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All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Chapter 5: Methods for Algal Protein Isolation and Proteome Analysis

Microalgae present promising feedstocks to produce renewable fuel and chemical intermediates, in part due to high storage carbon flux capacity to triacylglycerides or storage carbohydrates upon nutrient deprivation. However, the mechanism(s) governing deprivation-mediated carbon partitioning remain to be fully elucidated, limiting targeted strain engineering strategies in algal biocatalysts. Though genomic and transcriptomic analyses offer key insights into these mechanisms, active post-transcriptional regulatory mechanisms, ubiquitous in many microalgae, necessitate proteomic and post-translational (e.g., phospho- and nitroso-proteomic) analyses to more completely evaluate algal responsiveness to nutrient deprivation. Herein, we describe methods for isolating total algal protein and conducting proteomic, phosphoproteomic, and nitrosoproteomic analyses. We focus on methods deployed for the chlorophyte, Chlorella vulgaris, a model oleaginous alga with high flux to renewable fuel and chemical precursors.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
DOE OSTI2024

Implications of pond reliability on the techno-economic and life cycle environmental impacts of algal biofuels

Despite extensive research on algal bioproducts, there is limited understanding of how pond contamination affects their economics and environmental impacts. This work compared the costs and environmental impacts of algal biofuels across different pond failure scenarios. Pond failure was simulated by a reliability model based on pond mean-time-to-failure (MTTF). The reliability model was integrated with a process model to analyze the impacts of pond failure on the operations of algal farms and biorefineries. Process model outputs were used for techno-economic analysis and life cycle assessment to determine the minimum fuel selling price (MFSP), global warming potential (GWP), and freshwater consumption impacts of algal biofuels for five MTTF scenarios of 20, 54, 80,120, and 350 days, assuming an average mean-time-to-reset of 7 days. Results show that higher MTTFs reduce the cost and environmental impact of algal biofuels, but with diminishing returns. The average MFSPs for the 20-day, 54-day, and 350-day MTTF scenarios were $\$3.52$, $\$2.54$, and $\$2.10$ per liter of gasoline equivalent, respectively. The GWP for the same scenarios were 131, 96, and 83 g CO 2eq MJ –1 , respectively. This study highlights the significant impact of larger seed trains, required under low MTTFs, on the costs and greenhouse gas emissions of algal biofuels. Moreover, the work shows that algal biofuels fail to be cost-competitive with conventional fuels, even when productivities are increased from 17 to 35 g m –2 d –1 . Furthermore, this work is the first to explore the implications of pond failure on the sustainability of algal biofuels and provides valuable insights to algae farmers on how to reduce the costs and financial risks of algal cultivation through process design and pond management strategies.

09 BIOMASS FUELS↗
DOE OSTI2021

Development of the high-productivity marine microalga, Picochlorum renovo , as a photosynthetic protein secretion platform

Microalgae are remarkable in their ability to convert CO 2 and sunlight into renewable biomass and bioproducts. In this work, we have established a novel photosynthetic protein production platform via computational prediction and genetic engineering using native microalgal protein secretory signal peptides to achieve functional secretion of the fluorescent protein mCherry. Importantly, this work was conducted in the recently characterized alga Picochlorum renovo, an alga of industrial interest due to its rapid growth rate, tolerance to both high temperature and salinity, and genetically tractable nuclear and chloroplast genomes. Genomic queries allowed the identification of native secretory signal peptides, which were N-terminally fused to mCherry allowing for secretion into the culture supernatant. Further characterization revealed no impact on fitness, a production rate of 0.19 mg/L/day, and titer of 0.37 mg/L of transgenic mCherry protein in culture supernatant. These findings lay the foundation for applied genetic engineering approaches that could enable CO 2 -sequestering, sustainable photoproduction of industrially relevant enzymes at low cost.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Algal harvesting and water filtration

Systems for algae-based water filtration and algae harvesting are described herein. A flow-way comprises a channel and a divider positioned within the channel that separates the channel into sub-channels. The flow-way further includes an algal growth that grows on a sidewall of the channel and/or the divider. The algal growth can be established in the flow-way by positioning a pre-seeded mesh element within the channel, the pre-seeded mesh element having an algae culture growing thereon prior to positioning the pre-seeded mesh within the channel. A harvesting system comprises a hinged container that includes a straining screen. The hinged container is positioned downstream from a flow-way and receives water from the flow-way. Biomass in the water is strained by and accumulates on the screen. Weight of the accumulated biomass causes the hinged container to tip and deposit the accumulated biomass in a second container.

