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At least 235 records · Page 13

Co-expression of a β-d-xylosidase from Thermotoga maritima and a Family 10 xylanase from A. cellulolyticus significantly improves the xylan degrading activity of the Caldicellulosiruptor bescii exoproteome

Caldicellulosiruptor species are hyperthermophilic, Gram-positive, anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals and materials from plant biomass. Xylooligomers such as xylobiose and xylotriose that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose, are present in C. bescii fermentations after 90 h incubation and removal or breakdown of these types of xylooligomers is crucial to achieve high conversion of plant biomass to product. In previous studies the addition of exogenous β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here we report that the co-expression of a thermal stable β-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogeneous xylan.

59 BASIC BIOLOGICAL SCIENCES↗

From the bench to the reactor: engineered filamentous fungi for biochemical and biomaterial production

Filamentous fungi can convert a wide variety of naturally occurring chemical compounds, including organic biomass and waste streams, into a range of products. They have long been used for industrial organic acid production and food preparation. In this review, we will discuss production of products such as organic acids, lipids, small molecules, enzymes, materials, and foods, and highlight advances in metabolic and protein engineering, including CRISPR-Cas9-mediated strain improvements. We discuss to what extent these products are already being made on a commercial scale, as well as what is still required to make certain promising concepts industrially and commercially relevant. Despite significant progress, the systematic application of synthetic biology to filamentous fungi remains in its infancy, with many opportunities for discovery and innovation as new strains and genetic tools are developed. The integration of fungal biotechnology into circular and bio-based economies promises to address critical challenges in waste management, resource sustainability, and the development of new materials for terrestrial and extraterrestrial applications, but requires further developments in genetic engineering and process design.

09 BIOMASS FUELS↗

Data for Hydrothermal Pretreatment for Valorization of Genetically Engineered Bioenergy Crop for Lipid and Cellulosic Sugar Recovery

Lipids accumulated in the vegetative tissues of cellulosic feedstocks can be a potential raw material for biodiesel and bioethanol production. In this work, bagasse of genetically engineered sorghum was subjected to liquid hot-water pretreatment at 170, 180, and 190 °C for different reaction time. Under the optimal pretreatment condition (170 °C, 20 min), the residue was enriched in glucan (57.39 ± 2.63 % w/w) and xylan (13.38 ± 0.49 % w/w). The total lipid content of the pretreated residue was 6.81% w/w, similar to that observed in untreated bagasse (6.30% w/w). Pretreatment improved the enzymatic digestibility of bagasse, allowing a recovery of 79% w/w and 86% w/w of glucose and xylose, respectively. The pretreatment and enzymatic saccharification resulted in a 2-fold increase in total lipid in enzymatic residue compared to the original bagasse. Thus, pretreatment and enzymatic hydrolysis enabled high sugar recovery while concentrating triglycerides and free fatty acids in the residue.

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Data for "Integrated Green Biorefinery for the Production of Anthocyanins, Fermentable Sugars, and High Pure Lignin from Miscanthus × giganteus "

Miscanthus x giganteus (Mxg) is a promising perennial crop for producing natural colorants, renewable fuels, and bioproducts. However, natural recalcitrance and high pretreatment cost are major barriers to their complete conversion. In this study, a green processing method has been investigated for efficient recovery of natural pigments (anthocyanins), fermentable sugars, and pure lignin from Mxg genotypes using choline chloride-based natural deep eutectic solvents (NADES) systems. Interestingly, choline chloride: lactic acid (ChCl: LA) NADES-processed biomass resulted in 67.8 ± 2.1 μg g−1 of anthocyanins from dry biomass. A maximum of 87.4%–94.1% glucose yield was achieved after enzymatic saccharification. The effective extraction of lignin with high purity with higher β-aryl ether (βO4) bonds from advanced crops is crucial for lignin valorization. Notably, highly pure lignin (≈93.4% ± 1.4%) is achieved after low-temperature NADES pretreatment while retaining lignin’s native structure. 31P nuclear magnetic resonance demonstrated that total phenolics for ChCl: LA-lignin resulted in 1.20 mmol g−1 hydroxyls. The relative monolignol composition of syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) is 19.0, 65.7, and 14.3%, respectively, as evidenced by heteronuclear single quantum coherence analysis. This study provides a novel approach for obtaining high-purity lignin for catalytic depolymerization for oligomers and bifunctional monoaromatics production and leverages current cellulosic biorefinery technologies.

