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At least 865 records · Page 48
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DOE OSTI2025

Robust measurement of microbial reduction of graphene oxide nanoparticles using image analysis

ABSTRACT Shewanella oneidensis ( S. oneidensis ) has the capacity to reduce electron acceptors within a medium and is thus used frequently in microbial fuel generation, pollutant breakdown, and nanoparticle fabrication. Microbial fuel setups, however, often require costly or labor-intensive components, thus making optimization of their performance onerous. For rapid optimization of setup conditions, a model reduction assay can be employed to allow simultaneous, large-scale experiments at lower cost and effort. Since S. oneidensis uses different extracellular electron transfer pathways depending on the electron acceptor, it is essential to use a reduction assay that mirrors the pathways employed in the microbial fuel system. For microbial fuel setups that use nanoparticles to stimulate electron transfer, reduction of graphene oxide provides a more accurate model than other commonly used assays as it is a bulk material that forms flocculates in solutions with a large ionic component. However, graphene oxide flocculates can interfere with traditional absorbance-based measurement techniques. This study introduces a novel image analysis method for quantifying graphene oxide reduction, showing improved performance and statistical accuracy over traditional methods. A comparative analysis shows that the image analysis method produces smaller errors between replicates and reveals more statistically significant differences between samples than traditional plate reader measurements under conditions causing graphene oxide flocculation. Image analysis can also detect reduction activity at earlier time points due to its use of larger solution volumes, enhancing color detection. These improvements in accuracy make image analysis a promising method for optimizing microbial fuel cells that use nanoparticles or bulk substrates. IMPORTANCE Shewanella oneidensis ( S. oneidensis ) is widely used in reduction processes such as microbial fuel generation due to its capacity to reduce electron acceptors. Often, these setups are labor-intensive to operate and require days to produce results, so use of a model assay would reduce the time and expenses needed for optimization. Our research developed a novel digital analysis method for analysis of graphene oxide flocculates that may be utilized as a model assay for reduction platforms featuring nanoparticles. Use of this model reduction assay will enable rapid optimization and drive improvements in the microbial fuel generation sector.

Bennett, Danielle T. (ORCID:0009000188748827)↗
DOE OSTI2025

Engineering the hyperthermophilic archaeon Pyrococcus furiosus for 1-propanol production

ABSTRACT Society relies heavily on chemicals traditionally produced through the refinement of fossil fuels. The conversion of renewable biomass to value-added chemicals by microbes, particularly hyperthermophiles (T opt ≥80°C), offers a renewable alternative to this traditional approach. Herein, we describe the engineering of the hyperthermophilic archaeon Pyrococcus furiosus , which grows optimally (T opt ) at 100°C, for the conversion of sugar to 1-propanol. This was accomplished by constructing a hybrid metabolic pathway consisting of two native and seven heterologously produced enzymes to convert acetyl-CoA from carbohydrate metabolism to 1-propanol. A total of eleven foreign genes from two other organisms were utilized, one from the thermophilic bacterium Thermoanaerobacter sp. strain X514 and 10 from the thermoacidophilic archaeon Metallosphaera sedula, both of which grow optimally near 70°C. The recombinant P. furiosus strain produced 1-propanol at similar concentrations (up to ~1 mM) when incubated at 75°C to activate the gene products of Thermoanaerobacter sp. strain X514 and M. sedula and by initially incubating at 95°C for P. furiosus growth and then subsequently returning to 75°C to promote 1-propanol formation. Note that 1-propanol was not produced if the culture was grown only at 95°C. This work has the potential for future optimization through harnessing the genome-scale metabolic model of P. furiosus that was used herein to identify engineering targets to increase 1-propanol titer. IMPORTANCE As petroleum reserves become increasingly strained, the development of renewable alternatives to traditional chemical synthesis becomes more important. In this work, a high-temperature biological system for sugar to 1-propanol conversion was demonstrated by metabolic engineering of the hyperthermophilic archaeon Pyrococcus furiosus (T opt 100°C). The engineered strain produced 1-propanol by temperature shifting from 75°C to 95°C and then back to 75°C to accommodate the temperature ranges for native and foreign proteins associated with 1-propanol biosynthesis. Genome-scale metabolic modeling informed the carbon and reductant flux in the system, identified potential factors limiting 1-propanol production, and revealed potential optimization targets.

