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DOE OSTI2024

Evaluate Synergies of Using Hydrothermal Liquefaction and Anerobic Digestion Treatment Technologies for Wastewater Resource Recovery Facilities (CRADA 516 Final Report)

The research focuses on utilizing a new anaerobic digestion (AD) configuration to treat the aqueous by-product generated by hydrothermal liquefaction (HTL) of sewage sludge. This report found that for Anaerobic Digestion for HTL By-product, Anaerobic biofilms can degrade some HTL wastewater contaminants, but co-digestion is essential to address nutrient deficiencies and optimize performance. Without AD, toxicity of HTL aqueous streams may limit broader adoption in wastewater treatment plants (WWTPs). Great Lakes Water Authority (GLWA) used an innovative reactor design, involving a dynamic membrane anaerobic bioreactor to promote biofilm growth, improving contaminant degradation. The tree-like structure inside the reactor supports biofilm development with recirculation enhancing microbial activity. Overall, a 70% chemical oxygen demand (COD) removal was achieved, although nutrient supplementation is required for stability. The reactor achieved a diverse microbial community, including methanogens and bacteria capable of degrading phenols and aromatics.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2023

Insights into the physiological and genomic characterization of three bacterial isolates from a highly alkaline, terrestrial serpentinizing system

The terrestrial serpentinite-hosted ecosystem known as “The Cedars” is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < -550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars (‘Paeni-Cedars’), Alishewanella sp. BS5-314 (‘Ali-BS5-314’), and Anaerobacillus sp. CMMVII (‘Anaero-CMMVII’). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7–10 (minimum pH tested was 7), temperatures 20–40°C, and 0–3% NaCl concentration. The addition of 10–20 mM CaCl 2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10–12 and temperatures 10–40°C, with limited growth observed 1–5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9–12, with the addition of 0.01–1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1994

Factors influencing organic carbon preservation in marine sediments

The organic matter that escapes decomposition is buried and preserved in marine sediments, with much debate as to whether the amount depends on bottom-water O2 concentration. One group argues that decomposition is more efficient with O2, and hence, organic carbon will be preferentially oxidized in its presence, and preserved in its absence. Another group argues that the kinetics of organic matter decomposition are similar in the presence and absence of O2, and there should be no influence of O2 on preservation. A compilation of carbon preservation shows that both groups are right, depending on the circumstances of deposition. At high rates of deposition, such as near continental margins, little difference in preservation is found with varying bottom-water O2. It is important that most carbon in these sediments decomposes by anaerobic pathways regardless of bottom-water O2. Hence, little influence of bottom-water O2 on preservation would, in fact, be expected. As sedimentation rate drops, sediments deposited under oxygenated bottom water become progressively more aerobic, while euxinic sediments remain anaerobic. Under these circumstances, the relative efficiencies of aerobic and anaerobic decomposition could affect preservation. Indeed, enhanced preservation is observed in low-O2 and euxinic environments. To explore in detail the factors contributing to this enhanced carbon preservation, aspects of the biochemistries of the aerobic and anaerobic process are reviewed. Other potential influences on preservation are also explored. Finally, a new model for organic carbon decomposition, the "pseudo-G" model, is developed. This model couples the degradation of refractory organic matter to the overall metabolic activity of the sediment, and has consequences for carbon preservation due to the mixing together of labile and refractory organic matter by bioturbation.

Non-NASA Center↗

DOE OSTI2020

Tumor Microenvironment Targeting Nano–Bio Emulsion for Synergistic Combinational X-Ray PDT with Oncolytic Bacteria Therapy

Various cancer therapies have been developed, but tumor recurrence with incomplete tumor killing and remaining tumor cells/tissues is frequent in monotherapies. Herein, a nano–bio therapeutic emulsion formulated with multifunctional nanoscintillators and anaerobic Clostridium novyi-NT spores for synergistic image-guided combinational cancer therapy is reported. MRI visible nanoscintillators (NSs) are synthesized with a NaGdF 4 :Tb,Ce@NaGdF 4 core/shell structure for an image-guided X-ray photodynamic therapy (PDT) of the normoxic peripheral tumor. An anaerobic oncolytic bacterium (C. novyi-NT) therapy is combined to treat the hypoxic central tumor tissues. Photosensitizer-coated NSs (PS-NSs) and C. novyi-NT spores are emulsified with clinically available ethiodized oil (Lipiodol) to be the nano–bio therapeutic emulsion and injected into the tumor with computed tomography image guidance. The distribution of nano–bio therapeutic emulsion, including PS-NSs and anaerobic C. novyi-NT spores in the tumor site, is confirmed by both X-ray and T1-weighted magnetic resonance imaging. Following the image-guided X-ray PDT and anaerobic C. novyi- NT combination treatment, apoptotic cell death in cancer tissues, including both peripheral and central tumor regions, is significantly higher than in the control groups. Finally, this combination therapy approach using a nano–bio therapeutic emulsion is expected to overcome the limitations of conventional cancer therapy, resulting in increased cancer-therapeutic efficacy.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Groundwater-native Fe(II) oxidation prior to aeration with H 2 O 2 to enhance As(III) removal

