Influence of Long Term Irrigation with Pulp and Paper Mill Effluent on the Bacterial Community Structure and Catabolic Function in Soil

Microbial communities play a vital role in maintaining soil health. A multiphasic approach to assess the effect of pulp and paper mill effluent on both the structure and function of microbial soil communities is taken. Bacterial communities from agricultural soils irrigated with pulp and paper mill effluent were compared to communities form soils irrigated with well water. Samples were taken from fields in the state of Uttarakhand, India, where pulp and paper mill effluent has been used for irrigation for over 25 years. Comparisons of bacterial community structure were conducted using sequencing of the 16S rRNA gene from both isolates and clone libraries attained from the soil. Community-level physiological profiling was used to characterize the functional diversity and catabolic profile of the bacterial communities. The multiphasic approach using both physiological and molecular techniques proved to be a powerful tool in evaluating the soil bacterial community population and population differences therein. A significant and consistent difference in the population structure and function was found for the bacterial communities from soil irrigated with effluent in comparison to fields irrigated with well water. The diversity index parameters indicated that the microbial community in pulp and paper mill effluent irrigated fields were more diverse in both structure and function. This suggests that the pulp and paper mill effluent is not having a negative effect on the soil microbial community, but in fact may have a positive influence. In terms of soil health, this finding supports the continued use of pulp and paper mill effluent for irrigation. This is however only one aspect of soil health which was evaluated. Further studies on soil resistance and robustness could be undertaken to holistically evaluate soil health in this situation.     

Surface Attachment of Ammonia-Oxidizing Bacteria in Soil

Abstract Indigenous ammonia-oxidizing bacteria (AOB) in a clay loam soil were extremely difficult to release from soil particles compared to most heterotrophic bacteria; less than 1% of indigenous AOB (estimated as potential ammonia oxidation rate) were extractable by the dispersion-density-gradient centrifugation technique. This is at least 10-fold less than the extractability of heterotrophic bacteria. Urea applications to the same soil induced a 5-fold increase in the potential ammonia oxidation rate, and this resulted in a much higher percentage (8%) extractability of AOB. Thus, the newly grown AOB in the urea-treated soil were less strongly attached to the soil particles. The contrast suggests that the strong attachment of indigenous AOB is a gradual process taking place due to a long residence time (infrequent/slow cell division) compared to heterotrophic organisms. However, the contrast could also reflect differences in species composition of the original AOB community and those growing in response to urea inputs. Specific detection of AOB in extinction dilution cultures was done by PCR and sequencing of the products. Considerable diversity was found within the genus Nitrosospira, but severe problems with the specificity of the primers were observed. Two allegedly AOB specific PCR primers pairs were used: one specific for Nitrosospira (SPIRA) and one which should encompass all AOB within the β-Proteobacteria (GAOB). Only 33% of the cultures that gave PCR products with GAOB also gave products with the SPIRA primer pair, suggesting the presence of AOB other than Nitrosospira. However, the phylogeny based on the sequencing placed all the cultures in various clusters of the Nitrosospira clade, suggesting that the SPIRA primers do not match all members of the Nitrosospira genus. The cultures obtained from the urea-treated soil were different from the others in giving PCR products only with the SPIRA primers and not with the GAOB. Since sequencing also here confirmed the presence of Nitrosospira, these observations suggest that the GAOB primers do not match all AOB species. Received: 15 September 1999; Accepted: 8 November 1999; Online Publication: 28 April 2000     

Assessment of Pollution-Induced Microbial Community Tolerance to Heavy Metals in Soil Using Ammonia-Oxidizing Bacteria and Biolog Assay

This study attempted to investigate if the tolerance of soil bacterial communities in general, and autotrophic ammonia-oxidizing bacteria (AOB) in particular, evolved as a result of prolonged exposure to metals, and could be used as an indigenous bioindicator for soil metal pollution. A soil contaminated with copper, chromium, and arsenic (CCA) was mixed with an uncontaminated garden soil (GS3) to make five test soils with different metal concentrations. A modified potential ammonium oxidation assay was used to determine the metal tolerance of the AOB community. Tolerance to Cr, Cu, and As was tested at the beginning and after up to 13 months of incubation. Compared with the reference GS3 soil, the five CCA soils showed significantly higher tolerance to Cr no matter which form of Cr (Cr³⁺, CrO₄ ^2?, or Cr₂O₇ ^2?) was tested, and the Cr tolerance correlated with the total soil Cr concentration. However, the tolerance to Cu²⁺, As³⁺, and As⁵⁺ did not differ significantly between the GS3 soil and the five CCA soils. Community level physiological profiles using Biolog microtiter plates were also used to examine the chromate tolerance of the bacterial communities extracted after six months of exposure. Our results showed that the bacterial community tolerance was altered and increased as the soil Cr concentration was increased, indicating that the culturable microbial community and the AOB community responded in a similar manner.     

