Screening of phosphorus removing bacteria from activated sludge for biological phosphorus removal: starch-anoxic/aerobic alternation and acetate-anaerobic/aerobic alternation system
ZHOU Xu-hong1,2,3, YUAN Lin-jiang1,2,3, CHEN Xi4, YANG Rui1,2,3, ZHU Miao1,2,3, NAN Ya-ping1,2,3, HE Xiang-feng1,2,3, CHEN Yong1,2,3
1. School of Environmental and Municipal Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China; 2. Key Laboratory of Northwest Water Resource, Environment and Ecology, Ministry of Education, Xi'an University of Architecture and Technology, Xi'an 710055, China; 3. Shaanxi Key Laboratory of Environmental Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China; 4. School of Urban Planning and Municipal Engineering, Xi'an University of Engineering, Xi'an 710048, China
Abstract:In order to identify phosphorus accumulating organisms(PAOs) in situ from activated sludge. 4', 6-diamidino-2- phenylindole (DAPI) staining and flow cytometry fluorescence sorting (FACS) were used to sceen the PAOs from the two sludge at the anoxic/aerobic SBR system (R1) with starch as the only carbon source and the anaerobic/aerobic system (R2) with acetate as the only carbon source. The species of the sorted bacteria were identified by 16S rRNA high-throughput sequencing. The results showed that in the R1, biological phosphorus removal was carried out in both the anoxic periods and aerobic periods. The phosphorus uptake in the anoxic period was greater than that in the aerobic period. In the R2, the phosphorus release in the anaerobic period and a large amount of phosphorus absorption in the aerobic period occurs. Results of the in situ fluorescence staining showed that 106 bacteria with a relative purity of 85% with phosphorus-accumlating cell were sorted from the R1 and the R2. The sequencing results showed that in the R1, the dominant genera of PAOs in the anoxic periods were Halomonas (37.75%), unclassified Brucellaceae (14.15%), Pseudomonas (6.49%), unclassified Chlamydiales (0.027%) and Sphingopyxis (0.007%). The dominant PAOs in the aerobic periods were Halomonas (19.72%), unclassified Brucellaceae (14.62%), Pseudomonas (14.28%), unclassified Comamonadaceae (0.046%), unclassified Acidobacteria Gp3 (0.036%) and Ferruginibacter (0.026%). In the R1, unclassified Chlamydiales and Sphingopyxis only had phosphorus accumulation function under anoxic periods, while unclassified Comamonadaceae, unclassified Acidobacteria Gp3 and Ferruginibacter only had phosphorus accumulation function under aerobic periods. In the R2, the dominant PAOs were Dechloromonas (11.06%), unclassified Anaerolineaceae (9.29%), unclassified Bacteroidetes (7.44%), unclassified Gammaproteobacteria (7.34%) and Acinetobacter (0.31%). This means that in the R1, the bacteria involved in the phosphorus removal process include aerobic, anoxic and both aerobic and anoxic phosphorate accumulating bacteria three types. In the R2, the bacteria involved in the phosphorus removal process are only aerobic phosphorus accumulating bacteria.
Nicholls H A, Osborn D W. Bacterial stress: prerequisite for biological removal of phosphorus [J]. Water Pollution Control Federation, 1979,51(3):557-569.
[2]
Dorofeev A G, Nikolaev Y A, Mardanov A V, et al. Role of phosphateaccumulating bacteria in biological phosphorus removal from wastewater [J]. Applied Biochemistry and Microbiology, 2020, 56(1):1-14.
[3]
王亚东,王少坡,郑莎莎,等.生物除磷系统的聚磷微生物种群及其检测方法 [J]. 环境工程, 2015,24(2):21-26. Wang Y D, Wang S P, Zheng S S, et al Phosphorus accumulating microorganism population of biological phosphorus removal system and its detection method [J] Environmental engineering, 2015,24(2): 21-26.
[4]
Rao N N, Gómez-García M R, Kornberg A. Inorganic polyphosphate: essential for growth and survival [J]. Annual review of biochemistry, 2009,78:605-647.
[5]
李 冬,李晓莹,杨 杰,等.后置缺氧SBR短程反硝化除磷 [J]. 中国环境科学, 2017,37(8):2994-3001. Li D, Li X Y, Yang J, et al. Post-hypoxic SBR short-range denitrification for phosphorus removal [J]. China Environmental Science, 2017,37(8):2994-3001.
[6]
Nielsen P H, McIlroy S J, Albertsen M, et al. Re-evaluating the microbiology of the enhanced biological phosphorus removal process [J]. Current opinion in biotechnology, 2019,57:111-118.
