Progress 09/01/20 to 01/31/22

Outputs
Target Audience:Ultimately several target audiences will benefit from our work to develop game-changing GEMS PBR technology for scalable, algal CO2 capture and bioproduction at higher productivity and lower cost than in any existing system. These target audiences include: 1) Ethanol plants, breweries, distilleries, or others that emit clean CO2 streams, for added profitability by cost-effective algal CO2 capture and conversion to nutraceuticals, foods, or feeds. 2) Agricultural partners/customers interested in cost-effective algal biomass production for nutraceutical, food, or feed applications. 3) Agricultural partners/customers interested in using fallow land for algal bioproduction, or in supplementing income by integrating GEMS PBR tubes into field furrows. 4) CO2-emitting industries, such as power plants, cement factories, or foundries interested in cost-effective mitigation of their emissions. 5) Wastewater treatment plants interested in meeting new, stringent P and N effluent limits by efficient algal consumption of these enabled by profit-generating CO2 capture and bioproduction. 6) Any government agency, foundation, business, or homeowner interested in helping combat climate change by deploying GEMS PBRs for CO2 capture linked to biomass production. Efforts to reach target audiences during the Phase I project included: 1) Ethanol Plants, which are excellent first customers/partners for our production of high-value algal products on their clean fermentation CO2 and thin stillage. We have established an important connection with Neal Kemmet, President of Ace/Fox River Valley Ethanol for Phase II pilot testing at their Oshkosh, WI plant and a possible partnership for subsequent commercialization. Kemmet has provided a strong support letter for our Phase II application. 2) Other ethanol producers: A slide deck has been compiled to present the GEMS technology. This shows how GEMS PBRs can effectively use the clean CO2 and thin stillage waste streams from ethanol plants to capture and convert the CO2 to high-value algal biomass for feed, food, and nutraceutical feedstocks. We have contacted Jeff Broin, CEO of Poet, LLC, and will continue to seek partnerships with other ethanol producers. 3) Algal bioproduct, biofuel producers: Bruce Dannenberg, CEO of Phytonix and CyanobMega has expressed great interest in growing his algae in our GEMS PBRs and has provided a strong letter of support for our Phase II application. Phytonix has developed a butanol-producing cyanobacterium, and CyanoMega has developed a multi-amino-acid and omega-3 fatty acid producing alga. Both of these are of potentially great commercial interest, particularly if produced at high productivity and economically in GEMS PBRs. 4) Extensive work with Peter Jorgenson, a colleague with connections to Arizona Public Service (APS), and preparation of a slide deck presented to APS executives, for a proposed vertical GEMS (VGEMS) PBR pilot module at their 1060 MW RedHawk, natural gas power plant that emits ~1.68 metric tons CO2 per year. The proposed ~14,000 Liter VGEMS, pilot module consisting of four, vertical GEMS tubes would demonstrate the utility of a GEMS PBR system for profit-generating CO2 capture and biomass production. 5) Strong interest from Waupaca Foundry Inc. (WFI): Co-PI Toivo Kallas has previously done preliminary work with WFI to demonstrate the potential for growing algae on their flue gas CO2 emissions and a wastewater growth medium to convert these into monetizable products. Bryant Esch, Director of Environmental Engineering at WFI, has provided a strong letter for our Phase II application, expressing interest in GEMS PBR pilot tests at their plant. 6) PI Robert Falco's productive conversations with Gregory S. Guard, CEO of Clearas Water Recovery Inc., leading to a strong Phase II support letter, in which Guard expressed great interest in testing GEMS PBR tubes in their algal wastewater purification system. The GEMS technology is of particular interest because of its potential to enable larger diameter PBR tubes operating at low flow rates, thus resulting in both lower CAPEX and OPEX costs. 7) Extensive Zoom meetings and work with Larta advisor, Ken Hunt to develop a draft commercialization plan, that includes focused target audience and customer connections, for our Phase II application. We hope to pursue continued work with Larta on Techno Economic (TEA) and Life Cycle Analyses (LCA), market analyses, business development, and web site development enabled by Phase II Technology and Business Assistance (TABA) funding. 8) Several Zoom meetings with business colleagues Jeff Ebel and Leon Ostrowski of Midwest Wealth Ventures, LLC, and Bob Sylvest and Trez Wigfall of Opportunity Analysis, Inc., regarding work they can perform to aid us with market analyses, customer discovery, and business development. 9) Presentation of the GEMS technology to potential target audiences by PI Robert Falco at the 2020 Algae Biomass Organization (ABO) Algae Biomass Summit, Virtual Conference, (Algal growth and biomass yield in game-changing, Growth Enhancing Mixing Spectrum (GEMS) photobioreactors (PBRs). 10) Presentations of the GEMS technology to potential target audiences by Co-PI Toivo Kallas at the 2020 Midwest-Southeast Photosynthesis Virtual Conference, (Algal Bioproduction in Game-Changing, Growth Enhancing Mixing Spectrum (GEMS) Photobioreactors), and as part of the 2020 Carleton College Connects, Virtual Climate Change Panel (The potential and challenges of algal CO2 capture - Can Growth Enhancing Mixing Spectrum (GEMS) photobioreactors change the game?). Changes/Problems:Objective 1: Greater algal productivity in GEMS vs non-GEMS PBRs Objective 2: Test different GEMS geometries of helical pitch, depth, and flow Changes: 33 and 53 Liter PBRs were built rather than 20-L PBRs because: a) The advantage of the GEMS mixing increases with tube diameter and length. b) Culture circulation is from air pressure that drives the culture upward in vertical air-lift sections, followed by gravity flow through return tubes. The air-lift circulation is gentler on filamentous algae, requires less energy than liquid pumps in vertical PBRs, which maximize areal (per m2) productivity, and will be important for direct, air-CO2 capture. Problems: Monitoring and maintenance of constant culture flow rates, for consistent PBR comparisons: Several problems related to this were discovered and changes made to alleviate them. Flomec magnetic flow monitors were purchased and installed for precise flow monitoring in the diagonal downflow tube of each 53-Liter PBR. After initial inconsistent performance, stainless steel collars were installed around each Flomec for improved grounding that alleviated aberrant ground flow circuits. We are still examining the effects of Biofilm formation in the Flomecs, which may remain problematic and require a different type of flow meter. Culture flow rates were also greatly affected by culture foaming and evaporative losses, despite inclusion of an antifoam in the culture medium. If undetected, the culture liquid in the top horizontal tube could fall to a level