Archaeal 16S rRNA Research Articles

Distribution of methane production and methanogenic archaeal community structure across soil particle size fractions along a rice chronosequence

Methanogens, a phylogenetic group of archaea, are a key microbial community that mediate carbon (C) cycling and methane (CH4) production in submerged soils. In this study, we analyzed the potential of CH4 production and the archaeal community structure of different soil particle fractions in a chronosequence of rice (Oryza sativa L.) soil subjected to prolonged rice cultivation. The chronosequence of five age groups of a paddy soil formation was determined in a coastal area from East China, subjected to 50 to 700 years of rice cultivation. Undisturbed bulk soils were fractionated by low-energy ultra-sonication dispersion and centrifugation into four classes of particle size fractions (PSFs; coarse sand, fine sand, silt, and clay). The abundance and diversity of archaeal communities from each of the four fractions were analyzed by quantitative polymerase chain reaction and denaturing gradient gel electrophoresis, respectively. Among the size fractions, a high abundance of archaea and methanogens was found in both coarse sand- and clay-sized fractions. During prolonged rice cultivation, CH4 production potential (MPP) and methanogenic gene abundance increased in the soil cultivated for 100 years, and thereafter changed more or less in the soils cultivated for longer periods. Moreover, marked changes in gene abundance and community diversity were observed in coarse sand- and clay-sized fractions, but not in fine sand- and silt-sized fractions. Sequencing analysis of 61 selected archaeal 16S rRNA genes revealed that Methanocellales was an abundant group of methanogens in all sized fractions of soils across the chronosequence, whereas Methanosarcinaceae and Methanosaetaceae were less widespread in the clay fractions across the soils. This study indicated that methanogens and their activities can be heterogeneously distributed among soil PSFs, and their distribution and activities change in response to years of paddy cultivation that affect PSFs formation.

Microbial Population Change in Anaerobic Digestion during Copper Sulfate Inhibition and Recovery

Abstract. This study evaluated how feedstock with added copper sulfate (commonly used as an antimicrobial footbath solution in livestock operations) changed the performance and microbial populations of a dairy manure digester. High-throughput Illumina sequencing of the V4 region of the bacterial and the V6-V8 regions of the archaeal 16S rRNA gene in digester samples showed a significant change in the microbial community composition during addition of feedstock with copper sulfate, followed by a return to the original composition with regular feedstock. Bacterial genera that were suppressed during the inhibition period included Cloacamonas, Syntrophomonas, Butyrivibrio, and Caldicoprobacter, while the relative abundance of Acholeplasma, Desulfobulbus, Aminobacterium, Treponema, and YRC22 peaked. The dominant methanogen Methanosarcina, although initially resistant, was suppressed during inhibition, while Methanobrevibacter and Methanosphaera, prominent feedstock genera, increased. The return of the bacterial and archaeal communities to compositions similar to their previous steady states after inhibition highlights the digester’s microbial resilience and recovery potential. Keywords: . Anaerobic digestion, Copper sulfate inhibition, Illumina sequencing, Manure, Methanogens, Microbial populations, Microbiome.

Reduction in Methane Emissions From Acidified Dairy Slurry Is Related to Inhibition of Methanosarcina Species.

Liquid dairy manure treated with sulfuric acid was stored in duplicate pilot-scale storage tanks for 120 days with continuous monitoring of CH4 emissions and concurrent examination of changes in the structure of bacterial and methanogenic communities. Methane emissions were monitored at the site using laser-based Trace Gas Analyzer whereas quantitative real-time polymerase chain reaction and massively parallel sequencing were employed to study bacterial and methanogenic communities using 16S rRNA and methyl-coenzyme M Reductase A (mcrA) genes/transcripts, respectively. When compared with untreated slurries, acidification resulted in 69–84% reductions of cumulative CH4 emissions. The abundance, activity, and proportion of bacterial communities did not vary with manure acidification. However, the abundance and activity of methanogens (as estimated from mcrA gene and transcript copies, respectively) in acidified slurries were reduced by 6 and 20%, respectively. Up to 21% reduction in mcrA transcript/gene ratios were also detected in acidified slurries. Regardless of treatment, Methanocorpusculum predominated archaeal 16S rRNA and mcrA gene and transcript libraries. The proportion of Methanosarcina, which is the most metabolically-diverse methanogen, was the significant discriminant feature between acidified and untreated slurries. In acidified slurries, the relative proportions of Methanosarcina were ≤ 10%, whereas in untreated slurries, it represented up to 24 and 53% of the mcrA gene and transcript libraries, respectively. The low proportions of Methanosarcina in acidified slurries coincided with the reductions in CH4 emissions. The results suggest that reduction of CH4 missions achieved by acidification was due to an inhibition of the growth and activity of Methanosarcina species.

