长期施用猪粪对红壤完全氨氧化菌基因丰度的影响

完全氨氧化菌(comammox Nitrospira)的发现对硝化微生物的研究提出了新的挑战。大量研究表明完全氨氧化菌在陆地生态系统中广泛分布,但其在农田土壤中的分布规律及其对长期施用粪肥的响应尚不清楚。研究了长期施用猪粪对农田红壤完全氨氧化菌、氨氧化古菌(AOA)和氨氧化细菌(AOB)功能基因(amoA)丰度的影响,及其与土壤净硝化速率的关系。结果表明：与不施肥的对照处理相比,猪粪施用显著提高土壤有机质和养分含量,且随着猪粪的施用量增加而增加。同时,施用中量和高量猪粪显著提升土壤净硝化速率,增幅分别达到317%和416%。所有处理中,完全氨氧化菌丰度以进化枝A为主,进化枝B丰度极低,大多为非特异性扩增产物,但进化枝A的amoA丰度均低于氨氧化古菌和氨氧化细菌。长期施用高量猪粪显著提升进化枝A的amoA基因丰度,表明存在喜好富营养环境的完全氨氧化菌,而有效磷是最主要的影响因子。相关性分析表明,进化枝A的amoA丰度与净硝化速率呈显著正相关(P<0.01),而氨氧化古菌和氨氧化细菌则没有,表明进化枝A可能在长期施用粪肥的农田红壤硝化过程中发挥重要功能。综上所述,长期施用粪肥显著提高...     

Metagenomic recovery of two distinct comammox Nitrospira from the terrestrial subsurface

The recently discovered comammox process encompasses both nitrification steps, the aerobic oxidation of ammonia and nitrite, in a single organism. All known comammox bacteria are affiliated with Nitrospira sublineage II and can be grouped into two distinct clades, referred to as A and B, based on ammonia monooxygenase phylogeny. In this study, we report high-quality draft genomes of two novel comammox Nitrospira from the terrestrial subsurface, representing one clade A and one clade B comammox organism. The two metagenome-assembled genomes were compared with other representatives of Nitrospira sublineage II, including both canonical and comammox Nitrospira. Phylogenomic analyses confirmed the affiliation of the two novel Nitrospira with comammox clades A and B respectively. Based on phylogenetic distance and pairwise average nucleotide identity values, both comammox Nitrospira were classified as novel species. Genomic comparison revealed high conservation of key metabolic features in sublineage II Nitrospira, including respiratory complexes I–V and the machineries for nitrite oxidation and carbon fixation via the reductive tricarboxylic acid cycle. In addition, the presence of the enzymatic repertoire for formate and hydrogen oxidation in the Rifle clades A and B comammox genomes, respectively, suggest a broader distribution of these metabolic features than previously anticipated.     

Rapid nitrification involving comammox and canonical Nitrospira at extreme pH in saline-alkaline lakes

Nitrite-oxidizing bacteria (NOB) catalyse the second nitrification step and are the main biological source of nitrate. The most diverse and widespread NOB genus is Nitrospira, which also contains complete ammonia oxidizers (comammox) that oxidize ammonia to nitrate. To date, little is known about the occurrence and biology of comammox and canonical nitrite oxidizing Nitrospira in extremely alkaline environments. Here, we studied the seasonal distribution and diversity, and the effect of short-term pH changes on comammox and canonical Nitrospira in sediments of two saline, highly alkaline lakes. We identified diverse canonical and comammox Nitrospira clade A-like phylotypes as the only detectable NOB during more than a year, suggesting their major importance for nitrification in these habitats. Gross nitrification rates measured in microcosm incubations were highest at pH 10 and considerably faster than reported for other natural, aquatic environments. Nitrification could be attributed to canonical and comammox Nitrospira and to Nitrososphaerales ammonia-oxidizing archaea. Furthermore, our data suggested that comammox Nitrospira contributed to ammonia oxidation at an extremely alkaline pH of 11. These results identify saline, highly alkaline lake sediments as environments of uniquely strong nitrification with novel comammox Nitrospira as key microbial players.     

