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DOE OSTI2025

Characterization of Gas-Phase Native(-like) Proteins Using Structures for Lossless Ion Manipulations

High-resolution mobility-based ion separations in Structures for Lossless Ion Manipulations (SLIM) have been useful for ion mobility separations for a variety of molecular classes in the gas phase. Here, in this study, we present multipass SLIM separations for gas-phase proteins in their near-native state exhibiting charge-state-dependent arrival time distributions using carbonic anhydrase (29 kDa), alcohol dehydrogenase (148 kDa), and apo-transferrin (79 kDa). The experimental CCS values were obtained from calibration curves for the arrival times of Agilent Tune Mix ions. For multipass separations, the ATDs were converted to CCS values by deconvoluting the multipass arrival times into accurate single-pass values amenable to the single-pass calibration curves. Mass spectra of carbonic anhydrase (CA) showed three different charge states (z = 9+ to 11+). Their corresponding mobility peaks were baseline-separated by using 8-m single-pass separations. When compared to the corresponding drift tube ion mobility (DTIMS) measurements, the CCS values obtained from DTIMS and SLIM were in agreement within experimental error. Single-pass analysis of alcohol dehydrogenase (ADH) exhibits three predominant charge states (z = 23+ to 25+) with mobility overlap between adjacent charge states. The mobility peak resolution for ADH improved with multipass separations (up to 24-m path length). In addition, CCS distributions obtained for charge states z = 16+ to 18+ of apo-transferrin reveal a transition from a compact unimodal form (z = 18+ and 19+) to broader multimodal CCS distributions for z = 16+. For apo-transferrin, 40-m multipass separations were performed allowing for complete isolation of the selected mobility range corresponding to z = 17+, leading to selective isolation of a narrow arrival time window. The extended mobility separations provided minimal alterations to the structure of the proteins, and the experimentally derived CCS values showed minimal change as a function of the separation time or number of passes. Mobility-based ion separations for native-like proteins, using SLIM, open opportunities for native-IMS applications as well as other manipulations enabled by SLIM-like mobility-selective isolation and collection.

charge state distribution↗

DOE OSTI2022

Anatomy of Protein Electrospray Ionization Mass Spectra by Superconducting Tunnel Junction Mass and Energy Spectrometry

Cryogenic superconducting tunnel junction (STJ) detectors have the advantage of single-particle sensitivity, high quantum efficiency, low noise, and the ability to detect the time and relative impact energy of deposited ions. This makes them attractive for use in mass spectrometry (MS) and as a form of energy spectrometry. STJ cryodetectors have been coupled to time-offlight (TOF) mass spectrometers equipped with a matrix-assisted laser desorption ionization (MALDI) source and to an electrospray ionization (ESI) TOF mass spectrometer. Here, a lab-made linear ion trap (LIT) mass spectrometer system was coupled to an ESI source and a 16-channel Nb-STJ array with improved readout electronics. The goal was to investigate fundamentals of ESI-generated protein ions by further exploiting the advantage of resolving these ions in a third dimension of the relative energy deposited into the STJs. The proteins equine cytochrome c, bovine carbonic anhydrase, bovine serum albumin, and murine immunoglobulin G were studied using this ESI-LIT-STJ-MS instrument. Multiply charged monomers, multimers, and fragments from metastable ions were resolved from monomer peaks by differences in ion deposition energy even when these ions have the same mass-to-charge ratio as the corresponding monomer. The determination of fragment mass from metastable decomposition is accomplished without knowing the charge state of the fragment. The average charge state of the multimers is reduced with each addition of a protein which is a direct reflection of the surface area available for charging. Multiply charged in-source fragments have also been observed and distinguished in the mass spectrum of carbonic anhydrase by using the differences in the energy deposited in the STJs.

42 ENGINEERING↗

DOE OSTI2025

Contrast Matching Biopolymers: A SANS-Based Approach to Structural Characterization of Chitosan

Immobilizing enzymes in polysaccharide-based matrices has been shown to improve both their stability and their catalytic efficiency. Among available materials, chitosan was selected for its abundance, biocompatibility, and versatility in a range of applications. While the broader aim of this work is to study chitosan as a matrix for immobilizing carbonic anhydrase (CA) for CO₂ capture, this study focused on determining the contrast match point (CMP) of hydrogenated and deuterated chitosan in D₂O:H₂O mixtures to enable future structural investigations using small-angle neutron scattering (SANS). Previous work successfully synthesized deuterated chitosan from Rhizopus oryzae mycelia and assessed the degree of deuteration using Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). Building on this foundation, the current study determined the CMPs of both H-chitosan and D-chitosan by performing SANS measurements across a range of D₂O:H₂O solvent contrasts. The extracted CMPs for both forms correlated well with their respective calculated scattering length densities (SLDs), validating the experimental approach. These results establish a critical reference for selective contrast matching in future SANS studies of carbonic anhydrase immobilized in chitosan matrices, enabling detailed structural analysis of enzyme–matrix interactions.

