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

Abstract

Nitrous oxide (N O) reducing microorganisms may be key in the mitigation of N 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 N 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 (K ) for N O. We compared N O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N 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 N O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N O, defined by μ /K , in organisms with nosZ clade II.

O2 versus N2O respiration in a continuous microbial enrichment

“…Following the work presented in Conthe et al ( 2018b ), a microbial enrichment using acetate as a carbon and energy source and exogenous N 2 O as the sole electron acceptor was maintained under N 2 O-limiting conditions in a continuous culture at 20 °C, pH 7, and a dilution rate of 0.026 ± 0.001 h −1 . The reactor set-up, operation, sampling, and medium composition are described in detail in Conthe et al ( 2018b , c ).…”

“…Following the work presented in Conthe et al ( 2018b ), a microbial enrichment using acetate as a carbon and energy source and exogenous N 2 O as the sole electron acceptor was maintained under N 2 O-limiting conditions in a continuous culture at 20 °C, pH 7, and a dilution rate of 0.026 ± 0.001 h −1 . The reactor set-up, operation, sampling, and medium composition are described in detail in Conthe et al ( 2018b , c ). One hundred percent pure N 2 O gas diluted in Argon gas was fed to the chemostat at a total flow rate of 200 ml/min and the offgas from the reactor was recirculated at a rate of 700 ml/min, resulting in an incoming N 2 O concentration of roughly 0.30%.…”

“…Elemental and electron balances during steady state were set up as described in Conthe et al ( 2018a , b , c ). During the batch tests, the conversion rates (r, in mol h −1 ) for O 2 and N 2 O were calculated from the measured ingoing and outgoing gas composition and the argon supply rate (see Figures S2 – S6 and Tables S2 – S6 for details).…”

“…We explored O 2 versus N 2 O respiration in a continuous enrichment culture selected and grown with N 2 O as the sole electron acceptor and fully characterized—in terms of stoichiometry and community composition—in a previous study (Conthe et al 2018b ). The culture had been found to be composed of a relatively simple microbial community dominated by Dechlorobacter -like Betaproteobacteria .…”

Novel denitrifying bacteria Pseudomonas stutzeri strain D1 - from isolation to the biomass production

“…P. stutzeri is commonly isolated from groundwater, river, sea, wastewater [11][12][13], as well as from sludge and the ground [7,12]. Also, recent studies have been focused on enzyme systems and corresponding genes involved in biological denitrification processes in aerobic conditions [8,9,[14][15][16]. Generally, the reduction of nitrate to molecular nitrogen is catalyzed by the following enzymes: nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase [17].…”