Community Structure of Ammonia-Oxidizing Bacteria under Long-Term Application of Mineral Fertilizer and Organic Manure in a Sandy Loam Soil

The effects of mineral fertilizer (NPK) and organic manure on the community structure of soil ammonia-oxidizing bacteria (AOB) was investigated in a long-term (16-year) fertilizer experiment. The experiment included seven treatments: organic manure, half organic manure N plus half fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without fertilization). N fertilization greatly increased soil nitrification potential, and mineral N fertilizer had a greater impact than organic manure, while N deficiency treatment (PK) had no significant effect. AOB community structure was analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the amoA gene, which encodes the α subunit of ammonia monooxygenase. DGGE profiles showed that the AOB community was more diverse in N-fertilized treatments than in the PK-fertilized treatment or the control, while one dominant band observed in the control could not be detected in any of the fertilized treatments. Phylogenetic analysis showed that the DGGE bands derived from N-fertilized treatments belonged to Nitrosospira cluster 3, indicating that N fertilization resulted in the dominance of Nitrosospira cluster 3 in soil. These results demonstrate that long-term application of N fertilizers could result in increased soil nitrification potential and the AOB community shifts in soil. Our results also showed the different effects of mineral fertilizer N versus organic manure N; the effects of P and K on the soil AOB community; and the importance of balanced fertilization with N, P, and K in promoting nitrification functions in arable soils.     

Response of Microbial Community Composition and Function to Soil Climate Change

Soil microbial communities mediate critical ecosystem carbon and nutrient cycles. How microbial communities will respond to changes in vegetation and climate, however, are not well understood. We reciprocally transplanted soil cores from under oak canopies and adjacent open grasslands in a California oak–grassland ecosystem to determine how microbial communities respond to changes in the soil environment and the potential consequences for the cycling of carbon. Every 3 months for up to 2 years, we monitored microbial community composition using phospholipid fatty acid analysis (PLFA), microbial biomass, respiration rates, microbial enzyme activities, and the activity of microbial groups by quantifying ¹³C uptake from a universal substrate (pyruvate) into PLFA biomarkers. Soil in the open grassland experienced higher maximum temperatures and lower soil water content than soil under the oak canopies. Soil microbial communities in soil under oak canopies were more sensitive to environmental change than those in adjacent soil from the open grassland. Oak canopy soil communities changed rapidly when cores were transplanted into the open grassland soil environment, but grassland soil communities did not change when transplanted into the oak canopy environment. Similarly, microbial biomass, enzyme activities, and microbial respiration decreased when microbial communities were transplanted from the oak canopy soils to the grassland environment, but not when the grassland communities were transplanted to the oak canopy environment. These data support the hypothesis that microbial community composition and function is altered when microbes are exposed to new extremes in environmental conditions; that is, environmental conditions outside of their “life history” envelopes.     

Influences of Infaunal Burrows on the Community Structure and Activity of Ammonia-Oxidizing Bacteria in Intertidal Sediments

Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O₂ concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O₂ transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH₄⁺ consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH₄⁺ and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O₂ transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH₄⁺ consumption capacity. Consequently, the infaunal burrow became an important site for NH₄⁺ consumption in the intertidal sediment.     

Effects of Urban Green Infrastructure Designs on Soil Bacterial Community Composition and Function

Ecosystems - Green infrastructures (GIs) are environmental designs mainly used to treat urban stormwater runoff pollution. The influences of green infrastructure designs are associated with the...     

