Ammonia-oxidizing Crenarchaeota and nitrification inside the tissue of a colonial ascidian

Marine Crenarchaeota represent an abundant component of the oceanic microbiota that play an important role in the global nitrogen cycle. Here we report the association of the colonial ascidian Cystodytes dellechiajei with putative ammonia-oxidizing Crenarchaeota that could actively be involved in nitrification inside the animal tissue. As shown by 16S rRNA gene analysis, the ascidian-associated Crenarchaeota were phylogenetically related to Nitrosopumilus maritimus, the first marine archaeon isolated in pure culture that grows chemolithoautotrophically oxidizing ammonia to nitrite aerobically. Catalysed reporter deposition (CARD)-FISH revealed that the Crenarchaeota were specifically located inside the tunic tissue of the colony, where moreover the expression of amoA gene was detected. The amoA gene encodes the alpha-subunit of ammonia monooxygenase, which is involved in the first step of nitrification, the oxidation of ammonia to nitrite. Sequencing of amoA gene showed that they were phylogenetically related to amoA genes of N. maritimus and other putative ammonia-oxidizing marine Crenarchaeota. In order to track the suspected nitrification activity inside the ascidian colony under in vivo conditions, microsensor profiles were measured through the tunic tissue. Net NO(x) production was detected in the tunic layer 1200-1800 microm with rates of 58-90 nmol cm(-3) h(-1). Oxygen and pH microsensor profiles showed that the layer of net NO(x) production coincided with O(2) concentrations of 103-116 microM and pH value of 5.2. Together, molecular and microsensor data indicate that Crenarchaeota could oxidize ammonia to nitrite aerobically, and thus be involved in nitrification inside the ascidian tissue.     

Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(-), and NO(3)(-) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 microm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 microm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Redox control bioreactor: a unique biological water processor

The redox control bioreactor (RCB) is a new hollow fiber membrane bioreactor (HFMBR) design in which oxygen and hydrogen gases are provided simultaneously through separate arrays of juxtaposed hollow fiber (HF) membranes. This study applied the RCB for completely autotrophic conversion of ammonia to N(2) through nitrification with O(2) and denitrification using hydrogen as an electron donor (i.e., autohydrogentrophic denitrification). The hypothesis of this research was that efficient biofilm utilization of O(2) and H(2) at respective HFs would limit transport of these gases to bulk fluid, thereby enabling completely autotrophic ammonia conversion to N(2) through the co-occurrence of ammonia oxidation (O(2)-HF biofilms) and autohydrogenotrophic denitrification (H(2)-HF biofilms). A prototype RCB was fabricated and operated for 215 days on a synthetic, organic-free feedstream containing 217 mg L(-1) NH(4)(+)-N. When O(2) and H(2) were simultaneously supplied, the RCB achieved a steady NH(4)(+)-N removal flux of 5.8 g m(-2) day(-1) normalized to O(2)-HF surface area with a concomitant removal flux of 4.4 g m(-2) day(-1) (NO(3)(-))+NO(2)(-))-N based on H(2)-HF surface area. The significance of H(2) supply was confirmed by an increase in effluent NO(3)(-)-N when H(2) supply was discontinued and a decline in NO(3)(-)-N when H(2) supply was restarted. Increases in H(2) pressure caused decreased ammonia utilization, suggesting that excess H(2) interfered with nitrification. Microprobe profiling across radial transects revealed significant gradients in dissolved O(2) on spatial scales of 1 mm or less. Physiological and molecular analysis of biofilms confirmed that structurally and functionally distinct biofilms developed on adjacent, juxtaposed fibers.     

Spatial variability in nitrification rates and ammonia-oxidizing microbial communities in the agriculturally impacted Elkhorn Slough estuary, California

