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DOE OSTI2021

Transcriptional control of Clostridium autoethanogenum using CRISPRi

Gas fermentation by Clostridium autoethanogenum is a commercial process for the sustainable biomanufacturing of fuels and valuable chemicals using abundant, low-cost C1 feedstocks (CO and CO 2 ) from sources such as inedible biomass, unsorted and nonrecyclable municipal solid waste, and industrial emissions. Efforts toward pathway engineering and elucidation of gene function in this microbe have been limited by a lack of genetic tools to control gene expression and arduous genome engineering methods. To increase the pace of progress, here we developed an inducible CRISPR interference (CRISPRi) system for C. autoethanogenum and applied that system toward transcriptional repression of genes with ostensibly crucial functions in metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Harnessing photoautotroph-methanotroph interactions for biogas conversion to fuels and chemicals using binary consortia (Project Final Technical Report)

Industrial, municipal, and agricultural waste streams containing stranded organic carbon represent a significant and underutilized feedstock to produce fuels and chemicals. With anaerobic digestion deployed at large scales to capture organic waste streams, over 6 million tons of biogas are available today. However, the utilization of biogas represents a significant challenge due to its low pressure and presence of contaminants such as H 2 S, ammonia, and volatile organic carbon compounds. To tap into this immense potential, effective biotechnologies that co-utilize both CO 2 and CH 4 are needed. Recent studies demonstrated that, in nature, microbial communities have developed a highly efficient way to recover energy and capture carbon from both CH 4 and CO 2 through metabolic coupling of methane oxidation to oxygenic photosynthesis. Using two synthetic methanotroph – photoautotroph (M-P) co-cultures that exhibit stable growth under a broad range of cultivation conditions, in this project we proposed to harness the interspecies interactions within these cocultures for biogas conversion to fuels and chemicals. To facilitate this overarching objective, we aim to develop experimental and computational tools to gain qualitative and quantitative understandings on the interactions and dynamics of the coculture at both systems and molecular levels, and to validate our findings through experiments and mutant development. The fundamental understanding on the interactions and dynamics of the photoautotroph-methanotroph will lay the foundation for the design and optimization of synthetic binary consortia for production of fuels and chemicals from biogas. We expect the knowledge gained from this project may be generally applicable to other cross-feeding binary consortium, and the tools developed can be adapted to study the interactions and dynamics of other multi-organism platforms.

09 BIOMASS FUELS↗

DOE OSTI2023

Optimization of C. ljungdahlii for the Valorization of Combustion Off-Gas

One of the largest problems that faces the realization of the circular economy is the gap between waste streams and usable commodities. Synthetic biology enables a toolkit where this gap can begin to be addressed and closed. This work focuses on using synthetic biology techniques to optimize C. ljungdahlii for the valorization of combustion off-gas waste streams and navigates how to produce 3-hydroxybutyrate, a polymer precursor off of similar-composition syn-gas streams. If successful, these techniques can realize a carbon-negative solution to producing plastics.

3-hydroxybutyrate↗

NASA NTRS2018

Electrolyzer Exergy Analysis for an Environmental Control and Life Support System

An exergy based analysis of the Environmental Control and Life Support System (ECLSS) aboard the International Space Station (ISS) is conducted to assess its overall performance. Exergy is chosen as a measure of performance because it accounts for both the first and second laws of thermodynamics. The exergy efficiency of a system is first defined as the total exergy destroyed by the system relative to the total exergy input to the system. To determine the ECLSS exergy efficiency, the system is divided into constituent subsystems which in turn are divided into assemblies and components. Based on this system decomposition, exergy balances are derived for each assembly or component. Exergy balances and supporting calculations are implemented in MATLAB® code. The major subsystems of the ECLSS considered in this analysis include the Atmosphere Revitalization Subsystem (ARS), Atmosphere Control and Supply Subsystem (ACS), Temperature and Humidity Control Subsystem (THC), Water Recovery and Management Subsystem (WRM), and Waste Management Subsystem (WM). This paper focuses on the ARS and its constituent assemblies and components. Exergy efficiency of the ARS and its constituent assemblies and components is first presented. The Oxygen Generation Assembly (OGA), an assembly within the ARS, is then highlighted because the exergy destruction by the OGA is a large magnitude contributor to the overall exergy destruction of the ECLSS. The OGA produces oxygen to meet the crew's metabolic demand via water electrolysis in a proton exchange membrane (PEM) electrolyzer. The exergy destruction of the OGA's PEM electrolyzer is a function of the amount of oxygen produced, which determines the necessary current density and voltage drop across the PEM electrolyzer. In addition, oxygen production in the PEM electrolyzer requires deviation from the Nernst potential, presenting trade-offs between the exergy efficiency and critical life support functions. The results of parametric studies of PEM electrolyzer performance are presented with an emphasis on the impacts of polarization and operational conditions on exergy efficiency.

