WO2012024406A1 - Production of bio-based materials using photobioreactors with binary cultures - Google Patents

Abstract

A method, device and system for producing preselected products, (either finished products or preselected intermediary products) from biobased precursors. The principal features of the present invention include a method wherein a binary culture is incubated with a biobased precursor in a closed system to transform at least a portion of the biobased precursor to a preselected product. This improvement leads to significant savings in energy consumption and allows for the design of photobioreactors of any desired shape. The present invention also allows for the use of a variety of types of waste materials to be used as the organic starting material.

Description

PRODUCTION OF BIO-BASED MATERIALS USING

PHOTOBIOREACTORS WITH BINARY CULTURES

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with Government support under

Contract DE-AC0576RLO1830 awarded by the U.S. Department of Energy, The

Government has certain rights in the invention.

CLAIM TO PRIORITY

[0002] This application claims priority from US Patent Application No,

12/858,338 filed 17 August 2010, which is a continuation in part of US Patent

Application No. 12/555,631 filed 8 September 2009, which incorporates and claims priority to provisional patent application nos. 61/095,413 filed 9

September 2008 and 61/099,380 filed 23 September 2008. The present application also claims priority from Provisional Patent Application 61/467,608 filed 25

March, 2011.

BACKGROUND

[0003] Rising fuel prices and global climate change concerns have revived the interest in renewable sources of energy. Using solar energy to grow photosynthetic microorganisms is one of the most attractive ways to produce transportation fuels. Successful implementation of biodiesel via seed crops is one example of employing plant-based photosynthesis for fuel production. However, recent assessments of crop-based fuel economy showed that it can lead to food stock deficiency and drive lifecycle emissions of greenhouse gases up through increased land usage. Utilization of photosyntheti c microorganisms for primary biomass production has many advantages over growing crops. In particular, arid regions of the western U.S.,. for example, could be used for large-scale production excluding the competition with food-producing agriculture.

[0004] Cultivation of photoautotrophic microorganisms for metabolite and/or biomass production can be accomplished in various types of cultivation systems including open ponds and enclosed bioreactors. Each system has various advantages and limitations. Open ponds, for example, are designed to utilize natural sunlight while most of the enclosed bioreactor systems do require artificial illumination which results in additional energy expenditures. Open pond systems, however, are more prone to fouling by external contamination and are not suited to grow genetically modified organisms. In contrast, enclosed bioreactors provide highly controlled conditions, protection against external contamination, and higher growth rates and biomass/products yields while allowing use of genetically modified strains. Cultivation of photosynthetic organisms is also associated with several general problems which arise from the necessity to deliver CO2 into liquid medium and remove excess O2 produced as a result of photosynthesis in order to maintain desired growth conditions. The current practice is to continuously or periodically purge the system which adds significantly to the operating costs and results in frequent changes of cultivation conditions and reduction in efficiency. Removal of O2 by most other known methods such as by chemical catalysis is typically prohibitively costly. What is needed therefore is a solution that enables continuous operation under controlled conditions such as within an enclosed bioreactor without the need for venting as is required by the prior art. The present invention meets this need.

SUMMARY

[0005] The present invention is a method, device and system for producing preselected products, (either finished products or preselected intermediary products) from biobased precursors. The principal features of the present invention include a method wherein a culture consisting of two microorganisms (binary culture), is incubated with a biobased precursor in a closed system to transform at least a portion of the biobased precursor to a preselected product. However, a variety of other aspects including the partiailar systems and devices which enable the method of the present invention to be performed are also described and disclosed herein.

[0006] hi some embodiments the binary culture includes at least one oxygenic photoautotroph and at least one aerobic or facultative anaerobic chemoheterotroph. In other embodiments the pairings may include any set of organisms appropriately combined so that the rates of the growth of the two strains of organisms are maintained in an appropriate balance and the internal environment is maintained in a desired condition. This includes but is not limited to pairings of various strains of bacteria, algae, fungi and plant species and combinations thereof. Examples could include but are not limited to pairings of facultative aerobic and anaerobic organisms to produce a self- sustaining oxygen /carbon dioxide balance, as well as other combinations wherein the two organisms produce desired or required nutrients or growth enhancing materials.

[0007] In some embodiments the binary cu ltures are incubated in a closed chamber and artificially illuminated by variously placed light emitting diodes which are controlled by a control mechanism based upon interaction with a plurality of sensors within the chamber. In other applications the present invention may utilize natural sunlight or ambient light or combinations of ambient and directed light in order to obtain a desired effect. In one

embodiment of the invention, the system is completely artificially lighted and the emissions from the light emitting sources are coordinated bv interaction between a these light emitting sources preferably (light emitting diodes) having selected characteristics such as wavelength, frequency, intensity or other features and at least one sensor (preferably a plurality of sensors are utilized) that is located within the chamber by a computer program. In addition to the maintaining of optimal lighting conditions within the chamber, the temperature of the internal contents of the chamber can also be variously monitored and controlled to provide an optimal temperature for growth. A heat sink enables excess or unwanted heat to be removed from the chamber, while the light inside the chamber typically provides the heating means for raising the internal temperature to a desired value. In one embodiment of the invention, the heat sink is an integrated part of the cylindrical chamber, in other embodiments additional heat sinks may be attached to the cylinder in order to obtain a desired result. Preferably a cylinder having a height to width aspect ratio of at least 2 is considered optimal in such circumstances.