Groundwater contaminated with arsenic (As) must be treated prior to drinking, as human exposure to As at toxic levels can cause various diseases including cancer. Conventional aeration-filtration applied to anaerobic arsenite (As(III)) contaminated groundwater can remove As(III) by co-oxidizing native iron (Fe(II)) and As(III) with oxygen (O 2 ). However, the As(III) removal efficiency of conventional aeration can be low, in part, because of incomplete As(III) oxidation to readily-sorbed arsenate (As(V)). In this work, we investigated a new approach to enhance As(III) co-removal with native Fe(II) by the anaerobic addition of hydrogen peroxide (H 2 O 2 ) prior to aeration. Experiments were performed to co-oxidize Fe(II) and As(III) with H 2 O 2 (anaerobically), O 2 (aerobically), and by sequentially adding of H 2 O 2 and O 2 . Aqueous As(III) and As(V) measurements after the reaction were coupled with solid-phase speciation by Fe and As K-edge X-ray absorption spectroscopy (XAS). We found that complete anaerobic oxidation of 100 µM Fe(II) with 100 µM H 2 O 2 resulted in co-removal of 95% of 7 µM As(III) compared to 44% with 8.0-9.0 mg/L dissolved O 2 . Furthermore, we found that with 100 µM Fe(II), the initial Fe(II):H 2 O 2 ratio was a critical parameter to remove 7 µM As(III) to below the 10 µg/L (0.13 µM) WHO guideline, where ratios of 1:4 (mol:mol) Fe(II):H 2 O 2 led to As(III) removal matching that of 7 µM As(V). The improved As(III) removal with H 2 O 2 was found to occur partly because of the well-established enhanced efficiency of As(III) oxidation in Fe(II)+H 2 O 2 systems relatively to Fe(II)+O 2 systems. However, the XAS results unambiguously demonstrated that a large factor in the improved As(III) removal was also due to a systematic decrease in crystallinity, and thus increase in specific surface area, of the generated Fe(III) (oxyhydr)oxides from lepidocrocite in the Fe(II)+O 2 system to poorly-ordered Fe(III) precipitates in the Fe(II)+H 2 O 2 system. The combined roles of H 2 O 2 (enhanced As(III) oxidation and structural modification) can be easily overlooked when only aqueous species are measured, but this dual impact must be considered for accurate predictions of As removal in groundwater treatment.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Brockarchaeota, a novel archaeal phylum with unique and versatile carbon cycling pathways

Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, ‘Brockarchaeota’, named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1979

Energy from aquatic plant wastewater treatment systems

Water hyacinth (Eichhornia crassipes), duckweed (Spirodela sp. and Lemma sp.), water pennywort (Hydrocotyle ranunculoides), and kudzu (Pueraria lobata) were anaerobically fermented using an anaerobic filter technique that reduced the total digestion time from 90 days to an average of 23 days and produced 0.14-0.28 cu m CH4/kg (dry weight) (2.3-4.5 cu ft/lb) from mature filters. The anaerobic filter provided a large surface area for the anaerobic bacteria to establish and maintain an optimum balance of facultative, acid-forming, and methane-producing bacteria. Consequently the efficiency of the process was greatly improved over prior batch fermentations.

Wolverton, B. C.↗

NASA NTRS2001

Sulfate- and Sulfur-Reducing Bacteria as Terrestrial Analogs for Microbial Life on Jupiter's Satellite Io

