Molecular Screening of Nitrate Reductase Enzyme in Native Halophilic Bacteria of Iran

Journal of Cell and Molecular Research

مقاله 3، دوره 13، شماره 1 - شماره پیاپی 25، آذر 2021، صفحه 19-26   

نوع مقاله: Research Articles

شناسه دیجیتال (DOI): 10.22067/jcmr.2021.64301.0

نویسندگان

Saeedeh Sibevieh¹؛ Ensieh Salehghamari^{* 2}؛ Mohammad Ali Amoozegar³؛ Mohammad Reza Zolfaghari¹؛ Mohammad Soleimani⁴؛ Zohre Nasrollahzadeh⁵؛ Sara Eftekhari Yazdi³

¹Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran

²Department of Cell and Molecular Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

³Extremophiles Laboratory, Department of Microbiology, Faculty of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran

⁴Department of Microbiology, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran.

⁵Department of Cell and Molecular Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran,

چکیده

With the increased usage of nitrate fertilizers, the removal of their stable ionic and water-soluble end products is a challenge for human health. Several physicochemical methods have been examined for nitrate removal of water, but biological treatments are mostly preferred due to a higher efficiency and lower cost. To remove nitrogen from water, we investigated the potential of nitrate-reducing halophilic and halotolerant bacteria. A total of 50 strains from different saline and hypersaline environments of Iran, including the Incheboron wetland, Aran-Bidgol salt-lake, and Urmia endorheic salt-lake, were screened for nitrate reductase production. Among investigated bacteria, 60% and 19% of strains obtained from Urmia lake, and Incheboron wetland produced nitrate reductases, respectively. The nitrate reductase coding genes narG, and napA were analyzed in all strains with confirmed nitrate-reducing capacity. The napA gene was successfully amplified from a gram-negative halophilic strain, and the narG gene was detected in ten halophilic strains. Among nitrate-reducing isolates with the narG gene expression, the Kocuria rosea strain R3A34 showed the highest nitrate reductase production level. This strain was selected to optimize for its denitrifying activity. Results showed that 32°C, pH 7.0, NaCl 8% (w/v), and mannitol (as a carbon source) provide the optimal environmental conditions for the efficient production of nitrate reductase by the Kocuria rosea strain R3A34. As these are compatible with wastewaters conditions, this bacterium can be a proper candidate for bioremediation of wastewaters from nitrate pollutants.

کلیدواژه‌ها

Halophiles؛ Kocuria؛ napA؛ narG؛ Nitrate Reductase

مراجع

Argandona M., Martínez-Checa F., Llamas I., Arco Y., Quesada E., & Del Moral A. (2006). A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura. Extremophiles 10: 411-419.

Asamoto C. K., Rempfert K. R., Luu V. H., Younkin A. D. & Kopf S. H. (2021). Enzyme-Specific Coupling of Oxygen and Nitrogen Isotope Fractionation of the Nap and Nar Nitrate Reductases. Environmental Science and Technology, 55(8): 5537-5546.

Baggs E. M. (2011). Soil microbial sources of nitrous oxide: recent advances in knowledge, emerging challenges and future direction. Current Opinion in Environmental Sustainability 3: 321-327.

Baker S. C., Ferguson  S. J., Ludwig  B., Page  M. D., Richter O. M. H., & van Spanning  R. J. (1998). Molecular genetics of the genus paracoccus: metabolically versatile bacteria with bioenergetic flexibility. Microbiology and Molecular Biology Reviews  62:1046-1078.

Beeler E., & Singh O. V. (2016). Extremophiles as sources of inorganic bio-nanoparticles. World Journal of Microbiology and Biotechnology  32: 156-167.

Biswas  J., Bose P., Mandal  S., & Paul  A. K. (2018). Reduction of hexavalent chromium by a moderately halophilic bacterium, Halomonas smyrnensis KS802 under saline environment. Environmental Sustainability 1: 411-423.

