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At least 217 records · Page 12

Associative nitrogen fixation linked with three perennial bioenergy grasses in field and greenhouse experiments

Abstract Associative nitrogen (N 2 )‐fixation (ANF) by bacteria in the root‐zone of perennial bioenergy grasses has the potential to replace or supplement N fertilizer and support sustainable production of biomass, but its application in marginal ecosystems requires further evaluation. In this study, we first combined both greenhouse and field experiments, to explore the N 2 fixation effects of three temperate feedstocks Miscanthus × giganteus (giant miscanthus, Freedom), Panicum virgatum (switchgrass, Alamo), and Saccharum sp. (energycane, Ho 02‐147). In field studies across three growing seasons, plant and soil pools of candidate feedstocks were partially composed of N derived from the atmosphere (Ndfa). Energycane, giant miscanthus, and switchgrass were estimated to derive >30%, %Ndfa. Greenhouse studies were also performed to trace isotopically labeled 15 N 2 into plant biomass and soil pools. Evidence for Ndfa was detected in all three feedstock grasses (using reference 15 N of soil, chicory, and sorghum, δ 15 N~+7.0). Isotopically labeled 15 N 2 was traced into biomass (during grass elongation stage) and soil pools. Extrapolation of rates during the 24 hr labeling period to 50 days estimated 30%–55% of plant Ndfa, with the greatest Ndfa for energycane. The findings of the field natural abundance and greenhouse 15 N 2 feeding experiments provided complementary evidence that perennial bioenergy grasses have the potential to support relatively high rates of ANF, and accumulate diazotroph‐derived N into biomass when grown on non‐fertilized soil.

09 BIOMASS FUELS↗

The soybean β‐expansin gene GmINS1 contributes to nodule development in response to phosphate starvation

Abstract Legume biological nitrogen fixation (BNF) is the most important N source in agricultural ecosystems. Nodule organogenesis from the primordia to the development of mature nodules with the ability to fix N 2 largely determines BNF capacity. However, nodule growth is often limited by low phosphorus (P) availability, while the mechanisms underlying nodule development responses to P deficiency remain largely unknown. In this study, we found that nodule enlargement is severely inhibited by P deficiency, as reflected by the smaller individual nodule size from a soybean core collection in the field. Wide‐ranging natural diversity in nodule size was further identified in soybeans reared in low P soils, with the FC‐1 genotype outperforming FC‐2 in assessments of nodulation under low P conditions. Among β‐expansin members, GmINS1 expression is most abundantly enhanced by P deficiency in FC‐1 nodules, and its transcript level is further displayed to be tightly associated with nodule enlargement. Four single nucleotide polymorphisms discovered in the GmINS1 promoter distinguished the FC‐1 and FC‐2 genotypes and accounted for the differential expression levels of GmINS1 responses to P deficiency. GmINS1 overexpression led to increases in nodule size, infected cell abundance, and N 2 fixation capacity and subsequently promoted increases in N and P content, soybean biomass, and yield. Our findings provide a candidate gene for optimizing BNF capacity responses to low P stress in soybean molecular breeding programs.

Yang, Zhaojun↗

Examining Microalgae Growth with Respect to Cell Count and Salinity

In recent years, microalgae have been identified as potential source of biofuel due to their ability to grow abundantly in little time. As photosynthetic organisms, microalgae only need water, light, micronutrients, and carbon dioxide to yield algal biomass. Several applications for this biomass are being studied, such as the extraction of biofuel (Zhang et al. 2022) as well as wastewater treatment (Rude et al. 2022). Two specific microalgae species have been identified as uniquely prolific: Picochlorum celeri, a saltwater species, and Chlorella sorokiniana 1116, a freshwater species. Both species showcased survival in less-than-ideal conditions and showcased hardiness in their respective waters (Krishnan et al. 2021), making them popular specimen to be studied for a variety of applications. This project involved two components, the first component focusing on the development of a growth curve for P. celeri and the second studying varying salt concentration in both species.

