Enrichment Of Anammox Bacteria Research Articles

Analysis of the impact of reflux ratio on coupled partial nitrification–anammox for co-treatment of mature landfill leachate and domestic wastewater

A combined system of anaerobic–aerobic techniques was used for deep co-treatment of mature landfill leachate and domestic sewage, mixed at a ratio of 1:5, through partial nitrification–anammox. While ensuring synchronous removal of organic matter and nitrogen, we investigated changes in several factors under different reflux ratios (0%, 100% and 300%). High nitrification efficiency and a relatively high accumulation rate of NO2−-N were achieved, the latter through selective inhibition of bacteria by free ammonia. The results indicated that maintaining the reflux ratio within an optimum range contributes to the enrichment of anammox bacteria. In this study, the optimum ratio, which permitted the highest removal rates of chemical oxygen demand (COD), NH4+-N, and total nitrogen, was 300%. The COD level in the anammox reactor (ANAOR) decreased to lower than 70mg/L, resulting in no inhibitory effect on anammox and maximum enrichment of bacteria.

Vegetation effects on anammox spatial distribution and nitrogen removal in constructed wetlands treated with domestic sewage.

In this study, two horizontal subsurface-flow constructed wetlands (CWs) (planted and unplanted) were constructed and compared to investigate the effects of vegetation on nitrogen removal and anammox (anaerobic ammonium oxidation) spatial distribution and enrichment. Calamus (Acorus calamus L.), which has a large root system, was selected as the vegetation. Removal of total nitrogen from the planted wetland was much higher than that from the unplanted one. Radial oxygen loss from calamus provided the planted wetland with better oxygen restoration ability, benefitting ammonium removal in the CW, especially when anammox was inhibited under winter temperatures. Enrichment of anammox bacteria in planted wetlands was much greater than that in unplanted ones. The greatest enrichment of anammox bacteria occurred in the middle layer, which had a better anaerobic environment and moderate root system. The reduced rate of metabolism in plants during winter led to a sharp decrease in anammox bacteria copy numbers in the planted wetland. Under cold temperature, the degree of enrichment with anammox bacteria in the planted wetland was similar to or slightly superior to that in the unplanted wetland.

Enrichment of Anammox Bacteria by a Sequencing Fed Biofilm Batch Reactor (SFBFBR)

A sequencing fed biofilm batch reactor (SFBFBR) seeded with returning activated sludge of a WWTP was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the nitrogen removal characterization of the Anammox biofilm system. Initially, the operation period was controlled at 3 days and the mineral medium (30 mg/l ammonium, 30 mg/l nitrite, about 2 L) was supplied continuously to SFBFBR in the first 68 hours. After 44 days’ cultivation, ammonium and nitrite concentration were decreased simultaneously without COD and DO, which means the anammox activity presented in the reactor. From t=55 days, in order to further enrich anammox bacteria, the substrate load began to increase by reducing the operation period from 3 days to 1 day and increasing the ammonium and nitrite concentrations. At the end of the experiment, the reactor was able to treat nitrogen loading rates up to 200±10 mg N/(L.d). The ammonium and nitrite reacted in the stoichiometrical of 1:1.135.

Research on Startup Process and Influencing Factors of Half-Nitrosofication in CANON Reactor

The experimental study on startup process of half-nitrosofication for high ammonia nitrogen simulated wastewater has been accomplished with a reactor of completely autotrophic nitrogen removal over nitrite (CANON). The startup process and its influences of the concentration of influent , DO and pH were analyzed with the experimental results. The results show that the conversion rate of to is close to 55%, the accumulation rate of is over 95% and the rate of to steadily keeps as 1.02~1.24 under the condition of influent of 400 mg/L, pH of 7.6~8.2, DO of 0.95~1.3mg/L, HRT of 1.5d and water temperature of 17~27°C, which meet the environmental requirements for anaerobic ammonia oxidation bacteria growth, and half-nitrosofication was achieved in the CANON reactor, which create good conditions for further enrichment of anammox bacteria for the operation of the CANON reactor.

