Simultaneous nitrification and denitrification in a mixed methanotrophic culture

Simultaneous nitrification and denitrification using a mixed methanotrophic culture was investigated. When both NO₃ ^–-N (108 mg l^–1) and NH₃-N (59 mg l^–1) were added into batch reactors, nitrate removal was complete within 10 h at the rate of 47 mg NO₃ ^–-N g VSS^–1 day^–1 when dissolved oxygen (DO) concentration was maintained at 2 mg DO l^–1. Ammonia removal started simultaneously with nitrate removal at a slower rate of 14 NH₃-N g VSS^–1 day^–1. No significant accumulation of nitrite or nitrate during ammonia utilization suggested the occurrence of simultaneous nitrification and denitrification.     

Long-term patterns of invertebrate stream drift in an Australian temperate stream

1. Invertebrate stream drift was sampled bimonthly in the Acheron River, Victoria, Australia, over a period of 18 months. Replicated hourly samples were collected over a 25-h period on each sampling date. A total of 194 taxa were identified in the drift. However, total drift density was dominated by few taxa. 2. Some evidence was obtained for a seasonal pattern in drift: this was most pronounced in relative abundances of individual taxa and the composition of the drift, rather than in total drift densities. Most of the commonly collected taxa reflected the seasonal pattern of total drift. However, some of the common taxa did not. 3. A small number of taxa showed behavioral drift, with a nocturnal increase in drift densities. One species of ‘Baetis’ drifted in high densities over short periods of time around dusk and dawn. It did not drift in higher densities during the night than during the day. The results emphasize the need for drift studies to be more rigorously designed than is typically the case.     

Potential rates of nitrification and denitrification in an oligotrophic freshwater sediment system

Potential rates of nitrification and denitrification were measured in an oligotrophic sediment system. Nitrification potential was estimated using the CO oxidation technique, and potential denitrification was measured by the acetylene blockage technique. The sediments demonstrated both nitrifying and denitrifying activity. E_h, O₂, and organic C profiles showed two distinct types of sediment. One type was low in organic C, had high O₂ and E_h, and had rates of denitrification 1,000 times lower than the other which had high organic C, low O₂, and low E_h. Potential nitrification and denitrification rates were negatively correlated with E_h. This suggests that environmental heterogeneity in denitrifier and nitrifier populations in oligotrophic sediment systems may be assessed using E_h before sampling protocols for nitrification or denitrification rates are established. There was no correlation between denitrification and nitrification rates or between either of these processes and NH₄ ⁺ or NO₃ ^– concentrations. The maximum rate of denitrification was 0.969 nmole N cm^–3 hour^–1, and the maximum rate of nitrification was 23.6 nmole cm^–3 hour^–1, suggesting nitrification does not limit denitrification in these oligotrophic sediments. Some sediment cores had mean concentrations of 6.0 mg O₂/liter and still showed both nitrification and denitrification activity.     

Evaluation of denitrification in an urban stream receiving wastewater effluent

Urban streams often contain elevated concentrations of nitrogen (N) which can be amplified in systems receiving effluent from wastewater treatment plants (WWTP). In this study, we evaluated the importance of denitrification in a stream draining urban Greensboro, NC, USA, using two approaches: (1) natural abundance of ¹⁵N–NO₃⁻ in conjunction with background NO₃⁻–N concentrations along a 7 km transect downstream of a WWTP; and (2) C₂H₂ block experiments at three sites and at three habitat types within each site. Overall lack of a longitudinal pattern of δ¹⁵N–NO₃⁻ and NO₃⁻–N, combined with high concentrations of NO₃⁻–N suggested that other factors were controlling NO₃⁻–N flux in the study transect. However, denitrification did appear to be significant along one portion of the transect. C₂H₂ block experiments showed that denitrification rates were much higher downstream of the WWTP compared to upstream, and showed that denitrification rates were highest in erosional and depositional areas downstream of the WWTP and in erosional areas upstream of the plant. Thus, the combination of the two methods for evaluating denitrification provided more insight into the spatial dynamics of denitrification activity than either approach alone. Denitrification appeared to be a significant sink for NO₃⁻–N upstream of the WWTP, but not downstream. Approximately 46% of the total NO₃⁻–N load was removed via denitrification in the upstream, urban section of the stream, while only 2.3% of NO₃⁻–N was lost downstream of the plant. This result suggests that controlling NO₃⁻–N loading from the plant could result in considerable improvement of downstream water quality.     

