Archaeal Order Research Articles

Minimal and hybrid hydrogenases are active from archaea

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

Unraveling the phylogenomic diversity of Methanomassiliicoccales and implications for mitigating ruminant methane emissions

BackgroundMethanomassiliicoccales are a recently identified order of methanogens that are diverse across global environments particularly the gastrointestinal tracts of animals; however, their metabolic capacities are defined via a limited number of cultured strains.ResultsHere, we profile and analyze 243 Methanomassiliicoccales genomes assembled from cultured representatives and uncultured metagenomes recovered from various biomes, including the gastrointestinal tracts of different animal species. Our analyses reveal the presence of numerous undefined genera and genetic variability in metabolic capabilities within Methanomassiliicoccales lineages, which is essential for adaptation to their ecological niches. In particular, gastrointestinal tract Methanomassiliicoccales demonstrate the presence of co-diversified members with their hosts over evolutionary timescales and likely originated in the natural environment. We highlight the presence of diverse clades of vitamin transporter BtuC proteins that distinguish Methanomassiliicoccales from other archaeal orders and likely provide a competitive advantage in efficiently handling B12. Furthermore, genome-centric metatranscriptomic analysis of ruminants with varying methane yields reveal elevated expression of select Methanomassiliicoccales genera in low methane animals and suggest that B12 exchanges could enable them to occupy ecological niches that possibly alter the direction of H2 utilization.ConclusionsWe provide a comprehensive and updated account of divergent Methanomassiliicoccales lineages, drawing from numerous uncultured genomes obtained from various habitats. We also highlight their unique metabolic capabilities involving B12, which could serve as promising targets for mitigating ruminant methane emissions by altering H2 flow.

Rumen Methanogens Diversity in Native Cattle under Different Feeding Regimen using 16s Metagenomic Techniques

Background: Pulikulam cattle is an indigenous cattle breed native to south India, Understanding the rumen microbiome is an important step to develop effective strategies to minimize their enteric methane emission. The rumen microbiome of this breed has not been documented. Hence, a study was executed in Pulikulam cattle, fed different diets, to investigate the diversity of rumen methanogens using metagenomic techniques. Methods: Twenty recently calved Pulikulam cattle with mean body weight of 200 Kg were randomly distributed into four treatments, with five animals per treatment. The animals were fed their respective diets as per their treatment for 30 days, after which the rumen liquor was collected. DNA from the samples were extracted and subjected for 16s rRNA gene amplification using region specific primers. Result: Methanogens belonging to five orders, four classes and eleven families were identified. Order Methanomassiliicoccales was not present in treatment group fed with high energy diet. The methanogens affiliated to the order Methanomicrobiales were predominant in treatment 1 (14%) and treatment 2 (16.30%). Order methanococcales (15%) was predominant in treatment 3 and order Methanosarcinales (18%) was predominant in treatment 4. Archaeal order belonging to Thermoplasmatales were also identified in Pulikulam cattle fed different dietary regimen except in treatment 3. Archaeal family Methanothermaceae belonging to the order Methanobacteriales was identified in Pulikulam cattle. Methanocorpusculum labreanum was the predominant species in all the treatment groups. Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus solfataricus, Sulfolobus sp. A20 and Sulfolobus tokodaii were identified in treatment 1, which had higher dietary crude protein. It was concluded that methanogen diversity varied under different feeding regimen and predominant methanogen is varied in Pulikulam breed of cattle. M. labreanum was the predominant methanogen in Pulikulam cattle fed with different dietary regimen.

Metabolic versatility of Caldarchaeales from geothermal features of Hawai'i and Chile as revealed by five metagenome-assembled genomes.