Observations from the Voyager and Galileo spacecraft have revealed Jupiter's moon Io to be the most volcanically active body of our Solar System. The Galileo Near Infrared Imaging Spectrometer (NIMS) detected extensive deposits of sulfur compounds, elemental sulfur and SO2 frost on the surface of Io. There are extreme temperature variations on Io's surface, ranging from -130 C to over 2000 C at the Pillan Patera volcanic vent. The active volcanoes, fumaroles, calderas, and lava lakes and vast sulfur deposits on this frozen moon indicate that analogs of sulfur- and sulfate-reducing bacteria might inhabit Io. Hence Io may have great significance to Astrobiology. Earth's life forms that depend on sulfur respiration are members of two domains: Bacteria and Archaea. Two basic links of the biogeochemical sulfur cycle of Earth have been studied: 1) the sulfur oxidizing process (occurring at aerobic conditions) and 2) the process of sulfur-reduction to hydrogen sulfide (anaerobic conditions). Sulfate-reducing bacteria (StRB) and sulfur-reducing bacteria (SrRB) are responsible for anaerobic reducing processes. At the present time the systematics of StRB include over 112 species distributed into 35 genera of Bacteria and Archaea. Moderately thermophilic and mesophilic SrRB belong to the Bacteria. The hyperthermophilic SrRB predominately belong to the domain Archaea and are included in the genera: Pyrodictium, Thermoproteus, Pyrobaculum, Thermophilum, Desulfurococcus, and Thermodiscus. The StRB and SrRB use a wide spectrum of substrates as electron donors for lithotrophic and heterotrophic type nutrition. The electron acceptors for the StRB include: sulfate, thiosulfate, sulfite, sulfur, arsenate, dithionite, tetrathionate, sulfur monoxide, iron, nitrite, selenite, fumarate, oxygen, carbon dioxide, and chlorine-containing phenol compounds. The Sulfate- and Sulfur-reducing bacteria are widely distributed in anaerobic ecosystems, including extreme environments like hot springs, deepsea hydrothermal vents, soda and high salinity lakes, and cryo-environments. Furthermore, the StRB and SrRB have Astrobiological significance as these anaerobic extremophiles may represent the dominant relic life forms that inhabited our planet during the extensive volcanic activity in the Earth's early evolutionary period.

Pikuta, Elena V.↗

NASA NTRS2001

Benthic Marine Cyanobacterial Mat Ecosystems: Biogeochemistry and Biomarkers

Cyanobacterial mats are complete ecosystems that can include processes of primary production, diagenesis and lithification. Light sustains oxygenic photosynthesis, which in turn provides energy, organic matter and oxygen to the community. Due to both absorption and scattering phenomena, incident light is transformed with depth in the mat, both in intensity and spectral composition. Mobile photo synthesizers optimize their position with respect to this light gradient. When photosynthesis ceases at night, the upper layers of the mat become reduced and sulfidic. Counteracting gradients of oxygen and sulfide combine to provide daily-contrasting environments separated on a scale of a few mm. The functional complexity of mats, coupled with the highly proximal and ordered spatial arrangement of biota, offers the potential for a staggering number of interactions. At a minimum, the products of each functional group of microorganisms affect the other groups both positively and negatively. For example, cyanobacteria generate organic matter (potential substrates) but also oxygen (a toxin for many anaerobes). Anaerobic activity recycles nutrients to the photosynthesizers but also generates potentially toxic sulfide. The combination of benefits and hazards of light, oxygen and sulfide promotes the allocation of the various essential mat processes between light and dark periods, and to various depths in the mat. Observations of mats have produced numerous surprises. For example, obligately anaerobic processes can occur in the presence of abundant oxygen, highly reduced gases are produced in the presence of abundant sulfate, meiofauna thrive at high sulfide concentrations, and the mats' constituent populations respond to environmental changes in complex ways. While photosynthetic bacteria dominate the biomass and productivity of the mat, nonphotosynthetic, anaerobic processes constitute the ultimate biological filter on the ecosystem's emergent biosignatures, including those sedimentary textures, organic compounds, and minerals that enter the fossil record. The ability of cyanobacterial mats to channel abundant solar energy into the creation and maintenance of complex structures and processes has created a multitude of consequences, both for sedimentation and for the early evolution of our biosphere.

DesMarais, David J.↗

DOE OSTI2025

PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification

Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization.

42 ENGINEERING↗

DOE OSTI2022

Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na + ). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as N ε -acetyl-β-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Reinventing wastewater treatment plants: energy neutral treatment and enhanced fertilizer production through a novel resource recovery center

