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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.

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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Dispersion of sulfur creates a valuable new growth medium formulation that enables earlier sulfur oxidation in relation to iron oxidation in Acidithiobacillus ferrooxidans cultures

Abstract Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig‐sulfur, can be used to support the rapid antibiotic selection of plasmid‐transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig‐sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.

Inaba, Yuta↗
DOE OSTI2021

MCL1 inhibition enhances the efficacy of docetaxel against airway-derived squamous cell carcinoma cells

Highlights: • Authentic squamous cell carcinoma (SqCC) cell lines of the lung are sparse. • HARA lung SqCC cells depend highly on MCL1 for survival in vitro and in vivo. • MCL1-silenced HARA cells decline in number upon docetaxel (DTX) exposure. • High MCL1 mRNA expression is linked with shorter survival in lung SqCC patients. • MCL1 depletion + DTX is highly effective against Detroit 562 pharyngeal SqCC cells. MCL1 is an anti-apoptotic BCL2 family member that is often overexpressed in various malignant tumors. However, few reports have described the role of MCL1 in squamous cell carcinoma (SqCC) derived from airways including the lung. In this study, we examined whether MCL1 could be a novel druggable target for airway-derived SqCC, for which effective molecular targeted drugs are unavailable. We searched the Kaplan–Meier Plotter database and found that high MCL1 mRNA expression was significantly associated with shorter survival in patients with lower airway (lung) or upper airway (head and neck) derived SqCC. We also explored the Expression Atlas database and learned that authentic lung SqCC cell lines expressing both TP63 and KRT5 mRNA were extremely sparse among the publicly available “lung SqCC cell lines”, with an exception being HARA cells. HARA cells were highly dependent on MCL1 for survival, and MCL1-depleted cells were not able to grow, and even declined in number, upon docetaxel (DTX) exposure in vitro and in vivo. Similar in vitro experimental findings, including those in a 3D culture model, were also obtained using Detroit 562 pharyngeal SqCC cells. These findings suggested that combined treatment with MCL1 silencing plus DTX appears highly effective against airway-derived SqCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Iron acquisition and mineral transformation by cyanobacteria living in extreme environments

Iron is an essential micronutrient for most living organisms, including cyanobacteria. These microorganisms have been found in Earth's driest polar and non-polar deserts, including the Atacama Desert, Chile. Iron-containing minerals were identified in colonized rock substrates from the Atacama Desert, however, the interactions between microorganisms and iron minerals remain unclear. In the current study, we determined that colonized gypsum rocks collected from the Atacama Desert contained both magnetite and hematite phases. A cyanobacteria isolate was cultured on substrates consisting of gypsum with embedded magnetite nanoparticles. Transmission electron microscopy imaging revealed a significant reduction in the size of magnetite nanoparticles due to their dissolution, which occurred around the microbial biofilms. Concurrently, hematite was detected, likely from the oxidation of the magnetite nanoparticles. Higher cell counts and production of siderophores were observed in cultures with magnetite nanoparticles suggesting that cyanobacteria were actively acquiring iron from the magnetite nanoparticles. Magnetite dissolution and iron acquisition by the cyanobacteria was further confirmed using large bulk magnetite crystals, uncovering a survival strategy of cyanobacteria in these extreme environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

The Energy Costs of Dewatering Feedstock Microalgae Species Using Conventional Implementations of Ultrasonic and Crossflow Filtration Technologies

