DNA-based Stable Isotope Probing Research Articles

Different ammonia oxidizers are responsible for nitrification in two neutral paddy soils

Human intervention in agriculture, such as farming and fertilization, could result in major changes of ammonia availability that shapes niche-specific occupied communities of soil ammonia oxidizers. The aim of this study was to investigate nitrification and active ammonia oxidizers in N-rich and N-limited soils, which were induced by different management practices. Here, we used DNA-based stable isotope probing (DNA-SIP) microcosm to decipher active ammonia-oxidizing archaea (AOA) and bacteria (AOB) phylotypes involved in ammonia oxidation in the cultivated and uncultivated soils. The results showed that net nitrification rate in the cultivated soil was significantly higher than in the uncultivated soil (6.19 and 1.40 mg N kg−1dry soil d-1, respectively). Growth of soil AOB and AOA during incubation occurred in the cultivated soil, while in the uncultivated soil, only AOA significantly increased after 56 days. Combining DNA-SIP and sequencing results, we gathered evidence of ammonia oxidation by Nitrososphaera-like AOA, in addition to active AOB, within the classic Nitrosospira group in the cultivated soil. For the uncultivated soil, ammonia oxidation was driven by Nitrososphaera-like and Nitrosopumilus-like AOA, rather than AOB. Our results demonstrate the potential of ammonium concentration for shaping the communities of active AOA and AOB, which imply that ammonia-dependent “diversification” may be important strategies for niche differentiation of sympatric AOA/AOB under different field management conditions.

The dominance of non-halophilic archaea in autotrophic ammonia oxidation of activated sludge under salt stress: A DNA-based stable isotope probing study

Dynamics of nitrification activity, ammonia-oxidizing archaea (AOA) and bacteria (AOB) abundance and active ammonia oxidizers of activated sludge were explored under different salinities. Results showed that specific ammonium oxidation rates were significantly negative with increasing salinity. The responses of AOA and AOB populations to salt stress were distinct. AOA abundance decreased at moderate salinities (2.5, 5 and 7 g L−1) and increased at high salinities (10, 15, 20 and 30 g L−1), while AOB abundance showed opposite tendency. DNA-based stable isotope probing assays indicated AOA exclusively dominated active ammonia oxidation of test samples under different salinities. The active AOA communities retrieved were all non-halophilic and regulated by salinities. Candidatus Nitrosocosmicus exaquare and Ca. Nitrosocosmicus franklandus were the predominantly active AOA in both salt-free and salt-containing microcosms, while 13C-labeled Nitrososphaera viennensis and Ca. Nitrososphaera gargensis were only retrieved from the microcosms amended with 0 and 30 g L−1 salinity, respectively.

Stable-Isotope Probing-Enabled Cultivation of the Indigenous Bacterium Ralstonia sp. Strain M1, Capable of Degrading Phenanthrene and Biphenyl in Industrial Wastewater.

To identify and obtain the indigenous degraders metabolizing phenanthrene (PHE) and biphenyl (BP) from the complex microbial community within industrial wastewater, DNA-based stable-isotope probing (DNA-SIP) and cultivation-based methods were applied in the present study. DNA-SIP results showed that two bacterial taxa (Vogesella and Alicyclobacillus) were considered the key biodegraders responsible for PHE biodegradation only, whereas Bacillus and Cupriavidus were involved in BP degradation. Vogesella and Alicyclobacillus have not been linked with PHE degradation previously. Additionally, DNA-SIP helped reveal the taxonomic identity of Ralstonia-like degraders involved in both PHE and BP degradation. To target the separation of functional Ralstonia-like degraders from the wastewater, we modified the traditional cultivation medium and culture conditions. Finally, an indigenous PHE- and BP-degrading strain, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by enrichment of the 16S rRNA gene and distinctive dioxygenase genes in the DNA-SIP experiment. Our study has successfully established a program for the application of DNA-SIP in the isolation of the active functional degraders from an environment. It also deepens our insight into the diversity of indigenous PHE- and BP-degrading communities.IMPORTANCE The comprehensive treatment of wastewater in industrial parks suffers from the presence of multiple persistent organic pollutants (POPs), such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), which reduce the activity of activated sludge and are difficult to eliminate. Characterizing and applying active bacterial degraders metabolizing multiple POPs therefore helps to reveal the mechanisms of synergistic metabolism and to improve wastewater treatment efficiency in industrial parks. To date, SIP studies have successfully investigated the biodegradation of PAHs or PCBs in real-world habitats. DNA-SIP facilitates the isolation of target microorganisms that pose environmental concerns. Here, an indigenous phenanthrene (PHE)- and biphenyl (BP)-degrading strain in wastewater, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by DNA-SIP. Our study provides a routine protocol for the application of DNA-SIP in the isolation of the active functional degraders from an environment.