[0008] In one embodiment of the invention the binary culture includes at least one photoautotroph and at least one chemoheterotroph. In one example this may be selected from any of a variety species including but not limited to Shewanella species, Cyanothece species, Synechococcus species and other species appropriate for the particular necessities of a user. One of the advantages that the present invention provides is that unlike previously used methods for O2 removal/CO2- delivery, this method of cultivation does not need purging of a closed bioreactor with a defined gas phase. In contrast to most prior art practices for O2 removal/C02 delivery required for microalgae cultivation, the proposed approach accomplishes these tasks simultaneously by inclusion of a compatible heterotrophic microorganism. This method of cultivation does not require sparging of a closed bioreactor with air or other gas mixture/vigorous mixing to deliver CO2/remove produced O2 from liquid medium. This improvement leads to significant savings in energy consumption and allows for the design of photobioreactors of any desired shape to ensure optimal photoautotrophic culture illumination and space usage which will ultimately result in designing- more efficient processes with substantial increases in biomass production and/or product generation. Additionally, use of highly reduced organic compounds will help to consume externally added CO2 or a salt of carbonic acid without necessity to remove O2.

[0009] The present invention also allows for the use of a variety of types of waste materials to be used as the organic starting material. For example, biosludge produced from sewage water treatment plants or glycerol, a major dead-end by-product in biodiesel production can be utilized, in addition the present invention can be utilized for the production of microalgae biomass as feedstock for high-quality biofuels (biodiesel and biocrude) that require a minimum of post-production processing. The present invention also enables the design of light-driven processes for bio-Irk production and the production of organic fertilizers, animal feed, and other commodities including but not limited to vitamins, amino acids, antibiotics, or enzymes. The present invention solves the problems associated with the prior art by utilizing binary cultures of paired organism to produce self-sustaining interdependenci.es that foster the continued growth and development of the organism producing the desired biomass material within the closed system wherein the growth environment can be carefully monitored and maintained. Additionally, more than two cultures may be grown together as necessary. Such an approach provides a cost-efficient way to eliminate problems associated with the prior art methodologies.

[0010] In one embodiment of the invention the paired binary cultures are configured to provide CO₂ delivery and O₂ removal while creating high-value products by utilizing sun light, artificial light or their combination and organic matter (waste or renewables). In one exemplary embodiment a photoautotrophic organism such as a microalga or a cyanobacterium is paired with an aerobic or facultative anaerobic heterotrophic bacterium. The phototrophic oxygenic microorganisms can produce biofu els at a much higher productivity than land plants and can be cultivated in aquatic environments, including seawater, so as to not compete for resources with conventional agriculture. In most prior art systems high costs associated with increasing the mass transfer and by-product (O2) removal limit its use, however in the present embodiment these materials are consumed by an aerobic or a facultative anaerobic heterotrophic bacteria and the desired level of homeostasis within the closed chamber is maintained. This method can be employed by a variety of systems wherein binary cultures of paired organisms cooperatively co-exist to maintain a desired growth

environment depending upon the particular needs and necessities of a user.

[0011] The purpose of the foregoing abstract is to enable the United States

Patent and Trademark Office and the public generally, especially the scientists, engineers, and practitioners in the art who are not familiar with patent or legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The abstract is neither intended to define the invention of the application, which is measured by the claims, nor is it intended to be limiting as to the scope of the invention in any way.

[0012] Various advantages and novel features of the present invention are described herein and will become further readily apparent to those skilled in this art from the following detailed description. In the preceding and following descriptions We have shown and described only the preferred embodiment of the invention, by w^ray of illustration of the best mode contemplated for carrying out the invention. As will be realized, the invention is capable of modification in various respects without departing from the invention. Accordingly, the drawings and description of the preferred embodiment set forth hereafter are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a schematic view of the system and process of the present invention.

[0014] Figure 2 a is an exemplary view of one embodiment of a reactor system of the present invention.

[0015] Figure 2 b is an exemplary view of another embodiment of the reactor system of the present invention

[0016] Figures 3 and 4 are charts showing the independent growth phases of Synechococcus and Shewanella examples as compared to the binary culture.

[0017] Figures 5a and 5b shows the efficiency of the use of lactate in the various tested cultures.

[0018] Figure 6 shows the results of light testing of the Synechococcus culture.

[0019] Figures 7 and 8 show the quantity of electricity used for agitation and gas sparging per unit biomass.

[0020] Figures 9 and 10 show the results of astaxanthin production and nutrient reduction in one example of the invention.

[0021] Figure ϋ shows the growth of a M. alcaliphilum and

Synechococcus co-culture growth. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following description includes various preferred mode of various embodiments of the present invention. It will be clear from this description of the invention that the invention is not limited to these illustrated embodiments but that the invention also includes a variety of modifications and embodiments thereto. Therefore, the present description should be seen as illustrative and not limiting. While the invention is susceptible of various modifications and alternative constructions, it should be understood, that there is no intention to limit the invention to the specific form disclosed, but, on the contrary, the invention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention as defined in the claims.

[0023] As a proof of principle, we have used a binary culture of a photoautotrophic oxygenic cyanobacterium and a heterotrophic facultative anaerobic bacterium and cultivated them in a non-aerated photobioreactor with addition of organic carbon. During this process, the binary culture produced higher amounts of microalgal biomass without gas sparging (to remove O2 produced during photosynthesis) or additional CO2 injections. While specific examples are described hereafter and provided herein it is to be distinctly understood that the invention is not limited to these described configurations but that a variety of additional configurations and embodiments may be variously and alternatively configured according to the particular needs and necessities of the user. These binary culture systems of phototrophic organisms allow for efficient design and cost effective production processes for directing carbon and nutrients flow from CO2 and waste towards of biofuels: lipids, hydrocarbons and other preselected materials. The examples and descriptions provided herein should therefore be seen as illustrative in nature and not limiting.

