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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Chloroplast phosphatases LPPγ and LPPε1 facilitate conversion of extraplastidic phospholipids to galactolipids

Abstract Galactolipids comprise the majority of chloroplast membranes in plants, and their biosynthesis requires dephosphorylation of phosphatidic acid at the chloroplast envelope membranes. In Arabidopsis (Arabidopsis thaliana), the lipid phosphate phosphatases LPPγ, LPPε1, and LPPε2 have been previously implicated in chloroplast lipid assembly, with LPPγ being essential, as null mutants were reported to exhibit embryo lethality. Here, we show that lppγ mutants are in fact viable and that LPPγ, LPPε1, and LPPε2 do not appear to have central roles in the plastid pathway of membrane lipid biosynthesis. Redundant LPPγ and LPPε1 activity at the outer envelope membrane is important for plant development, and the respective lppγ lppε1 double mutant exhibits reduced flux through the ER pathway of galactolipid synthesis. While LPPε2 is imported and associated with interior chloroplast membranes, its role remains elusive and does not include basal nor phosphate limitation-induced biosynthesis of glycolipids. The specific physiological roles of LPPγ, LPPε1, and LPPε2 are yet to be uncovered, as does the identity of the phosphatidic acid phosphatase required for plastid galactolipid biosynthesis.

Cook, Ron (ORCID:0000000198356205)↗
DOE OSTI2024

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress

Abstract Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis (Arabidopsis thaliana) leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant trigalactosyldiacylglycerol1-1 sugar dependent 1-4 (tgd1-1 sdp1-4) that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we showed that a hyperaccumulation of the primary LD core lipid TAG is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress.

Plant Sciences↗
DOE OSTI2024

Crystal structure of vancomycin bound to the resistance determinant D -alanine- D -serine

Vancomycin is a glycopeptide antibiotic that for decades has been a mainstay of treatment for persistent bacterial infections. However, the spread of antibiotic resistance threatens its continued utility. In particular, vancomycin-resistant enterococci (VRE) have become a pressing clinical challenge. Vancomycin acts by binding and sequestering the intermediate Lipid II in cell-wall biosynthesis, specifically recognizing a D-alanine-D-alanine dipeptide motif within the Lipid II molecule. VRE achieve resistance by remodeling this motif to either D-alanine-D-lactate or D-alanine-D-serine; the former substitution essentially abolishes recognition by vancomycin of Lipid II, whereas the latter reduces the affinity of the antibiotic by roughly one order of magnitude. The complex of vancomycin bound to D-alanine-D-serine has been crystallized, and its 1.20 Å X-ray crystal structure is presented here. This structure reveals that the D-alanine-D-serine ligand is bound in essentially the same position and same pose as the native D-alanine-D-alanine ligand. The serine-containing ligand appears to be slightly too large to be comfortably accommodated in this way, suggesting one possible contribution to the reduced binding affinity. In addition, two flexible hydroxyl groups – one from the serine side chain of the ligand, and the other from a glucose sugar on the antibiotic – are locked into single conformations in the complex, which is likely to contribute an unfavorable entropic component to the recognition of the serine-containing ligand.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Towards oilcane: Engineering hyperaccumulation of triacylglycerol into sugarcane stems

Abstract Metabolic engineering to divert carbon flux from sucrose to oil in high biomass crop like sugarcane is an emerging strategy to boost lipid yields per hectare for biodiesel production. Sugarcane stems comprise more than 70% of the crops' biomass and can accumulate sucrose in excess of 20% of their extracted juice. The energy content of oils in the form of triacylglycerol (TAG) is more than twofold that of carbohydrates. Here, we report a step change in TAG accumulation in sugarcane stem tissues achieving an average of 4.3% of their dry weight (DW) in replicated greenhouse experiments by multigene engineering. The metabolic engineering included constitutive co‐expression of wrinkled1; diacylglycerol acyltransferase1‐2; cysteine‐oleosin; and ribonucleic acid interference‐suppression of sugar‐dependent1. The TAG content in leaf tissue was also elevated by more than 400‐fold compared to non‐engineered sugarcane to an average of 8.0% of the DW and the amount of total fatty acids reached about 13% of the DW. With increasing TAG accumulation an increase of 18:1 unsaturated fatty acids was observed at the expense of 16:0 and 18:0 saturated fatty acids. Total biomass accumulation, soluble lignin, Brix and juice content were significantly reduced in the TAG hyperaccumulating sugarcane lines. Overcoming this yield drag by engineering lipid accumulation into late stem development will be critical to exceed lipid yields of current oilseed crops.

