Simultaneous removal of crude oil and heavy metals by highly adapted bacterial strain Cutibacterium sp. NL2 isolated from Algerian oilfield

Investigating the ability of bacteria to simultaneously enhance hydrocarbon removal and reduce heavy metals’ toxicity is necessary to design more effective bioremediation strategies. A bacterium (NL2 strain) isolated from an Algerian oilfield was cultivated on crude oil as sole carbon and energy sources. Molecular analyses of the 16S rRNA gene sequence placed the strain within the Cutibacterium genera. This isolate was able to tolerate up to 60% of crude oil as sole carbon source. Chemical analyses (GC-MS) evidenced that strain NL2 was able to degrade 92.22% of crude oil (at optimal growing conditions: pH 10, 44 °C, 50 g L−1 NaCl, and 20% of crude oil (v/v) as sole carbon source) in only 7 days. NL2 isolate was also able to produce biosurfactants with reduction of surface tension of growing media (29.4 mN m−1). On the other hand, NL2 strain was able to tolerate high lead (Pb) and copper (Cu) concentrations (up to 60 mM). In fact, NL2 cultivated in the presence of 20% of crude oil, and 0.48 mM of Pb was able to reduce Pb concentration by a 41.36%. In turn, when cultivated on high Pb concentration (15 mM), the strain was able to remove 35.19% of it and 86.25% of crude oil, both in a time frame of 7 days. Our findings suggest that Cutibacterium strain NL2 is able to efficiently use and remove a wide range of crude oil substrates in presence of high Pb concentration. Accordingly, NL2 strain is of extreme interest from a biotechnological standpoint. (AU)

Exploring the Associations of Inflammatory and Oxidative Stress Biomarkers with Pancreatic Diseases: An Observational and Mendelian Randomisation Study.

Identifying biomarkers linked to pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP) is crucial for early detection, treatment, and prevention. Methods: Association analyses of 10 serological biomarkers involved in cell signalling (IFN-γ, IL-6, IL-8, IL-10), oxidative stress (superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities, total glutathione (GSH), malondialdehyde (MDA) levels), and intestinal permeability proteins (zonulin, I-FABP2) were conducted across PDAC (n = 12), CP (n = 21) and control subjects (n = 23). A Mendelian randomisation (MR) approach was used to assess causality of the identified significant associations in two large genetic cohorts (FinnGen and UK Biobank). Results: Observational results showed a downregulation of SOD and GPx antioxidant enzyme activities in PDAC and CP patients, respectively, and higher MDA levels in CP patients. Logistic regression models revealed significant associations between CP and SOD activity (OR = 0.21, 95% CI [0.05, 0.89], per SD), GPx activity (OR = 0.28, 95% CI [0.10, 0.79], per SD), and MDA levels (OR = 2.05, 95% CI [1.36, 3.08], per SD). MR analyses, however, did not support causality. Conclusions: These findings would not support oxidative stress-related biomarkers as potential targets for pancreatic diseases prevention. Yet, further research is encouraged to assess their viability as non-invasive tools for early diagnosis, particularly in pre-diagnostic CP populations.

Impact of bread diet on intestinal dysbiosis and irritable bowel syndrome symptoms in quiescent ulcerative colitis: A pilot study.

Gut microbiota may be involved in the presence of irritable bowel syndrome (IBS)-like symptomatology in ulcerative colitis (UC) patients in remission. Bread is an important source of dietary fiber, and a potential prebiotic. To assess the effect of a bread baked using traditional elaboration, in comparison with using modern elaboration procedures, in changing the gut microbiota and relieving IBS-like symptoms in patients with quiescent ulcerative colitis. Thirty-one UC patients in remission with IBS-like symptoms were randomly assigned to a dietary intervention with 200 g/d of either treatment or control bread for 8 weeks. Clinical symptomatology was tested using questionnaires and inflammatory parameters. Changes in fecal microbiota composition were assessed by high-throughput sequencing of the 16S rRNA gene. A decrease in IBS-like symptomatology was observed after both the treatment and control bread interventions as reductions in IBS-Symptom Severity Score values (p-value < 0.001) and presence of abdominal pain (p-value < 0.001). The treatment bread suggestively reduced the Firmicutes/Bacteroidetes ratio (p-value = 0.058). In addition, the Firmicutes/Bacteroidetes ratio seemed to be associated with improving IBS-like symptoms as suggested by a slight decrease in patient without abdominal pain (p-value = 0.059). No statistically significant differential abundances were found at any taxonomic level. The intake of a bread baked using traditional elaboration decreased the Firmicutes/Bacteroidetes ratio, which seemed to be associated with improving IBS-like symptoms in quiescent ulcerative colitis patients. These findings suggest that the traditional bread elaboration has a potential prebiotic effect improving gut health (ClinicalTrials.gov ID number of study: NCT05656391).

