环境科学  2019, Vol. 40 Issue (1): 336-342   PDF    
引用本文
崔有为, 金常林, 王好韩, 李晶. 碳源对O/A-F/F模式积累内源聚合物及反硝化的影响[J]. 环境科学, 2019, 40(1): 336-342.
CUI You-wei, JIN Chang-lin, WANG Hao-han, LI Jing. Effect of Carbon Sources on the Accumulation of Endogenous Polymers and Denitritation in the O/A-F/F Mode[J]. Environmental Science, 2019, 40(1): 336-342.
碳源对O/A-F/F模式积累内源聚合物及反硝化的影响
崔有为1, 金常林1, 王好韩1, 李晶2     
1. 北京工业大学环境与能源工程学院, 北京 100124;
2. 中国航空规划设计研究总院有限公司, 北京 100120
收稿日期: 2018-06-11; 修订日期: 2018-07-04
基金项目: 国家自然科学基金项目（51478011）
作者简介: 崔有为(1977~), 男, 博士, 教授, 主要研究方向为污水处理新技术, E-mail:cyw@bjut.edu.cn
摘要: 好氧/缺氧-盛宴/饥饿（O/A-F/F）选择模式能够在好氧段实现活性污泥积累内源聚合物的同时在缺氧段原位利用内源聚合物驱动反硝化.为了深入探究不同的碳源类型对O/A-F/F模式下内源聚合物积累和内源反硝化的影响，实验以乙酸和葡萄糖为主要碳源探究内源聚合物积累和内源反硝化特性以及富集的活性污泥菌群的结构和功能.结果表明，在O/A-F/F选择模式下，当进水化学需氧量（COD）为500 mg·L-1左右时，以乙酸为主要碳源系统（Ac-SBR）和以葡萄糖为主要碳源的系统（Gc-SBR）均能实现40 mg·L-1的硝酸盐氮的内源去除，且各系统均实现了部分短程反硝化.但Ac-SBR实现了更高的亚硝酸盐的积累.乙酸有利于内源聚羟基脂肪酸酯（PHA）积累并驱动内源反硝化过程，PHA产率为0.52，平均反硝化速率（DNR）为9.65 mg·（L·h）-1.Gc-SBR系统能够实现PHA和糖原（Gly）的同时积累，但Gly产率高于PHA产率，分别为0.36和0.17，DNR为4.35 mg·（L·h）-1.Gly是实现内源反硝化过程的主要驱动力，反硝化脱氮贡献率占总量的77%.16S rRNA高通量测序表明Proteobacteria门中的β-Proteobacteria在Ac-SBR中为优势菌纲，菌群丰度为40.56%，而在Gc-SBR中菌群丰度为18.05%.α-Proteobacteria可能在Gc-SBR中贡献了微生物的糖原积累.β-Proteobacteria、Unclassified Bacteroidetes和Lgnavibacteria在Ac-SBR中贡献了内源PHA积累.
关键词: 好氧/缺氧-盛宴/饥饿模式(O/A-F/F)      乙酸      葡萄糖      内源聚合物积累      内源反硝化     
Effect of Carbon Sources on the Accumulation of Endogenous Polymers and Denitritation in the O/A-F/F Mode
CUI You-wei1 , JIN Chang-lin1 , WANG Hao-han1 , LI Jing2     
1. College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China;
2. China Aviation Planning and Design Institute(Group) Co., Ltd., Beijing 100120, China
Abstract: To accumulate endogenous polymers during the aerobic phase, the aerobic/anoxic-feast/famine (O/A-F/F) selection mode can be used. It can also be used in situ for endogenous denitrification by activated sludge during the anoxic phase. To further explore the effect of carbon sources on the activated sludge accumulation of endogenous polymers and endogenous denitrification, this study used acetic and glucose as the main carbon sources to investigate the accumulation of endogenous polymers, endogenous denitrification, and the structure and function of enriched activated sludge. The results show that acetic (Ac-SBR) and glucose (Gc-SBR) as the main carbon source systems achieved a 40 mg·L-1 nitrate removal by endogenous denitrification when the influent chemical oxygen demand (COD) was~500 mg·L-1 in the O/A-F/F selection mode. Both the Ac-SBR and Gc-SBR achieved partial denitrification, but the nitrite accumulation of the Ac-SBR was higher than that of the Gc-SBR. Acetic is favorable for the accumulation of endogenous polyhydroxyalkanoate (PHA); PHA drives the endogenous denitrification. The yield of PHA was 0.52 and the denitrification rate (DNR) was 9.65 mg·(L·h)-1. The Gc-SBR system achieved the simultaneous accumulation of PHA and glycogen (Gly). The yield of Gly was higher than that of PHA and the DNR driven by Gly was 4.35 mg·(L·h)-1. The Gly was the main driving force to achieve endogenous denitrification and contributed to 77% of the total nitrogen removal. The 16S rRNA high-throughput sequencing analysis of activated sludge flora shows that the class of β-Proteobacteria in the Proteobacteria was dominant, with an abundance of 40.56% in the Ac-SBR. However, the abundance of β-Proteobacteria was only 18.05% in the Gc-SBR. The class of α-Proteobacteria contributes to glycogen accumulation in the Gc-SBR. The PHA can be accumulated by β-Proteobacteria, Unclassified Bacteroidetes, and Lgnavibacteria in the Ac-SBR.
Key words: aerobic/anoxic-feast/famine (O/A-F/F)      acetic      glucose      endogenous polymers accumulation      endogenous denitrification     