09 BIOMASS FUELS↗
DOE OSTI2024

The expression of genes encoding novel Sesame oleosin variants facilitates enhanced triacylglycerol accumulation in Arabidopsis leaves and seeds

Summary Triacylglycerols (TAG), accumulate within lipid droplets (LD), predominantly surrounded by OLEOSINs (OLE), that protect TAG from hydrolysis. We tested the hypothesis that identifying and removing degradation signals from OLE would promote its abundance, preventing TAG degradation and enhancing TAG accumulation. We tested whether mutating potential ubiquitin‐conjugation sites in a previously reported improved Sesamum indicum OLE ( SiO ) variant, o3‐3 Cys‐OLE ( SiCO herein), would stabilize it and increase its lipogenic potential. SiCOv1 was created by replacing all five lysines in SiCO with arginines. Separately, six cysteine residues within SiCO were deleted to create SiCOv2. SiCOv1 and SiCOv2 mutations were combined to create SiCOv3. Transient expression of SiCOv3 in Nicotiana benthamiana increased TAG by two‐fold relative to SiCO. Constitutive expression of SiCOv3 or SiCOv5 , containing the five predominant TAG‐increasing mutations from SiCOv3 , in Arabidopsis along with mouse DGAT2 ( mD ) increased TAG accumulation by 54% in leaves and 13% in seeds compared with control lines coexpressing SiCO and mD. Lipid synthesis rates increased, consistent with an increase in lipid sink strength that sequesters newly synthesized TAG, thereby relieving the constitutive BADC‐dependent inhibition of ACCase reported for WT Arabidopsis. These OLE variants represent novel factors for potentially increasing TAG accumulation in a variety of oil crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Disruption of starch biosynthesis and triacylglycerol degradation impairs growth but improves photosynthesis in Arabidopsis

Enhancing lipid accumulation by redirecting carbon from starch to triacylglycerol (TAG) in vegetative tissues is a promising strategy for developing high-energy-density crops for bioenergy production. However, our understanding of how starch and TAG metabolism interact and how this interaction affects growth and photosynthesis is incomplete. Here, we investigated the metabolic and physiological consequences of disrupting starch biosynthesis and TAG turnover in Arabidopsis thaliana by generating single, double, and triple mutants involving ADG1 (starch biosynthesis), TGD1 (lipid trafficking), and SDP1\\\\\\\\r\\\\\\\\n(TAG lipase). Unexpectedly, elimination of starch biosynthesis lowered TAG levels in the high-TAG-accumulating tgd1 mutant, primarily through enhanced TAG breakdown. This decline was completely reversed by sucrose supplementation, suggesting that TAG degradation was induced by carbon limitation.\\\\\\\\r\\\\\\\\nGenetic suppression of TAG turnover via SDP1 disruption in the starchless tgd1 adg1-1 background led to a nine-fold rise in leaf TAG accumulation in the tgd1 sdp1 adg1-1 triple mutant, together with enhanced photosynthetic performance. However, this metabolic reprogramming incurred growth penalties.\\\\\\\\r\\\\\\\\nOur results highlight the role of dynamic TAG turnover in maintaining metabolic balance and photosynthesis in starch-deficient backgrounds. These findings underscore the need for refined metabolic engineering strategies that coordinate TAG biosynthesis and degradation to optimize lipid accumulation in bioenergy crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media

Abstract Background The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Results Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets ( ACL1 and ACC1 , improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1 ) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1 Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. Conclusions The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1 , or the deletion of DGA1 combined with overexpression of ACC1 and ACL1 . Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The influence of dietary intake of omega-3 polyunsaturated fatty acids on the association between short-term exposure to ambient nitrogen dioxide and respiratory and cardiovascular outcomes among healthy adults