Geospatial variation in co‐occurrence networks of nitrifying microbial guilds

Microbial communities transform nitrogen (N) compounds, thereby regulating the availability of N in soil. The N cycle is defined by interacting microbial functional groups, as inorganic N‐products formed in one process are the substrate in one or several other processes. The nitrification pathway is often a two‐step process in which bacterial or archaeal communities oxidize ammonia to nitrite, and bacterial communities further oxidize nitrite to nitrate. Little is known about the significance of interactions between ammonia‐oxidizing bacteria (AOB) and archaea (AOA) and nitrite‐oxidizing bacterial communities (NOB) in determining the spatial variation of overall nitrifier community structure. We hypothesize that nonrandom associations exist between different AO and NOB lineages that, along with edaphic factors, shape field‐scale spatial patterns of nitrifying communities. To address this, we sequenced and quantified the abundance of AOA, AOB, and Nitrospira and Nitrobacter NOB communities across a 44‐hectare site with agricultural fields. The abundance of Nitrobacter communities was significantly associated only with AOB abundance, while that of Nitrospira was correlated to AOA. Network analysis and geostatistical modelling revealed distinct modules of co‐occurring AO and NOB groups occupying disparate areas, with each module dominated by different lineages and associated with different edaphic factors. Local communities were characterized by a high proportion of module‐connecting versus module‐hub nodes, indicating that nitrifier assemblages in these soils are shaped by fluctuating conditions. Overall, our results demonstrate the utility of network analysis in accounting for potential biotic interactions that define the niche space of nitrifying communities at scales compatible to soil management.     

A single Thaumarchaeon drives nitrification in deep oligotrophic Lake Constance

Ammonia released during organic matter mineralization is converted during nitrification to nitrate. We followed spatiotemporal dynamics of the nitrifying microbial community in deep oligotrophic Lake Constance. Depth-dependent decrease of total ammonium (0.01–0.84 μM) indicated the hypolimnion as the major place of nitrification with ¹⁵N-isotope dilution measurements indicating a threefold daily turnover of hypolimnetic total ammonium. This was mirrored by a strong increase of ammonia-oxidizing Thaumarchaeota towards the hypolimnion (13%–21% of bacterioplankton) throughout spring to autumn as revealed by amplicon sequencing and quantitative polymerase chain reaction. Ammonia-oxidizing bacteria were typically two orders of magnitude less abundant and completely ammonia-oxidizing (comammox) bacteria were not detected. Both, 16S rRNA gene and amoA (encoding ammonia monooxygenase subunit B) analyses identified only one major species-level operational taxonomic unit (OTU) of Thaumarchaeota (99% of all ammonia oxidizers in the hypolimnion), which was affiliated to Nitrosopumilus spp. The relative abundance distribution of the single Thaumarchaeon strongly correlated to an equally abundant Chloroflexi clade CL500-11 OTU and a Nitrospira OTU that was one order of magnitude less abundant. The latter dominated among recognized nitrite oxidizers. This extremely low diversity of nitrifiers shows how vulnerable the ecosystem process of nitrification may be in Lake Constance as Central Europe's third largest lake.     

Nitrifying bacterial growth inhibition in the presence of algae and cyanobacteria

Nitrifying bacteria, cyanobacteria, and algae are important microorganisms in open pond wastewater treatment systems. Nitrification involving the sequential oxidation of ammonia to nitrite and nitrate, mainly due to autotrophic nitrifying bacteria, is essential to biological nitrogen removal in wastewater and global nitrogen cycling. A continuous flow autotrophic bioreactor was initially designed for nitrifying bacterial growth only. In the presence of cyanobacteria and algae, we monitored both the microbial activity by measuring specific oxygen production rate (SOPR) for microalgae and cyanobacteria and specific oxygen uptake rate (SOUR) for nitrifying bacteria. The growth of cyanobacteria and algae inhibited the maximum nitrification rate by a factor of 4 although the ammonium nitrogen fed to the reactor was almost completely removed. Terminal restriction fragment length polymorphism (T‐RFLP) analysis indicated that the community structures of nitrifying bacteria remained unchanged, containing the dominant Nitrosospira, Nitrospira, and Nitrobacter species. PCR amplification coupled with cloning and sequencing analysis resulted in identifying Chlorella emersonii and an uncultured cyanobacterium as the dominant species in the autotrophic bioreactor. Notwithstanding their fast growth rate and their toxicity to nitrifiers, microalgae and cyanobacteria were more easily lost in effluent than nitrifying bacteria because of their poor settling characteristics. The microorganisms were able to grow together in the bioreactor with constant individual biomass fractions because of the uncoupled solids retention times for algae/cyanobacteria and nitrifiers. The results indicate that compared to conventional wastewater treatment systems, longer solids retention times (e.g., by a factor of 4) should be considered in phototrophic bioreactors for complete nitrification and nitrogen removal. Biotechnol. Bioeng. 2010;107: 1004–1011. © 2010 Wiley Periodicals, Inc.     

