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DOE OSTI2020

Prime Editing Technology and Its Prospects for Future Applications in Plant Biology Research

Many applications in plant biology requires editing genomes accurately including correcting point mutations, incorporation of single-nucleotide polymorphisms (SNPs), and introduction of multinucleotide insertion/deletions (indels) into a predetermined position in the genome. These types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems, which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit sequence. However, low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely inefficient. Another kind of technology known as base editing can perform precise editing; however, only certain types of modifications can be obtained, e.g., C/G-to-T/A and A/T-to-G/C. Recently, a new type of genome-editing technology, referred to as “prime editing,” has been developed, which can achieve various types of editing such as any base-to-base conversion, including both transitions (C→T, G→A, A→G, and T→C) and transversion mutations (C→A, C→G, G→C, G→T, A→C, A→T, T→A, and T→G), as well as small indels without the requirement for inducing double-stranded break in the DNA. Because prime editing has wide flexibility to achieve different types of edits in the genome, it holds a great potential for developing superior crops for various purposes, such as increasing yield, providing resistance to various abiotic and biotic stresses, and improving quality of plant product. In this review, we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Miscanthus × giganteus Responses to Nitrogen Fertilization and Harvest Timing in Illinois, USA

Renewable energy continues to be of interest in the USA due to concerns about the long-termed availability and the environmental impact of fossil fuels. Miscanthus × giganteus is a warm-season perennial bioenergy feedstock grass that has a long growing season, is high yielding, requires limited inputs, and will likely become an important renewable energy crop in the USA. This research studied the effects of N application rates and harvest timings on yields and biomass quality of a 4-year-old stand of M. × giganteus in Urbana, IL, USA. Plots received 0, 56, 112, 156, or 224 kg N ha –1 , and harvests were conducted at five dates between August and March in the 2009, 2010, and 2011 growing seasons. Miscanthus × giganteus production increased with N applications up to 112 kg ha –1 . Harvesting biomass before senescence, August through November, produced significantly more biomass compared to harvesting biomass after senescence. However, early harvest reduced biomass yields in the following years. Here, nitrogen fertilization compensated for the yield losses, but the yield gap between early and late harvest continually increased. Harvesting M. × giganteus after senescence optimized long-term productivity, reduced the need for N fertilization, and increased carbon and ash content in the harvested biomass, while harvesting before senescence not only removes N from the plant but also reduces remobilization of other nutrients and carbohydrates in autumn.

09 BIOMASS FUELS↗

DOE OSTI2020

Rice NIN-LIKE PROTEIN 4 plays a pivotal role in nitrogen use efficiency

Nitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavour in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the rice NIN-like protein 4 (OsNLP4) significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 plays a pivotal role in rice NUE and sheds light on crop NUE improvement.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Targeted mutagenesis with sequence–specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome–editing platforms employ sequence–specific nucleases (SSNs) to generate DNA double–stranded breaks at their target gene. Such DNA breaks are typically repaired via the error–prone nonhomologous end–joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end–use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN–mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass

Abstract Background The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. Results Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase 1-2 ( DGAT 1-2) and Oleosin 1 ( OLE 1) in combination with RNAi suppression of SUGAR-DEPENDENT 1 ( SDP 1) and Trigalactosyl diacylglycerol 1 ( TGD 1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson’s correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT 1-2, followed by the level of RNAi suppression for SDP 1. Conclusions This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

1-2↗

DOE OSTI2021

Overexpression of the rice BAHD acyltransferase AT10 increases xylan-bound p-coumarate and reduces lignin in Sorghum bicolor

Abstract Background The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results We obtained eight independent sorghum ( Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p -coumarate ( p CA), a higher amount of p -coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of p CA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions Expression of OsAT10 in sorghum leads to an increase of xylan-bound p CA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall p CA remains unchanged indicating that most p CA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound p CA and lignification in cell walls.

09 BIOMASS FUELS↗

DOE OSTI2026

Life cycle analysis of dedicated energy crops for fuel production in the United States

