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At least 739 records · Page 41

One Earth Energy FEED Process Design Basis

This report establishes the process design basis for the front-end engineering design (FEED) of a carbon capture and injection facility at One Earth Energy's (OEE) ethanol production plant in Gibson City, Illinois, developed as part of the Illinois Storage Corridor CarbonSAFE Phase III project. The facility is designed to compress and dehydrate up to approximately 458,000 metric tonnes of CO 2 per year, sourced directly from OEE's ethanol fermenters, for permanent injection into a saline aquifer approximately four miles from the plant. At normal operating conditions, the system will process 1,290 metric tonnes of CO 2 per day, assuming 355 operating days per year and an ethanol production rate of 160 million gallons per year. The proposed process trains a multistage centrifugal blower with a five-stage reciprocating compressor, delivering CO 2 to the injection wellhead at up to 1,500 psig. Triethylene glycol (TEG) dehydration, applied after the fourth compression stage, reduces water content to a target of 10 lb/MMscf, well within the 30 lb/MMscf injection limit. Beyond dehydration, no additional treatment is required; trace impurities including oxygen and nitrogen will remain in the injected stream. Key design considerations include the absence of spare cooling tower capacity at the site, necessitating new cooling infrastructure, and the need for a new electrical substation to support large motor loads. The facility is designed for continuous, largely unattended operation, monitored around the clock by existing OEE operations staff. This document serves as the foundational reference for all subsequent detailed engineering activities associated with the OEE CO 2 injection facility.

01 COAL, LIGNITE, AND PEAT↗

Developing a one-step, acid-free microbial process to recover and separate REE (CRADA Final Report)

The project aimed to develop and test microbial strains for increased bioleaching, bioconcentration, and/or bioaccumulation of REEs, and for enhanced separation of REEs from other metals and of individual REEs from one another. The team secured and tested unconventional REE feedstocks to evaluate strains/processes for bioleaching, bioconcentration, and bioaccumulation, including but not limited to wastewater, geothermal brines, red mud, and electronic waste. purified and chemically analyzed REE granules from the microbial platform; determined metabolite and biosynthetic impacts of growth with REEs and the composition of extracellular material(s) produced by platform strains; optimized the growth process with alternative carbon feedstocks to reduce the cost of the fermentation process; and scaled the process from 1 mL to 10 L.

36 MATERIALS SCIENCE↗

Process development and scale-up of value added molecules (CRADA Final Report)

In this project, a genetically modified Saccharomyces cerevisiae was used to produce Histamine via fermentation of dextrose. Zymergen provided a tech transfer document based on their 1 L studies, where they observed a titer of 0.9 g/L with this strain. The ABPDU staff performed two bench scale campaigns, with four 2 L reactors in each campaign, to carry out tech transfer and process optimization studies. Optimization was pursued by varying initial glucose concentration, feed initiation conditions, feed strategies, vitamin supplementation, inoculum size, etc. We chose a 2 L process condition, with 4% inoculum volume, continuous feed initiated after complete ethanol depletion, that yielded the most promising titers (2.41 g/L) for scale up to 300 L.

60 APPLIED LIFE SCIENCES↗

Metabacillus indicus EGFCL74

Chromosomal DNA of bacterium isolated from fermented cider (originally named m74) was sequenced on an Illumina NovaSeq platform 2x150bp. Raw paired end reads were trimmed and processed with BBDUK v39.01. Trimmed FASTQ files were uploaded to the KBase narrative. Within KBase, the paired-end reads were assembled using SPAdes v3.15.3 (output data file m74_SPAdes.Assembly). Completeness was evaluated with CheckM v1.0.18. The assembled genome was annotated using RAStk v1.073 (output data file m74_RAStkgenomeassembly). Comparison of the isolate genome to other available genomes was performed two ways. The InsertGenomeIntoSpeciesTree function placed the isolate in the same clade as Bacillus indicus (later renamed Metabacillus indicus). FastANI was then used to compare the average nucleotide identity of isolate (m74) to 3 available strains of Metabacillus indicus (ASM70993v2, ASM70875v2, 4-1317).