Background: Short-term exposure to ambient nitrogen dioxide (NO 2 ) is associated with adverse respiratory and cardiovascular outcomes. Supplementation of omega-3 polyunsaturated fatty acids (PUFA) has shown protection against exposure to fine particulate matter. This study aims to investigate whether habitual omega-3 PUFA intake differentially modify the associations between respiratory and cardiovascular responses and short-term exposure to ambient NO 2 . Methods: Sixty-two healthy participants were enrolled into low or high omega-3 groups based on their habitual omega-3 PUFA intake. Each participant was repeatedly assessed for lung function, blood lipids, markers of coagulation and fibrinolysis, vascular function, and heart rate variability (HRV) in up to five sessions, each separated by at least 7 days. This study was carried out in the Research Triangle area of North Carolina, USA between October 2016 and September 2019. Daily ambient NO 2 concentrations were obtained from an area air quality monitoring station on the day of outcome assessment (Lag0), 4 days prior (Lag1-4), as well as 5-day moving average (5dMA). The associations between short-term exposure to NO 2 and the measured indices were evaluated using linear mixed-effects models stratified by omega-3 levels and adjusted by covariates including relative humidity and temperature. Results: The average concentration of ambient NO 2 during the study periods was 5.3±3.8 ppb which was below the National Ambient Air Quality Standards (NAAQS). In the high omega-3 group, an interquartile range (IQR) increase in short-term NO 2 concentrations was significantly associated with increased lung function [e.g. 1.2% (95%CI: 0.2%, 2.2%) in FVC at lag1, 2.6% (95%CI: 0.4%, 4.8%) in FEV1 at 5dMA], decreased blood lipids [e.g. -2.6% (95%CI: -4.4%, -0.9%) in total cholesterol at lag2, -3.1% (95%CI: -6.1%, 0.0%) in HDL at 5dMA, and -3.1% (95%CI: -5.5%, -0.7%) in LDL at lag2], improved vascular function [e.g. 8.9% (95%CI: 0.6%, 17.2%) increase in FMD and 43.1% (95%CI: -79.8%, -6.3%) decrease in endothelin-1 at 5dMA], and changed HRV parameters [e.g. -7.2% (95%CI: -13.6%, -0.8%) in HFn and 13.4% (95%CI: 0.2%, 28.3%) in LF/HF ratio at lag3]. In the low omega-3 group, an IQR increase in ambient NO 2 was associated with elevations in coagulation markers (von Willebrand Factor, D-dimer) and a decrease in HRV (very-low frequency); however, null associations were observed between short-term NO 2 exposure and changes in lung function, blood lipids, and vascular function. Conclusions: The results in this study imply that dietary omega-3 PUFA consumption may offer respiratory and vascular benefits in response to short-term exposure of healthy adults to NO 2 levels below the NAAQS.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Rapid quantification of whole seed fatty acid amount, composition, and shape phenotypes from diverse oilseed species with large differences in seed size

Seed oils are widely used in the food, biofuel, and industrial feedstock industries, with their utility and value determined by total oil content and fatty acid composition. Current high throughput seed oil analysis methods either lack accuracy in total fatty acid profiling or require extensive labor for lipid extraction prior to derivatization to fatty acid methyl esters (FAME) and quantification by gas chromatography (GC). Alternatively, direct whole seed FAME production methods have been developed for the very small seeds in the model species Arabidopsis thaliana but these have generally not been adapted to larger seeds of most oilseed crops. High-throughput direct whole seed FAME production methods were optimized for seeds up to 5 mg each utilizing acid-catalyzed esterification. For the oilseed species Camelina sativa, Thlaspi avernse (pennycress), Cuphea viscosissima, and Brassica napus (var. Canola), the total seed fatty acid content and composition from direct seed esterification to FAME matched that of lipid extract derivatization demonstrating the accuracy of the methods. In combination with seed phenotyping using GridFree, this approach enabled the development of a rapid pipeline for simultaneous seed weight, count, size/shape phenotyping, and oil analysis. For the larger and tougher seeds produced by Limnanthes alba (Meadowfoam) and Cannabis sativa L. (hemp) the whole seed acid-based method proved insufficient, and prior laborious homogenization of seeds was required. Therefore, a rapid one-tube bead homogenization and base catalyzed-esterification method was developed. Base-derived fatty acid esterification cannot derivatize free fatty acids leading to slightly lower total seed fatty acid than acid-catalyzed methods, however the seed oil content and fatty acid composition that is valuable for screening large numbers of samples in research populations was accurately measured. New rapid whole seed fatty acid esterification and phenotyping protocols were developed to accurately assess oilseed lipid content. These methods are particularly valuable in oilseed research, breeding, and engineering applications where efficient analysis of large numbers of samples and accurate oil fatty acid profiling is essential. While having been developed for current and emerging oilseed crops, these methods also provide a foundation from which protocols might be established for new and emerging crop species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Nitrogen-dependent coordination of cell cycle, quiescence and TAG accumulation in Chlamydomonas