福州森林红壤细菌的菌群结构及其功能分析

福建地区是我国的第二大林区,土壤类型主要表现为红壤.对福州地区森林中因木材腐朽而形成的树洞中的土壤样品进行筛选,构建了一份具有强木质纤维素降解能力的酸性红壤的细菌16S rKNA基因文库,利用限制性片段长度多态性(RFLP)对随机克隆进行筛选;对该生态环境下的细菌菌群进行了系统发育分析.结果表明土壤pH偏酸性严重影响了细菌类群的多样性,酸杆菌门Acidobacteria(71.5%)和变形菌门Proteobacteria(24.1%)是该酸性土样中的优势菌群,其中酸杆菌门在该生态系统中占有绝对优势.变形菌菌群中的主要类群为光合细菌,固氮细菌和溶菌细菌.研究表明,在木质纤维素自然降解的过程中,存在于酸性树洞土壤中的细菌参与了碳素和氮素的固定与循环,并对其微生态的稳定起到重要作用.     

Spatiotemporal Stability of an Ammonia-Oxidizing Community in a Nitrogen-Saturated Forest Soil

Elevated levels of nitrogen input into various terrestrial environments in recent decades have led to increases in soil nitrate production and leaching. However, nitrifying potential and nitrifying activity tend to be highly variable over space and time, making broad-scale estimates of nitrate production difficult. This study investigates whether the high spatiotemporal variation in nitrate production might be explained by differences in the structure of ammonia-oxidizing bacterial communities in nitrogen-saturated coniferous forest soils. The diversity of ammonia-oxidizing bacteria of the β-subgroup Proteobacteria was therefore investigated using two different PCR-based approaches. The first targeted the 16S rRNA gene and involved temporal temperature gradient electrophoresis (TTGE) of specifically amplified PCR products, with subsequent band excision and nucleotide sequence determination. The second approach involved the cloning and sequencing of PCR-amplified amoA gene fragments. All recovered 16S rDNA sequences were closely related to the culture strain Nitrosospira sp. AHB1, which was isolated from an acid soil and is affiliated with Nitrosospira cluster 2, a sequence group previously shown to be associated with acid environments. All amoA-like sequences also showed a close affinity with this acid-tolerant Nitrosospira strain, although greater sequence variation could be detected in the amoA analysis. The ammonia-oxidizing bacterial community in the nitrogen-saturated coniferous forest soil was determined to be very stable, showing little variation between different organic layers and throughout the year, despite large differences in the total Bacterial community structure as determined by 16S rDNA DGGE community fingerprinting. These results suggest that environmental heterogeneity affecting ammonia oxidizer numbers and activity, and not ammonia oxidizer community structure, is chiefly responsible for spatial and temporal variation in nitrate production in these acid forest soils.     

Urease-Encoding Genes in Ammonia-Oxidizing Bacteria

Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the β-proteobacterium Nitrosospira sp. strain NpAV and the γ-proteobacterium Nitrosococcus oceani. In both organisms, all seven urease genes were contiguous: the three structural urease genes ureABC were preceded and succeeded by the accessory genes ureD and ureEFG, respectively. Green fluorescent protein reporter gene fusions revealed that the ure genes were under control of a single operon promoter upstream of the ureD gene in Nitrosococcus oceani. Southern analyses revealed two copies of ureC in the Nitrosospira sp. strain NpAV genome, while a single copy of the ure operon was detected in the genome of Nitrosococcus oceani. The ureC gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further ureC sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB, ureC genes were sequenced from several β-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from β-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other β-proteobacteria. Instead, it appears that urease in β-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of γ- and β-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for ureC from β-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for ure genes from autotrophic AOB.     

Diversity and Abundance of Ammonia-Oxidizing Bacteria and Ammonia-Oxidizing Archaea During Cattle Manure Composting

Ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) play important roles in nitrification in various environments. They may also be key communities for ammonia oxidation in composting systems, although few studies have discussed their presence. We investigated the relative diversity and abundance of AOB and AOA using cloning procedures, denaturing gradient gel electrophoresis analysis, and real-time PCR during several stages in the process of cattle manure composting. Our results revealed that the AOB community structure changed during the process. At the high-temperature stage (>60°C), a member of the Nitrosomonas europaea/eutropha cluster dominated while the uncultured Nitrosomonas spp. cluster appeared after the temperature decreased. Additionally, our analysis indicated that AOA sequences, which were classified into a soil/sediment cluster, were present after the temperature decreased during the composting process. At these stages, the number of the archaeal amoA gene copies (3.2 or 3.9?×?10⁷ copies per gram freeze-dried compost) was significantly higher than that of bacterial amoA gene copies (2.2–7.2?×?10⁶ copies per gram freeze-dried compost). Our results suggest that both AOB and AOA are actively involved in nitrification of composting systems.     