Members of the archaeal order Caldarchaeales (previously the phylum Aigarchaeota) are poorly sampled and are represented in public databases by relatively few genomes. Additional representative genomes will help resolve their placement among all known members of Archaea and provide insights into their roles in the environment. In this study, we analyzed 16S rRNA gene amplicons belonging to the Caldarchaeales that are available in public databases, which demonstrated that archaea of the order Caldarchaeales are diverse, widespread, and most abundant in geothermal habitats. We also constructed five metagenome-assembled genomes (MAGs) of Caldarchaeales from two geothermal features to investigate their metabolic potential and phylogenomic position in the domain Archaea. Two of the MAGs were assembled from microbial community DNA extracted from fumarolic lava rocks from Mauna Ulu, Hawai'i, and three were assembled from DNA obtained from hot spring sinters from the El Tatio geothermal field in Chile. MAGs from Hawai'i are high quality bins with completeness >95% and contamination <1%, and one likely belongs to a novel species in a new genus recently discovered at a submarine volcano off New Zealand. MAGs from Chile have lower completeness levels ranging from 27 to 70%. Gene content of the MAGs revealed that these members of Caldarchaeales are likely metabolically versatile and exhibit the potential for both chemoorganotrophic and chemolithotrophic lifestyles. The wide array of metabolic capabilities exhibited by these members of Caldarchaeales might help them thrive under diverse harsh environmental conditions. All the MAGs except one from Chile harbor putative prophage regions encoding several auxiliary metabolic genes (AMGs) that may confer a fitness advantage on their Caldarchaeales hosts by increasing their metabolic potential and make them better adapted to new environmental conditions. Phylogenomic analysis of the five MAGs and over 3,000 representative archaeal genomes showed the order Caldarchaeales forms a monophyletic group that is sister to the clade comprising the orders Geothermarchaeales (previously Candidatus Geothermarchaeota), Conexivisphaerales and Nitrososphaerales (formerly known as Thaumarchaeota), supporting the status of Caldarchaeales members as a clade distinct from the Thaumarchaeota.

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

Metagenomic insights into the environmental adaptation and metabolism of <i>Candidatus</i> Halarchaeoplasmatales, one archaeal order thriving in saline lakes

Expanding the phylogenetic distribution of cytochrome b-containing methanogenic archaea sheds light on the evolution of methanogenesis

Methane produced by methanogenic archaea has an important influence on Earth’s changing climate. Methanogenic archaea are phylogenetically diverse and widespread in anoxic environments. These microorganisms can be divided into two subgroups based on whether or not they use b-type cytochromes for energy conservation. Methanogens with b-type cytochromes have a wider substrate range and higher growth yields than those without them. To date, methanogens with b-type cytochromes were found exclusively in the phylum “Ca. Halobacteriota” (formerly part of the phylum Euryarchaeota). Here, we present the discovery of metagenome-assembled genomes harboring methyl-coenzyme M reductase genes reconstructed from mesophilic anoxic sediments, together with the previously reported thermophilic “Ca. Methylarchaeum tengchongensis”, representing a novel archaeal order, designated the “Ca. Methylarchaeales”, of the phylum Thermoproteota (formerly the TACK superphylum). These microorganisms contain genes required for methyl-reducing methanogenesis and the Wood-Ljundahl pathway. Importantly, the genus “Ca. Methanotowutia” of the “Ca. Methylarchaeales” encode a cytochrome b-containing heterodisulfide reductase (HdrDE) and methanophenazine-reducing hydrogenase complex that have similar gene arrangements to those found in methanogenic Methanosarcinales. Our results indicate that members of the “Ca. Methylarchaeales” are methanogens with cytochromes and can conserve energy via membrane-bound electron transport chains. Phylogenetic and amalgamated likelihood estimation analyses indicate that methanogens with cytochrome b-containing electron transfer complexes likely evolved before diversification of Thermoproteota or “Ca. Halobacteriota” in the early Archean Eon. Surveys of public sequence databases suggest that members of the lineage are globally distributed in anoxic sediments and may be important players in the methane cycle.

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.

A Rapid Targeted Gene Inactivation Approach in Sulfolobus islandicus.

