreted gaseous products

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(including ethylene) as a result of its photosynthetic activity. The formed gas bubbles displaced

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equal volume of culture through the effluent port into the sterile effluent bottle. The design of the

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side chimney ensured that the secreted gaseous products were separated from the liquid volume,

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and could, therefore, be easily extracted.

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At the end of each experimental run, the gaseous products were extracted from the SW-PBR and

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analyzed. Subsequently, the whole culture volume was removed from the bioreactor, centrifuged

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and subsequently resuspended in fresh medium. After two experimental runs in which the algae

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acclimatized to the new culture conditions, the procedure was repeated multiple times for each

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combination of density, incident light wavelength, and intensity. A new inoculum, which was

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taken from the semi-batch culture, was used to inoculate the SW-PBR for each new combination

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of experimental conditions.

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Gas Extraction and Analysis

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Gaseous products were extracted from the bioreactor upon completion of each experimental run.

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As the gas was removed through the septum at the effluent port, the liquid culture displaced

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earlier into the connected bottle was simultaneously pulled back into the bioreactor through the

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influent port. This prevented any dilution of the gaseous sample by the ambient air. The

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concentration of ethylene in the samples was analyzed using a gas chromatograph (GC). (See

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analysis procedure in earlier work28.)



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Measurement of Light Intensity

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Due to inaccessibility of the waveguides (except the top one) within the SW-PBR, it was not

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possible to measure the light emanating from each individual waveguide. However, our previous

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work28 had established that the light emitted by the waveguides within the SW-PBR was uniform

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across layers and contributed to the measurement obtained from the surface of the top waveguide.

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This could therefore be used to estimate the light intensity emanating from each waveguide by a

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single measurement from the surface of the top waveguide. From previous work28, it was found

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that the intensity at the top was given by:

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= a ∗

(1)

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Where I(n) is the light intensity at the top when n layers of the stack are exposed, a is the

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uniform light intensity emanating from each layer, and r is the fraction of light that is lost as it

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passes through one waveguide layer above. By fitting the measured data to the expression above

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the value of r was established experimentally to be 0.205. For this value of r, Equation (1) can be

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inverted to find the light intensity emanating from each waveguide as

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a = 0.227 ∗ 10

(2)

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where I(10) is the intensity measured from the surface of the top waveguide when all 10 layers

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are exposed. Thus, we measured the intensity from the surface of the top waveguide of an empty

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reactor and used equation (2) above to estimate the light intensity emanating from each

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individual waveguide.

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Statistical Data Analysis


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Statistical analysis on the collected data was performed using the software package R40. Separate

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general linear regression models were fit for ethylene production rates and PE of ethylene

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production. In both models, illumination wavelength, intensity, and culture OD were used as

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independent variables. The overall family-wise error rate (FWER) was controlled at 5% using R

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multivariate marginal models (mmm) package. The results of the regression analysis can be

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found in Supplementary Information (SI). The optimal values of OD and intensity for ethylene

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production were calculated assuming a multiplicative interaction term between OD and intensity.

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Standard errors (SE) for the optimal values were calculated using the simplified variance

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formula41.

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RESULTS

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Ethylene Production Rate Dependence on Light Wavelength and Intensity

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Experiments were conducted to evaluate and quantify the effect of wavelength and intensity of

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the incident light on the ethylene production rates of the genetically engineered strain of algae

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Synechocystis sp. PCC 6803 2x EFE in the SW-PBR. The tested wavelengths of the incident

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light were chosen to target the absorbtion peaks of the pigment chlorophyll - in red and blue

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spectrum of visible light – with experiments conducted at red (630 nm), deep red (660 nm), and

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blue (470 nm) wavelengths. Experiments could not be conducted at the peak situated around

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violet (~430 nm) due to the unavailability of a suitable illumination source at that wavelength.

