New insights into the coal-associated methane architect: the ancient archaebacteria.

Exploration and marketable exploitation of coalbed methane (CBM) as cleaner fuel has been started globally. In addition, incidence of methane in coal basins is an imperative fraction of global carbon cycle. Significantly, subsurface coal ecosystem contains methane forming archaea. There is a rising attention in optimizing microbial coal gasification to exploit the abundant or inexpensive coal reserves worldwide. Therefore, it is essential to understand the coalbeds in geo-microbial perspective. Current review provides an in-depth analysis of recent advances in our understanding of how methanoarchaea are distributed in coal deposits globally. Specially, we highlight the findings on coal-associated methanoarchaeal existence, abundance, diversity, metabolic activity, and biogeography in diverse coal basins worldwide. Growing evidences indicates that we have arrived an exciting era of archaeal research. Moreover, gasification of coal into methane by utilizing microbial methanogenesis is a considerable way to mitigate the energy crisis for the rising world population.

Soil prokaryotic and fungal biome structures associated with crop disease status across the Japan Archipelago.

Archaea, bacteria, and fungi in the soil are increasingly recognized as determinants of agricultural productivity and sustainability. A crucial step for exploring soil microbiomes with important ecosystem functions is to perform statistical analyses on the potential relationship between microbiome structure and functions based on comparisons of hundreds or thousands of environmental samples collected across broad geographic ranges. In this study, we integrated agricultural field metadata with microbial community analyses by targeting 2,903 bulk soil samples collected along a latitudinal gradient from cool-temperate to subtropical regions in Japan (26.1-42.8 °N). The data involving 632 archaeal, 26,868 bacterial, and 4,889 fungal operational taxonomic units detected across the fields of 19 crop plant species allowed us to conduct statistical analyses (permutational analyses of variance, generalized linear mixed models, randomization analyses, and network analyses) on the relationship among edaphic factors, microbiome compositions, and crop disease prevalence. We then examined whether the diverse microbes form species sets varying in potential ecological impacts on crop plants. A network analysis suggested that the observed prokaryotes and fungi were classified into several species sets (network modules), which differed substantially in association with crop disease prevalence. Within the network of microbe-to-microbe coexistence, ecologically diverse microbes, such as an ammonium-oxidizing archaeon, an antibiotics-producing bacterium, and a potentially mycoparasitic fungus, were inferred to play key roles in shifts between crop-disease-promotive and crop-disease-suppressive states of soil microbiomes. The bird's-eye view of soil microbiome structure will provide a basis for designing and managing agroecosystems with high disease-suppressive functions.IMPORTANCEUnderstanding how microbiome structure and functions are organized in soil ecosystems is one of the major challenges in both basic ecology and applied microbiology. Given the ongoing worldwide degradation of agroecosystems, building frameworks for exploring structural diversity and functional profiles of soil microbiomes is an essential task. Our study provides an overview of cropland microbiome states in light of potential crop-disease-suppressive functions. The large data set allowed us to explore highly functional species sets that may be stably managed in agroecosystems. Furthermore, an analysis of network architecture highlighted species that are potentially used to cause shifts from disease-prevalent states of agroecosystems to disease-suppressive states. By extending the approach of comparative analyses toward broader geographic ranges and diverse agricultural practices, agroecosystem with maximized biological functions will be further explored.

Physiological versatility of ANME-1 and Bathyarchaeotoa-8 archaea evidenced by inverse stable isotope labeling.

