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Novel waveguide reactor design for enhancing algal biofilm growth
Algal Research 12 (2015) 529–538
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Algal Research journal homepage: www.elsevier.com/locate/algal
Novel waveguide reactor design for enhancing algal biofilm growth Scott N. Genin a, J. Stewart Aitchison b, D. Grant Allen a,⁎ a b
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College St., Toronto, Ontario M5S 3E5, Canada The Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King's College Road, Toronto, Ontario M5S 3G4, Canada
a r t i c l e
i n f o
Article history: Received 21 May 2015 Received in revised form 11 September 2015 Accepted 21 October 2015 Available online xxxx Keywords: Algae biofilm Algae production Light waveguide Photobioreactor
a b s t r a c t Algal film photobioreactors have the potential to reduce dewatering costs and have a comparable productivity to raceway ponds. Current designs have focused on maximizing productivity and nutrient removal through material selection and design. However, fewer studies have focused on analyzing the effects of illumination and carbon dioxide (CO2) concentration on algal film growth. The transport of light in suspended photobioreactors is considered a serious problem due to cell shading, light scattering and non-uniform optical absorption. To enhance light delivery to algal biofilms, five different waveguide designs were developed Light emitting waveguides are capable of distributing a bright light source over a larger surface area. The light emission from these waveguide designs was characterized and the algal biofilm growth on waveguides was characterized in a parallel plate airlift reactor (17 L). The light intensity and CO2 concentration were varied in a 32 factorial design experiment to determine if there were any interaction effects. Light emission from the waveguides could be altered by changing the tapper angle or by notching the surfaces. Algal film growth kinetics on the waveguides were overall non-linear, but had linear regions of growth. The waveguides achieved an algal biofilm surface area productivity of 2.8 g/m2 day and an aerial productivity of 33.6 g/m2 day. Algal film productivity displayed saturation kinetics with respect to light intensity and CO2 concentration, but the interaction effect of light and CO2 on productivity is likely non-linear. Algal film biomass per photon consumed decreased with increasing light intensity when the reactor was not CO2 limited. The results indicate the potential for algal biofilms to be grown on light emitting waveguides which opens up the opportunity to explore new algal film photobioreactor configurations. © 2015 Published by Elsevier B.V.
1. Introduction Microalgae have been identified as a potential feedstock for both biofuels and biochemicals due to its high growth rate compared to terrestrial crops and high lipid content [1,2]. The production of biofuels and bioproducts from algae is possible, but commercialization is difficult [3] due to numerous challenges such as: insufficient supplies of low-cost concentrated CO2; high capital costs of photobioreactors [4], and low algal biomass concentration. Microalgae grown as a biofilm presents an opportunity to reduce dewatering costs since the biomass is more concentrated (90–150 g/L) [5,6,7] compared to suspended algae produced in raceway ponds (0.5–1 g/L) or photobioreactors (1–4 g/L) [1]. Algal biofilms are a mixed community of many different algae and bacteria species within a matrix of extracellular polymeric substances (EPS) [8,9]. EPS is a matrix of polysaccharides, proteins, glycoproteins, glycolipids, and extracellular DNA produced by the microorganisms that are imbedded in the biofilm [10]. Current research on algal film photobioreactor design focuses on rotating algal biofilm (RAB) systems [5,7,11] or on algal turf scrubbers ⁎ Corresponding author. E-mail addresses: [email protected] (S.N. Genin), [email protected] (J.S. Aitchison), [email protected] (D.G. Allen).
http://dx.doi.org/10.1016/j.algal.2015.10.013 2211-9264/© 2015 Published by Elsevier B.V.
[6,12,13,14]. These reactors are based on the principle of growing an algal biofilm on a rough attachment surface in which either the surface or the water is moving. These RAB systems show promise as their reported aerial biomass productivities are higher than those of raceway ponds [5,7]. The design considerations for these systems have been investigated from algal film productivity and nutrient removal standpoint, but have not investigated effects of light intensity and CO2 concentration on algal film growth. Recent studies by Schnurr et al. [15] and Ooms et al. [16] have provided new insight into new photobioreactor designs. In experiments by Schnurr et al. [15] algal biofilms were grown where light was incident through glass and the productivity was compared to algal biofilms which were grown in a conventional manner where light was incident from the water side. The results demonstrated that at the conditions tested, light direction did not impact algal biofilm growth. Ooms et al. [16] demonstrated the possibility of growing cyanobacteria in the evanescent field associated with an optical waveguide. While the biofilms only reached a thickness of 10–15 μm, the results showed by both these studies demonstrate the potential to grow algal biofilms on a light emitting surface. Growing an algal biofilm on a light emitting surface presents a series of potential advantages: when the attachment material and light source are integrated, the light is directly delivered to the algal biofilm without
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shading from suspended algae to produce a more compact bioreactor. However, the effects of CO2, illumination intensity and light direction on the growth kinetics of an algal biofilm on a light emitting surface have not been investigated. The objective of this study is to demonstrate the feasibility of growing algal biofilms on light emitting waveguides and determining the combined effects of varying light intensity and CO2 concentration. To complete this objective, five different acrylic waveguides and a parallel plate airlift (PPAL) reactor were designed and built. The parameters measured were biomass production, fatty acid methyl ester (FAME) content, total nitrogen, total phosphorous, temperature and pH. 2. Materials and methods 2.1. Waveguide design Five different waveguide designs were fabricated out of optically transparent cast acrylic with a top cross sectional area of 1.27 × 1.27 cm2 and a length of 25 cm (Fig. 1a–e). All sides of the waveguides were polished to remove imperfections in the surface after machining. Dimensions are provided in Table 1. The tapper and notches in the waveguides cause light to leak or scatter out of the waveguide. The taper in waveguide dimension along the direction of propagation prevents total internal reflection and as a result light leaks from the waveguide. The notches provide local scattering centers and cause light trapped in the waveguide to scatter into the bioreactor. Red Light Emitting Diodes (LED) (3.5 V and 700 mA, 635 ± 10 nm) provided by Pond Biofuels Inc. were mounted to the top of the waveguide. 2.2. Light intensity measurements The light emission from the waveguide was characterized by a photodiode (Thor Labs model #: FDS010) which was scanned along the direction of light propagation in the waveguide at a distance of 2.4 mm from the surface. The current and voltage provided to the LED by a Pyramid brand variable power supply (model #: PS-32lab) were measured using a volt and amp meter. The electrical input power to the LED was adjusted to control the light emission and was calibrated using a Newport Silicon photometer (model #: 818-SL) which was set to 635 nm, to confirm that over the electrical power range used in these experiments, the light emission was linearly correlated to the electrical input power. The photon flux from the LEDs at different input electrical power was measured using a Fieldscout Quantum
Light Meter (model #: 3415F) and a calibration curve of photon flux vs. electrical input power to the LED was generated. This calibration curve was used to estimate the photon flux emitted from the surfaces of the waveguide. 2.3. Reactor setup The PPAL reactor (Fig. 2) used in this study was designed to provide a constant shear force on each waveguide, adequate mixing, and nutrients. The reactor case was constructed of glass, with two vertical internal plates of cast acrylic which were secured by a silicone adhesive. The reactor was filled with 17 L of media and has the following dimensions: 41 × 20 × 25 cm3. Twenty-four waveguides were placed vertically in the down-comer of the PPAL for each run. The red LEDs were mounted to the top of each waveguide in four parallel circuits and power was supplied by a Pyramid brand variable power supply (model #: PS-32lab). 2.4. Reactor operation Past work by Irving and Allen [17] determined that the presence of wastewater is an important factor in enhancing the formation of an algae biofilm. To introduce EPS and bacteria, unsterile wastewater from Ashbridge's Bay Wastewater Treatment Facility, Toronto, ON, was blended with Fortified Bold's Basal Media (FBBM) [18] in a volumetric ratio of 1:2. FBBM was buffered to pH 6.8 and prepared to have the following concentration of nutrients: NaNO3 250 mg/L, CaCl2·2H2O 25 mg/L, MgSO4·7H2O 75 mg/L, K2HPO4 75 mg/L, KH2PO4 175 mg/L, NaCl 25 mg/L, Na 2EDTA 10 mg/L, FeSO 4 ·7H2 O 4.98 mg/L, H3BO 3 11.42 mg/L, Na 2 SiO3 ·7H2O 58 mg/L, ZnSO4 ·7H2 O 8.82 mg/L, MnCl2·4H2O 1.44 mg/L, Na2MoO3 0.70 mg/L, CuSO4·5H2O 1.57, and Co(NO3 ) 2·6H2 O 0.49 mg/L. The solution was sparged with air at 1 L/min in the reactor for 24 h before being inoculated. The inoculum contained seven algal species which were purchased from the Canadian Phycological Culture Centre (CPCC) or the Culture Collection of Algae and Protozoa (CCAP), which were selected because they were the same species previously used to inoculate the PPAL reactor in other experiments [19] and consistency between experiments was desired for a fair comparison between reactor setups. The species used in this experiment were: Scenedesmus obliquus (CPCC 157), Chlorella vulgaris (CPCC 147), Coccomyxa sp.(CPCC 508), Nannochloris sp. (CCAP 251/2), Nitschia palea (CPCC 160), Oocystis sp. (CPCC 9) and Oocystis polymorpha (CPCC 35) and were prepared by growing the individual cultures in flasks in a light incubator as described [19]. The
Fig. 1. Waveguide schematics for a) single wedge large notch, b) single wedge, c) double wedge, d) single wedge small notch, and e) double wedge small notch (side view).