Influence of Different Cultivars on Populations of Ammonia-Oxidizing Bacteria in the Root Environment of Rice

Comparisons of the activities and diversities of ammonia-oxidizing bacteria (AOB) in the root environment of different cultivars of rice (Oryza sativa L.) indicated marked differences despite identical environmental conditions during growth. Gross nitrification rates obtained by the ¹⁵N dilution technique were significantly higher in a modern variety, IR63087-1-17, than in two traditional varieties. Phylogenetic analysis based on the ammonium monooxygenase gene (amoA) identified strains related to Nitrosospira multiformis and Nitrosomonas europaea as the predominant AOB in our experimental rice system. A method was developed to determine the abundance of AOB on root biofilm samples using fluorescently tagged oligonucleotide probes targeting 16S rRNA. The levels of abundance detected suggested an enrichment of AOB on rice roots. We identified 40 to 69% of AOB on roots of IR63087-1-17 as Nitrosomonas spp., while this subpopulation constituted 7 to 23% of AOB on roots of the other cultivars. These results were generally supported by denaturing gradient gel electrophoresis of the amoA gene and analysis of libraries of cloned amoA. In hydroponic culture, oxygen concentration profiles around secondary roots differed significantly among the tested rice varieties, of which IR63087-1-17 showed maximum leakage of oxygen. The results suggest that varietal differences in the composition and activity of root-associated AOB populations may result from microscale differences in O₂ availability.     

Use of Aliphatic n-Alkynes To Discriminate Soil Nitrification Activities of Ammonia-Oxidizing Thaumarchaea and Bacteria

Ammonia (NH₃)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH₃ oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤20 μM concentrations of 1-alkynes C₈ and C₉. In contrast, NH₃ oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C₈ (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH₄⁺-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH₄⁺-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH₄⁺-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.     

Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 10⁵ cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH₄)₂SO₄ application. AOB populations in amended treatments increased from an initial density of approximately 4 × 10⁶ cells/g of dry soil to peak values (day 7) of 35 × 10⁶ and 66 × 10⁶ cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 10⁹ and 2.2 × 10⁹ cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 10⁶ to 38.0 × 10⁶ cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH₄⁺ h⁻¹ cell⁻¹ and decreased with time in both microcosms and the field. Growth yields were 5.6 × 10⁶, 17.5 × 10⁶, and 1.7 × 10⁶ cells/mol of NH₄⁺ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.     

Detection of Ammonia-Oxidizing Archaea in Fish Processing Effluent Treatment Plants

Ammonia oxidation is the rate limiting step in nitrification and thus have an important role in removal of ammonia in natural and engineered systems with participation of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, their relative distribution and activity in fish processing effluent treatment plants (FPETPs) though significant, is hitherto unreported. Presence of AOA in sludge samples obtained from FPETPs was studied by amplification and sequencing of thaumarchaeal ammonia monooxygenase subunit A (AOA-amoA) gene. Different primer sets targeting 16S rRNA and AOA-amoA gene were used for the detection of AOA in FPETPs. Phylogenetic analysis of the gene revealed that the AOA was affiliated with thaumarchaeal group 1.1a lineage (marine cluster). Quantitative real time PCR of amoA gene was used to study the copy number of AOA and AOB in FPETPs. The AOA-amoA and AOB-amoA gene copy numbers of sludge samples ranged from 2.2 × 10⁶ to 4.2 × 10⁸ and 1.1 × 10⁷ to 8.5 × 10⁸ mg⁻¹ sludge respectively. Primer sets Arch-amoAF/Arch-amoAR and 340F/1000R were found to be useful for the sensitive detection of AOA-amoA and Archaeal 16S rRNA genes respectively in FPETPs. Their presence suggests the widespread occurrence and possible usefulness in removing ammonia from FPETPs which is in line with reports from other waste water treatment plants.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0484-6) contains supplementary material, which is available to authorized users.     