Shifts of Hydrogen Metabolism From Methanogenesis to Propionate Production in Response to Replacement of Forage Fiber With Non-forage Fiber Sources in Diets in vitro.

The rumen microbial complex adaptive mechanism invalidates various methane (CH4) mitigation strategies. Shifting the hydrogen flow toward alternative electron acceptors, such as propionate, was considered to be a meaningful mitigation strategy. A completely randomized design was applied in in vitro incubation to investigate the effects of replacing forage fiber with non-forage fiber sources (NFFS) in diets on methanogenesis, hydrogen metabolism, propionate production and the methanogenic and bacterial community. There are two treatments in the current study, CON (a basic total mixed ration) and TRT (a modified total mixed ration). The dietary treatments were achieved by partly replacing forage fiber with NFFS (wheat bran and soybean hull) to decrease forage neutral detergent fiber (fNDF) content from 24.0 to 15.8%, with the composition and inclusion rate of other dietary ingredients remaining the same in total mixed rations. The concentrations of CH4, hydrogen (H2) and volatile fatty acids were determined using a gas chromatograph. The archaeal and bacterial 16S rRNA genes were sequenced by Miseq high-throughput sequencing and used to reveal the relative abundance of methanogenic and bacterial communities. The results revealed that the concentration of propionate was significantly increased, while the concentration of acetate and the acetate to propionate ratio were not affected by treatments. Compared with CON, the production of H2 increased by 8.45% and the production of CH4 decreased by 14.06%. The relative abundance of Methanomassiliicoccus was significantly increased, but the relative abundance of Methanobrevibacter tended to decrease in TRT group. At the bacterial phylum level, the TRT group significantly decreased the relative abundance of Firmicutes and tended to increase the relative abundance of Bacteroidetes. The replacement of forage fiber with NFFS in diets can affect methanogenesis by shifting the hydrogen flow toward propionate, and part is directed to H2 in vitro. The shift was achieved by a substitution of Firmicutes by Bacteroidetes, another substitution of Methanobrevibacter by Methanomassiliicoccus. Theoretical predictions of displacements of H2 metabolism from methanogenesis to propionate production was supported by the dietary intervention in vitro.

Response of freshwater sediment archaeal community to metal spill

Archaea play an important role in the biogeochemical cycling of elements in the environment. Heavy metals are ubiquitous pollutants in the environment. Previous studies have revealed a considerable influence of metal pollution on the archaeal community, but the short-term response of the archaeal community to metal pollution remains unclear. Hence, the present study investigated the short versus long-term responses of overall archaeal communities in freshwater sediments after exposure to accidental metal pollution caused by the discharge of heavy metal-containing wastewater from an indium-producing factory. Quantitative PCR was used to determine the archaeal abundance, while Illumina MiSeq sequencing was applied to characterize the diversity and structure of the archaeal community. The abundance (2.47 × 105–1.55 × 108 archaeal 16S rRNA gene copies per gram dry sediment), diversity (Shannon diversity index = 2.49–4.45) and structure of overall archaeal community illustrated a drastic temporal change. The archaeal communities mainly comprised the phyla Euryarchaeota, Thaumarchaeota and Bathyarchaeota. The exposure to metal pollution induced an increase in the proportion of Euryarchaeota but lowered the proportion of Thaumarchaeota. The accidental metal pollution exerted a profound impact on the archaeal community in freshwater sediment. This study could contribute our understanding of the short versus long-term response of archaeal communities to metal pollution.

Long-term investigation of microbial community composition and transcription patterns in a biogas plant undergoing ammonia crisis.