BACKGROUND: The trophic strategy is one key principle to categorize microbial lifestyles, by broadly classifying microorganisms based on the combination of their preferred carbon sources, electron sources, and electron sinks. Recently, a novel trophic strategy, i.e., chemoorganoautotrophy-the utilization of organic carbon as energy source but inorganic carbon as sole carbon source-has been specifically proposed for anaerobic methane oxidizing archaea (ANME-1) and Bathyarchaeota subgroup 8 (Bathy-8). RESULTS: To further explore chemoorganoautotrophy, we employed stable isotope probing (SIP) of nucleic acids (rRNA or DNA) using unlabeled organic carbon and 13C-labeled dissolved inorganic carbon (DIC), i.e., inverse stable isotope labeling, in combination with metagenomics. We found that ANME-1 archaea actively incorporated 13C-DIC into RNA in the presence of methane and lepidocrocite when sulfate was absent, but assimilated organic carbon when cellulose was added to incubations without methane additions. Bathy-8 archaea assimilated 13C-DIC when lignin was amended; however, their DNA was derived from both inorganic and organic carbon sources rather than from inorganic carbon alone. Based on SIP results and supported by metagenomics, carbon transfer between catabolic and anabolic branches of metabolism is possible in these archaeal groups, indicating their anabolic versatility. CONCLUSION: We provide evidence for the incorporation of the mixed organic and inorganic carbon by ANME-1 and Bathy-8 archaea in the environment. Video Abstract.

Light Control in Microbial Systems.

Light is a key environmental component influencing many biological processes, particularly in prokaryotes such as archaea and bacteria. Light control techniques have revolutionized precise manipulation at molecular and cellular levels in recent years. Bacteria, with adaptability and genetic tractability, are promising candidates for light control studies. This review investigates the mechanisms underlying light activation in bacteria and discusses recent advancements focusing on light control methods and techniques for controlling bacteria. We delve into the mechanisms by which bacteria sense and transduce light signals, including engineered photoreceptors and light-sensitive actuators, and various strategies employed to modulate gene expression, protein function, and bacterial motility. Furthermore, we highlight recent developments in light-integrated methods of controlling microbial responses, such as upconversion nanoparticles and optical tweezers, which can enhance the spatial and temporal control of bacteria and open new horizons for biomedical applications.

Microbial Diversity and Open Questions about the Deep Tree of Life.

In this perspective, we explore the transformative impact and inherent limitations of metagenomics and single-cell genomics on our understanding of microbial diversity and their integration into the Tree of Life. We delve into the key challenges associated with incorporating new microbial lineages into the Tree of Life through advanced phylogenomic approaches. Additionally, we shed light on enduring debates surrounding various aspects of the microbial Tree of Life, focusing on recent advances in some of its deepest nodes, such as the roots of bacteria, archaea, and eukaryotes. We also bring forth current limitations in genome recovery and phylogenomic methodology, as well as new avenues of research to uncover additional key microbial lineages and resolve the shape of the Tree of Life.

Increases in the soil ammonia oxidizing phylotypes and their rechange due to long-term irrigation with wastewater.

Wastewater irrigation is a common practice for agricultural systems in arid and semiarid zones, which can help to overcome water scarcity and contribute with nutrient inputs. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are key in the transformation of NH4+-N in soil and can be affected by variations in soil pH, EC, N and C content, or accumulation of pollutants, derived from wastewater irrigation. The objective of this study was to determine the changes in the ammonia oxidizing communities in agricultural soils irrigated with wastewater for different periods of time (25, 50, and 100 years), and in rainfed soils (never irrigated). The amoA gene encoding for the catalytic subunit of the ammonia monooxygenase was used as molecular reporter; it was quantified by qPCR and sequenced by high throughput sequencing, and changes in the community composition were associated with the soil physicochemical characteristics. Soils irrigated with wastewater showed up to five times more the abundance of ammonia oxidizers (based on 16S rRNA gene relative abundance and amoA gene copies) than those under rainfed agriculture. While the amoA-AOA: amoA-AOB ratio decreased from 9.8 in rainfed soils to 1.6 in soils irrigated for 100 years, indicating a favoring environment for AOB rather than AOA. Further, the community structure of both AOA and AOB changed during wastewater irrigation compared to rainfed soils, mainly due to the abundance variation of certain phylotypes. Finally, the significant correlation between soil pH and the ammonia oxidizing community structure was confirmed, mainly for AOB; being the main environmental driver of the ammonia oxidizer community. Also, a calculated toxicity index based on metals concentrations showed a correlation with AOB communities, while the content of carbon and nitrogen was more associated with AOA communities. The results indicate that wastewater irrigation influence ammonia oxidizers communities, manly by the changes in the physicochemical environment.

Rocky desertification succession alters soil microbial communities and survival strategies in the karst context.

