Archaeal 16S rRNA Gene Research Articles

Non-syntrophic Methanogenic Hydrocarbon Degradation by an Archaeal Species

Methanogenic hydrocarbon biodegradation alters the composition of many subsurface oil reservoirs (Jones et al. 2007). This process reduced the crude oil quality by removing alkanes and thus increasing the oil viscosity. The process has been described for syntrophic associations of hydrocarbon-degrading bacteria and methanogenic archaea (Zengler et al. 1999, Dolfing et al. 2007). However, recent culture-independent studies suggest that the archaeon ‘Candidatus Methanoliparum’ may combine alkane degradation and methanogenesis (Laso-Pérez et al. 2019, Borrel et al. 2019). Here we cultured Ca. Methanoliparum from a subsurface oil reservoir. To study this culture, situ hybridization, metagenomics and metatranscriptomics were combined with stable isotope probing and metabolite analyses for describing its functioning and assessing its potential role in reservoir chemistry. Incubated an anoxic oily sludge of the Shengli oilfield with sulfate-free medium, we established a methanogenic culture. This culture consumed various different long-chain alkanes, but also alkyl-benzenes and alkyl-cycloalkanes, and produced methane and CO2 as products (Fig. 1a-b). Our analyses revealed that our culture is dominated by a single archaeon, Ca. Methanoliparia (green). To study the specific turnover of n-alkanes, the cultures were supplemented with 1,2-13C-labelled or unlabelled n-hexadecane (Fig. 2). Within 100 days of incubation, both compounds were quantitatively converted into methane and carbon dioxide. In the 13C-labelling experiment, around 0.46 mmol of 13CH4 and around 0.15 mmol of 13CO2 were produced, which was equal to 85% to 92% of the stoichiometric conversion of the supplemented labelled hexadecane according to 4C16H34 + 30H2O -- 49CH4 + 15CO2 (Fig. 2a-d). We examined the functioning of Ca. Methanoliparum in the hexadecane-degrading culture using amplicon sequencing, metagenomics and metatranscriptomics. In the archaeal domain, the relative abundance of Ca. Methanoliparum in the hexadecane-degrading cultures comprised up to 75% of the total abundance according to analysis of archaeal 16S rRNA genes. Furthermore, Ca. Methanoliparum accounted for approximately 34–40% of the total microbial community as determined by metagenomic read recruitment estimation (Fig. 2e-f). We analysed the gene expression patterns of Ca. Methanoliparum during methanogenic hexadecane degradation (Fig. 3). The genes encoding the methanogenic hexadecane degradation pathway ranked among the top 10% to 25% of all Ca. M. thermophilum transcribed genes. Moreover, genes of Ca. M. thermophilum encoding ACR and MCR ranked among the top 2% of all transcribed genes within the whole community (Fig. 3b). The MAGs of Ca. M. thermophilum also showed the highest transcription among all described MAGs (Fig. 3c). These analyses indicate that Ca. M. thermophilum performs both the degradation of hexadecane and the formation of methane. We searched the cell extracts of the hexadecane-degrading cultures for hexadecyl-CoM formation using Q-Exactive Plus Orbitrap masss pectrometry. The unlabelled hexadecane culture contained a prominent mass peak of m/z = 365.21868 that matches the mass produced by synthesized authentic standard of hexadecyl-CoM. Fragmentation of both peaks yielded hexadecyl-thiol (m/z = 257.23080, C16H33S−), ethenesulfonate (m/z = 106.98074, C2H3SO3−) and bisulfite (m/z = 80.96510, HSO3−). Moreover, cultures supplied with 1,2-13C-hexadecane produced a peak at m/z = 367.22524 for 1,2-13C-hexadecyl-CoM and the fragment 259.23721 for 1,2-13C-hexadecyl-thiol, with a mass shift of 2 units compared with the unlabelled group. These analyses confirmed the activation of n-hexadecane as hexadecyl-CoM (Fig. 4). Here we demonstrate the activation of different hydrocarbon classes by ACRs of Ca. Methanoliparum, expanding the substrate range of this enzyme to an unforeseen number of compounds. Ca. Methanoliparum couples the degradation of long-chain alkanes and alkyl-substituted hydrocarbons to methane formation, proposed as alkylotrophy. Its metabolic pathways represent an additional mode of methanogenesis, adding to CO2 reduction, methylotrophy, methyl reduction, acetate fermentation and the recently reported methoxydotrophy. Ca. Methanoliparum grows in a wide temperature range, at least between 35 and 55 °C, covering the temperature range of most biodegraded oil reservoirs. Indeed, sequences of Ca. Methanoliparum are present in various anoxic hydrocarbon-rich environments worldwide. Thus, the demonstration of the unique features of Ca. Methanoliparum in hydrocarbon conversion may fundamentally change our view of crude oil transformation and biogeochemical processes in subsurface oil reservoirs. Future studies with Ca. Methanoliparia cultures will resolve the biochemical mechanisms of methanogenic hydrocarbon degradation in archaea, and will be helpful for the application of microbial-enhanced energyrecovery from depleted oil reservoirs.