[0G24] To prove this concept various experiments were performed. In one embodiment of the invention a binary culture of a photoautotrophic organism Cyanothece sp. strain ATCC 51142 was cultured in association with a facultative anaerobic heterotrophic bacterium Shewanella sp. strain W3-18-1 on defined mineral medium which was not supplemented with CO2, or bicarbonate salts as source of carbon. The mineral medium was supplemented with lactate as source of carbon and reducing equivalents to remove the O2. During this process, both organisms in this binary culture w^rere dependent on the metabolism of the other: Shewanella sp. W3-18- 1 consumed lactate and O2 and produced CO2 and

Cyanothece sp. ATCC 51142 incorporated the CO2 into the biomass and produced O2. During this process both cultures in the photobioreactor grew without air sparging or addition of supplemental CO2. In the presence of Shewanella sp. W3- 18-1, Cyanothece sp. ATCC 51142 was able to grOW at higher rates when compared to single-organism culture under identical conditions with sparging with CO2, and N₂.

[0025] In another embodiment of the invention an approach was utilized wherein binary photoautotroph-heterotroph cultures were used to spatially separate the processes of photosynthesis and photosynthate conversion into useful products (Fig, 1). This in particular allows for the cultivation of readily- engineered heterotrophic strains for major biotechnology products using CO2 and Sight instead of commodities such as glucose, sucrose, or other agricultural or synthetic feedstocks. Particularly, phototrophic oxygenic microorganisms that have been genetically or otherwise modified to produce and excrete a soluble organic compound(s) such as glycerol, lactate, pyruvate, acetate or any other organic compound that can be used by a heterotrophic organism as the sole source of carbon and energy to grow and/or synthesize a product of interest. Moreover, O2 as well as carbon and energy source(s) for the heterotrophic organism will be uniformly produced in the liquid culture by phototrophic component of a binary culture, ensuring absence of shock by periodic excess or deficiency of nutrients and oxidants that conventional types of cultivation usually suffer. An aerobic or facultative anaerobic heterotrophic organism will consume O2 produced as the resu lt of photosynthesis, thus dramatica lly decreasing mass transfer energy expenditure and simplifying photobioreactor design and operation. Additional CO2, produced by heterotroph will be again recycled by phototroph thus ensuring efficient utilization of carbon source(s). The binary culture approach also allows the utilization of various carbon sources ranging from CO2 from power plants to municipal wastes. Because of the robustness of the phototroph-heterotroph association, the binary cultivation provides a novel platform for the development of consolidated bio-processing methods leading to production of carbon-neutral products at reduced economic and energetic costs.

[0026] In one set of experiments one embodiment of the system, device and method of the present invention was compared against a prior art system, device and method. A description fol!ow^rs:

Materials and Methods:

Bioreactor:

[0027] A New Brunswick Scientific BioFlo 3000 bioreactor with a custom 7.5 Liter vessel was used with a 5.5 Liter working volume. The pH was held at 7.4 with 2 M NaOH and 2 M HCl when necessary. The temperature for all experiments was 30°C. The high agitation experiments were clone with 250 rpm, and the low agitation with 50 rpm. Sparging was done with pure air at 2.8 L/min (optimal condition) for the Sheioanella cultures, with 99% N?_ and 1% CO2 for Synechococcus sp. PCC 7002 cultures, or with no bubbling for the

Synechococcus 7002/Shewanella W3-18-1 co-cultures. Batches at 50 rpm agitation and 2.8 L/min aeration were also done with pure cultures of Synechococcus or Shewanella to show effects of low mass-transfer on a pure culture (suboptimal condition 1). Two batches at 50 rpm and 0.5 L/min aeration were done with Synechococcus (suboptimal condition 2).

Photobioreactor;

[0028] A custom photobioreactor enclosure (developed by Pacific

Northwest National Laboratory and shown in Fig. 2) was used for these experiments. This photobioreactor 10 includes a vessel 12 comprised of an outer cylinder 14 made of black anodized aluminum and an inner cylinderl6 made from borosilicate glass. The total height of the vessel 12 in this embodiment was set out at 19.5 inches. The inner diameter was 5.274 inches. The outer diameter (at the flange) was 6.5 inches. While these dimensions were provided in this instance, it is to be distinctly understood that the invention is not limited thereto. However, in the present embodiment it was found that a higher aspect ratio of height to diameter was more effective for enhancing growth within the chamber due to the enhanced ability to allow light to penetrate into the center of the chamber. ^'The bioreactor in this case had no additional ports placed upon the vessel 12. The anodized aluminum shell 14 acts as a mounting point for light emitting diodes (LED) 18 as a heat sink, and as a light shield for ambient light. The black anodized coating of the aluminum absorbs reflected light and acts as an efficient heat-transfer material.

