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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Postbiotic Gamma-Aminobutyric Acid and Camel Milk Intervention as Innovative Trends Against Hyperglycemia and Hyperlipidemia in Streptozotocin-Induced C57BL/6J Diabetic Mice

Diabetes is a serious disease that threatens human health worldwide. The study hypothesis is to investigate the novel trends that may aid in the prevention of diabetic complications. Camel milk was presented as traditional functional food, and Lactobacillus brevis KLDS 1.0727 and KLDS 1.0373 strains were shown to synthesize postbiotic Gamma-aminobutyric acid as a potential food additive, which can therapeutically intervene against hyperglycemia and hyperlipidemia in streptozotocin-induced C 57 BL/6J mice. During a four-week timeframe, body weight and postprandial blood glucose levels were monitored. Post-euthanasia, blood plasma was obtained to investigate hyperlipidemia, insulin concentrations, liver, and renal functions. The liver, pancreas, kidney, and spleen underwent histopathological examinations. The results demonstrated that KLDS 1.0727 and KLDS 1.0373 ( LAC S1 , LAC S2 ) and camel milk treatments all had a significant influence on hypoglycemic activity, as evidenced by reduced postprandial blood glucose levels. LAC S1 , LAC S2 , and camel milk therapy significantly reduced blood hypolipidemic, and some liver enzymes such as (alanine aminotransferase and aspartate transaminase) levels. Therefore, we recommend consuming camel milk regularly and expanding its use with fermented foods containing L. brevis , one of the probiotics capable of producing gamma-aminobutyric acid (GABA) as future food additives that can improve human health and reduce the prevalence of several diseases disorders.

Abdelazez, Amro↗
DOE OSTI2023

Aculeaxanthones A–E, new xanthones from the marine-derived fungus Aspergillus aculeatinus WHUF0198

Introduction Dimeric natural products are widespread in plants and microorganisms, which usually have complex structures and exhibit greater bioactivities than their corresponding monomers. In this study, we report five new dimeric tetrahydroxanthones, aculeaxanthones A−E ( 4−8 ), along with the homodimeric tetrahydroxanthone secalonic acid D ( 1 ), chrysoxanthones B and C ( 2 and 3 ), and 4−4’-secalonic acid D ( 9 ), from different fermentation batches of the title fungus. Methods A part of the culture was added to a total of 60 flasks containing 300 ml each of number II fungus liquid medium and culture 4 weeks in a static state at 28˚C. The liquid phase (18 L) and mycelia was separated from the fungal culture by filtering. A crude extract was obtained from the mycelia by ultrasound using acetone. To obtain a dry extract (18 g), the liquid phase combined with the crude extract were further extracted by EtOAc and concentrated in vacuo. The MIC of anaerobic bacteria was examined by a broth microdilution assay. To obtain MICs for aerobic bacteria, the agar dilution streak method recommended in Clinical and Laboratory Standards Institute document (CLSI) M07-A10 was used. Compounds 1−9 was tested against the Bel-7402, A-549 and HCT-116 cell lines according to MTT assay. Results and Discussion The structures of these compounds were elucidated on the base of 1D and 2D NMR and HR-ESIMS data, and the absolute configurations of the new xanthones 4−8 were determined by conformational analysis and time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations. Compounds 1–9 were tested for cytotoxicity against the Bel-7402, A549, and HCT-116 cancer cell lines. Of the dimeric tetrahydroxanthone derivatives, only compound 6 provided cytotoxicity effect against Bel-7402 cell line (IC50, 1.96 µM). Additionally, antimicrobial activity was evaluated for all dimeric tetrahydroxanthones, including four Gram-positive bacteria including Enterococcus faecium ATCC 19434, Bacillus subtilis 168, Staphylococcus aureus ATCC 25923 and MRSA USA300; four Gram-negative bacteria, including Helicobacter pylori 129, G27, as well as 26,695, and multi drug-resistant strain H. pylori 159, and one Mycobacterium M. smegmatis ATCC 607. However, only compound 1 performed activities against H. pylori G27, H. pylori 26695, H. pylori 129, H. pylori 159, S. aureus USA300, and B. subtilis 168 with MIC values of 4.0, 4.0, 2.0, 2.0, 2.0 and 1.0 μg/mL, respectively.

