Microaerobic methane-driven denitrification in a biotrickle bed - Investigating the active microbial biofilm community composition using RNA-stable isotope probing.

A microaerobic (2% O2 v/v) biotrickle bed reactor supplied continuously with 2% methane to drive nitrate removal (MAME-D) was investigated using 16S rDNA and rRNA amplicon sequencing in combination with RNA-stable isotope probing (RNA-SIP) to identify the active microorganisms. Methane removal rates varied from 500 to 1000 mmol m-3h-1 and nitrate removal rates from 25 to 58 mmol m-3h-1 over 55 days of operation. Biofilm samples from the column were incubated in serum bottles supplemented with 13CH4. 16S rDNA analysis indicated a simple community structure in which four taxa accounted for 45% of the total relative abundance (RA). Dominant genera included the methanotroph Methylosinus and known denitrifiers Nubsella and Pseudoxanthomonas; along with a probable denitrifier assigned to the order Obscuribacterales. The 16S rRNA results revealed the methanotrophs Methylocystis (15% RA) and Methylosinus (10% RA) and the denitrifiers Arenimonas (10% RA) and Pseudoxanthomonas (7% RA) were the most active genera. Obscuribacterales was the most active taxa in the community at 22% RA. Activity was confirmed by the Δ buoyant density changes with time for the taxa, indicating most of the community activity was associated with methane oxidation and subsequent consumption of methanotrophic metabolic intermediates by the denitrifiers. This is the first report of RNA stable isotope probing within a microaerobic methane driven denitrification system and the active community was markedly different from the full community identified via 16S-rDNA analysis.

Evaluation of pollution swapping phenomena from a woodchip denitrification wall targetting removal of nitrate in a shallow gravel aquifer.

Woodchip denitrification walls offer a potentially useful way for passive in situ remediation of groundwater nitrate pollution, yet because of the low redox state they induce on the subsurface environment there is an inherent risk they can promote pollution-swapping phenomena. We evaluated pollution-swapping phenomena associated with the first two operational years of a woodchip denitrification wall that is being trialled in a fast-flowing shallow gravel aquifer of quartzo-feldspathic mineralogy. Following burial of woodchip below the water table there was immediate export of dissolved organic carbon (DOC), phosphorus and ammonium into the groundwater. Under the low redox state sustained by labile DOC, the wall initially provided 100% nitrate removal at the expense of acute and localised pollution that occurred in the form of a plume of dissolved iron, manganese and arsenic that were mobilised from the aquifer sediments, in conjunction with methane gas emission. Within one year however, the reactivity of the woodchip wall subsided to support a steady state condition in which nitrate reduction was the terminal electron acceptor process with no measurable methane emission. Having initially functioned as a sink for the potent greenhouse gas nitrous oxide (N2O), evidence is that the woodchip wall is now exporting N2O, albeit at rates less than those associated with productive agricultural land.

RNA stable isotope probing and high-throughput sequencing to identify active microbial community members in a methane-driven denitrifying biofilm.

AIMS: Aerobic methane oxidation coupled to denitrification (AME-D) is a promising process for removing nitrate from groundwater and yet its microbial mechanism and ecological implications are not fully understood. This study used RNA stable isotope probing (RNA-SIP) and high-throughput sequencing to identify the micro-organisms that are actively involved in aerobic methane oxidation within a denitrifying biofilm. METHODS AND RESULTS: Two RNA-SIP experiments were conducted to investigate labelling of RNA and methane monooxygenase (pmoA) transcripts when exposed to 13 C-labelled methane over a 96-hour time period and to determine active bacteria involved in methane oxidation in a denitrifying biofilm. A third experiment was performed to ascertain the extent of 13 C labelling of RNA using isotope ratio mass spectrometry (IRMS). All experiments used biofilm from an established packed bed reactor. IRMS confirmed 13 C enrichment of the RNA. The RNA-SIP experiments confirmed selective enrichment by the shift of pmoA transcripts into heavier fractions over time. Finally, high-throughput sequencing identified the active micro-organisms enriched with 13 C. CONCLUSIONS: Methanotrophs (Methylovulum spp. and Methylocystis spp.), methylotrophs (Methylotenera spp.) and denitrifiers (Hyphomicrobium spp.) were actively involved in AME-D. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to use RNA-SIP and high-throughput sequencing to determine the bacteria active within an AME-D community.

