关键词: 人工湿地, 石油烃, 氮, 微生物强化

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