Abstract Microalgae hold great promises as sustainable cellular factories for the production of alternative fuels, feeds, and biopharmaceuticals for human health. While the biorefinery approach for fuels along with the coproduction of high-value compounds with industrial, therapeutic, or nutraceutical applications have the potential to make algal biofuels more economically viable, a number of challenges continue to hamper algal production systems at all levels. One such hurdle includes the metabolic trade-off often observed between the increased yields of desired products, such as triacylglycerols (TAG), and the growth of an organism. Initial genetic engineering strategies to improve lipid productivity in microalgae, which focused on overproducing the enzymes involved in fatty acid and TAG biosynthesis or inactivating competing carbon (C) metabolism, have seen some successes albeit at the cost of often greatly reduced biomass. Emergent approaches that aim at modifying the dynamics of entire metabolic pathways by engineering of pertinent transcription factors or signaling networks appear to have successfully achieved a balance between growth and neutral lipid accumulation. However, the biological knowledge of key signaling networks and molecular components linking these two processes is still incomplete in photosynthetic eukaryotes, making it difficult to optimize metabolic engineering strategies for microalgae. Here, we focus on nitrogen (N) starvation of the model green microalga, Chlamydomonas reinhardtii , to present the current understanding of the nutrient-dependent switch between proliferation and quiescence, and the drastic reprogramming of metabolism that results in the storage of C compounds following N starvation. We discuss the potential components mediating the transcriptional repression of cell cycle genes and the establishment of quiescence in Chlamydomonas, and highlight the importance of signaling pathways such as those governed by the target of rapamycin (TOR) and sucrose nonfermenting-related (SnRK) kinases in the coordination of metabolic status with cellular growth. A better understanding of how the cell division cycle is regulated in response to nutrient scarcity and of the signaling pathways linking cellular growth to energy and lipid homeostasis, is essential to improve the prospects of biofuels and biomass production in microalgae.

09 BIOMASS FUELS↗
DOE OSTI2023

Microbiome differences in sugarcane and metabolically engineered oilcane accessions and their implications for bioenergy production

Abstract Oilcane is a metabolically engineered sugarcane ( Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far. Here, we explore differences in the microbiome structure of different oilcane accessions and non-modified sugarcane. 16S SSU rRNA and ITS rRNA amplicon sequencing were performed to compare the characteristics of the microbiome structure from different plant compartments (leaf, stem, root, rhizosphere, and bulk soil) of four greenhouse-grown oilcane accessions and non-modified sugarcane. Significant differences were only observed in the bacterial microbiomes. In leaf and stem microbiomes, more than 90% of the entire microbiome of non-modified sugarcane and oilcane was dominated by similar core taxa. Taxa associated with Proteobacteria led to differences in the non-modified sugarcane and oilcane microbiome structure. While differences were observed between multiple accessions, accession 1566 was notable in that it was consistently observed to differ in its microbial membership than other accessions and had the lowest abundance of taxa associated with plant-growth-promoting bacteria. Accession 1566 is also unique among oilcane accessions in that it has the highest constitutive expression of the WRI 1 transgene. The WRI 1 transcription factor is known to contribute to significant changes in the global gene expression profile, impacting plant fatty acid biosynthesis and photomorphogenesis. This study reveals for the first time that genetically modified oilcanes associate with distinct microbiomes. Our findings suggest potential relationships between core taxa, biomass yield, and TAG in oilcane accessions and support further research on the relationship between plant genotypes and their microbiomes.

09 BIOMASS FUELS↗
DOE OSTI2024

Data for the Expression of Genes Encoding Novel Sesame Oleosin Variants Facilitates Enhanced Triacylglycerol Accumulation in Arabidopsis Leaves and Seeds

Triacylglycerols (TAG), accumulate within lipid droplets (LD), predominantly surrounded by OLEOSINs (OLE), that protect TAG from hydrolysis. We tested the hypothesis that identifying and removing degradation signals from OLE would promote its abundance, preventing TAG degradation and enhancing TAG accumulation. We tested whether mutating potential ubiquitin-conjugation sites in a previously reported improved Sesamum indicum OLE ( SiO ) variant, o3-3 Cys-OLE ( SiCO herein), would stabilize it and increase its lipogenic potential. SiCOv1 was created by replacing all five lysines in SiCO with arginines. Separately, six cysteine residues within SiCO were deleted to create SiCOv2. SiCOv1 and SiCOv2 mutations were combined to create SiCOv3. Transient expression of SiCOv3 in Nicotiana benthamiana increased TAG by two-fold relative to SiCO. Constitutive expression of SiCOv3 or SiCOv5 , containing the five predominant TAG-increasing mutations from SiCOv3 , in Arabidopsis along with mouse DGAT2 ( mD ) increased TAG accumulation by 54% in leaves and 13% in seeds compared with control lines coexpressing SiCO and mD . Lipid synthesis rates increased, consistent with an increase in lipid sink strength that sequesters newly synthesized TAG, thereby relieving the constitutive BADC-dependent inhibition of ACCase reported for WT Arabidopsis. These OLE variants represent novel factors for potentially increasing TAG accumulation in a variety of oil crops.

