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.     

Significance of biomass support particles in laboratory studies on microbial degradation of organic chemicals in aquifers

In degradation studies of xenobiotics in groundwater environments from where only water samples can be obtained (e.g. established deep groundwater wells, inhomogeneous formations as boulder aquifers, or consolidated aquifers), solids might be added as biomass support materials. The importance of biomass support materials as quartz sand, rock wool and crushed tiles on the degradation of 8 aromatic hydrocarbons (benzene, toluene, o-xylene, 1,2-dichlorobenzene (1,2-DCB), 1,4-dichlorobenzene (1,4-DCB), naphthalene, biphenyl and nitrobenzene) was investigated in experiments with groundwater collected from two aerobic aquifers (Vejen and Grindsted, Denmark). Experiments with only groundwater as well as groundwater suspensions with aquifer sediment were run as references. It was impossible to adjust pH to the desired level in the experiments with crushed tiles, where also substantial sorption of the test compounds and server clogging of filters used in sampling occurred, and this material was therefore useless as biomass support material. Presence of rock wool supported growth of bacteria and increased the degradation (in terms of rates and number of compounds degraded) compared with experiments with groundwater only. However, the degradation was less and the degradation patterns varied more than in the presence of aquifer sediment. Quartz sand gave the most promising results with respect to growth of bacteria, and the degradation patterns of most of the compounds were similar to those obtained in experiments including aquifer sediments, although the latter showed the most substantial degradation. This study suggests that in case aquifer sediment is not available, quartz sand should be added as biomass support material in studies on degradation of organic xenobiotics in groundwater environments.     

Occurrence of nitrifying bacteria and nitrification in Finnish drinking water distribution systems

Microbiological nitrification process may lead to chemical, microbiological and technical problems in drinking water distribution systems. Nitrification activity is regulated by several physical, and chemical, and operational factors. However, the factors affecting nitrification in the distribution systems in boreal region, having its specific environmental characteristics, are poorly known. We studied the occurrence and activity of nitrifying bacteria in 15 drinking water networks distributing water with very different origin and treatment practices. The waters included chloraminated surface water, chlorinated surface water, and non-disinfected groundwater. The networks were located in eight towns in different parts of Finland. Our results showed that nitrifying bacteria are common in boreal drinking water distribution systems despite their low temperature. Surprisingly high numbers and activities of nitrifiers were detected in pipeline sediment samples. The numbers of ammonia-oxidizing bacteria and their oxidation potentials were highest in chloraminated drinking water delivering networks, whereas the nitrite-oxidizing bacteria were present in the greatest numbers in those networks that used non-disinfected groundwater. The occurrence of nitrifying bacteria in drinking water samples correlated positively with the numbers of heterotrophic bacteria and turbidity, and negatively with the content of total chlorine. Although nitrifying bacteria grew well in drinking water distribution systems, the problems with nitrite accumulation are rare in Finland.     

Are meiofauna transient or resident in sand filters of marine aquariums?

A paradoxical situation was found in the sand filters of a cold marine mesocosm: meiofaunal masses which were large enough to inhibit the mineralization and nitrification processes coexisted with nitrogen cycling bacteria. To test whether the copepod-dominated meiofauna were resident and actively feeding or transient and carried passively through the sand filters, residence times (RTs) were measured for various meiofaunal groups in a newly started filter and in a long established one. Most meiofauna colonized the newly started filter in less than 6 h, but their RTs were less than 24 h. In contrast, RTs were 147d for halacarids, 291 d for harpacticoid copepods and 1228d for nematodes in the long established filter. Mesocosm periphyton. which occupied a large fraction of the mesocosm surface area and was characterized by high meiofaunal densities, was probably the main source of meiofauna in the sand filters. Pool sediments, consisting of gravel or sand, were second to periphyton and contributed hydrozoans and mesopsammic species to the filters. The small copepod Pseudonychocamptus proximus progressively replaced the large Tisbe furcata in sand filters during the fall of 1995 and was responsible for the large increase in meiofaunal biomass observed after spring 1996. This replacement was presumably facilitated by the copepod size selection process operated by the filters. Large copepods were retained by the surface layer of sand or brought up by the backwash water and then exit the mesocosm through the drain. High meiofaunal populations did not significantly affect nitrogen cycling bacteria in sand filters probably because meiofauna also fed on other abundant food sources which were carried in by the water flow.     

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.     