Rocky desertification is one of the most ecological problems in the karst context. Although extensive research has been conducted to explore how to restore and protect, the responses of soil fungi and archaea to rocky desertification succession remain limited. Here, four grades of rocky desertification in a karst ecosystem were selected, amplicon sequencing analysis was conducted to investigate fungal and archaeal community adaptation in response to rocky desertification succession. Our findings revealed that the diversity and community structure of fungi and archaea in soils declined with the aggravation of rocky desertification. As the rocky desertification succession intensified, microbial interactions shifted from cooperation to competition. Microbial survival strategies were K-strategist and r-strategist dominated in the early and late stages of succession, respectively. Additionally, the driving factors affecting microorganisms have shifted from vegetation diversity to soil properties as the intensification of rocky desertification. Collectively, our study highlighted that plant diversity and soil properties play important roles on soil microbiomes in fragile karst ecosystems and that environmental factors induced by human activities might still be the dominant factor exacerbating rocky desertification, which could significantly enrich our understanding of microbial ecology within karst ecosystems.

Tracking investigation of archaeal composition and methanogenesis function from parental to offspring pigs.

Archaea play a crucial role in microbial systems, including driving biochemical reactions and affecting host health by producing methane through hydrogen. The study of swine gut archaea has a positive significance in reducing methane emissions and improving feed utilization efficiency. However, the development and functional changes of archaea in the pig intestines have been overlooked for a long time. In this study, 54 fecal samples were collected from 36 parental pigs (18 boars and 18 pregnant/lactating sows), and 108 fecal samples from 18 offspring pigs during lactation, nursery, growing, and finishing stages were tracked and collected for metagenomic sequencing. We obtained 14 archaeal non-redundant metagenome-assembled genomes (MAGs). These archaea were classified as Methanobacteriota and Thermoplasmatota at the phylum level, and Methanobrevibacter, Methanosphaera, MX-02, and UBA71 at the genus level, involving hydrogenotrophic, methylotrophic, and acetoclastic pathways. The hydrogenotrophic pathway dominated the methanogenesis function, and the vast majority of archaea participated in it. Dietary changes profoundly affected the archaeal composition and methanogenesis function in pigs. The abundance of hydrogen-producing bacteria in parental pigs fed high-fiber diets was higher than that in offspring pigs fed low-fiber diets. The methanogenesis function was positively correlated with fiber decomposition functions and negatively correlated with the starch decomposition function. Increased abundance of sulfate reductase and fumarate reductase, as well as decreased acetate/propionate ratio, indicated that the upregulation of alternative hydrogen uptake pathways competing with methanogens may be the reason for the reduced methanogenesis function. These findings contribute to providing information and direction in the pig industry for the development of strategies to reduce methane emissions, improve feed efficiency, and maintain intestinal health.

A fast comparative genome browser for diverse bacteria and archaea.

Genome sequencing has revealed an incredible diversity of bacteria and archaea, but there are no fast and convenient tools for browsing across these genomes. It is cumbersome to view the prevalence of homologs for a protein of interest, or the gene neighborhoods of those homologs, across the diversity of the prokaryotes. We developed a web-based tool, fast.genomics, that uses two strategies to support fast browsing across the diversity of prokaryotes. First, the database of genomes is split up. The main database contains one representative from each of the 6,377 genera that have a high-quality genome, and additional databases for each taxonomic order contain up to 10 representatives of each species. Second, homologs of proteins of interest are identified quickly by using accelerated searches, usually in a few seconds. Once homologs are identified, fast.genomics can quickly show their prevalence across taxa, view their neighboring genes, or compare the prevalence of two different proteins. Fast.genomics is available at https://fast.genomics.lbl.gov.

Floristic changes and environmental drivers of soil fungi and archaea in different salt-tolerant plant communities in the intertidal habitat of coastal wetlands.

