Mixed Liquor Samples Research Articles

New insight into the irreversible membrane fouling in different pore-sized ultrafiltration ceramic membrane bioreactors (UCMBRs) for high-strength textile wastewater treatment

Despite great achievements in ceramic membrane bioreactor applications, membrane fouling, which decreases the permeability and separation performance of bioreactors and is associated with increased operational costs and energy consumption, remains a problem. The aim of this study was to expand our understanding of the fouling behavior in the long-term performance of ultrafiltration ceramic membrane bioreactors (UCMBRs) for high-strength textile wastewater reclamation. Using real textile wastewater effluent, the effects of ultrafiltration (UF) membrane pore sizes, cleaning strategies, and foulant distribution were systematically evaluated over more than three months of continuous operation. The results showed that UCMBR system achieved chemical oxygen demand and total nitrogen removal efficiencies as high as 91–95% and 39–43%, respectively. The high PN concentration can easily increase the viscosity of mixed liquor samples, contributing to a fouling layer on the membrane surface. In addition, the fouling layer formed on the surface of small-pore-sized ceramic UF membranes was not completely reversible but was difficult to eliminate by simple physical cleaning. Soluble extracellular polymeric substances, especially proteins and low molecular weight neutrals, remained, resulting in irreversible fouling on the UF membrane. However, saturated CO2 backwash showed great potential for enhancing the system through efficient fouling control without using environmentally unfriendly cleaning chemicals. The cake-intermediate and complete-standard models were suitable for explaining the fouling mechanism in the large- and small-pore-sized UF membranes, respectively.

Multi-year diagnosis of unpredictable fouling occurrences in a full-scale membrane bioreactor

The membrane bioreactor (MBR) at the Traverse City Regional Wastewater Treatment Plant has experienced sudden and unpredictable periods of substantial permeability decline since 2011. Early observations detected irregularly-shaped Gram-positive bacteria that correlated with plant upsets. Use of biomolecular techniques, such as DNA sequencing of laboratory isolates and the mixed liquor microbial community, and fluorescent in situ hybridization, identified the dispersed organisms as members of the genus Staphylococcus. However, Staphylococcus species were consistently present during normal operation and therefore were more likely to be an indicator of the upset, not the cause. The results suggest that these microorganisms are responding to specific influent wastewater constituents. We chemically analysed seven mixed liquor samples from periods of permeability decline in 2017 and 2018, and four samples from a period of normal operation. During upset conditions, the total carbohydrate content exceeded that of normal operation by 40%. Additionally, mixed liquor calcium concentrations were 65% above normal during the upset in 2017. It is hypothesized and supported through multivariate statistical analysis and estimation of specific resistance to filtration values that a calcium-intermediated polymer bridging mechanism with extracellular polymeric substance constituents is a major contributor to fouling and permeability disruptions in the Traverse City MBR.

Filamentous organisms degrade oxygen transfer efficiency by increasing mixed liquor apparent viscosity: Mechanistic understanding and experimental verification

Recent findings have demonstrated that activated sludge morphology significantly impacts oxygen transfer efficiency (OTE) in the activated sludge process. In this study, we developed a mechanistic understanding of this impact. Mixed liquor samples collected from a domestic wastewater treatment plant (WWTP) were blended with a bulking activated sludge from a bench scale reactor (BSR) cultured on synthetic wastewater to manipulate various morphological parameters such as the settled sludge volume (SV), the sludge volume index (SVI), and the specific filament length (SFL). The filaments that were present in the blended sludges consisted largely of Type 0041 and Type 021N, which are commonly found in WWTPs that treat domestic wastewater. Variations in sludge morphology, as quantified by settled sludge volume after 30 min (SV30), SVI, and SFL, systematically affected the mixed liquor apparent viscosity (μapp), which consequently impacted OTE. An increase in the SFL from 9.61 × 106 μm g−1 to 6.88 × 107 μm g−1 resulted in a 41.4% increase in apparent viscosity and a 24.6% decrease in volumetric mass transfer coefficient (KLa). A new parameter, named the ultimate settleability (SVULT), was developed by curve fitting the SV versus time data and found to relate with μapp through an expanded form of the Einstein Equation for the viscosity. Therefore, SVULT is a corollary for the particle volume fraction that incorporates effects of both the sludge morphology and mass concentration on μapp. Theoretical derivation revealed that an increase in SVULT resulted in an increase in μapp, which reduced oxygen transfer by increasing the air bubble size and reducing refreshment of the liquid at the gas-liquid interface. The KLa was found to be inversely proportional to μapp0.75 through fitting the experimental data with the theoretical model. Using a variance-based global sensitivity analysis, three operating parameters that have the most impact on oxygen transfer were identified: the power input per unit volume, the superficial gas flowrate, and the μapp.

