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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 Rewiring Yeast Metabolism for Producing 2,3-Butanediol and Two Downstream Applications: Techno-Economic Analysis and Life Cycle Assessment of Methyl Ethyl Ketone (MEK) and Agricultural Biostimulant Production

Rising concerns for sustainability and global climate change have driven the development of sustainable production pathways for biofuels and chemicals from lignocellulosic biomass via integrated biological and chemical processes. We constructed an engineered Saccharomyces cerevisiae capable of producing 2,3-butanediol (2,3-BDO) from glucose without accumulating ethanol and glycerol, which hinder downstream processing of 2,3-BDO, through extensive metabolic reprogramming. Specifically, we introduced heterologous 2,3-BDO biosynthetic enzymes and deleted the major isozymes of ethanol and glycerol biosynthetic enzymes. In addition, we introduced an NAD+ regenerating Pyruvate-Malate (PM) cycle and enhanced the NAD+ regenerating capability of the PM cycle to resolve the redox imbalance from the deletion of ethanol and glycerol production pathways. The resulting engineered yeast produced 109.9 g/L of 2,3-BDO with a productivity of 1.0 g/L/h and a yield of 0.36 g/g glucose in a fed-batch fermentation. We also conducted techno-economic analysis (TEA) and life cycle assessment (LCA) of the production of methyl ethyl ketone (MEK) through catalytic dehydration of 2,3-BDO. A TEA based on the experimental results indicated that the minimum product selling price (MPSP) was estimated to be $1.90/kg. Regarding cradle-to-grave LCA, 100-year global warming potential (GWP100) and fossil energy consumption (FEC) were found to be 0.37 kg CO2 eq/kg and 3.1 MJ/kg, respectively. These results demonstrated the feasibility of cost-competitive and sustainable bio-based MEK production via yeast fermentation. In addition, we explored the possibility of using the fermentation broth containing 2,3-BDO as a biostimulant inducing drought tolerance in plants. As a result, the yeast 2,3-BDO fermentation broth can induce drought tolerance in Arabidopsis thaliana without a complicated purification process.

Economics↗

Catalytic Upgrading of Renewable Feedstock (Final Technical Report)

The goal of this DOE-funded project was to investigate the fundamental science related to the development of homogeneous (de)hydrogenation catalysts in order to enable energy-relevant transformations of bio-relevant chemical feedstocks including ethanol. The primary focus was to improve the activity of ethanol upgrading catalysts, specifically informed through mechanistic studies, in order to enable rational design optimization strategies. Following in depth mechanistic studies, targeted reaction optimization approaches included ligand redesign to improve catalyst stability, developing new carbon-carbon bond forming reactions using ethanol as a precursor, and examining photochemically-mediated reactions, ultimately to integrate within other reactor designs such as continuous flow reactors. The high modularity of the catalyst components (ligands) has enabled the preparation and analyses of multiple catalyst precursors. These studies uncovered an unexpectedly beneficial substitution pattern of the ligand structure that led to the development of new catalysts that are the best in class for upgrading ethanol to butanol with a turnover number of 155,890 and a turnover frequency of 12,690 h –1 . In addition to upgrading ethanol to butanol, cascade reaction sequences were developed to form new C-C bonds using ethanol as a bio-relevant feedstock, providing access to platform chemicals from renewable sources. As part of the reaction discovery process, new mechanistic details were uncovered that provided insights into: a) catalyst speciation, b) decomposition pathways, c) carbon monoxide releasing pathways, and d) carbon-carbon and carbon hydrogen bond breaking pathways. Most of these outcomes were previously not known; however, they provide important directions for new catalyst design strategies. Finally, use of high throughput and in situ photochemical reaction analyses enabled detailed studies into changes to the catalyst structure upon irradiation. Irradiation was found to improve hydrogen transfer catalysis, by promoting a ligand dissociation event.