The use of microalgae as a feedstock for biofuels and other products continues to be explored, but large-scale biomass production faces persistent high costs. One major cost contributor is the energy required for harvesting and dewatering, a challenge resulting from the dilute nature of microalgae cultures as well as the properties of microalgae cells. This study investigated the energy demands of concentrating three diverse microalgae species (Nannochloropsis salina, Scenedesmus obliquus, and Chlorella luteoviridis) exhibiting different settling velocities, a key harvesting property, using conventional ultrasonic and crossflow membrane filtration. Membrane-free ultrasonic filtration removed 60-80% of the microalgae largely in steady-state operations, increasing concentrations 20-100 times, while consuming 1-6 kWh/m3 - similar to centrifugation. In contrast, crossflow membrane filtration retained nearly 100% of the microalgae, increasing concentrations over 20-fold, but consumed 158 kWh/m3 - over 10 times the energy of centrifugation. Both approaches exceeded the 10% energy target suggested in the 2010 National Algal Biofuel Technology Roadmap. This study also highlights potential sources of energy savings. For ultrasonic filtration, attention to an innovative, dynamic property of the ultrasonic chamber called the Energy Efficiency Factor, could lead to energy reductions by no more than 10-fold and improve the favorability of two of the microalgae species studied. For membrane filtration, a dead-end configuration could reduce energy consumption by no more than 1000-fold, making it favorable for all three species. This study not only highlights the need to further increase the energy content of microalgae cultures, but also the critical importance of continued development of ultrasonic filtration and other harvesting technologies to achieve low-cost, energy-sustainable algal biofuels.

09 BIOMASS FUELS↗
DOE OSTI2020

Outgrowth of erlotinib-resistant subpopulations recapitulated in patient-derived lung tumor spheroids and organoids

A model that recapitulates development of acquired therapeutic resistance is needed to improve oncology drug development and patient outcomes. To achieve this end, we established methods for the preparation and growth of spheroids from primary human lung adenocarcinomas, including methods to culture, passage, monitor growth, and evaluate changes in mutational profile over time. Primary lung tumor spheroids were cultured in Matrigel® with varying concentrations of erlotinib, a small molecule kinase inhibitor of epidermal growth factor receptor (EGFR) that is ineffective against KRAS mutant cells. Subtle changes in spheroid size and number were observed within the first two weeks of culture. Spheroids were cultured for up to 24 weeks, during which time interactions between different cell types, movement, and assembly into heterogeneous organoid structures were documented. Allele-specific competitive blocker PCR (ACB-PCR) was used to quantify low frequency BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA H1047R mutant subpopulations in tumor tissue residue (TR) samples and cultured spheroids. Mutant subpopulations, including multiple mutant subpopulations, were quite prevalent. Twelve examples of mutant enrichment were found in eight of the 14 tumors analyzed, based on the criteria that a statistically-significant increase in mutant fraction was observed relative to both the TR and the no-erlotinib control. Of the mutants quantified in erlotinib-treated cultures, PIK3CA H1047 mutant subpopulations increased most often (5/14 tumors), which is consistent with clinical observations. Thus, this ex vivo lung tumor spheroid model replicates the cellular and mutational tumor heterogeneity of human lung adenocarcinomas and can be used to assess the outgrowth of mutant subpopulations. Spheroid cultures with characterized mutant subpopulations could be used to investigate the efficacy of lung cancer combination therapies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Trace metal contents of autotrophic flagellates from contrasting open‐ocean ecosystems

Abstract Nano‐ and picophytoplankton are a major component of open‐ocean ecosystems and one of the main plankton functional types in biogeochemical models, yet little is known about their trace metal contents. In cultures of the picoeukaryote Ostreococcus lucimarinus , iron limitation reduced iron quotas by 68%, a fraction of the plasticity known in diatoms. In contrast, a commonly co‐occurring cyanobacterium, Prochlorococcus , showed variable iron contents with iron availability in culture. Synchrotron X‐ray fluorescence was used to measure single‐cell metal (Mn, Fe, Co, Ni, Zn) quotas of autotrophic flagellates (1.4–16.8‐ μ m diameter) collected from four ocean regions. Iron quotas were tightly constrained and showed little response to iron availability, similar to cultured Ostreococcus . Zinc quotas also did not vary with zinc availability but appeared to vary with phosphorus availability. These results suggest that macronutrient and metal availability may be equally important for controlling metal contents of small eukaryotic open‐ocean phytoplankton.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

The Influence of Bacteria on the Growth, Lipid Production, and Extracellular Metabolite Accumulation by Phaeodactylum tricornutum (Bacillariophyceae)

