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.     

Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity

Anammox bacteria are chemoautotrophic bacteria that oxidize ammonium with nitrite as the electron acceptor and with CO₂ as the main carbon source. The effects of inorganic carbon (IC) limitation on anammox bacteria were investigated using continuous feeding tests. In this study, a gel carrier with entrapped anammox sludge was used. It was clearly shown that the anammox activity deteriorated with a decrease in the influent IC concentration. The relationship between the influent IC concentration and the anammox activity was analyzed using Michaelis-Menten kinetics, and the apparent K_m was determined to be 1.2 mg-C/L. The activity could be recovered by adding IC to the influent. The consumption ratio of IC to ammonium was not constant and mainly depended on the influent ratio of the IC to ammonium concentrations (inf.IC/inf.NH₄-N). The results indicated that an inf.IC/inf.NH₄-N ratio of 0.2 in the anammox reactor was ideal for the anammox process using gel cubes.     

Lipid composition of activated sludge in a pilot plant for anaerobic ammonium oxidation

The lipid composition of the microbial community inhabiting activated sludge in a pilot reactor for the anaerobic oxidation of ammonium (anammox) at the Kur’yanovo Treatment Plant (Moscow) has been studied. The fatty acid composition is mostly based on common fatty acids C₁₄–C₁₈ (95%) with both normal and isomeric structures. The biomass of activated sludge was found to contain lipids with the so-called ladderane substances (ladder alcohols and fatty acids) that are common for anammox bacteria: C₂₀-[3]-lad-derane and C₂₀-[5]-ladderane alcohols and C₁₈- and C₂₀-[3]-ladderane and C₁₈- and C₂₀-[5]-ladderane acids. In addition, the native extract contained both simple and compound ethers of the above-mentioned substances with residues of phosphocholine, phosphoethanolamine, and phosphoglycerine. The spectra of the electron impact and tandem mass spectrometry of certain substances have been obtained and published for the first time.     

Long-term stability of partial nitritation of swine wastewater digester liquor and its subsequent treatment by Anammox

Partial nitritation using inhibition of free ammonia and free nitric acid is an effective technique for the treatment of high concentrations of ammonium in wastewaters. This technique was applied to the digester liquor of swine wastewater and the stability of its long-term operation was investigated. Partial nitritation was successfully maintained at a nitrogen loading rate (NLR) of 1.0 kg N m(-3)d(-1) for 120 days without acclimatization of nitrite oxidizing bacteria (NOB) to the inhibitory compounds (free ammonia and free nitric acid). The conversion efficiencies of NH(4)-N to NO(2)-N and to NO(3)-N were determined to be around 58% and <5%, respectively. After the establishment of partial nitritation, the influence of swine wastewater on the Anammox reaction was examined using continuous flow treatment experiments. Consistent nitrogen removal was achieved for 70 days at a nitrogen removal rate (NRR) of 0.22 kg N m(-3)d(-1) and the color of Anammox bacteria changed from red to greyish black. The NO(2)-N consumption and the NO(3)-N production increased concurrently and the Anammox reaction ratio was estimated to be 1:1.67:0.53, which is different from that reported previously (1:1.32:0.26).     

Engineering application of anaerobic ammonium oxidation process in wastewater treatment

Anaerobic ammonium oxidation (Anammox), a promising biological nitrogen removal process, has been verified as an efficient, sustainable and cost-effective alternative to conventional nitrification and denitrification processes. To date, more than 110 full-scale anammox plants have been installed and are in operation, treating industrial NH₄ ⁺-rich wastewater worldwide, and anammox-based technologies are flourishing. This review the current state of the art for engineering applications of the anammox process, including various anammox-based technologies, reactor selection and attempts to apply it at different wastewater plants. Process control and implementation for stable performance are discussed as well as some remaining issues concerning engineering application are exposed, including the start-up period, process disturbances, greenhouse gas emissions and especially mainstream anammox applications. Finally, further development of the anammox engineering application is proposed in this review.     

Applicability of one-stage partial nitritation and anammox in MBBR for anaerobically pre-treated municipal wastewater

Energy consumption of municipal wastewater treatment plants can be reduced by the anaerobic pre-treatment of the main wastewater stream. After this pre-treatment, nitrogen can potentially be removed by partial nitritation and anammox (PN/A). Currently, the application of PN/A is limited to nitrogen-rich streams (>500 mg L^?1) and temperatures 25–35 °C. But, anaerobically pretreated municipal wastewater is characterized by much lower nitrogen concentrations (20–100 mg L^?1) and lower temperatures (10–25 °C). We operated PN/A under similar conditions: total ammonium nitrogen concentration 50 mg L^?1 and lab temperature (22 °C). PN/A was operated for 342 days in a 4 L moving bed biofilm reactor (MBBR). At 0.4 mg O₂ L^?1, nitrogen removal rate 33 g N m^?3 day^?1 and 80 % total nitrogen removal efficiency was achieved. The capacity of the reactor was limited by low AOB activity. We observed significant anammox activity (40 g N m^?3 day^?1) even at 12 °C, improving the applicability of PN/A for municipal wastewater treatment.     