Hyperarid soil microbial community response to simulated rainfall.

The exceptionally long and protracted aridity in the Atacama Desert (AD), Chile, provides an extreme, terrestrial ecosystem that is ideal for studying microbial community dynamics under hyperarid conditions. Our aim was to characterize the temporal response of hyperarid soil AD microbial communities to ex situ simulated rainfall (5% g water/g dry soil for 4 weeks) without nutrient amendment. We conducted replicated microcosm experiments with surface soils from two previously well-characterized AD hyperarid locations near Yungay at 1242 and 1609 masl (YUN1242 and YUN1609) with distinct microbial community compositions and average soil relative humidity levels of 21 and 17%, respectively. The bacterial and archaeal response to soil wetting was evaluated by 16S rRNA gene qPCR, and amplicon sequencing. Initial YUN1242 bacterial and archaeal 16S rRNA gene copy numbers were significantly higher than for YUN1609. Over the next 4 weeks, qPCR results showed significant increases in viable bacterial abundance, whereas archaeal abundance decreased. Both communities were dominated by 10 prokaryotic phyla (Actinobacteriota, Proteobacteria, Chloroflexota, Gemmatimonadota, Firmicutes, Bacteroidota, Planctomycetota, Nitrospirota, Cyanobacteriota, and Crenarchaeota) but there were significant site differences in the relative abundances of Gemmatimonadota and Chloroflexota, and specific actinobacterial orders. The response to simulated rainfall was distinct for the two communities. The actinobacterial taxa in the YUN1242 community showed rapid changes while the same taxa in the YUN1609 community remained relatively stable until day 30. Analysis of inferred function of the YUN1242 microbiome response implied an increase in the relative abundance of known spore-forming taxa with the capacity for mixotrophy at the expense of more oligotrophic taxa, whereas the YUN1609 community retained a stable profile of oligotrophic, facultative chemolithoautotrophic and mixotrophic taxa. These results indicate that bacterial communities in extreme hyperarid soils have the capacity for growth in response to simulated rainfall; however, historic variations in long-term hyperaridity exposure produce communities with distinct putative metabolic capacities.

Influence of sedimentary deposition on the microbial assembly process in Arctic Holocene marine sediments.

The sea-level rise during the Holocene (11-0 ky BP) and its resulting sedimentation and biogeochemical processes may control microbial life in Arctic sediments. To gain further insight into this interaction, we investigated a sediment core (up to 10.7 m below the seafloor) from the Chuckchi Shelf of the western Arctic Ocean using metabarcoding-based sequencing and qPCR to characterize archaeal and bacterial 16S rRNA gene composition and abundance, respectively. We found that Arctic Holocene sediments harbor local microbial communities, reflecting geochemical and paleoclimate separations. The composition of bacterial communities was more diverse than that of archaeal communities, and specifically distinct at the boundary layer of the sulfate-methane transition zone. Enriched cyanobacterial sequences in the Arctic middle Holocene (8-7 ky BP) methanogenic sediments remarkably suggest past cyanobacterial blooms. Bacterial communities were phylogenetically influenced by interactions between dispersal limitation and environmental selection governing community assembly under past oceanographic changes. The relative influence of stochastic and deterministic processes on the bacterial assemblage was primarily determined by dispersal limitation. We have summarized our findings in a conceptual model that revealed how changes in paleoclimate phases cause shifts in ecological succession and the assembly process. In this ecological model, dispersal limitation is an important driving force for progressive succession for bacterial community assembly processes on a geological timescale in the western Arctic Ocean. This enabled a better understanding of the ecological processes that drive the assembly of communities in Holocene sedimentary habitats affected by sea-level rise, such as in the shallow western Arctic shelves.

