Resilience of the rhizosphere Pseudomonas and ammonia-oxidizing bacterial populations during phytoextraction of heavy metal polluted soil with poplar

We assessed the effects of phytoextraction on the dynamics of Pseudomonas spp. and ammonia-oxidizing bacterial populations in a heavy metal (HM) polluted soil. Hybrid poplars were grown in two-compartment root containers with a medium history (> 4 years) of HM pollution for 13 weeks. Bulk and poplar rhizosphere soils were analysed by denaturing gradient gel electrophoresis (DGGE) of Pseudomonas (sensu stricto) 16S rRNA and amoA gene fragments. DGGE patterns revealed that Pseudomonas and amoA-containing populations in the contaminated soils were markedly different from those in the uncontaminated soils. Pseudomonas and amoA profiles appeared to be stable over time in the bulk soils. In contrast, contaminated rhizosphere soils revealed a clear shift of populations with removal of HM becoming similar or at least shifted to the populations of the uncontaminated soils. The effect of phytoextraction was, however, not evident in the bulk samples, which still contained large amounts of HM. Cloning and sequencing of dominant DGGE bands revealed that Pseudomonas were phylogenetically related to the Pseudomonas fluorescens cluster and the amoA sequences to Nitrosospira spp. At the last sampling, major prominent band sequences from contaminated rhizosphere soils were identical to sequences obtained from uncontaminated rhizosphere soils, indicating that the populations were dominated by the same phylotypes. This study suggests that two taxonomically different populations are able to recover after the relief of HM stress by phytoextraction practices, whereas bulk microbial activities still remained depressed.     

Abundance and Diversity of Ammonia-Oxidizing Prokaryotes in the Root–Rhizosphere Complex of Miscanthus × giganteus Grown in Heavy Metal-Contaminated Soils

Mine wastes have been considered as a source of heavy metal (HM) contamination in the environment and negatively impact many important ecosystem services provided by soils. Plants like Miscanthus, which tolerate high HM concentrations in soil, are often used for phytoremediation and provide the possibility to use these soils at least for the production of energy crops. However, it is not clear if plant growth at these sites is limited by the availability of nutrients, mainly nitrogen, as microbes in soil might be affected by the contaminant. Therefore, in this study, we investigated in a greenhouse experiment the response of ammonia-oxidizing microbes in the root–rhizosphere complex of Miscanthus × giganteus grown in soils with different levels of long-term arsenic (As) and lead (Pb) contamination. Quantitative PCR of the ammonia monooxigenease gene (amoA) was performed to assess the abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) at two different points of plant growth. Furthermore, bulk soil samples before planting were analyzed. In addition, terminal restriction fragment length polymorphism (T-RFLP) analysis was used to investigate the diversity of archaeal amoA amplicons. Whereas high concentrations of As and Pb in soil (83 and 15?g/kg, respectively) resulted independent from plant growth in a clear reduction of AOA and AOB compared to the control soils with lower HM contents, in soils with contamination levels of 10?g/kg As and 0.2?g/kg Pb, only AOB were negatively affected in bulk soil samples. Diversity analysis of archaeal amoA genes revealed clear differences in T-RFLP patterns in response to the degree of HM contamination. Therefore, our results could clearly prove the different response patterns of AOA and AOB in HM-contaminated soils and the development of archaeal amoA phylotypes which are more tolerant towards HMs in soil samples from the areas that were impacted the most by mining waste, which could contribute to functional redundancy of ammonia-oxidizing microbes in soils and stability of nitrification pattern.     

Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the α-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.     

Interactions between plants and associated bacteria in soils contaminated with heavy metals

Interactions were studied between oat (Avena sativa) and two bacterial species, Bacillus subtilis and Pantoea agglomerans, in soils contaminated with heavy metals (HM), cadmium (50 mg/kg), and lead (200 mg/kg). Exposure to HM resulted in decreased (by 30–50%) length, mass, and ratio of shoot to root dimensions. Inoculation with bacteria lead to restoration and further enhancement of plant productivity, raising it above the level achieved via inoculation of oat in uncontaminated soils. It also reduced HM accumulation by plants. Pure cultures of P. agglomerans accumulate HM more intensively than those of B. subtilis (adsorbing activity was studied for both cells and extracellular metabolites). After the introduction of bacteria, lead, and cadmium content in soil decreased four- to fivefold and two- to threefold, respectively. Protection from HM is attributable to reorganizations in the populations of root-associated bacteria: cell number increases in the rhizoplane while decreasing in the rhizosphere.     

