【期刊】主流厌氧氨氧化颗粒污泥群落应对易降解碳源的自分层机制

图文摘要 | Graphical abstract

导读 | Introduction

厌氧氨氧化（anammox）作为一种高效经济的自养生物脱氮技术，应用于城市污水主流处理，可提高城市污水处理厂经济和环境效益，促进城市污水处理厂可持续发展。当前，主流anammox工艺面临着两大挑战：1）如何在低温和低氮浓度下实现稳定且有实际意义的氮去除率；2）如何在易降解有机物存在时实现对异养反硝化菌淘汰或抑制，确保anammox菌的的有效持留以抵消其低生长速率。先前的研究（Environ. Sci. Technol., 54 (2020) 12959-12966）表明厌氧氨氧化颗粒污泥系统在主流处理中具有很好的应用前景。然而，并未考虑前处理单元可溶性有机碳源的引入对系统性能的潜在影响。因此，本研究以两种模型碳源（葡萄糖和乙酸盐）为例，探讨了单糖和挥发性脂肪酸对主流anammox颗粒污泥系统的影响。

Anaerobic ammonium oxidation (anammox) process is an efficient and economical autotrophic biological nitrogen removal technology. The sustainability of WWTPs could be further improved if the anammox process could be applied in mainstream treatment due to its enormous economic and environmental benefits. At present, there are real and substantive challenges to mainstream anammox processes in nontropical areas: 1) the achievement of practically meaningful nitrogen removal rates and an acceptable stability at low temperatures and N concentrations and 2) the out-selection or suppression of heterotrophic denitrifiers with the organic matter input to ensure sufficient retention of the anammox biomass to offset a low anammox growth rate. Previous research (Environ. Sci. Technol., 54 (2020) 12959-12966) suggested a promising prospect of granule-based anammox systems in mainstream treatment. However, the inevitable introduction of soluble organic matter originating from pretreatment units was not considered in their experiments. Therefore, two model carbon sources (glucose and acetate) were selected to explore the influence of monosaccharides and volatile fatty acids on granule-based anammox systems under mainstream environment.

一、反应器的性能

Reactor performance

如图1所示，两个上流式厌氧污泥床（UASB）反应器（进水含有30 mg N L^-1 NH₄⁺-N和40 mg N L^-1 NO₂^--N）在15 ± 1°C的温度下连续运行了120天。在无外碳源添加的P0（COD/N=0）阶段，两个反应器中NH₄⁺-N、NO₂^--N和TN的去除效率分别为99.0 ± 0.9%, 93.8 ± 1.9%和84.3 ± 1.2%，均维持在较高水平。低浓度易降解有机物的引入（COD/N=0.5，P1阶段）只轻微降低了NH₄⁺-N的去除率，但显著提高了NO₂^--N和NO₃^--N的去除率。因此，低浓度易降解有机物的引入（COD/N≤0.5）不但没有改变anammox对TN去除的主导地位，而且还提高了反硝化脱氮贡献，系统成功实现深度脱氮（TN<10 mgN/L，图1d，60~80天）。值得注意的是，当易降解有机物的浓度增加到COD/N=1时（P2阶段）,反应器的NH₄⁺-N去除效率显著下降，抑制了反应器性能。

As shown in Fig. 1, two upflow anaerobic sludge blanket (UASB) reactors were operated continuously for 120 days at 15 ± 1 °C. In P0 (COD/N=0) without external carbon addition, the NH₄⁺-N, NO₂^--N and TN removal efficiencies in two reactors maintained at a high level. In P1 (COD/N=0.5), the addition of degradable carbon source specifically reduced the removal efficiency of NH₄⁺-N. At the same time, the removal efficiency of NO₂^--N and NO₃^--N was obviously improved. The addition of degradable carbon source (COD/N≤0.5) not only did not change the dominance of anammox for TN removal, but also increased the contribution of denitrification for TN removal, and the system successfully achieved deep nitrogen removal. In P2 (COD/N=1.0), the NH₄⁺-N removal efficiency further decline in two reactors, inhibiting the reactor performance.

图1 两个UASB反应器（G和A分别表示碳源为葡萄糖和乙酸）的长期运行性能

Fig. 1  Long-term operation of two UASB reactors (G and A represent glucose and acetate as the carbon source, respectively)

二、比厌氧氨氧化活性、比反硝化活性和胞外聚合物的变化

Variation in the SAA, SDA and EPS

低浓度葡萄糖和乙酸的引入（COD/N = 0.5）未对SAA产生明显影响，但略微提高了比反硝化活性（SDA）（图2（a）,2（b）和2（c））。易降解有机物（尤其是葡萄糖）浓度的进一步增加（COD/N = 1.0）降低了SAA（图2（a）），但显著提高了SDA（p < 0.05，t检验）。总得来讲，高浓度易降解有机物（无论是葡萄糖还是乙酸）限制了厌氧氨氧化活性，但大幅提高了以硝酸盐或亚硝酸盐为电子受体的反硝化活性（图2（b）和（c））。颗粒污泥胞外聚合物含量随着比反硝化活性的增加而下降，并且下降率远大于厌氧氨氧化活性的降低幅度，这表明易降解有机碳物的引入可能是促进了反硝化菌对胞外聚合物的降解（图2（d））。

