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Muconic acid production from algae hydrolysate as a high-value co-product of an algae biorefinery
Muconic acid is an attractive bio-derived product because it can be easily converted to adipic acid, a high-value and high-volume monomer used in the production of nylon and other valuable consumer plastics. As such, production of muconic acid from algae hydrolysate was explored to expand the suite of products available from algal biomass. Here we have established initial performance parameters and have shown that the range of substrates present in algae hydrolysate that are consumed by Pseudomonas putida includes at least glucose, mannose, glycerol, acetic acid, and lactic acid. We achieved complete utilization of these major carbon sources found in Scenedesmus acutus hydrolysate with a maximum muconic acid productivity of 0.25 g/L h. Final titer was 13.0 g/L (27% molar process yield) at approximately 50 h. In addition, algae hydrolysate does not require any additional nutrients to achieve these performance metrics. Techno-economic analysis showed the potential to support up to 150 algal biorefineries at this yield with significant greenhouse gas reductions.
Final Report for Phase I SBIR “Wastewater Grown Algae Feedstock Production and Processing to Bioproducts”
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An attached microalgae platform for recycling phosphorus through biologically mediated fertilizer formation and biomass cultivation
Nutrient management is a global challenge for protecting water bodies from eutrophication and for retaining and sustainably recycling phosphorus within the biosphere. This challenge is especially important for water resource recovery facilities (WRRFs) in jurisdictions that limit nutrient loads in plant effluent. A microalgae-based biofilm platform has been designed, constructed, and tested to remove phosphorus and nitrogen from anaerobic digester (AD) effluent filtrate through cultivating biomass and inducing the precipitation of a mineral called struvite (NH₄MgPO₄ · 6H₂O) using a rotating algae biofilm reactor (RABR). RABRs function by rotating a growth substratum through nutrient rich water and then into the atmosphere, immersing microalgae in both sunlight and water. Photosynthesis is utilized in RABR operation to enhance struvite formation by increasing the pH value within the biofilm through the uptake of carbon dioxide from solution. Measurements of pH trended higher with depth through the biofilm when exposed to light confirming the function of photosynthesis in increasing pH and, as a consequence, struvite formation. In addition, reducing RPM allows more time for water to evaporate through exposure of the biofilm to the atmosphere and provides a management option to exceed struvite solubility product. Struvite precipitation was predicted based on chemical analysis and MINTEQ modeling of ADE, was confirmed using Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy, and quantified by determining ash content. Increase in pH within the biofilm was confirmed as photosynthetic photon flux density increased. While struvite precipitation and removal from wastewater is conventionally accomplished through physicochemical methods, this research is the first report of struvite enhanced formation through algae biofilm-based photosynthesis processes.
Development of Biofuel and Bioenergy Processes for Photosynthetic CO 2 Assimilation (CRADA Final Report)
In order to fully exploit algae carbon assimilation for fuels, a preqrequisite is high photosynthetic efficiency and carbon conversion to fuel precursors. In the context of this collaborative project between NLR and EMRE we aim to address aspects of both the biological constraints around photosynthesis-driven carbon assimilation flux as well as elements that bridge the gap between laboratory cultivation and meaningful outdoor deployment, building on the unique capabilities of NLR and EMRE’s algae bioengineering teams. The goal of this work is to improve our understanding of key cellular biochemistry, mass cultivation, and biomass conversion constraints that presently limit the potential bio-oil yield. The proposed first phase of this work will focus on biomass growth improvements by testing hypotheses for alleviating identified metabolic and photophysiological bottlenecks. The results of this work could inform decisions by stakeholders (e.g., industry, community, investors) on the design and deployment of a sustainable biofuel supply chain from algae.
Barriers to Scale: Algae Crop Protection (Workshop Summary Report)
This report summarizes key takeaways from the 2021 Algae Crop Protection Workshop, including strategies to overcome the significant technical challenge of pests to the scaling and process intensification of the algae industry.