09 BIOMASS FUELS↗

FTIR instrumentation to monitor vapors from Shuttle tile waterproofing materials

The Space Shuttle Thermal Protection System (TPS) tiles and blankets are waterproofed using DimethylEthoxySilane (DMEX) in the Orbiter Processing Facilities (OPF). DMES has a Threshold Limit Value (TLV) for exposure of personnel to vapor concentration in air of 0.5 ppm. The OPF high bay cannot be opened for normal work after a waterproofing operation until the DMES concentration is verified by measurement to be below the TLV. On several occasions the high bay has been kept closed for up to 8 hours following waterproofing operations due to high DMES measurements. In addition, the Miran 203 and Miran 1 BX infrared analyzers calibrated at different wavelengths gave different readings under the same conditions. There was reason to believe that some of the high DMES concentration readings were caused by interference form water and ethanol vapors. The Toxic Vapor Detection Laboratory (TVDL) was asked to test the existing DMES instruments and identify the best qualified instrument. In addition the TVDL was requested to develop instrumentation to ensure the OPF high bay could be opened safely as soon as possible after a waterproofing operation. A Fourier Transform Infrared (FTIR) spectrophotometer instrument developed for an earlier project was reprogrammed to measure DMES vapor along with ethanol, water, and several common solvent vapors. The FTIR was then used to perform a series of laboratory and field tests to evaluate the performance of the single wavelength IR instruments in use. The results demonstrated that the single wavelength IR instruments did respond to ethanol and water vapors, more or less depending on the analytical IR wavelength selected. The FTIR was able to separate the responses to DMES, water and ethanol, and give consistent readings for the DMES vapor concentration. The FTIR was then deployed to the OPF to monitor real waterproofing operations. The FTIR was also used to measure the time for DMES to evaporate from TPS tile under a range of humidity conditions in controlled laboratory tests. The combination of laboratory and field tests with the FTIR instrument demonstrated superior sensitivity, ability to reject interference from water and ethanol vapors, ruggedness to be transported from the lab to the OPF and set up without special procedures or degradation of performance. The multiple component vapor analysis algorithm was developed at KSC and incorporates automatic baseline correction and shape fitting of the spectra. The analysis for DMES, TetraMethylDiSiloxane (TMDS), ethanol, methanol, isopropanol, and baseline parameters uses 161 points per sample at 4 cm(exp -1) resolution, and processes an eight scan sample every ten seconds. The standard deviation of the measurements is 0.013 ppm and the upper linear limit is 125 ppm DMES. Based on successful demonstration of capabilities we produced three mobile instrument carts to be used in each OPF to support future waterproofing operations. The design and building of the 'DMES Carts' were accomplished in Fiscal year 1995.

Mattson, C. B.↗

Techno-Economic Analysis and Life Cycle Assessment of Waste Lignin Fractionation and Valorization Using the ALPHA Process

Techno-Economic Analysis (TEA) and Life Cycle Assessment (LCA) were performed on the Aqueous Lignin Purification with Hot Agents (ALPHA) process, which is being investigated for the fractionation and purification of raw, bulk lignins recovered from cellulosic ethanol biorefineries or Kraft pulp mills. Here, ALPHA is proposed for the isolation of lignin from a corn stover-to-ethanol plant into purified low, medium, and high molecular weight (MW) fractions for producing polyurethane foam, activated carbon, and carbon fiber, respectively. A scenario analysis was conducted to determine the effect of ALPHA solvent choice on process economics and environmental performance. Solvent choice was found to have a significant impact on ALPHA, with a minimum selling price of 838/tonne with use of acetic acid vs 463/tonne with ethanol. Conversion of the lignin, processed with ethanol solvent, to high-value products yields 151 million/year in profit, which over 30 years results in a total net present value of 533 million. A life cycle assessment was conducted to determine the “gate-to-gate” greenhouse gas emissions and energy consumption of the lignin-based products compared to fossil-based equivalents. In conclusion, a value allocation scenario was conducted and it was determined that products generated using the ALPHA process with ethanol have similar or lower greenhouse gas emissions than the same products from fossil feedstocks.

