Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO₃⁻-N mg of mixed-liquor volatile suspended solids (MLVSS)⁻¹ h⁻¹ to a steady-state value of 0.06 mg of NO₃⁻-N mg of MLVSS⁻¹ h⁻¹ over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [¹³C]methanol to biomark the DNA of the denitrifiers. The extracted [¹³C]DNA and [¹²C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [¹³C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [¹²C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [¹⁴C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

Microzonation of Denitrification Activity in Stream Sediments as Studied with a Combined Oxygen and Nitrous Oxide Microsensor

Microzonation of denitrification was studied in stream sediments by a combined O₂ and N₂O microsensor technique. O₂ and N₂O concentration profiles were recorded simultaneously in intact sediment cores in which C₂H₂ was added to inhibit N₂O reduction in denitrification. The N₂O profiles were used to obtain high-resolution profiles of denitrification activity and NO₃⁻ distribution in the sediments. O₂ penetrated about 1 mm into the dark-incubated sediments, and denitrification was largely restricted to a thin anoxic layer immediately below that. With 115 μM NO₃⁻ in the water phase, denitrification was limited to a narrow zone from 0.7 to 1.4 mm in depth, and total activity was 34 nmol of N cm⁻² h⁻¹. With 1,250 μM NO₃⁻ in the water, the denitrification zone was extended to a layer from 0.9 to 4.8 mm in depth, and total activity increased to 124 nmol of N cm⁻² h⁻¹. Within most of the activity zone, denitrification was not dependent on the NO₃⁻ concentration and the apparent K_m for NO₃⁻ was less than 10 μM. Denitrification was the only NO₃⁻-consuming process in the dark-incubated stream sediment. Even in the presence of C₂H₂, a significant N₂O reduction (up to 30% of the total N₂O production) occurred in the reduced, NO₃⁻-free layers below the denitrification zone. This effect must be corrected for during use of the conventional C₂H₂ inhibition technique.     

Degradation and Fate of Carbon Tetrachloride in Unadapted Methanogenic Granular Sludge

The potential of granular sludge from upflow anaerobic sludge blanket (UASB) reactors for bioremediation of chlorinated pollutants was evaluated by using carbon tetrachloride (CT) as a model compound. Granular sludges cultivated in UASB reactors on methanol, a volatile fatty acid mixture, or sucrose readily degraded CT supplied at a concentration of 1,500 nmol/batch (approximately 10 μM) without any prior exposure to organohalogens. The maximum degradation rate was 1.9 μmol of CT g of volatile suspended solids⁻¹ day⁻¹. The main end products of CT degradation were CO₂ and Cl⁻, and the yields of these end products were 44 and 68%, respectively, of the initial amounts of [¹⁴C]CT and CT-Cl. Lower chlorinated methanes accumulated in minor amounts temporarily. Autoclaved (dead) sludges were capable of degrading CT at rates two- to threefold lower than those for living sludges, indicating that abiotic processes (mediated by cofactors or other sludge components) played an important role in the degradation observed. Reduced components in the autoclaved sludge were vital for CT degradation. A major part (51%) of the CT was converted abiotically to CS₂. The amount of CO₂ produced (23%) was lower and the amount of Cl⁻ produced (86%) was slightly higher with autoclaved sludge than with living sludge. Both living and autoclaved sludges could degrade chloroform. However, only living sludge degraded dichloromethane and methylchloride. These results indicate that reductive dehalogenation, which was mediated better by living sludge than by autoclaved sludge, is only a minor pathway for CT degradation. The main pathway involves substitutive and oxidative dechlorination reactions that lead to the formation of CO₂. Granular sludge, therefore, has outstanding potential for gratuitous dechlorination of CT to safe end products.     

