Assessing groundwater denitrification spatially is the key to targeted agricultural nitrogen regulation.

Globally, food production for an ever-growing population is a well-known threat to the environment due to losses of excess reactive nitrogen (N) from agriculture. Since the 1980s, many countries of the Global North, such as Denmark, have successfully combatted N pollution in the aquatic environment by regulation and introduction of national agricultural one-size-fits-all mitigation measures. Despite this success, further reduction of the N load is required to meet the EU water directives demands, and implementation of additional targeted N regulation of agriculture has scientifically and politically been found to be a way forward. In this paper, we present a comprehensive concept to make future targeted N regulation successful environmentally and economically. The concept focus is on how and where to establish detailed maps of the groundwater denitrification potential (N retention) in areas, such as Denmark, covered by Quaternary deposits. Quaternary deposits are abundant in many parts of the world, and often feature very complex geological and geochemical architectures. We show that this subsurface complexity results in large local differences in groundwater N retention. Prioritization of the most complex areas for implementation of the new concept can be a cost-efficient way to achieve lower N impact on the aquatic environment.

Linking denitrification and pesticide transformation potentials with community ecology and groundwater discharge in hyporheic sediments in a lowland stream.

Contamination of rivers by nitrate and pesticides poses a risk for aquatic ecosystems in lowland catchments that are often intensively used for agriculture. Here, the hyporheic zone, the streambed underneath the stream, plays a vital role due to its efficient self-purification capacity. The present study aims to evaluate the denitrification and transformation potential of 14 pesticides and three transformation products in the hyporheic sediment from a lowland stream with a high N load and by comparing an agricultural straightened section to a natural meandering part of the stream influenced by different groundwater discharges. Batch experiments were set up to evaluate the denitrification and pesticide transformation potentials in hyporheic sediment from two depths (5-15 cm (a) and 15-25 cm (b)). Our results revealed that (i) differences between the agricultural and natural sections of the river did not influence pollutant attenuation, (ii) both the nitrate and pesticide attenuation processes were more rapid in the upper "a" layer compared to the "b" layer due to higher microbial abundance, (iii) high groundwater discharge reduced the denitrification potential while pesticide transformation was unaffected, (iv) denitrification correlated with denitrifier abundance (nirK) in the "b" layer, while this correlation was not seen in the "a" layer, and (v) a microbial community with low diversity can explain limited transformation for the majority of tested pesticides. Overall, our results suggest that high groundwater discharge zones with reduced residence time in the hyporheic zone can be an important source of pesticides and nitrate to surface water.

N-Map: High-resolution groundwater N-retention mapping and modelling by integration of geophysical, geological, geochemical, and hydrological data.

A key aspect of protecting aquatic ecosystems from agricultural nitrogen (N) is to locate (i) farmlands where nitrate leaches from the bottom of the root zone and (ii) denitrifying zones in the aquifers where nitrate is removed before entering the surface water (N-retention). N-retention affects the choice of field mitigation measures to reduce delivered N to surface water. Farmland parcels associated with high N-retention gives the lowest impact of the targeted field measures and vice versa. In Denmark, a targeted N-regulation approach is currently implemented on small catchment scale (approx. 15 km2). Although this regulatory scale is much more detailed than what has been used previously, it is still so large that regulation for most individual fields will be either over- or under-regulated due to large spatial variation in the N-retention. The potential cost reduction for farmers is of up to 20-30% from detailed retention mapping at the field scale compared to the current small catchment scale. In this study, we present a mapping framework (N-Map) for differentiating farmland according to their N-retention, which can be used for improving the effectiveness of targeted N-regulation. The framework currently only includes N-retention in the groundwater. The framework benefits from the incorporation of innovative geophysics in hydrogeological and geochemical mapping and modelling. To capture and describe relevant uncertainties a large number of equally probable realizations are created through Multiple Point Statistical (MPS) methods. This allows relevant descriptions of uncertainties of parts of the model structure and includes other relevant uncertainty measures that affects the obtained N-retention. The output is data-driven high-resolution groundwater N-retention maps, to be used by the individual farmers to manage their cropping systems due to the given regulatory boundary conditions. The detailed mapping allows farmers to use this information in the farm planning in order to optimize the use of field measures to reduce delivered agricultural N to the surface water and thereby lower the costs of the field measures. From farmer interviews, however, it is clear that not all farms will have an economic gain from the detailed mapping as the mapping costs will exceed the potential economic gains for the farmers. The costs of N-Map is here estimated to 5-7 €/ha/year plus implementation costs at the farm. At the society level, the N-retention maps allow authorities to point out opportunities for a more targeted implementation of field measures to efficiently reduce the delivered N-load to surface waters.

