Bioremediation of oily hypersaline soil via autochthonous bioaugmentation with halophilic bacteria and archaea.

Kuwaiti hypersaline soil samples were contaminated with 5 % (w/w) weathered Kuwaiti light crude oil and bioaugmented with autochthonous halophilic hydrocarbonoclastic archaeal and bacterial strains, two each, individually and as consortia. Residual oil contents were determined, and microbial communities were analyzed by culture-dependent and culture-independent approaches initially and seasonally for one year. After one year of the bioremediation process, the mean oil degradation rate was similar across all treated soils including the controlled unbioaugmented one. Oil hydrocarbons were drastically reduced in all soil samples with values ranging from 82.7 % to 93 %. During the bioremediation process, the number of culturable oil-degrading bacteria increased to a range of 142 to 344 CFUx104 g-1 after 12 months of bioaugmentation. Although culture-independent analysis showed a high proportion of inoculants initially, none could be cultured throughout the bioremediation procedure. Within a year, microbial communities changed continually, and 33 species of halotolerant/halophilic hydrocarbonoclastic bacteria were isolated and identified belonged mainly to the three major bacterial phyla Actinobacteria, Proteobacteria, and Firmicutes. The archaeal phylum Halobacterota represented <1 % of the microbial community's relative abundance, which explains why none of its members were cultured. Improving the biodegradability of an already balanced environment by autochthonous bioaugmentation is more involved than just adding the proper oil degraders. This study emphasizes the possibility of a relatively large resistant population, a greater diversity of oil-degrading microorganisms, and the highly selective impacts of oil contamination on hypersaline soil bacterial communities.

Rice-fish coculture without phosphorus addition improves paddy soil nitrogen availability by shaping ammonia-oxidizing archaea and bacteria in subtropical regions of South China.

Rice-fish coculture (RFC), as a traditional agricultural strategy in China, can optimally utilize the scarce resource, especially in subtropical regions where phosphorus (P) deficiency limits agricultural production. However, ammonia-oxidizing archaea (AOA) and bacteria (AOB) are involved in the ammonia oxidation, but it remains uncertain whether their community compositions are related to the RFC combined with and without P addition that improves soil nitrogen (N) use efficiency. Here, a microcosm experiment was conducted to assess the impacts of RFC combined with and without inorganic P (0 and 50 mg P kg-1 as KH2PO4) addition on AOA and AOB community diversities, enzyme activities and N availability. The results showed that RFC significantly increased available N content without P addition compared with P addition. Moreover, RFC significantly increased urease activity and AOA shannon diversity, and reduced NAG activity and AOB shannon diversity without P addition, respectively. Higher diversity of AOA compared with that of AOB causes greater competition for resources and energy within their habitats, thereby resulting in lower network complexity. Our findings indicated that the abundances of AOA and AOB are influenced through the introduction of fish and/or P availability, of which AOB is linked to N availability. Overall, RFC could improve paddy soil N availability without P addition in subtropical region, which provides a scientific reference for promoting the practices that reduce N fertilizer application in RFC.

Enhancement of methane production from anaerobic digestion of Erigeron canadensis via O2-nanobubble water supplementation.

Malignant invasive Erigeron canadensis, as a typical lignocellulosic biomass, is a formidable challenge for sustainable and efficient resource utilization, however nanobubble water (NBW) coupled with anaerobic digestion furnishes a prospective strategy with superior environmental and economic effectiveness. In this study, influence mechanism of various O2-NBW addition times on methanogenic performance of E. canadensis during anaerobic digestion were performed to achieve the optimal pollution-free energy conversion. Results showed that supplementation of O2-NBW in digestion system could significantly enhance the methane production by 10.70-16.17%, while the maximum cumulative methane production reached 343.18 mL g-1 VS in the case of one-time O2-NBW addition on day 0. Furthermore, addition of O2-NBW was conducive to an increase of 2-90% in the activities of dehydrogenase, α-glucosidase and coenzyme F420. Simultaneously, both facultative bacteria and methanogenic archaea were enriched as well, further indicating that O2-NBW might be responsible for facilitating hydrolytic acidification and methanogenesis. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) cluster analysis, provision of O2-NBW enhanced the metabolism of carbohydrate and amino acid, translation as well as membrane transport of bacteria and archaea. This study might offer the theoretical guidance and novel insights for efficient recovery of energy from lignocellulosic biomass on account of O2-NBW adhibition in anaerobic digestion system, progressing tenor of carbon-neutral vision.

