Evaluation of autotrophic growth of ammonia-oxidizers associated with granular activated carbon used for drinking water purification by DNA-stable isotope probing

Nitrification is an important biological function of granular activated carbon (GAC) used in advanced drinking water purification processes. Newly discovered ammonia-oxidizing archaea (AOA) have challenged the traditional understanding of ammonia oxidation, which considered ammonia-oxidizing bacteria (AOB) as the sole ammonia-oxidizers. Previous studies demonstrated the predominance of AOA on GAC, but the contributions of AOA and AOB to ammonia oxidation remain unclear. In the present study, DNA-stable isotope probing (DNA-SIP) was used to investigate the autotrophic growth of AOA and AOB associated with GAC at two different ammonium concentrations (0.14 mg N/L and 1.4 mg N/L). GAC samples collected from three full-scale drinking water purification plants in Tokyo, Japan, had different abundance of AOA and AOB. These samples were fed continuously with ammonium and ¹³C-bicarbonate for 14 days. The DNA-SIP analysis demonstrated that only AOA assimilated ¹³C-bicarbonate at low ammonium concentration, whereas AOA and AOB exhibited autotrophic growth at high ammonium concentration. This indicates that a lower ammonium concentration is preferable for AOA growth. Since AOA could not grow without ammonium, their autotrophic growth was coupled with ammonia oxidation. Overall, our results point towards an important role of AOA in nitrification in GAC filters treating low concentration of ammonium.     

Ammonia-oxidizing archaea involved in nitrogen removal

Ammonia oxidation is critical to global nitrogen cycling and is often thought to be driven only by ammonia-oxidizing bacteria. The recent finding of new ammonia-oxidizing organisms belonging to the archaeal domain challenges this perception. Two major microbial groups are now believed to be involved in ammonia oxidation: chemolithotrophic ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Candidatus “Nitrosopumilus maritimus”, the first isolated ammonia-oxidizing archaeon from a tropical marine aquarium tank, representative of the ubiquitous marine group 1 Crenarchaeota, contains putative genes for all three subunits (amoA, amoB, and amoC) of ammonia monooxygenase, the key enzyme responsible for ammonia oxidation. In this article, important concepts of the nitrogen cycle, ammonia oxidation processes, ammonia-oxidizing organisms, and their physiology are described. AOA are found to thrive in various habitats including hot/thermal springs, marine and fresh waters, soils, and wastewater treatment systems, where they may outnumber their counterpart, AOB. Various molecular tools have been applied to study AOB and AOA and determine their abundance and community structure changes from natural and engineered systems. The presence of AOA in activated sludge opens new opportunities for elucidating its role of ammonia removal in wastewater treatment plants and wetlands. Several significant questions related to AOA research have been raised to evoke reader involvement for broadening future studies.     

Phosphorus limitation in nitrifying groundwater filters

Phosphorus limitation has been demonstrated for heterotrophic growth in groundwater, in drinking water production and distribution systems, and for nitrification of surface water treatment at low temperatures. In this study, phosphorus limitation was tested, in the Netherlands, for nitrification of anaerobic groundwater rich in iron, ammonium and orthophosphate. The bioassay method developed by Lehtola et al. (1999) was adapted to determine the microbially available phosphorus (MAP) for nitrification. In standardized batch experiments with an enriched mixed culture inoculum, the formation of nitrite and nitrate and ATP and the growth of ammonia-oxidizing bacteria (AOB; as indicated by qPCR targeting the amoA-coding gene) were determined for MAP concentrations between 0 and 100 μg PO₄-P L⁻¹. The nitrification and microbial growth rates were limited at under 100 μg PO₄-P L⁻¹ and virtually stopped at under 10 μg PO₄-P L⁻¹. In the range between 10 and 50 μg PO₄-P L⁻¹, a linear relationship was found between MAP and the maximum nitrification rate. AOB cell growth and ATP formation were proportional to the total ammonia oxidized. Contrary to Lehtola et al. (1999), biological growth was very slow for MAP concentrations less than 25 μg PO₄-P L⁻¹. No full conversion nor maximum cell numbers were reached within 19 days. In full-scale groundwater filters, most of the orthophosphate was removed alongside with iron. The remaining orthophosphate appeared to have only limited availability for microbial growth and activity. In some groundwater filters, nitrification was almost totally prevented by limitation of MAP. In batch experiments with filtrate water from these filters, the nitrification process could be effectively stimulated by adding phosphoric acid.     