Chow, Raymond↗

NASA NTRS1993

Kinetic Tetrazolium Microtiter Assay

Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

Pierson, Duane L.↗

NASA NTRS2004

Effects of Gravity on Cells, Tissues, and Organisms: Their Implications on Habitat and Human Support in Microgravity

This presentation will demonstrate that gravity plays a major role in advanced human life support in a closed habitat. The examples include, but are not limited to, control of purity in drinking water supplies (application of biocides), control of urine in space rodent habitats and operation of space septic tanks (waste management). Our goal is to understand and determine possible mechanisms that describe the process by which cells anchor to a substrate to form dynamic, vibrant communities of cells which influence human health in absence of gravity. The balance of all forces (mechanotransduction) acting on a cell will determine whether a cell thrives and multiplies or dies in a process called apoptosis and/or necrosis. The balance of forces are tightly coupled to the transport of nutrients and metabolic products (biochemotransduction) to and from the cell interface. We will highlight our effort to improve astronaut health by showing that microgravity life support systems have to be designed differently from those on Earth.

Kizito, John↗

NASA NTRS1987

Space Bioreactor Science Workshop

The first space bioreactor has been designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and a slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small (500 ml) bioreactor is being constructed for flight experiments in the Shuttle middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption, and control of low shear stress on cells. Applications of microcarrier cultures, development of the first space bioreactor flight system, shear and mixing effects on cells, process control, and methods to monitor cell metabolism and nutrient requirements are among the topics covered.

Morrison, Dennis R.↗

NASA NTRS1983

Prevention of metabolic alterations caused by suspension hypokinesia in leg muscles of rats

Rats were subjected to tail-cast suspension hypokinesia for 6 days with one leg immobilized in dorsal flexion by casting. Control animals were also tail-casted. The soleus, gastrocnemius and plantaris muscles of uncasted hypokinetic legs were smaller than control muscles. Dorsal flexion prevented atrophy of these muscles and caused the soleus to hypertrophy. The anterior muscles were unaffected by hypokinesia. The smaller size of the soleus of the uncasted leg relative to the dorsal flexed and weight bearing limbs correlated with slower protein synthesis and faster proteolysis. The capacity of this muscle to synthesize glutamine (gln), which carries nitrogenous waste from muscle was also measured. Although tissue homogenates showed higher activities of gln synthetase, the rate of de novo synthesis was not altered in intact muscle but the tissue ratio of gln/glutamate was decreased. Glutamate and ATP were not limiting for gln synthesis, but availability of ammonia may be a limiting factor for this process in hypokinesia.

Tischler, M. E.↗

NASA NTRS1980

The development of a Space Shuttle Research Animal Holding Facility

The ability to maintain the well being of experiment animals is of primary importance to the successful attainment of life sciences flight experiment goals. To assist scientists in the conduct of life sciences flight experiments, a highly versatile Research Animal Holding Facility (RAHF) is being developed for use on Space Shuttle/Spacelab missions. This paper describes the design of the RAHF system, which in addition to providing general housing for various animal species, approximating the environment found in ground based facilities, is designed to minimize disturbances of the specimens by vehicle and mission operations. Life-sustaining capabilities such as metabolic support and environmental control are provided. RAHF is reusable and is a modular concept to accommodate animals of different sizes. The basic RAHF system will accommodate a combination of 24 500-g rats or 144 mice or a mixed number of rats and mice. An alternative design accommodates four squirrel monkeys. The entire RAHF system is housed in a single ESA rack. The animal cages are in drawers which are removable for easy access to the animals. Each cage contains a waste management system, a feeding system and a watering system all of which will operate in zero or one gravity.