Effect of monensin feeding and withdrawal on populations of individual bacterial species in the rumen of lactating dairy cows fed high-starch rations

Population dynamics of ammonia-oxidizing bacteria (AOB) in a full-scale aerated submerged biofilm reactor for micropolluted raw water pretreatment was investigated using molecular techniques for a period of 1 year. The ammonia monooxygenase (amoA) gene fragments were amplified from DNA and RNA extracts of biofilm samples. Denaturing gradient gel electrophoresis (DGGE) profile based on the amoA messenger RNA approach exhibited a more variable pattern of temporal dynamics of AOB communities than the DNA-derived approach during the study. Phylogenetic analysis of excised DGGE bands revealed three AOB groups affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage, and an unknown Nitrosomonas group. The population size of betaproteobacterial AOB, quantified with 16S ribosomal RNA gene real-time polymerase chain reaction assay, ranged from 6.63 × 10⁵ to 2.67 × 10⁹ cells per gram of dry biofilm and corresponded to 0.23–1.8% of the total bacterial fraction. Quantitative results of amoA gene of the three specific AOB groups revealed changes in competitive dominance between AOB of the N. oligotropha lineage and N. communis lineage. Water temperature is shown to have major influence on AOB population size in the reactor by the statistic analysis, and a positive correlation between AOB cell numbers and ammonia removal efficiency is suggested (r = 0.628, P < 0.05).     

Applying a solute transfer model to phytoextraction: Zinc acquisition by Thlaspi caerulescens

Phytoextraction is the removal of metals from contaminated soils into harvested plant tissues. The rate of phytoextraction is governed by both soil and plant characteristics. Most effort has focused on identifying appropriate plants for phytoextraction, but the benefits from this effort will be marginal unless the metals are in phytoavailable forms in the rhizosphere. The concentration of a metal in the rhizosphere can be estimated using solute transfer models that incorporate: the metal concentration in the bulk soil solution, the buffer power of the soil, diffusion coefficient for the metal, water movement, root size and morphology, and the rate of entry of metal into the roots. Here a solute transfer model is developed to predict the concentration of Zn in the rhizosphere solution ([Zn]_ext) of Thlaspi caerulescens, a hyperaccumulator species that could be exploited for Zn phytoextraction. The model predicts that Zn accumulation by T. caerulescens is sub-optimal when the Zn concentration in the bulk soil solution is <27 M. Such a high [Zn]_ext is rare in contaminated agricultural soils, but is possible in the metalliferous substrates where T. caerulescens is endemic. Sensitivity analyses indicate that Zn diffusion is more important than transpiration-driven mass flow for Zn delivery to the root, implying that management of soil physical and hydrological properties will improve phytoextraction. Sensitivity analyses also imply that strategies to enhance the Zn absorption power of the root will not necessarily be successful for enhancing phytoextraction per se. Thus, research into enhancing Zn availability and mobility in soil will be as important as understanding and manipulating Zn uptake by plants. In general, such models can be used to identify constraints to efficient phytoextraction (whether plant or soil) and to determine whether commercial phytoextraction is feasible.     

Urease gene‐containing Archaea dominate autotrophic ammonia oxidation in two acid soils

The metabolic traits of ammonia‐oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA‐based stable isotope probing (SIP) and high‐throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea‐amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water‐amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time‐course incubations indicated that archaeal amoA genes were increasingly labelled by ¹³CO₂ in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the ¹³C‐DNA, and acetylene inhibition suggests that autotrophic growth of urease‐containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a‐associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.     

Rhizosphere microbial community and hexachlorocyclohexane degradative potential in contrasting plant species