Monochloramine cometabolism by Nitrosomonas europaea under drinking water conditions

Chloramine is widely used in United States drinking water systems as a secondary disinfectant, which may promote the growth of nitrifying bacteria because ammonia is present. At the onset of nitrification, both nitrifying bacteria and their products exert a monochloramine demand, decreasing the residual disinfectant concentration in water distribution systems. This work investigated another potentially significant mechanism for residual disinfectant loss: monochloramine cometabolism by ammonia-oxidizing bacteria (AOB).Monochloramine cometabolism was studied with the pure culture AOB Nitrosomonas europaea (ATCC 19718) in batch kinetic experiments under drinking water conditions. Three batch reactors were used in each experiment: a positive control to estimate the ammonia kinetic parameters, a negative control to account for abiotic reactions, and a cometabolism reactor to estimate the cometabolism kinetic constants. Kinetic parameters were estimated in AQUASIM with a simultaneous fit to all experimental data. The cometabolism reactors showed a more rapid monochloramine decay than in the negative controls, demonstrating that cometabolism occurs. Cometabolism kinetics were best described by a pseudo first order model with a reductant term to account for ammonia availability. Monochloramine cometabolism kinetics were similar to those of ammonia metabolism, and monochloramine cometabolism was a significant loss mechanism (30–60% of the observed monochloramine decay). These results suggest that monochloramine cometabolism should occur in practice and may be a significant contribution to monochloramine decay during nitrification episodes in drinking water distribution systems.     

Effect of temperature and disinfection strategies on ammonia-oxidizing bacteria in a bench-scale drinking water distribution system

The establishment of ammonia-oxidizing bacteria (AOB), a group of autotrophic microorganisms responsible for nitrification in chloraminated distribution systems, was studied in a bench-scale distribution system. The potential significance of temperature and disinfectant residual associated with chloramination in full-scale drinking water distribution systems was assessed. Biofilm development was primarily monitored using AOB abundance and nitrite concentrations. The bench-scale system was initially operated under typical North American summer (22 degrees C) and fall (12 degrees C) temperatures, representing optimal and less optimal growth ranges for these microorganisms. Additional experimentation investigated AOB establishment at a suboptimal winter distribution system temperature of 6 degrees C. The effect of chloramine residual on AOB establishment was studied at higher (0.2-0.6mg/L) and lower (0.05-0.1mg/L) ranges, using a 3:1 (w/w) chlorine:ammonia dosing ratio. Conditions were selected to represent those typically found in a North American distribution system, in areas of low flow and longer retention times, respectively. Finally, the effect of a free chlorine residual on an established nitrifying biofilm was briefly examined. Results clearly indicate that AOB development occurs at all examined temperatures, as well as at selected monochloramine residuals. The maintenance of a disinfectant residual was difficult at times, but was more inhibitory to the nitrifying biofilm than the lower temperature. It can be concluded from the data that nitrification may not be adequately inhibited during the winter months, which may result in more advanced stages of nitrification the following season. Free chlorination can be effective in controlling AOB activity in the short term, but may not prevent reestablishment of a nitrifying biofilm upon return to chloramination.     