Homologous recombination-based gene targeting is a powerful and classic reverse genetics approach to precisely elucidate in vivo gene functions in the organisms across all three domains of life. Gene function studies in Archaea, particularly for those flourishing in inhospitable natural environments that are anaerobic, usually hot, and acidic, have been a great challenge; however, this situation was recently overturned with the increasing availability of genetic manipulation systems in several cultivable archaeal species. In the present chapter, we describe a detailed procedure to rapidly generate gene disruption mutants in the hyperthermophilic crenarchaeon Sulfolobus islandicus via a recently developed Microhomology-Mediated Gene Inactivation (MMGI) approach. We highlight crucial experimental details required to be carefully considered when using the MMGI approach for genetic manipulations. We hope this highly reproducible procedure can supplement existing genetic tools for studying the biology of archaeal order Sulfolobales.

Continuous monocropping highly affect the composition and diversity of microbial communities in peanut (Arachis hypogaea L.)

Continuous cropping systems are the leading cause of decreased soil biological environments in terms of unstable microbial population and diversity index. Nonetheless, their responses to consecutive peanut monocropping cycles have not been thoroughly investigated. In this study, the structure and abundance of microbial communities were characterized using pyrosequencing-based approach in peanut monocropping cycles for three consecutive years. The results showed that continuous peanut cultivation led to a substantial decrease in soil microbial abundance and diversity from initial cropping cycle (T1) to later cropping cycle (T3). Peanut rhizosphere soil had Actinobacteria, Protobacteria, and Gemmatimonadetes as the major bacterial phyla. Ascomycota, Basidiomycota were the major fungal phylum, while Crenarchaeota and Euryarchaeota were the most dominant phyla of archaea. Several bacterial, fungal and archaeal taxa were significantly changed in abundance under continuous peanut cultivation. Bacterial orders, Actinomycetales, Rhodospirillales and Sphingomonadales showed decreasing trends from T1&gt;T2&gt;T3. While, pathogenic fungi Phoma was increased and beneficial fungal taxa Glomeraceae decreased under continuous monocropping. Moreover, Archaeal order Nitrososphaerales observed less abundant in first two cycles (T1&amp;T2), however, it increased in third cycle (T3), whereas, Thermoplasmata exhibit decreased trends throughout consecutive monocropping. Taken together, we have shown the taxonomic profiles of peanut rhizosphere communities that were affected by continuous peanut monocropping. The results obtained from this study pave ways towards a better understanding of the peanut rhizosphere soil microbial communities in response to continuous cropping cycles, which could be used as bioindicator to monitor soil quality, plant health and land management practices.

Identification and Enzymatic Analysis of an Archaeal ATP-Dependent Serine Kinase from the Hyperthermophilic Archaeon Staphylothermus marinus.

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O-phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk-SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here, we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk-SerK from Euryarchaeota (42 to 45% identical). Tk-serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli. All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity for l-Ser but not other compounds, including d-Ser, l-threonine, and l-homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as phosphate donors. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5'-triphosphates over triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis (Tk-SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk-SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

Intersection of Biotic and Abiotic Sulfur Chemistry Supporting Extreme Microbial Life in Hot Acid.

Microbial life on Earth exists within wide ranges of temperature, pressure, pH, salinity, radiation, and water activity. Extreme thermoacidophiles, in particular, are microbes found in hot, acidic biotopes laden with heavy metals and reduced inorganic sulfur species. As chemolithoautotrophs, they thrive in the absence of organic carbon, instead using sulfur and metal oxidation to fuel their bioenergetic needs, while incorporating CO2 as a carbon source. Metal oxidation by these microbes takes place extracellularly, mediated by membrane-associated oxidase complexes. In contrast, sulfur oxidation involves extracellular, membrane-associated, and cytoplasmic biotransformations, which intersect with abiotic sulfur chemistry. This novel lifestyle has been examined in the context of early aerobic life on this planet, but it is also interesting when considering the prospects of life, now or previously, on other solar bodies. Here, extreme thermoacidophily (growth at pH below 4.0, temperature above 55 °C), a characteristic of species in the archaeal order Sulfolobales, is considered from the perspective of sulfur chemistry, both biotic and abiotic, as it relates to microbial bioenergetics. Current understanding of the mechanisms involved are reviewed which are further expanded through recent experimental results focused on imparting sulfur oxidation capacity on a natively nonsulfur oxidizing extremely thermoacidophilic archaeon, Sulfolobus acidocaldarius, through metabolic engineering.