Ammonia oxidation-the microbial oxidation of ammonia to nitrite and the first step in nitrification-plays a central role in nitrogen cycling in coastal and estuarine systems. Nevertheless, questions remain regarding the connection between this biogeochemical process and the diversity and abundance of the mediating microbial community. In this study, we measured nutrient fluxes and rates of sediment nitrification in conjunction with the diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing betaproteobacteria (β-AOB). Sediments were examined from four sites in Elkhorn Slough, a small agriculturally impacted coastal California estuary that opens into Monterey Bay. Using an intact sediment core flowthrough incubation system, we observed significant correlations among NO(3)(-), NO(2)(-), NH(4)(+), and PO(4)(3+) fluxes, indicating a tight coupling of sediment biogeochemical processes. (15)N-based measurements of nitrification rates revealed higher rates at the less impacted, lower-nutrient sites than at the more heavily impacted, nutrient-rich sites. Quantitative PCR analyses revealed that β-AOB amoA (encoding ammonia monooxygenase subunit A) gene copies outnumbered AOA amoA gene copies by factors ranging from 2- to 236-fold across the four sites. Sites with high nitrification rates primarily contained marine/estuarine Nitrosospira-like bacterial amoA sequences and phylogenetically diverse archaeal amoA sequences. Sites with low nitrification rates were dominated by estuarine Nitrosomonas-like amoA sequences and archaeal amoA sequences similar to those previously described in soils. This is the first report measuring AOA and β-AOB amoA abundance in conjunction with (15)N-based nitrification rates in estuary sediments.     

Crenarchaeota and their role in the nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps

Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the "Franz-Josef-Quelle" (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (+/-4.5%) in the total 4',6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45 degrees C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation "thermal spring" cluster and which may be older than most AOA from soils on earth.     

Effect of soil ammonium concentration on N2O release and on the community structure of ammonia oxidizers and denitrifiers

The effect of ammonium addition (6.5, 58, and 395 microg of NH4+-N g [dry weight] of soil(-1)) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N2O release rates and a shift toward a higher contribution of nitrification to N2O release occurred after incubation for 5 days at 4 degrees C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected amoA sequences which probably belonged to Nitrosospira 16S rRNA clusters 3 and 4. Additional clones clustered with Nitrosospira sp. strains Ka3 and Ka4 and within an amoA cluster from unknown species. A Nitrosomonas-like amoA gene was detected in only one clone. In agreement with the amoA results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of nirK genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned nirK genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil nirK genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change.     

Analyses of spatial distributions of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms.

The vertical distribution of sulfate-reducing bacteria (SRB) in aerobic wastewater biofilms grown on rotating disk reactors was investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. To correlate the vertical distribution of SRB populations with their activity, the microprofiles of O(2), H(2)S, NO(2)(-), NO(3)(-), NH(4)(+), and pH were measured with microelectrodes. In addition, a cross-evaluation of the FISH and microelectrode analyses was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance of the probe SRB385-stained cells (approximately 10(9) to 10(10) cells per cm(3) of biofilm) were evenly distributed throughout the biofilm, even in the oxic surface. The probe SRB660-stained Desulfobulbus spp. were found to be numerically important members of SRB populations (approximately 10(8) to 10(9) cells per cm(3)). The result of microelectrode measurements showed that a high sulfate-reducing activity was found in a narrow anaerobic zone located about 150 to 300 microm below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S(0)) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 microm), which accounted for about 75% of the total S pool in the biofilm. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant (less than 1%) due to the relatively high sulfate reduction rate.     

Influence of nutrients, hexadecane, and temporal variations on nitrification and exopolysaccharide composition of river biofilms

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001. The reactors were supplemented with carbon (glucose), nitrogen (NH(4)Cl), phosphorus (KH(2)PO(4)), or combined nutrients (CNP), with or without hexadecane. The impact of these treatments on nitrification and on the exopolysaccharide composition of river biofilms was determined. The results showed that the biofilms had higher NH4(+) oxidation, NO3(-) production, and N2O production activities in fall 1999 than fall 2001 when grown with CNP but had higher activities in fall 2001 than fall 1999 when grown with individual nutrients. The exopolysaccharide amounts and proportions were generally higher in fall 1999 than fall 2001, as a consequence of the higher nutrient levels in the river water in the first year of this study. The addition of P and especially CNP stimulated NH4(+) oxidation by the biofilms, showing a P limitation in this river ecosystem. The presence of hexadecane negatively affected these activities and lowered the amounts of exopolysaccharides in CNP and P biofilms in fall 1999 but increased the biofilm activities and exopolysaccharide amounts in CNP biofilm in fall 2001. Antagonistic, synergistic, and independent effects between nutrients and hexadecane were also observed. This study demonstrated that the biofilm autotrophic nitrification activity in the South Saskatchewan River was limited by P, that this activity and the exopolysaccharide amounts and proportions were dependent on the nutrient concentrations in the river water, and suggested that exopolysaccharides may play a protective role for biofilm microorganisms against toxic pollutants.     

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.     