Biodiversity and population dynamics of microorganisms in a full-scale membrane bioreactor for municipal wastewater treatment

The total, ammonia-oxidizing, and denitrifying Bacteria in a full-scale membrane bioreactor (MBR) were evaluated monthly for over one year. Microbial communities were analyzed by denaturing gradient gel electrophoresis (DGGE) and clone library analysis of the 16S rRNA and ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes. The community fingerprints obtained were compared to those from a conventional activated sludge (CAS) process running in parallel treating the same domestic wastewater. Distinct DGGE profiles for all three molecular markers were observed between the two treatment systems, indicating the selection of specific bacterial populations by the contrasting environmental and operational conditions. Comparative 16S rRNA sequencing indicated a diverse bacterial community in the MBR, with phylotypes from the α- and β-Proteobacteria and Bacteroidetes dominating the gene library. The vast majority of sequences retrieved were not closely related to classified organisms or displayed relatively low levels of similarity with any known 16S rRNA gene sequences and thus represent organisms that constitute new taxa. Similarly, the majority of the recovered nosZ sequences were novel and only moderately related to known denitrifiers from the α- and β-Proteobacteria. In contrast, analysis of the amoA gene showed a remarkably simple ammonia-oxidizing community with the detected members almost exclusively affiliated with the Nitrosomonas oligotropha lineage. Major shifts in total bacteria and denitrifying community were detected and these were associated with change in the external carbon added for denitrification enhancement. In spite of this, the MBR was able to maintain a stable process performance during that period. These results significantly expand our knowledge of the biodiversity and population dynamics of microorganisms in MBRs for wastewater treatment.     

Diversity of nitrifying bacteria in full-scale chloraminated distribution systems

Chloramination for secondary disinfection of drinking water often promotes the growth of nitrifying bacteria in the distribution system due to the ammonia introduced by chloramine formation and decay. This study involved the application of molecular biology techniques to explore the types of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) present in several full-scale chloraminated systems. The results of AOB community characterization indicated the ubiquitous detection of representatives from the Nitrosomonas genus, with Nitrosospira constituting a negligible or small fraction of the AOB community in all but one sample. Cloning and sequencing demonstrated the presence of AOB representatives within the Nitrosomonas oligotropha cluster, a phylogenetic subgroup of AOB from which isolates demonstrate a high affinity for ammonia. For the NOB communities, Nitrospira were detected in most of the samples, while Nitrobacter were only detected in a few samples. These results provide insight into the types of AOB responsible for nitrification episodes in full-scale chloraminated systems, which should help direct future studies aimed at characterizing relevant AOB growth and inactivation properties. Furthermore, the detection of NOB in most of the samples suggests a need to evaluate the contribution of biological nitrite oxidation relative to chemical oxidation in these systems.     

Application of bacteriophages to selectively remove Pseudomonas aeruginosa in water and wastewater filtration systems

Water and wastewater filtration systems often house pathogenic bacteria, which must be removed to ensure clean, safe water. Here, we determine the persistence of the model bacterium Pseudomonas aeruginosa in two types of filtration systems, and use P. aeruginosa bacteriophages to determine their ability to selectively remove P. aeruginosa. These systems used beds of either anthracite or granular activated carbon (GAC), which were operated at an empty bed contact time (EBCT) of 45 min. The clean bed filtration systems were loaded with an instantaneous dose of P. aeruginosa at a total cell number of 2.3 (±0.1 [standard deviation]) × 10⁷ cells. An immediate dose of P. aeruginosa phages (1 mL of phage stock at the concentration of 2.7 × 10⁷ PFU (Plaque Forming Units)/mL) resulted in a reduction of 50% (±9%) and >99.9% in the effluent P. aeruginosa concentrations in the clean anthracite and GAC filters, respectively. To further evaluate the effects of P. aeruginosa phages, synthetic stormwater was run through anthracite and GAC biofilters where mixed-culture biofilms were present. Eighty five days after an instantaneous dose of P. aeruginosa (2.3 × 10⁷ cells per filter) on day 1, 7.5 (±2.8) × 10⁷ and 1.1 (±0.5) × 10⁷ P. aeruginosa cells/g filter media were detected in the top layer (close to the influent port) of the anthracite and GAC biofilters, respectively, demonstrating the growth and persistence of pathogenic bacteria in the biofilters. A subsequent 1-h dose of phages, at the concentration of 5.1 × 10⁶ PFU/mL and flow rate of 1.6 mL/min, removed the P. aeruginosa inside the GAC biofilters and the anthracite biofilters by 70% (±5%) and 56% (±1%), respectively, with no P. aeruginosa detected in the effluent, while not affecting ammonia oxidation or the ammonia-oxidizing bacterial community inside the biofilters. These results suggest that phage treatment can selectively remove pathogenic bacteria with minimal impact on beneficial organisms from attached growth systems for effluent quality improvement.     

