为了解决高温的煤化工废水生物脱氮效率不高的技术难题。
本研究从上海某能化集团有限公司的煤化工废水处理系统的活性污泥中筛选得到一株耐热氨氧化细菌A1和一株耐热反硝化细菌D1。
通过形态学观察、生理生化特征及16S rRNA基因序列分析,菌株A1初步鉴定为
本研究的结果可为高温煤化工废水的生物处理提供技术支撑及菌种储备,同时也为高温污水处理过程中N2O的释放规律提供理论参考。
To address the low efficiency of biological nitrogen removal from high-temperature coal chemical wastewater.
In this study, a mesophilic ammonia-oxidizing bacterial strain A1 and a mesophilic denitrifying bacterial strain D1 were isolated from the activated sludge of the coal chemical wastewater treatment system of an energy and chemical group Co. Ltd. in Shanghai.
Based on the morphological, physiological, and biochemical characteristics and 16S rRNA gene sequence, strain A1 was preliminarily identified as
This study can provide technological support and strain resources for the biological treatment of high-temperature coal chemical wastewater, and help to reveal the law of N2O release in the treatment of high-temperature wastewater.
现代煤化工是指煤炭通过清洁加工转化为燃料和化学品的过程,在过程中所产生的废水即为煤化工废水[
煤化工废水通过常规的氨氧化菌和反硝化细菌进行生物法处理,实际上游往往安装冷却设备或控温设备对废水先进行降温,但这无疑会大大增加企业的运行成本[
与此同时,在废水生物脱氮的过程中不可避免地会释放出污水处理副产物氧化亚氮(N2O)[
本研究以分离筛选自煤化工废水处理系统中的耐热氨氧化细菌和耐热反硝化细菌为对象,考察温度对其脱氮性能及N2O释放规律的影响,同时探究了耐热脱氮菌株构建共培养物的短程硝化反硝化脱氮及N2O释放的影响,为高温的煤化工氨氮废水的生物处理提供技术支撑及菌种储备,同时也为高温污水处理过程中N2O的释放规律提供理论参考。
(1) 上海某能化集团有限公司污水处理厂生化处理系统,其系统包括预处理和生化处理单元两部分。预处理单元主要作用是调节水质水量,生化处理单元为一体化AO处理装置。本研究筛选样品取自一体化AO处理装置的活性污泥,模拟废水按照一体化AO处理装置中的进水水质进行配置。
(2) 氨氧化菌富集培养基(g/L)[
(3) 反硝化菌富集培养基(g/L)[
(4) 模拟废水(g/L)[
以上培养基均在121 ℃、1.1×104 Pa条件下灭菌30 min,冷却备用[
取活性污泥样品在氨氧化菌固体培养基和反硝化菌固体培养基采用稀释涂布法于42 ℃、120 r/min培养。待菌落长出后,用接种环挑取单菌落利用连续划线分离法获得耐热纯化菌株[
取–80 ℃冻存菌株,冰浴解冻,参照文献预处理后采用ZEISS Gemini SEM 500场发射扫描电子显微镜观察菌体形态[
将活化的A1和D1菌液以20%和10%比例分别接入装有50 mL的氨氧化菌液体培养基的锥形瓶和10 mL的反硝化菌液体培养基的密闭血清瓶(预先除氧无菌处理)中,分别以不接入菌种的培养基作为空白对照组。其中锥形瓶中的NH4+-N初始浓度为100 mg/L,血清瓶中的NO2–-N初始浓度为180 mg/L,研究不同温度(25、30、35、40、42、45 ℃)条件下对菌株A1和D1脱氮能力的影响。以上处理均在120 r/min的摇床中培养,每隔一段时间取样分析NH4+-N和NO2–-N和顶空N2O的浓度。
将活化的菌液A1和D1 (
硝态氮采用紫外分光光度法、氨氮采用苯酚-次氯酸盐法、亚硝酸盐氮采用N-(1-萘基)-乙二胺分光光度法、蛋白质采用快速Lowry法[
式中,
菌株A1和D1的菌落和菌株形态特征如
菌株A1和D1菌落和菌株形态照片
Photographs of the strain A1 and D1 colony and strain morphology. A: flat chart, scale=2 mm; B: scanning electron microscopy, scale=0.2 μm; C: flat chart, scale=10 mm; D: scanning electron microscopy, scale=0.5 μm.
