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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Translation control tool for plants

The present invention provides for a system comprising (a) first polynucleotide encoding a first promoter operatively linked to an open reading frame (ORF) encoding an endoribonuclease, or an enzymatic active fragment thereof, which cleaves a cognition sequence (cog), and (b) a second polynucleotide encoding a second promoter operatively linked to a nucleotide sequence encoding (i) a cog linked to a coding sequence of interest (COI), (ii) a cog linked to the 3′ end of a first COI and the 5′ end of a second COI, or (iii) a cog locating within a LS (or targeting sequence) linked to a COI.

Liang, Yan↗
DOE OSTI2023

Environmental and Social Justice Considerations in a Circular Economy

A consideration of environmental justice (EJ) and social justice (SJ) is critical to minimize the impacts of technology deployment on local communities. SJ and EJ impacts occur in specific geographic locations but can cover a wide range of effects (e.g., air pollution, access to clean water, jobs, wages, and education), making it challenging to determine which metrics are appropriate to evaluate and which data are required for qualitative or quantitative analysis. This issue is only exacerbated when the technology in question is at an early technology readiness level (TRL) without community specific data. Here, we present a framework for evaluating the human health, local environment, and job implications of processes that are at early to middle TRLs. Using a case study on enzymatic polyethylene terephthalate (PET) recycling (middle TRL), we demonstrate how to qualitatively and quantitatively assess these EJ and SJ metrics for a circular economy context and how to communicate the results in a manner beneficial to both researchers and local communities.

circular economy↗
DOE OSTI2024

Plastic Parallel Pathways Platform- 4P Model

The U.S. generates 42 million metric tons of plastic waste each year - the most of any country - of which less than 9% is recycled. This represents an estimated loss of $2.3 billion and 3.4 EJ embodied energy per year. Plastic use reduction, reuse and recycling are thus increasingly important, but making informed policy and research decisions within this space can be challenging given the diverse range of available solutions. Various recycling and waste management options are available for plastics, such as conversion into energy (incineration, pyrolysis, gasification), conversion to lower- or higher-grade plastics (mechanical recycling, chemical or enzymatic depolymerization processes, dissolution) or up-cycling to feedstock chemicals (photo/electro/catalytic methods, pyrolysis, gasification). While many previous publications have investigated a subset of these options, there is currently no modelling platform that can quantitatively assess and compare the economic and environmental impacts of all these different plastic management pathways and their respective products simultaneously. To directly compare the benefits and disadvantages of plastic-to-x technologies, we propose a novel analysis framework: the Plastic Parallel Pathways Platform (4P). 4P will model a systems analysis framework for capturing plastic material flows processed via different waste management scenarios. The total economic cost and greenhouse gas emissions (as well as additional economic, energetic, and environmental metrics indicators) of a given scenario will be calculated enabling comparison to other scenarios, thereby informing decisions on plastic recycling pathways.

circular economy↗
DOE OSTI2024

Economical methods for performing oxidative catalytic pretreatment of plant biomass using a single-stage two oxidant process

An improved alkaline pretreatment of biomass is provided that is a single-stage, two oxidant alkaline oxidative pretreatment process. The process uses a homogenous catalyst with at least two oxidants (Hydrogen peroxide and enhanced levels of oxygen) in an alkaline environment to catalytically pretreat lignocellulosic biomass in a single-stage oxidation reaction. The provided single-stage alkaline-oxidative pretreatment improves biomass pretreatment and increase enzymatic digestibility to improve the economic feasibility of production of lignocellulose derived sugars.

Hegg, Eric L.↗
DOE OSTI2024

Method of adjusting the pH of a pretreatment solution using carbon dioxide useful for integrating saccharification and fermentation of a biomass

The present invention provides for a method of fermenting or saccharifying a biomass comprising: (a) (i) contacting a biomass comprising a polysaccharide, and an ionic liquid (IL) to form a first solution, or (ii) providing the first solution comprising the biomass and the IL, (b) contacting the first solution and carbon dioxide such that the first solution results in a lower pH, (c) introducing (i) an enzyme capable of enzymatically to breakdown at least one bond in the polysaccharide or a breakdown product of the polysaccharide, and/or (ii) a microorganism that capable of producing the enzyme and/or fermenting the polysaccharide or a breakdown product of the polysaccharide, such that the polysaccharide is at least partially broken down and the first solution is transformed into a second solution.

