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DOE OSTI2026

Convergent evolution of aerobic fermentation through divergent mechanisms acting on the same key glycolytic genes

Expression profiling by high throughput sequencing The model yeast Saccharomyces cerevisiae has evolved aerobic fermentation in part through a whole genome duplication and subsequent retention of glycolytic genes. To identify independent yeast lineages with a high glycolytic rate phenotype we developed an assay measuring extracellular acidification rates (ECAR) to evaluate the glycolytic rates across diverse yeast species. This assay identified a novel group of yeasts in the genus Saturnispora with rapid glycolytic rates. Through comparative approaches, we found that several glycolytic genes encoding hexokinases and enzymes in lower glycolysis had higher expression and modifications in promoter sequences in rapid ECAR species. Intriguingly, many of the upregulated genes are the same genes that are duplicated in S. cerevisiae. When the transcription factor required for their activation was deleted, the mutants had a slow glycolytic rate and more respiratory phenotype. This work shows how the aerobic fermentation phenotype has convergently evolved in Saturnispora through transcriptional rewiring. This divergent mechanism nevertheless impacts the same glycolytic genes, which suggests that there are evolutionary constraints on how aerobic fermentation can arise.

Horianopoulos, Linda C. [Department of Food Scienc↗

DOE OSTI2020

System and method for extracting ethanol from a fermentation broth

The present invention is directed to a system and method for producing an organic compound using fermentation wherein multiple components of the system are recycled within the system. The system and method allow for extraction of a high concentration of the organic compound from the fermentation broth in a continuous system that allows recycling of the biomass, aqueous fermentation broth and extraction solvents. The system and method are particularly well adapted for producing and extracting ethanol.

09 BIOMASS FUELS↗

DOE OSTI2023

Methods for the conversion of fermentable sugars to ethanol

Methods and systems for the enhancement of bioproduct process yields. The present invention provides systems and methods for improvement of the carbon conversion efficiency by yeast of an organic substrate such as a fermentable sugar to a fermentation product such as ethanol. Exemplary yield-increasing/growth-inhibitory compounds include tyrosol (TyrOH), 2-phenylethanol (PheOH), and tryptophol (TrpOH). Immobilization of the yeast, such as in a porous bead or pellet, can further increase yield. Exemplary immobilization included immobilization in calcium alginate beads. The system and methods taught herein demonstrate that product yield such as ethanol yield can be improved by adding yeast yield-increasing/growth-inhibitory molecules to reduce cell growth of yeast species, such as S. cerevisiae , suggesting a strategy to improve the yield of ethanol and other yeast fermentation products by manipulating native biological control systems.

Reardon, Kenneth F.↗

DOE OSTI2024

System and method for extracting ethanol from a fermentation broth

Abbas, Charles↗

DOE OSTI2020

Achieving high hydrogen productivities of 20 L/L-day via microbial electrolysis of corn stover fermentation products

Microbial electrolysis cells have the potential to generate renewable hydrogen from underused waste streams, however current devices have not reached very large productivity targets using real waste products at any scale. This study used a waste from fermented corn stover known as corn stover fermentation product to reach performance metrics that could be commercialized, if adequately scaled. Average current densities in MECs with mature biofilms reached 17.9 +/- 1.6 A/m 2 at an organic loading rate of 30 grams of chemical oxygen demand per liter of anode volume per day (g/L-day), reaching a maximum current density of 27.2 +/- 2.9 A/m 2 . Hydrogen productivities reached 20.2 +/- 2.0 L of H 2 per L anode volume per day (L/L-day). These represent the highest current densities and highest hydrogen productivities using a complex feedstock in a microbial electrolysis cell. Organic acids and sugars present in untreated substrate were converted at high percentages in MECs, with most above 90% conversion, at organic loading rates of 10, 20, and 30 g/L-day. The effect of periodic high liquid flow rates through the anode on performance was assessed. These tests, called pulsing, showed that hydrogen productivities and current densities increased most dramatically as flow was pulsed every hour at a baseline flow rate of 0.3 mL/min. These productivities show promise for high performance systems, if adequate scale up can be achieved.