09 BIOMASS FUELS↗

Soot Formation and Ignition Characteristics of Ethanol/Gasoline Blends in a Rapid Compression Machine

With the ever-increasing demand for sustainable energy, alcohol fuels have garnered interest for use in heavy duty engines. The significant infrastructure for ethanol production and blending of ethanol with gasoline make these fuels/fuel blends desirable candidates. However, development of heavy duty engine technology that is capable of burning alcohol fuels while retaining the advantages of traditional diesel combustion requires an improved understanding of the soot formation for these fuels under conditions relevant to mixing-controlled combustion. This work uses an extinction diagnostic to study the sooting tendency of ethanol and gasoline/ethanol blends ranging from E10 to E98 during ignition in a homogeneous environment. Experiments were conducted in a rapid compression machine (RCM) for compressed conditions of 20 ± 1 bar and an approximately constant temperature (± 10K) which was unique for each fuel. For a given soot volume fraction, a linear relationship was observed between ethanol content and the equivalence ratio in which that soot volume fraction was formed. Accounting for the oxygenated nature of ethanol, E85 and E98 fuels produced similar amounts of soot at a given Φ ox, , suggesting other factors outside of fuel oxygen content, such as fuel morphology, impact soot formation. Ignition delay data is reported for compressed pressures of 20 ± 1 bar and compressed temperatures ranging from 633 – 670 K for E10 and 771 – 789 K for E98. Varying pressures for E10 and E98 at conditions producing similar soot volume fractions demonstrated a linear dependence of soot formation on pressure, regardless of if the pressure considered was at top dead center or peak combustion pressure. Furthermore, the data gleaned from this work will be used to select soot models and chemical kinetic mechanisms for RCM simulations to ultimately model heavy duty engine technology with the studied fuels.

33 ADVANCED PROPULSION SYSTEMS↗

Structural Characterization and Dynamics of AdhE Ultrastructures from Clostridium thermocellum Show a Containment Strategy for Toxic Intermediates

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe; however, ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 A structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

09 BIOMASS FUELS↗

Organic contamination detection for isotopic analysis of water by laser spectroscopy

Hydrogen and oxygen stable isotope ratios (δ 2 H, δ 17 O, and δ 18 O values) are commonly used tracers of water. These ratios can be measured by isotope ratio infrared spectroscopy (IRIS). However, IRIS approaches are prone to errors induced by organic compounds present in plant, soil, and natural water samples. A novel approach using 17 O-excess values has shown promise for flagging spectrally contaminated plant samples during IRIS analysis. A systematic assessment of this flagging system is needed to prove it useful. Errors induced by methanol and ethanol water mixtures on measured IRIS and isotope ratio mass spectrometry (IRMS) results were evaluated. For IRIS analyses both liquid- and vapour-mode (via direct vapour equilibration) methods are used. The δ 2 H, δ 17 O, and δ 18 O values were measured and compared with known reference values to determine the errors induced by methanol and ethanol contamination. In addition, the 17 O-excess contamination detection approach was tested. This is a post-processing detection tool for both liquid and vapour IRIS triple-isotope analyses, utilizing calculated 17 O-excess values to flag contaminated samples. Organic contamination induced significant errors in IRIS results, not seen in IRMS results. Methanol caused larger errors than ethanol. Results from vapour-IRIS analyses had larger errors than those from liquid-IRIS analyses. The 17 O-excess approach identified methanol driven error in liquid- and vapour-mode IRIS samples at levels where isotope results became unacceptably erroneous. For ethanol contaminated samples, a mix of erroneous and correct flagging occurred with the 17 O-excess method. Here our results indicate that methanol is the more problematic contaminant for data corruption. The 17 O-excess method was therefore useful for data quality control. Organic contamination caused significant errors in IRIS stable isotope results. These errors were larger during vapour analyses than during liquid IRIS analyses, and larger for methanol than ethanol contamination. The 17 O-excess method is highly sensitive for detecting narrowband (methanol) contamination error in vapour and liquid analysis modes in IRIS.