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2.5. Sampling and analysis
Table 1 Waveguide specification. Angle of taper Number of Notch depth Notch spacing (degrees) notches (mm) (mm) Single wedge large notch Single wedge Double wedge Single wedge small notch Double wedge small notch
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1.88 1.88 1.11 1.88 1.11
6 0 0 167 334
5.0 0 0 0.25 0.25
7.5 0 0 0.2 0.2
reactor operated in batch mode for 48 h before the pumps were started to introduce fresh FBBM. The day the pumps were started was considered day zero, and the algal biomass on three waveguides was harvested from the reactor on days 0, 3, 5, 7, 10, 12, and 14. The cleaned waveguides were then placed back into the reactor. Suspended algae samples were also taken from the reactor in triplicate. Regrowth experiments were conducted by placing the waveguides back into the reactor after harvesting, then harvesting the front of all the waveguides on the final day (day 14). For the single wedge large notch waveguides, the reactor was operated under different light intensities and CO2 to compressed air ratios at a total flow rate of 1 L/min STP in a 32 factorial experimental design, where the light emission from the LED (1600 μmol/m2 s, 7200 μmol/m2 s, 12,600 μmol/m2 s) and CO2 partial pressures (atmospheric, 1%, 3%) were varied, with triplicate experiments conducted at the center point (1% CO2, 7200 μmol/m2 sec emitted from the LED) (Table 2). A dark control experiment, where the PPAL reactor was covered, was conducted to determine whether an algal biofilm could grow in the reactor conditions without the presence of light and at 1% CO2. For the single wedge, double wedge, single wedge small notch and double wedge small notch waveguides, the reactor was operated under the same light intensity (7200 μmol/m2 s) and CO 2 partial pressure (1%). A peristaltic pump (Cole Parmer Masterflex, model # 7520–35) was used to add and remove media from the reactor at a dilution rate of 0.90 day−1 to wash out the suspended algae.
The algal biomass on the waveguides was harvested and suspended in RO water. A vacuum filtration unit was used to filter the suspended algal mass through Supor®-450 47 mm filters with a pore size of 0.45 μm. The filters were baked at 103 °C for 4 h and weighed before and after filtration to measure the difference in dry mass. Algal film biomass harvested from the single wedge large notch waveguides on day 14 was freeze dried and the lipids were extracted using the Folch method [20] using 1:2 (v/v) chloroform to methanol as the solvent. The methanol phase and polar lipids solution were discarded to target the neutral lipids. The neutral lipids were then methylated using the FAME technique based on the Microbial Identification System, Microbial ID Inc. (MIDI Method) [21]. The samples were analyzed using gas chromatography (Perkin Elmer Clarus 680 GC) with a special performance capillary column (Hewlett Packard model # HP-5 MS, 30 m × 0.25 mm × 0.25 μm) and a flame ionization detector. Hexadecane (Sigma Aldrich #H6703) was used as the internal standard and olive oil was used as a calibration standard. The removal of nitrogen and phosphorous by the algal biofilms was evaluated at 1% CO2 partial pressure and 7200 μmol/m2 s. Nitrogen and phosphorous in the inlet and outlet streams were measured using Hach reagent kits for total nitrogen (TNT 827) and total phosphates (TNT 844). The total phosphorus concentration in the media was calculated based on the total phosphates. During the experiment, algal biofilms were harvested at the same intervals to determine growth. 2.6. Calculations Algal biofilm productivity is calculated by either using a single point measurement [5,6,7,14], a linear regression over the entire growth period [15,22] or through colonization time analysis [19]. In this study, productivity was calculated using the colonization time the method which removes bias that colonization can introduce into the productivity measurement using a linear regression on algal biofilm yield data which is above the confluence level as described in more detail by Genin et al. [19].
Fig. 2. Waveguide reactor schematic setup.
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Table 2 . Conditions for the factorial experimental design with varying light intensity and CO2 partial pressure.
ATM CO2 1% CO2 3% CO2
Photon flux emitted by LED: 1600 μmol/m2 s
Photon Flux emitted by LED: 7200 μmol/m2 s
Photon Flux emitted by LED: 12,600 μmol/m2 s
Single Single Single
Single Triplicate Single
Single Single Single
The percent of nitrogen and phosphorous removed was calculated using Eq. (1) below: Sin Sout 100 ¼ percent removal Sin
ð1Þ
where Sin and Sout are the concentration of the nutrients (nitrogen and phosphorous) in the inlet and outlet respectively. All productivity confidence intervals are the 95% confidence interval assuming a student-t distribution. The gram biomass per mol photon was calculated using the following formula: gBiomass Net waveguide algal f ilm productivity ¼ molphoton Total photon flux emitted from LED
ð2Þ
The total light emission from the waveguides was calculated by numerically integrating the light emission profiles on each side of the waveguide using the trapezoid method. 3. Results and discussion 3.1. Waveguide light emission profiles The results shown in Fig. 3a–e were normalized to an input electrical power of 0.67 W (at this power the red LED produces 7200 μmol/m2 s). For the single wedge large notch waveguide (Fig. 3a), the light emission from the front shows a regular cycle in light intensity peaks, which coincide with the notches in the waveguide. A smaller cycle of light intensity was also measured on the back. The light emission from the side of the waveguide decreased along the length of the waveguide and did not show a cyclical pattern in light intensity. The highest light intensity emitted from any waveguide was measured for the single wedge, large notch waveguide at 3320 μmol/m2 sec at a distance of 40–45 mm along the waveguide on the input surface. The light emission for the single wedge (Fig. 3b) and single wedge small notch waveguides (Fig. 3d) followed similar exponential decay trends for all sides. The light emission for the double wedge waveguide (Fig. 3c) was more uniform compared to the light emission from the other waveguides, but the light emission for the double wedge small notch waveguide followed an exponential decay after 50 mm, which is based on a line of best fit (Fig. 3e). The ratio of light emitted from each waveguide to the incident light intensity did not change with varying light intensity for all waveguide designs. The ratios are: 0.81, 0.89, 0.85, 0.68, and 0.83 for the single wedge large notch, single wedge, double wedge, single wedge small notch, and double wedge small notch waveguides respectively. These results demonstrate the ability to change the light emission profile of a waveguide by the addition of notches. 3.2. Reactor — suspended concentration The pH and temperature of the reactor were stable at 6.8 ± 0.3 and 23 ± 2 °C respectively throughout the duration of the experiments. During the experiment, the total suspended solids after day 3 remained below 0.025 g/L. In every experiment, it was observed that the total suspended solids (TSS) decreased from day 1 to day 3, and then remained
relatively stable afterwards. The average TSS did not increase with increasing CO2, but did increase from low light intensity (1600 μmol/m2 s) to medium (7200 μmol/m2 s), then plateaued at high (12,600 μmol/m2 s). The outlet nitrogen concentration was initially stable from day zero to day four (30–32 mgN/L), then decreased from day four to six, but afterwards remained between the values of 19–23 mgN/L for the remainder of the experiment (removal efficiency from day 6–13 was 40–49%). The outlet phosphorous concentration remained more consistent over the course of the experiment, ranging from 25 to 30 mgP/L (removal efficiency varied from 16 to 30%). 3.3. Growth kinetics of algal biofilms on light emitting waveguides The overall growth kinetics of the algal biofilms grown on the waveguide notches, back and sides were non-linear overall, but did have linear portions (Figs. 4a–d, and 5a–d). The growth appears to be linear and then is followed by a plateau or sloughing. This trend is consistent with data presented by Schnurr et al. [22], and Gross et al. [7]. Linear (as opposed to exponential) growth curves for microorganisms in conventional bioprocessing systems suggest chemical or mass transport limitations to growth. For the algal biofilms, this implies a nutrient, diffusion or lack of bulk nutrients, or a light limitation within the biofilm. Modeling of algal biofilms by Flora et al. [23] and Leihr et al. [24] shows the CO2 concentration within an algal biofilm drops to zero at a depth of 200 μm and that light intensity drops off to 6.1% of the initial light intensity. This implies that the fundamental growth kinetics of algal biofilms are likely limited by either inorganic carbon or light at the low light intensity conditions tested. At the medium and high light intensities, the algal biofilms are not entirely light limited, but since there are still regions of linear growth, this suggests that light saturation of algal biofilms also induces linear growth curves in algal biofilms. There also seems to be a photo inhibition effect when the CO2 partial pressure was atmospheric since the algal film biomass was less on day 14 when the incident photon flux into the waveguide was increased from 1600 μmol/m2 s to 7200 μmol/m2 s. In the dark control experiment (Fig. 4b), the algal biofilm yield was not statistically significantly different than zero, which implies that algal biofilms were not capable of growing in the reactor without light. Adding small notches to the waveguides appeared to increase the yield on the notched sides and decreased the yield on the non-notched sides (Fig. 5a–d).This implies that algal biofilms preferred to grow on the notched surface even though for the single wedge and single wedge with small notches the light emission profiles did not vary significantly which is in agreement with the results from Sathananthan et al. [25]. Sloughing of the algal biofilms on the back of the double wedge small notch was observed on days 12 and 14 (Fig. 5d). 3.4. Algal biofilm productivity The algal biofilm productivities, based on the area of the attachment material, on the front of the large notched waveguide ranged from 0.25–2.9 g/m2 day depending on conditions (Fig. 6a). The highest productivity observed in this study (2.9 g/m2 day) was at the conditions of 3% CO2 partial pressure and high light intensity (12,600 μmol/m2 s); however this was not statistically different compared to the following conditions: 1% CO2 with medium (7200 μmol/m2 s) and high light intensity (12,600 μmol/m2 s), and 3% CO2 at medium light intensity (7200 μmol/m2 s). These productivities were significantly higher than those reported by Christenson and Sims [5] (1.4 g/m2 day) and Genin et al. [19] (1.2 g/m2 day) for algal biofilms grown on acrylic (of which attachment material has been shown to play an important role in algal biofilm growth [5,19]). In the experiment by Genin et al. [19], the hydrodynamic and CO2 partial pressure conditions are the same as those reported in this paper. The surface area productivities at these conditions were also higher than for similar algal biofilms grown on other materials [7] (1–1.5 g/m2 day on cotton in a RAB reactor), and
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Fig. 3. Light emission profiles of the waveguides, a) single wedge large notch, b) single wedge, c) double wedge, d) single wedge small notch, and e) double wedge small notch.
