Effects of ultrasonic treatment on the ammonia-oxidizing bacterial (AOB) growth kinetics.

Ultrasound has in the past few decades found applications in a variety of disciplines including chemistry, medicine, physics, and to a much less extent microbiology. Our previous studies found that ultrasonic treatment increases the activity of ammonia-oxidizing bacteria (AOB) while suppressing nitrite-oxidizing bacteria (NOB), resulting in beneficial effects in wastewater treatment. In this study, the kinetic and microbiological features of nitrifying microorganisms in activated sludge intermittently treated with ultrasound were investigated to gain an improved understanding of the mechanism involved in ultrasound-induced stimulation of AOB kinetics. The nitrifying microorganisms were initially enriched over 100 days in a laboratory sequential batch reactor (SBR). Ultrasonic treatment of the sludge was then applied with the treatment time in each 12 h SBR cycle progressively increased from 4 to 24 min. Application of the treatment for 21 days led to a doubled maximum specific ammonia oxidation rate, and also the enhanced dominance of known AOB Nitrosomonas genus in the biomass. This stimulatory effect is well described by a modified enzyme catalyzed reaction model, showing a good linear relationship between the natural logarithm value of µmax,AOB and the applied ultrasonic energy density. This result suggests that ultrasonic treatment likely reduced the activation energy of key enzymes involved in ammonium oxidation.

A Physiological and Genomic Comparison of Nitrosomonas Cluster 6a and 7 Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas perform the first step in nitrification, ammonia oxidation, and are found in diverse aquatic and terrestrial environments. Nitrosomonas AOB were grouped into six defined clusters, which correlate with physiological characteristics that contribute to adaptations to a variety of abiotic environmental factors. A fundamental physiological trait differentiating Nitrosomonas AOB is the adaptation to either low (cluster 6a) or high (cluster 7) ammonium concentrations. Here, we present physiological growth studies and genome analysis of Nitrosomonas cluster 6a and 7 AOB. Cluster 6a AOB displayed maximum growth rates at ≤ 1 mM ammonium, while cluster 7 AOB had maximum growth rates at ≥ 5 mM ammonium. In addition, cluster 7 AOB were more tolerant of high initial ammonium and nitrite concentrations than cluster 6a AOB. Cluster 6a AOB were completely inhibited by an initial nitrite concentration of 5 mM. Genomic comparisons were used to link genomic traits to observed physiological adaptations. Cluster 7 AOB encode a suite of genes related to nitrogen oxide detoxification and multiple terminal oxidases, which are absent in cluster 6a AOB. Cluster 6a AOB possess two distinct forms of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for hydrogen or urea utilization. Several, but not all, cluster 6a AOB can utilize urea as a source of ammonium. Hence, although Nitrosomonas cluster 6a and 7 AOB have the capacity to fulfill the same functional role in microbial communities, i.e., ammonia oxidation, differentiating species-specific and cluster-conserved adaptations is crucial in understanding how AOB community succession can affect overall ecosystem function.

Energy efficient COD and N-removal from high-strength wastewater by a passively aerated GAO dominated biofilm.

Conventional aerobic treatment of high-strength wastewater is not economical due to excessively high energy requirement for compressed air supply. The use of passive aeration avoids the use of compressed air and enables energy efficient oxygen supply directly from the air. This study evaluates a passively aerated simultaneous nitrification and denitrification performing biofilm to treat concentrated wastewater. The biofilm reactor was operated > 5-months under alternating anaerobic/aerobic conditions. For 4-times concentrated wastewater, > 80% COD (2307 mg L-1 h-1) and > 60% N (60 mg L-1 h-1) was removed at a hydraulic retention time (HRT) of 7 h. A double application in the same reactor enabled > 95% COD and 85% N-removal, at an overall HRT of 14 h which is substantially shorter than what traditional activated sludge-based systems would require for the treatment of such concentrated feeds. Microbial community analysis showed Candidatus competibacter (27%) and nitrifying bacteria (Nitrosomonas, and Nitrospira) as key microbes involved in COD and N-removal, respectively.

Start-Up and Aeration Strategies for a Completely Autotrophic Nitrogen Removal Process in an SBR.