Dedicated energy crops are promising feedstocks to make biofuels including jet fuels. This study applies life cycle analysis (LCA) to estimate direct well-to-wake (WTW) greenhouse gas (GHG) emissions (g CO 2 e/MJ) for jet fuel derived from five energy crops-biomass sorghum, miscanthus, switchgrass, poplar, and willow-via Fischer-Tropsch-to-Jet (FTJ) and Ethanol-to-Jet (ETJ) pathways. The WTW boundary includes direct emissions from biomass production, fuel production, and fuel combustion. The R&D GREET model is expanded to conduct the LCA, using national average biomass yields and farming inputs from the 2023 Billion-Ton Study. In addition, this study estimates emissions from market-mediated effects, including induced land-use change, induced other crop (non-feedstock) production changes, and induced livestock production changes using global economic and emissions factor models. On a per-dry U.S. ton basis, cultivation and harvest emissions are lowest for willow (51,565 g CO 2 e) and highest for biomass sorghum (104,488 g CO 2 e). Per-acre results show similarly high emissions for sorghum and lowest values for poplar and willow. Direct WTW emissions are substantially lower for FTJ (biomass sorghum: 5.5; miscanthus: 10.3; switchgrass: 11.7; poplar: 11.9; and willow: 8.7 g CO 2 e/MJ) than ETJ (33.2; 33.8; 34.8; 36.2; and 31.7 g CO 2 e/MJ, respectively). When market-mediated emissions are included, miscanthus exhibits the lowest total emissions across energy crop pathways. Although results are sensitive to modeling assumptions, they indicate that high-yielding perennial and woody crops, particularly when planted on marginal land, could significantly reduce WTW emissions for bio-jet fuels by combining low direct emissions with soil carbon gains and favorable market-mediated effects.

Billion-Ton Study↗

DOE OSTI2024

Genetic Basis of Non–Photochemical Quenching and Photosystem II Efficiency Responses to Chilling in the Biomass Crop Miscanthus

Miscanthus holds a promise as a biocrop due to its high yield, perenniality and ability to grow on infertile soils. However, the current commercial biomass production of Miscanthus is mostly limited to a single sterile triploid clone of M. × giganteus. Nevertheless, parental species of M. × giganteus, Miscanthus sacchariflorus and Miscanthus sinensis contain vast genetic diversity for crop improvement. With M. sacchariflorus having a natural geographic distribution in cold-temperate northeast China and eastern Russia, we hypothesised that it has substantial variation in physiological response to chilling. Using a semi-high-throughput method, we phenotyped 209 M. sacchariflorus genotypes belonging to six genetic groups for non-photochemical quenching (NPQ) and photosystem II efficiency (ΦPSII) kinetics under warm and chilling treatments in three growing seasons. In response to the chilling treatment, all genetic groups exhibited an increase in NPQ induction rate indicating faster activation of NPQ in light. Notably, under chilling, the Korea/NE China/Russia 2x and N China 2x groups stood out for the highest NPQ rate in light and the highest steady-state NPQ in light. This NPQ phenotype may contribute adaptation to chilling during bright, cold mornings of spring and early autumn in temperate climates, when faster NPQ would better protect from oxidative stress. Such enhanced adaptation could expand the growing season and thus productivity at a given location or expand the range of economically viable growing locations to higher latitudes and altitudes. A genome-wide association study identified 126 unique SNPs associated with NPQ and ΦPSII traits. Among the identified candidate genes were enzymes involved in the ascorbate recycle and shikimate pathway, gamma-aminobutyric acid and cation efflux transporters. Identifying natural variation and genes involved in NPQ and ΦPSII kinetics considerably enlarges the toolbox for breeding and/or engineering Miscanthus with optimised photosynthesis under warm and chilling conditions for sustainable feedstock production for bioenergy.

09 BIOMASS FUELS↗

DOE OSTI2022

Agroecosystem model simulations reveal spatial variability in relative productivity in biomass sorghum and maize in Iowa, USA

Biomass sorghum (Sorghum bicolor L. Moench) is an annual C 4 grass that has emerged as a candidate bioenergy crop but has not been widely grown in the United States. Corn (Zea mays L.), another annual C 4 grass, has been produced on a large scale in the United States for biofuels. Iowa leads the nation in both corn and ethanol production. The high productivity of corn in Iowa creates the research question: could biomass sorghum be as or more productive than corn in the state in terms of bioenergy? Efforts to use crop models to fill the gaps left by field experimentation on biomass sorghum have also been limited thus far. To address our research question, we collected biophysical data on biomass sorghum grown in Iowa for two growing seasons, used them to develop a biomass sorghum module in an agroecosystem model (Agro-IBIS), and predict the potential performance of biomass sorghum across Iowa compared to maize. Despite dry conditions in 2019 and 2020, average biomass sorghum yields were 17.20 Mg ha –1 . By comparison, average corn aboveground biomass was higher in 2020 (22.02 Mg ha –1 ). Soil cores indicated average belowground biomass of 1.46 Mg ha –1 , with roots concentrated near the surface (73% of biomass above 50 cm in a 1 m core). When biomass sorghum model parameters were calibrated with measured values, model output was in close agreement with measured biomass (slope = 0.932, R 2 = 0.91) and evapotranspiration (slope = 0.757, R 2 = 0.64). Subsequent regional simulations revealed a notable latitudinal gradient in biomass sorghum yield, with a strong linear relationship between yield and seasonal growing degree-days (R 2 = 0.89). When these yields were compared to simulated corn aboveground biomass yields, only 3.4% of the state had biomass sorghum yields that were significantly higher than corn. This number was reduced to 0.3% when comparing the crops in terms of energy ethanol yield. Thus, we conclude biomass sorghum must be improved to be competitive with corn as an annual biofuel crop in Iowa. This study provides a baseline against which to compare advancements in sorghum breeding for biomass and stress tolerance in Iowa.