[0029] A door 20 on the aluminum shell permits the user to view the inner workings of the photobioreactor 10 as needed. Rubber fasteners 22 securely close the door during normal operation to prevent light from entering or exiting the photobioreactor. Foam rubber seals 24 and rubber gaskets 26 are used on mating surfaces of the photobioreactor 10 to keep it light-tight. The headplate 28 for the photobioreactor is configured to allow probes (to monitor various factors such as pH, DC), level/foaming, and temperature) 30, tubing 32 and an impeller shaft 34 for an agitator to reach the bottom of the vessel. (In this particular example an additional port was added for a CO2 probe for testing- purposes but such an addition is not required to allow for proper functioning of the photobioreactor. Similarly an exhaust condenser was included on this embodiment of the application (for testing purposes), in other applications various heat sinks 36 may be added. This is the only photobioreactor design that combines a large working volume, thorough mixing in one common chamber, optimal light delivery, LED lighting, selectable wavelength light, real-time light- intensity measurement and control, modeling of real outdoor lighting patterns and intensities, shielding from ambient room light, pH control, dissolved oxygen control, temperature control, level or foam control, gas mix control, off-gas monitoring, aseptic culturing conditions, and continuous-culture capability in one photobioreactor. This embodiment also provides various advantages in regulating mixed or binary cultures to ensure that appropriate conditions are maintained to support desired rates of growth and preserve and foster desired growth and/or material conversions within said system.

[0030] Electronic circuitry 38 directs power from a power supply 40 to the bioreactor 10. A custom control software called Biolume (developed by Derek Hopkins of PNNL) interacts with sensors 40 within photobioreactor and the interactive electronic circuitry to regulate the desired levels of light provided to the photobioreactor. For example in one embodiment of the invention the software controls maintains a specified light pattern by coordinating the flow of electricity to light sources (LEDs) based upon input from sensors 40 and a preselected program included within the device. This allows for light intensities to be varied over periods of time so as to provide lighting cycles that best enhance the growth for materials that are included therein. For example, in one embodiment of the invention light intensity and characteristics are adjusted over time to replicate the diurnal fluctuation in light intensity which occurs in natural environments. Depending upon the individual needs of the user any of a variety of characteristics may be varied to achieve the desired results. This alterations and fluctuation may include changes in intensity, wavelength, frequency, composition and other features as desired by a particular user. [0031] To ensure that appropriate lighting conditions are maintained input from the sensors are correlated against a desired standard and a self calibrating program enables the light output to be appropriately moderated so as to ensure that an appropriate desired result is obtained. For example, in one embodiment of the invention input from various sensors is compared to a desired output from a table of light intensities that outline a specified pattern. If the received information is not aligned with the targeted amount, modification is made so as to bring the light intensity within the system in line with the targeted values. This feedback loop with a self-calibrating feature also allows the electrical flow into the LEDs to be altered so as to compensate for wear and degradation of the LEDs over time.

[0032] In the experiments described below, the light control system was tuned to deliver an identical current to each LED in the system. In the demonstrated embodiment lighting for the photobioreactor is provided by 16 extremely high output illuminators at 630 ran, and 16 at 680 ran. Each illuminator contains 60 high efficiency InGaAlP diode chips (light emitting diodes) made by Marubeni Corporation (Japan). A total of 1,920 light emitting diodes are mounted to the inner wall of the aluminum shell. Each high output illuminator was positioned to be equidistant to adjacent illuminators. The cone of light emitted from each illuminator overlaps with adjacent i lluminators to provide even lighting to the reactor surface. In other applications the

wavelengths of the LEDs were modified so that half were blue and half were red, the integration of the light control module with the sensors in that application allowed for optimal growth conditions to be established and provided.

[0033] Six LI-COR Biosciences quantum sensors (for measuring photosynthetically active radiation) were used to measure light intensities within the photobioreactor. Three sensors measured incident light, and three measured transmitted light. The incident light sensors were mounted facing the LEDs, while the transmitted light sensors were mounted facing the center of the bioreactor. The light control software Biolume allows Proportional Integral Derivative (PID) control of incident or transmitted light intensity. The lighting can respond to a manual set point, or automatically adjust power levels to the LEDs to maintain a set point. In one application light intensity measurements are made by cycling power to just the 630 run LEDs, then to the 680 nm LEDs, then to both 630 and 680 nm LEDs. Sampling duration and frequency can be adjusted by the user of BioLume. Control of light-intensity can be clone from a large table of values allowing the user to reproduce "real outdoor" lighting intensities and timing or create custom lighting schemes. Lights can be turned on, off, or intensity corrected at any time. In the described application a 1 minute light sampling interval (with about 3 seconds total for light measurements) was demonstrated. PID control of light intensities allows the system to predict the future based on the past behavior.

[0034] The six quantum sensors were calibrated using a LI-250A Light

Meter Quantum/Radiometer/Photometer made by U-COR Biosciences. The reference light sensor was randomly moved along the inner glass wall of the photobioreactor for 15 seconds while the light meter averaged the light intensities seen during the 15 seconds. The 15 second moving-average light intensities were plotted against the signal produced by the light sensors at varying light intensities to produce a correlation plot. The least-squares best-fit equation was used to translate sensor-signal to light-intensity in

μ Einstein s/m²/sec.

[0035] A+ medium was used to support organism growth for these experiments and supplemented with lactate as needed. A-i- medium contained the following components (concentrations in mM): Tris (8.255 mM), Na2EDTA (0.0806 mM), KCl (8.0483 mM), CaCMHbO (1.8120 mM), MgSCWlrhO (20.2860 mM), KH2PO4 (0.3670 mM), NaCl (308.0082 mM), X l hCI (11.7540 mM-20.0 mM), Vitamin B12 (2.95xlQ-⁶ mM), ! i BO (0.5547 mM), MnCbMI L O (0.0218 mM),

Cu SO₄ *5H₂O (0.000012 mM). The pure Sheumnella cultures were given 45 mM lactate and consumed at most 27 mM of the lactate when grown to the highest density. Subsequent batches of the co-culture were given 25 mM lactate to avoid having excess lactate in the medium and were grown to lower biomass concentrations as measured by optical density. No lactate was added for batches of pure Synechococcus because preliminary experiments showed that lactate neither was consumed by Synechococcus nor affected its growth otherwise.

