Search NASA⌕ Search

SEARCH · Search NASA

Results for “16S rRNA gene”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Whole metagenome sequencing and 16S rRNA gene amplicon analyses reveal the complex microbiome responsible for the success of enhanced in-situ reductive dechlorination (ERD) of a tetrachloroethene-contaminated Superfund site

The North Railroad Avenue Plume (NRAP) Superfund site in New Mexico, USA exemplifies successful chlorinated solvent bioremediation. NRAP was the result of leakage from a dry-cleaning that operated for 37 years. The presence of tetrachloroethene biodegradation byproducts, organohalide respiring genera (OHRG), and reductive dehalogenase (rdh) genes detected in groundwater samples indicated that enhanced reductive dechlorination (ERD) was the remedy of choice. This was achieved through biostimulation by mixing emulsified vegetable oil into the contaminated aquifer. This report combines metagenomic techniques with site monitoring metadata to reveal new details of ERD. DNA extracts from groundwater samples collected prior to and at four, 23 and 39 months after remedy implementation were subjected to whole metagenome sequencing (WMS) and 16S rRNA gene amplicon (16S) analyses. The response of the indigenous NRAP microbiome to ERD protocols is consistent with results obtained from microcosms, dechlorinating consortia, and observations at other contaminated sites. WMS detects three times as many phyla and six times as many genera as 16S. Both techniques reveal abundance changes in Dehalococcoides and Dehalobacter that reflect organohalide form and availability. Methane was not detected before biostimulation but appeared afterwards, corresponding to an increase in methanogenic Archaea. Assembly of WMS reads produced scaffolds containing rdh genes from Dehalococcoides, Dehalobacter, Dehalogenimonas, Desulfocarbo, and Desulfobacula. Anaerobic and aerobic cometabolic organohalide degrading microbes that increase in abundance include methanogenic Archaea, methanotrophs, Dechloromonas, and Xanthobacter, some of which contain hydrolytic dehalogenase genes. Aerobic cometabolism may be supported by oxygen gradients existing in aquifer microenvironments or by microbes that produce O 2 via microbial dismutation. The NRAP model for successful ERD is consistent with the established pathway and identifies new taxa and processes that support this syntrophic process. This project explores the potential of metagenomic tools (MGT) as the next advancement in bioremediation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils

The advance of high-throughput molecular biology tools allows in-depth profiling of microbial communities in soils, which possess a high diversity of prokaryotic microorganisms. Amplicon-based sequencing of 16S rRNA genes is the most common approach to studying the richness and composition of soil prokaryotes. To reliably detect different taxonomic lineages of microorganisms in a single soil sample, an adequate pipeline including DNA isolation, primer selection, PCR amplification, library preparation, DNA sequencing, and bioinformatic post-processing is required. Besides DNA sequencing quality and depth, the selection of PCR primers and PCR amplification reactions arguably have the largest influence on the results. This study tested the performance and potential bias of two primer pairs, i.e., 515F (Parada)-806R (Apprill) and 515F (Parada)-926R (Quince) in the standard pipelines of 16S rRNA gene Illumina amplicon sequencing protocol developed by the Earth Microbiome Project (EMP), against shotgun metagenome-based 16S rRNA gene reads. The evaluation was conducted using five differently managed soils. We observed a higher richness of soil total prokaryotes by using reverse primer 806R compared to 926R, contradicting to in silico evaluation results. Both primer pairs revealed various degrees of taxon-specific bias compared to metagenome-derived 16S rRNA gene reads. Nonetheless, we found consistent patterns of microbial community variation associated with different land uses, irrespective of primers used. Total microbial communities, as well as ammonia oxidizing archaea (AOA), the predominant ammonia oxidizers in these soils, shifted along with increased soil pH due to agricultural management. In the unmanaged low pH plot abundance of AOA was dominated by the acid-tolerant NS-Gamma clade, whereas limed agricultural plots were dominated by neutral-alkaliphilic NS-Delta/NS-Alpha clades. This study stresses how primer selection influences community composition and highlights the importance of primer selection for comparative and integrative studies, and that conclusions must be drawn with caution if data from different sequencing pipelines are to be compared.