Electron shuttles enhance anaerobic oxidation of methane coupled to iron(III) reduction

Anaerobic oxidation of methane (AOM) has recently been coupled with the reduction of insoluble electron acceptors such as iron minerals. However, effects of electron shuttles (ESs) on this process and the underlying coupling mechanisms remain not well understood. Here, we evaluated AOM-coupled ferrihydrite reduction by a mixed culture in the absence and presence of ESs. The results showed that ESs (AQS, flavin, HA and AQDS) significantly enhanced the rate (up to 7.4 times) of AOM-dependent ferrihydrite reduction compared with the control. The enhancements were linearly related with the electron transfer capacity of ESs. Illumina high-throughput sequencing and DNA-based stable isotope probing revealed that the AOM-coupled iron reduction depended on the syntrophic interaction of Methanobacterium and the partner bacteria. Methanobacterium as the dominant microorganism, did not assimilate methane into its biomasses. However, it played a crucial role in the partial oxidation of methane into an intermediate (i.e. propionate), which was then assimilated by the partner bacteria (e.g. Cellulomonas, Desulfovibrio, Actinotalea, etc.) for ferrihydrite reduction. This work suggests that ESs in natural environments can mitigate the methane emissions by facilitating the AOM process and biogeochemical cycles of iron.

Autotrophic archaeal nitrification is preferentially stimulated by rice callus mineralization in a paddy soil

Previous studies suggest that organic carbon (C) and nitrogen (N) can stimulate soil nitrification, but whether autotrophic or heterotrophic nitrification is stimulated and who are active nitrifiers for the nitrification activity is still in debate. We elucidated which nitrification dominated and the active nitrifiers during the decomposition of rice callus. 15N-labeled callus and acetylene (C2H2) inhibition were used to explore the autotrophic or heterotrophic nitrification during the decomposition of callus and DNA-based stable isotope probing (SIP) and high-throughput sequencing were used to investigate the active nitrifiers. Autotrophic nitrification dominated the nitrification activity, driven by oxidation of ammonia (NH3) produced from mineralization of the callus-derived organic N. Callus significantly stimulated nitrification activity, which was paralleled by changes in the abundance and community composition of AOA. DNA-SIP further demonstrated that the active AOA outnumbered their bacterial counterparts in the 13C-DNA from the soil with callus amendment. Phylogenetic analysis revealed the functional importance of soil fosmid 29i4-like and 54d9-like AOA within soil group 1.1b during the active nitrification with callus cells. NH3 released from the mineralization of callus was the main substrate for autotrophic nitrification and preferentially stimulated the growth of AOA within group 1.1b in the paddy soil.

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.

Dependency of biological nitrogen fixation on organic carbon in acidic mine tailings under light and dark conditions

Several ecological restoration techniques have been used to enhance the levels of available organic matter in mine tailings. However, the effect of organic carbon on nitrogen fixation remains poorly understood. In this study, nitrogen fixation in two mine tailings treated with glucose was analyzed using 15N isotope labeling, pyrosequencing and DNA-based stable isotope probing (SIP) to identify nitrogen fixers and their activity under organic carbon addition (0.8 mg glucose-C/g soils). Following glucose addition, the total nitrogen content increased significantly (1.6–5.4% of initial N content), and the 15N content increased from 0.89 ± 0.20 mg·kg−1 to 37.08 ± 3.34 mg·kg−1 during a 28-day incubation period. In addition, glucose-stimulated nitrogen fixation in dark conditions was 8.53-fold (Shuimuchong) and 1.22-fold (Yangshanchong) higher than that in light conditions. At the same time, the addition of glucose led to distinctly different phylotypes of the bacterial communities and an increase in the abundance of the nifH gene in the two mine tailings. 13C-glucose SIP showed that the highest copy number of the 16S rRNA and nifH genes in the 13C-glucose-labeled treatment peaked in the heavy fraction, with a buoyant density of 1.737 g·mL−1. However, the pyrosequencing results revealed that the vast majority of the nifH genes were unique and belonged to yet uncultured bacteria, while the potentially known groups were associated with Azotobacter, acidophilic Leptospirillum and Pseudanabaena, accounting for 13.83% of all the total nifH genes. These results indicate that the addition of organic carbon may increase the nitrogen content by stimulating nitrogen fixers, highlighting the vital roles of organic carbon in increasing nitrogen accumulation during the restoration of mine tailings.