Kosgallana, Chathurika J [ORNL] (ORCID:00000002851↗

DOE OSTI2021

Diploid genomic architecture of Nitzschia inconspicua, an elite biomass production diatom

Abstract A near-complete diploid nuclear genome and accompanying circular mitochondrial and chloroplast genomes have been assembled from the elite commercial diatom species Nitzschia inconspicua . The 50 Mbp haploid size of the nuclear genome is nearly double that of model diatom Phaeodactylum tricornutum , but 30% smaller than closer relative Fragilariopsis cylindrus . Diploid assembly, which was facilitated by low levels of allelic heterozygosity (2.7%), included 14 candidate chromosome pairs composed of long, syntenic contigs, covering 93% of the total assembly. Telomeric ends were capped with an unusual 12-mer, G-rich, degenerate repeat sequence. Predicted proteins were highly enriched in strain-specific marker domains associated with cell-surface adhesion, biofilm formation, and raphe system gliding motility. Expanded species-specific families of carbonic anhydrases suggest potential enhancement of carbon concentration efficiency, and duplicated glycolysis and fatty acid synthesis pathways across cytosolic and organellar compartments may enhance peak metabolic output, contributing to competitive success over other organisms in mixed cultures. The N. inconspicua genome delivers a robust new reference for future functional and transcriptomic studies to illuminate the physiology of benthic pennate diatoms and harness their unique adaptations to support commercial algae biomass and bioproduct production.

09 BIOMASS FUELS↗

DOE OSTI2024

Final Report: Enhanced Algal Production of CA for Improved Atmospheric Delivery of CO2 To Ponds

Technologies that enable direct-air-capture (DAC) of CO2 and eliminate the need for a CO2 capture, storage, and distribution system would significantly reduce the cost of algal production, and greatly increase the volume of algae biomass that can be produced by enabling algae farms to be located anywhere. Such technologies include cultivation under high alkalinity/high pH conditions, which increase the driving force for CO2 absorption, and development of genetic tools and genetically engineered strains to decorate the surface of the algae with carbonic anhydrase (CA), enable secretion of CA by the algae, or more generally boost the performance of the carbon concentrating mechanism (CCM).

09 BIOMASS FUELS↗

NASA NTRSDate not supplied

CO2-Based Manufacturing System for Recombinant Protein Production

Space biomanufacturing is a potential In Situ Resource Utilization (ISRU) strategy to provide critical consumables and products while minimizing the launched mass for long-duration, deep space missions. On Earth, the primary biological conversion of CO 2 to biomass is through photosynthesis, and sugars from photosynthetic organisms are used as feedstocks for microbial biomanufacturing. The efficiency of non-biological reduction of CO 2 to organic molecules, such as acetate or ethanol, has greatly increased in recent years. We are designing a biomanufacturing system to rely on electrochemical CO 2 conversion products for carbon substrates to support microbial growth and production of recombinant proteins. The preliminary design includes a gas-permeable membrane bioreactor with dry salts that are rehydrated and mixed with the carbon source to support growth of bacteria or yeast. The system architecture has a partially automated bioprocessing system to concentrate biomass and purify recombinant protein. This system is designed to operate semi-autonomously with minimal crew intervention. The specific use-case scenario is to produce a thermal stable carbonic anhydrase to increase the efficiency of a proposed liquid amine CO 2 removal subsystem of an environmental control and life-support system (ECLSS) on Mars.

