Effects of Nitrogen Input on Community Structure of the Denitrifying Bacteria with Nitrous Oxide Reductase Gene (nosZ I): A Long-Term Pond Experiment

Excessive nitrogen (N) input is an important factor influencing aquatic ecosystems and has received increasing public attention in the past decades. It remains unclear, however, how N input affects the denitrifying bacterial communities that play a key role in regulating N cycles in various ecosystems. To test our hypothesis – that the abundance and biodiversity of denitrifying bacterial communities decrease with increasing N – we compared the abundance and composition of denitrifying bacteria having nitrous oxide reductase gene (nosZ I) from sediments (0-20 cm) in five experimental ponds with different nitrogen fertilization treatment (TN10, TN20, TN30, TN40, TN50) using quantitative PCR and pyrosequencing techniques. We found that: 1) N addition significantly decreased nosZ I gene abundance, 2) the Invsimpson and Shannon indices (reflecting biodiversity) first increased significantly along with the increasing N loading in TN10~TN40 followed by a decrease in TN50, 3) the beta diversity of the nosZ I denitrifier was clustered into three groups along the TN concentration levels: Cluster I (TN50), Cluster II (TN40), and Cluster III (TN10-TN30), 4) the proportions of Alphaproteobacteria and Betaproteobacteria in the high-N treatment (TN50) were significantly lower than in the lower N treatments (TN10-TN30). 5). The TN concentration was the most important factor driving the alteration of denitrifying bacteria assemblages. Our findings shed new light on the response of denitrification-related bacteria to long-term N loading at pond scale and on the response of denitrifying microorganisms to N pollution.

Nitrogen and Sulfur Additions Improved the Diversity of nirK- and nirS-Type Denitrifying Bacterial Communities of Farmland Soil

Anthropogenic nitrogen (N) and sulfur (S) deposition can change above- and belowground biodiversity, including soil microbial diversity. The diversity of denitrifying microorganisms is of great significance to the calculation of the global nitrogen cycle and nitrogen flux. For a long time, nirK and nirS have been used as the functional genes to study denitrifying microorganisms, and have gradually become molecular markers for studying the composition and diversity of denitrifying bacteria. Here, three-time exposures to N and S applications (7, 30, and 60 days), were independently established. Additionally, the abundance, diversity, and structure of nirK- and nirS-type denitrifying communities were examined by sequencing analyses in response to three treatments, namely, N and S (TN/S), sodium chloride (TNaCl) and deionized water (pH = 7.0) (CK). Our results suggest that TN/S led to higher electrical conductivity (EC), total nitrogen (TN), total organic carbon (TOC), nitrate nitrogen (NO3−-N), ammonium nitrogen (NH4+-N), and lower pH compared with TNaCl and CK, which affected the diversity of nirK- and nirS-type denitrifying bacterial communities. We also observed that the nirK-type denitrifying community demonstrated a higher sensitivity to N and S additions. Overall, our results are important for the understanding of nitrogen in soil and N2O emissions.

Effect of biochar particles applied in bedding course of the innovative permeable pavement on enhancing nitrogen removal

Coconut shell biochar and bamboo biochar were added to the bedding courses of permeable pavements to improve nitrogen removal efficiency. This was expected to be particularly effective for innovative permeable pavements that increase evaporation of stored rainwater. The effects of the rainfall intensity and ambient temperature on nitrogen removal were assessed. The NO3−-N removal rate for the pavements with biochar added and the blank controls were 48.6%–54.0% and 10.8%, respectively, at a rainfall intensity of 10 mm/h and temperatures of 22–28 °C. The total nitrogen removal rates for the pavements with biochar added and the blank controls were 52.6%–57.7% and 20%, respectively. Adding coconut shell and bamboo biochar improved denitrification without causing organic matter to be leached. Increasing the rainfall intensity and decreasing the temperature caused less nitrogen to be removed. The total nitrogen balance indicated that the innovative pavements and conventional pavements with the same biochar amendments removed 781.58 and 733.30 mg of nitrogen, respectively, suggesting that adding biochar improved the performances of the innovative pavements more than the conventional pavements. Quantitative polymerase chain reaction assays of bedding course samples indicated that adding biochar markedly increased the abundances of denitrifying microorganisms.