Soil extractable organic C and N contents, methanotrophic activity under warming and degradation in a Tibetan alpine meadow

The Tibetan alpine meadow ecosystem is an important part of the Eurasian grasslands and is experiencing intense warming at approximately three times the global warming rate and rapid degradation. However, little is known about the effect of warming and degradation and their interactions on ecosystem functions like soil carbon (C) and nitrogen (N) pools and methane (CH4) uptake in this region. Here, we selected a long-term simulated warming site in a Tibetan alpine meadow with different degradation levels. After 4 years of warming, we analyzed soil total C (TC) and total N (TN) contents, extractable organic C (EOC) and extractable organic N (EON) contents as well as methanotrophic activity, abundance and community structure. Soil EOC and EON contents were measured through hot water extraction, whereas methanotrophic activity was measured along a gradient of CH4 concentrations in laboratory incubations. Michaelis–Menten kinetics analysis [maximal rate of velocity (Vmax) and half-saturation constant (Km)] was used to quantify changes in methanotrophic activity among the treatments. Active methanotrophic communities in the natural soils were measured via DNA-based stable isotope probing (SIP). The results showed that warming significantly increased soil EON contents, whereas degradation significantly decreased soil TC and TN contents, and EOC and EON contents. Methanotrophic activity was significantly lower at different levels of degradation but no significant effects were observed under warming. Changes in soil methanotrophic abundance among the treatments followed the same trend, but warming and degradation had no interactive effects on methanotrophic activity and abundance. Active methanotrophic communities in the natural meadow soils were dominated by Methylosinus (a Type II methanotroph). In conclusion, our results indicate that soil C and N pools and CH4 oxidation capability were influenced more strongly by degradation than warming. However, warming may have an additional effect on the stability of these important ecosystem processes, regardless of degradation in this region.

Identification and Characterization of a Dominant Sulfolane-Degrading Rhodoferax sp. via Stable Isotope Probing Combined with Metagenomics

Sulfolane is an industrial solvent and emerging organic contaminant affecting groundwater around the world, but little is known about microbes capable of biodegrading sulfolane or the pathways involved. We combined DNA-based stable isotope probing (SIP) with genome-resolved metagenomics to identify microorganisms associated with sulfolane biodegradation in a contaminated subarctic aquifer. In addition to 16S rRNA gene amplicon sequencing, we performed shotgun metagenomics on the 13C-labeled DNA to obtain functional and taxonomic information about the active sulfolane-degrading community. We identified the primary sulfolane degrader, comprising ~85% of the labeled community in the amplicon sequencing dataset, as closely related to Rhodoferax ferrireducens strain T118. We obtained a 99.8%-complete metagenome-assembled genome for this strain, allowing us to identify putative pathways of sulfolane biodegradation. Although the 4S dibenzothiophene desulfurization pathway has been proposed as an analog for sulfolane biodegradation, we found only a subset of the required genes, suggesting a novel pathway specific to sulfolane. DszA, the enzyme likely responsible for opening the sulfolane ring structure, was encoded on both the chromosome and a plasmid. This study demonstrates the power of integrating DNA-SIP with metagenomics to characterize emerging organic contaminant degraders without culture bias and expands the known taxonomic distribution of sulfolane biodegradation.