Final Report for FE0032098: Improving the cost-effectiveness of algal CO2 utilization by synergistic integration with power plant and wastewater treatment operations
The overall goal of this project was to demonstrate an engineering-scale open raceway pond algae cultivation system (approximately 180 m²), including the integration of technologies that utilized carbon dioxide (CO₂) from a coal-fired power plant and wastewater-derived nutrient inputs for cost-effective and environmentally friendly biomass production. The key advantages associated with the innovative algae cultivation system and its integration with wastewater treatment functions, as described herein, had been demonstrated in previous bench- and pilot-scale work by the project team partners. This project combined those approaches to maximize practical benefits and available synergies, resulting in a significant reduction in the net cost of producing algal biomass products. The primary target algal species for the project was Spirulina, which served as a high-protein content ingredient for food and animal feed. Spirulina was selected because it had already been approved by the FDA, was in use as a food ingredient, and commanded prices of up to $30/kg. It had a typical protein content of 50–75%, comparable to other high-protein concentrates, featured high digestibility without requiring pretreatment, and offered a high conversion ratio in animal feed applications. In addition, Spirulina had a relatively high content of the blue pigment phycocyanin, which could be extracted as a high-value co-product prior to using the remaining biomass for nutritional purposes.
A long-term pilot-scale algal cultivation on power plant flue gas – Cultivation stability and biomass accumulation
A long-term culture of a selected freshwater microalgal assemblage on power plant flue gas was realized using a pilot-scale photobioreactor. Different culture conditions of light cycle, harvesting amount, pH stabilization, and sulfur addition were tested during a 12-month continuous operation at a natural gas fired power plant. The results concluded that the daily harvesting amount of 60% with no light-cycle and NH 4 NO 3 as the nitrogen source and pH stabilization agent, resulted in optimal cultivation performance. Biomass concentration and biomass productivity reached 1.07 ± 0.23 g L -1 and 0.43 ± 0.14 g L -1 d -1 , respectively. During the year-long cultivation period, the algal assemblage was relatively stable with average abundances of 88 ± 4% and 12 ± 4% for algae and bacteria, respectively, and no significant differences between individual case scenarios. The stable culture fostered a stable biomass composition. Protein, carbohydrate, and lipid contents of the algal biomass were 58.60 ± 2.41%, 19.5 ± 2.44%, and 9.70 ± 1.68%, respectively.
Prospects for Engineering Biophysical CO 2 Concentrating Mechanisms into Land Plants to Enhance Yields
Although cyanobacteria and algae represent a small fraction of the biomass of all primary producers, their photosynthetic activity accounts for roughly half of the daily CO 2 fixation that occurs on Earth. These microorganisms are able to accomplish this feat by enhancing the activity of the CO 2 -fixing enzyme Rubisco using biophysical CO 2 concentrating mechanisms (CCMs). Biophysical CCMs operate by concentrating bicarbonate and converting it into CO 2 in a compartment that houses Rubisco (in contrast with other CCMs that concentrate CO 2 via an organic intermediate, such as malate in the case of C 4 CCMs). This activity provides Rubisco with a high concentration of its substrate, thereby increasing its reaction rate. The genetic engineering of a biophysical CCM into land plants is being pursued as a strategy to increase crop yields. This review focuses on the progress toward understanding the molecular components of cyanobacterial and algal CCMs, as well as recent advances toward engineering these components into land plants.
Deep Learning Prediction of Protein Complex Structures
Proteins interact to form protein complex to carry out biological functions such as catalytic chemical reaction. Therefore, it is important to develop computational methods to predict protein-protein interaction and the structures of protein complexes to study and enhance protein function. In this project, we successfully developed several deep learning methods to predict inter-protein contacts and the reinforcement learning and optimization methods to reconstruct protein complex structures from predicted inter-chain contacts. The methods were integrated with the MULTICOM protein complex structure prediction system and applied to predict the complex structures of biomass production-related proteins of green algae. During the two and a half years of research and development, all the specific milestones of the project were achieved successfully. 16 publications/manuscripts were produced. 10 software tools were developed. A patent application was submitted. Our MULTICOM predictors leveraging some tools developed in this project were ranked among the top predictors in the 15th Critical Assessment of Techniques for Protein Structure Prediction (CASP15) in 2022.