Inhibitory Effects of Ammonia on Archaeal 16S rRNA Transcripts in Thermophilic Anaerobic Digester Sludge

High temperatures exacerbate the ammonia inhibition of anaerobic digestion coupled with methanogenesis. The inhibition of methane production by ammonia has been observed in other studies. However, the underlying mechanism is not well understood and requires further investigation. This study explored the effect of ammonia stress on archaeal 16S rRNA transcripts in thermophilic anaerobic digester sludge. Different ammonium concentrations were checked for their influence on the methanogenic rate and hydrogen accumulation. Quantitative PCR was used to compare the changes in total archaeal 16S rRNA expression. A Monte Carlo permutation test within redundancy analysis (RDA) was adopted for exploring the relationship between environmental variables and archaeal 16S rRNA and their transcripts. The results showed that with the increase in ammonium concentration, the methanogenic rate decreased and hydrogen accumulation occurred. The total archaeal 16S rRNA genes and transcripts copy numbers decreased significantly in treatments with higher ammonium concentrations (7 and 10 g NH4+-N/L), but did not change much at lower ammonia concentrations (3 g NH4+-N/L) compared with the 0 g NH4+-N/L treatment. The RDA analysis further revealed that most environmental variables, including ammonia and methane, except for formate, were significantly correlated with the community structure activity of archaeal 16S rRNA transcripts rather than the community structure of their genes. The composition of archaeal 16S rRNA transcripts showed that the hydrogenotrophic methanogen Methanothermobacter dominated the methanogenic community activity in all incubations. It exhibited sensitivity to ammonia stress and should be responsible for the methanogenic inhibition under thermophilic conditions. Our findings suggested that archaeal 16S rRNA transcripts, rather than 16S rRNA genes, are key indicators of ammonia stress and methanogenic activity.

In-depth metataxonomic investigation reveals low richness, high intervariability, and diverse phylotype candidates of archaea in the human urogenital tract

The urogenital microbiota is the potential principal factor in the pathophysiology of urinary tract infection and the protection of urinary tract health. Little is known about the urogenital archaeome although several reports have indicated that the archaeomes of various regions of the human body are associated with health. Accordingly, we aimed to determine the presence and diversity of archaeomes in the human urogenital tract. To explore the urogenital archaeome, voided urine specimens from 373 asymptomatic Korean individuals were used. No difference was observed in body mass index, age, or gender, according to presence of archaea. Analysis of archaeal 16S rRNA gene amplicons of archaea positive samples consisted of simple community structures, including diverse archaea, such as the phyla Methanobacteriota, Thermoproteota, and Halobacteriota. Asymptomatic individuals showed high participant-dependent intervariability in their urogenital archaeomes. The mean relative archaeal abundance was estimated to be 0.89%, and fluorescence in situ hybridisation micrographs provided evidence of archaeal cells in the human urogenital tract. In addition, the urogenital archaeome shared partial taxonomic compositional characteristics with those of the other body sites. In this study, Methanobacteriota, Thermoproteota, and Halobacteriota were suggested as inhabitants of the human urogenital tract, and a distinct human urogenital archaeome was characterised. These findings expand our knowledge of archaea-host associations in the human urogenital tract and may lead to novel insights into the role of archaea in urinary tract health.

Chemolithotrophic microbiome of buried soil layers following volcanic eruptions: A potential huge carbon sink

Soil burial processes create large pools of soil organic carbon that have been sequestered in the deep for long periods of time. This buried organic carbon is generally thought to be stable and resistant to decomposition, consisting of the remains of past plants and soil microbes. As a result, there is a lack of research on the role of microbiomes in the carbon transformation processes that may occur in the deep buried soil layers. In this study, we characterized the microbiomes and their metabolic potentials in organic soil layers buried following the volcanic eruption approximately 300 and 1,640 years ago, using bacterial and archaeal 16S rRNA gene sequencing. Both buried layers had comparable or even larger carbon and nitrogen contents and a microbial population as abundant and diverse as that of the surface layer. However, the organic soil layer buried 1,640 years ago showed a distinct microbiome from the surface layer, with a higher proportion of chemolithotrophic taxa, such as methanogens and NH3-oxidizing archaea. Additionally, this buried layer had a higher metabolic potential for fixing CO2 and synthesizing organic matter, while the surface community had a higher potential for degrading organic matter. These findings suggest that the buried organic soil in the deep may not simply be an accumulation of past organisms, but rather an active carbon sink driven by a chemolithotrophic microbiome that differs from the organotrophic microbiome of the surface soil.