Microorganisms are crucial elements of terrestrial ecosystems, which play significant roles in improving soil physicochemical properties, providing plant growth nutrients, degrading toxic and harmful chemicals, and biogeochemical cycling. Variations in the types and quantities of root exudates among different plants greatly alter soil physicochemical properties and result in variations in the diversity, structure, and function of soil microorganisms. Not much is understood about the differences of soil fungi and archaea communities for different plant communities in coastal wetlands, and their response mechanisms to environmental changes. In this study, fungal and archaea communities in soils of Suaeda salsa, Phragmites australis, and Spartina alterniflora in the intertidal habitat of coastal wetlands were selected for research. Soil fungi and archaea were analyzed for diversity, community structure, and function using high throughput ITS and 16S rRNA gene sequencing. The study revealed significant differences in fungi and archaea's diversity and community structure in the rhizosphere soil of three plant communities. At the same time, there is no significant difference in the functional groups. SOM, TP, AP, MC, EC and SOM, TN, TP, AP, MC, EC are the primary environmental determinants affecting changes in soil fungal and archaeal communities, respectively. Variations in the diversity, community structure, and ecological functions of fungi and archaea can be used as indicators characterizing the impact of external disturbances on the soil environment, providing a theoretical foundation for the effective utilization of soil microbial resources, thereby achieving the goal of environmental protection and health promotion.

Interplay of intracellular and trans-cellular DNA methylation in natural archaeal consortia.

DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano-sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous cTAG methylation, typical of Haloferax, was accompanied in this strain by methylation at four other motifs, including GDGcHC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGAaG methylation increased in highly expressed genes, while CcTTG and GTCGaGG methylation could be linked to restriction-modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self-restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo-ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host.

Comparative genomics reveals the adaptation of ammonia-oxidising Thaumarchaeota to arid soils.

Thaumarchaeota are predominant in oligotrophic habitats such as deserts and arid soils, but their adaptations to these arid conditions are not well understood. In this study, we assembled 23 Thaumarchaeota genomes from arid and semi-arid soils collected from the Inner Mongolia Steppe and the Qinghai-Tibet Plateau. Using a comparative genomics approach, integrated with 614 Thaumarchaeota genomes from public databases, we identified the traits and evolutionary forces that contribute to their adaptations to aridity. Our results showed that the newly assembled genomes represent an early diverging group within the lineage of ammonia-oxidising Thaumarchaeota. While the genomic functions previously identified in arid soil lineages were conserved across terrestrial, shallow-ocean and deep-ocean lineages, several traits likely contribute to Thaumarchaeota's adaptation to aridity. These include chlorite dismutase, arsenate reductase, V-type ATPase and genes dealing with oxidative stresses. The acquisition and loss of traits at the last common ancestor of arid soil lineages may have facilitated the specialisation of Thaumarchaeota in arid soils. Additionally, the acquisition of unique adaptive traits, such as a urea transporter, Ca2+ :H+ antiporter, mannosyl-3-phosphoglycerate synthase and phosphatase, DNA end-binding protein Ku and phage shock protein A, further distinguishes arid soil Thaumarchaeota. This study provides evidence for the adaptations of Thaumarchaeota to arid soil, enhancing our understanding of the nitrogen and carbon cycling driven by Thaumarchaeota in drylands.

Environmental filtering governs consistent vertical zonation in sedimentary microbial communities across disconnected mountain lakes.

Subsurface microorganisms make up the majority of Earth's microbial biomass, but ecological processes governing surface communities may not explain community patterns at depth because of burial. Depth constrains dispersal and energy availability, and when combined with geographic isolation across landscapes, may influence community assembly. We sequenced the 16S rRNA gene of bacteria and archaea from 48 sediment cores across 36 lakes in four disconnected mountain ranges in Wyoming, USA and used null models to infer assembly processes across depth, spatial isolation, and varying environments. Although we expected strong dispersal limitations across these isolated settings, community composition was primarily shaped by environmental selection. Communities consistently shifted from domination by organisms that degrade organic matter at the surface to methanogenic, low-energy adapted taxa in deeper zones. Stochastic processes-like dispersal limitation-contributed to differences among lakes, but because these effects weakened with depth, selection processes ultimately governed subsurface microbial biogeography.

Expanded phylogeny of extremely halophilic archaea shows multiple independent adaptations to hypersaline environments.