Applications of a UV optical nitrate sensor in a surface water/groundwater quality field study.

Examples of the utility of UV optical nitrate sensors are provided for two field applications, investigating nitrate pollution in a lowland, peri-urban catchment. In one application, rapid, in-stream longitudinal nitrate surveys were made in summer and winter, by fixing an optical nitrate sensor operating in continuous measurement mode to a kayak that was paddled along 10 km of the mainstem of the low-order stream in under 4 h. Nitrate concentrations ranged between 3.45 and 6.39 mg NO3-N/L. Nitrate hot-spots and cool-spots were mapped and found to relate to point discharges from spring-fed tributaries and land drains. Effective nitrate removal (dN/dx = - 0.08 mg N/L/km), inferred to be from assimilation reactions, was evident in the summer dataset, but not the winter nitrate dataset. In a second application, the optical sensor was configured with appropriate technology to establish an autonomous and fully automated nitrate monitoring station. The station makes daily nitrate measurements of surface water, and groundwater, sampled from a cluster of four multi-level wells. Quarterly maintenance of the nitrate sensor has proven sufficient to keep measurement errors under 5%. Most nitrate variation has been recorded at or near the water table where concentrations have ranged between 3.47 and 5.88 mg NO3-N/L, and annual maxima have occurred in late winter/spring, which coincides with when most nitrate leaching occurs from agricultural land. Seasonal nitrate patterns are not evident in groundwater sampled from 8-m depth, or deeper. High-frequency monitoring has revealed that some infra-season, short-term variability also occurs in shallow groundwater nitrate, driven by storm events, and which on occasion results in a temporary inversion of the groundwater nitrate-depth profile.

Eigenmodels to forecast groundwater levels in unconfined river-fed aquifers during flow recession.

Low-land alluvial gravel aquifers are formed from, and tend to be recharged, by rivers. These interconnected river - groundwater systems can be highly dynamic with groundwater levels following the seasonality of the hydrological regime of the river. The associated groundwater resources are regularly under stress during summer periods when abstractive demand is high and recharge is low. Predicting lead-times for critical groundwater levels allows for a more flexible and adaptive groundwater management. An eigenmodel approach is proposed here as a way of making such predictions, fast and efficiently. The eigenmodel is a mathematical concept that represents the hydraulic function of a groundwater aquifer as a set of conceptual linear reservoirs, arranged in-series. River recharge, land surface recharge, and groundwater abstraction for irrigation are considered as model forcings. The eigenmodel approach is demonstrated on three wells of the unconfined Wairau Aquifer in the Marlborough District of New Zealand, which are used for water resources management. Individual eigenmodels were calibrated to historic data and predictive uncertainty bounds were determined by Markov chain Monte Carlo sampling. Hindcasting of past recession periods showed a low predictive error of the models and a good coverage of the predictive uncertainty bounds. The main advantage of the approach is a 4-orders of magnitude higher computational efficiency compared to a numerical benchmark model. This allows for probabilistic simulation in operational forecasting of groundwater levels. The framework is implemented as a web application for 30-day operational forecasts that comprises automatic data downloads and model input generation, stochastic simulation, uncertainty estimation, visualization, and daily updates on a website.

The effects of denitrification parameterization and potential benefits of spatially targeted regulation for the reduction of N-discharges from agriculture.