O'Quinn, Hailey C. (ORCID:0009000274865755)↗
DOE OSTI2022

Bulk and Spatially Resolved Extracellular Metabolome of Free-Living Nitrogen Fixation

Free-living nitrogen-fixing bacteria contribute significantly to terrestrial nitrogen availability; however, the forms of nitrogen contributed by this process are poorly understood. This is in part because of inherent challenges to studying soil microorganisms in situ , such as vast differences in scale between microorganism and ecosystem and complexities of the soil system (e.g., opacity, chemical complexity).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Host-Associated Rhizobial Fitness: Dependence on Nitrogen, Density, Community Complexity, and Legume Genotype

Legume crops establish beneficial associations with rhizobial bacteria that perform biological nitrogen fixation, providing nitrogen to plants without the economic and greenhouse gas emission costs of chemical nitrogen inputs. Here, we examine the influence of three environmental factors that vary in agricultural fields on strain relative fitness in nodules.

Burghardt, Liana T.↗
DOE OSTI2024

Leptothrix ochracea genomes reveal potential for mixotrophic growth on Fe(II) and organic carbon

ABSTRACT Leptothrix ochracea creates distinctive iron-mineralized mats that carpet streams and wetlands. Easily recognized by its iron-mineralized sheaths, L. ochracea was one of the first microorganisms described in the 1800s. Yet it has never been isolated and does not have a complete genome sequence available, so key questions about its physiology remain unresolved. It is debated whether iron oxidation can be used for energy or growth and if L. ochracea is an autotroph, heterotroph, or mixotroph. To address these issues, we sampled L. ochracea -rich mats from three of its typical environments (a stream, wetlands, and a drainage channel) and reconstructed nine high-quality genomes of L. ochracea from metagenomes. These genomes contain iron oxidase genes cyc2 and mtoA, showing that L. ochracea has the potential to conserve energy from iron oxidation. Sox genes confer potential to oxidize sulfur for energy. There are genes for both carbon fixation (RuBisCO) and utilization of sugars and organic acids (acetate, lactate, and formate). In silico stoichiometric metabolic models further demonstrated the potential for growth using sugars and organic acids. Metatranscriptomes showed a high expression of genes for iron oxidation; aerobic respiration; and utilization of lactate, acetate, and sugars, as well as RuBisCO, supporting mixotrophic growth in the environment. In summary, our results suggest that L. ochracea has substantial metabolic flexibility. It is adapted to iron-rich, organic carbon-containing wetland niches, where it can thrive as a mixotrophic iron oxidizer by utilizing both iron oxidation and organics for energy generation and both inorganic and organic carbon for cell and sheath production. IMPORTANCE Winogradsky's observations of L. ochracea led him to propose autotrophic iron oxidation as a new microbial metabolism, following his work on autotrophic sulfur-oxidizers. While much culture-based research has ensued, isolation proved elusive, so most work on L. ochracea has been based in the environment and in microcosms. Meanwhile, the autotrophic Gallionella became the model for freshwater microbial iron oxidation, while heterotrophic and mixotrophic iron oxidation is not well-studied. Ecological studies have shown that Leptothrix overtakes Gallionella when dissolved organic carbon content increases, demonstrating distinct niches. This study presents the first near-complete genomes of L. ochracea , which share some features with autotrophic iron oxidizers, while also incorporating heterotrophic metabolisms. These genome, metabolic modeling, and transcriptome results give us a detailed metabolic picture of how the organism may combine lithoautotrophy with organoheterotrophy to promote Fe oxidation and C cycling and drive many biogeochemical processes resulting from microbial growth and iron oxyhydroxide formation in wetlands.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Thermal adaptation of soil microbial growth traits in response to chronic warming