Microbial community similarity and dissimilarity inside and across full-scale activated sludge processes for simultaneous nitrification and denitrification.

Simultaneous nitrification and denitrification under low dissolved oxygen conditions is an energy-saving modification of the activated sludge process to achieve efficient nitrogen removal. Geographically distinct full-scale treatment plants are excellent platforms to address the links of microbial community with operating parameters. Mixed liquor samples were collected from a sequencing batch reactor plant, oxidation ditch plant, and step-feed activated sludge plant. Next-Generation Sequencing of the samples showed that the microbial communities were similar at the phylum level among the plants, being dominated by Proteobacteria. Microbial composition of functional groups was similar between the react fill and react phases of the sequencing batch reactors, among four sequencing batch reactors, and among four oxidation ditches. Nitrospira was the only identified genus of autotropic nitrifying bacteria with a relative abundance of 2.2-2.5% in the oxidation ditches and 0.4-0.7% at the other plants. Heterotrophic nitrifying-aerobic denitrifying bacteria were dominated by Dechloromonas with a relative abundance of 0.4-1.0%. Microbial community composition and nitrogen removal mechanisms were related to overall level and local zonation of dissolved oxygen, mixed liquor suspended solids concentration, nitrogen and organic loadings, and solids retention time. Low dissolved oxygen and low organic and nitrogen loadings favored growth of Nitrospira.

The use of respirometry as a tool for the diagnosis of waste water treatment plants. A real case study in Southern Spain

The influence of operating conditions of wastewater treatment plants in microbial composition of activated sludge should be controlled to evaluate the efficiency of the plant. Respirometry has been widely applied as a useful tool for a better understanding of the processes taking place in activated sludge system. In the present study, respirometry tests have provided relevant information about possible faults in the plant and potential improvements. With this aim, experimental tests were applied to a real wastewater treatment plant serving an equivalent population of 730,000. To evaluate the sludge activated process, the samples of mixed liquor studied were: those collected from inlet and outlet flow of reactor system and the laboratory prepared samples from mixing those from the influent/effluent wastewater to/from reactor with the returned activated sludge. The comparison of reference values with experimental results of factor charge (0.9–1.5), organic loading rate (>0.5 gBODgMLVSS−1 d−1) and specific oxygen uptake rate in mixed liquor for outlet samples from reactor (mainly > 10 mgO2 g-1 h−1) obtained from respirometry tests revealed that the plant was operated at low efficiency and at overload. These conditions have a direct influence on the efficiency of biological treatment and the secondary clarifier operation. The sedimentation of activated sludge was evaluated through the determination of the sludge volume index during 10 months obtaining an average value of 208 mL g−1. Respirometry tests also revealed that the decrease of biomass activity was associated with the wastewater instead of with the biomass.

The nitrogen removal of autotrophic and heterotrophic bacteria in aerobic granular reactors with different feast/famine ratio

Aerobic granular sludge was cultivated in three column reactors, which had been operated for 120 days under different feast/famine ratio (1:7, 1:11, 1:15). The composition of total bacteria was analyzed by testing oxygen uptake rates of mixed liquor samples taken from the reactors and calculating according to activated sludge model. The results revealed that long famine phase favored the growth of heterotrophic bacteria. The heterotrophic bacteria accounts for 49.80, 53.37, 91.39% of total bacteria respectively in R1, R2 and R3. The heterotrophic nitrification was also observed in all the reactors, which accounts for 58.62, 58.33, 61.54% of total nitrification respectively in R1, R2 and R3. A novel nitrogen-removal pathway involving simultaneous nitrification-denitrification by heterotrophic nitrification bacteria was proposed. The results revealed that microbial system consisted of heterotrophic ammonia oxidizing bacteria showed stronger capacity of simultaneous nitrification-denitrification.