Davis, Ryan Wesley↗
DOE OSTI2021

Techno-economic and life-cycle assessment of fuel production from mixotrophic Galdieria sulphuraria microalgae on hydrolysate

Outdoor photoautotrophic algal growth is limited by light attenuation and attendant respiratory CO 2 losses during dark periods, limiting its productivity potential and carbon use efficiency. Developing a system that leverages mixotrophic growth (combining the benefits of both heterotrophic and photoautotrophic growth) has the potential to dramatically improve the total productivity and economics of the system. However, it is unknown if the productivity gains offset the added costs of outdoor mixotrophic cultivation using cellulosic hydrolysate as the feedstock. In this study, corn stover-derived cellulosic sugars were evaluated as the mixotrophic organic carbon source for the cultivation of Galdieria sulphuraria, which can metabolize both glucose and xylose from corn stover hydrolysate. A techno-economic analysis (TEA) and a life-cycle assessment (LCA) were conducted based on a detailed engineering process model for both glass helical photobioreactor and covered pond cultivation platforms, coupled with downstream conversion and upgrading to renewable diesel through hydrothermal liquefaction. Results show the minimum biomass selling price for cultivation in the photobioreactor design assuming a productivity of 1.575 kg m -3 day -1 and a substrate yield of 0.57 g g -1 is $2869 per dry metric ton. The costs are dramatically reduced in the covered pond design which assumes a productivity of 0.8 kg m -3 day -1 and a substrate yield of 0.7 g g -1 , 921 dollars per dry metric ton. Expanding the system boundary to include downstream processing results in a minimum fuel selling price of 8.24 dollars and 3.32 dollars dm 3 GE -1 for the photobioreactor and covered pond systems, respectively. Finally, life-cycle results demonstrate a global warming potential of 339 and 9.1 gCO 2 -eq. MJ -1 on a well-to-wheels basis and a net energy ratio of 2.21 and 0.25 MJ MJ -1 for the photobioreactor and covered pond systems, respectively. Discussion focuses on a required co-product selling price as a function of biomass diversion to meet economic parity with conventional fuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Reliability metrics and their management implications for open pond algae cultivation

The prevalence of contaminating organisms in outdoor algae cultivation, with the often-associated dramatic crop failures, necessitates the need for metrics describing production system reliability. Standard metrics for algae cultivation reliability are critically needed to be able to map improvements in operational parameters, but do not currently exist. In this work, we present a set of standard metrics including mean time to failure (MTTF) and mean time between failures (MTBF) as the basis for the calculation of pond failure rate (FR) and reliability coefficient (RC). Metrics associated with the numbers of contaminating organisms such as abundance ratio (AR), prevalence of infection (PI), and mean intensity of infection (MII) are also relevant. These metrics, based on measured experimental values of outdoor pond performance during the Algae Testbed Public-Private Partnership (ATP3) Unified Field Studies (UFS), provide a basis for quantifying pond failure and provide insight into potential pond management and contaminant mitigation strategies. From these reliability metrics applied to this dataset, we are able to link operational parameters, such as harvest frequency and inoculum source, to pond reliability for different algae strains and seasons, and from an assessment of AR, provide contamination thresholds beyond which a culture may be unrecoverable. Ultimately, the implementation of widely-practiced and simple-to-calculate algae pond reliability metrics calculated from rapid and easy to collect data or observations will reduce risk and uncertainty in large-scale algae deployment and aid in the development of integrated pest management (IPM) strategies.