[0036] Shewanella W3-I8-1 was grown in a 5.5 I, batch at 250 rpm with 2.8

L/min sparging with air. Most of the culture was removed and replaced with fresh medium before growing the cells to the same final optical density.

Reproducibility of the duplicate batches was shown before decreasing the agitation speed to 50 rpm. Growth rates and biomass yields were analyzed at the lower agitation speed.

[0037] Synechococcus sp. PCC 7002 was grown in batch phase for three replicate batches at 250 rpm and 2.8 L/min sparging rate with 99% N-J. and 1% CO2. Reproducibility of replicate cultures was evaluated and then the agitation was decreased to 50 rpm for replicate batches. The effects of 50 rpm agitation on growth-rate and biomass yield were evaluated and then the culture was allowed to grow to late log-phase for replicate batches using 0.5 L/min bubbling. The Sate log-phase batches were done to show that all other samples were considered mid-log phase. The biomass yield and growth-rate were determined and then the culture was diluted with fresh medium in preparation for co-culture growth with Shewanella W3-18-1. Lactate was added to the medium to act as a source of carbon and energy for Shewanella and carbon for Synechococcus.

[0038] Shewanella W3- 18-1 and Synechococcus 7002 were grown together in batch-phase. Shewanella W3-18-1 used lactate to produce CO2 needed by Synechococcus 7002. In turn, Synechococcus produced O2 needed by Shewanella W3-18-1 to oxidize lactate. After diluting the cu lture with fresh medium, the dissolved CO2 concentration in the medium was very low as measured by a dissolved CO2 probe. To speed up growth of the co-culture on the 1^st batch after inoculation of both species, a small amount of sodium bicarbonate was added (about 0.5 m!VI). Subsequent batches of co-culture did not require

supplementation with bicarbonate.

Results

[0039] Synechococcus grew at the same rate at 250 rpm and at 50 rpm agitation as long as 2.8 L/min gas-addition was used. However, the growth-rate decreased slightly when 50 rpm agitation and only 0.5 L/min aeration was used. Mass transfer through the high-aspect-ratio reactor (described in paragraph 23) was much more efficient than would he expected of a lower aspect ratio reactor with a shorter path-length for gas-exchange. The impact of mass transfer changes (agitation and aeration) have been much more obvious in a Sower aspect ratio reactor as our previous experiments with Shewanella showed. The maximum growth-rate of pure Synechococcus was nearly the same as the co- culture of Synechococcus and Shewanella. This is because the co-culture is rate- limited by the growth of Synechococcus as the result of both strains tight metabolic coupling. The lag phase of growth of Synechococcus was longer than the lag phase for the co-culture (see Fig. 3), and Shewanella W3-18-1 grew faster than the co-culture or pure Synechococcus (see Fig. 4) as cyanobacteria general ly has lower growth rates than aerobically grown heterotrophs under optimal conditions. However, Shewanella did not use lactate as efficiently as the co- culture (see Fig. 5a). The co-culture used lactate 25% more efficiently than Shewanella alone because Synechococcus used CO2 that was produced by pure Shewanella cultures. Analysis of culture filtrates for organic acids revealed that lactate was not used fully by coculture. Acetate and sometimes formate

(products of partial lactate oxidation by Shewanella) were found in the coculture environment. When growth yield was calculated per mole of carbon used we found that co-culture converts carbon to biomass 3.2 times better than the pure Shewanella culture (see Fig. 5b). [0040] Shewanella grew much better than expected at lower agitation (50 rpm). Again,, this is due to the unusually high aspect ratio of the used bioreactor. The 50 rpm cu ltu re did become 02-limited as was indicated by the red color of the culture due to the production and extracellular localization of cytochromes and accumulation of acetate. The biomass yield of the 50 rpm culture of

Shewanella was much lower than the 250 rpm culture by ash-free dry weight. Shewanella produced about 0.8 g/L of ash-free dry weight in a 12 hour growth period with 250 rpm (see Fig. 3). This biomass concentration exceeded what was produced by the co-culture or Synechococcus in 30 hours (Fig. 3).

[0041] The co-culture of Shewanella and Synechococcus grew at about the same maximum growth rate as pure Synechococcus (on an ash-free dry weight basis). However, the co-culture used light 2.5 times more effectively than the pure Synechococcus culture (see Fig. 6). In other words, the biomass yield as ash- free dry weight for a given amount of light was 2.5 times as high for the co- culture as the pure Synechococcus culture. Obviously, the growth rate of the coculture was limited by growth rate of Synechococcus as the result of tight metabolic coupling of two species.

[0042] The packed cell volume analysis showed that a co-culture of

Shewanella and Synechococcus had a stable ratio of about 1 :1 by cell volume. The amount of Synechococcus was always slightly higher that Shewanella, with

Synechococcus to Shewanella ratios from 1.03:1 to 1.3:1. A packed cell volume sample that was 43.45% (+/'- 0.76) Shewanella and 56.55% (+/- 0.76) Synechococcus by volume was shown to have a percent by mass of 43.51% Shewanella and 56.49% Synechococcus. This means that the density of the cell pellets in g/^'L is equal for Synechococcus and Shewanella. We can therefore relate the packed cell volume analysis directly to cell mass (ash free dry weight) by multiplying the fractional-share of the cell-volume by the combined ash-free dry weight.