59 BASIC BIOLOGICAL SCIENCES↗

Sustainable Herbaceous Energy Crop Production in the Southeast United States

This project aims to develop a comprehensive assessment of the economic viability and environment sustainability of energycane and biomass sorghum for bioenergy development in the southeast United States. Data on biomass production, economics, and environmental sustainability of energycane and biomass sorghum were collected across four states (Texas, Louisiana, Georgia, and Florida) in multi-year field experiments. Comprehensive data analyses were conducted to address 13 subject areas

09 BIOMASS FUELS↗

Biotic countermeasures that rescue Nannochloropsis gaditana from a Bacillus safensis infection

The natural assemblage of a symbiotic bacterial microbiome (bacteriome) with microalgae in marine ecosystems is now being investigated as a means to increase algal productivity for industry. When algae are grown in open pond settings, biological contamination causes an estimated 30% loss of the algal crop. Therefore, new crop protection strategies that do not disrupt the native algal bacteriome are needed to produce reliable, high-yield algal biomass. Bacteriophages offer an unexplored solution to treat bacterial pathogenicity in algal cultures because they can eliminate a single species without affecting the bacteriome. To address this, we identified a highly virulent pathogen of the microalga Nannochloropsis gaditana, the bacterium Bacillus safensis, and demonstrated rescue of the microalgae from the pathogen using phage. 16S rRNA amplicon sequencing showed that phage treatment did not alter the composition of the bacteriome. It is widely suspected that the algal bacteriome could play a protective role against bacterial pathogens. To test this, we compared the susceptibility of a bacteriome-attenuated N. gaditana culture challenged with B. safensis to a N. gaditana culture carrying a growth-promoting bacteriome. We showed that the loss of the bacteriome increased the susceptibility of N. gaditana to the pathogen. Transplanting the microalgal bacteriome to the bacteriome-attenuated culture reconstituted the protective effect of the bacteriome. Finally, the success of phage treatment was dependent on the presence of beneficial bacteriome. This study introduces two synergistic countermeasures against bacterial pathogenicity in algal cultures and a tractable model for studying interactions between microalgae, phages, pathogens, and the algae microbiome.

60 APPLIED LIFE SCIENCES↗

Strategies to achieve high productivity, high conversion, and high yield in yeast fermentation of algal biomass hydrolysate

The conversion of carbohydrates in biomass via fermentation is an important component of an overall strategy to decarbonize the production of fuels and chemicals. Owing to the cost and resources required to produce biomass hydrolysates, the economic and environmental sustainability of these fermentation processes requires that they operate with high yields, sugar conversion, and productivity. Immobilized-cell technology in a continuous bioprocess can achieve significantly higher volumetric productivities than is possible from standard batch fermentation using free cells. Here, we demonstrate approaches for improvement of ethanol yield from algal hydrolysates and a mock hydrolysate medium. Saccharomyces cerevisiae was immobilized in alginate and incorporated into a two-column immobilized cell reactor system. Furthermore, the yeast quorum-sensing molecule, 2-phenylethanol, was added to improve ethanol yield by restricting growth and diverting sugar to ethanol. The bioreactor system could achieve high ethanol volumetric productivity (>20 g/ Lreactor ·h) and high glucose conversion (>99%) in mock hydrolysate, while the addition of 0.2% 2-phenylethanol resulted in 4.9% higher ethanol yield. With an algal hydrolysate of <10 g/L sugar, the ethanol volumetric productivity reached 9.8 g/ Lreactor ·h, and the addition of 0.2% 2-phenylethanol increased the ethanol yield by up to 7.4%. These results demonstrate the feasibility of novel strategies to achieve sustainability goals in biomass conversions.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers

ABSTRACT The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway inCaldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C 5 and C 6 sugars. In addition, C. besciigrows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO.C. besciinatively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway,C. besciiwas engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present inC. bescii,the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model ofC. bescii. IMPORTANCE Lignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacteriumCaldicellulosiruptor besciiwas successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling ofC. besciiwas applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.

Biotechnology & Applied Microbiology↗

Photosynthesis and rhizome carbohydrate concentrations of switchgrass grown from reserve-depleted rhizomes

A long-standing question in perennial grass breeding and physiology is whether yield improvement strategies could compromise winter survival. Perennial grasses rely on the pool of carbohydrates accumulated in storage organs from the previous growing season for winter maintenance and spring regrowth. Yield improvement strategies could reduce winter survival if they increase biomass and grain yields at the expense of carbon allocation to storage. Therefore, it is crucial to better understand the dependence of regrowth on storage reserves. We experimentally depleted switchgrass ( Panicum virgatum L.) rhizome reserves by storing rhizomes for two weeks at 5 °C (control treatment) and 25 °C (reserve-depleted treatment).

bioenergy↗

Data for Multisite Field Evaluation of Oil Accumulation and Agronomic Performance in Grain and Sweet Sorghums Engineered for Lipid Hyperaccumulation