09 BIOMASS FUELS↗
DOE OSTI2022

Advances in engineering algae for biofuel production

While algae demonstrate potential as a sustainable fuel source, low productivities limit the economic realization of algal biofuels. High-throughput strain engineering, omics-informed genome-scale modeling, and microbiome engineering are key technologies for enabling algal biofuels. High-throughput strain engineering efforts generate improved traits, including high biomass productivity and lipid content, in diverse algal species. Genome-scale models, constructed with the aid of omics data, provide insight into metabolic limitations and guide rational algal strain engineering efforts. As outdoor cultivation systems introduce exogenous organisms, microbiome engineering seeks to eliminate harmful organisms and introduce beneficial species. In conclusion, optimizing algal biomass production and lipid content using these technologies may overcome the productivity barrier for the commercialization of algal biofuels.

09 BIOMASS FUELS↗
DOE OSTI2022

The landscape of Chlamydomonas histone H3 lysine 4 methylation reveals both constant features and dynamic changes during the diurnal cycle

Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. Here, we interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G 1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G 0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04

Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments - high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO 2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Altogether, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequencebased methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Feasibility of Algal Biochar, a Byproduct of Biofuel Production, as a Supplemental Cementitious Material

Algal biochar, as the solid residue of biofuel production from algal biomass, is reported to explore disposition options, aiming to lessen the liability or obstacles to biofuel production processes. However, landfills and open combustion lead to adverse environmental impacts. One way to add value to such wastes is to use them as admixtures in cementitious construction materials. This study aims to investigate the feasibility of algae-derived biochar as supplementary cementitious materials (SCM) at different water contents and mixture ratios. Algal biochar-cement composites were prepared with different algal biochar content as well as different water-to-cement (w/c) ratios, and the surface area, morphology, elemental, and mineralogical composition were characterized. To compensate for the high-water absorption of algal biochar, a small concentration of a superplasticizer was used since higher w/c ratios negatively impact strength. The mechanical performance of algal biochar-cement composites is compared with control composites using commercial silica fume as a typical commercial SCM. The findings suggest that algal biochar is a promising candidate to replace commercial SCM, like silica fume, since algal biochar-cement composites can reach comparable compressive strength and Young’s modulus to commercial pozzolan-cement materials with the same w/c ratio, though at later curing times, 33 days. Although the tensile strength of algal biochar-cement composites is statistically similar at 7 days, it is significantly lower at later curing times, and further investigation is required to improve this property. Algal biochar-based cement binders showed comparable embodied carbon to silica fume-based cement binders based on a cradle-to-gate lifecycle analysis. However, the ability of algal biochar to absorb large volumes of CO 2 over short periods of time, as measured in this study, makes this novel SCM an excellent alternative to reduce the embodied carbon of concrete structures cradle-to-grave at 1/10 of the cost. In conclusion, valorization of algae-derived solid waste provides great potential to reduce embodied carbon and brings credit to biofuel production and concrete-based construction.

algae-derived↗
DOE OSTI2023

Towards an Integrated Process for CO 2 Capture and Utilization: Cultivation of Scenedesmus acutus Using Gaseous CO 2 and NH 3

Integrating CO 2 scrubbing from flue gas with its utilization in algae cultivation represents a potential means of lowering the cost of CO 2 capture. Towards this goal, this study sought to assess the feasibility of using gaseous a CO 2 /NH 3 stream, derived from CO 2 capture using aqueous ammonia, as a C- and N-source for algae cultivation. Scenedesmus acutus was cultured in 800 mL photobioreactors using gaseous CO 2 /NH 3 in mole ratios varying from 7 to 18. Excellent growth of Scenedesmus acutus was observed, the average growth rate for CO 2 /NH 3 = 10 of 0.171 ± 0.015 g/L·day exceeding that obtained using 1% CO 2 /N 2 and urea as the N-source (0.099 ± 0.28 g/L·day). Under optimal growth conditions (CO 2 /NH 3 mole ratio of 10), CO 2 utilization ranged from 57% to 72%, while the NH 3 utilization was >90%. The CO 2 /NH 3 feed rate was also found to exert a significant effect on algae productivity, excessive feed rates leading to accumulation of NH 3 in the culture at concentrations that were toxic to the algae. Consequently, to avoid the toxic effects of high NH 3 concentrations (>2.0 mM), it proved necessary to balance the NH 3 supply with the algae growth rate so that excessive NH 3 accumulation was prevented. This indicates that for practical applications, a CO 2 /NH 3 feed control strategy would be required that takes into account the ammonium ion concentration in solution and the pH so as to avoid significant concentrations of free NH 3 . Furthermore, analysis of the harvested biomass revealed a high protein (≥ 47 wt%) and a low ash content (< 3.6 wt%), suggesting it would be well suited for use as animal feed or as a feedstock for the production of bioplastics.