The start-up and performance of the completely autotrophic nitrogen removal via nitrite (CANON) process were examined in a sequencing batch reactor (SBR) with intermittent aeration. Initially, partial nitrification was established, and then the DO concentration was lowered further, surplus water in the SBR with high nitrite was replaced with tap water, and continuous aeration mode was turned into intermittent aeration mode, while the removal of total nitrogen was still weak. However, the total nitrogen (TN) removal efficiency and nitrogen removal loading reached 83.07% and 0.422 kgN/(m3·d), respectively, 14 days after inoculating 0.15 g of CANON biofilm biomass into the SBR. The aggregates formed in SBR were the mixture of activated sludge and granular sludge; the volume ratio of floc and granular sludge was 7 : 3. DNA analysis showed that Planctomycetes-like anammox bacteria and Nitrosomonas-like aerobic ammonium oxidization bacteria were dominant bacteria in the reactor. The influence of aeration strategies on CANON process was investigated using batch tests. The result showed that the strategy of alternating aeration (1 h) and nonaeration (1 h) was optimum, which can obtain almost the same TN removal efficiency as continuous aeration while reducing the energy consumption, inhibiting the activity of NOB, and enhancing the activity of AAOB.

The effects of salinity on nitrification using halophilic nitrifiers in a Sequencing Batch Reactor treating hypersaline wastewater.

With annual increases in the generation and use of saline wastewater, the need to avoid environmental problems such as eutrophication is critical. A previous study identified ways to start up a halophilic sludge domesticated from estuarine sediments to remove nitrogen from wastewater with a salinity of 30 g/L. This investigation expands that work to explore the impact of salinity on nitrogen removal. This study demonstrated that the mixed halophilic consortia removed nitrogen from wastewater with a salinity of 30-85 g/L. A kinetic analysis showed that halophilic nitrifiers selected based on hypersalinity were characterized by low Ks, µmax and specific ammonium oxidization rates. This explains the decrease in ammonium removal efficiency in the high salinity operational phases. Salinity inhibited ammonia oxidizing bacteria (AOB) activity, as well as the number of dominant AOB, but did not significantly affect the AOB dominant species. Three most dominant AOB lineages in the halophilic sludge were Nitrosomonas marina, Nitrosomonas europaea, and Nitrosococcus mobilis. Nitrosomonas europaea and Nitrosococcus mobilis were mainly affected by salinity, while nitrite accumulation and ammonia loading played the key role in determining the abundance of Nitrosococcus mobilis and Nitrosococcus europaea. The study contributes insights about shifts in halophilic nitrifying bacterial populations.

Process Performance and Bacterial Community Structure Under Increasing Influent Disturbances in a Membrane-Aerated Biofilm Reactor.

The membrane-aerated biofilm reactor (MABR) is a promising municipal wastewater treatment process. In this study, two cross-flow MABRs were constructed to explore the carbon and nitrogen removal performance and bacterial succession, along with changes of influent loading shock comprising flow velocity, COD, and NH4-N concentrations. Redundancy analysis revealed that the function of high flow velocity was mainly embodied in facilitating contaminants diffusion and biosorption rather than the success of overall bacterial populations (p > 0.05). In contrast, the influent NH4-N concentration contributed most to the variance of reactor efficiency and community structure (p < 0.05). Pyrosequencing results showed that Anaerolineae, and Beta- and Alphaproteobacteria were the dominant groups in biofilms for COD and NH4-N removal. Among the identified genera, Nitrosomonas and Nitrospira were the main nitrifiers, and Hyphomicrobium, Hydrogenophaga, and Rhodobacter were the key denitrifiers. Meanwhile, principal component analysis indicated that bacterial shift in MABR was probably the combination of stochastic and deterministic processes.

Development and Quality Control of Nanohexaconazole as an Effective Fungicide and Its Biosafety Studies on Soil Nitifiers.

The study was aimed to develop a nano form of an existing fungicide for improving plant protection and reducing crop losses caused by fungal pathogens. The protocol for the preparation and estimation of nanohexaconazole was developed. Technically pure hexaconazole was converted into its nanoform using polyethyleneglycol-400 (PEG) as the surface stabilizing agent. Nanohexaconazole was characterized using Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS) studies. The average particle size of nanohexaconazole was about 100 nm. An analytical method was also developed for quality control of the nanofungicide by GLC fitted with flame ionization detector. Its limit of detection was 2.5 ppm. Fungicidal potential of nanohexaconazole was better in comparison to that of conventional hexaconazole. Hydrolytic and thermal stability studies confirmed its stability at par with the conventional formulation of fungicide. Impact of nanohexaconazole on soil nitrifiers was tested in vitro and there were no significant adverse effect in their numbers observed as compared to conventional registered formulation, proving the safety of the nanofungicide.

Nitrogen removal and microbial communities in a three-stage system simulating a riparian environment.