Feedstock Production↗
DOE OSTI2022

Data for Metabolic Engineering of Rhodotorula toruloides IFO0880 Improves C16 and C18 Fatty Alcohol Production from Synthetic Media

The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Conversion↗
DOE OSTI2023

High wax ester and triacylglycerol biosynthesis potential in coastal sediments of Antarctic and Subantarctic environments

The wax ester (WE) and triacylglycerol (TAG) biosynthetic potential of marine microorganisms is poorly understood at the microbial community level. The goal of this work was to uncover the prevalence and diversity of bacteria with the potential to synthesize these neutral lipids in coastal sediments of two high latitude environments, and to characterize the gene clusters related to this process. Homolog sequences of the key enzyme, the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) were retrieved from 13 metagenomes, including subtidal and intertidal sediments of a Subantarctic environment (Ushuaia Bay, Argentina), and subtidal sediments of an Antarctic environment (Potter Cove, Antarctica). The abundance of WS/DGAT homolog sequences in the sediment metagenomes was 1.23 ± 0.42 times the abundance of 12 single-copy genes encoding ribosomal proteins, higher than in seawater (0.13 ± 0.31 times in 338 metagenomes). Homolog sequences were highly diverse, and were assigned to the Pseudomonadota, Actinomycetota, Bacteroidota and Acidobacteriota phyla. The genomic context of WS/DGAT homologs included sequences related to WE and TAG biosynthesis pathways, as well as to other related pathways such as fatty-acid metabolism, suggesting carbon recycling might drive the flux to neutral lipid synthesis. These results indicate the presence of abundant and taxonomically diverse bacterial populations with the potential to synthesize lipid storage compounds in marine sediments, relating this metabolic process to bacterial survival.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Simulated Night- Shift Schedule Disrupts the Plasma Lipidome and Reveals Early Markers of Cardiovascular Disease Risk

Introduction: The circadian system coordinates daily rhythms in lipid metabolism, storage and utilization. Disruptions of internal circadian rhythms due to altered sleep/wake schedules, such as in night-shift work, have been implicated in increased risk of cardiovascular disease and metabolic disorders. To determine the impact of a night-shift schedule on the human blood plasma lipidome, an in-laboratory simulated shift work study was conducted. Methods: Fourteen healthy young adults were assigned to 3 days of either a simulated day or night-shift schedule, followed by a 24-h constant routine protocol with fixed environmental conditions, hourly isocaloric snacks, and constant wakefulness to investigate endogenous circadian rhythms. Blood plasma samples collected at 3-h intervals were subjected to untargeted lipidomics analysis. Results: More than 400 lipids were identified and quantified across 21 subclasses. Focusing on lipids with low between-subject variation per shift condition, alterations in the circulating plasma lipidome revealed generally increased mean triglyceride levels and decreased mean phospholipid levels after night-shift relative to day-shift. The circadian rhythms of triglycerides containing odd chain fatty acids peaked earlier during constant routine after night-shift. Regardless of shift condition, triglycerides tended to either peak or be depleted at 16:30 h, with chain-specific differences associated with the direction of change. Discussion: The simulated night-shift schedule was associated with altered temporal patterns in the lipidome. This may be premorbid to the elevated cardiovascular risk that has been found epidemiologically in night-shift workers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

CO 2 to Bioplastics: Beneficial Re-use of Carbon Emissions from Coal-fired Power Plants using Microalgae