菌株A1和D1的16S rRNA基因全长分别为1 375 bp和1 429 bp,将16S rRNA基因序列在GenBank数据库作BLAST比对,并根据比对的结果构建系统发育树。菌株A1归于
菌株A1和D1系统发育树
Strains A1 and D1 phylogenetic trees. Bar 0.005 0 and 0.001 0 at the bottom is the sequence divergence. The numbers of GenBank were shown in parentheses.
为了考察温度对菌株A1和D1脱氮特性的影响,试验了24 h内在不同温度分别为25、30、35、40、42 ℃的条件下菌株A1和D1的氮素去除情况。结果如
温度对菌株A1和D1脱氮特性的影响
Effect of temperature on nitrogen removal characteristics of the strain A1 and D1. A: ammonia nitrogen conversion rate of the strain A1; B: nitrite nitrogen reduction rate of the strain D1. Values represents means±SD. Three biological repeats were performed.
如
不同温度条件下菌株D1的亚硝酸盐氮还原情况及N2O得率
Nitrite reduction and N2O yield rate of the strain D1 at different temperatures. A:
为了明确C/N对该共培养物短程硝化反硝化脱氮及N2O释放效应的影响,在C/N分别为1:1、2:1、3:1和4:1条件下监测了196 h内的氮素去除情况。结果如
不同C/N条件下短程硝化反硝化脱氮及N2O释放效应
Effects of shortcut nitrification-denitrification on nitrogen removal and N2O release under different C/N ratios. A: C/N=1:1; B: C/N=2:1; C: C/N=3:1; D: C/N=4:1.
为了明确pH对该共培养物短程硝化反硝化脱氮及N2O释放效应的影响,在pH分别为6.0、7.0、8.0、9.0和10.0条件下监测了220 h内的氮素去除情况。结果如
不同pH条件下短程硝化反硝化脱氮及N2O释放效应
Effects of shortcut nitrification-denitrification on nitrogen removal and N2O release under different pH conditions. A: pH=6; B: pH=7; C: pH=8; D: pH=9; E: pH=10.
(1) 本文从煤化工废水处理系统的活性污泥中筛选得到1株耐热氨氧化细菌A1和1株耐热反硝化细菌D1,菌株A1鉴定为
(2) 耐热菌株A1和D1均具有较强的脱氮能力,其最适生长温度分别为42 ℃和40 ℃。
(3) 由耐热菌株A1和D1构建的共培养物在42 ℃高温条件下,处理NH4+-N浓度为100 mg/L模拟废水的最佳pH为9.0–10.0,最佳C/N为2:1,氮素去除率达 > 99.0%,最大N2O得率高达51.3%。
(4) 菌株A1和D1能在42 ℃的高温条件下生存和脱氮,说明其具有一定的耐热特性,对环境温度适应范围较广,可为高温的煤化工氨氮废水的生物处理提供技术支撑及菌种储备,同时也为高温污水处理过程中N2O的释放规律提供理论参考。
孙双印. 煤化工行业发展趋势及问题分析. 河南化工, 2016, 33(4): 11–13.
Sun SY. Development trend and problem analysis of coal chemical industry.
Coal Processing & Comprehensive Utilization, 2016(2): 1–3. (in Chinese)]]>
郑平, 胡宝兰, 徐向阳. 新型生物脱氮理论与技术. 北京: 科学出版社, 2004.
郑辉. 生态环境部正式发布《2018中国生态环境状况公报》. 水处理技术, 2019, 45(6): 105.
Zheng H. The ministry of ecological environment officially issued the 《2018 China ecological environment bulletin》.
Guo L. Ecology. Doing battle with the green monster of Taihu Lake.
Zhao Q, Han HJ, Xu CY, Zhuang HF, Fang F, Zhang LH. Effect of powdered activated carbon technology on short-cut nitrogen removal for coal gasification wastewater.
Gao JQ, Gao D, Liu H, Cai J, Zhang JQ, Qi ZL. Biopotentiality of high efficient aerobic denitrifier
张苗, 黄少斌. 高温好氧反硝化菌的分离鉴定及其反硝化性能研究. 环境科学, 2011, 32(1): 259–265.
Zhang M, Huang SB. Identification and Denitrification Characteristics of a Thermophilic Aerobic Denitrifier.
李誉琦, 马佩钰, 刘涵, 王灵芝, 高仁玲, 李慧娟. 一株耐高温亚硝酸盐型反硝化细菌的鉴定及脱氮特性. 生物技术通报, 2019, 35(9): 194–201.
Li YQ, Ma PY, Liu H, Wang LZ, Gao RL, Li HJ. Identification and denitrification characters of a high-temperature-resistant nitrite-denitrifying bacterium.