60 APPLIED LIFE SCIENCES↗

Multifunctional landscapes for dedicated bioenergy crops lead to low-carbon market-competitive biofuels

Switchgrass is a promising feedstock for cellulosic biorefineries, due to its ability to maintain comparatively high biomass yields across a wide range of soil and climatic conditions. However, there is an incomplete understanding of the economic and environmental tradeoffs associated with cultivating switchgrass on low-productivity land for conversion to biofuels. This study surveys prior literature and demonstrates a new integrated assessment framework, including agroecosystem, ecosystem services valuation, technoeconomic, and life-cycle assessment models, to quantify and contextualize the economic and environmental impacts of switchgrass cultivation on marginal land with downstream conversion to biofuels. Monetizing and incorporating the value of ecosystem services, such as improved water quality benefits from nitrate and sediment reductions, climate change mitigation benefits from CO 2 emission reduction, and recreational and pollination benefits from increased biodiversity, the modeled multifunctional landscape reduces the ethanol production cost by 33.3–58.9 cents/$\scriptsize{L}$-gasoline-equivalent ($\$$1.3–2.2/gge). Planting switchgrass in low productivity land improves soil health, resulting in the carbon footprint reduction credit of 12.8–20.2 gCO 2e /MJ. For an improved switchgrass-to-ethanol conversion pathway with the maximum benefits from ecosystem services, the minimum ethanol selling price and carbon footprint of ethanol, respectively, could reach to 31 cents/$\scriptsize{L}$-gasoline-equivalent (47% reduction relative to average gasoline price) and 3 gCO 2e /MJ (97% reduction relative to gasoline). In conclusion, this low carbon renewable ethanol leads to substantial State and/or Federal policy incentives (~$\$$1/$\scriptsize{L}$-gasoline-equivalent) providing a large benefit to biorefinery operators, farmers, and the public as a whole.

09 BIOMASS FUELS↗

Surface Nanostructure Control and Thermodynamic Stability Analysis of Femtosecond Laser-Ablated CuCoMn 1.75 NiFe 0.25 Nanoparticles

Surface nanostructure control is the key to functionalizing nanomaterials. This paper presents a characterization with thermodynamic stability analysis of CuCoMn 1.75 NiFe 0.25 high-entropy alloy (HEA) nanoparticles synthesized by femtosecond laser ablation in ethanol and liquid nitrogen (LN2). Using multimodal electron microscopy and spectroscopy, we examine phase, particle size, defect structure, chemical distribution, and surface composition and relate them to HEA stability. Elemental distributions are uniform in both media, but LN2 produces smaller particles with a narrower size distribution and mainly single- or few-domain interiors, whereas ethanol yields larger particles built from 2–4 nm crystallites with domain aggregation. Edge defects appear in both but energy-dispersive X-ray spectroscopy (EDS) is broadly uniform with local fluctuations in ethanol. X-ray photoelectron spectroscopy (XPS), supported by an attenuation model, indicates an ∼1 nm oxide overlayer that suppresses Mn 2p intensity; correcting for it returns Mn toward the bulk value. UV–NIR and photoluminescent spectra independently support a thin oxide shell. Composition-based thermodynamic descriptors place LN2 closer to bulk mixing parameters, while ethanol raises ΔH_mix and lowers Ω. Cooling simulations are consistent (LN2 ∼ 0.1 μs quench, ethanol ∼1 μs). In conclusion, these results connect solvent-controlled kinetics and thermodynamics to crystalline state and surface chemistry, informing surface control of HEA nanoparticles.

Femtosecond Laser Ablation↗

Modelling drug adsorption in metal–organic frameworks: the role of solvent

Solvent plays a key role in biological functions, catalysis, and drug delivery. Metal–organic frameworks (MOFs) due to their tunable functionalities, porosities and surface areas have been recently used as drug delivery vehicles. To investigate the effect of solvent on drug adsorption in MOFs, we have performed integrated computational and experimental studies in selected biocompatible MOFs, specifically, UiO-AZB, HKUST-1 (or CuBTC) and NH 2 -MIL-53(Al). The adsorption of three drugs, namely, 5-fluorouracil (5-FU), ibuprofen (IBU), and hydroxyurea (HU) were performed in the presence and absence of the ethanol. Our computational predictions, at 1 atmospheric pressure, showed a reasonable agreement with experimental studies performed in the presence of ethanol. We find that in the presence of ethanol the drug molecules were adsorbed at the interface of solvent and MOFs. Moreover, the computationally calculated adsorption isotherms suggested that the drug adsorption was driven by electrostatic interactions at lower pressures (<10 –4 Pa). Our computational predictions in the absence of ethanol were higher compared to those in the presence of ethanol. The MOF–adsorbate interaction (U HA ) energy decreased with decrease in the size of a drug molecule in all three MOFs at all simulated pressures. At high pressure the interaction energy increases with increase in the MOFs pore size as the number of molecules adsorbed increases. Thus, our research shows the important role played by solvent in drug adsorption and suggests that it is critical to consider solvent while performing computational studies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Supplementing biofuel mandates with a carbon mitigation policy can lead to water quality co-benefits