comparable to the maximum values reported by Schnurr et al. [15] (2.1–2.8 g/m2 day on glass in a horizontal plate reactor). Algal biofilm productivity on the large notch waveguide front was significantly higher than growth on the sides or back of the waveguide (Fig. 4a). This observation was consistent across all conditions tested for the large notch waveguide. This is in contrast to algal biofilms grown on the single, double wedge waveguides, and double wedge waveguides with small notches (Fig. 7), where the growth on the front, sides and back of the waveguides were similar even though the light emission
from the sides of the waveguides is statistically significantly lower than the front or the back (Fig. 3b and c). The algal biofilm productivity on front, back, sides and regrowth of the single wedge (0.82 g/m2 day, 0.81 g/m2 day, 0.71 g/m2 day, 0.86 g/m 2 day respectively) and on the double wedge waveguide (0.57 g/m2 day, 0.61 g/m2 day, 0.69 g/m2 day, 0.68 g/m2 day respectively) were not statistically different (Fig. 7). The light intensity emitted from the front and back surfaces of the double edge waveguides was ~ 2–3 time larger than that emitted from the side surfaces. (Fig. 3b and c). It
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Fig. 4. Algal growth kinetics on the large notch waveguides: a) growth kinetics on the various faces of the waveguide at 1% CO2 and 7200 μmol/m2 s light emitted by single LED, b) growth kinetics on the front with CO2 = 1%, c) growth kinetics on the front with CO2 = ATM, and d) growth kinetics on the front with CO2 = 3%.
is possible that the light intensity emitted from the sides is sufficient enough to saturate the algal biofilm. Algae grown in suspension under white light becomes light saturated between 400 and 500 μmol/m2 s
[26] which is about 6.6 times less than the highest photon flux emitted from the waveguides (3320 μmol/m2 s for the single wedge large notch waveguide). Previous results from Johnson and Wen [14] and
Fig. 5. Algal biofilm growth kinetics on a) single wedge waveguides, b) double wedge waveguides, c) single wedge small notches and d) double wedge small notches (7200 μmol/m2 s emitted by single LED, CO2 = 1%).
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Fig. 6. a) Algal biofilm productivity on the front of the single wedge large notch waveguide at different CO2 partial pressures and incident photon fluxes emitted by the LEDs, b) algal biofilm productivity per single wedge large notch waveguide at different conditions (95% confidence intervals shown).
Christenson and Sims [5] demonstrated that regrowth on materials improved algal biofilm productivity, but this result was not replicated in this study possibly due to the difference in materials used or the vertical
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orientation of the waveguides. Genin et al. [19] demonstrated that acrylic was a poor material to grow algal biofilms on, which had the lowest revised productivity out of the materials tested (glass, cellulose acetate, polycarbonate, polystyrene, acrylic, and silicone rubber). The algal film productivity on the front of the single wedge small notched waveguides (1.12 g/m2 day) was statistically significantly higher than the productivity on the back (0.44 g/m2 day) and the side (0.33 g/m2 day) (Fig. 7). The light emission from the single wedge small notched waveguides was similar to the light emission from the single wedge waveguides (Fig. 3b and d). The improvement in algal film productivity from the front of the single wedge small notch waveguide compared to the back, sides and the front of the single wedge waveguide may be the result of improved attachment of the algal biofilm. Sathananthan et al. [25] saw a similar trend, where embossing acrylic with microstructures in a parallel plate airlift reactor resulted in a 70% increase in algal film productivity. The increases in algal film growth on the front of the single wedge small notch waveguide appear to have come at the expense of growth on the back and sides of the waveguide. At the operation conditions of 1% CO2 and medium light intensity (7200 μmol/m2 s), the algal biofilm productivities on the front, back and side of the waveguide were 2.8 g/m2 day, 0.26 g/m2 day and 0.57 g/m2 day respectively, which yields an overall algal film productivity of 0.0562 g/day on a single waveguide. With 24 waveguides in the reactor, the net reactor productivity was 0.13 g/day and when considering the aerial foot print of the reactor, the productivity was 1.59 g/m2 day. If the waveguide aerial foot print is considered, the aerial productivity of the waveguide was 33.6 g/m2 day. It would be unreasonable to assume that the ideal waveguide reactor would contain waveguides which were not spaced apart, so if a reactor was designed in which the waveguides were spaced 1.27 cm apart, and the productivity obtained in this study was transferable to the other reactor, the aerial productivity of the reactor could be 14.4 g/m2 day, which is comparable to the results obtained by Christenson and Sims [5] (21–30 g/m2 day). This implies that important design parameters for scaling up a waveguide algal film photobioreactor would be waveguide size, packing, light emission and CO2 concentration. The total algal film productivity per waveguide was not statistically significantly different except between the single wedge large notch, single wedge, double wedge and double wedge small notch at the 95% confidence level (Table 3). For all waveguide reactor runs at 7200 μmol/m2 sec and 1% CO2 partial pressure, the suspended productivity was not statistically significantly different from the total biofilm productivity (defined as the algal film productivity on the waveguides only). The suspended productivity was statistically significantly lower in the waveguide reactors than the productivity in the parallel plate air lift reactor by Genin et al. [19] (suspended productivity of 0.72 g/day). The total reactor productivity from the single wedge waveguide reactor
Fig. 7. Algal biofilm productivity on single wedge, double wedge, single wedge small notch and double wedge small notch, on each side and including regrowth (CO2 = 1%, 7200 μmol/m2 s light emitted by LED, 95% confidence intervals shown).
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Table 3 . Suspended and attached estimation of productivities in the reactor at the following conditions: 7200 μmol/m2 s incident photon flux into the waveguides, 1% partial pressure of CO2. (95% confidence intervals shown). Waveguide type
Productivity per waveguide (g/day)
Total biofilm productivity (g/day)
Suspended productivity (g/day)
Total reactor productivity
Biofilm productivity percentage of total
Single wedge large notch Single wedge Double wedge Single wedge small notch Double wedge small notch
0.0057 ± 0.001 0.0071 ± 0.001 0.0055 ± 0.002 0.0038 ± 0.001 0.0038 ± 0.002
0.14 ± 0.02 0.17 ± 0.03 0.13 ± 0.05 0.09 ± 0.03 0.09 ± 0.05
0.19 ± 0.03 0.20 ± 0.04 0.20 ± 0.04 0.11 ± 0.02 0.14 ± 0.03
0.33 ± 0.05 0.37 ± 0.07 0.33 ± 0.09 0.20 ± 0.05 0.23 ± 0.08
42% 46% 39% 45% 39%
was statistically significantly greater than the productivity from the single wedge small notch waveguide reactor. The light emission profiles from these two waveguides are similar, so this suggests that the notched surface had a significant impact on the productivity. 3.5. Substrate and light saturation Algal biofilms grown at atmospheric CO2 (0.04%) demonstrated a statistically significant decrease in productivity from low light intensity (0.52 g/m2 day) to medium (0.21 g/m2 day) and high light intensity (0.23 g/m2 day) as shown in Fig. 6a. Increasing the light intensity from low (1600 μmol/m2 s) to medium (7200 μmol/m2 s) at 1% and 3% CO2 resulted in statistically significant increases in algal biofilm productivity from 0.61 g/m2 day to 2.8 g/m2 day for 1% CO2 and 1.2 g/m2 day to 2.8 g/m 2 day for 3% CO 2. Increasing the light intensity to high (12,600 μmol/m 2 s) did not improve the productivity of the algal biofilms at 1% and 3% CO2. A multiple linear regression on the algal biofilm productivity does not yield a statistically significant correlation for the front (P = 0.097), back (P = 0.163), or sides (P = 0.559) which implies algal film productivity on the waveguide is a non-linear function of CO2 partial pressure and light intensity or the number of data points is insufficient. A modeling study by Liehr et al. [24] on the effects of light intensity and CO2 on algal biofilm growth would imply that the relationship is highly non-linear, which is in agreement with the results of this study. The results from Fig. 6a and b show that algal biofilm productivity increases with light intensity and CO2 concentration, but experience a saturation effect with respect to CO2 and light intensity. Increasing the CO2 partial pressure from atmospheric (0.04%) to 1% improved algal biofilm productivity at medium and high light intensities. This is counter to what Gross et al. [7] observed with their RAB system, where an increase in CO2 partial pressure beyond atmospheric did not result in an increase in productivity, but is consistent with Blanken et al. [11]. In both studies, the algal biofilms were partially exposed to the atmosphere. Modeling studies by Liehr et al. (1990) and Flora et al. [23] showed algal biofilms experienced inorganic carbon diffusion limitations at inorganic carbon concentrations in water expected at atmospheric CO2 partial pressure. Previous studies on the influence of light intensity on algal biofilm growth are limited. A study by Schnurr et al. [15] demonstrated that a
decrease in light intensity from 100 μmol/m2 s to 50 μmol/m2 s resulted in a decrease in productivity from 1.7 g/m2 day to 0.9 g/m2 day. The study did not examine light intensities or the possibility of light saturation. Planktonic algal cultures will become light saturated and increasing the light intensity beyond the light saturation point does not improve algal growth [26,27,28] and past the saturation light intensity, there can be a decrease in algal cell growth [29]. When the algal biofilms were grown at 1% and 3% CO2, increasing light intensity past the saturation point did not improve algal film growth, but did not decrease it either, whereas increasing light intensity when atmospheric CO2 was sparged in, algal film productivity decreased. This implies that when algal biofilms are carbon starved, increasing light intensity past the light saturation point will hinder growth rates; whereas non-carbon starved biofilms will not see a decrease or increase in growth rates. This suggests that increasing CO2 concentration can offset the effects that high light intensity has on photo inhibition of algal biofilms. Pope [30] observed that for suspended microalgae inhibition of photosynthesis (CO2 fixation and growth) increases with increasing light intensity beyond the light saturation point, but that increased CO2 concentrations can decrease the effect that high light intensities have on photo inhibition. There is evidence in the literature which demonstrates that the photosynthetic carbon metabolism is required to prevent photoinhibition in algae species [31]. 3.6. Photon to biomass conversion The grams of biomass produced per mol of photon emitted from the LEDs decreased with increasing inlet photon flux (Fig. 8).The highest biomass to photon ratio (0.074 gbiomass/molphoton) occurred at 1600 μmol/m2 s and 1% CO2, while the lowest biomass to photon ratio (0.003 gbiomass/molphoton) occurred at atmospheric CO2 and photon flux of 12,600 μmol/m2 s, which was statistically significantly different than the biomass to photon ratio with the same CO2 conditions and lower photon flux of 7200 μmol/m2 s. The gram biomass per mol photon ratio was not statistically significantly different for the CO2 partial pressures at the same photon flux (1600 μmol/m2 s). This implies that the photon to biomass conversion is most efficient at low light intensities and when the biofilm is not carbon limited. This is consistent with the fundamental theories outlined by Osborne and Geider [32] and Melis [26] and the results on suspended algae which were reported by Melis
Fig. 8. Biomass photon conversion ratios from algal biofilms grown on the single wedge large notch waveguide (95% confidence intervals are shown).