Biomanufacturing↗

NASA NTRSDate not supplied

Development of a Space Compatible Biomanufacturing System

Recombinant↗

DOE OSTI2023

Biocatalytic Membranes for Carbon Capture and Utilization

Innovative carbon capture technologies that capture CO 2 from large point sources and directly from air are urgently needed to combat the climate crisis. Likewise, corresponding technologies are needed to convert this captured CO 2 into valuable chemical feedstocks and products that replace current fossil-based materials to close the loop in creating viable pathways for a renewable economy. Biocatalytic membranes that combine high reaction rates and enzyme selectivity with modularity, scalability, and membrane compactness show promise for both CO 2 capture and utilization. This review presents a systematic examination of technologies under development for CO 2 capture and utilization that employ both enzymes and membranes. CO 2 capture membranes are categorized by their mode of action as CO 2 separation membranes, including mixed matrix membranes (MMM) and liquid membranes (LM), or as CO 2 gas–liquid membrane contactors (GLMC). Because they selectively catalyze molecular reactions involving CO 2 , the two main classes of enzymes used for enhancing membrane function are carbonic anhydrase (CA) and formate dehydrogenase (FDH). Small organic molecules designed to mimic CA enzyme active sites are also being developed. CO 2 conversion membranes are described according to membrane functionality, the location of enzymes relative to the membrane, which includes different immobilization strategies, and regeneration methods for cofactors. Parameters crucial for the performance of these hybrid systems are discussed with tabulated examples. Progress and challenges are discussed, and perspectives on future research directions are provided.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Eight Up-Coming Biotech Tools to Combat Climate Crisis

Biotechnology has a high potential to substantially contribute to a low-carbon society. Several green processes are already well established, utilizing the unique capacity of living cells or their instruments. Beyond that, the authors believe that there are new biotechnological procedures in the pipeline which have the momentum to add to this ongoing change in our economy. Eight promising biotechnology tools were selected by the authors as potentially impactful game changers: (i) the Wood–Ljungdahl pathway, (ii) carbonic anhydrase, (iii) cutinase, (iv) methanogens, (v) electro-microbiology, (vi) hydrogenase, (vii) cellulosome and, (viii) nitrogenase. Some of them are fairly new and are explored predominantly in science labs. Others have been around for decades, however, with new scientific groundwork that may rigorously expand their roles. In the current paper, the authors summarize the latest state of research on these eight selected tools and the status of their practical implementation. We bring forward our arguments on why we consider these processes real game changers.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1992

Histochemical discrimination of fibers in regenerating rat infraorbital nerve

In rat dorsal root ganglia, histochemical staining of carbonic anhydrase (CA) and cholinesterase (CE) yields a reciprocal pattern of activity: Sensory processes are CA positive and CE negative, whereas motor processes are CA negative and CE positive. In rat infraorbital nerve (a sensory peripheral nerve), we saw extensive CA staining of nearly 100% of the myelinated axons. Although CE reactivity in myelinated axons was extremely rare, we did observe CE staining of unmyelinated autonomic fibers. Four weeks after transection of infraorbital nerves, CA-stained longitudinal sections of the proximal stump demonstrated 3 distinct morphological zones. A fraction of the viable axons retained CA activity to within 2 mm of the distal extent of the stump, and the stain is capable of resolving growth sprouts being regenerated from these fibers. Staining of unmyelinated autonomic fibers in serial sections shows that CE activity was not retained as far distally as is the CA sensory staining.

NASA Discipline Musculoskeletal↗

DOE OSTI2026

Hybrid biophysical systems for atmospheric CO 2 capture

Negative emissions technologies will be essential for limiting anthropogenic global temperature increases to 2 °C in the later years of the 21st century. Carbonic anhydrase (CA) metalloenzymes catalyze the otherwise slow conversion of CO 2 into carbonic acid (H 2 CO 3 ), suggesting their utility in the rapid hydration and downstream capture of dissolved CO 2 in aqueous media for a variety of CO 2 capture methods, such as thermal and pH swings and mineralization. The possibility of driving the rapid capture of CO 2 by catalyzing the CO 2 hydration bottleneck carries real potential for realizing efficient direct air capture (DAC) and direct ocean capture (DOC) systems. However, scaled application of CAs will be dependent on some way of economically sourcing the enzymes at volumes relevant to scaled DAC/DOC operations. In this perspective, we consider the prospect of catalyzing CO 2 hydration using a CA that is bound to the outer membrane of a cyanobacterial host, engineered constructs we call CyCAMs. Coupling extracellular CA expression to a marine, photosynthetic cyanobacterial host results in a CO 2 hydration catalyst that is continuously refreshed during the bacterial growth cycle, without the need for enzyme purification or harvesting steps. In the case of marine cyanobacteria, sunlight and the solutes native to seawater provide the respective energy, nutrients and sources of inorganic carbon essential to maintaining the bacterial population. We consider the potential impact of CA-mediated CO 2 hydration on a variety of DAC processes, with CO 2 mineralization explored as a primary use case.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Evaluating the economic impacts of using enzymatic membranes to optimize bicarbonate production and delivery to open raceway ponds