The organochlorine 1,2,3,4,5,6 hexachlorocyclohexane (HCH) is a broad-spectrum insecticide that was used on a large-scale worldwide. The soil–plant–microbe system and its influence on HCH biodegradation are evaluated. A greenhouse experiment was designed to evaluate HCH dissipation and several microbial parameters among rhizosphere and bulk soil of two contrasting plants, Cytisus striatus (Hill) Rothm and Holcus lanatus L. Plants were grown for 180 days in three treatments: uncontaminated soil (control), uncontaminated soil inoculated with soil (3% w/w) from a HCH-contaminated site (INOC), and uncontaminated soil inoculated with soil (3% w/w) from the HCH-contaminated site and artificially contaminated to obtain 100 mg HCH kg⁻¹ dry soil (100HCH-INOC). At harvest, plant biomass, soil water-extractable organic C, pH and Cl concentration, rhizosphere microbial densities (total heterotrophs, ammonifiers, amylolytics) and C substrate utilization patterns, and degradation of α-, β-, δ- and γ-HCH isomers were determined in bulk and rhizosphere soils. Soil solution Cl concentration was determined every 30 days throughout the entire growth period. Results demonstrate that both Cytisus striatus and Holcus lanatus can grow in soils with up to 100 mg HCH kg⁻¹. An enhanced degradation of α-HCH, but not β- or δ-HCH, was observed in the rhizosphere. Significant changes in the microbial densities were observed between bulk and rhizosphere soils of Cytisus, and an increase in C source utilization indicated changes in community level physiological profiles (CLPP) in the rhizosphere of this species when grown in contaminated soils. HCH dissipation was also greater in soils planted with this species. In accordance, increases in soil extractable C, Cl concentration and acidity were greater at the rhizosphere of Cytisus. Concentration of Cl in soil solutions also indicates greater HCH dechlorination in soils planted with Cytisus than Holcus. Results suggest that phytostimulation of bacteria present or added to soil is a promising approach to cleaning HCH-contaminated sites, and especially for biodegradation of α-HCH.     

Effects of Long-Term exposure to Heavy Metals upon Rhizosphere Bacteria from Baia Mare Area (Maramureş County,Romania)

Abstract

The aim of this study was to investigate the extent of heavy metal (HM) pollution and its effect on microorganisms from rhizosphere soil in Baia Mare area (Maramure? County, Romania). Two sites with different contamination degrees were included in the study: one with a long history of mining activities and one within a drinking water safeguard zone. Rhizosphere soil samples were characterized with respect to physico-chemical parameters and the Cd, Cu, Pb and Zn contents. Native bacteria were investigated for HM tolerance and biofilm formation under toxic exposure by the microdilution assay. The most resistant strains were identified and the minimum inhibitory concentrations for HMs were determined. Cd, Cu, Pb and Zn exceeded the intervention threshold in Bozânta tailings site, while Pb content exceeded the intervention level within the area of the drinking water treatment plant. Cd showed a very high potential ecological risk in Bozânta area. The long-term exposure to HMs contributed to the selection of HM-tolerant and weakly adherent strains. Biofouling was significantly reduced under the influence of copper ions. Arthrobacter, Rhodococcus and Acidovorax strains with exceptional resistant profiles were isolated from the tailings site, indicating the important role of native microorganisms in rhizosphere ecosystems of contaminated sites.     

Diversity of amoA and pmoA Genes in Extremely Saline Alkaline Soils of the Former Lake Texcoco

Abstract

Ammonia-oxidizing bacteria (AOB) and aerobic methane oxidizing-bacteria (MOB) were studied in three extreme soils of the former Lake Texcoco, Mexico, with pH ranging from 8.5 to 10.5 and electrolytic conductivity (EC) from 0.67 to 84.76 dS m^?1, and in two arable soils. Soil DNA was extracted with three different methods and total DNA was used as a template to amplify the pmoA and amoA functional genes and subsequently sequenced by pyrosequencing. The amoA gene sequences clustered as uncultured AOB dominated in the Texcoco soils, while Nitrosospira was dominant in the arable soils. Sequences of MOB associated with Nitrosococcus-rel (Type I) dominated (>85%) in the Texcoco soils, but they were more diverse in the arable soils, for example, JR2, JR3, Methylocaldum USC-g (Type I), USC-a (Type II) and gp23 (pxmA). Aerobic methane oxidizing-bacteria and AOB microbial diversity were significantly related to EC and pH (p?<?0.05). As such, the lower MOB and AOB microbial diversity in the Texcoco soil compared to the arable soil was determined by its higher EC and pH.     