ABSTRACT Adaptation of soil microbes due to warming from climate change has been observed, but it remains unknown what microbial growth traits are adaptive to warming. We studied bacterial isolates from the Harvard Forest Long-Term Ecological Research site, where field soils have been experimentally heated to 5°C above ambient temperature with unheated controls for 30 years. We hypothesized that Alphaproteobacteria from warmed plots have (i) less temperature-sensitive growth rates; (ii) higher optimum growth temperatures; and (iii) higher maximum growth temperatures compared to isolates from control plots. We made high-throughput measurements of bacterial growth in liquid cultures over time and across temperatures from 22°C to 37°C in 2–3°C increments. We estimated growth rates by fitting Gompertz models to the growth data. Temperature sensitivity of growth rate, optimum growth temperature, and maximum growth temperature were estimated by the Ratkowsky 1983 model and a modified Macromolecular Rate Theory (MMRT) model. To determine evidence of adaptation, we ran phylogenetic generalized least squares tests on isolates from warmed and control soils. Our results showed evidence of adaptation of higher optimum growth temperature of bacterial isolates from heated soils. However, we observed no evidence of adaptation of temperature sensitivity of growth and maximum growth temperature. Our project begins to capture the shape of the temperature response curves, but illustrates that the relationship between growth and temperature is complex and cannot be limited to a single point in the biokinetic range. IMPORTANCE Soils are the largest terrestrial carbon sink and the foundation of our food, fiber, and fuel systems. Healthy soils are carbon sinks, storing more carbon than they release. This reduces the amount of carbon dioxide released into the atmosphere and buffers against climate change. Soil microbes drive biogeochemical cycling and contribute to soil health through organic matter breakdown, plant growth promotion, and nutrient distribution. In this study, we determined how soil microbial growth traits respond to long-term soil warming. We found that bacterial isolates from warmed plots showed evidence of adaptation of optimum growth temperature. This suggests that increased microbial biomass and growth in a warming world could result in greater carbon storage. As temperatures increase, greater microbial activity may help reduce the soil carbon feedback loop. Our results provide insight on how atmospheric carbon cycling and soil health may respond in a warming world.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

ABSTRACT Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds ( p- coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p -coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+ p -coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+ p -coumarate) condition compared to when repression was absent ( p -coumarate or glucose only). IMPORTANCE A newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Anaerobic benzene oxidation in Geotalea daltonii involves activation by methylation and is regulated by the transition state regulator AbrB

ABSTRACT Benzene is a widespread groundwater contaminant that persists under anoxic conditions. The aim of this study was to more accurately investigate anaerobic microbial degradation pathways to predict benzene fate and transport. Preliminary genomic analysis of Geotalea daltonii strain FRC-32, isolated from contaminated groundwater, revealed the presence of putative aromatic-degrading genes. G. daltonii was subsequently shown to conserve energy for growth on benzene as the sole electron donor and fumarate or nitrate as the electron acceptor. The hbs gene, encoding for 3-hydroxybenzylsuccinate synthase (Hbs), a homolog of the radical-forming, toluene-activating benzylsuccinate synthase (Bss), was upregulated during benzene oxidation in G. daltonii , while the bss gene was upregulated during toluene oxidation. Addition of benzene to the G. daltonii whole-cell lysate resulted in toluene formation, indicating that methylation of benzene was occurring. Complementation of σ 54 - (deficient) E. coli transformed with the bss operon restored its ability to grow in the presence of toluene, revealing bss to be regulated by σ 54 . Binding sites for σ 70 and the transition state regulator AbrB were identified in the promoter region of the σ 54 -encoding gene rpoN, and binding was confirmed. Induced expression of abrB during benzene and toluene degradation caused G. daltonii cultures to transition to the death phase. Our results suggested that G. daltonii can anaerobically oxidize benzene by methylation, which is regulated by σ 54 and AbrB. Our findings further indicated that the benzene, toluene, and benzoate degradation pathways converge into a single metabolic pathway, representing a uniquely efficient approach to anaerobic aromatic degradation in G. daltonii . IMPORTANCE The contamination of anaerobic subsurface environments including groundwater with toxic aromatic hydrocarbons, specifically benzene, toluene, ethylbenzene, and xylene, has become a global issue. Subsurface groundwater is largely anoxic, and further study is needed to understand the natural attenuation of these compounds. This study elucidated a metabolic pathway utilized by the bacterium Geotalea daltonii capable of anaerobically degrading the recalcitrant molecule benzene using a unique activation mechanism involving methylation. The identification of aromatic-degrading genes and AbrB as a regulator of the anaerobic benzene and toluene degradation pathways provides insights into the mechanisms employed by G. daltonii to modulate metabolic pathways as necessary to thrive in anoxic contaminated groundwater. Our findings contribute to the understanding of novel anaerobic benzene degradation pathways that could potentially be harnessed to develop improved strategies for bioremediation of groundwater contaminants.