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Experiments were carried out at five intensity levels with a culture of OD730 10. Experiments at

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blue (470 nm) wavelength were discontinued after ethylene production was found to be

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negligible (results not shown). 11 ACS Paragon Plus Environment


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Ethylene production rates measured for each of the intensities at red (630 nm) and deep red (660

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nm) are shown in Figure 2. Ethylene production rates were dependent on light intensity, with

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maximum production at 69 µE m-2 s-1 for both red and deep red illumination. The concave nature

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of the curve (p-value for regression coefficient associated with quadratic term in intensity was

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below 0.001, see SI) suggests that at lower intensities the algae were photolimited due to the

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dominant self-shading effects, while at high light intensities the ethylene production decreased

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due to photoinhibition. In literature, microalgal flux tolerance has been measured to be

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approximately 200-400 µE m-2 s-1 for full solar spectrum22, which agrees well with the intensity

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values found here for photoinhibition at red wavelengths (which are used 5 times more

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efficiently than full solar spectrum34). However, there was no statistically significant difference

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between the red and the deep red illumination sources indicating that the algae were capable of

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utilizing either of the wavelengths equally efficiently (p-value of 0.92 for the regression

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coefficient associated with wavelength, which was subsequently removed from the model).

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Ethylene Production Rate Dependence on Culture Density (OD730)

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We hypothesized that the short light-path (1 mm) in the SW-PBR should be capable of

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supporting considerably higher densities of algae culture, thus further increasing the volumetric

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production rates. Experiments were, therefore, conducted with higher density algal cultures at

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OD730 20 and OD730 30. Since the earlier experiments indicated no significant difference between

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red and deep red illumination sources, experiments with high culture densities were only carried


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out with the red (630 nm) light source. Three levels of light intensity were tested to determine the

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optimal operation point at high culture densities (Figure 3).

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The maximal ethylene production rate for the OD730 20 was nearly 30% higher - 937 µg L-1 h-1

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as compared to 715 µg L-1 h-1 observed for OD730 10 culture, demonstrating that the SW-PBR

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was capable of supporting high culture densities. The maximal production rate for OD730 20 was

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found at an intensity of 86 µE m-2 s-1. This is higher than that observed for OD730 10 (69 µE m-2

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s-1), which is indicative of a greater shading effects in the denser culture.

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A similar functional dependence on light intensity was observed for OD730 30, although the

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production rates were lower than those for OD730 20, but greater than those for OD730 10 (p-value

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< 0.001) (Figure 3). Moreover, at OD730 30 the culture density decreased (data not shown) over

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the course of the experiment, suggesting that light attentuation due to absorbtion by the algae

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was too substantial to penetrate even through the small 1-mm light-path. This indicates that the

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maximum carrying capacity of the SW-PBR, with a 2-mm spacing between the stacks, lies

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between an OD730 20 and OD730 30 (equivalent to cell dry weight (CDW) of 9 - 14 g L-1).

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Photosynthetic Efficiency

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Besides volumetric productivity, another important consideration is the PE exhibited by the algae

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inside the SW-PBR. PE is defined as the ratio of the amount of energy stored by the algae to the

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amount of photon energy absorbed. PE can be defined in terms of: (1) total biomass produced, or 13 ACS Paragon Plus Environment


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(2) product, i.e., ethylene, secreted. In both cases, as a conservative assumption, the entire light

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incident on the algae (i.e., the light emanating from the surface of an individual waveguide) was

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assumed to be absorbed completely. The PEs for the above described experiments were

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calculated in terms of lower heating value of the ethylene produced. There was no significant

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difference observed in the PEs between red (630nm) and deep red (660 nm) illumination sources

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at all five intensities of the incident light (p-value of 0.57 for the regression coefficient associated

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with wavelength, which was subsequently removed from the model) (Figure 4). Although the

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production rates peaked at intermediate intensities at both wavelengths, PE monotonically

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decreased with increasing intensity (p-value < 0.001). This implies that even though a rise in

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number of incident photons led to more photons being utilized by the algae – thus improving

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volumetric production rates – they were overall utilized less efficiently. A similar monotonically

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decreasing trend with an increase in light intensity was also observed for higher OD cultures

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(Figure 5).

