Pathways of Carbon Assimilation and Ammonia Oxidation Suggested by Environmental Genomic Analyses of Marine Crenarchaeota

Marine Crenarchaeota represent an abundant component of oceanic microbiota with potential to significantly influence biogeochemical cycling in marine ecosystems. Prior studies using specific archaeal lipid biomarkers and isotopic analyses indicated that planktonic Crenarchaeota have the capacity for autotrophic growth, and more recent cultivation studies support an ammonia-based chemolithoautotrophic energy metabolism. We report here analysis of fosmid sequences derived from the uncultivated marine crenarchaeote, Cenarchaeum symbiosum, focused on the reconstruction of carbon and energy metabolism. Genes predicted to encode multiple components of a modified 3-hydroxypropionate cycle of autotrophic carbon assimilation were identified, consistent with utilization of carbon dioxide as a carbon source. Additionally, genes predicted to encode a near complete oxidative tricarboxylic acid cycle were also identified, consistent with the consumption of organic carbon and in the production of intermediates for amino acid and cofactor biosynthesis. Therefore, C. symbiosum has the potential to function either as a strict autotroph, or as a mixotroph utilizing both carbon dioxide and organic material as carbon sources. From the standpoint of energy metabolism, genes predicted to encode ammonia monooxygenase subunits, ammonia permease, urease, and urea transporters were identified, consistent with the use of reduced nitrogen compounds as energy sources fueling autotrophic metabolism. Homologues of these genes, recovered from ocean waters worldwide, demonstrate the conservation and ubiquity of crenarchaeal pathways for carbon assimilation and ammonia oxidation. These findings further substantiate the likely global metabolic importance of Crenarchaeota with respect to key steps in the biogeochemical transformation of carbon and nitrogen in marine ecosystems.     

深渊沉积物中盐单胞菌(Halomonas sp.)NyZ771菌株的分离培养及其降解芳香酸的研究

【背景】海洋是地球上最大的碳库,也是地球生物最大的栖息地。在这个庞大的生态系统中拥有多种多样的微生物,它们在全球碳循环中扮演了重要的角色。海斗深渊(海平面6 000 m以下的海域)由于高静水压和表层沉积汇集了大量有机质,形成了包含丰富生物资源的特殊生境。【目的】从马里亚纳海沟海斗深渊沉积物样品中分离培养能够以芳香酸为唯一碳源和能源生长的微生物,并研究其降解特性。【方法】通过模拟原位高压环境富集培养和常压条件下芳香酸选择性分离培养获得深渊来源的纯培养细菌,并根据形态学观察和16S rRNA基因序列系统发育分析进行种属鉴定,利用不同芳香酸进行培养和生物转化,通过HPLC和LC/MS鉴定芳香酸代谢中间产物。【结果】从马里亚纳海沟6 300 m沉积物样本中分离获得了一株盐单胞菌(Halomonas sp.)NyZ771。该菌株能够利用苯甲酸和4-羟基苯甲酸作为唯一碳源生长。其代谢4-羟基苯甲酸的中间产物鉴定为原儿茶酸。【结论】从深渊沉积物样本分离得到一株能降解苯甲酸和4-羟基苯甲酸的盐单胞菌NyZ771,丰富了深渊来源的微生物资源,为今后研究深渊中微生物的芳香酸降解及海洋微生物驱动的碳循环提供了一定的理论基础。     

Single Cells within the Puerto Rico Trench Suggest Hadal Adaptation of Microbial Lineages

Hadal ecosystems are found at a depth of 6,000 m below sea level and below, occupying less than 1% of the total area of the ocean. The microbial communities and metabolic potential in these ecosystems are largely uncharacterized. Here, we present four single amplified genomes (SAGs) obtained from 8,219 m below the sea surface within the hadal ecosystem of the Puerto Rico Trench (PRT). These SAGs are derived from members of deep-sea clades, including the Thaumarchaeota and SAR11 clade, and two are related to previously isolated piezophilic (high-pressure-adapted) microorganisms. In order to identify genes that might play a role in adaptation to deep-sea environments, comparative analyses were performed with genomes from closely related shallow-water microbes. The archaeal SAG possesses genes associated with mixotrophy, including lipoylation and the glycine cleavage pathway. The SAR11 SAG encodes glycolytic enzymes previously reported to be missing from this abundant and cosmopolitan group. The other SAGs, which are related to piezophilic isolates, possess genes that may supplement energy demands through the oxidation of hydrogen or the reduction of nitrous oxide. We found evidence for potential trench-specific gene distributions, as several SAG genes were observed only in a PRT metagenome and not in shallower deep-sea metagenomes. These results illustrate new ecotype features that might perform important roles in the adaptation of microorganisms to life in hadal environments.     