Biodegradation of trace sulfonamide antibiotics accelerated by substrates across oxic to anoxic conditions during column infiltration experiments.

Frequent occurrence of trace organic contaminants in aquatic environments, such as sulfonamide antibiotics in rivers receiving reclaimed water, is concerning. Natural attenuation by soil and sediment is increasingly relied upon. In the case of riverbank filtration for water purification, the reliability of antibiotic attenuation has been called into question due to incomplete understanding of their degradation processes. This study investigated influence of substrates and redox evolution along infiltration path on biotransformation of sulfonamides. Eight sand columns (length: 28 cm) with a riverbed sediment layer at 3-8 cm were fed by groundwater-sourced tap water spiked with 1 µg/L of sulfadiazine (SDZ), sulfamethazine (SMZ), and sulfamethoxazole (SMX) each, with or without amendments of dissolved organic carbon (5 mg-C/L of 1:1 yeast and humics) or ammonium (5 mg-N/L). Two flow rates were tested over 120 days (0.5 mL/min and 0.1 mL/min). Iron-reducing conditions persisted in all columns for 27 days during the initial high flow period due to respiration of sediment organics, evolving to less reducing conditions until the subsequent low flow period to resume more reducing conditions. With surplus substrates, the spatial and temporal patterns of redox conditions differentiated among columns. The removal of SDZ and SMZ in effluents was usually low (15 ± 11%) even with carbon addition (14 ± 9%), increasing to 33 ± 23% with ammonium addition. By contrast, SMX removal was higher and more consistent among columns (46 ± 21%), with the maximum of 64 ± 9% under iron-reducing conditions. When sulfonamide removal was compared between columns for the same redox zones during infiltration, their enhancements were always associated with the availability of dissolved or particulate substrates, suggesting co-metabolism. Manipulation of the exposure time to optimal redox conditions with substrate amendments, rather than to simply prolong the overall residence time, is recommended for nature-based solutions to tackle target antibiotics.

Degradation potential of MCPA, metolachlor and propiconazole in the hyporheic sediments of an agriculturally impacted river.

Hyporheic sediments are influenced by physical, biological, and chemical processes due to the interactions with river water and has been shown to play an important role in the environmental fate of pesticides. Therefore, this study evaluated the bacterial degradation potential of MCPA, metolachlor and propiconazole in hyporheic sediments sampled along a 20 km long stretch of an agriculturally impacted river dominated primarily by water losing conditions. Water physicochemical parameters in the river and nearby groundwater wells were assessed along with pesticide sorption to sediments and bacterial community composition. Degradation and mineralisation batch experiments were set up from six locations (five water losing, one water gaining) using environmentally relevant concentrations of pesticides (10 µg kg-1). Highly variable DT50 values from 11 to 44 days for MCPA, 11-27 days for metolachlor (MTC) and 60-147 days for propiconazole were calculated based on ~140 day studies. Degradation of MTC led to accumulation of the transformation products MOA and MESA in batch experiments. Noteworthy, MESA was detected in the groundwater wells adjacent to the part of the river impacted by losing conditions suggesting that degradation processes in hyporheic sediments may lead to the formation of transformation products (TP) leaching towards groundwater. Further, from propiconazole was identified a persistent transformation product being different from 1,2,4-triazole. Specific calculated DT50 values could not the linked to bacterial diversity. However, generally all sediment samples were characterised by high bacterial diversity, where approximately 80% of the relative sequence abundances were < 1%, which may increase the likelihood of finding contaminant-degrading genes, thereby explaining the general high contaminant-degrading activity. The studied sediments revealed a high potential to degrade pesticides despite only being exposed to low diffuse pollutant concentrations that is similar to calculated DT50 values in agricultural soils.