Agro-IBIS↗

DOE OSTI2024

Imitating the “breeder's eye”: Predicting grain yield from measurements of non‐yield traits

Abstract Plant breeding relies on information gathered from field trials to select promising new crop varieties for release to farmers and to develop genomic prediction models that can enhance the efficiency of genetic improvement in future breeding cycles. However, generating the genetic marker data required to apply genomic prediction at the early stages of a breeding program remains costly for many public‐sector breeding programs as well as for many plant breeders operating in developing countries. As the pace of climate change intensifies, the time lag of developing and deploying new crop varieties requires plant breeders to make selection decisions without knowing the future environments those crop varieties will encounter in farmers’ fields. Therefore, both lower cost and higher accuracy methods for prediction of crop performance are essential for creating and maintaining resilient agricultural systems in the latter half of the 21 st century. To address this challenge, we conducted linked yield trials of 752 public maize ( Zea mays ) genotypes in two distinct environments. We developed and trained a phenotypic prediction model to predict yield from manually scored plant traits. The phenotypic prediction approach we employed outperformed genomic prediction in predicting yields in a second environment, with 8.7%–63% higher R 2 and 4%–13% less root mean square error than the genomic prediction. The phenotypic prediction has the potential to be applied to a wider range of breeding programs, including those that lack the resources to genotype large populations, such as programs in the developing world, breeding programs for specialty crops, and public sector programs.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Techno‐economic feasibility analysis of engineered energycane‐based biorefinery co‐producing biodiesel and ethanol

Abstract High feedstock cost and low oil yields per unit of land from temperate oilseed crops limit the growth of commercial‐scale biodiesel production. Recently, highly productive crops, such as sugarcane and energycane, have been engineered to accumulate triacylglycerides (TAGs) that allow the production of far more industrial vegetable oil than previously possible. A proof‐of‐concept suggests that biodiesel production from engineered energycane will be possible. However, before making efforts for scale‐up, it is critical to understand the commercial feasibility and economic competitiveness of this process. This study performs techno‐economic analysis of a unique biorefinery processing energycane to co‐produce biodiesel and ethanol. Comprehensive process simulation models were developed for two scenarios: (i) biodiesel from TAGs and ethanol from fermentation of sugars in juice and (ii) biodiesel from TAGs and ethanol from fermentation of sugars in juice and hydrolysis of carbohydrates in bagasse. Based on the target levels, the analysis was performed for energycane containing 0%, 5%, and 7.7% TAGs (d.b.). The biodiesel from engineered energycane was found economically viable and competitive to soybean biodiesel. Although the capital investment is higher compared to the soybean biodiesel plant, the biodiesel production costs ($0.66–$0.9/L) were lower than soybean biodiesel ($0.91/L). Biorefinery‐scenario‐1 processing energycane containing 7.7% TAG produces biodiesel with profitability (IRR 7.84) slightly lower than soybean biodiesel (IRR 8.3), but yields five times of biodiesel per unit land and is self‐sustainable for energy requirements. The surplus electricity can displace fossil electricity and provide environmental benefits. Monte Carlo simulation indicated that biorefinery is profitable with a 29%–65% probability (NPV > 0) which is largely controlled by feedstock composition and biodiesel market price. It is important to note that energycane can be grown on the marginal rainfed lands in S.E. USA, where soybean would not be viable. Biodiesel from engineered energycane would therefore be complementary to soydiesel in the United States.