[0043] The presence of products of incomplete lactate oxidation described above was the consequence of Shewanella growing faster than Synechococcus and therefore it exhausted O2 faster than Synechococcus could produce it. As the result, Shewanella growth was limited by O2. Under O2 limitation Shew^ranella is known to convert part of lactate into acetate and additionally can accumulate some formate; both these compounds cannot be used by Synechococcus, therefore decreasing its growth and O2 production rate. Hence, the process may become self-fading unless some external O2 or C02/T>icarbonate is added. This conclusion is supported by the following experiments. Uncontrolled batches of Shewanella, Synechococcus, and binary co-cultures of these bacteria were grown at room temperature (23-24°C) in sterile Roux bottles (total volume 1 L) without gas sparging and mixing of cultural liquid. Bottles were illuminated with cool white light at 55 pEinsteins/m²/sec, In the first set of experiments 0.6 L of A+ medium (supplemented with 9 mM lactate in case of pure Shewanella or binary cultures) was added, therefore leaving 0.4 1 of air present in the headspace. Both pure cultures did not produce significant growth for more than 250 hours, whereas binary culture fully used lactate for 120 hours. Biomass yield of co- culture w^ras 6 times higher than yield of pure Shevjanella produced in controlled aerated bioreactor (see Fig. 3a) and was 40 g of ash-free dry weight/mol of lactate carbon. Toward the end of active growth of binary culture most of the cells precipitated and formed a pellet on the bottom of the Roux bottle. This precipitation can be used as the way to concentrate biomass in the process of growth by proper cultivation chambers design without necessity to centrifuge whole fermentation broth, therefore potentially leading to significant additional energy savings on downstream processing. Additionally, packed cell volume analysis showed that co-culture of Synechococcus and Shewanella grown with additional O2 influx from the bottle headspace had a ratio 5:1 by cell volume, which means that Synechococcus made 5 times more biomass per lactate used that it did in binary culture bioreactor runs without external O2 influx.

[0044] In the second set of experiments the Roux bottles were filled with

960 ml of media. These experiments yielded no growth for pure Synechococcus and Shewanella cultures; result for binary culture was comparable with those obtained in photobioreactor experiments. The overa ll binary culture growth limitation with O2 caused by higher Shewanella growth rate and incomplete lactate oxidation can be overcome as follow. (1) Our previous experiments with Shewanella showed that deletion of ack.A gene encoding for acetate kinase abolished incomplete lactate oxidation and acetate excretion under O2 limitation. All lactate was converted into CO2 and biomass, their ratio depended on the level of O₂ supply. (2) Alternatively, addition of CO2 or bicarbonate will enhance specific rate of O2 production by cyanobacteria in the initial stage of cultivation and as the result increase robustness of binary culture and final biomass production,, yield, and degree of organic carbon source assimilation by the binary culture.

[0045] The graph of electricity used (KWhrs) for illumination, sparging, and agitation per gram of ash-free dry weight (see Fig. 6) show's that

Synechococcus required the greatest investment for the smallest return; whereas, Shewanella produced the greatest amount of biomass for the smallest input of electricity . This is because Shewanella did not require illumination, which was one of the costliest expenditures for electricity in this experiment. Use of sunlight will significantly reduce energy expenditures on bina ry culture illumination as well as optimization of lighting conditions and overall process design. The amount of electricity used for agitation and sparging per unit biomass (grams of ash-free dry weight) was highest for pure Synechococcus and lowest for the co- culture {57 times lower} (see Fig. 7). It should be noted that experiments conducted in Roux bottles used neither sparging nor mixing therefore potentially driving energy expenditures for these purposes almost to nothing. Since Shewanella required both bubbling and agitation, it required about 7 times more electricity per unit biomass as the co-culture. Thus, binary cultures not only provide advantage in relation to efficiency because venting or sparging is no longer needed. There are also substantial cost improvements because there is no need for aerating the mixtures and off-gas purification, the last absolutely necessarv in the industrial environments.

[0046] Unlike other methods described in the prior art the proposed approach accomplishes CO2, delivery /O?. removal simultaneously with the process of biomass/product biosynthesis. This method of cultivation does not need sparging of a closed bioreactor with air or other gas mixture/vigorous mixing to deliver CCVrernove produced O2 from liquid medium. This improvement leads to significant savings on energy consumption to accomplish these tasks. It also allows designing photobioreactors of any desired shape to ensure optimal illumination and space usage. Use of highly reduced organic compounds (for example wasted fats) in some cases will help to consume externally added CO2 or bicarbonate without necessity to remove O2 As source of organic matter many types of waste materials can be used, for example biosludge produced from sewage w^rater treatment plants or starch solutions generated as the resu lt of industrial food processing that are typically wasted. Some of these materials may also be a cheap source of other nu trients such as N, P, and 8 sources.

[0047] hi addition to the organisms previously described, a variety of other organism pairings are envisioned. These include pairs of Synechococcus sp. PCC6038 + Shewanella oneidensis MR-1 and Cyanothece ATCC 51142 + Shewanella sp. W3-18-1. Other possible candidates from phototrophic side include microorganisms that are able to grow autotrophically by using C02 as the source of carbon, lieht as the source of energy, and water as source of electron, i.e. carry water splitting and produce oxygen: Examples of such organism include but are not limited to various types of cyanobacteria: nitrogen fixing, both single ceil (e.g. Cyanothece, some species belonging to Synechococcus) and filamentous (e.g. Trichodesmium, Anabaena, Nostoc), and non-nitrogen fixing belonging to single cell (e.g. Synechocystis, some species belonging to Synechococcus) and filamentous e.g. Arthrospira (former Spirulina) as well as various genetically modified strains of these organisms. Other examples include microalgae: e.g. Haematococcus pluvialis , Clamydomonas etc., as well as various genetically modified strains. Any heterotrophic obligatively aerobic or facultative anaerobic microorganism, belonging to archaea, bacteria, or eukaryotes, that are able to oxidize organic compounds to CO2 using O2 as electron acceptor, and use organic compounds for growth and/or biosynthesis of product of interest may also be utilized examples of such materials include: E. coll, Corynebacterium glutamicum, Saccharomyces cerevisiae and the like.