亚热带米槠天然林土壤氨氧化微生物对模拟氮沉降的响应

我国亚热带地区是全球氮沉降的热点区域。氮沉降会影响氨氧化微生物的丰度和群落结构,进而改变土壤微生物驱动的养分循环。目前对新近发现的完全氨氧化菌认识不足,极大地制约了对森林土壤氨氧化微生物响应氮沉降的整体认识。本研究以福建省三明市辛口镇格氏栲自然保护区长期模拟氮沉降处理土壤为研究对象,利用实时定量PCR方法,研究氨氧化微生物(包括氨氧化细菌AOB、氨氧化古菌AOA和完全氨氧化菌comammox Nitrospira),尤其是完全氨氧化菌的amoA基因丰度。模拟氮沉降处理包括:不添加N(CK)、低氮(添加40 kg N·hm^-2·a^-1,LN)和高氮(添加80 kg N·hm^-2·a^-1,HN)。结果表明: 8年的氮添加降低了土壤pH值和有机碳含量,提高了土壤硝态氮含量。供试土壤的AOB丰度低于检测限,无法获得目的片段。高氮处理显著提高了AOA丰度,但对完全氨氧化菌clade A和clade B丰度无显著影响。两种氮添加处理均降低了完全氨氧化菌/AOA值,表明氮添加降低了完全氨氧化菌在亚热带森林土壤氨氧化微生物类群中的相对竞争力。针对完全氨氧化菌clade A和clade B的扩增都存在非特异性产物,表明针对森林土壤的高特异性和覆盖度设计引物的必要性。Clade A和clade B丰度与总氮和铵态氮含量呈显著正相关,clade B丰度还与有机碳含量呈显著正相关。总之,模拟氮沉降提高了AOA在亚热带米槠天然林土壤硝化过程中的相对重要性,这些发现可为该地区应对全球变化和氮沉降的风险评估提供理论依据。     

Ciliate grazing on <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrospira</Emphasis><Emphasis Type="Italic">moscoviensis</Emphasis>: Is selectivity a factor for the nitrogen cycle in natural aquatic systems?

Ciliated protists are important predators of bacteria in many aquatic habitats, including sediments. Since, many biochemical transformations within the nitrogen cycle are performed by bacteria, ciliates could have an indirect impact on the nitrogen cycle through selective grazing on nitrogen-transforming bacteria. As a case study, we examined ciliate grazing on nitrifying bacteria of the genera Nitrosomonas and Nitrospira. All experiments were designed as in vitro-experiments with cultures of different bacteria and ciliate species. The nitrifying bacteria used in our experiments were Nitrosomonas europaea [Winogradsky 1892] and Nitrospira moscoviensis [Ehrich 2001]. The ciliates comprised of four species that are known as efficient bacterivores and common members of the protist community in aquatic systems: Paramecium aurelia [Müller 1773], Euplotes octocarinatus [Carter 1972], Tetrahymena pyriformis [Ehrenberg 1830] and Cyclidium glaucoma [Müller 1786]. Our experimental approach, using a combination of DAPI and FISH staining, was successful in allowing the observation of ingestion of specific bacteria and their detection within ciliate food vacuoles. However, the ciliates in this study showed no significant selective grazing. No food preferences for a any bacterial taxon or any size class or morphotype were detected. Correlation with time between ciliate abundance and bacterial abundance or biovolume, using log transformed growth rates of ciliates and bacteria, showed no significant results. On the bacterial side, neither an active defence mechanism of the nitrifying bacteria against ciliate grazing, such as changes in morphology, nor competition for resources were observed. These results suggest that in our in vitro-experiments grazing by ciliates has no influence on abundance and growth of nitrifying bacteria and nitrification.     