内源反硝化工艺[1~3]是污水处理中广泛应用的生物脱氮工艺, 该工艺具有节省碳源, 降低CO2释放量和污泥产量, 提高系统脱氮能力的优点[4].内源反硝化脱氮能力取决于内源聚合物的积累量[4], 因此如何将污水中有机物最大效率地转化为内源积累物是关键.盛宴饥饿选择机制(F/F)被报道是一种高效积累内源聚合物的方法[5~8].最近的研究表明在好氧/缺氧交替F/F选择模式(O/A-F/F)下活性污泥能迁移外源有机物转换为内源聚合物并在缺氧段实现内源反硝化脱氮[9], 这为实现在好氧条件下迁移有机物进入到缺氧进行反硝化提供了理论保障.微生物的内源积累受外源碳类型的影响, 同时碳源类型影响了微生物菌群结构和功能, 从而影响内源反硝化脱氮效果[10, 11].但是, O/A-F/F选择模式下碳源对内源聚合物的积累和内源反硝化的影响研究还未见报道.为了更好地理解内源聚合物的积累及内源反硝化脱氮, 本研究采用乙酸和葡萄糖为两种主要碳源, 在O/A-F/F模式下探究它们对内源聚合物积累和内源反硝化的影响, 从生物功能和生物组成层面比较了微生物的特性.碳源的影响探究有利于更好地实现各类废水内源聚合物的积累及内源反硝化脱氮.

1 材料与方法 1.1 废水和接种污泥

实验采用北京工业大学家属楼的生活污水作为实验用水, 生活污水经过化粪池处理.生活污水水质指标如表 1所示.在生活污水中投加乙酸钠或葡萄糖使进水后反应器内初始COD浓度在500 mg·L-1左右, 乙酸和葡萄糖分别在各自系统中的COD占比为80%左右.实验污泥取自北京某污水处理厂.活性污泥COD的去除, 硝化, 脱氮性能良好.两个实验反应器接种污泥浓度均在3 000 mg·L-1左右.

表 1 生活污水水质条件 Table 1 Quality of the sewage wastewater

1.2 反应器的运行

实验采用2个相同的序批式生物反应器(SBR), 分别为以乙酸为主要碳源的SBR(Ac-SBR)和以葡萄糖为主要碳源的SBR(Gc-SBR), SBR有效容积8 L.控制好氧和缺氧时间使F/F=0.1.由于好氧段硝化不足, 在缺氧段投加0.289 g硝酸钾来维持缺氧环境.各SBR周期运行包括进水5 min, 盛宴(好氧)期和饥饿期(缺氧)根据动态调整.好氧盛宴段时间采用监控溶解氧(DO)跃升高于3 mg·L-1作为曝气结束的信号.好氧结束后开始缺氧搅拌, 以F/F=0.1确定缺氧时间.沉淀1 h, 排水5 min, 闲置1 h.每天运行2个周期, 排水比为0.5, 污泥龄(SRT)20d, 温度控制在(28±2)℃.活性污泥经过O/A-F/F选择模式连续运行3个SRT选择富集后, 连续运行数据作为有效数据进行比较分析.