Stability Assessment of the Rumen Bacterial and Archaeal Communities in Dairy Cows Within a Single Lactation and Its Association With Host Phenotype.

Better characterization of changes in the rumen microbiota in dairy cows over the lactation period is crucial for understanding how microbial factors may potentially be interacting with host phenotypes. In the present study, we characterized the rumen bacterial and archaeal community composition of 60 lactating Holstein dairy cows (33 multiparous and 27 primiparous), sampled twice within the same lactation with a 122 days interval. Firmicutes and Bacteroidetes dominated the rumen bacterial community and showed no difference in relative abundance between samplings. Two less abundant bacterial phyla (SR1 and Proteobacteria) and an archaeal order (Methanosarcinales), on the other hand, decreased significantly from the mid-lactation to the late-lactation period. Moreover, between-sampling stability assessment of individual operational taxonomic units (OTUs), evaluated by concordance correlation coefficient (C-value) analysis, revealed the majority of the bacterial OTUs (6,187 out of 6,363) and all the 79 archaeal OTUs to be unstable over the investigated lactation period. The remaining 176 stable bacterial OTUs were mainly assigned to Prevotella, unclassified Prevotellaceae, and unclassified Bacteroidales. Milk phenotype-based screening analysis detected 32 bacterial OTUs, mainly assigned to unclassified Bacteroidetes and Lachnospiraceae, associated with milk fat percentage, and 6 OTUs, assigned to Ruminococcus and unclassified Ruminococcaceae, associated with milk protein percentage. These OTUs were only observed in the multiparous cows. None of the archaeal OTUs was observed to be associated with the investigated phenotypic parameters, including methane production. Co-occurrence analysis of the rumen bacterial and archaeal communities revealed Fibrobacter to be positively correlated with the archaeal genus vadinCA11 (Pearson r = 0.76) and unclassified Methanomassiliicoccaceae (Pearson r = 0.64); vadinCA11, on the other hand, was negatively correlated with Methanobrevibacter (Pearson r = –0.56). In conclusion, the rumen bacterial and archaeal communities of dairy cows displayed distinct stability at different taxonomic levels. Moreover, specific members of the rumen bacterial community were observed to be associated with milk phenotype parameters, however, only in multiparous cows, indicating that dairy cow parity could be one of the driving factors for host–microbe interactions.

Temperature and elemental sulfur shape microbial communities in two extremely acidic aquatic volcanic environments.

Aquatic environments of volcanic origin provide an exceptional opportunity to study the adaptations of microorganisms to early planet life conditions. Here, we characterized the prokaryotic communities and physicochemical properties of seepage sites at the bottom of the Poas Volcano crater and the Agrio River, two geologically related extremely acidic environments located in Costa Rica. Both locations hold a low pH (1.79-2.20) and have high sulfate and iron concentrations (Fe = 47-206mg/L, SO42- = 1170-2460mg/L), but significant differences in their temperature (90.0-95.0 ºC in the seepages at Poas Volcano, 19.1-26.6 ºC in Agrio River) and in the elemental sulfur content. Based on the analysis of 16S rRNA gene sequences, we determined that Sulfobacillus spp. represented more than half of the sequences in Poas Volcano seepage sites, while Agrio River was dominated by Leptospirillum and members of the archaeal order Thermoplasmatales. Both environments share some chemical characteristics and part of their microbiota, however, the temperature and the reduced sulfur are likely the main distinguishing features, ultimately shaping their microbial communities. Our data suggest that in the Poas Volcano-Agrio River system there is a common metabolism but with specialization of species that adapt to the physicochemical conditions of each environment.