Effects of free cyanide on microbial communities and biological carbon and nitrogen removal performance in the industrial activated sludge process

The changes in process performance and microbial communities under free cyanide (CN⁻) were investigated in a lab-scale activated sludge process treating industrial wastewater. The performance of phenol degradation did not appear to be adversely affected by increases in CN⁻ concentrations. In contrast, CN⁻ was found to have an inhibitory effect on SCN⁻ biodegradation, resulting in the increase of TOC and COD concentrations. Nitratation also appeared to be inhibited at CN⁻ concentrations in excess of 1.0 mg/L, confirming that nitrite-oxidizing bacteria (NOB) is more sensitive to the CN⁻ toxicity than ammonia oxidizing bacteria (AOB). After CN⁻ loads were stopped, SCN⁻ removal, denitrification, and nitrification inhibited by CN⁻ were recovered to performance efficiency of more than 98%. The AOB and NOB communities in the aerobic reactor were analyzed by terminal restriction fragment length (T-RFLP) and quantitative real-time PCR (qPCR). Nitrosomonas europaea lineage was the predominant AOB at all samples during the operation, but an obvious change was observed in the diversity of AOB at the shock loading of 30 and 50 mg/L CN⁻, resulting in Nitrosospira sp. becoming dominant. We also observed coexisting Nitrospira and Nitrobacter genera for NOB. The increase of CN⁻ loading seemed to change the balance between Nitrospira and Nitrobacter, resulting in the high dominance of Nitrobacter over Nitrospira. Meanwhile, through using the qPCR, it was observed that the nitrite-reducing functional genes (i.e., nirS) were dominant in the activated sludge of the anoxic reactor, regardless of CN⁻ loads.     

Assessment of nitrification in groundwater filters for drinking water production by qPCR and activity measurement

In groundwater treatment for drinking water production, the causes of nitrification problems and the effectiveness of process optimization in rapid sand filters are often not clear. To assess both issues, the performance of a full-scale groundwater filter with nitrification problems and another filter with complete nitrification and pretreatment by subsurface aeration was monitored over nine months. Quantitative real-time polymerase chain reaction (qPCR) targeting the amoA gene of bacteria and archaea and activity measurements of ammonia oxidation were used to regularly evaluate water and filter sand samples. Results demonstrated that subsurface aeration stimulated the growth of ammonia-oxidizing prokaryotes (AOP) in the aquifer. Cell balances, using qPCR counts of AOP for each filter, showed that the inoculated AOP numbers from the aquifer were marginal compared with AOP numbers detected in the filter. Excessive washout of AOP was not observed and did not cause the nitrification problems. Ammonia-oxidizing archaea grew in both filters, but only in low numbers compared to bacteria. The cell-specific nitrification rate in the sand and backwash water samples was high for the subsurface aerated filter, but systematically much lower for the filter with nitrification problems. From this, we conclude that incomplete nitrification was caused by nutrient limitation.     

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic-aerobic sequencing batch reactor

The structure dynamic of ammonia-oxidizing bacteria (AOB) community and the distribution of AOB and nitrite-oxidizing bacteria (NOB) in granular sludge from an anaerobic-aerobic sequencing batch reactor (SBR) were investigated. A combination of process studies, molecular biotechniques and microscale techniques were employed to identify and characterize these organisms. The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge. Along with granules formation, the AOB diversity declined due to the selection pressure imposed by process conditions. Denaturing gradient gel electrophoresis (DGGE) and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling-washing out action. Furthermore, DGGE analysis revealed that larger granules benefit more AOB species surviving in the reactor. In the SBR were various size granules coexisted, granule diameter affected the distribution range of AOB and NOB. Small and medium granules (d < 0.6 mm) cannot restrict oxygen mass transfer in all spaces of the sludge. Larger granules (d > 0.9 mm) can result in smaller aerobic volume fraction and inhibition of NOB growth. All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.     

