Use of lectins to in situ visualize glycoconjugates of extracellular polymeric substances in acidophilic archaeal biofilms

Biofilm formation and the production of extracellular polymeric substances (EPS) by meso‐ and thermoacidophilic metal‐oxidizing archaea on relevant substrates have been studied to a limited extent. In order to investigate glycoconjugates, a major part of the EPS, during biofilm formation/bioleaching by archaea on pyrite, a screening with 75 commercially available lectins by fluorescence lectin‐binding analysis (FLBA) has been performed. Three representative archaeal species, Ferroplasma acidiphilum DSM 28986, Sulfolobus metallicus DSM 6482^T and a novel isolate Acidianus sp. DSM 29099 were used. In addition, Acidianus sp. DSM 29099 biofilms on elemental sulfur were studied. The results of FLBA indicate (i) 22 lectins bound to archaeal biofilms on pyrite and 21 lectins were binding to Acidianus sp. DSM 29099 biofilms on elemental sulfur; (ii) major binding patterns, e.g. tightly bound EPS and loosely bound EPS, were detected on both substrates; (iii) the three archaeal species produced various EPS glycoconjugates on pyrite surfaces. Additionally, the substratum induced different EPS glycoconjugates and biofilm structures of cells of Acidianus sp. DSM 29099. Our data provide new insights into interactions between acidophilic archaea on relevant surfaces and also indicate that FLBA is a valuable tool for in situ investigations on archaeal biofilms.     

Thermodynamic characterization of proton‐ionizable functional groups on the cell surfaces of ammonia‐oxidizing bacteria and archaea

The ammonia‐oxidizing archaeon Nitrosopumilus maritimus strain SCM1 (strain SCM1), a representative of the Thaumarchaeota archaeal phylum, can sustain high specific rates of ammonia oxidation at ammonia concentrations too low to sustain metabolism by ammonia‐oxidizing bacteria (AOB). One structural and biochemical difference between N. maritimus and AOB that might be related to the oligotrophic adaptation of strain SCM1 is the cell surface. A proteinaceous surface layer (S‐layer) comprises the outermost boundary of the strain SCM1 cell envelope, as opposed to the lipopolysaccharide coat of Gram‐negative AOB. In this work, we compared the surface reactivities of two archaea having an S‐layer (strain SCM1 and Sulfolobus acidocaldarius) with those of four representative AOB (Nitrosospira briensis, Nitrosomonas europaea, Nitrosolobus multiformis, and Nitrosococcus oceani) using potentiometric and calorimetric titrations to evaluate differences in proton‐ionizable surface sites. Strain SCM1 and S. acidocaldarius have a wider range of proton buffering (approximately pH 10–3.5) than the AOB (approximately pH 10–4), under the conditions investigated. Thermodynamic parameters describing proton‐ionizable sites (acidity constants, enthalpies, and entropies of protonation) are consistent with these archaea having proton‐ionizable amino acid side chains containing carboxyl, imidazole, thiol, hydroxyl, and amine functional groups. Phosphorous‐bearing acidic functional groups, which might also be present, could be masked by imidazole and thiol functional groups. Parameters for the AOB are consistent with surface structures containing anionic oxygen ligands (carboxyl‐ and phosphorous‐bearing acidic functional groups), thiols, and amines. In addition, our results showed that strain SCM1 has more reactive surface sites than the AOB and a high concentration of sites consistent with aspartic and/or glutamic acid. Because these alternative boundary layers mediate interaction with the local external environment, these data provide the basis for further comparisons of the thermodynamic behavior of surface reactivity toward essential nutrients.     