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Further, PE was found to be dependent on culture density, with the regression model indicating

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that PE is concave in OD (p-value for regression coefficient associated with quadratic term in

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OD was below 0.01). The initial increase in PE can be attributed to a larger portion of the

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provided photons being utilized by the greater number of algae in a denser culture. However, on

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further increase of culture density the light distribution within a stack becomes suboptimal with

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the algae near the waveguide incapable of utilizing all provided photons and the algae in the

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interior of the stack being limited due to strong self-shading.


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In general, the photosynthetic efficiencies associated with ethylene production were low, with

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maximum values around 0.125% (Figure 4). The low efficiencies are primarily due to the

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current state of genetic modification of the organism, which utilizes the majority of the fixed

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carbon for biomass production while only a limited portion is channeled towards ethylene

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synthesis30. Consequently only a small fraction of the energy is stored in ethylene molecules.

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With this consideration, we also calculated PE of biomass production under maximal ethylene

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production conditions (i.e., for OD730 20 at 630 nm and 86 µE m-2 s-1). The biomass

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accumulation rate was ~0.2 g L-1 h-1 (corresponding to an OD730 change of 1.39 units over 2.5

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hours), nearly 200 times more than ethylene production rate, leading to a PE of light to biomass

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conversion of ~15% (heat of combustion for Synechocystis sp. PCC 6803 is 0.026 J µg-1). This

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value is two to threefold higher than the PE published for conventional closed PBR42. The

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improvement can be mainly attributed to the use of red light, which minimizes energy losses in

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photosystems I and II.

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DISCUSSION

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Optimization of Short Light-path stacked PBR Varying Wavelength and Intensity

317 318

The results described above allow performance optimization of the SW-PBR. Two illumination

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variables, namely wavelength and intensity of the supplied light, were investigated. The

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experiments showed that incident light of both red (630 nm) and deep red (660 nm) wavelengths

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is readily utilized by Synechocystis sp. PCC 6803 2x EFE for growth and ethylene production.

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The obtained results are consistent with earlier studies showing algal growth under red

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light1,31,33,34. From an overall efficiency point of view, the use of red light is preferable due to



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lower energy dissipation per photon captured in photosystem I or II. It is estimated that

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photosynthesis driven by red light is five times more energy efficient than under broad-spectrum

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solar light34. As no significant differences were found in growth and ethylene production under

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red and deep red light, the latter should be preferred to improve photosynthetic efficiencies,

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albeit the achievable difference is only about 5%.

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Light energy required per unit volume increases with the density of the culture. In conventional

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PBRs this is achieved by supplying high light intensities at the reactor surface, with

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photoinhibition as an unwanted side effect. Due to fast light attenuation along the light path,

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intensities in the interior of the reactor rapidly become too low to support growth (i.e., result in

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photolimitation13,20–23). A high surface-to-volume ratio is, therefore, desirable to uniformly

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distribute light across the culture volume. This is realized in the SW-PBR by using the stacked

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waveguide architecture combined with thin light-paths. The highest production rate of 937 µg L-1

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h-1 was attained at OD730 of 20 and an intensity of 86 µE m-2 s-1. This is a nearly two times

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improvement over the production rates of 510 µg L-1 h-1 reported earlier28, and a fourfold

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improvement over the maximal production rate of 244 µg L-1 h-1 achieved in a conventional flat-

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plate PBR (3 cm light-path; illuminated from one side at 200 µE m-2 s-1)28.

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Even though the volumetric production rate was concave in nature, as confirmed by the

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regression model, the production levels around the maximal production rate varied little with

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intensity. This was further borne out in the regression model by the low coefficient values of the

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quadratic term in intensity, which is indicative of small differentials around the optimum (See 16 ACS Paragon Plus Environment

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SI). Therefore, economic considerations could dictate operation at a lower intensity to achieve

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higher PE, since the latter decreases with increasing intensity22. This would reduce the area

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required for solar collectors, lowering the capital investment costs.