Gas sparging is the most commonly used method for delivering CO2 to algal cultures in Open Raceway Ponds (ORPs). While this method is well-tested and effective, these systems experience large amounts of outgassing to the surrounding atmosphere and often achieve carbon utilization efficiencies (CUE) lower than 30%. With such large losses, system are overdesigned to provide CO2 gas in extreme excess, resulting in increased capital and operational expenses. This research is focused on the development, testing, and implementation of a hydrogel membrane seeded with the enzyme carbonic anhydrase and designed to convert gaseous CO2 into aqueous bicarbonate and deliver this inorganic carbon (iC) directly to the algae pond. Despite increased capital expenses for the module, bicarbonate storage tank, and added pumps and infrastructure, preliminary results suggest that the membrane module has the potential to reduce the minimum biomass selling price by 35% by significantly reducing the operational expenditure for CO2.

09 BIOMASS FUELS↗

DOE OSTI2024

Modeling bacterial microcompartment architectures for enhanced cyanobacterial carbon fixation

The carboxysome is a bacterial microcompartment (BMC) which plays a central role in the cyanobacterial CO 2 -concentrating mechanism. These proteinaceous structures consist of an outer protein shell that partitions Rubisco and carbonic anhydrase from the rest of the cytosol, thereby providing a favorable microenvironment that enhances carbon fixation. The modular nature of carboxysomal architectures makes them attractive for a variety of biotechnological applications such as carbon capture and utilization. In silico approaches, such as molecular dynamics (MD) simulations, can support future carboxysome redesign efforts by providing new spatio-temporal insights on their structure and function beyond in vivo experimental limitations. However, specific computational studies on carboxysomes are limited. Fortunately, all BMC (including the carboxysome) are highly structurally conserved which allows for practical inferences to be made between classes. Here, we review simulations on BMC architectures which shed light on (1) permeation events through the shell and (2) assembly pathways. These models predict the biophysical properties surrounding the central pore in BMC-H shell subunits, which in turn dictate the efficiency of substrate diffusion. Meanwhile, simulations on BMC assembly demonstrate that assembly pathway is largely dictated kinetically by cargo interactions while final morphology is dependent on shell factors. Overall, these findings are contextualized within the wider experimental BMC literature and framed within the opportunities for carboxysome redesign for biomanufacturing and enhanced carbon fixation.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Bioreactor Development for CO2-Based In Situ Resource Utilization Manufacturing

Sustainable long-duration manned missions on both the Moon and Mars will require in situ resource utilization (ISRU). Carbon dioxide (CO2) has great potential as a harvestable resource, making up 95% of the atmosphere on Mars and being produced as respiratory waste in spacecraft and future planetary habitats. Through ISRU, biomanufacturing has the capability to produce a near limitless array of products from local space resources, which include pharmaceuticals, bioplastics, chemical feedstocks, and industrial enzymes. Here, a CO2-based ISRU recombinant protein bioreactor and associated biomanufacturing organisms were designed to produce a highly stable carbonic anhydrase (CA). Initial work characterized candidate organisms for growth on acetate and formic acid, carbon substrates that can be synthesized via electrochemical conversion of CO2. To improve growth on the CO2 producing substrate formic acid and for direct integration of ISRU CO2, a synthetic Calvin-Benson-Bassam cycle was designed for use in Cyberlindnera jadinii and Escherichia coli. Genetic modifications in E. coli will be facilitated by a tailored CRISPR/Cas9 and λ red recombineering two-vector system. For expression of CA, a blue light regulated T7 promoter was employed for dynamic and small molecule free induction. Efficient bioproduction through a fed-batch exponential feeding strategy was determined via mass balance calculations from ISRU substrates to biomass and CA yield. Flux balance analysis was used to model ISRU substrate metabolism and metabolic pathway engineering in candidate organisms under cultivation strategy conditions for both metabolism reconstruction and pathway design optimization. Finally, a small-scale, disposable bag bioreactor concept for use in the NASA Bioculture System infrastructure was designed to enable CO2-based CA production in reduced-gravity environments.