SummaryAmmonia caused disturbance of biogas production is one of the most frequent incidents in regular operation of biogas reactors. This study provides a detailed insight into the microbial community of a mesophilic, full‐scale biogas reactor (477 kWh h−1) fed with maize silage, dried poultry manure and cow manure undergoing initial process disturbance by increased ammonia concentration. Over a time period of 587 days, the microbial community of the reactor was regularly monitored on a monthly basis by high‐throughput amplicon sequencing of the archaeal and bacterial 16S rRNA genes. During this sampling period, the total ammonia concentrations varied between 2.7 and 5.8 g l−1 [NH 4 +–N]. To gain further inside into the active metabolic pathways, for selected time points metatranscriptomic shotgun analysis was performed allowing the quantification of marker genes for methanogenesis, hydrolysis and syntrophic interactions. The results obtained demonstrated a microbial community typical for a mesophilic biogas plant. However in response to the observed changing process conditions (e.g. increasing NH 4 + levels, changing feedstock composition), the microbial community reacted highly flexible by changing and adapting the community composition. The Methanosarcina‐dominated archaeal community was shifted to a Methanomicrobiales‐dominated archaeal community in the presence of increased ammonia conditions. A similar trend as in the phylogenetic composition was observed in the transcription activity of genes coding for enzymes involved in acetoclastic methanogenesis and syntrophic acetate oxidations (Codh/Acs and Fthfs). In accordance, Clostridia simultaneously increased under elevated ammonia concentrations in abundance and were identified as the primary syntrophic interaction partner with the now Methanomicrobiales‐dominated archaeal community. In conclusion, overall stable process performance was maintained during increased ammonia concentration in the studied reactor based on the microbial communities’ ability to flexibly respond by reorganizing the community composition while remaining functionally stable.

Petroleum hydrocarbon rich oil refinery sludge of North-East India harbours anaerobic, fermentative, sulfate-reducing, syntrophic and methanogenic microbial populations

BackgroundSustainable management of voluminous and hazardous oily sludge produced by petroleum refineries remains a challenging problem worldwide. Characterization of microbial communities of petroleum contaminated sites has been considered as the essential prerequisite for implementation of suitable bioremediation strategies. Three petroleum refinery sludge samples from North Eastern India were analyzed using next-generation sequencing technology to explore the diversity and functional potential of inhabitant microorganisms and scope for their on-site bioremediation.ResultsAll sludge samples were hydrocarbon rich, anaerobic and reduced with sulfate as major anion and several heavy metals. High throughput sequencing of V3-16S rRNA genes from sludge metagenomes revealed dominance of strictly anaerobic, fermentative, thermophilic, sulfate-reducing bacteria affiliated to Coprothermobacter, Fervidobacterium, Treponema, Syntrophus, Thermodesulfovibrio, Anaerolinea, Syntrophobacter, Anaerostipes, Anaerobaculum, etc., which have been well known for hydrocarbon degradation. Relatively higher proportions of archaea were detected by qPCR. Archaeal 16S rRNA gene sequences showed presence of methanogenic Methanobacterium, Methanosaeta, Thermoplasmatales, etc. Detection of known hydrocarbon utilizing aerobic/facultative anaerobic (Mycobacterium, Pseudomonas, Longilinea, Geobacter, etc.), nitrate reducing (Gordonia, Novosphigobium, etc.) and nitrogen fixing (Azovibrio, Rhodobacter, etc.) bacteria suggested niche specific guilds with aerobic, facultative anaerobic and strict anaerobic populations. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) predicted putative genetic repertoire of sludge microbiomes and their potential for hydrocarbon degradation; lipid-, nitrogen-, sulfur- and methane- metabolism. Methyl coenzyme M reductase A (mcrA) and dissimilatory sulfite reductase beta-subunit (dsrB) genes phylogeny confirmed methanogenic and sulfate-reducing activities within sludge environment endowed by hydrogenotrophic methanogens and sulfate-reducing Deltaproteobacteria and Firmicutes members.ConclusionRefinery sludge microbiomes were comprised of hydrocarbon degrading, fermentative, sulfate-reducing, syntrophic, nitrogen fixing and methanogenic microorganisms, which were in accordance with the prevailing physicochemical nature of the samples. Analysis of functional biomarker genes ascertained the activities of methanogenic and sulfate-reducing organisms within sludge environment. Overall data provided better insights on microbial diversity and activity in oil contaminated environment, which could be exploited suitably for in situ bioremediation of refinery sludge.