that prevents flow from this 4" diameter section into the subsequent 2" diameter, diagonal downflow tube. Effectively the transition from the 4" to the 2" created a dam that blocked culture flow at low liquid levels. Several measures were tested to alleviate these problems. Ultimately, the following solutions worked well, although they may need further refinement and testing: The oscillation and surge phenomenon has been alleviated by replacing the 4" to 2" diameter junction that connects the top, horizontal tube and the diagonal down tube with a 4" diameter junction. This and a level top tube have eliminated the oscillation and resulting culture medium surge, as indicated by steady flow readings from the Flomec flow monitors. Design and installation of S-traps on the gas outflows of the PBRs has effectively eliminated most of the foaming. After shifting focus to scale-up and testing of the 300-L vertical (VGEMS) and reference (VREF) PBRs, most of the foam trap work has been focused on those. Foaming from those PBRs has been solved for most conditions as described below. Leakage and/or breakage of the clear, flexible plastic tubing used in the PBRs, resulting in flow stoppage or complete culture loss. The incentive for use of plastic tubing, rather than glass or clear PVC, is compelling because even that made of top-quality Exxon 1012HJ resin costs ~$0.10 to $0.20 per ft of 6" diameter versus $40/ft for clear PVC. Many types of plastic tubing are available with very different properties such as transparency and breakage resistance. Unfortunately, supply chain issues, and minimum order barriers, have greatly limited access to these for testing. We have begun to explore metallocene catalyzed Linear Low-Density Polyethylene, lay-flat tubing (mLLDPE). Once the right mLLDPE is found, the cost per foot of a GEMS PBR can be reduced by a factor of 100x over rigid clear tubing. For example, Exxon Mobil Exceed brand, performance polymers, like their 1012HJ resin, have high tensile strength and very high rupture resistance. Since we seek a combination of properties such as tensile strength, dart drop impact strength, Secant Modulus, and elongation, that must be optimized, we need to test several formulations. However, a minimum order of any of these resins made to our specifications requires 'gambling' $5,000 or more per order, which was not possible in Phase I, but is an important goal of Phase II. In the meantime, we found that a plastic-coated wire mesh, very effectively supports the clear, flexible plastic tubes in the vertical PBRs, as discussed below. Challenges in maintaining optimal temperatures (38°C to 40°C for Synechococcus PCC 11901 cyanobacteria, used in Phase I) and light intensities in the SCF lab. This was originally designed as a warehouse with limited insulation. We were unable to adequately cool this lab during hot summer months in AZ. This, plus heat from the LEDs, generated temperatures of 45°C or higher, which inhibited algal growth. One solution for AZ, and installations in any hot climate, is to use a thermophilic alga. Scott Miller (Univ. Montana) has kindly provided for such tests a thermophilic Synechococcus, that grows in the 40°C to 55°C range. This strain is being maintained in our Bio-lab. An alternative, for testing a variety of algae, including commercially important ones, is to upgrade the SCF lab air conditioning. We have requested funds for this in our Phase II budget. Maintenance of constant CO2/air mixtures with equal flow and distribution to PBRs. Alicat mass flow controllers (MFCs) were purchased to enable constant CO2/air mixtures and flow rates into the PBRs. These worked very well but problems arose with water vapor condensation in the gas lines or culture backflow into the MFCs. This was solved with appropriate drier and check valve installations. CO2/air-line leakages or blockages at various junctions resulted in line ruptures and disruption of CO2/air flow into the PBRs. Some problems, including clogging, also occurred with the spargers that introduce CO2/air microbubbles into the PBRs. These problems have been addressed with check valves and improved sparger fixtures. Objective 3: GEMS PBR scale-up Objective 4: Enhanced productivity in scaled-up GEMS versus control PBRs Changes: We scaled to 300-L vertical, air-lift GEMS (VGEMS) and control (VREF) PBRs instead of a 500-L horizontal design, because: a) Smaller footprints and thus higher areal productivity in vertical PBRs, b) Importance of air-lift designs for air-CO2 capture, c) Easy scale up of the modular VGEMS by increasing the number of modules, d) Gentler circulation of sensitive filamentous algae, and e) For Phase I, the prohibitive cost of 8" diameter fittings for a 500-L system relative to 6" diameters for 300-Liters. Problems: As with the 53-L PBRs, tube leakage and rupture: This has been nicely solved by using a plastic-coated wire mesh to coat and support a single layer of transparent, flexible plastic PBR tubing. A further improvement would be use of a robust mLLDPE tubing that would alleviate the wire mesh and care required for its installation. Foaming, evaporative culture loss, and flow stoppage: These linked problems were discussed above for the 53-L PBRs and remained problematic for the 300-L VPBRs. Foaming from these PBRs under most conditions has been solved by directing the PBR exhaust through a deep, liquid-filled, S-trap connected to a vertical, 4' stainless-steel outflow tube filled with a stainless-steel wool matrix, surrounded on the outside with coiled copper tubing cooled by chilled water circulation. This is an important advance for trouble-free, commercial operations without messy foaming and evaporative loss. Variations of it should be applicable to any PBR. Microbial contamination: that became evident in the 300-L VGEMS PBR, was accompanied by increased foaming, and correlated with growth inhibition of the Synechococcus cyanobacteria. A treatment of ammonia addition to the PBR, raised the pH, did not impede the cyanobacteria, appeared to inhibit the contaminants, resulted in their disappearance as evidenced by microscopic observation, and allowed resumption of algal growth. The contamination and growth inhibition challenge is solvable, one solution may be at hand, and this and others will be further investigated in PhaseII. What opportunities for training and professional development has the project provided?The principal team members of the SCF/AABT, USDA-NIFA Phase I SBIR project include Robert Falco, PI, CEO of SolarClean Fuels, LLC, former Michigan State University Professor of Mechanical Engineering with deep expertise in fluid dynamics and turbulent flows; Toivo Kallas, Co-PI, Managing Director of Algoma Algal Biotechnology, LLC, and Bio-Research Director of SCF, a Distinguished Professor Emeritus of Microbial Genetics and Biotechnology at the University of Wisconsin Oshkosh, with deep expertise in photosynthesis, algal cultivation, and bioengineering; Ron Will, former Chief Design Engineer at Subaru; Rudy Rivas, a skilled union contractor experienced in plumbing and high-pressure systems, and subsequently a builder; Travis Smith, a part-time consultant