P915 Uncovering the role of Archaea in the pathogenesis of Inflammatory bowel disease

Abstract Background Microbial dysbiosis is a major factor that contributes to the course of Inflammatory Bowel Disease (IBD), a complex class of relapsing-remitting chronic inflammatory diseases. The body's overall homeostasis is significantly influenced by the human microbiota, which is a diverse population of bacteria, viruses, fungi, protists, and archaea. Recent research shows that archaea can affect both health and disease, by directly interacting with the host as well as other members of the microbial community. Although the role of bacteria, viruses, and fungi has already been studied, the archaeome remains the under-discovered part of the gut microbiota, especially in the context of IBD. Methods In this study, we profiled the gut archaea using archaeal 16S rRNA gene region in a cohort of pediatric patients with different forms of IBD including Ulcerative colitis (UC), Crohn’s disease (CD), unclassified IBD (IBDU) and healthy controls. We determined the compositional, diversity, and taxonomic alterations of archaea in pediatric patients with IBD and compared to healthy controls. Results Euryarchaeota, and Crenarchaeota, were the major archaeal phyla identified in our samples, other minor phyla identified were Halobacterota and Thermoplasmatota. At the genus level, Methanobrevibacter and Methanosphaera were the most abundant archaea, respectively. Overall, we did not observe any significant difference in the archeal alpha diversity within the various groups. There was however a significant difference between the overall community structure of the archaea between controls and IBD patients (p<0.05). Conclusion A thorough understanding of the role played by the archaea during the pathogenesis of IBD may open the door to the identification of new processes and, ultimately, the development of creative therapeutic targets that can complement the currently used treatments for IBD patients.

Microbial diversity across compartments in an aquaponic system and its connection to the nitrogen cycle

Aquaponics combines hydroponic crop production with recirculating aquaculture. These systems comprise various compartments (fish tank, biofilter, sump, hydroponic table, radial flow settler and anaerobic digester), each with their own specific environmental pressures, which trigger the formation of unique microbial communities. Triplicated aquaponic systems were used to investigate the microbial community composition during three lettuce growing cycles. The sampling of individual compartments allowed community patterns to be generated using amplicon sequencing of bacterial and archaeal 16S rRNA genes. Nitrifying bacteria were identified in the hydroponic compartments, indicating that these compartments may play a larger role than previously thought in the system's nitrogen cycle. In addition to the observed temporal changes in community compositions within the anaerobic compartment, more archaeal reads were obtained from sludge samples than from the aerobic part of the system. Lower bacterial diversity was observed in fresh fish feces, where a highly discrete gut flora composition was seen. Finally, the most pronounced differences in microbial community compositions were observed between the aerobic and anaerobic loops of the system, with unique bacterial compositions in each individual compartment.

Laboratory-Controlled Experiments Reveal Microbial Community Shifts during Sediment Resuspension Events.

In freshwater ecosystems, dynamic hydraulic events (floods or dam maintenance) lead to sediment resuspension and mixing with waters of different composition. Microbial communities living in the sediments play a major role in these leaching events, contributing to organic matter degradation and the release of trace elements. However, the dynamics of community diversity are seldom studied in the context of ecological studies. Therefore, we carried out laboratory-induced leaching experiments, using sediments from the Villerest dam reservoir (Villerest, France). To assess whole microbial community diversity, we sequenced the archaeal and bacterial 16S rRNA genes using Illumina MiSeq. Our results suggest that the degree of dissolved oxygen found in the water during these resuspension episodes influenced community dynamics, with anoxic waters leading to drastic shifts in sedimentary communities compared to oxic waters. Furthermore, the release of microbial cells from sediments to the water column were more favorable to water colonization when events were caused by oxic waters. Most of the bacteria found in the sediments were chemoorganotrophs and most of the archaea were methanogens. Methylotrophic, as well as archaeal, and bacterial chemoorganotrophs were detected in the leachate samples. These results also show that organic matter degradation occurred, likely participating in carbonate dissolution and the release of trace elements during freshwater resuspension events.

Characterizing the vaginal microbiome in patients with high grade cervical dysplasia (277)

