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.

Differential expression of clade I and II N2O reductase genes in denitrifying Thauera linaloolentis 47LolT under different nitrogen conditions.

Nitrous oxide (N2O) is a potent greenhouse gas and its reduction to dinitrogen gas by the N2O reductase (encoded by the nosZ gene) is the only known biological N2O sink. Within the nosZ phylogeny there are two major clades (I and II), which seem to have different ecological niches. However, physiological differences of nosZI and nosZII expression that may impact emissions of N2O are not well understood. Here, we evaluated the differential expression of nosZI and nosZII, both present in Thauera linaloolentis strain 47LolT, in response to N2O concentration and the presence of the competing electron acceptor nitrate (NO3-). Different N2O levels had a negligible effect on the expression of both nosZ clades. Interestingly, nosZII expression was strongly upregulated in the absence of NO3-, while nosZI expression remained constant across the conditions tested. Thus, NO3- possibly inhibited nosZII expression, which suggests that N2O mitigation mediated by nosZII can be restricted due to the presence of NO3- in the environment. This is the first study demonstrating differential expression of nosZI and nosZII genes under the same physiological conditions and their implications for N2O emission under varying environmental conditions in terms of NO3- availability.

Habitat diversity and type govern potential nitrogen loss by denitrification in coastal sediments and differences in ecosystem-level diversities of disparate N2O reducing communities.

In coastal sediments, excess nitrogen is removed primarily by denitrification. However, losses in habitat diversity may reduce the functional diversity of microbial communities that drive this important filter function. We examined how habitat type and habitat diversity affects denitrification and the abundance and diversity of denitrifying and N2O reducing communities in illuminated shallow-water sediments. In a mesocosm experiment, cores from four habitats were incubated in different combinations, representing ecosystems with different habitat diversities. We hypothesized that habitat diversity promotes the diversity of N2O reducing communities and genetic potential for denitrification, thereby influencing denitrification rates. We also hypothesized that this will depend on the identity of the habitats. Habitat diversity positively affected ecosystem-level diversity of clade II N2O reducing communities, however neither clade I nosZ communities nor denitrification activity were affected. The composition of N2O reducing communities was determined by habitat type, and functional gene abundances indicated that silty mud and sandy sediments had higher genetic potentials for denitrification and N2O reduction than cyanobacterial mat and Ruppia maritima meadow sediments. These results indicate that loss of habitat diversity and specific habitats could have negative impacts on denitrification and N2O reduction, which underpin the capacity for nitrogen removal in coastal ecosystems.

Expression of nirK and nirS genes in two strains of Pseudomonas stutzeri harbouring both types of NO-forming nitrite reductases.

Reduction of nitrite to nitric oxide in denitrification is catalysed by two different nitrite reductases, encoded by nirS or nirK. Long considered mutually exclusive and functionally redundant in denitrifying bacteria, we show expression of both genes co-occurring in Pseudomonas stutzeri. The differential expression patterns between strain AN10 and JM300 in relation to oxygen and nitrate and their different denitrification phenotypes, with AN10 reducing nitrate more rapidly and accumulating nitrite, suggest that nirS and nirK can have different roles. Dissimilar gene arrangements and transcription factors in the nir gene neighbourhoods could explain the observed differences in gene expression and denitrification activity.

Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N2 O respiring bacteria.

Nitrous oxide (N2 O) reducing microorganisms may be key in the mitigation of N2 O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2 O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (Ks ) for N2 O. We compared N2 O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2 O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N2 O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2 O, defined by µmax /Ks , in organisms with nosZ clade II.

Life on N2O: deciphering the ecophysiology of N2O respiring bacterial communities in a continuous culture.

Reduction of the greenhouse gas N2O to N2 is a trait among denitrifying and non-denitrifying microorganisms having an N2O reductase, encoded by nosZ. The nosZ phylogeny has two major clades, I and II, and physiological differences among organisms within the clades may affect N2O emissions from ecosystems. To increase our understanding of the ecophysiology of N2O reducers, we determined the thermodynamic growth efficiency of N2O reduction and the selection of N2O reducers under N2O- or acetate-limiting conditions in a continuous culture enriched from a natural community with N2O as electron acceptor and acetate as electron donor. The biomass yields were higher during N2O limitation, irrespective of dilution rate and community composition. The former was corroborated in a continuous culture of Pseudomonas stutzeri and was potentially due to cytotoxic effects of surplus N2O. Denitrifiers were favored over non-denitrifying N2O reducers under all conditions and Proteobacteria harboring clade I nosZ dominated. The abundance of nosZ clade II increased when allowing for lower growth rates, but bacteria with nosZ clade I had a higher affinity for N2O, as defined by µmax/Ks. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N2O respiration under growth-limited conditions.

Design and evaluation of primers targeting genes encoding NO-forming nitrite reductases: implications for ecological inference of denitrifying communities.

The detection of NO-forming nitrite reductase genes (nir) has become the standard when studying denitrifying communities in the environment, despite well-known amplification biases in available primers. We review the performance of 35 published and 121 newly designed primers targeting the nirS and nirK genes, against sequences from complete genomes and 47 metagenomes from three major habitats where denitrification is important. There were no optimal universal primer pairs for either gene, although published primers targeting nirS displayed up to 75% coverage. The alternative is clade-specific primers, which show a trade-off between coverage and specificity. The test against metagenomic datasets showed a distinct performance of primers across habitats. The implications of clade-specific nir primers choice and their performance for ecological inference when used for quantitative estimates and in sequenced-based community ecology studies are discussed and our phylogenomic primer evaluation can be used as a reference along with their environmental specificity as a guide for primer selection. Based on our results, we also propose a general framework for primer evaluation that emphasizes the testing of coverage and phylogenetic range using full-length sequences from complete genomes, as well as accounting for environmental range using metagenomes. This framework serves as a guideline to simplify primer performance comparisons while explicitly addressing the limitations and biases of the primers evaluated.

Habitat partitioning of marine benthic denitrifier communities in response to oxygen availability.

Denitrification is of global significance for the marine nitrogen budget and the main process for nitrogen loss in coastal sediments. This facultative anaerobic respiratory pathway is modular in nature and the final step, the reduction of nitrous oxide (N2 O), is performed by microorganisms with a complete denitrification pathway as well as those only capable of N2 O reduction. Fluctuating oxygen availability is a significant driver of denitrification in sediments, but the effects on the overall N2 O-reducing community that ultimately controls the emission of N2 O from marine sediments is not well known. To investigate the effects of different oxygen regimes on N2 O reducing communities, coastal marine surface sediment was incubated in microcosms under oxic, anoxic or oscillating oxygen conditions in the overlying water for 137 days. Quantification of the genetic potential for denitrification, anammox and respiratory ammonification indicated that denitrification supported nitrogen removal in these sediments. Furthermore, denitrifiers with a complete pathway were identified as the dominant community involved in N2 O reduction, rather than organisms that are only N2 O reducers. Specific lineages within each group were associated with different oxygen regimes suggesting that oxygen availability in the overlying water is associated with habitat partitioning of N2 O reducers in coastal marine surface sediments.