Using image processing for determination of settled sludge volume

Determination of the sludge volume index is key to describing the settling characteristics of sludge in the aeration process of wastewater treatment plants (WWTPs). The two core components of this calculation are the settled sludge volume (SSV) and suspended solids. While the measurement procedure for SSV is generally defined by national or international standards, in practice a wide variety of vessel sizes and shapes are used by operators to monitor WWTP performance. Furthermore, differences in how these tests are carried out can lead to poor data, inefficient WWTP operation and a lack of comparable metrics for WWTP operational monitoring. Thus, there is a requirement to improve operational performance of WWTPs to meet the increasingly stringent legislation regarding discharge limits. The aim of this study was to utilise a novel image-processing system (AutoSSV) to (i) determine its efficacy in describing SSV and (ii) measure and compare different methodologies for measurement of SSV. The AutoSSV system was tested using samples from various WWTPs and the results compared to those determined by standard manual measurement. Both standard and modified settlement tests were conducted on 30 mixed liquor samples, with modified settlement tests consistently resulting in lower SSV measurements. Results from the study showed a strong correlation between the SSV measurements provided by the AutoSSV system and results obtained from current manual measurement methods. The proposed technique would help to standardise the measurement in practice and increase the frequency of monitoring, particularly in small-scale rural WWTPs where there may not be permanent operators on site, and thus provide sufficient performance monitoring for efficient and effective operation.

Virus Type-Specific Removal in a Full-Scale Membrane Bioreactor Treatment Process.

We investigated removal of noroviruses, sapoviruses, and rotaviruses in a full-scale membrane bioreactor (MBR) plant by monitoring virus concentrations in wastewater samples during two gastroenteritis seasons and evaluating the adsorption of viruses to mixed liquor suspended solids (MLSS). Sapoviruses and rotaviruses were detected in 25% of MBR effluent samples with log reduction values of 3- and 2-logs in geometric mean concentrations, respectively, while noroviruses were detected in only 6% of the samples. We found that norovirus and sapovirus concentrations in the solid phase of mixed liquor samples were significantly higher than in the liquid phase (P<0.01, t test), while the concentration of rotaviruses was similar in both phases. The efficiency of adsorption of the rotavirus G1P[8] strain to MLSS was significantly less than norovirus GI.1 and GII.4 and sapovirus GI.2 strains (P<0.01, t test). Differences in the adsorption of viruses to MLSS may cause virus type-specific removal during the MBR treatment process as shown by this study.

Nitric Oxide Production Interferes with Aqueous Dissolved Oxygen Sensors

Abstract It was observed in previous studies that optical dissolved oxygen (DO) sensors were measuring values up to 1 mg O2/L in anoxic mixed liquor samples containing nitrite (NO2−). Based on these observations of false DO measurements it was hypothesized that NO2−, N2O, or NO were interfering with the DO sensors. A variety of DO probes were tested for interference while measuring NO2−, N2O, and NO. It was concluded that NO causes a positive inference with some models of optical DO probes. In bench-scale denitrification tests, 25 mg/L of NO2− led to the production of enough NO to cause a DO sensor reading of 1 mgO2/L. These findings are important for any wastewater treatment process that is utilizing online DO measurements in the presence of NO such as shortcut nitrogen removal processes.

Rheology and Microbiology of Sludge from a Thermophilic Aerobic Membrane Reactor

A thermophilic aerobic membrane reactor (TAMR) treating high-strength COD liquid wastes was submitted to an integrated investigation, with the aim of characterizing the biomass and its rheological behaviour. These processes are still scarcely adopted, also because the knowledge of their biology as well as of the physical-chemical properties of the sludge needs to be improved. In this paper, samples of mixed liquor were taken from a TAMR and submitted to fluorescent in situ hybridization for the identification and quantification of main bacterial groups. Measurements were also targeted at flocs features, filamentous bacteria, and microfauna, in order to characterize the sludge. The studied rheological properties were selected as they influence significantly the performances of membrane bioreactors (MBR) and, in particular, of the TAMR systems that operate under thermophilic conditions (i.e., around 50°C) with high MLSS concentrations (up to 200 gTS L−1). The proper description of the rheological behaviour of sludge represents a useful and fundamental aspect that allows characterizing the hydrodynamics of sludge suspension devoted to the optimization of the related processes. Therefore, in this study, the effects on the sludge rheology produced by the biomass concentration, pH, temperature, and aeration were analysed.