59 BASIC BIOLOGICAL SCIENCES↗

Integrative Modeling and Analysis of Fungal Central Carbon Metabolism

Over a thousand fungal genomes have been sequenced, yet manually curated genome-scale metabolic models (GEMs) are available for only a limited number of species. Moreover, these models have often been developed independently, leading to inconsistencies in namespaces, compartment definitions, and pathway representations that hinder comparative analysis, the systematic reuse of prior curation efforts, and the integration of consolidated metabolic knowledge. Here, we present the Consolidated Fungal Core Metabolism Model (CFCMM), constructed by integrating thirteen published fungal models spanning Ascomycota, Mucoromycota, and both Crabtree-positive and Crabtree-negative yeasts. We harmonized metabolites and reactions into a non-redundant shared ModelSEED ontological space, standardized compartmentalization, and refined gene–protein–reaction (GPR) rules. Using pathway-level visualization and systematic gap detection, we further improved the integrated network through literature-guided curation to correct stoichiometry, stereospecificity, and pathway architecture. Orthologous protein family reconstruction and functional annotation workflows were used to validate and inform GPR associations, with particular emphasis on ambiguous enzyme superfamilies and membrane-associated components. Using the resulting CFCMM, we built high-quality central carbon core models for each fungus and performed flux balance analysis to quantify ATP-yield variation under aerobic and anaerobic conditions, explicitly evaluating scenarios driven by differences in electron transport chain (ETC) composition. Simulations reproduced the expected fermentative yield of approximately 2 mmol ATP per mmol glucose under anaerobic conditions and separated the thirteen fungi into two bioenergetic groups under aerobic respiration based on Complex I status, with predicted yields of approximately 30 versus 22 mmol ATP per mmol glucose. Forcing flux through the alternative oxidase bypass further reduced ATP yields to approximately 12 and 4 mmol ATP per mmol glucose in Complex I-containing and Complex I-lacking fungi, respectively. Collectively, this work provides a manually curated, ModelSEED-consistent, and extensible fungal core metabolic template, deployed in DOE KBase as a resource for automated reconstruction of central carbon core models from any sequenced fungal genome. In addition, the CFCMM provides modular components for developing GEMs with more accurate energy predictions and enables robust comparative analyses of fungal bioenergetics and core metabolic diversity

59 BASIC BIOLOGICAL SCIENCES↗

Yield, chemistry, and TEA/LCA data for 84 switchgrass (Panicum virgatum L.) variants

Switchgrass yield, chemistry, technoeconomic assessment (TEA), and life cycle assessment (LCA) data referenced in the manuscript "Economics and Sustainability impacts of yield and composition variation in bioenergy crops: Panicum virgatum L.", Happs, et. al. (manuscript in revision at ACS Sustainable Chemistry & Engineering). These results are based on analysis of yield data generated from a set of switchgrass common garden experiments established by the Center for Bioenergy Innovation (CBI; https://cbi.ornl.gov/). These data include estimates of commercial-scale yields and characterization of biomass chemistry (fermentable carbohydrate fraction) for 84 different switchgrass variants from a genome-wide association study population, as well as estimates of minimum fuel selling price and various life-cycle footprint metrics for cellulosic ethanol produced from that switchgrass biomass.

switchgrass, biofuels↗

Higher_wood_density_lowers_feedstock_cost_and_has_minimal_impact_on_biomass_conversion_to_biofuels

Poplar and other woody feedstocks have the potential to provide up to 200 million tons of biomass per year that could be converted to liquid fuels. Most forestry strategies that aim at increasing biomass productivity per hectare rely on short rotation plantations of fast-growing varieties. The improvement of wood density as a key trait itself has largely been overlooked. We evaluated natural variation in wood density across a population of genetically diversePopulus trichocarpatrees grown in a common garden. Wood density varies greatly within this population but is heritable higher wood density was not systematically associated with reduced growth, challenging assumptions of a trade-off between wood density and biomass accumulation. Furthermore, denser wood led to significant improvements throughout the supply chain, including, lowering biomass production and transportation costs. Higher density not correlate to changes in biomass composition. Density did not impact bioconversion in the two feedstock-to-fuel pipelines tested (pretreatment by ionic liquids or soaking in aqueous ammonia, and fermentation to ethanol) on a representative subset of poplars. These findings highlight wood density as a promising breeding target for accelerating the development of high-yielding, conversion-efficient bioenergy crops and as an avenue for increasing land-use efficiency and reducing biomass transportation cost. This data set contain three datasets.

