High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 ± 0.1) and low bulk ammonium concentrations (9.3 ± 3.3 mg·liter⁻¹), a very high nitrification rate of 5.6 g of N oxidized·liter⁻¹·day⁻¹ was achieved. The specific nitrification rate (0.55 g of N·g of biomass⁻¹·day⁻¹) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 ± 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (¹⁵N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass·g of N oxidized⁻¹. Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.     

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor

Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (± 0.19) kg-N m⁻³ d⁻¹ than the reactor initiated as the partial nitrifying reactor (0.23 (± 0.16) kg-N m⁻³ d⁻¹). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor.     

Bioaugmentation as a tool for improving the start-up and stability of a pilot-scale partial nitrification biofilm airlift reactor

The effectiveness of bioaugmentation in the improvement of the start-up of a biofilm airlift reactor to perform partial nitrification was investigated. Two identical biofilm airlift reactors were inoculated. The non-bioaugmented reactor (NB-reactor) was inoculated with conventional activated sludge, whereas the bioaugmented reactor (B-reactor) was seeded with the same conventional activated sludge but bioaugmented with nitrifying activated sludge from a pilot plant performing full nitritation under stable conditions (100% oxidation of influent ammonium to nitrite). The fraction of specialized nitrifying activated sludge in the inoculum of the B-reactor was only 6% (measured as dry matter). To simplify comparison of the results, operational parameters were equivalent for both reactors. Partial nitrification was achieved significantly faster in the B-reactor, showing a very stable operation. The results obtained by fluorescence in situ hybridization assays showed that the specialized nitrifying biomass added to the B-reactor remained in the biofilm throughout the start-up period.     

Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor

Biological nitrogen removal (BNR) based on partial nitrification and denitrification via nitrite is a cost-effective alternate to conventional nitrification and denitrification (via nitrate). The goal of this study was to investigate the microbial ecology, biokinetics, and stability of partial nitrification. Stable long-term partial nitrification resulting in 82.1 +/- 17.2% ammonia oxidation, primarily to nitrite (77.3 +/- 19.5% of the ammonia oxidized) was achieved in a lab-scale bioreactor by operation at a pH, dissolved oxygen and solids retention time of 7.5 +/- 0.1, 1.54 +/- 0.87 mg O(2)/L, and 3.0 days, respectively. Bioreactor ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) populations were most closely related to Nitrosomonas europaea and Nitrobacter spp., respectively. The AOB population fraction varied in the range 61 +/- 45% and was much higher than the NOB fraction, 0.71 +/- 1.1%. Using direct measures of bacterial concentrations in conjunction with independent activity measures and mass balances, the maximum specific growth rate (micro(max)), specific decay (b) and observed biomass yield coefficients (Y(obs)) for AOB were 1.08 +/- 1.03 day(-1), 0.32 +/- 0.34 day(-1), and 0.15 +/- 0.06 mg biomass COD/mg N oxidized, respectively. Corresponding micro(max), b, and Y(obs) values for NOB were 2.6 +/- 2.05 day(-1), 1.7 +/- 1.9 day(-1), and 0.04 +/- 0.02 mg biomass COD/mg N oxidized, respectively. The results of this study demonstrate that the highly selective partial nitrification operating conditions enriched for a narrow diversity of rapidly growing AOB and NOB populations unlike conventional BNR reactors, which host a broader diversity of nitrifying bacteria. Further, direct measures of microbial abundance enabled not only elucidation of mixed community microbial ecology but also estimation of key engineering parameters describing bioreactor systems supporting these communities.     

Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater

Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.     