Wastewater treatment plants (WWTPs) are typically energy intensive, mainly due to the secondary treatment processes such as activated sludge (AS) for treatment of organics as well as nutrients like nitrogen. Nitrogen removal presents a big problem for WWTPs. The main form of nitrogen in wastewater is ammonium, and an AS process uses oxygen to convert ammonium into nitrite and nitrate which is then converted to nitrogen through denitrification process. During anaerobic digestion (AD), organic nitrogen gets degraded, resulting in an effluent stream (centrate) with a high nitrogen content, mostly in the form of ammonium. This contributes 15-30% of total nitrogen to the wastewater influent which further increases energy consumption for aeration. The project aims to transform this conventional municipal WWTPs into energy-neutral, resource-recovering facilities by integrating three core technologies: • Cloth Media Filtration (CMF) to replace conventional primary sedimentation (CPS) and increase the diversion of organics from the energy intensive secondary treatment to AD. This results in reduced energy demand for aeration in the secondary process while simultaneously increasing the biogas production in the anaerobic digesters. • Anerobic Digester to increase biogas and ammonia production. • Membrane Evaporation (ME) to recover ammonia from AD centrate and produce marketable fertilizer. The benefits of proposed WWTP process modifications were evaluated using techno economic analysis (TEA) and life cycle assessment (LCA). For CMF portion of the research a statistical analysis was employed to develop data-driven tools that could be used to enhance and optimize its performance in terms of energy savings and effluent quality. The main objective of this project is to reduce the energy demand for secondary treatment at municipal WWTPs by at least 50%, increase anaerobic digester (AD) biogas and ammonia production by 100% and 120%, respectively, and recover 90% of ammonia from the AD. Integrated CMF, AD, and ME was shown to work synergistically toward achieving these decarbonization targets through energy-positive treatment and fertilizer recovery techniques.

42 ENGINEERING↗

DOE OSTI2022

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression

Methyl-coenzyme M reductase (MCR) is an archaeal enzyme that catalyzes the final step of methanogenesis and the first step in the anaerobic oxidation of methane, the energy metabolisms of methanogens and anaerobic methanotrophs (ANME), respectively. Variants of MCR, known as alkyl-coenzyme M reductases, are involved in the anaerobic oxidation of short-chain alkanes including ethane, propane, and butane as well as the catabolism of long-chain alkanes from oil reservoirs. MCR is a dimer of heterotrimers (encoded by mcrABG) and requires the nickel-containing tetrapyrrole prosthetic group known as coenzyme F430. MCR houses a series of unusual post-translational modifications within its active site whose identities vary depending on the organism and whose functions remain unclear. Methanogenic MCRs are encoded in a highly conserved mcrBDCGA gene cluster, which encodes two accessory proteins, McrD and McrC, that are believed to be involved in the assembly and activation of MCR, respectively. The requirement of a unique and complex coenzyme, various unusual post-translational modifications, and many remaining questions surrounding assembly and activation of MCR largely limit in vitro experiments to native enzymes with recombinant methods only recently appearing. Production of MCRs in a heterologous host is an important step toward developing optimized biocatalytic systems for methane production as well as for bioconversion of methane and other alkanes into value-added compounds. This review will first summarize MCR catalysis and structure, followed by a discussion of advances and challenges related to the production of diverse MCRs in a heterologous host.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Zymomonas mobilis oxidative stress transcriptomics

Zymomonas mobilis is an important bioenergy organism that has potential to produce biofuels, including ethanol, in high volumes. Here we examined the response of Zymomonas mobilis to various oxidative stresses using genome-scale transcriptomics data. We first examined the transcrpit abundance in WT aerobic growth compared to aerobic grown in paraquat, which forms superoxide. Under anaerobic growth conditions we compared WT Zymomonas mobilis with strains grown in media lacking iron as well as strains lacking iron that were treated with the iron chelator DIP before collection. Finally we examined transcript abundance in cells lacking ZMO_0422 (Rrf2 family transcription factor homolog) and ZMO_1411 (Fur homolog) grown under anaerobic conditions. Overall design: Transcriptomic analysis of WT, a deletion of ZMO_0422, and a deletion of ZMO_1411 in Zymomonas mobilis ZM4 under aerobic and anaerobic growth conditions along with various oxidative stresses: Paraquate addition, No Iron, and hydrogen peroxide addition.

aerobic↗

DOE OSTI2024

An efficient cre‐based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum

Abstract Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single‐carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis‐independent recombinase‐assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum . We then integrated a non‐ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum , which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Sanford, Patrick A.↗

DOE OSTI2025

Environmental controls on the kinetics of iron-sulfur cluster nucleation and nanoparticle formation