Simultaneous removal of crude oil and heavy metals by highly adapted bacterial strain Cutibacterium sp. NL2 isolated from Algerian oilfield.

Investigating the ability of bacteria to simultaneously enhance hydrocarbon removal and reduce heavy metals' toxicity is necessary to design more effective bioremediation strategies. A bacterium (NL2 strain) isolated from an Algerian oilfield was cultivated on crude oil as sole carbon and energy sources. Molecular analyses of the 16S rRNA gene sequence placed the strain within the Cutibacterium genera. This isolate was able to tolerate up to 60% of crude oil as sole carbon source. Chemical analyses (GC-MS) evidenced that strain NL2 was able to degrade 92.22% of crude oil (at optimal growing conditions: pH 10, 44 °C, 50 g L-1 NaCl, and 20% of crude oil (v/v) as sole carbon source) in only 7 days. NL2 isolate was also able to produce biosurfactants with reduction of surface tension of growing media (29.4 mN m-1). On the other hand, NL2 strain was able to tolerate high lead (Pb) and copper (Cu) concentrations (up to 60 mM). In fact, NL2 cultivated in the presence of 20% of crude oil, and 0.48 mM of Pb was able to reduce Pb concentration by a 41.36%. In turn, when cultivated on high Pb concentration (15 mM), the strain was able to remove 35.19% of it and 86.25% of crude oil, both in a time frame of 7 days. Our findings suggest that Cutibacterium strain NL2 is able to efficiently use and remove a wide range of crude oil substrates in presence of high Pb concentration. Accordingly, NL2 strain is of extreme interest from a biotechnological standpoint.

Chromosome-level genome assembly reveals homologous chromosomes and recombination in asexual rotifer Adineta vaga.

Bdelloid rotifers are notorious as a speciose ancient clade comprising only asexual lineages. Thanks to their ability to repair highly fragmented DNA, most bdelloid species also withstand complete desiccation and ionizing radiation. Producing a well-assembled reference genome is a critical step to developing an understanding of the effects of long-term asexuality and DNA breakage on genome evolution. To this end, we present the first high-quality chromosome-level genome assemblies for the bdelloid Adineta vaga, composed of six pairs of homologous (diploid) chromosomes with a footprint of paleotetraploidy. The observed large-scale losses of heterozygosity are signatures of recombination between homologous chromosomes, either during mitotic DNA double-strand break repair or when resolving programmed DNA breaks during a modified meiosis. Dynamic subtelomeric regions harbor more structural diversity (e.g., chromosome rearrangements, transposable elements, and haplotypic divergence). Our results trigger the reappraisal of potential meiotic processes in bdelloid rotifers and help unravel the factors underlying their long-term asexual evolutionary success.

In vitro Prebiotic Effect of Bread-Making Process in Inflammatory Bowel Disease Microbiome.