349 350

Demonstration of High Culture Density and Areal Yields

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We demonstrated that the carrying capacity of the current SW-PBR lies between OD730 of 20 and

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30, corresponding to CDW of 9 to 14 g L-1. In addition, due to the stackable design, high areal

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yields between 115 and 173 g m-2 have been achieved. This represents a seven to tenfold

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improvement in biomass density and a fourfold improvement in areal density compared to the

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control conventional flat-plate PBR28. High volumetric productivities are advantageous both if

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algae are used as a biocatalyst secreting biofuel or biofuel precursors (as supported in the SW-

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PBR) or directly as high-energy biomass. The downstream processing of the biomass requires

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energy intensive dewatering (up to 1 kWh m-3

359

reduced by harvesting dense cultures. Thus, production of high density cultures, such as

360

achievable in SW-PBR, will be key determinants for economic viability2.

43

), for which the cost per unit product can be

361 362

Integrated System Design

363

The current SW-PBR is an intermediate step in the development of a full-grown technology.

364

Here, we have first optimized the illumination parameters. A second bottleneck in PBR

365

development is appropriate gas exchange. Sufficient CO2 needs to be provided as a carbon

366

source for cell growth while O2 must be removed to avoid toxic concentrations6,44. High gas

367

exchange rates are of particular importance in high density cultures due to the high volumetric

368

reaction rates catalyzed by these cultures. Integration of SW-PBR with membrane-based gas 17 ACS Paragon Plus Environment


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369

transfer, such as achieved through hollow fibers6,44–47, is a promising way to improve

370

productivity by alleviating inhibitions possibly caused by oxygen toxicity and carbon depletion.

371

Alleviating carbon limitation by providing a continuous CO2 supply would arguably further

372

increase the ethylene production rates, especially at higher light intensities and higher cell

373

densities. Hollow fibers would further increase yields by preventing culture displacement due to

374

gas production. In addition, the present reactor design supports secreted gas products, further

375

necessitating adequate gas exchange to enable extraction of said products. The ability to extract

376

ethylene through hollow fibers has already been successfully tested in preliminary experiments

377

(results not shown).

378 379

In conventional systems most mixing energy is required to circulate cells across the light

380

gradient and guarantee adequate gas transfer. The SW-PBR as described here (i.e., using a

381

combination of slab waveguides and short light-path for optimal light delivery, and integrated

382

with hollow fibers for gas exchange) could reduce the amount of mixing required. As mixing

383

contributes 13 to 52% of total construction and operation costs of conventional closed PBR27,

384

decrease in mixing requirements is key to make large-scale production of biofuels cost-effective7.

385

Further research and development of scaled-up SW-PBR systems would be necessary to quantify

386

the energy and cost savings attainable.

387 388

Similar to using visible light for operation, the ultraviolet regime of the light spectrum can be

389

used for disinfection of the SW-PBR when required. The efficacy of such treatment via etched

390

slab waveguides has also been recently demonstrated48. Further improvements in efficiency can 18 ACS Paragon Plus Environment

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391

be obtained by using flashing light of frequencies greater than 1 Hz. This approach has been

392

proven to increase photosynthetic rates and energy efficiencies49,50.

393

The presented work forms a first step in the optimization of the earlier developed stacked

394

photobioreactor with internal illumination through slab waveguides. After optimizing only

395

illumination, we observed fourfold ethylene production rates and up to tenfold higher biomass

396

densities compared to a conventional flat-plate PBR. We suggested further improvements,

397

including incorporation of hollow fibers for efficient gas transfer, and integration of the stacked

398

PBR with solar collectors and spectral splitting technology to achieve integrated, holistic systems

399

for future algal biofuel production.

400 401

ACKNOWLEDGEMENTS

402

This work was supported by the Advanced Research Project Agency – Energy (DE-

403

AR0000312). This work was performed in part at the Cornell NanoScale Facility, a

404

member of the National Nanotechnology Infrastructure Network, which is supported by

405

the National Science Foundation (Grant ECCS-0335765). We thank Jianping Yu, PhD

406

(NREL, Golden, CO) for providing the Synechocystis sp. PCC 6803 2x EFE algal strain.

407

We thank Rose Harmon for assistance with gas analyses.