Investigating the effect of supplementing different levels of Isochrysis galbana on in vitro rumen fermentation parameters.

This study aimed to investigate the effect of supplementing Isochrysis galbana (I. galbana) at levels of 0 (control), 1, 2, 3, 4, and 5 (g/100 g DM) of the diet on the gas production kinetics, methane production, rumen fermentation parameters, and relative microbial population in vitro. Supplementation of I. galbana at high level (5 g/100 g DM) caused a significant decrease in total gas production (p < 0.05). High supplementation rates (4 and 5 g/100 g DM) decreased CH4 production relative to the control by 18.4% and 23.2%, respectively. Although rumen ammonia nitrogen (N-NH3) and total volatile fatty acids (VFA) concentrations were affected by dietary treatments, but the VFA profile did not changed. The relative proportion of protozoa and methanogenic archaea as well as Anaerovibrio lipolytica, Prevotella spp., Ruminococcus flavefaciens, and Fibrobacter succinogenes were decreased significantly as a result of microalgae supplementation. However, the relative abundance of Ruminococcus albus, Butyrivibrio fibrisolvens and Selenomonas ruminantium were significantly increased (p < 0.05), related to the control group. As well, the pH was not affected by dietary treatments. It was concluded that I. galbana reduced in vitro CH4 production and methanogenic archaea that its worth to be investigated further in in vivo studies.

Functional and structural responses of a halophilic consortium to oily sludge during biodegradation.

Biotreatment of oily sludge and the involved microbial communities, particularly in saline environments, have been rarely investigated. We enriched a halophilic bacterial consortium (OS-100) from petroleum refining oily sludge, which degraded almost 86% of the aliphatic hydrocarbon (C10-C30) fraction of the oily sludge within 7 days in the presence of 100 g/L NaCl. Two halophilic hydrocarbon-degrading bacteria related to the genera Chromohalobacter and Halomonas were isolated from the OS-100 consortium. Hydrocarbon degradation by the OS-100 consortium was relatively higher compared to the isolated bacteria, indicating potential synergistic interactions among the OS-100 community members. Exclusion of FeCl2, MgCl2, CaCl2, trace elements, and vitamins from the culture medium did not significantly affect the hydrocarbon degradation efficiency of the OS-100 consortium. To the contrary, hydrocarbon biodegradation dropped from 94.1 to 54.4% and 5% when the OS-100 consortium was deprived from phosphate and nitrogen sources in the culture medium, respectively. Quantitative PCR revealed that alkB gene expression increased up to the 3rd day of incubation with 11.277-fold, consistent with the observed increments in hydrocarbon degradation. Illumina-MiSeq sequencing of 16 S rRNA gene fragments revealed that the OS-100 consortium was mainly composed of the genera Halomonas, Idiomarina, Alcanivorax and Chromohalobacter. This community structure changed depending on the culturing conditions. However, remarkable changes in the community structure were not always associated with remarkable shifts in the hydrocarbonoclastic activity and vice versa. The results show that probably synergistic interactions between community members and different subpopulations of the OS-100 consortium contributed to salinity tolerance and hydrocarbon degradation.

Trophic relationships between protists and bacteria and fungi drive the biogeography of rhizosphere soil microbial community and impact plant physiological and ecological functions.

Rhizosphere microorganisms play a vital role in enhancing plant health, productivity, and the accumulation of secondary metabolites. Currently, there is a limited understanding of the ecological processes that control the assembly of community. To address the role of microbial interactions in assembly and for functioning of the rhizosphere soil microbiota, we collected rhizosphere soil samples from Anisodus tanguticus on the Tibetan Plateau spanning 1500 kilometers, and sequenced the bacteria, fungi, archaea, and protist communities. We observed a significant but weak distance-decay relationship in the microbial communities of rhizosphere soil. Our comprehensive analysis of spatial, abiotic, and biotic factors showed that trophic relationships between protists and bacteria and fungi predominantly influenced the alpha and beta diversity of bacterial, fungal, and protistan communities, while abiotic factors had a greater impact on archaeal communities, including soil pH, available phosphorus, total phosphorus and mean annual temperature. Importantly, microbial interactions had a more significant influence on Anisodus tanguticus physiological and ecological functions compared to individual microorganisms. Network analyses revealed that bacteria occupy a central position of the co-occurrence network and play a crucial role of connector within this community. The addition of protists increased the stability of bacterial, fungal, and archaeal networks. Overall, our findings indicate that trophic relationships play an important role in assembly and for functioning of the rhizosphere soil microbiota. Bacterial communities serve as a crucial link between different kingdoms of microorganisms in the rhizosphere community. These findings help us to fully harness the beneficial functions of rhizosphere microorganisms for plants and achieve sustainable use of biological resources.