Bullows, James E.↗
DOE OSTI2024

Evidence for autotrophic growth of purple sulfur bacteria using pyrite as electron and sulfur source

ABSTRACT Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that Allochromatium vinosum (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures (“py”’) demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various c - and b -type cytochromes was observed in “py,” pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells’ photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(−), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis. IMPORTANCE Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet’s history.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Community Structure of Arbuscular Mycorrhizal Fungi in Soils of Switchgrass Harvested for Bioenergy

We assessed the different species of beneficial fungi living in agricultural fields of switchgrass, a large grass grown for biofuels, using high-resolution DNA sequencing. Contrary to our expectations, the fungi were not greatly affected by fertilization. However, we found a positive relationship between plant productivity and the number of families of beneficial fungi at one site. Furthermore, we sequenced many species that could not be identified with existing reference databases. One group of fungi was highlighted in an earlier study for being widely distributed but of unknown taxonomy. We discovered that this group belonged to a family called Pervetustaceae , which may benefit switchgrass in stressful environments. To produce higher-yielding switchgrass in a more sustainable manner, it could help to study these undescribed fungi and the ways in which they may contribute to greater switchgrass yield in the absence of fertilization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath

The importance of RubisCO and CO 2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO 2 and RubisCO are essential for M. capsulatus Bath growth. 13 CO 2 tracing experiments supported that RubisCO mediates CO 2 fixation and that a noncanonical Calvin cycle is active in this organism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Methanogens acquire and bioaccumulate nickel during reductive dissolution of nickelian pyrite

ABSTRACT Nickel (Ni) is a key component of the active site metallocofactors of numerous enzymes required for methanogenesis, including [NiFe]-hydrogenase, carbon monoxide dehydrogenase, and methyl CoM reductase, leading to a high demand for Ni among methanogens. However, methanogens often inhabit euxinic environments that favor the sequestration of nickel as metal-sulfide minerals, such as nickelian pyrite [(Ni,Fe)S 2 ], that have low solubilities and that are not considered bioavailable. Recently, however, several different model methanogens ( Methanosarcina barkeri , Methanococcus voltae , Methanococcus maripaludis ) were shown to reductively dissolve pyrite (FeS 2 ) and to utilize dissolution products to meet iron and sulfur biosynthetic demands. Here, using M. barkeri Fusaro, and laboratory-synthesized (Ni,Fe)S 2 that was physically isolated from cells using dialysis membranes, we show that trace nickel (<20 nM) abiotically solubilized from the mineral can support methanogenesis and limited growth, roughly fivefold less than the minimum concentration known to support methanogenesis. Furthermore, when provided direct contact with (Ni,Fe)S 2 , M. barkeri promoted the reductive dissolution of (Ni,Fe)S 2 and assimilated solubilized nickel, iron, and sulfur as its sole source of these elements. Cells that reductively dissolved (Ni,Fe)S 2 bioaccumulated approximately fourfold more nickel than those grown with soluble nickel and sulfide but had similar metabolic coupling efficiencies. While the mechanism for Ni uptake in archaeal methanogens is not known, homologs of the bacterial Nik uptake system were shown to be ubiquitous across methanogen genomes. Collectively, these observations indicate that (Ni,Fe)S 2 is bioavailable in anoxic environments and that methanogens can convert this mineral into nickel-, iron-, and sulfur-containing metalloenzymes to support methanogenesis and growth. IMPORTANCE Nickel is an essential metal, and its availability has changed dramatically over Earth history due to shifts in the predominant type of volcanism in the late Archean that limited its availability and an increase in euxinic conditions in the early Proterozoic that favored its precipitation as nickel sulfide minerals. Observations presented herein indicate that the methanogen, Methanosarcina barkeri , can acquire nickel at low concentration (<20 nM) from soluble and mineral sources. Furthermore, M. barkeri was shown to actively reduce nickelian pyrite; use dissolution products to meet their iron, sulfur, and nickel demands; and bioaccumulate nickel. These data help to explain how M. barkeri (and possibly other methanogens and anaerobes) can acquire nickel in contemporary and past anoxic or euxinic environments.