Stimulating denitrification in a stream mesocosm with elemental sulfur as an electron donor

The heavy use of fertilizers in agricultural lands can result in significant nitrate (NO₃⁻) loadings to the aquatic environment. We hypothesized that biological denitrification in agricultural ditches and streams could be enhanced by adding elemental sulfur (S^o) to the sediment layer, where it could act as a biofilm support and electron donor. Using a bench-scale stream mesocosm with a bed of S^o granules, we explored NO₃⁻ removal fluxes as a function of the effluent NO₃⁻ concentrations. With effluent NO₃⁻ ranging from 0.5 mg N L⁻¹ to 4.1 mg N L⁻¹, NO₃⁻ removal fluxes ranged from 228 mg N m⁻² d⁻¹ to 708 mg N m⁻² d⁻¹. This is as much as 100 times higher than for agricultural drainage streams. Sulfate (SO₄²⁻) production was high due to aerobic sulfur oxidation. Molecular studies demonstrated that the S^o amendment selected for Thiobacillus species, and that no special inoculum was required for establishing a S^o-based autotrophic denitrifying community. Modeling studies suggested that denitrification was diffusion limited, and advective flow through the bed would greatly enhance NO₃⁻ removal fluxes. Our results indicate that amendment with S^o is an effective means to stimulate denitrification in a stream environment. To minimize SO₄²⁻ production, it may be better to place S^o deeper in the sediment layer.     

Particulate organic matter quality influences nitrate retention and denitrification in stream sediments: evidence from a carbon burial experiment

Organic carbon supply is linked to nitrogen transformation in ecosystems. However, the role of organic carbon quality in nitrogen processing is not as well understood. We determined how the quality of particulate organic carbon (POC) influenced nitrogen transformation in stream sediments by burying identical quantities of varying quality POC (northern red oak (Quercus rubra) leaves, red maple (Acer rubrum) leaves, red maple wood) in stream mesocosms and measuring the effects on nitrogen retention and denitrification compared to a control of combusted sand. We also determined how POC quality affected the quantity and quality of dissolved organic carbon (DOC) and dissolved oxygen concentration in groundwater. Nitrate and total dissolved nitrogen (TDN) retention were assessed by comparing solute concentrations and fluxes along groundwater flow paths in the mesocosms. Denitrification was measured by in situ changes in N₂ concentrations (using MIMS) and by acetylene block incubations. POC quality was measured by C:N and lignin:N ratios and DOC quality was assessed by fluorescence excitation emission matrix spectroscopy. POC quality had strong effects on nitrogen processing. Leaf treatments had much higher nitrate retention, TDN retention and denitrification rates than the wood and control treatments and red maple leaf burial resulted in higher nitrate and TDN retention rates than burial of red oak leaves. Leaf, but not wood, burial drove pore water to severe hypoxia and leaf treatments had higher DOC production and different DOC chemical composition than the wood and control treatments. We think that POC quality affected nitrogen processing in the sediments by influencing the quantity and quality of DOC and redox conditions. Our results suggest that the type of organic carbon inputs can affect the rates of nitrogen transformation in stream ecosystems.     

Effect of organic loading on nitrification and denitrification in a marine sediment microcosm