Dark aerobic sulfide oxidation by anoxygenic phototrophs in anoxic waters

Anoxygenic phototrophic sulfide oxidation by green and purple sulfur bacteria (PSB) plays a key role in sulfide removal from anoxic shallow sediments and stratified waters. Although some PSB can also oxidize sulfide with nitrate and oxygen, little is known about the prevalence of this chemolithotrophic lifestyle in the environment. In this study, we investigated the role of these phototrophs in light-independent sulfide removal in the chemocline of Lake Cadagno. Our temporally resolved, high-resolution chemical profiles indicated that dark sulfide oxidation was coupled to high oxygen consumption rates of ~9 μM O₂·h⁻¹. Single-cell analyses of lake water incubated with ¹³CO₂ in the dark revealed that Chromatium okenii was to a large extent responsible for aerobic sulfide oxidation and it accounted for up to 40% of total dark carbon fixation. The genome of Chr. okenii reconstructed from the Lake Cadagno metagenome confirms its capacity for microaerophilic growth and provides further insights into its metabolic capabilities. Moreover, our genomic and single-cell data indicated that other PSB grow microaerobically in these apparently anoxic waters. Altogether, our observations suggest that aerobic respiration may not only play an underappreciated role in anoxic environments but also that organisms typically considered strict anaerobes may be involved.     

Colonization of rice roots with methanogenic archaea controls photosynthesis‐derived methane emission

The methane emitted from rice fields originates to a large part (up to 60%) from plant photosynthesis and is formed on the rice roots by methanogenic archaea. To investigate to which extent root colonization controls methane (CH₄) emission, we pulse‐labeled rice microcosms with ¹³CO₂ to determine the rates of ¹³CH₄ emission exclusively derived from photosynthates. We also measured emission of total CH₄ (¹²⁺¹³CH₄), which was largely produced in the soil. The total abundances of archaea and methanogens on the roots and in the soil were analysed by quantitative polymerase chain reaction of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase respectively. The composition of archaeal and methanogenic communities was determined with terminal restriction fragment length polymorphism (T‐RFLP). During the vegetative growth stages, emission rates of ¹³CH₄ linearly increased with the abundance of methanogenic archaea on the roots and then decreased during the last plant growth stage. Rates of ¹³CH₄ emission and the abundance of methanogenic archaea were lower when the rice was grown in quartz‐vermiculite with only 10% rice soil. Rates of total CH₄ emission were not systematically related to the abundance of methanogenic archaea in soil plus roots. The composition of the archaeal communities was similar under all conditions; however, the analysis of mcrA genes indicated that the methanogens differed between the soil and root. Our results support the hypothesis that rates of photosynthesis‐driven CH₄ emission are limited by the abundance of methanogens on the roots.     

High diversity of ammonia‐oxidizing archaea in permanent and seasonal oxygen‐deficient waters of the eastern South Pacific

The community structure of putative aerobic ammonia‐oxidizing archaea (AOA) was explored in two oxygen‐deficient ecosystems of the eastern South Pacific: the oxygen minimum zone off Peru and northern Chile (11°S–20°S), where permanent suboxic and low‐ammonium conditions are found at intermediate depths, and the continental shelf off central Chile (36°S), where seasonal oxygen‐deficient and relatively high‐ammonium conditions develop in the water column, particularly during the upwelling season. The AOA community composition based on the ammonia monooxygenase subunit A (amoA) genes changed according to the oxygen concentration in the water column and the ecosystem studied, showing a higher diversity in the seasonal low‐oxygen waters. The majority of the archaeal amoA genotypes was affiliated to the uncultured clusters A (64%) and B (35%), with Cluster A AOA being mainly associated with higher oxygen and ammonium concentrations and Cluster B AOA with permanent oxygen‐ and ammonium‐poor waters. Q‐PCR assays revealed that AOA are an abundant community (up to 10⁵amoA copies ml^?1), while bacterial amoA genes from β proteobacteria were undetected. Our results thus suggest that a diverse uncultured AOA community, for which, therefore, we do not have any physiological information, to date, is an important component of the nitrifying community in oxygen‐deficient marine ecosystems, and particularly in rich coastal upwelling ones.     