Human associated Archaea: a neglected microbiome worth investigating.

The majority of research in the field of human microbiota has predominantly focused on bacterial and fungal communities. Conversely, the human archaeome has received scant attention and remains poorly studied, despite its potential role in human diseases. Archaea have the capability to colonize various human body sites, including the gastrointestinal tract, skin, vagina, breast milk, colostrum, urinary tract, lungs, nasal and oral cavities. This colonization can occur through vertical transmission, facilitated by the transfer of breast milk or colostrum from mother to child, as well as through the consumption of dairy products, organic produce, salty foods, and fermented items. The involvement of these microorganisms in diseases, such as periodontitis, might be attributed to their production of toxic compounds and the detoxification of growth inhibitors for pathogens. However, the precise mechanisms through which these contributions occur remain incompletely understood, necessitating further studies to assess their impact on human health.

The oral microbiome: diversity, biogeography and human health.

The human oral microbiota is highly diverse and has a complex ecology, comprising bacteria, microeukaryotes, archaea and viruses. These communities have elaborate and highly structured biogeography that shapes metabolic exchange on a local scale and results from the diverse microenvironments present in the oral cavity. The oral microbiota also interfaces with the immune system of the human host and has an important role in not only the health of the oral cavity but also systemic health. In this Review, we highlight recent advances including novel insights into the biogeography of several oral niches at the species level, as well as the ecological role of candidate phyla radiation bacteria and non-bacterial members of the oral microbiome. In addition, we summarize the relationship between the oral microbiota and the pathology of oral diseases and systemic diseases. Together, these advances move the field towards a more holistic understanding of the oral microbiota and its role in health, which in turn opens the door to the study of novel preventive and therapeutic strategies.

Study on the accelerated biodegradation of PAHs in subsurface soil via coupled low-temperature thermally treatment and electron acceptor stimulation based on metagenomic sequencing.

In situ anoxic bioremediation is a sustainable technology to remediate PAHs contaminated soils. However, the limited degradation rate of PAHs under anoxic conditions has become the primary bottleneck hindering the application of this technology. In this study, coupled low-temperature thermally treatment (<50 °C) and EA biostimulation was used to enhance PAH removal. Anoxic biodegradation of PAHs in soil was explored in microcosms in the absence and presence of added EAs at 3 temperatures (15 °C, 30 °C, and 45 °C). The influence of temperature, EA, and their interaction on the removal of PAHs were identified. A PAH degradation model based on PLSR analysis identified the importance and the positive/negative role of parameters on PAH removal. Soil archaeal and bacterial communities showed similar succession patterns, the impact of temperature was greater than that of EA. Soil microbial community and function were more influenced by temperature than EAs. Close and frequent interactions were observed among soil bacteria, archaea, PAH-degrading genes and methanogenic genes. A total of 15 bacterial OTUs, 1 PAH-degrading gene and 2 methanogenic genes were identified as keystones in the network. Coupled low-temperature thermally treatment and EA stimulation resulted in higher PAH removal efficiencies than EA stimulation alone and low-temperature thermally treatment alone.

Biochar amendment reassembles microbial community in a long-term phosphorus fertilization paddy soil.