Pyrite oxidization accelerates bacterial carbon sequestration in copper mine tailings

Abstract. Polymetallic mine tailings have great potential as carbon sequestration tools to stabilize atmospheric CO2 concentrations. However, previous studies focused on carbonate mineral precipitation, whereas the role of autotrophic bacteria in mine tailing carbon sequestration has been neglected. In this study, carbon sequestration in two samples of mine tailings treated with FeS2 was evaluated using 13C isotope, pyrosequencing and DNA-based stable isotope probing (SIP) analyses to identify carbon fixers. Mine tailings treated with FeS2 exhibited a higher percentage of 13C atoms (1.76±0.06 % for Yangshanchong and 1.36±0.01 % for Shuimuchong) than did controls over a 14-day incubation, which emphasized the role of autotrophs in carbon sequestration with pyrite addition. Pyrite treatment also led to changes in the composition of bacterial communities, and several autotrophic bacteria increased, including Acidithiobacillus and Sulfobacillus. Furthermore, pyrite addition increased the relative abundance of the dominant genus Sulfobacillus by 8.86 % and 5.99 % in Yangshanchong and Shuimuchong samples, respectively. Furthermore, DNA SIP results indicated a 8.20–16.50 times greater gene copy number for cbbL than cbbM in 13C-labeled heavy fractions, and a Sulfobacillus-like cbbL gene sequence (cbbL-OTU1) accounted for 30.11 %–34.74 % of all cbbL gene sequences in 13C-labeled heavy fractions of mine tailings treated with FeS2. These findings highlight the importance of the cbbL gene in bacterial carbon sequestration and demonstrate the ability of chemoautotrophs to sequester carbon during sulfide mineral oxidation in mine tailings. This study is the first to investigate carbon sequestration by autotrophic bacteria in mine tailings through the use of isotope tracers and DNA SIP.

Stable isotope probing of active methane oxidizers in rice field soils from cold regions

DNA-based stable isotope probing (DNA-SIP) was employed to establish direct link between methane oxidation activity and the taxonomic identity of active methanotrophs in three rice field soils from Jian-San-Jiang (one baijiang origin soil, JB and one meadow origin soil, JM) and Qing-An (meadow origin soil, QA) districts in Northeastern China. Following microcosm incubation under 1% v/v13CH4 condition, soil organic 13C atom percent significantly increased from background 1.08 to 1.21% in average, indicating the biomass synthesis supported by methanotrophy. Real-time PCR analysis of methanotroph-specific biomarker pmoA genes of the buoyant density for DNA gradient, following the ultracentrifugation of the total DNA extracted from SIP microcosms, indicated an enrichment of methanotroph genomes in 13C-labeled DNA. It suggested propagation of microbial methane oxidizers in soils. High-throughput sequencing of 16S rRNA and pmoA genes from 13C-labeled DNA further revealed a diverse guild of both type I and II methanotrophs in all three soils. Specifically, Methylobacter-affiliated type I methanotrophs dominated the methanotrophic activity in JB and JM soils, whereas Methylocystis-affiliated type II methanotrophs dominated QA soil. This implied the physiological diversification of soil methanotrophs that might be due to constant environmental fluctuations in paddies.

Bacterial communities involved directly or indirectly in the anaerobic degradation of cellulose

To determine bacterial communities involved, directly or indirectly, in the anaerobic degradation of cellulose, we conducted a microcosm experiment with soil treated with 13C-cellulose, 12C-cellulose, or without cellulose with analyses of DNA-based stable isotope probing (DNA-SIP), real-time quantitative PCR, and high-throughput sequencing. Firmicutes, Actinobacteria, Verrucomicrobia, and Fibrobacteres were the dominant bacterial phyla-degrading cellulose. Generally, bacteria possessing gene-encoding enzymes involved in the degradation of cellulose and hemicellulose were stimulated. Phylotypes affiliated to Geobacter were also stimulated by cellulose, probably due to their role in electron transfer. Nitrogen-fixing bacteria were also detected, probably due to the decreased N availability during cellulose degradation. High-throughput sequencing showed the presence of bacteria not incorporating 13C and probably involved in the priming effect caused by the addition of cellulose to soil. Collectively, our findings revealed that a more diverse microbial community than expected directly and indirectly participated in anaerobic cellulose degradation.