A combined lipidomic and 16S rRNA gene amplicon sequencing approach reveals archaeal sources of intact polar lipids in the stratified Black Sea water column

Archaea are important players in marine biogeochemical cycles, and their membrane lipids are useful biomarkers in environmental and geobiological studies. However, many archaeal groups remain uncultured and their lipid composition unknown. Here, we aim to expand the knowledge on archaeal lipid biomarkers and determine the potential sources of those lipids in the water column of the euxinic Black Sea. The archaeal community was evaluated by 16S rRNA gene amplicon sequencing and by quantitative PCR. The archaeal intact polar lipids (IPLs) were investigated by ultra‐high‐pressure liquid chromatography coupled to high‐resolution mass spectrometry. Our study revealed both a complex archaeal community and large changes with water depth in the IPL assemblages. In the oxic/upper suboxic waters (<105 m), the archaeal community was dominated by marine group (MG) I Thaumarchaeota, coinciding with a higher relative abundance of hexose phosphohexose crenarchaeol, a known marker for Thaumarchaeota. In the suboxic waters (80–110 m), MGI Nitrosopumilus sp. dominated and produced predominantly monohexose glycerol dibiphytanyl glycerol tetraethers (GDGTs) and hydroxy‐GDGTs. Two clades of MGII Euryarchaeota were present in the oxic and upper suboxic zones in much lower abundances, preventing the detection of their specific IPLs. In the deep sulfidic waters (>110 m), archaea belonging to the DPANN Woesearchaeota, Bathyarchaeota, and ANME‐1b clades dominated. Correlation analyses suggest that the IPLs GDGT‐0, GDGT‐1, and GDGT‐2 with two phosphatidylglycerol (PG) head groups and archaeol with a PG, phosphatidylethanolamine, and phosphatidylserine head groups were produced by ANME‐1b archaea. Bathyarchaeota represented 55% of the archaea in the deeper part of the euxinic zone and likely produces archaeol with phospho‐dihexose and hexose‐glucuronic acid head groups.     

The Variation of Microbial Communities in a Depth Profile of Peat in the Gahai Lake Wetland Natural Conservation Area

The Gahai Lake wetland natural conservation area in northwestern China includes peatland that has been accumulating over hundreds of years and is seldom disturbed by industry. Bacteria and archaea in peat soil, which is a reservoir for carbon and water, may influence its ecological function. The objective of this study was to obtain a clearer understanding of peat microbial ecology and its relationship to the environmental conditions of this area. Hence, the microbial community of the peatland ecosystem was investigated by sequencing bacterial and archaeal DNA extracted from samples collected at different peat depths. Results showed that in all samples the dominant bacterial phyla were Proteobacteria (relative abundance 0.39 ± 0.12) and Chloroflexi (0.16 ± 0.09), while the dominant archaeal phyla were Miscellaneous Crenarchaeotic Group (MCG) (0.62 ± 0.21) and Euryarchaeota (0.27 ± 0.16). The diversity and microbial community structure at deeper depths (90 and 120 cm below the peat surface) significantly differ from that at shallower depths (10, 30 and 50 cm deep). In contrast to the shallow layers, the deeper layers became more abundant in the bacterial phyla Chloroflexi, Bacteroidetes, Atribacteria, Aminicenantes, Chlorobi, TA06, Caldiserica and Spirochaetae; and in the archaeal phyla MCG and Miscellaneous Euryarchaeotic Group (MEG). This study revealed a significant shift in microbial community in peat between 50 cm and 90 cm deep, as probably influenced by the oxygen supply at different depths. Furthermore, new insights into the microbial taxa were obtained, thus providing a baseline for future studies of this peat ecosystem.     

Record of archaeal activity at the serpentinite‐hosted Lost City Hydrothermal Field