New Insights into the Ecology and Physiology of Methanomassiliicoccales from Terrestrial and Aquatic Environments.

Members of the archaeal order Methanomassiliicoccales are methanogens mainly associated with animal digestive tracts. However, environmental members remain poorly characterized as no representatives not associated with a host have been cultivated so far. In this study, metabarcoding screening combined with quantitative PCR analyses on a collection of diverse non-host-associated environmental samples revealed that Methanomassiliicoccales were very scarce in most terrestrial and aquatic ecosystems. Relative abundance of Methanomassiliicoccales and substrates/products of methanogenesis were monitored during incubation of environmental slurries. A sediment slurry enriched in Methanomassiliicoccales was obtained from a freshwater sample. It allowed the reconstruction of a high-quality metagenome-assembled genome (MAG) corresponding to a new candidate species, for which we propose the name of Candidatus ‘Methanomassiliicoccus armoricus MXMAG1’. Comparison of the annotated genome of MXMAG1 with the published genomes and MAGs from Methanomassiliicoccales belonging to the 2 known clades (‘free-living’/non-host-associated environmental clade and ‘host-associated’/digestive clade) allowed us to explore the putative physiological traits of Candidatus ‘M. armoricus MXMAG1’. As expected, Ca. ‘Methanomassiliicoccus armoricus MXMAG1’ had the genetic potential to produce methane by reduction of methyl compounds and dihydrogen oxidation. This MAG encodes for several putative physiological and stress response adaptations, including biosynthesis of trehalose (osmotic and temperature regulations), agmatine production (pH regulation), and arsenic detoxication, by reduction and excretion of arsenite, a mechanism that was only present in the ‘free-living’ clade. An analysis of co-occurrence networks carried out on environmental samples and slurries also showed that Methanomassiliicoccales detected in terrestrial and aquatic ecosystems were strongly associated with acetate and dihydrogen producing bacteria commonly found in digestive habitats and which have been reported to form syntrophic relationships with methanogens.

Biogas Production and Microbial Communities in the Anaerobic Digestion of Sewage Sludge Under Hydrothermal Pretreatment with Air and a Catalyst

Hydrothermal pretreatment (HTP) of sewage sludge (SS) has been shown to improve the subsequent biogas production by anaerobic digestion (AD), but the effect of catalysts on HTP performance was less explored. This study intended to investigate the SS pretreatment by wet air oxidation (WAO) with the addition of K2CO3 as a catalyst on the performance of methane production by AD. WAO was found to improve the solubilization of SS, the soluble chemical oxygen demand, dissolved organic carbon, and total dissolved nitrogen. The methane yield from WAO increased from 202 mL/gVSin with no catalyst added to 277 mL/gVSin with 10 wt% of K2CO3 added at 180 °C with 30 min of residence time. Under this pretreatment condition, the highest methane production rate could achieve 15.8 mL/gVSin day, and the percentage of methane reached 73%. The structure of the microbial community involved in the AD was affected by the residence time, working gas, and catalyst of the HTP process. The results showed that Bacteroidetes, Bacteroidia, and SC103 were the dominant phylum, class, and genus of bacteria, respectively, of almost all of the samples. In addition, the most abundant archaeal order was Methanosarcinales, while Methanosaeta was the dominant archaeal genus of most of the samples. However, Methanosarcina largely increased the relative abundance, corresponding to the amount of K2CO3 catalyst used. The findings in this study demonstrated the potential use of K2CO3 during WAO of SS and implied the link between shift of methanogen community and the enhanced methane yield in AD.

Life in hot acid: a genome-based reassessment of the archaeal order Sulfolobales.