Adding low concentration glucose and acetate (COD/N = 0.5) did not reduce the SAA, but slightly promoted denitrification activity (Fig. 2a, 2b and 2c). However, the SAA significantly decreased with a further increase in glucose and acetate addition (COD/N = 1.0), while the SDA significantly increased (p<0.05, t-test). Further increase of degradable organics (especially glucose) (COD/N=1.0) reduced SAA (Fig. 2a) but significantly increased SDA (Fig. 2b and 2c). The content of extracellular polymers (EPS) in granular sludge decreased with the increase of SDA, and the decline percentage was far greater than that of SAA, which indicated that the introduction of easily degradable organic carbon might promote the degradation of EPS by denitrifying bacteria.

图2 污泥的比厌氧氨氧化活性（a）。以亚硝酸盐为电子受体的比反硝化活性（b）。硝酸盐作为电子受体的比反硝化活性（c）。多糖（PS）和蛋白质（PN）的含量以及PS/PN（d）的比值。

Fig. 2 The specific anammox activity of anammox granular sludge (a). The specific denitrification activity with the nitrite as electron acceptors (b). The specific denitrification activity with the nitrate as electron acceptors (c). The contents of polysaccharides (PS) and proteins (PN) in anammox granular sludge and the ratio of PS/PN (d).

三、主流anammox系统的微生物特性

Microbial characteristics of the mainstream anammox system

隶属于浮霉菌门的Ca. Kuenenia是颗粒污泥系统中的优势厌氧氨氧化菌。易降解有机物对Ca. Kuenenia丰度的影响因有机物类型而异，低浓度乙酸（COD/N = 0.5）的存在促进了Ca. Kuenenia丰度的增加，而葡萄糖的引入（COD/N = 0.5~1.0）抑制了Ca. Kuenenia的增殖。易降解有机物的类型对反硝化菌的富集作用也因有机物类型而异：葡萄糖添加的导致Rhodobacter和Zoogloea反硝菌被富集，而乙酸钠的添加促进了Flavobacterium、Pseudomonas和Thauera的富集。有趣的是，在P2阶段，通过分析反应器不同高度的污泥样品发现Ca. Kuenenia主要在反应器底部富集（图3），但反硝化菌主要分布在反应器上部。反应器中出现自养和异养菌随空间高度变化的自分层现象：Ca. Kuenenia的相对丰度随着反应器高度的增加呈现下降趋势，而反硝化菌的相对丰度随反应器高度变化的趋势恰恰相反。

Ca. Kuenenia is a key functional bacterium in the anammox system affiliated with the phylum Planctomycetes. The effect of degradable organic matter on Ca. Kuenenia abundance varies by organic matter type. The presence of low concentration acetate (COD/N=0.5) promotes the relative abundance of Ca. Kuenenia, while the introduction of glucose (COD/N=0.5~1.0) inhibits the relative abundance of Ca. Kuenenia. The enrichment of denitrifying bacteria also depends on the type of organic matter. Rhodobacter and Zoogloea were enriched with the addition of glucose, while Flavobacterium, Pseudomonas and Thauera were enriched with the addition of acetate. Notably, Ca. Kuenenia was mainly enriched at the bottom of the reactor by analyzing sludge samples at different heights of the reactor in P2, while denitrifying bacteria were mainly distributed at the top of the reactor (Fig. 3). Self-stratification of autotrophic and heterotrophic bacteria was observed with space height changes in the reactor. The relative abundance of Ca. Kuenenia decreased with the increase in reactor height, while the relative abundance of denitrifying bacteria increased with reactor height.

图3 高通量测序结果显示Ca.Kuenenia和主要反硝化细菌的相对丰度

Fig. 3 Relative abundance of the anammox bacterium Ca. Kuenenia and the dominant denitrifying bacteria revealed by high-throughput sequencing profiles.