408 409

Supporting Information Available

410

Supporting Information (SI) provides the results of the regression analysis for ethylene

411

production rates and PE. This information is available free of charge via the Internet at

412

http://pubs.acs.org. 19 ACS Paragon Plus Environment


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REFERENCES:

415 416 417

(1)

Zhao, Y.; Wang, J.; Zhang, H.; Yan, C.; Zhang, Y. Effects of various LED light wavelengths and intensities on microalgae-based simultaneous biogas upgrading and digestate nutrient reduction process. Bioresour. Technol. 2013, 136, 461–468.

418

(2)

Yusuf, C. Biodiesel from microalgae. Biotechnol. Adv. 2007, 25, 294–306.

419 420

(3)

Chen, C.-Y.; Yeh, K.-L.; Aisyah, R.; Lee, D.-J.; Chang, J.-S. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour. Technol. 2011, 102, 71–81.

421 422 423

(4)

Rodolfi, L.; Chini Zittelli, G.; Bassi, N.; Padovani, G.; Biondi, N.; Bonini, G.; Tredici, M. R. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 2009, 102, 100–112.

424 425

(5)

Posten, C.; Schaub, G. Microalgae and terrestrial biomass as source for fuels--a process view. J. Biotechnol. 2009, 142, 64–69.

426 427

(6)

Greenwell, H. C.; Laurens, L. M. L.; Shields, R. J.; Lovitt, R. W.; Flynn, K. J. Placing microalgae on the biofuels priority list: a review of the technological challenges. J. R. Soc. Interface 2010, 7, 703–726.

428

(7)

Wijffels, R. H.; Barbosa, M. J. An outlook on microalgal biofuels. Science 2010, 329, 796–799.

429 430

(8)

Yun, Y.-S.; Lee, S. B.; Park, J. M.; Lee, C.-I.; Yang, J.-W. Carbon Dioxide Fixation by Algal Cultivation Using Wastewater Nutrients. J. Chem. Technol. Biotechnol. 1997, 69, 451–455.

431 432 433

(9)

Pires, J. C. M.; Alvim-Ferraz, M. C. M.; Martins, F. G.; Simões, M. Carbon dioxide capture from flue gases using microalgae: Engineering aspects and biorefinery concept. Renew. Sustain. Energy Rev. 2012, 16, 3043–3053.

434 435

(10)

Mata, T. M.; Martins, A. a.; Caetano, N. S. Microalgae for biodiesel production and other applications: A review. Renew. Sustain. Energy Rev. 2010, 14, 217–232.

436 437

(11)

Janssen, M.; Tramper, J.; Mur, L. R.; Wijffels, R. H. Enclosed outdoor photobioreactors: light regime, photosynthetic efficiency, scale-up, and future prospects. Biotechnol. Bioeng. 2003, 81, 193–210.

438 439

(12)

Wijffels, R. H.; Barbosa, M. J.; Eppink, M. H. M. Microalgae for the production of bulk chemicals and biofuels. Biofuels Bioprod. Biorefining 2010, 4, 287–295.

440 441

(13)

Grobbelaar, J. U. Factors governing algal growth in photobioreactors: the “open” versus “closed” debate. J. Appl. Phycol. 2008, 21, 489–492.

442 443

(14)

Ogbonna, J. C.; Soejima, T.; Tanaka, H. An integrated solar and artificial light system for internal illumination of photobioreactors. J. Biotechnol. 1999, 70, 289–297.

444 445 446

(15)

Olivieri, G.; Salatino, P.; Marzocchella, A. Advances in photobioreactors for intensive microalgal production: configurations, operating strategies and applications. J. Chem. Technol. Biotechnol. 2014, 89, 178–195.

447 448

(16)

Soman, A.; Shastri, Y. Optimization of novel photobioreactor design using computational fluid dynamics. Appl. Energy 2015, 140, 246–255.



449 450

(17)

Grobbelaar, J. U. Microalgal biomass production: challenges and realities. Photosynth. Res. 2010, 106, 135– 144.

451 452

(18)

Brennan, L.; Owende, P. Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products. Renewable and Sustainable Energy Reviews, 2010, 14, 557–577.