焦化废水工业处理装置和实验室装置硝化菌群的分子比较

反应器的群落结构分析有助于对工业装置的故障原因进行诊断。为了解决某焦化废水处理装置硝化功能低下的故障,构建了一套相似的实验室装置作为参照系统,该装置的硝化功能良好。通过工业装置和实验室装置好氧池生物膜16SrDNA克隆文库的比较,分析了它们之间硝化菌群的组成差异。实验室装置克隆文库的构成说明Nitrosomonas europaea-Nitrosoccus mobilis类群和Nitrospira属Ⅰ亚区系分别是该工艺条件下优势的氨氧化菌和亚硝酸氧化菌,但工业装置的克隆文库中却没有找到任何与硝化菌序列相近的克隆,这说明工业装置中硝化菌的多度较低。进一步使用Taqman荧光探针实时定量PCR测定了样品中Nitrospira属的多度,实验室装置中Nitrospira属16S rDNA的拷贝数达到3.4×106个/微克基因组DNA,而工业装置的测定值不到实验室装置的1/300。这些试验结果都表明工业装置好氧池微生物群落中缺少适当的硝化菌群是造成其硝化能力低下的重要原因。提高菌群中Nitrosomonas属和Nitrospira属的多度是解决工业装置硝化能力低下的关键。     

Influence of dissolved oxygen concentration on nitrite accumulation in a biofilm airlift suspension reactor

The biofilm airlift suspension (BAS) reactor can treat wastewater at a high volumetric loading rate combined with a low sludge loading. Two BAS reactors were operated, with an ammonium load of 5 kg N/(m(3) d), in order to study the influence of biomass and oxygen concentration on the nitrification process. After start-up the nitrifying biomass in the reactors gradually increased up to 30 g VSS/L. Due to this increased biomass concentration the gas-liquid mass transfer coefficient was negatively influenced. The resulting gradual decrease in dissolved oxygen concentration (over a 2-month period) was associated with a concomitantly nitrite build-up. Short term experiments showed a similar relation between dissolved oxygen concentration (DO) and nitrite accumulation. It was possible to obtain full ammonium conversion with approximately 50% nitrate and 50% nitrite in the effluent. The facts that (i) nitrite build up occurred only when DO dropped, (ii) the nitrite formation was stable over long periods, and (iii) fully depending on DO levels in short term experiments, led to the conclusion that it was not affected by microbial adaptations but associated with intrinsic characteristics of the microbial growth system. A simple biofilm model based on the often reported difference of oxygen affinity between ammonium and nitrite oxydizers was capable of adequately describing the phenomena.Measurements of biomass density and concentration are critical for the interpretation of the results, but highly sensitive to sampling procedures. Therefore we have developed an independent method, based on the residence time of Dextran Blue, to check the experimental methods. There was a good agreement between procedures.The relation between biomass concentration, oxygen mass transfer rate and nitrification in a BAS reactor is discussed. (c) 1997 John Wiley & Sons, Inc.     

Nitrification at Low pH by Aggregated Chemolithotrophic Bacteria

A study was performed to gain insight into the mechanism of acid-tolerant, chemolithotrophic nitrification. Microorganisms that nitrified at pH 4 were enriched from two Dutch acid soils. Nitrate production in the enrichment cultures was indicated to be of a chemolithoautotrophic nature as it was (i) completely inhibited by acetylene at a concentration as low as 1 μmol/liter and (ii) strongly retarded under conditions of carbon dioxide limitation. Electron microscopy of the enrichment cultures showed the presence of bacteria that were morphologically similar to strains of known chemolithotrophic nitrifying genera. Many of the enriched bacteria, in particular those that were identified as ammonium oxidizers, were aggregated. Filtration experiments indicated that aggregated cells were able to nitrify at low pH, whereas single cells were not. It is hypothesized that cells inside the aggregates are protected against the toxicity of nitrous acid. Nitrification by aggregated chemolithoautotrophic bacteria may be the dominating process of nitrate formation in many acid soils as it does not appear to depend on the existence of microsites of high pH (acid-sensitive autotrophic nitrification) or on the availability of organic carbon (heterotrophic nitrification).     

Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.     