(NiFe)S2↗
DOE OSTI2024

The military gear microbiome: risk factors surrounding the warfighter

ABSTRACT Combat extremity wounds are highly susceptible to contamination from surrounding environmental material. This bioburden could be partially transferred from materials in immediate proximity to the wound, including fragments of the uniform and gear. However, the assessment of the microbial bioburden present on military gear during operational conditions of deployment or training is relatively unexplored. Opportunistic pathogens that can survive on gear represent risk factors for infection following injury, especially following combat blasts, where fibers and other materials are embedded in wounded tissue. We utilized 16S rRNA sequencing to assess the microbiome composition of different military gear types (boot, trouser, coat, and canteen) from two operational environments (training in Hawai’i and deployed in Indonesia) across time (days 0 and 14). We found that microbiome diversity, stability, and composition were dependent on gear type, training location, and sampling timepoint. At day 14, species diversity was significantly higher in Hawai’i samples compared to Indonesia samples for boot, coat, and trouser swabs. In addition, we observed the presence of potential microbial risk factors, as opportunistic pathogenic species, such as Acinetobacter , Pseudomonas, and Staphylococcus , were found to be present in all sample types and in both study sites. These study outcomes will be used to guide the design of antimicrobial materials and uniforms and for infection control efforts following combat blasts and other injuries, thereby improving treatment guidance during military training and deployment. IMPORTANCE Combat extremity wounds are vulnerable to contamination from environments of proximity to the warfighter, leading to potential detrimental outcomes such as infection and delayed wound healing. Therefore, microbial surveillance of such environments is necessary to aid the advancement of military safety and preparedness through clinical diagnostics, treatment protocols, and uniform material design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Novel, active, and uncultured hydrocarbon-degrading microbes in the ocean

ABSTRACT Given the vast quantity of oil and gas input to the marine environment annually, hydrocarbon degradation by marine microorganisms is an essential ecosystem service. Linkages between taxonomy and hydrocarbon degradation capabilities are largely based on cultivation studies, leaving a knowledge gap regarding the intrinsic ability of uncultured marine microbes to degrade hydrocarbons. To address this knowledge gap, metagenomic sequence data from the Deepwater Horizon (DWH) oil spill deep-sea plume was assembled to which metagenomic and metatranscriptomic reads were mapped. Assembly and binning produced new DWH metagenome-assembled genomes that were evaluated along with their close relatives, all of which are from the marine environment (38 total). These analyses revealed globally distributed hydrocarbon-degrading microbes with clade-specific substrate degradation potentials that have not been reported previously. For example, methane oxidation capabilities were identified in all Cycloclasticus . Furthermore, all Bermanella encoded and expressed genes for non-gaseous n -alkane degradation; however, DWH Bermanella encoded alkane hydroxylase, not alkane 1-monooxygenase. All but one previously unrecognized DWH plume member in the SAR324 and UBA11654 have the capacity for aromatic hydrocarbon degradation. In contrast, Colwellia were diverse in the hydrocarbon substrates they could degrade. All clades encoded nutrient acquisition strategies and response to cold temperatures, while sensory and acquisition capabilities were clade specific. These novel insights regarding hydrocarbon degradation by uncultured planktonic microbes provides missing data, allowing for better prediction of the fate of oil and gas when hydrocarbons are input to the ocean, leading to a greater understanding of the ecological consequences to the marine environment. IMPORTANCE Microbial degradation of hydrocarbons is a critically important process promoting ecosystem health, yet much of what is known about this process is based on physiological experiments with a few hydrocarbon substrates and cultured microbes. Thus, the ability to degrade the diversity of hydrocarbons that comprise oil and gas by microbes in the environment, particularly in the ocean, is not well characterized. Therefore, this study aimed to utilize non-cultivation-based ‘omics data to explore novel genomes of uncultured marine microbes involved in degradation of oil and gas. Analyses of newly assembled metagenomic data and previously existing genomes from other marine data sets, with metagenomic and metatranscriptomic read recruitment, revealed globally distributed hydrocarbon-degrading marine microbes with clade-specific substrate degradation potentials that have not been previously reported. This new understanding of oil and gas degradation by uncultured marine microbes suggested that the global ocean harbors a diversity of hydrocarbon-degrading bacteria, which can act as primary agents regulating ecosystem health.