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537
Table 4 Reactor productivity comparison. Reactor
Material
Surface area productivity g/m2 day
Lipid content (w/w)
Light source
Reference
Waveguide reactor PPAL Flat plate RAB Bench scale RAB Turf scrubber
Acrylic Cellulose acetate Glass Cotton Cotton rope Concrete
2.9 2.1 2.8 3.51 2.5 0.7
12–15% 6–8% 5–10% 7–8% 11.2–12.4% 20–25%
Red LEDs White LEDS Red LEDs White Light White light White light
This study Genin et al. [19] Schnurr et al. [22] Gross et al. [7] Christenson and Sims [5] Ozkan et al. [6]
et al. [27]. The photon to biomass conversion ratio can be a helpful metric for understanding the light efficiency of algal film photobioreactors. 3.7. Lipid content The neutral lipid content for the algal biofilms grown on the waveguides ranged from 12 to 15% (w/w) and was not statistically significantly different at the 95% confidence level depending on the CO2 partial pressure or light intensity. The results are higher than those reported by Johnson and Wen [14] (6–9% w/w) and Genin et al. [19] (6–8% w/w), but are similar to those found by Schnurr et al. [22] (5–10% w/w) and by Christenson and Sims [5] (11–12% w/w). For the conditions of 1% CO2 partial pressure and 7200 μmol/m2 s, the lipid productivity for algal biofilms grown in the notch was 0.34–0.42 g/m2 day; aerial reactor productivity was 0.19–0.24 g/m2 day, and the waveguide aerial productivity was 4–5 g/m2 day. The aerial reactor productivity is lower than those of terrestrial crops (0.25 g/m2 day) and values reported by Christenson and Sims [5] (2.2–2.5 g/m2 day). The results in this study suggest that the lipid content of algal films may not be affected by light intensity which is consistent with the results presented by Ho et al. [33] for S. Obliquus grown in suspension; however Guedes et al. [34] showed a decrease in lipid content of Pavlova lutheri with increasing light intensity. 3.8. Comparison Table 4 compares the algal film productivities, attachment material, light source and FAME content of the reactor in this study to other algal film photobioreactors reported in literature. RAB reactors such as the ones designed by Christenson and Sims [5] and Gross et al. [7] continue to have the highest reported aerial productivity (20–31 g/m2 day and 10.5 g/m2 day respectively), but direct comparisons can be difficult, because attachment material, light intensity, algal culture composition, reactor configuration, and nutrient loads can impact algal biofilm productivity [5,19,21]. When examining surface area productivity, the single wedge large notch waveguide (2.8 g/m2 day) performs just as well as reactors developed by Christenson and Sims [5] (2.5 g/m2 day), Schnurr et al. [15] and Gross et al. [7]. The cost to manufacture the waveguides was relatively expensive compared to the cost of the cast acrylic with the small notched waveguides being the most expensive due to the precision required to machine the small notch. This indicates that light emitting waveguides present a promising approach to the production of algal biomass. 4. Conclusions In this paper we demonstrate that it is possible to grow algal biofilms on light emitting waveguides, and the design of the waveguides impacts the light emitted from the waveguides, which in turn, impacts the algal biofilm productivity. The highest measured surface area productivity recorded was on front of the single wedge large notch waveguides which was 2.9 g/m2 day, a FAME productivity of 0.34–0.42 g/m2 day, a nitrogen removal efficiency of 40–49% and a phosphorus removal efficiency of 15–30%. Algal biofilm productivity showed a dependency on light intensity and CO2 concentration, however algal biofilms became light and CO2 saturated. The interaction effects that light and
CO2 have on algal biofilm productivity are complex. The negative effect of increasing light intensity beyond the saturation point that has on algal biofilm growth can be reduced by increasing the CO2 concentration. Algal biofilms grown on the light emitting waveguides did not show changes in lipid content with the various light and CO2 conditions tested. The results indicate the potential for algal biofilms to be grown on light emitting waveguides which opens up the opportunity to explore new algal film photobioreactor configurations. Acknowledgments The authors are grateful to the Natural Sciences and Engineering Research Council's Strategic Grant's program (#STPGP 380795-2009) and to the Ontario Graduate Scholarships program for funding this research. References [1] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294–306. [2] T.M. Mata, A.A. Martins, N.S. Caetano, Microalgae for biodiesel production and other applications: a review, Renew. Sust. Energ. Rev. 14 (2010) 217–232. [3] Y. Chisti, J. Yan, Algal biofuels—a status report, Appl. Energy 88 (2011) 3277–3279. [4] R. Pate, G. Kilse, B. Wu, Resource demand implications for US algae biofuels production scale-up, Appl. Energy 88 (10) (2011) 3377–3388. [5] L.B. Christenson, R.C. Sims, Rotating algal biofilm reactor and spool harvester for wastewater treatment with biofuels by-products, Biotechnol. Bioeng. 109 (7) (2012) 1674–1684. [6] A. Ozkan, K. Kinney, L. Katz, H. Berberoglu, Reduction water and energy requirement of algae cultivation using an algae biofilm photobioreactor, Bioresour. Technol. 114 (2012) 542–548. [7] M. Gross, W. Henry, C. Michael, Z. Wen, Development of a rotating algal biofilm growth system for attached microalgae growth with in situ biomass harvest, Bioresour. Technol. 150 (2013) 195–201. [8] Y. Hodoki, Bacteria biofilm encourages algal immigration onto substrata in lotic systems, Hydrobiologia 539 (2005) 27–34. [9] J.R. Lawrence, T.R. Neu, G.D.W. Swerhone, Application of multiple parameter imaging for the quantification of algal, bacterial and exopolymer components of microbial biofilms, J. Microbiol. Methods 32 (1998) 253–261. [10] H.-C. Flemming, T.R. Neu, D.J. Wozniak, The EPS matrix: the “House of Biofilm Cells”, J. Bacteriol. 189 (22) (2007) 7945–7947. [11] W. Blanken, M. Janssen, M. Cuaresma, Z. Libor, T. Bhaiji, R.H. Wijffels, Biofilm growth of Chlorella sorokiniana in a rotating biological contactor based photobioreactor, Biotechnol. Bioeng. 111 (12) (2014) 2436–2445. [12] R.J. Craggs, W.H. Adey, K.R. Jenson, M.S. St. John, F.B. Green, W.J. Oswald, Phosphorus removal from wastewater using an algal turf scrubber, Water Sci. Technol. 33 (7) (1996) 191–198. [13] W. Mulbry, S. Kondrad, C. Pizarro, E. Kebede-Westhead, Treatment of dairy manure effluent using freshwater algae: algal productivity and recovery of manure nutrients using pilot-scale algal turf scrubbers, Bioresour. Technol. 99 (2008) 8137–8142. [14] M.B. Johnson, Z. Wen, Development of an attached microalgal growth system for biofuel production, Appl. Microbiol. Biotechnol. 85 (2010) 525–534. [15] P.J. Schnurr, G. Espie, D.G. Allen, The effect of light direction and suspended cell concentrations on algal biofilm growth rates, Appl. Mircrobiol. Biotechnol. 98 (20) (2014) 8553–8562. [16] M.D. Ooms, V.J. Sieben, S.C. Pierobon, E.E. Jung, M. Kalontarov, D. Erickson, D. Sinton, Evanescent photosynthesis: exciting cyanobacteria in a surface-confiled light field, Phys. Chem. Chem. Phys. 14 (2012) 4817–4823. [17] T.E. Irving, D.G. Allen, Species and material considerations in the formation and development of microalgal biofilms, Appl. Microbiol. Biotechnol. 92 (2011) 283–294. [18] H.C. Bold, The morphology of Chlamydomonas chlamydogma, sp. Nov, Bull. Torrey Bot. Club 76 (2) (1949) 101–108. [19] S.N. Genin, J.S. Aitchison, D.G. Allen, Design of algal film photobioreactors: material surface energy effects on algal film productivity, colonization and lipid content, Bioresour. Technol. 155 (2014) 136–143. [20] J. Folch, M. Lees, G.H.S. Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1956) 497–509. [21] I. Smid, M. Salfinger, Microbial identification by computer-aided gas–liquid chromatography, Diagn. Microbiol. Infect. Dis. 19 (2) (1994) 81–88.
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[22] P.J. Schnurr, G. Espie, D.G. Allen, Algae biofilm growth and the potential to stimulate lipid accumulation through nutrient starvation, Bioresour. Technol. 136 (2013) 337–344. [23] J.R.V. Flora, M.T. Suidan, P. Biswas, G.D. Sayles, Modeling algal biofilms: role of carbon, light, cell surface charge, and ionic species, Water Environ. Res. 67 (1) (1995) 87–94. [24] S.K. Leihr, T.S. Makram, J.W. Eheart, A modeling study of carbon and light limitation in algal biofilms, Biotechnol. Bioeng. 35 (1990) 233–243. [25] S. Sathananthan, S.N. Genin, J.S. Aitchison, D.G. Allen, Micro-structured surfaces for algal biofilm growth, Proc. SPIE 8923, Micro/Nano Materials, Devices, and Systems 2013, p. 892350. [26] A. Melis, Solar energy conversion efficiencies in photosynthesis: minimizing the chlorophyll antennae to maximize efficiency, Plant Sci. 177 (2009) 272–280. [27] A. Melis, J. Neidhardt, J.R. Benemann, Dunaliella salina (Chlorophyta) with small chlorophyll antenna sizes exhibit higher photosynthetic productivities and photon use efficiencies than normally pigmented cells, J. Appl. Phycol. 10 (1999) 515–525. [28] J.H. Steele, Notes on some theoretical problems in production ecology, in: Goldman (Ed.), Primary Production in Aquatic Environments, University of California Press, Berkeley, CA, 1965.
