PHÂN LẬP VÀ TUYỂN CHỌN NHÓM VI KHUẨN OXY HOÁ AMMONIA BẢN ĐỊA CÓ HOẠT TÍNH CAO TẠI QUẢNG NINH, HẢI PHÒNG

Ammonia trong môi trường nước là một trong những vấn đề chính dẫn đến thiệt hại trong nuôi trồng thuỷ sản. Ammonia là chất độc đối với tất cả các động vật có xương sống, ammonia là nguyên nhân gây co giật, hôn mê và tử vong. Quá trình phân lập và tuyển chọn các vi khuẩn nitrate hóa bản địa được hy vọng là giải pháp làm giảm nồng độ ammonia và cải thiện chất lượng nước. Nghiên cứu này đã phân lập và xác định được các vi khuẩn ở Quảng Ninh, Hải Phòng để khử ammonia. Vi khuẩn phân lập trong môi trường chọn lọc được định danh bằng phương pháp hoá sinh và phân tích trình tự gen 16S rRNA. Kết quả đã phân lập và tuyển chọn được 8 loài vi khuẩn nitrate có tiềm năng ứng dụng xử lý nước trong nuôi trồng thuỷ sản. Bằng phần mềm BLAST trên NCBI, trình tự gen 16S rRNA của các vi khuẩn nitrate phân lập được so sánh với các chủng vi khuẩn trên GenBank. Kết quả cho thấy, tỷ lệ tương đồng đều đạt hơn 96,71% so với các trình tự gốc trên ngân hàng gen thế giới. Các loài vi khuẩn nitrate hóa được phân lập bao gồm: Nitrosomonas marina, Nitrosomonas nitrosa, Nitrosomonas aesturii... trong đó có 2 loài vi khuẩn đặc trưng nhất là Nitrosomonas europaea và Nitrobacter Winogradskyi.

Transcriptomic Response of Nitrosomonas europaea Transitioned from Ammonia- to Oxygen-Limited Steady-State Growth

ABSTRACT Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea. Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea’s sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria. IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Transcriptomic response of Nitrosomonas europaea transitioned from ammonia- to oxygen-limited steady-state growth

Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g. by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea. Under oxygen-limited conditions, growth yield was reduced and ammonia to nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea’s sNOR with regards to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other AOB.ImportanceNitrification is a ubiquitous, microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments increasing the eutrophication of downstream aquatic ecosystems and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their response to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here we investigate the physiology of the best characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Effects of Exogenous N-Acyl-Homoserine Lactone as Signal Molecule on Nitrosomonas Europaea under ZnO Nanoparticle Stress

Despite the adverse effects of emerging ZnO nanoparticles (nano-ZnO) on wastewater biological nitrogen removal (BNR) systems being widely documented, strategies for mitigating nanoparticle (NP) toxicity impacts on nitrogen removal have not been adequately addressed. Herein, N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) was investigated for its effects against nano-ZnO toxicity to a model nitrifier, Nitrosomonas europaea. The results indicated that AHL-attenuated nano-ZnO toxicity, which was inversely correlated with the increasing dosage of AHL from 0.01 to 1 µM. At 0.01 µM, AHL notably enhanced the tolerance of N. europaea cells to nano-ZnO stress, and the inhibited cell proliferation, membrane integrity, ammonia oxidation rate, ammonia monooxygenase activity and amoA gene expression significantly increased by 18.2 ± 2.1, 2.4 ± 0.9, 58.7 ± 7.1, 32.3 ± 1.7, and 7.3 ± 5.9%, respectively, after 6 h of incubation. However, increasing the AHL dosage compromised the QS-mediated effects and even aggravated the NPs’ toxicity effects. Moreover, AHLs, at all tested concentrations, significantly increased superoxide dismutase activity, indicating the potential of QS regulations to enhance cellular anti-oxidative stress capacities when facing NP invasion. These results provide novel insights into the development of QS regulation strategies to reduce the impact of nanotoxicity on BNR systems.