Comparison between mixed liquors of two side-stream membrane bioreactors treating wastewaters from waste management plants with high and low solids anaerobic digestion

In the last years, biological treatment plants for the previously separated organic fraction from municipal solid wastes (OFMSW) have gained importance. In these processes a liquid effluent (liquid fraction from the digestate and leachate from composting piles), which has to be treated previously to its discharge, is produced. In this paper, the characteristics of the mixed liquor from two full-scale membrane bioreactors treating the effluents of two OFMSW treatment plants have been evaluated in view to study their influence on membrane fouling in terms of filterability. For that, the mixed liquor samples have been ultrafiltrated in an UF laboratory plant. Besides, the effect of the influent characteristics to MBRs and the values of the chemical and physical parameters of the mixed liquors on the filterability have been studied. Results showed that the filterability of the mixed liquor was strongly influenced by the soluble microbial products in the mixed liquors and the influent characteristics to MBR. Permeate flux of MBR mixed liquor treating the most polluted wastewater was considerable the lowest (around 20 L/m2 h for some samples), what was explained by viscosity and soluble microbial products concentration higher than those measured in other MBR mixed liquor.

Effects of salinity build-up on the performance and bacterial community structure of a membrane bioreactor

This study investigated the effects of salinity increase on bacterial community structure in a membrane bioreactor (MBR) for wastewater treatment. The influent salt loading was increased gradually to simulate salinity build-up in the bioreactor during the operation of a high retention-membrane bioreactor (HR-MBR). Bacterial community diversity and structure were analyzed using 454 pyrosequencing of 16S rRNA genes of MBR mixed liquor samples. Results show that salinity increase reduced biological performance but did not affect microbial diversity in the bioreactor. Unweighted UniFrac and taxonomic analyses were conducted to relate the reduced biological performance to the change of bacterial community structure. In response to the elevated salinity condition, the succession of halophobic bacteria by halotolerant/halophilic microbes occurred and thereby the biological performance of MBR was recovered. These results suggest that salinity build-up during HR-MBR operation could be managed by allowing for the proliferation of halotolerant/halophilic bacteria.

Removal properties of human enteric viruses in a pilot-scale membrane bioreactor (MBR) process

In order to evaluate removal properties of human enteric viruses from wastewater by a membrane bioreactor (MBR), influent, anoxic and oxic mixed liquor, and membrane effluent samples were collected in a pilot-scale anoxic-oxic MBR process for 16 months, and concentrations of enteroviruses, norovirus GII, and sapoviruses were determined by real-time PCR using murine norovirus as a process control. Mixed liquor samples were separated into liquid and solid phases by centrifugation, and viruses in the bulk solution and those associated with mixed liquor suspended solids (MLSS) were quantified. Enteroviruses, norovirus GII, and sapoviruses were detected in the influent throughout the sampling period (geometrical mean, 4.0, 3.1, and 4.4 log copies/mL, respectively). Enterovirus concentrations in the solid phase of mixed liquor were generally lower than those in the liquid phase, and the mean log reduction value between influent and anoxic mixed liquor was 0.40 log units. In contrast, norovirus GII and sapovirus concentrations in the solid phase were equal to or higher than those in the liquid phase, and higher log reduction values (1.3 and 1.1 log units, respectively) were observed between influent and anoxic mixed liquor. This suggested that enteroviruses were less associated with MLSS than norovirus GII and sapoviruses, resulting in lower enterovirus removal in the activated sludge process. Enteroviruses and norovirus GII were detected in the MBR effluent but sapoviruses were not in any effluent samples. When MLSS concentration was reduced to 50–60% of a normal operation level, passages of enteroviruses and norovirus GII through a PVDF microfiltration membrane were observed. Since rejection of viruses by the membrane was not related to trans-membrane pressure which was monitored as a parameter of membrane fouling, the results indicated that adsorption to MLSS plays an important role in virus removal by an MBR, and removal properties vary by viruses reflecting different adsorptive behavior to MLSS. Our observations suggested that sapoviruses are more associated with MLSS and removed more efficiently than enteroviruses and norovirus GII.

Study of 16 Portuguese activated sludge systems based on filamentous bacteria populations and their relationships with environmental parameters.