First Molecular Identification of Symbiotic Archaea in a Sponge Collected from the Persian Gulf, Iran.

Background:Marine sponges are associated with numerically vast and phylogenetically diverse microbial communities at different geographical locations. However, little is known about the archaeal diversity of sponges in the Persian Gulf. The present study was aimed to identify the symbiotic archaea with a sponge species gathered from the Persian Gulf, Iran.Methods:Sponge sample was collected from a depth of 3 m offshore Bushehr, Persian Gulf, Iran. Metagenomic DNA was extracted using a hexadecyl trimethyl ammonium bromide (CTAB) method. The COI mtDNA marker was used for molecular taxonomy identification of sponge sample. Also, symbiotic archaea were identified using the culture-independent analysis of the 16S rRNA gene and PCR- cloning.Results:In this study, analysis of multilocus DNA marker and morphological characteristics revealed that the sponge species belonged to Chondrilla australiensis isolate PG_BU4. PCR cloning and sequencing showed that all of the sequences of archaeal 16S rRNA gene libraries clustered into the uncultured archaeal group.Conclusion:The present study is the first report of the presence of the genus of Chondrilla in the Persian Gulf. Traditional taxonomy methods, when used along with molecular techniques, could play a significant role in the accurate taxonomy of sponges. Also, the uncultured archaea may promise a potential source for bioactive compounds. Further functional studies are needed to explore the role of the sponge-associated uncultured archaea as a part of the marine symbiosis.

Preservation of microbial DNA in marine sediments: insights from extracellular DNA pools.

Marine sediments harbour extracellular DNA (exDNA) not associated with currently living organisms. Including this exDNA in genetic surveys may distort abundance and diversity estimates of living prokaryotic communities. We separately extract exDNA and intracellular DNA (inDNA) from 11 horizons in a 10-m deep sediment core from Aarhus Bay (Denmark) that spans > 9000 years of Holocene sedimentation. We compare depth profiles of bacterial and archaeal 16S rRNA gene compositions to those of macrofaunal activity (bioturbation), sulfate and methane concentrations, sediment age and lithology. Among these variables, bioturbation shows the strongest relationship with the two DNA pools. In bioturbated surface sediments, the majority of Operational Taxonomic Units (OTUs) present in exDNA is absent from inDNA, thus belonging to microorganisms that were not alive at the time of sampling. Below the bioturbation zone, the two DNA pools display a much higher phylogenetic similarity. At all depths, the majority of exDNA and inDNA sequences show highest sequence similarities to sediment microorganisms, a finding that is additionally supported by separate analyses on low- and high-molecular weight exDNA. Our results indicate that in Aarhus Bay the vast majority of prokaryotic exDNA is turned over, thus not contributing to a genetic archive of past environmental change.

Dependency of DNA extraction efficiency on cell concentration confounds molecular quantification of microorganisms in groundwater.

Quantification of microbes in water systems is essential to industrial practices ranging from drinking water and wastewater treatment to groundwater remediation. While quantification using DNA-based molecular methods is precise, the accuracy is dependent on DNA extraction efficiencies. We show that the DNA yield is strongly impacted by the cell concentration in groundwater samples (r = -0.92, P < 0.0001). This has major implications for industrial applications using quantitative polymerase chain reaction (qPCR) to determine cell concentrations in water, including bioremediation. We propose a simple normalization method using a DNA recovery ratio, calculated with the total cell count and DNA yield. Application of this method to enumeration of bacteria and archaea in groundwater samples targeting phylogenetic markers (16S rRNA) demonstrated an increased goodness of fit after normalization (7.04 vs 0.94 difference in Akaike's information criteria). Furthermore, normalization was applied to qPCR quantification of functional genes and combined with DNA sequencing of archaeal and bacterial 16S rRNA genes to monitor changes in abundance of methanogenic archaea and sulphate-reducing bacteria in groundwater. The integration of qPCR and DNA sequencing with appropriate normalization enables high-throughput quantification of microbial groups using increasingly affordable and accessible techniques. This research has implications for microbial ecology and engineering research as well as industrial practice.