[7]
Qiu G, Zuniga-Montanez R, Law Y, et al. Polyphosphate- accumulating organisms in full-scale tropical wastewater treatment plants use diverse carbon sources [J]. Water research, 2019,149:496- 510.
[8]
Fuhs G W, Chen M. Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater [J]. Microbial Ecology, 1975,2(2):119-138.
[9]
Wagner M, Erhart R, Manz W, et al. Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge [J]. Applied and Environmental Microbiology, 1994,60(3):792- 800.
[10]
Cloete T E, Steyn P L. The role of Acinetobacter as a phosphorus removing agent in activated sludge [J]. Water Research, 1988,22(8): 971-976.
[11]
Meganck M, Malnou D, Le Flohic P, et al. The importance of the acidogenic microflora in biological phosphorus removal [J]. Water science and technology, 1985,17(11/12):199-212.
[12]
Nakamura K, Masuda K, Mikami E. Isolation of a new type of polyphosphate accumulating bacterium and its phosphate removal characteristics [J]. Journal of fermentation and bioengineering, 1991, 71(4):258-263.
[13]
Santos M M, Lemos P C, Reis M A M, et al. Glucose metabolism and kinetics of phosphorus removal by the fermentative bacterium Microlunatus phosphovorus [J]. Applied and Environmental Microbiology, 1999,65(9):3920-3928.
[14]
Stante L, Cellamare C M, Malaspina F, et al. Biological phosphorus removal by pure culture of Lampropedia spp [J]. Water research, 1997,31(6):1317-1324.
[15]
任世英,肖 天.聚磷菌体内多聚物的染色方法 [J]. 海洋科学, 2005, 29(1):59-63. Ren S Y, Xiao T. Staining method of polymer in polyphosphate accumulating bacteria [J]. Marine science, 2005,29(1):59-63.
[16]
Cai T M, Guan L B, Chen L W, et al. Enhanced biological phosphorus removal with P seudomonas putida GM6 from activated sludge [J]. Pedosphere, 2007,17(5):624-629.
[17]
Terashima M, Yama A, Sato M, et al. Culture-Dependent and -Independent Identification of Polyphosphate-Accumulating Dechloromonas spp. Predominating in a Full-Scale Oxidation Ditch Wastewater Treatment Plant [J]. Microbes and Environments, 2016, 31(4):449-455.
[18]
周明璟,纪树兰,崔丹红,等.厌氧/好氧交替快速筛选聚磷菌及其生理特性的研究 [J]. 中国环境科学, 2012,32(10):1838-1844. Zhou M J, Ji S L, Cui D H, et al. Rapid Screening of Phosphorus Accumulating Bacteria and Their Physiological Characteristics by Alternating Anaerobic/Aerobic [J]. China Environmental Science, 2012,32(10):1838-1844.
[19]
张立成,李艳美,袁雅姝,等.亚硝化反硝化聚磷菌的筛选及生化特性分析 [J]. 中国给水排水, 2013,29(11):77-80. Zhang L C, Li Y M, Yuan Y S, et al. Screening and biochemical characteristic analysis of nitrosating and denitrifying phosphorus accumulating bacteria [J]. China Water Supply and Drainage, 2013, 29(11):77-80.
[20]
李 慧,刘丹丹,陈文清.反硝化聚磷菌的筛选及脱氮除磷特性 [J]. 环境工程, 2016,34(4):25-28. Li H, Liu D D, Chen W Q. Screening of denitrifying phosphorus- accumulating bacteria and their characteristics of nitrogen and phosphorus removal [J]. Environmental Engineering, 2016,34(4):25- 28.
[21]
郝晓地,陈 峤,刘然彬.Tetrasphaera聚磷菌研究进展及其除磷能力辨析 [J]. 环境科学学报, 2020,40(3):741-753. Hao X D, Chen Q, Liu R B. Research Progress of Tetrasphaera Phosphorus Accumulating Bacteria and Analysis of Phosphorus Removal Ability [J]. Journal of Environmental Science, 2020,40(3): 741-753.
[22]
Qian W, Linjiang Y, Xi C, et al. Biological phosphorus removal and its mechanism in anoxic/aerobic continuous flow system with different carbon sources [J]. Chinese Journal of Environmental Engineering, 2021,15(3):954-961.
[23]
马 放,杨菲菲,李 昂,等.1株高效反硝化聚磷菌的生物学特性研究 [J]. 环境科学, 2011,32(9):2710-2715. Ma F, Yang F F, Li A, et al. Biological characteristics of a high- efficiency denitrifying phosphorus accumulating bacteria [J]. Environmental Science, 2011,32(9):2710-2715.