[29] I. Baroli, A. Melis, Photoinhibition and repair in Dunaliella salina acclimated to different growth irradiances, Planta 198 (1995) 640–646. [30] D.H. Pope, Effects of light intensity, oxygen concentration, and carbon dioxide concentrations on photosynthesis in algae, Microb. Ecol. 2 (1) (1975) 1–16. [31] S.B. Powles, Photoinhibition of photosynthesis induced by visible light, Annu. Rev. Plant Physiol. 35 (1984) 15–44. [32] B.A. Osborne, R.J. Geider, The minimum photon requirement for photosynthesis, New Phytol. 106 (1987) 631–644. [33] S.-H. Ho, C.-Y. Chen, J.-S. Chang, Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedemus obliquus, Bioresour. Technol. 113 (2012) 244–252. [34] A.C. Guedes, L.A. Meireles, H.M. Amaro, F.X. Malcata, Changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity, J. Am. Oil Chem. Soc. 87 (7) (2010) 791–801.
Contents lists available at ScienceDirect
Algal Research journal homepage: www.elsevier.com/locate/algal
Novel waveguide reactor design for enhancing algal biofilm growth Scott N. Genin a, J. Stewart Aitchison b, D. Grant Allen a,⁎ a b
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College St., Toronto, Ontario M5S 3E5, Canada The Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King's College Road, Toronto, Ontario M5S 3G4, Canada
a r t i c l e
i n f o
Article history: Received 21 May 2015 Received in revised form 11 September 2015 Accepted 21 October 2015 Available online xxxx Keywords: Algae biofilm Algae production Light waveguide Photobioreactor
a b s t r a c t Algal film photobioreactors have the potential to reduce dewatering costs and have a comparable productivity to raceway ponds. Current designs have focused on maximizing productivity and nutrient removal through material selection and design. However, fewer studies have focused on analyzing the effects of illumination and carbon dioxide (CO2) concentration on algal film growth. The transport of light in suspended photobioreactors is considered a serious problem due to cell shading, light scattering and non-uniform optical absorption. To enhance light delivery to algal biofilms, five different waveguide designs were developed Light emitting waveguides are capable of distributing a bright light source over a larger surface area. The light emission from these waveguide designs was characterized and the algal biofilm growth on waveguides was characterized in a parallel plate airlift reactor (17 L). The light intensity and CO2 concentration were varied in a 32 factorial design experiment to determine if there were any interaction effects. Light emission from the waveguides could be altered by changing the tapper angle or by notching the surfaces. Algal film growth kinetics on the waveguides were overall non-linear, but had linear regions of growth. The waveguides achieved an algal biofilm surface area productivity of 2.8 g/m2 day and an aerial productivity of 33.6 g/m2 day. Algal film productivity displayed saturation kinetics with respect to light intensity and CO2 concentration, but the interaction effect of light and CO2 on productivity is likely non-linear. Algal film biomass per photon consumed decreased with increasing light intensity when the reactor was not CO2 limited. The results indicate the potential for algal biofilms to be grown on light emitting waveguides which opens up the opportunity to explore new algal film photobioreactor configurations. © 2015 Published by Elsevier B.V.
1. Introduction Microalgae have been identified as a potential feedstock for both biofuels and biochemicals due to its high growth rate compared to terrestrial crops and high lipid content [1,2]. The production of biofuels and bioproducts from algae is possible, but commercialization is difficult [3] due to numerous challenges such as: insufficient supplies of low-cost concentrated CO2; high capital costs of photobioreactors [4], and low algal biomass concentration. Microalgae grown as a biofilm presents an opportunity to reduce dewatering costs since the biomass is more concentrated (90–150 g/L) [5,6,7] compared to suspended algae produced in raceway ponds (0.5–1 g/L) or photobioreactors (1–4 g/L) [1]. Algal biofilms are a mixed community of many different algae and bacteria species within a matrix of extracellular polymeric substances (EPS) [8,9]. EPS is a matrix of polysaccharides, proteins, glycoproteins, glycolipids, and extracellular DNA produced by the microorganisms that are imbedded in the biofilm [10]. Current research on algal film photobioreactor design focuses on rotating algal biofilm (RAB) systems [5,7,11] or on algal turf scrubbers ⁎ Corresponding author. E-mail addresses: [email protected] (S.N. Genin), [email protected] (J.S. Aitchison), [email protected] (D.G. Allen).
http://dx.doi.org/10.1016/j.algal.2015.10.013 2211-9264/© 2015 Published by Elsevier B.V.
[6,12,13,14]. These reactors are based on the principle of growing an algal biofilm on a rough attachment surface in which either the surface or the water is moving. These RAB systems show promise as their reported aerial biomass productivities are higher than those of raceway ponds [5,7]. The design considerations for these systems have been investigated from algal film productivity and nutrient removal standpoint, but have not investigated effects of light intensity and CO2 concentration on algal film growth. Recent studies by Schnurr et al. [15] and Ooms et al. [16] have provided new insight into new photobioreactor designs. In experiments by Schnurr et al. [15] algal biofilms were grown where light was incident through glass and the productivity was compared to algal biofilms which were grown in a conventional manner where light was incident from the water side. The results demonstrated that at the conditions tested, light direction did not impact algal biofilm growth. Ooms et al. [16] demonstrated the possibility of growing cyanobacteria in the evanescent field associated with an optical waveguide. While the biofilms only reached a thickness of 10–15 μm, the results showed by both these studies demonstrate the potential to grow algal biofilms on a light emitting surface. Growing an algal biofilm on a light emitting surface presents a series of potential advantages: when the attachment material and light source are integrated, the light is directly delivered to the algal biofilm without
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shading from suspended algae to produce a more compact bioreactor. However, the effects of CO2, illumination intensity and light direction on the growth kinetics of an algal biofilm on a light emitting surface have not been investigated. The objective of this study is to demonstrate the feasibility of growing algal biofilms on light emitting waveguides and determining the combined effects of varying light intensity and CO2 concentration. To complete this objective, five different acrylic waveguides and a parallel plate airlift (PPAL) reactor were designed and built. The parameters measured were biomass production, fatty acid methyl ester (FAME) content, total nitrogen, total phosphorous, temperature and pH. 2. Materials and methods 2.1. Waveguide design Five different waveguide designs were fabricated out of optically transparent cast acrylic with a top cross sectional area of 1.27 × 1.27 cm2 and a length of 25 cm (Fig. 1a–e). All sides of the waveguides were polished to remove imperfections in the surface after machining. Dimensions are provided in Table 1. The tapper and notches in the waveguides cause light to leak or scatter out of the waveguide. The taper in waveguide dimension along the direction of propagation prevents total internal reflection and as a result light leaks from the waveguide. The notches provide local scattering centers and cause light trapped in the waveguide to scatter into the bioreactor. Red Light Emitting Diodes (LED) (3.5 V and 700 mA, 635 ± 10 nm) provided by Pond Biofuels Inc. were mounted to the top of the waveguide. 2.2. Light intensity measurements The light emission from the waveguide was characterized by a photodiode (Thor Labs model #: FDS010) which was scanned along the direction of light propagation in the waveguide at a distance of 2.4 mm from the surface. The current and voltage provided to the LED by a Pyramid brand variable power supply (model #: PS-32lab) were measured using a volt and amp meter. The electrical input power to the LED was adjusted to control the light emission and was calibrated using a Newport Silicon photometer (model #: 818-SL) which was set to 635 nm, to confirm that over the electrical power range used in these experiments, the light emission was linearly correlated to the electrical input power. The photon flux from the LEDs at different input electrical power was measured using a Fieldscout Quantum
Light Meter (model #: 3415F) and a calibration curve of photon flux vs. electrical input power to the LED was generated. This calibration curve was used to estimate the photon flux emitted from the surfaces of the waveguide. 2.3. Reactor setup The PPAL reactor (Fig. 2) used in this study was designed to provide a constant shear force on each waveguide, adequate mixing, and nutrients. The reactor case was constructed of glass, with two vertical internal plates of cast acrylic which were secured by a silicone adhesive. The reactor was filled with 17 L of media and has the following dimensions: 41 × 20 × 25 cm3. Twenty-four waveguides were placed vertically in the down-comer of the PPAL for each run. The red LEDs were mounted to the top of each waveguide in four parallel circuits and power was supplied by a Pyramid brand variable power supply (model #: PS-32lab). 2.4. Reactor operation Past work by Irving and Allen [17] determined that the presence of wastewater is an important factor in enhancing the formation of an algae biofilm. To introduce EPS and bacteria, unsterile wastewater from Ashbridge's Bay Wastewater Treatment Facility, Toronto, ON, was blended with Fortified Bold's Basal Media (FBBM) [18] in a volumetric ratio of 1:2. FBBM was buffered to pH 6.8 and prepared to have the following concentration of nutrients: NaNO3 250 mg/L, CaCl2·2H2O 25 mg/L, MgSO4·7H2O 75 mg/L, K2HPO4 75 mg/L, KH2PO4 175 mg/L, NaCl 25 mg/L, Na 2EDTA 10 mg/L, FeSO 4 ·7H2 O 4.98 mg/L, H3BO 3 11.42 mg/L, Na 2 SiO3 ·7H2O 58 mg/L, ZnSO4 ·7H2 O 8.82 mg/L, MnCl2·4H2O 1.44 mg/L, Na2MoO3 0.70 mg/L, CuSO4·5H2O 1.57, and Co(NO3 ) 2·6H2 O 0.49 mg/L. The solution was sparged with air at 1 L/min in the reactor for 24 h before being inoculated. The inoculum contained seven algal species which were purchased from the Canadian Phycological Culture Centre (CPCC) or the Culture Collection of Algae and Protozoa (CCAP), which were selected because they were the same species previously used to inoculate the PPAL reactor in other experiments [19] and consistency between experiments was desired for a fair comparison between reactor setups. The species used in this experiment were: Scenedesmus obliquus (CPCC 157), Chlorella vulgaris (CPCC 147), Coccomyxa sp.(CPCC 508), Nannochloris sp. (CCAP 251/2), Nitschia palea (CPCC 160), Oocystis sp. (CPCC 9) and Oocystis polymorpha (CPCC 35) and were prepared by growing the individual cultures in flasks in a light incubator as described [19]. The
Fig. 1. Waveguide schematics for a) single wedge large notch, b) single wedge, c) double wedge, d) single wedge small notch, and e) double wedge small notch (side view).
