Loss in soil microbial diversity constrains microbiome selection and alters the abundance of N-cycling guilds in barley rhizosphere

Plant roots are shaping microbial communities that are distinct from the surrounding soil. These root-associated microbial communities can have both positive and negative effects on the host nutrient acquisition and thereby growth, yet how loss of soil microbial diversity will constrain the plant microbiome selection is relatively unknown. In this study, we manipulated the soil microbial community using a removal-by-dilution approach to examine how microbial diversity modulates microbiome selection in barley, including microbial guilds involved in nitrogen (N) cycling processes causing N loss, and its consequences for plant performance. We found that microbial diversity loss reduced the barley's ability to recruit specific microorganisms from the soil and only members of the Alphaproteobacteria and Bacteroidetes were enriched in both rhizosphere and root-associated compartments irrespective of dilution level. Loss in soil microbial diversity and the presence of plants affected the N-cycling communities, with the abundance of nitrous oxide reducers being 2-4 times higher in both barley compartments in the lower diversity soils. In these soils, the low abundance of bacterial ammonia oxidizers (close or below detection level in the barley compartments) was concomitant with an increase in leaf greenness (ca. 12%), an indicator of the plant N status. The reduction in soil microbial diversity was thus coupled to a change in functional traits of rhizosphere and root-associated communities, with consequences for plant performance. This work contributes to our understanding of plant-microbe interactions, which is needed to steer the crop microbiome towards increased N-use efficiency while minimizing negative environmental impact

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

Denitrifying and nitrous oxide reducing genotypes

Denitrification is a biogeochemical process of major importance for nitrogen loss from ecosystems. This four-step pathway is modular, as organisms can have different subsets and variants of the genes involved in each step. The last step is the only known biological sink of nitrous oxide, a potent greenhouse gas and ozone depleting substance. The aim of this thesis was to assess whether specific environmental conditions favour certain denitrifying and nitrous oxide reducing genotypes. The effects of nitrogen, carbon and oxygen availabilities, as well as habitat type and diversity were examined in studies of denitrifying and nitrous oxide reducing microorganisms in pure cultures, enrichment cultures and natural communities from coastal sediments. By utilizing molecular techniques and directly targeting functional genes encoding for nitrite and nitrous oxide reductases, this work explores the link between genetic potential and functionality of denitrifying and nitrous oxide reducing microbial communities. Microorganisms harbouring genes for complete denitrification dominated in coastal marine sediments, irrespective of oxygen regime and habitat type, which suggests they have an important role not only for nitrogen removal, but also nitrous oxide reduction in coastal ecosystems. However, oxygen affected the nitrous oxide reducing communities. The results indicate niche differentiation between nitrous oxide reducers in relation to oxic/anoxic conditions, as specific lineages within the nitrous oxide reductase gene phylogeny were favoured by certain oxygen regimes. In enrichment cultures, complete denitrifiers were competitive nitrous oxide reducers and could outcompete organisms only capable of nitrous oxide reduction when subjected to carbon and nitrous oxide limitation. For denitrifiers, functional difference between the genes nirS and nirK encoding two structurally different nitrite reductases within the same organism was observed, corroborating that closely related and almost identical genotypes differ in their denitrification activity. Furthermore, primers targeting nitrite reducing communities harbouring either nirS or nirK were re-evaluated and clade-specific primers are suggested. Finally, a framework for primer evaluation using metagenomes is suggested. These results highlight the importance of accounting for the genetic potential of denitrifying and nitrous oxide reducing communities to better understand overall ecosystem constraints and how environmental factors might control this potential

Loss in soil microbial diversity constrains microbiome selection and alters the abundance of N-cycling guilds in barley rhizosphere

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Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N₂O respiring bacteria

Nitrous oxide (N2O) reducing microorganisms may be key in the mitigation of N2O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2O 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 N2O. We compared N2O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2O 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 N2O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2O, defined by μmax/Ks, in organisms with nosZ clade II.BT/Environmental Biotechnolog

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.BT/Environmental Biotechnolog