16S rRNA gene amplicon Illumina sequencing↗
DOE OSTI2020

Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps

Efforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgonians Eunicea flexuosa and Gorgonia ventalina, and the scleractinian Porites panamensis. The 20-bp PNA clamp was designed using DNA from E. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads in E. flexuosa samples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction of E. flexuosa DNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian, G. ventalina, by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral, P. panamensis, by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities.

16S rRNA gene sequencing↗
DOE OSTI2020

A Genus Definition for Bacteria and Archaea Based on a Standard Genome Relatedness Index

In recent decades, the taxonomy of Bacteria and Archaea , and therefore genus designation, has been largely based on the use of a single ribosomal gene, the 16S rRNA gene, as a taxonomic marker. We propose an approach to delineate genera that excludes the direct use of the 16S rRNA gene and focuses on a standard genome relatedness index, the average nucleotide identity. Our findings are of importance to the microbiology community because the emergent properties of Bacteria and Archaea that are identified in this study will help assign genera with higher taxonomic resolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Protection and enrichment: how two different carbonaceous biofilm supports improve methane yield from encapsulated anaerobic microorganisms

Encapsulating anaerobic microorganisms allows for the separation of the solids retention time from the hydraulic retention time during anaerobic wastewater treatment. The harsh chemistries involved in the process of encapsulation can have adverse effects on microorganisms for anaerobic digestion, especially methanogens, and can lead to lower methane yields after encapsulation. Improving the survival and maintaining activity of anaerobic communities during encapsulation will likely be the key to improving methane yield. In this study, we investigated the encapsulation of biomass grown as biofilms on two carbonaceous materials, biochar and powdered activated carbon (PAC), to improve methane yield. Microorganisms grown as biofilms on biochar and PAC were encapsulated in polyethylene glycol (PEG) and incubated for 10 days. After 10 days, the unamended control capsules produced 81.6 ± 5.4 μmol of methane, while PAC-amended capsules produced 129.8 ± 1.9 μmol and biochar-amended capsules produced 432.96 ± 20.8 μmol methane, with the differences being statistically significant (p < 0.05). In biochar, a higher relative abundance of methanogens led to increased methane production capacity. The ratio of the methyl coenzyme M reductase (mcrA) genes to total 16S rRNA genes in the encapsulated biochar-supported biofilms was significantly higher than that in the encapsulated unsupported (p = 4.9 × 10 −5 ) and the PAC-supported biofilms (p = 0.012). Biochar-supported biofilms also had higher methane output per mcrA or 16S rRNA gene copy number. For the PAC, biofilms were protected from ammonium persulfate (APS), a powerful oxidant used in the encapsulation process. PAC removed 92% of dissolved APS, reducing exposure of the methanogens to this chemical. Unfortunately, this removal of APS compromised capsule stability, limiting the amount of PAC that could be added to the capsules. Furthermore, amendments that improve survival and activity of methanogens should be used in the capsules instead of those that protect methanogens by interfering with encapsulant polymerization chemistry.

Resource recovery↗
DOE OSTI2022

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria . We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 10 5 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria , representing a candidate order ( Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O 2 , suggesting a microaerophilic lifestyle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Tectonic and geological setting influence hot spring microbiology

Hydrothermal systems form at divergent and convergent boundaries of lithospheric plates and within plates due to weakened crust and mantle plumes, playing host to diverse microbial ecosystems. Little is known of how differences in tectonic setting influence the geochemical and microbial compositions of these hydrothermal ecosystems. Here, for this work, coordinated geochemical and microbial community analyses were conducted on 87 high-temperature (>65°C) water and sediment samples from hot springs in Yellowstone National Park, Wyoming, USA ( n = 41; mantle plume setting), Iceland ( n = 41, divergent boundary), and Japan ( n = 5; convergent boundary). Region-specific variation in geochemistry and sediment-associated 16S rRNA gene amplicon sequence variant (ASV) composition was observed, with 16S rRNA gene assemblages being nearly completely distinguished by region and pH being the most explanatory parameter within regions. Several low abundance ASVs exhibited cosmopolitan distributions across regions, while most high-abundance ASVs were only identified in specific regions. The presence of some cosmopolitan ASVs across regions argues against dispersal limitation primarily shaping the distribution of taxa among regions. Rather, the results point to local tectonic and geologic characteristics shaping the geochemistry of continental hydrothermal systems that then select for distinct microbial assemblages. These results provide new insights into the co-evolution of hydrothermal systems and their microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Description of Pegethrix niliensis sp. nov., a Novel Cyanobacterium from the Nile River Basin, Egypt: A Polyphasic Analysis and Comparative Study of Related Genera in the Oculatellales Order