Oil sorghum (OS) has been developed by engineering grain (TX430) and sweet (Ramada) genetic backgrounds to accumulate triacylglycerols (TAG) in vegetative tissues as an energy-dense feedstock for sustainable aviation fuel (SAF) and other biofuels. This study evaluated two TX430 OS lines (TxHO-2, TxHO-3) and two Ramada OS lines (RmHO-1, RmHO-2) alongside wild-type (WT) lines in NE and IL over 2 years (2023–2024) to quantify genotype × environment effects on agronomic performance and TAG accumulation. Across four environments, TX430 OS lines showed average TAG concentrations of 15.0 g kg−1 in leaves and 12.8 g kg−1 in stems, approximately 19-fold higher than WT. Ramada OS lines accumulated 26.1 g kg−1 in leaves and 12.3 g kg−1 in stems, approximately 25-fold and 13-fold increases over WT, respectively. OS lines in TX430 exhibited an 18% reduction in biomass (8.4 vs. 9.9 Mg ha−1 for WT), while Ramada OS lines had similar WT biomass (18.3 vs. 19.9 Mg ha−1 for WT). Among TX430 OS lines, TxHO-2 achieved the highest TAG yield (190 kg ha−1), while RmHO-1 led the Ramada lines (335 kg ha−1) due to higher biomass and similar TAG concentration. Enhanced TAG accumulation increased N, P, and K removal in TX430 lines but not in Ramada lines. Structural carbohydrate and ash concentration were unaffected. Overall, results confirm vegetative lipid accumulation as a viable strategy for high-biomass sorghum, supporting its potential as a dual-purpose feedstock for SAF. Future work should focus on minimizing biomass yield penalties and improving nutrient use efficiency in oil sorghum systems.

Agronomy↗

Can Multiple Ions in an Ionic Liquid Improve the Biomass Pretreatment Efficacy?

we report over the last few decades, efforts to transition the global production of fuels and chemicals toward renewable carbon feedstocks have accelerated. A large portion of these efforts have focused on valorization of one of the most abundant renewable carbon sources, lignocellulose. Pretreatment of lignocellulose is the first critical step in this process. In this study, novel ionic liquid (IL) systems consisting of multiple ions known to be effective at biomass pretreatment were tested on woody and grassy biomass. Molecular simulations and experimental results established the synergistic advantages of combining specific individual components in these systems. For pine (woody) biomass, pretreatment with the combination of imidazolium, cholinium, acetate, and lysinate ions achieved 80% glucose and 70% xylose yields at high biomass loading. For sorghum biomass, an IL system comprising cholinium, lysinate, and palmitate ions not only enabled a 98% glucose yield but was also found to be biocompatible in a one-pot configuration, producing the biofuel precursor bisabolene using an engineered strain of the yeast Rhodosporidium toruloides.

09 BIOMASS FUELS↗

Data from: Long-term yields in annual and perennial bioenergy crops in the Midwestern USA

Data sets relating to the manuscript “Long-term yields in annual and perennial bioenergy crops in the Midwestern USA” published in Global Change Biology Bioenergy. Field data, including annual peak biomass and harvest yields from maize/soy, miscanthus, switchgrass, and prairie field trials from 2008-2018 are included. Peak and harvest biomass for fertilized and unfertilized miscanthus are included from 2014-2018.

bioenergy↗

Miscanthus: An Environmental Choice for Marginal Lands

Miscanthus x giganteus, or Giant Miscanthus, is a tall, perennial grass originally from Asia. It is considered a sterile hybrid that has been bred to not produce viable seeds. Instead, rhizomes are planted to establish the crop. Here, the rhizome is an underground modified stem mass that puts out shoots and roots supporting new growth each year. This perennial crop takes two to three years to reach maturity, but established stands can reach upwards of 12 ft. tall with a very thick canopy (Pyter, 2007). Miscanthus has shown potential for high dry matter yields compared to other biomass crops, which could in turn lead to more efficient land use especially when applied to a bioenergy and bioproduct supply chain. In one study, switchgrass (Panicum virgatum) yielded 10.3 Mg/ha of biomass on average, while miscanthus yielded 22.4 Mg/ha (Heaton et al., 2004). Additionally, miscanthus production has been associated with many environmental benefits, including benefits to water quality, nutrient load removal, soil stabilization, and soil health (Bhardwaj et al., 2011; Hussain et al., 2019). Many of the possible practical and environmental benefits that are associated with miscanthus production may be attractive to stakeholders who are interested in alternative land use options, particularly in areas considered to be marginal in productivity.