Enrichment of an anammox bacterial community from a flooded paddy soil

This study describes the enrichment of anammox bacteria in a column simulating oxygen limited flooded paddy soils, which are important man-made ecosystems that receive substantial amounts of fixed nitrogen. The upper 50 cm of the paddy soil, containing a high amount of ammonium [1.6-10.4 mmol N kg (dry weight)(-1)], was selected as the inoculum for anammox enrichment. After 18 months of incubation with freshwater from the paddy soil ecosystem, the enrichment culture consumed approximately 4 mmol ammonium l(-1) day(-1) and 5 mmol nitrite l(-1) day(-1). The maximum specific anammox activity of the culture was 35.7 μmol N g (dry weight)(-1) h(-1). Fluorescence in situ hybridization indicated that anammox cells constituted 50% ± 10% of the enrichment culture. The phylogenetic analyses of 16S rRNA and the diagnostic hydrazine synthase (hzsA) genes showed that two dominant anammox species were enriched from paddy soil. The enriched Candidatus Anammoxoglobus-like organisms showed a 16S rRNA gene similarity of 97.5-99.2% to Candidatus Anammoxoglobus propionicus and the Candidatus Jettenia-like organisms showed 92.1-93.1% 16S rRNA gene identity to Candidatus Jettenia asiatica. Real-time quantitative PCR of hzsA gene suggested that up to 10(10) copies g (dry weight)(-1) of soil anammox bacteria were present in the enrichment culture.

Modeling of Simultaneous Partial Nitrification, Anammox and Denitrification Process In a Single Reactor

One sequencing batch reactor (SBR) seeded with anaerobic granular sludge was started-up to enrich simultaneous partial nitrification and anaerobic ammonia oxidation (Anammox) bacteria. The SBR could be operated successfully through first enriching Anammox bacteria under strictly anaerobic condition during the first stage and then controlling the dissolved oxygen (DO) in the reactor between 0.3 and 0.6 mg/L during the second and third stage, and eventually the maximum total nitrogen removal rate could reach 63.7%. Besides, a dynamic mathematical model including nitrification, Anammox, COD oxidation and denitrification was developed for the simulation of the performance of biological processes. The dynamic mathematical model was extended with two parts: model correction factors of DO, and Haldane kinetics for Anammox. Subsequently, a set of experiments and simulations were carried out to analyze the effects of substrate nitrite and DO on SBR system, and to validate the model. Research results indicated that biological processes could be inhibited by a certain nitrite concentration 20~30mg/L, and high or low DO concentration was not in favor of operation of SBR system. In addition, the simulation results were consistent with the experimental results, which illuminated that the mathematical model was appropriate for the modeling of simultaneous partial nitrification, Anammox and denitrification process in a single reactor, thus it could estimate the impact of specific parameters and predict the efficiency of system.

Anammox start-up in sequencing batch biofilm reactors using different inoculating sludge

In this study, anammox bacteria were rapidly enriched in sequencing batch biofilm reactors (SBBRs) with different inoculations. The activated sludge taken from a sequencing batch reactor was used and inoculated to SBBR1, while SBBR2 was seeded with stored anaerobic sludge from an upflow anaerobic fixed bed (2-year stored at 5-15 °C). Nitrogen removal performance, anammox activity, biofilm characteristics and variation of the microbial community were evaluated. The maximum total nitrogen loading rate (NLR) of SBBR1 gradually reached to 1.62 kgN/(m³/day) with a removal efficiency higher than 88% and the NLR of SBBR2 reached to 1.43 kgN/(m³/day) with a removal efficiency of 86%. SBBR2 was more stable compared to SBBR1. These results, combined with molecular techniques such as scanning electron microscope, fluorescence in situ hybridization, and terminal restriction fragment length polymorphism, indicated that different genera of anammox bacteria became dominant. This research also demonstrates that SBBR is a promising bioreactor for starting up and enriching anammox bacteria.