biomass analytics↗

Data for Pilot-Scale Processing of Miscanthus x giganteus for Recovery of Anthocyanins Integrated with Production of Microbial Lipids and Lignin-Rich Residue

Chemical-free hydrothermal pretreatment of Miscanthus x giganteus (Mxg) at the lab scale using high liquid-to-solid ratios resulted in the recovery of anthocyanins and enhanced enzymatic digestibility of residual biomass. In this study, the process is scaled up by using a continuous hydrothermal pretreatment reactor operated at a low liquid-to-solid ratio (50 % w/w solids) as an important step towards commercialization. Anthocyanin yield was 70 % w/w at the pilot scale (50 kg of Mxg), compared to the 94 % w/w yield achieved at the lab scale (0.5 g of Mxg). The pretreated biomass was subsequently refined mechanically using a disc mill to increase the accessibility of cellulose by cellulases. Enzymatic saccharification of the pretreated and disc-milled residue yielded 238 g/L sugar concentration by operating in fed-batch mode at 50 % w/v solids content. Two strains of Rhodosporidium toruloides were evaluated for converting the hydrolysate sugars into microbial lipids, and strain Y-6987 had the highest lipid titer (11.0 g/L). Further, the residue left after enzymatic saccharification was determined to be enriched 1.7-fold in the lignin content. This lignin-rich residue has value as a feedstock for the production of sustainable aviation fuel precursors and other high-value lignin-based chemicals. Hence the proposed biorefinery based on Mxg creates an opportunity for generating revenue from multiple high-value products. As the demand for biofuels and biobased products is rising, the biorefinery products from Mxg would create a niche in the industrial sector.

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Data for Variability in Structural Carbohydrates, Lipid Composition, and Cellulosic Sugar Production from Industrial Hemp Varieties

The aim of this study was to determine carbohydrate recovery from hemp for ethanol production and quantify biodiesel from TAG (triacylglycerol) present in hemp. The structural composition of five different hemp varieties (Seward County-SC, York County-YC, Loup County-LC, 19 m96136-19 m, and CBD Hemp-CBD) were analyzed. Concentration of glucan and xylan ranged between 32.63 to 44.52% and 10.62 to 15.48% respectively. The biomass was then pretreated with Liquid hot water followed by disk milling and then hydrolyzed enzymatically to yield monomeric sugars. High glucose (63-85%) and xylose (73-88%) recovery was achieved. Lipids were extracted from hemp using hexane and isopropanol and then transesterified to produce biodiesel. Approximately, 50% of total fatty acids in SC, LC, and CBD hemp were linoleic acid. Palmitic acid was present between 32 to 50% in varieties YC and 19 m. Highest TAG concentration at 25% of total lipids was observed in CBD hemp. The analysis on lipid composition and high sugar recovery demonstrates hemp as a potential bioenergy crop for ethanol and biodiesel coproduction.

