Abstract:

With the development of urbanization and industrialization, nitrogen and petroleum hydrocarbons have become two dominant contaminants in urban surface runoff. These pollutants originate from multiple sources, including vehicle exhaust emissions, excessive agricultural fertilizer application, industrial wastewater discharge, atmospheric deposition, and so on. Nitrogen and petroleum hydrocarbons in urban surface runoff poses serious threats to both ecological security and human health. Therefore, it is urgent to develop efficient and environmentally friendly remediation methods to remove combined pollution. Constructed wetlands (CWs) have gained increasing attention as a sustainable bioremediation technology due to their low cost, high efficiency, and environmentally friendly. CWs can simulate the self-purification functions of natural wetlands through the synergistic actions of substrates, water, plants, and microorganisms. However, research on the simultaneous removal of nitrogen and petroleum hydrocarbons in CWs remains limited. The underlying synergistic removal mechanisms of CWs are still largely unexplored. This study used biochar-immobilized microorganisms to enhance removal of nitrogen and petroleum hydrocarbon in CWs. Furthermore, this study used metagenomic and metabolomic approaches to reveal the mechanistic coupling between nitrogen metabolism and petroleum hydrocarbon degradation. These provided a theoretical basis for the application of CWs in treating combined pollution. The biochar-immobilized microorganisms were made up of TX3 (Acinetobacter sp.) and nitrifying bacteria (Nitrobacter) with biochar as a carrier and sodium alginate as the embedding matrix. Laboratory-scale CWs (L=0.71 m, W=0.48 m, H=0.39 m) were constructed and filled with coarse gravel (Φ=20 mm). Iris sibirica had the ability to remove both nitrogen and petroleum hydrocarbons. Six experimental groups were designed: PBCN (plants and biochar-immobilized microorganisms and hexadecane and nitrogen), PCN (plants and hexadecane and nitrogen), PC (plants and hexadecane), PN (plants and nitrogen), BCN (biochar-immobilized microorganisms and hexadecane and nitrogen), and CN (hexadecane and nitrogen). The HRT was set to 7 days. The initial influent concentrations were: hexadecane of 350 mg·L⁻¹, NH₄⁺-N of 15 mg·L⁻¹, NO₃⁻-N of 10 mg·L⁻¹. Water samples were collected daily from the outlet, and pollutant concentrations were measured according to standard national methods(HJ 970—2018 for petroleum oils, HJ 535－2009 for NH₄⁺-N, HJ/T 346－2007 for NO₃⁻-N, and HJ 636—2012 for TN). Metagenomic and metabolomic analyses were performed by sent to Majorbio (Shanghai, China). The data were analyzed through the majorbio choud platform (cloud.majorbio.com). The results showed that PBCN group achieved significantly higher removal efficiencies compared to the control groups. Specifically, the effluent concentration of hexadecane in the PBCN group was 65.8 mg·L⁻¹, corresponding to a removal rate of 81.3%, while the effluent TN concentration was 5.64 mg·L⁻¹, with a removal rate of 77.4%. The PCN group showed hexadecane and TN removal rates of 73.2% and 68.2%. The addition of biochar-immobilized microorganisms increased the removal rates of hexadecane and TN by 8.11% and 9.24%. The presence of plants also contributed to pollutant removal. Microbial community analysis at the phylum level revealed that biochar-immobilized microorganisms significantly altered the community structure. In PBCN group, the relative abundance of Pseudomonadota (formerly Proteobacteria) reached 35.7%, which was notably higher than the 27.8% observed in PCN group. Pseudomonadota is known to contain numerous genera involved in nitrification, denitrification, and petroleum hydrocarbons degradation. Other phyla with important ecological functions included Cyanobacteriota (involved in nitrogen fixation), Bacteroidota (involved in short-cut denitrification), Bacillota (involved in dissimilatory nitrate reduction to ammonium, DNRA), Planctomycetota (involved in anaerobic ammonia oxidation), and Nitrospirota (involved in nitrification). At the genus level, several functional taxa were enriched in the PBCN group, including Lysobacter (which has been reported to enhance the removal of nitrogen and petroleum hydrocarbons in contaminated soil), Leptolyngbya (favorable for alkane degradation), Bradyrhizobium (involved in nitrogen cycling), and Steroidobacter (involved in nitrification and denitrification). These compositional changes suggest that biochar-immobilized microorganisms promoted the proliferation of functional microorganisms that are jointly responsible for nitrogen and petroleum hydrocarbon removal. Metagenomic analysis identified key functional enzymes involved in nitrogen metabolism and hexadecane degradation. For nitrogen metabolism, the main enzymes included EC 4.2.1.1, EC 6.3.1.2, EC1.4.1.13, and EC1.7.2.1. These enzymes are involved in the conversion of NH₄⁺-N to glutamine (Gln) and then to glutamate (Glu), as well as in denitrification and DNRA pathways. For hexadecane degradation, the key enzymes included EC 1.14.15.3, EC 1.1.1.1, EC 1.2.1.10, and a series of acyl-CoA dehydrogenases involved in β-oxidation: EC 1.3.8.1, EC 1.3.8.7, EC 1.3.8.8, and EC 1.3.8.9. The β-oxidation pathway also involved EC 4.2.1.17 and EC 1.1.1.35, which generate FADH₂ and NADH. Correlation heatmap analysis between nitrogen metabolism and hexadecane degradation enzymes showed that the positive correlations between alkane hydroxylase, alcohol dehydrogenase, aldehyde dehydrogenase and nitrogen metabolism enzymes in PBCN group were more numerous and significant compared to PCN group. This indicates that biochar-immobilized microorganisms accelerated the initial conversion of hexadecane to hexadecanoic acid to enhance the coupling between nitrogen and hydrocarbon metabolism. In PCN group, the correlations between β-oxidation enzymes (acyl-CoA dehydrogenases) and denitrification enzymes (e.g., EC 1.7.5.1, EC 1.7.2.1, EC 1.7.2.4) were stronger. A novel synergistic degradation pathway was proposed. In this pathway, hexadecane was first oxidized to hexadecanoic acid via the alkane hydroxylase (AlkB) system. Hexadecanoic acid then entered the β-oxidation pathway, where it was sequentially broken down into acetyl-CoA. During β-oxidation, acyl-CoA dehydrogenases (EC 1.3.8.1, EC 1.3.8.7, EC 1.3.8.8, EC 1.3.8.9) reduce FAD to FADH₂, while enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) reduce NAD⁺ to NADH. Both FADH₂ and NADH serve as electron donors for the denitrification process, which reduces NO₃⁻ to NO₂⁻, NO, N₂O, and finally N₂. Meanwhile, in the DNRA pathway, nitrate reductase (EC 1.7.5.1) and nitrite reductase (EC 1.7.2.2) convert NO₃⁻ to NH₄⁺. This NH₄⁺ were then assimilated into glutamine (Gln) by glutamine synthetase (EC 6.3.1.2) and subsequently converted to glutamate (Glu) by glutamate synthase (EC 1.4.1.13 or EC 1.4.7.1). Glutamate enters the glutamate metabolism pathway and is deaminated by glutamate dehydrogenases (EC 1.4.1.2, EC 1.4.1.3, EC 1.4.1.4) to produce 2-Oxoglutaric acid (2-Og). 2-Og is a key intermediate of the tricarboxylic acid (TCA) cycle. When 2-Og entered the TCA cycle, it was metabolized to generate ATP. The ATP promoted the expression of alkane hydroxylase and other hexadecane-degrading enzymes to accelerate hexadecane biodegradation. This pathway demonstrated that the carbon skeleton and energy derived from petroleum hydrocarbon degradation can be efficiently channeled into nitrogen assimilation and energy metabolism, forming a closed-loop synergistic degradation system.