Combining reverse osmosis and microbial degradation for remediation of drinking water contaminated with recalcitrant pesticide residue.

Groundwater contamination by recalcitrant organic micropollutants such as pesticide residues poses a great threat to the quality of drinking water. One way to remediate drinking water containing micropollutants is to bioaugment with specific pollutant degrading bacteria. Previous attempts to augment sand filters with the 2,6-dichlorobenzamide (BAM) degrading bacterium Aminobacter niigataensis MSH1 to remediate BAM-polluted drinking water initially worked well, but the efficiency rapidly decreased due to loss of degrader bacteria. Here, we use pilot-scale augmented sand filters to treat retentate of reverse osmosis treatment, thus increasing residence time in the biofilters and potentially nutrient availability. In a first pilot-scale experiment, BAM and most of the measured nutrients were concentrated 5-10 times in the retentate. This did not adversely affect the abundances of inoculated bacteria and the general prokaryotic community of the sand filter presented only minor differences. On the other hand, the high degradation activity was not prolonged compared to the filter receiving non-concentrated water at the same residence time. Using laboratory columns, it was shown that efficient BAM degradation could be achieved for >100 days by increasing the residence time in the sand filter. A slower flow may have practical implications for the treatment of large volumes of water, however this can be circumvented when treating only the retentate water equalling 10-15% of the volume of inlet water. We therefore conducted a second pilot-scale experiment with two inoculated sand filters receiving membrane retentate operated with different residence times (22 versus 133 min) for 65 days. While the number of MSH1 in the biofilters was not affected, the effect on degradation was significant. In the filter with short residence time, BAM degradation decreased from 86% to a stable level of 10-30% degradation within the first two weeks. The filter with the long residence time initially showed >97% BAM degradation, which only slightly decreased with time (88% at day 65). Our study demonstrates the advantage of combining membrane filtration with bioaugmented filters in cases where flow rate is of high importance.

Upscaling of Denitrification Rates from Point to Catchment Scales for Modeling of Nitrate Transport and Retention.

The spatial and temporal variability of denitrification makes it challenging to integrate conceptual, process-based understandings of nitrate transport and retention into numerical modeling at the catchment scale, although it is critical for the realism and predictive power of the model. In this study, we propose a novel approach where the conceptual understandings of the spatial structure of denitrification zones and the corresponding representative denitrification rates are transformed into a form that can be integrated into a multi-point statistical simulation framework. This is done by constructing a denitrification training image (TI) coupled to a geophysically based TI of the hydrogeological structure. The field observations and laboratory analyses of denitrification rates and the chemistry of water and sediment revealed that the study catchment's subsurface can be characterized by three zones: (1) the oxic zone with no nitrate reduction; (2) the slow-denitrification zone (mean of ln-transformed rate = -1.19 ± 0.52 mg N L-1 yr-1); and (3) the high-denitrification zone (mean of ln-transformed rate = 3.86 ± 1.96 mg N L-1 yr-1). The underlying controls on the spatial distribution of these zones and the representativeness of denitrification rates were investigated. Then, a TI illustrating the subsurface structure of the denitrification zone was constructed by synthesizing the results of these geochemical interpretations and the hydrogeology TI.

Redox-dependent biotransformation of sulfonamide antibiotics exceeds sorption and mineralization: Evidence from incubation of sediments from a reclaimed water-affected river.

Trace levels of sulfonamide antibiotics are ubiquitous in reclaimed water, yet environmental pathways to completely remove those chemicals are not well understood when such water is used to restore flows in dried rivers. This study investigated sulfonamide sorption-desorption, biodegradation, and mineralization processes with seven sediments from a reclaimed water-dominant river. Batch experiments were conducted under oxic and anoxic (nitrate-reducing) conditions, and each removal process of sulfamethazine, sulfadiazine, and sulfamethoxazole (SMX) was evaluated individually at environmentally relevant concentrations (≤ 10 µg/L). Over 28 days, 44 ± 32% of sulfonamides were biodegraded, while the full mineralization to carbon dioxide was < 1%. Around 5% of sulfonamides were removed via sediment sorption, with a positive correlation with sediment organic contents. Detailed investigation of SMX biodegradation revealed that although its transformation appeared to be faster in anoxic than oxic tests by day 2, it reversed over 28 days with a longer apparent half-life in anoxic tests (69 ± 25 days) than that in oxic tests (12 ± 11 days). This is attributed to the formation of reversible metabolites at denitrifying conditions, such as DesAmino-SMX of which the production was affected by nitrite concentrations. Despite measurements of three frequently reported metabolites, > 70% biotransformation products remained unknown in this study. The findings highlight the persistency of sulfonamides and their derivatives, with research needed to further elucidate degradation mechanisms and to perform risk assessment of reclaimed water reuse.