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2.5. Sampling and analysis
Table 1 Waveguide specification. Angle of taper Number of Notch depth Notch spacing (degrees) notches (mm) (mm) Single wedge large notch Single wedge Double wedge Single wedge small notch Double wedge small notch
531
1.88 1.88 1.11 1.88 1.11
6 0 0 167 334
5.0 0 0 0.25 0.25
7.5 0 0 0.2 0.2
reactor operated in batch mode for 48 h before the pumps were started to introduce fresh FBBM. The day the pumps were started was considered day zero, and the algal biomass on three waveguides was harvested from the reactor on days 0, 3, 5, 7, 10, 12, and 14. The cleaned waveguides were then placed back into the reactor. Suspended algae samples were also taken from the reactor in triplicate. Regrowth experiments were conducted by placing the waveguides back into the reactor after harvesting, then harvesting the front of all the waveguides on the final day (day 14). For the single wedge large notch waveguides, the reactor was operated under different light intensities and CO2 to compressed air ratios at a total flow rate of 1 L/min STP in a 32 factorial experimental design, where the light emission from the LED (1600 μmol/m2 s, 7200 μmol/m2 s, 12,600 μmol/m2 s) and CO2 partial pressures (atmospheric, 1%, 3%) were varied, with triplicate experiments conducted at the center point (1% CO2, 7200 μmol/m2 sec emitted from the LED) (Table 2). A dark control experiment, where the PPAL reactor was covered, was conducted to determine whether an algal biofilm could grow in the reactor conditions without the presence of light and at 1% CO2. For the single wedge, double wedge, single wedge small notch and double wedge small notch waveguides, the reactor was operated under the same light intensity (7200 μmol/m2 s) and CO 2 partial pressure (1%). A peristaltic pump (Cole Parmer Masterflex, model # 7520–35) was used to add and remove media from the reactor at a dilution rate of 0.90 day−1 to wash out the suspended algae.
The algal biomass on the waveguides was harvested and suspended in RO water. A vacuum filtration unit was used to filter the suspended algal mass through Supor®-450 47 mm filters with a pore size of 0.45 μm. The filters were baked at 103 °C for 4 h and weighed before and after filtration to measure the difference in dry mass. Algal film biomass harvested from the single wedge large notch waveguides on day 14 was freeze dried and the lipids were extracted using the Folch method [20] using 1:2 (v/v) chloroform to methanol as the solvent. The methanol phase and polar lipids solution were discarded to target the neutral lipids. The neutral lipids were then methylated using the FAME technique based on the Microbial Identification System, Microbial ID Inc. (MIDI Method) [21]. The samples were analyzed using gas chromatography (Perkin Elmer Clarus 680 GC) with a special performance capillary column (Hewlett Packard model # HP-5 MS, 30 m × 0.25 mm × 0.25 μm) and a flame ionization detector. Hexadecane (Sigma Aldrich #H6703) was used as the internal standard and olive oil was used as a calibration standard. The removal of nitrogen and phosphorous by the algal biofilms was evaluated at 1% CO2 partial pressure and 7200 μmol/m2 s. Nitrogen and phosphorous in the inlet and outlet streams were measured using Hach reagent kits for total nitrogen (TNT 827) and total phosphates (TNT 844). The total phosphorus concentration in the media was calculated based on the total phosphates. During the experiment, algal biofilms were harvested at the same intervals to determine growth. 2.6. Calculations Algal biofilm productivity is calculated by either using a single point measurement [5,6,7,14], a linear regression over the entire growth period [15,22] or through colonization time analysis [19]. In this study, productivity was calculated using the colonization time the method which removes bias that colonization can introduce into the productivity measurement using a linear regression on algal biofilm yield data which is above the confluence level as described in more detail by Genin et al. [19].
Fig. 2. Waveguide reactor schematic setup.
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Table 2 . Conditions for the factorial experimental design with varying light intensity and CO2 partial pressure.
ATM CO2 1% CO2 3% CO2
Photon flux emitted by LED: 1600 μmol/m2 s
Photon Flux emitted by LED: 7200 μmol/m2 s
Photon Flux emitted by LED: 12,600 μmol/m2 s
Single Single Single
Single Triplicate Single
Single Single Single
The percent of nitrogen and phosphorous removed was calculated using Eq. (1) below: Sin Sout 100 ¼ percent removal Sin
ð1Þ
where Sin and Sout are the concentration of the nutrients (nitrogen and phosphorous) in the inlet and outlet respectively. All productivity confidence intervals are the 95% confidence interval assuming a student-t distribution. The gram biomass per mol photon was calculated using the following formula: gBiomass Net waveguide algal f ilm productivity ¼ molphoton Total photon flux emitted from LED
ð2Þ
The total light emission from the waveguides was calculated by numerically integrating the light emission profiles on each side of the waveguide using the trapezoid method. 3. Results and discussion 3.1. Waveguide light emission profiles The results shown in Fig. 3a–e were normalized to an input electrical power of 0.67 W (at this power the red LED produces 7200 μmol/m2 s). For the single wedge large notch waveguide (Fig. 3a), the light emission from the front shows a regular cycle in light intensity peaks, which coincide with the notches in the waveguide. A smaller cycle of light intensity was also measured on the back. The light emission from the side of the waveguide decreased along the length of the waveguide and did not show a cyclical pattern in light intensity. The highest light intensity emitted from any waveguide was measured for the single wedge, large notch waveguide at 3320 μmol/m2 sec at a distance of 40–45 mm along the waveguide on the input surface. The light emission for the single wedge (Fig. 3b) and single wedge small notch waveguides (Fig. 3d) followed similar exponential decay trends for all sides. The light emission for the double wedge waveguide (Fig. 3c) was more uniform compared to the light emission from the other waveguides, but the light emission for the double wedge small notch waveguide followed an exponential decay after 50 mm, which is based on a line of best fit (Fig. 3e). The ratio of light emitted from each waveguide to the incident light intensity did not change with varying light intensity for all waveguide designs. The ratios are: 0.81, 0.89, 0.85, 0.68, and 0.83 for the single wedge large notch, single wedge, double wedge, single wedge small notch, and double wedge small notch waveguides respectively. These results demonstrate the ability to change the light emission profile of a waveguide by the addition of notches. 3.2. Reactor — suspended concentration The pH and temperature of the reactor were stable at 6.8 ± 0.3 and 23 ± 2 °C respectively throughout the duration of the experiments. During the experiment, the total suspended solids after day 3 remained below 0.025 g/L. In every experiment, it was observed that the total suspended solids (TSS) decreased from day 1 to day 3, and then remained
relatively stable afterwards. The average TSS did not increase with increasing CO2, but did increase from low light intensity (1600 μmol/m2 s) to medium (7200 μmol/m2 s), then plateaued at high (12,600 μmol/m2 s). The outlet nitrogen concentration was initially stable from day zero to day four (30–32 mgN/L), then decreased from day four to six, but afterwards remained between the values of 19–23 mgN/L for the remainder of the experiment (removal efficiency from day 6–13 was 40–49%). The outlet phosphorous concentration remained more consistent over the course of the experiment, ranging from 25 to 30 mgP/L (removal efficiency varied from 16 to 30%). 3.3. Growth kinetics of algal biofilms on light emitting waveguides The overall growth kinetics of the algal biofilms grown on the waveguide notches, back and sides were non-linear overall, but did have linear portions (Figs. 4a–d, and 5a–d). The growth appears to be linear and then is followed by a plateau or sloughing. This trend is consistent with data presented by Schnurr et al. [22], and Gross et al. [7]. Linear (as opposed to exponential) growth curves for microorganisms in conventional bioprocessing systems suggest chemical or mass transport limitations to growth. For the algal biofilms, this implies a nutrient, diffusion or lack of bulk nutrients, or a light limitation within the biofilm. Modeling of algal biofilms by Flora et al. [23] and Leihr et al. [24] shows the CO2 concentration within an algal biofilm drops to zero at a depth of 200 μm and that light intensity drops off to 6.1% of the initial light intensity. This implies that the fundamental growth kinetics of algal biofilms are likely limited by either inorganic carbon or light at the low light intensity conditions tested. At the medium and high light intensities, the algal biofilms are not entirely light limited, but since there are still regions of linear growth, this suggests that light saturation of algal biofilms also induces linear growth curves in algal biofilms. There also seems to be a photo inhibition effect when the CO2 partial pressure was atmospheric since the algal film biomass was less on day 14 when the incident photon flux into the waveguide was increased from 1600 μmol/m2 s to 7200 μmol/m2 s. In the dark control experiment (Fig. 4b), the algal biofilm yield was not statistically significantly different than zero, which implies that algal biofilms were not capable of growing in the reactor without light. Adding small notches to the waveguides appeared to increase the yield on the notched sides and decreased the yield on the non-notched sides (Fig. 5a–d).This implies that algal biofilms preferred to grow on the notched surface even though for the single wedge and single wedge with small notches the light emission profiles did not vary significantly which is in agreement with the results from Sathananthan et al. [25]. Sloughing of the algal biofilms on the back of the double wedge small notch was observed on days 12 and 14 (Fig. 5d). 3.4. Algal biofilm productivity The algal biofilm productivities, based on the area of the attachment material, on the front of the large notched waveguide ranged from 0.25–2.9 g/m2 day depending on conditions (Fig. 6a). The highest productivity observed in this study (2.9 g/m2 day) was at the conditions of 3% CO2 partial pressure and high light intensity (12,600 μmol/m2 s); however this was not statistically different compared to the following conditions: 1% CO2 with medium (7200 μmol/m2 s) and high light intensity (12,600 μmol/m2 s), and 3% CO2 at medium light intensity (7200 μmol/m2 s). These productivities were significantly higher than those reported by Christenson and Sims [5] (1.4 g/m2 day) and Genin et al. [19] (1.2 g/m2 day) for algal biofilms grown on acrylic (of which attachment material has been shown to play an important role in algal biofilm growth [5,19]). In the experiment by Genin et al. [19], the hydrodynamic and CO2 partial pressure conditions are the same as those reported in this paper. The surface area productivities at these conditions were also higher than for similar algal biofilms grown on other materials [7] (1–1.5 g/m2 day on cotton in a RAB reactor), and
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Fig. 3. Light emission profiles of the waveguides, a) single wedge large notch, b) single wedge, c) double wedge, d) single wedge small notch, and e) double wedge small notch.
