Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.     

Contribution of Crenarchaeota and Bacteria to autotrophy in the North Atlantic interior

Marine Crenarchaeota are among the most abundant groups of prokaryotes in the ocean and recent reports suggest that they oxidize ammonia as an energy source and inorganic carbon as carbon source, while other studies indicate that Crenarchaeota use organic carbon and hence, live heterotrophically. We used catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) to determine the crenarchaeal and bacterial contribution to total prokaryotic abundance in the (sub)tropical Atlantic. Bacteria contributed ～50% to total prokaryotes throughout the water column. Marine Crenarchaeota Group I (MCGI) accounted for ～5% of the prokaryotes in subsurface waters (100 m depth) and between 10 and 20% in the oxygen minimum layer (250–500 m depth) and deep waters (North East Atlantic Deep Water). The fraction of both MCGI and Bacteria fixing inorganic carbon, determined by combining microautoradiography with CARD‐FISH (MICRO‐CARD‐FISH), decreased with depth, ranging from ～30% in the oxygen minimum zone to < 10% in the intermediate waters (Mediterranean Sea Outflow Water, Antarctic Intermediate Water). In the deeper water masses, however, MCGI were not taking up inorganic carbon. Using quantitative MICRO‐CARD‐FISH to determine autotrophy activity on a single cell level revealed that MCGI are incorporating inorganic carbon (0.002–0.1 fmol C cell^?1 day^?1) at a significantly lower rate than Bacteria (0.01–0.6 fmol C cell^?1 day^?1). Hence, it appears that MCGI contribute substantially less to autotrophy than Bacteria. Taking the stoichiometry of nitrification together with our findings suggests that MCGI might not dominate the ammonia oxidation step in the mesopelagic waters of the ocean to that extent as the reported dominance of archaeal over bacterial amoA would suggest.     

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.     

Phylogenetic Diversity of the Archaeal Component in Microbial Mats on Coral-like Structures Associated with Methane Seeps in the Black Sea

With the use of molecular ecology methods, the archaeal component of microbial mats on coral-like structures associated with methane seeps occurring at a depth of about 200 m in the Black Sea was investigated without the isolation of pure cultures. Using archaea-specific 16S rDNA–targeted oligonucleotide primes, long fragments of genes were amplified, cloned, and sequenced and their phylogenetic analysis was carried out. It was shown that archaea in microbial mats on coral-like structures are represented by two dominant phylotypes that belong to the kingdoms Crenarchaeota and Euryarchaeota and are not specifically related to any described archaeal species. The possible role of the revealed archaea in the process of anaerobic methane oxidation is discussed.     

Latitudinal distribution of prokaryotic picoplankton populations in the Atlantic Ocean

Members of the prokaryotic picoplankton are the main drivers of the biogeochemical cycles over large areas of the world's oceans. In order to ascertain changes in picoplankton composition in the euphotic and twilight zones at an ocean basin scale we determined the distribution of 11 marine bacterial and archaeal phyla in three different water layers along a transect across the Atlantic Ocean from South Africa (32.9°S) to the UK (46.4°N) during boreal spring. Depth profiles down to 500 m at 65 stations were analysed by catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) and automated epifluorescence microscopy. There was no obvious overall difference in microbial community composition between the surface water layer and the deep chlorophyll maximum (DCM) layer. There were, however, significant differences between the two photic water layers and the mesopelagic zone. SAR11 (35 ± 9%) and Prochlorococcus (12 ± 8%) together dominated the surface waters, whereas SAR11 and Crenarchaeota of the marine group I formed equal proportions of the picoplankton community below the DCM (both ～15%). However, due to their small cell sizes Crenarchaeota contributed distinctly less to total microbial biomass than SAR11 in this mesopelagic water layer. Bacteria from the uncultured Chloroflexi‐related clade SAR202 occurred preferentially below the DCM (4–6%). Distinct latitudinal distribution patterns were found both in the photic zone and in the mesopelagic waters: in the photic zone, SAR11 was more abundant in the Northern Atlantic Ocean (up to 45%) than in the Southern Atlantic gyre (～25%), the biomass of Prochlorococcus peaked in the tropical Atlantic Ocean, and Bacteroidetes and Gammaproteobacteria bloomed in the nutrient‐rich northern temperate waters and in the Benguela upwelling. In mesopelagic waters, higher proportions of SAR202 were present in both central gyre regions, whereas Crenarchaeota were clearly more abundant in the upwelling regions and in higher latitudes. Other phylogenetic groups such as the Planctomycetes, marine group II Euryarchaeota and the uncultured clades SAR406, SAR324 and SAR86 rarely exceeded more than 5% of relative abundance.     

Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

Abundance and activity of Chloroflexi-type SAR202 bacterioplankton in the meso- and bathypelagic waters of the (sub)tropical Atlantic

The contribution of Chloroflexi-type SAR202 cells to total picoplankton and bacterial abundance and uptake of D- and L-aspartic acids (Asp) was determined in the different meso- and bathypelagic water masses of the (sub)tropical Atlantic (from 35 degrees N to 5 degrees S). Fluorescence in situ hybridization (FISH) revealed that the overall abundance of SAR202 was < or = 1 x 10(3) cells ml(-1) in subsurface waters (100 m layer), increasing in the mesopelagic zone to 3 x 10(3) cells ml(-1) and remaining fairly constant down to 4000 m depth. Overall, the percentage of total picoplankton identified as SAR202 increased from < 1% in subsurface waters to 10-20% in the bathypelagic waters. On average, members of the SAR202 cluster accounted for about 30% of the Bacteria in the bathypelagic waters, whereas in the mesopelagic and subsurface waters, SAR202 cells contributed < 5% to total bacterial abundance. The ratio of D-Asp : L-Asp uptake by the bulk picoplankton community increased from the subsurface layer (D-Asp : L-Asp uptake ratio approximately 0.03) to the deeper layers reaching a ratio of approximately 1 at 4000 m depth. Combining FISH with microautoradiography to determine the proportion of SAR202 cells taking up D-Asp versus L-Asp, we found that approximately 30% of the SAR202 cells were taking up L-Asp throughout the water column while D-Asp was essentially not taken up by SAR202. This D-Asp : L-Asp uptake pattern of SAR202 cells is in contrast to that of the bulk bacterial and crenarchaeal community in the bathypelagic ocean, both sustaining a higher fraction of D-Asp-positive cells than L-Asp-positive cells. Thus, although the Chloroflexi-type SAR202 constitutes a major bathypelagic bacterial cluster, it does not contribute to the large fraction of d-Asp utilizing prokaryotic community in the meso- and bathypelagic waters of the North Atlantic, but rather utilizes preferentially L-amino acids.     

Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.     

象山港网箱养殖区沉积物的古菌空间分布

对象山港网箱养殖区及其周边沉积物中古菌群落的空间分布进行研究,应用基于16S rRNA基因的T-RFLP(末端限制性片段多态性分析)技术分析象山港网箱养殖区及其周边不同深度沉积物中古菌的群落结构和多样性,并构建克隆文库进行系统发育学分析。测定沉积物各项理化因子,通过PCA和RDA分析了古菌群落分布及其与环境因子之间的关系。结果表明,泉古菌是港口沉积物中的优势古菌群,占古菌群落的50%以上。网箱养殖区沉积物的古菌群落结构较非养殖区简单,多样性降低。非养殖区古菌群落随深度呈现有规律的变化。营养盐类和pH是造成养殖区域古菌群落结构区别于非养殖区域的主要环境因素。     

Temporal Changes in Archaeal Diversity and Chemistry in a Mid-Ocean Ridge Subseafloor Habitat

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-μm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.     