453 454

(19)

Kunjapur, A. M.; Eldridge, R. B. Photobioreactor Design for Commercial Biofuel Production from Microalgae. Ind. Eng. Chem. Res. 2010, 49, 3516–3526.

455 456

(20)

Long, S. P.; Humphries, S. Photoinhibition of Photosynthesis in Nature. Annu. Rev. Plant Mol. Biol. 1994, 45, 633–662.

457 458 459

(21)

Grima, E. M.; Femtidez Sevilla, J. M.; Sánchez Pérez, J. A.; García Camacho, F. A study on simultaneous photolimitation and photoinhibition in dense microalgal cultures taking into account incident and averaged irradiances. J. Biotechnol. 1996, 45, 59–69.

460 461

(22)

Carvalho, A. P.; Silva, S. O.; Baptista, J. M.; Malcata, F. X. Light requirements in microalgal photobioreactors: an overview of biophotonic aspects. Appl. Microbiol. Biotechnol. 2011, 89, 1275–1288.

462 463 464

(23)

Richmond, A.; Cheng-Wu, Z.; Zarmi, Y. Efficient use of strong light for high photosynthetic productivity: interrelationships between the optical path, the optimal population density and cell-growth inhibition. Biomol. Eng. 2003, 20, 229–236.

465 466

(24)

Molina Grima, E.; Fernández, F. G. A.; Garcı́a Camacho, F.; Chisti, Y. Photobioreactors: light regime, mass transfer, and scaleup. J. Biotechnol. 1999, 70, 231–247.

467 468 469

(25)

Beal, C. M.; Stillwell, A. S.; King, C. W.; Cohen, S. M.; Berberoglu, H.; Bhattarai, R. P.; Connelly, R. L.; Webber, M. E.; Hebner, R. E. Energy Return on Investment for Algal Biofuel Production Coupled with Wastewater Treatment. Water Environ. Res. 2012, 84, 692–710.

470 471

(26)

Jegathese, S.; Farid, M. Microalgae as a Renewable Source of Energy: A Niche Opportunity. J. Renew. Energy 2014, Article ID 430203. doi:10.1155/2014/430203

472 473

(27)

Norsker, N.-H.; Barbosa, M. J.; Vermuë, M. H.; Wijffels, R. H. Microalgal production--a close look at the economics. Biotechnol. Adv. 2011, 29, 24–27.

474 475

(28)

Jung, E. E.; Jain, A.; Voulis, N.; Doud, D. D. R.; Angenent, L. T.; Erickson, D. Stacked optical waveguide photobioreactor for high density algal cultures. Bioresour. Technol. 2014, 171, 495–499.

476 477

(29)

Kleinegris, D. M. M.; Janssen, M.; Brandenburg, W. a; Wijffels, R. H. Two-phase systems: potential for in situ extraction of microalgal products. Biotechnol. Adv. 2011, 29, 502–507.

478 479

(30)

Ungerer, J.; Tao, L.; Davis, M.; Ghirardi, M.; Maness, P.-C.; Yu, J. Sustained photosynthetic conversion of CO2 to ethylene in recombinant cyanobacterium Synechocystis 6803. Energy Environ. Sci. 2012, 5, 8998.

480 481 482

(31)

Matthijs, H. C.; Balke, H.; van Hes, U. M.; Kroon, B. M.; Mur, L. R.; Binot, R. a. Application of lightemitting diodes in bioreactors: flashing light effects and energy economy in algal culture (Chlorella pyrenoidosa). Biotechnol. Bioeng. 1996, 50, 98–107.

483 484

(32)

Katsuda, T.; Lababpour, A.; Shimahara, K.; Katoh, S. Astaxanthin production by Haematococcus pluvialis under illumination with LEDs. Enzyme Microb. Technol. 2004, 35, 81–86.


Page 22 of 30

Page 23 of 30


485 486

(33)

Wang, C.-Y.; Fu, C.-C.; Liu, Y.-C. Effects of using light-emitting diodes on the cultivation of Spirulina platensis. Biochem. Eng. J. 2007, 37, 21–25.