NREL's Capabilities in Biorefinery Analysis
NREL remains at the forefront of expertise in conducting process, techno-economic, and life cycle analyses to connect research with future commercial process integration, a critical step in the scale-up of biomass conversion technologies. Technology areas covered include lignocellulosic biorefineries, algae, CO2 utilization, waste-to-energy, aviation and marine fuels, refinery modeling and analysis, and the circular economy.
Development of integrated screening, cultivar optimization, and verification research (DISCOVR): A coordinated research-driven approach to improve microalgal productivity, composition, and culture stability for commercially viable biofuels production
To address major knowledge gaps and barriers to the commercial development of algal biomass for biofuels and co-products, a collaborative consortium, Development of Integrated Screening, Cultivar Optimization, and Verification Research (DISCOVR), was established in 2016. Funded by the U.S. Department of Energy (DOE) Bioenergy Technologies Office (BETO), this consortium constitutes a partnership between four DOE national laboratories - Pacific Northwest National Laboratory (PNNL), Los Alamos National Laboratory (LANL), the National Renewable Energy Laboratory (NREL), and Sandia National Laboratories (SNL) - and the Arizona Center for Algae Technology and Innovation (AzCATI) at Arizona State University. To address the barriers of strain selection for achieving high seasonal productivities with a suitable composition and culture resilience, a tiered strain down-selection pipeline is implemented. At Tier I, the temperature and salinity tolerance of strains is determined in flask cultures; at Tier II, the areal biomass productivity and composition is determined in climate-simulation photobioreactors; at Tier III, the productivity and culture stability are determined in outdoor raceways. The top performing strains move forward to long-term testing at the algae testbed site at AzCATI to generate annual biomass productivity data. Concurrent to the strain down-selection in the DISCOVR pipeline, hypotheses for increasing biomass productivity, shifting biomass composition to enhance intrinsic value, and improving culture stability and resistance to pests are also tested. Techno-economic analyses are carried out to determine whether promising findings from laboratory studies or proposed modifications in outdoor pond cultivation conditions translate into reductions in the minimum biomass selling price (MBSP). Notably, in the three years following the launch of DISCOVR, annual biomass productivity has increased from 11.7 to 17.6 g m -2 day -1 , resulting in an MBSP decrease from 824 to 611 $ ton -1 .
DISCOVR strain screening pipeline – Part III: Strain evaluation in outdoor raceway ponds
Not Available
Development of a kinetic model to describe six types of symbiotic interactions in a formate utilizing microalgae-bacteria cultivation system
This study investigated an algae/formate-utilizing-bacteria system that has been developed for carbon capture. This photomixotrophic consortium consumed formate to support bacterial growth so that the resulting respiration CO 2 could be simultaneously used by algae to avoid CO 2 gas-lipid mass transfer limitation. To understand biomass growth and population interactions in this unique system, a kinetic model has been developed to describe algae and bacteria multiplication, formate and nitrogen utilizations, CO 2 mass transfer, O 2 generation and consumption, lighting condition and shading effect. The simulation indicated that this ecosystem could form six types of interactions (mutualism, commensalism, parasitism, neutralism, amensalism, and competition) depending on the light intensity and nutrient availability. Furthermore, the simulation of both batch culture and chemostat, along with parameter sensitivity tests and experimental observations, offered insights into optimal applications of algae-bacteria consortium based photobiorefinery.