Bacterial and archaeal phylogenetic diversity associated with swine sludge from an anaerobic treatment lagoon

Over the last decades, the demand for pork products has increased significantly, along with concern about suitable waste management. Anaerobic-lagoon fermentation for swine-sludge stabilization is a good strategy, although little is known about the microbial communities in the lagoons. Here, we employed a cloning- and sequencing-based analysis of the 16S rRNA gene to characterize and quantify the prokaryotic community composition in a swine-waste-sludge anaerobic lagoon (SAL). DNA sequence analysis revealed that the SAL library harbored 15 bacterial phyla: Bacteroidetes, Cloroflexi, Proteobacteria, Firmicutes, Deinococcus-Thermus, Synergystetes, Gemmatimonadetes, Chlorobi, Fibrobacteres, Verrucomicrobia and candidates division OP5, OP8, WWE1, KSB1, WS6. The SAL library was generally dominated by carbohydrate-oxidizing bacteria. The archaeal sequences were related to the Crenarchaeota and Euryarchaeota phyla. Crenarchaeota predominated in the library, demonstrating that it is not restricted to high-temperature environments, being also responsible for ammonium oxidation in the anaerobic lagoon. Euryarchaeota sequences were associated with the hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales). Quantitative PCR analysis revealed that the number of bacterial cells was at least three orders of magnitude higher than the number of archaeal cells in the SAL. The identified prokaryotic diversity was ecologically significant, particularly the archaeal community of hydrogenotrophic methanogens, which was responsible for methane production in the anaerobic lagoon. This study provided insight into the archaeal involvement in the overall oxidation of organic matter and the production of methane. Therefore, the treatment of swine waste in the sludge anaerobic lagoon could represent a potential inoculum for the start-up of municipal solid-waste digesters.     

454 pyrosequencing analysis on microbial diversity of an expanded granular sludge bed reactor treating high NaCl and nitrate concentration wastewater

The treatment of high-salinity, high-nitrate wastewater was investigated in a single expanded granular sludge bed reactor. Complete denitrification was achieved when nitrate concentration was as high as 6,000 mg N/L and the salinity of influent reached 11% NaCl at liquid up-flow velocity of 3.0 m/h, hydraulic retention time of, 24 h and the C/N molar ratio of 2.0. Furthermore, 454-pyrosequencing technology was used to analyze archaea bacterial diversity under high salinity and high nitrate conditions. Results showed that the total number of effective sequences was 5749 consisting of 5678 bacterial sequences and 71 archaea sequences after denoising and filtering out chimeras, which could be affiliated to 5 phylogenetic groups, including Proteobacteria, Firmicutes, Euryarchaeota, Crenarchaeota and unclassified phylum. Although Proteobacteria was the dominant microbial population, two archaea phylogenetic groups-Crenarchaeota and Euryarchaeota were observed in this study.     

Robustness of archaeal populations in anaerobic co-digestion of dairy and poultry wastes

The objective of this study is to investigate the responses of methanogen populations to poultry waste addition by comparing the archaeal microbial populations in continuous anaerobic digesters with or without the addition of poultry waste as a co-substrate. Poultry waste was characterized as an organic/nitrogen-rich substrate for anaerobic digestion. Supplementing dilute dairy waste with poultry waste for anaerobic co-digestion to increase organic loading rate by 50% resulted in improved biogas production. Elevated ammonia derived from poultry waste did not lead to process inhibition at the organic loadings tested, demonstrating the feasibility of the anaerobic co-digestion of dairy and poultry wastes for improved treatment efficiency. The stability of the anaerobic co-digestion process was linked to the robust archaeal microbial community, which remained mostly unchanged in community structure following increases in organic loading and ammonia levels. Surprisingly, Crenarchaeota archaeal populations, instead of the Euryarchaeota methanogens, dominated the archaeal communities in the anaerobic digesters. The ecological functions of these abundant non-methanogen archaeal populations in anaerobic digestion remain to be identified.     