[24]
Sun L, Zhao X, Zhang H, et al. Biological characteristics of a denitrifying phosphorus-accumulating bacterium [J]. Ecological engineering, 2015,81(2015):82-88.
[25]
Kong Y, Nielsen J L, Nielsen P H. Identity and ecophysiology of uncultured actinobacterial polyphosphate-accumulating organisms in full-scale enhanced biological phosphorus removal plants [J]. Applied and environmental microbiology, 2005,71(7):4076-4085.
[26]
蒋志云,韦佳敏,缪新年,等.ABR-MBR工艺反硝化除磷微生物群落特征分析 [J]. 环境工程学报, 2019,13(7):1653-1661. Jiang Z Y, Wei J M, Miao X N, et al. Characteristic analysis of microbial community for denitrification and phosphorus removal by ABR-MBR process [J]. Chinese Journal of Environmental Engineering, 2019,13(7):1653-1661.
[27]
Kong Y, Xia Y, Nielsen P H. Activity and identity of fermenting microorganisms in full-scale biological nutrient removing wastewater treatment plants [J]. Environmental microbiology, 2008,10(8):2008- 2019.
[28]
Crocetti G R, Hugenholtz P, Bond P L, et al. Identification of polyphosphate-accumulating organisms and design of 16S rRNAdirected probes for their detection and quantitation [J]. Applied and environmental microbiology, 2000,66(3):1175-1182.
[29]
Hammes F, Berney M, Wang Y, et al. Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes [J]. Water research, 2008,42(1/2):269-277.
[30]
Wang H G, Huang H, Liu R L, et al. Investigation on polyphosphate accumulation in the sulfur transformation-centric EBPR (SEBPR) process for treatment of high-temperature saline wastewater [J]. Water research, 2019,167:115138.
[31]
Luo D, Yuan L, Liu L, et al. Biological phosphorus removal in anoxic-aerobic sequencing batch reactor with starch as sole carbon source [J]. Water Science and Technology, 2017,75(1):28-38.
[32]
国家环保局本书编委会.水和废水监测分析方法 [M]. 北京:水和废水监测分析方法, 1989. The State Environmental Protection Administration's book editorial committee. Water and wastewater monitoring and analysis methods [M]. Beijing. Water and wastewater monitoring and analysis methods, 1989.
[33]
李夕耀,彭永臻,王淑莹,等.聚磷菌胞内多聚物的分析检测方法 [J]. 四川环境, 2009,28(2):106-111. Li X Y, Peng Y Z, Wang S Y, et al. Analysis and detection method of intracellular polymers of phosphorus accumulating bacteria [J]. Sichuan Environment, 2009,28(2):106-111.
[34]
Huo X, Yuan L, Wang Q, et al. Nitrate Promotes Phosphorus Removal in the Anoxic–Aerobic Sequencing Batch Reactor with Starch as Sole Carbon Source [J]. Environmental Engineering Science, 2021,38(2): 66-73.
[35]
葛艳辉,赵 林,周 艳.聚磷菌胞内聚合物的染色条件优化及染色方法比较 [J]. 环境科学与技术, 2014,37(2):1-6. Ge Y H, Zhao L, Zhou Y. Optimization of dyeing conditions and comparison of dyeing methods for intracellular polymers of phosphorus accumulating bacteria [J]. Environmental Science and Technology, 2014,37(2):1-6.
[36]
Wang H G, Biswal B K, Mao Y P, et al. Multiple-cycle operation of sulphur-cycle-enhanced biological phosphorus removal to maintain stable performance at high temperatures [J]. Bioresource technology, 2019,289:121736.
[37]
Terashima M, Kamagata Y, Kato S. Rapid enrichment and isolation of polyphosphate accumulating organisms through 4’6-diamidino-2- phenylindole (DAPI) staining with fluorescence-activated cell sorting (FACS) [J]. Frontiers in microbiology, 2020,11(2015):793.
[38]
任世英,张宇红,张武昌,等.海洋聚磷菌Halomonas YSR-3的除磷特性研究 [J]. 高技术通讯, 2008,18(7):743-747. Ren S Y, Zhang Y H, Zhang W C, et al. Study on Phosphorus Removal Characteristics of Marine Phosphorus Accumulating Bacteria Halomonas YSR-3 [J]. High-Tech Communications, 2008,18(7):743- 747.
[39]
Miyauchi R, Oki K, Aoi Y, et al. Diversity of nitrite reductase genes in “Candidatus Accumulibacter phosphatis”-dominated cultures enriched by flow-cytometric sorting [J]. Applied and environmental microbiology, 2007,73(16):5331-5337.