Quantifying potential nitrogen removal by denitrification in stream sediments at a regional scale

Potential denitrification rates were measured using the acetylene block method, in sediments collected from streams in the sub-tropical, south-east Queensland region of Australia. Our aim was to estimate how much nitrogen could be removed from lotic systems by denitrification at the regional scale. Denitrification measured at 65 sites in August and September from a catchment of 22700 km(2) was extrapolated to all streams and rivers in the region based on the sediment area available for denitrification. Denitrification rates ranged between 4 and 950 mumol N m(-2) h(-1), with most sites having rates below 150 mumol N m(-2) h(-1). Based on these results, the current study estimates that a total of 305 t of nitrogen could be denitrified per year from all streams and rivers in the region, representing 6% of the total annual nitrogen load from surrounding land use. During baseflow conditions, when nitrogen loads to streams are low, the proportion of nitrogen removed through denitrification would be substantially higher, in some cases removing 100% of the nitrogen load. It is proposed that denitrification is an important process maintaining low concentrations of dissolved inorganic nitrogen under baseflow conditions and is therefore likely to enhance nitrogen limitation of primary production in this region.

Nitrogen removal from sludge digester liquids by nitrification/denitrification or partial nitritation/anammox: environmental and economical considerations

In wastewater treatment plants with anaerobic sludge digestion, 15-20% of the nitrogen load is recirculated to the main stream with the return liquors from dewatering. Separate treatment of this ammonium-rich digester supernatant significantly reduces the nitrogen load of the activated sludge system. Two biological applications are considered for nitrogen elimination: (i) classical autotrophic nitrification/heterotrophic denitrification and (ii) partial nitritation/autotrophic anaerobic ammonium oxidation (anammox). With both applications 85-90% nitrogen removal can be achieved, but there are considerable differences in terms of sustainability and costs. The final gaseous products for heterotrophic denitrification are generally not measured and are assumed to be nitrogen gas (N-2). However, significant nitrous oxide (N2O) production can occur at elevated nitrite concentrations in the reactor. Denitrification via nitrite instead of nitrate has been promoted in recent years in order to reduce the oxygen and the organic carbon requirements. Obviously this achievement turns out to be rather disadvantageous from an overall environmental point of view. On the other hand no unfavorable intermediates are emitted during anaerobic ammonium oxidation. A cost estimate for both applications demonstrates that partial nitritation/anammox is also more economical than classical nitrification/denitrification. Therefore autotrophic nitrogen elimination should be used in future to treat ammonium-rich sludge liquors.

A novel wastewater treatment process: Simultaneous nitrification, denitrification and phosphorus removal

Simultaneous nitrification and denitrification (SND) via the nitrite pathway and anaerobic-anoxic enhanced biological phosphorus removal (EBPR) are two processes that can significantly reduce the COD demand for nitrogen and phosphorus removal. The combination of these two processes has the potential of achieving simultaneous nitrogen and phosphorus removal with a minimal requirement for COD. A lab-scale sequencing batch reactor (SBR) was operated in alternating anaerobic-aerobic mode with a low dissolved oxygen concentration (DO, 0.5 mg/L) during the aerobic period, and was demonstrated to accomplish nitrification, denitrification and phosphorus removal. Under anaerobic conditions, COD was taken up and converted to polyhydroxyalkanoates (PHA), accompanied with phosphorus release. In the subsequent aerobic stage, PHA was oxidized and phosphorus was taken up to less than 0.5 mg/L at the end of the cycle. Ammonia was also oxidised during the aerobic period, but without accumulation of nitrite or nitrate in the system, indicating the occurrence of simultaneous nitrification and denitrification. However, off-gas analysis found that the final denitrification product was mainly nitrous oxide (N2O) not N-2. Further experimental results demonstrated that nitrogen removal was via nitrite, not nitrate. These experiments also showed that denitrifying glycogen.-accumulating organisms rather than denitrifying polyphosphate-accumulating organisms were responsible for the denitrification activity.

Using nitrogen stable isotope ratios (delta N-15) of macroalgae to determine the effectiveness of sewage upgrades: changes in the extent of sewage plumes over four years in Moreton Bay, Australia

Nitrogen loading to aquatic ecosystems from sewage is recognised worldwide as a growing problem. The use of nitrogen stable isotopes as a means of discerning sewage nitrogen in the environment has been used annually by the Ecosystem Health Monitoring Program in Moreton Bay (Australia) since 1997 when the technique was first developed. This (sewage plume mapping) technique, which measures the delta(15)N isotopic signature of the red macroalga Catenella nipae after incubation in situ, has demonstrated a large reduction in the magnitude and spatial extent of sewage nitrogen within Moreton Bay over the past 5 years. This observed reduction coincides with considerable upgrades to the nitrogen removal efficacy at several sewage treatment plants within the region. This paper describes the observed changes and evaluates whether they can be attributed to the treatment upgrades. (c) 2004 Published by Elsevier Ltd.

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 [C-13] methanol to biomark the DNA of the denitrifiers. The extracted [C-13]DNA and [C-12]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [C-13]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 [C-12]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 [C-14] 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.

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [C-13]acetate was used in SIP to label the DNA of the denitrifiers. The [C-13]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the C-13 library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking Up [C-14] acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the waste-water industry to enhance denitrification.

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation' Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO2- uptake than NH4 uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO2- from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal. (c) 2006 Elsevier Inc. All rights reserved.

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3) d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L-1. Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosonlonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662. (c) 2005 Elsevier Ltd. All rights reserved.