Samples of young, outer surfaces of brucite–carbonate deposits from the ultramafic‐hosted Lost City hydrothermal field were analyzed for DNA and lipid biomarker distributions and for carbon and hydrogen stable isotope compositions of the lipids. Methane‐cycling archaeal communities, notably the Lost City Methanosarcinales (LCMS) phylotype, are specifically addressed. Lost City is unlike all other hydrothermal systems known to date and is characterized by metal‐ and CO₂‐poor, high pH fluids with high H₂ and CH₄ contents resulting from serpentinization processes at depth. The archaeal fraction of the microbial community varies widely within the Lost City chimneys, from 1–81% and covaries with concentrations of hydrogen within the fluids. Archaeal lipids include isoprenoid glycerol di‐ and tetraethers and C₂₅ and C₃₀ isoprenoid hydrocarbons (pentamethylicosane derivatives – PMIs – and squalenoids). In particular, unsaturated PMIs and squalenoids, attributed to the LCMS archaea, were identified for the first time in the carbonate deposits at Lost City and probably record processes exclusively occurring at the surface of the chimneys. The carbon isotope compositions of PMIs and squalenoids are remarkably heterogeneous across samples and show highly ¹³C‐enriched signatures reaching δ¹³C values of up to +24.6‰. Unlike other environments in which similar structural and isotopic lipid heterogeneity has been observed and attributed to diversity in the archaeal assemblage, the lipids here appear to be synthesized solely by the LCMS. Some of the variations in lipid isotope signatures may, in part, be due to unusual isotopic fractionation during biosynthesis under extreme conditions. However, we argue that the diversity in archaeal abundances, lipid structure and carbon isotope composition rather reflects the ability of the LCMS archaeal biofilms to adapt to chemical gradients in the hydrothermal chimneys and possibly to perform either methanotrophy or methanogenesis using dissolved inorganic carbon, methane or formate as a function of the prevailing environmental conditions.     

Bacterial and Archaeal Community Structure in the Surface Diatom Sediments of Deep Freshwater Lake Baikal (Eastern Siberia)

Diatom sediment records of large lakes can be used to decipher the history of ancient phytoplankton. The upper layer of the sediment is an important area of remineralization of the sedimenting phytoplankton biomass. It hosts a bacterial community different from those of both the water column and deeper sediment layers. In this work, we analyzed the structure and diversity of the communities of Bacteria and Archaea in the surface sediment core containing valves of diatoms, the major producers in Lake Baikal. Pyrosequencing of the bacterial V3–V4 region of the 16 S ribosomal RNA (rRNA) and archaeal V1–V3 16 S rRNA gene regions yielded 29,168 and 36,997 reads, respectively. In total, we have identified 33 bacterial phyla; uncultured Actinobacteria were the most abundant in the upper layers, while lower sediment was dominated by Firmicutes and Alphaproteobacteria. The composition of the archaeal community changed with depth, but was generally dominated by Crenarchaeota from the classes Marine Group I and Miscellaneous Crenarchaeotic Group, as well as Euryarchaeota from the class Thermoplasmata. These dominant bacterial and archaeal taxa are presumed to participate in the destruction of buried organic matter, which eventually leads to degradation of the diatom valves.     

Epsilonproteobacteria Represent the Major Portion of Chemoautotrophic Bacteria in Sulfidic Waters of Pelagic Redoxclines of the Baltic and Black Seas

Recent studies have indicated that chemoautotrophic Epsilonproteobacteria might play an important role, especially as anaerobic or microaerophilic dark CO₂-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO₂-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of Epsilonproteobacteria to dark CO₂ fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [¹⁴C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 × 10^{5 14}CO₂-fixing prokaryotic cells ml⁻¹ were enumerated in the redoxcline of the central Baltic Sea and up to 9 × 10^{4 14}CO₂-fixing cells ml⁻¹ were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance. ¹⁴CO₂-incorporating cells belonged exclusively to the domain Bacteria. Among these, members of the Epsilonproteobacteria were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the Sulfurimonas subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO₂-fixing group. In conclusion, Epsilonproteobacteria were found to be mainly responsible for chemoautotrophic activity in the dark CO₂ fixation maxima of the Black Sea and central Baltic Sea redoxclines. These Epsilonproteobacteria might be relevant in similar habitats of the world's oceans, where high dark CO₂ fixation rates have been measured.     

