厭氧氨氧化菌的培養及其除氮效率評估

Abstract

厭氧氨氧化菌自上世紀末被發現以來，由於其低有機物需求量、低溫室氣體產出、污泥產量低的優勢，被視為代替現有污水廠除氮使用的反硝化菌法的潜在方法。 近二十年來，關於它的研究及應用已有初步進展，但是在實際應用中仍然面臨種種難題，比如該菌生長有嚴苛的環境條件限制、生長繁殖速度極其緩慢、在汙水處理廠中的低溫環境難以維持活躍、以及難以檢測對比其與反硝化菌的除氮貢獻高低等問題。 本研究使用序批式活性污泥法（Sequencing Batch Reactor，SBR），在接種了來自兩個不同汙水處理廠污泥後，在適宜條件下進行培養，隨後對污泥進行16S rRNA基因的全長測序以及改為15氮-亞硝酸根的穩定同位素示踪法進行厭氧氨氧化菌產氣量和貢獻率測試，探討在實驗室層面的情况下，厭氧氨氧化菌貢獻的檢測方法改良及培養效果。 在其中一個接種某食品廠和某污水廠的混合污泥的反應器B培養350天后，使用16S rRNA基因的全長定序檢測到厭氧氨氧化菌豐度達到3.264%，其優勢種為Candidatus Jettenia屬中一個尚未被歸類的種，而另一個接種某污水廠污泥的反應器A中僅檢測到0.172%，優勢種為Candidatus Brocadia屬種一個尚未被歸類的種。 然而，在厭氧氨氧化菌特有基因肼合成酶A/B/C及肼脫氫酶的宏基因組分析中，反應器A中的基因豐度高達15.7%，反而遠遠高於反應器B的3.26%，推測可能分析程式中有未被發現的問題，也可能是其中未被詳細研究的一個Fimbriimonadaceae屬中的未被分類的種可能攜帶ANAMMOX關鍵代謝的基因組，具體原因需要進一步純化厭氧氨氧化菌后，對該菌全基因組定序后進行確認。 在穩定同位素示踪法示踪法中，為類比利用厭氧氨氧化菌無需額外有機碳添加的優勢的使用場景，本實驗使用15氮-亞硝酸根替代原實驗中的15氮-硝酸根，以避免反硝化菌在缺乏有機物環境下無法將硝酸根轉化成亞硝酸根使厭氧氨氧化作用缺乏底物，從而導致厭氧氨氧化作用被低估的潜在問題。 而結果表明，在該情况下，厭氧氨氧化菌在反應器A、B污泥中分別達到了99.09%、99.61%的貢獻占比，其除氮率分別為1.21±0.57、3.05±0.42毫克每天每升污泥基質混合物（mg/L/h）。 而這一結果與A、B瓶進行的進出水量測離子色譜-質量平衡法有顯著差异。 在改進方法後量測兩個垃圾掩埋場污水廠厭氧池採集的污泥，得到 0.36±0.18、3.10±2.55毫克每天每升污泥基質混合物（mg/L/h）的反應速率，以揮發性懸浮固體VSS計為0.088 ± 0.044 毫克氮每毫克揮發性懸浮固體每小時（mg-N/mg-VSS/h）與0.721 ± 0.593 毫克氮每毫克揮發性懸浮固體每小時（mg-N/mg-VSS/h）及99.26%、89.21%的貢獻占比。

Since the end of the last century, anaerobic ammonium-oxidizing (ANAMMOX) bacteria have been regarded as a potential alternative to the denitrification method used in existing wastewater treatment plants for nitrogen removal, owing to their advantages of minimal organic demand, low production of greenhouse gas (nitrous oxide), and minimal sludge yield. Over the past two decades, research and application have made initial progress. However, the practical implementation of it still faces several obstacles, including stringent environmental constraints for bacterial growth, extremely slow growth and reproduction rates, difficulty maintaining bacterial activity in the low-temperature environment of wastewater treatment plants, and challenges in detecting and comparing their nitrogen removal contributions relative to denitrifying bacteria. This study employed a Sequencing Batch Reactor (SBR) system with two reactors, A and B. After inoculation with sludge from two distinct wastewater treatment plants, cultivation was conducted under optimized conditions. Subsequently, full-length 16S rRNA gene sequencing was performed on the sludge. Additionally, the contribution percentage and gas production rate of ANAMMOX bacteria were assessed using the stable isotope tracer method with 15N-nitrite instead of the commonly used 15N-nitrate. This approach aimed to refine detection methods for ANAMMOX bacteria contributions and evaluate cultivation efficiency under lab-level conditions. After 350 days of cultivation in Reactor B inoculated with mixed sludge from a food processing plant and a wastewater treatment plant, full-length 16S rRNA gene sequencing detected an ANAMMOX bacteria abundance of 3.264%, with the dominant species being an unclassified species of the genus Candidatus Jettenia. In contrast, reactor A sludge, which is from a wastewater treatment plant, detected only 0.172% ANAMMOX bacteria, dominated by an unclassified species within the genus Candidatus Brocadia. However, in metagenomic analysis of ANAMMOX-specific genes (hydrazine synthase subunit A/B/C and hydrazine dehydrogenase, HzsA/B/C and Hdh), gene abundance in Reactor A reached 15.7%, significantly higher than 3.26% in Reactor B. This suggests potential issues with the analysis software, or the presence of an unclassified species within the Fimbriimonadaceae family that may contain key ANAMMOX metabolic genes. The cause requires further purification of the ANAMMOX bacteria followed by genome sequencing to confirm. In the stable isotope tracing experiment, to simulate an optimal application circumstance for ANAMMOX bacteria, which does not require additional organic carbon supplementation, this study replaced 15N-nitrate with 15N-nitrite to prevent denitrifying bacteria from failing to convert nitrate to nitrite in an organic-carbon-depleted environment, which would deprive the ANAMMOX reaction of substrate, potentially leading to underestimation of its contribution. Results indicate that under these conditions, ANAMMOX bacteria contributed 99.09% and 99.61% to nitrogen removal in reactors A and B sludge, respectively, achieving nitrogen removal rates of 1.21 ± 0.57 and 3.05 ± 0.42 milligrams per day per liter of sludge substrate mixture (mg/L/h). This result differs significantly from the ion chromatography-mass balance measurements of influent and effluent conducted in bottles A and B. After refining the method, measurements of sludge collected from the anaerobic tanks at two different wastewater treatment plants yielded reaction rates of 0.36 ± 0.18 and 3.10 ± 2.55 mg/L/h, which could also be expressed as 0.088 ± 0.044 mg-N/mg-VSS/h and 0.721 ± 0.593 mg-N/mg-VSS/h, respectively, with contribution rates of 99.26% and 89.21%.