In this paper, we examine the filamentous cyanobacterial strain NILCB16 and describe it as a new species within the genus Pegethrix. The original population was sampled from a mat growing in an irrigation canal in the Nile River, Egypt. Initially classified under Plectonema or Planktolyngbya, the strain is a potential producer of the toxins microcystin and β-N-Methylamino-L-Alanine (BMAA). Additionally, we reviewed the taxonomic relationships between the Oculatellales genera. To describe the new species, we conducted a polyphasic study, encompassing 16S rRNA gene phylogenetic analyses performed using both Maximum Likelihood and Bayesian methods, sequence identity (p-distance) analysis, 16S-23S ITS secondary structures, and morphological and habitat comparisons. The phylogenetic analysis revealed that strain NILCB16 clustered within the Pegethrix clade with strong phylogenetic support, but in a distinct position from other species in the genus. The strain shared a maximum 16S rRNA gene identity of 97.3% with P. qiandaoensis and 96.1% with the type species, P. bostrychoides. Morphologically, NILCB16 can be differentiated from other species in the genus by its lack of false branching. Our phylogenetic analyses also show that Pegethrix, Cartusia, Elainella, and Maricoleus are clustered with strong phylogenetic support. They exhibit high 16S rRNA gene identity and are morphologically indistinguishable, suggesting they could potentially be merged into a single genus in the future.

Hentschke, Guilherme Scotta (ORCID:000000034396024↗
DOE OSTI2025

Soil microbial ecology and microbiome-metabolite linkages improve understanding of ecosystem states along terrestrial-aquatic interfaces

These data are from Bandopadhyay et al., "Soil microbial ecology and microbiome-metabolite linkages improve understanding of ecosystem states along terrestrial-aquatic interfaces". This study aims to understand the soil microbial ecology along terrestrial-aquatic interfaces of a freshwater and estuarine region and how it relates to organic matter. We analyzed soil microbial (16S rRNA gene) and organic matter (Fourier-transform ion cyclotron resonance mass spectrometry, FTICR-MS) composition from upland (forested), transition (stressed forest), and wetland positions at three sites in each of the Lake Erie (freshwater) and Chesapeake Bay (estuarine) regions. This dataset includes 16S rRNA gene amplicon data (only processed file types included here) and organic matter composition from FTICR-MS data (raw and processed files included here) from upland (forested), transition (stressed forest), and wetland positions at three sites in each of the Lake Erie and Chesapeake Bay regions. These sites are part of the COMPASS-FME project (https://compass.pnnl.gov/FME/COMPASSFME). File formats and software needed to access files: 16S rRNA gene amplicon data: These files follow the format reported here https://ess-dive.gitbook.io/amplicon-sequencing-reporting-format#updates-in-v1.0.1. As per this format, there are four file types reported: 1. Taxon tables (also called sequence-by-sample or OTU (operational taxonomic unit)/ESV (exact sequence variant) tables) : available in a .txt file format and accessible using TextEdit or MS Excel. 2. Representative sequences (also called consensus sequences) : available in a .fasta format and accessible using TextEdit. 3. Sequencing metadata : available in a MS Excel workbook file format and CSV file format 4. Bioinformatic metadata : available in a MS Excel workbook file format and CSV file format FTICR-MS data: 1. Raw data converted to a processed file with intensities of the peaks in the given samples : available in a MS Excel CSV file format 2. Processed file used in analyses and visualizations (appended as icr_long_) : available in a MS Excel CSV file format 3. Metadata file for ICR features (appended as icr_meta) : available in a MS Excel CSV file format

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Dissecting the dominant hot spring microbial populations based on community-wide sampling at single-cell genomic resolution

With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek's Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Expanded diversity of novel hemoplasmas in rare and undersampled Neotropical bats

