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.     

氯胺消毒给水管网中的硝化作用及其控制

介绍了氯胺消毒管网中的硝化作用及其控制方法。研究表明,在采用氯胺消毒的给水管网中,存在着硝化反应发生的可能性。硝化作用会造成出水亚硝酸盐含量升高、消毒剂含量降低、异养菌繁殖等危害,水中的氨氮是引起该问题的主要原因。国外对氯胺消毒管网中硝化作用的研究主要集中在硝化作用与水质的相互影响方面,但对硝化作用成因的研究还不系统,部分研究的结论还属于推断性结论。目前我国部分城市给水管网中已经出现硝化现象,但还没有对氯胺消毒产生的硝化作用进行系统研究,指出在我国开展给水管网中硝化作用的研究是非常必要的。     

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.     

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.     

Soft deposits, the key site for microbial growth in drinking water distribution networks

In this project we studied the microbiological quality of soft pipeline deposits removed from drinking water distribution networks during mechanical cleaning. Drinking water and deposit samples were collected from 16 drinking water distribution networks located at eight towns in different parts of Finland. Soft pipeline deposits were found to be the key site for microbial growth in the distribution networks. The microbial numbers in the soft deposits were significantly higher than numbers in running water. The highest microbial numbers were detected in the main deposit pushed ahead by the first swab. The deposits contained high numbers of heterotrophic bacteria, actinomycetes and fungi. Also coliform bacteria were often isolated from deposit samples. Manganese and copper in the deposits correlated negatively with the numbers of heterotrophic bacteria. After a year, the viable microbial numbers in the new deposits were almost as high as in the old deposits before the first mechanical cleaning. The bacterial biomass production was higher in the new than in the old deposits.     

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.     

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.     

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.     

Evidence of soluble microbial products accelerating chloramine decay in nitrifying bulk water samples

The discovery of a microbially derived soluble product that accelerates chloramine decay is described. Nitrifying bacteria are believed to be wholly responsible for rapid chloramine loss in drinking water systems. However, a recent investigation showed that an unidentified soluble agent significantly accelerated chloramine decay. The agent was suspected to be either natural organic matter (NOM) or soluble microbial products (SMPs). A laboratory scale reactor was fed chloraminated reverse osmosis (RO) treated water to eliminate the interference from NOM. Once nitrification had set in, experiments were conducted on the reactor and feed waters to determine the identity of the component. The study showed the presence of SMPs released by microbes in severely nitrified waters. Further experiments proved that the SMPs significantly accelerated chloramine decay, probably through catalytic reaction. Moreover, application of common protein denaturing techniques stopped the reaction implying that the compound responsible was likely to be a protein. This significant finding will pave the way for better control of chloramine in the distribution systems.     

氯胺消毒的配水系统中硝化作用的研究进展

对氯胺消毒的配水系统中的硝化现象及其危害,以及近年来国内外有关硝化作用的影响因素和检测方法等方面的研究进展进行了综述.探讨了分子生物学分析技术在对硝化细菌进行结构分析和定量评估中的应用,以及在硝化细菌培养过程中尚待解决的问题.     

Microbial community changes with decaying chloramine residuals in a lab-scale system

When chloramine is used as a disinfectant, managing an acceptable “residual” throughout the water distribution systems particularly once nitrification has set in is challenging. Managing chloramine decay prior to the onset of nitrification through effective control strategies is important and to-date the strategies developed around nitrification has been ineffective. This study aimed at developing a more holistic knowledge on how decaying chloramine and nitrification metabolites impact microbial communities in chloraminated systems. Five lab-scale reactors (connected in series) were operated to simulate a full-scale chloraminated distribution system. Culture independent techniques (cloning and qPCR) were used to characterise and quantify the mixed microbial communities in reactors maintaining a residual of high to low (2.18–0.03 mg/L). The study for the first time associates chloramine residuals and nitrification metabolites to different microbial communities. Bacterial classes Solibacteres, Nitrospira, Sphingobacteria and Betaproteobacteria dominated at low chloramine residuals whereas Actinobacteria and Gammaproteobacteria dominated at higher chloramine residuals. Prior to the onset of nitrification bacterial genera Pseudomonas, Methylobacterium and Sphingomonas were found to be dominant and Sphingomonas in particular increased with the onset of nitrification. Nitrosomonas urea, oligotropha, and two other novel ammonia-oxidizing bacteria were detected once the chloramine residuals had dropped below 0.65 mg/L. Additionally nitrification alone failed to explain chloramine decay rates observed in these reactors. The finding of this study is expected to re-direct the focus from nitrifiers to heterotrophic bacteria, which the authors believe could hold the key towards developing a control strategy that would enable better management of chloramine residuals.     