Pentachlorophenol alters the acetate-assimilating microbial community and redox cycling in anoxic soils

Acetate is a common electron donor that can drive microbial reductive processes in anaerobic environments. Apart from acting as a terminal electron acceptor, chlorinated organic pollutant of pentachlorophenol (PCP) has antimicrobial properties but little is known about its effect on anaerobic microbial populations during bioremediation. To elucidate the effect of PCP on the anaerobic microbial community, DNA-based stable isotope probing was performed using 13C-acetate as a substrate in two depths (0–20 cm and 80–100 cm) of mangrove soils. The addition of PCP had little influence on Fe(III) reduction, but dramatically inhibited the SO42− reduction, resulting in a high emission of CH4 under anoxic conditions. Correspondingly, PCP significantly affected the composition of prokaryotic 13C-labeled OTUs (Operational Taxonomic Units) at both soil depths. Members of Clostridiales (Firmicutes) and Burkholderiales (Betaproteobacteria) were dominant 13C-acetate utilizers and potential PCP degraders. The PCP addition decreased sulfate reduction process through inhibition of classical sulfate- and sulfur-reducing bacteria belonging to families of Desulfarculaceae, Desulfobulbaceae and Desulfobacteraceae. Our study showed that indigenous microbial communities associated with terminal electron-accepting pathways (e.g. Fe3+ and SO42− reduction) changed differently during PCP dechlorination. These findings suggest a great impact of chlorinated pollutants on the soil biogeochemical processes.

The complex interactions between novel DEHP-metabolising bacteria and the microbes in agricultural soils

The indigenous microorganisms with the ability of metabolising di-(2-ethylhexyl) phthalate (DEHP) in agricultural soils and their interactions with non-degrading microbes were revealed by DNA-based stable isotope probing coupled with molecular ecological network. Aside from the previously reported DEHP degraders (family Planococcaceae and genus Sphingobacterium), five OTUs representing bacteria affiliated with genus Brevundimona, class Spartobacteria, genus Singulisphaera, genus Dyella and class Ktedonobacteria were linked with DEHP biodegradation. The analysis of the constructed ecological network based on soil microbial communities demonstrated the negative relationships between DEHP degraders and the dominant family Oxalobacteraceae in soils. Additionally, two cultivable bacteria isolated from the same soils, Rhizobium-1 and Ensifer-1, had strong capabilities in degrading DEHP but their involvement in in situ DEHP degradation was questioned, as their DNA was not labelled with 13C from DEHP. These findings provide deeper understanding on the indigenous DEHP-degrading communities and will benefit the remediation of phthalate esters contaminated soils.

DNA-Based Stable Isotope Probing.

Microbiomes on Earth are often considered the most heterogeneous biological entities, but their vital roles in driving global biogeochemical cycles often remain elusive. DNA-based stable isotope probing (DNA-SIP) provides a powerful means to establish a direct link between biogeochemical processes and the taxonomic identities of active microorganisms involved in the processes. Combined with high-throughput sequencing, it significantly aids in deciphering ecophysiological functions of active microorganisms at the level of microbial communities. DNA-SIP relies solely on the propagation of targeted microbial communities, during which the entire genomes of daughter cells are synthesized and increasingly 13C-labeled. This growth on 13C-labeled substrate in association with cell division provides solid evidence for the functional importance and metabolic potential of targeted microorganisms. The essential prerequisite for a successful DNA-SIP experiment is the identification, with confidence, of isotopically enriched 13C-DNA, of which the amount is generally too low to allow for the direct measurement of 13C atomic percent of nucleic acid. The 13C labeling can be readily identified in the fractionated DNA by quantification of functional genes specific to the known targeted microorganisms, and by high-throughput sequencing of the total microbial communities via 16S rRNA genes without prior knowledge of which microorganisms are 13C-labeled (i.e., highly enriched in the heavy fractions relative to 12C (natural isotope abundance) control treatments). In this chapter, the protocol for obtaining DNA highly enriched in heavy isotope is presented using diazotrophic methanotrophs in a paddy soil as a case study.

Plant Nutrient Resource Use Strategies Shape Active Rhizosphere Microbiota Through Root Exudation.