1.3 计算

(1) 比基质摄取速率

(1)

式中, ΔCOD为外源基质降解量, mg·L-1; Δt为缺氧反硝化时间, h; VSS为污泥浓度, g·L-1; qs为比基质的摄取速率(以COD/VSS计), mg·(h·g)-1.

(2) 污泥中PHA质量分数

(2)

式中, PHA为胞内PHA单位细胞质量分数, %; PHA污泥为称取污泥中PHA质量, mg; VSS为称取的污泥质量, mg.

(3) 系统污泥中PHA的浓度

(3)

式中, PHA为碳浓度, mmol·L-1.

(4) 污泥中Gly的质量分数

(4)

式中, Gly胞内为胞内Gly单位细胞干重的质量分数, %; Gly污泥为称取污泥中的Gly质量, mg.

(5) 污泥中的Gly浓度

(5)

式中, Gly为碳浓度, mmol·L-1.

(6) 缺氧饥饿阶段的内源消耗速率

(6)

式中, ΔPolymer1为内源PHA和Gly的碳总浓度产量, mmol·L-1; Δt为缺氧反硝化时间, h; rp为内源消耗速率, mmol·(L·h)-1.

(7) 反硝化速率

(7)

式中, ΔNOx--N为缺氧反硝化去除的NO3--N和NO2--N浓度(NOx--N=NO2--N+NO3--N), mg·L-1; Δt为缺氧反硝化时间, h; DNR为反硝化脱氮速率, mg·(h·L)-1.

(8) 内源聚合物产率

(8)

式中, Y为Ac-SBR或Gc-SBR中PHA或Gly产率; ΔPolymer2为PHA或Gly碳浓度产量, mmol·L-1; ΔS为外源基质降解碳浓度, mmol·L-1.

VSS、PHA和Gly的分子式分别是CH1.8O0.5N0.2[12]、CH1.5O0.5[13]和CH2O. VSS、PHA和Gly以每碳摩尔计时的相对分子质量分别是24.6、21.5和30 mg·mmol-1.

1.4 分析方法

根据国标法[14]对COD、NO3--N、NO2--N和MLVSS进行检测. PHA检测成分包含聚-β-羟基丁酸(PHB)和聚-β-羟基戊酸(PHV). PHA测量采用内标法进行气相色谱分析[15]. Gly测量采用蒽酮法[16].

1.5 微生物群体分析

实验中采集了Ac-SBR和Gc-SBR两种碳源条件下驯化稳定的活性污泥(连续运行60 d以后), 污泥样品在-20℃保存.使用E.Z.N.A.®固体DNA试剂盒(Omega Bi-TEK, Norcross, GA, 美国)从样品中提取微生物DNA, 随后采用扩增细菌16S核糖体RNA基因的V4-V5区且带条形码的引物经PCR扩增.每个样品设3个平行样, 将对应的3个PCR扩增产物混匀为1个样本; 胶回收阳性克隆条带用Tris盐酸进行洗脱, 根据2.0%琼脂糖电泳对各阳性胶回收产物的初步定量结果, 采用蓝色荧光定量系统(QuantiFluorTM-ST, Promega, 美国)定量检测待测样PCR产物后, 将各待测样的PCR产物根据测序量要求进行混合.根据标准方案, 在Illumina MiSeq平台上将纯化扩增子整合[17, 18].原始序列被保存到NCBI数据库, 存档序列为SRP2905300.

2 结果与讨论 2.1 Ac-SBR和Gc-SBR周期反应比较

通过驯化活性污泥, 系统达到了稳定状态. 图 1分别是以乙酸为主要碳源和以葡萄糖为主要碳源时各系统的周期动态变化.外源有机碳源在1h左右的好氧盛宴期被利用, Ac-SBR和Gc-SBR的qs分别为342 mg·(h·g)-1和151 mg·(h·g)-1. qs的差异主要是由于Ac-SBR和Gc-SBR中不同VSS导致的. Ac-SBR的VSS为1221 mg·L-1, 而Gc-SBR的VSS为2 990 mg·L-1.伴随着外源有机质的消耗, 内源聚合物逐渐增加. Ac-SBR和Gc-SBR的内源积累碳浓度分别为6.14 mmol·L-1和7.25 mmol·L-1.在缺氧饥饿期, 伴随着内源的降解, 缺氧段初期投加的40 mg·L-1的NO3--N几乎被完全去除, 外源COD缺氧段保持不变, 表明反硝化碳源来自内源聚合物. Ac-SBR在缺氧段前4 h基本完成大部分NOx--N的去除, rp为1.55 mmol·(L·h)-1, DNR为9.65 mg·(L·h)-1. Gc-SBR对NOx--N的去除较慢, rp为0.86 mmol·(L·h)-1, DNR为4.35 mg·(L·h)-1.