09 BIOMASS FUELS↗

DOE OSTI2021

Contrasting long-term temperature trends reveal minor changes in projected potential evapotranspiration in the US Midwest

Abstract Warming generally leads to increased evaporative demand, altering the amount of water needed for growing crops. For the Midwest, some studies have suggested that reaching yield targets by 2050 will not be possible without additional precipitation or large expansion of irrigation. Here, we show that this claim is not supported by the historical summer climate trends, which indicate that the warming of daily average temperatures is largely driven by increases in minimum temperatures, while maximum temperatures have decreased. This has translated into a net decrease in vapor pressure deficit (VPD) and potential evapotranspiration (PET). With the increasing rainfall, this suggests that crop water deficits have likely become less frequent in the region despite the warming climate. By projecting these trends into 2050 and ancillary use of a crop model, we estimate minor changes in PET that would have minimal effects on corn yields (<6%) under persistence of these trends.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Registration of ‘Independence’ switchgrass

Switchgrass (Panicum virgatum L.), a valuable forage and bioenergy crop, is established more easily than other native perennial warm-season grasses, but its establishment is still slower than that of annual crops. Vigorous switchgrass establishment is crucial for achieving its full potential yield and for effectively competing with weeds for water and nutrient availability. To satisfy this demand, ‘Independence’ (Reg. no. CV-295, PI 704577) switchgrass was developed at the University of Illinois at Urbana-Champaign. Independence was selected for establishment vigor, winter survivorship, and high biomass yield for two cycles from ‘Kanlow’. Here, it is characterized by rapid establishment, robust seedling growth, and the capacity to achieve peak production by the second year. Independence is well adapted to USDA hardiness zones 5b–7b. In field experiments conducted from 2016 to 2017, averaged over seven locations and all years, Independence annually yielded 13 Mg ha –1 of biomass, outperforming ‘Cave-in-Rock’ by 31%, ‘Liberty’ by 15%, ‘Shawnee’ by 42%, ‘Summer’ by 81%, and ‘Sunburst’ by 129%. In wet marginal sites in Illinois from 2020 to 2023, Independence exhibited an average biomass yield of 12 Mg ha –1 , outperforming Shawnee by 31%, Liberty by 27%, and Kanlow by 19%, indicating its potential use on less productive land for annual crops. Independence was publicly released by the University of Illinois at Urbana-Champaign in October 2021.

09 BIOMASS FUELS↗

DOE OSTI2022

From Farm to Flight: CoverCress as a Low Carbon Intensity Cash Cover Crop for Sustainable Aviation Fuel Production. A Review of Progress Towards Commercialization

Thlaspi arvense L. (Field Pennycress; pennycress) is being converted into a winter-annual oilseed crop that confers cover crop benefits when grown throughout the 12 million-hectares U.S. Midwest. To ensure a fit with downstream market demand, conversion involves not only improvements in yield and maturity through traditional breeding, but also improvements in the composition of the oil and protein through gene editing tools. The conversion process is similar to the path taken to convert rapeseed into Canola. In the case of field pennycress, the converted product that is suitable as a rotational crop is called CoverCress™ as marketed by CoverCress Inc. or golden pennycress if marketed by others. Off-season integration of a CoverCress crop into existing corn and soybean hectares would extend the growing season on established croplands and avoid displacement of food crops or ecosystems while yielding up to 1 billion liters of seed oil annually by 2030, with the potential to grow to 8 billion liters from production in the U.S. Midwest alone. The aviation sector is committed to carbon-neutral growth and reducing emissions of its global market, which in 2019 approached 122 billion liters of consumption in the U.S. and 454 billion liters globally. The oil derived from a CoverCress crop is ideally suited as a new bioenergy feedstock for the production of drop-in Sustainable Aviation Fuel (SAF), renewable diesel, biodiesel and other value-added coproducts. Through a combination of breeding and genomics-enabled mutagenesis approaches, considerable progress has been made in genetically improving yield and other agronomic traits. With USDA-NIFA funding and continued public and private investments, improvements to CoverCress germplasm and agronomic practices suggest that field-scale production can surpass 1,680 kg ha -1 (1,500 lb ac -1 ) in the near term. At current commodity prices, economic modeling predicts this level of production can be profitable across the entire supply chain. Two-thirds of the grain value is in oil converted to fuels and chemicals, and the other one-third is in the meal used as an animal feed, industrial applications, and potential plant-based protein products. In addition to strengthening rural communities by providing income to producers and agribusinesses, cultivating a CoverCress crop potentially offers a myriad of ecosystem services. The most notable service is water quality protection through reduced nutrient leaching and reduced soil erosion. Biodiversity enhancement by supporting pollinators’ health is also a benefit. While the efforts described herein are focused on the U.S., cultivation of a CoverCress crop will likely have a broader application to regions around the world with similar agronomic and environmental conditions.