Extremely halophilic archaea (Haloarchaea, Nanohaloarchaeota, Methanonatronarchaeia and Halarchaeoplasmatales) thrive in saturating salt concentrations where they must maintain osmotic equilibrium with their environment. The evolutionary history of adaptations enabling salt tolerance remains poorly understood, in particular because the phylogeny of several lineages is conflicting. Here we present a resolved phylogeny of extremely halophilic archaea obtained using improved taxon sampling and state-of-the-art phylogenetic approaches designed to cope with the strong compositional biases of their proteomes. We describe two uncultured lineages, Afararchaeaceae and Asbonarchaeaceae, which break the long branches at the base of Haloarchaea and Nanohaloarchaeota, respectively. We obtained 13 metagenome-assembled genomes (MAGs) of these archaea from metagenomes of hypersaline aquatic systems of the Danakil Depression (Ethiopia). Our phylogenomic analyses including these taxa show that at least four independent adaptations to extreme halophily occurred during archaeal evolution. Gene-tree/species-tree reconciliation suggests that gene duplication and horizontal gene transfer played an important role in this process, for example, by spreading key genes (such as those encoding potassium transporters) across extremely halophilic lineages.

Impact of salinity and organic matter on the ammonia-oxidizing archaea and bacteria in treating hypersaline industrial wastewater: amoA gene abundance and ammonia removal contributions.

Studies published recently proposed that ammonia-oxidizing archaea (AOA) may be beneficial for hypersaline (salinity > 50 g NaCl L-1) industrial wastewater treatment. However, knowledge of AOA activity in hypersaline bioreactors is limited. This study investigated the effects of salinity, organic matter, and practical pickled mustard tuber wastewater (PMTW) on AOA and ammonia-oxidizing bacteria (AOB) in two sequencing batch biofilm reactors (SBBRs). Results showed that despite observed salinity inhibition (p < 0.05), both AOA and AOB contributed to high ammonia removal efficiency at a salinity of 70 g NaCl L-1 in the two SBBRs. The ammonia removal efficiency of SBBR2 did not significantly differ from that of SBBR1 in the absence of organic matter (p > 0.05). Batch tests and quantitative real-time PCR (qPCR) reveal that salinity and organic matter inhibition resulted in a sharp decline in specific ammonia oxidation rates and amoA gene copy numbers of AOA and AOB (p < 0.05). AOA demonstrated higher abundance and more active ammonia oxidation activity in hypersaline and high organic matter environments. Salinity was positively correlated with the potential ammonia oxidation contribution of AOA (p < 0.05), resulting in a potential transition from AOB dominance to AOA dominance in SBBR1 as salinity levels rose. Moreover, autochthonous AOA in PMTW promoted the abundance and ammonia oxidation activities of AOA in SBBR2, further elevating the nitrification removal efficiency after feeding the practical PMTW. AOA demonstrates greater tolerance to the challenging hypersaline environment, making it a valuable candidate for the treatment of practical industrial wastewater with high salinity and organic content.

Blue-light irradiation induced partial nitrification.

The role of ray radiation from the sunlight acting on organisms has long-term been investigated. However, how the light with different wavelengths affects nitrification and the involved nitrifiers are still elusive. Here, we found more than 60 % of differentially expressed genes (DEGs) in nitrifiers were observed under irradiation of blue light with wavelengths of 440-480 nm, which were 13.4 % and 20.3 % under red light and white light irradiation respectively. Blue light was more helpful to achieve partial nitrification rather than white light or red light, where ammonium oxidization by ammonia-oxidizing archaea (AOA) with the increased relative abundance from 8.6 % to 14.2 % played a vital role. This was further evidenced by the enhanced TCA cycle, reactive oxygen species (ROS) scavenge and DNA repair capacity in AOA under blue-light irradiation. In contrast, nitrite-oxidizing bacteria (NOB) was inhibited severely to achieve partial nitrification, and the newly discovered encoded blue light photoreceptor proteins made them more sensitive to blue light and hindered cell activity. Ammonia-oxidizing bacteria (AOB) expressed genes for DNA repair capacity under blue-light irradiation, which ensured their tiny impact by light irradiation. This study provided valuable insights into the photosensitivity mechanism of nitrifiers and shed light on the diverse regulatory by light with different radiation wavelengths in artificial systems, broadening our comprehension of the nitrogen cycle on earth.

