PVA-alginate immobilized cells for anaerobic ammonium oxidation (anammox) process

The feasibility of an anaerobic ammonium oxidation (anammox) process combined with a cell-immobilization technique for autotrophic nitrogen removal was investigated. Anammox biomass was cultivated from local activated sludge and achieved significant anammox activity in 6 months. The development of a mature anammox biomass was confirmed by fluorescence in situ hybridization (FISH) analysis and off-line activity measurements. The abundance fraction of the anammox bacteria determined by FISH analysis was estimated by software. The anaerobic ammonia oxidizers occupied almost half of the total cells. Additionally, the anammox biomass was granulated as spherical gel beads of 3-4 mm in diameter by using a cell-immobilization technique. The nitrogen removal activity was proved to be successfully retained in the beads, with about 80% of nitrogenous compounds (NH(4) (+), NO(2) (- )and total nitrogen) removed after 48 h. These results offer a promising technique for the preservation of anammox microorganisms, the protection of them against the unfavorable surroundings, and the prevention of biomass washout towards the implementation of sustainable nitrogen elimination biotechnology. This is the first report on the immobilization of anammox biomass as gel beads.     

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".     

Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor

The ammonium-oxidizing microbial community was investigated in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor that was operated for about 1 year with high anaerobic ammonium oxidation activity (up to 0.8 kg NH(4)(+)-N m(-3) day(-1)). A Planctomycetales-specific 16S rRNA gene library was constructed to analyse the diversity of the anaerobic ammonium-oxidizing bacteria (AnAOB). Most of the specifically amplified sequences (15/16) were similar to each other (> 99%) but were distantly related to all of the previously recognized sequences (< 94%), with the exception of an unclassified anammox-related clone, KSU-1 (98%). An ammonia monooxygenase (amoA) gene library was also analysed to investigate the diversity of 'aerobic' ammonium-oxidizing bacteria (AAOB) from the beta-Proteobacteria. Most of the amoA gene fragments (53/55) clustered in the Nitrosomonas europaea-Nitrosococcus mobilis group which has been reported to prevail under oxygen-limiting conditions. The quantitative results from real-time polymerase chain reaction (PCR) amplification showed that the dominant AnAOB comprised approximately 50% of the total bacterial 16S rRNA genes in the reactor, whereas the AAOB of beta-Proteobacteria represented only about 3%. A large fragment (4008 bp) of the rRNA gene cluster of the dominant AnAOB (AS-1) in this reactor sludge was sequenced and compared with sequences of other Planctomycetales including four anammox-related candidate genera. The partial sequence of hydrazine-oxidizing enzyme (hzo) of dominant AnAOB was also identified using new designed primers. Based on this analysis, we propose to tentatively name this new AnAOB Candidatus'Jettenia asiatica'.     

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Ecological characteristics of anaerobic ammonia oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is the microbial conversion of ammonium and nitrite to dinitrogen gas. The functional microbes of anammox reaction are anammox bacteria, which were discovered in a wastewater treatment system for nitrogen removal. Anammox bacteria are prevalent in anoxic ecosystems and play an important role in both biological nitrogen cycle and nitrogen pollution control. In this paper, we reviewed the investigation on ecological characteristics of anammox bacteria, and tried to figure out their complicated intraspecies and interspecies relationships. As for intraspecies relationship, we focused on the quorum sensing system, a cell density-dependent phenomenon. As for interspecies relationship, we focused on the synergism and competition of anammox bacteria with other microorganisms for substrate and space. Finally, we discussed the great influence of environmental factors (e.g., dissolved oxygen, organic matters) on the constitution, structure and function of anammox bacteria community.     

Evaluation of inhibitory effects of heavy metals on anaerobic ammonium oxidation (anammox) by continuous feeding tests

To facilitate the application of anaerobic ammonium oxidation (anammox) to a nitrogen removal process, the effects of heavy metals (Ni, Cu, Co, Zn, and Mo) on anammox bacteria entrapped in gel carriers were examined by conducting continuous feeding tests for each metal. The results show that all anammox activities decreased by more than 10 % when influent concentrations of Ni, Cu, Co, Zn, and Mo were 5, 5, 5, 10, and 0.2 mg/L, respectively. It was observed that the effects of Ni, Cu, Co, and Zn on anammox activity were reversible and that of Mo on anammox activity was irreversible. Anammox activity was not affected when influent containing mixed Ni, Cu, Co, and Zn (0.5 mg/L) was fed into the reactor.     