09 BIOMASS FUELS↗

DOE OSTI2025

Upstream considerations for gas fermentation processes

Gas fermentation enables the production of fuels, chemicals, and foods from gaseous carbon sources and could serve as a technology for valorizing carbon that may otherwise be emitted to the atmosphere. In this review, we focus on upstream feedstock considerations: the supply of carbon and the supply of electrical power. Electrical power serves a dual role, providing both process energy and biochemical redox potential (via hydrogen or reduced intermediates). We define gas fermentation as bioprocesses involving gaseous feedstocks metabolized by microbes, distinct from microbial electrosynthesis. Trends in CO 2 point sources and low-carbon electricity systems are analyzed, highlighting opportunities and challenges for future deployment. This review synthesizes current knowledge and identifies key R&D priorities for process integration at industrial scale.

09 BIOMASS FUELS↗

DOE OSTI2024

Volatile fatty acid extraction from fermentation broth using a hydrophobic ionic liquid and in situ enzymatic esterification

Efficient recovery of volatile fatty acids (VFAs) from fermentation broth is a challenge due to low VFA titers and thus limits the commercialization of VFA production using biological routes. Liquid–liquid extraction using hydrophobic ionic liquids (ILs) shows great promise for the extraction and esterification of hydrophilic VFAs. In this study, several ILs were evaluated to select a water-immiscible and efficient extraction solvent. The selected IL, trihexyltetradecyl phosphonium dibutylphosphate ([P 666,14 ][DBP]), gave a cumulative VFA extraction of around 842.8 mg per g IL. The predicted excess enthalpy (H E ) and logarithmic activity coefficients ln(γ) using the COSMO-RS model were validated with the experimentally obtained VFA recovery from fermentation broth. To understand the extraction mechanism of VFAs, quantum theory of atoms in molecules (QTAIM) and noncovalent interaction (NCI) were performed. The results suggest that long chain fatty acids exhibit strong van der Waals interaction with the DBP anion leading to higher VFA extraction. The enzymatic esterification of VFAs with ethanol in [P 666,14 ][DBP] was optimized using the Box–Behnken response surface design of experiment. Under the optimized conditions, up to 83.7% of hexanoic acid was converted to ethyl esters, while other shorter chain VFAs have lower conversion efficiency (38.3–63.2%).

09 BIOMASS FUELS↗

DOE OSTI2024

High temperatures and low soil moisture synergistically reduce switchgrass yields from marginal field sites and inhibit fermentation

‘Marginal lands’ are low productivity sites abandoned from agriculture for reasons such as low or high soil water content, challenging topography, or nutrient deficiency. To avoid competition with crop production, cellulosic bioenergy crops have been proposed for cultivation on marginal lands, however on these sites they may be more strongly affected by environmental stresses such as low soil water content. In this study we used rainout shelters to induce low soil moisture on marginal lands and determine the effect of soil water stress on switchgrass growth and the subsequent production of bioethanol. Five marginal land sites that span a latitudinal gradient in Michigan and Wisconsin were planted to switchgrass in 2013 and during the 2018–2021 growing seasons were exposed to reduced precipitation under rainout shelters in comparison to ambient precipitation. The effect of reduced precipitation was related to the environmental conditions at each site and biofuel production metrics (switchgrass biomass yields and composition and ethanol production). During the first year (2018), the rainout shelters were designed with 60% rain exclusion, which did not affect biomass yields compared to ambient conditions at any of the field sites, but decreased switchgrass fermentability at the Wisconsin Central–Hancock site. In subsequent years, the shelters were redesigned to fully exclude rainfall, which led to reduced biomass yields and inhibited fermentation for three sites. When switchgrass was grown in soils with large reductions in moisture and increases in temperature, the potential for biofuel production was significantly reduced, exposing some of the challenges associated with producing biofuels from lignocellulosic biomass grown under drought conditions.

