Elucidation of the microbial N-cycle in the subsurface: key microbial players and processes

Despite the high relevance of anaerobic ammonia oxidation (anammox) for reactive nitrogen removal from marine systems, its relative importance compared to denitrification has only rarely been studied in aquifers. Comparative investigations were carried out in two superimposed aquifers in the Hainich region (Thuringia, Germany) (i) to evaluate the genetic potential for anammox and denitrification in groundwater (ii) to assess the potential relevance of anammox versus denitrification by activity measurements and (iii) to investigate the groundwater-originated consortiums capacity to use inorganic electron donor for complete denitrification. Groundwater samples were taken from eight groundwater wells differed in oxygen availabilty. Abundances of nitrite reductase genes (nirS) for denitrifiers as determined by quantitative PCR ranged from 2.6 x 103 to 8.2 x 107 genes L-1 groundwater while abundances of hydrazine synthase genes (hzsA) indicative of anammox bacteria ranged from 6.9 x 102 to 2.1 x 107 genes L-1 groundwater. Functional gene transcript analysis and ladderane phospholipid provided further evidence for the presence of active anammox bacteria, mostly related to Candidatus Brocadia fulgida. Despite similar estimated abundances of denitrifiers and anammox bacteria, anammox rates (4.7 nmol N2 L-1 d-1) were about eight times higher than denitrification rates (0.6 nmol N2 L-1 d-1). Moreover, a chemolithoautotrophic denitrifying consortium was maintained in bicarbonate buffered thiosulfate-nitrate medium under argon atmosphere along with H2 and CO2 (3% headspace volume). This consortium could oxidize S2O32- and H2 completely while simultaneously reducing nitrate to nitrite and further on to dinitrogen gas, confirmed using Raman gas spectroscopy, following the conversion of 15N-nitrate to 30N2. These findings suggest a high relevance of chemolithoautotrophic processes for reactive nitrogen removal in oligotrophic limestone aquifers at Hainich, Germany

Activity and electron donor preference of two denitrifying bacterial strains identified by Raman gas spectroscopy

Human activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H2, O2, CO2, 14N2, 15N2 and 15N2O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes. © 2021, The Author(s)

Nitrogen Loss from Pristine Carbonate-Rock Aquifers of the Hainich Critical Zone Exploratory (Germany) Is Primarily Driven by Chemolithoautotrophic Anammox Processes

Despite the high relevance of anaerobic ammonium oxidation (anammox) for nitrogen loss from marine systems, its relative importance compared to denitrification has less been studied in freshwater ecosystems, and our knowledge is especially scarce for groundwater. Surprisingly, phospholipid fatty acids (PLFA)-based studies identified zones with potentially active anammox bacteria within two superimposed pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory (CZE; Germany). We found anammox to contribute an estimated 83% to total nitrogen loss in suboxic groundwaters of these aquifer assemblages at rates of 3.5–4.7 nmol L−1 d−1, presumably favored over denitrification by low organic carbon availability. Transcript abundances of hzsA genes encoding hydrazine synthase exceeded nirS and nirK transcript abundances encoding denitrifier nitrite reductase by up to two orders of magnitude, providing further support of a predominance of anammox. Anammox bacteria, dominated by groups closely related to Cand. Brocadia fulgida, constituted up to 10.6% of the groundwater microbial community and were ubiquitously present across the two aquifer assemblages with indication of active anammox bacteria even in the presence of 103 μmol L−1 oxygen. Co-occurrence of hzsA and amoA gene transcripts encoding ammonia mono-oxygenase suggested coupling between aerobic and anaerobic ammonium oxidation under suboxic conditions. These results clearly demonstrate the relevance of anammox as a key process driving nitrogen loss from oligotrophic groundwater environments, which might further be enhanced through coupling with incomplete nitrification

Multiple, Compounding Disturbances in a Forest Ecosystem: Fire Increases Susceptibility of Soil Edaphic Properties, Bacterial Community Structure, and Function to Change with Extreme Precipitation Event

The intensity and frequency of ecosystem disturbances are shifting with climate change, and multiple disturbances in close succession have the potential to compound their independent effects and strongly alter ecosystem structure and function. In this paper, we examine the effects of an extreme precipitation event on a montane forest landscape that was previously decimated by wildfire (37 months prior) relative to an unburned site in the same ecosystem. We assessed responses in soil edaphic properties, bacterial community composition and assembly, and soil enzyme activities involved in carbon (C) and nitrogen (N) acquisition. Our research reveals that previously burned landscapes are susceptible to a subsequent extreme precipitation event via significant increases in soil pH where unburned soils are not. Beta- and Delta-proteobacteria associated with early succession increased and shifts were observed in N- vs. C-acquiring extracellular enzymes within burned soils after the extreme precipitation event. Finally, we connected variation in ecological selective pressures on bacterial communities associated with pH change to these differences in microbial mediated soil enzyme activity. Thus, this research demonstrates how multiple, compounding disturbances drive distinct changes relative to systems experiencing a single disturbance and suggests that changes in bacterial community assembly process with disturbance may underlie this response

Activity and electron donor preference of two denitrifying bacterial strains identified by Raman gas spectroscopy

Human activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H₂, O₂, CO₂, ¹⁴N₂, ¹⁵N₂ and ¹⁵N₂O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes

Thiosulfate- and hydrogen-driven autotrophic denitrification by a microbial consortium enriched from groundwater of an oligotrophic limestone aquifer

Despite its potentially high relevance for nitrate removal in freshwater environments limited in organic carbon, chemolithoautotrophic denitrification has rarely been studied in oligotrophic groundwater. Using thiosulfate and H2 as electron donors, we established a chemolithoautotrophic enrichment culture from groundwater of a carbonate-rock aquifer to get more insight into the metabolic repertoire, substrate turnover, and transcriptional activity of subsurface denitrifying consortia. The enriched consortium was dominated by representatives of the genus Thiobacillus along with denitrifiers related to Sulfuritalea hydrogenivorans, Sulfuricella denitrificans, Dechloromonas sp. and Hydrogenophaga sp., representing the consortium's capacity to use multiple inorganic electron donors. Microcosm experiments coupled with Raman gas spectroscopy demonstrated complete denitrification driven by reduced sulfur compounds and hydrogen without formation of N2O. The initial nitrate/thiosulfate ratio had a strong effect on nosZ transcriptional activity and on N2 formation, suggesting similar patterns of the regulation of gene expression as in heterotrophic denitrifiers. Sequence analysis targeting nirS and nosZ transcripts identified Thiobacillus denitrificans-related organisms as the dominant active nirS-type denitrifiers in the consortium. An additional assessment of the nirS-type denitrifier community in the groundwaterclearly confirmed the potential for sulfur- and hydrogen-dependent chemolithoautotrophic denitrification as important metabolic feature widely spread among subsurface denitrifiers at the Hainich Critical Zone Exploratory