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This paper examines some of the issues underlying potential robotic rescue devices (RRD) in the context where autonomous or manned rotorcraft deployment of such robotic systems is a crucial attribute for their success in supporting future disaster relief and emergency response (DRER) missions. As a part of this discussion, work related to proof-of-concept prototyping of two notional RRD systems is summarized.
In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4 PPH-4.1 phosphatase and ATR ATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4 PPH-4.1 , ATR ATL-1 , and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.
Firefighters want to go to work, do their job well, and go home alive and uninjured. For their most important job, saving lives, firefighters want protective equipment that will allow more extended and effective time at fire scenes in order to perform victim search and rescue. A team, including engineers at NASA JSC and firefighters from Houston, has developed a list of problem areas for which NASA technology and know-how can recommend improvements for firefighter suits and gear. Prototypes for solutions have been developed and are being evaluated. This effort will spin back to NASA as improvements for lunar and planetary suits.
With relevant chemical space growing larger and larger by the day, the ability to extend computational tractability over that larger space is of paramount importance in virtually all fields of science. The solution we aim to provide here for this issue is in the form of the generalized many-body expansion for building density matrices (GMBE-DM) based on the set-theoretical derivation with overlapping fragments, through which the energy can be obtained by a single Fock build. In combination with the purification scheme and the truncation at the one-body level, the DM-based GMBE(1)-DM-P approach shows both highly accurate absolute and relative energies for medium-to-large size water clusters with about an order of magnitude better than the corresponding energy-based GMBE(1) scheme. Simultaneously, GMBE(1)-DM-P is about an order of magnitude faster than the previously proposed MBE-DM scheme [F. Ballesteros and K. U. Lao, J. Chem. Theory Comput. 18, 179 (2022)] and is even faster than a supersystem calculation without significant parallelization to rescue the fragmentation method. For even more challenging systems including ion–water and ion–pair clusters, GMBE(1)-DM-P also performs about 3 and 30 times better than the energy-based GMBE(1) approach, respectively. In addition, this work provides the first overlapping fragmentation algorithm with a robust and effective binning scheme implemented internally in a popular quantum chemistry software package. Thus, GMBE(1)-DM-P opens a new door to accurately and efficiently describe noncovalent clusters using quantum mechanics.
The 1991-1992 senior Mechanical and Aerospace Engineering Design class continued work on the post landing configurations for the Assured Crew Return Vehicle (ACRV) and the Emergency Egress Couch (EEC). The ACRV will be permanently docked to Space Station Freedom fulfilling NASA's commitment of Assured Crew Return Capability in the event of an accident or illness aboard Space Station Freedom. The EEC provides medical support and a transportation surface for an incapacitated crew member. The objective of the projects was to give the ACRV Project Office data to feed into their feasibility studies. Four design teams were given the task of developing models with dynamically and geometrically scaled characteristics. Groups one and two combined efforts to design a one-fifth scale model for the Apollo Command Module derivative, an on-board flotation system, and a lift attachment point system. This model was designed to test the feasibility of a rigid flotation and stabilization system and to determine the dynamics associated with lifting the vehicle during retrieval. However, due to priorities, it was not built. Group three designed a one-fifth scale model of the Johnson Space Center (JSC) benchmark configuration, the Station Crew Return Alternative Module (SCRAM) with a lift attachment point system. This model helped to determine the flotation and lifting characteristics of the SCRAM configuration. Group four designed a full scale EEC with changeable geometric and geometric and dynamic characteristics. This model provided data on the geometric characteristics of the EEC and on the placement of the CG and moment of inertia. It also gave the helicopter rescue personnel direct input to the feasibility study. Section 1 describes in detail the design of a one-fifth scale model of the Apollo Command Module Derivative (ACMD) ACRV. The objective of the ACMD Configuration Model Team was to use geometric and dynamic constraints to design a one-fifth scale working model of the Apollo Command Module Derivative (ACMD) configuration with a Lift Attachment Point (LAP) System. This model was required to incorporate a rigidly mounted flotation system and the egress system designed the previous academic year. The LAP system was to be used to determine the dynamic effects of locating the lifting points at different locations on the vehicle. The team was then to build and test the model; however, due to priorities, this did not occur. To better simulate the ACMD after a water landing, the nose cone section was removed and the deck area exposed. The areas researched during the design process were construction, center of gravity and moment of inertia, and lift attachment points.
