Active Soil Nitrifying Communities Revealed by In Situ Transcriptomics and Microcosm-Based Stable-Isotope Probing.

Long-term nitrogen field fertilization often results in significant changes in nitrifying communities that catalyze a key step in the global N cycle. However, whether microcosm studies are able to inform the dynamic changes in communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) under field conditions remains poorly understood. This study aimed to evaluate the transcriptional activities of nitrifying communities under in situ conditions, and we found that they were largely similar to those of 13C-labeled nitrifying communities in the urea-amended microcosms of soils that had received different N fertilization regimens for 22 years. High-throughput sequencing of 16S rRNA genes and transcripts suggested that Nitrosospira cluster 3-like AOB and Nitrososphaera viennensis-like AOA were significantly stimulated in N-fertilized fresh soils. Real-time quantitative PCR demonstrated that the significant increase of AOA and AOB in fresh soils upon nitrogen fertilization could be preserved in the air-dried soils. DNA-based stable-isotope probing (SIP) further revealed the greatest labeling of Nitrosospira cluster 3-like AOB and Nitrosospira viennensis-like AOA, despite the strong advantage of AOB over AOA in the N-fertilized soils. Nitrobacter-like nitrite-oxidizing bacteria (NOB) played more important roles than Nitrospira-like NOB in urea-amended SIP microcosms, while the situation was the opposite under field conditions. Our results suggest that long-term fertilization selected for physiologically versatile AOB and AOA that could have been adapted to a wide range of substrate ammonium concentrations. It also provides compelling evidence that the dominant communities of transcriptionally active nitrifiers under field conditions were largely similar to those revealed in 13C-labeled microcosms.IMPORTANCE The role of manipulated microcosms in microbial ecology has been much debated, because they cannot entirely represent the in situ situation. We collected soil samples from 20 field plots, including 5 different treatments with and without nitrogen fertilizers for 22 years, in order to assess active nitrifying communities by in situ transcriptomics and microcosm-based stable-isotope probing. The results showed that chronic N enrichment led to competitive advantages of Nitrosospira cluster 3-like AOB over N. viennensis-like AOA in soils under field conditions. Microcosm labeling revealed similar results for active AOA and AOB, although an apparent discrepancy was observed for nitrite-oxidizing bacteria. This study suggests that the soil microbiome represents a relatively stable community resulting from complex evolutionary processes over a large time scale, and microcosms can serve as powerful tools to test the theory of environmental filtering on the key functional microbial guilds.

[Ammonia Oxidation in a Neutral Purple Soil Measured by the 13C-DNA-SIP Method].

Increasing evidence suggests that ammonia oxidation in acidic soils is primarily catalyzed by ammonia-oxidizing archaea (AOA), while ammonia-oxidizing bacteria (AOB) drive ammonia oxidation in neutral and alkaline soils in which AOA overwhelmingly outnumber AOB. Therefore, neutral purple soil with a pH of 7.2 was selected to study the composition of the active ammoxidation microbial community with a stable isotope nucleic acid probe technique combined with cloning sequencing. Results showed that the nitrification rate was 9.68 mg·(kg·d)-1, and AOA and AOB were abundant in neutral purple soils. By using DNA-based stable isotope probing (SIP), we gathered strong evidence of archaeal ammonia oxidation by AOA and AOB. Phylogenetic analysis indicated that the Nitrosospira Cluster 3a.1 AOB was dominant in terms of quantity at 0 days, and the Nitrosospira Cluster 3a.2 only accounted for a small part. After 56 days of cultivation, the Nitrosospira Cluster 3a.2 replaced the Nitrosospira Cluster 3a.1 as the active AOB that dominated ammonia oxidation. The AOA that predominated quantitatively at day 0 was Nitrososphaera Subcluster 9, but after cultivation this became Nitrososphaera Subcluster 3.2/3.3. Thus, the community structure of AOA and AOB changed. Active autotrophic nitrification was found in this neutral purple soil. Sequencing analysis of the 13C-labeled DNA provided robust evidence that both archaea and bacteria played important roles in the nitrification and not all ammonia oxidizers in native soil were active in the nitrification. Phylogenetic analysis clearly showed that the dominant active archaea and bacteria during the incubation were affiliated with Nitrososphaera Subcluster 3.2/3.3 within the soil group 1.1b lineage and Nitrosospira Cluster 3a.2, respectively, which were different from the dominant ammonia oxidizers at the beginning of the incubation. These results suggest that the community structure of ammonia oxidizers can shift quickly upon changes in the substrate availability in soils.

Profile Changes in the Soil Microbial Community When Desert Becomes Oasis.