09 BIOMASS FUELS↗

DOE OSTI2025

Natural variation in growth and yield to waterlogging across climate-diverse pennycress accessions

Many winter annual crops, such as pennycress ( Thlaspi arvense L.), are subjected to heavy precipitation events during their growing season. Therefore, it is essential to identify pennycress accessions with natural variation in flooding resilience. We used climate modeling data to assess spring soil moisture levels in the geographic origins of 471 natural pennycress accessions. We selected 34 accessions with variation in predicted soil moisture and tested survivability under prolonged waterlogging at the rosette stage. It took seven weeks for the first accessions to die, indicating that pennycress is hardy to prolonged waterlogging at the vegetative stage. Furthermore, we chose ‘susceptible’ and ‘tolerant’ accessions to waterlog for one week at the reproductive stage, the growth stage aligned with spring rainfall. Six accessions had significantly reduced seed weight at maturity and two had minimal impacts on growth and seed yield after waterlogging and can be further explored for adaptive traits.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Building on success in systems design of high yielding low‐input energycanes for marginal lands

Energycane and Miscanthus are highly sustainable and productive perennial grasses that produce a large annual crop of shoot biomass. Theoretical analysis of metabolism has suggested that these crops could be engineered to accumulate 20% of their harvestable biomass as oil. Given their much higher biomass yields than soybean and canola, this would yield several times more oil per acre. This could be converted to biodiesel or sustainable aviation fuel (SAF) by well‐established technologies. Energycane is well suited to the southeast quarter of the contiguous 48 states and Miscanthus to the northeast quarter. The objective of this award was to provide the foundation for converting these crops to oilcanes from DNA constructs and transformation to field trials and pilot plant development of oil extraction. Genetic engineering focused on development and implementation of improved constructs to increase oil levels, and increased photosynthesis to support the additional energy needed for oil production. The specific progress in each of our subtasks follows together with the products arising.

09 BIOMASS FUELS↗

DOE OSTI2026

Feedstock Choices for Advancing the Bioeconomy in the US and Policy Implications

Biomass feedstocks will be critical for scaling up the bioeconomy in the United States to meet multiple demands for biofuels, sustainable aviation fuel, bioproducts, and biochemicals. There is a wide range of choices of feedstocks, including a variety of dedicated energy crops and crop residues. There are substantial differences in the yields, costs of production, and carbon intensity of these feedstocks and for each feedstock across locations. These feedstocks also differ in the trade-offs they offer among multiple environmental impacts. We discuss the economic factors that will influence the production of these feedstocks and the implications of alternative biofuel and low-carbon policies for the mix of feedstocks that will be incentivized.

Khanna, Madhu (ORCID:0000000349944451)↗

DOE OSTI2021

Grazer-induced changes in molecular signatures of cyanobacteria

Algal biomass production is an emerging renewable source of fuels, nutrients, manufacturing materials, and pharmaceuticals. Industrial-scale production is predominantly performed in open raceway ponds that are inexpensive to build, operate, and maintain compared to closed bioreactors. However, these open pond systems suffer from increased opportunities for biological contamination from predators, pathogens, and competitors, which result in reduced biomass quality and yields and often the complete destruction of the crop over a short period of time. Early detection of contaminants is a necessary step of integrated pest management for triggering and informing interventions to prevent crop losses. To develop a sensitive method of detection utilizing mass spectrometry (MS), we used three methods – imaging mass spectrometry (imaging MS), liquid chromatography MS/MS (LC-MS/MS) combined with molecular networking, and gas chromatography MS/MS (GC–MS/MS) – to observe and identify molecular signatures from a model predator-prey system of the heterolobosean amoeba HGG1 preying on the filamentous cyanobacterium Anabaena sp. PCC 7120. Imaging MS enabled the association of molecules with the crop, the predator, or the activity of protozoan grazing, while LC-MS/MS-based molecular networking identified a subset of the grazing-specific signals as the chlorophyll breakdown products pyropheophytin, pheophorbide A, and pyropheophorbide. Application of MS techniques to other amoebacyanobacterial predator-prey pairs allowed categorization of grazing signatures as universal or specific to the species of prey, predator, or the prey-predator pairing, indicating that MS-based techniques can distinguish crops from competitors and potentially identify predators. Finally, GCMS/MS was shown to be capable of monitoring novel volatile organic compounds (VOCs), including those predicted to be released through chlorophyll breakdown, in the headspace over algal cultures under predation. Furthermore, these results demonstrate that the combination of multiple MS technologies creates a predictive framework to identify and catalog relevant molecular signatures for informing crop protection strategies.

09 BIOMASS FUELS↗