Influence of Shrub Encroachment on the Soil Microbial Community Composition of Remnant Hill Prairies

Hill prairies are remnant grasslands perched on the bluffs of major river valleys, and because their steep slopes make them unsuitable for traditional row crop agriculture, they have some of the lowest levels of anthropogenic disturbance of any prairie ecosystems in the Midwestern USA. However, many decades of fire suppression have allowed for shrub encroachment from the surrounding forests. While shrub encroachment of grasslands can modify soil respiration rates and nutrient storage, it is not known whether shrubs also alter the community composition of soil microorganisms. We conducted transect sampling of nine different hill prairie remnants showing varying degrees of shrub encroachment, and we used DNA-based community profiling (automated ribosomal intergenic spacer analysis) to characterize the composition of bacterial and fungal communities in the open prairie habitat, the shrub-encroached border, and the surrounding forest. While both bacterial and fungal communities showed statistically significant variation across these habitats, their predominant patterns were different. Bacterial communities of forest soils were distinct from those of the open prairie and the shrub-encroached areas, while fungal communities of the open prairie were distinct from those of the forest and the shrub-encroached border. Shrub encroachment significantly altered the community composition of soil fungal communities. Furthermore, fungal communities of heavily encroached prairie remnants more closely resembled those of the surrounding forest than those of lightly encroached prairies. Thus, shrub encroachment can cause soil fungi to shift from a “grassland” community to a “woody” community, with potential consequences for soil processes and plant-microbe interactions.     

Effect of Lake Trophic Status and Rooted Macrophytes on Community Composition and Abundance of Ammonia-Oxidizing Prokaryotes in Freshwater Sediments

Communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in freshwater sediments and those in association with the root system of the macrophyte species Littorella uniflora, Juncus bulbosus, and Myriophyllum alterniflorum were compared for seven oligotrophic to mesotrophic softwater lakes and acidic heathland pools. Archaeal and bacterial ammonia monooxygenase alpha-subunit (amoA) gene diversity increased from oligotrophic to mesotrophic sites; the number of detected operational taxonomic units was positively correlated to ammonia availability and pH and negatively correlated to sediment C/N ratios. AOA communities could be grouped according to lake trophic status and pH; plant species-specific communities were not detected, and no grouping was apparent for AOB communities. Relative abundance, determined by quantitative PCR targeting amoA, was always low for AOB (<0.05% of all prokaryotes) and slightly higher for AOA in unvegetated sediment and AOA in association with M. alterniflorum (0.01 to 2%), while AOA accounted for up to 5% in the rhizospheres of L. uniflora and J. bulbosus. These results indicate that (i) AOA are at least as numerous as AOB in freshwater sediments, (ii) aquatic macrophytes with substantial release of oxygen and organic carbon into their rhizospheres, like L. uniflora and J. bulbosus, increase AOA abundance; and (iii) AOA community composition is generally determined by lake trophy, not by plant species-specific interactions.Oxygen release from the roots of macrophyte species such as Littorella uniflora (L.) Asch. (shore weed), Lobelia dortmanna L. (water lobelia), and Glyceria maxima (Hartm.) Holmb. (reed sweet grass) stimulates nitrification and coupled nitrification-denitrification in the rhizosphere compared to that in unvegetated sediment (2, 36, 40). These interactions are of high ecological relevance especially in oligotrophic systems, since enhanced nitrogen loss due to rhizosphere-associated denitrification can retard natural eutrophication and succession of plant communities (1). While the microbial communities involved in coupled nitrification-denitrification have been well studied in rice paddy soils (7, 11), less information is available for natural freshwater sediments, especially those from oligotrophic lakes (2, 26).The first key step of coupled nitrification-denitrification, the oxidation of ammonia to nitrite, is catalyzed by two groups of prokaryotes—the ammonia-oxidizing bacteria (AOB) (24) and the only recently recognized ammonia-oxidizing archaea (AOA) (22). For both groups, the gene encoding the alpha-subunit of ammonia monooxygenase (amoA) has been widely used as a functional marker to analyze their community compositions (15, 25); recent studies demonstrated the ubiquity of AOA and their predominance over AOB in a broad range of environments (32, 38). AOA, but not AOB, were also strongly enriched in the rhizosphere of the freshwater macrophyte Littorella uniflora in a mesotrophic Danish lake, suggesting that AOA were primarily responsible for increased rates of nitrification in the rhizosphere of this plant species (19). Moreover, ammonia oxidizer communities differed between rhizosphere and unvegetated sediment, indicating a plant-specific effect on AOA and AOB community composition. The objectives of this study were therefore to test whether (i) AOA generally predominate over AOB in freshwater sediments and especially in macrophyte rhizospheres and (ii) macrophytes have species-specific effects on abundance and community composition of AOA and AOB in rhizosphere sediments and on root surfaces.To address these questions, two shallow heathland pools and five lakes in Denmark and Germany, ranging from low-pH and dystrophic sites to neutral-pH and oligotrophic and mesotrophic sites, were chosen, and three macrophyte species—Littorella uniflora, Juncus bulbosus L. (bulbous rush), and Myriophyllum alterniflorum DC. (alternate water milfoil)—were selected as model systems. These plant species differ in nitrogen nutrition, extent of radial oxygen loss, and lifestyle, presumably resulting in differential, plant species-specific effects on rhizosphere- and root-associated AOA and AOB communities. L. uniflora prefers nitrate as the nitrogen source, while J. bulbosus prefers ammonium (41, 45); oxygen release is high to moderate from the roots of L. uniflora and J. bulbosus (9, 12) but is minor from the roots of M. alterniflorum (M. Herrmann, P. Stief, and A. Schramm, unpublished results); L. uniflora and J. bulbosus remain photosynthetically active throughout the year, while only the below-ground parts of M. alterniflorum are retained during winter.Rhizosphere sediments and roots from each plant species were sampled from three different sites per species, and unvegetated sediment was obtained from all seven sites. The comparison of samples from these different sites and compartments (rhizosphere, root surface, unvegetated sediment) allowed an evaluation of the importance of plant species relative to that of environmental conditions related to lake trophic status and pH on ammonia oxidizer communities.     