Anoxic, sulfidic conditions have been prevalent since the early Proterozoic and favor aqueous iron-sulfur (FeS aq ) clusters as a major fraction of the soluble, reduced iron and sulfur pool. FeS aq cluster formation and nucleation is driven by the high affinity between ferrous iron (Fe(II)) and sulfide (HS − ), ultimately yielding particles that precipitate as iron sulfide minerals. FeS aq clusters were recently shown to be bioavailable sources of iron and sulfur for a variety of anaerobes, yet little is known of the factors that influence the kinetics of their formation and nucleation. Here we apply computational and spectroscopic approaches to investigate the dynamics of FeS aq nucleation, cluster growth, precipitation, and redissolution as a function of Fe(II)/HS − concentration, temperature, and pH. Experiments were conducted under excess HS − to mimic euxinic conditions common to contemporary anaerobic aquatic ecosystems and those of the Proterozoic. Density functional theory calculations reveal the key role of water oxygen-iron interactions in stabilizing small FeS aq clusters and promoting solubility. Dynamic light scattering revealed a concentration-dependent increase in the kinetics of FeS aq nucleation and cluster aggregation. Increasing temperature promoted FeS aq cluster nucleation and aggregation while also enhancing dissolution. Alkaline pH also promoted FeS aq nucleation and cluster aggregation. At 25 °C, pH 7.0, and at reactant concentrations of 30 µM, FeS aq clusters < 10 nm in diameter remained in solution for > 2 h. These results underscore the importance of temperature, pH, and reactant concentration in the kinetics of FeS aq nucleation and cluster growth that, in turn, influence their bioavailability in anaerobic ecosystems.

Aquatic ecosystems↗

DOE OSTI2026

Quantitative DNA Stable Isotope Probing Identifies Active Microorganisms Assimilating Volatile Fatty Acids in Full-Scale Enhanced Biological Phosphorus Removal Processes

Enhanced biological phosphorus removal (EBPR) systems often rely on exogenous carbon sources, such as volatile fatty acids (VFAs), to achieve higher P removal. Here, we employed DNA quantitative stable isotope probing (qSIP) using two VFAs, acetate and propionate, in cyclic anaerobic/aerobic incubations to assess their effects on P cycling and microbial activity with biomass from two full-scale EBPR water resource-recovery facilities that utilize VFA addition. We found that anaerobic VFA uptake preferences differed within known groups of PAOs, such as Candidatus Accumulibacter and Tetrasphaera-affiliated members (e.g., Ca. Phosphoribacter), between the two biomasses. The combination of qSIP with metagenomics identified isotopically labeled phages that were linked to active PAOs, highlighting their potential roles in modulating EBPR community composition and activity. The highest levels of anaerobic labeling from acetate were in genomes belonging to Saccharimonadales and Rickettsiales, which are generally host-associated with bacteria and eukaryotes, respectively. Furthermore, this finding highlights the possibility of cross-feeding between PAO hosts and their parasites or predators, as well as the role of so-far uncharacterized organisms participating in carbon cycling under EBPR conditions. Collectively, these results expand our understanding of the ecological interactions involved in communities anaerobically uptaking VFAs and cycling P that are central to EBPR.

Polyphosphate accumulating organisms↗

DOE OSTI2023

Molecular Nature of Mineral-Organic Associations within Redox-Active Mountainous Floodplain Sediments

Floodplains are critical terrestrial-aquatic interfaces that act as hotspots of organic carbon (OC) cycling, regulating ecosystem carbon storage as well as export to riverine systems. Within floodplain sediments, regular flooding and textural gradients interact to create dynamic redox conditions. While anaerobic protection of OC upon burial is a well-recognized carbon storage mechanism in redox-active floodplain sediments, the impact of protective mineral-organic associations is relatively unknown. Here we determined the quantitative importance and chemical composition of mineral-organic associations along well-defined redox gradients emerging from textural variations and depth within meander sediments of the subalpine East River watershed (Gothic, CO). Here we characterized mineral-organic associations using a combination of sequential extractions, physical fractionation, and high-resolution mass spectrometry. Across the meander, we found that mineral-associated OC constitutes a significant fraction of total OC, and that extractable iron (Fe) and aluminum (Al) phases as well as high-density isolates were strongly correlated with total OC content, suggesting that mineral-organic associations are quantitatively important for floodplain sediment OC protection. Our mass spectrometry results showed OC associated with increasingly ordered Fe and Al phases are relatively enriched in low-molecular weight, oxidized, aromatic compounds. Surprisingly, however, total OC content showed weak or no correlation with indicators of anaerobic protection, such as relatively bioavailable OC pools (water-extractable and particulate OC) or the molecular weight and oxidation state of OC. Overall, this work highlights that protection of OC bound to reactive mineral phases-in addition to anaerobic protection-can play a quantitatively important role in controlling soil carbon storage in redox-active floodplain sediments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