轮作模式对冬小麦土壤氨氧化微生物群落多样性和组成的影响

农田温室气体减排已成为农业绿色发展的重要内容,驱动温室气体氧化亚氮(N₂O)产生的氨氧化微生物受到了研究者们的广泛关注。为探究轮作模式对土壤氨氧化微生物群落的影响,基于田间定位试验,研究了夏红小豆-冬小麦、夏绿豆-冬小麦、夏花生-冬小麦、夏大豆-冬小麦和夏玉米-冬小麦5种轮作模式中冬小麦根际和非根际土壤氨氧化古菌(AOA)和氨氧化细菌(AOB)的群落组成和多样性变化特征。结果表明:与夏玉米-冬小麦模式相比,豆禾轮作模式增加了根际土中有机碳和硝态氮含量,以及非根际土中全氮和铵态氮含量。豆禾轮作模式降低了非根际土壤中AOA群落的ACE指数和Chao1指数,并显著降低根际土中AOB群落的ACE指数和Chao1指数(P<0.05)。豆禾轮作显著增加AOA群落中泉古菌门(Crenarchaeota)和AOB群落中变形菌门(Proteobacteria)某些类群的相对丰度(P<0.05)。根际土中豆禾轮作模式与麦玉模式的AOA群落结构发生明显分离,而非根际土中豆禾轮作模式与麦玉模式的AOB群落发生分离(P<0.05)。研究结果表明:豆禾轮作种植改变了AOA和AOB的群落结构和多样性,土壤pH值和速效氮含量是驱动AOA和AOB群落结构变化的重要因子,且根际与非根际土壤中氨氧化微生物存在生态位分离。     

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation‐sensitive amplified polymorphism

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper . MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.     

重庆紫色水稻土中“全程”和“半程”氨氧化微生物的垂直分异

【目的】系统评估全程氨氧化细菌(complete ammonia oxidizing bacteria, Comammox bacteria)、半程氨氧化细菌(AOB)和古菌(AOA)在典型水稻土剖面的垂直分异规律。2015年发现的"全程"氨氧化细菌(Comammox Nitrospira)可将氨分子一步氧化为硝酸盐,实现硝化作用。而经典的"半程"氨氧化细菌(AOB)或古菌(AOA)将氨分子氧化为亚硝酸盐后,再由系统发育完全不同的硝化细菌将其氧化为硝酸盐。全程氨氧化细菌实现了一步硝化全过程,根本改变了学术界对2类微生物分步硝化的经典认知,但相关研究仍处于初步阶段。【方法】选择重庆北碚地区2017年典型水稻土并采集5、10、20和40 cm不同深度土壤(剖面采样点的上下误差不超过1cm),提取水稻土总DNA后,利用标靶功能基因amoA,通过实时荧光定量PCR技术分析全程氨氧化细菌(Comammox)、半程氨氧化细菌(AOB)和古菌(AOA)在水稻土不同深度的数量变异规律。【结果】半程氨氧化细菌AOB和古菌AOA均随土壤深度增加呈显著下降趋势。然而,全程氨氧化细菌的两大类微生物则表现出相反的规律,Comammox Clade A的丰度随着土壤剖面的加深而显著增加(P0.05),但Clade B并未有类似规律。Clade A在水稻土不同层次的土层中均比Clade B高出1个数量级,在5 cm和40 cm处的最低和最高值分别为3.42×10~7、8.46×10~7 copies/g。AOA与AOB的丰度大致相当,5cm剖面处数量最高分别为1.23×10~7、1.83×10~5copies/g,但其平均丰度远低于全程氨氧化细菌,Comammox与AOA、AOB amoA功能基因拷贝数之比为10–2000。【结论】全程氨氧化细菌(Comammox bacteria)广泛分布于水稻土不同土层中,且数量远高于"半程"氨氧化细菌和古菌,意味着Comammox可能在水稻土硝化作用中起重要作用。     

Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars

Ammonia-oxidizing bacteria (AOB) populations were studied on the root surface of different rice cultivars by PCR coupled with denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). PCR-DGGE of the ammonium monooxygenase gene (amoA) showed a generally greater diversity on root samples compared to rhizosphere and unplanted soil. Sequences affiliated with Nitrosomonas spp. tended to be associated with modern rice hybrid lines. Root-associated AOB observed by FISH were found within a discrete biofilm coating the root surface. Although the total abundance of AOB on root biofilms of different rice cultivars did not differ significantly, there were marked contrasts in their population structure, indicating selection of Nitrosomonas spp. on roots of a hybrid cultivar. Observations by FISH on the total bacterial community also suggested that different rice cultivars support different bacterial populations even under identical environmental conditions. The presence of active AOB in the root environment predicts that a significant proportion of the N taken up by certain rice cultivars is in the form of NO₃ ^–-N produced by the AOB. Measurement of plant growth of hydroponically grown plants showed a stronger response of hybrid cultivars to the co-provision of NH₄ ⁺ and NO₃ ^–. In soil-grown plants, N use efficiency in the hybrid was improved during ammonium fertilization compared to nitrate fertilization. Since ammonium-fertilized plants actually receive a mixture of NH₄ ⁺ and NO₃ ^– with ratios depending on root-associated nitrification activity, these results support the advantage of co-provision of ammonium and nitrate for the hybrid cultivar.     