Illuminating microbial species-specific effects on organic matter remineralization in marine sediments

Marine microorganisms play a fundamental role in the global carbon cycle by mediating the sequestration of organic matter in ocean waters and sediments. A better understanding of how biological factors, such as microbial community composition, influence the lability and fate of organic matter is needed. Here, we explored the extent to which organic matter remineralization is influenced by species-specific metabolic capabilities. We carried out aerobic time-series incubations of Guaymas Basin sediments to quantify the dynamics of carbon utilization by two different heterotrophic marine isolates (Vibrio splendidus 1A01; Pseudoalteromonas sp. 3D05). Continuous measurement of respiratory CO₂ production and its carbon isotopic compositions (¹³C and ¹⁴C) shows species-specific differences in the rate, quantity and type of organic matter remineralized. Each species was incubated with hydrothermally-influenced versus unimpacted sediments, resulting in a ~2-fold difference in respiratory CO₂ yield across the experiments. Genomic analysis indicated that the observed carbon utilization patterns may be attributed in part to the number of gene copies encoding for extracellular hydrolytic enzymes. Our results demonstrate that the lability and remineralization of organic matter in marine environments is not only a function of chemical composition and/or environmental conditions, but also a function of the microorganisms that are present and active.     

Isolation of an aerobic sulfur oxidizer from the SUP05/Arctic96BD-19 clade

Bacteria from the uncultured SUP05/Arctic96BD-19 clade of gamma proteobacterial sulfur oxidizers (GSOs) have the genetic potential to oxidize reduced sulfur and fix carbon in the tissues of clams and mussels, in oxygen minimum zones and throughout the deep ocean (>200 m). Here, we report isolation of the first cultured representative from this GSO clade. Closely related cultures were obtained from surface waters in Puget Sound and from the deep chlorophyll maximum in the North Pacific gyre. Pure cultures grow aerobically on natural seawater media, oxidize sulfur, and reach higher final cell densities when glucose and thiosulfate are added to the media. This suggests that aerobic sulfur oxidation enhances organic carbon utilization in the oceans. The first isolate from the SUP05/Arctic96BD-19 clade was given the provisional taxonomic assignment ‘Candidatus: Thioglobus singularis'', alluding to the clade''s known role in sulfur oxidation and the isolate''s planktonic lifestyle.     

Pathways of Carbon Assimilation and Ammonia Oxidation Suggested by Environmental Genomic Analyses of Marine Crenarchaeota

Marine Crenarchaeota represent an abundant component of oceanic microbiota with potential to significantly influence biogeochemical cycling in marine ecosystems. Prior studies using specific archaeal lipid biomarkers and isotopic analyses indicated that planktonic Crenarchaeota have the capacity for autotrophic growth, and more recent cultivation studies support an ammonia-based chemolithoautotrophic energy metabolism. We report here analysis of fosmid sequences derived from the uncultivated marine crenarchaeote, Cenarchaeum symbiosum, focused on the reconstruction of carbon and energy metabolism. Genes predicted to encode multiple components of a modified 3-hydroxypropionate cycle of autotrophic carbon assimilation were identified, consistent with utilization of carbon dioxide as a carbon source. Additionally, genes predicted to encode a near complete oxidative tricarboxylic acid cycle were also identified, consistent with the consumption of organic carbon and in the production of intermediates for amino acid and cofactor biosynthesis. Therefore, C. symbiosum has the potential to function either as a strict autotroph, or as a mixotroph utilizing both carbon dioxide and organic material as carbon sources. From the standpoint of energy metabolism, genes predicted to encode ammonia monooxygenase subunits, ammonia permease, urease, and urea transporters were identified, consistent with the use of reduced nitrogen compounds as energy sources fueling autotrophic metabolism. Homologues of these genes, recovered from ocean waters worldwide, demonstrate the conservation and ubiquity of crenarchaeal pathways for carbon assimilation and ammonia oxidation. These findings further substantiate the likely global metabolic importance of Crenarchaeota with respect to key steps in the biogeochemical transformation of carbon and nitrogen in marine ecosystems.     

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.     