Rare earth elements (REEs) are essential to modern technologies, yet their high physical and chemical similarity makes separation of individual REEs difficult and environmentally taxing. Metalloproteins offer a promising alternative for selective REE binding, as they tend to have high metal ion affinity and specificity. Lanmodulin (LanM), in particular, has arisen as a potential candidate for REE separation as it exhibits picomolar affinity for elements in the REE family. Prior work has shown that the single point mutation D9N can shift LanM’s preference away from lanthanides toward actinides, motivating efforts to tune selectivity of LanM through targeted mutagenesis. Here, we tested the hypothesis that introducing selective aspartic acid to glutamic acid substitutions in the metal coordinating EF hands of LanM would impose steric constraints that would drive LanM affinity away from larger ions, such as La3+, to smaller ions, such as Y3+. To test this hypothesis, a combination of computational and experimental approaches were employed to evaluate the signal mutations LanM D5E and LanM D3E and the double mutants LanM D1ED5E and LanM D3ED9E. Surprisingly, increasing the number of mutations within the metal center did not enhance affinity for smaller REEs, or decrease affinity for larger ions. Only the single point mutation LanM D5E weakened La3+ binding by one order of magnitude relative to LanM wild type (WT), and pairing it with a second mutation to produce LanM D1ED5E drove La3+ affinity to be stronger than that seen for LanM WT. The D3E mutation alone prevented proper expression and folding, but paring it with D9E to produce LanM D3ED9E rescued expression and yielded La3+ affinities comparable to LanM WT. All variants that expressed (LanM D5E, LanM D1ED5E, LanM D3ED9E) displayed Y3+ affinities comparable to LanM WT. Overall, these results highlight the tunability of LanM’s metal-binding environment but also expose current limitations in predicting structural responses to point mutations within a protein sequence. This work establishes a foundation that can be used for refining computational and experimental strategies to engineer metalloproteins with tailored REE selectivity.
Six Apollo missions landed on the Moon from 1969-72, returning to Earth 382 kg of lunar rock, soil, and core samples-among the best documented and preserved samples on Earth that have supported a robust research program for 45 years. From mission planning through sample collection, preliminary examination, and subsequent research, strict protocols and procedures are followed for handling and allocating Apollo subsamples. Even today, 100s of samples are allocated for research each year, building on the science foundation laid down by the early Apollo sample studies and combining new data from today's instrumentation, lunar remote sensing missions and lunar meteorites. Today's research includes advances in our understanding of lunar volatiles, lunar formation and evolution, and the origin of evolved lunar lithologies. Much sample information is available to researchers at curator.jsc.nasa.gov. Decades of analyses on lunar samples are published in LPSC proceedings volumes and other peer-reviewed journals, and tabulated in lunar sample compendia entries. However, for much of the 1969-1995 period, the processing documentation, individual and consortia analyses, and unpublished results exist only in analog forms or primitive digital formats that are either inaccessible or at risk of being lost forever because critical data from early investigators remain unpublished. We have initiated several new efforts to rescue some of the early Apollo data, including unpublished analytical data. We are scanning NASA documentation that is related to the Apollo missions and sample processing, and we are collaborating with IEDA to establish a geochemical database called Moon DB. To populate this database, we are working with prominent lunar PIs to organize and transcribe years of both published and unpublished data. Other initiatives include micro-CT scanning of complex lunar samples to document their interior structure (e.g. clasts, vesicles); linking high-resolution scans of Apollo film products to samples; and new procedures for systematic high resolution photography of samples before additional processing, enabling detailed 3D reconstructions of the samples. All of these efforts will provide comprehensive access to Apollo samples and support better curation of the samples for decades to come.