Biofuel mandates can impact the environment in multiple ways that may be positive or negative, including affecting life-cycle greenhouse gas (GHG) emissions by displacing fossil fuels, affecting soil carbon stocks due to accompanying land use change, and water quality due to changes in fertilizer requirements and the mix of crops used as feedstocks. To achieve desired environmental outcomes in the presence of a biofuel mandate, additional policy instruments must be adopted to supplement the mandate. We develop an integrated and spatially explicit ecosystem-economic modeling framework to analyze the cost-effectiveness of alternative policies to achieve desired targets for GHG emissions reduction from the agricultural and fuel sectors in the USA and nitrate leaching reduction in the Gulf of Mexico below the levels that would be achieved by a corn ethanol and/or a cellulosic ethanol mandate in the USA. We find that while a corn ethanol mandate lowers GHG emissions, it increases nitrate leaching due to the expansion of corn production; a cellulosic ethanol mandate lowers both GHG emissions and nitrate leaching relative to a corn ethanol mandate, but the additional carbon and nitrate prices are needed to achieve anticipated GHG reduction and nitrate reduction targets. We also find that accompanying a biofuel mandate with a GHG reduction target alone leads to substantial nitrate reduction co-benefits, but a nitrate reduction target alone is less effective in reducing GHG emissions. Combining a GHG standard with a nitrate standard can achieve GHG and nitrate reduction targets at lower carbon and nitrate prices as compared to implementing each of these policies independently. Furthermore, our findings show that disregarding policy co-benefits can overestimate the GHG and nitrate prices needed to achieve policy targets and higher policy costs.

09 BIOMASS FUELS↗

Lipid droplet-associated proteins in alcohol-associated fatty liver disease: A proteomic approach

The earliest manifestation of alcohol-associated liver disease (ALD) is steatosis characterized by deposition of fat in specialized organelles called lipid droplets (LDs). While alcohol administration causes a rise in LD numbers in the hepatocytes, little is known regarding their characteristics that allow their accumulation and size to increase. The aim of the present study is to gain insights into underlying pathophysiological mechanisms by investigating the ethanol-induced changes in hepatic LD proteome as a function of LD size. Adult male Wistar rats (180–200 g BW) were fed with ethanol liquid diet for 6 weeks. At sacrifice, large-, medium-, and small-sized hepatic LD subpopulations (LD1, LD2, and LD3, respectively) were isolated and subjected to morphological and proteomic analyses. Morphological analysis of LD1-LD3 fractions of ethanol-fed rats clearly demonstrated that LD1 contained larger LDs compared with LD2 and LD3 fractions. Our preliminary results from principal component analysis showed that the proteome of different-sized hepatic LD fractions was distinctly different. Proteomic data analysis identified over 2000 proteins in each LD fraction with significant alterations in protein abundance among the three LD fractions. Among the altered proteins, several were related to fat metabolism, including synthesis, incorporation of fatty acid, and lipolysis. Ingenuity pathway analysis revealed increased fatty acid synthesis, fatty acid incorporation, LD fusion, and reduced lipolysis in LD1 compared to LD3. Overall, the proteomic findings indicate that the increased level of protein that facilitates fusion of LDs combined with an increased association of negative regulators of lipolysis dictates the generation of large-sized LDs during the development of alcohol-associated hepatic steatosis. Several significantly altered proteins were identified in different-sized LDs isolated from livers of ethanol-fed rats. Ethanol-induced increases in specific proteins that hinder LD lipid metabolism led to the accumulation and persistence of large-sized LDs in the liver.