Diffuse nitrate leaching from agricultural areas is a major environmental problem in many parts of the world. Understanding where in a catchment nitrate is removed is key for designing effective land use management strategies that protect water quality, while minimizing the impact on economic development. In this study we assess the effects of spatially targeted nitrate leaching regulation in a basin with limited knowledge of the complexity of chemical heterogeneity. Three alternative nitrate reactivity spatial parameterizations were incorporated in a catchment-scale flow and transport model and used to evaluate the effectiveness of four possible spatially targeted regulation options. Our findings confirm that denitrification parameterization cannot be numerically determined based on model inversion alone. Detailed field based characterization using physical and geochemical methods should be considered and incorporated in the numerical inversion scheme. We also demonstrate that there are potential benefits of implementing spatially targeted regulation compared to spatially uniform regulation. Focusing regulation in areas where nitrate residence time is short, such as riparian zones or areas with low natural N-reduction, results in greater reduction of N-discharges through groundwater. Significantly improved efficiencies can be expected when delineation of management zones considers the chemical heterogeneity and groundwater flow paths. These improved efficiencies are achieved by adopting management rules that regulate land use in discharge sensitive areas, where leaching changes contribute the most to the catchment nitrate discharges. In our case study, regulation in discharge sensitive zones was twice as efficient compared to other management options.

Efficacy of Coral Sand for Removal of and Bacteriophage under Saturated Flow Conditions.

Knowledge of how effectively microbes are transported through porous media is useful for water resource/wastewater management. Despite much research having been done to characterize microbial contaminant transport through various sedimentary materials, very little study has been made on coral sand, such as constitutes the primary substrate of many Pacific atolls. We conducted a set of laboratory column experiments as a preliminary examination of how effective coral sand is at attenuating model pathogens J6-2 and MS2 bacteriophage (phage) under saturated flow conditions mildly representative of field conditions at the Bonriki freshwater lens, South Tarawa, Kiribati. The very poorly sorted gravelly sand coral substrate tested proved very effective at attenuating the bacterial tracer, and spatial removal rates of between 0.02 and 0.07 log cm were determined for J6-2. The ability to determine precise removal rates for MS2 phage was compromised by the use of a plastic apparatus, although the evidence weights toward coral sand being less effective at attenuating MS2 phage than it is . Further research is required to fully assess the ability of coral sand to remove pathogens and to explore how this medium could be engineered into cost-effective water/wastewater treatment solutions on Pacific atolls. The phage data from this work highlight the limitations of using plastic apparatus in experiments targeted at characterizing the fate and transport of viruses.

Application of the re-circulating tracer well test method to determine nitrate reaction rates in shallow unconfined aquifers.

Five re-circulating tracer well tests (RCTWTs) have been conducted in a variety of aquifer settings, at four sites across New Zealand. The tests constitute the first practical assessment of the two-well RCTWT methodology described by Burbery and Wang (Journal of Hydrology, 2010; 382:163-173) and were aimed at evaluating nitrate reaction rates in situ. The performance of the RCTWTs differed significantly at the different sites. The RCTWT method performed well when it was applied to determine potential nitrate reaction rates in anoxic, electro-chemically reductive, nitrate-free aquifers of volcanic lithology, on the North Island, New Zealand. Regional groundwater flow was not fast-flowing in this setting. An effective first-order nitrate reaction rate in the region of 0.09 d(-1) to 0.26 d(-1) was determined from two RCTWTs applied at one site where a reaction rate of 0.37 d(-1) had previously been estimated from a push-pull test. The RCTWT method performed poorly, however, in a fast-flowing, nitrate-impacted fluvio-glacial gravel aquifer that was examined on the South Island, New Zealand. This setting was more akin to the hypothetical physiochemical problem described by Burbery and Wang (2010). Although aerobic conditions were identified as the primary reason for failure to measure any nitrate reaction in the gravel aquifer, failure to establish significant interflow in the re-circulation cell due to the heterogeneous nature of the aquifer structure, and natural variability exhibited in nitrate contaminant levels of the ambient groundwater further contributed to the poor performance of the test. Our findings suggest that in practice, environmental conditions are more complex than assumed by the RCTWT methodology, which compromises the practicability of the method as one for determining attenuation rates in groundwater based on tracing ambient contaminant levels. Although limited, there appears to be a scope for RCTWTs to provide useful information on potential attenuation rates when reactants are supplemented to the aquifer system under examination.