Annual Pattern of Denitrification and Nitrate Ammonification in Estuarine Sediment

The seasonal variation and depth distribution of the capacity for denitrification and dissimilatory NO₃⁻ reduction to NH₄⁺ (NO₃⁻ ammonification) were studied in the upper 4 cm of the sediment of Norsminde Fjord estuary, Denmark. A combination of C₂H₂ inhibition and ¹⁵N isotope techniques was used in intact sediment cores in short-term incubations (maximum, 4 h). The denitrification capacity exhibited two maxima, one in the spring and one in the fall, whereas the capacity for NO₃⁻ ammonification was maximal in the late summer, when sediments were progressively reduced. The denitrification capacity was always highest in the uppermost 1 cm of the sediment and declined with depth. The NO₃⁻ ammonification was usually higher with depth, but the maximum activity in late summer was observed within the upper 1 cm. The capacity for NO₃⁻ incorporation into organic material was investigated on two occasions in intact sediment cores and accounted for less than 5% of the total NO₃⁻ reduction. Denitrification accounted for between 13 and 51% of the total NO₃⁻ reduction, and NH₄⁺ production accounted for between 4 and 21%, depending on initial rates during the time courses. Changes of the rates during the incubation were observed in the late summer, which reflected synthesis of denitrifying enzymes. This time lag was eliminated in experiments with mixed sediment because of preincubation with NO₃⁻ and alterations of the near-environmental conditions. The initial rates obtained in intact sediment cores therefore reflect the preexisting enzyme content of the sediment.     

Anaerobic Ammonium Oxidation Measured in Sediments along the Thames Estuary, United Kingdom

Until recently, denitrification was thought to be the only significant pathway for N₂ formation and, in turn, the removal of nitrogen in aquatic sediments. The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment. By using a combination of ¹⁵N-labeled NH₄⁺, NO₃⁻, and NO₂⁻ (and ¹⁴N analogues), production of ²⁹N₂ and ³⁰N₂ was measured in anaerobic sediment slurries from six sites along the Thames estuary. The production of ²⁹N₂ in the presence of ¹⁵NH₄⁺ and either ¹⁴NO₃⁻ or ¹⁴NO₂⁻ confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO₂⁻. Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO₂⁻ or indirect reduction, following the initial reduction of NO₃⁻. Whether NO₂⁻ was directly present at 800 μM or it accumulated at 3 to 20 μM (from the reduction of NO₃⁻), the rate of ²⁹N₂ formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO₂⁻. We observed a shift in the significance of anaerobic ammonium oxidation to N₂ formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast. The relative importance of anaerobic ammonium oxidation was positively correlated (P < 0.05) with sediment organic content. This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system.     

Microelectrode Measurements of Nitrate Gradients in the Littoral and Profundal Sediments of a Meso-Eutrophic Lake (Lake Vechten, The Netherlands)

NO₃⁻ concentration profiles were measured in the sediments of a meso-eutrophic lake with a newly developed microelectrode. The depth of penetration of NO₃⁻ varied from only 1.3 mm in organic-rich profundal silty sediments to 5 mm in organic-poor littoral sandy sediments. The thickness of the zone of denitrification in the organic-rich sediments was <500 μm. Oxygen profiles measured simultaneously revealed that the zone of denitrification was directly adjacent to the aerobic zone. The results demonstrate high denitrification rates (0.26 to 1.31 mmol m⁻² day⁻¹) at in situ nitrate concentrations in the overlying water (0.030 mmol liter⁻¹) and limitation of denitrification by nitrate availability.     

Capacity for Denitrification and Reduction of Nitrate to Ammonia in a Coastal Marine Sediment

The capacity for dissimilatory reduction of NO₃⁻ to N₂ (N₂O) and NH₄⁺ was measured in ¹⁵NO₃⁻-amended marine sediment. Incubation with acetylene (7 × 10⁻³ atmospheres [normal]) caused accumulation of N₂O in the sediment. The rate of N₂O production equaled the rate of N₂ production in samples without acetylene. Complete inhibition of the reduction of N₂O to N₂ suggests that the “acetylene blockage technique” is applicable to assays for denitrification in marine sediments. The capacity for reduction of NO₃⁻ by denitrification decreased rapidly with depth in the sediment, whereas the capacity for reduction of NO₃⁻ to NH₄⁺ was significant also in deeper layers. The data suggested that the latter process may be equally as significant as denitrification in the turnover of NO₃⁻ in marine sediments.     