Abstract The effects of organic additions on nitrification and dentrification were examined in sediment microcosms. The organic material, heat killed yeast, had a C/N ratio of 7.5 and was added to sieved, homogenized sediments. Four treatments were compared: no addition (control), 30 g dry weight (dw) m⁻² mixed throughout the 10 cm sediment column (30M), 100 g dw m⁻² mixed throughout sediments (100M), and 100 g dw m⁻² mixed into top 1 cm (100S). After the microcosms had been established for 7–11 days, depth of O₂ penetration, sediment-water fluxes and nitrification rates were measured. Nitrification rates were measured using three different techniques: N-serve and acetylene inhibition in intact cores, and nitrification potentials in slurris. Increased organic additions decreased O₂ penetration from 2.7 to 0.2 mm while increasing both O₂ consumption, from 30 to 70 mmol O₂ m⁻² d⁻¹, and NO₃⁻ flux into sediments. Nitrification rates in intact cores were similar for the two methods. Highest rates occurred in the 30M treatment, while the lowest rate was measured in the 100S treatment. Total denitrification rates (estimated from nitrification and nitrate fluxes) increased with increased organic addition, because of the high concentrations of NO₃⁻ (40 μM) in the overlaying water. The ratio of nitrification: denitrification was used as an indication of the importance of nitrification as the NO₃⁻ supply for denitrificaion. This ratio decreased from 1.55 to 0.05 iwth increase organic addition.     

Spatial distribution of denitrifying activity in a stream draining an agricultural catchment

SUMMARY 1. A field study was made of the spatial distribution of denitrification activity in the sediment of the River Dorn, Oxfordshire, England.
2. An assay of denitrifying enzyme activity was used to examine the distribution of denitrification with depth in cores of sediment representative of the types found in the stream. The maximum activity recorded in a predominantly silt sediment core was 5 times greater than that recorded in a sandy gravel core. In both fine sand and silt cores, peaks in denitrifier enzyme activity were shown to correspond to the limit of the nitrate diffusion front. At this depth the redox potential dropped rapidly from + 300 mV to 0 or less. Denitrifying enzyme activity in the stream water was negligible.
3. In situ denitrification activity (I DA) measurements were carried out in an 800 m reach of the Dorn using the acetylene inhibition technique on small sediment cores. Concurrent measurements were also made of stream depth and velocity, nitrate concentration in the interstitial water, and the wet bulk density, loss on ignition, mineraliz- able carbon and total nitrogen contents of the sediment. Mineralizable carbon was the variable which showed the best correlation with I DA. Highest IDAs were associated with accumulations of fine-grained sediment at meander bends. Mean IDAs measured under flood conditions were significantly higher ( P <0.05) than those measured under baseflow. It was estimated that denitrification reduced the nitrate load in the River Dorn by 15% under summer baseflow conditions     

Role of nitrification and denitrification for NO metabolism in soil

Release and uptake of NO was measured in a slightly alkaline (pH 7.8) and an acidic (pH 4.7) cambisol. In the alkaline soil under aerobic conditions, NO release was stimulated by ammonium and inhibited by nitrapyrin. Nitrate accumulated simultaneously and was also inhibited by nitrapyrin.¹⁵NO was released after fertilization with¹⁵NH₄NO₃ but not with NH₄ ¹⁵NO₃. The results indicate that in aerobic alkaline cambisol NO was mainly produced during nitrification of ammonium. The results were different under anaerobic conditions and also in the acidic cambisol. There, NO release was stimulated by nitrate and not by ammonium, and was inhibited by chlorate and not by nitrapyrin indicating that NO production was exclusively due to reduction of nitrate. The results were confirmed by¹⁵NO being released mainly from NH₄ ¹⁵NO₃ rather than from¹⁵NH₄NO₃. The observed patterns of NO release were explained by the NO production processes being stimulated by either ammonium or nitrate in the two different soils, whereas the NO consumption processes being only stimulated by nitrate. NO release was larger than N₂O release, but both were small compared to changes in concentrations of soil ammonium or nitrate.(*request for offprints)     

Analysis of an upflow bioreactor system for nitrogen removal via autotrophic nitrification and denitrification

The upflow bioreactor system without biomass-liquid separation unit was evaluated for its efficacy in sustaining autotrophic nitrification and denitrification (AND). The bioreactor system was capable of sustaining AND by means of carefully controlled oxygenation to achieve the maximum NH(4)(+)-N removal rate of 0.054 g N gVSS(-1) day(-1) (38% removal efficiency) at the oxygen influx and nitrogen loading rate of 3.68 mg O(2) h(-1) L-bioreactor(-1) and 182 mg N day(-1) L-bioreactor(-1), respectively. Additional nitrogen removal was achieved in a two-stage bioreactor configuration due to endogenous denitrification under long mean cell residence time. Quiescent conditions maintained in the bioreactor provided stable hydrodynamic environments for the chemoautotrophic biomass matrix, which revealed porous, loosely-structured, and mat-like architecture. More than 95% of the total biomass holdup (1.3-1.5 g VSS) was retained, thereby producing low biomass washout rate ( approximately 40 mg VSS day(-1)) with VSS < 11 mg VSSL(-1) in the effluent.     

