Ammonia oxidation is the first step of nitrification carried out by ammonia-oxidizing Archaea (AOA) and Bacteria (AOB). Lake Superior and Erie are part of the Great Lakes system differing in trophic ...status with Lake Superior being oligotrophic and Lake Erie meso- to eutrophic. Sediment samples were collected from both lakes and used to characterize abundance and diversity of AOA and AOB based on the ammonia monooxygenase (amoA) gene. Diversity was accessed by a pyro-sequencing approach and the obtained sequences were used to determine the phylogeny and alpha and beta diversity of the AOA and AOB populations. In Lake Erie copy numbers of bacterial amoA genes were in the same order of magnitude or even higher than the copy numbers of the archaeal amoA genes, while in Lake Superior up to 4 orders of magnitude more archaeal than bacterial amoA copies were detected. The AOB detected in the samples from Lake Erie belonged to AOB that are frequently detected in freshwater. Differences were detected between the phylogenetic affiliations of the AOA from the two lakes. Most sequences detected in Lake Erie clustered in the Nitrososphaera cluster (Thaumarchaeal soil group I.1b) where as most of the sequences in Lake Superior were found in the Nitrosopumilus cluster (Thaumarchaeal marine group I.1a) and the Nitrosotalea cluster. Pearson correlations and canonical correspondence analysis (CCA) showed that the differences in abundance and diversity of AOA are very likely related to the sampling location and thereby to the different trophic states of the lakes.
The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their ...natural environment. An inoculum is sandwiched between semipermeable (0.03-μm-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and BACTEROIDETES: However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and ACIDOBACTERIA: Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.
Ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas perform the first step in nitrification, ammonia oxidation, and are found in diverse aquatic and terrestrial environments. Nitrosomonas ...AOB were grouped into six defined clusters, which correlate with physiological characteristics that contribute to adaptations to a variety of abiotic environmental factors. A fundamental physiological trait differentiating Nitrosomonas AOB is the adaptation to either low (cluster 6a) or high (cluster 7) ammonium concentrations. Here, we present physiological growth studies and genome analysis of Nitrosomonas cluster 6a and 7 AOB. Cluster 6a AOB displayed maximum growth rates at ≤ 1 mM ammonium, while cluster 7 AOB had maximum growth rates at ≥ 5 mM ammonium. In addition, cluster 7 AOB were more tolerant of high initial ammonium and nitrite concentrations than cluster 6a AOB. Cluster 6a AOB were completely inhibited by an initial nitrite concentration of 5 mM. Genomic comparisons were used to link genomic traits to observed physiological adaptations. Cluster 7 AOB encode a suite of genes related to nitrogen oxide detoxification and multiple terminal oxidases, which are absent in cluster 6a AOB. Cluster 6a AOB possess two distinct forms of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for hydrogen or urea utilization. Several, but not all, cluster 6a AOB can utilize urea as a source of ammonium. Hence, although Nitrosomonas cluster 6a and 7 AOB have the capacity to fulfill the same functional role in microbial communities, i.e., ammonia oxidation, differentiating species-specific and cluster-conserved adaptations is crucial in understanding how AOB community succession can affect overall ecosystem function.
Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a ...common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.
Microorganisms in the environment do not exist as the often-studied pure cultures but as members of complex microbial communities. Characterizing the interactions within microbial communities is ...essential to understand their function in both natural and engineered environments. In this study, we investigated how the presence of a nitrite-oxidizing bacterium (NOB) and heterotrophic bacteria affect the growth and proteome of the chemolithoautotrophic ammonia-oxidizing bacterium (AOB) Nitrosomonas sp. strain Is79. We investigated Nitrosomonas sp. Is79 in co-culture with Nitrobacter winogradskyi, in co-cultures with selected heterotrophic bacteria, and as a member of the nitrifying enrichment culture G5-7. In batch culture, N. winogradskyi and heterotrophic bacteria had positive effects on the growth of Nitrosomonas sp. Is79. An isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was used to investigate the effect of N. winogradskyi and the co-cultured heterotrophic bacteria from G5-7 on the proteome of Nitrosomonas sp. Is79. In co-culture with N. winogradskyi, several Nitrosomonas sp. Is79 oxidative stress response proteins changed in abundance, with periplasmic proteins increasing and cytoplasmic proteins decreasing in abundance. In the presence of heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation pathway increased, while the abundance of proteins related to amino acid synthesis and metabolism decreased. In summary, the proteome of Nitrosomonas sp. Is79 was differentially influenced by the presence of either N. winogradskyi or heterotrophic bacteria. Together, N. winogradskyi and heterotrophic bacteria reduced the oxidative stress for Nitrosomonas sp. Is79, which resulted in more efficient metabolism.