Co-occurring Synechococcus ecotypes occupy four major oceanic regimes defined by temperature,macronutrients and iron

Marine picocyanobacteria, comprised of the genera Synechococcus and Prochlorococcus, are the most abundant and widespread primary producers in the ocean. More than 20 genetically distinct clades of marine Synechococcus have been identified, but their physiology and biogeography are not as thoroughly characterized as those of Prochlorococcus. Using clade-specific qPCR primers, we measured the abundance of 10 Synechococcus clades at 92 locations in surface waters of the Atlantic and Pacific Oceans. We found that Synechococcus partition the ocean into four distinct regimes distinguished by temperature, macronutrients and iron availability. Clades I and IV were prevalent in colder, mesotrophic waters; clades II, III and X dominated in the warm, oligotrophic open ocean; clades CRD1 and CRD2 were restricted to sites with low iron availability; and clades XV and XVI were only found in transitional waters at the edges of the other biomes. Overall, clade II was the most ubiquitous clade investigated and was the dominant clade in the largest biome, the oligotrophic open ocean. Co-occurring clades that occupy the same regime belong to distinct evolutionary lineages within Synechococcus, indicating that multiple ecotypes have evolved independently to occupy similar niches and represent examples of parallel evolution. We speculate that parallel evolution of ecotypes may be a common feature of diverse marine microbial communities that contributes to functional redundancy and the potential for resiliency.     

Ammonia-oxidizing archaea release a suite of organic compounds potentially fueling prokaryotic heterotrophy in the ocean

Ammonia-oxidizing archaea (AOA) constitute a considerable fraction of microbial biomass in the global ocean, comprising 20%–40% of the ocean's prokaryotic plankton. However, it remains enigmatic to what extent these chemolithoautotrophic archaea release dissolved organic carbon (DOC). A combination of targeted and untargeted metabolomics was used to characterize the exometabolomes of three model AOA strains of the Nitrosopumilus genus. Our results indicate that marine AOA exude a suite of organic compounds with potentially varying reactivities, dominated by nitrogen-containing compounds. A significant fraction of the released dissolved organic matter (DOM) consists of labile compounds, which typically limit prokaryotic heterotrophic activity in open ocean waters, including amino acids, thymidine and B vitamins. Amino acid release rates corresponded with ammonia oxidation activity and the three Nitrosopumilus strains predominantly released hydrophobic amino acids, potentially as a result of passive diffusion. Despite the low contribution of DOC released by AOA (~0.08%–1.05%) to the heterotrophic prokaryotic carbon demand, the release of physiologically relevant metabolites could be crucial for microbes that are auxotrophic for some of these compounds, including members of the globally abundant and ubiquitous SAR11 clade.     

Ecological succession among iron-oxidizing bacteria

Despite over 125 years of study, the factors that dictate species dominance in neutrophilic iron-oxidizing bacterial (FeOB) communities remain unknown. In a freshwater wetland, we documented a clear ecological succession coupled with niche separation between the helical stalk-forming Gallionellales (for example, Gallionella ferruginea) and tubular sheath-forming Leptothrix ochracea. Changes in the iron-seep community were documented using microscopy and cultivation-independent methods. Quantification of Fe-oxyhydroxide morphotypes by light microscopy was coupled with species-specific fluorescent in situ hybridization (FISH) probes using a protocol that minimized background fluorescence caused by the Fe-oxyhydroxides. Together with scanning electron microscopy, these techniques all indicated that Gallionellales dominated during early spring, with L. ochracea becoming more abundant for the remainder of the year. Analysis of tagged pyrosequencing reads of the small subunit ribosomal RNA gene (SSU rRNA) collected during seasonal progression supported a clear Gallionellales to L. ochracea transition, and community structure grouped according to observed dominant FeOB forms. Axis of redundancy analysis of physicochemical parameters collected from iron mats during the season, plotted with FeOB abundance, corroborated several field and microscopy-based observations and uncovered several unanticipated relationships. On the basis of these relationships, we conclude that the ecological niche of the stalk-forming Gallionellales is in waters with low organic carbon and steep redoxclines, and the sheath-forming L. ochracea is abundant in waters that contain high concentrations of complex organic carbon, high Fe and Mn content and gentle redoxclines. Finally, these findings identify a largely unexplored relationship between FeOB and organic carbon.     