The LunaNet Interoperability Specification (LNIS) is a set of standards currently under development by NASA, ESA, and JAXA, which define a common, interoperable set of services and interfaces for lunar communication and navigation. The LNIS includes specifications for the GNSS-like Augmented Forward Signal (AFS). The LANS (Lunar Augmented Navigation Service) will be comprised of Multiple LunaNet Service Provider (LNSP) nodes broadcasting the AFS, such as NASA’s LCRNS (Lunar Communications Relay and Navigation Systems), ESA’s Moonlight LCNS (Lunar Communication and Navigation Services) and the Japan LNSS (Lunar Navigation Satellite System). The LANS will provide a GNSS-like capability enabling orbiting and surface users in lunar space (such as Artemis) to estimate their position, velocity and time as described in Giordano et al., (2023). Initial capabilities will focus on providing service to the lunar South pole region. The specification of AFS defines two orthogonal signal components on a single carrier, with the in-phase component (AFS-I) being a lower-chip-rate data channel tailored for applications where low SWaP is critical (e.g., IoT devices or search and rescue), and the quadrature component (AFS-Q) being a high-chip-rate data-less pilot signal for high-precision, robust lunar navigation and positioning applications. An initial description of AFS was provided in the LNIS, (2023), and initial analysis results were shown in Dafesh, et al., (2024). In this work, we provide rationale for updates to the LNIS that define key aspects of the signal including the primary spreading code designs for the data and pilot channels, and a three-tiered overlay code approach for the pilot channel that provides flexible signal acquisition alternatives, rapid time dissemination and robust frame Sync. The paper also describes a robust data sync word that is designed to enable frame Sync. for low-SWaP receivers that only use the I channel, as well as a low-density parity check code (LDPC) data message encoding design and interleaving definition. The work further describes the impact of the updated AFS design in terms of improved acquisition performance, interference resistance, navigation message capabilities and rapid absolute time dissemination for users able to access clock and ephemeris data over an external network. The cross-correlation and synchronization performance of the AFS design is also compared to potential alternatives, further providing rationale for the final signal design configuration.
The LunaNet Interoperability Specification (LNIS) is a set of standards currently under development by NASA, ESA, and JAXA, which define a common, interoperable set of services and interfaces for lunar communication and navigation. The LNIS includes specifications for the GNSS-like Augmented Forward Signal (AFS). The LANS (Lunar Augmented Navigation Service) will be comprised of Multiple LunaNet Service Provider (LNSP) nodes broadcasting the AFS, such as NASA’s LCRNS (Lunar Communications Relay and Navigation Systems), ESA’s Moonlight LCNS (Lunar Communication and Navigation Services) and the Japan LNSS (Lunar Navigation Satellite System). The LANS will provide a GNSS-like capability enabling orbiting and surface users in lunar space (such as Artemis) to estimate their position, velocity and time as described in Giordano et al., (2023). Initial capabilities will focus on providing service to the lunar South pole region. The specification of AFS defines two orthogonal signal components on a single carrier, with the in-phase component (AFS-I) being a lower-chip-rate data channel tailored for applications where low SWaP is critical (e.g., IoT devices or search and rescue), and the quadrature component (AFS-Q) being a high-chip-rate data-less pilot signal for high-precision, robust lunar navigation and positioning applications. An initial description of AFS was provided in the LNIS, (2023), and initial analysis results were shown in Dafesh, et al., (2024). In this work, we provide rationale for updates to the LNIS that define key aspects of the signal including the primary spreading code designs for the data and pilot channels, and a three-tiered overlay code approach for the pilot channel that provides flexible signal acquisition alternatives, rapid time dissemination and robust frame Sync. The paper also describes a robust data sync word that is designed to enable frame Sync. for low-SWaP receivers that only use the I channel, as well as a low-density parity check code (LDPC) data message encoding design and interleaving definition. The work further describes the impact of the updated AFS design in terms of improved acquisition performance, interference resistance, navigation message capabilities and rapid absolute time dissemination for users able to access clock and ephemeris data over an external network. The cross-correlation and synchronization performance of the AFS design is also compared to potential alternatives, further providing rationale for the final signal design configuration.
Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery.