Characteristics of archaea and bacteria in rice rhizosphere along a mercury gradient

Several strains of archaea have the ability to methylate or resist mercury (Hg), and the paddy field is regarded to be conducive to Hg methylation. However, our knowledge of Hg-methylating or Hg-resistant archaea in paddy soils is very limited so far. Therefore, the distribution of archaea and bacteria in the rhizosphere (RS) and bulk soil (BS) of the rice growing in Xiushan Hg-mining area of southwest China was investigated. Bacterial and archaeal 16S rRNA gene amplicon sequencing of the rice rhizosphere along the Hg gradient was conducted. THg concentrations in RS were significantly higher than that in BS at site S1 and S2, while MeHg concentrations in RS was always higher than that in BS, except S6. Bacterial species richness estimates were much higher than that in archaea. The bacterial α-diversity in high-Hg sites was significant higher than that in low-Hg sites based on ACE and Shannon indices. At the genus level, Thiobacillus, Xanthomonas, Defluviicoccus and Candidatus Nitrosoarchaeum were significantly more abundant in the rhizosphere of high-Hg sites, which meant that strains in these genera might play important roles in response to Hg stress. Hg-methylating archaea in the paddy field could potentially be affiliated to strains in Methanosarcina, but further evidence need to be found. The results provide reference to understand archaeal rhizosphere community along an Hg gradient paddy soils.

Benthic archaea as potential sources of tetraether membrane lipids in sediments across an oxygen minimum zone

Abstract. Benthic archaea comprise a significant part of the total prokaryotic biomass in marine sediments. Recent genomic surveys suggest they are largely involved in anaerobic processing of organic matter, but the distribution and abundance of these archaeal groups are still largely unknown. Archaeal membrane lipids composed of isoprenoid diethers or tetraethers (glycerol dibiphytanyl glycerol tetraether, GDGT) are often used as archaeal biomarkers. Here, we compare the archaeal diversity and intact polar lipid (IPL) composition in both surface (0–0.5 cm) and subsurface (10–12 cm) sediments recovered within, just below, and well below the oxygen minimum zone (OMZ) of the Arabian Sea. Archaeal 16S rRNA gene amplicon sequencing revealed a predominance of Thaumarchaeota (Marine Group I, MG-I) in oxygenated sediments. Quantification of archaeal 16S rRNA and ammonia monoxygenase (amoA) of Thaumarchaeota genes and their transcripts indicated the presence of an active in situ benthic population, which coincided with a high relative abundance of hexose phosphohexose crenarchaeol, a specific biomarker for living Thaumarchaeota. On the other hand, anoxic surface sediments within the OMZ and all subsurface sediments were dominated by archaea belonging to the Miscellaneous Crenarchaeota Group (MCG), the Thermoplasmatales and archaea of the DPANN (superphylum grouping Micrarchaeota, Diapherotrites, Aenigmarchaeota, Nanohaloarchaeota, Parvarchaeota, Nanoarchaeota, Pacearchaeota and Woesearchaeota). Members of the MCG were diverse, with a dominance of subgroup MCG-12 in anoxic surface sediments. This coincided with a high relative abundance of IPL GDGT-0 with an unknown polar head group. Subsurface anoxic sediments were characterized by higher relative abundance of GDGT-0, -2 and -3 with dihexose IPL types, GDGT-0 with a cyclopentanetetraol molecule and hexose, as well as the presence of specific MCG subgroups, suggesting that these groups could be the biological sources of these archaeal lipids.

Diversity and abundance of arsenic methylating microorganisms in high arsenic groundwater from Hetao Plain of Inner Mongolia, China.