Inflammatory bowel disease (IBD), including its two main categories (Crohn's disease and ulcerative colitis), has been linked both to gut microbiota and to diet. Bread is a daily food that has a potential capacity as a prebiotic. Our aim was to evaluate different bread-making processes and their effect on fecal colonic microbiota in IBD patients. The microbial composition of several sourdoughs and dough samples was analyzed by high-throughput sequencing of 16S and 18S rRNA genes. Three types of bread, which followed different bread-making processes, were in vitro digested and incubated with feces from IBD patients. Changes in gut microbiota were assessed by a quantitative polymerase chain reaction using specific bacterial sequence targets. Short-chain fatty acid production was also analyzed by gas chromatography. Lactobacillus sanfranciscensis was the dominant lactic acid bacteria species found in sourdough and bread doughs prepared using sourdough, whereas Saccharomyces cerevisiae was the most dominant yeast in all groups, especially in bread doughs before baking. Differences in microbial composition in raw bread doughs were more related to the type of dough and elaboration than to fermentation time lengths. The analysis of in vitro fecal incubations with bread conditions revealed an increase in most bacterial groups analyzed and short-chain fatty acid production, both in Crohn's disease and ulcerative colitis samples. Most remarkable increases in short-chain fatty acid production mirrored higher abundances of Roseburia species. The potential prebiotic properties observed were mainly obtained when using a high quantity of bread, regardless of bread type. Overall, this study highlights the bacterial dynamics within the bread-making process and the potential prebiotic effect in IBD patients.

The possible occurrence of iron-dependent anaerobic methane oxidation in an Archean Ocean analogue.

In the ferruginous and anoxic early Earth oceans, photoferrotrophy drove most of the biological production before the advent of oxygenic photosynthesis, but its association with ferric iron (Fe3+) dependent anaerobic methane (CH4) oxidation (AOM) has been poorly investigated. We studied AOM in Kabuno Bay, a modern analogue to the Archean Ocean (anoxic bottom waters and dissolved Fe concentrations > 600 µmol L-1). Aerobic and anaerobic CH4 oxidation rates up to 0.12 ± 0.03 and 51 ± 1 µmol L-1 d-1, respectively, were put in evidence. In the Fe oxidation-reduction zone, we observed high concentration of Bacteriochlorophyll e (biomarker of the anoxygenic photoautotrophs), which co-occurred with the maximum CH4 oxidation peaks, and a high abundance of Candidatus Methanoperedens, which can couple AOM to Fe3+ reduction. In addition, comparison of measured CH4 oxidation rates with electron acceptor fluxes suggest that AOM could mainly rely on Fe3+ produced by photoferrotrophs. Further experiments specifically targeted to investigate the interactions between photoferrotrophs and AOM would be of considerable interest. Indeed, ferric Fe3+-driven AOM has been poorly envisaged as a possible metabolic process in the Archean ocean, but this can potentially change the conceptualization and modelling of metabolic and geochemical processes controlling climate conditions in the Early Earth.

Microbacterium algeriense sp. nov., a novel actinobacterium isolated from Algerian oil production waters.

A non-motile, straight-rod-shaped, Gram-stain-positive and facultative anaerobic bacterium (i.e., strain G1T) was isolated from production waters from an Algerian oilfield. Growth was observed in the presence of 0.3-3.5 % (w/v) NaCl, at 20-50 °C and at pH 6.0-9.0. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that strain G1T belonged to the genus Microbacterium. Strain G1 T was closely related to Microbacterium oxydans (DSM 20578T) and Microbacterium maritypicum (DSM 12512T) with 99.8 % sequence similarity and to Microbacterium saperdae (DSM 20169T) with 99.6 % sequence similarity. Strain G1 T contained MK9, MK10, MK11, MK12 and MK13 as respiratory quinones, and phosphatidylglycerol, diphosphatidylglycerol and glycolipid as the major polar lipids. The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The estimated DNA G+C content was 69.57 mol% based on its draft genome sequence. Genome annotation of strain G1T predicted the presence of 3511 genes, of which 3483 were protein-coding and 47 were tRNA genes. The DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) values between strain G1T and M. oxydans (DSM 20578T) and M. maritypicum (DSM 12512T) were in both cases far below the respective species boundary thresholds (27.5 and 28.0 % for DDH; and 84.40 and 84.82% for ANI, respectively). Based on the data presented above, strain G1T was considered to represent a novel species for which the name Microbacterium algeriense is proposed with the type strain G1T (=DSM 109018T=LMG 31276T).