Biological iron oxidation by Gallionella spp. in drinking water production under fully aerated conditions

Iron oxidation under neutral conditions (pH 6.5-8) may be a homo- or heterogeneous chemically- or a biologically-mediated process. The chemical oxidation is supposed to outpace the biological process under slightly alkaline conditions (pH 7-8). The iron oxidation kinetics and growth of Gallionella spp. - obligatory chemolithotrophic iron oxidizers - were assessed in natural, organic carbon-containing water, in continuous lab-scale reactors and full-scale groundwater trickling filters in the Netherlands. From Gallionella cell numbers determined by qPCR, balances were made for all systems. The homogeneous chemical iron oxidation occurred in accordance with the literature, but was retarded by a low water temperature (13 °C). The contribution of the heterogeneous chemical oxidation was, despite the presence of freshly formed iron oxyhydroxides, much lower than in previous studies in ultrapure water. This could be caused by the adsorption of natural organic matter (NOM) on the iron oxide surfaces. In the oxygen-saturated natural water with a pH ranging from 6.5 to 7.7, Gallionella spp. grew uninhibited and biological iron oxidation was an important, and probably the dominant, process. Gallionella growth was not even inhibited in a full-scale filter after plate aeration. From this we conclude that Gallionella spp. can grow under neutral pH and fully aerated conditions when the chemical iron oxidation is retarded by low water temperature and inhibition of the autocatalytic iron oxidation.     

Accumulation and fate of green fluorescent labeled Escherichia coli in laboratory-scale drinking water biofilters

Biological filters combining microbial activity and rapid sand filtration are used in drinking water treatment plants for enhanced biodegradable organic matters (BOM) removal. Biofilms formed on filter media comprised of bacteria enclosed in a polymeric matrix are responsible for the adsorption of BOM and attachment of planktonic microorganisms. This study investigated the removal of Escherichia coli cells injected into laboratory-scale biofilters and the role of biofilm in retaining the injected E. coli. Green fluorescent protein was used as a specific marker to detect and quantify E. coli in the biofilms. About 35% of the total injected E. coli cells were observed in the filter effluents, when initial cell concentrations were measured at 7.4 x 10(6) CFU/mL and 1.6 x 10(7) CFU/mL in two separate experiments. The results from real-time PCR and plate count analysis indicated that 95% of the E. coli retained inside the filters were either non-viable or could not be recovered by colony counting techniques. Injected cells were unevenly distributed inside the filter with more than 70% located at the top 1/5 of the filter. Images obtained from an epifluorescent microscope showed that E. coli cells were embedded inside the biofilm matrix and presented mainly as microcolonies intertwined with other microorganisms, which was consistent with findings from standard plate count methods and qPCR.     

Effect of plants and filter materials on bacteria removal in pilot-scale constructed wetlands

Due to the lack of testing units or appropriate experimental approaches, only little is known about the removal of bacteria in constructed wetlands. However, improved performance in terms of water sanitation requires a detailed understanding of the ongoing processes. Therefore, we analyzed the microbial diversity and the survival of Enterobacteriaceae in six pilot-scale constructed wetland systems treating domestic wastewater: two vertical sand filters, two vertical expanded clay filters and two horizontal sand filters (each planted and unplanted). Samples were taken from the in- and outflow, from the rhizosphere, and from the bulk soil at various depths. Colony-forming units of heterotrophic bacteria and coliforms were analyzed and the removal of bacteria between the in- and outflow was determined to within 1.5-2.5 orders of magnitude. To access the taxon-specific biodiversity of potential pathogens in the filters and to reduce the complexity of the analysis, specific primers for Enterobacteriaceae were developed. While performing PCR-SSCP analyses, a pronounced decrease in diversity from the inflow to the outflow of treated wastewater was observed. No differences were observed between the bulk soil of planted and unplanted vertical filters. Some bands appeared in the rhizosphere that were not present in the bulk soil, indicating the development of specific communities stimulated by the plants. The fingerprinting of the rhizosphere of plants grown on sand or expanded clay exhibited many differences, which show that different microbial communities exist depending on the soil type of the filters. The use of the taxon-specific primers enabled us to evaluate the fate of the Enterobacteriaceae entering the wetlands and to localize harboring in the rhizosphere. The most abundant bands of the profiles were sequenced: Pantoea agglomerans was found in nearly all samples from the soil but not in the effluent, whereas Citrobacter sp. could not be removed by the horizontal unplanted sand and vertical planted expanded clay filters. These results show that the community in wetland system is strongly influenced by the filtration process, the filter material and the plants.     