Here, to examine the impact of heterotrophic bacteria on microalgal physiology, we co-cultured the diatom Phaeodactylum tricornutum with six bacterial strains to quantify bacteria-mediated differences in algal biomass, total intracellular lipids, and for a subset, extracellular metabolite accumulation. A Marinobacter isolate significantly increased algal cell concentrations, dry biomass, and lipid content compared to axenic algal cultures. Two other bacterial strains from the Bacteroidetes order, of the genera Algoriphagus and Muricauda, significantly lowered P. tricornutum biomass, leading to overall decreased lipid accumulation. These three bacterial co-cultures (one mutualistic, two competitive) were analyzed for extracellular metabolites via untargeted liquid chromatography mass spectrometry to compare against bacteria-free cultures. Over 80% of the extracellular metabolites differentially abundant in at least one treatment were in higher concentrations in the axenic cultures, in agreement with the hypothesis that the co-cultured bacteria incorporated algal-derived organic compounds for growth. Furthermore, the extracellular metabolite profiles of the two growth-inhibiting cultures were more similar to one another than the growth-promoting co-culture, linking metabolite patterns to ecological role. Our results show that algal–bacterial interactions can influence the accumulation of intracellular lipids and extracellular metabolites, and suggest that utilization and accumulation of compounds outside the cell play a role in regulating microbial interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Egr1 is a 3D matrix–specific mediator of mechanosensitive stem cell lineage commitment

While extracellular matrix (ECM) mechanics strongly regulate stem cell commitment, the field’s mechanistic understanding of this phenomenon largely derives from simplified two-dimensional (2D) culture substrates. Here, we found a 3D matrix–specific mechanoresponsive mechanism for neural stem cell (NSC) differentiation. NSC lineage commitment in 3D is maximally stiffness sensitive in the range of 0.1 to 1.2 kPa, a narrower and more brain-mimetic range than we had previously identified in 2D (0.75 to 75 kPa). Transcriptomics revealed stiffness-dependent up-regulation of early growth response 1 (Egr1) in 3D but not in 2D. Egr1 knockdown enhanced neurogenesis in stiff ECMs by driving β-catenin nuclear localization and activity in 3D, but not in 2D. Mechanical modeling and experimental studies under osmotic pressure indicate that stiff 3D ECMs are likely to stimulate Egr1 via increases in confining stress during cell volumetric growth. To our knowledge, Egr1 represents the first 3D-specific stem cell mechanoregulatory factor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF)

Assessing the structure of living microbial cell membranes is a challenging analytical goal. The cell membrane is defined by its transverse structure, an approximately 5 nm-thick selectively permeable bilayer that serves many important cellular functions. Compositionally complex, dynamic, and organized in both the transverse and lateral dimensions, understanding the cell membrane structure—and the role that structure plays in cellular function, communication, and environmental sensing is an active scientific effort. Previously, we have devised a novel isotopic labeling approach for membrane lipids to enable direct in vivo structural studies of the cell membrane in the Gram-positive bacterium, Bacillus subtilis , using small-angle neutron scattering. This was accomplished through a genetic inhibition of fatty acid (FA) degradation (Δ fadN ) and a chemical inhibition of FA biosynthesis using cerulenin, an irreversible inhibitor of type II fatty acid synthases. Here, we improve upon the previous system by introducing a dCas9/sgRNA- fabF complex that blocks transcription of the essential fabF gene when under xylose induction. This leads to greater sensitivity to cerulenin in the mutant strain (JEBS102) and more robust cell growth when supplementary FAs are introduced to the culture medium. A subtle change in FA uptake is noted when compared to the prior labeling strategy. This is seen in the gas chromatography/mass spectrometry (GC/MS) data as a higher ratio of n 16:0 to a 15:0, and manifests in an apparent increase in the membrane thickness determined via neutron scattering. This represents an improved method of isotopic labeling for the cell membrane of Bacillus subtilis; enabling improved investigations of cellular uptake and utilization of FAs, cell membrane structure and organization as a phenotypic response to metabolic and environmental changes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