Objectives: The influence of the vaginal microbiome on risk for cervical dysplasia and cancer is unknown. It is hypothesized that the microbiome can impact the infectivity of human papillomavirus, and some bacteria may even have pro-oncogenic effects. The impact of dysplasia and cancer treatment on the microbiome is also unknown. The objective of this study was to characterize the vaginal microbiome of patients with high-grade cervical dysplasia and determine whether microbiome phylotypes changed after the cervical loop electrosurgical excision procedure (LEEP). Methods: Pre-menopausal women undergoing LEEP for high-grade cervical dysplasia were recruited. Vaginal swabs were collected prior to the LEEP procedure and six weeks post-procedure. DNA was extracted, and the hypervariable V4 region of the bacterial and archaeal 16S rRNA gene was amplified and sequenced using Illumina next-generation sequencing. Sequenced reads were processed and quality-filtered using AdapterRemoval and clustered into operational taxonomic units (OTUs) using a closed-reference OTU-picking strategy implemented in QIIME. A 97% identity threshold was used, and taxonomy was assigned using the EZTaxon database. The resulting OTU table was rarefied to 10,000 sequences. Conclusions: Our findings are consistent with existing data demonstrating an association between an L. iners-dominant microbiome and severe cervical dysplasia. Additionally, a more diverse and less Lactobacillus-dominated vaginal microbiome has been shown to be more prevalent amongst HPV-positive women. In contrast to published data, our findings, while based on a small number of cases, did not demonstrate consistent alterations in vaginal microbiota after the LEEP procedure. We plan to collect more longitudinal data on the persistence and progression of dysplasia in association with vaginal microbiomes to better understand the relationship between these two entities. Objectives: The influence of the vaginal microbiome on risk for cervical dysplasia and cancer is unknown. It is hypothesized that the microbiome can impact the infectivity of human papillomavirus, and some bacteria may even have pro-oncogenic effects. The impact of dysplasia and cancer treatment on the microbiome is also unknown. The objective of this study was to characterize the vaginal microbiome of patients with high-grade cervical dysplasia and determine whether microbiome phylotypes changed after the cervical loop electrosurgical excision procedure (LEEP). Methods: Pre-menopausal women undergoing LEEP for high-grade cervical dysplasia were recruited. Vaginal swabs were collected prior to the LEEP procedure and six weeks post-procedure. DNA was extracted, and the hypervariable V4 region of the bacterial and archaeal 16S rRNA gene was amplified and sequenced using Illumina next-generation sequencing. Sequenced reads were processed and quality-filtered using AdapterRemoval and clustered into operational taxonomic units (OTUs) using a closed-reference OTU-picking strategy implemented in QIIME. A 97% identity threshold was used, and taxonomy was assigned using the EZTaxon database. The resulting OTU table was rarefied to 10,000 sequences. Conclusions: Our findings are consistent with existing data demonstrating an association between an L. iners-dominant microbiome and severe cervical dysplasia. Additionally, a more diverse and less Lactobacillus-dominated vaginal microbiome has been shown to be more prevalent amongst HPV-positive women. In contrast to published data, our findings, while based on a small number of cases, did not demonstrate consistent alterations in vaginal microbiota after the LEEP procedure. We plan to collect more longitudinal data on the persistence and progression of dysplasia in association with vaginal microbiomes to better understand the relationship between these two entities.

Factors shaping the abundance and diversity of the gut archaeome across the animal kingdom

Archaea are common constituents of the gut microbiome of humans, ruminants, and termites but little is known about their diversity and abundance in other animals. Here, we analyse sequencing and quantification data of archaeal and bacterial 16S rRNA genes from 250 species of animals covering a large taxonomic spectrum. We detect the presence of archaea in 175 animal species belonging to invertebrates, fish, amphibians, birds, reptiles and mammals. We identify five dominant gut lineages, corresponding to Methanobrevibacter, Methanosphaera, Methanocorpusculum, Methanimicrococcus and “Ca. Methanomethylophilaceae”. Some archaeal clades, notably within Methanobrevibacter, are associated to certain hosts, suggesting specific adaptations. The non-methanogenic lineage Nitrososphaeraceae (Thaumarchaeota) is frequently present in animal samples, although at low abundance, but may have also adapted to the gut environment. Host phylogeny, diet type, fibre content, and intestinal tract physiology are major drivers of the diversity and abundance of the archaeome in mammals. The overall abundance of archaea is more influenced by these factors than that of bacteria. Methanogens reducing methyl-compounds with H2 can represent an important fraction of the overall methanogens in many animals. Together with CO2-reducing methanogens, they are influenced by diet and composition of gut bacteria. Our results provide key elements toward our understanding of the ecology of archaea in the gut, an emerging and important field of investigation.

Diverse Bathyarchaeotal Lineages Dominate Archaeal Communities in the Acidic Dajiuhu Peatland, Central China.