Arsenic methylation is regarded as an effective way of arsenic detoxification. Current knowledge about arsenic biomethylation in high arsenic groundwater remains limited. In the present study, 16 high arsenic groundwater samples from deep wells of the Hetao Plain were investigated using clone library and quantitative polymerase chain reaction (qPCR) analyses of arsM genes as well as geochemical analysis. The concentrations of methylated arsenic (including monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) varied from 2.40 to 16.85 μg/L. Both bacterial and archaeal arsenic methylating populations were detected in the high arsenic aquifer. They were dominated by Proteobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Methanomicrobia and a large unidentified group. The abundances of predominant populations were correlated positively to either total organic carbon or total arsenic and arsenite concentrations. The arsM gene abundances in high arsenic groundwater ranged from below detection to 5.71 × 106 copies/L and accounted for 0-3.32‰ of total bacterial and archaeal 16S rRNA genes. The arsM gene copies in high arsenic groundwater showed closely positive correlations with methylated arsenic concentrations. The overall results implied that arsenic methylating microorganisms were abundant and diverse in high arsenic groundwater. This was the first study of arsenic methylating microbial communities in high arsenic groundwater aquifers and might provide useful information for arsenic bioremediation in groundwater systems.

Canola Root-Associated Microbiomes in the Canadian Prairies.

Canola is one of the most economically important crops in Canada, and the root and rhizosphere microbiomes of a canola plant likely impact its growth and nutrient uptake. The aim of this study was to determine whether canola has a core root microbiome (i.e., set of microbes that are consistently selected in the root environment), and whether this is distinct from the core microbiomes of other crops that are commonly grown in the Canadian Prairies, pea, and wheat. We also assessed whether selected agronomic treatments can modify the canola microbiome, and whether this was associated to enhanced yield. We used a field experiment with a randomized complete block design, which was repeated at three locations across the canola-growing zone of Canada. Roots and rhizosphere soil were harvested at the flowering stage of canola. We separately isolated total extractable DNA from plant roots and from adjacent rhizosphere soil, and constructed MiSeq amplicon libraries for each of 60 samples, targeting bacterial, and archaeal 16S rRNA genes and the fungal ITS region. We determined that the microbiome of the roots and rhizosphere of canola was consistently different from those of wheat and pea. These microbiomes comprise several putative plant-growth-promoting rhizobacteria, including Amycolatopsis sp., Serratia proteamaculans, Pedobacter sp., Arthrobacter sp., Stenotrophomonas sp., Fusarium merismoides, and Fusicolla sp., which correlated positively with canola yield. Crop species had a significant influence on bacterial and fungal assemblages, especially within the roots, while higher nutrient input or seeding density did not significantly alter the global composition of bacterial, fungal, or archaeal assemblages associated with canola roots. However, the relative abundance of Olpidium brassicae, a known pathogen of members of the Brassicaceae, was significantly reduced in the roots of canola planted at higher seeding density. Our results suggest that seeding density and plant nutrition management modified the abundance of other bacterial and fungal taxa forming the core microbiomes of canola that are expected to impact crop growth. This work helps us to understand the microbial assemblages associated with canola grown under common agronomic practices and indicates microorganisms that can potentially benefit or reduce the yield of canola.

Methanogens in the Antarctic Dry Valley permafrost.

Polar permafrost is at the forefront of climate change, yet only a few studies have enriched the native methane-producing microbes that might provide positive feedbacks to climate change. Samples Ant1 and Ant2, collected in Antarctic Miers Valley from permafrost sediments, with and without biogenic methane, respectively, were evaluated for methanogenic activity and presence of methanogens. After a one-year incubation of both samples under anaerobic conditions, methane production was observed only at room temperature in microcosm Ant1 with CO2/H2 (20/80) as carbon and energy sources and was monitored during the subsequent 10 years. The concentration of methane in the headspace of microcosm Ant1 changed from 0.8% to a maximum of 45%. Archaeal 16S rRNA genes from microcosm Ant1 were related to psychrotolerant Methanosarcina lacustris. Repeated efforts at achieving a pure culture of this organism were unsuccessful. Metagenomic reads obtained for the methane-producing microcosm Ant1 were assembled and resulted in a 99.84% complete genome affiliated with the genus Methanosarcina. The metagenome assembled genome contained cold-adapted enzymes and pathways suggesting that the novel uncultured Methanosarcina sp. Ant1 is adapted to sub-freezing conditions in permafrost. This is the first methanogen genome reported from the 15 000 years old permafrost of the Antarctic Dry Valleys.

