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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Reshaping the 2‐Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides

Abstract Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2‐pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value‐added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high‐value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen‐bond interactions play key roles in tuning the selectivity, efficiency and yield.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, in this study, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2021

n-Butanol production by Rhodopseudomonas palustris TIE-1

Anthropogenic carbon dioxide (CO 2 ) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO 2 to biofuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO 2 . However, oxygen generation during oxygenic photosynthesis adversely affects biofuel production efficiency. To produce n-butanol (biofuel) from CO 2 , here we introduce an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieve n-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produce the highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produces n-butanol using CO 2 , solar panel-generated electricity, and light with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Upcycling plastic waste into polyhydroxyalkanoates with high carbon conversion via CO 2 plasma-enabled deconstruction

Plastic deconstruction into fermentable intermediates is a key step for microbial bio-upcycling into value-added products. In this study, CO 2 plasma deconstruction was used as an electrified route to convert polyethylene into oxygenated intermediates and liquid (OIL). The resulting OIL was rich in fatty acids, fatty alcohols, and hydrocarbons, making it a suitable feedstock for medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida NRRL B-14688 and Pseudomonas resinovorans NRRL B-2649. Compared with batch fermentation and monocultures, fed-batch co-cultivation markedly improved biomass formation and PHA accumulation, likely due to complementary substrate utilization, particularly hydrocarbon conversion by P. resinovorans. Using virgin polyethylene-derived OIL (Vir-OIL), the co-culture achieved 40.11% mcl-PHA content and about 18% PHA yield based on total OIL fed. More importantly, OIL produced from post-consumer single-use plastic films (PCR-OIL) was directly fermented and well supported the cell growth and PHA accumulation, achieving 38.11% PHA content and about 14.7% PHA yield. Based on emulsified OIL fractions, PHA yields for Vir-OIL and PCR-OIL were comparable (∼28%). PHA granules were extracted from PCR-OIL-grown cells with high recovery (88.25%) and purity (94.8%). Five monomers were identified in the polymer, including 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD), and 3-hydroxytetradecanoate (3HTD), with 3HO (44.28%) and 3HD (40.60%) as the dominant units. The polymer exhibited moderate molecular weight and narrow dispersity (M n = 65.4 kDa, M w = 92.1 kDa, Đ = 1.40) and low crystallinity (T m ≈ 76.6 °C, X c ≈ 15.5%). These characteristics indicate elastomer-like behavior, making the material suitable for flexible applications such as films, coatings, adhesives, and blend modifiers. Overall, this study establishes a CO 2 plasma-assisted route for generating fermentable polyethylene-derived intermediates and demonstrates that fed-batch co-culture fermentation can effectively funnel plastic-derived carbon into mcl-PHA.

42 ENGINEERING↗
DOE OSTI2024

Extracellular Charge Transport in Microbial Redox Chains: Linking the Living and Non-Living Worlds

The fundamental process of electron transfer (ET) within and between molecules dictates all biological energy conversion strategies, including respiration and photosynthesis. This project resulted in a comprehensive physics-based understanding of the mechanisms and limits of redox networks that mediate and regulate electron transport through microbial metabolic pathways, with special emphasis on environmental microbes that can acquire energy by catalyzing anodic or cathodic reactions on solid-state electrodes (extracellular electron transfer, or EET). By performing electron transfer to/from electrodes, such microbes may be used as biocatalysts for converting the energy stored in diverse chemical fuels to electricity, or vice versa (microbial electrosynthesis), in renewable energy technologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Predominance of Methanomicrobiales and diverse hydrocarbon–degrading taxa in the Appalachian coalbed biosphere revealed through metagenomics and genome–resolved metabolisms