Agreement between Theory and Measurement in Quantification of Ammonia-Oxidizing Bacteria

Autotrophic ammonia-oxidizing bacteria (AOB) are of vital importance to wastewater treatment plants (WWTP), as well as being an intriguing group of microorganisms in their own right. To date, corroboration of quantitative measurements of AOB by fluorescence in situ hybridization (FISH) has relied on assessment of the ammonia oxidation rate per cell, relative to published values for cultured AOB. Validation of cell counts on the basis of substrate transformation rates is problematic, however, because published cell-specific ammonia oxidation rates vary by over two orders of magnitude. We present a method that uses FISH in conjunction with confocal scanning laser microscopy to quantify AOB in WWTP, where AOB are typically observed as microcolonies. The method is comparatively simple, requiring neither detailed cell counts or image analysis, and yet it can give estimates of either cell numbers or biomass. Microcolony volume and diameter were found to have a log-normal distribution. We were able to show that virtually all (>96%) of the AOB biomass occurred as microcolonies. Counts of microcolony abundance and measurement of their diameter coupled with a calibration of microcolony dimensions against cell numbers or AOB biomass were used to determine AOB cell numbers and biomass in WWTP. Cell-specific ammonia oxidation rates varied between plants by over three orders of magnitude, suggesting that cell-specific ammonia oxidation is an important process variable. Moreover, when measured AOB biomass was compared with process-based estimates of AOB biomass, the two values were in agreement.     

Linking Diversity and Stable Isotope Fractionation in Ammonia-Oxidizing Bacteria

The link between similarity in amino acid sequence for ammonia monooxygenase (AMO) and isotopic discrimination for ammonia oxidation ( l AMO ) was investigated in g -subdivision ammonia-oxidizing bacteria. The isotope effects for ammonia oxidation in pure cultures of the nitrifying strains Nitrosomonas marina , Nitrosomonas C-113a, Nitrosospira tenuis , Nitrosomonas europaea , and Nitrosomonas eutropha ranged from 14.2 to 38.2. The differences in isotope effects could not be readily explained by differential rates of ammonia oxidation, transport of NH 4 + , or accumulation of NH 2 OH or N 2 O among the strains. The major similarities and differences observed in l AMO are, however, paralleled by similarities and differences in amino acid sequences for the f -subunit of AMO (AmoA). Robust differences in l AMO among nitrifying bacteria may be expected to influence the stable isotopic signatures of nitrous oxide (N 2 O) produced in various environments.