APPLICATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on U.S. Provisional Patent Application Serial No. 61/413,685 filed on November 15, 2010, the content of which is relied upon and incorporated herein by reference in its entirety, and the benefit of priority under 35 U.S.C. §119(e) is hereby claimed.

Government Sponsorship

N/A.

BACKGROUND

1. Field of the Invention

[0002] Embodiments of the invention relate generally to photobioreactors and associated photobioreaction methods and applications. More particularly, embodiments of the invention are directed to optofluidic photobioreactor apparatuses and associated methods and applications, in which light is delivered to a photosynthetically-active entity (e.g., bacteria, algae, or other photosynthetic microorganism) through the evanescent radiation field from the surface of a waveguide adjacent the microorganism. Non-limiting embodied applications of the invention pertain to the delivery of said evanescent radiation to said microorganisms to directly or indirectly produce fuels, chemicals, and/or biomass such as, but not limited to, algae, that can be further processed to produce chemicals such as, but not limited to, fuel.

2. Technical Background

[0003] The conversion of solar energy to fuel through the cultivation of photosynthetic algae and cyanobacteria relies critically on light delivery to

microorganisms. Conventional direct irradiation of a bulk suspension leads to nonuniform light distribution within a strongly absorbing culture, and related inefficiencies.

[0004] Growing concern over global climate change and the rising cost of fossil fuels has led to substantial investment and research into alternative fuel sources. For this reason, bioenergy approaches have been developed to produce fuels such as ethanol, methanol, hydrogen and diesel. In order to compete with fossil fuels, however, producing biofuels require large feedstock volumes of inexpensive biomass. Although many feedstocks have been explored, including used cooking oil, food crops, and bio wastes, most suffer from low net energy benefit, poor energy density, large footprint requirements and/or insufficient availability. Alternatively, microalgae, which exhibit high growth rates and oil content compared to higher plants and have and

have the ability to grow in a range of diverse environments, have been used to produce biofuels. In particular, cyanobacteria use solar energy to convert carbon dioxide and water into biofuel, making possible a near carbon neutral petrochemical alternative.

[0005] Cost-effective biofuel production from cyanobacteria is directly linked to the density of cultures within a photobioreactor and its overall volume. Currently, the simplest strategy for cultivation of large volumes of microalgae is an open racetrack- style pond exposed to ambient air and sunlight. However, due to issues related to insufficient light distribution, temperature control, nutrient delivery, contamination, and water consumption, pond operations run at low cell densities. As a result, pond strategies suffer from poor areal productivity and low overall power density.

Consequently, fully enclosed photobioreactors have been designed to provide precise control over the cultivation environment and maintain growth conditions. However, a central problem common to both open and closed cultivation strategies remains the efficient delivery of light to the microorganisms. As cultures increase in both volume and density, it becomes increasingly difficult for light to be distributed evenly to the individual bacteria. In current reactors, areas near the exposed surface tend to be overexposed, resulting in photoinhibition, and large interior regions are effectively in darkness. Flowing, dilute solutions must be employed to circulate bacteria through regions with productive light levels, placing a fundamental limit on culture density and overall power density of this technology.

[0006] A variety of photobioreactor strategies have been developed to provide more effective light distribution to cells by spatially diluting the light over a larger surface area. One strategy is to use light guides to channel light into the reactor volume and subsequently scatter the light into the media. Our reported approach employed cylindrical glass light distributors inserted into a culture tank to assist in distributing light. Sunlight harvested from arrays of Fresnel lenses was channeled to the reactor via optical fiber. Another reported approach used side-lit optical fibers inserted into the culture tank to improve light delivery. Another used optical fibers inserted into the culture chambers with the goal of scattering light collected from external solar collectors into the culture. Although these early studies indicate that increasing control and irradiated surface area within a photobioreactor can improve productivity, these technologies do not escape the fundamental limitation posed by the overexposure and shadowing issues accompanying direct irradiation of bulk cultures.

[0007] An evanescent field is a nearfield standing wave having an intensity that exhibits exponential decay with distance from the boundary at which the wave was formed. Evanescent waves are formed when waves traveling in a medium (e.g., an optical waveguide) via total internal reflection strike the boundary at an angle greater than the critical angle. Evanescent field phenomena are well known in the art.