[0048] Another embodiment of a photobioreactor is shown in Figure 2B.

In this embodiment the photobioreactor has several alterations and

modifications over the design previously discussed. First, this new design is made through an extruded process which allows for a more cost effective design and allows for enhanced efficiencies related to both cost and function. In addition, this new⁷ design also includes integral heat sinks and ports for making the various lighting and harnesses for the lighting. This new design also offers space for twice as many LEDs as the previous design because less space is used for the round heat-sinks. Cooling fans are automatically controlled by a temperature dependant programmable logic controller to increase the longevity of LEDs.

[0049] The present invention core forms an economically attractive way to create a variety of biobased products including but not limited to biomass; H2; organic fertilizers; biodiesel and biocrude oil; ethano!; α ID 1Π O a CI ds; vitamins; antibiotics; polysaccharides and fine chemicals, for example D- and L- lactate as polylactates precursor. These methodolgies may also be utilized in a variety of other different ways including but not limited to biosludge and other organic wastes utilization in economically sound way. In addition other embodiments may provide other possibilities and potentials such as the ability to regulate CO2 production/O₂ removal or CO2 consurnption/Cb removal at desired rates.

[0050] In other applications and embodiments the methods and materials described herein have been combined to create value added products in a variety of ways. In one embodiment of the invention wastewater generated from a food processing facility was treated with a binary culture in a co-cultivation approach to simultaneously remove of C/N/P nutrients and produce a high-value biomass. This technology has the potential to significantly reduce operating expenses in waste treatment while also generating revenue through high-value products. In this set of experiments, starch-containing wastewater was passed through a co-cultivation of a green alga (Haematococcus pluvialis) and a heterotrophic bacterium (Bacillus subtilis). In this process, Bacillus subtilis degraded the organic carbon (starch) present in the in wastewater to C02 and low molecular organic acids (e.g., acetate, lactate). The resulting products (i.e., C02, organic acids) and other nutrient (N/P) are subsequently used by Haematococcus pluvialis to synthesize biomass which contains high-value carotenoid product (astaxanthin).

[0051] This co-cultivation process is an innovative yet simple solution. While heterotrophic bacteria (Bacillus subtilis) still degrade and consume available carbohydrates, the necessary oxygen for this aerobic respiration is provided by green algae (Haematococcus pluvialis). In turn, the green algae consume the carbon dioxide produced, and they also remove nutrients like nitrogen and phosphorus from the wastewater and incorporate thera into their algal biomass This exchange of vital biological gases (02, C02) reduces the mass transfer requirements

(aeration and agitation) for cultivation of either organism separately. The cost savings would be significant— mass transfer (aeration and agitiation) accounts for ~50% of cultivation electricity costs), Furthermore, some green algae (e.g.

Haematococcus pluvia!is) naturally accumulate high-value products (e.g., astaxanthin) in their biomass. This high-value biomass was then harvested following wastewater treatment, resulting in a source of revenue for the process. In this set of experiments the previously described co-culture was grown for 14 days under constant illumination and without aeration. In that period of time, over 70% of the COD and 33% of the Total N & P were removed under non- optimized conditions. The final biomass density was 2.2 g/L, 63% of which was H. phivialis, and the astaxanthin content was 0.6% under non-optimized conditions. Based on these results, a techno-economic analysis estimated that the payback period was between 1-3 years for a full-scale process, depending on the performance at scale. Examples of these production charts showing astaxanthin production and nutrient reduction are shown in figures 9 and 10.

[0052] In another embodiment of the invention a closed co-culture technique was utilized to produce value-added commodities by co-culturing oxygenic phototrophic and heterotrophic microorganisms, in this embodiment of the invention a methane-oxidizing bacteria (MOB) Methylomicrobium aicaliphilum 20Z and cyanobacteria Synechococais sp. PCC 7002 were co-cultured in il luminated batches containing mineral medium and methane (CHU) as the sole source of carbon for both strains. Since MOB cannot grow by oxidizing CH4 without O2 and cyanobacteria cannot utilize CH4 as carbon source, the growth of the binary culture occurred due to tight metabolic coupling of these bacteria. Synechococais sp. PCC 7002 consumed CO₂ produced by MOB and generated O2, which was consumed by M. alcaliphilum 20Z and resulted in CO2 production C H ₄ is the most reduced organic compound and therefore to be used for biomass synthesis it typically has to be oxidized by O2 to the level of formaldehyde. Since MOB spend more than 50% of CHU carbon for biomass synthesis,, providing additional CO2 would be advantageous to balance co-culture growth and maintain growth rate at maximum level. To prove this, batches of pure M.

alcaliphilum 20Z and binary M. alcaliphilum 20Z-Synechococcus PCC7002 cultures were grown using LED-illuminated photobioreactor described above. In addition the enclosed system was modified to allow for gas consumption measurements. We found that M. alcaliphilum 20Z and Synechococais PCC7002 can be successfully grow^rn in a co-culture at pPI 8.3 with recycling of headspace gases starting with pure methane (see Fig.11^'). However, supplying the co- culture with pure CH4 leads to growth limitation with O2 due to CH4 and CO2 consumption stoichiometry. As a result, the M. alcaliphilum 20Z- Synechococais PC.C7002 co-culture grew slower than the pure M. alcaliphilum 20Z and produced less biomass .