Abundance and community composition of ammonia-oxidizing archaea and bacteria in two different zones of Lake Taihu

The abundance and community composition of ammonia-oxidizing archaea and ammonia-oxidizing bacteria in the surface sediments of 2 different zones (Meiliang Bay and Eastern Lake Taihu) of Lake Taihu were investigated using real-time quantitative polymerase chain reaction and clone libraries. The amoA gene copy numbers in the surface sediment of Meiliang Bay ranged from 4.91?× 10(5) to 8.65?× 10(6) copies/g dry sediment for the archaeal amoA gene and from 3.74?× 10(4) to 3.86?× 10(5) copies/g dry sediment for the bacterial amoA gene, which were significantly higher than those of Eastern Lake Taihu (P?< 0.05). Concentrations of ammonia (NH(4)(+)), total nitrogen, organic matter, and pH of the sediments exhibited significantly negative correlations with the abundance of ammonia-oxidizing archaea or ammonia-oxidizing bacteria (P?< 0.05 or P?< 0.01, respectively). The potential nitrification rates show remarkable correlations with the copy numbers of the archaeal amoA gene. Diversity of the archaeal amoA gene in Eastern Lake Taihu was significantly higher than that of Meiliang Bay, whereas the bacterial amoA gene diversity was comparable for the 2 lake zones. The data obtained in this study would be useful to elucidate the role of ammonia-oxidizing archaea and ammonia-oxidizing bacteria in the nitrogen cycle of freshwater ecosystems.     

Plant growth-promoting archaea trigger induced systemic resistance in Arabidopsis thaliana against Pectobacterium carotovorum and Pseudomonas syringae

Archaea have inhabited the earth for a long period of time and are ubiquitously distributed in diverse environments. However, few studies have focused on the interactions of archaea with other organisms, including eukaryotes such as plants, since it is difficult to cultivate sufficient numbers of archaeal cells for analysis. In this study, we investigated the interaction between soil archaea and Arabidopsis thaliana. We demonstrate for the first time that soil archaea promote plant growth and trigger induced systemic resistance (ISR) against the necrotrophic bacterium Pectobacterium carotovorum subsp. carotovorum SCC1 and biotrophic bacterium Pseudomonas syringae pv. tomato DC3000. Ammonia-oxidizing archaeon Nitrosocosmicus oleophilus MY3 cells clearly colonized the root surface of Arabidopsis plants, and increased resistance against both pathogenic species via the salicylic acid-independent signalling pathway. This mechanism of bacterial resistance resembles that underlying soil bacteria- and fungi-mediated ISR signalling. Additionally, volatile emissions from N. oleophilus MY3 were identified as major archaeal determinants that elicit ISR. Our results lay a foundation for archaea–plant interactions as a new field of research.     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia‐oxidizing archaea (AOA) rather than marine ammonia‐oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium‐oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory‐scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C₁₈ anammox [3]‐ladderane fatty acid was highly abundant in the oxygen‐limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.     

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.     

AMMONIUM STIMULATION OF DARK CARBON FIXATION AS AN INDICATOR OF NITROGEN DEFICIENCY IN PHYTOPLANKTON: POTENTIAL ERRORS CAUSED BY AMMONIUM-OXIDIZING BACTERIA1

Stimulation of dark fixation of carbon by NH₄⁺ is often used as an indicator of phytoplankton N deficiency. This assay is based on the influence of available NH₄⁺ on anaplerotic CO₂ fixation by algae. However, carbon fixation by chemoautotrophic NH₄⁺-oxidizing bacteria may also be stimulated by NH₄⁺ enrichment, a process that can mask the algal response in natural communities. NH₄⁺ addition enhanced dark carbon fixation up to 300%, relative to unamended controls, in organisms collected on a 0.7-μm retention filter in oligotrophic Flathead Lake, Montana, but the effect was not detectable in the presence of nitrapyrin, an inhibitor of NH₄⁺-oxidizing bacteria. Dark carbon fixation was enhanced with addition of NH₄⁺ in organisms retained on 2-μm filters (which should allow passage of most bacteria). NH₄⁺ stimulated dark carbon fixation in N-deficient axenic cultures of Chlamydomonas reinhardtii Dang but not in N-replete cultures in both the presence and absence of nitrapyrin. Application of nitrapyrin or size fractionation treatments, to separate the processes of dark carbon fixation by nitrifiers and phytoplankton, may improve the efficacy of assays using NH₄⁺ stimulation of dark carbon fixation to specifically indicate N deficiency in natural algal communities.