Enrichment of autotrophic anaerobic ammonium-oxidizing consortia from various wastewaters

The option for biological nitrogen removal has recently been broadened with the description of simultaneous nitrification/denitrification, anaerobic ammonium oxidation (ANAMMOX) and the concept of CANON (completely autotrophic nitrogen removal over nitrite). An autotrophic anaerobic ammonium oxidation (AAAO) consortium was successfully selected and enriched from municipal treatment plant sludges in Sydney, Australia, but not from industrial coke-oven wastewater sludges. Chemolithoautotrophic basic salt (CLABS) medium was used in the selection of AAAO organisms and chloramphenicol was added to the initial stage of selection to eliminate denitrifiers. Two different temperatures, 37degreesC and 55degreesC, were used in the selection of mesophilic and thermophilic consortia, respectively. Thermophilic AAAO organisms were not selected at 55degreesC. Mesophilic AAAO activities, however, were evident in both batch and continuous cultures, whereby ammonium was consumed concurrently with a decrease of nitrite, giving a ratio of 1:1-1:1.3 in ammonium removal rate over nitrite consumption rate. A continuous-mode mesophilic fixed-bed reactor was established to enrich the AAAO consortium. After 1 year, biofilms, pinkish in color, had developed on the support media and side wall of the feed-line tubing. Ammonium and nitrite consumption increased from similar to15 mg to 60 mg d(-1) L-1 over a period of 243 days. Later, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) techniques revealed that the dominant cell type in the AAAO consortium had a similar morphology and 16S rDNA sequence homology to that of the recently described ANAMMOX organism, Brocadia anammoxidans.

Integration of sulphate reduction, autotrophic denitrification and nitrification to achieve low-cost excess sludge minimisation for Hong Kong sewage

An integrated anaerobic-aerobic treatment system of sulphate-laden wastewater was proposed here to achieve low sludge production, low energy consumption and effective sulphide control. Before integrating the whole system, the feasibility of autotrophic denitrification utilising dissolved sulphide produced during anaerobic treatment of sulphate rich wastewater was studied here. An upflow anaerobic sludge blanket reactor was operated to treat sulphate-rich synthetic wastewater (TOC = 100 mg/L and sulphate = 500 mg/L) and its effluent with dissolved sulphide and external nitrate solution were fed into an anoxic biofilter. The anaerobic reactor was able to remove 77-85% of TOC at HRT of 3 h and produce 70-90 mg S/L sulphide in dissolved form for the subsequent denitrification. The performance of anoxic reactor was stable, and the anoxic reactor could remove 30 mg N/L nitrate at HRT of 2 h through autotrophic denitrification. Furthermore, sulphur balance for the anoxic filter showed that more than 90% of the removed sulphide was actually oxidised into sulphate, thereby there was no accumulation of sulphur particles in the filter bed. The net sludge productions were approximately 0.15 to 0.18 g VSS/g COD in the anaerobic reactor and 0.22 to 0.31 g VSS/g NO3--N in the anoxic reactor. The findings in this study will be helpful in developing the integrated treatment system to achieve low-cost excess sludge minimisation.

Tracing the fate of N-15-enriched feed in an intensive shrimp system

The fate of N-15-nitrogen-enriched formulated feed fed to shrimp was traced through the food web in shallow, outdoor tank systems (1000 1) stocked with shrimp. Triplicate tanks containing shrimp water with and without sediment were used to identify the role of the natural biota in the water column and sediment in processing dietary nitrogen (N). A preliminary experiment demonstrated that N-15-nitrogen-enriched feed products could be detected in the food web. Based on this, a 15-day experiment was conducted. The ammonium (NH4+) pool in the water column became rapidly enriched (within one day) with N-15-nitrogen after shrimp were fed N-15-enriched feed. By day 15, 6% of the added N-15-nitrogen was in this fraction in the 'sediment' tanks compared with 0.4% in the 'no sediment' tanks. The particulate fraction in the water column, principally autotrophic nanoflagellates, accounted for 4-5% of the N-15-nitrogen fed to shrimp after one day. This increased to 16% in the 'no sediment' treatment, and decreased to 2% in the 'sediment' treatment by day 15. It appears that dietary N was more accessible to the phytoplankton community in the absence of sediment. The difference is possibly because a proportion of the dietary N was buried in the sediment in the 'sediment' treatment, making it unavailable to the phytoplankton. Alternatively, the dietary N was retained in the NH4+ pool in the water column since phytoplankton growth, and hence, N utilization was lower in the 'sediment' treatment. The lower growth of phytoplankton in the 'sediment' treatment appeared to be related to higher turbidity, and hence, lower light availability for growth. The percentage N-15-nitrogen detected in the sediment was only 6% despite the high capacity for sedimentation of the large biomass of plankton detritus and shrimp waste. This suggests rapid remineralization of organic waste by the microbial community in the sediment resulting in diffusion of inorganic N sources into the water column. It is likely that most of the dietary N will ultimately be removed from the tank system by water discharges. Our study showed that N-15-nitrogen derived from aquaculture feed can be processed by the microbial community in outdoor aquaculture systems and provides a method for determining the effect of dietary N on ecosystems. However, a significant amount of the dietary N was not retained by the natural biota and is likely to be present in the soluble organic fraction. (C) 2002 Elsevier Science B.V. All rights reserved.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria

Recently, two fresh water species, 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis', and one marine species, 'Candidatus Scalindua sorokinii', of planctomycete anammox bacteria have been identified. 'Candidatus Scalindua sorokinii' was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle-the anammoxosome-in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Improved nitrogen removal by application of new nitrogen-cycle bacteria

In order to meet increasingly stringent European discharge standards, new applications and control strategies for the sustainable removal of ammonia from wastewater have to beimplemented. In this paper we discuss anitrogen removal system based on the processesof partial nitrification and anoxic ammoniaoxidation (anammox). The anammox process offers great opportunities to remove ammonia in fully autotrophic systems with biomass retention. No organic carbon is needed in such nitrogenremoval system, since ammonia is used a selectron donor for nitrite reduction. The nitrite can be produced from ammonia in oxygen-limited biofilm systems or in continuous processes without biomass retention. For successful implementation of the combined processes, accurate biosensors for measuring ammonia and nitrite concentrations, insight inthe complex microbial communities involved, and new control strategies have to be developed and evaluated.

Nitrifying microbial community analysis of nitrite accumulating biofilm reactor by fluorescence in situ hybridization

Biological nitrogen removal via nitrite pathway in wastewater treatment is very important especially in the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulations were carried out in a biofilm reactor. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.2 kg N/m3/d. Nitrite accumulation was achieved by the selective inhibition of nitrite oxidizers by free ammonia and oxygen limitation. Nitrite oxidation activity was recovered as soon as the inhibition factor was removed. Fluorescence in situ hybridization studies of the nitrite accumulating biofilm system have shown that genus Nitrosomonas which is specifically hybridized with probe NSM 156 was the dominant nitrifying bacteria while Nitrospira was less abundant than those of normal nitrification systems. Further FISH analysis showed that the combinations of Nitrosomonas and Nitrospira cells were identified as important populations of nitrifying bacteria in an autotrophic nitrifying biofilm system.

Nitrification of high strength ammonia wastewtaer treatment - process selection is the major factor.

Biological nitrogen removal via the nitrite pathway in wastewater treatment is very important in Saving the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulation were carried out in a biofilm airlift reactor with autotrophic nitrifying biofilm. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.5 to 3.5 kg N/m3.d. Nitrite accumulation was stably achieved by the selective inhibition of nitrite oxidizers with free ammonia and dissolved oxygen limitation. Stable 100% conversion to nitrite could also be achieved even under the absence of free ammonia inhibition on nitrite oxidizers. Batch ammonium oxidation and nitrite oxidation with nitrite accumulating nitrifying biofilm showed that nitrite Oxidation was completely inhibited when free ammonia is higher than 0.2 mg N/L. However, nitrite oxidation activity was recovered as soon as the free ammonia concentration was below the threshold level when dissolved oxygen concentration was not the limiting factor. Fluorescence in situ hybridization analysis of cryosectioned nitrite accumulating nitrifying biofilm showed that the β-subclass of Proteobacteria, where ammonia oxidizers belong, was distributed outside the biofilm whereas the α-subclass of Proteobacteria, where nitrite oxidizers belong, was found mainly in the inner part of the biofilm. It is likely that dissolved oxygen deficiency or limitation in the inner part of the nitrifying biofilm, where nitrite oxidizers exist, is responsible for the complete shut down of the nitrite oxidizers activity under the absence of free ammonia inhibition.

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.

Mechanisms of acetohydroxyacid synthases

Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

Diurnal polyp expansion behavior in stony corals may enhance carbon availability for symbionts photosynthesis

Photosynthesis of zooxanthellate stony corals may be limited by inorganic carbon at high irradiances. We demonstrated that oxygen consumption of expanded corals is higher than that of contracted corals in both night-expanding and day-expanding corals. It is assumed that at the single-polyp level, the expansion of tentacles increases the surface area for solute exchange with the surrounding water, which may alleviate potential carbon limitation and excess oxygen levels in the tissue under high irradiance. We investigated this hypothesis using stable carbon isotope (613 C) analysis of coral species from the Red Sea exhibiting different morphologies. delta C-13 ratios in zooxanthellae of branched coral colonies with small polyp size that extend their tentacles during daytime (diurnal morphs) showed lower delta C-13 values in their zooxanthellae - 13.83 +/- 1.45 parts per thousand, compared to corals from the same depth with large polyps, which are usually massive and expand their tentacles only at night (nocturnal morphs). Their algae delta C-13 was significantly higher, averaging - 11.33 +/- 0.59 parts per thousand. Carbon isotope budget of the coral tissue suggests that branched corals are more autotrophic, i.e., that they depend on their symbionts for nutrition compared to massive species, which are more heterotrophic and depend on plankton predation. (c) 2005 Elsevier B.V. All rights reserved.