Jagow, R. B.↗

NASA NTRS1994

Life support systems analysis and technical trades for a lunar outpost

The NASA/JPL life support systems analysis (LISSA) software tool was used to perform life support system analysis and technology trades for a Lunar Outpost. The life support system was modeled using a chemical process simulation program on a steady-state, one-person, daily basis. Inputs to the LiSSA model include metabolic balance load data, hygiene load data, technology selection, process operational assumptions and mission parameter assumptions. A baseline set of technologies has been used against which comparisons have been made by running twenty-two cases with technology substitutions. System, subsystem, and technology weights and powers are compared for a crew of 4 and missions of 90 and 600 days. By assigning a weight value to power, equivalent system weights are compared. Several less-developed technologies show potential advantages over the baseline. Solid waste treatment technologies show weight and power disadvantages but one could have benefits associated with the reduction of hazardous wastes and very long missions. Technology development towards reducing the weight of resupplies and lighter materials of construction was recommended. It was also recommended that as technologies are funded for development, contractors should be required to generate and report data useful for quantitative technology comparisons.

Ferrall, J. F.↗

NASA NTRS2012

Evaluation of Sorbents for Acetylene Separation in Atmosphere Revitalization Loop Closure

State-of-the-art carbon dioxide reduction technology uses a Sabatier reactor to recover water from metabolic carbon dioxide. In order to maximize oxygen loop closure, a byproduct of the system, methane, must be reduced to recover hydrogen. NASA is currently exploring a microwave plasma methane pyrolysis system for this purpose. The resulting product stream of this technology includes unreacted methane, product hydrogen, and acetylene. The hydrogen and the small amount of unreacted methane resulting from the pyrolysis process can be returned to the Sabatier reactor thereby substantially improving the overall efficiency of the system. However, the acetylene is a waste product that must be removed from the pyrolysis product. Two materials have been identified as potential sorbents for acetylene removal: zeolite 4A, a commonly available commercial sorbent, and HKUST-1, a newly developed microporous metal. This paper provides an explanation of the rationale behind acetylene removal and the results of separation testing with both materials

Abney, Morgan B.↗

NASA NTRS2011

Evaluation of Sorbents for Acetylene Separation in Atmosphere Revitalization Loop Closure

Abney, Morgan B.↗

NASA NTRS2009

Providing Oxygen for the Crew of a Lunar Outpost

Oxygen (O2) is obviously essential for human space missions, but it is important to examine all the different ways it will be used and the potential sources that it may come from. This effort will lead to storage and delivery requirements and help to determine the optimum architecture from an overall systems engineering point of view. Accounting for all the oxygen in a Lunar Outpost mission includes meeting the metabolic needs of the crew while in the surface Habitat, leakage through the Habitat or Pressurized Rover walls, recharge of the space suit backpack and emergency situations. Current plans indicate that both primary and secondary O2 bottles for the space suit will be filled to a pressure of 20.7 MPa (3000 psia). Other uses of O2 require much lower pressure. Sources of O2 at a Lunar Outpost include compressed or liquefied O2 brought along specifically for life support, scavenged O2 from the Lander propulsion system, recovered O2 from waste water or exhaled carbon dioxide and O2 mined from the moon itself. Previously, eight technology options were investigated to provide the high pressure space suit O2. High pressure O2 storage was treated as the baseline technology and compared to the other seven. The other seven were cryogenic storage followed by high pressure vaporization, scavenging liquid oxygen (LOX) from Lander followed by vaporization, LOX delivery followed by sorption compression, low pressure water electrolysis followed by mechanical compression, high pressure water electrolysis, sharing a high pressure electrolyzer with a regenerative fuel cell power system, and making use of In- Situ Resource Utilization (ISRU). This system-level analysis was conducted by comparing equivalent system mass of the eight technologies in open and closed loop life support architectures. The most promising high pressure O2 generation technologies were recommended for development. Updates and an expansion of the earlier study have been made and the results are reported in this paper. Examples of recent analyses include feasibility of recovering space suit purge O2 in a Pressurized Rover and using sub-critical LOX for primary supply while using high-pressure gaseous O2 as the secondary supply for the space-suit. Preliminary analysis on scavenging LOX from the Lander and delivering it to the Habitat has also been incorporated into this integrated lunar fluids analysis