Biomass Analytics↗

Data from Technical and Economic Feasibility of an Integrated Ethanol and Anthocyanin Coproduction Process Using Purple Corn Stover

The coproduction of high-value anthocyanin extract in the cellulosic ethanol process would diversify the co-product market, increase revenue, and potentially improve the economics of the process. The high anthocyanin concentration in the cob and structural carbohydrates in residual stover make purple corn stover an attractive source for anthocyanin and ethanol coproduction. This study aimed to develop simulation models for processes integrating ethanol production and anthocyanin extraction using purple corn stover, to evaluate their techno-economic feasibility, and to compare their performance with the conventional ethanol production process using corn stover. The annual ethanol production for plants processing 2000 MT dry feedstock / day was 148.6 million L/year for the integrated processes compared with 222.6 million L/year for the conventional process. Anthocyanin production in the modified processes using dilute acid-based and water-based anthocyanin extraction processes was 1779 and 1099 MT/year, respectively. Capital investments for the integrated processes ($448.1 to $443.8 million) were higher than the conventional process ($371.9 million). Due to high revenue from anthocyanin extract, the ethanol production cost for the integrated process using acid-based anthocyanin extraction ($0.36/L) was 34.5% lower than conventional ethanol production ($0.55/L). The ethanol production cost for the integrated process using water-based anthocyanin extraction ($0.68/L) was higher than conventional ethanol production due to low ethanol and anthocyanin yields. The minimum ethanol selling price for the integrated process using acid-based anthocyanin extraction ($0.65/L) was also lower than the conventional process ($0.72/L), indicating an improvement in economic performance.

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Data from High Solids Loading Biorefinery for the Production of Cellulosic Sugars from Bioenergy Sorghum

A novel process applying high solids loading in chemical-free pretreatment and enzymatic hydrolysis was developed to produce sugars from bioenergy sorghum. Hydrothermal pretreatment with 50% solids loading was performed in a pilot scale continuous reactor followed by disc refining. Sugars were extracted from the enzymatic hydrolysis at 10% to 50% solids content using fed-batch operations. Three surfactants (Tween 80, PEG 4000, and PEG 6000) were evaluated to increase sugar yields. Hydrolysis using 2% PEG 4000 had the highest sugar yields. Glucose concentrations of 105, 130, and 147 g/L were obtained from the reaction at 30%, 40%, and 50% solids content, respectively. The maximum sugar concentration of the hydrolysate, including glucose and xylose, obtained was 232 g/L. Additionally, the glucose recovery (73.14%) was increased compared to that of the batch reaction (52.74%) by using two-stage enzymatic hydrolysis combined with fed-batch operation at 50% w/v solids content.

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Data for Harnessing the Potential of Oilcane Waste Mud for Recovering Biobased Waxes

Oilcane is an engineered sugarcane with the ability to hyper-accumulate vegetative lipids. It is processed to obtain juice and bagasse as a potential substrate for the production of biofuels and biochemicals. The juice comprises solid particles that are separated as waste mud before the fermentation of the juice. In this study, the oilcane waste mud (OWM) generated from 1000 liters of oilcane juice was quantified and evaluated as a potential resource for recovering biobased waxes. Hexane and ethyl acetate were evaluated as two different solvents for extracting waxes from OWM followed by its purification using acetone. The extracted biobased wax samples were characterized for their chemical and thermal profiles which were then compared with commercial natural waxes. Detailed mass balance shows that 53.6 ± 2.6 kg (dry basis) of solid OWM gets generated upon processing 1000 L (~1068 kg) of oilcane juice. Hexane and ethyl acetate led to a crude wax yield of 25.6 ± 0.2% and 16.6 ± 0.4% (wt/wt, dry basis) respectively from OWM at the end of 8 h. The relative purification of the wax samples was reported in the range of 58%–65% (wt/wt). The purified OWM wax has a melting point of 74.7°C. The waste mud was valorized as a source of biobased waxes with characteristic chemical and thermal profiles comparable to commercial natural waxes (carnauba and beeswax). Considering the decline in the supply of petroleum wax in the future coupled with the switch to “greener” alternative products by consumers, OWM could be a valuable source of natural wax in the industrial sector reducing the dependence on petroleum waxes. Eventually, recovering biobased wax as a co-product from OWM would bring in an additional stream of revenue leading to the development of a zero-waste biorefinery based on bioenergy crops.