[0008] In view of the problems and shortcomings identified above and known in the art, the embodied invention provides solutions and advantageous approaches that will benefit and advance the state of the art in this and related technologies.

SUMMARY OF THE INVENTION

[0009] An embodiment of the invention is directed to an optofluidic

photobioreactor. The bioreactor includes an optical waveguide having an input, characterized by an evanescent optical field confined along an outer surface of the optical waveguide that is produced by radiation propagating in the optical waveguide, and a selected photosynthetic microorganism disposed substantially within the evanescent field. As will be more fully understood from the detailed description below, the evanescent field advantageously extends from the waveguide for a distance on the order of the thickness (i.e., minor diameter) of the photosynthetic microorganism that is being irradiated by the evanescent field, thus the microorganism will be understood to be disposed substantially within the evanescent field. According to various non- limiting, exemplary aspects:

- the optical waveguide is an unclad optical fiber having a diameter, d, where 10 μιη < d < 100 μιη, and an input end; - the optical waveguide is a multi-mode optical fiber;

- the photobioreactor further includes two or more optical waveguides disposed in a side-by-side array configuration and having a center-to-center intra-waveguide separation, D, where d < D < 1.5d;

- the photobioreactor further includes a photobioreactor enclosure having an input and an output, inside of which the two or more optical waveguides are disposed, wherein the photobioreactor enclosure is characterized by a plurality of optically-dark fluid channels created by a void space surrounding the plurality of optical waveguides;

- the optical waveguide further comprises a prism waveguide;

- the photobioreactor further includes means for inputting light to the input of the optical waveguide;

- the means for inputting light to the input of the optical waveguide comprises a laser output directly input to a prism waveguide;

- the means for inputting light to the input of the optical waveguide comprises solar radiation channeled to the input of the optical waveguide;

- the photobioreactor includes a liquid microorganism-nutrient media disposed in a void space of the photobioreactor;

- the photobioreactor includes a controller operably connected to the photobioreactor enclosure;

- the photosynthetic microorganism is at least one of a bacterium and algae;

- the photosynthetic microorganism is a cyanobacterium;

- the cyanobacterium is Synechococcus;

- the cyanobacterium is Synechococcus elongatus;

- the photosynthetic microorganism is a genetically-engineered, direct biofuel- producing microorganism;

- the photobioreactor includes a microfluidic chip in or on which a plurality of the optical waveguides are disposed;

- the microfluidic chip is in the form of a high aspect ratio (thin) sheet;

- the high aspect ratio (thin) sheet is corrugated;

- the optical waveguide is a sheet waveguide;

- the sheet waveguide is corrugated; - the selected photosynthetic microorganism is in the form of an adsorbed single layer of the microorganism;

- the photobioreactor includes an artificial adhesive disposed intermediate the outer surface of the waveguide and the selected photosynthetic microorganism such that the microorganism is purposefully adhered to the outer surface of the waveguide.

[0010] An embodiment of the invention is directed to a method for optically exciting a photosynthetic microorganism for generating a biofuel, a biofuel pre-cursor, or a biomass from the optically-excited photosynthetic microorganism. The method include the steps of providing an optical waveguide having an outer surface; inputting optical radiation to the optical waveguide; propagating the optical radiation in the optical waveguide; generating an evanescent optical field adjacent the outer surface of the optical waveguide from the optical radiation propagating in the optical waveguide; providing a photosynthetic microorganism within the evanescent optical field of the optical waveguide; and driving photosynthesis in the microorganism by irradiating at least a portion of a thylakoid membrane of the photosynthetic microorganism with the evanescent optical field. According to various non- limiting, exemplary aspects:

- the step of providing an optical waveguide further comprises providing a prism waveguide;

- the step of inputting optical radiation to the optical waveguide further comprises directly injecting light from a laser into a prism waveguide in a manner to propagate the light by total internal reflection;

- injecting light in a wavelength range 600 nm < λ < 700 nm;

- the step of providing a photosynthetic microorganism within the evanescent optical field of the optical waveguide further comprises providing the photosynthetic microorganism adjacent the outer surface of the optical waveguide in a region extending not more than about five microns (5 μιη) from the outer surface of the optical waveguide;