Boon N  et al. (2018). Microbiome analysis and-omics studies of microbial denitrification processes in wastewater treatment: recent advances. Science China Life Sciences  61: 753-761.

Boujida  N., Palau  M., Charfi  S., Manresa À., Senhaji  N. S., Abrini  J., & Miñana-Galbis  D. (2019). Marinobacter maroccanus sp. nov., a moderately halophilic bacterium isolated from a saline soil. International Journal of Systematic and Evolutionary Microbiology 69: 227-234.

Cyplik  P., Grajek  W., Marecik  R., & Kroliczak  P. (2007). Effect of macro/micro nutrients and carbon source over the denitrification rate of Haloferax denitrificans archaeon. Enzyme and microbial technology 40: 212-220.

Duan J., Fang  H., Su  B., Chen  J., & Lin  J. (2015). Characterization of a halophilic heterotrophic nitrification–aerobic denitrification bacterium and its application on treatment of saline wastewater. Bioresource technology  179: 421-428.

Eltarahony M., Zaki S., & Abd-El-Haleem D. (2020). Aerobic and anaerobic removal of lead and mercury via calcium carbonate precipitation mediated by statistically optimized nitrate reductases. Science Report 10: 4029.

Gao  H., Yang  Z. K., Barua  S., Reed  S. B., Romine  M. F., Nealson  K. H., ... & Zhou  J. (2009). Reduction of nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA. The ISME journal  3: 966-976.

He T., Xie  D., Li  Z., Ni  J., & Sun  Q. (2017). Ammonium stimulates nitrate reduction during simultaneous nitrification and denitrification process by Arthrobacter arilaitensis Y-10. Bioresource technology  239: 66-73.

Javaheri-Kermani M., & Asoodeh  A. (2019). A novel beta-1, 4 glucanase produced by symbiotic Bacillus sp. CF96 isolated from termite (Anacanthotermes). International journal of biological macromolecules 131: 752-759.

Klatte  T., Evans  L., Whitehead  R. N., & Cole  J. A. (2011). Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA 39: 321-326.

Kroneck  P. M. (2018). Walking the seven lines: binuclear copper A in cytochrome c oxidase and nitrous oxide reductase. JBIC Journal of Biological Inorganic Chemistry  23: 27-39.

Kushner  D. J. (1988). Physiology of halophilic eubacteria. Halophilic bacteria. 109-138.

Li  R., Zi  X., Wang  X., Zhang  X., Gao  H., & Hu  N. (2013). Marinobacter hydrocarbonoclasticus NY-4, a novel denitrifying, moderately halophilic marine bacterium. SpringerPlus 3: 1-9.

Luvizotto  D. M., Araujo  J. E., Silva  M. D. C. P., Dias  A. C., Kraft  B., Tegetmeye  H., ... & Andreote  F. D. (2019). The rates and players of denitrification, dissimilatory nitrate reduction to ammonia (DNRA) and anaerobic ammonia oxidation (anammox) in mangrove soils. Anais da Academia Brasileira de Ciências  91: 1-14.

Ma  Y., Zille , J. L., & Kent  A. D. (2019). An evaluation of primers for detecting denitrifiers via their functional genes. Environmental microbiology 21: 1196-1210.

Marmur  J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. Journal of molecular biology 3:  208-IN1.

Li  R., Zi  X., Wang  X., Zhang  X., Gao  H., & Hu  N. (2013). Marinobacter hydrocarbonoclasticus NY-4, a novel denitrifying, moderately halophilic marine bacterium. SpringerPlus  2: 1-9.

Paniagua-Michel  J., & Fathepure  B. Z. (2018). Microbial consortia and biodegradation of petroleum hydrocarbons in marine environments. In Microbial Action on Hydrocarbons Springer, Singapore. 1-20.

Pastorelli  R., Piccolo  R., Simoncini  S., & Landi  S. (2013). New primers for denaturing gradient gel electrophoresis analysis of nitrate-reducing bacterial community in soil. Pedosphere  23: 340-349.