Nitrate reduction mechanisms and rates in an unconfined eogenetic karst aquifer in two sites with different redox potential

Abstract

This study integrates push‐pull tracer tests (PPTT) with microbial characterization of extracted water via quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR (RT‐qPCR) of selected functional N transformation genes to quantify nitrate reduction mechanisms and rates in sites with different redox potential in a karst aquifer. PPTT treatments with nitrate (AN) and nitrate‐fumarate (ANC) were executed in two wells representing anoxic and oxic geochemical end‐members. Oxic aquifer zero‐order nitrate loss rates (mmol L−1 h−1) were similar for AN and ANC treatment, ranging from 0.03 ± 0.01 to 0.05 ± 0.01. Anoxic aquifer zero‐order nitrate loss rates ranged from 0.03 ± 0.02 (AN) to 0.13 ± 0.02 (ANC). Microbial characterization indicates mechanisms influencing these rates were dissimilatory nitrate reduction to ammonium (DNRA) at the anoxic site with AN treatment, assimilatory reduction of nitrate to ammonium (ANRA) with ANC treatment in the water column at both sites, and additional documented nitrate reduction that occurred in unsampled biofilms. With carbon treatment, total numbers of microbes (16S rRNA genes) significantly increased (fourteenfold to thirtyfold), supporting stimulated growth with resulting ANRA. Decreased DNRA gene concentrations (nrfA DNA) and increased DNRA activity ratio (nrfA‐cDNA/DNA) supported the assertion that DNRA occurred in the anoxic zone with AN and ANC treatment. Furthermore, decreased DNRA gene copy numbers at the anoxic site with ANC treatment suggests that DNRA microbes in the anoxic site are chemolithoautotrophic. Increased RT‐qPCR denitrification gene expression (nirK and nirS) was not observed in water samples, supporting that any observed NO3‐N loss due to denitrification may be occurring in unsampled microbial biofilms.

Spatially distributed denitrification in a karst springshed

“…This mixing could effectively decouple the local measured redox state from the conditions where denitrification occurred. Alternatively, local reduction zones in microbial biofilms on aquifer substrates may have created favourable conditions where transformations could occur locally despite high DO concentrations in the bulk aquifer, as was observed by Biesterfeld, Farmer, Figueroa, Parker, and Russell () and Henson et al ().…”

“…The available number of sample locations may have prevented detection of a significant correlation. However, in direct injection experiments in Wells 9 and 16, unexplained nitrate losses were observed under oxic and anoxic conditions with and without carbon (Henson et al, ). Thus, reducing conditions in aquifer samples may not be required to facilitate denitrification.…”

“…Evidence of denitrification was observed where reactant availability (e.g., nitrate and DOC) was both high (Sites 10 and 5) and low (Sites 8 and 14). Recent research (Heffernan et al, ; Henson et al, ) suggests that chemolithoautotrophic denitrification may be occurring in the low DOC waters of the UFA using solid‐phase alternate electron donors (e.g., iron and manganese). High iron and manganese concentrations observed at the site with the highest [N 2 ] Den value further support this possibility.…”

“…This assumed that areal rates of N inputs were equivalent and of identical isotopic source signatures and that denitrification was the only significant pathway (i.e., Dissimilatory Nitrate Reduction to Ammonia (DNRA) and direct nitrate assimilation are negligible). Although some evidence of DNRA in the UFA has been documented (Henson, Huang, Graham, & Ogram, ), denitrification was assumed to have the most significant effect on δ 15 N‐NO 3 . These assumptions are consistent with those of Heffernan et al () and supported by (a) relatively homogenous springshed land use with fertilizer application to crops and improved pasture as the dominant source of N (Katz et al, ) and (b) low spatially distributed and spring ammonium measurements (Table S2).…”

Solute Reactive Tracers for Hydrogeological Applications: A Short Review and Future Prospects

“…Their main purpose is to determine microbial metabolic activity (natural attenuation processes) and/or to assess redox conditions. Numerous redox-sensitive tracers have been applied for laboratory and field scale investigations, such as inorganic electron acceptors (e.g., O 2 , NO 3 − , SO 4 2− , CO 3 2− ) [106][107][108][109][110][111][112][113][114][115][116], organic electron donors (e.g., low-molecular weight alcohols and sugars [117] and benzoate [118][119][120]), or the organic electron acceptor resazurin [8,[121][122][123][124][125][126][127][128].…”