Wu, Jun↗
DOE OSTI2023

Biological methane production and accumulation under sulfate-rich conditions at Cape Lookout Bight, NC

Introduction: Anaerobic oxidation of methane (AOM) is hypothesized to occur through reverse hydrogenotrophic methanogenesis in marine sediments because sulfate reducers pull hydrogen concentrations so low that reverse hydrogenotrophic methanogenesis is exergonic. If true, hydrogenotrophic methanogenesis can theoretically co-occur with sulfate reduction if the organic matter is so labile that fermenters produce more hydrogen than sulfate reducers can consume, causing hydrogen concentrations to rise. Finding accumulation of biologically-produced methane in sulfate-containing organic-rich sediments would therefore support the theory that AOM occurs through reverse hydrogenotrophic methanogenesis since it would signal the absence of net AOM in the presence of sulfate. Methods: 16S rRNA gene libraries were compared to geochemistry and incubations in high depth-resolution sediment cores collected from organic-rich Cape Lookout Bight, North Carolina. Results: We found that methane began to accumulate while sulfate is still abundant (6–8 mM). Methane-cycling archaea ANME-1, Methanosarciniales, and Methanomicrobiales also increased at these depths. Incubations showed that methane production in the upper 16 cm in sulfate-rich sediments was biotic since it could be inhibited by 2-bromoethanosulfonoic acid (BES). Discussion: We conclude that methanogens mediate biological methane production in these organic-rich sediments at sulfate concentrations that inhibit methanogenesis in sediments with less labile organic matter, and that methane accumulation and growth of methanogens can occur under these conditions as well. Our data supports the theory that H2 concentrations, rather than the cooccurrence of sulfate and methane, control whether methanogenesis or AOM via reverse hydrogenotrophic methanogenesis occurs. We hypothesize that the high amount of labile organic matter at this site prevents AOM, allowing methane accumulation when sulfate is low but still present in mM concentrations

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metagenome-assembled genomes provide insight into the metabolic potential during early production of Hydraulic Fracturing Test Site 2 in the Delaware Basin

Demand for natural gas continues to climb in the United States, having reached a record monthly high of 104.9 billion cubic feet per day (Bcf/d) in November 2023. Hydraulic fracturing, a technique used to extract natural gas and oil from deep underground reservoirs, involves injecting large volumes of fluid, proppant, and chemical additives into shale units. This is followed by a “shut-in” period, during which the fracture fluid remains pressurized in the well for several weeks. The microbial processes that occur within the reservoir during this shut-in period are not well understood; yet, these reactions may significantly impact the structural integrity and overall recovery of oil and gas from the well. To shed light on this critical phase, we conducted an analysis of both pre-shut-in material alongside production fluid collected throughout the initial production phase at the Hydraulic Fracturing Test Site 2 (HFTS 2) located in the prolific Wolfcamp formation within the Permian Delaware Basin of west Texas, USA. Specifically, we aimed to assess the microbial ecology and functional potential of the microbial community during this crucial time frame. Prior analysis of 16S rRNA sequencing data through the first 35 days of production revealed a strong selection for a Clostridia species corresponding to a significant decrease in microbial diversity. Here, we performed a metagenomic analysis of produced water sampled on Day 33 of production. This analysis yielded three high-quality metagenome-assembled genomes (MAGs), one of which was a Clostridia draft genome closely related to the recently classified Petromonas tenebris. This draft genome likely represents the dominant Clostridia species observed in our 16S rRNA profile. Annotation of the MAGs revealed the presence of genes involved in critical metabolic processes, including thiosulfate reduction, mixed acid fermentation, and biofilm formation. These findings suggest that this microbial community has the potential to contribute to well souring, biocorrosion, and biofouling within the reservoir. Our research provides unique insights into the early stages of production in one of the most prolific unconventional plays in the United States, with important implications for well management and energy recovery.

natural gas↗
DOE OSTI2025

Limited effects of tannin supplementation on the dairy cattle fecal microbiome with modulation of metabolites