Onset of severe nitrification in mildly nitrifying chloraminated bulk waters and its relation to biostability

Triggers of severe nitrification in distribution systems are still not clearly understood. Recently, the biostability concept was proposed to explain the chloramine residual below which signs of nitrification would be seen. To improve understanding, mildly nitrifying bulk water samples (nitrite less than 0.010mg-N/L) from Sydney Water distribution systems were incubated at constant temperatures and periodically analysed for nitrogenous compounds and total chlorine. Total ammoniacal nitrogen in the sample was between 0.25 and 0.35mg-N/L. Severe nitrification was triggered when chloramine residuals dropped below about 0.4mg/L - the critical threshold residual. In 45 such samples, the critical threshold residual was 0.2-0.65mg/L. The biostability concept was found to be useful in explaining the residual below which net growth of microorganisms begins. However, this alone could not predict the critical threshold residual. Different means of overcoming this problem are discussed. One of these is the use of the microbial decay factor method, since microbiologically assisted chloramine decay in the samples studied was found to be mostly the result of ammonia-oxidising bacterial activity. Nitrite levels in winter were found to be poor indicators of nitrifying status. Overall the results were found to be useful in controlling nitrification and to obtain early warning of severe nitrification.     

Removal of soft deposits from the distribution system improves the drinking water quality

Deterioration in drinking water quality in distribution networks represents a problem in drinking water distribution. These can be an increase in microbial numbers, an elevated concentration of iron or increased turbidity, all of which affect taste, odor and color in the drinking water. We studied if pipe cleaning would improve the drinking water quality in pipelines. Cleaning was arranged by flushing the pipes with compressed air and water. The numbers of bacteria and the concentrations of iron and turbidity in drinking water were highest at 9 p.m., when the water consumption was highest. Soft deposits inside the pipeline were occasionally released to bulk water, increasing the concentrations of iron, bacteria, microbially available organic carbon and phosphorus in drinking water. The cleaning of the pipeline decreased the diurnal variation in drinking water quality. With respect to iron, only short-term positive effects were obtained. However, removing of the nutrient-rich soft deposits did decrease the microbial growth in the distribution system during summer when there were favorable warm temperatures for microbial growth. No Norwalk-like viruses or coliform bacteria were detected in the soft deposits, in contrast to the high numbers of heterotrophic bacteria.     

Occurrence of filamentous fungi and yeasts in three different drinking water sources

In order to determine the occurrence of fungi in different drinking water sources and capture variability in terms of matrix composition and seasonal effects, surface water, spring water, and groundwater samples were collected in numerous sampling events. The occurrence and significance of fungi detected in the different water sources are reported and discussed in terms of colony-forming units per millilitre and by the identification of the most frequently detected isolates, at the species level, based on morphology and other phenotypic characters. All the samples were also analyzed in terms of total coliforms and Escherichia coli that are widely monitored bacteria considered as microbiology indicators of water quality. All the groundwater samples showed significantly lower levels of total coliforms, E. coli, and fungi compared to the surface and spring water samples. No significant correlations were found between the levels of fungi detected in all the matrices and the physico-chemical parameters and bacteria regularly monitored by drinking water utilities. Fifty-two fungi isolates were identified in this study, most of which have never been described to occur in water sources. The results obtained show that fungi occur widely in drinking water sources and that further studies should be conducted to address their biodegradation potential as well as if the drinking water treatment processes currently used are effective in removing these organisms and the potential secondary metabolites produced.     

Nitrification of ammonia in wastewater: Field observations and laboratory studies

Complete nitrification of ammonia in partially treated domestic wastewater was demonstrated in a mixed culture chemostat. The optimal detention time of water in the chemostat for maximal nitrification was 6–7 days. In the field, a significant number of nitrifying bacteria was present in a wastewater reservoir, as well as in samples of inflowing water from the preceding anaerobic overloaded oxidation pond.     

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.     

Automatic sampling equipment and bod test nitrification

The influence of automatic sampling equipment on BOD test nitrification in non-nitrified final effluent was evaluated. Samples were tested for BOD, carbonaceous BOD, nitrogenous oxygen demand and concentration of nitrifying bacteria. Biofilms inside the equipment were tested for nitrification potential. A sampler utilizing continuous circulation of final effluent was found to support attached growth of nitrifying bacteria and was associated with relatively high effluent nitrogenous oxygen demand. The effluent nitrogenous oxygen demand and nitrification potential of attached growth were significantly less with a unit that aspirated effluent on an intermittent basis, purging the sample line with air before and after sampling. Peak nitrifier counts in samples from the continuous flow equipment exceeded those in samples from the intermittent flow equipment.     