Biomass Analytics↗

Data from Biodiesel Production from Engineered Sugarcane Lipids under Uncertain Feedstock Compositions: Process Design and Techno-Economic Analysis

In this study, different process schemes were designed and evaluated for biodiesel production from engineered cane lipids with uncertain fatty acid compositions. Four different process schemes were compared under (i) thermal glycerolysis and (ii) enzymatic glycerolysis approaches. These schemes were based on the biodiesel yield and economic indicators such as the net present value (NPV) and the minimum selling price (MSP) of biodiesel. A scheme with polar lipid separation under thermal glycerolysis resulted in the maximum NPV ($96.5 million) and minimum MSP ($1107/ton biodiesel), respectively. Through local sensitivity analysis, it was concluded that the cane lipid percentage is the most significant factor influencing process economics. A conjoint analysis of the lipid procurement price and cane lipid percent suggested that 15% cane lipids with a low lipid procurement price ($0.536/kg) results in a positive NPV. When the cane lipid price is higher (>$0.80/kg), a 20% lipid content should be considered to achieve a positive NPV. At 20% cane lipids, the worst-case and best-case scenarios were evaluated by analyzing the interplay of the three most important parameters, The best-case scenario revealed that the minimum NPV under any process scheme could yield more than $100 million (or MSP: $0.80/L), and the worst-case analysis showed that losses incurred by the plant could be as high as $80 million (MSP: $1.36/L). A Monte Carlo simulation indicated that there is a 70% chance of the plant being profitable (NPV > 0).

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Data from Response Surface Methodology Guided Adsorption and Recovery of Free Fatty Acids from Oil Using Resin

The presence of free fatty acids interferes with the conversion of plant oils to biodiesel. Four strong and weak base resins were evaluated for the removal of free fatty acids (FFA) from oil. Amberlite FPA 51 showed the highest adsorption capacity of FFA. A resin concentration above 3% could enable a higher percentage FFA adsorption. The adsorption process fitted a pseudo-first-order kinetic model and achieved equilibrium in approximately 8 h. A full factorial design was used to optimize the resin and FFA concentrations at a fixed temperature (40° C). A ratio of resin to fatty acid concentrations above 1.875 was sufficient for 70% adsorption and the amount adsorbed continued to increase with further added resin. A two-step washing of resin using hexane and ethanol recovered approximately 67.55% ± 4.05% of the initially added fatty acid. The resin that was used was regenerated with 5% NaOH and re-used for a minimum of three consecutive cycles. However, the adsorption capacity diminished to 75% of the initial cycle in cycles 2 and 3. Thus, the work presents a resin-based process for deacidification of oil to reduce fatty acid content of oil for biodiesel production.

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Data for Impact of Non-Irrigation on 1G and 2G Bioethanol Potential of Oilcane Feedstock: A Field to Fuel Pipeline Study

This study evaluates the bioethanol potential in response to irrigation (IR) and non-irrigation (NIR) of oilcane (OC) during a seasonal drought prior harvest. The juice was extracted through mechanical pressing of stems and fermented by Ethanol Red® yeast to produce first-generation bioethanol. Hydrothermal pretreatment followed by enzymatic hydrolysis of bagasse was performed to produce monomeric sugars from structural carbohydrates. The hydrolysates were fermented with engineered yeast for second-generation bioethanol production. The irrigated oilcane juice (276.3 ± 8.9 g/L) constitutes higher sugar concentrations than non-irrigated oilcane juice (236.5 ± 2.2 g/L). The enzymatic hydrolysis of IR-OC and NIR-OC pretreated bagasse yielded similar concentrations of 247.5 ± 2.22 and 249.7 ± 4.98 g/L fermentable sugars. Industry-relevant bioethanol titers of ≥99 g/L and ≥75 g/L were achieved from juice and hydrolysates, respectively. Therefore, the non-irrigation regime did not impact the 1G and 2G bioethanol titers. However, the overall bioethanol yield can be lower due to the reduction of stem yield (8 %) per hectare.