- the step of providing a photosynthetic microorganism within the evanescent optical field of the optical waveguide further comprises providing an adsorbed layer of the microorganism on the outer surface of the optical waveguide;

- providing a plurality of the optical waveguides disposed in a side-by-side array configuration;

- providing a photobioreactor enclosure having an input and an output, inside of which a plurality of optical waveguides are disposed, wherein the photobioreactor enclosure is characterized by a plurality of optically-dark fluid channels created by a void space surrounding the plurality of optical waveguides;

- providing a microorganism nutrient media in the plurality of optically-dark fluid channels; and

- harvesting a biofuel, a biofuel pre-cursor, or a biomass from the photobioreactor;

- the step of providing a photosynthetic microorganism further comprises providing a free-floating microorganism media within a photobioreactor enclosure;

- inputting solar optical radiation to the optical waveguide;

- providing a controller and controlling a parameter of the optical radiation input to the optical waveguide;

- providing a suitable photosynthetic microorganism and directly harvesting a biofuel from the photobioreactor;

- providing a suitable photosynthetic microorganism and harvesting a biofuel precursor from the photobioreactor;

- providing a suitable photosynthetic microorganism and harvesting a biomass from the photobioreactor.

[0011] Additional features and advantages of the invention will be set forth in the detailed description that follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the invention as described herein according to the detailed description which follows, the claims, as well as the appended drawings.

[0012] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary of the invention, and are intended to provide an overview or framework for understanding the nature and character of the invention as it is claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate various embodiments of the invention, and together with the description serve to explain the principles and operation of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1 schematically illustrates in perspective view an array of optical waveguides in the form of unclad optical fibers and a layer of algae disposed on the outer surface of each of the fibers, according to an illustrative embodiment of the invention;

[0014] Fig. 2 is a schematic overview of a photobioreactor system utilizing the array of optical waveguides shown in Fig. 1, according to an illustrative embodiment of the invention;

[0015] Fig. 3 is a schematic view in cross section showing the evanescent field generated from an optical waveguide as illustrated in Fig. 1 and a Synechococcus elongatus cyanobacterium (algae) disposed on the surface of the waveguide being irradiated by the evanescent field, according to an illustrative embodiment of the invention;

[0016] Fig. 4 shows a schematic top view of the photobioreactor enclosure containing the array of optical fibers with adsorbed photosynthetic bacteria and the dark or void regions (fluid channels) where the evanescent field does not penetrate, according to an illustrative embodiment of the invention;

[0017] Fig. 5 is a photo of a bench-top optical fiber-based photobioreactor according to an exemplary embodiment of the invention;

[0018] Fig. 6 illustrates excitation process and apparatus for photosynthetic bacteria with an evanescent light field; (A) a schematic cross sectional illustration showing evanescent coupling of a photosynthetic bacterium on the surface of a waveguide. The characteristic decay of the light intensity, as plotted at the right of the figure, is on the order of the cell minor diameter size; (B) a schematic illustration of an experimental setup to generate the evanescent wave at the surface of a prism optical waveguide. The input beam is Gaussian and the evanescent field resulting from total internal reflection is elliptical in shape, as shown;

[0019] Fig. 7A: image of cyanobacteria growth pattern resulting from direct irradiation, showing distinct regions of photoinhibition (centre), growth, surrounded by negligible growth; B: plot correlating radial growth intensity to laser light intensity. Outlying peaks beyond 1.2 mm are artifacts of the imaging setup and do not correspond to growth. FWHM thresholds on the growth region correspond to radiant light intensities of 66 W/m ² and 12 W/m ² , shown as upper and lower bounds, respectively, according to illustrative aspects of the invention;

[0020] Fig. 8 graphically shows a spectral power distribution of daylight and an absorption spectrum of PSII plotted with measured absorbance of the S. elongatus culture. The photoinhibition threshold at λ = 633 nn measured from experiments in Fig. 7 (66 W/m ² ) correlates to established photoinhibition intensities of white light exposure, when related through the absorption spectrum shown, according to an illustrative aspect of the invention;