The riparian zone is an active interface for nitrogen removal, in which nitrogen transformations by microorganisms have not been valued. In this study, a three-stage system was constructed to simulate the riparian zone environments, and nitrogen removal as well as the microbial community was investigated in this 'engineered riparian system'. The results demonstrated that stage 1 of this system accounted for 41-51 % of total nitrogen removal. Initial ammonium loading and redox potential significantly impacted the nitrogen removal performances. Stages 1 and 2 were both composed of an anoxic/oxic (A/O) zone and an anaerobic column. The A/O zone removed most of the ammonium load (6.8 g/m(2)/day), while the anaerobic column showed a significant nitrate removal rate (11.1 g/m(2)/day). Molecular biological analysis demonstrated that bacterial diversity was high in the A/O zones, where ammonium-oxidizing bacteria and nitrite-oxidizing bacteria accounted for 8.42 and 3.32 % of the bacterial population, respectively. The denitrifying bacteria Acidovorax sp. and the nitrifying bacteria Nitrosospira/Nitrosomonas were the predominant microorganisms in this engineered riparian system. This three-stage system was established to achieve favorable nitrogen removal and the microbial community in the system was also retained. This investigation should deepen our understanding of biological nitrogen removal in engineered riparian zones.

Biological and technical study of a partial-SHARON reactor at laboratory scale: effect of hydraulic retention time.

This study was on the technical and biological characteristics of a partial-SHARON submerged-filter bioreactor of 3 L. The main focus was the influence of the hydraulic retention time (HRT) on biofilms. For this purpose, we used molecular tools based on the partial 16S rRNA genes. The results showed that the HRT may affect the nitrification processes of a bioreactor using synthetic wastewater containing 600 mg/L of ammonia. It was found that an HRT of 0.5 day transformed 100 % of the ammonium into nitrite. However, when the HRT was decreased to 0.4 day, there was a significant reduction (35 %) in the quantity of ammonia transformed, which confirmed the complexity of the system operation. Moreover, a PCR-TGGE approach highlighted the differences observed. The results obtained showed that an HRT of 0.5 day reduced bacterial biodiversity in the biofilms, which were mainly formed by Nitrosomonas and Diaphorobacter. In contrast, an HRT of 0.4 day facilitated the formation of heterogeneous biofilms formed by nitrifying bacteria, such as Nitrosomonas sp., Nitrosospira sp., and Nitrosovibrio sp.).

Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process.

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1 week at a nitrogen loading rate of 0.46 kg-N/m(3)/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m(3)/day at a nitrogen loading rate of 1.0 kg-N/m(3)/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m(3)/day at a nitrogen loading rate of 0.93 kg-N/m(3)/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5-6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.

Fluctuation of microbial activities after influent load variations in a full-scale SBR: recovery of the biomass after starvation.

Due to variations in the production levels, a full-scale sequencing batch reactor (SBR) for post-treatment of tannery wastewater was exposed to low and high ammonia load periods. In order to study how these changes affected the N-removal capacity, the microbiology of the reactor was studied by a diverse set of techniques including molecular tools, activity tests, and microbial counts in samples taken along 3 years. The recover capacity of the biomass was also studied in a lab-scale reactor operated with intermittent aeration without feeding for 36 days. The results showed that changes in the feeding negatively affected the nitrifying community, but the nitrogen removal efficiencies could be restored after the concentration stress. Species substitution was observed within the nitrifying bacteria, Nitrosomonas europaea and Nitrobacter predominated initially, and after an ammonia overload period, Nitrosomonas nitrosa and Nitrospira became dominant. Some denitrifiers, with nirS related to Alicycliphilus, Azospirillum, and Marinobacter nirS, persisted during long-term reactor operation, but the community fluctuated both in composition and in abundance. This fluctuating community may better resist the continuous changes in the feeding regime. Our results showed that a nitrifying-denitrifying SBR could be operated with low loads or even without feeding during production shut down periods.

Analysis of nitrifying bacterial communities in aerobic biofilm reactors with different DO conditions using molecular techniques.

In order to assess the relationship between the dissolved oxygen (DO) concentration and the characteristics of nitrifying bacterial communities in an aerobic biofilm reactor, molecular techniques including denaturing gradient gel electrophoresis (DGGE)/cloning based on PCR targeting 16S rRNA and the amoA gene and fluorescence in situ hybridisation (FISH) were conducted. The D-1, D-2, D-3 and D-4 reactors with different DO concentrations (1, 3, 5 and 7 mg/L, respectively) were set up in the thermostat and acclimated. The optimal DO concentration with stable nitrification efficiency was above 5.0 mg/L. As was shown by the results of DGGE and cloning, the community of ammonia-oxidising bacteria (AOB) and the ratio of Nitrosomonas sp. changed only slightly despite their differing nitrification efficiencies. The results of FISH indicated that higher DO concentrations resulted in an increase in AOB and nitrite-oxidising bacteria (NOB), and a reduction in heterotrophic microorganisms. The INT-dehydrogenase activity (DHA) test demonstrated that the activity of AOB decreased with reductions in the DO concentration. This means that the DO concentration does not influence the community of AOB, but rather the activity of AOB. In the relationship between the attached biomass and the nitrification efficiency, only the active biomass affected the nitrification efficiencies.