This study investigates the effect of biochar amendment on microbial community structure and soil nutrient status in paddy soil that has been fertilized for an extended period of time, shedding light on sustainable agricultural practices. A 90-day incubation period revealed that biochar amendment, as opposed to long-term fertilization, significantly influenced the physicochemical properties and microbial composition of the soil. The microcosm experiment conducted using six treatments analyzed soil samples from a long-term rice ecosystem. We employed microbial biomarkers (phospholipid fatty acids, PLFAs; isoprenoid and branched glycerol dialkyl glycerol tetraethers, iGDGTs and brGDGTs; DNA) to assess microbial biomass and community structure. Biochar addition led to a decrease in PLFA biomass (15-32%) and archaeal iGDGT abundance (14-43%), while enhancing bacterial brGDGT abundance by 15-77%. Intact biochar increased archaeal and bacterial diversity, though fungal diversity remained unchanged. However, acid-washed biochar did not result in a uniform microbial diversity response. The abundance of various microbial taxa was changed by biochar amendment, including Crenarchaeota, Proteobacteria, Nitrospira, Basidiomycota, Halobacterota, Chloroflexi, Planctomycetota, and Ascomycota. Soil NH4+-N was found as the primary environmental factor impacting the composition of archaea, bacteria, and fungus in this study. These findings imply that the addition of biochar has a quick influence on the structure and activity of microbial communities, with fungi possibly having a critical role in acid paddy soil. This study contributes valuable knowledge for developing sustainable agricultural practices that promote healthy soil ecosystems. KEY POINTS: • Biochar type and phosphorus fertilization demonstrated an interactive effect on the diversity of archaea, but no such effect was observed for bacteria and fungi. • Soil fungi contribute to approximately 20% of the total phospholipid fatty acid (PLFA) content. • Biochar, especially acid-washed rice straw biochar, increases glucose metabolism in bacteria and archaea and decreases saprophytic fungi.

Cas13d-mediated multiplex RNA targeting confers a broad-spectrum resistance against RNA viruses in potato.

CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immune mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13d has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However a vast number of different viruses can potentially infect the same host plant resulting in mixed infection, thus necessitating the generation of crops with broad-spectrum resistance to multiple viruses. Here we report the repurposing of CRISPR/Cas13d coupled with an endogenous tRNA-processing system (polycistronic tRNA-gRNA, PTG) to target the multiple potato RNA viruses. Expression of Cas13d and four different gRNAs were observed in transgenic potato lines expressing the Cas13d/PTG construct. We show that the Cas13d/PTG transgenic plants exhibit resistance to either PVY, PVS, PVX or PLRV alone or two/three viruses simultaneously by reducing viral accumulation in plant cells. In sum, our findings provide an efficient strategy for engineering crops that can simultaneously resist infection by multiple RNA viruses.

Candidatus Alkanophaga archaea from Guaymas Basin hydrothermal vent sediment oxidize petroleum alkanes.

Methanogenic and methanotrophic archaea produce and consume the greenhouse gas methane, respectively, using the reversible enzyme methyl-coenzyme M reductase (Mcr). Recently, Mcr variants that can activate multicarbon alkanes have been recovered from archaeal enrichment cultures. These enzymes, called alkyl-coenzyme M reductase (Acrs), are widespread in the environment but remain poorly understood. Here we produced anoxic cultures degrading mid-chain petroleum n-alkanes between pentane (C5) and tetradecane (C14) at 70 °C using oil-rich Guaymas Basin sediments. In these cultures, archaea of the genus Candidatus Alkanophaga activate the alkanes with Acrs and completely oxidize the alkyl groups to CO2. Ca. Alkanophaga form a deep-branching sister clade to the methanotrophs ANME-1 and are closely related to the short-chain alkane oxidizers Ca. Syntrophoarchaeum. Incapable of sulfate reduction, Ca. Alkanophaga shuttle electrons released from alkane oxidation to the sulfate-reducing Ca. Thermodesulfobacterium syntrophicum. These syntrophic consortia are potential key players in petroleum degradation in heated oil reservoirs.

Selenite bioreduction by a consortium of halophilic/halotolerant bacteria and/or yeasts in saline media.

Selenium oxyanions are released into environments by natural and anthropogenic activities and are present in agricultural and glass manufacturing wastewater in several locations worldwide. Excessive amounts of this metalloid have adverse effects on the health of living organisms. Halophilic and halotolerant microorganisms were selected for selenium oxyanions remediation due to presence of significant amount of salt in selenium-containing wastewater. Effects of aeration, carbon sources, competitive electron acceptors, and reductase inhibitors were investigated on SeO32- bio-removal. Additionally, NO3--containing wastewater were exploited to investigate SeO32- remediation in synthetic agricultural effluents. The results showed that the SeO32- removal extent is maximum in aerobic conditions with succinate as a carbon source. SO42- and PO43- do not significantly interfere with SeO32- reduction, while WO42- and TeO32- decrease the SeO32- removal percentage (up to 35 and 37%, respectively). Furthermore, NO3- had an adverse effect on SeO32- biotransformation by our consortia. All consortia reduced SeO32- in synthetic agricultural wastewaters with a 45-53% removal within 120 h. This study suggests that consortia of halophilic/halotolerant bacteria and yeasts could be applied to treat SeO32--contaminated drainage water. In addition, sulphates, and phosphates do not interfere with selenite bioreduction by these consortia, which makes them suitable candidates for the bioremediation of selenium-containing wastewater.