A survey in 16 activated sludge wastewater treatment plants (WWTP) was conducted to contribute to the knowledge of the environmental parameters that determine the composition of the filamentous community. A total of 128 samples of mixed liquor from municipal WWTP were collected during 2 years, and 22 filamentous morphotypes were identified. The most frequent and abundant filamentous bacteria were, in both cases and by this order, type 0041/0675, type 0092, Microthrix parvicella and 1851, nocardioforms and Haliscomenobacter hydrossis. Concerning dominance, type 1851 was the most frequently dominant morphotype, followed by M. parvicella and types 0092 and 0041/0675. These were also, and by this order, the dominant morphotypes during bulking occurrences. Significant correlations were obtained between the abundance of filamentous bacteria and environmental parameters, but multivariate statistical analysis only confirmed the correlation between type 0092 and Sludge Volume Index (SVI), emphasizing the association of this filament with bulking. The discussion of the results in light of published works was complicated by the random use of terms such as frequency, abundance, and dominance with different and often unclear meanings. This reinforces the need of clarifying these terms when discussing the causes of filamentous overgrowth in WWTP.

Modification of oxygen transfer rates in activated sludge with its characteristic changes by the addition of organic polyelectrolyte

The present study focused on the influence of the apparent viscosity of activated sludge on mixed liquor oxygen transfer rates, and examined whether both could be modified by the addition of an organic polyelectrolyte. Batch experiments carried out using mixed liquor samples in a stirred tank reactor revealed that α-factors, which incorporate any difference in oxygen mass transfer coefficient values between clean water and mixed liquor samples, showed exponential relationships when plotted against increases in mixed liquor apparent viscosities and sludge mixed liquid suspended solids values. Furthermore, oxygen mass transfer coefficients increased exponentially with increases in the Reynolds number, a parameter which reflects liquid turbulence levels. Oxygen mass transfer rates of five sludge samples collected from four full scale wastewater treatment plants (WWTPs) also increased by about 10% after flocculation, while with a sludge sample collected from the lab scale reactor, it decreased by about 5%. One likely explanation is that the apparent viscosities of the WWTPs and the lab-reactor sludges decreased and increased respectively after flocculation. Such changes probably reflect changes in the volume fraction of mixed liquor suspended solids as indicated by changes in sludge volume indices, particle size values and endogenous respiration rates before and after flocculation. These findings assist in developing an understanding of how oxygen mass transfer characteristics may be affected by activated sludge suspensions, and hence assist in reducing the operational costs of WWTPs.

Monitoring Bacterial Diversity in a full-scale Municipal Wastewater Treatment plant in Dubai by Fluorescence in situ hybridization Technique

In this study, the bacterial diversity in the activated sludge system of a full-scale municipal wastewater treatment plant in Dubai was monitored over a period of one year using ribosomal RNA (rRNA) targeted oligonucleotide probes for a defined phylogenetic group of bacteria by the Fluorescence in situ hybridization (FISH) technique. The largest fraction of the bacterial community in the sludge samples belonged to the gamma-subgroup of proteobacteria (25%) followed by gram positive bacteria of high G+C content(16%), gram positive bacteria with low G+C content (9 %), beta-proteobacteria (8%) and alpha-proteobacteria(5 %) with respect to the population percentages stained by DAPI (4,6-diamino-2-phenylindole).A specific nocardioform actinomycete, simultaneously targeted by both HGC69a and MNP1 probes, was predominantly found throughout the study period in all activated sludge mixed liquor samples. The nocardioform actinomycetes group members were detected in both branched and single cell morphotypes. Most of the previously published genus and species specific probes failed to hybridize to the sludge samples. In conclusion, the overall bacterial community populations detected by the sub-group specific 16S rRNA targeted oligonucleotide probes in FISH technique remained almost constant throughout the period of study irrespective of treatment plant conditions.

Detection and distribution of putative virulence associated genes inAeromonasspecies from freshwater and wastewater treatment plant

The detection of genes responsible for Aeromonas virulence is a vital tool in establishing the potential pathogenicity of the bacteria, as these virulence genes may act alone or in synergy in the establishment of infections. Freshwater and wastewater mixed liquor samples were collected from Kat river and Fort Beaufort wastewater treatment plant in the Eastern Cape Province of South Africa. Polymerase chain reaction was utilized for the amplification of the different genes coding for virulence. All virulence associated genes screened (alt, lip, fla, aer, ast, hlyA) were detected in at least one Aeromonas isolates. In fresh water sample, virulence genes were distributed as follows: lip (67%), aer (43%), alt (33%), fla (62%), ast (10%), and hlyA (86%), while in wastewater samples the occurrence were as follows: lip (92%), aer (21%), alt (54%), fla (83%), ast (29%), and hlyA (88%). The presence of these virulence genes in environmental Aeromonas isolates is of concern to public health as these organisms are potential pathogens in the environment and the virulence determinants could be transferred to aquatic organisms and humans by one mechanism or the other.