Effects of Cultivation Years in Protected Vegetable Crop Fields On The Structure and Abundance of Soil nosZ Denitrifying Microbial Community

The study of the changes in the structure and quantity of the denitrifying microbial community in the protected vegetable crop field is crucial for evaluating the soil quality of long-term protected cultivation and improving the understanding of nitrogen conversion. In this study, quantitative fluorescent PCR was used, with the help of the Illumina Miseq high-throughput sequencing platform, aiming at the nosZ gene, to study the effects of protected cultivation plots 3 a, 5 a, and 7 a, and the outdoor cultivation plot (ck) on the structure and quantity of soil denitrifying microbial community. The results demonstrated that the abundance of nosZ gene in ck was significantly higher than the other treatments, and was 1.32, 1.45 and 1.69 times those of Group 3 a, 5 a and 7 a, respectively. As the cultivation year increased, the abundance of nosZ gene was decreased, and the α-diversity index was decreased. The Chao1 index and ACE index of ck were the highest. At the phylum level, the dominant group was Proteobacteria. While at the genus level, the dominant groups were Bradyrhizobium and Achromobacter. The relative abundances of Proteobacteria and Bradyrhizobium were decreased with the increase of protected cultivation time. The principal component analysis (PCA) results showed that the community structure of nosZ varied greatly with the increase of planting time. The community structures of Group 3 a and 5 a were similar, but the community structure of Group 7 a was very different from Group 3 a and 5 a. The soil available potassium (AK), ammonium nitrogen and nitrate nitrogen were the primary factors affecting the copy number, α-diversity and community structure of nosZ denitrifying microorganisms. As a conclusion, it can be seen that long-term cultivation of vegetable crops in a protected environment significantly reduced the copy number of nosZ denitrifying microorganisms and had a significant impact on the community structure.

Effects of Organic Fertilizers on the Soil Microorganisms Responsible for N2O Emissions: A Review

The use of organic fertilizers constitutes a sustainable strategy to recycle nutrients, increase soil carbon (C) stocks and mitigate climate change. Yet, this depends largely on balance between soil C sequestration and the emissions of the potent greenhouse gas nitrous oxide (N2O). Organic fertilizers strongly influence the microbial processes leading to the release of N2O. The magnitude and pattern of N2O emissions are different from the emissions observed from inorganic fertilizers and difficult to predict, which hinders developing best management practices specific to organic fertilizers. Currently, we lack a comprehensive evaluation of the effects of OFs on the function and structure of the N cycling microbial communities. Focusing on animal manures, here we provide an overview of the effects of these organic fertilizers on the community structure and function of nitrifying and denitrifying microorganisms in upland soils. Unprocessed manure with high moisture, high available nitrogen (N) and C content can shift the structure of the microbial community, increasing the abundance and activity of nitrifying and denitrifying microorganisms. Processed manure, such as digestate, compost, vermicompost and biochar, can also stimulate nitrifying and denitrifying microorganisms, although the effects on the soil microbial community structure are different, and N2O emissions are comparatively lower than raw manure. We propose a framework of best management practices to minimize the negative environmental impacts of organic fertilizers and maximize their benefits in improving soil health and sustaining food production systems. Long-term application of composted manure and the buildup of soil C stocks may contribute to N retention as microbial or stabilized organic N in the soil while increasing the abundance of denitrifying microorganisms and thus reduce the emissions of N2O by favoring the completion of denitrification to produce dinitrogen gas. Future research using multi-omics approaches can be used to establish key biochemical pathways and microbial taxa responsible for N2O production under organic fertilization.

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.