[0021] Fig. 9 illustrates the theoretical light intensity distribution in the evanescent light field, and corresponding predicted growth patterns: A: plot of the penetration depth as a function of incident laser angle for a glass-media interface. Penetration depth is defined as the location where field intensity drops e ^"2 , or 87%, from that at the surface. The dashed line indicates a penetration depth of 1 μιη occurring at 9i ₂ = 9c + 0.074°, and the geometry of S. elongatus is shown inset for reference; B: surface plot of evanescent field, 1 μιη from the surface, with power intensity plotted to indicate the photoinhibited, growth, and negligible-growth regions, based on thresholds measured for radiant light. Based on these values an elliptical ring pattern of growth is predicted, as shown by the useful portion of the power spectrum shown in (green) shaded region 2. The vertical line plot indicates the useful light intensity decay with distance, according to illustrative aspects of the invention; and

[0022] Fig. 10 illustrates the growth of photosynthetic bacteria using evanescent light: A-C: Images of cyanobacteria growth patterns resulting from evanescent excitation at the glass-media interface for incident light powers of 1 mW, 0.5 mW, and 0.25 mW, respectively. The elliptical growth patterns correspond to the evanescent field geometry, and show distinct regions of photoinhibition (centre), and growth, surrounded by negligible growth; D-F: Corresponding growth profiles for each light power with the corresponding evanescent field intensities plotted at the surface, 1 μιη above the surface, and as a 5 μιη average. The power range determined from the direct radiation experiments (Fig. 7) is shown by the red band for reference. The full-width at half maximum indicating growth onset is observed at 1 μιη intensity levels of 79 + 10 W/m ² , and observed at 60 + 8 W/m ² for the 5 μιη average light intensity. These values bracket the 66 W/m ² threshold determined for radiant light at this wavelength, according to illustrative aspects of the invention.

DETAILED DESCRIPTION

[0023] Reference will now be made in detail to the present exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0024] Fig. 1 schematically illustrates in perspective view an array of optical waveguides 100 in the form of unclad optical fibers 101 having a diameter, d, where 10 μιη < d < 100 μιη, and a single layer of algae 151 disposed on the outer surface of each of the fibers. By propagating light in the optical fibers, an exponentially decaying optical field (referred to as an evanescent field) 303 is created over the surface of the optical fiber. Fig. 3 is a schematic view in cross section showing the evanescent field 303, and a Synechococcus elongatus cyanobacterium (algae) 351 adsorbed on the surface 107 of the waveguide 101 being irradiated by the evanescent field. In a non- limiting alternative aspect, the bacteria may be artificially attached to the waveguide surface with a suitable adhesive material.

[0025] Fig. 2 illustrates a photobioreactor system 200 in which solar radiation is collected (202) and coupled into the optical fibers than run through the reactor volume in a reactor enclosure 207. The photosynthetic mechanics of the cyanobacteria is contained in the thylakoid membranes (~ 100 nm) that surround the cyanobacteria, as illustrated in the inset 212. The evanescent field extends up to about 1 μιη from the unclad fiber surface (Fig. 3) and provides the proper light intensity, Io (see 214, Fig. 2), to the bacteria. Due to the exponential decay of the evanescent field, as illustrated by the graph inset 214, a dark region 218 occurs in the interstitial space between the fibers, which serve as fluid channels (see also Fig. 4). These fluid channels can be used for transport of C0 ₂ and media to the bacteria, and for the collection of produced biofuel. The use of the evanescent field allows for the optimum utilization of light by the bacteria and for enhanced volume utilization for the reactor as a whole.

[0026] Fig. 4 shows a schematic top view of the photobioreactor enclosure 207 containing the array of optical fibers 101 with adsorbed photosynthetic bacteria 151 and the dark or void regions (fluid channels) 218 where the evanescent field 303 does not penetrate. The fibers have a center-to-center intra-waveguide separation, D, where d <

D < \.5d.

[0027] Fig. 2 further illustrates a control component 237 that enables parameters including, but not limited to, wavelength, intensity, and duty cycle of light in the fibers to be tuned with precision such that the bacteria are optimally exposed.