with excellent instrumentation and programming skills; and Joshua Navigato, a skilled young plumber and handyman. The Falco/Kallas partnership uniquely combines key areas of expertise in fluid-systems engineering and algal biology for innovative algal bioreactor designs and bioproduction solutions. All members of the team have learned from each other and experienced professional development. PI Falco has gained experience in algal microbiology/physiology, culture maintenance, propagation, and growth assessment at small and large scales. Kallas in turn has gained valuable insights into photobioreactor design, construction, fluid dynamics, and troubleshooting. All other team members have benefitted from each other's skills and experiences in similar ways. For example, options and solutions for automated PBR monitoring and data collection have been gained from Smith, and Navigato has learned the basics of algal culture media preparation and growth measurements, in addition to his invaluable support of PBR construction, maintenance, and troubleshooting work. How have the results been disseminated to communities of interest?To date, results have been disseminated to communities of interest primarily through individual or small group Zoom meetings as well as conference presentations to larger audiences. Covid severely restricted our ability to meet directly with target audiences of interest and to interact with colleagues and potential partners or customers by direct attendance of professional or commercial conferences. Efforts to disseminate results are directly related to our efforts to reach target audiences as outlined in the Target Audience Section. These efforts include: 1) A Zoom conference with Neal Kemmet (President of Ace/Fox River Valley Ethanol, Oshkosh and Stanley, Wisconsin) and his engineering staff, and several email communications, to work out an agreement for Phase II pilot testing of VGEMS PBRs on their clean fermentation CO2 and thin stillage at their Oshkosh plant. Kemmet has provided a strong support letter for our Phase II application. 2) A slide deck has been compiled to present the GEMS technology to Ace and other ethanol producers. This shows how GEMS PBRs can effectively use the clean CO2 and thin stillage waste streams from ethanol plants to capture and convert the CO2 to high-value algal biomass for feed, food, and nutraceutical feedstocks. We have contacted Jeff Broin, CEO of Poet, LLC, and will continue to seek partnerships with other ethanol producers. 3) An important connection by PI Falco with Bruce Dannenberg, CEO of Phytonix and of CyanoMega, who expresses great interest in growing his algae in our GEMS PBRs and has provided a strong letter of support for our Phase II application. Phytonix has developed a butanol-producing cyanobacterium, and CyanoMega has developed a multi-amino-acid and omega-3 fatty acid producing alga. Both of these are of potentially great commercial interest, particularly if produced at high productivity and economically in GEMS PBRs. 4) Numerous meetings with Peter Jorgenson, a colleague with connections to Arizona Public Service (APS), and preparation of a slide deck presented to APS executives, for a proposed VGEMS PBR pilot installation at their 1060 MW RedHawk, natural gas power plant that emits ~1.68 metric tons CO2 per year. 5) Several communications with Bryant Esch, Director of Environmental Engineering at Waupaca Foundry Inc. (WFI). Co-PI Kallas has previously done work with WFI to demonstrate the potential for growing algae on their flue gas CO2 emissions and a wastewater growth medium to convert these into monetizable products. Esch, has provided a strong letter for our Phase II application, expressing interest in GEMS PBR pilot tests at their plant. 6) PI Robert Falco's productive conversations with Gregory S. Guard, CEO of Clearas Water Recovery Inc., leading to a strong Phase II support letter, in which Guard expressed great interest in testing GEMS PBR tubes in their algal wastewater purification system. The GEMS technology is of particular interest because of its potential to enable larger diameter PBR tubes operating at low flow rates, thus resulting in both lower CAPEX and OPEX costs. 7) Extensive Zoom meetings and work with Larta advisor, Ken Hunt to develop a draft commercialization plan, that includes focused target audience and customer connections, for our Phase II application. We hope to pursue continued work with Larta on Techno Economic (TEA) and Life Cycle Analyses (LCA), market analyses, business development, and web site development enabled by Phase II Technology and Business Assistance (TABA) funding. 8) Several Zoom meetings with business colleagues Jeff Ebel and Leon Ostrowski of Midwest Wealth Ventures, LLC, and Bob Sylvest and Trez Wigfall of Opportunity Analysis, Inc., regarding work they could perform to aid us with market analyses, customer discovery, and business development. 9) Presentation of the GEMS technology to potential target audiences by PI Robert Falco at the 2020 Algae Biomass Organization (ABO) Algae Biomass Summit, Virtual Conference, (Algal growth and biomass yield in game-changing, Growth Enhancing Mixing Spectrum (GEMS) photobioreactors (PBRs). 10) Presentations of the GEMS technology to potential target audiences by Co-PI Toivo Kallas at the 2020 Midwest-Southeast Photosynthesis Virtual Conference, ("Algal Bioproduction in Game-Changing, Growth Enhancing Mixing Spectrum (GEMS) Photobioreactors"); and part of the 2020 Carleton College Connects, Virtual Climate Change Panel ("The potential and challenges of algal CO2 capture - Can Growth Enhancing Mixing Spectrum (GEMS) photobioreactors change the game?"); and May 5, 2022 virtual presentation to the Department of Molecular Microbiology, Madurai Kamaraj University, Madurai, Tamil Nadu, India ("The potential and challenges of algal carbon capture, bioproducts, and fuels,") 11) Upcoming presentation ("Action Plans -- & The Potential for Algae - Carbon Capture and Bioproduction,") by Co-PI Kallas as part of a panel on Sustainable Endeavors to Combat Climate Change, at Carleton College, multi-year reunions, Northfield, MN, August 6, 2022. 12) Abstract submitted by Falco and Kallas ("Results Showing Scale-up Potential of a New Modified Tubular Photobioreactor Technology") for presentation at the Algae Biomass Summit, Virtual Conference, October 3-28, 2022. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 - Show greater algal productivity in GEMS vs non-GEMS PBRs. Experiments were done with Synechococcus PCC 11901 cyanobacteria, which can grow to high biomass yields. We believe, however, that GEMS PBRs will be advantageous for enhanced growth of any algae. Experiments in GEMS vs control PBRs were first conducted in 33-Liter PBRs under natural sunlight, ambient temperature, and air CO2 in Los Alamos, NM; and subsequently in 53?L PBRs in the SolarClean Fuels' lab in Fountain Hills, AZ. The 33- and 53-L PBRs were chosen rather than 20-Liters because: a) The advantage of the GEMS mixing (that brings algae to and from the PBR tube walls for optimal photon usage) for enhanced algal productivity increases as the tube diameter and length increases. b) Culture circulation in both the 33 and 53?L PBRs is provided by air pressure that drives the culture upward in vertical air-lift sections, followed by gravity flow through down-sloping or horizontal tubes to return to the air-lift section. The air-lift circulation is gentler on filamentous algae, requires less energy than liquid pumps in vertical PBR configurations, which maximize areal (per m2) productivity, and will be important for direct, air-CO2 capture applications. In the Los Alamos experiments, under ambient, day-night conditions (~4°C to 25°C), the 33-L GEMS PBR produced a 2 to 2.5-fold more biomass in a 175-hour run than the otherwise identical, non-GEMS, control PBR. In the 53-L PBRs, the GEMS also outperformed the non-GEMS control. In the best run to date, a GEMS PBR reached a cell density of ~15 OD750nm (~3.4 grams Dry Weight/Liter, gDW/L) compared to ~5 OD750nm (~1.1 gDW/L) for the best non-GEMS run - i.e. 3-fold greater biomass yield. Four 53-L PBRs were built, three with different GEMS geometries, and one non-GEMS control. All were identical, except for their GEMS vs non-GEMS configurations. All operated with concentrated CO2 inputs, temperatures ~30°C to 40°C (depending on outside, AZ temperatures), and with LED illumination up to 2300 µmol photons m-2 sec-1 (~full sunlight). Maximum biomass yields in the GEMS PBRs were comparable to those obtained under similar conditions in 500 mL lab cultures. Initial growth rates in all 4 PBRs (doubling times, tD, ~7 hours), in low density cultures up to ~20 hours of growth, approached those obtained under comparable conditions in lab cultures. Under these conditions neither photon nor nutrient availability is limiting. Initial growth rates in the 53-L GEMS and control PBRs were also similar up to ~60 hours of growth and cell densities of ~4OD750nm (~0.9 gDW/L). At these still low, initial, cell densities, neither light nor nutrient availability is limiting. The GEMS advantage became evident at higher cell densities. Several technical pitfalls occurred that prevented consistent and comparable culture flow rates needed for consistent productivity evaluations. These challenges are being addressed, and once fully alleviated, we believe we will be able to consistently show at least 3-fold greater biomass yields in the GEMS vs. non-GEMS PBRs. Objective 2 - Test different GEMS PBR geometries. Three air-lift, 53-L GEMS and one non-GEMS, control PBR were built. These are identical except for their GEMS or non-GEMS configurations. Each has an ~7 ft, 4.5" diameter, vertical air-lift section connected at the top to an ~2 ft horizontal section containing two exhaust ports for gas release. This section connects to a 2" diameter, diagonal downflow tube, connected at ground level to a 4.5" diameter, ~10 ft horizontal tube that connects back to the air-lift section. The long vertical and horizontal sections are made of flexible, transparent plastic tubing, surrounded by LEDs that provide up to full intensity sunlight illumination at the PBR surface. A central compressor provides up to 140 psi of driving, air-lift air for each PBR. Air inflow and culture flow rates are controlled by a 10-turn valve at the base of each air-lift section. Flomec, magnetic, liquid-flow meters in the diagonal downflow tubes are used to monitor and equalize culture flow rates. Minimum flow rates that still generate GEMS mixing are an important goal for minimal energy use. CO2/air mixtures are introduced via spargers at the junctions of the downflow and ground-level tubes. Gas mixtures and flow rates are controlled by a pair of Mass Flow Controllers (MFCs) and separate MFCs control the CO2/air flow rate into each PBR. GEMS 1 has a "baseline" helical groove pitch. GEMS 2 has a tighter pitch at the same depth. GEMS 3 has the same pitch as GEMS 1 but a shallower depth. The impact of flow rates on GEMS mixing and algal productivity was investigated only qualitatively because of problems controlling precise flow rates as discussed in the Changes/Problems Section. Available data show 15% to 20% greater productivity in GEMS 1 than in the GEMS 2 or 3 pitch and depth configurations. All three GEMS outperformed the non-GEMS control, with 60% to 80% greater productivity in all of the GEMS relative to the non-GEMS control PBR in a 200-hour run. The GEMS 1 helical pitch and depth configuration was used in the scaled-up, 300-Liter vertical, VGEMS PBR. Objective 3 - GEMS PBR scale-up. We decided to initially test scale-up to a 300-L vertical, air-lift GEMS (VGEMS) rather than the proposed 500-L horizontal design, because: a) Smaller footprints and thus higher areal productivity in vertical PBRs, b) Importance of air-lift designs for air-CO2 capture, c) Easy scale up of the modular VGEMS design by increasing the number of modules, d) Gentler circulation of sensitive filamentous algae, and e) For Phase I, the prohibitive cost of 8" diameter fittings for a 500-L system relative to 6" diameters for 300-Liters. A 300-L VGEMS and otherwise identical 300-L non-GEMS control PBR were built and tested with dye polymer to confirm GEMS mixing. Both are ~17 ft tall and have central 6.6" diameter air-lift tubes flanked by two 6.6" diameter downflow tubes. CO2/air mixtures are introduced via spargers at the base of the downflow tubes resulting in counter-current flow of rising gas bubbles against downward culture flow for maximum CO2 utilization. The transparent tubes are surrounded by LEDs that provide up to full sunlight illumination. Objective 4 - Show greater productivity in scaled-up GEMS relative to controls. Initial data showed growth in the 300-L VGEMS comparable to that in the 53-L GEMS and ~2x faster than in the 300-L control PBR. Because of technical problems and unexpected microbial contamination of inocula and PBR cultures, we were unable to run further, parallel VGEMS and control PBR tests, until these issues are resolved. Subsequent VGEMS runs showed less rapid growth and a best yield of ~6.6OD750nm, which is still ~40% higher than the ~4.1 OD750nm in the 300-L control PBR. A treatment of the PBRs with ammonia appears to have solved the contamination problem as discussed in the Changes/Problems section. Once resolved, we believe the VGEMS PBRs can be scaled to much larger dimensions, and will be able to grow any algae to much higher yields, at least 2x greater than in the control PBRs or any other algal mass cultivation system. Work to achieve this is ongoing and an important objective of Phase II. Objective 5 - Prepare Phase I Final Reports and Phase II proposal. We were granted a no-cost extension of Phase I to January 31, 2022. Our Phase II proposal has been submitted. The SF-425 Federal Final Financial Report has been submitted, and the Final REEport and Technical Reports are being prepared. Additional accomplishments: We have established a well-equipped Microbiology lab for media preparation, growth and maintenance of algal stock and inoculum cultures, low-temperature storage, and analyses. And work has been initiated toward automated monitoring, data acquisition, and feedback regulation of PBRs. This is a continuing objective of Phase II.