[0053] The follow-up experiments revealed that bicarbonate addition substantially improves the growth of M. alcaliphilum 20Z- Synechococcus PCC7002 co-culture. In parti.cu.lar, the co-culture, grown on CHU in the presence of bicarbonate, generated 3.75-fold more biomass than axenic M. alcaliphilum 20Z grown on a 1:1 mix of CHVair with the same agitation and gas flow rate.

Application of bicarbonate also significantly improved the efficiency of methane assimilation during the headspace recycling. Bicarbonate additions to the bioreactor confirmed its positive effect on the co-culture growth at the same level of mass-transfer as the exhaustion of bicarbonate in the medium led to almost immediate growth rate decrease. Figure 11 shows a growth chart of M.

alcaliphilum 20Z and Synechococcus PCC7002 co-culture growth in LED-PBR. Cultures were grown in A+ mineral medium at agitation 150 rpm, pH 8.3, and gas flow⁷ rate at 0.127 L/L/min. Light control was set to keep transmitted light at 10 pEinsteins/m²/^'sec. M. alcaliphilum 20Z- Synechococcus PCC7002 binary culture sustained active bacterial growth significantly longer than pure M. alcaliphilum 20Z culture when gas recycling was applied. In contrast to most prior art practices for MOB production, this application of binary cultivation eliminated the necessity to use highly explosive CFWair mixes by substitution of O?. with CO2 or bicarbonate. Addition of inorganic carbon increased overall growth and biomass production rates in the binary cultures through stimulation of photosynthetic O2 evolution, which occurs throughout the entire reactor space_., therefore eliminating dissolved O2 irregularities. Obtained results can be used to calculate and build an illuminated air-lift column bioreactor system which allows for complete gas utilization and results in almost zero greenhouse gas emission. Finally, given the growth stimulation by CO2, these results allow developing a robust and easily controlled process for co-cultivation of MOB and oxygenic phototrophs for production of biofuels, feed protein and other value- added, commodities

[0054] This configuration provides a variety of advantages over the prior art methodologies particularly in regard to systems which utilize methane as a carbon source. Methane is an abundant component in both natural gas and biogas streams. Furthermore, utilization of biogas made from agricultural, municipal, and landfill wastes would produce a benefit by generating a useful material from a feedstock of uncertain and variable composition. In the past, large-scale production of materials using MOBs was hampered because of (i) high gas mass-transfer energy spending to dissolve CH₄ and 0₂ in media, (ii) explosiveness of CH₄/O? mixes, and (in) need for sophisticated controls to keep dissolved CH₄ and 0₂ at ratios favorable for microbial growth. However, the present invention provides a means for overcoming these issues and establishes a credible methodology for moving forward in the future. In the preceding descriptions we have shown and described only several preferred embodiments of the invention, by way of illustration of the modes contemplated for carrying out the invention. As will be realized, the invention is capable of modification in various respects without departing from the invention. Accordingly, the drawings and description of the preferred embodiment set forth hereafter are to be regarded as illustrative in nature, and not as restrictive.

[0055] While various preferred embodiments of the invention are shown and described, it is to be distinctly understood that this invention is not limited thereto but may be variously embodied to practice within the scope of the following claims. From the foregoing description, it will be apparent that various changes may be made without departing from the spirit and scope of the invention as defined by the following claims.

Claims

What is claimed is: 1. A photobioreactor system for obtaining biotechnology products comprising a binary culture made up of a combination of a prototrophic organism and a heterotrophic organism said photobioreactor having a chamber with a height to diameter ration greater than two, an integrated heat sink and an artificial lighting system comprised of light emitting diodes (LEDs) arranged in a spaced configuration.

2. The photobioreactor system of claim 1 wherein light emitted from said LEDs is the only light within said system.

3. The photobioreactor system of claim 2 wherein said LED's are controlled by a system based upon inputs from sensors surrounding said chamber.

4. A method for simultaneously generating algal biomass carotenoids and reducing nutrient loads in starch wastewater characterized by incubation of said nutrient loads with a co-culture of at least two heretogenous organisms in a closed photobioreactor system bioreactor.

5. The method of claim 4 wherein said heterogenous organisms are Bacillus subtilis and Haematococcus pluvialis.

6. The method of claim 4 wherein said closed photobioreactor system has a chamber with a height to diameter ration greater than two and lit by an artificial lighting system comprised of light emitting diodes (LEDs) arranged in a spaced configuration.

7. A method for producing a biofuel characterized by the step of:

incubating a co-culture of a methane-oxidizing phototrophic

microorganism and a heterotrophic microorganism in a controlled illuminated environment containing a mineral medium sparged by methane (CH4).

8. The method of claim 7 wherein said mineral medium contains

bicarbonate.

9. The method of claim 7 wherein the co-culture comprise a

methylomicrobium bacteria (MOB) species and a cyanobacteria Synechococcus

10. The method of claim 9 wherein the co-culture comprise

Methylomicrobium alcaliphilum 20Z and cyanobacteria Synechococcus sp. FCC 7002.

11. The method of claim 10 wherein the co-culture is incubated at a pH of 8.3.

12. The method of claim l lfurther comprising the step of recycling headspace gasses.