09 BIOMASS FUELS↗

DOE OSTI2021

Stepping on the Gas to a Circular Economy: Accelerating Development of Carbon-Negative Chemical Production from Gas Fermentation

Owing to rising levels of greenhouse gases in our atmosphere and oceans, climate change poses significant environmental, economic, and social challenges globally. Technologies that enable carbon capture and conversion of greenhouse gases into useful products will help mitigate climate change by enabling a new circular carbon economy. Gas fermentation using carbon-fixing microorganisms offers an economically viable and scalable solution with unique feedstock and product flexibility that has been commercialized recently. Here, we review the state of the art of gas fermentation and discuss opportunities to accelerate future development and rollout. We discuss the current commercial process for conversion of waste gases to ethanol, including the underlying biology, challenges in process scale-up, and progress on genetic tool development and metabolic engineering to expand the product spectrum. We emphasize key enabling technologies to accelerate strain development for acetogens and other nonmodel organisms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Mixed Acid Fermentation of Carbohydrate-Rich Dairy Manure Hydrolysate

Dairy manure (DM) is an abundant agricultural residue that is largely composed of lignocellulosic biomass. The aim of this study was to investigate if carbon derived from DM fibers can be recovered as medium-chain fatty acids (MCFAs), which are mixed culture fermentation products of economic interest. DM fibers were subjected to combinations of physical, enzymatic, chemical, and thermochemical pretreatments to evaluate the possibility of producing carbohydrate-rich hydrolysates suitable for microbial fermentation by mixed cultures. Among the pretreatments tested, decrystalization dilute acid pretreatment (DCDA) produced the highest concentrations of glucose and xylose, and was selected for further experiments. Bioreactors fed DCDA hydrolysate were operated. Acetic acid and butyric acid comprised the majority of end products during operation of the bioreactors. MCFAs were transiently produced at a maximum concentration of 0.17 mg COD MCFAs /mg COD Total . Analyses of the microbial communities in the bioreactors suggest that lactic acid bacteria, Megasphaera , and Caproiciproducens were involved in MCFA and C4 production during DCDA hydrolysate metabolism.

09 BIOMASS FUELS↗

DOE OSTI2023

Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense

Genetically engineering microorganisms to produce chemicals has changed the industrialized world. The budding yeast Saccharomyces cerevisiae is frequently used in industry due to its genetic tractability and unique metabolic capabilities. S. cerevisiae has been engineered to produce novel compounds from diverse sugars found in lignocellulosic biomass, including pentose sugars, like xylose, not recognized by the organism. Engineering high flux toward novel compounds has proved to be more challenging than anticipated since simply introducing pathway components is often not enough. Several studies show that the rewiring of upstream signaling is required to direct products toward pathways of interest, but doing so can diminish stress tolerance, which is important in industrial conditions. As an example of these challenges, we reviewed S. cerevisiae engineering efforts, enabling anaerobic xylose fermentation as a model system and showcasing the regulatory interplay’s controlling growth, metabolism, and stress defense. Enabling xylose fermentation in S. cerevisiae requires the introduction of several key metabolic enzymes but also regulatory rewiring of three signaling pathways at the intersection of the growth and stress defense responses: the RAS/PKA, Snf1, and high osmolarity glycerol (HOG) pathways. The current studies reviewed here suggest the modulation of global signaling pathways should be adopted into biorefinery microbial engineering pipelines to increase efficient product yields.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Data from: High temperatures and low soil moisture synergistically reduce switchgrass yields from marginal field sites and inhibit fermentation