Bacterial degradation of microcystin toxins within a biologically active sand filter

Microcystin toxins are a problem for water authorities as they are recalcitrant to conventional water treatment. In this study, biological sand filtration was assessed in laboratory column experiments for its ability to remove two microcystin analogues, microcystin-LR and microcystin-LA. A lag period of 3 days was evident prior to the commencement of degradation. Contact times were varied during the experiment; however, no microcystin was detected in the effluent after 4 days, even under conditions similar to those of a rapid sand filter. Removals of microcystin through the sand filters were shown to be primarily through biological degradation processes. Using polymerase chain reaction (PCR), biofilm, extracted from one of the sand filters that had effectively removed the microcystins, was shown to contain bacteria with the mlrA gene. Detection of this gene provided additional evidence that biological degradation of microcystin was the primary removal mechanism.     

Extracellular enzyme activities during slow sand filtration in a water recharge plant

Activities of the extracellular enzymes beta-glucosidase and phosphatase and bacterial densities were investigated during the filtration process at several sites in a groundwater recharge plant at the Ruhr river (Hengsen recharge plant in Schwerte. Germany). Low numbers of microorganisms and low levels of activity in this type of habitat, compared to most surface waters, caused methodological problems when determining microbial activity. In this study, fluorigenic model substrates, which enable hydrolytic rates as low as 1 nmol (L x h)(-1) to be measured, were used to determine extracellular enzyme activities. Highest activities were determined in surface water (107 nmol (L x h)(-1) for beta-glucosidase and 252 nmol (L x h)(-1) for phosphatase). which decreased during the filtration process in the gravel prefilter and the main sand filter until the end of subsurface flow (1.6 nmol (L x h)(-1) and 6.8 nmol (L x h)(-1), respectively). Similarly, bacterial numbers decreased from 3.4 x 10(6) to 0.29 x 10(6) cells mL(-1). These data showed that microbial activity within the prefilter and the shallow layers of the sand filter had the greatest impact on water quality. In addition to its involvement in the continuous purification of surface water, the microbial community in the sand filter probably acts as a biological buffer against ephemeral increases in the loads of organic matter and nutrients in the recharge plant.     

Biofiltration for removal of BOM and residual ammonia following control of bromate formation

Nitrification was developed within a biological filter to simultaneously remove biodegradable organic matter (BOM) and residual ammonia added to control bromate formation during the ozonation of drinking water. Testing was performed at pilot-scale using three filters containing sand and anthracite filter media. BOM formed during ozonation (e.g., assimilable organic carbon (396-572 microg/L), formaldehyde (11-20 microg/L), and oxalate (83-145 microg/L)) was up to 70% removed through biofiltration. Dechlorinated backwash water was required to develop the nitrifying bacteria needed to convert the residual ammonia (0.1-0.5 mg/L NH(3)-N) to nitrite and then to nitrate. Chlorinated backwash water resulted in biofiltration without nitrification. Deep-bed filtration (empty-bed contact time (EBCT) = 8.3 min) did not enhance the development of nitrification when compared with shallow-bed filtration (EBCT = 3.2 min). Variable filtration rates between 4.8 and 14.6 m/h (2 and 6 gpm/sf) had minimal impact on BOM removal. However, conversion of ammonia to nitrite was reduced by 60% when increasing the filtration rate from 4.8 to 14.6 m/h. The results provide drinking water utilities practicing ozonation with a cost-effective alternative to remove the residual ammonia added for bromate control.     

Effect of pumping on the spatio-temporal distribution of microbial communities in a water well field

A water well field adjacent to the North Saskatchewan River (City of North Battleford, Saskatchewan, Canada) with a history of rapid deterioration of both well water quality and yield was selected to study the spatial and temporal distribution of subsurface microbial communities and their response to water pumping. A range of conventional cultural, microscopic and molecular techniques, including confocal laser scanning microscopy (CLSM), Biolog, qPCR and Denaturing Gradient Gel Electrophoresis (DGGE), was used during this study. Redox data and water and sediment chemistry showed that the aquifer was anoxic and harbored substantial amounts of Fe and Mn. CLSM analyses of incubated coupons indicated extensive biofilm growth in the zone immediately surrounding the well and was coincident with reduced water well yield. PCR screening and qPCR analyses showed that the potential for iron- and sulfate-reducing activity increased with proximity to the well. Bacterial communities inhabiting the zone closest to the well showed the greatest changes and differences in metabolic activities and composition as revealed by PCA (Principal Components Analysis) of the Biolog and DGGE data. The sequence analysis of all the samples revealed that Sulfuricurvum spp., Methylobacter spp., Geobacter spp. and Rhodobacter spp. were most commonly detected in this aquifer. Overall the findings demonstrated that the microbial numbers, metabolic activities and the community composition changed in response to water pumping but effects did not extend beyond 1-2 m zone from the well.     