Tannins are plant secondary metabolites that bind organic carbon (C) and nitrogen (N), potentially altering substrate bioavailability for enteric fermentation in ruminants. This interaction may reduce greenhouse gas (GHG) emissions and influence nitrogen partitioning. Given tannins' resistance to ruminal degradation and persistence through the gastrointestinal tract, this study investigated the effects of a tannin-based feed additive on fecal microbial diversity, fecal chemical composition, and GHG emissions. Twenty-four early- to mid-lactation dairy cows were randomized to receive either a tannin-based feed additive (TRT; containing condensed and hydrolyzable tannins from Schinopsis quebracho-colorado [Schltdl.]) or a control diet (CON) for 64 days. Cows were blocked by parity, dry matter intake, milk yield, body weight, and days in milk. Fecal samples were collected on days 0, 16, 32, and 64 and analyzed using 16S rRNA gene amplicon sequencing. Fecal C, N, and indole-3-lactate were measured, and GHG emissions (N2O, CH4, CO2) were assessed via 14-day laboratory incubation. A total of 1,538 amplicon sequence variants were identified, with Firmicutes as the dominant phylum. Fecal phylogenetic diversity showed a significant treatment × day interaction (p < 0.01), with TRT cows exhibiting reduced microbial diversity from day 16 to 64. Fecal C and N concentrations were significantly lower (p < 0.01) in TRT cows on day 16, while indole-3-lactate levels were higher on day 64 (p = 0.02). GHG emissions did not differ significantly between treatments. The tannin-based feed additive influenced fecal microbial community structure and select chemical parameters but did not significantly affect GHG emissions from feces. These findings suggest that dietary tannins may modulate gut microbial ecology with minimal impact on downstream manure-related emissions.

Klein, Matthew L↗
DOE OSTI2025

In anaerobic reactors the microbial community structure depends on feed type, with no “keystone” species tied to COD removal

Two-stage anaerobic digestion (AD) systems provide treatment for high strength wastewater with high stability and performance. Encapsulation technology can intensify AD to facilitate the separation of the solids retention time from the hydraulic retention time (HRT), offering lower HRTs, smaller reactors, and high effluent quality. To support successful deployment, however, the encapsulated community must contain all the needed microorganisms for successful treatment and be flexible enough to treat a variety of wastewaters. Here, a two-stage system was investigated in which microbial cultures were enriched on various high-strength wastewaters in suspended flow-through systems to determine how feed type influenced performance and microbial community structure. The hypothesis was that specific genera, or so-called “keystone species” would positively correlate to organic carbon degradation for a given feed, enabling construction of a well-functioning community for encapsulation. Results showed that the number of total bacteria (as 16S rRNA gene copies) did not correlate to soluble chemical oxygen demand (sCOD) removal, indicating that the community structure and/or members were important for good performance. Results also showed that feed type strongly influenced carbon removal and microbial community structure for 1st-stage fermenting communities, but not 2nd-stage methanogenic communities. In this study, the “core” community members were defined as organisms common to all of either the 1st- or 2nd-stage reactors irrespective of the feed they received and were present in at least 50% of the samples throughout the entire experiment. “Unique” community members were specific to a single feed, and hence, only present in either the 1st- or 2nd-stage reactors receiving that feed. In both 1st- and 2nd-stage communities, only one core genera and no unique genera were positively and significantly correlated to sCOD removal. Verification experiments performed with encapsulated communities showed that organisms identified in flow-through system and correlated with carbon degradation, though not significantly, seemed to be important for performance. Our results suggest that one cannot construct a community containing specific populations in lieu of enrichment. Nevertheless, a single diverse encapsulated anaerobic community should provide good (>80%) carbon removal when fed a variety of influents, if time is provided for enrichment after deployment.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