Functional robustness and gene pools of a wastewater nitrification reactor: comparison of dispersed and intact biofilms when stressed by low oxygen and low pH

The functional robustness of biofilms in a wastewater nitrification reactor, and the gene pools therein, were investigated. Nitrosomonas and Nitrosospira spp. were present in similar amounts (cloning-sequencing of ammonia-oxidizing bacteria 16S rRNA gene), and their estimated abundance (1.1 x 10(9) cells g(-1) carrier material, based on amoA gene real-time PCR) was sufficient to explain the observed nitrification rates. The biofilm also had a diverse community of heterotrophic denitrifying bacteria (cloning-sequencing of nirK). Anammox 16S rRNA genes were detected, but not archaeal amoA. Dispersed biofilms (DB) and intact biofilms (IB) were incubated in gas-tight reactors at different pH levels (4.5 and 5.5 vs. 6.5) while monitoring O(2) depletion and concentrations of NO, N(2)O and N(2) in the headspace. Nitrification was severely reduced by suboptimal O(2) concentrations (10-100 microM) and low pH (IB was more acid tolerant than DB), but the N(2)O/NO(3)(-) product ratio of nitrification remained low (<10(-3)). The NO(2)(-) concentrations during nitrification were generally 10 times higher in DB than in IB. Transient NO and N(2)O accumulation at the onset of denitrification was 10-10(3) times higher in DB than in IB (depending on the pH). The contrasting performance of DB and IB suggests that the biofilm structure, with anoxic/micro-oxic zones, helps to stabilize functions during anoxic spells and low pH.     

Identification of novel small molecule enhancers of protein production by cultured mammalian cells

Shortcut nitrogen removal, that is, removal via formation and reduction of nitrite rather than nitrate, has been observed in membrane-aerated biofilms (MABs), but the extent, the controlling factors, and the kinetics of nitrite formation in MABs are poorly understood. We used a special MAB reactor to systematically study the effects of the dissolved oxygen (DO) concentration at the membrane surface, which is the biofilm base, on nitrification rates, extent of shortcut nitrification, and microbial community structure. The focus was on anoxic bulk liquids, which is typical in MAB used for total nitrogen (TN) removal, although aerobic bulk liquids were also studied. Nitrifying MABs were grown on a hollow-fiber membrane exposed to 3 mg N/L ammonium. The MAB intra-membrane air pressure was varied to achieve different DO concentrations at the biofilm base, and the bulk liquid was anoxic or with 2 g m(-3) DO. With 2.2 and 3.5 g m(-3) DO at the biofilm base, and with an anoxic bulk-liquid, the ammonium fluxes were 0.75 and 1.0 g N m(-2) day(-1), respectively, and nitrite was the main oxidized nitrogen product. However, with membrane DO of 5.5 g m(-3), and either zero or 2 g m(-3) DO in the bulk, the ammonium flux was around 1.3 g N m(-2) day(-1), and nitrate flux increased significantly. For all experiments, the cell density of ammonium oxidizing bacteria (AOB) was relatively uniform throughout the biofilm, but the density of nitrite oxidizing bacteria (NOB) decreased with decreasing biofilm DO. Among NOB, Nitrobacter spp. were dominant in biofilm regions with 2 g m(-3) DO or greater, while Nitrospira spp. were dominant in regions with less than 2 g m(-3) DO. A biofilm model, including AOB, Nitrobacter spp., and Nitrospira spp., was developed and calibrated with the experimental results. The model predicted the greatest extent of nitrite formation (95%) and the lowest ammonium oxidation flux (0.91 g N m(-2) day(-1)) when the membrane DO was 2 g m(-3) and the bulk liquid was anoxic. Conversely, the model predicted the lowest extent of nitrite formation (40%) and the highest ammonium oxidation flux (1.5 g N m(-2) day(-1)) when the membrane-DO and bulk-DO were 8 g m(-3) and 2 g m(-3), respectively. The estimated kinetic parameters for Nitrospira spp., revealed a high affinity for nitrite and oxygen. This explains the dominance of Nitrospira spp. over Nitrobacter spp. in regions with low nitrite and oxygen concentrations. Our results suggest that shortcut nitrification can effectively be controlled by manipulating the DO at the membrane surface. A tradeoff is made between increased nitrite accumulation at lower DO, and higher nitrification rates at higher DO.     