A novel double-membrane system for simultaneous nitrification and denitrification in a single tank

A novel biological treatment system, which contains two types of membrane modules in a single tank, was developed for simultaneous nitrification and denitrification. Both of the modules were fed with the substrates on the tube side of the silicone tubes by diffusing them to the biofilms which form on the surface of the tubes. One module was fed with methanol for denitrification and the other one was fed with pure oxygen for nitrification. As a result, the interference of organic carbon on nitrification, and that of oxygen on denitrification, were both hindered by the diffusion barriers (biofilms), thereby allowing two different niches for nitrifiers and denitrifiers to coexist in a single tank. Besides saving space and the amount of alkalinity required for nitrification, this system also produced low residual chemical oxygen demand (COD) and high nitrogen removal rates (2.9-3.4 gN m-2 d-1 of surface area of membrane).     

Efficiency of doxorubicin handling by isolated hepatocytes is a valuable indicator for restored cell function

Pig liver is a possible source of hepatocytes for extracorporeal bio-artificial liver devices. In order to evaluate recovered hepatocyte function following enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour drug doxorubicin within intracellular acidic compartments. Doxorubicin is a naturally fluorescent molecule. Thus, the process of drug concentration within hepatocytes can be visualized in living conditions by fluorescence microscopy. Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers. Immediately after isolation and at fixed culture times, cells were incubated with 0.1mM doxorubicin in Hanks' balanced salt solution for 10min at 37°C in 5% CO₂-humidified atmosphere and observed by fluorescence microscopy. Parallel electron microscopy was performed to compare fluorescence data with general cell morphology. To monitor lysosomal acidification capacity, the fluorescent pH-sensitive vital dye LysoSensor-Blue was used. Doxorubicin fluorescence showed different patterns of nuclear and cytoplasmic staining, according to the time allowed for cell recovery and the culture method. In particular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a punctate perinuclear and pericanalicular fluorescence detectable in fully recovered hepatocyte monolayers. This study indicates that the doxorubicin-fluorescence test may be considered a simple and rapid procedure for assessing hepatocyte functional condition. It may provide valuable and real time guidelines for judging the correct way these cells are to be collected, preserved and utilized for clinical purposes.     

Soil microbial diversity, community structure and denitrification in a temperate riparian zone

Nitrate (NO ₃ ^– ) removal in riparian zones bordering agricultural areas occurs via plant uptake, microbial immobilisation and bacterial denitrification. Denitrification is a desirable mechanism for removal because the bacterial conversion of NO ₃ ^– to N gases permanently removes NO ₃ ^– from the watershed. A field and laboratory study was conducted in riparian soils adjacent to Carroll Creek, Ontario, Canada, to assess the spatial distribution of denitrification relative to microbial community structure and microbial functional diversity. Soil samples were collected in March, June, and August 1997 at varying soil depths and distances from the stream. Denitrification measurements made using the acetylene block technique on intact soil cores were highly variable and did not show any trends with riparian zone location. Microbial community composition and functional diversity were determined using sole carbon source utilization (SCSU) on Biolog® GN microplates. Substrate richness, evenness and diversity (Shannon index) were greatest within the riparian zone and may also have been influenced by a rhizosphere effect. A threshold relationship between denitrification and measures of microbial community structure implied minimum levels of richness, evenness and diversity were required for denitrification.     