Plant strategies for soil nutrient uptake have the potential to strongly influence plant–microbiota interactions, due to the competition between plants and microorganisms for soil nutrient acquisition and/or conservation. In the present study, we investigate whether these plant strategies could influence rhizosphere microbial activities via root exudation, and contribute to the microbiota diversification of active bacterial communities colonizing the root-adhering soil (RAS) and inhabiting the root tissues. We applied a DNA-based stable isotope probing (DNA-SIP) approach to six grass species distributed along a gradient of plant nutrient resource strategies, from conservative species, characterized by low nitrogen (N) uptake, a long lifespans and low root exudation level, to exploitative species, characterized by high rates of photosynthesis, rapid rates of N uptake and high root exudation level. We analyzed their (i) associated microbiota composition involved in root exudate assimilation and soil organic matter (SOM) degradation by 16S-rRNA-based metabarcoding. (ii) We determine the impact of root exudation level on microbial activities (denitrification and respiration) by gas chromatography. Measurement of microbial activities revealed an increase in denitrification and respiration activities for microbial communities colonizing the RAS of exploitative species. This increase of microbial activities results probably from a higher exudation rate and more diverse metabolites by exploitative plant species. Furthermore, our results demonstrate that plant nutrient resource strategies have a role in shaping active microbiota. We present evidence demonstrating that plant nutrient use strategies shape active microbiota involved in root exudate assimilation and SOM degradation via root exudation.

Response of symbiotic and asymbiotic nitrogen-fixing microorganisms to nitrogen fertilizer application

Biological nitrogen fixation (BNF) plays an important role in nitrogen cycling by transferring atmospheric dinitrogen to the soil. BNF is performed by symbiotic and asymbiotic nitrogen-fixing microorganisms. However, the abundance, activity, and community structure of diazotrophs under different nitrogen fertilizer application rates and how root exudates influence diazotrophs remain unclear. 15N-N2 and 13C-CO2 labeling, DNA-based stable isotope probing (SIP), and molecular biological techniques were used in this study. The abundance, activity, and structure of symbiotic and asymbiotic diazotrophs under different nitrogen fertilizer applications in paddy soil were investigated. We found that the nitrogen fixation capacity in milk vetch (Astragalus sinicus L.) and nifH gene abundance in the root nodules were significantly higher in the low-nitrogen treatment than in the control (zero) and high-nitrogen treatments. Nitrogen-fixing bacteria were abundant in the soils with a high biodiversity. Soil nifH gene sequences were dominated by α-, β-, and δ-proteobacteria, as well as by Cyanobacteria. The relative abundance of α-proteobacteria was lower, and the relative abundance of Cyanobacteria was higher under high nitrogen. Incubation of soil with 13CO2 and subsequent DNA-SIP analysis demonstrated that OTU65 from α-proteobacteria was relatively more abundant in heavy fractions of the 13C-labeled soils than that in the unlabeled soils, indicating that α-proteobacteria may prefer rhizodeposition carbon to other organic carbon. However, OTU24 and OTU73 from δ-proteobacteria had relatively high abundances in light fractions both in the 13C-labeled and unlabeled samples, indicating that δ-proteobacteria may prefer other soil organic carbon to rhizodeposition carbon. The results suggested that soil N availability and rhizodeposition strongly modified the microbial communities of nitrogen-fixing bacteria. Moderate nitrogen application increased the symbiotic biological N fixing activity in the Astragalus sinicus L. rhizosphere. The BNF activity in the legume-rhizobia system is regulated by the exchange of organic C and N nutrient between the host plant and N-fixing bacteria.

Temporal heterogeneity and temperature response of active ammonia-oxidizing microorganisms in winter in full-scale wastewater treatment plants

Ammonia-oxidizing microorganisms (AOMs) play important roles in nitrogen removal, but their metabolic activity in winter months in wastewater treatment plants (WWTPs) remains unclear. Here, two full-scale WWTPs were selected, and a total of 48 DNA-based stable isotope probing (DNA-SIP) microcosms of activated sludge samples at different winter months (November-January) and temperatures (13 °C, 25 °C and 37 °C) were constructed to evaluate the temporal heterogeneity and temperature response of active AOMs. The results provided direct evidence that the temporal heterogeneity of AOMs’ activity was not obvious in winter months. In general, ammonia-oxidizing archaea (AOA) and bacteria (AOB) co-participated in the active ammonia oxidation in three winter months at in situ temperature (13 °C) in two WWTPs, and AOA were the dominant active AOMs, even though AOB outnumbered AOA in seed sludge. In contrast, higher peaks of AOA in “heavy” fractions at 25 °C and 37 °C also provided compelling evidence for the dominant contribution of AOA to active ammonia oxidation under higher temperatures. The Illumina sequencing of 13C-DNA obtained from the DNA-SIP microcosms at 13 °C in three winter months further suggest that Candidatus Nitrososphaera evergladensis and Candidatus Nitrosocosmicus exaquare were the dominant active AOA in two WWTPs, while the active AOB were dominated by Nitrosomonas oligotropha. At higher temperatures, active AOA shifted to Ca. N. evergladensis and Candidatus Nitrososphaera gargensis, and Nitrosomonas marina, Nitrosomonas europaea and Nitrosomonas communis dominated in the active AOB. Overall, AOA rather than AOB dominated the active ammonia oxidation in winter months and at higher temperatures in the full-scale WWTPs tested.