The order Sulfolobales was one of the first named Archaeal lineages, with globally distributed members from terrestrial thermal acid springs (pH< 4; T> 65°C). The Sulfolobales represent broad metabolic capabilities, ranging from lithotrophy, based on inorganic iron and sulfur biotransformations, to autotrophy, to chemoheterotrophy in less acidophilic species. Components of the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle, as well as sulfur oxidation, are nearly universally conserved, although dissimilatory sulfur reduction and disproportionation (Acidianus, Stygiolobus and Sulfurisphaera) and iron oxidation (Acidianus, Metallosphaera, Sulfurisphaera, Sulfuracidifex and Sulfodiicoccus) are limited to fewer lineages. Lithotrophic marker genes appear more often in highly acidophilic lineages. Despite the presence of facultative anaerobes and one confirmed obligate anaerobe, oxidase complexes (fox, sox, dox and a new putative cytochrome bd) are prevalent in many species (even facultative/obligate anaerobes), suggesting a key role for oxygen among the Sulfolobales. The presence of fox genes tracks with a putative antioxidant OsmC family peroxiredoxin, an indicator of oxidative stress derived from mixing reactive metals and oxygen. Extreme acidophily appears to track inversely with heterotrophy but directly with lithotrophy. Recent phylogenetic re-organization efforts are supported by the comparative genomics here, although several changes are proposed, including the expansion of the genus Saccharolobus.

Thermophilic Anaerobic Digestion of Arundo donax cv. Lvzhou No. 1 for Biogas Production: Structure and Functional Analysis of Microbial Communities

Thermophilic anaerobic digestion (TAD) is an efficient method for biogas production. In this study, the TAD of Arundo donax cv. Lvzhou No. 1 (ADL-1, a new kind of energy crop with high cold tolerance) at different growth stages was carried out, in order to investigate the relationship between microbial community structure and its function during the fermentation process. The results showed that the most optimal growth period of ADL-1 was 3 months, regarding the yield of biogas production. The TAD process lasted for 10 days with cumulative biogas and methane yields of 312.7 mL/g VS and 231 mL/g VS, respectively. The degradation rates of hemicellulose, cellulose, and lignin were 41.78%, 27.99%, and 14.46%, respectively. The high-throughput sequencing of 16S rRNA gene amplicons revealed that the most abundant bacterial phylum in TAD was Firmicutes with three dominant genera of Tepidiphilus, Sedimentibacter, and Gelria. Also, the main archaeal order was Methanomicrobiales, in which Methanoculleus and Methanosarcina were detected as dominant genera. Therefore, this article reveals the dynamic changes of structure and function of microbial communities during TAD of ADL-1, providing the theoretical basis for the development of energy crops with cold tolerance as potential biogas feedstock.

Evidences of aromatic degradation dominantly via the phenylacetic acid pathway in marine benthic Thermoprofundales.

Thermoprofundales (Marine Benthic Group D archaea, MBG-D) is a newly proposed archaeal order and widely distributed in global marine sediment, and the members in the order may play a vital role in carbon cycling. However, the lack of pure cultures of these oeganisms has hampered the recognition of their catabolic roles. Here, by constructing high-quality metagenome-assembled genomes (MAGs) of two new subgroups of Thermoprofundales from hydrothermal sediment and predicting their catabolic pathways, we here provide genomic evidences that Thermoprofundales are capable of degrading aromatics via the phenylacetic acid (PAA) pathway. Then, the gene sequences of phenylacetyl-CoA ligase (PCL), a key enzyme for the PAA pathway, were searched in reference genomes. The widespread distribution of PCL genes among 14.9% of archaea and 75.9% of Thermoprofundales further supports the importance of the PAA pathway in archaea, particularly in Thermoprofundales where no ring-cleavage dioxygenases were found. Two PCLs from Thermoprofundales MAGs, PCLM8-3 and PCLM10-15 , were able to convert PAA to phenylacetyl-CoA (PA-CoA) in vitro, demonstrating the involvement of Thermoprofundales in aromatics degradation through PAA via CoA activation. Their acid tolerance (pH 5-7), high-optimum temperatures (60°C and 80°C), thermostability (stable at 60°C and 50°C for 48 h) and broad substrate spectra imply that Thermoprofundales are capable of transforming aromatics under extreme conditions. Together with the evidence of in situ transcriptional activities for most genes related to the aromatics pathway in Thermoprofundales, these genomic, and biochemical evidences highlight the essential role of this ubiquitous and abundant archaeal order in the carbon cycle of marine sediments.