Key role of overlooked twilight zone towards climate buffering

<p>The twilight zone of the oceans layering between the bottom of the sunlit ocean and 1000 m depth, is one of the largest continuous ecosystems on the Earth, yet remains least explored. While the sunlit ocean is well-studied for its major role in sequestering CO<sub>2 </sub>from the atmosphere, the role of twilight zone in CO<sub>2 </sub>sequestration remains a mystery. The twilight zone of the Arabian Sea, north-western part of the Indian Ocean inarguably possesses an active nitrogen‐cycle owing to abundant chemoautotrophic (anammox, nitrite oxidising, nitrifying) microorganisms and heterotrophic (denitrifying) microorganisms. However, these microorganisms with ramifications for the nitrogen cycle, incentivize the carbon cycle. Since chemoautotrophy is a light-independent autotrophic process, a significant amount of dissolved CO<sub>2</sub> may be assimilated rather than released in the Arabian Sea twilight zone by these organisms. With this supposition, we commenced the expedition in the off-shore and the central Arabian Sea during winter monsoon (Dec-Jan 2019) to measure carbon fixation rates in its sunlit and twilight zone using <sup>13</sup>C tracer incubation technique. The sunlit zone and twilight zone carbon fixation rates ranged from 6.8 to 40 mmol C m<sup>-2</sup> d<sup>-1</sup> and 0.4 to1.6 mmol C m<sup>-2</sup> d<sup>-1</sup>, respectively. The twilight zone carbon fixation did not vary spatially much, unlike sunlit zone which showed a sharp decreasing trend of carbon fixation from northern to the southern Arabian Sea. Notably, the twilight zone contribution to water column carbon fixation ranged from 2 to 10% during the study period. This study corroborates that the twilight zone forms an integral component of the carbon cycle; implying, the overlooked twilight zone can significantly contribute CO<sub>2</sub> drawdown. Therefore, the role of twilight zone towards climate buffering is bigger than previously assumed, demanding a review of its role in the current paradigm of the Earth’s climate.</p>

The influence of the denitrifying strain of Staphylococcus carnosus No. 5304 on the content of nitrates in the technology of yogurt production

Contamination of food with nitrates is a generally recognized problem. Milk is the basis for the production of many milk mixtures for baby food, and children are considered to be the most vulnerable category to the harmful influence of nitrates. The purpose of the search was to investigate the denitrification of milk with different amounts of nitrates by the denitrifying microorganisms of Staphylococcus carnosus in the technology of production of sour-milk products. The denitrification process of S. carnosus milk in the amount of 103 CFU.cm-3 was found to reduce the nitrate content by an average of 88.0 ±3.9 mg.kg-1 and in the samples of the first group was 10.3 ±2.4 mg.kg-1, the second 110.7 ±4.1 and the third 214.5 ±6.3 mg.kg-1, respectively. In the search of the denitrification process of S. carnosus milk in the amount of 104 CFU.cm-3, was found that in the ready yogurt in the samples of the first group the amount of nitrates was 1.1 ±0.1 mg.kg-1, in the second group 56.4 ±3.5 mg.kg-1, and in the third 159.5 ±4.1 mg.kg-1 respectively. In the search of the denitrification process of S. carnosus milk in the amount of 105 CFU.cm-3, was found that nitrates were practically absent in the samples of the first group, the second group did not exceed 10 mg.kg-1, and the third was 107.4 ±3.9 mg.kg-1. Therefore, received data indicate the possibility of using strain S. carnosus No. 5304 for denitrification of milk with a high content of nitrates in the technology of production of fermented milk products, in particular yogurt.

The removal performance of nitrates in the novel 3D-BERS with GAC and diversity of immobilized microbial communities treating nitrate-polluted water: Effects of pH and COD/NO3--N ratio

In this work, a three-dimensional bioelectrochemical reactor system (3D-BERs) with granular activated carbon (GAC) was utilized to study the feasibility of simultaneous removal of nitrates by autotrophic-heterotrophic denitrification process under different pH levels. In this present study, it was found that when the influent COD/ NO3--N ratio ranged between 1.5 and 3.5, both autotrophic and heterotrophic denitrifying microorganisms played an important role in denitrification. The experimental results demonstrated that the highest removal efficiency of nitrates under the optimum COD/NO3--N ratio of 1.5 (98.62%) was achieved with an initial pH of 7.5 ± 0.4. Likewise, when the COD/NO3--N ratio of 3.5, the nitrates removal efficiency (81.12%) was achieved with an initial pH of 8.2 ± 0.3, respectively. Batch denitrification processes followed zero-order kinetics at various NO3--N concentrations obtained. The bacterial community structure and relative abundance of bacteria changed at the level of genes and the phylum of immobilized GAC particles. Moreover, the diversity of bacterial composition enhanced the removal of NO3--N at the inner surface (IS), and bottom surface (BS) of immobilized GAC carriers were Gammaproteobacteria, Bacilli, Proteobacteria, and Thauera. In general, this technique is more effective for enhancing the denitrification process in the 3D-BER system.