The discovery of archaea: from observed anomaly to consequential restructuring of the phylogenetic tree.

Observational and experimental discoveries of new factual entities such as objects, systems, or processes, are major contributors to some advances in the life sciences. Yet, whereas discovery of theories was extensively deliberated by philosophers of science, very little philosophical attention was paid to the discovery of factual entities. This paper examines historical and philosophical aspects of the experimental discovery by Carl Woese of archaea, prokaryotes that comprise one of the three principal domains of the phylogenetic tree. Borrowing Kuhn's terminology, this discovery of a major biological entity was made during a 'normal science' project of building molecular taxonomy for prokaryotes. Unexpectedly, however, an observed anomaly instigated the discovery of archaea. Substantiation of the existence of the new archaeal entity and consequent reconstruction of the phylogenetic tree prompted replacement of a long-held model of a prokarya and eukarya bipartite tree of life by a new model of a tripartite tree comprising of bacteria, archaea, and eukarya. This paper explores the history and philosophical implications of the progression of Woese's project from normal science to anomaly-instigated model-changing discovery. It is also shown that the consequential discoveries of RNA splicing and of ribozymes were similarly prompted by unexpected irregularities during normal science activities. It is thus submitted that some discoveries of factual biological entities are triggered by unforeseen observational or experimental anomalies.

Functional Roles and Genomic Impact of Miniature Inverted-Repeat Transposable Elements (MITEs) in Prokaryotes.

Prokaryotic genomes are dynamic tapestries that are strongly influenced by mobile genetic elements (MGEs), including transposons (Tn's), plasmids, and bacteriophages. Of these, miniature inverted-repeat transposable elements (MITEs) are undoubtedly the least studied MGEs in bacteria and archaea. This review explores the diversity and distribution of MITEs in prokaryotes and describes what is known about their functional roles in the host and involvement in genomic plasticity and evolution.

Potential directions for future development of mainstream partial nitrification-anammox processes: Ammonia-oxidizing archaea as novel functional microorganisms providing nitrite.

The application of ammonia-oxidizing archaea (AOA)-based partial nitrification-anammox (PN-A) for mainstream wastewater treatment has attracted research interest because AOA can maintain higher activity in low-temperature environments and they have higher affinity for oxygen and ammonia-nitrogen compared with ammonia-oxidizing bacteria (AOB), thus facilitating stabilized nitrite production, deep removal of low-ammonia, and nitrite-oxidizing bacteria suppression. Moreover, the low affinity of AOA for ammonia makes them more tolerant to N-shock loading and more efficiently integrated with anaerobic ammonium oxidation (anammox). Based on the limitations of the AOB-based PN-A process, this review comprehensively summarizes the potential and significance of AOA for nitrite supply, then gives strategies and influencing factors for replacing AOB with AOA. Additionally, the methods and key influences on the coupling of AOA and anammox are explored. Finally, this review proposes four AOA-based oxygen- or ammonia-limited autotrophic nitritation/denitrification processes to address the low effluent quality and instability of mainstream PN-A processes.

The significant contribution of comammox bacteria to nitrification in a constructed wetland revealed by DNA-based stable isotope probing.

The discovery of Comammox bacteria (CMX) has changed our traditional concept towards nitrification, yet its role in constructed wetlands (CWs) remains unclear. This study investigated the contributions of CMX and two canonical ammonia-oxidizing microorganisms, ammonia-oxidizing bacteria (AOB) and archaea to nitrification in four regions (sediment, shoreside, adjacent soil, and water) of a typical CW using DNA-based stable isotope probing. The results revealed that CMX not only widely occurred in sediment and shoreside zones with high abundance (5.08 × 104 and 6.57 × 104 copies g-1 soil, respectively), but also actively participated in ammonia oxidation, achieving ammonia oxidation rates of 1.43 and 2.00 times that of AOB in sediment and shoreside, respectively. Phylogenetic analysis indicated that N. nitrosa was the dominant and active CMX species. These findings uncovered the crucial role of CMX in nitrification of sediment and shoreside, providing a new insight into nitrogen cycle of constructed wetlands.