Macroinvertebrate communities in streams with contrasting water sources in the Japanese Alps

Alpine streams are typically fed from a range of water sources including glacial meltwater, snowmelt, groundwater flow, and surface rainfall runoff. These contributions are projected to shift with climate change, particularly in the Japanese Alps where snow is expected to decrease, but rainfall events increase. The overarching aim of the study was to understand the key variables driving macroinvertebrate community composition in groundwater and snowmelt‐fed streams (n = 6) in the Kamikochi region of the northern Japanese Alps (April–December 2017). Macroinvertebrate abundance, species richness, and diversity were not significantly different between the two stream types. Community structure, however, was different between groundwater and snowmelt‐fed streams with macroinvertebrate taxa specialized for the environmental conditions present in each system. Temporal variation in the abundance, species richness, and diversity of macroinvertebrate communities was also significantly different between groundwater and snowmelt streams over the study period, with snowmelt streams exhibiting far higher levels of variation. Two snowmelt streams considered perennial proved to be intermittent with periodic drying of the streambed, but the macroinvertebrates in these systems rebounded rapidly after flows resumed with no reduction in taxonomic diversity. These same streams, nevertheless, showed a major reduction in diversity and abundance following periods of high flow, indicating floods rather than periodic drying was a major driver of community structure. This conclusion was also supported from functional analyses, which showed that the more variable snowmelt streams were characterized by taxa with resistant, rather than resilient, life‐history traits. The findings demonstrate the potential for significant turnover in species composition with changing environmental conditions in Japanese alpine stream systems, with groundwater‐fed streams potentially more resilient to future changes in comparison to snowmelt‐fed streams.     

Geochemical factors impacting nitrifying communities in sandy sediments

Sandy sediment beaches covering 70% of non-ice-covered coastlines are important ecosystems for nutrient cycling along the land-ocean continuum. Subterranean estuaries (STEs), where groundwater and seawater meet, are hotspots for biogeochemical cycling within sandy beaches. The STE microbial community facilitates biogeochemical reactions, determining the fate of nutrients, including nitrogen (N), supplied by groundwater. Nitrification influences the fate of N, oxidising reduced dissolved inorganic nitrogen (DIN), making it available for N removal. We used metabarcoding of 16S rRNA genes and quantitative PCR (qPCR) of ammonia monooxygenase (amoA) genes to characterise spatial and temporal variation in STE microbial community structure and nitrifying organisms. We examined nitrifier diversity, distribution and abundance to determine how geochemical measurements influenced their distribution in STEs. Sediment microbial communities varied with depth (p-value = 0.001) and followed geochemical gradients in dissolved oxygen (DO), salinity, pH, dissolved inorganic carbon and DIN. Genetic potential for nitrification in the STE was evidenced by qPCR quantification of amoA genes. Ammonia oxidiser abundance was best explained by DIN, DO and pH. Our results suggest that geochemical gradients are tightly linked to STE community composition and nitrifier abundance, which are important to determine the fate and transport of groundwater-derived nutrients to coastal waters.     

Nitrification in intertidal sponge <Emphasis Type="Italic">Cinachyrella cavernosa</Emphasis>

The nitrification process in different sections of the sponges remains unresolved, despite several studies on the nitrogen cycling pathways in the tissues of temperate and Arctic sponges. In this study, the abundance, diversity and activity of the associated nitrifying organisms in intracellular, intercellular, extracellular and cortex of a tropical intertidal sponge, Cinachyrella cavernosa, were investigated using most probable number, next-generation sequencing and incubation method, respectively. The nitrification rate and the abundance of nitrifying bacteria showed significant difference among different sections. The nitrification rate in C. cavernosa was 2–12× higher than the reported values in other sponge species from temperate and Arctic regions. Nitrification rate in sponge cortex was 2× higher than in intercellular and extracellular sections. Ammonium and nitrite oxidisers ranged from 10³ to 10⁴ CFU g^?1 in the sponge with a high number of ammonium and nitrite oxidisers in the cortex. Nitrifiers belonging to Nitrosomonas, Nitrospira, Nitrospina, Nitrobacter and Nitrosopumilus were present in different sections of the sponge, with nitrifying archaea dominating the intracellular section and nitrifying bacteria dominating other sections. This study reports for the first time the nitrification inside the sponge cells. The study also suggests that the intertidal sponge, C. cavernosa, harbours metabolically active nitrifiers in different sections of the sponge body with different rates of nitrification. Thus, nitrifiers play an important role in ammonia detoxification within the sponge and also contribute to the nitrogen budget of the coastal ecosystem.     