Howe, Kathryn L.↗
DOE OSTI2023

Production of carotenoids from aromatics and pretreated lignocellulosic biomass by Novosphingobium aromaticivorans

ABSTRACT Carotenoids are lipophilic compounds found in the membranes of various organisms. Individual carotenoids are also commodity chemicals, produced industrially for use as food additives, nutritional supplements, cosmetics, and pharmaceuticals. The alphaproteobacterium Novosphingobium aromaticivorans has previously been established as a potential platform microbe for converting aromatic compounds derived from lignocellulosic plant biomass into valuable extracellular products. Here, we show that N. aromaticivorans DSM 12444 cells naturally produce the carotenoid nostoxanthin, and we construct a set of gene deletion mutants that accumulate β-carotene, lycopene, or zeaxanthin, which are predicted intermediates in nostoxanthin biosynthesis as well as commodity chemicals. We also show that a mutant strain heterologously expressing a CrtW protein accumulates the carotenoid astaxanthin. When grown on vanillate as the carbon source, we find that the levels of carotenoids are not significantly affected by O 2 concentration in the tested range of 5% to 21% O 2 . We also show that these carotenoids are produced at comparable levels when strains are grown in liquor from alkaline pretreated sorghum biomass [sorghum alkaline pretreatment liquor (APL)], which contains a mixture of aromatics. Finally, we construct strains that produce zeaxanthin, β-carotene, or astaxanthin concurrently with 2-pyrone-4,6-dicarboxylic acid, a potential building block for biodegradable polymers, when grown in sorghum APL. Combined, our results show that N. aromaticivorans can simultaneously produce valuable intracellular and extracellular commodities when grown in the presence of either pure aromatics or pretreated lignocellulosic biomass. IMPORTANCE There is economic and environmental interest in generating commodity chemicals from renewable resources, such as lignocellulosic biomass, that can substitute for chemicals derived from fossil fuels. The bacterium Novosphingobium aromaticivorans is a promising microbial platform for producing commodity chemicals from lignocellulosic biomass because it can produce these from compounds in pretreated lignocellulosic biomass, which many industrial microbial catalysts cannot metabolize. Here, we show that N. aromaticivorans can be engineered to produce several valuable carotenoids. We also show that engineered N. aromaticivorans strains can produce these lipophilic chemicals concurrently with the extracellular commodity chemical 2-pyrone-4,6-dicarboxylic acid when grown in a complex liquor obtained from alkaline pretreated lignocellulosic biomass. Concurrent microbial production of valuable intra- and extracellular products can increase the economic value generated from the conversion of lignocellulosic biomass-derived compounds into commodity chemicals and facilitate the separation of water- and membrane-soluble products.

09 BIOMASS FUELS↗
DOE OSTI2020

Magnetotactic Bacteria Accumulate a Large Pool of Iron Distinct from Their Magnetite Crystals

ABSTRACT Magnetotactic bacteria (MTB) are ubiquitous aquatic microorganisms that form intracellular nanoparticles of magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) in a genetically controlled manner. Magnetite and greigite synthesis requires MTB to transport a large amount of iron from the environment. Most intracellular iron was proposed to be contained within the crystals. However, recent mass spectrometry studies suggest that MTB may contain a large amount of iron that is not precipitated in crystals. Here, we attempted to resolve these discrepancies by performing chemical and magnetic assays to quantify the different iron pools in the magnetite-forming strain Magnetospirillum magneticum AMB-1, as well as in mutant strains showing defects in crystal precipitation, cultivated at various iron concentrations. All results show that magnetite represents at most 30% of the total intracellular iron under our experimental conditions and even less in the mutant strains. We further examined the iron speciation and subcellular localization in AMB-1 using the fluorescent indicator FIP-1, which was designed for the detection of labile Fe(II). Staining with this probe suggests that unmineralized reduced iron is found in the cytoplasm and associated with magnetosomes. Our results demonstrate that, under our experimental conditions, AMB-1 is able to accumulate a large pool of iron distinct from magnetite. Finally, we discuss the biochemical and geochemical implications of these results. IMPORTANCE Magnetotactic bacteria (MTB) produce iron-based intracellular magnetic crystals. They represent a model system for studying iron homeostasis and biomineralization in microorganisms. MTB sequester a large amount of iron in their crystals and have thus been proposed to significantly impact the iron biogeochemical cycle. Several studies proposed that MTB could also accumulate iron in a reservoir distinct from their crystals. Here, we present a chemical and magnetic methodology for quantifying the iron pools in the magnetotactic strain AMB-1. Results showed that most iron is not contained in crystals. We then adapted protocols for the fluorescent Fe(II) detection in bacteria and showed that iron could be detected outside crystals using fluorescence assays. This work suggests a more complex picture for iron homeostasis in MTB than previously thought. Because iron speciation controls its fate in the environment, our results also provide important insights into the geochemical impact of MTB.

59 BASIC BIOLOGICAL SCIENCES↗