Bathyarchaeota are believed to have roles in the carbon cycle in marine systems. However, the ecological knowledge of Bathyarchaeota is limited in peatland ecosystems. Here, we investigated the vertical distribution of Bathyarchaeota community structure using quantitative PCR and high-throughput sequencing technology of ribosomal 16S rRNA gene integrated with detailed chemical profiling in the Dajiuhu Peatland, central China. Eight archaeal phyla were observed in peat samples, which mainly composed of Bathyarchaeota with a mean relative abundance about 88%, followed by Thaumarchaeota (9%). Bathyarchaeota were further split into 17 subgroups, and some subgroups showed habitat specificity to peat horizons with distinct lithological and physicochemical properties, for example, Bathy-6 and Bathy-15 had preference for the acrotelm, Bathy-5b, Bathy-16, and Bathy-19 were enriched in the catotelm, Bathy-5a, Bathy-8, and Bathy-11 were specific for the clay horizon. This spatial distribution pattern of archaeal communities along peat profile was mainly influenced by water content as indicated by RDA ordination and permutational MANOVA, whereas organic matter content exclusively affected Bathyarchaeota distribution along the peat profile significantly. The abundance of archaeal 16S rRNA genes ranged from 105 to 107 copies per gram dry sediment, and the highest archaeal biomass was observed in the periodically oxic mesotelm horizon with more dynamic archaeal interaction relationship as indicated by the network analysis. Bathyarchaeota dominated the archaeal interaction network with 82% nodes, 96% edges, and 71% keystone species. Our results provide an overview of the archaeal population, community structure, and relationship with environmental factors that affect the vertical distribution of archaeal communities and emphasize the ecology of bathyarchaeotal lineages in terrestrial peatland ecosystems.

Metagenomic insights into the environmental adaptation and metabolism of Candidatus Haloplasmatales, one archaeal order thriving in saline lakes.

The KTK 4A-related Thermoplasmata thrives in the sediment of saline lakes; however, systematic research on its taxonomy, environmental adaptation and metabolism is lacking. Here, we detected this abundant lineage in the sediment of five artificially separated ponds (salinity 7.0%-33.0%) within a Chinese soda-saline lake using culture-independent metagenomics and archaeal 16S rRNA gene amplicons. The phylogenies based on the 16S rRNA gene, and 122 archaeal ubiquitous single-copy proteins and genome-level identity analyses among the metagenome-assembled genomes demonstrate this lineage forming a novel order, Candidatus Haloplasmatales, comprising four genera affiliated with the identical family. Isoelectric point profiles of predicted proteomes suggest that most members adopt the energetically favourable 'salt-in' strategy. Functional prediction indicates the lithoheterotrophic nature with the versatile metabolic potentials for carbohydrate and organic acids as well as carbon monoxide and hydrogen utilization. Additionally, hydrogenase genes hdrABC-mvhADG are linked with incomplete reductive citrate cycle genes in the genomes, suggesting their functional connection. Comparison with the coupling of HdrABC-MvhADG and methanogenesis pathway provides new insights into the compatibility of laterally acquired methanogenesis with energy metabolism in the related order Methanomassiliicoccales. Globally, our research sheds light on the taxonomy, environmental adaptative mechanisms, metabolic potentials and evolutional significance of Ca. Haloplasmatales.

Methane production potential of pulp mill sludges: microbial community and substrate constraints.

Sludges from pulp and paper mills represent a major ecological and environmental cost, and anaerobic digestion represents a method of waste reduction and energy recovery for these mills. This study compared methane production potential and microbial communities across 11 primary- and biosludges from five pulp and paper mills using various mill processes. We measured methane production from sludges in anaerobic batch reactor experiments over 64 days. Sludges were incubated with and without added substrate to test for organic substrate limitation versus inhibition of methanogens. Initial microbial communities and changes to community composition were determined using Illumina MiSeq for metabarcoding of bacterial and archaeal 16S rRNA genes. Mean methane production potential varied greatly between sludges (0.002-79mL CH4 g-1 TS). Among primary sludges, kraft mill sludge produced more methane than other mill types. For these other mills, biosludge produced more methane than primary sludge, which had evidence of methanogen inhibition. Microbial communities and diversity were influenced by the initial community composition, and high methane production was only seen in sludges with high diversity. A number of sludges innately produced substantial methane and may be targets for further modelling and larger scale testing of anaerobic digestion.

Bacterial, archaeal, and fungal community structure and interrelationships of deep-sea shrimp intestine and the surrounding sediment

Invertebrate shrimp are one of the dominant benthic macrofaunae in the deep-sea environment. The microbiota of shrimp intestine can contribute to the adaptation of their host. The impact of surrounding sediment on intestinal microbiota has been observed in cultured shrimp species, but needs to be further investigated in deep-sea shrimp. The characterization of bacterial, archaeal, and fungal community structure and their interrelationships is also limited. In this study, wild-type deep-sea shrimp and the surrounding sediment were sampled. Shrimp individuals incubated in a sediment-absent environment were also used in this study. Microbial community structure of the shrimp intestine and sediment was investigated through amplicon sequencing targeting bacterial 16S rRNA genes, archaeal 16S rRNA genes, and fungal ITS genes. The results demonstrate distinct differences in community structure between shrimp intestine and the surrounding sediment and between surface and deep (5 mbsf) sediment. The composition of the intestinal microbiota in shrimp living without sediment was different from that of wild-type shrimp, indicating that the presence or absence of sediment could influence the shrimp intestinal microbiota. Carbohydrate metabolism, energy metabolism (carbon fixation, methane metabolism, nitrogen metabolism, and sulfur metabolism), amino acid metabolism, and xenobiotic biodegradation were the most commonly predicted microbial functionalities and they interacted closely with one another. Overall, this study provided comprehensive insights into bacterial, archaeal, and fungal community structure of deep-sea shrimp intestine as well as potential ecological interactions with the surrounding sediment. This study could update our understanding of the microbiota characteristics in shrimp and sediment in deep-sea ecosystems.