Hemotropic mycoplasmas are emerging as a model system for studying bacterial pathogens in bats, but taxonomic coverage of sampled host species remains biased. We leveraged a long-term field study in Belize to uncover novel hemoplasma diversity in bats by analyzing 80 samples from 19 species, most of which are infrequently encountered. PCR targeting the partial 16S rRNA gene found 41% of bats positive for hemoplasmas. Phylogenetic analyses found two novel host shifts of hemoplasmas, four entirely new hemoplasma genotypes, and the first hemoplasma detections in four bat species. One of these novel hemoplasmas (from Neoeptesicus furinalis) shared 97.6% identity in the partial 16S rRNA gene to a human hemoplasma (Candidatus Mycoplasma haemohominis). Additional analysis of the partial 23S rRNA gene allowed us to also designate two novel hemoplasma species, in Myotis elegans and Phyllostomus discolor, with the proposed names Candidatus Mycoplasma haematomyotis sp. nov. and Candidatus Mycoplasma haematophyllostomi sp. nov., respectively. Our analyses show that additional hemoplasma diversity in bats can be uncovered by targeting rare or undersampled host species.

16S rRNA gene↗
DOE OSTI2024

Transfer of beef bacterial communities onto food-contact surfaces

Introduction Food spoilage and pathogenic bacteria on food-contact surfaces, especially biofilm-forming strains, can transfer to meats during processing. The objectives of this study were to survey the bacterial communities of beef cuts that transfer onto two commonly used food-contact surfaces, stainless steel (SS) and high-density polyethylene (HDPE) and identify potentially biofilm-forming strains. Methods Top round, flank, chuck, and ground beef were purchased from 3 retail stores. SS and HDPE coupons (approximately 2cm × 5cm) were placed on beef portions (3h, 10°C), after which, the coupons were submerged halfway in PBS (24h, 10°C). Bacteria from the beef cuts and coupon surfaces ( n = 3) were collected, plated on tryptic soy agar plates and incubated (5 days, 25°C). Bacterial isolates were identified by 16S rRNA gene amplicon sequencing and assayed for biofilm formation using a crystal violet binding (CV) assay (72h, 10°C). Additionally, beef and coupon samples were collected for bacterial community analysis by 16S rRNA gene amplicon sequencing. Results and discussion Sixty-one of 972 beef isolates, 29 of 204 HDPE isolates, and 30 of 211 SS isolates were strong biofilm-formers (Absorbance>1.000 at 590 nm in the CV assay). Strong-binding isolates identified were of the genera Pseudomonas , Acinetobacter , Psychrobacter , Carnobacterium , and Brochothrix . Coupon bacterial communities among stores and cuts were distinct ( p < 0.001, PERMANOVA), but there was no distinction between the communities found on HDPE or SS coupons ( p > 0.050, PERMANOVA). The bacterial communities identified on the coupons may help determine the communities capable of transferring and colonizing onto surfaces, which can subsequently cross-contaminate foods.

Guron, Giselle K. P.↗
DOE OSTI2020

Organic amendments change soil organic C structure and microbial community but not total organic matter on sub-decadal scales

Organic C has many benefits for soil, but it is depleted by tillage and crop harvest, and especially so for biofuel crops. Accordingly, strategies such as partially retaining stover or planting a cover crop can help ameliorate the negative effect of C removal. We used a long-term field experiment to study the impacts of stover retention and planting cover crop on soil organic matter (SOM), its extractable components and the soil microbial community. SOM chemical composition characterization was determined by electrospray ionization (ESI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in sequential water, methanol (MeOH), and chloroform (CHCl3) extracts. The characteristics of the soil bacterial community were measured by phospholipid fatty acid (PLFA), real-time quantitative PCR, and 16S rRNA gene sequence. The variations in total SOM content, total microbial biomass, and bacterial population were slight among treatments, but SOM chemical compounds, arbuscular mycorrhizal fungi (AMF) biomass, and bacterial structure changed significantly, and especially so in the coupled application of stover retention and cover crop. Specifically, stover retention enriched more lignin-like compounds in soil, whereas cover crop enriched more condensed hydrocarbons, and had more compounds with an aromaticity index (AI) >0.5. The bacterial community was not altered by the cover crop, but the corn stover retention increased the relative abundances of Myxococcales (Deltaproteobacteria) and decreased that of Actinobacteria. Redundancy analysis (RDA) further unveiled that the bacterial community in the stover treatments had significantly positive association with CHCl3-extracted chemical classes, i.e. unsaturated hydrocarbon and lipid with the coupled application (stover and cover crop), and lignin and protein with the single corn stover treatment. Taken together, our study shows how different C addition practices influence the molecular composition of SOM and the structure of soil microbial communities.