60 APPLIED LIFE SCIENCES↗

Sustained Ability of a Natural Microbial Community to Remove Nitrate from Groundwater

Abstract Microbial‐mediated nitrate removal from groundwater is widely recognized as the predominant mechanism for nitrate attenuation in contaminated aquifers and is largely dependent on the presence of a carbon‐bearing electron donor. The repeated exposure of a natural microbial community to an electron donor can result in the sustained ability of the community to remove nitrate; this phenomenon has been clearly demonstrated at the laboratory scale. However, in situ demonstrations of this ability are lacking. For this study, ethanol (electron donor) was repeatedly injected into a groundwater well (treatment) for six consecutive weeks to establish the sustained ability of a microbial community to remove nitrate. A second well (control) located upgradient was not injected with ethanol during this time. The treatment well demonstrated strong evidence of sustained ability as evident by ethanol, nitrate, and subsequent sulfate removal up to 21, 64, and 68%, respectively, as compared to the conservative tracer (bromide) upon consecutive exposures. Both wells were then monitored for six additional weeks under natural (no injection) conditions. During the final week, ethanol was injected into both treatment and control wells. The treatment well demonstrated sustained ability as evident by ethanol and nitrate removal up to 20 and 21%, respectively, as compared to bromide, whereas the control did not show strong evidence of nitrate removal (5% removal). Surprisingly, the treatment well did not indicate a sustained and selective enrichment of a microbial community. These results suggested that the predominant mechanism(s) of sustained ability likely exist at the enzymatic‐ and/or genetic‐levels. The results of this study demonstrated the in situ ability of a microbial community to remove nitrate can be sustained in the prolonged absence of an electron donor.

54 ENVIRONMENTAL SCIENCES↗

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗

Non-Toxic Reaction Control System for the Reusable First Stage Vehicle

This paper presents the Boeing Reusable Space Systems vision of a Reaction Control System (RCS) for the Reusable First Stage (RFS) being considered as a replacement for the Solid Rocket Booster for the Space Shuttle. The requirement is to,achieve reliable vehicle control during the upper atmospheric portion of the RFS trajectory while enabling more efficient ground operations, unhindered by constraints caused by operating with highly toxic RCS propellants. Boeing's objective for this effort is to develop a safer, more efficient and environmentally friendly RCS design approach that is suitable for the RFS concept of operations, including a low cost, efficient turnaround cycle. The Boeing RCS concept utilizes ethanol and liquid oxygen in place of the highly toxic, suspected carcinogen, ozone- depleting mono-methyl-hydrazine and highly toxic nitrogen tetroxide. The Space Shuttle Upgrade program, under the leadership of the NASA Johnson Space Flight Center, is currently developing liquid oxygen and ethanol (ethyl alcohol) technology for use as non-toxic orbital maneuvering system (OMS) and RCS. The development of this liquid oxygen and ethanol technology for the Space Shuffle offers a significant leverage to select much of the same technology for the RFS program. There are significant design and development issues involved with bringing this liquid oxygen and ethanol technology to a state of maturity suitable for an operational RCS, The risks associated with a new LOX and Ethanol RCS are mitigated by maintaining kerosene and hydrogen peroxide RCS technology as an alternative. These issues, presented within this paper, include managing the oxygen supply and achieving reliable ignition in the short pulse mode of engine operation. Performance, reliability and operations requirements are presented along with a specific RCS design concept to satisfying these requirements. The work reported in this paper was performed under NASA Marshall Space Flight Center Contract to define Reusable First Stage design concepts for the Space Shuttle.