Coalbed deposits are a unique subsurface environment and represent an underutilized resource for methane generation. Microbial communities extant in coalbed deposits are responsible for key subsurface biogeochemical cycling and could be utilized to enhance methane production in areas where existing gas wells have depleted methane stores, or in coalbeds that are unmined, or conversely be utilized for mitigation of methane release. Here we utilize metagenomics and metagenome-assembled genomes to identify extant microbial lineages and genome-resolved microbial metabolisms of coalbed produced water, which has not yet been explored in the Appalachian Basin. Our analyses resulted in the recovery of over 40 metagenome-assembled genomes (MAGs) from eight coalbed methane wells. The most commonly identified taxa among samples were hydrogenotrophic methanogens from the order Methanomicrobiales and these dominant MAGs were highly similar to one another. Conversely, low-abundance coalbed bacterial populations were taxonomically and functionally diverse, mostly belonging to a variety of Proteobacteria classes, and encoding various hydrocarbon solubilization and degradation pathways. Further, the data presented herein provides novel insights into Appalachian Basin coalbed microbial ecology, and our findings provide new perspectives on underrepresented Methanocalculus species and low-relative abundance bacterial assemblages in coalbed environments, and their potential roles in stimulation or mitigation of methane release.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Chelator-mediated Fenton post-treatment enhances methane yield from lignocellulosic residues via microbial community modulation

Advancing biomethane production from anaerobic digestion (AD) is essential for building a more reliable and resilient bioenergy system. However, incomplete conversion of lignocellulose-rich agricultural waste remains a key limitation, often leaving energy-dense residues in the digestate by-product. In this study, we introduce a novel application of chelator-mediated Fenton (CMF) post-treatment to recover untapped biomethane potential from these recalcitrant residues, representing a significant departure from conventional pre-treatment strategies. By systematically varying pH, iron-chelator concentration, and hydrogen peroxide dosage, we identified reaction conditions (pH 6–8, 5 mM Fe 2+ -dihydroxybenzene, 3–4 wt.% H 2 O 2 ) that enhanced lignocellulose deconstruction and increased dissolved organic carbon (DOC) availability for methanogenesis. CMF post-treatment led to up to a tenfold increase in biomethane potential compared to untreated controls. Microbial community analysis revealed enrichment of cellulolytic species, suggesting enhanced hydrolytic activity as a driver of improved conversion. Application of the CMF post-treatment method to isolated poplar lignin further demonstrated its versatility for diverse lignocellulosic substrates. These findings position CMF post-treatment as a promising strategy to enhance AD efficiency and valorize digestate.

Martinez, Daniella Victoria [Sandia National Labor↗
DOE OSTI2021

Integration of CO2 Electrolysis with Microbial Syngas Upgrading to Rewire the Carbon Economy

This project has worked towards demonstrating and understanding the conversion of waste CO2 into value added fuels and chemicals. If successful, our proposed technology will incentivize CO2 capture and can add carbon efficiency to biorefineries. We have integrated two conversion technologies to demonstrate a novel approach of combining electrolytic CO2 reduction with biocatalytic CO upgrading. Industrial partners 3M and Dioxide Materials have recently demonstrated electrolytic conversion of CO2 and water to syngas using inexpensive renewable energy. Concurrently, supporting partner Lanzatech has demonstrated industrial scale CO fermentation using steel mill waste CO as a substrate. This presentation will highlight the investment BETO has made to establish a CO2 electrolysis test station and CO fermentation at NREL. We will also discuss the process of increasing the size of the electrolyzers to meet fermentation needs, along with the investigation into the affect CO2 concentration and contaminates have on electrolyzer efficiency, lifetime, and specificity. Additionally, we have begun to examine the downstream impact of electrolyzer off-gas CO:H2 ratios have on the metabolism of C. autoethanologen. Experimental results along input from our industrial partners have led to a baseline techno-economic and life cycle analyses. These results enabled the identification of key cost drivers and carbon intensity of the process. We have used these metrics to identify technology gaps to iteratively inform the process optimization and potential deployment siting to achieve economically viable, sustainable conversion of biopower-derived flue gases to fuels and chemical intermediates.

bioenergy↗
DOE OSTI2021

Conversion of S-lignin compounds to useful intermediates

The present disclosure relates to a genetically modified microbial cell that includes a genetic modification resulting in the expression of a vanillate demethylase, where the microbial cell is capable of metabolizing at least one S-lignin decomposition molecule including at least one of syringate and/or 3-O-methyl gallate, and the genetically modified microbial cell is capable of producing gallate. In some embodiments of the present disclosure, the vanillate demethylase may include VanAB.