Production and Loss of Nitric Oxide from Denitrification in Anaerobic Brookston Clay

Nitric oxide, nitrous oxide, and nitrite ion production was measured in a Brookston clay column undergoing anaerobic denitrification. A flow system method was used whereby argon carrier gas continuously stripped soil gases from the column, allowing steady-state rates to be obtained. Over several days the temporal change in rates of these gases and NO₂⁻ followed a pattern of increase and decay which may be expected of a reaction proceeding by several consecutive steps. The method permits observation of the relatively large net production rate of NO, which is normally not observed in static systems based on head space analysis of gaseous denitrification products. In the first several hours after the onset of anoxic conditions, the net rate of NO production, f_NO, increased sharply to a maximum (~1 × 10⁻¹⁰ mol of N/g of soil per min), paralleling the rapid increase in NO₂⁻ level, and then followed a more gradual decline extending over approximately 45 h. A similar but less pronounced pattern was observed for N₂O, with net rates of production being considerably less than for NO. The ratio [NO-N]/[N₂O-N] decreased with time from ~2.5 at 6 h to ~2.0 at 45 h. Estimated rates of N₂ production appeared to be initially high, decreased rapidly within a few hours, and then gradually increased with time after the establishment of anaerobic conditions.     

Simultaneous Measurement of Denitrification and Nitrogen Fixation Using Isotope Pairing with Membrane Inlet Mass Spectrometry Analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N₂ gas species (²⁸N₂, ²⁹N₂, and ³⁰N₂) were monitored during incubation experiments after the addition of ¹⁵NO₃⁻. Formulas were developed to estimate the production (denitrification) and consumption (N₂ fixation) of N₂ gas from the fluxes of the different isotopic forms of N₂. Proportions of the three isotopic forms produced from ¹⁵NO₃⁻ and ¹⁴NO₃⁻ agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 μg-atoms of N m⁻² h⁻¹. They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N₂ production rates due to denitrification could be separated from high N₂ consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 μg-atoms of N m⁻² h⁻¹) were lower than nitrogen fixation rates (average, 60 μg-atoms of N m⁻² h⁻¹). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N₂ isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Nitrate Reduction in a Groundwater Microcosm Determined by 15N Gas Chromatography-Mass Spectrometry

Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO₃⁻-N liter⁻¹. Traces of ¹⁵NO₃⁻ were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day⁻¹. Both assimilatory and dissimilatory reduction rates were estimated from analyses of ¹⁵N₂, ¹⁵N₂O, ¹⁵NH₄⁺, and ¹⁵N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N₂ and N₂O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO₃⁻ and NH₄⁺ were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their [methyl-³H]thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g⁻¹ day⁻¹. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N₂, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO₃⁻ was reduced to NH₄⁺. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month.     

Impact of Bacterial NO3? Transport on Sediment Biogeochemistry

Experiments demonstrated that Beggiatoa could induce a H₂S-depleted suboxic zone of more than 10 mm in marine sediments and cause a divergence in sediment NO₃⁻ reduction from denitrification to dissimilatory NO₃⁻ reduction to ammonium. pH, O₂, and H₂S profiles indicated that the bacteria oxidized H₂S with NO₃⁻ and transported S⁰ to the sediment surface for aerobic oxidation.     

Selenate Reduction to Elemental Selenium by Anaerobic Bacteria in Sediments and Culture: Biogeochemical Significance of a Novel, Sulfate-Independent Respiration