As emerging flight operations become more prevalent and increasingly automated and distributed, the capabilities for managing safety of vehicles and operations will also need to evolve. To address this challenge, the National Academies has envisioned an In-Time Aviation Safety Management System (IASMS) capability for a wide range of aviation operations including current commercial operations as well as new entrants envisioned with advanced air mobility (AAM). The suite of IASMS services, functions, and capabilities (SFCs) would be implemented in a federated approach and would address trends as well as individual operations. Through predictive modeling and data analysis, IASMS is envisioned to identify arising risks so that they can be mitigated, in-time, before a safety incident occurs. IASMS and its requisite set of SFCs must leverage a wide range of information to perform. To better understand these new needs, FSF worked with the aviation and humanitarian communities to develop and validate scenarios that include traditional aviation operations and UAS operations intermingled as they are deployed for disaster management and first responder (DMFR) situations. The three scenarios developed include: • Post Natural disaster response, such as a hurricane, involving multiple parties utilizing traditional aviation and UAS to support rescue operations, surveil damage, and locate survivors needing assistance. • Wildfire fighting in remote locations with traditional aircraft for transport and fire-retardant delivery combined with UAS for surveillance of fire locations as well as to track individual firefighter locations. • Medical Operations and AAM in Urban Environments including passenger-carrying helicopters and AAM vehicles, medical missions (such as transport of radio-pharmaceuticals), and other UAS delivery operations (such as the delivery of defibrillators). Each scenario was developed and validated by representatives with expertise in humanitarian operations, urban and rural emergency response, air traffic management, UAS operations, and traditional flight operations. The scenario definitions address roles and responsibilities of individual actors, the appropriate utilization of UAS, and the actions taken by those actors to appropriately manage risks associated with the mission and environment. The risks to aviation traffic and to people on the ground explored included potential risks arising from incompatibilities in calculating reference altitudes (eg, differing uses of AGL, MSL, barometric, or GPS-derived values), loss of command and control (C2) communications, rapid changes in weather and winds, and physical interference. For each risk, IASMS SFCs were postulated in the context of monitoring services, risk assessment capabilities, and identifying appropriate mitigation strategies. The identified SFC capabilities were envisioned from known services postulated for IASMS and for UTM. For these unique environments, IASMS SFCs are needed to address conditions such as hazardous payloads, micro-climates and urban canyons, and the need to keep uninvolved air traffic out of the area where DMFR operations are being conducted. The second phase of analysis focused on inferring the specific information needs and the SFCs for IASMS, utilizing a structure of 16 information classes to organize requirements. For each of the risks identified in the workshops, it was postulated what data sources would be necessary to monitor critical aspects of the risk (eg, surrounding air traffic, ground population, terrain, etc). to be directly measured as well as data that would be derived, which implies additional SFCs for different actors to understand what information would likely be exchanged between parties. For an IASMS to be effective, additional research is needed to develop the advanced algorithms that can address the increasingly autonomous and complex operations in differing environments and to develop means of identifying unknown risks. Looking at these scenarios highlighted a number of research issues. These include the ability to quickly "cordon off" airspace thru temporary flight restrictions (TFRs) or other means, developing clear definitions to enable automation-based algorithms for prioritizing operations, defining airspace density metrics, standardization of altitude reporting, and establishing a basis for safety data metrics definition and collection. This paper seeks to outline the development of an IASMS in the context of the DMFR scenarios and resulting demonstrations. Utilizing this contextual approach, NASA will generate recommendations for an assured safety framework for AAM operations that enables AAM operations to safely access the NAS.
Establishing a basis for certification of autonomous systems using trust and trustworthiness is the focus of Autonomy Teaming and TRAjectories for Complex Trusted Operational Reliability (ATTRACTOR). The Human-Machine Interface (HMI) team is working to capture and utilize the multitude of ways in which humans are already comfortable communicating mission goals and translate that into an intuitive mission planning interface. Several input/output modalities (speech/audio, typing/text, touch, and gesture) are being considered and investigated in the context human-machine teaming for the ATTRACTOR design reference mission (DRM) of Search and Rescue or (more generally) intelligence, surveillance, and reconnaissance (ISR). The first of these investigations, the Human Informed Natural-language GANs Evaluation (HINGE) data collection effort, is aimed at building an image description database to train a Generative Adversarial Network (GAN). In addition to building an image description database, the HMI team was interested if, and how, modality (spoken vs. written) affects different aspects of the image description given. The results will be analyzed to better inform the designing of an interface for mission planning.
The Group Task Force was chartered by the Administrator of NASA to recommend 'a policy outlining the criteria, the design standards, and the pricing model to guide NASA in assessing the responsibilities for government and nongovernment Satellite Rescue and Repair Missions.' Criteria for accepting such missions, risks, and benefits to all sectors of our economy involved in satellite services, adequacy of planning and training, and the impact on NASA's primary mission were reviewed. The Group began by asking a more fundamental question; is satellite rescue and repair a logical element of NASA's mission? Factors considered were: (1) the probability of rescue or repair opportunities arising; (2) the economic justification for such attempts; (3) the benefits to NASA, both from such ad hoc learning experiences in space operations and the impact on the public perception of NASA; (4) the effect of such unanticipated missions on NASA's scheduled activities; (5) any potential effect on NASA's technical capability to work in space; and (6) any potential effect on U.S. economic competitiveness.
