A Dual Enrichment Strategy Provides Soil- and Digestate-Competent Nitrous Oxide-Respiring Bacteria for Mitigating Climate Forcing in Agriculture.

Manipulating soil metabolism through heavy inoculation with microbes is feasible if organic wastes can be utilized as the substrate for growth and vector as a fertilizer. This, however, requires organisms active in both digestate and soil (generalists). Here, we present a dual enrichment strategy to enrich and isolate such generalists among N2O-respiring bacteria (NRB) in soil and digestates, to be used as an inoculum for strengthening the N2O-reduction capacity of soils. The enrichment strategy utilizes sequential batch enrichment cultures alternating between sterilized digestate and soil as substrates, with each batch initiated with limited O2 and unlimited N2O. The cultures were monitored for gas kinetics and community composition. As predicted by a Lotka-Volterra competition model, cluster analysis identified generalist operational taxonomic units (OTUs) which became dominant, digestate/soil-specialists which did not, and a majority that were gradually diluted out. We isolated several NRBs circumscribed by generalist OTUs. Their denitrification genes and phenotypes predicted a variable capacity to act as N2O-sinks, while all genomes predicted broad catabolic capacity. The latter contrasts with previous attempts to enrich NRB by anaerobic incubation of unsterilized digestate only, which selected for organisms with a catabolic capacity limited to fermentation products. The two isolates with the most promising characteristics as N2O sinks were a Pseudomonas sp. with a full-fledged denitrification-pathway and a Cloacibacterium sp. carrying only N2O reductase (clade II), and soil experiments confirmed their capacity to reduce N2O-emissions from soil. The successful enrichment of NRB with broad catabolic spectra suggests that the concept of dual enrichment should also be applicable for enrichment of generalists with traits other than N2O reduction. IMPORTANCE N2O emissions from farmed soils are a major source of climate forcing. Here, denitrifying bacteria act as both source and sink for N2O, determined by regulatory traits or the absence of genes coding for the enzymes producing or reducing N2O. One approach to reducing emissions is to amend large numbers of N2O-reducing bacteria (NRB) to soil. This was shown to be feasible by growing NRB to high densities in organic wastes and then applying them as fertilizers. The effect on N2O emissions, however, was transient because the isolated NRBs were unsuited to soil. Here, we have developed an enrichment strategy selecting for organisms with generalist lifestyles, tolerant of rapid environmental changes. This was used to isolate robust NRBs that grow both in digestate and when amended to soils. This strategy opens an avenue for obtaining not just robust NRBs to reduce N2O emissions, but any organism destined for application to complex environments.

Genotypic and phenotypic characterization of hydrogenotrophic denitrifiers.

Stimulating litho-autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO3 - , but it often entails accumulation of the cytotoxic intermediate NO2 - and the greenhouse gas N2 O. To explore if these high NO2 - and N2 O concentrations are caused by differences in the genomic composition, the regulation of gene transcription or the kinetics of the reductases involved, we isolated hydrogenotrophic denitrifiers from a polluted aquifer, performed whole-genome sequencing and investigated their phenotypes. We therefore assessed the kinetics of NO2 - , NO, N2 O, N2 and O2 as they depleted O2 and transitioned to denitrification with NO3 - as the only electron acceptor and hydrogen as the electron donor. Isolates with a complete denitrification pathway, although differing intermediate accumulation, were closely related to Dechloromonas denitrificans, Ferribacterium limneticum or Hydrogenophaga taeniospiralis. High NO2 - accumulation was associated with the reductases' kinetics. While available, electrons only flowed towards NO3 - in the narG-containing H. taeniospiralis but flowed concurrently to all denitrification intermediates in the napA-containing D. denitrificans and F. limneticum. The denitrification regulator RegAB, present in the napA strains, may further secure low intermediate accumulation. High N2 O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N2 O reductase expression.

Complete Genome Sequences of Hydrogenotrophic Denitrifiers.