comparable to the maximum values reported by Schnurr et al. [15] (2.1–2.8 g/m2 day on glass in a horizontal plate reactor). Algal biofilm productivity on the large notch waveguide front was significantly higher than growth on the sides or back of the waveguide (Fig. 4a). This observation was consistent across all conditions tested for the large notch waveguide. This is in contrast to algal biofilms grown on the single, double wedge waveguides, and double wedge waveguides with small notches (Fig. 7), where the growth on the front, sides and back of the waveguides were similar even though the light emission
from the sides of the waveguides is statistically significantly lower than the front or the back (Fig. 3b and c). The algal biofilm productivity on front, back, sides and regrowth of the single wedge (0.82 g/m2 day, 0.81 g/m2 day, 0.71 g/m2 day, 0.86 g/m 2 day respectively) and on the double wedge waveguide (0.57 g/m2 day, 0.61 g/m2 day, 0.69 g/m2 day, 0.68 g/m2 day respectively) were not statistically different (Fig. 7). The light intensity emitted from the front and back surfaces of the double edge waveguides was ~ 2–3 time larger than that emitted from the side surfaces. (Fig. 3b and c). It
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Fig. 4. Algal growth kinetics on the large notch waveguides: a) growth kinetics on the various faces of the waveguide at 1% CO2 and 7200 μmol/m2 s light emitted by single LED, b) growth kinetics on the front with CO2 = 1%, c) growth kinetics on the front with CO2 = ATM, and d) growth kinetics on the front with CO2 = 3%.
is possible that the light intensity emitted from the sides is sufficient enough to saturate the algal biofilm. Algae grown in suspension under white light becomes light saturated between 400 and 500 μmol/m2 s
[26] which is about 6.6 times less than the highest photon flux emitted from the waveguides (3320 μmol/m2 s for the single wedge large notch waveguide). Previous results from Johnson and Wen [14] and
Fig. 5. Algal biofilm growth kinetics on a) single wedge waveguides, b) double wedge waveguides, c) single wedge small notches and d) double wedge small notches (7200 μmol/m2 s emitted by single LED, CO2 = 1%).
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Fig. 6. a) Algal biofilm productivity on the front of the single wedge large notch waveguide at different CO2 partial pressures and incident photon fluxes emitted by the LEDs, b) algal biofilm productivity per single wedge large notch waveguide at different conditions (95% confidence intervals shown).
Christenson and Sims [5] demonstrated that regrowth on materials improved algal biofilm productivity, but this result was not replicated in this study possibly due to the difference in materials used or the vertical
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orientation of the waveguides. Genin et al. [19] demonstrated that acrylic was a poor material to grow algal biofilms on, which had the lowest revised productivity out of the materials tested (glass, cellulose acetate, polycarbonate, polystyrene, acrylic, and silicone rubber). The algal film productivity on the front of the single wedge small notched waveguides (1.12 g/m2 day) was statistically significantly higher than the productivity on the back (0.44 g/m2 day) and the side (0.33 g/m2 day) (Fig. 7). The light emission from the single wedge small notched waveguides was similar to the light emission from the single wedge waveguides (Fig. 3b and d). The improvement in algal film productivity from the front of the single wedge small notch waveguide compared to the back, sides and the front of the single wedge waveguide may be the result of improved attachment of the algal biofilm. Sathananthan et al. [25] saw a similar trend, where embossing acrylic with microstructures in a parallel plate airlift reactor resulted in a 70% increase in algal film productivity. The increases in algal film growth on the front of the single wedge small notch waveguide appear to have come at the expense of growth on the back and sides of the waveguide. At the operation conditions of 1% CO2 and medium light intensity (7200 μmol/m2 s), the algal biofilm productivities on the front, back and side of the waveguide were 2.8 g/m2 day, 0.26 g/m2 day and 0.57 g/m2 day respectively, which yields an overall algal film productivity of 0.0562 g/day on a single waveguide. With 24 waveguides in the reactor, the net reactor productivity was 0.13 g/day and when considering the aerial foot print of the reactor, the productivity was 1.59 g/m2 day. If the waveguide aerial foot print is considered, the aerial productivity of the waveguide was 33.6 g/m2 day. It would be unreasonable to assume that the ideal waveguide reactor would contain waveguides which were not spaced apart, so if a reactor was designed in which the waveguides were spaced 1.27 cm apart, and the productivity obtained in this study was transferable to the other reactor, the aerial productivity of the reactor could be 14.4 g/m2 day, which is comparable to the results obtained by Christenson and Sims [5] (21–30 g/m2 day). This implies that important design parameters for scaling up a waveguide algal film photobioreactor would be waveguide size, packing, light emission and CO2 concentration. The total algal film productivity per waveguide was not statistically significantly different except between the single wedge large notch, single wedge, double wedge and double wedge small notch at the 95% confidence level (Table 3). For all waveguide reactor runs at 7200 μmol/m2 sec and 1% CO2 partial pressure, the suspended productivity was not statistically significantly different from the total biofilm productivity (defined as the algal film productivity on the waveguides only). The suspended productivity was statistically significantly lower in the waveguide reactors than the productivity in the parallel plate air lift reactor by Genin et al. [19] (suspended productivity of 0.72 g/day). The total reactor productivity from the single wedge waveguide reactor
Fig. 7. Algal biofilm productivity on single wedge, double wedge, single wedge small notch and double wedge small notch, on each side and including regrowth (CO2 = 1%, 7200 μmol/m2 s light emitted by LED, 95% confidence intervals shown).
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Table 3 . Suspended and attached estimation of productivities in the reactor at the following conditions: 7200 μmol/m2 s incident photon flux into the waveguides, 1% partial pressure of CO2. (95% confidence intervals shown). Waveguide type
Productivity per waveguide (g/day)
Total biofilm productivity (g/day)
Suspended productivity (g/day)
Total reactor productivity
Biofilm productivity percentage of total
Single wedge large notch Single wedge Double wedge Single wedge small notch Double wedge small notch
0.0057 ± 0.001 0.0071 ± 0.001 0.0055 ± 0.002 0.0038 ± 0.001 0.0038 ± 0.002
0.14 ± 0.02 0.17 ± 0.03 0.13 ± 0.05 0.09 ± 0.03 0.09 ± 0.05
0.19 ± 0.03 0.20 ± 0.04 0.20 ± 0.04 0.11 ± 0.02 0.14 ± 0.03
0.33 ± 0.05 0.37 ± 0.07 0.33 ± 0.09 0.20 ± 0.05 0.23 ± 0.08
42% 46% 39% 45% 39%
was statistically significantly greater than the productivity from the single wedge small notch waveguide reactor. The light emission profiles from these two waveguides are similar, so this suggests that the notched surface had a significant impact on the productivity. 3.5. Substrate and light saturation Algal biofilms grown at atmospheric CO2 (0.04%) demonstrated a statistically significant decrease in productivity from low light intensity (0.52 g/m2 day) to medium (0.21 g/m2 day) and high light intensity (0.23 g/m2 day) as shown in Fig. 6a. Increasing the light intensity from low (1600 μmol/m2 s) to medium (7200 μmol/m2 s) at 1% and 3% CO2 resulted in statistically significant increases in algal biofilm productivity from 0.61 g/m2 day to 2.8 g/m2 day for 1% CO2 and 1.2 g/m2 day to 2.8 g/m 2 day for 3% CO 2. Increasing the light intensity to high (12,600 μmol/m 2 s) did not improve the productivity of the algal biofilms at 1% and 3% CO2. A multiple linear regression on the algal biofilm productivity does not yield a statistically significant correlation for the front (P = 0.097), back (P = 0.163), or sides (P = 0.559) which implies algal film productivity on the waveguide is a non-linear function of CO2 partial pressure and light intensity or the number of data points is insufficient. A modeling study by Liehr et al. [24] on the effects of light intensity and CO2 on algal biofilm growth would imply that the relationship is highly non-linear, which is in agreement with the results of this study. The results from Fig. 6a and b show that algal biofilm productivity increases with light intensity and CO2 concentration, but experience a saturation effect with respect to CO2 and light intensity. Increasing the CO2 partial pressure from atmospheric (0.04%) to 1% improved algal biofilm productivity at medium and high light intensities. This is counter to what Gross et al. [7] observed with their RAB system, where an increase in CO2 partial pressure beyond atmospheric did not result in an increase in productivity, but is consistent with Blanken et al. [11]. In both studies, the algal biofilms were partially exposed to the atmosphere. Modeling studies by Liehr et al. (1990) and Flora et al. [23] showed algal biofilms experienced inorganic carbon diffusion limitations at inorganic carbon concentrations in water expected at atmospheric CO2 partial pressure. Previous studies on the influence of light intensity on algal biofilm growth are limited. A study by Schnurr et al. [15] demonstrated that a
decrease in light intensity from 100 μmol/m2 s to 50 μmol/m2 s resulted in a decrease in productivity from 1.7 g/m2 day to 0.9 g/m2 day. The study did not examine light intensities or the possibility of light saturation. Planktonic algal cultures will become light saturated and increasing the light intensity beyond the light saturation point does not improve algal growth [26,27,28] and past the saturation light intensity, there can be a decrease in algal cell growth [29]. When the algal biofilms were grown at 1% and 3% CO2, increasing light intensity past the saturation point did not improve algal film growth, but did not decrease it either, whereas increasing light intensity when atmospheric CO2 was sparged in, algal film productivity decreased. This implies that when algal biofilms are carbon starved, increasing light intensity past the light saturation point will hinder growth rates; whereas non-carbon starved biofilms will not see a decrease or increase in growth rates. This suggests that increasing CO2 concentration can offset the effects that high light intensity has on photo inhibition of algal biofilms. Pope [30] observed that for suspended microalgae inhibition of photosynthesis (CO2 fixation and growth) increases with increasing light intensity beyond the light saturation point, but that increased CO2 concentrations can decrease the effect that high light intensities have on photo inhibition. There is evidence in the literature which demonstrates that the photosynthetic carbon metabolism is required to prevent photoinhibition in algae species [31]. 3.6. Photon to biomass conversion The grams of biomass produced per mol of photon emitted from the LEDs decreased with increasing inlet photon flux (Fig. 8).The highest biomass to photon ratio (0.074 gbiomass/molphoton) occurred at 1600 μmol/m2 s and 1% CO2, while the lowest biomass to photon ratio (0.003 gbiomass/molphoton) occurred at atmospheric CO2 and photon flux of 12,600 μmol/m2 s, which was statistically significantly different than the biomass to photon ratio with the same CO2 conditions and lower photon flux of 7200 μmol/m2 s. The gram biomass per mol photon ratio was not statistically significantly different for the CO2 partial pressures at the same photon flux (1600 μmol/m2 s). This implies that the photon to biomass conversion is most efficient at low light intensities and when the biofilm is not carbon limited. This is consistent with the fundamental theories outlined by Osborne and Geider [32] and Melis [26] and the results on suspended algae which were reported by Melis
Fig. 8. Biomass photon conversion ratios from algal biofilms grown on the single wedge large notch waveguide (95% confidence intervals are shown).