Interstitial water profiles of SeO₄²⁻, SeO₃²⁻, SO₄²⁻, and Cl⁻ in anoxic sediments indicated removal of the seleno-oxyanions by a near-surface process unrelated to sulfate reduction. In sediment slurry experiments, a complete reductive removal of SeO₄²⁻ occurred under anaerobic conditions, was more rapid with H₂ or acetate, and was inhibited by O₂, NO₃⁻, MnO₂, or autoclaving but not by SO₄²⁻ or FeOOH. Oxidation of acetate in sediments could be coupled to selenate but not to molybdate. Reduction of selenate to elemental selenium was determined to be the mechanism for loss from solution. Selenate reduction was inhibited by tungstate and chromate but not by molybdate. A small quantity of the elemental selenium precipitated into sediments from solution could be resolublized by oxidation with either nitrate or FeOOH, but not with MnO₂. A bacterium isolated from estuarine sediments demonstrated selenate-dependent growth on acetate, forming elemental selenium and carbon dioxide as respiratory end products. These results indicate that dissimilatory selenate reduction to elemental selenium is the major sink for selenium oxyanions in anoxic sediments. In addition, they suggest application as a treatment process for removing selenium oxyanions from wastewaters and also offer an explanation for the presence of selenite in oxic waters.     

Effects of Carbon Substrates on Nitrite Accumulation in Freshwater Sediments

The contribution of the biochemical pathways nitrification, denitrification, and dissimilatory NO₃⁻ reduction to NH₄⁺ (DNRA) to the accumulation of NO₂⁻ in freshwaters is governed by the species compositions of the bacterial populations resident in the sediments, available carbon (C) and nitrogen (N) substrates, and environmental conditions. Recent studies of major rivers in Northern Ireland have shown that high NO₂⁻ concentrations found in summer, under warm, slow-flowing conditions, arise from anaerobic NO₃⁻ reduction. Locally, agricultural pollutants entering rivers are important C and N sources, providing ideal substrates for the aquatic bacteria involved in cycling of N. In this study a range of organic C compounds commonly found in agricultural pollutants were provided as energy sources in 48-h incubation experiments to investigate if the chemical compositions of the pollutants affected which NO₃⁻ reduction pathway was followed and influenced subsequent NO₂⁻ accumulation. Carbon stored within the sediments was sufficient to support DNRA and denitrifier populations, and the resulting NO₂⁻ peak (80 μg of N liter⁻¹ [approximate]) observed at 24 h was indicative of the simultaneous activities of both bacterial groups. The value of glycine as an energy source for denitrification or DNRA appeared to be limited, but glycine was an important source of additional N. Glucose was an efficient substrate for both the denitrification and DNRA pathways, with a NO₂⁻ peak of 160 μg of N liter⁻¹ noted at 24 h. Addition of formate and acetate stimulated continuous NO₂⁻ production throughout the 48-h period, caused by partial inhibition of the denitrification pathway. The formate treatment resulted in a high NO₂⁻ accumulation (1,300 μg of N liter⁻¹ [approximate]), and acetate treatment resulted in a low NO₂⁻ concentration (<100 μg of N liter⁻¹).     

Adaptation of Denitrifying Populations to Low Soil pH

Natural denitrification rates and activities of denitrifying enzymes were measured in an agricultural soil which had a 20-year past history of low pH (pH ca. 4) due to fertilization with acid-generating ammonium salts. The soil adjacent to this site had been limed and had a pH of ca. 6.0. Natural denitrification rates of these areas were of similar magnitude: 158 ng of N g⁻¹ of soil day⁻¹ for the acid soil and 390 ng of N g⁻¹ of soil day⁻¹ at the neutral site. Estimates of in situ denitrifying enzyme activity were higher in the neutral soil, but substantial enzyme activity was also detected in the acid soil. Rates of nitrous oxide reduction were very low, even when NO₃⁻ and NO₂⁻ were undetectable, and were ca. 400 times lower than the rates of N₂O production from NO₃⁻. Denitrification rates measured in slurries of the acid and neutral soil showed distinctly different pH optima (pH 3.9 and pH 6.3) which were near the pH values of the two soils. This suggests that an acid-tolerant denitrifying population had been selected during the 20-year period of low pH.     