Key words: constructed wetlands, petroleum hydrocarbon, nitrogen, microbial enhancement

摘要：

随着现代化城市建设的发展，机动车尾气排放、农业化肥过量使用以及大气沉降等污染源，使得城市地表径流中存在大量的氮与石油烃等污染物，对城市路域生态系统和人类健康构成严重威胁，探究高效去除地表径流中氮与石油烃复合污染的方法迫在眉睫。人工湿地（CWs）因其绿色友好及对污染物的高效降解等特点，近年来受到了国内外研究者的关注。本研究通过固定化微生物提高CWs对氮与石油烃的去除效果。固定化微生物强化的CWs中十六烷的出水值为65.8 mg·L⁻¹、去除率为81.3%，总氮的出水值为5.64 mg·L⁻¹、去除率为77.4%，较对照组对十六烷、总氮的去除率分别提高8.11%、9.24%，这是由于固定化微生物改善了CWs中的微生物群落结构。利用宏基因组学和代谢组学分析CWs中氮与石油烃的协同去除机理，发现2-酮戊二酸（2-Og）作为三羧酸循环（TCA循环）的中间产物连接氮代谢与石油烃降解。固定化微生物加速十六烷转化为十六烷酸，十六烷酸为厚壁菌门（Bacillota）或其他异养微生物提供碳源，参与异化硝酸盐还原为铵（DNRA）的过程。十六烷酸通过β-氧化途径生成辅酶FADH₂和NADH为反硝化过程提供电子供体，DNRA产生的NH₄⁺在功能基因的作用下合成Gln和Glu，进入谷氨酸代谢生成2-Og进入TCA循环，产生的ATP有利于促进十六烷的进一步降解。

关键词: 人工湿地, 石油烃, 氮, 微生物强化