Antibiotics as a silent driver of climate change? A case study investigating methane production in freshwater sediments

Methane (CH4) is the second most important greenhouse gas after carbon dioxide (CO2) and is inter alia produced in natural freshwater ecosystems. Given the rise in CH4 emissions from natural sources, researchers are investigating environmental factors and climate change feedbacks to explain this increment. Despite being omnipresent in freshwaters, knowledge on the influence of chemical stressors of anthropogenic origin (e.g., antibiotics) on methanogenesis is lacking. To address this knowledge gap, we incubated freshwater sediment under anaerobic conditions with a mixture of five antibiotics at four levels (from 0 to 5000 µg/L) for 42 days. Weekly measurements of CH4 and CO2 in the headspace, as well as their compound-specific δ13C, showed that the CH4 production rate was increased by up to 94% at 5000 µg/L and up to 29% at field-relevant concentrations (i.e., 50 µg/L). Metabarcoding of the archaeal and eubacterial 16S rRNA gene showed that effects of antibiotics on bacterial community level (i.e., species composition) may partially explain the observed differences in CH4 production rates. Despite the complications of transferring experimental CH4 production rates to realistic field conditions, the study indicated that chemical stressors contribute to the emissions of greenhouse gases by affecting the methanogenesis in freshwaters.

Methane emissions may be driven by hydrogenotrophic methanogens inhabiting the stem tissues of poplar.

Living trees in forests emit methane (CH4 ) from their stems. However, the magnitudes, patterns, drivers, origins, and biogeochemical pathways of these emissions remain poorly understood. We measured insitu CH4 fluxes in poplar stems and soils using static chambers and investigated the microbial communities of heartwood and sapwood by sequencing bacterial 16S, archaeal 16S, and fungal ITS rRNA genes. Methane emissions from poplar stems occurred throughout the sampling period. The mean CH4 emission rate was 2.7 mg m-2 stem d-1 . Stem CH4 emission rate increased significantly with air temperature, humidity, soil water content, and soil CH4 fluxes, but decreased with increasing sampling height. The CO2 reduction and methylotrophic methanogenesis were the major methanogenic pathways in wood tissues. The dominant methanogen groups detected in stem tissues were Methanobacterium, Methanobrevibacter, Rice Cluster I, Methanosarcina, Methanomassiliicoccus, Methanoculleus, and Methanomethylophilaceae. In addition, three methanotrophic genera were identified in the heartwood and sapwood - Methylocystis, Methylobacterium, and Paracoccus. Overall, stem CH4 emissions can originate directly from the internal tissues or co-occur from soils and stems. The co-existence of methanogens and methanotrophs within heartwood and sapwood highlights a need for future research in the microbial mechanisms underlying stem CH4 exchange with the atmosphere.

Influence of Environmental Factors on the Variability of Archaeal Communities in a Karst Wetland.

Archaea are ubiquitous and play an important role in elemental cycles in Earth’s biosphere; but little is known about their diversity, distribution, abundance, and impact in karst environments. The present study investigated the effect of environmental factors on the variability of archaeal communities in the sediment of the Huixian karst wetland, the largest karst wetland in South China. Sediment cores were obtained from four sampling sites with different water depths and macrophyte inhabitants in both the winter of 2016 and the summer of 2018. The community analysis was based on PacBio sequencing and quantitative PCR of the archaeal 16S rRNA gene. The results showed that Euryarchaeota (57.4%) and Bathyarchaeota (38.7%) were dominant in all the samples. Methanogenic Methanosarcinales (25.1%) and Methanomicrobiales (13.7%), and methanotrophic archaea ANME-2d (9.0%) were the dominant Euryarchaeota; MCG-11 (16.5%), MCG-6 (9.1%), and MCG-5b (5.5%) were the dominant Bathyarchaeota. The community composition remained stable between summer and winter, and the vertical distributions of the archaeal phyla conformed to two patterns among the four sampling sites. In the winter samples, the archaeal 16S rRNA gene abundance was approximately 1.0E+10 copies/g of wet sediment and the Shannon index was 7.3±5, which were significantly higher than in the summer samples and in other karst environments. A correlation analysis showed that the moisture content and pH were the factors that mostly affected the archaeal communities. The prevalence of nitrate in the summer may be a key factor causing a significant decrease in archaeal abundance and diversity. Two features specific to karst environments, calcium-richness and weak alkalescence of the water supplies, may benefit the prevalence of bathyarchaeotal subgroups MCG-11, MCG-5b, and MCG-6. These results suggest that in karst wetlands, most of the archaea belong to clades that have significant roles in carbon turnover; their composition remains stable, but their abundance and diversity vary significantly from season to season.