Candidatus Abditibacter,a novel genus within the Cryomorphaceae,thriving in the North Sea

Coastal phytoplankton blooms are frequently followed by successive blooms of heterotrophic bacterial clades. The class Flavobacteriia within the Bacteroidetes has been shown to play an important role in the degradation of high molecular weight substrates that become available in the later stages of such blooms. One of the flavobacterial clades repeatedly observed over the course of several years during phytoplankton blooms off the coast of Helgoland, North Sea, is Vis6. This genus-level clade belongs to the family Cryomorphaceae and has been resistant to cultivation to date. Based on metagenome assembled genomes, comparative 16S rRNA gene sequence analyses and fluorescence in situ hybridization, we here propose a novel candidate genus Abditibacter, comprising three novel species Candidatus Abditibacter vernus, Candidatus Abditibacter forsetii and Candidatus Abditibacter autumni. While the small genomes of the three novel photoheterotrophic species encode highly similar gene repertoires, including genes for degradation of proteins and algal storage polysaccharides such as laminarin, two of them – Ca. A. vernus and Ca. A. forsetii – seem to have a preference for spring blooms, while Ca. A. autumni almost exclusively occurs in late summer and autumn.     

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.     

Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean

Methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. We report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d⁻¹ (∼10 nmol l⁻¹d⁻¹). On average, coastal upwelling waters were 11 times greater than open ocean northern temperate (NT) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (EU) waters; suggesting that all upwelling waters upon reaching the surface (⩽20 m), contain a microbial population that uses a relatively high amount of carbon (0.3–10 nmol l⁻¹d⁻¹), derived from methanol, to support their growth. In open ocean Atlantic regions, microbial uptake of methanol into biomass was significantly lower, ranging between 0.04–0.68 nmol l⁻¹d⁻¹. Microbes in the Mauritanian coastal upwelling used up to 57% of the total methanol for assimilation of the carbon into cells, compared with an average of 12% in the EU, and 1% in NT and gyre waters. Several methylotrophic bacterial species were identified from open ocean Atlantic waters using PCR amplification of mxaF encoding methanol dehydrogenase, the key enzyme in bacterial methanol oxidation. These included Methylophaga sp., Burkholderiales sp., Methylococcaceae sp., Ancylobacter aquaticus, Paracoccus denitrificans, Methylophilus methylotrophus, Methylobacterium oryzae, Hyphomicrobium sp. and Methylosulfonomonas methylovora. Statistically significant correlations for upwelling waters between methanol uptake into cells and both chlorophyll a concentrations and methanol oxidation rates suggest that remotely sensed chlorophyll a images, in these productive areas, could be used to derive total methanol biological loss rates, a useful tool for atmospheric and marine climatically active gas modellers, and air–sea exchange scientists.     

Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes ‘Gramella forsetii' KT0803

Members of the phylum Bacteroidetes are abundant in many marine ecosystems and are known to have a pivotal role in the mineralization of complex organic substrates such as polysaccharides and proteins. We studied the decomposition of the algal glycans laminarin and alginate by ‘Gramella forsetii'' KT0803, a bacteroidetal isolate from North Sea surface waters. A combined application of isotope labeling, subcellular protein fractionation and quantitative proteomics revealed two large polysaccharide utilization loci (PULs) that were specifically induced, one by alginate and the other by laminarin. These regulons comprised genes of surface-exposed proteins such as oligomer transporters, substrate-binding proteins, carbohydrate-active enzymes and hypothetical proteins. Besides, several glycan-specific TonB-dependent receptors and SusD-like substrate-binding proteins were expressed also in the absence of polysaccharide substrates, suggesting an anticipatory sensing function. Genes for the utilization of the beta-1,3-glucan laminarin were found to be co-regulated with genes for glucose and alpha-1,4-glucan utilization, which was not the case for the non-glucan alginate. Strong syntenies of the PULs of ‘G. forsetii'' with similar loci in other Bacteroidetes indicate that the specific response mechanisms of ‘G. forsetii'' to changes in polysaccharide availability likely apply to other Bacteroidetes. Our results can thus contribute to an improved understanding of the ecological niches of marine Bacteroidetes and their roles in the polysaccharide decomposition part of carbon cycling in marine ecosystems.     