Environmental Parameters Regulating Gaseous Nitrogen Losses from Two Forested Ecosystems via Nitrification and Denitrification

Gaseous N losses from disturbed and reference forested watersheds at the Coweeta Hydrologic Laboratory in western North Carolina were studied by in situ N₂O diffusion measurements and laboratory incubations throughout a 10-month period. Soil temperature, percent base saturation, and water-filled pore space accounted for 43% of the variation in in situ N₂O diffusion measurements. Laboratory incubations distinguished the gaseous N products of nitrification and denitrification. Nitrifying activity, ambient NO₃⁻, and nitrification N₂O were positively correlated with percent base saturation. However, differences between watersheds in soil N substrate caused by presence of leguminous black locust in the disturbed watershed were confounded with differences in soil acidity. Denitrification was most strongly affected by soil moisture, which in turn was determined by precipitation events and slope position. Gaseous N losses from well-drained midslope and toeslope landscape positions appeared to be minor relative to other N transformations. Favorable conditions for denitrification occurred at a poorly drained site near the stream of the disturbed watershed. Laboratory incubations revealed high rates of NO₃⁻ reduction in these soils. We speculate that the riparian zone is a major site of depletion of NO₃⁻ from the soil solution via denitrification.     

Anaerobic Growth and Denitrification among Different Serogroups of Soybean Rhizobia

We screened soybean rhizobia originating from three germplasm collections for the ability to grow anaerobically in the presence of NO₃⁻ and for differences in final product formation from anaerobic NO₃⁻ metabolism. Denitrification abilities of selected strains as free-living bacteria and as bacteroids were compared. Anaerobic growth in the presence of NO₃⁻ was observed in 270 of 321 strains of soybean rhizobia. All strains belonging to the 135 serogroup did not grow anaerobically in the presence of NO₃⁻. An investigation with several strains indicated that bacteria not growing anaerobically in the presence of NO₃⁻ also did not utilize NO₃⁻ as the sole N source aerobically. An exception was strain USDA 33, which grew on NO₃⁻ but failed to denitrify. Dissimilation of NO₃⁻ by the free-living cultures proceeded without the significant release of intermediate products. Nitrous oxide reductase was inhibited by C₂H₂, but preceding steps of denitrification were not affected. Final products of denitrification were NO₂⁻, N₂O, or N₂; serogroups 31, 46, 76, and 94 predominantly liberated NO₂⁻, whereas evolution of N₂ was prevalent in serogroups 110 and 122, and all three were formed as final products by strains belonging to serogroups 6 and 123. Anaerobic metabolism of NO₃⁻ by bacteroid preparations of Bradyrhizobium japonicum proceeded without delay and was evident by NO₂⁻ accumulation irrespective of which final product was formed by the strain as free-living bacteria. Anaerobic C₂H₂ reduction in the presence of NO₃⁻ was observed in bacteroid preparations capable of NO₃⁻ respiration but was absent in bacteria that were determined to be deficient in dissimilatory nitrate reductase.     

Members of the Family Comamonadaceae as Primary Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)-Degrading Denitrifiers in Activated Sludge as Revealed by a Polyphasic Approach

The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23 strains of PHBV-degrading denitrifiers were isolated from activated sludges from different sewage treatment plants. 16S ribosomal DNA (rDNA) sequence comparisons showed that 20 of the isolates were identified as members of the family Comamonadaceae, a major group of β-Proteobacteria. When the sludges from different plants were acclimated with PHBV under denitrifying conditions in laboratory scale reactors, the nitrate removal rate increased linearly during the first 4 weeks and reached 20 mg NO₃⁻-N h⁻¹ g of dry sludge⁻¹ at the steady state. The bacterial-community change in the laboratory scale sludges during the acclimation was monitored by rRNA-targeted fluorescence in situ hybridization and quinone profiling. Both approaches showed that the population of β-Proteobacteria in the laboratory sludges increased sharply during acclimation regardless of their origins. 16S rDNA clone libraries were constructed from two different acclimated sludges, and a total of 37 clones from the libraries were phylogenetically analyzed. Most of the 16S rDNA clones were grouped with members of the family Comamonadaceae. The results of our polyphasic approach indicate that β-Proteobacteria, especially members of the family Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Our data provide useful information for the development of a new nitrogen removal system with solid biopolymer as an electron donor.