The complete genome of 2,6-dichlorobenzamide (BAM) degrader Aminobacter sp. MSH1 suggests a polyploid chromosome, phylogenetic reassignment, and functions of plasmids.

Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.

Combined removal of organic micropollutants and ammonium in reactive barriers developed for managed aquifer recharge.

Groundwater is an important drinking water resource. To ensure clean drinking water, managed aquifer recharge (MAR) could be an attractive solution when recharging with treated wastewater. The installation of reactive barriers, e.g. with compost or other organic materials at MAR facilities, may improve pollutant removal. To link pollutant transformation processes and microbiology in reactive barriers, we simulated infiltration through different sand-compost mixtures using laboratory columns with depth-specific sampling of water and barrier material. We also evaluated the effect of inoculation with activated sludge. Our focus was on the simultaneous removal of organic micropollutants and nitrogen species, with parallel monitoring of the development of microbial communities. During 17 weeks of operation, the columns were fed with synthetic wastewater containing five organic micropollutants (1-2 µg/L each) and ammonium (2 mg N/L). Unique communities developed in the columns in relation to barrier material, with high effects of compost addition and minor effect of inoculation. Removal of the micropollutant paracetamol (acetaminophen) occurred in all columns, while sulfamethoxazole was only removed in columns with 50% compost. By contrast, limited removal was observed for sulfadiazine, carbamazepine and diuron, with the latter two displaying transient removal, attributed sorption. Oxygen was depleted within the top few cm of the columns when compost was present, but this was sufficient to remove all ammonium through nitrification. The fate of accumulated nitrate at deeper layers depended on the fraction of compost, with more compost leading to removal of nitrate by denitrification, but also by dissimilatory nitrate reduction to ammonium, hampering the overall nitrogen removal efficiency. Introducing compost as reactive barrier in MAR facilities has a large effect on the microbial communities and processes, but whether it will provide overall cleaner water to the underlying aquifer is uncertain and will depend very much on the type of pollutant.

History and Sources of Co-Occurring Pesticides in an Abstraction Well Unraveled by Age Distributions of Depth-Specific Groundwater Samples.

When groundwater-based drinking water supply becomes contaminated, the timing and source of contamination are obvious questions. However, contaminants often have diffuse sources and different contaminants may have different sources even in a single groundwater well, making these questions complicated to answer. Age dating of groundwater has been used to reconstruct contaminant travel times to wells; however, critics have highlighted that groundwater flow is often complex with mixing of groundwater of different ages. In drinking water wells, where water is typically abstracted from a large depth interval, such mixing is even more problematic. We present a way to overcome some of the obstacles in identifying the source and age of contaminants in drinking water wells by combining depth-specific sampling with age tracer modeling, particle tracking simulations, geological characterization, and contaminant properties. This multitool approach was applied to a drinking water well, where bentazon and dichlorprop contamination was found to have different pollutant sources and release histories, even though both pesticides can be associated with the same land use. Bentazon was derived from recent application to a golf course, while dichlorprop was derived from agricultural use more than 30 years ago. The advantages, limitations, and pitfalls of the proposed course of action are then further discussed.

A novel hybrid concept for implementation in drinking water treatment targets micropollutant removal by combining membrane filtration with biodegradation.