Xu, Jiangbin↗
DOE OSTI2023

Sphingobium lignivorans sp. nov., isolated from river sediment downstream of a paper mill

Here, a bacterial isolate, B1D3A T , was isolated from river sediment collected from the Hiwassee River near Calhoun, TN, by enrichment culturing with a model 5–5' lignin dimer, dehydrodivanillate, as its sole carbon source. B1D3A T was also shown to utilize several model lignin-derived monomers and dimers as sole carbon sources in a variety of minimal media. Cells were Gram-stain-negative, aerobic, motile, rod-shaped and formed yellow/cream-coloured colonies on rich agar. Optimal growth occurred at 30°C, pH 7–8, and in the absence of NaCl. The major fatty acids of B1D3A T were C 18:1 ω7c and C 17:1 ω6c. The predominant hydroxy fatty acids were C 14: 0 2-OH and C 15:0 2-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine and sphingoglycolipid. B1D3A T contained spermidine as the only major polyamine. The major isoprenoid quinone was Q-10 with minor amounts of Q-9 and Q-11. The genomic DNA G+C content of B1D3A T was 65.6mol%. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 49 core, universal genes defined by Clusters of Orthologous Groups gene families indicated that B1D3A T was a member of the genus Sphingobium. B1D3A T was most closely related to Sphingobium sp. SYK-6, with a 100% 16S rRNA gene sequence similarity. B1D3A T showed 78.1–89.9%average nucleotide identity and 19.5–22.2% digital DNA–DNA hybridization identity with other type strains from the genus Sphingobium. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain B1D3A T should be classified as representing a novel species of the genus Sphingobium, for which the name Sphingobium lignivorans sp. nov. is proposed. The type strain is strain B1D3A T (ATCC TSD-279 T =DSM 111877 T ).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A standardized quantitative analysis strategy for stable isotope probing metagenomics

ABSTRACT Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg , an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, “who is eating what?” and “who is active?” within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism’s taxonomic identity and genome composition while providing quantitative estimates of the microorganism’s isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Examination of coal combustion management sites for microbiological and chemical signatures of groundwater impacts

Coal combustion accounts for 40% of the world’s electricity and generates more than a billion tons of coal combustion products (CCP) annually, half of which end up in landfills and impoundments. CCP contain mixtures of chemicals that can be mobile in the environment and impact the quality of surface water and potable groundwater. In this investigation, water samples from 14 coal combustion management sites across 4 physiographic regions in the United States, paired with background and down-gradient groundwater samples, were analyzed for water chemistry and microbiology. The objective was to determine if microbiology data alone, or supported by chemistry data, could reliably differentiate source waters and identify sites where CCP is known or expected to be influencing groundwater. Two percent of the total amplicons showed genus level conservation across CCP management sites, regions, and sample types; corresponding to ubiquitous, facultatively aerobic proteobacterial taxa that are generally recognized for the potential to respire using different terminal electron acceptors. Ordination plots did not reveal significant differences ( p > 0.05) in 16S rRNA gene amplicon diversity by CCP management site, water sample types, or physiographic regions. Contrastingly, chemistry distinguished sample types by standard water quality metrics (total dissolved solids, Ca:SO 4 ratio), alkali earth metals (K, Na, Li), selenium, boron, and fluoride. A focused evaluation of 16S rRNA gene amplicons for a subset of CCP management sites revealed microbiological features and chemical drivers (F, Ca, temperature) that positively identified the single CCP management site confirmed to have groundwater impacted by CCP leachate. At this site, 9 genera (>0.5% relative abundance) were exclusive to CCP porewater and downgradient groundwater. Inferred metabolisms for these taxa indicates potential for N and S biogeochemical transformations and 1-C metabolism that are consistent with a reducing environment, as evidenced by low ORP and depleted SO 4 2− . This research contributes to a growing understanding of conditions where these data types, analyses, and interpretation methods could be applied for distinguishing influence from CCP on the surrounding environment, as well as practical limitations.

01 COAL, LIGNITE, AND PEAT↗