In vitro toxicity assessment of uranium particulates on different human lung epithelial cell models

Inhalation of uranium aerosols produced via human activities such as mining can pose a threat to human respiratory systems. Uranium oxide particulates emit short-range alpha particles that elicit DNA and direct damage, beyond associated physiochemical heavy-metal toxicity, to internal epithelial tissues. The availability of reliable in vitro models to study radiation exposure can greatly enhance our ability to understand and combat the biological impacts of exposure. However, the toxicological effects of alpha emissions and/or the oxidation states of uranium particulates vary across different human lung epithelial cell models and have not been systematically compared. We have endeavored to address this limitation by comparing impacts in three different human lung cell models: primary human bronchial and tracheal epithelial cells, primary human small airway epithelial cells, and human adenocarcinoma alveolar basal epithelial cells. Other studies have mainly investigated the toxicity of depleted uranium. Here, we compared the exposure of uranium oxide particulates (U 3 O 8 and UO 3 ) of different enrichment states on the chosen cell systems. Each cell model was exposed to 0.1, 1, 10, 50, 100, and 500 µg/mL of depleted U 3 O 8 , highly-enriched U 3 O 8 , and natural UO 3 particulates for 24 hours in submerged monolayer cultures. We compared viability and superoxide dismutase activity results across cell lines and uranium enrichment/ oxidative states. The results showed that 1) the oxide state of the particulates affected cell viability, implying that uranium’s different oxidation states contribute to different toxicological responses, and 2) each cell model reacts differently when exposed to uranium oxides, which may provide insights into the mechanistic processes associated with the exposure of radiological particulates on different biological systems. For instance, increased uranium enrichment corresponds to increased toxicity for the primary cells, but not for the immortalized cells. Our study shows that a holistic approach that incorporates similarities between model systems and types of radionuclides is required to truly develop empirical solutions for radiation exposure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Photobiological production of high-value pigments via compartmentalized co-cultures using Ca-alginate hydrogels

Abstract Engineered cyanobacterium Synechococcus elongatus can use light and CO 2 to produce sucrose, making it a promising candidate for use in co-cultures with heterotrophic workhorses. However, this process is challenged by the mutual stresses generated from the multispecies microbial culture. Here we demonstrate an ecosystem where S. elongatus is freely grown in a photo-bioreactor (PBR) containing an engineered heterotrophic workhorse (either β-carotene-producing Yarrowia lipolytica or indigoidine-producing Pseudomonas putida ) encapsulated in calcium-alginate hydrogel beads. The encapsulation prevents growth interference, allowing the cyanobacterial culture to produce high sucrose concentrations enabling the production of indigoidine and β-carotene in the heterotroph. Our experimental PBRs yielded an indigoidine titer of 7.5 g/L hydrogel and a β-carotene titer of 1.3 g/L hydrogel, amounts 15–22-fold higher than in a comparable co-culture without encapsulation. Moreover, 13 C-metabolite analysis and protein overexpression tests indicated that the hydrogel beads provided a favorable microenvironment where the cell metabolism inside the hydrogel was comparable to that in a free culture. Finally, the heterotroph-containing hydrogels were easily harvested and dissolved by EDTA for product recovery, while the cyanobacterial culture itself could be reused for the next batch of immobilized heterotrophs. This co-cultivation and hydrogel encapsulation system is a successful demonstration of bioprocess optimization under photobioreactor conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