Zhuge YY, Shen XY, Liu YD, Shapleigh J, Li W. Application of acidic conditions and inert-gas sparging to achieve high-efficiency nitrous oxide recovery during nitrite denitrification.
Kampschreur MJ, Temmink H, Kleerebezem R, Jetten MSM, Van Loosdrecht MCM. Nitrous oxide emission during wastewater treatment.
Zhuang JL, Zhou YY, Liu YD, Li W. Flocs are the main source of nitrous oxide in a high-rate anammox granular sludge reactor: insights from metagenomics and fed-batch experiments.
Li W, Zhuang JL, Zhou YY, Meng FG, Da Kang, Zheng P, Shapleigh JP, Liu YD. Metagenomics reveals microbial community differences lead to differential nitrate production in anammox reactors with differing nitrogen loading rates.
Takaya N, Catalan-Sakairi MAB, Sakaguchi Y, Kato I, Zhou ZM, Shoun H. Aerobic denitrifying bacteria that produce low levels of nitrous oxide.
汤默然, 李茹莹. 异养硝化-好氧反硝化菌株的分离筛选及复配菌剂对河水的净化效果. 环境科学学报, 2021, 41(7): 2657–2663.
Tang MR, Li RY. Isolation of heterotrophic nitrification-aerobic denitrification bacteria strains and purification of river water by mixed cultures.
Pseudomonas stutzeri T13的脱氮特性及机制研究. 哈尔滨工业大学博士学位论文, 2021. ]]>
王秀杰, 王维奇, 李军, 李芸, 张彦灼, 孙艺齐, 王思宇, 卞伟. 异养硝化菌
Wang XJ, Wang WQ, Li J, Li Y, Zhang YZ, Sun YQ, Wang SY, Bian W. Isolation and identification of a heterotrophic nitrifier,
黄雪娇, 杨冲, 倪九派, 李振轮. 1株高效去除氨氮的红假单胞菌的分离鉴定及特性. 环境科学, 2016, 37(6): 2276–2283.
Huang XJ, Yang C, Ni JP, Li ZL. Isolation, identification and characteristics of a
东秀珠, 蔡妙英. 常见细菌系统鉴定手册. 北京: 科学出版社, 2001.
布坎南, 吉本斯. 伯杰细菌鉴定手册. 8版. 北京: 科学出版社, 1984.
蔡茜, 何腾霞, 冶青, 李振轮. 耐冷嗜碱蒙氏假单胞菌H97的鉴定及其好氧反硝化特性. 环境科学, 2018, 39(7): 3314–3320.
Cai X, He TX, Ye Q, Li ZL. Identification and characterization of a hypothermic alkaliphilic aerobic denitrifying bacterium
国家环境保护总局《水和废水监测分析方法》编委会. 水和废水监测分析方法(第四版). 北京: 中国环境出版社, 2002.
Wang JP, Li LZ, Liu YD, Li W. A review of partial nitrification in biological nitrogen removal processes: from development to application.
董莲华, 杨金水, 袁红莉. 氨氧化细菌的分子生态学研究进展. 应用生态学报, 2008, 19(6): 1381–1388.
Dong LH, Yang JS, Yuan HL. Research advances in molecular ecology of ammonia oxidizing bacteria.
Wang JP, Liu YD, Li W. Model-based assessment of nitritation using formic acid as a selective inhibitor.
Wang JP, Liu YD, Meng FG, Li W. The short- and long-term effects of formic acid on rapid nitritation start-up.
Wang XJ, Ye CS, Zhang ZJ, Guo Y, Yang RL, Chen SH. Effects of temperature shock on N2O emissions from denitrifying activated sludge and associated active bacteria.
Reino C, Van Loosdrecht MCM, Carrera J, Pérez J. Effect of temperature on N2O emissions from a highly enriched nitrifying granular sludge performing partial nitritation of a low-strength wastewater.
Pan YT, Ni BJ, Lu HJ, Chandran K, Richardson D, Yuan ZG. Evaluating two concepts for the modelling of intermediates accumulation during biological denitrification in wastewater treatment.
Wang JP, Liu YD, Meng FG, Li W. The short- and long-term effects of formic acid on rapid nitritation start-up.
Li W, Cai ZY, Duo ZJ, Lu YF, Gao KX, Abbas G, Zhang M, Zheng P. Heterotrophic ammonia and nitrate bio-removal over nitrite (hanbon): performance and microflora.