Linking 16S rRNA Gene Classification to amoA Gene Taxonomy Reveals Environmental Distribution of Ammonia-Oxidizing Archaeal Clades in Peatland Soils.

A highly resolved taxonomy for ammonia-oxidizing archaea (AOA) based on the alpha subunit of ammonia monooxygenase (amoA) was recently established, which uncovered novel environmental patterns of AOA, challenging previous generalizations. However, many microbiome studies target the 16S rRNA gene as a marker; thus, the usage of this novel taxonomy is currently limited. Here, we exploited the phylogenetic congruence of archaeal amoA and 16S rRNA genes to link 16S rRNA gene classification to the novel amoA taxonomy. We screened publicly available archaeal genomes and contigs for the co-occurring amoA and 16S rRNA genes and constructed a 16S rRNA gene database with the corresponding amoA clade taxonomy. Phylogenetic trees of both marker genes confirmed congruence, enabling the identification of clades. We validated this approach with 16S rRNA gene amplicon data from peatland soils. We succeeded in linking 16S rRNA gene amplicon sequence variants belonging to the class Nitrososphaeria to seven different AOA (amoA) clades, including two of the most frequently detected clades (Nitrososphaerales γ and δ clades) for which no pure culture is currently available. Water status significantly impacted the distribution of the AOA clades as well as the whole AOA community structure, which was correlated with pH, nitrate, and ammonium, consistent with previous clade predictions. Our study emphasizes the need to distinguish among AOA clades with distinct ecophysiologies and environmental preferences, for a better understanding of the ecology of the globally abundant AOA. IMPORTANCE The recently established phylogeny of amoA provides a finer resolution than previous studies, allowing clustering of AOA beyond the order level and thus revealing novel clades. While the 16S rRNA gene is mostly appreciated in microbiome studies, this novel phylogeny is in limited use. Here, we provide an alternative path to identifying AOA with this novel and highly resolved amoA taxonomy by using 16S rRNA gene sequencing data. We constructed a 16S rRNA gene database with the associated amoA clade taxonomy based on their phylogenetic congruence. With this database, we were able to assign 16S rRNA gene amplicons from peatland soils to different AOA clades, with a level of resolution provided previously only by amoA phylogeny. As 16S rRNA gene amplicon sequencing is still widely employed in microbiome studies, our database may have a broad application for interpreting the ecology of globally abundant AOA.

New insights in bacterial and eukaryotic diversity of microbial mats inhabiting exploited and abandoned salterns at the Ré Island (France)

In order to understand the effect of human practices on microbial mats organisation, the study aimed to investigate the biodiversity within microbial mats from exploited and abandoned salterns. Despite several attempts, archaeal 16S rRNA gene fragment sequences were not obtained, indicating that microbial mats were probably dominated by Bacteria with very low abundance of Archaea (< 1%). Thus, the study compared the bacterial and meiofaunal diversity of microbial mats from abandoned and exploited salterns. The higher salinity (101 ± 3.7 psu vs. 51.1 ± 0.7 psu; Welch t-test p < 0.05) of the exploited site maintained lower bacterial diversity in comparison to the abandoned site where the salinity gradient was no longer maintained. However, the microbial mats exhibited similar bacterial class composition while the eukaryotic diversity was significantly higher in the exploited saltern. The abandoned saltern was dominated by sulfate-reducing bacteria and Nematoda, while the exploited saltern was characterized by the presence of halophilic bacteria belonging to Marinobacter, Salinivibrio and Rhodohalobacter genera, and the larger abundance of Hypotrichia (ciliates). Such bacterial and eukaryotic diversity difference might be explained by human actions for salt recovery in exploited salterns such as scraping the surface of microbial mat and increasing salinity renewing the microbial mat each year. Such action decreases the bacterial diversity changing the food web structure that favour the presence of a larger diversity of eukaryotic organisms. Our study provides new insights on microbial mat communities inhabiting salterns, especially the consequences of abandoning saltern exploitation.