‘Marginal lands’ are low productivity sites abandoned from agriculture for reasons such as low or high soil water content, challenging topography, or nutrient deficiency. To avoid competition with crop production, cellulosic bio- energy crops have been proposed for cultivation on marginal lands, however on these sites they may be more strongly affected by environmental stresses such as low soil water content. In this study we used rainout shelters to in- duce low soil moisture on marginal lands and determine the effect of soil water stress on switchgrass growth and the subsequent production of bioetha- nol. Five marginal land sites that span a latitudinal gradient in Michigan and Wisconsin were planted to switchgrass in 2013 and during the 2018–2021 growing seasons were exposed to reduced precipitation under rainout shelters in comparison to ambient precipitation. The effect of reduced precipitation was related to the environmental conditions at each site and biofuel produc- tion metrics (switchgrass biomass yields and composition and ethanol pro- duction). During the first year (2018), the rainout shelters were designed with 60% rain exclusion, which did not affect biomass yields compared to ambient conditions at any of the field sites, but decreased switchgrass fermentability at the Wisconsin Central–Hancock site. In subsequent years, the shelters were redesigned to fully exclude rainfall, which led to reduced biomass yields and inhibited fermentation for three sites. When switchgrass was grown in soils with large reductions in moisture and increases in temperature, the potential for biofuel production was significantly reduced, exposing some of the chal- lenges associated with producing biofuels from lignocellulosic biomass grown under drought conditions.

bioethanol↗

DOE OSTI2026

OxyR regulates the oxidative stress response in Zymomonas mobilis during oxic growth and anoxic biofuel fermentation

The bacterium Zymomonas mobilis is widely studied for its potential as an industrial biofuel producer. Anoxic fermentation by Z. mobilis in lignocellulosic hydrolysate can generate bioethanol from renewable plant biomass. In this study, we deleted a gene from the Z. mobilis genome encoding a homolog of OxyR, a transcription factor that activates an oxidative stress response in bacteria to reduce reactive oxygen species (ROS). Deletion of this transcription factor inhibited growth of Z. mobilis in oxic, but not anoxic, conditions in laboratory media. RNA-Sequencing was perfromed on wild-type Z. mobilis (ZM4) and ∆oxyR in both oxic and anoxic conditions in rich media (ZRMG). This study reveals the indirect regulon of OxyR in Z. mobilis, which is important for both oxic growth and anoxic biofuel fermentation.

bioenergy↗

DOE OSTI2022

Adsorptive separation and recovery of triacetic acid lactone from fermentation broth

Triacetic acid lactone (TAL), an emerging bioprivileged molecule, can be produced microbially and further chemically upgraded to several high-value chemicals. In this work, several acidic and basic ion-exchange resins and activated charcoal were evaluated for their ability to adsorb microbially produced TAL. Activated charcoal and a weak base resin, Dowex 66, showed similar TAL adsorption capacity of 0.18 ± 0.002 g/g. At 15% w/v activated charcoal, about 98% of TAL present in fermentation broth could be adsorbed. Further, ethanol washing allowed recovery of 72% of adsorbed TAL. A biorefinery producing TAL from sucrose was designed, simulated, and evaluated (through technoeconomic analysis) under uncertainty, for an estimated TAL minimum product selling price (MPSP) of $\$4.27$/kg ($\$3.71$–4.94/kg; 5th–95th percentiles) given the current state of technology and $\$2.83$/kg ($\$2.46$–3.29/kg) following potential near-term improvements to fermentation. This work provides an adsorptive process for the recovery of microbially produced TAL that can be upgraded chemically to a range of industrial products.

09 BIOMASS FUELS↗

DOE OSTI2022

Plant biomass fermentation by the extreme thermophile Caldicellulosiruptor bescii for co-production of green hydrogen and acetone: Technoeconomic analysis

A variety of chemical and biological processes have been proposed for conversion of sustainable low-cost feedstocks into industrial products. Here, a biorefinery concept is formulated, modeled, and analyzed in which a naturally (hemi)cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii, is metabolically engineered to convert the carbohydrate content of lignocellulosic biomasses (i.e., soybean hulls, transgenic poplar) into green hydrogen and acetone. Experimental validation of C. bescii fermentative performance demonstrated 82% carbohydrate solubilization of soybean hulls and 55% for transgenic poplar. A detailed technical design, including equipment specifications, provides the basis for an economic analysis that establishes metabolic engineering targets. This robust industrial process leveraging metabolically engineered C. bescii yields 206 kg acetone and 25 kg H 2 per metric ton of soybean hull, or 174 kg acetone and 21 kg H 2 per metric ton transgenic poplar. Beyond this specific case, the model demonstrates industrial feasibility and economic advantages of thermophilic fermentation.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗

DOE OSTI2020

Pollution to products: recycling of ‘above ground’ carbon by gas fermentation

Climate crisis and rapid population growth are posing some of the most pressing challenges to mankind and have intensified the need for the deployment of carbon recycling and carbon capture and utilization (CCU) technologies. Gas fermentation offers a solution using carbon-fixing chemolithoautotrophic microorganisms. After a decade of scale up, the technology has recently been commercialized with the first plant operating successfully since 2018 and additional units under construction. Gas fermentation offers unique feedstock and product flexibility when compared to other available gas-to-liquid technologies. Advancements in process technology and synthetic biology enable a broad range of feedstocks including emissions from industry or syngas generated from any biomass resource to be converted into a wide range of molecules realizing a circular economy.

09 BIOMASS FUELS↗

DOE OSTI2026

A mathematical design framework for membrane pre-concentration in energy-efficient recovery of fermentation products

Due to the dilute nature of products manufactured via fermentation and cell-free bioprocessing, dewatering is a common unit operation in downstream processing (DSP) for bioproduct recovery, but it is typically energy intensive. To improve DSP energy efficiency for bio-based small molecules, integrating high-pressure membrane pre-concentration is a promising process option. However, this approach is typically constrained by a tradeoff between concentration factor (CF) and product recovery (PR), namely increasing the CF typically results in greater product loss, and vice versa. Here we developed a model that enables process design guidelines to: (i) identify scenarios in which the additional energy consumption and product loss from membrane pre-concentration are justified for use in DSP, and (ii) determine the optimal CF that minimizes process specific energy consumption. We compared the energy consumption of high-pressure membrane-integrated processes to evaporation-only processes and applied the model to an experimental case study for the separation and purification of butyric acid from Clostridium tyrobutyricum fermentation using an in situ product recovery (ISPR) process. The model estimated that integrating a tangential-flow reverse osmosis (RO) pre-concentration unit could reduce process energy consumption up to 45%. The use of advanced membrane pre-concentration technologies, such as negative rejection membranes and organic solvent reverse osmosis (OSRO), have the potential to further reduce the overall process specific energy consumption up to 96%, projected based on modeling. Overall, membrane pre-concentration, especially when strategically integrated prior to an evaporation step with optimized process conditions, holds significant potential for improving DSP energy efficiency, particularly in applications requiring substantial solvent removal for product recovery from dilute mixtures.

09 BIOMASS FUELS↗

DOE OSTI2025

Sustainable Triacetic Acid Lactone Production from Sugarcane by Fermentation and Crystallization

Triacetic acid lactone (TAL) has the potential to serve as a bioderived platform chemical for commercial products including sorbic acid and recyclable polydiketoenamine plastics. In this study, we leveraged BioSTEAM to design, simulate, and evaluate (via techno-economic analysis, TEA, and life cycle assessment, LCA) TAL production from sugarcane. We experimentally characterized TAL solubility, calibrated solubility models, and designed a process to separate TAL from fermentation broths by crystallization. The biorefinery could produce TAL at a minimum product selling price (MPSP) of $\$3.73$–5.86·kg –1 (5th–95th percentiles; baseline at $\$4.60$·kg –1 ) and a carbon intensity (CI) of 5.31 [2.60–8.71] kg CO 2 -eq·kg –1 , which could enable financially viable, low-CI production of sorbic acid and polydiketoenamines. To drive down costs and CI, we explored the theoretical fermentation space (titer, yield, productivity combinations), operation scheduling and capacity expansion strategies (e.g., integrated sorghum processing), and potential separation improvements (mitigating TAL loss through pH control). Advancements in key design and technological parameters could further reduce MPSP by 51% to $\$2.26$·kg –1 [$\$1.97$–2.80·kg –1 ] and CI by 43% to 3.05 [1.91–4.15] kg CO 2 -eq·kg –1 . This research highlights the ability of agile TEA-LCA to screen promising designs, navigate sustainability trade-offs, prioritize research needs, and chart quantitative roadmaps to advance bioproducts and biofuels.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