Insights into the mechnisms of the differential abilities of two N2O reducers, Pseudomonas sp., isolated from paddy soil

Microbial reduction of nitrous oxide (N 2 O) in soil plays an important role in mitigating N 2 O emission, and it is the only known biological process for N 2 O sink. However, it is not clear about the mechnisms of differential N 2 O reducing function of N 2 O reducers. In this study, the N 2 O reducing activities and nosZ gene transcript abundance of two N 2 O reducers named P. veronii DM15 (DM15) and P. frederiksbergensis DM22 (DM22) were determined under varied temperature and oxygen concentration conditions, as well as the whole genomes were sequenced by Illumina sequencing. The results showed that DM15 generally exhibited significantly higher abilities in N 2 O reduction than DM22 in regardless of low or high temperature and aerobic or anaerobic conditions. Coincidently, DM15 expressed significantly more nosZ gene transcripts under above environments. Genomic analysis further revealed that DM15 possessed about 30% more transcription related genes than DM22 and the nos cluster of the former contained a transcriptional regulator gene of dnr which not found in that of the later. Additionally, the nos genes of DM15 possessed obviously higher expression potentials (CAI value). In conclusion, the transcriptional regulation of nos gene region would be a crucial factor in determining the differences of N 2 O reducing abilities of the two N 2 O reducing isolates.

Effect of straw incorporation and nitrification inhibitor on nitrous oxide emission in various cropland soils and its microbial mechanism

Nitrification inhibitor and straw incorporation are widely used to improve crop nitrogen use efficiency in agricultural soil, but their effects on nitrous oxide (N2O) emission across different soil types and the microbial mechanisms remain less understood. In this study, we used controlled experiment and DNA-based molecular analysis to study how nitrification inhibitor (dicyandiamide, DCD) and straw incorporation affect soil nitrogen balance, N2O emission and microbial nitrifiers/denitrifers in three distinct agricultural soils (the black, fluvo-aquic and red soils) across China. Both DCD and straw incorporation improved nitrogen balance by increasing NH4+ and decreasing NO3- in all soils. DCD tended to decrease N2O emission from all soils especially the Fluvo-aquic one, while straw incorporation reduced N2O emission only in the fluvo-aquic soil but increased N2O emission in the other two especially the red soil (by ~600%). T-RFLP analysis revealed that the denitriers community structure are distinct among the three soils, but was not strongly affected by DCD or straw incorporation. qPCR analysis revealed that DCD or straw incorporation had no significant effect on nitrifier abundance, but increased nitrous oxide reductase nosZ gene abundance in the black/fluvo-aquic soil rather than the red soil. Structural equational modelling further confirmed that, when accounting for treatments and soil properties, nosZ gene abundance is the only biological factor significantly determined N2O emission in different soil types. Taken together, our work advanced the knowledge on the agricultural practices and N2O emission in cropland soils, and suggested that straw incorporation may not be a good choice for the red and black soil areas; management practices should be used as per soil type to balance between nitrogen use efficiency and N2O emission.

Groundwater Nitrate Removal Performance of Selected Pseudomonas Strains Carrying nosZ Gene in Aerobic Granular Sequential Batch Reactors

Four granular sequencing batch reactors (GSBRs) were inoculated with four denitrifying Pseudomonas strains carrying nosZ to study the process of granule formation, the operational conditions of the bioreactors, and the carbon concentration needed for nitrate removal. The selected Pseudomonas strains were P. stutzeri I1, P. fluorescens 376, P. denitrificans Z1, and P. fluorescens PSC26, previously reported as denitrifying microorganisms carrying the nosZ gene. Pseudomonas denitrificans Z1 produced fluffy, low-density granules, with a decantation speed below 10 m h−1. However, P. fluorescens PSC26, P. stutzeri I1, and P. fluorescens 376 formed stable granules, with mean size from 7 to 15 mm, related to the strain and carbon concentration. P. stutzeri I1 and P. fluorescens 376 removed nitrate efficiently with a ratio in the range of 96%, depending on the source and concentration of organic matter. Therefore, the findings suggest that the inoculation of GSBR systems with denitrifying strains of Pseudomonas spp. containing the nosZ gene enables the formation of stable granules, the efficient removal of nitrate, and the transformation of nitrate into nitrogen gas, a result of considerable environmental interest to avoid the generation of nitrous oxide.