Enrichment of anammox bacteria from mudflat sediments collected in Tokyo Bay

We established an enrichment culture of anammox bacteria by seeding a batch reactor with sediments from the Banzu mudflat in Tokyo Bay. The reactor was intermittently fed with an anammox substrate containing NH4Cl, NaNO2 and artificial seawater (3.03% salinity) for more than 800 days. An anammox enrichment culture capable of simultaneous reduction of approximately equivalent amounts of ammonium nitrogen and nitrite nitrogen was established. Batch assays of the enrichment culture revealed that anammox activity was similar for all batches at salinities ranging from 1.01 to 3.03%, with lower activity observed at a salinity of 0.02%. We identified three 16S rRNA genes in DNA fragments amplified from the enriched culture. One of these genes, mudflat sediment clone_K01 was highly homologous with Candidatus “Kuenenia stuttgartiensis” (identity 97%) being the most abundant (85 out of isolated 94 clones). In most of the studies conducted to date, anammox species belonging to the Candidatus “Scalindua” genus have been found to be the dominant species in enrichment cultures obtained from estuarine and marine environments. The results reported here are unusual, in that, a freshwater anammox bacterium, Candidatus K. stuttgartiensis, was the dominant bacterium in a sediment sample from a marine environment. Key words: Anammox, mudflat sediment, salinity, Candidatus “Kuenenia stuttgartiensis”,Candidatus “Scalindua wagneri”

Enrichment of anammox bacteria from three sludge sources for the startup of monosodium glutamate industrial wastewater treatment system

Three activated sludges from a landfill leachate treatment plant (S1), a municipal sewage treatment plant (S2) and a monosodium glutamate (MSG) wastewater treatment plant (S3) were used as inocula to enrich anaerobic ammonium oxidation (anammox) bacteria for the startup of MSG industrial wastewater treatment system. After 360 days of cultivation using MSG wastewater, obvious anammox activity was observed in all three cultures. The maximum specific anammox activities of cultures S1, S2 and S3 were 0.11 kg N kg −1 VSS day −1, 0.09 kg N kg −1 VSS day −1 and 0.16 kg N kg −1 VSS day −1, respectively. Brownish-red anammox granules having diameters in the range of 0.2–1.0 mm were visible in cultures S1 and S2, and large red granules having diameters in the range of 0.5–2.5 mm were formed in culture S3 after 420 days of cultivation. Phylogenetic analysis of 16S rRNA genes showed that Kuenenia organisms were the dominant anammox species in all three cultures. The copy numbers of 16S rRNA genes of anammox bacteria in cultures S1, S2 and S3 were 6.8 × 10 7 copies mL −1, 9.4 × 10 7 copies mL −1 and 7.5 × 10 8 copies mL −1, respectively. The results of this study demonstrated that anammox cultivation from conventional activated sludges was highly possible using MSG wastewater. Thus the anammox process has possibility of applying to the nitrogen removal from MSG wastewater.

Impact of reactor configuration on anammox process start-up: MBR versus SBR

Anaerobic ammonium oxidation (anammox) is an energy saving biological nitrogen removal process which was limited to slow growth rate of anammox bacteria during start-up period. This study investigated the start-up of anammox process by a laboratory sequential batch reactor (SBR) for 218days and subsequently modified the reactor as a membrane bioreactor (MBR) for 178days. Modification of a SBR as MBR with installation of an external membrane module resulted in acceleration of specific anammox activity by 19 times. The acceleration of specific anammox activity with MBR was further confirmed by starting-up another MBR for a 242day period. Molecular microbial analyses showed that Candidatus “Brocadia anammoxidans” and Candidatus “Kuenenia stuttgartiensis” were the dominant species in the inocula and biomass developed in the reactor. The start-up with MBR appeared to be more effective than SBR for the enrichment of anammox bacteria due to high sludge retention property of MBR configuration.

Research Progress on the Anammox Technology

With the continuing phenomenon of eutrophication, the pollution of nitrogen has caused wide concern. Lack of organic carbon sources is always considered to be a key problem for nitrogen removal efficiency and cost when conventional biological nitrogen removal process was used to the treatment of ammonium-rich wastewater, but the anaerobic ammonium oxidation（ANAMMOX）can solve this problem .Anammox has so many advantages that it has good prospects in the biological nitrogen removal in wastewater. Anammox bacteria grows slowly, has a long generation time, and is susceptible to the environmental conditions. DO, Temperature, pH and organic can impact the process of anammox, so it is necessary to take careful methods to cultivate Anammox biomass. Depending on the purpose of improve water quality, appropriate treatment reactors and seed sludge should be selected to start ANAMMON process. The discovery of anaerobic ammonia oxidation, the reaction mechanism, influence application, the enrichment of anammox bacteria, physiological and biochemical characteristics of anammox bacteria, the start of anaerobic ammonia oxidation are reviewed in this paper.