Effects of selected pharmaceutically active compounds on treatment performance in sequencing batch reactors mimicking wastewater treatment plants operations

The impact of four pharmaceutically active compounds (PhACs) introduced both individually and in mixtures was ascertained on the performance of laboratory-scale wastewater treatment sequencing batch reactors (SBRs). When introduced individually at concentrations of 0.1, 1 and 10 μM, no significant differences were observed with respect to chemical oxygen demand (COD) and ammonia removal. Microbial community analyses reveal that although similarity index values generally decreased over time with an increase in PhAC concentrations as compared to the controls, no major microbial community shifts were observed for total bacteria and ammonia-oxidizing bacteria (AOB) communities. However, when some PhACs were introduced in mixtures, they were found to both inhibit nitrification and alter AOB community structure. Ammonia removal decreased by up to 45% in the presence of 0.25 μM gemfibrozil and 0.75 μM naproxen. PhAC mixtures did not however affect COD removal performance suggesting that heterotrophic bacteria are more robust to PhACs than AOB. These results highlight that the joint action of PhACs in mixtures may have significantly different effects on nitrification than the individual PhACs. This phenomenon should be further investigated with a wider range of PhACs so that toxicity effects can more accurately be predicted.     

Nitrogen removal from wastewater using membrane aerated microbial fuel cell techniques

Nitrogen removal mainly relies on sequential nitrification and denitrification in wastewater treatment. Microbial fuel cells (MFCs) are innovative wastewater treatment techniques for pollution control and energy generation. In this study, bench-scale wastewater treatment systems using membrane-aerated MFC (MAMFC) and diffuser-aerated MFC (DAMFC) techniques were constructed for simultaneous removal of carbonaceous and nitrogenous pollutants and electricity production from wastewater. During 210 days of continuous flow operation, when the dissolved oxygen (DO) in the cathodic compartment was kept at 2 mg/L, both reactors demonstrated high COD removal (>99%) and high ammonia removal (>99%) but low nitrogen removal (<20%). When a lower DO (0.5 mg/L) was maintained after day 121, both the MFC-based reactors still had excellent COD removal (>97%). However, the nitrogen removal of MAMFC (52%) was 2-fold higher than that of DAMFC (24%), indicating an enhanced performance of denitrification after DO reduction in the cathodic compartment of the MAMFC. Meanwhile, terminal restriction fragment length polymorphism (T-RFLP) analysis of ammonia-oxidizing bacteria (AOB) population in the MAMFC indicated the diversity of AOB with equally important Nitrosospira and Nitrosomonas species present in the cathodic biofilm after DO reduction. The average voltage output in the MAMFC was significantly higher than that in DAMFC under both DO conditions. The results suggest that MAMFC systems have the potential for wastewater treatment with improved nitrogen removal and electricity production.     

Influence of manganese and ammonium oxidation on the removal of 17 alpha-ethinylestradiol (EE2)

Flow-through reactors with manganese oxides were examined for their capacity to remove 17α-ethinylestradiol (EE2) at μg L⁻¹ and ng L⁻¹ range from synthetic wastewater treatment plant (WWTP) effluent. The mineral MnO₂ reactors removed 93% at a volumetric loading rate (B_V) of 5 μg EE2 L⁻¹ d⁻¹ and from a B_V of 40 μg EE2 L⁻¹ d⁻¹ on, these reactors showed 75% EE2 removal. With the biologically produced manganese oxides, only 57% EE2 was removed at 40 μg EE2 L⁻¹ d⁻¹. EE2 removal in the ng L⁻¹ range was 84%. The ammonium present in the influent (10 mg N L⁻¹) was nitrified and ammonia-oxidizing bacteria (AOB) were found to be of prime importance for the degradation of EE2. Remarkably, EE2 removal by AOB continued for a period of 4 months after depleting NH₄⁺ in the influent. EE2 removal by manganese-oxidizing bacteria was inhibited by NH₄⁺. These results indicate that the metabolic properties of nitrifiers can be employed to polish water containing EE2 based estrogenic activity.     