Ewert, Michael K.↗

DOE OSTI2024

Genome reduction improves octanoic acid production in scale down bioreactors

Microorganisms in large-scale bioreactors are exposed to heterogeneous environmental conditions due to physical mixing constraints. Nutritional gradients can lead to transient expression of energetically wasteful stress responses and as a result, can reduce the titres, rates and yields of a bioprocess at larger scales. To what extent these process parameters are impacted is often unknown and therefore bioprocess scale-up comes with major risk. Designing platform strains to account for these intermittent stresses before introducing synthesis pathways is one strategy for de-risking bioprocess development. For example, Escherichia coli strain RM214 is a derivative of wild-type MG1655 that has had several genes and whole operons removed from its genome based on their metabolic cost. In this study, we engineered E. coli strain RM214 (referred to as WG02) to produce octanoic acid from glycerol in batch-flask and fed-batch bioreactor cultivations and compared it to an octanoic acid-producing E. coli MG1655 (WG01). In batch flask cultivations, the two strains performed similarly. However, in carbon limited fed-batch bioreactor cultivations, WG02 provided a greater than 22% boost to biomass compared to WG01 while maintaining similar titres of octanoic acid. Reducing the biomass accumulation of WG02 with nitrogen limited fed-batch cultivation resulted in a 16% improvement in octanoic acid titre over WG01. Finally, in a scale-down system consisting of a stirred tank reactor (representing a well-mixed zone) and plug flow reactor (representing an intermittent carbon starvation zone), WG02 again improved octanoic acid titre by almost 18% while maintaining similar biomass concentrations as WG01.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Developing a Carbon Negative Biorefinery for Organic Waste Valorization

Production of bio-based chemicals have become increasingly attractive as efforts to meet carbon neutrality goals expand. Diverse organic waste feedstocks can be valorized via arrested anaerobic digestion and chain elongation to produce important key intermediates, such as medium chain carboxylic acids. Our work aims to develop a carbon negative biorefinery that funnels multiple organic waste feedstocks into a chemically consistent stream of carboxylic acids that are then upgraded to exemplary carbon negative products. The pairing of a hydrolysis reactor with a chain elongation reactor will allow each biological step to be optimized to improve the ability to valorize a variety of organic waste streams. Specifically, this work has so far been aimed at screening for potential chain elongating organisms to produce VFAs and MCCAs of interest. Four chain elongating organisms were tested for their chain elongation potential with diverse single and mixed substrates. So far, Megasphaera elsdenii and Actinobacillus succinogenes have been tested to determine potential differences in titer as well as product speciation due to variations in pH. To do this, each organism was tested under 3 different substrate combinations with pH maintained at either 5.5, 6, or 7. With better understanding of their metabolic needs and optimal operating conditions, these chain elongating organisms could provide a valuable option to facilitate the chain elongation necessary to produce precursor molecules. In addition to optimizing the bioconversion steps, downstream processing of carboxylic acids is also a key component for the overall feasibility of the process. Our group previously developed a downstream in-situ product recovery (ISPR) process for continuously recovering bio-based carboxylic acids from fermentation broth. The ISPR includes: (i) a solid-liquid separation as a cell retention device, (ii) a liquid-liquid extraction (LLE) to selectively extract the desired bio-based acids, and (iii) a distillation to obtain the neat product. The integrated process was demonstrated at bench-scale and is now scaled up for pilot-scale operations. A more cost-efficient membrane-based emulsion separation is introduced for LLE in downstream separation process with greatly promoted mass transfer, leading to -2800 times smaller needed membrane area than membrane contactors to achieve the same butyric acid extraction rate.