Monitoring Microbial Diversity of a Full-Scale Municipal Wastewater Treatment Plant in Dubai

In this study, fluorescence in situhybridization (FISH) technique was employedfor monitoring the microbial community in the activated sludge system of a full-scale municipal wastewater treatment plant in Dubai over a period of one year. A total of 96 activated sludge mixed liquor samples were characterized using ribosomal RNA (16S and 23S rRNA) targeted oligonucleotide probes for defined phylogenetic groups of bacteria. Several filamentous and non-filamentous bacteriawere predominantly found throughout the study period in all activated sludge mixed liquor samples. The bacterial species belonging to High G+C group were detected in both branched and single cell morphotypes. The previously published genus and species specific probes detected several of members belonging to archaea, sulphate reducing bacteria, ammonia-oxidizing bacteria, nitrobacter, nitrospira including halophilic and halotolerantnitrosomonasspp.In conclusion, the overall microbial community populations detected by the sub-group and species specific 16S and 23S rRNA targeted oligonucleotide probes found to be quite diverse and varied seasonally.

Fate and effect of naphthenic acids on oil refinery activated sludge wastewater treatment systems

Naphthenic acids (NAs) are a complex group of alkyl-substituted acyclic, monocyclic and polycyclic carboxylic acids present in oil sands process waters, crude oil, refinery wastewater and petroleum products. Crude oil, desalter brine, influent, activated sludge mixed liquor and effluent refinery samples were received from six United States refineries. The total acid number (TAN) of the six crudes tested ranged from 0.12 to 1.5 mg KOH/g crude oil and correlated to the total NA concentration in the crudes. The total NA concentration in the desalter brine, influent, activated sludge mixed liquor and effluent samples ranged from 4.2 to 40.4, 4.5 to 16.6, 9.6 to 140.3 and 2.8 to 11.6 mg NA/L, respectively. The NAs in all wastewater streams accounted for less than 16% of the total COD, indicating that many other organic compounds are present and that NAs are a minor component in refinery wastewaters. Susceptibility tests showed that none of the activated sludge heterotrophic microcosms was completely inhibited by NAs up to 400 mg/L. Growth inhibition ranging from 10 to 59% was observed in all microcosms at and above 100 mg NA/L. NAs chronically-sorbed to activated sludge mixed liquor biomass and powdered activated carbon (PAC) were recalcitrant and persistent. More than 80% of the total NAs remained in the solid phase at the end of the 10-day desorption period (five successive desorption steps). Throughout a 90-day incubation period, the total NA concentration decreased by 33 and 51% in PAC-free and PAC-containing mixed liquor microcosms, respectively. The lower molecular weight fraction of NAs was preferentially degraded in both mixed liquors. The persistence of the residual, higher molecular weight NAs is likely a combination of molecular recalcitrance and decreased bioavailability when chronically-sorbed to the biomass and/or PAC.

하수처리장에서의 암모니아 전환 미생물군의 생태학적 연구

The overall goal of this study was to characterize and quantify ammonia-oxidizing bacteria (AOB) in four different full-scale sequence batch reactor (SBR) wastewater treatment plants. Also, this study focused on assessing the occurrence of the alternative ammonia-oxidizing microbes such as anammox (anaerobic ammonia oxidation) bacteria (AMX) and ammonia-oxidizing archaea (AOA) in these systems. Based on total AOB numbers and the estimated cell density in the mixed liquor samples, AOB constituted 0.3 - 1.8% of the total bacterial population in the four WWTPs. Based on clone library, Nitrosomonas ureae-like AOB were dominant in plant A and B, while plant C and D had Nitrosomonas nitrosa-like AOB as major AOB group. The four different AMX primer sets targeting AMX 16S rRNA gene produced PCR amplicons distantly related to Chlamydia and Planctomycetales group bacteria. However, it was not clear these groups of bacteria perform anammox reaction in the SBR plants. Also, molecular evidence of AOA was found in one of the SBR plants, with a sequence located in the deep branch of the sediment creanarchaeota group.