[0028] For the direct conversion of C0 ₂ to biofuel, genetically modified S.

elongatus SA665, produced at UCLA, has shown the ability to directly convert C0 ₂ to isobutyraldehyde, which may be converted to isobutanol (a gasoline substitute), at area- wise efficiencies comparable or greater to current biofuel production strategies.

[0029] An estimate of achievable density for the illustrated optofluidic bioreactor architecture is useful for comparison with current systems. A hexagonal packing of the optical fibers as shown in Figure 4 would provide an absorbed layer of fuel producing Synechococcus Elongatus occupying 10% of the total volume, while the remaining 90% of the volume is required for optics and fluid. A density of 10% active bacteria by reactor volume is 4500-fold greater than demonstrated tube photobioreactors known in the art and eight orders of magnitude greater than that of a comparable pond reactor. This scaling suggests that the output of a one-story 5680 m ² tube facility could be matched by a table-sized (2.5 m ³ ) optofluidic reactor 500-1 according to the embodied invention as shown in Fig. 5. While such a reactor would require a separate photocollector and associated irradiated area, the separation of reactor with collection additionally offers the ability to tailor the light spectrum to the photosynthetically active range (400-700 nm); cycle light at an optimal frequency for photosynthetic activity (typically 100 Hz); collect incident solar light at high angles as required outside of equatorial regions; and control the reactor temperature making operation feasible in colder climates where fuel demand is high, such as North America and Europe.

Detailed Exemplary Embodiment [0030] According to another exemplary embodiment and aspects thereof, Fig. 6A and 6B, respectively, illustrate the evanescent excitation process 600-1 of a bacterium 651 and a prism waveguide-based photobioreactor 600-2 used to generate the evanescent excitation field 303. As more particularly illustrated in Fig. 6B, a prism waveguide 627 is shown in cross section. Monochromatic red (λ = 633 nm) light 606 provided by a HeNe laser 605 is directly injected into the side of the prism waveguide. The evanescent field 303 is generated at the interface surface 617 of a glass slide 642 where the light is totally internally reflected. Total internal reflection, and the corresponding evanescent field, result when the light is incident at the glass-media interface at angles greater than the critical angle; i.e., Q _i2 > 9 _C . Reflecting a circular cross-section input beam creates an elliptical evanescent field profile on the prism surface at the point of reflection, as shown in the inset 653 in Fig. 6B. It will be appreciated by those skilled in the art that there are alternative ways to generate an evanescent field; however, the illustrated approach is simple and provides an evanescent light field distribution that can be reliably described with theory in all three dimensions.

[0031] In the experimental set-up of Fig. 6B, cavities to contain a bacteria culture solution were fabricated by moulding PDMS (Sylgard(R) 184 Elastomer Kit, Dow Corning) around a poly(methylmethacrylate) (PMMA) master to create cylindrical cavities 10 mm in diameter and 4.75 mm deep (0.373 mL). These culture cavities were bonded to the surface of a 1 mm thick BK7 glass microscope slide using oxygen plasma treatment.

[0032] Cells of the wild type S. elongatus (ATCC 33912) cyanobacteria were used to demonstrate the embodied invention. Cells were cultured under optimal conditions of 32-36 °C and under continuous irradiation of 50-75 uE-m ^"2 - s ^_1 using fluorescent lamps. The stock culture was kept at a constant cell density (in the exponential growth phase) by regularly diluting the culture with fresh BGl 1 cyanobacteria growth medium (Sigma Aldrich C3061) to maintain a constant optical density of 0.2 at 750 nm (OD ₇₅₀ ). The OD75 ₀ was determined using a broad spectrum halogen light source (Thorlabs OSL1) and spectrometer (Edmond brcl 12e) and normalized to the OD ₇₅₀ of fresh BGl 1 growth media. Samples of this culture were used in our experiments. [0033] Once mounted to the glass plates and inoculated (dead end filling via syringe injection), the cultures were placed on the top faces of right angle BK7 prisms (Thorlabs PS908L-A), as shown in Fig. 6. Optical contact was achieved using an index matched immersion oil (Leica 11513 859). Light was coupled to the chamber from a helium neon laser (632.8 nm Thorlabs HRR020) directed toward the prism by reflecting it off a broadband dielectric mirror (Thorlabs CM1-4E; not shown) mounted to a precision rotation mount (Thorlabs CRM1P; not shown). The incident angle at the glass media interface was adjusted by changing the angle of the mirror in the rotation mount. The prism/culture assembly was mounted to a sliding stage (not shown), which allowed the laser beam to be maintained in the center of the culture chamber as the angle of incidence was varied. The prism assembly was aligned such that the reflection of the beam leaving the prism did not pass through the culture. This ensured that optical excitation of the bacteria was solely due to the evanescent field where the beam was totally internally reflected at the glass-bacteria culture interface.