Sun, Jian↗
DOE OSTI2025

Unveiling Feedstock Variability: Insights into Corn Stover Conversion - Part I: Physicochemical Properties and Self-Degradation

Transforming agricultural waste into biofuels and bioproducts is crucial to advancing a low-carbon bioeconomy. However, the inherent variability in the composition and quality introduces uncertainties in the conversion efficiency and poses challenges in process development. Through integrating a high-throughput conversion system, material characterization techniques, and advanced data analysis tools, this study investigates the variability of corn stover and its subsequent impacts on carbohydrate conversion. The findings reveal that indoor storage substantially reduces the moisture and ash content and soil contamination, while other properties remain largely unchanged. Self-degradation due to microbial activity during storage decreases the carbohydrate content of corn stover but enhances glucose and xylose yields. A negative correlation is observed between sugar yields and lignin content across samples with varying ash and moisture content. The inhibitory effect of lignin diminishes in self-degraded samples likely due to the disrupted cell wall structure. Although self-degradation slightly increases cellulose crystallinity, no strong correlation was observed between the crystallinity and sugar yield. Hot water pretreatment under mild conditions effectively mitigates inherent variability, consistently improving the sugar yield from corn stover by up to 50%. By elucidating the feedstock variability and its impact on convertibility, these findings offer valuable insights into appropriate feedstock handling and management, highlighting potential strategies to address variability challenges.

09 BIOMASS FUELS↗
DOE OSTI2020

Moving from residual lignocellulosic biomass into high–value products: Outcomes from a long–term international cooperation

Major progress in the bioprocessing of lignocellulose to fuels and value-added chemicals has created the possibility of a low carbon-footprint economy. However, the current complexity and associated costs of lignocellulose conversion result in a higher price for ethanol than for fossil fuels. The cost of cellulosic ethanol production will be lowered by further progress in development of biorefinery technology that produces both ethanol and high-value chemicals with bio-based products that are beginning to penetrate consumer markets in the USA, Brazil, and worldwide. The cost-effectiveness of low carbon-footprint bioproducts will benefit from advances in supplying large amounts of biomass solids to the biorefinery. We describe here outcomes from a successful long-term international cooperation between the Laboratory of Renewable Resources Engineering (LORRE) at Purdue University in the United States and Brazil's Agricultural Research Corporation (EMBRAPA) and Federal University of Sâo Carlos (UFSCar), which has contributed practical pathways to enhance the biorefinery concept. Furthermore, this paper gives an overview of developments that address fundamental knowledge of lignocellulosic biomass pretreatment hydrolysis under optimized operational conditions and bioreactor configurations, and the science and engineering that contributes to the effective production of fuel and ethanol and value-added products from biomass.

09 BIOMASS FUELS↗
DOE OSTI2023

Decontamination Strategies to Increase Fuel and Product Conversion Yields of Municipal Solid Waste

Since the implementation of China’s Green Fence and National Sword policies, many areas lost a market for heavily contaminated municipal solid waste (MSW). Approximately 17.2 million tons of paper and 26.9 million tons of plastic were landfilled in the United States in 2018. This non-recyclable waste that would normally be landfilled can be converted into fuels and chemicals, but common contaminants that are present in non-recyclable waste can negatively affect low and high-temperature conversion yields. In addition, MSW is a heterogeneous feedstock whose composition can vary widely across geographical areas and socio-economic statuses. This presents a unique challenge for providing a consistent feedstock to biorefineries around the world. Techniques that are commonly used in other industries, in addition to pre-processing methods used for traditional biomass, can be used to increase conversion yields. Drawing on established techniques, as well as experimental decontamination strategies, non-recyclable MSW could represent a viable alternative to agricultural residues for biofuel production. Although many of the techniques explored in this chapter have been used extensively in other industries, the feedstock composition needs to be carefully considered when choosing prospective decontamination methods. It is likely that there is not a “one-size-fits-all” decontamination method for MSW. In conclusion, a pre-processing and decontamination plan will rely heavily on the composition of the individual waste stream.