Groundwater extracted for drinking water production is commonly treated by aeration and sand filtration. However, this simple treatment is typically unable to remove pesticide residues. As a solution, bioaugmentation of sand filter units (i.e., the addition of specific degrader strains) has been proposed as an alternative "green" technology for targeted pesticide removal. However, the introduced degraders are challenged by (i) micropollutant levels of target residue, (ii) the oligotrophic environment and (iii) competition and predation by the native microorganisms, leading to loss of population and degradation potential. To overcome these challenges, we propose the introduction of a novel hybrid treatment step to the overall treatment process in which reverse osmosis filtration and biodegradation are combined to remove a target micropollutant. Here, the reverse osmosis produces a concentrated retentate that will act as a feed to a dedicated biofilter unit, intended to promote biodegradation potential and stability of an introduced degrader. Subsequently, the purified retentate will be re-mixed with the permeate from reverse osmosis, for re-mineralization and downstream consumption. In our study, we investigated the effect of reverse osmosis retentates on the degradation potential of an introduced degrader. This paper provides the first promising results of this hybrid concept using the 2,6-dichlorobenzamide (BAM)-degrading bacteria Aminobacter sp. MSH1 in batch experiments, spiked with radiolabeled BAM. The results showed an increased degradation potential of MSH1 in retentate waters versus untreated water. Colony-forming units and qPCR showed a stable MSH1 population, despite higher concentrations of salts and metals, and increased growth of native bacteria.

3D characterization of the subsurface redox architecture in complex geological settings.

Nitrogen (N) leaching caused by agricultural activities is one of the major threats to the aquatic ecosystems and public health. Moving from the agricultural soils through the subsurface and reemerging to the surface water, N undergoes various biogeochemical reactions along pathways in the subsurface, which occur heterogeneously in space and time. Thus to improve our understanding on the fate and distribution of N in the aquatic environment, detailed knowledge about the subsurface hydrogeological and biogeochemical conditions, especially the redox conditions, are essential. In this study, 3D information of the redox conditions termed the redox architecture was investigated in two Danish catchments with intensive agriculture underlain by glacial deposits. Towed transient electromagnetic (tTEM) resistivity was interpreted which reveals the subsurface geological structures at a few hectare scale. These geophysical data were integrated with sediment and water chemistry for the redox architecture interpretations. The top soils of both catchments are characterized as clay-till, but the tTEM showed that the subsurface hydrogeological structures are distinctively different. We identified three types of redox architectures in the studied catchments: 1) a planar redox architecture with a single redox interface; 2) a geological-window redox architecture with local complexity; and 3) a glaciotectonic-thrusted redox architecture with high complexity. The baseflow N load at the catchment outlets reflect the contributions of N via oxic pathways through the complex redox architectures of the subsurface. We conclude that in some landscapes, the redox architecture cannot be simplified as a single interface that roughly follows the terrain; hence, thorough investigations of the structural heterogeneity of the local redox architectures will be necessary to improve simulations of N evolution along pathways and quantifications of N attenuation under various mitigation scenarios.

Successional trophic complexity and biogeographical structure of eukaryotic communities in waterworks' rapid sand filters.

As groundwater-fed waterworks clean their raw inlet water with sand filters, a variety of pro- and eukaryotic microbial communities develop on these filters. While several studies have targeted the prokaryotic sand filter communities, little is known about the eukaryotic communities, despite the obvious need for knowledge of microorganisms that get in contact with human drinking water. With a new general eukaryotic primer set (18S, V1-V3 region), we performed FLX-454 sequencing of material from 21 waterworks' sand filters varying in age (3-40 years) and geographical location on a 250 km east-west axis in Denmark, and put the data in context of their previously published prokaryotic communities. We find that filters vary highly in trophic complexity depending on age, from simple systems with bacteria and protozoa (3-6 years) to complex, mature systems with nematodes, rotifers and turbellarians as apex predators (40 years). Unlike the bacterial communities, the eukaryotic communities display a clear distance-decay relationship that predominates over environmental variations, indicating that the underlying aquifers feeding the filters harbor distinct eukaryotic communities with limited dispersal in between. Our findings have implications for waterworks' filter management, and offer a window down to the largely unexplored eukaryotic microbiology of groundwater aquifers.

Preferential flow paths shape the structure of bacterial communities in a clayey till depth profile.