图 1 Ac-SBR和Gc-SBR的周期动态变化 Fig. 1 Dynamic change of the Ac-SBR and Gc-SBR per cycle

2.2 碳源对内源聚合物积累的影响

内源PHA和Gly的细胞含量的周期变化过程见图 2.在Ac-SBR系统中, PHA在好氧盛宴期大量积累, 在缺氧段被消耗, 但是周期内Gly含量没有明显变化.这说明Gly并未积累和参与内源反硝化脱氮过程.但是, 在Gc-SBR系统中, PHA和Gly在好氧盛宴期均大量积累, 且在缺氧段均被消耗.值得关注的是, 缺氧反硝化过程中, 内源PHA在缺氧前1 h内优先被消耗降低至最低水平, 该过程去除了33%的NO3--N.而后胞内Gly继续降解用于反硝化脱氮过程.由于Gly积累含量较高, 以Gly为内碳源将剩余的77%NO3--N反硝化去除.

图 2 Ac-SBR和Gc-SBR的周期PHA和Gly动态变化 Fig. 2 PHA and Gly dynamic change of Ac-SBR and Gc-SBR per cycle

通过对30个稳定周期的最大PHA和Gly积累细胞干重含量(PHAmax和Glymax)和产率(YPHA和YGly)的统计分析发现, 在Ac-SBR系统中[图 3(a)], PHAmax为14.01%±1.02%, YPHA为0.52±0.03, 而在Gc-SBR系统中[图 3(b)], PHAmax为2.32%±0.51%, YPHA为0.17±0.03.在Ac-SBR系统中, Glymax为5.52%±0.04%, YGly几乎为0.在Gc-SBR系统中, Glymax为24.98%±1.05%, YGly为0.36±0.04, YGly是YPHA的2.11倍. Gc-SBR系统中Glymax是导致胞内聚合物PHA和Gly总内源积累量基数(>20 mmol·L-1)显著高于Ac-SBR系统(< 10 mmol·L-1)的主要原因(图 1).前人研究表明, F/F机制下乙酸做碳源有利于PHA的积累[19~21], 而在以葡萄糖为碳源时微生物积累Gly[22].在以葡萄糖为主要碳源的废水中, 更多的碳源用于糖原的积累导致PHA积累能力低[23].

图 3 Ac-SBR和Gc-SBR的PHA和Gly最大细胞干重和产率 Fig. 3 PHA and Gly maximum dry cell weight and yield for Ac-SBR and Gc-SBR

2.3 碳源对内源反硝化的影响

传统反硝化过程难以实现NO2--N的积累[24], 有研究指出短程反硝化将NO3--N首先还原为NO2--N, 再将NO2--N还原为N2, 有利于与厌氧氨氧化工艺结合降低能耗[25]. Ac-SBR和Gc-SBR系统内源反硝化过程均出现NO2--N积累(图 4). Ac-SBR系统中NO2--N积累达到19 mg·L-1, NO2--N积累率为47.5%. Gc-SBR系统NO2--N积累达11 mg·L-1, NO2--N积累率为27.5%.本研究表明内源反硝化在O/A-F/F选择模式下有利于实现NO2--N的积累, 乙酸较葡萄糖更有利于NO2--N的积累.这可能是由于在Ac-SBR系统中硝酸盐还原酶相比亚硝酸盐还原酶竞争电子受体NO3--N的能力更强, 从而导致了更多的NO2--N积累[26].本研究同时发现, 在进水COD和进水NO3--N浓度等条件相同时, 在以乙酸为碳源启动内源反硝化过程中反硝化脱氮速率快.表明PHA在反硝化脱氮过程中可能更容易被利用, 而Gly不易被降解.Zhu等[13]的研究也发现, 内源反硝化脱氮过程中内源PHA被优先降解, PHA被降解至最低水平后Gly开始降解, 与本研究结果一致.