[0034] Laser beam power into and out of the prism was measured using a photodiode power sensor (Thorlabs S120C) and measured once at the beginning of the experiment and once at the end. The entire experimental apparatus was optically isolated in enclosures made from 5 mm thick hardboard (Thorlabs TB4). These chambers were kept at a constant temperature of 32-36 °C for optimal cell growth rates for the duration of the evanescent growth experiments using a 950 W enclosure fan heater (CR030599, OMEGA Engineering Inc., USA).

Experimental Results and Discussion

[0035] Cell cultures were first placed under direct laser light exposure, to establish the effectiveness of using monochromatic red (λ = 633 nm) at growing S. elongatus, and measure cell response to direct radiation light. The beam from a Helium-Neon (HeNe) laser was passed through a culture cavity perpendicular the bottom glass slide (Fig. 6B) and the culture was left to grow for 72 hours (under conditions described above). This type of direct irradiation experiment was done for various laser powers, yielding consistent results to those shown in Fig. 7. Fig. 7A shows a typical growth ring pattern 700-1, where the effect on growth from the three distinct intensity regions 712 (center), 714 (mid), 716 (outer) is evident as shown. There is a bleached (yellowish orange in color view) region 712 in the center, a growth region 714 (green in color view) and a negligible growth outer region 716. To quantify growth in a radial profile, the image was filtered for green intensity and integrated in circumference. The resulting radial growth profile is plotted with the laser intensity profile in Fig. 7B. The threshold electric field intensities (low and high) between regions were determined from the intersection of the full width at half the maximum (FWHM) growth locations and the incident light power profile. The resulting threshold values of 66 W/m ² and 12 W/m ² (shown in Fig. 7B by the rectangle within the growth peak) indicate the productive growth intensities of S. elongatus under direct irradiation at λ = 633 nm.

[0036] Relating these direct irradiation experiment results to known growth characteristics of S. elongatus requires determining the daylight equivalent power of red light at λ = 633 nm. To do so, we compared radiometric measurements of daylight to optimal intensity ranges published in literature. At high light intensities, the rate of radiation induced damage to the cell's photosystems exceeds the cell's ability to repair itself and the result is a sharp decrease in photosynthetic activity, or photoinhibition. High light conditions that approach saturating intensities are reported as a

Photosynthetic Photon Flux Density (PPFD) on the order of 150 uE-m ^"2 - s ^_1 to 500 μΕ-π ² ^ ^"1 in the Photosynthetically Active Radiation (PAR) wavelength range (400- 700 nm), or 10% -25% of full daylight. To convert the photosynthetic photon flux density to radiometric units (i.e. W/m ² ), the optical power of full daylight was measured at 635 nm to be 1.37 W/m ² (48°25 ^' 43" N, 123°21 ^' 56" W). The spectral power distribution of normal daylight 806 was then calculated from this set point and the relative spectral power distribution defined by CIE Standard Illuminant D65, as shown in Fig. 8. Total full daylight irradiance of photosynthetically active radiation was calculated to be 472 W/m ² , which corresponds to a photosynthetic photon flux density of 2137 uE-m ^"2 - s ^_1 . This value was independently confirmed by a QSR-2100