Seasonal comparison of potential denitrification rates and nitrogen functional genes with sediment depths in the wetland, Korea

<p>Wetlands provide not only habitats for a wide range of organisms but also ecological functions of degrading and removing pollutants from water body through a variety of physical, chemical and biological processes. Seasonal variation including recent increases in the frequency of floods and droughts have affected the hydrological environment of wetlands. Furthermore, these effects may result in changes in redox conditions in the nitrogen biogeochemical process in wetland sediments. Therefore, in this study, the potential denitrification rate and denitrification-related gene quantitative analysis was performed to investigate seasonal nitrogen dynamics of wetland sediments associated with surface and groundwater interactions in Baekseok reservoir wetlands (Gunsan-si, Jeollabuk Province, Korea). Sediment from two different sites (i.e., PA and PB) in wetland were collected with different depths in June and December 2019 to investigate seasonal effects on denitrification with sediment depths. Potential denitrification rate experiments were performed using the acetylene inhibition technique, and denitrification-related gene quantification was performed by qPCR analysis. As a result of potential denitrification rate, PA sites ranged from 2.67–3.27 ng N<sub>2</sub>O/g/hr and 3.13–15.13 ng N<sub>2</sub>O/g/hr in June and December, respectively. PB sites ranged from 2.43-6.30 ng N<sub>2</sub>O/g/hr and 5.47-6.30 ng N<sub>2</sub>O/g/hr. Overall, higher levels were observed at 0–10 cm, with higher denitrification rates in December than in June. The qPCR analysis showed that the narG, nirS and nosZ gene copy number ranges for the PA site in June showed 1.82 x 10<sup>6</sup> – 6.15 x 10<sup>7</sup> copies/g, and in December 7.71 x 10<sup>5</sup> – 5.97 x 10<sup>8</sup> copies/g. The narG, nirS and nosZ gene copy number ranges for the PB site in June showed 3.53 x 10<sup>5</sup> – 3.86 x 10<sup>8</sup> copies/g, and in December, 1.24 x 10<sup>6</sup> – 3.47 x 10<sup>8</sup> copies/g. Overall, both sites had higher copy numbers in December than in June, corresponding to an increase in potential denitrification rate in December.</p>

Biochar Enhances Nitrous Oxide Reduction in Acidic but Not in Near-Neutral pH Soil

We quantified nitrous oxide (N2O) fluxes and total denitrification (N2O + N2) in an acidic (Ferralsol) and a near-neutral pH soil (Cambisol) to determine whether biochar’s alkalinization effect could be the mechanism inducing potential reductions in N2O fluxes. In Ferralsol, decreases in N2O emissions and in the N2O to N2O + N2 ratio were observed in both biochar and lime treatments. In Cambisol, neither biochar nor lime decreased N2O emissions, despite significantly increasing soil pH. The abundance and community structure of nosZ gene-bearing microorganisms indicated that gene abundances did not explain biochar effects, but a higher diversity of nosZ gene-bearing microorganisms correlated to lower total denitrification. Overall, our results suggest that biochar’s potential to decrease N2O emissions, through soil alkalinization, may be more effective in acidic soils.

A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase fromPseudomonas stutzeriin transgenic plants

Wan, S., Greenham, T., Goto, K., Mottiar, Y., Johnson, A. M., Staebler, J. M., Zaidi, M. A., Shu, Q. and Altosaar, I. 2014. A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase from Pseudomonas stutzeri in transgenic plants. Can. J. Plant Sci. 94: 1013–1025. As a stable greenhouse gas, nitrous oxide (N2O) plays a significant role in stratospheric ozone destruction. The primary anthropogenic N2O source is the use of nitrogen in agriculture. Currently, the annual N2O emissions from this soil–plant–microbial system is more than 2.6 Tg (1 Tg=1 million metric tonnes) of N2O-N globally. So it is important to explore some innovative and effective biology-based strategies for N2O mitigation. If shown to be effective in field trails as well as laboratory-scale experiments, such GMO plants could help guide international policies on adaptation to climate change. The bacterial enzyme nitrous oxide reductase (N2OR) is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. To “scrub” the N2O emissions, bacterial N2OR was heterologously expressed in plants. Structurally, the enzyme N2OR is encoded by nosZ, but its biosynthesis and assembly in prokaryotes require the products of several nos genes, including a putative ABC-type transporter encoded by nosDFY, and the copper chaperone NosL for biogenesis of the metal centre. We have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other nos genes were co-expressed under the control of a root-specific promoter (rolD) and a constitutive promoter (d35S). The nosZ gene from Pseudomonas stutzeri heterologously expressed in tobacco produced active recombinant N2OR. The positive results in the preliminary proof-of-principle experiments indicated that plants heterologously expressing N2OR could mitigate emissions at the source before N2O reaches the stratosphere or troposphere.