BIOMASS FUELS↗

DOE OSTI2021

High-Performance Piezo-Electrocatalytic Sensing of Ascorbic Acid with Nanostructured Wurtzite Zinc Oxide

We report nanostructured piezoelectric semiconductors offer unprecedented opportunities for high-performance sensing in numerous catalytic processes of biomedical, pharmaceutical, and agricultural interests, leveraging piezocatalysis that enhances the catalytic efficiency with the strain-induced piezoelectric field. Here, we design and demonstrate for the first time a cost-efficient, high-performance piezo-electrocatalytic sensor for detecting L-ascorbic acid (AA), a critical chemical for many organisms, metabolic processes, and medical treatments. We prepared ZnO nanorods and nanosheets to characterize and compare their efficacy for the piezo-electrocatalysis of AA. The electrocatalytic efficacy of AA was significantly boosted by the piezoelectric polarization induced in the nanostructured semiconducting ZnO catalysts. We elucidated the charge transfer between the strained ZnO nanostructures and AA to reveal the mechanism for the related piezo-electrocatalytic process. The low-temperature synthesis of high-quality ZnO nanostructures allows the low-cost, scalable production and integration directly into wearable electrocatalytic sensors whose performance could be boosted by otherwise wasted mechanical energy from the working environment, e.g., the human-generated mechanical signals.

30 DIRECT ENERGY CONVERSION↗

DOE OSTI2021

Genomic and functional analyses of fungal and bacterial consortia that enable lignocellulose breakdown in goat gut microbiomes

Abstract The herbivore digestive tract is home to a complex community of anaerobic microbes that work together to break down lignocellulose. These microbiota are an untapped resource of strains, pathways and enzymes that could be applied to convert plant waste into sugar substrates for green biotechnology. We carried out more than 400 parallel enrichment experiments from goat faeces to determine how substrate and antibiotic selection influence membership, activity, stability and chemical productivity of herbivore gut communities. We assembled 719 high-quality metagenome-assembled genomes (MAGs) that are unique at the species level. More than 90% of these MAGs are from previously unidentified herbivore gut microorganisms. Microbial consortia dominated by anaerobic fungi outperformed bacterially dominated consortia in terms of both methane production and extent of cellulose degradation, which indicates that fungi have an important role in methane release. Metabolic pathway reconstructions from MAGs of 737 bacteria, archaea and fungi suggest that cross-domain partnerships between fungi and methanogens enabled production of acetate, formate and methane, whereas bacterially dominated consortia mainly produced short-chain fatty acids, including propionate and butyrate. Analyses of carbohydrate-active enzyme domains present in each anaerobic consortium suggest that anaerobic bacteria and fungi employ mostly complementary hydrolytic strategies. The division of labour among herbivore anaerobes to degrade plant biomass could be harnessed for industrial bioprocessing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Progress Towards a New Model Chemolithoautotrophic Host (CRADA Final Report)

Some of the largest scale chemical processes for the production of important commodity chemicals and fuels involve an H 2 -containing gas mixture as an intermediate [e.g. ammonia, urea, methanol, Fischer-Tropsch (F-T) diesel, and hydrotreatment of crude petroleum to refined fuels]. The successful development of a model H 2 -oxidizing chemoautotrophic host could expand the range of fuels and chemicals produced from H 2 and CO 2 intermediates in the chemical, oil, and gas sectors in the near term, as well as from renewable and waste-derived sources of these gases that are expected to greatly expand in coming years. Among non-photosynthetic bacteria that can utilize H 2 and CO 2 , Cupriavidus necator (C. necator, formerly Ralstonia eutropha), is the best studied. C. necator is an excellent microbial host for the production of a variety of chemicals because it grows extremely quickly to very high cell densities autotrophically on H 2 and CO 2 , is genetically tractable, and has the ability to accumulate polymers, such as polyhydroxybutyrate, at industrial levels. Despite having great potential as a platform bioproduction host, genetic tools are limited, making metabolic engineering of this organism slow and laborious. In this CRADA project, we developed a number of genetic tools for C. necator: 1) Improvement of C. necator genetic transformation efficiency 2) Integration of heterologous genes into the C. necator chromosome and development of promoter library 3) Development of graded RBSs to control heterologous protein expression 4) Use of RBSs to demonstrate fatty alcohol production in C. necator 5) Demonstration of CRISPR-Cas9 gene editing in C. necator

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