03 NATURAL GAS↗
DOE OSTI2022

Comparative performance and technoeconomic analyses of two microalgae harvesting systems evaluated at a commercially relevant scale

Using a commercially scalable system designed for processing thousands of liters a day, here we evaluated the factors that affected the performance, energy consumption and capital/operating costs of both flocculation-based and filtration-based algal harvesting systems over a 16 month period. Coagulation efficiency was the primary driver of harvest efficiency in the flocculation-based method, while cell lysis was important for the filtration-based method. Culture (algae) age differentially influenced harvest efficiency through changes in cell fragility and stickiness. The average energy consumption of the flocculation-based method was 0.389 kWh/m 3 , while that for the filtration-based method was 4.343 kWh/m 3 . The average harvesting cost of the filtration-based method was 5.35 $\$$/m 3 , while for flocculation-based method it was 4.52 $\$$/m 3 . The concentration factor of filtration-based and flocculation-based method were 770–1086 and 407–448, respectively. For both harvesting methods, labor costs dominated and ranged from 55.84%–67.94% of total cost. Further system automation is a potential method to lower the harvesting cost. The filtration-based harvesting method could produce a better quality of algal biomass with higher concentration factors and less ash content, but needed more energy input, as compared with a flocculation-based method. This study highlights the importance of algal culture status to successful harvesting, and it also provides insight into developing more efficient harvesting technology with lower energy and capital cost.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Results of the International Energy Agency Bioenergy Round Robin on the Analysis of Heteroatoms in Biomass Liquefaction Oils

A round robin study evaluating the analysis of biomass liquefaction oils (BLOs) from fast pyrolysis and hydrothermal liquefaction (HTL) was performed, involving fifteen laboratories in seven countries in order to assess the current status of analytical techniques for the determination of nitrogen, sulfur, and chlorine content in BLOs and evaluate potential differences in origin (i.e. fast pyrolysis vs HTL). The BLOs were produced from a range of feedstocks including pine, mixed softwoods, forest residues, micro-algae, miscanthus, and wheat straw to cover a variety in nitrogen, sulfur, and chlorine content and speciation. Nine samples were distributed, comprised of eight separate BLOs and one blind duplicate produced by five producers. The samples were analyzed for water, carbon, hydrogen, nitrogen, sulfur, and chlorine content. No analytical test method was mandated; laboratories were encouraged to utilize whichever method they determined would be most applicable, relying on the existing body of BLO literature as a guide. The results of this round robin study are presented in this paper. The results of the carbon, hydrogen, and water measurements as reference analyses had relative standard deviations (2.9%, 3.5, and 5.6%, respectively) that were comparable to those found in past round robin studies on fast pyrolysis bio-oil. The analysis of nitrogen, sulfur, and chlorine showed higher levels of variability. Laboratories mostly chose the same method for water, carbon, hydrogen and nitrogen determination whereas there were a variety of methods chosen for sulfur and chlorine determination. The results suggest that specific analytical methods for the determination of nitrogen, sulfur, and chlorine should be further refined to ensure reproducible and accurate results for BLO analysis due to their importance on emissions, material selection, and catalyst activity.

Biomass, IEA Bioenergy, pyrolysis, hydrothermal li↗
DOE OSTI2021

Ultraviolet radiation pre-treatment of wastewater, improving its utility for algal cultivation

The present invention relates to a process of remediating wastewaters, preferably agricultural wastewaters resulting from animal production or contained animal feeding operation sites. The water is treated to promote assimilation of nutrients into algal biomass, which can be harvested and sold, and the resultant wastewater is then purified. According to the invention, short wavelength UV radiation (less than 280 nm wavelength) is used to pretreat wastewater, with the dose determined by absorbance of the water, not by bacterial load. Pretreated water exhibits changes in chromophoric dissolved organic matter that allows for improved and increased algae production by as much as 88%.