The Manned Maneuvering Unit (MMU), a self-contained zero-gravity backpack designed for astronaut extravehicular activity, is discussed with reference to the system requirements and characteristics, and potential near-term and future uses. Attention is given to the MMU man-machine interfaces, propulsion capability, attitude control, crew restraint hardware, donning, doffing, activation, and deactivation. Specific applications discussed include: spacecraft inspection and servicing, assembly of large space systems, payload deployment/retrieval, and crew rescue.
In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c"' and dm' clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b" and b"' (2b and 3b in traditional nomenclature) die in normal development; the b" clone can be rescued to assume the normal c" fate if micromere c" or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b" does not normally die, and contributes a subset of the cells arising exclusively from c" in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c"' makes no definitive contribution, whereas micromere dm' gives rise to cells equivalent to those arising from c"' and dm' in H. robusta (Sacramento).
It is shown that communication satellites can provide significant economic benefits to shipping company operations and alleviate the problems of distress alerting, search and rescue, and losses due to collisions, groundings, and heavy weather damage. Emphasis is placed upon both the application of space technology to minimize present service deficiencies and the costs and benefits of this new application. It is concluded that while maritime service satellites appear to be viable technically and economically, there is a substantial amount of analysis, definition, and development work yet to be accomplished.
Accurate lunar navigation and timing knowledge provides for the development of safety-critical services in the cislunar and lunar surface domain. Currently under development, the Goddard Space Flight Center’s (GSFC) Search and Rescue Mission Office is investigating and integrating search and rescue (SAR) capability into planned and future lunar communication and navigation interfaces. Lunar Search and Rescue (LunaSAR) development has a stated end-goal for assured, reliable, and timely indication of distress events for a wide variety of lunar surface users, including government-sponsored, commercial, and international users. LunaSAR performance requirements are modelled after the current terrestrial Cospas-Sarsat distress notification system, leveraging an internationally robust global navigation satellite system (GNSS) ecosystem as a core element of survivor locating capability. This presentation will discuss NASA’s work to develop user-focused distress messaging capabilities including infusion of example sensor data for triggering of automated distress alerts coupled with location-tagging. Additionally, the presentation will examine overall message structures, rotating fields for use in bi-directional distress messaging, and specific use cases based on NASA’s lunar exploration and lunar communication relay architectures. Modelling and simulation of LunaSAR use by individual lunar explorers will be discussed, based on notional industry and government design reference missions and mission considerations. Results from GSFC-funded Internal Research and Development (IRAD) efforts will be detailed, including successful distress message formulation simulating the ingestion of example legacy space suit telemetry fields. Hardware-in-the-loop testing using high-reliability software defined radio (SDR) modules serve as an example of IRAD successes and the framework for technical requirements. Architectural development and technical evolution from 2020 to 2021 included alignment of LunaSAR distress waveforms with ongoing NASA LunaNet interoperability development, as well as engagement with NASA Lunar Spectrum authorities for allocation of UHF-band distress frequencies on the lunar surface. S-Band and UHF-band transmission characteristics will be detailed, along with band-specific applications of each emission type. Additionally, examples of ingestion and formatting of GNSS signals (using historical terrestrial National Marine Electronics Association-formatted GNSS data) will be detailed, underscoring lunar user needs for a common lunar GNSS receiver output message framework. Maturity and ability to support evolving lunar exploration goals has been demonstrated and will be detailed, with maturity gaps such as position, navigation, and timing (PNT) and lunar reference frames identified within the context of distress message generation. Provision of LunaSAR services for lunar surface users represents a new era of ensured safety for lunar explorers and builds off of forty years of the Cospas-Sarsat program, underscoring the importance of lunar GNSS for safety-critical applications and growing interest in safe, reliable lunar surface operations. Enabled by new GNSS systems being developed by government and industry partners, NASA will continue to evolve and integrate lunar GNSS types into distress message generation, with a focus on compact and efficient message transmission over various lunar communication links. When fielded, LunaSAR will be the first dedicated search and rescue notification system employed on another celestial body. Robust lunar navigation and timing services form the core of LunaSAR capabilities, allowing for system syncing with time-dominant sensors, and high-accuracy location of those in distress while engaged in lunar surface activities.
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.