Preferential flow paths in subsurface soils serve as transport routes for water, dissolved organic matter and oxygen. Little is known about bacterial communities in flow paths or in subsoils below ∼4 m. We compared communities from preferential flow paths (biopores, fractures and sand lenses) with those in adjacent matrix sediments of clayey till from the plough layer to a depth of 6 m. 16S rRNA gene-targeted community analysis showed bacterial communities of greater abundance and diversity in flow paths than in matrix sediments at all depths. Deep fracture communities contained a higher relative abundance of aerobes and plant material decomposers like Nitrospirae, Acidobacteria and Planctomycetes than adjacent matrix sediments. Similarly, analyses of the relative abundances of archaeal amoA, nirK and dsrB genes indicated transition from aerobic to anaerobic nitrogen and sulphur cycling at greater depth in preferential flow paths than in matrix sediments. Preferential flow paths in the top 260 cm contained more indicator operational taxonomic units from the plough layer community than the matrix sediments. This study indicates that the availability of oxygen and organic matter and downward transport of bacteria shape bacterial communities in preferential flow paths, and suggests that their lifestyles differ from those of bacteria in matrix communities.

Biodegradation of marine oil spills in the Arctic with a Greenland perspective.

New economic developments in the Arctic, such as shipping and oil exploitation, bring along unprecedented risks of marine oil spills. Microorganisms have played a central role in degrading and reducing the impact of the spilled oil during past oil disasters. However, in the Arctic, and in particular in its pristine areas, the self-cleaning capacity and biodegradation potential of the natural microbial communities have yet to be uncovered. This review compiles and investigates the current knowledge with respect to environmental parameters and biochemical constraints that control oil biodegradation in the Arctic. Hereby, seawaters off Greenland are considered as a case study. Key factors for biodegradation include the bioavailability of hydrocarbons, the presence of hydrocarbon-degrading bacteria and the availability of nutrients. We show how these key factors may be influenced by the physical oceanographic conditions in seawaters off Greenland and other environmental parameters including low temperature, sea ice, sunlight regime, suspended sediment plumes and phytoplankton blooms that characterize the Arctic. Based on the acquired insights, a first qualitative assessment of the biodegradation potential in seawaters off Greenland is presented. In addition to the most apparent Arctic characteristics, such as low temperature and sea ice, the impact of typical Arctic features such as the oligotrophic environment, poor microbial adaptation to hydrocarbon degradation, mixing of stratified water masses, and massive phytoplankton blooms and suspended sediment plumes merit to be topics of future investigation.

Groundwater contamination with 2,6-dichlorobenzamide (BAM) and perspectives for its microbial removal.

The pesticide metabolite 2,6-dichlorobenzamide (BAM) is very persistent in both soil and groundwater and has become one of the most frequently detected groundwater micropollutants. BAM is not removed by the physico-chemical treatment techniques currently used in drinking water treatment plants (DWTP); therefore, if concentrations exceed the legal threshold limit, it represents a sizeable problem for the stability and quality of drinking water production, especially in places that depend on groundwater for drinking water. Bioremediation is suggested as a valuable strategy for removing BAM from groundwater by deploying dedicated BAM-degrading bacteria in DWTP sand filters. Only a few bacterial strains with the capability to degrade BAM have been isolated, and of these, only three isolates belonging to the Aminobacter genus are able to mineralise BAM. Considerable effort has been made to elucidate degradation pathways, kinetics and degrader genes, and research has recently been presented on the application of strain Aminobacter sp. MSH1 for the purification of BAM-contaminated water. The aim of the present review was to provide insight into the issue of BAM contamination and to report on the current status and knowledge with regard to the application of microorganisms for purification of BAM-contaminated water resources. This paper discusses the prospects and challenges for bioaugmentation of DWTP sand filters with specific BAM-degrading bacteria and identifies relevant perspectives for future research.

Adhesion to sand and ability to mineralise low pesticide concentrations are required for efficient bioaugmentation of flow-through sand filters.

