Optimizations of extraction and activity determination methods of nitric oxide reductase in activated sludge
YANG Rui1,2,3, YUAN Lin-jiang1,2,3, WANG Gang1,2,3, YUAN Lin-jie1,2,3, YOU Xiao-long1,2,3, NIU Wan-xia1,2,3, YU Li-ping1,2,3
1. School of Environment and Municipal Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China; 2. Key Laboratory of Environmental Engineering of Shaanxi Province, Xi'an 710055, China; 3. Key Laboratory of Northwest Water Resources and Environmental Ecology Ministry of Education, Xi'an 710055, China
Abstract:Orthogonal experiments were carried out to study the effects of different fragmentation methods (e.g. fragmentation by ultrasonic, fragmentation by low temperature and high-pressure) on the catalytic activity of nitric oxide reductase (nor). The release of intracellular soluble protein, nucleic acid (DNA) and the catalytic activity of nor were analyzed under different ultrasonic intensity and frequency, breaking pressure and frequency, and the amount of lysate addition. According to the catalytic characteristics of enzymes in activated sludge under different experimental conditions, the method for measuring the catalytic activity of nor was improved. According to the results, the parameters of nor extraction by ultrasonic fragmentation were suggested to set at 100times (ultrasonic times), 500W (ultrasonic intensity), and 0.1mL (amount of lysate addition). The parameters of nor extraction by low temperature and high-pressure fragmentation were suggested to set at 4times (breaking times), 50Mpa (breaking pressure), and 0.1mL (amount of lysate addition). The release of intracellular soluble protein, DNA and the maximum catalytic activity of nor by low temperature and high-pressure fragmentation were higher than that by ultrasonic fragmentation. Last but not least, the end time for activity determination of nor was suggested at 15min.
Czepiel P, Crill P, Harriss R. Nitrous oxide emissions from municipal wastewater treatment[J]. Environmental Science & Technology, 1995, 29(9):2352-2356.
[2]
Kampschreur M J, Temmink H, Kleerebezem R, et al. Nitrous oxide emission during wastewater treatment[J]. Water Research, 2009, 43(17):4093-4103.
[3]
刘秀红,杨庆,吴昌永,等.不同污水生物脱氮工艺中N2O释放量及影响因素[J]. 环境科学学报, 2006,(12):1940-1947.Liu X H, Yang Q, Wu C Y, et al. N2O emissions from different biological nitrogen removal processes and factors affecting N2O product[J]. Acta Scientiae Circumstantiae, 2006,(12):1940-1947.
[4]
白雪,张荣兵,顾剑,等.大型污水处理厂污泥膨胀原因分析及其控制方法[J]. 中国给水排水, 2011,27(23):31-35.Bai X, Zhang RB, Gu J, et al. Cause analysis and control of sludge bulking in large-scale WWTP[J]. China Water & Wastewater, 2011, 27(23):31-35.
[5]
庞子山,况勋华,朱俊,等.三峡库区污水处理厂的污泥膨胀及恢复运行[J]. 中国给水排水, 2009,25(10):95-98.Pang ZH, Kuang XH, Zhu J, et al. Research on sludge bulking and recovering of wastewater treatment plant in Three Gorges Reservoir Area[J]. China Water & Wastewater, 2009,25(10):95-98.
[6]
Caranto J D, Vilbert A C, Lancaster K M. Nitrosomonas europaea cytochrome P460is a direct link between nitrification and nitrous oxide emission[J]. Proceedings of the National Academy of Sciences, 2016,113(51):14704.
[7]
Cavazos A R, Taillefert M, Tang Y, et al. Kinetics of nitrous oxide production from hydroxylamine oxidation by birnessite in seawater-ScienceDirect[J]. Marine Chemistry, 2018,202:49-57.
[8]
彭永臻.活性污泥膨胀机理、成因及控制[M]. 北京:科学出版社, 2012.Peng Y Z. Mechanism, causes and control of activated sludge bulking[M]. Beijing:Science Press, 2012.
[9]
Casey T G, Wentzel M C, Ekama G A, et al. A hypothesis for the causes and control of anoxic-aerobic (AA) filament bulking in nutrient removal activated sludge systems[J]. Social Studies of Science, 1994, 42(5):709-31.
[10]
Zumft W G. Cell biology and molecular basis of denitrification[J]. Microbiology and Molecular biology reviews, 1997,61(4):533-616.
[11]
Kastrau D H W, Heiss B, Kroneck P M H, et al. Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex. Phospholipid requirement, electron paramagnetic resonance and redox properties[J]. Febs Journal, 2010,222(2):293-303.
[12]
Heiss B, Frunzke K, Zumft W G. Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri[J]. Journal of Bacteriology, 1989,171(6):3288-3297.
[13]
Burnell J N, John P, Whatley F R. The reversibility of active sulphate transport in membrane vesicles of Paracoccus denitrificans[J]. Biochemical Journal, 1975,150(3):527-536.
[14]
Carr G J, Ferguson S J. The nitric oxide reductase of Paracoccus denitrificans[J]. Biochemical Journal, 1990,269(2):423.
[15]
Hino T, Matsumoto Y, Nagano S, et al. Structural basis of biological N2O generation by bacterial nitric oxide reductase[J]. Science, 2010, 330(6011):1666.
[16]
Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976,72(1/2):248-254.
[17]
Burton K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid[J]. Biochemical Journal, 1956,62(2):315.
[18]
He Z X, Yuan L J, Wei Y N, et al. N2O emission and hydroxylamine oxidase (HAO) activity in a nitrogen removal process based on activated sludge with three COD/NH4+ Ratios[J]. Water Environment Research, 2017,89(5):387.
[19]
Yang R, Yuan LJ, Wang R, et al. New insight on the regulation of N2O production in aerobic condition:An N2O metabolic perspective based on enzymatic analysis of nitrous oxide reductase[J]. Journal of Water Process Engineering, 2021,41(2021):102090.
[20]
刘国诠.生物工程下游技术[M]. 北京:化学工业出版社, 2011.Liu G Q. Downstream Bioengineering Technology[M]. Beijing:Chemical Industry Press, 2012.
[21]
Zhang PY, Zhang GM, Wang W, et al. Ultrasonic treatment of biological sludge:floc disintegration, cell lysis and inactivation[J]. Bioresour Technol, 2007,98(1):207-210.
[22]
Rosen M J, Kunjappu J T. Surfactants and interfacial phenomena[M]. John Wiley & Sons, 2012.