Rewetting the hyper-arid Atacama Desert soil reactivates a carbon-starved microbial decomposer community and also triggers archaeal metabolism.

Extreme environmental conditions make soils of the hyper-arid Atacama Desert one of the most hostile habitats for life on the planet. During the short intervals of moisture availability that occur, it remains unresolved how soil microorganisms physiologically respond to such dramatic environmental changes. Therefore, we simulated a precipitation event - without (H2O) and with (H2O + C) labile carbon (C) supplementation - and investigated the responses in microbial communities (using phospholipid fatty acids (PLFAs) and archaeal glycerol dialkyl glycerol tetraether (GDGTs)) and physiology (by means of respiration, bacterial and fungal growth and C-use efficiency (CUE)) during a five-day incubation. We demonstrated that bacterial and fungal growth does occur in these extreme soils following rewetting, albeit at 100-10,000-fold lower rates compared to previously studied soil systems. C supplementation increased levels of bacterial growth and respiration responses by 5- and 50-fold, respectively, demonstrating a C-limited microbial decomposer community. While the microbial CUE following rewetting was c. 14 %, the addition of labile C during rewetting resulted in a substantial reduction (c. 1.6 %). Consistent with these interpretations, the PLFA composition clearly shifted from saturated towards more unsaturated and branched PLFAs, which could arise from (i) a physiological adaptation of the cell membrane to changing osmotic conditions or (ii) a community composition shift. Significant increases in total PLFA concentrations were solely found with H2O + C addition. Contrary to other recent studies, we found evidence for a metabolically active archaeal community in these hyper-arid soils upon rewetting. We conclude that (i) microorganisms in this extreme soil habitat can be activated and grow within days following rewetting, (ii) available C is the limiting factor for microbial growth and biomass gains, and (iii) that an optimization of tolerating the extreme conditions while maintaining a high CUE comes at the expense of very poor resource-use efficiency during high resource availability.

Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.

Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components.

Efficacy of rice husk and halophilic bacterial biofilms in the treatment of saline water.

Water salinity causes less production of agricultural productivity, low economic returns, soil destructions, less sustainability, and reduction in the germination rate. The current study was aimed to understand the combined potential of halophilic bacteria and rice husk in treating water salinity. In total, 10 halophilic bacterial isolates were isolated from Khewra Mines, Pakistan. Bacterial isolates were characterized by biochemical tests. 16S rRNA gene sequencing identified the isolate SO 1 as Bacillus safensis (accession number ON203008) being the promising halophilic bacteria tolerating upto 3 M NaCl concentration. Next, rice husk was used as carbon source for bacterial biofilm formation, growth and propagation. For saline water treatment, the experimental setting comprising glass wool, rice husk and artificial sea water (3 M) was set. B. safensis biofilm was developed in test samples to desaline the saline water containing 3 M NaCl concentration. Following NaCl decline, flame photometric analysis was used to check the desalination extent of treated saline water. Results showed decreased sodium level in sea water in the presence of rice husk and glass wool. The eluted water used for the germination of Zea mays seeds showed improved growth performance. Also, decreased photosynthetic pigments (chlorophyll "a" = 18.99, and chlorophyll "b" = 10.65), sugar contents (0.7593), and increased carotenoid (1526.91), protein contents (0.4521) were noted compared to control. This eco-friendly approach for bioremediation of salt-affected soils to optimize crop yields under stress through halophilic bacteria and rice husk may overcome the problem of the reduced yield of cash crops/agriculture and water shortage by salinity.

Response of gene abundance of ammonia-oxidizing microorganisms and denitrifying microorganisms to nitrogen and phosphorus addition in subtropical forest.

