Unraveling the role of formate in improving nitrogen removal via coupled partial denitrification-anammox

doi:10.1007/s11783-024-1872-8

Front. Environ. Sci. Eng.

2024, Vol. 18

Issue (9) : 112 https://doi.org/10.1007/s11783-024-1872-8

Unraveling the role of formate in improving nitrogen removal via coupled partial denitrification-anammox

Wanlu Zhu¹, Rui Xiao²(

), Min Xu¹, Wenbo Chai¹, Wenlong Liu³, Zhengyu Jin⁴, David Ikumi⁵, Huijie Lu^1,⁶(

)

¹. Key Laboratory of Environmental Remediation and Ecological Health (Ministry of Education), College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
². China National Nuclear Corporation, Beijing Research Institute of Chemical Engineering and Metallurgy, Beijing 100029, China
³. College of Environment, Zhejiang University of Technology, Hangzhou 310014, China
⁴. Key Laboratory of Ecology and Environment in Minority Areas, National Ethnic Affairs Commission, College of Life & Environmental Sciences, Minzu University of China, Beijing 100081, China
⁵. Water Research Group, Department of Civil Engineering, University of Cape Town, Rondebosch 7700, South Africa
⁶. Key Laboratory of Water Pollution Control and Environmental Safety, Hangzhou 310058, China

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Abstract

● Formate addition led to more abundant and active anammox bacteria in community.

● FISH–NanoSIMS identified Ca. Brocadia and Desulfobacillus as main formate utilizers.

● Anammox bacteria were key players in formate uptake and partial denitrification.

● Formate was assimilated by Ca. Brocadia via the Wood–Ljungdahl and rGly pathways.

● Desulfobacillus could provide necessities e.g., folate to support Ca. Brocadia growth.

The addition of traditional carbon sources (e.g., acetate) could favor heterotrophic overgrowth in partial denitrification coupled with anammox (PD–A) systems, thus hindering the performance and stability of this novel wastewater nitrogen removal technology. Therefore, it is necessary to develop an effective, environmentally friendly, and inexpensive alternative. This study demonstrated the potential of formate to enhance the performance and community stability of PD–A under mainstream conditions. In a laboratory-scale biofilm reactor, formate addition (COD/NO₃^––N = 1.75) improved nitrogen removal efficiency (from 72.1 ± 3.5% to 81.7 ± 2.7%), EPS content (from 106.3 ± 8.1 to 163.0 ± 15.5 mg/gVSS) and increased anammox bacteria growth (predominantly Candidatus Brocadia, from 29.5 ± 0.7% to 34.5 ± 5.4%) while maintaining stable heterotrophs dominated by methylotrophic Desulfobacillus. FISH–NanoSIMS revealed a formate uptake using Ca. Brocadia and Desulfobacillus, with Ca. Brocadia being the major contributor to partial nitrate reduction to nitrite. Desulfobacillus can synthesize diverse hydrophobic amino acids and provide key nutrients for Ca. Brocadia. To achieve comparable nitrogen removal, the cost of the formate-driven PD–A process should be 11.2% lower than that of acetate. These results greatly enrich our understanding of C1 metabolism represented by formate in anammox communities and its application in the context of coupling partial denitrification–anammox toward enhanced nitrogen removal in global wastewater treatment systems.

Keywords Formate Mixotrophic growth Partial denitrification-anammox Metabolic interaction FISH–NanoSIMS

Corresponding Author(s): Rui Xiao,Huijie Lu

Issue Date: 14 August 2024

Cite this article:

Wanlu Zhu,Rui Xiao,Min Xu, et al. Unraveling the role of formate in improving nitrogen removal via coupled partial denitrification-anammox[J]. Front. Environ. Sci. Eng., 2024, 18(9): 112.

URL:

https://academic.hep.com.cn/fese/EN/10.1007/s11783-024-1872-8
https://academic.hep.com.cn/fese/EN/Y2024/V18/I9/112

Fig.1 (a) Performance of the laboratory-scale MBBR in two phases; (b) SAA, SDA, and EPS content of biofilm samples in two phases. Error bars represent standard deviations from biological triplicate.

Fig.2 Abundances of the top 10 populations in the biofilm community in Phase I (Day 60) and II (Day 90 and Day 150). Bubble size and number denote the relative abundance (%) of populations as calculated based on metagenomic data. Bubble color indicates functional classifications. Taxonomy is assigned at the lowest level possible. The three columns at each time point represent biological replicates.

Tab.1 Comparisons of different carbon sources for PD–A under mainstream conditions

Fig.3 FISH–NanoSIMS images indicating ¹³C–formate and ¹⁵N–nitrate uptake by AnAOB, Desulfobacillus, and other bacteria at the single-cell level. (a) Representative FISH images of samples collected at t = 0, 10, 20, and 30 h of incubation (left column). Red: AnAOB (probe: Amx568), Blue: Desulfobacillus (probe: PRO405). Small squares denote AnAOB–enriched regions that are enlarged in the NanoSIMS images (right columns). Scale bar: 2 μm; Color bar: carbon isotope ratios (¹³C/¹²C), nitrogen isotope ratios (¹⁵N/¹⁴N) and ¹²C counts for AnAOB. (b) Correlations between ¹³C atom% and ¹⁵N atom% for AnAOB, Desulfobacillus, and other bacteria. Error bars indicate standard deviations from multiple single–cell scans. Symbol colors represent samples collected at t = 0 (light), 10, 20, and 30h (dark).

Fig.4 (a) Relative abundances of the nitrogen metabolic genes narG, nirS, and hzo and the anammox 16S rRNA gene in the two phases (Day 60 and Day 90); (b) Relative expression levels of the nitrogen metabolic genes narG, nirS, and hzo and the anammox 16S rRNA gene. Error bars represent standard deviations of biological triplicates according to RT–qPCR assays. Statistically significant differences between the two phases are indicated by * p < 0.05. (c) Relative expression levels of the nitrate reductase genes narG and napA in the top 8 bacterial groups (Day 150).

Fig.5 Expression levels of biosynthesis pathways of the selected amino acids (blue) and cofactors (orange) in the top 10 abundant populations (Phase II, Day 150). Detailed information of key genes involved in amino acid biosynthesis pathways can be found in Table S3. TPM values were calculated based on the metatranscriptomic data. Lines inside circles indicate absent or incomplete synthetic pathways.

Fig.6 Key metabolic pathways, gene expression, and metabolic exchanges between Ca. Brocadia and Desulfobacillus. Pathway color codes: yellow, Wood–Ljungdahl pathway in Ca. Brocadia; blue, rGly pathway in Ca. Brocadia; orange, roTCA cycle in Desulfobacillus; purple, CBB cycle in Desulfobacillus. Expression levels (log₂ TPM) of key genes were visualized as colored dots placed adjacent to the gene names (averaged for biological triplicates).

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