Anammox bacteria enrichment and characterization from municipal activated sludge

A sustainable option for nitrogen removal is the anaerobic ammonium-oxidizing (anammox) process in which ammonium is oxidized to nitrogen gas with nitrite as electron acceptor. Application of this process, however, is limited by the availability of anammox biomass. In this study, two Brocadia-like anammox phylotypes were successfully enriched, detected and identified from an activated sludge taken from a domestic wastewater treatment plant (Minas Gerais, Brazil) employing a Sequencing Batch Reactor (SBR). The dominant phylotype was closely related to 'Candidatus Brocadia sinica', but one clone seemed to represent a novel species for which we propose the name 'Candidatus Brocadia brasiliensis'. Based on Fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 52.7% (±15.6) anammox bacteria after 6 months of cultivation. The cultivation process can be divided into three phases: phase 1 (approximately 25 days) was characterized by heterotrophic denitrification metabolism, phase 2 was the propagation phase and phase 3 (from the 87th day onwards), in which significant anammox activity was detected. A long-term performance of the SBR showed a near perfect removal of nitrite based on the influent NO(2)(-)-N concentration of 61-95 mg L(-1). The average ammonia removal efficiency was 90% with the influent NH(4)(+)-N concentration of 55-82 mg L(-1). Therefore, anammox cultivation and enrichment from activated sludge was possible under a controlled environment within 3 months.

ANAMMOX process start up and stabilization with an anaerobic seed in Anaerobic Membrane Bioreactor (AnMBR)

ANaerobic AMMonium OXidation (ANAMMOX) process, an advanced biological nitrogen removal alternative to traditional nitrification – denitrification removes ammonia using nitrite as the electron acceptor without oxygen. The feasibility of enriching anammox bacteria from anaerobic seed culture to start up an Anaerobic Membrane Bioreactor (AnMBR) for N-removal is reported in this paper. The Anammox activity was established in the AnMBR with anaerobic digester seed culture from a Sewage Treatment Plant in batch mode with recirculation followed by semi continuous process and continuous modes of operation. The AnMBR performance under varying Nitrogen Loading Rates (NLR) and HRTs is reported for a year, in terms of nitrogen transformations to ammoniacal nitrogen, nitrite and nitrate along with hydrazine and hydroxylamine. Interestingly ANAMMOX process was evident from simultaneous Amm-N and nitrite reduction, consistent nitrate production, hydrazine and hydroxylamine presence, notable organic load reduction and bicarbonate consumption.

Enrichment of Anammox bacteria in seed sludges from different wastewater treating processes and start-up of Anammox process

Three seed sludges (sludge A, B and C) were collected from various wastewater treatment processes. Then they were inoculated in Reactor A, B and C in parallel. After 67days of operation, only Reactor A successfully started up the Anaerobic ammonia oxidation (Anammox) process, while the other two reactors failed. The Anammox activity was observed within 49days and a final nitrogen removal rate of 0.19g NH4+–NL−1d−1 and 0.21g NO2−–NL−1d−1 was achieved after 95days. In Reactor A, Fluorescence in situ hybridization (FISH) analysis confirmed that Anammox bacteria became the dominant population on day 90, and Scanning electron microscope (SEM) observation also showed that short rod and spherical bacteria predominated in the cultivated sludge. FISH analysis also disclosed that sludge A had aboriginal Anammox bacteria but the other two have no signal of Anammox bacteria. From a new micro-ecological viewpoint, the wastewater treating process, which sludge A was taken from, could provide Anammox ecological niche, but the other two processes could not. The results indicated that selection of the seed sludge from the special wastewater treating process providing Anammox ecological niches made the Anammox start-up more effectively and quickly.