Keith, E. L.↗

Non-Toxic Reaction Control System for the Reusable First Stage Vehicle

This paper presents the Boeing Reusable Space Systems vision of a Reaction Control System (RCS) for the Reusable First Stage (RFS) being considered as a replacement for the Solid Rocket Booster for the Space Shuttle. The requirement is to achieve reliable vehicle control during the upper atmospheric portion of the RFS trajectory while enabling more efficient ground operations, unhindered by constraints caused by operating with highly toxic RCS propellants. Boeing's objective for this effort is to develop a safer, more efficient and environmentally friendly RCS design approach that is suitable for the RFS concept of operations, including a low cost, efficient turnaround cycle. The Boeing RCS concept utilizes ethanol and liquid oxygen in place of the highly toxic, suspected carcinogen, ozone-depleting mono-methyl-hydrazine and highly toxic nitrogen tetroxide. The Space Shuttle Upgrade program, under the leadership of the NASA Johnson Space Flight Center, is currently developing liquid oxygen and ethanol (ethyl alcohol) technology for use as non-toxic orbital maneuvering system (OMS) and RCS. The development of this liquid oxygen and ethanol technology for the Space Shuttle offers a significant leverage to select much of the same technology for the RFS program. There are significant design and development issues involved with bringing this liquid oxygen and ethanol technology to a state of maturity suitable for an operational RCS. The risks associated with a new LOX and Ethanol RCS are mitigated by maintaining kerosene and hydrogen peroxide RCS technology as an alternative. These issues, presented within this paper, include managing the oxygen supply and achieving reliable ignition in the short pulse mode of engine operation. Performance, reliability and operations requirements are presented along with a specific RCS design concept to satisfying these requirements. The work reported in this paper was performed under NASA Marshall Space Flight Center Contract Number NAS8-97272 to define Reusable First Stage design concepts for the Space Shuttle.

Keith, E. L.↗

Effects of Potassium Permanganate Oxidation on Subsurface Microbial Activity

In situ chemical oxidation has the potential for degrading large quantities of organic contaminants and can be more effective and timely than traditional ex situ treatment methods. However, there is a need to better characterize the potential effects of this treatment on natural processes. This study focuses on potential inhibition to anaerobic dechlorination of trichloroethene (TCE) in soils from a large manufacturing facility as a result of in situ oxidation using potassium permanganate (KMn04)Previous microcosm studies established that natural attenuation occurs on-site and that it is enhanced by the addition of ethanol to the system. A potential remediation scheme for the site involves the use of potassium permanganate to reduce levels of TCE in heavily contaminated areas, then to inject ethanol into the system to "neutralize" excess oxidant and enhance microbial degradation. However, it is currently unknown whether the exposure of indigenous microbial populations to potassium permanganate may adversely affect biological reductive dechlorination by these microorganisms. Consequently, additional microcosm studies were conducted to evaluate this remediation scheme and assess the effect of potassium permanganate addition on biological reductive dechlorination of TCE. Samples of subsurface soil and groundwater were collected from a TCE-impacted area of the site. A portion of the soil was pretreated with nutrients and ethanol to stimulate microbial activity, while the remainder of the soil was left unamended. Soil/groundwater microcosms were prepared in sealed vials using the nutrient-amended and unamended soils, and the effects of potassium permanganate addition were evaluated using two permanganate concentrations (0.8 and 2.4 percent) and two contact times (1 and 3 weeks). TCE was then re-added to each microcosm and TCE and dichloroethene (DCE) concentrations were monitored to determine the degree to which microbial dechlorination occurred following chemical oxidation. Evidence of microbial degradation was generally detected within four weeks after TCE addition. Increases in DCE concentrations were consistent with decreases in TCE. The concentration of TCE in the nutrient-amended samples exposed to 2.4% KMnO4 for one week degraded somewhat more slowly than the samples exposed to the 0.8% KMnO4. The rates of degradation did not correlate with the length of KMn04 exposure for the nutrient-amended microcosms. Microbial degradation of TCE in the unamended microcosms was generally similar to that observed in the nutrient-amended microcosms. One treatment condition (unamended, one week exposure, 2.4% KMnO4) was exposed to elevated levels of ethanol and showed little evidence of degradation. It is suspected that the high levels of ethanol were toxic to the microorganisms. The results of the study indicate that exposure of indigenous soil and groundwater microbial populations to KMnO4 at concentrations of 0.8 to 2.4% do not impair the ability of the microbial populations to dechlorinate TCE. Consequently, the combination of chemical oxidation followed by enhanced biological reductive dechlorination appears to be a viable remedial strategy for highly-impacted subsurface areas of the site.

Rowland, Martin A.↗