Settleability and kinetics of a nitrifying sludge in a sequencing batch reactor

A physiological study of a nitrifying sludge was carried out in a sequencing batch reactor (SBR). Pseudo steady-state nitrification conditions were obtained with an ammonium removal efficiency of 99% +/- 1% and 98% +/- 2% conversion of NH4+-N to NO3 - -N. The rate of biomass production was negligible (1.3 +/- 0.1 mg microbial protein-N.L(-1).d(-1)). The sludge presented good settling properties with sludge volume index values lower than 20 mL.g(-1) and an exopolymeric protein/carbohydrate ratio of 0.53 +/- 0.34. Kinetic results indicated that the nitrifying behavior of the sludge changed with the number of cycles. After 22 cycles, a decrease in the specific rate of NO3- -N production coupled with an increase in the NO2- -N accumulation were observed. These results showed that the activity of the nitrite oxidizing bacteria decreased at a longer operation time. Ammonia oxidizing bacteria were found to exhibit the best stability. After 4 months of operation, the specific rates of NH4+-N consumption and NO3- -N production were 1.72 NH4+-N per microbial protein-N per hour (g.g(-1).h(-1)) and 0.54 NO3- -N per microbial protein-N per hour (g.g(-1).h(-1)), respectively.     

Nitrifying bacterial growth inhibition in the presence of algae and cyanobacteria

Nitrifying bacteria, cyanobacteria, and algae are important microorganisms in open pond wastewater treatment systems. Nitrification involving the sequential oxidation of ammonia to nitrite and nitrate, mainly due to autotrophic nitrifying bacteria, is essential to biological nitrogen removal in wastewater and global nitrogen cycling. A continuous flow autotrophic bioreactor was initially designed for nitrifying bacterial growth only. In the presence of cyanobacteria and algae, we monitored both the microbial activity by measuring specific oxygen production rate (SOPR) for microalgae and cyanobacteria and specific oxygen uptake rate (SOUR) for nitrifying bacteria. The growth of cyanobacteria and algae inhibited the maximum nitrification rate by a factor of 4 although the ammonium nitrogen fed to the reactor was almost completely removed. Terminal restriction fragment length polymorphism (T‐RFLP) analysis indicated that the community structures of nitrifying bacteria remained unchanged, containing the dominant Nitrosospira, Nitrospira, and Nitrobacter species. PCR amplification coupled with cloning and sequencing analysis resulted in identifying Chlorella emersonii and an uncultured cyanobacterium as the dominant species in the autotrophic bioreactor. Notwithstanding their fast growth rate and their toxicity to nitrifiers, microalgae and cyanobacteria were more easily lost in effluent than nitrifying bacteria because of their poor settling characteristics. The microorganisms were able to grow together in the bioreactor with constant individual biomass fractions because of the uncoupled solids retention times for algae/cyanobacteria and nitrifiers. The results indicate that compared to conventional wastewater treatment systems, longer solids retention times (e.g., by a factor of 4) should be considered in phototrophic bioreactors for complete nitrification and nitrogen removal. Biotechnol. Bioeng. 2010;107: 1004–1011. © 2010 Wiley Periodicals, Inc.     

Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm

A biofilm from a nitrifying pilot-scale sequencing batch reactor was investigated for effects of varying process conditions on its microscale activity and structure. Microsensor measurements of oxygen, substrates and products of nitrification were applied under incubation at different ammonium and oxygen concentrations which reflected various situations during a treatment cycle. A high net N loss was observed under high ammonium (HA) concentrations in contrast to low ones. Additionally, results indicated inhibition of nitrite-oxidizing bacteria (NOB), but not of ammonia-oxidizing bacteria (AOB) by free ammonia under HA conditions. Diversity, spatial distribution, and abundance of nitrifying bacteria as analysed by fluorescence in situ hybridization (FISH) revealed six different nitrifying populations with heterogeneous distributions. Nitrosococcus mobilis formed conspicuous microcolonies locally surrounded by cells of the dominating N. europaea/eutropha-related AOB population. A third less abundant population was affiliated to N. oligotropha. Nitrite-oxidizing bacteria of the genera Nitrobacter and Nitrospira (with at least two distinct populations) showed a large scale heterogeneity in their distribution. Nitrospira spp. were also found in deeper inactive layers where they might persist rather than thrive, and act as seed population when detached. Results of functional and structural analyses are discussed with respect to specific niches of individual populations in this system.     