Determinants of sulphur chemolithoautotrophy in the extremely thermoacidophilic Sulfolobales.

Species in the archaeal order Sulfolobales thrive in hot acid and exhibit remarkable metabolic diversity. Some species are chemolithoautotrophic, obtaining energy through the oxidation of inorganic substrates, sulphur in particular, and acquiring carbon through the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) CO2 -fixation cycle. The current model for sulphur oxidation in the Sulfolobales is based on the biochemical analysis of specific proteins from Acidianus ambivalens, including sulphur oxygenase reductase (SOR) that disproportionates S° into H2 S and sulphite (SO3 2- ). Initial studies indicated SOR catalyses the essential first step in oxidation of elemental sulphur, but an ancillary role for SOR as a 'recycle' enzyme has also been proposed. Here, heterologous expression of both SOR and membrane-bound thiosulphate-quinone oxidoreductase (TQO) from Sulfolobus tokodaii 'restored' sulphur oxidation capacity in Sulfolobus acidocaldarius DSM639, but not autotrophy, although earlier reports indicate this strain was once capable of chemolithoautotrophy. Comparative transcriptomic analyses of Acidianus brierleyi, a chemolithoautotrophic sulphur oxidizer, and S. acidocaldarius DSM639 showed that while both share a strong transcriptional response to elemental sulphur, S. acidocaldarius DSM639 failed to upregulate key 3-HP/4-HB cycle genes used by A. brierleyi to drive chemolithoautotrophy. Thus, the inability for S. acidocaldarius DSM639 to grow chemolithoautotrophically may be rooted more in gene regulation than the biochemical capacity.

Subsurface processes influence oxidant availability and chemoautotrophic hydrogen metabolism in Yellowstone hot springs.

The geochemistry of hot springs and the availability of oxidants capable of supporting microbial metabolisms are influenced by subsurface processes including the separation of hydrothermal fluids into vapor and liquid phases. Here, we characterized the influence of geochemical variation and oxidant availability on the abundance, composition, and activity of hydrogen (H2 )-dependent chemoautotrophs along the outflow channels of two-paired hot springs in Yellowstone National Park. The hydrothermal fluid at Roadside East (RSE; 82.4°C, pH 3.0) is acidic due to vapor-phase input while the fluid at Roadside West (RSW; 68.1°C, pH 7.0) is circumneutral due to liquid-phase input. Most chemotrophic communities exhibited net rates of H2 oxidation, consistent with H2 support of primary productivity, with one chemotrophic community exhibiting a net rate of H2 production. Abundant H2 -oxidizing chemoautotrophs were supported by reduction in oxygen, elemental sulfur, sulfate, and nitrate in RSW and oxygen and ferric iron in RSE; O2 utilizing hydrogenotrophs increased in abundance down both outflow channels. Sequencing of 16S rRNA transcripts or genes from native sediments and dilution series incubations, respectively, suggests that members of the archaeal orders Sulfolobales, Desulfurococcales, and Thermoproteales are likely responsible for H2 oxidation in RSE, whereas members of the bacterial order Thermoflexales and the archaeal order Thermoproteales are likely responsible for H2 oxidation in RSW. These observations suggest that subsurface processes strongly influence spring chemistry and oxidant availability, which in turn select for unique assemblages of H2 oxidizing microorganisms. Therefore, these data point to the role of oxidant availability in shaping the ecology and evolution of hydrogenotrophic organisms.