Pesticide-polluted drinking water may be remediated by inoculating waterworks sand filters with specific degrading bacteria. However, degradation efficiency is often hampered by the poor adhesion behaviour of the introduced bacteria. The phenoxy acid herbicide 4-chloro-2-methyl-phenoxy-acetic acid (MCPA) is a widespread groundwater contaminant. The aim of this study was to investigate whether specific surface characteristics of MCPA-degrading bacteria could be linked to their degrading capabilities in sand filters. Four MCPA degraders with different taxonomic affiliations and original habitats (Sphingomonas sp. PM2, Sphingomonas sp. ERG5, Burkholderia sp. TFD34, Cupriavidus sp. TFD38) were characterised with regard to their motility, cell surface hydrophobicity, biofilm formation, adhesion behaviour and ability to mineralise MCPA. Strains PM2 and ERG5 were non-motile and hydrophobic, whilst strains TFD34 and TFD38 were motile and less hydrophobic. All the strains except ERG5 showed low biofilm formation on polystyrene, although it was significantly higher on glass. PM2 was the most efficient MCPA degrader as it displayed no lag phase and reached >50 % mineralisation at all concentrations (0.0016-25 mg L-1). PM2 adhered significantly better to sand than the other strains. No link was found between motility, biofilm formation and the ability to adhere to sand. PM2 completely removed MCPA for 14 days when inoculated in sand columns with a constant inlet of 1 mg L-1 MCPA. These results demonstrate that besides the ability to degrade the contaminant, surface hydrophobicity and adherence abilities are significant parameters controlling sustained degradation in flow-through sand columns and must be considered when selecting bacteria for bioaugmentation.

Surface Colonization and Activity of the 2,6-Dichlorobenzamide (BAM) Degrading Aminobacter sp. Strain MSH1 at Macro- and Micropollutant BAM Concentrations.

Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM) as a C and N source and is a potential catalyst for biotreatment of BAM-contaminated groundwater in filtration units of drinking water treatment plants (DWTPs). The oligotrophic environment of DWTPs including trace pollutant concentrations, and the high flow rates impose challenges for micropollutant biodegradation in DWTPs. To understand how trace BAM concentrations affect MSH1 surface colonization and BAM degrading activity, MSH1 was cultivated in flow channels fed continuously with BAM macro- and microconcentrations in a N- and C-limiting medium. At all BAM concentrations, MSH1 colonized the flow channel. BAM degradation efficiencies were concentration-dependent, ranging between 70 and 95%. Similarly, BAM concentration affected surface colonization, but at 100 µg/L BAM and lower, colonization was similar to that in systems without BAM, suggesting that assimilable organic carbon and nitrogen other than those supplied by BAM sustained colonization at BAM microconcentrations. Comparison of specific BAM degradation rates in flow channels and in cultures of suspended freshly grown cells indicated that starvation conditions in flow channels receiving BAM microconcentrations resulted into MSH1 biomasses with 10-100-times reduced BAM degrading activity and provided a kinetic model for predicting BAM degradation under continuous C and N starvation.

Protozoa graze on the 2,6-dichlorobenzamide (BAM)-degrading bacterium Aminobacter sp. MSH1 introduced into waterworks sand filters.

Groundwater contamination by pesticide residues often leads to the closure of drinking water wells, making the development of new techniques to remediate drinking water resources of considerable interest. Pesticide-degrading bacteria were recently added to a waterworks sand filter in an attempt to remediate pesticide-polluted drinking water. The density of the introduced bacteria, however, decreased rapidly, which was partly attributed to predation by protozoa in the sand filter. This study investigated the effects of indigenous sand filter protozoa on the population density and degradation efficiency of degrader bacteria introduced into sand from a waterworks sand filter. The 2,6-dichlorobenzamide (BAM)-degrading bacterium Aminobacter sp. MSH1 was used as a model organism. The introduction of MSH1 at high cell densities was followed by a >1000-fold increase in the protozoan population size and at the same time a 29 % reduction in Aminobacter cell numbers. The protozoan population in the systems that had MSH1 added at a lower density only increased 50-fold, and a decrease in Aminobacter numbers was not detectable. Furthermore, a reduction in the number of Aminobacter and in BAM degradation efficiency was seen in flow-through sand filter columns inoculated with MSH1 and fed BAM-contaminated water, when comparing sand columns containing the indigenous microbial filter community, i.e. containing protozoa, to columns with sterilised sand. These results suggest that degrader bacteria introduced into waterworks sand filters are adversely affected by grazing from the indigenous protozoa, reducing the size of the degrader population and the sand filter degradation efficiency.