Hydrogenotrophic denitrifiers are important bacteria for nitrate removal in wastewater and aquifers. Here, we report the complete genome sequences of three hydrogenotrophic denitrifiers, namely, Dechloromonas denitrificans strain D110, Ferribacterium limneticum strain F76, and Hydrogenophaga taeniospiralis strain H3, all of which were isolated from a nitrate-polluted aquifer in Bavaria (Germany).

Strategies to Overcome Intermediate Accumulation During in situ Nitrate Remediation in Groundwater by Hydrogenotrophic Denitrification.

Bioremediation of polluted groundwater is one of the most difficult actions in environmental science. Nonetheless, the clean-up of nitrate polluted groundwater may become increasingly important as nitrate concentrations frequently exceed the EU drinking water limit of 50 mg L-1, largely due to intensification of agriculture and food production. Denitrifiers are natural catalysts that can reduce increasing nitrogen loading of aquatic ecosystems. Porous aquifers with high nitrate loading are largely electron donor limited and additionally, high dissolved oxygen concentrations are known to reduce the efficiency of denitrification. Therefore, denitrification lag times (time prior to commencement of microbial nitrate reduction) up to decades were determined for such groundwater systems. The stimulation of autotrophic denitrifiers by the injection of hydrogen into nitrate polluted regional groundwater systems may represent a promising remediation strategy for such environments. However, besides high costs other drawbacks, such as the transient or lasting accumulation of the cytotoxic intermediate nitrite or the formation of the potent greenhouse gas nitrous oxide, have been described. In this article, we detect causes of incomplete denitrification, which include environmental factors and physiological characteristics of the underlying bacteria and provide possible mitigation approaches.

Dechloromonas and close relatives prevail during hydrogenotrophic denitrification in stimulated microcosms with oxic aquifer material.

Globally occurring nitrate pollution in groundwater is harming the environment and human health. In situ hydrogen addition to stimulate denitrification has been proposed as a remediation strategy. However, observed nitrite accumulation and incomplete denitrification are severe drawbacks that possibly stem from the specific microbial community composition. We set up a microcosm experiment comprising sediment and groundwater from a nitrate polluted oxic oligotrophic aquifer. After the microcosms were sparged with hydrogen gas, samples were taken regularly within 122 h for nitrate and nitrite measurements, community composition analysis via 16S rRNA gene amplicon sequencing and gene and transcript quantification via qPCR of reductase genes essential for complete denitrification. The highest nitrate reduction rates and greatest increase in bacterial abundance coincided with a 15.3-fold increase in relative abundance of Rhodocyclaceae, specifically six ASVs that are closely related to the genus Dechloromonas. The denitrification reductase genes napA, nirS and clade I nosZ also increased significantly over the observation period. We conclude that taxa of the genus Dechloromonas are the prevailing hydrogenotrophic denitrifiers in this nitrate polluted aquifer and the ability of hydrogenotrophic denitrification under the given conditions is species-specific.

A comparison of dynamic tertiary and competition models for describing the fate of Listeria monocytogenes in Minas fresh cheese during refrigerated storage.

This study compares dynamic tertiary and competition models for L. monocytogenes growth as a function of intrinsic properties of a traditional Brazilian soft cheese and the inhibitory effect of lactic acid bacteria (LAB) during refrigerated storage. Cheeses were prepared from raw or pasteurized milk with or without the addition of selected LAB with known anti-listerial activity. Cheeses were analyzed for LAB and L. monocytogenes counts, pH and water activity (aw) throughout cold storage. Two approaches were used to describe the effect of LAB on L. monocytogenes: a Huang-Cardinal model that considers the effect of pH and aw variation in a dynamic kinetic analysis framework; and microbial competition models, including Lotka-Volterra and Jameson-effect variants, describing the simultaneous growth of L. monocytogenes and LAB. The Jameson-effect with γ and the Lotka-Volterra models produced models with statistically significant coefficients that characterized the inhibitory effect of selected LAB on L. monocytogenes in Minas fresh cheese. The Huang-Cardinal model [pH] outperformed both competition models. Taking aw change into account did not improve the fit quality of the Huang-Cardinal [pH] model. These models for Minas soft cheese should be valuable for future microbial risk assessments for this culturally important traditional cheese.