[Inhibition investigation of aniline on nitrification process in batch and continuous running reactors].

Inhibition effect of aniline on nitrifier was investigated in suspended sludge batch reactors and a three-phase fluidized bed. The tested nitrifier and aniline degradation bacteria were obtained from excess sludge of a treatment plant for aniline wastewater. The results showed that aniline had produced obvious inhibition on nitrifier in suspended sludge batch reactors. Barely when aniline concentration was less than 3 mg/L, had nitrifier gradually recovered their ability. But the recovered time prolonged with the augment of initial aniline concentration. Feasible hydraulic retention time (HRT) was a key operating factor to remove aniline and ammonia simultaneously in fluidized bed reactor. When aniline concentration in influent was 200 mg/L and HRT was 10 h, aniline concentration was 6.58 mg/L and nitrifying rate achieved 84.95% in the fluidized bed reactor. Three-phase fluidized biofilm nitrifying reactor was superior to suspended sludge nitrifying reactor in resisting aniline toxicity and has practical value in denitrifying treatment of wastewater containing toxic organic chemicals.

[Stability of short-cut nitrification using immobilized ammonia-oxidizing bacteria].

The ammonia-oxidizing bacteria were immobilized by copolymer with cell proliferation technology. The effects of NH(4+) -N load, HRT, free ammonia (FA) and organic matter on short-cut nitrification process were studied. The results showed that when influent NH(4+) -N load were 100 mg/L, 150 mg/L and 200 mg/L respectively, effluent NH(4+) -N concentration was less than 10 mg/L. When the system run for 3 h, 6 h and 12 h, corresponding to influent NH(4+) -N concentration of 25.8 mg/L, 51.1 mg/L and 93.3 mg/L respectively, NH(4+) -N concentration was low in effluent with high short-cut nitrification efficiency. The HRT could be adjusted to optimize the system operation with variation of influent NH(4+) -N concentration. The results also indicated that the ammonia-oxidizing bacteria were restrained while free ammonia concentration was over 9 mg/L. The activity of the ammonia-oxidizing bacteria could be enhanced under existence of low-molecular-weight organic compounds, but the short-cut nitrification efficiency was little affected. In addition, the short-cut nitrification and denitrification could be realized with existence of organic compounds during the experiment.

[Nitrification performance and microbial community analysis in carbon membrane-aerated biofilm reactor].

A carbon membrane-aerated biofilm reactor was developed to treat nitrogenous inorganic wastewater. Influent NH; -N concentrations and HRT were changed to investigate nitrification performance of reactor,oxygen utilization and NH4+ -N's removal loading. Biofilm's surface characteristics and dominant bacteria of nitrifier were analyzed. The results show that under the conditions of intra-membrane pressure of 0.017 MPa, influent NH4+ -N of 50 mg/L and HRT of 8 h NH4+ -N removal efficiency reaches 96% and effluent average nitrite is 17 mg/L, which benefits short-cut nitrification to a certain extent. The bacteria within biofilm consume all oxygen supplied through carbon membrane. The maximum specific removal rate of NH4+ -N is 9.7 g/(m2 x d), which is limited by the amount of bacteria grown onto carbon membrane's surface. Fluorescent in situ hybridization analysis indicates that within the biofilm Nitrosomonas and Nitrosospira are main ammonia-oxidizing bacteria and occupy about 19% and 21% of the total bacteria number, respectively. The Nitrobacter are not observed and Nitrospira are dominant nitrite-oxidizing bacteria, the fraction of which is 20% of total bacteria.

Stoichiometric and kinetic characterisation of Nitrosomonas sp. in mixed culture by decoupling the growth and energy generation processes.

A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosomonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO2 were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14-0.16 mgN mgCOD(biomass)(-1)h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH 7.

Effects of hydroxylamine on microbial community structure and function of autotrophic nitrifying biofilms determined by in situ hybridization and the use of microelectrodes.