(Biospherical Instruments Inc.) light meter measurement of 2100-2300 μΕ-π ² ^ ^"1 . Also shown in Fig. 8, is the absorption spectrum 804 for Photosystem II (Sugiura M & Inoue Y (1999) Highly Purified Thermo-Stable Oxygen-Evolving Photosystem Π Core Complex from the Thermophilic Cyanobacterium Synechococcus elongatus Having His-Tagged CP43, Plant Cell Physiol. 40(12): 1219-1231). Photosystem Π (PSH) is the link in the photosynthetic pathway most susceptible to light induced damage and is the first point of failure in high light environments. The characteristic shape of the published absorption spectrum can also be observed in the measured absorption spectrum of the sample culture 802 (OD ₇₅₀ of 0.37), most notably at the -450 nm, 630 nm and 670 nm peaks as plotted Fig. 8. Absorption in the red region of the spectrum contributes most significantly to photosynthesis while absorption of lower wavelengths is due to the presence of molecules not directly involved in the electron transport process. Under normal daylight conditions, S. elongatus PSII absorbs 30 W/m ² of red light (600 nm < λ < 700 nm) determined by weighing the spectral power distribution for daylight by the absorption spectrum of PSII and integrating across the red portion of the spectrum. In order to deliver an equivalent amount of energy using monochromatic laser light at λ = 633 nm, the ability of PSII to absorb at that wavelength needs to be considered to determine the appropriate corresponding laser power. In this case, PSII absorbs approximately 13% of 633 nm light, which requires a laser power of 230 W/m ² to simulate full daylight conditions. The threshold measured in the direct irradiation experiments, 66 W/m ² , therefore suggests that -28% of full daylight is the upper limit for our cultures before severe photoinhibition occurs. This value agrees well with the upper bounds of what are considered high- light conditions as reported in the literature.

[0037] The light intensity distribution in an evanescent light field varies both in the plane of the surface and depth-wise into the media. Established theory was applied to describe the evanescent electric field intensity and used to correlate field strength to experimental growth results. Fig. 9A shows the penetration depth 904 (y-axis) of the evanescent light field as a function of incident angle (x-axis). Here, the penetration depth is quantified as the location where the field intensity drops e ^"2 , or 87%, of the peak intensity at the surface. The geometry of S. elongatus is shown inset (909) in Fig. 9A for reference, and the dashed line 910 indicates a penetration depth of about 1 μιη, which occurs at an angle of incidence of Q _i2 = 9c + 0.074°. As shown, the penetration depth of the evanescent field is a strong function of incident angle, with values corresponding to the inherent lengthscale of the bacterium occurring only near the critical angle (below 0.5° past critical). [0038] Fig. 9B shows the predicted evanescent field intensity in the plane, and the characteristic oval shape for an incident 0.5 mm diameter Gaussian beam at λ = 633 nm. The intensity values indicated correspond to the evanescent light intensity at 1 μιη from the glass-media interface, with an incident angle of 62° (θ ₁₂ = 9 _C + 0.5°) and penetration depth of 400 nm. Based on the above-determined threshold light intensity for the red light employed here (66 W/m ² , at 633 nm), the expected growth regions can be predicted based on the calculated evanescent field intensity. As shown in Fig. 9B, in region 1 the evanescent field intensity exceeds the red component of 10% daylight and would be expected to lead to photoinhibition in a radiant light system. This analysis would predict an elliptical ring pattern of growth, as shown by the useful portion of the power spectrum, region 2 (green shading in color diagram). The vertical line plot indicates the useful light intensity decay with distance. Relatively intense growth is expected near the inside boundary where useful light intensities are high, and growth rates would decay with the light intensity outward. Although the sharpness of the inside edge of the growth profile is an artifact of the threshold boundary condition, the model provides the predicted pattern of growth for a photosynthetic microorganism cultured in this evanescent field.