Aerobic ammonia-oxidizing microorganisms play an important role in the global nitrogen cycle, converting ammonia to nitrite. In their natural environment, they coexist and interact with nitrite oxidizers, which convert nitrite to nitrate, and with heterotrophic microorganisms. The presence of nitrite oxidizers and heterotrophic bacteria has a positive influence on the growth of the ammonia oxidizers. Here, we present a study investigating the effect of nitrite oxidizers and heterotrophic bacteria on the proteome of a selected ammonia oxidizer in a defined culture to elucidate how these two groups improve the performance of the ammonia oxidizer. The results show that the presence of a nitrite oxidizer and heterotrophic bacteria reduced the stress for the ammonia oxidizer and resulted in more efficient energy generation. This study contributes to our understanding of microbe-microbe interactions, in particular between ammonia oxidizers and their neighboring microbial community.
The majority of environmental microorganisms cannot be grown by traditional techniques. Here we employed, and contrasted with conventional plating, an alternative approach based on cultivation of ...microorganisms inside diffusion chambers incubated within natural samples, followed by subculturing in petri dishes. Using this approach, we isolated microorganisms from subsurface sediments from the Field Research Center (FRC) in Oak Ridge, TN. The sediments were acidic and highly contaminated with uranium, heavy metals, nitrate, and organic pollutants. Phylogenetic analysis of 16S rRNA gene sequences revealed clear differences between diversity of isolates obtained by the diffusion chamber approach and those obtained by conventional plating. The latter approach led to isolation of members of the Alpha- and Gammaproteobacteria, Actinobacteria, and VERRUCOMICROBIA: Isolates obtained via the diffusion chamber approach represented the Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria, Firmicutes, and BACTEROIDETES: Notably, one-third of the isolates obtained by the new method were closely related to species known from previous molecular surveys conducted in the FRC area. Since the initial growth of microorganisms inside diffusion chambers occurred in the presence of the environmental stress factors, we expected the isolates we obtained to be tolerant of these factors. We investigated the physiologies of selected isolates and discovered that the majority were indeed capable of growth under low pH and/or high concentrations of heavy metals and nitrate. This indicated that in contrast to conventional isolation, the diffusion chamber-based approach leads to isolation of species that are novel, exhibit tolerance to extant environmental conditions, and match some of the species previously discovered by molecular methods.
Ammonia-oxidizing microorganisms (AOM) generate their energy by the oxidation of ammonia (NH(3)) to nitrite (NO(2)(-)). This process can be carried out by ammonia-oxidizing bacteria (AOB) as well as ...by the recently discovered ammonia-oxidizing archaea (AOA). In the past, AOB were enriched in batch cultures, often in the presence of rather high concentrations of NH(4)(+). Here, we describe methods to enrich, isolate, and investigate the basic physiology of AOB and AOA with emphasis on those that are adapted to low NH(4)(+) concentrations. The methods described include enrichment of AOA and AOB in batch cultures and of AOB in continuous cultures, the isolation of AOA by serial dilution and AOB by pour plates or dilution to extinction, and techniques to determine growth and activity of the AOA and AOB. Finally, we incorporated a section with Appendix about the identification of AOA and AOB as well as the measurement of the different inorganic nitrogen species.
The approach of growing microorganisms in situ, or in a simulated natural environment is appealing, and different versions of it have been described by several groups. The major difficulties with ...these approaches are that they are not selective for actinomycetes — a group of gram-positive bacteria well known as a rich source of antibiotics. In order to efficiently access actinomycetes, a trap for specifically capturing and cultivating these microorganisms in situ has been developed, based on the ability of these bacteria to form hyphae and penetrate solid environments. The trap is formed by two semi-permeable membranes (0.2–0.6 μm pore-size bottom membrane and 0.03 μm pore-size top membrane) glued to a plastic washer with sterile agar or gellan gum inside. The trap is placed on top of soil, and filamentous microorganisms selectively penetrate into the device and form colonies. Decreasing the size of the pores of the lower membrane to 0.2 μm restricted penetration of fungi. The trap produced more filamentous actinobacteria, and a higher variety of them, as compared to a conventional Petri dish cultivation from the same soil sample. Importantly, the trap cultivation resulted in the isolation of unusual and rare actinomycetes.
Linear α-olefins, such as 1-hexene and 1-octene, are important comonomers in the production of linear low-density polyethylene (LLDPE). The conventional method of producing 1-hexene and 1-octene is ...by oligomerization of ethylene, which yields a wide spectrum of linear α-olefins (LAOs). While there exists several processes for producing 1-hexene via ethylene trimerization, a similar route for the selective production of 1-octene has so far been elusive. We now, for the first time, report an unprecedented ethylene tetramerization reaction that produces 1-octene in selectivities exceeding 70%, using an aluminoxane-activated chromium/((R2)2P)2NR catalyst system.
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