Biomass Analytics↗

Data for Scale-up of Microbial Lipid and Bioethanol Production from Oilcane

Microbial oils are a sustainable biomass-derived substitute for liquid fuels and vegetable oils. Oilcane, an engineered sugarcane with superior feedstock characteristics for biodiesel production, is a promising candidate for bioconversion. This study describes the processing of oilcane stems into juice and hydrothermally pretreated lignocellulosic hydrolysate and their valorization to ethanol and microbial oil using Saccharomyces cerevisiae and engineered Rhodosporidium toruloides strains, respectively. A bioethanol titer of 106 g/L was obtained from S. cerevisiae grown on oilcane juice in a 3 L fermenter, and a lipid titer of 8.8 g/L was obtained from R. toruloides grown on oilcane hydrolysate in a 75 L fermenter. Oil was extracted from the R. toruloides cells using supercritical CO2, and the observed fatty acid profile was consistent with previous studies on this strain. These results demonstrate the feasibility of pilot-scale lipid production from oilcane hydrolysate as part of an integrated bioconversion strategy.

Bioproducts↗

Data for "Evaluating the industrial potential of emerging biomass pretreatment technologies in bioethanol production and lipid recovery from transgenic sugarcane"

The selection of pretreatment methods is critical to achieving high product yields during bioconversion of lignocellulosic biomass. Hydrothermal, soaking-in-aqueous ammonia, and ionic liquid pretreatment methods are viable candidates for minimizing sugar decomposition, permitting the effective hydrolysis of structural carbohydrates, and producing a fermentable substrate suitable for achieving industrial ethanol titers and yields. In this study, the effect of these three pretreatment methods on non-modified sugarcane cultivar CP88-1762 and two transgenic lipid-accumulating sugarcane lines, oilcane 1565 and oilcane 1566, were investigated and compared in terms of lipid recovery, sugar yield, and ethanol yields within the lignocellulosic biomass conversion pipeline. Fed-batch enzymatic hydrolysis at high solid loading yielded hydrolysates capable of supporting industrial bioethanol titers across all conditions. The highest sugar yields were obtained on ammonia-pretreated biomass hydrolysate (253.73 g L−1), followed by hydrothermally pretreated hydrolysate (213.10 g L−1) and ionic liquid-pretreated hydrolysate (154.20 g L−1). Commercially viable ethanol titers of 100.62 g L−1, 64.47 g L−1, and 52.95 g L−1 were achieved from ammonia, hydrothermal, and ionic liquid pretreated hydrolysate with the corresponding ethanol productivities of 2.08 g L−1 h−1, 0.53 g L−1 h−1, and 0.36 g L−1 h−1. The lower acetic acid concentration in ammonia-pretreated hydrolysate may have enhanced its fermentability relative to the hydrothermal pretreatment condition, as indicated by the differences in ethanol titer and productivity. Lower sugar yields and ethanol productivities under the ionic liquid conditions likely resulted from the inhibitory effect of cholinium lysinate. Oilcane 1565 and oilcane 1566 bagasse accumulated over 16- and 3 times higher lipids than the non-modified sugarcane CP88-1762. The total fatty acid content in the oilcane samples was reduced in ammonia and ionic liquid-pretreated bagasse relative to the hydrothermal pretreatment condition. While all pretreatment techniques tested are industrially viable, the observed differences in titer, productivity, and lipid content indicate that careful selection and validation of upstream processing methods can contribute to improved economic and environmental outcomes.

biomass analytics↗

Data for Conversion of High-Solids Hydrothermally Pretreated Bioenergy Sorghum to Lipids and Ethanol Using Yeast Cultures

