In situ incubation of iron(II)-bearing minerals and Fe(0) reveals insights into metabolic flexibility of chemolithotrophic bacteria in a nitrate polluted karst aquifer.

Groundwater nitrate pollution is a major reason for deteriorating water quality and threatens human and animal health. Yet, mitigating groundwater contamination naturally is often complicated since most aquifers are limited in bioavailable carbon. Since metabolically flexible microbes might have advantages for survival, this study presents a detailed description and first results on our modification of the BacTrap© method, aiming to determine the prevailing microbial community's potential to utilize chemolithotrophic pathways. Our microbial trapping devices (MTDs) were amended with four different iron sources and incubated in seven groundwater monitoring wells for ∼3 months to promote growth of nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOxB) in a nitrate-contaminated karst aquifer. Phylogenetic analysis based on 16S rRNA gene sequences implies that the identity of the iron source influenced the microbial community's composition. In addition, high throughput amplicon sequencing revealed increased relative 16S rRNA gene abundances of OTUs affiliated to genera such as Thiobacillus, Rhodobacter, Pseudomonas, Albidiferax, and Sideroxydans. MTD-derived enrichments set up with Fe(II)/nitrate/acetate to isolate potential NRFeOxB, were dominated by e.g., Acidovorax spp., Paracoccus spp. and Propionivibrio spp. MTDs are a cost-effective approach for investigating microorganisms in groundwater and our data not only solidifies the MTD's capacity to provide insights into the metabolic flexibility of the aquifer's microbial community, but also substantiates its metabolic potential for anaerobic Fe(II) oxidation.

Streambed microbial communities in the transition zone between groundwater and a first-order stream as impacted by bidirectional water exchange.

The input of nitrate and other agricultural pollutants in higher-order streams largely derives from first-order streams. The streambed as the transition zone between groundwater and stream water has a decisive impact on the attenuation of such pollutants. This reactivity is not yet well understood for lower-order agricultural streams, which are often anthropogenically altered and lack the streambed complexity allowing for extensive hyporheic exchange. Reactive hot spots in such streambeds have been hypothesized as a function of hydrology, which controls the local gaining (groundwater exfiltration) or losing (infiltration) of stream water. However, streambed microbial communities and activities associated with such reactive zones remain mostly uncharted. In this study, sediments of a first-order agriculturally impacted stream in southern Germany were investigated. Along with a hydraulic dissection of distinct gaining and losing reaches of the stream, community composition and the abundance of bacterial communities in the streambed were investigated using PacBio long-read sequencing of bacterial 16S rRNA gene amplicons, and qPCR of bacterial 16S rRNA and denitrification genes (nirK and nirS). We show that bidirectional water exchange between groundwater and the stream represents an important control for sediment microbiota, especially for nitrate-reducing populations. Typical heterotrophic denitrifiers were most abundant in a midstream net losing section, while up- and downstream net gaining sections were associated with an enrichment of sulfur-oxidizing potential nitrate reducers affiliated with Sulfuricurvum and Thiobacillus spp. Dispersal-based community assembly was found to dominate such spots of groundwater exfiltration. Our results indicate a coupling of N- and S-cycling processes in the streambed of an agricultural first-order stream, and a prominent control of microbiology by hydrology and hydrochemistry in situ. Such detailed local heterogeneities in exchange fluxes and streambed microbiomes have not been reported to date, but seem relevant for understanding the reactivity of lower-order streams.

Nitrate Removal by a Novel Lithoautotrophic Nitrate-Reducing, Iron(II)-Oxidizing Culture Enriched from a Pyrite-Rich Limestone Aquifer.