54 ENVIRONMENTAL SCIENCES↗

Data for Development of Vegetative Oil Sorghum: From Lab-to-Field

Biomass crops engineered to accumulate energy-dense triacylglycerols (TAG or ‘vegetable oils’) in their vegetative tissues have emerged as potential feedstocks to meet the growing demand for renewable diesel and sustainable aviation fuel (SAF). Unlike oil palm and oilseed crops, the current commercial sources of TAG, vegetative tissues, such as leaves and stems, only transiently accumulate TAG. In this report, we used grain (Texas430 or TX430) and sugar-accumulating ‘sweet’ (Ramada) genotypes of sorghum, a high-yielding, environmentally resilient biomass crop, to accumulate TAG in leaves and stems. We initially tested several gene combinations for a ‘push-pull-protect’ strategy. The top TAG-yielding constructs contained five oil transgenes for a sorghum WRINKLED1 transcription factor (‘push’), a Cuphea viscosissima diacylglycerol acyltransferase (DGAT; ‘pull’), a modified sesame oleosin (‘protect’) and two combinations of specialized Cuphea lysophosphatidic acid acyltransferases and medium-chain acyl-acyl carrier protein thioesterases. Though intended to generate oils with medium-chain fatty acids, engineered lines accumulated oleic acid-rich oil to amounts of up to 2.5% DW in leaves and 2.0% DW in stems in the greenhouse, 36-fold and 49-fold increases relative to wild-type (WT) plants, respectively. Under field conditions, the top-performing event accumulated TAG to amount to 5.5% DW in leaves and 3.5% DW in stems, 78-fold and 58-fold increases, respectively, relative to WT TX430. Transcriptomic and fluxomic analyses revealed potential bottlenecks for increased TAG accumulation. Overall, our studies highlight the utility of a lab-to-field pipeline coupled with systems biology studies to deliver high vegetative oil sorghum for SAF and renewable diesel production.

Biofuels↗

Yield and Survival of 19 Cultivars of Willow (Salix spp.) Biomass Crops over Eight Rotations

This study reveals patterns of yield and survival of short-rotation coppice (SRC) willow cultivars over eight rotations (1993–2019). Cultivars fell into four broad categories: commercial, released, stable, and decline. SV1, the singular cultivar that advanced to commercial deployments, had first-rotation yields of 8.9 Mg ha−1 a−1, peaking at 15.2 Mg ha−1 a−1 by the fourth. Mean yields from rotations 2–8 were still 36% above first-rotation yields, confirming the commercial potential for this cultivar over 26 years. The released group (four cultivars) had stable yields over six rotations (approximately 3 to 7 Mg ha−1 a−1), rising to match commercial yields (10 Mg ha−1 a−1) between the sixth and eighth rotation. Most of the cultivars were in the stable group that had relatively consistent yields over time. First-rotation yields in this group were approximately 5 Mg ha−1 a−1, and average yield increased by 23% for rotations 2–8. The two cultivars in the decline group were impacted by disease and browsing that lowered survival and growth. These findings are crucial for understanding willow systems’ potential over their full lifespan as a bioenergy crop, which is a crucial input into yield, economic, and environmental models.

Santucci, Shane↗

Harvest and nitrogen effects on bioenergy feedstock quality of grass-legume mixtures on Conservation Reserve Program grasslands

Perennial grass mixtures established on Conservation Reserve Program (CRP) lands can be an important source of feedstock for bioenergy production. This study aimed to evaluate management practices for optimizing the quality of bioenergy feedstock and stand persistence of grass-legume mixtures under diverse environments. A 5-year field study (2008–2012) was conducted to assess the effects of two harvest timings (at anthesis vs after complete senescence) and three nitrogen (N) rates (0, 56, 112 kg N ha -1 ) on biomass chemical compositions (i.e., cell wall components, ash, volatiles, total carbon, and N contents) and the feedstock energy potential, examined by the theoretical ethanol yield (TEY) and the total TEY (i.e., the product of biomass yield and TEY, L ha -1 ), of cool-season mixtures in Georgia and Missouri and a warm-season mixture in Kansas. The canonical correlation analysis (CCA) was used to investigate the effect of vegetative species transitions on feedstock quality. Although environmental variations (mainly precipitation) greatly influenced the management effect on chemical compositions, the delayed harvest after senescence generally improved feedstock quality. In particular, the overall cell wall concentrations and TEY of the warm-season mixtures increased by approximately 7%. Additional N supplies improved the total TEY of both mixtures by ~1.6–4.2 L ha -1 per 1.0 kg N ha -1 input but likely lowered the feedstock quality, particularly for the cool-season mixture. The cell wall concentrations of cool-season mixture reduced by approximately 3%–6%. The CCA results indicated that the increased legume compositions (under low N input) likely enhanced lignin but reduced ash concentrations. This field research demonstrated that with proper management, grass-legume mixtures on CRP lands can provide high-quality feedstock for bioenergy productions.