Processes of organic carbon in mangrove ecosystems

In addition to carbon accumulation in plants, processes of organic carbon in mangrove ecosystems include origins of sediment organic carbon, carbon fluxes between mangroves and their adjacent systems (coastal waters and atmosphere), and cycling processes. Sediment organic carbon originates from suspending solids in coastal waters, mangrove plants and benthic algae. In mangroves with low organic carbon content in sediments, tidal seawater is the main origin of sediment organic carbon, while in mangroves with high sediment organic carbon contents, sediment organic carbon mainly originates from mangrove plants. Due to tidal flush, there is large material exchange between mangrove ecosystems and their adjacent coastal waters. In China, exports of organic carbon in litter falls and dissolved organic carbon from mangroves to their adjacent coastal waters have not been documented. Processes of mangrove litter falls, including production, decomposition, export and animal consumption, determine linkages among organic carbon among mangrove plants, secondary production and coastal ocean. Consumers especially benthic animals may influence organic carbon in mangrove ecosystems, because (1) their consumption rates are high, and their selective feeding on some food sources will change the relative quantities of export, bury and mineralization of organic carbon from different origins; (2) their consumption is much more than assimilation, resulting in the changes in sizes, forms and qualities of non-assimilated organic matters, and then the changes in availability of export, consumption or mineralization of organic carbon. Respiration and sulfate reduction are important mineralization processes of organic carbon in mangrove sediments. Mineralization rates of organic carbon in mangrove sediments are influenced by quantities, activities and particle sizes of organic matters, and other factors such as forest ages, root activities and animal burrowing activities. Researches on processes of mangrove organic carbon should be based on open systems, and ecological processes of organic carbon should be coupled with vegetation restoration.     

Processes of organic carbon in mangrove ecosystems

In addition to carbon accumulation in plants, processes of organic carbon in mangrove ecosystems include origins of sediment organic carbon, carbon fluxes between mangroves and their adjacent systems (coastal waters and atmosphere), and cycling processes. Sediment organic carbon originates from suspending solids in coastal waters, mangrove plants and benthic algae. In mangroves with low organic carbon content in sediments, tidal seawater is the main origin of sediment organic carbon, while in mangroves with high sediment organic carbon contents, sediment organic carbon mainly originates from mangrove plants. Due to tidal flush, there is large material exchange between mangrove ecosystems and their adjacent coastal waters. In China, exports of organic carbon in litter falls and dissolved organic carbon from mangroves to their adjacent coastal waters have not been documented. Processes of mangrove litter falls, including production, decomposition, export and animal consumption, determine linkages among organic carbon among mangrove plants, secondary production and coastal ocean. Consumers especially benthic animals may influence organic carbon in mangrove ecosystems, because (1) their consumption rates are high, and their selective feeding on some food sources will change the relative quantities of export, bury and mineralization of organic carbon from different origins; (2) their consumption is much more than assimilation, resulting in the changes in sizes, forms and qualities of non-assimilated organic matters, and then the changes in availability of export, consumption or mineralization of organic carbon. Respiration and sulfate reduction are important mineralization processes of organic carbon in mangrove sediments. Mineralization rates of organic carbon in mangrove sediments are influenced by quantities, activities and particle sizes of organic matters, and other factors such as forest ages, root activities and animal burrowing activities. Researches on processes of mangrove organic carbon should be based on open systems, and ecological processes of organic carbon should be coupled with vegetation restoration.     

Microbial community composition and hydrochemistry of underexplored geothermal waters in Croatia

In Croatia, a variety of geothermal springs with a wide temperature range and varied hydrochemical conditions exist, and they may harbor different niches for the distribution of microbial communities. In this study, 19 different sites, mainly located in central and eastern Croatia, were selected for primary characterization of spring hydrochemistry and microbial community composition. Using 16S rRNA gene amplicon sequencing, it was found that the bacterial communities that dominated most geothermal waters were related to Proteobacteria and Campylobacteria, while most archaeal sequences were related to Crenarchaeota. At the genus level, the prokaryotic community was highly site-specific and was often dominated by a single genus, including sites dominated by Hydrogenophilus, Sulfuricurvum, Sulfurovum, Thiofaba and Nitrospira, while the most abundant archaeal genera were affiliated to the ammonia-oxidizing archaea, Candidatus Nitrosotenuis and Candidatus Nitrososphaera. Whereas the microbial communities were overall highly location-specific, temperature, pH, ammonia, nitrate, total nitrogen, sulfate and hydrogen sulfide, as well as dissolved organic and inorganic carbon, were the abiotic factors that significantly affected microbial community composition. Furthermore, an aquifer-type effect was observed in the community composition, but there was no pronounced seasonal variability for geothermal spring communities (i.e. the community structure was mainly stable during the three seasons sampled). These results surprisingly pointed to stable and geographically unique microbial communities that were adapted to different geothermal water environments throughout Croatia. Knowing which microbial communities are present in these extreme habitats is essential for future research. They will allow us to explore further the microbial metabolisms prevailing at these geothermal sites that have high potential for biotechnological uses, as well as the establishment of the links between microbial community structure and the physicochemical environment of geothermal waters.