Identification of novel small molecule enhancers of protein production by cultured mammalian cells

Shortcut nitrogen removal, that is, removal via formation and reduction of nitrite rather than nitrate, has been observed in membrane-aerated biofilms (MABs), but the extent, the controlling factors, and the kinetics of nitrite formation in MABs are poorly understood. We used a special MAB reactor to systematically study the effects of the dissolved oxygen (DO) concentration at the membrane surface, which is the biofilm base, on nitrification rates, extent of shortcut nitrification, and microbial community structure. The focus was on anoxic bulk liquids, which is typical in MAB used for total nitrogen (TN) removal, although aerobic bulk liquids were also studied. Nitrifying MABs were grown on a hollow-fiber membrane exposed to 3 mg N/L ammonium. The MAB intra-membrane air pressure was varied to achieve different DO concentrations at the biofilm base, and the bulk liquid was anoxic or with 2 g m(-3) DO. With 2.2 and 3.5 g m(-3) DO at the biofilm base, and with an anoxic bulk-liquid, the ammonium fluxes were 0.75 and 1.0 g N m(-2) day(-1), respectively, and nitrite was the main oxidized nitrogen product. However, with membrane DO of 5.5 g m(-3), and either zero or 2 g m(-3) DO in the bulk, the ammonium flux was around 1.3 g N m(-2) day(-1), and nitrate flux increased significantly. For all experiments, the cell density of ammonium oxidizing bacteria (AOB) was relatively uniform throughout the biofilm, but the density of nitrite oxidizing bacteria (NOB) decreased with decreasing biofilm DO. Among NOB, Nitrobacter spp. were dominant in biofilm regions with 2 g m(-3) DO or greater, while Nitrospira spp. were dominant in regions with less than 2 g m(-3) DO. A biofilm model, including AOB, Nitrobacter spp., and Nitrospira spp., was developed and calibrated with the experimental results. The model predicted the greatest extent of nitrite formation (95%) and the lowest ammonium oxidation flux (0.91 g N m(-2) day(-1)) when the membrane DO was 2 g m(-3) and the bulk liquid was anoxic. Conversely, the model predicted the lowest extent of nitrite formation (40%) and the highest ammonium oxidation flux (1.5 g N m(-2) day(-1)) when the membrane-DO and bulk-DO were 8 g m(-3) and 2 g m(-3), respectively. The estimated kinetic parameters for Nitrospira spp., revealed a high affinity for nitrite and oxygen. This explains the dominance of Nitrospira spp. over Nitrobacter spp. in regions with low nitrite and oxygen concentrations. Our results suggest that shortcut nitrification can effectively be controlled by manipulating the DO at the membrane surface. A tradeoff is made between increased nitrite accumulation at lower DO, and higher nitrification rates at higher DO.     

Community composition of ammonia-oxidizing bacteria and archaea in soils under stands of red alder and Douglas fir in Oregon

This study determined nitrification activity and nitrifier community composition in soils under stands of red alder (Alnus rubra) and Douglas fir (Pseudotsuga menziesii) at two sites in Oregon. The H.J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon, has low net N mineralization and gross nitrification rates. Cascade Head Experimental Forest, in the Coast Range, has higher net N mineralization and nitrification rates and soil pH is lower. Communities of putative bacterial [ammonia-oxidizing bacteria (AOB)] and archaeal [ammonia-oxidizing archaea (AOA)] ammonia oxidizers were examined by targeting the gene amoA, which codes for subunit A of ammonia monooxygenase. Nitrification potential was significantly higher in red alder compared with Douglas-fir soil and greater at Cascade Head than H.J. Andrews. Ammonia-oxidizing bacteria amoA genes were amplified from all soils, but AOA amoA genes could only be amplified at Cascade Head. Gene copy numbers of AOB and AOA amoA were similar at Cascade Head regardless of tree type (2.3-6.0 x 10(6)amoA gene copies g(-1) of soil). DNA sequences of amoA revealed that AOB were members of Nitrosospira clusters 1, 2 and 4. Ammonia-oxidizing bacteria community composition, determined by terminal restriction fragment length polymorphism (T-RFLP) profiles, varied among sites and between tree types. Many of the AOA amoA sequences clustered with environmental clones previously obtained from soil; however, several sequences were more similar to clones previously recovered from marine and estuarine sediments. As with AOB, the AOA community composition differed between red alder and Douglas-fir soils.     