Publications

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Ultimately several target audiences will benefit from our work to develop game-changing GEMS PBR technology for scalable, algal CO2 capture and bioproduction at higher productivity and lower cost than in any existing system. These target audiences include: 1) Agricultural partners/customers interested in using fallow land for algal bioproduction, or in supplementing income by integrating GEMS PBR tubes into field furrows. 2) Agricultural partners/customers interested in cost-effective algal biomass production for nutraceutical, food, or feed applications. 3) CO2-emitting industries, such as power plants, cement factories, or foundries interested in cost-effective mitigation of their emissions. 4) Ethanol plants, breweries, distilleries, or others that emit clean CO2 streams, for added profitability by cost-effective algal CO2 capture and conversion to nutraceuticals, foods, or feeds. 5) Wastewater treatment plants interested in meeting new, stringent P and N effluent limits by efficient algal consumption of these enabled by profit-generating CO2 capture and bioproduction. 6) Any government agency, foundation, business, or homeowner interested in helping combat climate change by deploying GEMS PBRs for CO2 capture linked to biomass production. Efforts to reach target audiences during year 1 of the Phase I project included: 1) Extensive work with Peter Jorgenson, a colleague with connections to Arizona Public Service (APS), and preparation of a slide deck presented to APS executives, for a proposed vertical GEMS (VGEMS) PBR pilot module at their 1060 MW RedHawk, natural gas power plant that emits ~1.68 metric tons CO2 per year. The proposed ~14,000 Liter VGEMS, pilot module consisting of four, vertical GEMS tubes would demonstrate the utility of a GEMS PBR system for profit-generating CO2 capture and biomass production. 2) PI Robert Falco's productive conversations with Gregory S. Guard, CEO of Clearas Water Recovery Inc., leading to a strong Phase II support letter, in which Guard expressed great interest in testing GEMS PBR tubes in their algal wastewater purification system. The GEMS technology is of particular interest because of its potential to enable larger diameter PBR tubes operating at low flow rates, thus resulting in both lower CAPEX and OPEX costs. 3) A slide deck compiled to present the GEMS technology to ethanol plants. This shows how GEMS PBRs can effectively use the clean CO2 and thin stillage waste streams from these plant to capture and convert the CO2 to high-value algal biomass for feed, food, and nutraceutical feedstocks. We plan to contact Jeff Broin, CEO of Poet, LLC, as one of our first partner/customer targets. Our patent attorney has advised us to await submission of a utility patent on the vertical GEMS (VGEMS) innovations, as well as international PCT (Patent Control Treaty) filings before entering into discussions with the ethanol plants. 4) Several Zoom meetings with business colleagues Jeff Ebel and Leon Ostrowski of Midwest Wealth Ventures, LLC, and Bob Sylvest and Trez Wigfall of Opportunity Analysis, Inc., regarding work they can perform to aid us with customer discovery and business development in preparation for and during Phase II. 5) Extensive Zoom meetings and work with Larta advisor, Ken Hunt to develop a draft commercialization plan, that includes focused target audience and customer connections, for our Phase II application. We intend to continue working with Mr. Hunt on business development, enabled as part of the Phase II Technology and Business Assistance (TABA) funding. 6) Presentation of the GEMS technology to potential target audiences by PI Robert Falco at the 2020 Algae Biomass Organization (ABO) Algae Biomass Summit, Virtual Conference, (Algal growth and biomass yield in game-changing, Growth Enhancing Mixing Spectrum (GEMS) photobioreactors (PBRs). 7) Presentations of the GEMS technology to potential target audiences by Co-PI Toivo Kallas at the 2020 Midwest-Southeast Photosynthesis Virtual Conference, (Algal Bioproduction in Game-Changing, Growth Enhancing Mixing Spectrum (GEMS) Photobioreactors), and as part of the 2020 Carleton College Connects, Virtual Climate Change Panel (The potential and challenges of algal CO2 capture - Can Growth Enhancing Mixing Spectrum (GEMS) photobioreactors change the game?). Changes/Problems: Changes: 33 and 53 Liter PBRs were constructed for the initial experiments rather than 20 Liter units because: The advantage of the GEMS mixing technology (that brings algae to and from the PBR tube walls for optimal photon usage) for enhanced algal productivity increases as the tube diameter and length increases. Culture circulation in both the 33 and 53 L PBRs is provided by air pressure that drives cultures upward in vertical air-lift sections. The air-lift mechanism requires less energy than liquid pumps in vertical PBR designs, which maximize areal (per m2) productivity, will be important for any direct, air-CO2 capture application, and is gentler than liquid pumping on high-value, filamentous algae such as Spirulina species. Problems: 1. Monitoring and maintenance of constant culture flow rates, to obtain consistent and reliable comparisons from the PBRs, was a major problem. Several problems related to this were discovered and changes made to alleviate them. Flomec magnetic flow monitors, that were not in the original USDA budget, were purchased and installed for precise flow monitoring in the diagonal downflow tube of each 53-Liter PBR. After initial inconsistent performance, stainless steel collars were installed around each Flomec for improved grounding and alleviation of aberrant ground flow circuits. We are still examining the effects of Biofilm formation in the Flomecs, which may still be a problem and may indicate the need for some other flow meters to be installed. 2. Culture flow rates were also greatly affected by culture foaming and evaporative losses. If undetected (e.g. during night-time periods), the culture liquid level in the top horizontal tube could fall to a level that would prevent flow from this 4" diameter section into the subsequent 2" diameter diagonal downflow tube. Effectively the transition from the 4" to the 2" created a dam that blocked culture flow if the liquid level fell too low. Several measures were tested to alleviate foaming and the accompanying evaporation: Installation of ~30" high, 2" diameter PVC tube extensions connected to the short PVC tubes protruding from the gas release ports at the top of the top, horizontal section. This had only a small impact. Most of the foam simply pushed out of the tops of these extensions. Installation of a condenser in one each of the two PVC extensions at the top of each PBR. The condenser consisted of a section packed with aluminum wool through which cooling water circulated. This prevented foam and probably most evaporation from escaping through the Al wool condenser. However, considerable foam with evaporative loss still escaped through the second 30" extension