Effects of hydroxylamine (NH2OH), an intermediate of NH4+ oxidation, on microbial community structure and function of two autotrophic nitrifying biofilms fed with and without NH2OH were analyzed by a 16S rRNA approach and the use of microelectrodes. In the NH2OH-added biofilm, partial oxidation of NH4+ to NO2- was observed, whereas complete oxidation of NH4+ to NO3- was achieved in the control biofilm. In situ hybridization results revealed that no nitrite-oxidizing bacteria (NOB) hybridized with any specific probes were detected in the NH2OH-added biofilm. Thus, the addition of low concentrations of NH2OH (250 microM) completely inhibited the growth of NOB. Phylogenetic analysis of 16S rDNA indicated that the ammonia-oxidizing bacteria (AOB) detected in both biofilms were closely related to Nitrosomonas europaea, and that the clone sequences from both biofilm libraries have more than 99% similarity to each other. However, in situ hybridization results revealed that the addition of NH2OH changed the form of growth pattern of the dominant Nitrosomonas spp. from dense clusters mode to single scattered cells mode. Microelectrode measurements revealed that the average NH4+ consumption rate calculated in the NH2OH-added biofilm was two times higher than that in the control biofilm. This clearly demonstrated that the oxidation of NH4+ was stimulated by NH2OH addition.

High nitrification rate at low pH in a fluidized bed reactor with chalk as the biofilm carrier.

A typical steady state bulk pH of about 5 was established in a nitrifying fluidized bed with chalk as the only buffer agent. In spite of the low pH, high rate nitrification was observed with the nitrification kinetic parameters in the chalk reactor similar to those of biological reactors operating at pH>7. Various methods were used to determine the reasons for high rate nitrification at such low pH including (i) determination of bacterial species, (ii) microsensor measurements in the biofilm, and (iii) comparison of nitrification performance at low pH with a non-chalk fluidized bed reactor. Fluorescence in situ hybridization (FISH) using existing 16S rRNA-targeted oligonucleotide probes showed common nitrifying bacteria in the low pH chalk reactor. The prevalent nitrifying bacteria were identified in the Nitrosomonas oligotropha, Nitrosomonas europeae/eutropha, Nitrosospira and Nitrospira related groups, all well known nitrifiers. Microelectrode measurements showed that the pH in the biofilm was low and similar to that of the bulk pH. Finally, reactor performance using a non-chalk biofilm carrier (sintered glass) with the same bacterial inoculum also showed high rate nitrification below pH 5. The results suggest that inhibition of nitrification at low pH is highly overestimated.

A two-stage SBR process for removal of organic substrate and nitrogen via nitrite-type nitrification-denitrification.

A two-stage SBR process (TSSBR) was applied for the treatment of wastewater with high strength COD and nitrogen. Most of organic substrate was removed in the first stage SBR reactor (SBR1) under the aerobic condition. Subsequently the second stage SBR reactor (SBR2) firstly was operated under the aerobic condition for simultaneous nitrite-type nitrification and removal of a small amount of residual organic substrate. Nitrification was controlled to the nitrite-type nitrification. Then denitrification in which the wastewater was used as external carbon sources occurred in SBR2 under the anoxic condition. The experimental results indicated that two kinds of biomass with the different function were cultured in a TSSBR system, which was beneficial to increase the proportion of Nitrosomonas in the microorganism of SBR2. It avoided the inhibition of the high organic loading to nitrification and C/N was not the limiting factor affecting the nitrogen removal efficiency. The rate of COD degradation and nitrification was improved, 8 kg COD/kg MLSS/d and 0.25 kg NH4+-N/kg MLSS/d, respectively. The final effluent COD concentration further reduced because the organic substrate that was left in SBR1 effluent was removed in SBR2. Consequently, when compared to a single SBR process, a two-stage SBR (TSSBR) not only improved the treatment efficiency, but also saved the operational cost.

Ammonium and hydroxylamine uptake and accumulation in Nitrosomonas.

Starved cells of Nitrosomonas europaea and further ammonia oxidizers were able to rapidly accumulate ammonium and hydroxylamine to an internal concentration of about 1 and 0.8 M, respectively. In kinetic studies, the uptake/accumulation rates for ammonium [3.1 mmol (g protein)(-1) min(-1)] and hydroxylamine [4.39 mmol (g protein)(-1) min(-1)] were determined. The uptake and accumulation process of ammonium and hydroxylamine was not coupled to ammonia or hydroxylamine oxidation and nitrite was not produced. In the presence of uncouplers the ammonium accumulation was completely inhibited, indicating an active, membrane-potential-driven transport mechanism. When the external ammonium or hydroxylamine pool was depleted, the internal ammonium and hydroxylamine was consumed within 12 h or 20 min, respectively. The binding of ammonium/ammonia was correlated with an energized membrane system, and hydroxylamine may bind to the hydroxylamine oxidoredutase.