Macrophyte beds contribute disproportionately to benthic invertebrate abundance and biomass in a sand plains stream

Macrophyte beds have been shown to influence organic matter retention and nutrient processing in streams. Less is known about the extent to which plant beds contribute to abundance, biomass, and diversity of macroinvertebrate assemblages in low-order streams. We measured aquatic invertebrate abundance, biomass, and diversity associated with plant beds and sand/gravel patches in a low-gradient second-order stream in the Central Sand Plains of Wisconsin, USA from March to October. Invertebrate abundance and biomass were higher on average in plant beds (2,552 m⁻² and 1,575 mg m⁻²) than in sand/gravel patches (893 m⁻² and 486 mg m⁻²). Although sand/gravel habitat was over three times more abundant than plant beds in the study reach, plant beds and sand/gravel patches contributed similarly to invertebrate abundance and biomass at the whole-reach scale. The abundance and biomass of invertebrates associated with plant beds decreased from spring to autumn. Non-insect invertebrates in the plant beds increased in relative abundance as the year progressed. Shannon–Weiner diversity and taxa richness of invertebrates were higher in the plant beds than in the sand/gravel habitat. Our results suggest that plant beds can represent hot spots for invertebrate abundance and production in low-gradient streams, and have implications for stream management and restoration in these types of ecosystems. Handling editor: S. I. Dodson     

The integration of methanogenesis with shortcut nitrification and denitrification in a combined UASB with MBR

A combined system consisting of an up-flow anaerobic sludge blanket (UASB) and an aerobic membrane bioreactor (MBR) was operated at 28-30 degrees C and pH 7.8-8.1 for the treatment of low-strength synthetic wastewater enriched with organic carbon and NH4Cl. The MBR slurry was recirculated into the UASB with a ratio of 50-800%. It was found that nitrite was able to accumulate steadily during the nitrification step in the MBR at a low TOC/NH4+-N ratio. The mixed liquid containing NOX(-)-N in the MBR was recirculated to the UASB, and denitrification rather than methanogenesis became the preferred pathway. Whereas, the less carbon requirement for denitrification via nitrite rather than nitrate allowed methanogenesis to proceed simultaneously in the same reactor. The combination of membrane filtration and partial nitrification in the MBR with simultaneous denitrification and methanogenesis in the UASB could stably reach 98% TOC removal and 48.1-82.8% TN removal with recirculation ratio increasing from 50% to 800%.     

Simultaneous nitrification and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor

Nitrogen and carbon components in domestic modified wastewater were completely removed by simultaneous nitrification and denitrification using a membrane-aerated biofilm reactor where biofilm was fixed on a hollow-fiber membrane. To measure the spatial distribution of pH, ammonium and nitrate ions and to observe microbes inside the biofilm fixed on the membrane, microelectrodes and the fluorescence in situ hybridization (FISH) method were applied. Due to plug flow in the vertical direction (from the bottom to the top of the reactor), ammonium nitrogen was gradually removed and negligible nitrate nitrogen was detected throughout the reactor. FISH revealed that ammonia-oxidizing bacteria were mainly distributed inside the biofilm and other bacteria, which included denitrifying bacteria, were mainly distributed outside the biofilm and over the suspended sludge. In order to characterize bacterial activity in the vertical direction of the reactor, nitrification rates at lower, central and upper points were calculated using microelectrode data. The nitrification rate at the lower point was 7 and 125 times higher than those at the central and upper points, respectively. These results show that the removal of carbon and nitrogen compounds was accomplished efficiently by using various kinds of bacteria distributed vertically and horizontally in a single reactor.     

Spatio-temporal variation of periphyton biomass and accumulation in a temperate spring-fed stream

Spatio-temporal variation of plant populations often can demonstrate synchronous patterns, particularly within highly connected landscapes. Periphyton biomass (chlorophyll a) and net accumulation were measured at five sites in a spring-fed fourth-order stream located in central Pennsylvania with a mixed land-uses watershed (Spring Creek, USA) to characterize longitudinal variation within the stream. Samples were collected at three-week intervals over one year to describe seasonal patterns of periphyton biomass and net production (n = 17 per site). Spring Creek periphyton biomass and net accumulation increased dramatically from the headwaters to downstream (range 10–1,000 mg/m²). The downstream reaches had exceptionally large algal biomass (chlorophyll a > 300 mg/m²) and potential for rapid turnover. Varying degrees of seasonality were observed among the sites, with upstream sites showing more temporal variation but no distinct seasonal pattern. Despite this, large-scale disturbances within the watershed seem to promote synchrony among sites throughout the stream as reflected by close correlations in chlorophyll values (Pearson correlation coefficient r > 0.50).