Sand creep as a factor in land subsidence during groundwater level recovery in the southern Yangtze River delta, China

A number of areas worldwide are suffering from the land subsidence, including the southern Yangtze River delta, the most developed area in China. Land subsidence is mainly induced by excessive groundwater pumping and has caused numerous problems, for example, flooding, structural cracking, and ground fissuring. Although several countermeasures have been adopted to mitigate the land subsidence problem in the southern Yangtze River delta, commonly using groundwater recharge, land subsidence is still developing even when groundwater levels are rising. The observation data of land subsidence in each stratum show that the deformation of the pumped aquifer is even greater than that in the adjacent aquitards when groundwater levels are recovering. Laboratory test results on the aquifer sand in Shanghai and Changzhou (two cities in the studied area, which have the most sufficient observation data about the land subsidence and groundwater level developing with time) proved that sand creep deformation is significant and is partially responsible for the land subsidence without groundwater level drawdown. Considering the difficulty and cost of collecting borehole samples from deep pumped aquifers to obtain the essential parameters for the existing sand creep calculation models, a simplified method was then proposed to calculate the sand creep deformation in the pumped aquifer. The relationship between the sand creep rate and the time can be recognized as linear using double logarithmic coordinates and the slope can be assumed to be one. In the proposed method, the laboratory test data are not necessary. Finally, case histories from Shanghai and Changzhou were used to verify the effectiveness of the proposed method.     

Effect of synthetic iron colloids on the microbiological NH(4)(+) removal process during groundwater purification

Subsurface aeration is used to oxidise Fe in situ in groundwater that is used to make drinking water potable. In a groundwater system with pH>7 subsurface aeration results in non-mobile Fe precipitate and mobile Fe colloids. Since originally the goal of subsurface aeration is to remove iron in situ, the formation of non-mobile iron precipitate, which facilitates the metal's removal, is the desired result. In addition to this intended effect, subsurface aeration may also strongly enhance the microbiological removal of ammonium (NH(4)(+)) in the purification station. Mobile iron colloids could be the link between subsurface aeration and the positive effect on the NH(4)(+) removal process. Therefore, the objective of this study was to assess whether synthetic iron colloids could improve the NH(4)(+) removal process. The effect of synthetic iron colloids on the NH(4)(+) removal process was studied using an artificial purification set-up on a laboratory scale. Columns that purified groundwater with or without added synthetic iron colloids were set up in duplicate. The results showed that the NH(4)(+) removal was significantly ( alpha = 0.05 ) increased in columns treated with the synthetic iron colloids. Cumulative after 4 months about 10% more NH(4)(+) was nitrified in the columns that was treated with the groundwater containing synthetic iron colloids. The results support the hypothesis that mobile iron colloids could be the link between subsurface aeration and the positive effect on the NH(4)(+) removal process.     

Estimating nitrifying biomass in drinking water filters for surface water treatment

The objective of this work is to estimate active nitrifying biomass and its main influencing factors in low-loaded biofilters based on operational data. An analytical approach based on balancing growth, decay and biomass removed by backwashing is proposed. The method is developed and applied in pilot-scale rapid sand filters for drinking water treatment. Decay rate was measured directly in the filter for different temperatures. To assess the amount of active biomass in backwash water, a technique based on respiration measurements was used. Backwash losses increased overproportional with balanced biomass in the filter. The impact of both parameters on active biomass is quantified exemplarily for a given constant nitrification rate.     

黄河下游悬河段傍河开采地下水的试验研究——以郑州市区黄河滩地为例

黄河下游频频出现的断流现象给沿岸城市的供水造成严重威胁 ,本文根据近年在郑州市北郊黄河滩地供水水文地质勘探中 ,几组大型抽水试验所获得的地质信息 ,论证了傍河地带的地下水资源潜力 ;细颗粒含水地层中的成井技术 ;傍河开采地下水可能诱发的水环境和地质环境问题。文章的结论是 :黄河下游沿岸地带 ,地下水资源十分丰富 ,大规模开采傍河地带的地下水所诱发的环境负效应十分有限 ,具有建立傍河地下水源地的优越条件。     

Evaluation of quantitative real time PCR for the measurement of Helicobacter pylori at low concentrations in drinking water