图 4 Ac-SBR和Gc-SBR周期NO3--N和NO2--N的反硝化去除 Fig. 4 NO3--N and NO2--N denitrification nitrogen removal for Ac-SBR and Gc-SBR per cycle

2.4 碳源对微生物菌群影响

对Ac-SBR和Gc-SBR驯化泥样进行16S rRNA MiSeq焦磷酸测序, 分析丰度>1%的种属.在门水平比较两种碳源条件下的微生物(图 5), 本研究结果显示, Proteobacteria菌门在Ac-SBR和Gc-SBR中均为优势菌群, 菌群丰度分别为49.11%和28.99%.有研究指出, Proteobacteria菌门微生物具有一定的PHA积累能力[27, 28]. TM7为Gc-SBR中的优势菌群, 比例为36.78%, 该菌属在Ac-SBR中未被发现, 葡萄糖对TM7丰度的增加有明显的促进作用. Bacteroidetes在Ac-SBR和Gc-SBR中菌群丰度为31.51%和16.40%.研究认为Bacteroidetes菌门微生物具有积累PHA和反硝化脱氮功能[29~31]. Chlorobi在Ac-SBR和Gc-SBR中菌群丰度为8.69%和1.54%.此外, GN02、Lentisphaerae、Firmicutes、Spirochaetes在Ac-SBR中菌群丰度为2.85%、2.72%、2.39%和1.44%, 但Gc-SBR中菌群丰度均 < 1%. Firmicutes菌门微生物也具有一定的PHA积累能力[27, 28]. Actinobacteria、OD1和Chloroflexi在Ac-SBR中菌群丰度均 < 1%, 但在Gc-SBR中菌群丰度为5.05%、3.59%和2.84%.

图 5 门水平比较Ac-SBR和Gc-SBR系统的种群结构 Fig. 5 Comparison of the Ac-SBR with the Gc-SBR microbial community structure at the phylum level

在纲水平(图 6), Proteobacteria门中的β-Proteobacteria在Ac-SBR中为优势菌纲, 菌群丰度为40.56%, 而在Gc-SBR中菌群丰度仅为18.05%. γ-Proteobacteria, α-Proteobacteria和δ-Proteobacteria菌纲在Ac-SBR和Gc-SBR中分别为2.89%和3.2%、1.72%和4.67%及1.02%和2.23%, 其中, α-Proteobacteria被报道具有一定的糖原积累能力[32]. Bacteroidetes菌门中的Unclassified Bacteroidetes在Ac-SBR中菌群丰度为13.21%, 远高于Gc-SBR中的丰度3.75%.在Ac-SBR中的Saprospirae、Lgnavibacteria和Flavobacteriia菌纲丰度高于Gc-SBR, 分别为10%和6.13%、8.52%和1.29%及2.63%和低于1%. Bacteroidia菌纲在Ac-SBR和Gc-SBR丰度相似, 分别为5.13%和5.93%. β-Proteobacteria、Unclassified Bacteroidetes和Lgnavibacteria在Ac-SBR中的高丰度可能是其PHA积累量高于Gc-SBR系统生物学原因.

图 6 纲水平比较Ac-SBR和Gc-SBR系统的种群结构 Fig. 6 Comparison of the Ac-SBR with the Gc-SBR microbial community structure at the class level

3 结论

(1) 本研究表明O/A-F/F选择模式可以实现污水中有机物以内源积累的形式迁移到缺氧段进行内源反硝化.乙酸促进PHA的积累, 葡萄糖有利于Gly的积累.

(2) 在O/A-F/F选择模式下碳源类型影响内源反硝化过程和内源反硝化速率.乙酸较葡萄糖作为碳源更有利于反硝化过程中NO2--N的积累, PHA做为胞内碳源驱动内源反硝化的速率较Gly高.

(3) Gc-SBR系统中TM7为优势菌属, α-Proteobacteria可能是积累Gly的菌属. β-Proteobacteria、Unclassified Bacteroidetes和Lgnavibacteria的高度富集是Ac-SBR中PHA积累量高于Gc-SBR的生物学原因.

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