Fluctuation of microbial activities after influent load variations in a full-scale SBR: recovery of the biomass after starvation

Due to variations in the production levels, a full-scale sequencing batch reactor (SBR) for post-treatment of tannery wastewater was exposed to low and high ammonia load periods. In order to study how these changes affected the N-removal capacity, the microbiology of the reactor was studied by a diverse set of techniques including molecular tools, activity tests, and microbial counts in samples taken along 3 years. The recover capacity of the biomass was also studied in a lab-scale reactor operated with intermittent aeration without feeding for 36 days. The results showed that changes in the feeding negatively affected the nitrifying community, but the nitrogen removal efficiencies could be restored after the concentration stress. Species substitution was observed within the nitrifying bacteria, Nitrosomonas europaea and Nitrobacter predominated initially, and after an ammonia overload period, Nitrosomonas nitrosa and Nitrospira became dominant. Some denitrifiers, with nirS related to Alicycliphilus, Azospirillum, and Marinobacter nirS, persisted during long-term reactor operation, but the community fluctuated both in composition and in abundance. This fluctuating community may better resist the continuous changes in the feeding regime. Our results showed that a nitrifying–denitrifying SBR could be operated with low loads or even without feeding during production shut down periods.     

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (K_{m(app)_NH3} ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (K_{m(app)_NO2}- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of “Ca. N. kreftii” indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.Subject terms: Bacterial genomics, Environmental microbiology, Bacterial physiology     

Seasonal diversity and dynamics of haptophytes in the Skagerrak,Norway, explored by high‐throughput sequencing

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high‐throughput sequencing. From outer Oslofjorden, S Norway, nano‐ and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta‐specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA‐sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP‐3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in‐depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters.     

Internal Porosity of Mineral Coating Supports Microbial Activity in Rapid Sand Filters for Groundwater Treatment

A mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. The coating changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity, and abundance of microbiota. This study reveals that a mineral coating can positively affect the colonization and activity of microbial communities in rapid sand filters. To understand this effect, we investigated the abundance, spatial distribution, colonization, and diversity of all and of nitrifying prokaryotes in filter material with various degrees of mineral coating. We also examined the physical and chemical characteristics of the mineral coating. The amount of mineral coating correlated positively with the internal porosity, the packed bulk density, and the biologically available surface area of the filter material. The volumetric NH₄⁺ removal rate also increased with the degree of mineral coating. Consistently, bacterial 16S rRNA and amoA abundances positively correlated with increased mineral coating levels. Microbial colonization could be visualized mainly within the outer periphery (60.6 ± 35.6 μm) of the mineral coating, which had a thickness of up to 600 ± 51 μm. Environmental scanning electron microscopic (E-SEM) observations suggested an extracellular polymeric substance-rich matrix and submicron-sized bacterial cells. Nitrifier diversity profiles were similar irrespective of the degree of mineral coating, as indicated by pyrosequencing analysis. Overall, our results demonstrate that mineral coating positively affects microbial colonization and activity in rapid sand filters, most likely due to increased volumetric cell abundances facilitated by the large surface area of internal mineral porosity accessible for microbial colonization.     

Shifts between Nitrospira‐ and Nitrobacter‐like nitrite oxidizers underlie the response of soil potential nitrite oxidation to changes in tillage practices

Despite their role in soil functioning, the ecology of nitrite‐oxidizing bacteria, NOB, and their response to disturbances such as those generated by agricultural practices are scarcely known. Over the course of 17 months, we surveyed the potential nitrite oxidation, PNO, the abundance of the Nitrobacter‐ and Nitrospira‐like NOB (by quantitative PCR) and the community structure of the Nitrobacter‐like NOB (by PCR‐DGGE and cloning‐sequencing targeting the nxrA gene) in soils for four treatments: after establishment of tillage on a previously no‐tillage system, after cessation of tillage on a previously tillage system, and on control tillage and no‐tillage systems. Key soil variables (moisture, organic carbon content and gross mineralization – i.e. ammonification – measured by the ¹⁵N dilution technique) were also surveyed. PNO was always higher for the no‐tillage than tillage treatments. Establishment of tillage led to a strong and rapid decrease in PNO whereas cessation of tillage did not change PNO even after 17 months. PNO was strongly and positively correlated to the abundance of Nitrobacter‐like NOB and was also strongly related to gross mineralization, a proxy of N‐availability; in contrast, PNO was weakly and negatively correlated to the abundance of Nitrospira‐like NOB. Selection of a dominant population was observed under no‐tillage, and PNO was loosely correlated to the community structure of Nitrobacter‐like NOB. Our results demonstrate that Nitrobacter‐like NOB are the key functional players within the NOB community in soils with high N availability and high activity level, and that changes in PNO are due to shifts between Nitrospira‐like and Nitrobacter‐like NOB and to a weaker extent by shifts of populations within Nitrobacter‐like NOB.