Beckham, Gregg Tyler↗
DOE OSTI2023

Conversion of S-lignin compounds to useful intermediates

The present disclosure relates to a genetically modified microbial cell that includes a first genetic modification resulting in the expression of an exogenous vanillate demethylase, such that the microbial cell is capable of metabolizing an S-lignin decomposition product and producing 2-pyrone-4,6-dicarboxylate (PDC).

Johnson, Christopher W.↗
DOE OSTI2025

Integrating catalytic fractionation and microbial funneling to produce 2-pyrone-4,6-dicarboxylic acid and ethanol

Replacing biorefinery designs that use stepwise fractionation, depolymerization, and conversion processes with designed tandem process steps can greatly reduce a biorefinery's operating costs and environmental impact. Reductive Catalytic Fractionation (RCF) is a highly efficient lignin-first approach that combines biomass fractionation and lignin depolymerization to generate a hydrogenolysis oil and pulp. The oil is composed of a complex mixture of phenolic monomers, dimers, and oligomers. Intergrating this chemical deconstruction process with microbial funneling of phenolics can simplify the product mixture and make high-value products. We applied RCF to poplar biomass in a biomass-to-bioproduct processing chain in which the phenolics were funneled to 2-pyrone-4,6-dicarboxylic acid (PDC) by an engineered strain of Novosphingobium aromaticivorans DSM12444. The pulp was enzymatically digested and the glucose and xylose was funneled to ethanol by an engineered strain of Saccharomyces cerevisiae GLBRCY945. By combining biomass fractionation and lignin depolymerization we removed a costly processing step that directly translated into a 29% reduction in the minimum selling price of PDC. This work combines experimentation with process modeling of an integrated biorefinery design to show how utilizing tandem process steps can significantly reduce operating expenses and environmental impact of upgrading lignocellulosic biomass to a portfolio of high value products.

Sener, Canan [Great Lakes Bioenergy Research Cente↗
DOE OSTI2022

Data for Reshaping the 2-Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides

Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2-pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value-added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high-value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen-bond interactions play key roles in tuning the selectivity, efficiency, and yield.

Conversion↗
DOE OSTI2023

Plastispheres as hotspots of microbially-driven methylmercury production in paddy soils

Microplastics (MPs) as emerging contaminants have accumulated extensively in agricultural ecosystems and are known to exert important effects on biogeochemical processes. However, how MPs in paddy soils influence the conversion of mercury (Hg) to neurotoxic methylmercury (MeHg) remains poorly understood. Here, in this study, we evaluated the effects of MPs on Hg methylation and associated microbial communities in microcosms using two typical paddy soils in China (i.e., yellow and red soils). Results showed that the addition of MPs significantly increased MeHg production in both soils, which could be related to higher Hg methylation potential in the plastisphere than in the bulk soil. We found significant divergences in the community composition of Hg methylators between the plastisphere and the bulk soil. In addition, the plastisphere had higher proportions of Geobacterales in the yellow soil and Methanomicrobia in the red soil compared with the bulk soil, respectively; and plastisphere also had more densely connected microbial groups between non-Hg methylators and Hg methylators. These microbiota in the plastisphere are different from those in the bulk soil, which could partially account for their distinct MeHg production ability. Our findings suggest plastisphere as a unique biotope for MeHg production and provide new insights into the environment risks of MP accumulation in agricultural soils.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Comparison of metagenomes from fermentation of various agroindustrial residues suggests a common model of community organization

The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Biofuels for a sustainable future

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.