CBI↗

A taxogenomic view of the genus Torulaspora : an expansion from ten to twenty-two species

The yeast genus Torulaspora (subphylum Saccharomycotina, family Saccharomycetaceae) is mostly known from its type species, T. delbrueckii, a frequent colonizer of wine and sourdough bread fermentations. The genus currently contains 10 species that are typically found in various natural terrestrial environments in temperate and tropical climates. Here we employ taxogenomic analyses to investigate a large collection of Torulaspora strains obtained in multiple surveys we carried out in Asia, Australasia, North America, South America, and Europe, and to which we added several strains maintained in culture collections. Our analyses detected twelve novel species that are formally described here, thereby more than doubling the species diversity of Torulaspora. We also sketch a genotype-phenotype map for the genus and show how the complex relationship between key genes and the physiological traits they control both between and within species. This remarkable increase in the number of species in the genus Torulaspora highlights how limited the current inventory of fungal taxa is. It also shows how integrated taxogenomic approaches can foster the assessment of species circumscriptions in fungi.

59 BASIC BIOLOGICAL SCIENCES↗

Microbial Membranes: A Challenge for Bioenergy

The production of bioenergy and bioproducts from lignocellulosic biomass promises to contribute significantly to the establishment of a clean, green economy. However, obstacles remain to making this industry economically viable. Among these is achieving high production rates and titers of target molecules through fermentation: microorganisms used in bioprocesses are inhibited by the toxic effects of biomass pretreatment and biofuel solvents which degrade the overall function of biological membranes. Here, we review historical research on the structure of the plasma membrane before detailing some progress on understanding the effects of amphiphilic solvents relevant to biofuel production on microbial membranes at the nanoscale.

09 BIOMASS FUELS↗

Preservation of Microalgae, Lignocellulosic Biomass Blends by Ensiling to Enable Consistent Year-Round Feedstock Supply for Thermochemical Conversion to Biofuels

Seasonal variation in microalgae productivity is a significant barrier to economical production of algae biofuels and chemicals. Summer production can be 3–5 times higher than in the winter resulting in uneven feedstock supplies at algae biorefineries. A portion of the summer production must be preserved for conversion in the winter in order to maintain a biorefinery running at capacity. Ensiling, a preservation process that utilizes lactic acid fermentation to limit microbial degradation, has been demonstrated to successfully stabilize algae biomass (20% solids) and algae-lignocellulosic blends (40% algae-60% lignocellulosic biomass, dry basis) for over 6 months, resulting in fuel production cost savings with fewer emissions. Preservation of algae as blends could be beneficial to biorefineries that utilize thermochemical approaches to fuel production as co-processing of algae and lignocellulosic biomass has been observed to enhance biocrude yield and improve oil quality. This study conducts a resource assessment of biomass residues in the southern United States to identify materials available during peak algae productivity and in sufficient quantity to meet the algae storage needs of an algae biofuel industry. Eight feedstocks met the quantity threshold but only three, distillers grains, haylage, and yard waste, were also available in season. Storage experiments utilizing both freshwater and marine strains of microalgae – Scenedesmus acutus, Chlorella vulgaris, Chlorella zofingiensis, Nannochloropsis gaditana, and Porphyridium purpureum – and yard waste were conducted for 30 days. Storage losses were less than 10% in all but one case, and the pH of all but one blend was reduced to less than 4.7, indicating that yard waste is a suitable feedstock for blending with algae prior to storage. To better understand whether the benefits to conversion realized by processing blends might be affected by storage, elemental analysis and bomb calorimetry of pre- and post-storage algae-yard waste blends were conducted to characterize changes occurring during storage. Storing algae biomass as blends with lignocellulosic biomass could be an effective method of mitigating seasonal variability in algae biomass production while retaining the synergistic effect of co-processing algae blends in thermochemical conversion.