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Table 4 Reactor productivity comparison. Reactor
Material
Surface area productivity g/m2 day
Lipid content (w/w)
Light source
Reference
Waveguide reactor PPAL Flat plate RAB Bench scale RAB Turf scrubber
Acrylic Cellulose acetate Glass Cotton Cotton rope Concrete
2.9 2.1 2.8 3.51 2.5 0.7
12–15% 6–8% 5–10% 7–8% 11.2–12.4% 20–25%
Red LEDs White LEDS Red LEDs White Light White light White light
This study Genin et al. [19] Schnurr et al. [22] Gross et al. [7] Christenson and Sims [5] Ozkan et al. [6]
et al. [27]. The photon to biomass conversion ratio can be a helpful metric for understanding the light efficiency of algal film photobioreactors. 3.7. Lipid content The neutral lipid content for the algal biofilms grown on the waveguides ranged from 12 to 15% (w/w) and was not statistically significantly different at the 95% confidence level depending on the CO2 partial pressure or light intensity. The results are higher than those reported by Johnson and Wen [14] (6–9% w/w) and Genin et al. [19] (6–8% w/w), but are similar to those found by Schnurr et al. [22] (5–10% w/w) and by Christenson and Sims [5] (11–12% w/w). For the conditions of 1% CO2 partial pressure and 7200 μmol/m2 s, the lipid productivity for algal biofilms grown in the notch was 0.34–0.42 g/m2 day; aerial reactor productivity was 0.19–0.24 g/m2 day, and the waveguide aerial productivity was 4–5 g/m2 day. The aerial reactor productivity is lower than those of terrestrial crops (0.25 g/m2 day) and values reported by Christenson and Sims [5] (2.2–2.5 g/m2 day). The results in this study suggest that the lipid content of algal films may not be affected by light intensity which is consistent with the results presented by Ho et al. [33] for S. Obliquus grown in suspension; however Guedes et al. [34] showed a decrease in lipid content of Pavlova lutheri with increasing light intensity. 3.8. Comparison Table 4 compares the algal film productivities, attachment material, light source and FAME content of the reactor in this study to other algal film photobioreactors reported in literature. RAB reactors such as the ones designed by Christenson and Sims [5] and Gross et al. [7] continue to have the highest reported aerial productivity (20–31 g/m2 day and 10.5 g/m2 day respectively), but direct comparisons can be difficult, because attachment material, light intensity, algal culture composition, reactor configuration, and nutrient loads can impact algal biofilm productivity [5,19,21]. When examining surface area productivity, the single wedge large notch waveguide (2.8 g/m2 day) performs just as well as reactors developed by Christenson and Sims [5] (2.5 g/m2 day), Schnurr et al. [15] and Gross et al. [7]. The cost to manufacture the waveguides was relatively expensive compared to the cost of the cast acrylic with the small notched waveguides being the most expensive due to the precision required to machine the small notch. This indicates that light emitting waveguides present a promising approach to the production of algal biomass. 4. Conclusions In this paper we demonstrate that it is possible to grow algal biofilms on light emitting waveguides, and the design of the waveguides impacts the light emitted from the waveguides, which in turn, impacts the algal biofilm productivity. The highest measured surface area productivity recorded was on front of the single wedge large notch waveguides which was 2.9 g/m2 day, a FAME productivity of 0.34–0.42 g/m2 day, a nitrogen removal efficiency of 40–49% and a phosphorus removal efficiency of 15–30%. Algal biofilm productivity showed a dependency on light intensity and CO2 concentration, however algal biofilms became light and CO2 saturated. The interaction effects that light and
CO2 have on algal biofilm productivity are complex. The negative effect of increasing light intensity beyond the saturation point that has on algal biofilm growth can be reduced by increasing the CO2 concentration. Algal biofilms grown on the light emitting waveguides did not show changes in lipid content with the various light and CO2 conditions tested. The results indicate the potential for algal biofilms to be grown on light emitting waveguides which opens up the opportunity to explore new algal film photobioreactor configurations. Acknowledgments The authors are grateful to the Natural Sciences and Engineering Research Council's Strategic Grant's program (#STPGP 380795-2009) and to the Ontario Graduate Scholarships program for funding this research. References [1] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294–306. [2] T.M. Mata, A.A. Martins, N.S. Caetano, Microalgae for biodiesel production and other applications: a review, Renew. Sust. Energ. Rev. 14 (2010) 217–232. [3] Y. Chisti, J. Yan, Algal biofuels—a status report, Appl. Energy 88 (2011) 3277–3279. [4] R. Pate, G. Kilse, B. Wu, Resource demand implications for US algae biofuels production scale-up, Appl. Energy 88 (10) (2011) 3377–3388. [5] L.B. Christenson, R.C. Sims, Rotating algal biofilm reactor and spool harvester for wastewater treatment with biofuels by-products, Biotechnol. Bioeng. 109 (7) (2012) 1674–1684. [6] A. Ozkan, K. Kinney, L. Katz, H. Berberoglu, Reduction water and energy requirement of algae cultivation using an algae biofilm photobioreactor, Bioresour. Technol. 114 (2012) 542–548. [7] M. Gross, W. Henry, C. Michael, Z. Wen, Development of a rotating algal biofilm growth system for attached microalgae growth with in situ biomass harvest, Bioresour. Technol. 150 (2013) 195–201. [8] Y. Hodoki, Bacteria biofilm encourages algal immigration onto substrata in lotic systems, Hydrobiologia 539 (2005) 27–34. [9] J.R. Lawrence, T.R. Neu, G.D.W. Swerhone, Application of multiple parameter imaging for the quantification of algal, bacterial and exopolymer components of microbial biofilms, J. Microbiol. Methods 32 (1998) 253–261. [10] H.-C. Flemming, T.R. Neu, D.J. Wozniak, The EPS matrix: the “House of Biofilm Cells”, J. Bacteriol. 189 (22) (2007) 7945–7947. [11] W. Blanken, M. Janssen, M. Cuaresma, Z. Libor, T. Bhaiji, R.H. Wijffels, Biofilm growth of Chlorella sorokiniana in a rotating biological contactor based photobioreactor, Biotechnol. Bioeng. 111 (12) (2014) 2436–2445. [12] R.J. Craggs, W.H. Adey, K.R. Jenson, M.S. St. John, F.B. Green, W.J. Oswald, Phosphorus removal from wastewater using an algal turf scrubber, Water Sci. Technol. 33 (7) (1996) 191–198. [13] W. Mulbry, S. Kondrad, C. Pizarro, E. Kebede-Westhead, Treatment of dairy manure effluent using freshwater algae: algal productivity and recovery of manure nutrients using pilot-scale algal turf scrubbers, Bioresour. Technol. 99 (2008) 8137–8142. [14] M.B. Johnson, Z. Wen, Development of an attached microalgal growth system for biofuel production, Appl. Microbiol. Biotechnol. 85 (2010) 525–534. [15] P.J. Schnurr, G. Espie, D.G. Allen, The effect of light direction and suspended cell concentrations on algal biofilm growth rates, Appl. Mircrobiol. Biotechnol. 98 (20) (2014) 8553–8562. [16] M.D. Ooms, V.J. Sieben, S.C. Pierobon, E.E. Jung, M. Kalontarov, D. Erickson, D. Sinton, Evanescent photosynthesis: exciting cyanobacteria in a surface-confiled light field, Phys. Chem. Chem. Phys. 14 (2012) 4817–4823. [17] T.E. Irving, D.G. Allen, Species and material considerations in the formation and development of microalgal biofilms, Appl. Microbiol. Biotechnol. 92 (2011) 283–294. [18] H.C. Bold, The morphology of Chlamydomonas chlamydogma, sp. Nov, Bull. Torrey Bot. Club 76 (2) (1949) 101–108. [19] S.N. Genin, J.S. Aitchison, D.G. Allen, Design of algal film photobioreactors: material surface energy effects on algal film productivity, colonization and lipid content, Bioresour. Technol. 155 (2014) 136–143. [20] J. Folch, M. Lees, G.H.S. Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1956) 497–509. [21] I. Smid, M. Salfinger, Microbial identification by computer-aided gas–liquid chromatography, Diagn. Microbiol. Infect. Dis. 19 (2) (1994) 81–88.
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