tube. Finally, design and installation of S-traps joined via flexible tubing to the top of each 30" extension. The S-trap tubes connected to downward, rigid 2" PVCs whose perforated ends were submerged in ~2 gallons of water in a 5 gallon bucket. A drain tube connected to the bucket at the 2 gallon level, returned culture fluid back to the PBR whenever the liquid level rose to this level in the bucket. Initially, the S-trap design created some problems of its own, namely: The closed S-trap system at first created significant backpressure that restricted the air-lift speed and the culture circulation rate. And, inadvertently resulted in a subtle tilting of the top horizontal tube creating a larger dam at the junction of the 4" diameter top section and the 2" diameter diagonal down tube, exacerbating a fluid oscillation in this section that resulted in periodic surges of culture into the down tube and oscillating fast and slow culture flow rates that disrupted GEMS mixing. These unanticipated consequences of the S-trap system have now been alleviated. The oscillation and surge phenomenon has been alleviated by replacing the 4" to 2" diameter junction that connects the top, horizontal tube and the diagonal down tube with a 4" diameter junction. This and a level top tube have eliminated the oscillation and resulting culture surge, as indicated by steady flow rate readings from the Flomec flow monitors. At the same time, the modified S-trap appears to working effectively to eliminate essentially all foaming and evaporative culture loss. 3. Leakage and/or breakage of the flexible plastic tubing that was used in all illuminated sections of the PBRs, resulting in flow stoppage or complete culture loss. The incentive for use of plastic tubing, rather that glass or clear PVC, is compelling because even that made of top quality Exxon 1012HJ resin costs ~$0.10 to $0.20 per ft of 6" diameter vs. $40/ft for clear PVC. Many types of plastic tubing are available with very different properties such as transparency and breakage resistance. Unfortunately, supply chain issues during Covid, as well as minimum order hurdles, have greatly limited access to these for testing. We have begun to explore the use of metallocene catalyzed Linear Low Density Polyethylene lay-flat tubing (mLLDPE). Once the right mLLDPE material is obtained, the cost per foot of a GEMS PBR can be reduced by a factor of 100x over rigid clear tubing. We have discovered some formulations that could easily be the basis of good GEMS PBRs. For example, Exxon Mobil Exceed brand, performance polymers, like their 1012HJ resin, have high tensile strength and very high rupture resistance. Since we seek a combination of properties such as tensile strength, dart drop impact strength, Secant Modulus and elongation, that must be optimized, we need to test a number of combinations. However, getting a minimum order of any of their resins made to our specifications requires 'gambling' $5,000 or more per order, which has not been possible within the Phase I budget. 4. Challenges in maintaining optimal temperatures (38°C to 40°C for Synechococcus PCC 11901 cyanobacteria, blue-green algae, being used for the Phase I tests) and light intensities during different seasons in the SCF main lab. This space was originally designed for use as a warehouse with little insulation. We are unable to adequately cool this lab during 4 summer months. This, together with heat from the high-intensity LED illumination, results in temperatures of 45°C or higher, which inhibits the growth of our current algal test strain. As a solution for next summer, our colleague Scott Miller (University of Montana) and his student, Chris Pierpont, have kindly provided us with a thermophilic Synechococcus strain that grows well in the 40°C to 55°C range. This strain is now being maintained in our Bio-lab in Arizona. A less appealing alternative is to upgrade the air conditioning to a 15 ton unit. We currently do not have the funds for this could be a significant OPEX cost with a significant carbon footprint for any commercial operation. 5. Maintenance of optimal light intensity as a function of temperature. We initially used fluorescent lights to help maintain culture temperature in the winter, but these provided suoptimal illumination. These have now been replaced with 7 LED tubes per each section, providing illumination up to full intensity sunlight. 6. Maintenance of constant CO2/air mixtures and equal distribution to PBRs. Alicat mass flow controllers (MFCs) were purchased to enable constant CO2/air mixtures and constant CO2/air flow rates into the PBRs. These worked very well but over time problems arose with water vapor condensation in these gas lines or culture backflow into the MFCs. This is being addressed with driers and check valves installations. CO2/air-line leakages or blockages at various junctions resulted in some cases in line ruptures and disruption of CO2/air flow into the PBRs. Some problems including clogging also occurred with the spargers that introduce CO2/air bubbles into the PBRs. These problems have been addressed. What opportunities for training and professional development has the project provided?The principal team members of the SCF/AABT, USDA-NIFA Phase I SBIR project include Robert Falco, PI, CEO of SolarClean Fuels, LLC, former Michigan State University Professor of Mechanical Engineering with deep expertise in fluid dynamics and turbulent flows; Toivo Kallas, Co-PI, Managing Director of Algoma Algal Biotechnology, LLC, and Bio-Research Director of SCF, a Distinguished Professor Emeritus of Microbial Genetics and Biotechnology at the University of Wisconsin Oshkosh, with deep expertise in photosynthesis, algal cultivation, and bioengineering; Ron Will, former Chief Design Engineer at Subaru; Rudy Rivas, a skilled union contractor experienced in plumbing and high-pressure systems; Travis Smith, a part-time consultant with excellent instrumentation and programming skills; and Joshua Navigato, a skilled young plumber and handyman. The Falco/Kallas partnership uniquely combines key areas of expertise in fluid-systems engineering and algal biology for innovative algal bioreactor designs and bioproduction solutions. All members of the team have learned from each other and experienced professional development. PI Falco has gained experience in algal strains, culture maintenance, propagation, and growth assessment at small and large scales. Kallas in turn has gained valuable insights into photobioreactor design, construction, fluid dynamics, and troubleshooting. All other team members have benefitted from each other's skills and experiences in similar ways. For example, options and solutions for automated PBR monitoring and data collection have been gained from Smith, and Navigato has learned the basics of algal culture media preparation and growth measurements. How have the results been disseminated to communities of interest?To date, results have been disseminated to communities of interest primarily through individual or small group Zoom meetings as well as