The Role of Sugar Signaling in Regulating Plant Fatty Acid Synthesis

Photosynthates such as glucose, sucrose, and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism. Such sugars provide substrates for de novo fatty acid (FA) biosynthesis. However, compared with the well-defined examples of sugar signaling in starch and anthocyanin synthesis, until recently relatively little was known about the role of signaling in regulating FA and lipid biosynthesis. Recent research progress shows that trehalose 6-phosphate and 2-oxoglutarate (2-OG) play direct signaling roles in the regulation of FA biosynthesis by modulating transcription factor stability and enzymatic activities involved in FA biosynthesis. Specifically, mechanistic links between sucrose non-fermenting−1–related protein kinase 1 (SnRK1)–mediated trehalose 6-phosphate (T6P) sensing and its regulation by phosphorylation of WRI1 stability, diacylglycerol acyltransferase 1 (DGAT1) enzyme activity, and of 2-OG–mediated relief of inhibition of acetyl-CoA carboxylase (ACCase) activity by protein PII are exemplified in detail in this review.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Field performance of switchgrass plants engineered for reduced recalcitrance

Switchgrass ( Panicum virgatum L.) is a promising perennial bioenergy crop that achieves high yields with relatively low nutrient and energy inputs. Modification of cell wall composition for reduced recalcitrance can lower the costs of deconstructing biomass to fermentable sugars and other intermediates. We have engineered overexpression of OsAT10 , encoding a rice BAHD acyltransferase and QsuB , encoding dehydroshikimate dehydratase from Corynebacterium glutamicum , to enhance saccharification efficiency in switchgrass. These engineering strategies demonstrated low lignin content, low ferulic acid esters, and increased saccharification yield during greenhouse studies in switchgrass and other plant species. In this work, transgenic switchgrass plants overexpressing either OsAT10 or QsuB were tested in the field in Davis, California, USA for three growing seasons. No significant differences in the content of lignin and cell wall-bound p -coumaric acid or ferulic acid were detected in transgenic OsAT10 lines compared with the untransformed Alamo control variety. However, the transgenic overexpressing QsuB lines had increased biomass yield and slightly increased biomass saccharification properties compared to the control plants. This work demonstrates good performance of engineered plants in the field, and also shows that the cell wall changes in the greenhouse were not replicated in the field, emphasizing the need to validate engineered plants under relevant field conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

GRIK phosphorylates and activates KIN10 which also promotes its degradation

The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (a) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to low sugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Impact of irradiance and inorganic carbon availability on heterologous sucrose production in Synechococcus elongatus PCC 7942

Cyanobacteria have been proposed as a potential alternative carbohydrate feedstock and multiple species have been successfully engineered to secrete fermentable sugars. To date, the most productive cyanobacterial strains are those designed to secrete sucrose, yet there exist considerable differences in reported productivities across different model species and laboratories. In this study, we investigate how cultivation conditions (specifically, irradiance, CO 2 , and cultivator type) affect the productivity of sucrose-secreting Synechococcus elongatus PCC 7942. We find that S. elongatus produces the highest sucrose yield in irradiances far greater than what is often experimentally utilized, and that high light intensities are tolerated by S. elongatus , especially under higher density cultivation where turbidity may attenuate the effective light experienced in the culture. By increasing light and inorganic carbon availability, S. elongatus cscB / sps produced a total of 3.8 g L -1 of sucrose and the highest productivity within that period being 47.8 mg L -1 h -1 . This study provides quantitative description of the impact of culture conditions on cyanobacteria-derived sucrose that may assist to standardize cross-laboratory comparisons and demonstrates a significant capacity to improve productivity via optimizing cultivation conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Morpho-physiological and transcriptomic responses of field pennycress to waterlogging

Field pennycress (Thlaspi arvense) is a new biofuel winter annual crop with extreme cold hardiness and a short life cycle, enabling off-season integration into corn and soybean rotations across the U.S. Midwest. Pennycress fields are susceptible to winter snow melt and spring rainfall, leading to waterlogged soils. The objective of this research was to determine the extent to which waterlogging during the reproductive stage affected gene expression, morphology, physiology, recovery, and yield between two pennycress lines (SP32-10 and MN106). In a controlled environment, total pod number, shoot/root dry weight, and total seed count/weight were significantly reduced in SP32-10 in response to waterlogging, whereas primary branch number, shoot dry weight, and single seed weight were significantly reduced in MN106. This indicated waterlogging had a greater negative impact on seed yield in SP32-10 than MN106. We compared the transcriptomic response of SP32-10 and MN106 to determine the gene expression patterns underlying these different responses to seven days of waterlogging. The number of differentially expressed genes (DEGs) between waterlogged and control roots were doubled in MN106 (3,424) compared to SP32-10 (1,767). Functional enrichment analysis of upregulated DEGs revealed Gene Ontology (GO) terms associated with hypoxia and decreased oxygen, with genes in these categories encoding proteins involved in alcoholic fermentation and glycolysis. Additionally, downregulated DEGs revealed GO terms associated with cell wall biogenesis and suberin biosynthesis, indicating suppressed growth and energy conservation. Interestingly, MN106 waterlogged roots exhibited significant stronger regulation of these genes than SP32-10, displaying a more robust transcriptomic response overall. Together, these results reveal the reconfiguration of cellular and metabolic processes in response to the severe energy crisis invoked by waterlogging in pennycress.