Molecular characterization of microbial populations in groundwater sources and sand filters for drinking water production

In full-scale drinking water production from groundwater, subsurface aeration is an effective means of enhancing the often troublesome process of nitrification. Until now the exact mechanism, however, has been unknown. By studying the microbial population we can improve the understanding of this process. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments of bacteria, archaea and ammonia-oxidizing bacteria was used to characterize the microbial populations in raw groundwater and trickling filters of an active nitrifying surface aerated system and an inactive non-surface aerated system. Only in the active filter were nitrifying microorganisms found above the detection limit of the method. In ammonia oxidation in this groundwater filter both bacteria and archaea played a role, while members belonging to the genus Nitrospira were the only nitrite-oxidizing species found. The subsurface aerated groundwater did not contain any of the nitrifying organisms active in the filter above the detection limit, but did contain Gallionella species that might play a major role in iron oxidation in the filter.     

Distributions and activities of ammonia oxidizing bacteria and polyphosphate accumulating organisms in a pumped-flow biofilm reactor

The spatial distributions and activities of ammonia oxidizing bacteria (AOB) and polyphosphate accumulating organisms (PAOs) were investigated for a novel laboratory-scale sequencing batch pumped-flow biofilm reactor (PFBR) system that was operated for carbon, nitrogen and phosphorus removal. The PFBR comprised of two 16.5 l tanks (Reactors 1 and 2), each with a biofilm module of 2 m² surface area. To facilitate the growth of AOB and PAOs in the reactor biofilms, the influent wastewater was held in Reactor 1 under stagnant un-aerated conditions for 6 h after feeding, and was then pumped over and back between Reactors 1 and 2 for 12 h, creating aerobic conditions in the two reactors during this period; as a consequence, the biofilm in Reactor 2 was in an aerobic environment for almost all the 18.2 h operating cycle. A combination of micro-sensor measurements, molecular techniques, batch experiments and reactor studies were carried out to analyse the performance of the PFBR system. After 100 days operation at a filtered chemical oxygen demand (COD_f) loading rate of 3.46 g/m² per day, the removal efficiencies were 95% COD_f, 87% TN_f and 74% TP_f. While the PFBR microbial community structure and function were found to be highly diversified with substantial AOB and PAO populations, about 70% of the phosphorus release potential and almost 100% of the nitrification potential were located in Reactors 1 and 2, respectively. Co-enrichment of AOB and PAOs was realized in the Reactor 2 biofilm, where molecular analyses revealed unexpected microbial distributions at micro-scale, with population peaks of AOB in a 100–250 μm deep sub-surface zone and of PAOs in the 0–150 μm surface zone. The micro-distribution of AOB coincided with the position of the nitrification peak identified during micro-sensor analyses. The study demonstrates that enrichment of PAOs can be realized in a constant or near constant aerobic biofilm environment. Furthermore, the findings suggest that when successful co-enrichment of AOB and PAOs occur in biofilm environments, such as in the PFBR system, they do so at different zone depths in the biofilm.     

Occurrence of nitrifiers and diversity of ammonia-oxidizing bacteria in developing drinking water biofilms