Evaluation of in vitro ruminal fermentation of ensiled fruit byproducts and their potential for feed use.

ObjectiveEnsiling of tannin-rich fruit byproducts (FB) involves quantitative and qualitative changes in the tannins, which would consequently change the rumen fermentation characteristics. This study aimed to evaluate whether ensiled FBs are effective in mitigating methane emission from ruminants by conducting in vitro assessments.MethodsFruit byproducts (grape pomace, wild grape pomace, and persimmon skin) were collected and subjected to four-week ensiling by Lactobacillus buchneri inoculant. A defined feed component with or without FB samples (both fresh and ensiled material) were subjected to in vitro anaerobic culturing using rumen fluid sampled from beef cattle, and the fermentation parameters and microbial populations were monitored.ResultsReduced methane production and a proportional change in total volatile fatty acids (especially enhanced propionate proportion) was noted in bottles containing the FBs compared with that in the control (without FB). In addition, we found lower gene copy number of archaeal 16S rRNA and considerably higher levels of one of the major fibrolytic bacteria (Fibrobacter succinogenes) in the bottles containing FBs than in the control, particularly, when it was included in a forage-based feed. However, in the following cultivation experiment, we observed that FBs failed to exhibit a significant difference in methane production with or without polyethylene glycol, implying that tannins in the FBs may not be responsible for the mitigation of methane generation.ConclusionThe results of the in vitro cultivation experiments indicated that not only the composition but also ensiling of FBs affected rumen fermentation patterns and the degree of methane generation. This is primarily because of the compositional changes in the fibrous fraction during ensiling as well as the presence of readily fermented substrates, whereas tannins in these FBs seemed to have little effect on the ruminal fermentation kinetics.

Key Role of Alphaproteobacteria and Cyanobacteria in the Formation of Stromatolites of Lake Dziani Dzaha (Mayotte, Western Indian Ocean).

Lake Dziani Dzaha is a thalassohaline tropical crater lake located on the “Petite Terre” Island of Mayotte (Comoros archipelago, Western Indian Ocean). Stromatolites are actively growing in the shallow waters of the lake shores. These stromatolites are mainly composed of aragonite with lesser proportions of hydromagnesite, calcite, dolomite, and phyllosilicates. They are morphologically and texturally diverse ranging from tabular covered by a cauliflower-like crust to columnar ones with a smooth surface. High-throughput sequencing of bacterial and archaeal 16S rRNA genes combined with confocal laser scanning microscopy (CLSM) analysis revealed that the microbial composition of the mats associated with the stromatolites was clearly distinct from that of the Arthrospira-dominated lake water. Unicellular-colonial Cyanobacteria belonging to the Xenococcus genus of the Pleurocapsales order were detected in the cauliflower crust mats, whereas filamentous Cyanobacteria belonging to the Leptolyngbya genus were found in the smooth surface mats. Observations using CLSM, scanning electron microscopy (SEM) and Raman spectroscopy indicated that the cauliflower texture consists of laminations of aragonite, magnesium-silicate phase and hydromagnesite. The associated microbial mat, as confirmed by laser microdissection and whole-genome amplification (WGA), is composed of Pleurocapsales coated by abundant filamentous and coccoid Alphaproteobacteria. These phototrophic Alphaproteobacteria promote the precipitation of aragonite in which they become incrusted. In contrast, the Pleurocapsales are not calcifying but instead accumulate silicon and magnesium in their sheaths, which may be responsible for the formation of the Mg-silicate phase found in the cauliflower crust. We therefore propose that Pleurocapsales and Alphaproteobacteria are involved in the formation of two distinct mineral phases present in the cauliflower texture: Mg-silicate and aragonite, respectively. These results point out the role of phototrophic Alphaproteobacteria in the formation of stromatolites, which may open new perspective for the analysis of the fossil record.