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.     

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.     

Sulfate-reducing anaerobic ammonium oxidation as a potential treatment method for high nitrogen-content wastewater

After sulfate-reducing ammonium oxidation (SRAO) was first assumed in 2001, several works have been published describing this process in laboratory-scale bioreactors or occurring in the nature. In this paper, the SRAO process was performed using reject water as a substrate for microorganisms and a source of NH(4) (+), with SO(4) (2-) being added as an electron acceptor. At a moderate temperature of 20°C in a moving bed biofilm reactor (MBBR) sulfate reduction along with ammonium oxidation were established. In an upflow anaerobic sludge blanket reactor (UASBR) the SRAO process took place at 36°C. Average volumetric TN removal rates of 0.03?kg-N/m3/day in the MBBR and 0.04?kg-N/m3/day in the UASBR were achieved, with long-term moderate average removal efficiencies, respectively. Uncultured bacteria clone P4 and uncultured planctomycete clone Amx-PAn30 were detected from the biofilm of the MBBR, from sludge of the UASBR uncultured Verrucomicrobiales bacterium clone De2102 and Uncultured bacterium clone ATB-KS-1929 were found also. The stoichiometrical ratio of NH(4) (+) removal was significantly higher than could be expected from the extent of SO(4) (2-) reduction. This phenomenon can primarily be attributed to complex interactions between nitrogen and sulfur compounds and organic matter present in the wastewater. The high NH(4) (+) removal ratio can be attributed to sulfur-utilizing denitrification/denitritation providing the evidence that SRAO is occurring independently and is not a result of sulfate reduction and anammox. HCO(3) (-) concentrations exceeding 1,000?mg/l were found to have an inhibiting effect on the SRAO process. Small amounts of hydrazine were naturally present in the reaction medium, indicating occurrence of the anammox process. Injections of anammox intermediates, hydrazine and hydroxylamine, had a positive effect on SRAO process performance, particularly in the case of the UASBR.     

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.     

Identification of anammox bacteria in a full-scale deammonification plant making use of anaerobic ammonia oxidation

The existence of anaerobic ammonia-oxidizing (anammox) bacteria was postulated in the late 1970s. Approximately 20 years later, these lithotrophic members of the nitrogen cycle were identified as deep-branching members of the planctomycetes. Recently, full-scale implementation of biological deammonification was successfully achieved in the DEMON reactor at the wastewater treatment plant in Strass, Austria. The sludge of this reactor contains red granules and brownish flocs that can be physically separated. The two fractions yielded different banding patterns in denaturing gradient gel electrophoresis of PCR products obtained with primer sets targeting the 16S rRNA genes of planctomycetes. Comparative analysis of partial sequences of almost full-length 16S rRNA gene clones obtained from the granules and flocs confirms the differences in the community composition of the two fractions. The sequences retrieved from the red granules were 93% similar to those of Candidatus Brocadia anammoxidans, a bacterium known to catalyze the anaerobic ammonia oxidation.     

Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary

Summary Anaerobic ammonium oxidation (anammox) specific PCR method was developed to examine diversity and distribution of anammox bacteria in sediments collected from three different sites at Cape Fear River Estuary, North Carolina, where environmental parameters vary greatly over the year. Abundance and activities of anammox bacteria in these sediments were measured using the quantitative PCR (Q-PCR) method and ¹⁵N isotope tracer incubations. Different anammox bacterial communities composed with Brocadia , Kuenenia , Jettenia or Scalindua were found among sites along the estuarine gradient. Seasonal variations of anammox community structures were observed along the estuary based on terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. Correlation analysis suggested that salinity variation influenced the diversity and distribution of different anammox bacteria in the estuary. Q-PCR assays of anammox bacteria showed temporal and spatial variations of their abundances, which were highly correlated to salinity variation. ¹⁵N isotope tracer incubations measured different anammox rates and its per cent contribution to total N₂ production among sites. The highest anammox rate was found at the site where Scalindua organisms dominated with the highest anammox bacterial abundance. Thus, we demonstrated a biogeographical distribution of diverse anammox bacteria influenced by salinity, and provide evidence to link anammox abundance and activities in estuarine sediments.