This project sought to address the technical and economic barriers to carbon dioxide (CO 2 ) capture and utilization using microalgae. Specifically, a dual photobioreactor (PBR)/open raceway pond (ORP) cultivation system was evaluated with respect to capital and operational costs, productivity, and culture health, and compared to an ORP-only cultivation system. In the dual (or “hybrid”) system, a cyclic flow PBR was used to provide inoculum for two open raceway ponds after each harvest, with the hypothesis that this PBR + pond system should result in improved performance compared to traditional raceway ponds. Two other raceway ponds were operated conventionally as a control, the experiments being performed at Duke Energy’s East Bend Station in northern KY using coal-derived flue gas as the CO 2 source. Although algae productivity achieved was mediocre due to the fact that the experiments had to be performed late in the growing season when climatic conditions were not optimal for high growth, the hybrid cultivation system showed a higher level of algae productivity (statistically significant) compared to the traditional ponds. In order to realize the maximum value of the algal biomass produced, fractionation of the biomass was examined. Bioplastic compounding and material characterization was subsequently performed using three feed stocks: whole biomass, lipid-extracted biomass and the proteinaceous residue from full fractionation. In general, the whole and lipid-extracted biomass gave similar results in terms of the properties of the resulting bioplastics, while the proteinaceous residue showed promise for the production of a polybutylene adipate terephthalate (PBAT) blend. Finally, sustainability assessment of a putative biorefinery system was conducted, including techno-economic and life cycle impact assessment. Nine different production scenarios were considered, comprising combinations of the three different biomass growth architectures (ORP, PBR and dual PBR-ORP systems) coupled with three different algae biomass processing pathways (drying only, lipid extraction and fractionation). Results show that the minimum selling price of the bioplastic feed stock (BPFS) is within the realm of economic competition with prices as low as $970 USD tonne -1 . Additionally, life cycle impact assessment results indicate drastic improvements in performance of the produced BPFS, with reductions in greenhouse gas emissions ranging between 67 and 116% compared to a petroleum based plastic feedstock.

01 COAL, LIGNITE, AND PEAT↗
DOE OSTI2020

Conceptual Basis and Techno-Economic Modeling for Integrated Algal Biorefinery Conversion of Microalgae to Fuels and Products (2019 NREL TEA Update: Highlighting Paths to Future Cost Goals via a New Pathway for Combined Algal Processing)

The report documents the conceptual basis for a new potential Combined Algal Processing design strategy which may allow more flexibility in accommodating different algal biomass feedstock compositions, by enabling upgrading of both protein and carbohydrates in a single step, without a strict requirement for either component to be in soluble or monomeric form, while maintaining effective wet lipid extraction techniques to enable high lipid recoveries. In light of previously-established constraints around algal biomass costs (which are significantly higher than lignocellulosic terrestrial biomass), the present CAP processing strategy reflects an integrated biorefinery concept producing both fuels and value-added chemical coproducts as a means to improve profitability and generate coproduct revenues to help drive down the minimum fuel selling price (MFSP) towards economically viable levels. Namely, this report highlights an integrated CAP biorefinery process and associated technical targets that would be required to achieve U.S. Department of Energy target MFSP goals of $2.5/gallon gasoline equivalent by 2030. This is accomplished by a process involving low-cost seasonal storage of algal biomass during high-growth seasons, rapid flash hydrolysis pretreatment of the biomass, solvent extraction of pretreated biomass, cleanup and fractionation of lipids into triglyceride and free fatty acid fractions, and a series of thermochemical conversion steps to upgrade carbohydrates and protein to hydrocarbon fuels. These steps include mild oxidative treatment (MOT), a process originally investigated at NREL for upgrading lignin, followed by catalytic ketonization and hydrotreating of MOT products to fuels. Isolated triglycerides are sent to a coproduct train, with the base case focused on upgrading to polyurethane foams as a high-value, high-market-volume coproduct.

09 BIOMASS FUELS↗
DOE OSTI2023

Development of Production Strains of Chlamydomonas Using Breeding Technologies

Chlamydomonas is a well-studied strain with excellent breeding capability. It can produce recombinant proteins in the raceway in the greenhouse. Increasing the pH of the culture resulted in a very stable cultivation process and cultures did not crash. This strain was not tolerant of high UV levels at KAF. This is can be probably easily overcome by breeding with a light tolerant strain. Overall Chlamydomonas is a very promising strain for production of recombinant proteins not only in the lab but also outdoors. It has excellent breeding capability and its progeny expressed the introduced protein for a long tiem. Chlamydomonas sp. produced lipids but overall lipid yield was low with highest lipid content of 15%. Chlamydomonas is a genus with a great potential for production of various specific proteins. It can be potentially cultivated outdoors and should be further investigated for production of specific molecules.

09 BIOMASS FUELS↗