四、功能基因的转录反应

Transcriptional response of the functional genes

分别由hzsA、hdh和nirS基因编码的联胺合成酶、联胺脱氢酶和一氧化二氮还原酶在anammox的代谢过程中起着举足轻重的作用。hzsA和hdh的绝对丰度随着易降解有机物浓度的增加显著降低（图4a），与Ca. Kuenenia相对丰度的变化趋势一致。反硝化基因的绝对丰度随着易降解有机物浓度的增加而增加，与反硝化菌的活性和微生物群落一致（图4b）。

Hydrazine synthetase, hydrazine dehydrogenase and nitrous oxide reductase, encoded by hzsA, hdh and nirS, respectively, play a pivotal role in the biological process of anammox. The absolute abundance of hzsA and hdh decreased significantly with the increase of degradable organic matter (Fig. 4a), which showed the same trend as the relative abundance of Ca. Kuenenia. The absolute abundance of the denitrification genes increased with increasing organic matter, which was consistent with the activity and microbial community of the denitrifying bacteria (Fig. 4b).

图4 Anammox基因 (a) 和反硝化基因在mRNA水平上的表达 (b)

Fig. 4 Expression of anammox gene (a) and denitrification gene at mRNA level (b).

总结和展望 | Conclusion and Prospection

当进水中易降解有机物浓度在COD/N=0.5时，anammox颗粒污泥反应器能够稳定长期运行。当COD/N比增加到1.0时，anammox功能基因的表达被明显抑制，而反硝化功能基因的表达被显著促进，由于厌氧氨氧化作用被抑制导致氨氮去除率下降，使得反应器总氮去除率明显下降。此外，微生物群落沿反应器高度呈现自分层现象，有利于颗粒中的anammox菌的自持留，而絮体中的反硝化菌自流失，可增强厌氧氨氧化颗粒污泥系统在主流条件下应对易降解有机物冲击的鲁棒性。在实际工程应用中，厌氧氨氧化前处理单元的易降解有机物含量无法精确控制。因此，当rbCOD/N>0.5时，选择性分离并排放反硝化菌对维持反应器中anammox菌的主导地位至关重要。主流颗粒污泥anammox系统中内在的生物质自分离特性为选择性排出异养反硝化菌提供了优势。后续研究应根据实际废水易降解有机物的浓度开发选择性定量排泥策略。

The stable long-term operation of anammox reactor performance with the introduction of two model carbon sources (glucose and acetate) was achieved at a COD/N ratio of 0.5. The expression of anammox functional gene was significantly inhibited, while the expression of denitrification functional gene was significantly promoted at a COD/N ratio of 1.0. Therefore, the TN removal rate of the reactor decreased significantly due to the inhibition of anammox. Besides, the microbial community showed the self-stratification along the reactor height, which was conducive to retain the anammox bacteria in granules but washed out the denitrifying bacteria in flocs. The results can enhance the robustness of anammox granular sludge system to respond to the impact of easily degradable organics under mainstream conditions. In practical engineering applications, the amount of organic matter from pretreatment units cannot be accurately controlled. Therefore, it is crucial to selectively separate and discharge denitrifying bacteria for the maintain the dominant role of anammox bacteria when rbCOD/N is over 0.5. The inherent biomass self-segregation property in the granule-based mainstream anammox systems offers advantages for the selective discharge of heterotrophic denitrifying bacteria. Subsequent research should develop the selective and quantitative discharge strategy according to the concentration of easily biodegradable organics in the actual wastewater.

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https://www.sciencedirect.com/

science/article/pii/

S0048969722052901

本文内容来自ELSEVIER旗舰期刊Sci Total Environ第851卷发表的论文：

Cheng Y.F., Zhang Z.Z., Ma W.J., Li G.F., Huang B.C., Fan N.S., Jin R.C., 2022. Response of the mainstream anammox process to the biodegradable carbon sources in the granule-based systems: The difference in self-stratification of the microbial community. Sci Total Environ 851, 158191. 

https://doi.org/10.1016/j.scitotenv.2022.158191

第一作者：程雅菲  硕士

杭州师范大学

生命与环境科学学院

在杭州师范大学获得硕士学位，现为复旦大学环境科学与工程系博士。主要研究方向为新型生物脱氮工艺的开发、典型新兴污染物对生物脱氮工艺的影响及调控技术、废弃生物质资源化利用。以第一作者或共同作者在Science of the Total Environment、Environmental Science & Technology、Journal of Hazardous Materials、Bioresource Technology等国际期刊发表论文10篇。

通讯作者：金仁村  教授

杭州师范大学

生命与环境科学学院

杭州师范大学生命与环境科学学院教授、博导，中国微生物学会环境微生物学专委会委员、中国生态学学会微生物生态学专委会委员、浙江省环境科学学会常务理事、美国水环境协会会刊Water Environ. Res.副主编，浙江省“杰出青年基金”获得者，浙江省“万人计划”青年拔尖人才、浙江省“中青年学科带头人”、浙江省“151人才”、杭州市“131人才”(重点资助层次)。主要研究方向为水污染控制工程、修复生态学、环境微生物学，主持了多项国家自然科学基金项目等。在有重要影响力的环境科学国际学术期刊发表论文200余篇，获授权发明专利70余项。

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