Variance in bacterial communities, potential bacterial carbon sequestration and nitrogen fixation between light and dark conditions under elevated CO2 in mine tailings

This study is the first to show the response of bacterial communities with primary carbon and nitrogen fixers to elevated CO2 (eCO2) in light and dark conditions based on 6 months of culture growth. Carbon sequestration and nitrogen fixation were analyzed by 13C and 15N isotope labeling using 13C-labeled CO2 and 15N-labeled N2, followed by pyrosequencing and DNA-based stable isotope probing (SIP) to identify carbon fixers and nitrogen fixers. The results indicated that eCO2 decreased the Chao 1 richness, and the eCO2-light treatment exhibited the highest Shannon diversity. In addition, eCO2 (in either light or dark conditions) greatly increased the relative abundances of bacteria belonging to the classes Betaproteobacteria and Alphaproteobacteria. The 13C atom % in the mine tailings increased from 1.108 to 1.84 ± 0.11 under light conditions and 1.52 ± 0.17 under dark conditions after 6 months of culture growth. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I-coding gene (cbbL) copy numbers were 164.30-fold and 40.36-fold higher than RubisCO form II-coding gene (cbbM) copy numbers in the heavy fractions with a buoyant density of 1.7388 g·mL−1 relative to the buoyant density gradients of DNA fractions obtained under eCO2-light and eCO2-dark treatment, respectively. The Proteobacteria-like cbbL genes were dominant in the carbon fixers. In addition, the 15N atom % in the mine tailings increased from 0.366 to 0.454 ± 0.021 in light conditions and 0.437 ± 0.018 in dark conditions. Furthermore, uncultured nitrogen-fixing bacteria were the dominant nitrogen fixers in light conditions, and bacteria harboring the Bradyrhizobium-like nifH and Leptospirillum-like nifH genes were the dominant nitrogen fixers in dark conditions. These first data for a mine tailing ecosystem are inconsistent with those obtained for a range of other ecosystems, in which the effects of CO2 were limited to several nonphotoautotrophic communities and different nitrogen fixers.

The more important role of archaea than bacteria in nitrification of wastewater treatment plants in cold season despite their numerical relationships

Nitrification failure of wastewater treatment plants (WWTPs) in cold season calls into investigations of the functional ammonia-oxidizing microorganisms (AOMs). In this study, we report the abundance of ammonia-oxidizing archaea (AOA), bacteria (AOB) and complete ammonia-oxidizing (comammox) Nitrospira in 23 municipal WWTPs in cold season, and explore the correlations between AOMs abundance and their relative contribution to nitrification. The copy numbers of AOA and AOB amoA gene ranged from 2.42 × 107 to 2.47 × 109 and 5.54 × 106 to 3.31 × 109 copies/g sludge, respectively. The abundance of amoA gene of Candidatus Nitrospira inopinata, an important strain of comammox Nitrospira, was stable with averaged abundance of 8.47 × 106 copies/g sludge. DNA-based stable isotope probing (DNA-SIP) assays were conducted with three typical WWTPs in which the abundance of AOA was lower than, similar to and higher than that of AOB, respectively. The results showed that considerable 13C-assimilation by AOA was detected during active nitrification in all WWTPs, whereas just a much lesser extent of 13C-incorporation by AOB and comammox Nitrospira was found in one WWTP. High-throughput sequencing with 13C-labeled DNA also showed the higher reads abundance of AOA than AOB and comammox Nitrospira. Nitrososphaera viennensis was the dominant active AOA, while Nitrosomonas oligotropha and Nitrosomonas europaea were identified as active AOB. The results obtained suggest that AOA, rather than AOB and comammox Nitrospira, dominate ammonia oxidation in WWTPs in cold season despite the numerical relationships of AOMs.