Chemical inhibition of nitrification in activated sludge

Conventional aerobic nitrification was adversely affected by single pulse inputs of six different classes of industrially relevant chemical toxins: an electrophilic solvent (1-chloro-2,4-dinitrobenzene, CDNB), a heavy metal (cadmium), a hydrophobic chemical (1-octanol), an uncoupling agent (2,4-dinitrophenol, DNP), alkaline pH, and cyanide in its weak metal complexed form. The concentrations of each chemical source that caused 1 5, 25, and 50% respiratory inhibition of a nitrifying mixed liquor during a short-term assay were used to shock sequencing batch reactors containing nitrifying conventional activated sludge. The reactors were monitored for recovery over a period of 30 days or less. All shock conditions inhibited nitrification, but to different degrees. The nitrate generation rate (NGR) of the shocked reactors recovered overtime to control reactor levels and showed that it was a more sensitive indicator of nitrification inhibition than both initial respirometric tests conducted on unexposed biomass and effluent nitrogen species analyses. CDNB had the most severe impact on nitrification, followed by alkaline pH 11, cadmium, cyanide, octanol, and DNP. Based on effluent data, cadmium and octanol primarily inhibited ammonia-oxidizing bacteria (AOB) while CDNB, pH 11,and cyanide inhibited both AOB and nitrite-oxidizing bacteria (NOB). DNP initially inhibited nitrification but quickly increased the NGR relative to the control and stimulated nitrification after several days in a manner reflective of oxidative uncoupling. The shocked mixed liquor showed trends toward recovery from inhibition for all chemicals tested, but in some cases this reversion was slow. These results contribute to our broader effort to identify relationships between chemical sources and the process effects they induce in activated sludge treatment systems.     

Sensitivity analysis of a biofilm model describing a one-stage completely autotrophic nitrogen removal (CANON) process.

A mathematical model for nitrification and anaerobic ammonium oxidation (ANAMMOX) processes in a single biofilm reactor (CANON) was developed. This model describes completely autotrophic conversion of ammonium to dinitrogen gas. Aerobic ammonium and nitrite oxidation were modeled together with ANAMMOX. The sensitivity of kinetic constants and biofilm and process parameters to the process performance was evaluated, and the total effluent concentrations were, in general, found to be insensitive to affinity constants. Increasing the amount of biomass by either increasing biofilm thickness and density or decreasing porosity had no significant influence on the total effluent concentrations, provided that a minimum total biomass was present in the reactor. The ANAMMOX process always occurred in the depth of the biofilm provided that the oxygen concentration was limiting. The optimal dissolved oxygen concentration level at which the maximum nitrogen removal occurred is related to a certain ammonium surface load on the biofilm. An ammonium surface load of 2 g N/m2. d, associated with a dissolved oxygen concentration level of 1.3 g O2/m3 in the bulk liquid and with a minimum biofilm depth of 1 mm seems a proper design condition for the one-stage ammonium removal process. Under this condition, the ammonium removal efficiency is 94% (82% for the total nitrogen removal efficiency) (30 degrees C). Better ammonium removal could be achieved with an increase in the dissolved oxygen concentration level, but this would strongly limit the ANAMMOX process and decrease total nitrogen removal. It can be concluded that a one-stage process is probably not optimal if a good nitrogen effluent is required. A two-stage process like the combined SHARON and ANAMMOX process would be advised for complete nitrogen removal.     

The importance of autotrophic versus heterotrophic oxidation of atmospheric ammonium in forest ecosystems with acid soil

Abstract The role of autotrophic and heterotrophic nitrifying microorganisms in the oxidation of atmospheric ammonium in two acid and one calcareous location of a Dutch woodland area was investigated. In soil slurries nitrate formation was completely inhibited by acetylene, a specific inhibitor of autotrophic ammonium-oxidizing bacteria. A survey of nitrifiers in the forest soils showed that both autotrophic ammonium- and nitrite-oxidizing bacteria were present in high numbers and evidence was obtained that autotrophic bacteria are able to nitrify below pH 4. These results show that autotrophic nitrifying bacteria may account for most of the nitrification in the examined soils. To assess the contribution of heterotrophic nitrifiers, about 200 strains of heterotrophic bacteria and 21 morphologically distinct fungal strains were isolated from the acid soil locations and tested for their ability to nitrify. Only one Penicillium strain produced nitrate in test media, but its nitrate formation when added to acid soils was poor. These findings indicate that in the investigated soil heterotrophs are of minor importance in the oxidation of atmospheric ammonium.     