Photoferrotrophy, deposition of banded iron formations, and methane production in Archean oceans.

Banded iron formation (BIF) deposition was the likely result of oxidation of ferrous iron in seawater by either oxygenic photosynthesis or iron-dependent anoxygenic photosynthesis-photoferrotrophy. BIF deposition, however, remains enigmatic because the photosynthetic biomass produced during iron oxidation is conspicuously absent from BIFs. We have addressed this enigma through experiments with photosynthetic bacteria and modeling of biogeochemical cycling in the Archean oceans. Our experiments reveal that, in the presence of silica, photoferrotroph cell surfaces repel iron (oxyhydr)oxides. In silica-rich Precambrian seawater, this repulsion would separate biomass from ferric iron and would lead to large-scale deposition of BIFs lean in organic matter. Excess biomass not deposited with BIF would have deposited in coastal sediments, formed organic-rich shales, and fueled microbial methanogenesis. As a result, the deposition of BIFs by photoferrotrophs would have contributed fluxes of methane to the atmosphere and thus helped to stabilize Earth's climate under a dim early Sun.

Draft Genome Sequence of the Pelagic Photoferrotroph Chlorobium phaeoferrooxidans.

Here, we report the draft genome sequence of Chlorobium phaeoferrooxidans, a photoferrotrophic member of the genus Chlorobium in the phylum Chlorobi This genome sequence provides insight into the metabolic capacity that underpins photoferrotrophy within low-light-adapted pelagic Chlorobi.

Molecular and carbon isotopic characterization of an anaerobic stable enrichment culture containing Dehalobacterium sp. during dichloromethane fermentation.

Biodegradation of dichloromethane (DCM) under reducing conditions is of major concern due to its widespread detection in contaminated groundwaters. Here, we report an anaerobic enrichment culture derived from a membrane bioreactor operating in an industrial wastewater treatment plant, capable of fermenting DCM and the brominated analogue dibromomethane (DBM). Comparative analysis of bacterial 16S rDNA-DGGE profiles from fresh liquid medium inoculated with single colonies picked from serial dilution-to-extinction agar vials showed that cultures degrading DCM contained a predominant band belonging to Dehalobacterium, however this band was absent in cultures unable to degrade DCM. Analysis of the microbial composition of the enrichment by bacterial 16S rRNA gene amplicon paired-end sequencing confirmed the presence of Dehalobacterium together with three additional phylotypes belonging to Acetobacterium, Desulfovibrio, and Wolinella, representing all four operational taxonomic units >99.9% of the retrieved sequences. The carbon isotopic fractionation (ε) determined for DCM degradation in this culture was -27±2‰. This value differs from the ε previously reported for the DCM-fermentative bacteria Dehalobacter (-15.5±1.5‰) but they are both significantly different from those reported for facultative methylotrophic organisms (ranging from -45 to -61‰). This significant difference in the ε allows differentiating between hydrolytic transformation of DCM via glutathione-dependent dehalogenases and fermentation pathway. CAPSULE: The carbon isotopic fractionation of dichloromethane by an enriched Dehalobacterium-containing culture has significant potential to monitor biodegradation of DCM in groundwaters.

Pelagic photoferrotrophy and iron cycling in a modern ferruginous basin.

Iron-rich (ferruginous) ocean chemistry prevailed throughout most of Earth's early history. Before the evolution and proliferation of oxygenic photosynthesis, biological production in the ferruginous oceans was likely driven by photoferrotrophic bacteria that oxidize ferrous iron {Fe(II)} to harness energy from sunlight, and fix inorganic carbon into biomass. Photoferrotrophs may thus have fuelled Earth's early biosphere providing energy to drive microbial growth and evolution over billions of years. Yet, photoferrotrophic activity has remained largely elusive on the modern Earth, leaving models for early biological production untested and imperative ecological context for the evolution of life missing. Here, we show that an active community of pelagic photoferrotrophs comprises up to 30% of the total microbial community in illuminated ferruginous waters of Kabuno Bay (KB), East Africa (DR Congo). These photoferrotrophs produce oxidized iron {Fe(III)} and biomass, and support a diverse pelagic microbial community including heterotrophic Fe(III)-reducers, sulfate reducers, fermenters and methanogens. At modest light levels, rates of photoferrotrophy in KB exceed those predicted for early Earth primary production, and are sufficient to generate Earth's largest sedimentary iron ore deposits. Fe cycling, however, is efficient, and complex microbial community interactions likely regulate Fe(III) and organic matter export from the photic zone.

