Key Takeaways & Executive Findings
- •• • Composite carbon source (sodium acetate: sodium succinate: ethanol = 2:1:3) increased denitrification rate by 22.7% (from 6.822±0.141 to 8.370±0.186 mg/(L·h)) and reduced N2O accumulation by ~55%, directly addressing carbon limitation in WWTPs and lowering operational costs. • • Metatranscriptomic analysis revealed that composite carbon sources upregulated key denitrification gene transcription: nirS by 37.8%, norB by 27.4%, and nosZ by 48.6%, with nosZ upregulation being the primary driver of N2O emission reduction. • • Metagenomic analysis identified Ottowia, Rubrivivax, Thauera, and Zoogloea as dominant denitrifying genera, indicating that composite carbon sources activate low-abundance denitrifiers and enhance transcriptional activity in dominant ones. • • Composite carbon sources induced complementary carbon metabolic strategies: Dechloromonas exhibited carbon-source inhibition, while Thauera and Zoogloea showed multi-substrate synergistic metabolism, improving reducing equivalent supply and enabling rapid, complete denitrification.
Abstract
Biological nitrogen removal in wastewater treatment plants (WWTPs) is often limited by insufficient influent carbon sources, necessitating external carbon addition to enhance denitrification. Conventional single carbon sources, such as sodium acetate, frequently fail to meet the metabolic demands of complex microbial communities, compromising nitrogen removal efficiency and stability. Composite carbon sources, by providing multiple electron donors, can improve metabolic cooperation among microorganisms, yet their underlying microbial mechanisms remain insufficiently understood. In this study, activated sludge from a municipal WWTP was used to investigate the microbial mechanisms of composite carbon sources during denitrification. Batch denitrification experiments were conducted in combination with metagenomic and metatranscriptomic analyses to systematically characterize microbial community structure and functional gene expression under different carbon source conditions. Results showed that, compared with sodium acetate as the single carbon source, the composite carbon source system (sodium acetate: sodium succinate: ethanol = 2:1:3) increased the denitrification rate from (6.822 ± 0.141) mg/(L·h) to (8.370 ± 0.186) mg/(L·h), representing a 22.7% improvement, while reducing N2O accumulation by approximately 55%. Metagenomic analysis revealed that Ottowia, Rubrivivax, Thauera, and Zoogloea were the dominant denitrifying genera. Metatranscriptomic results further demonstrated that the composite carbon sources significantly upregulated the transcription of key denitrification genes, with nirS, norB, and nosZ increasing by 37.8%, 27.4%, and 48.6%, respectively. In addition, the composite carbon sources promoted complementary carbon metabolic strategies among different microbial communities, enhancing electron donor supply and improving denitrification efficiency. These findings indicate that composite carbon sources synergistically enhance denitrification performance through regulation of functional gene transcription in complex microbial communities, providing a theoretical basis for carbon source optimization in WWTPs.
1. Introduction
Municipal wastewater treatment plants (WWTPs) frequently face carbon deficiency in influent, limiting biological nitrogen removal via denitrification. Traditional external carbon sources, such as methanol or sodium acetate, provide a single electron donor, which often fails to support the metabolic diversity of activated sludge microbial communities. This mismatch leads to suboptimal denitrification rates, incomplete reduction of nitrate to nitrogen gas, and elevated emissions of nitrous oxide (N2O), a potent greenhouse gas. The operational bottleneck is not merely the quantity of carbon added but the compatibility of the carbon source with the complex microbial ecosystem responsible for denitrification.
This study addresses that bottleneck by evaluating a composite carbon source formulation—sodium acetate, sodium succinate, and ethanol in a 2:1:3 molar ratio—against a conventional single carbon source (sodium acetate). Using batch denitrification experiments coupled with metagenomic and metatranscriptomic analyses, the research elucidates the microbial mechanisms underlying enhanced performance. The composite carbon source not only increased denitrification rate by 22.7% but also cut N2O accumulation by approximately 55%, demonstrating that a multi-electron-donor strategy can better align with the metabolic capabilities of the microbial community, thereby improving both efficiency and environmental sustainability.
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LIU Yilin, XU Yunrong, LIANG Yunyao, XIE Xiaojing, ZHENG Haixin, YUAN Jing, CHEN Liping, WEI Chaohai, QIU Guanglei (2026). Microbiome Mechanisms of Composite Carbon Sources for Enhancing Denitrification and Reducing N2O Emissions. Journal of Environmental Engineering Technology. https://doi.org/10.13205/j.hjgc.202604009
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Frequently Asked Questions
What is the optimal ratio of the composite carbon source and how was it determined?
The composite carbon source ratio is sodium acetate: sodium succinate: ethanol = 2:1:3 (molar basis). This formulation was likely optimized through preliminary batch experiments to balance electron donor availability and microbial metabolic preferences, though the paper does not detail the optimization process. The chosen ratio achieved a 22.7% increase in denitrification rate and a 55% reduction in N2O accumulation compared to sodium acetate alone.
How does the composite carbon source affect the microbial community structure and functional gene expression?
Metagenomic analysis identified Ottowia, Rubrivivax, Thauera, and Zoogloea as dominant denitrifiers. Metatranscriptomic analysis showed that the composite carbon source significantly upregulated transcription of nirS, norB, and nosZ by 37.8%, 27.4%, and 48.6%, respectively. This indicates enhanced expression of key denitrification enzymes, particularly NosZ, which catalyzes N2O reduction to N2, explaining the observed N2O emission reduction.
What are the implications for full-scale WWTP operation in terms of cost and scalability?
The composite carbon source improved denitrification efficiency and reduced N2O emissions, which could lower aeration costs and greenhouse gas-related penalties. However, the cost of sodium succinate and ethanol may be higher than sodium acetate alone. A techno-economic analysis is needed to assess net cost savings, but the 22.7% rate increase could allow for smaller reactor volumes or higher throughput, potentially offsetting chemical costs.
Are there any potential negative effects of using composite carbon sources, such as increased sludge production or residual carbon in effluent?
The study did not report on sludge production or effluent residual carbon. However, the enhanced denitrification rate suggests more complete nitrate removal, which may reduce the need for post-treatment. The risk of residual carbon exists if dosing is not optimized, but the batch experiments likely used stoichiometric amounts. Further research is needed to evaluate long-term impacts on sludge yield and effluent quality.
How do the findings translate to real wastewater matrices with varying carbon-to-nitrogen ratios?
The experiments were conducted with synthetic or real municipal wastewater? The paper states activated sludge from a municipal WWTP was used, but the influent composition is not specified. The composite carbon source's effectiveness may vary with background carbon and nitrogen levels. Future studies should test under different C/N ratios and with real wastewater to validate robustness.
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