09 BIOMASS FUELS↗
DOE OSTI2021

Spatial variation in soil microbial processes as a result of woody encroachment depends on shrub size in tallgrass prairie

Aims: As woody plants encroach into grassland ecosystems, we expect that altered plant-soil interactions will lead to changes in the microbial processes that affect carbon storage and nutrient cycling. Specifically, this research aimed to address how (1) soil chemistry, (2) microbial nutrient demand, and (3) the rate and source of potential soil C mineralization vary spatially under individual woody shrubs of varying size within a mesic grassland. Methods: Here, we collected soil samples from the center, the midpoint between the center and edge, the edge, and the shrub-grass ecotone of multiple Cornus drummondii shrubs across a shrub-size gradient in infrequently burned tallgrass prairie. Results: We found total soil carbon and total soil nitrogen increased with shrub size in every location but the edge. Microbial demand for nitrogen also increased as shrubs increased in size. Across all shrub sizes and sampling locations, potential soil carbon mineralization rates were higher when microbes broke down proportionally more shrub-derived (C 3 ) organic matter than grass-derived (C 4 ) organic matter. Conclusions: Our results suggest that the spatio-temporal context of woody encroachment is critical for understanding its impact on belowground microbial processes. In this ecosystem, a longer period of occupancy by woody plants increases potentially mineralizable carbon.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Chapter 4 - Pretreatment Technologies for Lignocellulosic Biomass Refineries

Pretreatment is a critical step in processing lignocellulosic biomass into biofuel and bioproducts and is considered the energy and cost center of the biomass conversion process. Although a large number of pretreatment technologies have been developed, not all the techniques are viable on a commercial scale at this stage. Moreover, the technology choice and process conditions depend highly on the type of biomass and the overall biorefinery scheme. This chapter provides an overview of different pretreatment methods, including their mechanism, important process parameters, current status, and challenges. The opportunities associated with new technologies and approaches are presented. Emphasis is placed on low-severity thermal pretreatment technologies. The chapter also provides a brief discussion of the challenges to achieving the economic viability of the technologies on a commercial scale.

BIOMASS FUELS↗
DOE OSTI2026

Limited proteolysis of pea protein to promote aggregation/gelation: role of enzyme concentration and molecular characteristics

Limited proteolysis promotes aggregation/gelation of plant proteins, but its relationship with the molecular characteristics of hydrolysates remains poorly understood. In here, response surface methodology was employed to evaluate the effects of protein concentration, Alcalase level, and hydrolysis time on the storage modulus (G′) of pea protein hydrolysates (PPH). 7.5% protein, 1.65% Alcalase, and a 6-min reaction time resulted in the highest relative increase in G′ upon heating PPH, whereas both higher (3.55%) and lower (0.3%) Alcalase levels led to less increment. Increasing the Alcalase level increased the degree of hydrolysis while decreasing the molecular weight, surface hydrophobicity, and total sulfhydryl and disulfide bond contents of PPH. Confocal laser scanning microscopy and X-ray scattering analyses found PPH produced with 1.65% Alcalase formed interconnected aggregates with dual structural domains, but not at other Alcalase conditions. These results demonstrate the role of controlled proteolysis in governing the aggregation/gelation behavior of PPH.

X-ray scattering↗
DOE OSTI2025

Protocol for engineering poly(ethylene terephthalate) hydrolases via directed evolution using a high-throughput screening assay

Poly(ethylene terephthalate) (PET) hydrolases, which depolymerize PET to its monomers, have gained attention for their potential to facilitate bio-industrial recycling of this waste plastic. Here, we present a protocol for screening large, random mutagenesis enzyme libraries simultaneously for enhanced activity, solubility, and stability. We outline steps for library construction, screening using plate-based split GFP and model substrate assays, and determination of enzyme thermostability. We then detail procedures for validation assays on PET substrates and characterization of final variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

H-cluster Intermediates and Catalytic Properties of Clostridium pasteurianum [FeFe]-Hydrogenase III