09 BIOMASS FUELS↗
DOE OSTI2026

Urea-to-Ammonia Conversion at Proteus mirabilis Modified Pt–Ni/BDD Electrodes

Efficient wastewater recycling technologies are essential for long-duration space missions and sustainable water management on Earth. Here, a bioelectrochemical system integrating Proteus mirabilis with a platinum–nickel-modified boron-doped diamond electrode (Pt–Ni/BDDE) for urea-to-ammonia conversion in synthetic urine is presented. Immobilized P. mirabilis catalyzes enzymatic ureolysis, converting urea into ammonia, which is subsequently oxidized electrochemically, and no direct electrochemical urea oxidation is observed. Cyclic voltammetry (CV) of P. mirabilis on Pt–Ni/BDDE in 0.1 M urea and synthetic urine revealed a broad anodic oxidation peak at approximately 0.55–0.75 V vs Ag/AgCl (sat. KCl), corresponding to ammonia oxidation. Control experiments using bare BDDE and Pt–Ni/BDDE in synthetic urine showed no oxidation peak, establishing that the bioelectrocatalytic response originates exclusively from the bioelectrode interface. Chronoamperometry studies revealed that immobilization potential and time critically influenced bacterial adhesion and electrochemical response, with optimal conditions yielding a maximum current density of 0.0055 mA·cm –2 . These quantitative results establish that microbial ureolysis can be efficiently coupled with advanced electrode materials for urea-to-ammonia conversion, offering a promising self-sustaining strategy for urine processing and water recovery in closed-loop life support systems.

Ammonia↗
DOE OSTI2023

Electrons to Molecules by Engineering and Evolution: Biological Upgrading of Formate by Cupriavidus necator

Waste carbon from industrial point sources can be captured, stored, and/or transformed using electrochemical conversion or "electrons to molecules" technologies using low-cost renewable electricity. One such process involves electrocatalytic reduction of CO2 to generate formate/formic acid, a C1 carboxylic acid. Formate is a promising potential feedstock for microbial upgrading, as it is water soluble and can be consumed as the sole source of carbon and energy by some microbial species, such as the soil bacterium Cupriavidus necator. Here we will present progress toward improving C. necator as a host for biological conversion of formate to value-added products. Using the power of adaptive laboratory evolution, we were able to isolate mutants of C. necator with significantly faster growth rates on formate. We then sequenced the genomes of these strains, elucidated the metabolic role of the mutations we found, and then used these insights to build rationally engineered strains that outperform even the best evolved isolates. These results highlight the utility of "genome streamlining" as a route for generating platform strains with potential industrial applications.

adaptive laboratory evolution↗
DOE OSTI2020

Biotic degradation of acetonitrile. Limitations, controls and conversion rates

This document has been prepared to support Washington River Protection Solutions (WRPS) in their evaluation of technologies to remediate organic compounds in the Liquid Effluent Retention Facility (LERF)/Effluent Treatment Facility (ETF) during direct feed low activity waste (DFLAW) operations at Hanford. This report summarizes the results of a review of the available literature describing aerobic and anaerobic biological treatment of acetonitrile and proposes a test matrix to evaluate microbial degradation rates of acetonitrile. Numerous studies describe acetonitrile degradation by pure culture strains of bacteria, fungi, and yeast; however, the purpose of this review was to provide estimations of degradation under conditions that more closely approximate the Liquid Effluent Retention Facility (LERF) basins at the Hanford Site. To this end, documented limitations, controls, and conversion rates are summarized for mixed and natural microbial communities being maintained or operated as a biological treatment for wastewater processing. Based on the literature evaluation, the inherent capacity and rates of microbial degradation of acetonitrile are sufficiently high that if applied in the LERF basins, acetonitrile removal would be achieved. To be clear, though, published studies have not been conducted under conditions that approximate or that could be directly related to the LERF basins. Thus, a focused test matrix the encompasses LERF relevant conditions to specifically measure microbial degradation of acetonitrile is proposed and the results would provide the strong technical justification for use of this low-cost efficient biological process at LERF during DFLAW operations and beyond.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