09 BIOMASS FUELS↗

Biofilm mitigation in hybrid chemical-biological upcycling of waste polymers

Accumulation of plastic waste in the environment is a serious global issue. To deal with this, there is a need for improved and more efficient methods for plastic waste recycling. One approach is to depolymerize plastic using pyrolysis or chemical deconstruction followed by microbial-upcycling of the monomers into more valuable products. Microbial consortia may be able to increase stability in response to process perturbations and adapt to diverse carbon sources, but may be more likely to form biofilms that foul process equipment, increasing the challenge of harvesting the cell biomass. To better understand the relationship between bioprocess conditions, biofilm formation, and ecology within the bioreactor, in this study a previously-enriched microbial consortium (LS1_Calumet) was grown on (1) ammonium hydroxide-depolymerized polyethylene terephthalate (PET) monomers and (2) the pyrolysis products of polyethylene (PE) and polypropylene (PP). Bioreactor temperature, pH, agitation speed, and aeration were varied to determine the conditions that led to the highest production of planktonic biomass and minimal formation of biofilm. The community makeup and diversity in the planktonic and biofilm states were evaluated using 16S rRNA gene amplicon sequencing. Results showed that there was very little microbial growth on the liquid product from pyrolysis under all fermentation conditions. When grown on the chemically-deconstructed PET the highest cell density (0.69 g/L) with minimal biofilm formation was produced at 30°C, pH 7, 100 rpm agitation, and 10 sL/hr airflow. Results from 16S rRNAsequencing showed that the planktonic phase had higher observed diversity than the biofilm, and that Rhodococcus, Paracoccus, and Chelatococcus were the most abundant genera for all process conditions. Biofilm formation by Rhodococcus sp. And Paracoccus sp. Isolates was typically lower than the full microbial community and varied based on the carbon source. Ultimately, the results indicate that biofilm formation within the bioreactor can be significantly reduced by optimizing process conditions and using pure cultures or a less diverse community, while maintaining high biomass productivity. The results of this study provide insight into methods for upcycling plastic waste and how process conditions can be used to control the formation of biofilm in bioreactors.

36 MATERIALS SCIENCE↗

Synergistic Improvement of Carbohydrate and Lignin Processability by Biomimicking Biomass Processing

The sustainability and economic feasibility of modern biorefinery depend on the efficient processing of both carbohydrate and lignin fractions for value-added products. By mimicking the biomass degradation process in white-rote fungi, a tailored two-step fractionation process was developed to maximize the sugar release from switchgrass biomass and to optimize the lignin processability for bioconversion. Biomimicking biomass processing using Formic Acid: Fenton: Organosolv (F 2 O) and achieved high processability for both carbohydrate and lignin. Specifically, switchgrass pretreated by the F 2 O process had 99.6% of the theoretical yield for glucose release. The fractionated lignin was also readily processable by fermentation via Rhodococcus opacus PD630 with a lipid yield of 1.16 g/L. Scanning electron microscope analysis confirmed the fragmentation of switchgrass fiber and the cell wall deconstruction by the F 2 O process. 2D-HSQC NMR further revealed the cleavage of aryl ether linkages (β-O-4) in lignin components. These results revealed the mechanisms for efficient sugar release and lignin bioconversion. The F 2 O process demonstrated effective mimicking of natural biomass utilization system and paved a new path for improving the lignin and carbohydrate processability in next generation lignocellulosic biorefinery.

09 BIOMASS FUELS↗

Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose

Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca ) fused with endocellulase Cel5A (from T. fusca ) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning -14 to +6. These enzymes were recombinantly expressed in E. coli , purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (~3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.

09 BIOMASS FUELS↗

A Streamlined Strategy for Biohydrogen Production with Halanaerobium hydrogeniformans , an Alkaliphilic Bacterium

Biofuels are anticipated to enable a shift from fossil fuels for renewable transportation and manufacturing fuels, with biohydrogen considered attractive since it could offer the largest reduction of global carbon budgets. Currently, lignocellulosic biohydrogen production remains inefficient with pretreatments that are heavily fossil fuel-dependent. However, bacteria using alkali-treated biomass could streamline biofuel production while reducing costs and fossil fuel needs. An alkaliphilic bacterium, Halanaerobium hydrogeniformans, is described that is capable of biohydrogen production at levels rivaling neutrophilic strains, but at pH 11 and hypersaline conditions. H. hydrogeniformans ferments a variety of 5- and 6-carbon sugars derived from hemicellulose and cellulose including cellobiose, and forms the end products hydrogen, acetate, and formate. Further, it can also produce biohydrogen from switchgrass and straw pretreated at temperatures far lower than any previously reported and in solutions compatible with growth. Hence, this bacterium can potentially increase the efficiency and efficacy of biohydrogen production from renewable biomass resources.