conference presentations to larger audiences. Covid has severely restricted our ability to meet directly with target audiences of interest and to interact with colleagues and potential partners or customers by direct attendance of professional or commercial conferences. Efforts to disseminate results are directly related to our efforts to reach target audiences as outlined in the Target Audience Section. These efforts include: Meetings with Peter Jorgenson, a colleague with connections to Arizona Public Service (APS), and preparation of a slide deck presented to APS executives, for a proposed vertical GEMS (VGEMS) PBR pilot module at their 1060 MW RedHawk, natural gas power plant. PI Falco's conversations with Gregory S. Guard, CEO of Clearas Water Recovery Inc., leading to a strong Phase II support letter, in which Guard expressed great interest in testing GEMS PBR tubes in their algal wastewater purification system. A slide deck compiled to present the GEMS technology to ethanol plants. Meetings with ethanol producers await approval from our patent attorney who has advised us to await submission of a utility patent on the vertical GEMS (VGEMS) system, as well as international PCT (Patent Control Treaty) filings before entering into these discussions. Zoom meetings with business colleagues Jeff Ebel and Leon Ostrowski of Midwest Wealth Ventures, LLC, and Bob Sylvest and Trez Wigfall of Opportunity Analysis, Inc., regarding work to aid us with customer discovery and business development. Zoom meetings and work with Larta advisor, Ken Hunt to develop a commercialization plan, that includes focused target audience and customer connections. Presentation by PI Falco at the 2020 Algae Biomass Organization (ABO) Algae Biomass Summit, Virtual Conference, (Algal growth and biomass yield in game-changing, Growth Enhancing Mixing Spectrum (GEMS) photobioreactors (PBRs)). Presentations by Co-PI Kallas at the 2020 Midwest-Southeast Photosynthesis Virtual Conference, (Algal Bioproduction in Game-Changing, Growth Enhancing Mixing Spectrum (GEMS) Photobioreactors), and as part of the 2020 Carleton College Connects, Virtual Climate Change Panel (The potential and challenges of algal CO2 capture - Can Growth Enhancing Mixing Spectrum (GEMS) photobioreactors change the game?). What do you plan to do during the next reporting period to accomplish the goals?As discussed in the Accomplishments Section and further discussed below in the Changes/Problems Section, several technical problems were encountered, primarily with aspects of the design, materials, and operational procedures of the 53-Liter PBRs. These problems were exacerbated by Covid-imposed restrictions or limitations on work schedules, supply chains, and outreach efforts. Problems of design, operation, and to some extent, materials, have either been mostly resolved or are in the process of being resolved. In the next reporting period up to the termination of the Phase I period, we expect: To collect further and better data from the optimized 53-Liter PBRs to firmly establish at least a 2?fold to 3-fold productivity (biomass yield) advantage in the best GEMS PBR relative to the non-GEMS control. To finish construction of the 300-Liter vertical GEMS (VGEMS) and corresponding, non-GEMS control PBR. To run algal growth experiments in the 300-Liter PBRs to determine whether the VGEMS will show at least a 2?fold to 3-fold productivity (biomass yield) advantage relative to the otherwise identical, non-GEMS, 300-Liter vertical control PBR. To establish contact with ethanol producers and/or other potential partners/customers in support of our Phase II applications and pilot-scale GEMS PBRs at their sites. Specifically: Objective 1): Demonstrate comparable algal growth rates and biomass yields in a 20-Liter GEMS PBR relative to 500 mL lab-cultures, and at least 2x (and perhaps up to 5x) greater productivity relative to an otherwise identical, 20-Liter, smooth-tube PBR. As discussed further in the Changes/Problems Section, several technical problems occurred that prevented consistent and comparable culture flow rates that are essential for productivity evaluations in GEMS vs non-GEMS PBRs. These include: Flomec, magnetic flow monitor grounding problems resulting in erratic readings. Culture oscillation problems in the top horizontal tube resulting in surges of culture liquid into the diagonal downflow tube and alternating extreme high and low flow rates that disrupted the cross-tube, GEMS mixing. Leakage or complete breakage of the flexible plastic tube sections resulting in flow stoppage and culture loss. This arose from supply chain problems and/or prohibitive costs of large bulk purchases needed to acquire small amounts of better tubing for initial tests. Challenges in maintaining optimal temperatures and light intensities during different seasons in the SCF main lab, which was previously used as a warehouse. Challenges in maintaining constant and uninterrupted flow of CO2/air mixtures into the PBRs. Foaming and culture fluid evaporation from the gas exhaust port in the top, horizontal tubes, resulting variously in alter culture flow rates or complete stoppage. These challenges have been or are being addressed, and once fully alleviated, we believe we will be able to consistently demonstrate at least 2 to 3-fold greater biomass yields in the GEMS PBRs. Objective 2): Test several different configurations of helical pitch, depth, and flow rates in 20-Liter GEMS PBRs to determine optimal designs for highest productivity of specific algal strains. The same problems that prevented consistent and comparable culture flow rate for algal growth and yield comparisons in the GEMS vs. non-GEMS control PBRs under Objective 1 above, prevented consistent comparisons of helical pitch, depth, and flow rate impacts on algal productivity among the three different, 53-Liter GEMS configurations that were built. As under Objective 1, these problems are being addressed, and once fully alleviated, final comparisons of the best performing GEMS configuration will be possible. Objective 3): Build a 500-Liter GEMS PBR based on the best 20-Liter design. Demonstrate scalable algal productivity in 500-Liter GEMS within 20% of that observed in the best 20-Liter GEMS. Objective 4): Demonstrate at least 2x (and perhaps up to 5x) greater algal productivity in a 500-Liter GEMS PBR relative to an otherwise identical, 500-Liter, smooth-tube PBR. As discussed under Accomplishments, Objective 3, we opted to build a 300-Liter vertical GEMS (VGEMS) PBR and an otherwise identical, 300-Liter, non-GEMS, control PBR, instead of the projected, 500-Liter, horizontal PBRs. As further discussed, These PBRs have been built, are in the final stages of construction testing, and algal productivity experiments in them will be initiated in the coming weeks. Lessons learned from pitfalls encountered with the 53-Liter PBRs are invaluable and have been applied to the 300-Liter units, and we look forward to seeing data from these larger PBRs where the advantage of GEMS mixing should become even more apparent.