Autotrophic biological nitrogen removal from saline wastewater under low DO

AbstractBACKGROUND: This study was conducted to investigate the feasibility and performance of nitrogen removal through the complete autotrophic nitrogen removal over nitrite (CANON) process for saline wastewater in a continuous reactor, and to characterize microorganisms in the sludge from the reactor using DNA‐based techniques.RESULTS: The nitrogen removal experiment in the reactor was operated over five phases for 286 days treating a synthetic sewage of 1.2% salinity at 21–25 °C. At dissolved oxygen (DO) concentrations of 0.5–1.0 mg L−1 and in the presence of glucose, NO2− was accumulated, indicating the activity of ammonia‐oxidizing bacteria (AOB). At DO concentration of 0.5 mg L−1 without organic substrate, the anaerobic ammonium oxidation (Anammox) process was the major pathway responsible for nitrogen removal, with a total nitrogen removal of 70% and an ammonium conversion efficiency of 96%. A maximum ammonium removal rate of 0.57 kg‐N m−3 d−1 was achieved during the experimental period. The concentrations of AOB and Anammox bacteria were monitored over the operation of reactor using quantitative real‐time polymerase chain reaction (qRT‐PCR).CONCLUSION: In this study, autotrophic nitrogen removal process was achieved under salinity condition in a one‐reactor system. An over 100 fold increase of AOB was found due to the increased supply of ammonium at the beginning, then AOB concentration decreased temporarily in correspondence with the decreased DO, and the AOB resumed their concentration at the last phase. The Anammox bacteria abundance was about 150 fold higher than that at the beginning, indicating the successful enrichment of Anammox bacteria in the reactor. Copyright © 2010 Society of Chemical Industry

Enrichment and biofilm formation of Anammox bacteria in a non-woven membrane reactor

An innovative reactor configuration for Anammox enrichment by connecting a non-woven membrane module with an anaerobic reactor was developed in this study. The Anammox non-woven membrane reactor (ANMR) exhibited high biomass retention ability through the formation of aggregates in the reactor and biofilm on the interior surface of the non-woven membrane. No fouling problems occurred on the membrane after the development of mature biofilms. After 8 months of operation, the nitrogen loading rate (NLR) and nitrogen removal rate (NRR) reached 1263 mg N/l/d and 1047.5 mg N/l/d, respectively, with a maximum specific ammonium consumption (SAC) of 51 nmol/mg protein/min. At steady state, the average ammonium and nitrite removal efficiencies were 90.9% and 95.0%, respectively. Morphological observation of Anammox aggregates and biofilm showed a high degree of compactness. Also, enrichment of Anammox bacteria was quantified by real-time polymerase chain reaction (PCR) analysis as 97.7%.

Distribution of anammox bacteria in domestic WWTPs and their enrichments evaluated by real-time quantitative PCR

Enrichment of anaerobic ammonium oxidation (anammox) bacteria using five activated sludges in three domestic wastewater treatment plants (WWTPs) were processed in a short term of 70 days and evaluated by real-time quantitative PCR (RTQ-PCR). Before the enrichment, building phylogenetic trees of Planctomycetes phylum in four reactors of sequencing batch reactor (SBR), anoxic and oxic reactors of anaerobic–anoxic–oxic (A2O) process, and rotating biological contactor (RBC) revealed six groups of distantly relative genera of Planctomyces, Pirellula, Gemmata, Isophaera, Candidatus and putative anammox bacteria. All clones of Candidatus sp. were affiliated with anammox bacteria and the majority of anammox clones were related to Planctomycete KSU-1 (AB057453). The discovery of anammox bacteria in raw activated sludges provided a partial rationale for the utilization of activated sludge as a seeding source of the anammox process. To verify the activity of anammox bacteria in the activated sludges, enrichment cultivations were conducted using SBRs. The enrichment of anammox bacteria resulted in the significant anammox activity of three samples. Quantification of 16S rRNA gene of anammox bacteria using RTQ-PCR showed the highest concentration of anammox bacteria of 2.48 ± 0.22 × 109 copies of 16S rRNA gene/mg-volatile suspended solids (VSS), which was the same order of magnitude as that of the referential granular anammox sludge, 6.23 ± 0.59 × 109 copies of 16S rRNA gene/mg-VSS, taken from an anammox upflow anaerobic sludge blanket (UASB) reactor. The doubling time of anammox bacteria enriched in this study was 1.18 days. The growth yield of anammox bacteria enriched in this study was 4.75 ± 0.57 × 106 copies of 16S rRNA gene/mg of ammonium- and nitrite-nitrogen, which was similar to 4.50 ± 0.61 × 106 copies of 16S rRNA gene/mg of ammonium- and nitrite-nitrogen for the referential anammox sludge. Substrate uptake rates of three successful enrichments at the end of the enrichment were comparable to those of granular and suspended anammox sludges. Rapid enrichment of anammox bacteria using activated sludge could offer an alternative method for obtaining a large volume of seeding anammox sludge.