Chemical and biological interactions during nitrate and goethite reduction by Shewanella putrefaciens 200

Although previous research has demonstrated that NO(3)(-) inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO(3)(-) biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO(3)(-) and synthetic, high-surface-area goethite. Results showed that the presence of NO(3)(-) inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO(3)(-) reduction, a 10-fold decrease in the rate of NO(2)(-) reduction, and a 20-fold increase in the amounts of N(2)O produced. Nitrogen stable isotope experiments that utilized delta(15)N values of N(2)O to distinguish between chemical and biological reduction of NO(2)(-) revealed that the N(2)O produced during NO(2)(-) or NO(3)(-) reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO(3)(-) reduction produces NO(2)(-) and Fe(II), which then abiotically react to reduce NO(2)(-) to N(2)O with the subsequent oxidation of Fe(II) to Fe(III).     

Nitrification in terrestrial hot springs of Iceland and Kamchatka

Archaea have been detected recently as a major and often dominant component of the microbial communities performing ammonia oxidation in terrestrial and marine environments. In a molecular survey of archaeal ammonia monooxygenase (AMO) genes in terrestrial hot springs of Iceland and Kamchatka, the amoA gene encoding the alpha-subunit of AMO was detected in a total of 14 hot springs out of the 22 investigated. Most of these amoA-positive hot springs had temperatures between 82 and 97 degrees C and pH range between 2.5 and 7. In phylogenetic analyses, these amoA genes formed three independent lineages within the known sequence clusters of marine or soil origin. Furthermore, in situ gross nitrification rates in Icelandic hot springs were estimated by the pool dilution technique directly on site. At temperatures above 80 degrees C, between 56 and 159 mumol NO(3)(-) L(-1) mud per day was produced. Furthermore, addition of ammonium to the hot spring samples before incubation yielded a more than twofold higher potential nitrification rate, indicating that the process was limited by ammonia supply. Our data provide evidence for an active role of archaea in nitrification of hot springs in a wide range of pH values and at a high temperature.     

High-rate nitrification at low pH in suspended- and attached-biomass reactors

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 +/- 0.1) and low bulk ammonium concentrations (9.3 +/- 3.3 mg.liter(-1)), a very high nitrification rate of 5.6 g of N oxidized.liter(-1).day(-1) was achieved. The specific nitrification rate (0.55 g of N.g of biomass(-1).day(-1)) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 +/- 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (15N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass.g of N oxidized(-1). Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.     

Abundance, diversity, and activity of ammonia-oxidizing prokaryotes in the coastal Arctic ocean in summer and winter

Ammonia oxidation, the first step in nitrification, is performed by certain Beta- and Gammaproteobacteria and Crenarchaea to generate metabolic energy. Ammonia monooxygenase (amoA) genes from both Bacteria and Crenarchaea have been found in a variety of marine ecosystems, but the relative importance of Bacteria versus Crenarchaea in ammonia oxidation is unresolved, and seasonal comparisons are rare. In this study, we compared the abundance of betaproteobacterial and crenarchaeal amoA genes in the coastal Arctic Ocean during summer and winter over 2 years. Summer and winter betaproteobacterial amoA clone libraries were significantly different, although the gene sequences were similar to those found in temperate and polar environments. Betaproteobacterial and crenarchaeal amoA genes were 30- to 115-fold more abundant during the winter than during the summer in both years of the study. Archaeal amoA genes were more abundant than betaproteobacterial amoA genes in the first year, but betaproteobacterial amoA was more abundant than archaeal amoA the following year. The ratio of archaeal amoA gene copies to marine group I crenarchaeal 16S rRNA genes averaged 2.9 over both seasons and years, suggesting that ammonia oxidation was common in Crenarchaea at this location. Potential nitrification rates, as well as the total amoA gene abundance, were highest in the winter when competition with phytoplankton was minimal and ammonium concentrations were the highest. These results suggest that ammonium concentrations were important in determining the rates of ammonia oxidation and the abundance of ammonia-oxidizing Betaproteobacteria and Crenarchaea.     

Decreased N2O reduction by low soil pH causes high N2O emissions in a riparian ecosystem