Feris, Kevin↗
DOE OSTI2021

Mapping the Path Forward to Next Generation Algal Technologies: Workshop on Understanding the Rules of Life and Complexity in Algal Systems

Algal technology offers enormous potential as a source of renewable biomass and bioproducts for fuels, biochemicals, nutrients, and numerous other applications. While tremendous opportunities exist, significant challenges are also present in harnessing and utilizing this valuable and potentially sustainable bioproduction platform. Funded by a joint partnership between the Bioenergy Technologies Office (BETO) at the Department of Energy (DOE) and the National Science Foundation (NSF), the recent virtual workshop, "Understanding the Rules of Life: Complexity in Algal Systems Workshop," was organized to bring together thought leaders from academia, national laboratories and other government agencies, and industry to identify key challenges and propose research strategies to overcome these barriers. Two parallel sessions were undertaken: 1) algae strain and toolkit development and 2) algal ecosystem and microbiome dynamics, in order to advance algae engineering and cultivation efforts. In each session, attendees discussed the most pressing challenges from the perspective of model systems and omics analysis. Themes such as genetic engineering toolboxes, translation from the laboratory to outdoor cultivation, functional genomics, metabolomics, algal ecology, consortial design, microbiome dynamics, mathematical models, and data analytics emerged from the discussion. Overall, this workshop has served to elucidate the key hurdles and many opportunities present as we continue to advance algal science and technology from the laboratory and into the commercial realm.

09 BIOMASS FUELS↗
DOE OSTI2023

An algal-bacterial symbiotic system of carbon fixation using formate as a carbon source

The commercial implementation of algal cultures for efficient capture of CO 2 from fossil-derived flue gases is not a reality yet due to several major challenges, particularly low gas-liquid mass transport efficiency along with loss of productivity due to pests. This study has explored an algal-bacterial symbiotic system to utilize formate as a carbon source. The algal-bacterial assemblage, after a 400-day adaptive evolution using the formate medium, has demonstrated a new capability to assimilate both formate and CO 2 to promote biomass production. The formate tolerant culture system also addresses CO 2 mass transfer limitation under high light conditions. Continuous cultivation of the assemblage on formate led to a carbon capture efficiency of 90 % with a biomass concentration of 0.92 g L -1 and biomass productivity of 0.31 g L -1 day -1 , and the system did not require CO 2 aeration. In addition, 13 C isotope tracing, proteomics, and microbial community analysis reveal a uniquely evolved community for formate utilizations and robust biomass synthesis, in which Chlorella sorokiniana is a dominant phototroph companying with highly diverse bacterial subpopulations. In the presence of formate, evidence has shown that Chlorella is able to form mutualism with formate utilizing bacteria in semi-continuous cultures, leading to a highly effective photomixotrophic metabolism. Furthermore, this study demonstrates a new route of using electrochemical-derived formate to support mutualistic algae-bacteria biorefinery while the formate as an alternative carbon source could repel pests for outdoor algal cultivations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Advanced Algal Biofoundries for the Production of Polyurethane Precursors (CRADA Final Report)

The primary goal of the BEEPs project was to develop a process that could accelerate the development of algae as bioproduction platforms, from initial chemical product concept to an economically viable market supply. Under this program we elected to develop strains of algae that could generate polyurethane precursors, while simultaneously developing basic genetic tools to enable improved algal production systems. This program was specifically designed to incorporate National Laboratories as a means to utilize the expertise and facilities for new bioproduction platforms.

09 BIOMASS FUELS↗
DOE OSTI2020

VITAL-Viral InhibiTors from ALgae: Generating Extracts for Antiviral Activity Assays

Algae from marine and extreme environments may provide novel compounds with antiviral activity for use in therapeutic drug development. We established a first effort combining experience and cultures used in biofuel research and from the Culture Collection of Microbes from Extreme Environments at PNNL. We generated a novel library of fractionated, algae-derived compounds for guiding research on small molecules in the environment and for the treatment of SARS-CoV-2 and other infectious diseases.

09 BIOMASS FUELS↗