487 488

(34)

Gordon, J. M.; Polle, J. E. W. Ultrahigh bioproductivity from algae. Appl. Microbiol. Biotechnol. 2007, 76, 969–975.

489 490

(35)

Fan, L.-H.; Zhang, Y.-T.; Zhang, L.; Chen, H.-L. Evaluation of a membrane-sparged helical tubular photobioreactor for carbon dioxide biofixation by Chlorella vulgaris. J. Memb. Sci. 2008, 325, 336–345.

491 492 493 494

(36)

Li, Y.; Zhou, W.; Hu, B.; Min, M.; Chen, P.; Ruan, R. R. Effect of light intensity on algal biomass accumulation and biodiesel production for mixotrophic strains Chlorella kessleri and Chlorella protothecoide cultivated in highly concentrated municipal wastewater. Biotechnol. Bioeng. 2012, 109, 2222– 2229.

495 496 497

(37)

Ruangsomboon, S. Effect of light, nutrient, cultivation time and salinity on lipid production of newly isolated strain of the green microalga, Botryococcus braunii KMITL 2. Bioresour. Technol. 2012, 109, 261– 265.

498 499 500

(38)

Cheirsilp, B.; Torpee, S. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour. Technol. 2012, 110, 510–516.

501 502

(39)

Sorokin, C.; Krauss, R. W. The Effects of Light Intensity on the Growth Rates of Green Algae. Plant Physiol. 1958, 33 , 109–113.

503

(40)

R Core Team. R: A language and environment for statistical computing, 2014.

504 505

(41)

Ku, H. H. Notes on the Use of Propagation of Error Formulas. J. Res. Natl. Bur. Stand. (1934). 1966, 70C, 263–273.

506 507

(42)

Tredici, M.; Zittelli, G. Efficiency of sunlight utilization: tubular versus flat photobioreactors. Biotechnol. Bioeng. 1998, 57, 187–197.

508 509

(43)

Molina Grima, E.; Belarbi, E.-H.; Acién Fernández, F. G.; Robles Medina, a; Chisti, Y. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 2003, 20, 491–515.

510 511

(44)

Carvalho, A. P.; Meireles, L. a; Malcata, F. X. Microalgal reactors: a review of enclosed system designs and performances. Biotechnol. Prog. 2006, 22, 1490–1506.

512 513 514

(45)

Kumar, A.; Yuan, X.; Sahu, A. K.; Dewulf, J.; Ergas, S. J.; Van Langenhove, H. A hollow fiber membrane photo-bioreactor for CO2 sequestration from combustion gas coupled with wastewater treatment: a process engineering approach. J. Chem. Technol. Biotechnol. 2010, 85, 387–394.

515 516 517

(46)

Ferreira, B. S.; Fernandes, H. L.; Reis, A.; Mateus, M. Microporous hollow fibres for carbon dioxide absorption: Mass transfer model fitting and the supplying of carbon dioxide to microalgal cultures. J. Chem. Technol. Biotechnol. 1998, 71, 61–70.

518 519

(47)

Drexler, I. L. C.; Yeh, D. H. Membrane applications for microalgae cultivation and harvesting: a review. Rev. Environ. Sci. Bio/Technology 2014, 13, 487–504.

520 521

(48)

Doud, D. F. R.; Jain, A.; Ahsan, S. S.; Erickson, D.; Angenent, L. T. In Situ UV Disinfection of a Waveguide-Based Photobioreactor. Environ. Sci. Technol. 2014, 48, 11521–11526.



522 523 524

(49)

Grobbelaar, J.; Nedbal, L.; Tichý, V. Influence of high frequency light/dark fluctuations on photosynthetic characteristics of microalgae photoacclimated to different light intensities and implications for mass algal cultivation. J. Appl. Phycol. 1996, 8, 335–343.

525 526

(50)

Grobbelaar, J. U. Upper limits of photosynthetic productivity and problems of scaling. J. Appl. Phycol. 2008, 21, 519–522.