Glucose and xylose are the major sugars present in cellulosic hydrolysates. The cellulosic sugars can be used for the production of platform chemicals. In this study, productions of lipid and ethanol by yeasts were compared for concentrated bioenergy sorghum syrup. Bioenergy sorghum was hydrothermally pretreated at 50% w/w solids in a continuous industrial reactor and sequentially mechanically refined using a burr mill to improve biomass accessibility for hydrolysis. Fed-batch enzymatic hydrolysis was conducted with 50% w/v solids loading and cellulase cocktail (50 FPU/g biomass) to achieve 230 g/L sugar concentration. Various strains of Rhodosporidium toruloides were evaluated for converting sugars into lipids, and strain Y-6987 had the highest lipid titer (9.2 g/L). The lipid titer was improved to 19.0 g/L by implementing a two-stage culture scheme, where the first stage was optimized for yeast growth and the second for lipid production. For ethanol production, the engineered Saccharomyces cerevisiae SR8ΔADH6 was used to coferment glucose and xylose. Ethanol fermentation was optimized for media nutrients (YP, YNB/urea, and urea), cellulosic sugar concentration, and sulfite conditioning to maximize the ethanol concentration from sorghum syrups. Fermentation of 70% v/v concentrated hydrolysate conditioned with sulfite produces 50.1 g/L ethanol from 141 g/L of sugars.

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Production of Bioproducts from Electrochemically-Generated C1 Intermediates

The overarching objective of the project was to develop an integrated process that converts carbon dioxide (CO 2 ) into the platform chemical isopropanol (IPA) using a combination of two key technologies: CO 2 electrolysis and gas fermentation. The final goal was to demonstrate the full process at bench scale with a minimum carbon conversion efficiency of 37% and to evaluate the economics and environmental impact of scaling up the electrochemical-biochemical process.

30 DIRECT ENERGY CONVERSION↗

2023 Project Peer Review Report

The Bioenergy Technologies Office (BETO) within the U.S. Department of Energy’s Office of Energy Efficiency and Renewable Energy supports the research, development, and demonstration (RD&D) of technologies aimed at mobilizing domestic renewable carbon resources for the reduction of greenhouse gas emissions across the U.S. economy. BETO systematically prioritizes RD&D into technology opportunities across a range of emerging scientific breakthroughs and technology readiness levels in the subprogram areas illustrated in Figure 1. This approach supports a diverse portfolio while developing the most promising and widely applicable technologies, testing technologies as integrated processes, and demonstrating integrated processes to support scale-up. These technologies will use a broad variety of renewable carbon resources to produce increasing volumes of biofuels and bioproducts. More information on BETO’s mission, goals, and strategic approaches can be found in the Bioenergy Technologies Office Multi-Year Program Plan. The biennial Peer Review process enables external stakeholders to provide feedback on the responsible use of taxpayer funding and develop recommendations for the most efficient and effective ways to accelerate the development of a bioenergy industry. This report includes the results of the Project Peer Review meeting held on April 3–7, 2023, in Denver, Colorado.

09 BIOMASS FUELS↗

HT Model Dataset

This website contains the dataset that was used for writing the manuscript "HT Model: Using the Molecular Transformer for predicting hydrotreating reactions" (PNNL-SA-186589) The dataset includes a collection of hydrotreating reactions compiled from 41 peer-reviewed literature sources. These sources contain experimental data related to hydrotreating reactions. These reactions involve the reaction of chemical compounds with hydrogen gas in the presence of a catalyst to remove heteroatoms or to convert specific functional groups. The dataset contains reactions both with and without reaction conditions. Reaction conditions refer to the specific parameters under which the reaction takes place, such as temperature and pressure. For each reaction, the dataset includes both SMILES and SELFIES representations. SMILES (Simplified Molecular Input Line Entry System) and SELFIES (SELF-referencIng Embedded Strings) are two popular notations used to represent chemical structures in a compact and standardized format. The dataset was created with the aim of training a predictive model, specifically using the Molecular Transformer architecture.

bioprocessing, hydrotreating, deep learning algori↗