Quantification of harmful nitrous oxide (N(2)O) emissions from soils is essential for mitigation measures. An important N(2)O producing and reducing process in soils is denitrification, which shows deceased rates at low pH. No clear relationship between N(2)O emissions and soil pH has yet been established because also the relative contribution of N(2)O as the denitrification end product decreases with pH. Our aim was to show the net effect of soil pH on N(2)O production and emission. Therefore, experiments were designed to investigate the effects of pH on NO(3)(-) reduction, N(2)O production and reduction and N(2) production in incubations with pH values set between 4 and 7. Furthermore, field measurements of soil pH and N(2)O emissions were carried out. In incubations, NO(3)(-) reduction and N(2) production rates increased with pH and net N(2)O production rate was highest at pH 5. N(2)O reduction to N(2) was halted until NO(3)(-) was depleted at low pH values, resulting in a built up of N(2)O. As a consequence, N(2)O:N(2) production ratio decreased exponentially with pH. N(2)O reduction appeared therefore more important than N(2)O production in explaining net N(2)O production rates. In the field, a negative exponential relationship for soil pH against N(2)O emissions was observed. Soil pH could therefore be used as a predictive tool for average N(2)O emissions in the studied ecosystem. The occurrence of low pH spots may explain N(2)O emission hotspot occurrence. Future studies should focus on the mechanism behind small scale soil pH variability and the effect of manipulating the pH of soils.     

Effects of temperature and fertilizer on activity and community structure of soil ammonia oxidizers

We investigated the effect of temperature on the activity of soil ammonia oxidizers caused by changes in the availability of ammonium and in the microbial community structure. Both short (5 days) and long (6.5, 16 and 20 weeks) incubation of an agricultural soil resulted in a decrease in ammonium concentration that was more pronounced at temperatures between 10 and 25 degrees C than at either 4 degrees C or 30-37 degrees C. Consistently, potential nitrification was higher between 10 and 25 degrees C than at either 4 degrees C or 37 degrees C. However, as long as ammonium was not limiting, release rates of N2O increased monotonously between 4 and 37 degrees C after short-term temperature adaptation, with nitrification accounting for about 35-50% of the N2O production between 4 and 25 degrees C. In order to see whether temperature may also affect the community structure of ammonia oxidizers, we studied moist soil during long incubation at low and high concentrations of commercial fertilizer. The soil was also incubated in buffered (pH 7) slurry amended with urea. Communities of ammonia oxidizers were assayed by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the alpha subunit of ammonia monooxygenase. We found that a polymerase chain reaction (PCR) system using a non-degenerated reverse primer (amoAR1) gave the best results. Community shifts occurred in all soil treatments after 16 weeks of incubation. The community shifts were obviously influenced by the different fertilizer treatments, indicating that ammonium was a selective factor for different ammonia oxidizer populations. Temperature was also a selective factor, in particular as community shifts were also observed in the soil slurries, in which ammonium concentrations and pH were better controlled. Cloning and sequencing of selected DGGE bands indicated that amoA sequences belonging to Nitrosospira cluster 1 were dominant at low temperatures (4-10 degrees C), but were absent after long incubation at low fertilizer treatment. Sequences of Nitrosospira cluster 9 could only be detected at low ammonium concentrations, whereas those of Nitrosospira cluster 3 were found at most ammonium concentrations and temperatures, although individual clones of this cluster exhibited trends with temperature. Obviously, ammonia oxidizers are able to adapt to soil conditions by changes in the community structure if sufficient time (several weeks) is available.     

Impact of nitrate on the structure and function of bacterial biofilm communities in pipelines used for injection of seawater into oil fields

We studied the impact of NO(3)(-) on the bacterial community composition, diversity, and function in in situ industrial, anaerobic biofilms by combining microsensor profiling, (15)N and (35)S labeling, and 16S rRNA gene-based fingerprinting. Biofilms were grown on carbon steel coupons within a system designed to treat seawater for injection into an oil field for pressurized oil recovery. NO(3)(-) was added to the seawater in an attempt to prevent bacterial H(2)S generation and microbially influenced corrosion in the field. Microprofiling of nitrogen compounds and redox potential inside the biofilms showed that the zone of highest metabolic activity was located close to the metal surface, correlating with a high bacterial abundance in this zone. Upon addition, NO(3)(-) was mainly reduced to NO(2)(-). In biofilms grown in the absence of NO(3)(-), redox potentials of <-450 mV at the metal surface suggested the release of Fe(2+). NO(3)(-) addition to previously untreated biofilms induced a decline (65%) in bacterial species richness, with Methylophaga- and Colwellia-related sequences having the highest number of obtained clones in the clone library. In contrast, no changes in community composition and potential NO(3)(-) reduction occurred upon subsequent withdrawal of NO(3)(-). Active sulfate reduction was below detection levels in all biofilms, but S isotope fractionation analysis of sulfide deposits suggested that it must have occurred either at low rates or episodically. Scanning electron microscopy revealed that pitting corrosion occurred on all coupons, independent of the treatment. However, uniform corrosion was clearly mitigated by NO(3)(-) addition.