09 BIOMASS FUELS↗

Review: The Next Steps in Crop Improvement: Adoption of Emerging Strategies to Identify Bottlenecks in Sugar Flux

We report sugar allocation in plants is the fundamental process that transports sugar from source to sink tissues and has a dramatic impact on crop yields. Controlling sugar allocation is required to increase crop yields, as well as biomass for biofuel production. Successful examples have demonstrated that genetic engineering of sugar partitioning offers a promising strategy to achieve this goal. However, improvement has thus far been limited by gaps in understanding of the underlying mechanisms controlling the allocation of sugars. The dynamics of sugar partitioning are minimally predictable under different conditions, between species, or in response to abiotic stresses. Here, we discuss four methodologies that have not been sufficiently exploited for the identification of bottlenecks in sugar flux. Furthermore, we suggest how these strategies can be used and combined to provide the insight needed to maximize crop yields or biomass, especially under conditions of environmental stress.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Quorum-sensing molecules increase ethanol yield from Saccharomyces cerevisiae

ABSTRACT One strategy to increase the yield of desired fermentation products is to redirect substrate carbon from biomass synthesis. Nongenetic approaches to alter metabolism may have advantages of general applicability and simple control. The goal of this study was to identify and evaluate chemicals for their ability to inhibit the growth of Saccharomyces cerevisiae while allowing ethanol production with higher yields. Eight potential growth-inhibitory chemicals were screened for their ability to reduce cell growth in 24-well plates. Effective chemicals were then evaluated in cultivations to identify those that simultaneously reduced biomass yield and increased ethanol yield. The yeast quorum-sensing molecules 2-phenylethanol, tryptophol and tyrosol were found to increase the ethanol yield of S. cerevisiae JAY 270. These molecules were tested with seven other yeast strains and ethanol yields of up to 15% higher were observed. The effects of 2-phenylethanol and tryptophol were also studied in bioreactor fermentations. These findings demonstrate for the first time that the ethanol yield can be improved by adding yeast quorum-sensing molecules to reduce the cell growth of S. cerevisiae, suggesting a strategy to improve the yield of ethanol and other yeast fermentation products by manipulating native biological control systems.

Huang, Xing-Feng (ORCID:0000000251165177)↗

Comparison of the efficacy of a biocompatible and a distillable solvent for pretreatment of mixed bioenergy feedstocks

Biomass deconstruction is a crucial step in the production of lignocellulosic biofuels and bioproducts. However, identifying and selecting an optimal pretreatment solvent that enhances enzymatic saccharification while being cost-efficient and ensuring sustainability remains a challenge. In this study, we compare the effectiveness of the biocompatible ionic liquid cholinium lysinate ([Ch][Lys]) against the distillable solvent ethanolamine, when used for the pretreatment of mixed bioenergy feedstocks, including poplar, switchgrass, and sorghum. [Ch][Lys] was used at a concentration of 10 % wt. in a one-pot configuration without biomass washing and ethanolamine was used in a concentrated form and removed with a vacuum oven, before performing enzymatic hydrolysis and microbial conversion. Our results show that ethanolamine pretreatment consistently enhances the glucose yield across various biomass types compared to [Ch][Lys], with improvements ranging from 22.0 % to 52.7 %. The highest combined sugar release was observed when the three feedstocks were combined in equal amounts and pretreated with ethanolamine, achieving a glucose yield of 84.6 % and a xylose yield of 76.6 %. Additionally, ethanolamine exhibited exceptional solvent recovery efficiency, with removal efficiencies exceeding 99.8 % at 120 °C across all feedstocks, highlighting its advantage over non-distillable solvents. While both solvents produced biocompatible hydrolysates, the hydrolysates prepared using ethanolamine resulted in higher bioproduct formation. These findings highlight the industrial relevance of selecting recyclable and biocompatible solvents for scalable, sustainable, and cost-effective bioenergy production. This study contributes to the development of economically viable and scalable pretreatment technologies for bioenergy applications using different feedstocks and process configurations.

Rahman, Maksudur↗