Nitrate removal in oligotrophic environments is often limited by the availability of suitable organic electron donors. Chemolithoautotrophic bacteria may play a key role in denitrification in aquifers depleted in organic carbon. Under anoxic and circumneutral pH conditions, iron(II) was hypothesized to serve as an electron donor for microbially mediated nitrate reduction by Fe(II)-oxidizing (NRFeOx) microorganisms. However, lithoautotrophic NRFeOx cultures have never been enriched from any aquifer, and as such, there are no model cultures available to study the physiology and geochemistry of this potentially environmentally relevant process. Using iron(II) as an electron donor, we enriched a lithoautotrophic NRFeOx culture from nitrate-containing groundwater of a pyrite-rich limestone aquifer. In the enriched NRFeOx culture that does not require additional organic cosubstrates for growth, within 7 to 11 days, 0.3 to 0.5 mM nitrate was reduced and 1.3 to 2 mM iron(II) was oxidized, leading to a stoichiometric NO3-/Fe(II) ratio of 0.2, with N2 and N2O identified as the main nitrate reduction products. Short-range ordered Fe(III) (oxyhydr)oxides were the product of iron(II) oxidation. Microorganisms were observed to be closely associated with formed minerals, but only few cells were encrusted, suggesting that most of the bacteria were able to avoid mineral precipitation at their surface. Analysis of the microbial community by long-read 16S rRNA gene sequencing revealed that the culture is dominated by members of the Gallionellaceae family that are known as autotrophic, neutrophilic, and microaerophilic iron(II) oxidizers. In summary, our study suggests that NRFeOx mediated by lithoautotrophic bacteria can lead to nitrate removal in anthropogenically affected aquifers. IMPORTANCE Removal of nitrate by microbial denitrification in groundwater is often limited by low concentrations of organic carbon. In these carbon-poor ecosystems, nitrate-reducing bacteria that can use inorganic compounds such as Fe(II) (NRFeOx) as electron donors could play a major role in nitrate removal. However, no lithoautotrophic NRFeOx culture has been successfully isolated or enriched from this type of environment, and as such, there are no model cultures available to study the rate-limiting factors of this potentially important process. Here, we present the physiology and microbial community composition of a novel lithoautotrophic NRFeOx culture enriched from a fractured aquifer in southern Germany. The culture is dominated by a putative Fe(II) oxidizer affiliated with the Gallionellaceae family and performs nitrate reduction coupled to Fe(II) oxidation leading to N2O and N2 formation without the addition of organic substrates. Our analyses demonstrate that lithoautotrophic NRFeOx can potentially lead to nitrate removal in nitrate-contaminated aquifers.

In-situ mass spectrometry improves the estimation of stream reaeration from gas-tracer tests.

The estimation of gas-exchange rates between streams and the atmosphere is of great importance for the fate of volatile compounds in rivers. For dissolved oxygen, this exchange process is called reaeration, and its accurate and precise estimation is essential for the quantification of metabolic rates. A common method for the determination of gas-exchange rates is through artificial gas-tracer tests with a proxy gas. We present the implementation of a portable gas-equilibrium membrane inlet mass spectrometer (GE-MIMS) to record concentrations of krypton and propane injected as tracer compound in the context of a gas-tracer test. The field-compatible GE-MIMS uses signals of atmospheric measurements for concentration standardization, and allows recording the dissolved-gas concentrations at a high temporal resolution, leading to overall low measurement uncertainty. Furthermore, the in-situ approach avoids loss of gas during the steps of sampling, transport, storage, and analysis required for ex-situ gas measurements. We compare obtained gas-exchange rate coefficients, reaeration and derived metabolic rates from the in-situ measurements to results obtained from head-space sampling of propane followed by laboratory analysis, and find much lower uncertainties with the in-situ method.

Impact of non-idealities in gas-tracer tests on the estimation of reaeration, respiration, and photosynthesis rates in streams.

Estimating respiration and photosynthesis rates in streams usually requires good knowledge of reaeration at the given locations. For this purpose, gas-tracer tests can be conducted, and reaeration rate coefficients are determined from the decrease in gas concentration along the river stretch. The typical procedure for analysis of such tests is based on simplifying assumptions, as it neglects dispersion altogether and does not consider possible fluctuations and trends in the input signal. We mathematically derive the influence of these non-idealities on estimated reaeration rates and how they are propagated onto the evaluation of aerobic respiration and photosynthesis rates from oxygen monitoring. We apply the approach to field data obtained from a gas-tracer test using propane in a second-order stream in Southwest Germany. We calculate the reaeration rate coefficients accounting for dispersion as well as trends and uncertainty in the input signals and compare them to the standard approach. We show that neglecting dispersion significantly underestimates reaeration, and results between sections cannot be compared if trends in the input signal of the gas tracer are disregarded. Using time series of dissolved oxygen and the various estimates of reaeration, we infer respiration and photosynthesis rates for the same stream section, demonstrating that the bias and uncertainty of reaeration using the different approaches significantly affects the calculation of metabolic rates.

Dating of 'young' groundwaters using environmental tracers: advantages, applications, and research needs.

Many problems related to groundwater supply and quality, as well as groundwater-dependent ecosystems require some understanding of the timescales of flow and transport. For example, increased concern about the vulnerabilities of 'young' groundwaters (less than ~1000 years) to overexploitation, contamination, and land use/climate change effects are driving the need to understand flow and transport processes that occur over decadal, annual, or shorter timescales. Over the last few decades, a powerful suite of environmental tracers has emerged that can be used to interrogate a wide variety of young groundwater systems and provide information about groundwater ages/residence times appropriate to the timescales over which these systems respond. These tracer methods have distinct advantages over traditional approaches providing information about groundwater systems that would likely not be obtainable otherwise. The objective of this paper is to discuss how environmental tracers are used to characterise young groundwater systems so that more researchers, water managers, and policy-makers are aware of the value of environmental tracer approaches and can apply them in appropriate ways. We also discuss areas where additional research is required to improve ease of use and extend quantitative interpretations of tracer results.