ERF-VII↗
DOE OSTI2026

Context-dependent coordination of TOR and SnRK1 signaling under carbon and nitrogen perturbations

Target of rapamycin (TOR) and sucrose non-fermenting 1–related protein kinase 1 (SnRK1) are conserved regulators of plant growth and metabolism and are often portrayed as functionally antagonistic under nutrient limitation. However, how this relationship operates across different nutrient contexts remains poorly defined. Here, we generated an Arabidopsis dual-reporter line that enables simultaneous monitoring of TOR and SnRK1 activities and profiled their dynamics under carbon and nitrogen perturbations. We found that TOR and SnRK1 activities overall exhibit a negative relationship during the transition from carbon starvation to carbon abundance; however, their temporal dynamics during that transition do not support a strictly inverse correlation. Under dark conditions, TOR activity is gradually repressed, while SnRK1 is initially repressed in the early hours and subsequently activated during extended darkness. During nitrogen starvation, TOR activity is progressively repressed, whereas SnRK1 is activated during early hours and then becomes repressed. In vitro , recombinant SnRK1α1 directly inhibits the activity of immunoprecipitated TOR (IP-TOR), whereas IP-TOR does not directly affect SnRK1α1 activity. Together, these results support a nutrient-dependent model in which TOR and SnRK1 are coordinated primarily by cellular metabolic status.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Phenotypic Evaluation of Saccharum spp. Genotypes during the Plant-Cane Crop for Biomass Production in Northcentral Mississippi

Saccharum is relatively new to 33° N latitude. S. spontaneum readily hybridizes with commercial sugarcane (Saccharum spp.) and lends cold tolerance and greater yield to the hybrid progeny, called energycane. Since 2007, there have been numerous new hybrid and backcross energycane genotypes developed but there is a paucity of information about them. Twenty energycane genotypes were tested in the first season of growth from cane propagules (plant cane; PC) against Ho 02-113 (a control) for two site-years in northcentral Mississippi. Grand (exponential) growth continued into October. The prevailing paradigm is that tonnage is what matters. Except for percentage cellulose, all factors tested (dry matter yield, extractable juice volume, °Brix, theoretical ethanol from fermentation, theoretical ethanol from cellulose, and total theoretical ethanol) were greater from the second site-location compared to the first. Dry matter yield (DMY) and total theoretical ethanol yield (TTEY) were moderately correlated. Over the two years of this test only Ho 14-9213 exceeded in mean DMY of Ho 02-113. Sixteen of the 19 test genotypes in this test equaled or exceeded the mean TTEY of Ho 02-113.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Grape ASR Regulates Glucose Transport, Metabolism and Signaling

To decipher the mediator role of the grape Abscisic acid, Stress, Ripening (ASR) protein, VvMSA, in the pathways of glucose signaling through the regulation of its target, the promoter of hexose transporter VvHT1, we overexpressed and repressed VvMSA in embryogenic and non-embryogenic grapevine cells. The embryogenic cells with organized cell proliferation were chosen as an appropriate model for high sensitivity to the glucose signal, due to their very low intracellular glucose content and low glycolysis flux. In contrast, the non-embryogenic cells displaying anarchic cell proliferation, supported by high glycolysis flux and a partial switch to fermentation, appeared particularly sensitive to inhibitors of glucose metabolism. By using different glucose analogs to discriminate between distinct pathways of glucose signal transduction, we revealed VvMSA positioning as a transcriptional regulator of the glucose transporter gene VvHT1 in glycolysis-dependent glucose signaling. The effects of both the overexpression and repression of VvMSA on glucose transport and metabolism via glycolysis were analyzed, and the results demonstrated its role as a mediator in the interplay of glucose metabolism, transport and signaling. The overexpression of VvMSA in the Arabidopsis mutant abi8 provided evidence for its partial functional complementation by improving glucose absorption activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum

Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

EMS-Induced Mutagenesis of Clostridium carboxidivorans for Increased Atmospheric CO 2 Reduction Efficiency and Solvent Production

Clostridium carboxidivorans (P7) is one of the most important solvent-producing bacteria capable of fermenting syngas (CO, CO 2 , and H 2 ) to produce chemical commodities when grown as an autotroph. This study aimed to develop ethyl methanesulfonate (EMS)-induced P7 mutants that were capable of growing in the presence of CO 2 as a unique source of carbon with increased solvent formation and atmospheric CO 2 reduction to limit global warming. Phenotypic analysis including growth and end product characterization of the P7 wild type (WT) demonstrated that this strain grew better at 25 °C than 37 °C when CO 2 served as the only source of carbon. In the current study, 55 mutagenized P7-EMS mutants were developed by using 100 mM and 120 mM EMS. Interestingly, using a forward genetic approach, three out of the 55 P7-EMS mutants showed a significant increase in ethanol, butyrate, and butanol production. The three P7-EMS mutants presented on average a 4.68-fold increase in concentrations of ethanol when compared to the P7-WT. Butyric acid production from 3 P7-EMS mutants contained an average of a 3.85 fold increase over the levels observed in the P7-WT cultures under the same conditions (CO 2 only). In addition, one P7-EMS mutant presented butanol production (0.23 ± 0.02 g/L), which was absent from the P7-WT under CO 2 conditions. Most of the P7-EMS mutants showed stability of the obtained end product traits after three transfers. Most importantly, the amount of reduced atmospheric CO 2 increased up to 8.72 times (0.21 g/Abs) for ethanol production and up to 8.73 times higher (0.16 g/Abs) for butyrate than the levels contained in the P7-WT. Additionally, to produce butanol, the P7-EMSIII-J mutant presented 0.082 g/Abs of CO 2 reduction. This study demonstrated the feasibility and effectiveness of employing EMS mutagenesis in generating solvent-producing anaerobic bacteria mutants with improved and novel product formation and increased atmospheric CO 2 reduction efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Effect of CO 2 Concentration on the Microbial Activity of Orenia metallireducens (Strain Z6) in Surface Inert Materials

Carbon dioxide (CO 2 ) sequestration has garnered widespread attention as a key strategy for mitigating CO 2 emissions and combating the greenhouse effect. However, the mechanisms underlying the interactions between CO 2 , widespread siliceous minerals and biological processes remain unclear. The present study explored the potential impacts of different CO 2 concentrations on microbial activity, environmental conditions and their feedback on the fate of CO 2 . A total of 20 experimental conditions was created, with the variables including different natural and synthetic siliceous minerals (e.g., quartz sand and a type of commercial glass beads), the presence or absence of the iron-reducing microorganism Orenia metallireducens (strain Z6) and varying CO 2 concentrations (0%, 20%, 50%, 100%) in the presence of ferrihydrite and pyruvate. Geochemical, microbial and mineralogical analyses revealed that elevated CO 2 concentrations significantly inhibited microbial Fe(III) reduction and pyruvate metabolism. Interestingly, compared to cultures without mineral amendments or those with glass beads alone, the addition of quartz sand enabled strain Z6 to better withstand the environmental stress caused by elevated CO 2 , promoting pyruvate fermentation and iron reduction. In addition to an increased pH, the formation of siderite, hematite and vivianite was also observed in the bioactive systems. Although both glass beads and quartz sand were primarily composed of silica, differences in the mineral structure, elemental composition and acid neutralization capacity rendered quartz sand more chemically active and unexpectedly led to greater CO 2 sequestration.

CO2 stress↗