[0039] Evanescent light based excitation of the culture was performed using the experimental setup shown in Fig. 6B. Three laser powers were employed (1 mW, 0.5 mW, 0.25 mW) with incident laser angles of 62° (9j ₂ = 9 _C + 0.5°), and total internal reflection was ensured by measuring the output intensity. Each experiment was performed in triplicate and the cultures were exposed to the evanescent field for 72 hours. Figs. lOA-C show substantial bacteria growth in response to the evanescent light field at the surface of the glass-media interface. The growth patterns showed the elliptical shape mirroring the evanescent light field intensity, and delineate the three characteristic regions (photoinhibition, growth, negligible-growth), providing data on the onset of growth under evanescent light. As the laser power was reduced (Figs. 10A to C), the radial distribution moved inward, consistent with the change in the light intensity profile. To relate the observed growth to the evanescent field intensity, the images were filtered for green intensity, scaled along the axis of the beam, and integrated to provide growth profiles. Figs. 10D-F show the growth profiles for each light power with the corresponding evanescent field intensities plotted at the surface, 1 μηι above the surface, and as a 5 μηι average. Due to rapidly decaying nature of the evanescent field, the surface intensity is much higher than that at 1 μιη above the surface, which is also similar to the average intensity over the first 5 μιη (both 1 μιη and 5 μιη are relevant lengthscales of this rod-shaped bacteria). The power range determined from the direct radiation experiments is shown by the red band 1090 for reference. The onset of growth occurs at a radial location where the evanescent light intensity - as measured at 1 μιη and as a 5 μιη average - drops to a value corresponding to the threshold of 66 W/m ² , established from direct radiation experiments. As the total intensity of light is decreased (Figs. lOA-C and D-F), the location of the onset intensity moves inward, and remains consistent with the predicted power curves.

Specifically, the full- width at half maximum, indicating growth onset, is observed at 1 μιη intensity levels of 79 + 10 W/m ² , and observed at 60 + 8 W/m ² for the 5 μιη average light intensity. These results both demonstrate growth of photosynthetic bacteria using evanescent light, and provide metrics for their successful cultivation within this unique light field.

[0040] The growth patterns shown in Fig. 10 show some downbeam bias, that is, growth intensity increases with distance from the laser source. When the cells interact with the evanescent field near the surface, some of the light is absorbed and utilized, while some of the light is scattered. The light will be scattered preferentially in the direction of the beam. With the present experimental setup, this scattered light would contribute to higher growth rates, and thicker biofilms, on the downbeam side of the ring pattern. This effect was noticed in most cases with downbeam growth biases of 1%, 8%, and 15% for the 0.25 mW, 0.50 mW, 1.0 mW cases plotted in Fig. 10.

Although the extent of this bias varied between trials, and some trials showed negligible, and even a small upbeam bias, the effect was in general small and in all cases less than 15%. While it is likely that downbeam bias and secondary scattering effects influence growth, the relative symmetry of the growth patterns indicates that the downbeam scattering effect is minor. [0041] The additional effect of light penetration depth was investigated using incident light at larger angles past critical (9c < < 9c + 5). At angles greater than 0.5° over critical (as plotted in Fig. 10), however, only faint growth rings were observed. We attribute the lack of growth at larger angles to the change in penetration depth, which diminishes rapidly with increasing incident angle, as shown in Fig. 9A. Specifically, the penetration depth corresponds to the minor-dimension of the rod- shaped bacterium (1 μιη) only at angles less than 9c + 0.074°. These results are thus consistent with the observed evanescent growth patterns in that the penetration depth approached the cell diameter only at small angles away from critical.

[0042] We have thus demonstrated an evanescent- light based approach to deliver light on the lengthscale of cyanobacteria for photosynthesis. In addition to

demonstrating cultivation of bacteria in the evanescent field, analysis of the growth pattern provides guidelines for determining appropriate evanescent light based exposure conditions from known radiant light response. Growth can be predicted based on these metrics both with respect to light penetration depth and light intensity. In the context of photobioreactor technology, this approach to light distribution differs from

conventional approaches of bulk irradiation in that it offers a means of controlling the energy delivered to individual cells rather than bulk cultures. A photobioreactor architecture based on the embodied evanescent light delivery approach could take several forms. For example, one approach could be to leverage the low cost and relative ubiquity of fiber optic technology in a reactor with a dense network of bacteria- absorbed fibers, or fabric, in a large media vessel. Paired with recent advances in the genetic modification of cyanobacteria for direct production of fuels such as ethanol or isobutyraldehyde/isobutanol, such a strategy presents new opportunities for solar fuel generation.

[0043] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0044] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having,"

"including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. The term "connected" is to be construed as partly or wholly contained within, attached to, or joined together, even if there is something intervening.

[0045] The recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0046] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate embodiments of the invention and does not impose a limitation on the scope of the invention unless otherwise claimed.

[0047] No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0048] It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit and scope of the invention. There is no intention to limit the invention to the specific form or forms disclosed, but on the contrary, the intention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention, as defined in the appended claims. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.