An orthogonal metabolic framework for one-carbon utilization

Metabolic engineering often entails concurrent engineering of substrate utilization, central metabolism, and product synthesis pathways to maximize the conversion of a carbon substrate into desired product(s). Here, we report an alternative approach using synthetic pathways for C1 bioconversion that are orthogonal to the host metabolic network, thus minimizing interdependency on native metabolism and enabling more efficient biocatalysts. Here, the engineered pathways are based on formyl-CoA elongation (FORCE) reactions catalyzed by the enzyme 2-hydroxyacyl-CoA lyase (HACL) and generate multi-carbon products directly from C1 elongation units in the form of formyl-CoA. Herein, we use thermodynamic and stoichiometric analyses to evaluate different FORCE pathway variants, including aldose elongation, α-reduction, and aldehyde elongation. Promising variants were further prototyped using cell-free systems (purified enzymes and cell extracts) as well as resting and growing cultures of non-methylotrophic bacterium Escherichia coli. We demonstrate that C1 substrates formate, formaldehyde, and methanol can be used as inputs for FORCE pathways and that FORCE reactions can serve as a platform for varied product synthesis including glycolate, ethylene glycol, ethanol, and glycerate. Furthermore, the orthogonal FORCE pathways have the potential to be integrated with host metabolism for synthetic methylotrophy by the production of native growth substrates as demonstrated in a two-strain culture system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

In situ diversity of metabolism and carbon use efficiency among soil bacteria

The central carbon (C) metabolic network harvests energy to power the cell and feed biosynthesis for growth. In pure cultures, bacteria use some but not all of the network’s major pathways, such as glycolysis and pentose phosphate and Entner-Doudoroff pathways. However, how these pathways are used in microorganisms in intact soil communities is unknown. Here, we analyzed the incorporation of 13 C from glucose isotopomers into phospholipid fatty acids. We showed that groups of Gram-positive and Gram-negative bacteria in an intact agricultural soil used different pathways to metabolize glucose. They also differed in C use efficiency (CUE), the efficiency with which a substrate is used for biosynthesis. Our results provide experimental evidence for diversity among microbes in the organization of their central carbon metabolic network and CUE under in situ conditions. These results have important implications for our understanding of how community composition affects soil C cycling and organic matter formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Glutathione Synthetase Overexpression in Acidithiobacillus ferrooxidans Improves Halotolerance of Iron Oxidation

Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

In Situ Optical Quantification of Extracellular Electron Transfer Using Plasmonic Metal Oxide Nanocrystals**

Extracellular electron transfer (EET) is a critical form of microbial metabolism that enables respiration on a variety of inorganic substrates, including metal oxides. However, quantifying current generated by electroactive bacteria has been predominately limited to biofilms formed on electrodes. To address this, we developed a platform for quantifying EET flux from cell suspensions using aqueous dispersions of infrared plasmonic tin-doped indium oxide nanocrystals. Furthermore, tracking the change in optical extinction during electron transfer enabled quantification of current generated by planktonic Shewanella oneidensis cultures. Using this method, we differentiated between starved and actively respiring cells, cells of varying genotype, and cells engineered to differentially express a key EET gene using an inducible genetic circuit. Overall, our results validate the utility of colloidally stable plasmonic metal oxide nanocrystals as quantitative biosensors in aqueous environments and contribute to a fundamental understanding of planktonic S. oneidensis electrophysiology using simple in situ spectroscopy.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli

Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics in high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here, we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct Escherichia coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

O-GlcNAc transferase Ogt regulates embryonic neuronal development through modulating Wnt/β-catenin signaling

Abstract Ogt-mediated O-GlcNAcylation is enriched in the nervous system and involves in neuronal development, brain function and neurological diseases. However, the roles of Ogt and O-GlcNAcylation in embryonic neurogenesis have remained largely unknown. Here, we show that Ogt is highly expressed in embryonic brain, and Ogt depletion reduces the proliferation of embryonic neural stem cells and migration of new born neurons. Ogt depletion in cultured hippocampal neurons impairs neuronal maturation, including reduced dendritic numbers and length, and immature development of spines. Mechanistically, Ogt depletion decreases the activity of Wnt/β-catenin signaling. Ectopic β-catenin rescues neuronal developmental deficits caused by Ogt depletion. Ogt also regulates human cortical neurogenesis in forebrain organoids derived from induced pluripotent stem cells. Our findings reveal the essential roles and mechanisms of Ogt-mediated O-GlcNAc modification in regulating mammalian neuronal development.

Shen, Hui↗
DOE OSTI2023

Proposed minimal standards for description of methanogenic archaea

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA–DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

Microbiology↗