We studied the population dynamics of nitrifying bacteria during the development of biofilms up to 233 or 280 days on polyvinylchloride pipes connected to two full-scale drinking water distribution networks supplying processed and chloraminated surface water. The numbers of nitrifiers in biofilms were enumerated at intervals of 10–64 days by the most probable number (MPN) method at waterworks and at several study sites in distribution network areas. The numbers of nitrifiers increased towards the distal sites. The highest detected MPN counts of ammonia-oxidizing bacteria (AOB) for study areas 1 and 7 were 500 MPN cm⁻² and 1.0×10⁶ MPN cm⁻², and those of nitrite-oxidizing bacteria (NOB) 96 MPN cm⁻² and 2.2×10³ MPN cm⁻², respectively. The diversity of AOB was determined by PCR amplifying, cloning and sequencing the partial ammonia monooxygenase (amoA) gene of selected biofilm samples presenting different biofilm ages. The PCR primers used, A189 and A682, also amplified a fragment of particulate methane monooxygenase (pmoA) gene of methane-oxidizing bacteria. The majority of biofilm clones (24 out of 30 studied) contained Nitrosomonas amoA-like sequences. There were only two pmoA-like sequences of Type I methanotrophs, and four sequences positioned in amoA/pmoA sequence groups of uncultured bacteria. From both study area very similar or even completely identical Nitrosomonas amoA-like sequences were obtained despite of high difference in AOB numbers. The results show that the conditions in newly formed biofilms in drinking water distribution systems favor the growth of Nitrosomonas-type AOB.     

Development of biomass in a drinking water granular active carbon (GAC) filter

Indigenous bacteria are essential for the performance of drinking water biofilters, yet this biological component remains poorly characterized. In the present study we followed biofilm formation and development in a granular activated carbon (GAC) filter on pilot-scale during the first six months of operation. GAC particles were sampled from four different depths (10, 45, 80 and 115 cm) and attached biomass was measured with adenosine tri-phosphate (ATP) analysis. The attached biomass accumulated rapidly on the GAC particles throughout all levels in the filter during the first 90 days of operation and maintained a steady state afterward. Vertical gradients of biomass density and growth rates were observed during start-up and also in steady state. During steady state, biomass concentrations ranged between 0.8-1.83 x 10⁻⁶ g ATP/g GAC in the filter, and 22% of the influent dissolved organic carbon (DOC) was removed. Concomitant biomass production was about 1.8 × 10¹² cells/m²h, which represents a yield of 1.26 × 10⁶ cells/μg. The bacteria assimilated only about 3% of the removed carbon as biomass. At one point during the operational period, a natural 5-fold increase in the influent phytoplankton concentration occurred. As a result, influent assimilable organic carbon concentrations increased and suspended bacteria in the filter effluent increased 3-fold as the direct consequence of increased growth in the biofilter. This study shows that the combination of different analytical methods allows detailed quantification of the microbiological activity in drinking water biofilters.     

Mechanisms of N2O production in biological wastewater treatment under nitrifying and denitrifying conditions

Nitrous oxide (N₂O) is an important greenhouse gas and a major sink for stratospheric ozone. In biological wastewater treatment, microbial processes such as autotrophic nitrification and heterotrophic denitrification have been identified as major sources; however, the underlying pathways remain unclear. In this study, the mechanisms of N₂O production were investigated in a laboratory batch-scale system with activated sludge for treating municipal wastewater. This relatively complex mixed population system is well representative for full-scale activated sludge treatment under nitrifying and denitrifying conditions.Under aerobic conditions, the addition of nitrite resulted in strongly nitrite-dependent N₂O production, mainly by nitrifier denitrification of ammonia-oxidizing bacteria (AOB). Furthermore, N₂O is produced via hydroxylamine oxidation, as has been shown by the addition of hydroxylamine. In both sets of experiments, N₂O production was highest at the beginning of the experiment, then decreased continuously and ceased when the substrate (nitrite, hydroxylamine) had been completely consumed. In ammonia oxidation experiments, N₂O peaked at the beginning of the experiment when the nitrite concentration was lowest. This indicates that N₂O production via hydroxylamine oxidation is favored at high ammonia and low nitrite concentrations, and in combination with a high metabolic activity of ammonia-oxidizing bacteria (at 2 to 3 mgO₂/l); the contribution of nitrifier denitrification by AOB increased at higher nitrite and lower ammonia concentrations towards the end of the experiment.Under anoxic conditions, nitrate reducing experiments confirmed that N₂O emission is low under optimal growth conditions for heterotrophic denitrifiers (e.g. no oxygen input and no limitation of readily biodegradable organic carbon). However, N₂O and nitric oxide (NO) production rates increased significantly in the presence of nitrite or low dissolved oxygen concentrations.