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FIGURES

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Cover Slip

Etched Glass Slide

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 is coupled into the waveguides, which transport and evenly distribute the light inside the reactor. The photomask prevents uncoupled light from entering the reactor. The side chimney connects the layers and allows collection of the produced gas. The influent port is used to inoculate the reactor before operation and collect displaced liquid during operation. The produced gas is extracted through the effluent port. (b) Schematic of the SW-PBR with 10 waveguides. (c) Schematic of the waveguide structure. A thin cover slip is attached on top of an etched glass slide. 530 25 ACS Paragon Plus Environment


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Figure 2. Volumetric ethylene production rates in the SW-PBR achieved by Synechocystis sp.

PCC 6803 2x EFE algal strain (OD730 of 10). The figure shows results obtained at two different wavelengths (630 nm and 660 nm) and five light intensity levels (35 µE m-2 s-1 to 104 µE m-2 s1

). The flags indicate standard errors.

532 533


Page 27 of 30


534

Figure 3. Volumetric ethylene production rates in the SW-PBR achieved 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 µE m-2 s-1 to 104 µE m-2 s-1). The flags indicate standard errors. 535 536



Page 28 of 30

Figure 4. Conversion efficiency from light incident on the algal culture to energy contained in

ethylene secreted by Synechocystis sp. PCC 6803 2x EFE (OD730 10). The figure shows results obtained at two different wavelengths (630 nm and 660 nm) and five light intensity levels (35 µE m-2 s-1 to 104 µE m-2 s-1). The flags indicate standard errors. 537 538 539 540 541 542 543


Page 29 of 30


Figure 5. Conversion efficiency from light incident on the algal culture to energy contained in

ethylene secreted by Synechocystis sp. PCC 6803 2x EFE at 630 nm. The figure shows results obtained at three culture densities (OD730 of 10, 20 and 30) and five light intensity levels (35 µE m-2 s-1 to 104 µE m-2 s-1). The flags indicate standard errors. 544 545



546

ABSTRACT ART

547 548 549 550 551 552 553 554 555 556 557 558 559 560


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Process energy comparison for the production and harvesting of algal biomass as a biofuel feedstock.

A novel suspended-solid phase photobioreactor to improve biomass production and separation of microalgae.

Strategies for the production of cell wall-deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production.

Characterization of Nizimuddinia zanardini macroalgae biomass composition and its potential for biofuel production.

Growing duckweed for biofuel production: a review.

Comparative energetics and kinetics of autotrophic lipid and starch metabolism in chlorophytic microalgae: implications for biomass and biofuel production.

Modifying plants for biofuel and biomaterial production.

Stacked optical waveguide photobioreactor for high density algal cultures.

Fiber-optic differential absorption sensor for accurately monitoring biomass in a photobioreactor.

The influence of light intensity and photoperiod on duckweed biomass and starch accumulation for bioethanol production.

Isolation, identification and characterization of Paenibacillus polymyxa CR1 with potentials for biopesticide, biofertilization, biomass degradation and biofuel production.

Plant biotechnology for lignocellulosic biofuel production.

Evaluation of batch and semi-continuous culture of Porphyridium purpureum in a photobioreactor in high latitudes using Fourier Transform Infrared spectroscopy for monitoring biomass composition and metabolites production.

A fiber-optic sensor for accurately monitoring biofilm growth in a hydrogen production photobioreactor.

Integrated strategic and tactical biomass-biofuel supply chain optimization.

Selection of microalgae for biodiesel production in a scalable outdoor photobioreactor in north China.

Aeration and mass transfer optimization in a rectangular airlift loop photobioreactor for the production of microalgae.

Design and construction of a photobioreactor for hydrogen production, including status in the field.

Cultivation of Desmodesmus subspicatus in a tubular photobioreactor for bioremediation and microalgae oil production.

Cultivation of Chlorella protothecoides with urban wastewater in continuous photobioreactor: biomass productivity and nutrient removal.

Biomass and carotenoid production in photosynthetic bacteria wastewater treatment: effects of light intensity.

Tailoring lignin biosynthesis for efficient and sustainable biofuel production.

Potential of lipid metabolism in marine diatoms for biofuel production.

Growth kinetics of Chlorococcum humicola - A potential feedstock for biomass with biofuel properties.