Diversity of freshwater Epsilonproteobacteria and dark inorganic carbon fixation in the sulphidic redoxcline of a meromictic karstic lake.

Sulfidic redoxclines are a suitable niche for the growth and activity of different chemo- and photolithotrophic sulphide-oxidizing microbial groups such as the Epsilonproteobacteria and the green sulfur bacteria (GSB). We have investigated the diversity, abundance and contribution to inorganic carbon uptake of Epsilonproteobacteria in a meromictic basin of Lake Banyoles. CARD-FISH counts revealed that Epsilonproteobacteria were prevalent at the redoxcline in winter (maximum abundance of 2 × 10(6) cells mL(-1), ≈60% of total cells) but they were nearly absent in summer, when GSB bloomed. This seasonal trend was supported by 16S rRNA gene pyrotag datasets, which revealed that the epsilonproteobacterial community was mainly composed of a member of the genus Arcobacter. In situ incubations using NaH(14)CO3 and MAR-CARD-FISH observations showed that this population assimilated CO2 in the dark, likely being mainly responsible for the autotrophic activity at the redoxcline in winter. Clone libraries targeting the aclB gene provided additional evidence of the potential capacity of these epsilonproteobacteria to fix carbon via rTCA cycle. Our data reinforce the key role of Epsilonproteobacteria in linking carbon and sulphur cycles, extend their influence to freshwater karstic lakes and raise questions about the actual contribution of chemolithotrophy at their redoxcline and euxinic water compartments.

Vertical Distribution of Functional Potential and Active Microbial Communities in Meromictic Lake Kivu.

The microbial community composition in meromictic Lake Kivu, with one of the largest CH4 reservoirs, was studied using 16S rDNA and ribosomal RNA (rRNA) pyrosequencing during the dry and rainy seasons. Highly abundant taxa were shared in a high percentage between bulk (DNA-based) and active (RNA-based) bacterial communities, whereas a high proportion of rare species was detected only in either an active or bulk community, indicating the existence of a potentially active rare biosphere and the possible underestimation of diversity detected when using only one nucleic acid pool. Most taxa identified as generalists were abundant, and those identified as specialists were more likely to be rare in the bulk community. The overall number of environmental parameters that could explain the variation was higher for abundant taxa in comparison to rare taxa. Clustering analysis based on operational taxonomic units (OTUs at 0.03 cutoff) level revealed significant and systematic microbial community composition shifts with depth. In the oxic zone, Actinobacteria were found highly dominant in the bulk community but not in the metabolically active community. In the oxic-anoxic transition zone, highly abundant potentially active Nitrospira and Methylococcales were observed. The co-occurrence of potentially active sulfur-oxidizing and sulfate-reducing bacteria in the anoxic zone may suggest the presence of an active yet cryptic sulfur cycle.

Bacterial community composition in three freshwater reservoirs of different alkalinity and trophic status.

In order to investigate the factors controlling the bacterial community composition (BCC) in reservoirs, we sampled three freshwater reservoirs with contrasted physical and chemical characteristics and trophic status. The BCC was analysed by 16S rRNA gene amplicon 454 pyrosequencing. In parallel, a complete dataset of environmental parameters and phytoplankton community composition was also collected. BCC in the analysed reservoirs resembled that of epilimnetic waters of natural freshwater lakes with presence of Actinobacteria, Alpha- and Betaproteobacteria, Cytophaga-Flavobacteria-Bacteroidetes (CFB) and Verrucomicrobia groups. Our results evidenced that the retrieved BCC in the analysed reservoirs was strongly influenced by pH, alkalinity and organic carbon content, whereas comparatively little change was observed among layers in stratified conditions.

Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing.

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species.

Active bacteria and archaea cells fixing bicarbonate in the dark along the water column of a stratified eutrophic lagoon.

We studied the carbon dioxide fixation activity in a stratified hypereutrophic karstic lagoon using a combination of fingerprinting techniques targeting bacterial and archaeal 16S rRNA genes, functional gene cloning [the acetyl-CoA carboxylase (accC)], and isotopic labelling ((14)C-bicarbonate) coupled to single-cell analyses [microautoradiography combined with catalyzed reported deposition-FISH (MAR-CARD-FISH)]. The microbial planktonic community was dominated by bacteria with maximal abundances of archaea just below the oxic/anoxic transition zone (7% of total cells). In situ incubations with radiolabelled bicarbonate showed maximal photoassimilation activity in the oxic epilimnion, whereas dark CO(2) fixation was consistently observed throughout the water column, with a maximum at the oxic/anoxic interface (8.6 mg C m(-3) h(-1)). The contributions of light and dark carbon fixation activities in the whole water column were 69% and 31% of the total C incorporated, respectively. MAR-CARD-FISH incubations corroborated these results and revealed that the highest fraction of bacterial and archaeal cells actively uptaking bicarbonate in the light was found at the surface. The bacterial community was mainly composed of green sulfur bacteria (Chlorobi) and members of the Betaproteobacteria and the Bacteroidetes. The archaeal assemblage was composed of phylotypes of the Miscellaneous Crenarchaeotic Group and a few methanogens. Clone libraries of the accC gene showed an absolute dominance of bacterial carboxylases. Our results suggest that the dark carbon fixation activity measured was mainly related to CO(2) incorporation by heterotrophs rather than to the activity of true chemoautotrophs.

Maintenance of previously uncultured freshwater archaea from anoxic waters under laboratory conditions.

Culture conditions for the maintenance of previously uncultured members of the Archaea thriving in anoxic water layers of stratified freshwater lakes are described. The proposed enrichment conditions, based on the use of defined medium composition and the maintenance of anoxia, have been proven effective for the maintenance of the archaeal community with virtually no changes over time for periods up to 6 months as revealed by a PCR-DGGE analysis. Phylotypes belonging to groups poorly represented in culture collections such as the Deep-Sea Hydrothermal Vent Euryarchaeota (DHVE) and the Miscellaneous Crenarchaeotic Group (MCG) were maintained and selectively enriched when compared to the correspondent indigenous planktonic archaeal community.

Vertical distribution of ammonia-oxidizing crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo).

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4',6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.

High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study.

We surveyed the archaeal assemblage in a stratified sulfurous lake (Lake Vilar, Banyoles, Spain) over 5 consecutive years to detect potential seasonal and interannual trends in the free-living planktonic Archaea composition. The combination of different primer pairs and nested PCR steps revealed an unexpectedly rich archaeal community. Overall, 140 samples were analyzed, yielding 169 different 16S rRNA gene sequences spread over 14 Crenarchaeota (109 sequences) and six Euryarchaeota phylogenetic clusters. Most of the Crenarchaeota (98% of the total crenarchaeotal sequences) affiliated within the Miscellaneous Crenarchaeota Group (MCG) and were related to both marine and freshwater phylotypes. Euryarchaeota mainly grouped within the Deep Hydrothermal Vent Euryarchaeota (DHVE) cluster (80% of the euryarchaeotal sequences) and the remaining 20% distributed into three less abundant taxa, most of them composed of soil and sediment clones. The largest fraction of phylotypes from the two archaeal kingdoms (79% of the Crenarchaeota and 54% of the Euryarchaeota) was retrieved from the anoxic hypolimnion, indicating that these cold and sulfide-rich waters constitute an unexplored source of archaeal richness. The taxon rank-frequency distribution showed two abundant taxa (MCG and DHVE) that persisted in the water column through seasons, plus several rare ones that were only detected occasionally. Differences in richness distribution and seasonality were observed, but no clear correlations were obtained when multivariate statistical analyses were carried out.