We conducted a nitrogen (N) and phosphorus (P) addition experiment in Qianjiangyuan National Park in 2015, to investigate the response of ammonia-oxidizing microorganisms and denitrifying microorganisms. There were four treatments, including N addition (N), P addition (P), NP, and control (CK). Soil samples were collected in April (wet season) and November (dry season) of 2021. The abundance of amoA gene of ammonia-oxidizing microorganisms (i.e., ammonia-oxidizing archaea, AOA; ammonia-oxidizing bacteria, AOB; comammox) and denitrifying microbial genes (i.e., nirS, nirK, and nosZ) were determined using quantitative PCR approach. The results showed that soil pH was significantly decreased by long-term N addition, while soil ammonium and nitrate contents were significantly increased. Soil available P and total P contents were significantly increased with the long-term P addition. The addition of N (N and NP treatments) significantly increased the abundance of AOB-amoA gene in both seasons, and reached the highest in the N treatment around 8.30×107 copies·g-1 dry soil. The abundance of AOA-amoA gene was significantly higher in the NP treatment than that in CK, with the highest value around 1.17×109 copies·g-1 dry soil. There was no significant difference in N-related gene abundances between two seasons except for the abundance of comammox-amoA. Nitrogen addition exerted significant effect on the abundance of AOB-amoA, nirK and nosZ genes, especially in wet season. Phosphorus addition exerted significant effect on the abundance of AOA-amoA and AOB-amoA genes in both seasons, but did not affect denitrifying gene abundances. Soil pH, ammonium, nitrate, available P, and soil water contents were the main factors affecting the abundance of soil N-related functional genes. In summary, the response of soil ammonia-oxidizing microorganisms and denitrifying microorganisms was more sensitive to N addition than to P addition. These findings shed new light for evaluating soil nutrient availability as well as their response mechanism to global change in subtropical forests.

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.

Cobalamin producers and prokaryotic consumers in the Northwest Atlantic.

Cobalamin availability can influence primary productivity and ecological interactions in marine microbial communities. The characterization of cobalamin sources and sinks is a first step in investigating cobalamin dynamics and its impact on productivity. Here, we identify potential cobalamin sources and sinks on the Scotian Shelf and Slope in the Northwest Atlantic Ocean. Functional and taxonomic annotation of bulk metagenomic reads, combined with analysis of genome bins, were used to identify potential cobalamin sources and sinks. Cobalamin synthesis potential was mainly attributed to Rhodobacteraceae, Thaumarchaeota, and cyanobacteria (Synechococcus and Prochlorococcus). Cobalamin remodelling potential was mainly attributed to Alteromonadales, Pseudomonadales, Rhizobiales, Oceanospirilalles, Rhodobacteraceae, and Verrucomicrobia, while potential cobalamin consumers include Flavobacteriaceae, Actinobacteria, Porticoccaceae, Methylophiliaceae, and Thermoplasmatota. These complementary approaches identified taxa with the potential to be involved in cobalamin cycling on the Scotian Shelf and revealed genomic information required for further characterization. The Cob operon of Rhodobacterales bacterium HTCC2255, a strain with known importance in cobalamin cycling, was similar to a major cobalamin producer bin, suggesting that a related strain may represent a critical cobalamin source in this region. These results enable future inquiries that will enhance our understanding of how cobalamin shapes microbial interdependencies and productivity in this region.

The Overlooked Microbiome-Considering Archaea and Eukaryotes Using Multiplex Nanopore-16S-/18S-rDNA-Sequencing: A Technical Report Focusing on Nasopharyngeal Microbiomes.

In contrast to bacteria, microbiome analyses often neglect archaea, but also eukaryotes. This is partly because they are difficult to culture due to their demanding growth requirements, or some even have to be classified as uncultured microorganisms. Consequently, little is known about the relevance of archaea in human health and diseases. Contemporary broad availability and spread of next generation sequencing techniques now enable a stronger focus on such microorganisms, whose cultivation is difficult. However, due to the enormous evolutionary distances between bacteria, archaea and eukaryotes, the implementation of sequencing strategies for smaller laboratory scales needs to be refined to achieve as a holistic view on the microbiome as possible. Here, we present a technical approach that enables simultaneous analyses of archaeal, bacterial and eukaryotic microbial communities to study their roles in development and courses of respiratory disorders. We thus applied combinatorial 16S-/18S-rDNA sequencing strategies for sequencing-library preparation. Considering the lower total microbiota density of airway surfaces, when compared with gut microbiota, we optimized the DNA purification workflow from nasopharyngeal swab specimens. As a result, we provide a protocol that allows the efficient combination of bacterial, archaeal, and eukaryotic libraries for nanopore-sequencing using Oxford Nanopore Technologies MinION devices and subsequent phylogenetic analyses. In a pilot study, this workflow allowed the identification of some environmental archaea, which were not correlated with airway microbial communities before. Moreover, we assessed the protocol's broader applicability using a set of human stool samples. We conclude that the proposed protocol provides a versatile and adaptable tool for combinatorial studies on bacterial, archaeal, and eukaryotic microbiomes on a small laboratory scale.