A rapid DNA extraction and quantitative, real time polymerase chain reaction (QRTPCR) analysis method targeting the ureA gene of Helicobacter pylori was evaluated for the measurement of these organisms on membrane filters at levels that might be expected to be found in drinking water samples. No interference was seen from high levels of background organisms and related, non-target species were detected at approximately 4-5 log(10) lower levels of sensitivity than H. pylori by this assay. A standard curve was generated for the method from analyses of filters containing known numbers of added H. pylori cells. Cell numbers on these filters were determined by staining with a species-specific fluorescent antibody and solid phase cytometry analyses. The mean detection sensitivity of the method was 10 H. pylori cells per filter with a 95% confidence sensitivity of 40 cells and a 95% confidence precision interval of +/-0.57 log(10) based on duplicate analyses of the samples. One liter drinking water samples from several locations in the US were inoculated with the same H. pylori cell suspensions used to generate the standard curve and gave measurements that were consistent with the standard curve suggesting that these sample matrices produced no interference in the method. This method may be useful for the rapid screening of drinking water for H. pylori.     

BIOFILM FORMATION IN GRANULAR-BED FILTERS

A high microbiological quality of drinking water must be ensured to protect public health. The filtration techniques that are used in treating drinking water play an important role; however, a biofilm can form on granular-media filters and the accumulated bacteria can slough off and enter the filtered water.
The aim of this research was to examine (a) the potential for biofilm formation and detachment from filter sand, and (b) the effect of different backwash regimes on biofilm removal. During the operation of the filter, bacteria became attached to the sand media, particularly in the top 30 mm of the filter bed. A water-only backwash at 20% and 40% bed expansion demonstrated poor removal of biofilm throughout the depth of the bed. Collapse-pulsing is a more efficient method and results in a reduction in the number of bacteria in the filtered water.     

Molecular comparison of the sampling efficiency of four types of airborne bacterial samplers

In the present study, indoor and outdoor air samples were collected using four types of air samplers often used for airborne bacterial sampling. These air samplers included two solid impactors (BioStage and RCS), one liquid impinger (BioSampler), and one filter sampler with two kinds of filters (a gelatin and a cellulose acetate filter). The collected air samples were further processed to analyze the diversity and abundance of culturable bacteria and total bacteria through standard culture techniques, denaturing gradient gel electrophoresis (DGGE) fingerprinting and quantitative polymerase chain reaction (qPCR) analysis.The DGGE analysis indicated that the air samples collected using the BioStage and RCS samplers have higher culturable bacterial diversity, whereas the samples collected using the BioSampler and the cellulose acetate filter sampler have higher total bacterial diversity. To obtain more information on the sampled bacteria, some gel bands were excised and sequenced. In terms of sampling efficiency, results from the qPCR tests indicated that the collected total bacterial concentration was higher in samples collected using the BioSampler and the cellulose acetate filter sampler.In conclusion, the sampling bias and efficiency of four kinds of air sampling systems were compared in the present study and the two solid impactors were concluded to be comparatively efficient for culturable bacterial sampling, whereas the liquid impactor and the cellulose acetate filter sampler were efficient for total bacterial sampling.     

Organic carbon removal and nitrification of high strength wastewaters using stratified sand filters

The current practice of spray irrigation of dairy parlour wastewaters is laborious and time consuming. Intermittent sand filtration systems may offer an alternative to spray irrigation when designed to remove organic carbon, nitrogen, phosphorus, coliforms and viruses from such wastewaters to allow discharge of the final effluent directly into receiving waters without damage to the environment. In this study two instrumented stratified sand filter columns (0.425 and 0.9 m deep, and both 0.3 m in diameter) were intermittently loaded for 439 days with synthetic dairy parlour washings at a number of hydraulic and organic loading rates. At a biochemical oxygen demand (BOD) loading of 22 g m(-2) d(-1), over 92% of the BOD and suspended solids in the wastewater was removed in the two filters and nitrification was complete. The 0.9 m column had a sustained ability to adsorb the influent phosphorus during the study period; however, the phosphorus adsorption capacity of the 0.425 m column began to decrease after approximately 30 days. Biomass, comprising hydrated extracellular polymers (exopolymers) and living and dead cells, accumulated in the 0.9 m column; it was assessed by sodium bromide tracer studies and by variations in the sand volumetric water contents using time domain reflectometry (TDR). The biomass growth increased the retention time of the wastewater in the filter media, and occurred mainly at the top of the first sand layer. Intermittent stratified sand filters appear to offer an effective and sustainable treatment process for the removal of BOD from high-strength wastewaters, and for the complete nitrification of ammonium.