Competition and coexistence of aerobic ammonium- and nitrite-oxidizing bacteria at low oxygen concentrations

In natural and man-made ecosystems nitrifying bacteria experience frequent exposure to oxygen-limited conditions and thus have to compete for oxygen. In several reactor systems (retentostat, chemostat and sequencing batch reactors) it was possible to establish co-cultures of aerobic ammonium- and nitrite-oxidizing bacteria at very low oxygen concentrations (2–8 μM) provided that ammonium was the limiting N compound. When ammonia was in excess of oxygen, the nitrite-oxidizing bacteria were washed out of the reactors, and ammonium was converted to mainly nitrite, nitric oxide and nitrous oxide by Nitrosomonas-related bacteria. The situation could be rapidly reversed by adjusting the oxygen to ammonium ratio in the reactor. In batch and continuous tests, no inhibitory effect of ammonium, nitric oxide or nitrous oxide on nitrite-oxidizing bacteria could be detected in our studies. The recently developed oxygen microsensors may be helpful to determine the kinetic parameters of the nitrifying bacteria, which are needed to make predictive kinetic models of their competition.     

Assessment of the positive effect of salinity on the nitrogen removal performance and microbial composition during the start-up of CANON process

In this study, a non-woven rotating biological contactor reactor was operated for the start-up of completely autotrophic nitrogen removal over nitrite (CANON) process. In this perfectly attached growth system, nitrite oxidizing was identified, which interfered with the nitrogen removal performance. Batch tests indicated that 10 g NaCl per liter salinity was a preferable definite level to stand out ammonium-oxidizing activity and anammox activity, and selectively suppress nitrite-oxidizing activity under oxygen-limited conditions. Reactor operation showed that the maximum TN removal rate was increased from 425 mg N l(-1) day(-1) to 637 mg N l(-1) day(-1) after the addition of 10 g NaCl per liter salinity on analogous technological parameters. Microbiological community analysis revealed that bacteria strains similar to the genus Nitrospira sp. were specialized nitrite oxidizers existing in CANON reactor, which were then eliminated under salinity exposure for their no salinity-tolerant relative. However, anammox bacteria belonging to Planctomycetes and some aerobic ammonium oxidizers belonging to Nitrosomonas could be highly enriched under this oxygen-limited salinity conditions. Salinity-contained high ammonium wastewater will be so considered as suitable influent for CANON process in further industrial application.     

Simultaneous nitrification and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor

Nitrogen and carbon components in domestic modified wastewater were completely removed by simultaneous nitrification and denitrification using a membrane-aerated biofilm reactor where biofilm was fixed on a hollow-fiber membrane. To measure the spatial distribution of pH, ammonium and nitrate ions and to observe microbes inside the biofilm fixed on the membrane, microelectrodes and the fluorescence in situ hybridization (FISH) method were applied. Due to plug flow in the vertical direction (from the bottom to the top of the reactor), ammonium nitrogen was gradually removed and negligible nitrate nitrogen was detected throughout the reactor. FISH revealed that ammonia-oxidizing bacteria were mainly distributed inside the biofilm and other bacteria, which included denitrifying bacteria, were mainly distributed outside the biofilm and over the suspended sludge. In order to characterize bacterial activity in the vertical direction of the reactor, nitrification rates at lower, central and upper points were calculated using microelectrode data. The nitrification rate at the lower point was 7 and 125 times higher than those at the central and upper points, respectively. These results show that the removal of carbon and nitrogen compounds was accomplished efficiently by using various kinds of bacteria distributed vertically and horizontally in a single reactor.