09 BIOMASS FUELS↗

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria . We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 10 5 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria , representing a candidate order ( Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O 2 , suggesting a microaerophilic lifestyle.

59 BASIC BIOLOGICAL SCIENCES↗

Identification and genetic characterization of mitochondrial citrate transporters in Aspergillus niger

Aspergillus niger is a major cell factory for citric acid production, and the process of citrate export from mitochondria to cytoplasm is predicted to be one of rate-limiting steps in citric acid accumulation. Currently, the mitochondrial citrate transporters (Ctps) in A. niger are not fully characterized. Here, six putative Ctp encoding genes ( ctpA to ctpF ) were identified based on their homology with a mitochondrial citrate transporter Sc Ctp1 from Saccharomyces cerevisiae . Disruption of individual ctpA to ctpF caused varying degrees of decline in citric acid accumulation at different fermentation stages, whereas a mutant strain S1696 with disruption of all six ctp s showed complete loss of citiric acid production. S1696 also exhibited delayed growth, reduced conidia formation, and decreased pigmentogenesis. Exogenous addition of citrate partially restored the conidia formation and pigmentogenesis in S1696 mutant. Reintroduction of individual ctp s ( ctpA to ctpF ) into S1696 at the amyA locus showed that ctpA , ctpB , and ctpD restored the citric acid titers to 88.5, 93.8, and 94.6% of the parent strain, respectively. Additionally, the formation of conidia and pigment production was partially restored after reintroduction of ctpA , ctpB , or ctpD . Overexpression of respective ctpA , ctpB , and ctpD in the parent strain resulted in increases in citric acid accumulation by 32.8, 19.3, and 24.2%, respectively. These results demonstrate that CtpA, CtpB, and CtpD play important roles in citric acid transport across the mitochondrial membrane and function in a redundant manner. Enhancement of citric acid transport process can serve as a target for boosting citric acid accumulation in A. niger .

Cao, Wei↗

Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions

The cyanobacterium Synechocystis sp.PCC 6803 possesses a bidirectional NiFe-hydrogenase, HoxEFUYH. It functions to produce hydrogen under dark, fermentative conditions and photoproduces hydrogen when dark-adapted cells are illuminated. Unexpectedly, we found that the deletion of the large subunit of the hydrogenase (HoxH) in Synechocystis leads to an inability to grow on arginine and glucose under continuous light in the presence of oxygen. This is surprising, as the hydrogenase is an oxygen-sensitive enzyme. In wild-type (WT) cells, thylakoid membranes largely disappeared, cyanophycin accumulated, and the plastoquinone (PQ) pool was highly reduced, whereas ΔhoxH cells entered a dormant-like state and neither consumed glucose nor arginine at comparable rates to the WT. Hydrogen production was not traceable in the WT under these conditions. We tested and could show that the hydrogenase does not work as an oxidase on arginine and glucose but has an impact on the redox states of photosynthetic complexes in the presence of oxygen. It acts as an electron valve as an immediate response to the supply of arginine and glucose but supports the input of electrons from arginine and glucose oxidation into the photosynthetic electron chain in the long run, possibly via the NDH-1 complex. Despite the data presented in this study, the latter scenario requires further proof. The exact role of the hydrogenase in the presence of arginine and glucose remains unresolved. In addition, a unique feature of the hydrogenase is its ability to shift electrons between NAD(H), NADP(H), ferredoxin, and flavodoxin, which was recently shown in vitro and might be required for fine-tuning. Taken together, our data show that Synechocystis> depends on the hydrogenase to metabolize organic carbon and nitrogen in the presence of oxygen, which might be an explanation for its prevalence in aerobic cyanobacteria.

59 BASIC BIOLOGICAL SCIENCES↗

Editorial: Microorganisms for Consolidated 2nd Generation Biorefining

In the last few decades, lignocellulosic biomass has attracted substantial interest as a feedstock for fermentative production of fuels and other commodity chemicals due to its wide availability and low cost. However, lignocellulose has innate complexity and recalcitrance to biodegradation. In natural environments, effective plant biomass decay is obtained by synergistic activity of complex microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