Nitrogen dynamics and removal in a horizontal flow biofilm reactor for wastewater treatment

A horizontal flow biofilm reactor (HFBR) designed for the treatment of synthetic wastewater (SWW) was studied to examine the spatial distribution and dynamics of nitrogen transformation processes. Detailed analyses of bulk water and biomass samples, giving substrate and proportions of ammonia oxidising bacteria (AOB) and nitrite oxidising bacteria (NOB) gradients in the HFBR, were carried out using chemical analyses, sensor rate measurements and molecular techniques. Based on these results, proposals for the design of HFBR systems are presented.The HFBR comprised a stack of 60 polystyrene sheets with 10-mm deep frustums. SWW was intermittently dosed at two points, Sheets 1 and 38, in a 2 to 1 volume ratio respectively. Removals of 85.7% COD, 97.4% 5-day biochemical oxygen demand (BOD₅) and 61.7% TN were recorded during the study.In the nitrification zones of the HFBR, which were separated by a step-feed zone, little variation in nitrification activity was found, despite decreasing in situ ammonia concentrations. The results further indicate significant simultaneous nitrification and denitrification (SND) activity in the nitrifying zones of the HFBR. Sensor measurements showed a linear increase in potential nitrification rates at temperatures between 7 and 16 °C, and similar rates of nitrification were measured at concentrations between 1 and 20 mg NH₄-N/l. These results can be used to optimise HFBR reactor design. The HFBR technology could provide an alternative, low maintenance, economically efficient system for carbon and nitrogen removal for low flow wastewater discharges.     

A quantitative measure of nitrifying bacterial growth

Nitrifying bacteria convert ammonia (NH3) to nitrate (NO3-) in a nitrification reaction. Methods to quantitatively separate the growth rate of these important bacterial populations from that of the dominant heterotrophic bacteria are important to our understanding of the nitrification process. The changing concentration of ammonia is often used as an indirect measure of nitrification but ammonification processes generate ammonia and confound this approach while heterotrophs remove nitrate via denitrification. Molecular probe methods can tell us what proportion of the microbial community is nitrifying bacteria but not their growth rate. The technique proposed here was able to quantify the growth rate of the nitrifying bacterial populations amidst complex ecological processes. The method incubates [methyl-3H] thymidine with water samples in the presence and absence of an inhibitor of nitrification-thiourea. The radioactively labeled DNA in the growing bacteria was extracted. The rate of incorporation of the label into the dividing bacterial DNA was used to determine bacterial growth rate. Total bacterial community growth rates in full-scale and pilot-scale fixed-film nitrifying reactors and an activated sludge reactor were 2.1 x 10(8), 4.1 x 10(8) and 0.4 x 10(8)cell ml(-1)d(-1), respectively; the growth rate of autotrophic-nitrifying bacteria was 0.7 x 10(8), 2.6 x 10(8) and 0.01 x 10(8)cell ml(-1)d(-1), respectively. Autotrophic-nitrifying bacteria contributed 30% and 60% of the total bacterial community growth rate in the nitrifying reactors whereas only 2% was observed in the activated sludge reactor that was not designed to nitrify. The rates of ammonia loss from the nitrifying reactors corresponded to the rate of growth of the nitrifying bacteria. This method has the potential to more often identify factors that enhance or limit nitrifying processes in both engineered and natural aquatic environments.     

Characterization of nitrifying granules produced in an aerobic upflow fluidized bed reactor

Since nitrification is the rate-determining step in the biological nitrogen removal from wastewater, many research studies have been conducted on the immobilization of nitrifying bacteria. In this research, granulation of nitrifying bacteria in an aerobic upflow fluidized bed (AUFB) reactor in a nitrification process for inorganic wastewater containing 500 g/m³ of NH₄⁺-N was investigated. It was observed that spherical, pseudocubic and elliptical granules with a diameter of 346 μm were produced at the bottom of the reactor after 300 days. Denaturing gradient gel electrophoresis analysis revealed that Nitrosomonas-like bacteria were the dominant ammonia-oxidizing species in the granules. Many colonies of Nitrosomonas-like bacteria were found in the outer part of the granules based on the spatial distribution analysis by fluorescence in situ hybridization. By stepwise reduction of the hydraulic retention time, the ammonia removal rate of the AUFB reactor containing these nitrifying granules finally reached 1.5 kg-N/m³/day. Results suggested that the use of granules realizes the retention of a large amount of nitrifying bacteria in the reactor, which guarantees a highly efficient nitrification.