The conversion of virgin desert into oasis farmland creates two contrasting types of land-cover. During oasis formation with irrigation and fertilizer application, however, the changes in the soil microbial population, which play critical roles in the ecosystem, remain poorly understood. We applied high-throughput pyrosequencing to investigate bacterial and archaeal communities throughout the profile (0-3 m) in an experimental field, where irrigation and fertilization began in 1990 and cropped with winter wheat since then. To assess the effects of cultivation, the following treatments were compared with the virgin desert: CK (no fertilizer), PK, NK, NP, NPK, NPKR, and NPKM (R: straw residue; M: manure fertilizer). Irrigation had a greater impact on the overall microbial community than fertilizer application. The greatest impact occurred in topsoil (0-0.2 m), e.g., Cyanobacteria (25% total abundance) were most abundant in desert soil, while Actinobacteria (26%) were most abundant in oasis soil. The proportions of extremophilic and photosynthetic groups (e.g., Deinococcus-Thermus and Cyanobacteria) decreased, while the proportions of R-strategy (e.g., Gammaproteobacteria including Xanthomonadales), nitrifying (e.g., Nitrospirae), and anaerobic bacteria (e.g., Anaerolineae) increased throughout the oasis profile. Archaea occurred only in oasis soil. The impact of fertilizer application was mainly reflected in the non-dominant communities or finer taxonomic divisions. Oasis formation led to a dramatic shift in microbial community and enhanced soil enzyme activities. The rapidly increased soil moisture and decreased salt caused by irrigation were responsible for this shift. Furthermore, difference in fertilization and crop growth altered the organic carbon contents in the soil, which resulted in differences of microbial communities within oasis.

Long-term nitrogen fertilization of paddy soil shifts iron-reducing microbial community revealed by RNA-(13)C-acetate probing coupled with pyrosequencing.

Iron reduction is an important biogeochemical process in paddy soils, yet little is known about the microbial coupling between nitrogen and iron reduction. Here, we investigated the shift of acetate-metabolizing iron-reducers under long-term nitrogen fertilization using (13)C-acetate-based ribosomal RNA (rRNA)-stable isotope probing (SIP) and pyrosequencing in an incubation experiment, and the shift of putative iron-reducers in original field samples were investigated by 16S rRNA gene-based pyrosequencing. During SIP incubations, in the presence of iron(III) oxyhydroxides, more iron(II) formation and less methane production were detected in nitrogen-fertilized (N) compared with non-fertilized (NF) soil. In (13)C-rRNA from microcosms amended with ferrihydrite (FER), Geobacter spp. were the important active iron-reducers in both soils, and labeled to a greater extent in N (31% of the bacterial classified sequences) than NF soils (11%). Pyrosequencing of the total 16S rRNA transcripts from microcosms at the whole community level further revealed hitherto unknown metabolisms of potential FER reduction by microorganisms including Pseudomonas and Solibacillus spp. in N soil, Dechloromonas, Clostridium, Bacillus and Solibacillus spp. in NF soil. Goethite (GOE) amendment stimulated Geobacter spp. to a lesser extent in both soils compared with FER treatment. Pseudomonas spp. in the N soil and Clostridium spp. in the NF soil may also be involved in GOE reduction. Pyrosequencing results from field samples showed that Geobacter spp. were the most abundant putative iron-reducers in both soils, and significantly stimulated by long-term nitrogen fertilization. Overall, for the first time, we demonstrate that long-term nitrogen fertilization promotes iron(III) reduction and modulates iron-reducing bacterial community in paddy soils.

[Ex-situ remediation of PAHs contaminated site by successive methyl-beta-cyclodextrin enhanced soil washing].

Polycyclic aromatic hydrocarbon (PAH) polluted sites caused by abandoned coking plants have attracted great attentions. This study investigated the feasibility of using methyl-beta-cyclodextrin (MCD) solution to enhance ex situ soil washing for extracting PAHs. Treatment with elevated temperature (50 degrees C) in combination with ultrasonication (35 kHz, 30 min) at 100 g x L(-1) was effective. It was found that 96.7% +/- 2.4% of 3-ring PAH, 89.7% +/- 3.2% of 4-ring PAH, 76.3% +/- 2.2% of 5 (+6)-ring PAH and 91.3% +/- 3.1% of total PAHs were removed from soil after five successive washing cycles. The desorption kinetics of PAHs from contaminated soil was determined before and after successive washings. The 400 h Tenax extraction of PAHs from soil was decreasing gradually with increasing washing times. Furthermore, the F(r), F(sl), k(r), k(sl) and k(vl) were significantly lower than those of CK (P < 0.01). Therefore, considering the removal efficiency and potential environmental risk after soil )ashing, successive washing three times was selected as a reasonable parameter. These results have practical implications for site risk assessment and cleanup strategies.