Impacts of Fungal Disease on Algal Biofuel Systems: Using Life Cycle Assessment to Compare Control Strategies
While climate change has incentivized attention on sustainable fuel sources, algae has positioned itself as a both promising and problematic biofuel feedstock. Diseases such as fungal pathogens cause costly algal feedstock crashes, but the life cycle assessments (LCAs) used to analyze the viability of algal feedstocks for biofuel have yet to consider the impact of disease on life cycle metrics. Here, we incorporate a disease model into a well-documented LCA for algal biorefineries to compare two sustainability metrics, energy return on investment (EROI) and global warming potential (GWP). We begin by showing that failure to consider disease leads to overly optimistic LCA metric outputs. Then, we compare two leading control strategies of disease chemical and biological. Our analyses show that biological engineering of a multispecies consortium of algae has a greater positive impact on LCA metrics than chemical control of the fungal pathogen using a fungicide. We expand how and when bi-cultures might advantageously exhibit the “dilution effect” whereby differentially susceptible species exhibit compensatory dynamics that stabilize feedstock production. Furthermore, our results emphasize the impact of disease and suggest that multispecies consortia of algae can be biologically engineered to reduce greenhouse gas emissions and improve the economic viability of biofuel.
BETO 2021 Peer Review - 1.3.5.270 - Rewiring Algal Carbon Energetics for Renewables (RACER)
Critically needed improvements in biomass and biofuel intermediate productivity can be made by addressing fundamental inefficiencies in algal carbon conversion efficiency (CCE) to biofuel intermediates. Algae photosynthesis is, at best, able to convert 5-7% of incident light energy to biomass, while conversion to fuel intermediates falls 15-25% short of its maximum potential due to inefficiencies along the pathways. Recent progress in the Rewiring Algal Carbon Energetics (RACER) project consortium focused on a means to address the above inefficiencies in a pathway from algal biomass to a trifecta of fuel intermediates, ethanol, 2,3-butane diol, lipids and green biocrude. This project engineered a production-relevant algal species Desmodesmus armatus (SE 00107), to demonstrate biomass productivity improvements, with a doubling of the fuel intermediate yields. The new algae biorefinery paradigm embodied in RACER opens opportunities for algae engineering beyond efforts typically targeted solely at lipid content or improved light harvesting efficiency. Parallel approaches showed improved CCE through elimination of wasted energy during photosynthesis and increased carbon flux to transitory carbohydrate storage in the cells. Outdoor operation and nutrient management strategies with improvements in pretreatment, fermentation and extraction in a Combined Algal Processing approach showed a 40% reduction in MFSP, with a combined biofuel productivity of > 3700 gal/acre.
Algae Asphalt to Enhance Pavement Sustainability and Performance at Subzero Temperatures
This paper evaluates the potential of algae-derived biobinders as sustainable alternatives for pavement construction. It specifically examines the physicochemical and rheological properties of biomodified binders and their potential to offset carbon emissions when used as partial replacements for conventional petroleum-based asphalt binders. Biosequestration of CO 2 using microalgal cell factories is a promising way of recycling CO 2 into biomass via photosynthesis. Our study demonstrates that incorporating algae-derived binders into asphalt can significantly reduce carbon emissions. Each 1% increase in algae-based biobinder leads to an approximate 4.5% decrease in net carbon emissions. This indicates that a blend containing about 22% biobinder has the potential to achieve carbon neutrality. Blends with higher proportions may even result in net-negative emissions, highlighting a promising strategy for environmentally responsible road construction. In terms of performance, the study shows that certain algae-derived biobinders significantly enhance the cracking resistance of asphalt, particularly under subzero temperatures, by improving its stress-relief capacity. A key contribution of this work is the introduction of polarizability as a novel molecular-level parameter for assessing the compatibility of algae-derived bio-oils with asphalt. By capturing the electronic responsiveness of bio-oil molecules, polarizability serves as a predictive indicator of their interaction potential with asphalt components, providing a new dimension for evaluating the binder performance at the molecular scale. Among the tested materials, the biobinder derived from Haematococcus pluvialis demonstrated particularly strong improvements in resistance to permanent deformation under repeated loading conditions analogous to traffic-induced stress, as well as enhanced resistance to moisture-induced damage. In conclusion, these findings advance the chemistry-driven design of biomass-based binders and highlight a promising pathway toward the development of low-carbon, high-performance, and sustainable infrastructure materials.