Higher Abundance of Ammonia Oxidizing Archaea than Ammonia Oxidizing Bacteria and Their Communities in Tibetan Alpine Meadow Soils under Long-term Nitrogen Fertilization

Increasing usage of nitrogen fertilizer for food production has resulted in severely environmental problems of nutrients enrichment. This study aimed to examine the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to a long-term nitrogen fertilization in Tibetan alpine meadow. The abundance and composition of both AOB and AOA were assessed using quantitative real-time PCR, cloning and sequencing techniques based on amoA gene under different fertilization gradient (0, 30, 60, 90, and 120 g m^?2 year^?1). Our results showed that, abundances of AOA amoA genes (ranging from 1.48 × 10⁹ to 2.00 × 10⁹ copies per gram of dry soil) were significantly higher than those of AOB amoA genes (1.25 × 10⁷ to 2.62 × 10⁸ copies per gram of dry soil) under fertilization scenario. The abundance of AOB amoA genes increased with increasing nitrogen fertilization, whereas fertilization had little effect on AOA abundance. Sequences of clone libraries of the different treatments revealed that AOB communities were dominated by representatives of Cluster 4, constituting 48.94–64.44% in each clone library. Sequences of Clusters 9, 1 and 2 were prevalent in soils under higher fertilization. All archaeal amoA sequences recovered were affiliated with the soil/sediment clade and marine sediment clade, and no significant difference was observed on the community structure among different fertilization treatments. Variations in the AOB community structure and abundance were linked to ammonium-N and soil pH induced by different fertilization treatments. These results showed that the abundance and structure of the AOB community respond to the fertilization gradient, not AOA.     

Mediating N2O emissions from municipal solid waste landfills: Impacts of landfill operating conditions on community structure of ammonia-oxidizing bacteria in cover soils

Chemolithotrophic ammonia-oxidizing bacteria (AOB) can produce N₂O, a highly potent greenhouse gas. Denaturing gradient gel electrophoresis (DGGE) analyses of the ammonia monooxygenase structural gene (amoA) and 16S rDNA gene were used to investigate the AOB community structure in the cover soils of municipal solid waste (MSW) landfills under three operating conditions: (a) MSW with soil cover, (b) MSW with soil cover, irrigation piping and vegetation, and (c) MSW covered with high-density polyethylene (HDPE) liner, soil cover, irrigation piping and vegetation. AOB species in MSW cover soils were significantly distinguished by the operation of HDPE liner isolation. The community structures of the Nitrosomonas europaea-like AOB species dominated in soils without HDPE liner isolation, whether vegetation and irrigation with landfill leachate existed or not, whereas Nitrospira-like AOB species dominated in soils with HDPE liner isolation. Lower N₂O flux from the soils with HDPE liner isolation would be partially related to these special community structures.     

Community composition of ammonia-oxidizing bacteria and archaea in rice field soil as affected by nitrogen fertilization

Little information is available on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in flooded rice soils. Consequently, a microcosm experiment was conducted to determine the effect of nitrogen fertilizer on the composition of AOB and AOA communities in rice soil by using molecular analyses of ammonia monooxygenase gene (amoA) fragments. Experimental treatments included three levels of N (urea) fertilizer, i.e. 50, 100 and 150 mg N kg⁻¹ soil. Soil samples were operationally divided into four fractions: surface soil, bulk soil deep layer, rhizosphere and washed root material. NH₄⁺-N was the dominant form of N in soil porewater and increased with N fertilization. Cloning and sequencing of amoA gene fragments showed that the AOB community in the rice soil consisted of three major groups, i.e. Nitrosomonas communis cluster, Nitrosospira cluster 3a and cluster 3b. The sequences related to Nitrosomonas were predominant. There was a clear effect of N fertilizer and soil depth on AOB community composition based on terminal restriction fragment length polymorphism fingerprinting. Nitrosomonas appeared to be more abundant in the potentially oxic or micro-oxic fractions, including surface soil, rhizosphere and washed root material, than the deep layer of anoxic bulk soil. Furthermore, Nitrosomonas increased relatively in the partially oxic fractions and that of Nitrosospira decreased with the increasing application of N fertilizer. However, AOA community composition remained unchanged according to the denaturing gradient gel electrophoresis analyses.     

Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments

Chesapeake Bay, the largest estuary in North America, encompasses a wide range of nutrient loading and trophic levels from the rivers and upper Bay to the sea, providing an ideal natural environment in which to explore relationships between functional diversity, physical/chemical complexity and ecosystem function (e.g. nitrification). In this study, amoA gene fragments (encoding subunit A of the key nitrification enzyme, ammonia monooxygenase) were PCR‐amplified from DNA extracted from sediment cores collected at five stations spanning gradients of salinity, ammonium, nitrate, oxygen and organic carbon along the Bay and Choptank River, a subestuary of the Bay. Phylogenetic analysis of ～30 amoA clones from each station revealed extensive diversity within the β‐Proteobacteria group of ammonia‐oxidizing bacteria (AOB), with the vast majority of sequences falling into coherent phylogenetic clusters distinct from sequences of cultivated AOB. Over 70% of the clones fell into two major phylogenetic clusters that appear to represent novel groups of Nitrosomonas‐like and Nitrosospira‐like amoA sequences that may be specific to estuarine and marine environments. Rarefaction analysis, estimators of genetic variation and dissimilarity indices all revealed differences in the relative amoA‐based diversity and/or richness among most of the stations, with the highest diversity at the North Bay station and the lowest at the mesohaline stations. Although salinity appears to play a role, no single physical or chemical parameter entirely explains the pattern of diversity along the estuary, suggesting that a complex combination of environmental factors may shape the overall level of AOB diversity in this dynamic environment.     

Abundance,Composition and Activity of Ammonia Oxidizer and Denitrifier Communities in Metal Polluted Rice Paddies from South China

While microbial nitrogen transformations in soils had been known to be affected by heavy metal pollution, changes in abundance and community structure of the mediating microbial populations had been not yet well characterized in polluted rice soils. Here, by using the prevailing molecular fingerprinting and enzyme activity assays and comparisons to adjacent non-polluted soils, we examined changes in the abundance and activity of ammonia oxidizing and denitrifying communities of rice paddies in two sites with different metal accumulation situation under long-term pollution from metal mining and smelter activities. Potential nitrifying activity was significantly reduced in polluted paddies in both sites while potential denitrifying activity reduced only in the soils with high Cu accumulation up to 1300 mg kg⁻¹. Copy numbers of amoA (AOA and AOB genes) were lower in both polluted paddies, following the trend with the enzyme assays, whereas that of nirK was not significantly affected. Analysis of the DGGE profiles revealed a shift in the community structure of AOA, and to a lesser extent, differences in the community structure of AOB and denitrifier between soils from the two sites with different pollution intensity and metal composition. All of the retrieved AOB sequences belonged to the genus Nitrosospira, among which species Cluster 4 appeared more sensitive to metal pollution. In contrast, nirK genes were widely distributed among different bacterial genera that were represented differentially between the polluted and unpolluted paddies. This could suggest either a possible non-specific target of the primers conventionally used in soil study or complex interactions between soil properties and metal contents on the observed community and activity changes, and thus on the N transformation in the polluted rice soils.     

Rhizosphere Microbial Densities and Trace Metal Tolerance of the Nickel Hyperaccumulator Alyssum Serpyllifolium Subsp. Lusitanicum

In this study we determine culturable microbial densities (total heterotrophs, ammonifiers, amylolytics and cellulolytics) and bacterial resistance to Co, Cr, and Ni in bulk and rhizosphere soils of three populations of the Ni-hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum and the excluder Dactylis glomerata from ultramafic sites (two populations in Northeast (NE) Portugal (Samil (S), Morais (M)) and one population in Northwest (NW) Spain (Melide (L)). The relationship between bioavailable metal concentrations (H₂O-soluble) and microbial densities were analysed. Significant differences in microbial densities and metal-resistance were observed between the two species and their three populations. The hyperaccumulator showed higher microbial densities (except cellulolytics) and a greater rhizosphere effect, but this was only observed in S and M populations. These populations of A. serpyllifolium also showed selective enrichment of Ni-tolerant bacteria at the rhizosphere where Ni solubility was enhanced (densities of Ni-resistant bacteria were positively correlated with H₂O-soluble Ni). These rhizobacteria could solubilise Ni in the soil and potentially improve phytoextraction strategies.