Impacts
What was accomplished under these goals? Objective 1 All algal growth experiments to date have been done with Synechococcus PCC 11901, a fast-growing cyanobacterium that can grow to high biomass yields. We believe, however, that GEMS PBRs will be advantageous for enhanced growth of any algae. Experiments in GEMS vs control PBRs were first conducted in 33 Liters PBRs under natural sunlight, ambient temperature, and air CO2 in Los Alamos, NM in September 2020; and subsequently in 53 Liter PBRs in the SolarClean Fuels' lab in Fountain Hills, AZ. The 33 and 53 Liter PBRs were chosen rather than 20 Liter units because: a) The advantage of the GEMS mixing technology (that brings algae to and from the PBR tube walls for optimal photon usage) for enhanced algal productivity increases as the tube diameter and length increases. b) Culture circulation in both the 33 and 53 L PBRs is provided by air pressure that drives the culture upward in vertical air-lift sections, followed by gravity flow through down-sloping and horizontal tubes to return to the air-lift section. This air-lift mechanism is gentler on filamentous algae, requires less energy than liquid pumps in vertical PBR configurations, which maximize areal (per m2) productivity, and will be important for any direct, air-CO2 capture application. In the Los Alamos experiments, most importantly, under ambient, day-night conditions (temperatures ~4°C to 25°C), the 33-Liter GEMS PBR produced a 2 to 2.5-fold greater biomass yield over a 175 hour run than the otherwise identical, non-GEMS, 33-Liter control PBR. In the 53 Liter PBRs, the GEMS PBRs also outperformed the non-GEMS control. The advantage of GEMS mixing became most evident at higher cell densities. In the best runs to date, one of the GEMS PBRs reached a cell density of ~15 OD750nm (~3.4 grams Dry Weight/Liter, gDW/L) compared to ~5 OD750nm (~1.1 gDW/L) for the best non-GEMS run - i.e. 3-fold greater biomass yield. Four 53-Liter PBRs were built, three with different GEMS geometries, and one non-GEMS control. All were identical with the exception of the GEMS vs non-GEMS configurations. All operated with concentrated CO2 inputs, temperatures of ~30°C to 40°C (depending on outside, AZ temperatures), and ultimately with LED illumination up to ~2300 µmol photons m-2 sec-1 (~full sunlight). Maximum biomass yields of ~15 OD750nm (~3.4 gDW/L) in the GEMS PBRs were comparable to those obtained under comparable conditions in 500 mL lab cultures. As expected, initial growth rates in all 4 PBRs (doubling times, tD, ~7 hours) in low density cultures up to ~20 hours of growth approached those obtained in under comparable conditions in 500 mL laboratory cultures. Under these conditions neither photon nor nutrient availability is limiting. Similarly, as expected, initial growth rates and biomass yields in the 53 Liter GEMS and the 53 Liter control PBR were similar up to ~60 hours of growth and cell densities of ~4OD750nm (~0.9 gDW/L). At these still low, initial, cell densities, neither light nor nutrient availability are limiting, and there is no advantage of GEMS mixing. As stated, the GEMS advantage became evident at cell densities beyond this point. As discussed in the Changes/Problems Section, several technical pitfalls occurred that prevented consistent and comparable culture flow rates that are essential for consistent productivity evaluations in GEMS vs non-GEMS PBRs. These challenges are being addressed, and once fully alleviated, we believe we will be able to consistently show at least 3-fold greater biomass yields in the GEMS vs. non-GEMS PBRs. Objective 2 Three air-lift, 53-L GEMS and one non-GEMS, control PBR were constructed. These are identical with the exception of their GEMS or non-GEMS configurations. Each has an ~7 ft, 4.5" diameter, vertical air-lift section connected at the top to an ~2 ft horizontal section containing two exhaust ports for gas release. This section connects to a 2" diameter, diagonal downflow tube connects in turn, at ground level to a 4.5" diameter, ~10 ft horizontal tube that connects back to the air-lift section. The 7 ft vertical and 10 ft horizontal sections are made of flexible, transparent plastic tubing, each surrounded by seven, white-light LED tubes that provide up to ~2300 µmol photons m-2 sec-1 (~full sunlight) at the PBR surface. A central compressor provides up to 140 psi of driving, air-lift air for each PBR. The air inflow and culture flow rates in each PBR are controlled by a 10-turn valve at the base of the air-lift section. Flomec magnetic, liquid flow meters located in the diagonal downflow tube of each PBR are used to monitor and equalize culture flow rates. Maintaining equal flow rates in the 4 PBRs is critical for good comparisons and monitoring. To achieve the minimum flow rates that generate GEMS mixing is also a goal (thus, using the lowest necessary energy input). CO2/air mixtures are introduced via spargers at the junction of the downflow and ground-level section of each PBR. CO2/air mixtures and flow rates are controlled by a pair of Mass Flow Controllers (MFCs) and separate MFCs control the rate of CO2/air flow into each PBR. GEMS 1 has a "standard" helical groove pitch, based on flow experiments with marker dyes. GEMS 2 has a tighter pitch at the same depth. GEMS 3 has the same pitch as GEMS 1 but a shallower depth. The impact of flow rates on GEMS mixing and algal productivity was investigated only qualitatively because of problems encountered in controlling precise flow rates as discussed in the Changes/Problems Section. Based on available data, GEMS 1 showed 15% to 20% greater productivity than the GEMS 2 or 3 pitch and depth configurations. All three GEMS configurations outperformed the non-GEMS control. For example, 60% to 80% greater productivity in all of the GEMS PBRs relative to the non-GEMS control in a run of 200 hours. The GEMS 1 helical pitch and depth configuration is being used in the 300 Liter vertical GEMS (VGEMS) PBR currently under final construction. Objective 3 We decided to initially test scale-up to a 300 Liter vertical, air-lift GEMS (VGEMS) rather than a 500 Liter horizontal design, because of: a) Smaller areal footprint and thus higher productivity per m2 in vertical PBRs, b) The importance of air-lift designs for air-CO2 capture/conversion applications c) Ready scale up of the modular VGEMS design to any size by simply increasing the number of modules, d) Gentler circulation of sensitive filamentous algae, and e) For Phase I work, the prohibitive cost of 8" diameter fittings for a 500-L system relative to 6" diameters for 300-Liters. A 300-Liter VGEMS and otherwise identical 300-Liter non-GEMS control PBR have been constructed and tested with water and dye polymer to confirm GEMS mixing. Both are ~17 ft tall and have central 6" diameter air-lift tubes flanked by two 6" diameter downflow tubes. CO2/air mixtures are introduced via spargers at the base of the downflow tubes resulting in counter-current flow of rising gas bubbles against downward culture flow, enabling variable residence times of the gas, for maximum CO2 utilization. The vertical, transparent tubes are all surrounded by LED tubes that provide up to full sunlight illumination. Objective 4 As discussed in Objective 3, 300 Liter VGEMS and non-GEMS control PBRs have been built, but data on relative algal productivity are not yet available. Objective 5 We have been granted a no-cost extension of Phase I to January 31, 2022. The Phase I Final Report and Phase II proposal will be submitted by the anticipated, February 2022 Phase II application deadline. Additional accomplishments that were discussed in the proposal but not specifically included in the objectives list: We have established a well-equipped Bio-lab for growth and maintenance of algal stock and inoculum cultures, and low-temperature storage. Work has been done but not completed toward automated monitoring and data acquisition from PBRs.

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