Biomanufacturing, Pathway Engineering, Flux Balanc↗

NASA NTRSDate not supplied

Bioreactor Development for CO2-Based In Situ Resource Utilization Manufacturing

Sustainable long-duration manned missions on both the Moon and Mars will require in situ resource utilization (ISRU). Carbon dioxide (CO2) has great potential as a harvestable resource, making up 95% of the atmosphere on Mars and being produced as respiratory waste in spacecraft and future planetary habitats. Through ISRU, biomanufacturing has the capability to produce a near limitless array of products from local space resources. Here, a CO2-based ISRU recombinant protein producing bioreactor and associated biomanufacturing organisms were designed to produce a highly stable carbonic anhydrase (CA). Candidate organisms were selected by growth characterization on acetate and formic acid, carbon substrates that are synthesized via electrochemical conversion of CO2. To improve growth on the CO2 producing substrate formic acid and for direct integration of ISRU CO2, a synthetic Calvin-Benson-Bassam cycle was designed for use in Escherichia coli. Multiplex genetic modification in E. coli was facilitated by a tailored CRISPR/Cas9 and λ red recombineering two-vector system. For expression of CA, a blue light regulated T7 promoter was employed for dynamic and small molecule free induction. Efficient bioproduction through a fed-batch exponential feeding strategy was determined via mass balance analysis from ISRU substrates to biomass and CA yield. Flux balance analysis was used to model ISRU substrate metabolism and metabolic pathway engineering in candidate organisms under cultivation strategy conditions for both metabolism reconstruction and pathway design optimization. Finally, a small-scale, disposable bag bioreactor for use in the NASA Bioculture System infrastructure was designed to enable CO2-based CA biomanufacturing in reduced-gravity environments.

biomanufacturing↗

DOE OSTI2025

Role of Histidine‐Containing Peptoids in Accelerating the Kinetics of Calcite Growth

Carbonate mineralization, the conversion of CO 2 into stable, thermodynamically favorable carbonate minerals, offers a promising strategy for permanent and environmentally friendly carbon storage, with minimal risk of long-term leakage and minimal monitoring requirements. Drawing inspiration from carbonic anhydrase (CA), a family of zinc-containing metalloenzymes that catalyze the hydration of CO 2 to bicarbonate and promote carbonate precipitation, a class of histidine-containing peptoids was designed that is capable of coordinating with Zn 2+ ions to act as CA mimetics for accelerating calcite step growth. In situ atomic force microscopy (AFM) measurements reveal that these peptoids significantly enhance step advancement, with a more pronounced effect observed when combined with Zn 2+ ions and under higher calcium-to-carbonate activity ratios, indicating that peptoids facilitate the incorporation of CO 3 2− ions at step edges. Solution NMR and 3D atomic force microscopy (3D AFM) analyses show that the coordination of peptoids with Zn 2+ promotes both the deprotonation of HCO 3 − to CO 3 2− and restructures the interfacial hydration layers of calcite, collectively lowering the activation barrier for step growth. These findings establish a design framework for sequence-defined polymers to regulate carbonate mineralization, offering promising applications in CO 2 capture and long-term storage.

CO2 mineralization↗

DOE OSTI2026

Breakthrough Zn(II) Catalyst for Direct Air Capture Employing CO 2 Hydration

Direct air capture (DAC) represents a vital technology for atmospheric CO₂ remediation, but few studies have tested catalysts at dilute atmospheric CO₂ concentrations. Inspired by the carbonic anhydrase metalloenzyme, we report a catalytic DAC strategy employing robust zinc(II) enzyme mimics that enable efficient CO₂ sequestration pathways. A catalyst-mediated CO₂ hydration cycle in aqueous sorbents facilitates accelerated capture from dilute atmospheric air, thereby addressing the kinetic limitations observed in carbonate-based systems. Our developed complexes [ZnC1] and [ZnC2] enhance capture rates up to two-fold at millimolar concentrations and improve CO₂ mass transfer by 40-60% in 1 M K₂CO₃ sorbent under ambient conditions. These bench-stable, earth-abundant zinc catalysts operate effectively under dilute CO₂ concentrations, overcoming the kinetic limitations of conventional carbonate-based sorbents. As a result, mechanistic studies support a biomimetic catalytic cycle that facilitates rapid CO₂ conversion, demonstrating that catalyst-assisted DAC can enable energy-efficient, scalable carbon capture technologies.

Atmospheric chemistry↗

DOE OSTI2022

Role of carboxysomes in cyanobacterial CO 2 assimilation: CO 2 concentrating mechanisms and metabolon implications

Many carbon-fixing organisms have evolved CO 2 concentrating mechanisms (CCMs) to enhance the delivery of CO 2 to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O 2 . These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called ‘carboxysome’ in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. Further, the cyanobacterial CCM is capable of increasing CO 2 around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO 2 . The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin–Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO 2 fixations. Research on CCM-associated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.

59 BASIC BIOLOGICAL SCIENCES↗