Linking chloride mass balance infiltration rates with chlorofluorocarbon and SF6 groundwater dating in semi-arid settings: potential and limitations.

In the framework of the investigation of enrichment processes of nitrate in groundwater of the Kalahari of Botswana near Serowe, recharge processes were investigated. The thick unsaturated zone extending to up to 100 m of mostly unconsolidated sediments and very low recharge rates pose a serious challenge to study solute transport related to infiltration and recharge processes, as this extends past the conventional depths of soil scientific investigations and is difficult to describe using evidence from the groundwater due to the limitations imposed by available tracers. To determine the link between nitrate in the vadose zone and in the uppermost groundwater, sediment from the vadose zone was sampled up to a depth of 15-20 m (in one case also to 65 m) on several sites with natural vegetation in the research area. Among other parameters, sediment and water were analysed to determine chloride and nitrate concentration depth profiles. Using the chloride mass balance method, an estimation of groundwater infiltration rates produced values of 0.2-4 mm a(-1). The uncertainty of these values is, however, high. Because of the extreme thickness of the vadose zone, the travel time in the unsaturated zone might reach extreme values of up to 500 years and more. For investigations using groundwater, we applied the chlorofluorocarbons CFC-113, CFC-12, sulphur hexafluoride (SF(6)) and tritium to identify potential recharge, and found indications for some advective transport of the CFCs and SF(6), which we accounted for as constituting potential active localised recharge. In our contribution, we show the potential and limitations of the applied methods to determine groundwater recharge and coupled solute transport in semi-arid settings, and compare travel time ranges derived from soil science and groundwater investigations.

Elevated nitrate levels in the groundwater of the Gaza Strip: distribution and sources.

Seven years of monitoring groundwater in the Gaza Strip has shown that nitrate was and still is a major groundwater pollutant. The objectives of this research were to study the distribution of NO(3)(-) in the groundwater of the Gaza Strip and to identify the sources of NO(3)(-) in the Gaza aquifer system by assessing nitrogen and oxygen isotopes. The most recent samples collected in 2007 showed 90% of the wells having NO(3)(-) concentrations that are several times higher than the WHO standards of 50 mg/L. Potential NO(3)(-) source materials in Gaza are animal manure N, synthetic NH(4) based fertilizers, and wastewater/sludge. The average concentrations of N in the sludge, manure and soil of Gaza were 2.9%, 1% and 0.08%, respectively. The range in delta(15)N of solid manure samples was +7.5 to +11.9 per thousand. The range in delta(15)N of sludge samples was +4.6 to +7.4 per thousand, while four brands of synthetic fertilizers commonly used in Gaza had delta(15)N ranging from +0.2 to +1.0 per thousand. Sludge amended soil had delta(15)N ranging from +2.0 to +7.3 per thousand. For both delta(18)O and delta(15)N, the ranges of groundwater NO(3)(-) were -0.1 to +9.3 per thousand and +3.2 to 12.8 per thousand, respectively. No significant bacterial denitrification is taking place in the Gaza Strip aquifer. Nitrate was predominantly derived from manure and, provided delta(15)N of sludge represents the maximum delta(15)N of human waste, to a lesser extent from septic effluents/sludge. Synthetic fertilizers were a minor source.

Sources and transport of selected organic micropollutants in urban groundwater underlying the city of Halle (Saale), Germany.

In this study, we used isotopic (delta18O, delta2H, delta34S-SO4) and chemical tracers (boron) to assess the sources and transport processes of the micropollutants carbamazepine, galaxolide, and bisphenol A in groundwater underlying the city of Halle (Saale), Germany. Their ubiquitous presence in urban groundwater results from a combination of local river water infiltration, sewer exfiltration, and urban stormwater recharge. Attenuation during transport with infiltrating river water increased from carbamazepine (0-60%) to galaxolide (60-80%) in accordance with their increasing sorption affinity and decreasing recalcitrance against biodegradation. Distinctly higher attenuation during transport was found for carbamazepine (85-100%) and galaxolide (95-100%) if micropollutants originated from sewer exfiltration. Most likely, this is related to higher contents of organic matter and higher transit times of the respective flow paths. Although attenuation undoubtedly also affects the transport of bisphenol A, quantification is limited due to additional contributions from the urban stormwater recharge. As a consequence, micropollutant loads in groundwater indicate that groundwater discharge may dominate the export of bisphenol A from urban areas.