[FeFe]-Hydrogenases are structurally diverse enzymes that catalyze reversible H2 activation at a catalytic cofactor or H-cluster. The H-cluster is a [4Fe-4S] cubane linked by a cysteine thiolate to a diiron subsite containing unique CO, CN-, and dithiomethylamine ligands. The established H-cluster resting state of [4Fe-4S]2+-[FeII-FeI], or Hox, functions in H2 binding and oxidation, or by proton-coupled reduction initiates H2 evolution. In contrast, in Clostridium pasteurianum [FeFe]-hydrogenase III (CpIII) the resting state of the H-cluster is fully oxidized, [4Fe-4S]2+-[FeII-FeII], or Hox+1. To begin to understand if Hox+1 has a role in the mechanism of CpIII, we determined the spectroscopic and redox properties of CpIII H-cluster states under catalytic conditions. CpIII poised in Hox+1 and either equilibrated under 1 atm of H2 or reduced with sodium dithionite, resulted in a mixture of reduced states including Hox (Em8 = -407 mV), Htrans-like [4Fe-4S]+-[FeII-FeII] (Em8 = -418 mV), Hred [4Fe-4S]+-[FeII-FeI], and HredH+ [4Fe-4S]2+-[FeI-FeI] (Em8 = -455-480 mV). Under H2 the population of the Htrans-like state was >20-fold higher than Hox, implicating a role in CpIII catalysis. Unlike other enzymes, there was no spectral evidence of fully reduced states, such as HsredH+ ([4Fe-4S]+-[FeI-FeI]) or Hhyd ([4Fe-4S]+-[FeII-FeII]-H-). Thus, while the H-cluster states of CpIII encompass most of the catalytic intermediates, it is either unable to form HsredH+ and Hhyd, or these states are highly destabilized in CpIII. Thus, these results demonstrate that catalytic intermediates of reduced CpIII differ from the typical intermediates of other catalytic [FeFe]-hydrogenases and may explain the catalytic preference for H2 production.

08 HYDROGEN↗
DOE OSTI2019

Enhanced Softwood Cellulose Accessibility by H 3 PO 4 Pretreatment: High Sugar Yield without Compromising Lignin Integrity

Softwood lignocellulose is a potential feedstock for the production of biofuels and bioproducts. However, the highly cross-linked nature of softwood lignocellulose restricts enzyme access to its sugars. Thus, harsh pretreatment conditions (180–280 °C) and/or high enzyme loading are required to unlock sugars. These requirements negatively affect the economic viability of softwoods in biorefineries. Here we show that H 3 PO 4 pretreatment of pine and Douglas fir under a mild reaction temperature (50 °C) and atmospheric pressure enabled a high (~80%) glucan digestibility with low enzyme loading (5 filter paper units (FPU)/g glucan). The dissolution and regeneration of softwoods disrupted the hydrogen bonding between cellulose chains, thereby increasing the cellulose accessibility to cellulase (CAC) values by ~38-fold (from ~0.4 to 15 m 2 /g biomass). Examination of H 3 PO 4 -pretreated softwoods by cross-polarization/magic angle spin (CP/MAS), 13 C- nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR) revealed that breaking of the orderly hydrogen bonding of crystalline cellulose caused the increase in CAC (higher than 11 m 2 /g biomass), which, in turn, was responsible for the high glucan digestibility of pretreated softwoods. The H 3 PO 4 pretreatment process was feedstock independent. Finally, 2D 13 C– 1 H heteronuclear single quantum coherence (HSQC) NMR showed that the lignin was depolymerized but not condensed; thus, the lignin can be available for producing high-value products.

09 BIOMASS FUELS↗
DOE OSTI2023

Ice Recrystallization Inhibition Activity of Soy Protein Hydrolysates

Identifying and developing ice recrystallization inhibitors from sustainable food proteins such as soy protein isolate (SPI) can lead to practical applications in both pharmaceutical and food industries. Here, the objective of this study was to investigate the ice recrystallization inhibition (IRI) activity of SPI hydrolysates, and this was achieved by using an IRI activity-guided fractionation approach and relating IRI activity to interfacial molecular activity measured by vibrational sum frequency generation (VSFG). In addition, the impact of molecular weight (MW) and enzyme specificity was analyzed using three different proteases (Alcalase, trypsin, and pancreatin) and varying hydrolysis times. Using preparative chromatography, hydrolysates from each enzyme treatment were fractionated into five different MW fractions (F1–F5), which were then characterized by high-performance liquid chromatography (HPLC). All SPI hydrolysates had IRI activity, resulting in a 57–29% ice crystal diameter reduction when compared to native SPI. The F1 fraction (of 4–14 kDa) was most effective among all tested hydrolysates, while the lower MW peptide fractions lacked activity. One sample (SPI-ALC 20-F1) had a 52% reduction of ice crystal size at a lower concentration of 2% compared to the typical 4% used. SFG showed a difference in H-bonding and hydrophobic interactions of the molecules on the water/air interface, which may be linked to IRI activity. This study demonstrates for the first time the ability of SPI hydrolysates to inhibit ice crystal growth and the potential application of SFG to study molecular interaction at the interface that may help illustrate the mechanism of action.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