Diversity and distribution of Archaea in global estuarine ecosystems

Estuarine ecosystem is a unique geographical transitional zone between freshwater and seawater, harboring a wide range of microbial communities including Archaea. Although a large number of Archaea have been detected in such ecosystem, the global patterns in archaeal diversity and distribution are extremely scarce. To bridge this gap, we carried out a comprehensive survey of archaeal communities using ca. 4000 publicly available archaeal 16S rRNA gene sequences (>300 bp) collected from 24 estuaries in different latitude regions. These sequences were divided into 1450 operational taxonomic units (OTUs) at 97% identity, suggesting a high biodiversity that increased gradually from the high- to low-latitude estuaries. Phylogenetic analysis showed that estuarine ecosystem was a large biodiversity pool of Archaea that was mainly composed of 12 phyla. Among them, the predominant groups were Bathyarchaeota, Euryarchaeota and Thaumarchaeota. Interestingly, archaeal distribution demonstrated a geographical differentiation in that Thaumarchaeota was dominated in the low-latitude estuaries, Bathyarchaeota in the mid-latitude estuaries, and Euryarchaeota in the high-latitude estuaries, respectively. Furthermore, the majority of the most abundant 20 OTUs demonstrated an overrepresented or underrepresented distribution pattern in some specific estuaries or latitude regions while a few were evenly distributed throughout the estuaries. This pattern indicates a potential selectivity of geographical distribution. In addition, the analysis of environmental parameters suggested that latitude would be one of the major factors driving the distribution of archaeal communities in estuarine ecosystem. This study profiles a clear framework on the diversity and distribution of Archaea in the global estuarine ecosystem and explores the general environmental factors that influence these patterns. Our findings constitute an important part of the exploration of the global ecology of Archaea.

Archaea are prominent members of the prokaryotic communities colonizing common forest mushrooms.

In this study, the abundance and composition of prokaryotic communities associated with the inner tissue of fruiting bodies of Suillus bovinus, Boletus pinophilus, Cantharellus cibarius, Agaricus arvensis, Lycoperdon perlatum, and Piptoporus betulinus were analyzed using culture-independent methods. Our findings indicate that archaea and bacteria colonize the internal tissues of all investigated specimens and that archaea are prominent members of the prokaryotic community. The ratio of archaeal 16S rRNA gene copy numbers to those of bacteria was >1 in the fruiting bodies of four out of six fungal species included in the study. The largest proportion of archaeal 16S rRNA gene sequences belonged to thaumarchaeotal classes Terrestrial group, Miscellaneous Crenarchaeotic Group (MCG), and Thermoplasmata. Bacterial communities showed characteristic compositions in each fungal species. Bacterial classes Gammaproteobacteria, Actinobacteria, Bacilli, and Clostridia were prominent among communities in fruiting body tissues. Bacterial populations in each fungal species had different characteristics. The results of this study imply that fruiting body tissues are an important habitat for abundant and diverse populations of archaea and bacteria.

Composition of Microbial Communities in Sediments from Southern Baikal Containing Fe/Mn Concretions

Massive parallel sequencing of the 16S rRNA gene fragments was used to investigate the composition and diversity of microbial communities in sediments from Southern Baikal to a depth of 9 cm with 1-cm step. In the layers from the sediment surface to the lower border of oxygen penetration (2 cm), organotrophic bacteria with high similarity to the heterotrophic species Luteolibacter luojiensis constituted the largest fraction of the community. In the formation zone of Fe/Mn crusts (3–5 cm), Proteobacteria and Actinobacteria predominated in the community, while the share of Cyanobacteria was considerable. The lower reduced layers showed an increased contribution of the Bacteroidetes, while the shares of the taxa predominant in the higher layers remained significant. Analysis of archaeal 16S rRNA gene amplicons revealed predominance of the soil and aquatic Thaumarchaeota (Marine Group I lineage), which are involved in anaerobic ammonium oxidation, practically in all sediment layers. The buried oxidized layer (6–7 cm), where members of the uncultured Marine Benthic Group D lineage of the order Thermoplasmatales (Euryarchaeota) predominated, was an exception in this regard. Small numbers of archaea of the Baikal-1 lineages (below 1%) were observed in the communities from the 6–7 and 7–8 cm layers, while the archaea involved in anaerobic methane oxidation (including the ANME-2d group) were not detected.