Microbes mediating the sulfur cycle in the Atlantic Ocean and their link to chemolithoautotrophy

Only about 10%–30% of the organic matter produced in the epipelagic layers reaches the dark ocean. Under these limiting conditions, reduced inorganic substrates might be used as an energy source to fuel prokaryotic chemoautotrophic and/or mixotrophic activity. The aprA gene encodes the alpha subunit of the adenosine-5′-phosphosulfate (APS) reductase, present in sulfate-reducing (SRP) and sulfur-oxidizing prokaryotes (SOP). The sulfur-oxidizing pathway can be coupled to inorganic carbon fixation via the Calvin–Benson–Bassham cycle. The abundances of aprA and cbbM, encoding RuBisCO form II (the key CO₂ fixing enzyme), were determined over the entire water column along a latitudinal transect in the Atlantic from 64°N to 50°S covering six oceanic provinces. The abundance of aprA and cbbM genes significantly increased with depth reaching the highest abundances in meso- and upper bathypelagic layers. The contribution of cells containing these genes also increased from mesotrophic towards oligotrophic provinces, suggesting that under nutrient limiting conditions alternative energy sources are advantageous. However, the aprA/cbbM ratios indicated that only a fraction of the SOP is associated with inorganic carbon fixation. The aprA harbouring prokaryotic community was dominated by Pelagibacterales in surface and mesopelagic waters, while Candidatus Thioglobus, Chromatiales and the Deltaproteobacterium_SCGC dominated the bathypelagic realm. Noticeably, the contribution of the SRP to the prokaryotic community harbouring aprA gene was low, suggesting a major utilization of inorganic sulfur compounds either as an energy source (occasionally coupled with inorganic carbon fixation) or in biosynthesis pathways.     

Archaeal Diversity in Waters from Deep South African Gold Mines

A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of Euryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated.     

Protist Community Grazing on Prokaryotic Prey in Deep Ocean Water Masses

Oceanic protist grazing at mesopelagic and bathypelagic depths, and their subsequent effects on trophic links between eukaryotes and prokaryotes, are not well constrained. Recent studies show evidence of higher than expected grazing activity by protists down to mesopelagic depths. This study provides the first exploration of protist grazing in the bathypelagic North Atlantic Deep Water (NADW). Grazing was measured throughout the water column at three stations in the South Atlantic using fluorescently-labeled prey analogues. Grazing in the deep Antarctic Intermediate water (AAIW) and NADW at all three stations removed 3.79% ± 1.72% to 31.14% ± 8.24% of the standing prokaryote stock. These results imply that protist grazing may be a significant source of labile organic carbon at certain meso- and bathypelagic depths.     

Environmental shaping of sponge associated archaeal communities

Background

Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood.

Methodology/Principal Findings

We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA) gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA) generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum.

Conclusion/Significance

The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition of their associated archaeal communities, thereby improving their fitness in impacted environments.     

Genomic signatures of fifth autotrophic carbon assimilation pathway in bathypelagic Crenarchaeota

Marine Crenarchaeota, ubiquitous and abundant organisms in the oceans worldwide, remain metabolically uncharacterized, largely due to their low cultivability. Identification of candidate genes for bicarbonate fixation pathway in the Cenarchaeum symbiosum A was an initial step in understanding the physiology and ecology of marine Crenarchaeota. Recent cultivation and genome sequencing of obligate chemoautotrophic Nitrosopumilus maritimus SCM1 were a major breakthrough towards understanding of their functioning and provide a valuable model for experimental validation of genomic data. Here we present the identification of multiple key components of 3-hydroxipropionate/4-hydroxybutyrate cycle, the fifth pathway in carbon fixation, found in data sets of environmental sequences representing uncultivated superficial and bathypelagic Crenarchaeota from Sargasso sea (GOS data set) and KM3 (Mediterranean Sea) and ALOHA (Atlantic ocean) stations. These organisms are likely to use acetyl-CoA/propionyl-CoA carboxylase(s) as CO₂-fixing enzyme(s) to form succinyl-CoA, from which one molecule of acetyl-CoA is regenerated via 4-hydroxybutyrate cleavage and another acetyl-CoA to be the pathway product. The genetic distinctiveness and matching sympatric abundance imply that marine crenarchaeal genotypes from the three different geographic sites share similar ecophysiological properties, and therefore may represent fundamental units of marine ecosystem functioning. To couple results of sequence comparison with the dark ocean primary production, dissolved inorganic carbon fixation rates were measured at KM3 Station (3000 m depth, Eastern Mediterranean Sea), i.e. at the same site and depth used for metagenomic library construction.