Anaerobic ammonium oxidation (anammox) in different natural ecosystems

Anammox (anaerobic ammonium oxidation), which is a reaction that oxidizes ammonium to dinitrogen gas using nitrite as the electron acceptor under anoxic conditions, was an important discovery in the nitrogen cycle. The reaction is mediated by a specialized group of planctomycete-like bacteria that were first discovered in man-made ecosystems. Subsequently, many studies have reported on the ubiquitous distribution of anammox bacteria in various natural habitats, including anoxic marine sediments and water columns, freshwater sediments and water columns, terrestrial ecosystems and some special ecosystems, such as petroleum reservoirs. Previous studies have estimated that the anammox process is responsible for 50% of the marine nitrogen loss. Recently, the anammox process was reported to account for 9-40% and 4-37% of the nitrogen loss in inland lakes and agricultural soils respectively. These findings indicate the great potential for the anammox process to occur in freshwater and terrestrial ecosystems. The distribution of different anammox bacteria and their contribution to nitrogen loss have been described in different natural habitats, demonstrating that the anammox process is strongly influenced by the local environmental conditions. The present mini-review summarizes the current knowledge of the ecological distribution of anammox bacteria, their contribution to nitrogen loss in various natural ecosystems and the effects of major influential factors on the anammox process.     

Structural identification of ladderane and other membrane lipids of planctomycetes capable of anaerobic ammonium oxidation (anammox)

The membrane lipid composition of planctomycetes capable of the anaerobic oxidation of ammonium (anammox), i.e. Candidatus'Brocadia anammoxidans' and Candidatus'Kuenenia stuttgartiensis', was shown to be composed mainly of so-called ladderane lipids. These lipids are comprised of three to five linearly concatenated cyclobutane moieties with cis ring junctions, which occurred as fatty acids, fatty alcohols, alkyl glycerol monoethers, dialkyl glycerol diethers and mixed glycerol ether/esters. The highly strained ladderane moieties were thermally unstable, which resulted in breakdown during their analysis with GC. This was shown by isolation of a thermal product of these ladderanes and subsequent analysis with two-dimensional NMR techniques. Comprehensive MS and relative retention time data for all the encountered ladderane membrane lipids is reported, allowing the identification of ladderanes in other bacterial cultures and in the environment. The occurrence of ladderane lipids seems to be limited to the specific phylogenetic clade within the Planctomycetales able to perform anammox. This was consistent with their proposed biochemical function, namely as predominant membrane lipids of the so-called anammoxosome, the specific organelle where anammox catabolism takes place in the cell.     

Ladderane lipid distribution in four genera of anammox bacteria

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C(18) and C(20) ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C(20) alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of "Candidatus Kuenenia stuttgartiensis", suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C(27) hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.     

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

Biological treatment of ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium oxidation (anammox) in a pilot plant

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 would significantly reduce the nitrogen load of the activated sludge system. Some years ago, a novel biological process was discovered in which ammonium is converted to nitrogen gas under anoxic conditions with nitrite as the electron acceptor (anaerobic ammonium oxidation, anammox). Compared to conventional nitrification and denitrification, the aeration and carbon-source demand is reduced by over 50 and 100%, respectively. The combination of partial nitritation to produce nitrite in a first step and subsequent anaerobic ammonium oxidation in a second reactor was successfully tested on a pilot scale (3.6 m(3)) for over half a year. This report focuses on the feasibility of nitrogen removal from digester effluents from two different wastewater treatment plants (WWTPs) with the combined partial nitritation/anammox process. Nitritation was performed in a continuously stirred tank reactor (V=2.0 m(3)) without sludge retention. Some 58% of the ammonium in the supernatant was converted to nitrite. At 30 degrees C the maximum dilution rate D(x) was 0.85 d(-1), resulting in nitrite production of 0.35 kg NO(2)-N m(-3)(reactor) d(-1). The nitrate production was marginal. The anaerobic ammonium oxidation was carried out in a sequencing batch reactor (SBR, V=1.6 m(3)) with a nitrogen elimination rate of 2.4 kg N m(-3)(reactor) d(-1) during the nitrite-containing periods of the SBR cycle. Over 90% of the inlet nitrogen load to the anammox reactor was removed and the sludge production was negligible. The nitritation efficiency of the first reactor limited the overall maximum rate of nitrogen elimination.     

The bacterial diversity in an anaerobic ammonium-oxidizing (anammox) reactor community

An anaerobic ammonium-oxidation (anammox) reactor was operated for more than 500 days and the anammox activity of the biomass in the reactor reached 0.58 kg N_total/kg VSS d. The removal ratios of NO₂⁻-N to NH₄⁺-N in both reactor and activity tests were nearly 1.1. The bacterial diversity in the reactor was investigated by analysis of 16S rRNA gene clone libraries and quantitative real-time PCR (qPCR). The analysis showed that more than half of the clones in the library were affiliated to recognized filamentous bacteria. The previously recognized anammox bacterium (AnAOB) Candidatus Kuenenia stuttgartiensis was only detected by using a Planctomycetes-specific 16S rRNA gene primer set. However, at least two different types of AnAOB were detected by the primer set targeting the hydrazine-oxidizing enzyme gene (hzo). The aerobic ammonium-oxidizing bacteria (AAOB) Nitrosomonas europaea–eutropha group, which is widely detected in oxygen-limited environments, was also found in this reactor. The result of qPCR indicated that AnAOB comprised 16% of the community population while AAOB comprised less than 1% in the reactor.