Microbial diversity of anammox bacteria enriched from different types of seed sludge in an anaerobic continuous-feeding cultivation reactor

Enrichment of anammox bacteria from three types of seed sludge, sewage, digester, and nitrification sludges, was conducted using a nonwoven fabric carrier for immobilizing the anammox bacteria, and the microbial diversity of the enriched anammox culture was investigated. About four months later, simultaneous removals of ammonium and nitrite, and production of a small amount of nitrate, which is unique to the anammox reaction, were observed in all 3 sludge reactors. Results of 16S rRNA gene analysis indicated that anammox bacteria were cultivated and diversified in each sludge type. Moreover, the microbial diversity of anammox bacteria was higher in the enriched culture from sewage sludge compared to the other two types of seed sludge. Bacillus sp. coexisted in the anammox culture cultivated from sewage sludge. These results suggest that differences in the anammox community in the enriched culture were caused by differences in the type of seed sludge.

Development of batch-culture enrichment coupled to molecular detection for screening of natural and man-made environments in search of anammox bacteria for N-removal bioreactors systems

This investigation probed for anammox bacterial populations that would be suitable to start-up a biological reactor for N-removal. Samples of sludge and sediments from different environments were screened and used as inoculum for enrichment of anammox bacteria in batch cultures. Enrichments were monitored in order to detect anammox bacteria or their potential activity. Candidatus “Brocadia anammoxidans” was successfully enriched, detected, and identified in five of the twelve batch cultures. Furthermore, this organism was retrieved for the first time from a brackish environment. Wide-range primers used in several Polymerase Chain Reaction (PCR) attempts were unable to successfully amplify the 16S rRNA sequence from anammox populations, but were used to search for hypothetical ecological partners of anammox. A nested PCR approach with specific primers was also employed since conventional PCR was unable to amplify anammox DNA from the inocula of the successful enrichments. However, it was impossible to obtain optimal results with the different strategies utilized to improve PCR performance (higher annealing temperature or more specific primers), and only the primer set Amx368F-Amx820R resulted in an acceptable balance between both specificity and sensitivity. Although the enrichment process is relatively slow and requires lengthy incubation periods, it proved to be useful as the molecular analyses were not sensitive enough to detect anammox in the original samples or even after short enrichment periods. Therefore, a batch enrichment procedure coupled with molecular techniques was an appropriate approach to achieve successful inocula for starting-up anammox biological reactor.

Effect of inorganic carbon on anaerobic ammonium oxidation enriched in sequencing batch reactor

The present lab-scale research reveals the enrichment of anaerobic ammonium oxidation microorganism from methanogenic anaerobic granular sludge and the effect of inorganic carbon (sodium bicarbonate) on anaerobic ammonium oxidation. The enrichment of anammox bacteria was carried out in a 7.0-L sequencing batch reactor (SBR) and the effect of bicarbonate on anammox was conducted in a 3.0-L SBR. Research results, especially the biomass, showed first signs of anammox activity after 54 d cultivation with synthetic wastewater, when the pH was controlled between 7.5 and 8.3, the temperature was 35 degrees C. The anammox activity increased as the influent bicarbonate concentration increased from 1.0 to 1.5 g/L, and then, was inhibited as the bicarbonate concentration approached 2.0 g/L. However, the activity could be restored by the reduction of bicarbonate concentration to 1.0 g/L, as shown by rapid conversion of ammonium, and nitrite and nitrate production with normal stoichiometry. The optimization of the bicarbonate concentration in the reactor could increase the anammox rate up to 66.4 mgN/(L x d).