Higher N₂O production in sequencing batch reactors compared to continuous stirred tank reactors: effect of feast-famine cycles

Abstract

● N₂O emissions from a denitrifying SBR were 23 times higher than that of the CSTR.

● Feast famine conditions in SBR uniquely lead to producing undesirable levels of N₂O.

● An MAG closely related to previously identified Thauera is likely a major N₂O driver.

● Post-transcriptional regulation may be linked to higher N₂O production in SBR.

Nitrous oxide (N₂O) is a potent greenhouse gas that can be emitted during the biological treatment of wastewater. In this study, a comparison of the long-term performance characteristics and N₂O production of sequencing batch reactors (SBR) and continuous stirred tank reactors (CSTR) during nitrite-based denitrification was undertaken. It was found that both reactors had NO₂⁻-N removal efficiencies over 99.9 %, but the N₂O-N emissions from the SBR reached ~2.3 % of the removal nitrite-N load, while in the CSTR it never exceeded 0.1 %. High frequency sampling during one operation cycle of the SBR demonstrated that the N₂O accumulation ratio was ~0.1 % during the feast period, increased to ~1.9 % in the first five hours of the famine period, and then gradually reached ~2.3 % at the end of famine. Batch experiments showed that limiting extracellular electron donor is required for N₂O accumulation in cells from the SBR-famine period and that cells from the CSTR do not accumulate N₂O when either nitrite or carbon is limiting. Another notable difference in the two reactor communities was the high level of accumulation of intracellular granules, most likely polyhydroxybutyrate (PHB), in cells during the feast period in the SBR. Metagenome assembly and binning found that one genome (PRO1), which is a Thauera, accounted for over half the metagenomic reads in both reactors. Neither shifts in gene regulation nor community composition explained the observed differences in reactor performance suggesting some post-transcriptional regulation obligatorily linked to antecedent conditions underly increased N₂O production in the SBR.

Graphical abstract

Keywords

Denitrifying N ₂O mitigation / SBR / CSTR / Meta-omics / PHB

References

[1] Anburajan P , Naresh Kumar A , Sabapathy P C , Kim G B , Cayetano R D , Yoon J J , Kumar G , Kim S H . (2019). Polyhydroxy butyrate production by Acinetobacter junii BP25, Aeromonas hydrophila ATCC 7966, and their co-culture using a feast and famine strategy. Bioresource Technology, 293: 122062

[2] APHA (1998). Standard Methods for the Examinations of Water and Wastewater. Washington, DC: American Public Health Association

[3] Arkin A P , Cottingham R W , Henry C S , Harris N L , Stevens R L , Maslov S , Dehal P , Ware D , Perez F , Canon S . . (2018). KBase: The United States department of energy systems biology knowledgebase. Nature Biotechnology, 36(7): 566–569

[4] Bergaust L , Shapleigh J , Frostegård A , Bakken L . (2008). Transcription and activities of NO_x reductases in Agrobacterium tumefaciens: the influence of nitrate, nitrite and oxygen availability. Environmental Microbiology, 10(11): 3070–3081

[5] Bhattacharjee A S , Wu S , Lawson C E , Jetten M S M , Kapoor V , Domingo J W S , McMahon K D , Noguera D R , Goel R . (2017). Whole-community metagenomics in two different anammox configurations: process performance and community structure. Environmental Science & Technology, 51(8): 4317–4327

[6] Bollon J , Filali A , Fayolle Y , Guerin S , Rocher V , Gillot S . (2016). N₂O emissions from full-scale nitrifying biofilters. Water Research, 102: 41–51

[7] Buchfink B , Xie C , Huson D H . (2015). Fast and sensitive protein alignment using DIAMOND. Nature Methods, 12(1): 59–60

[8] Colpa D I , Zhou W , Wempe J P , Tamis J , Stuart M C A , Krooneman J , Euverink G W . (2020). Thauera aminoaromatica MZ1T identified as a polyhydroxyalkanoate-producing bacterium within a mixed microbial consortium. Bioengineering (Basel, Switzerland), 7(1): 19

[9] Conthe M , Lycus P , Arntzen M O , Ramos da Silva A , Frostegård Å , Bakken L R , Kleerebezem R , van Loosdrecht M C M . (2019). Denitrification as an N₂O sink. Water Research, 151: 381–387

[10] Conthe M , Wittorf L , Kuenen J G , Kleerebezem R , van Loosdrecht M C M , Hallin S . (2018). Life on N₂O: deciphering the ecophysiology of N₂O respiring bacterial communities in a continuous culture. ISME Journal, 12(4): 1142–1153

[11] Corsino S F , Campo R , Di Bella G , Torregrossa M , Viviani G . (2016). Study of aerobic granular sludge stability in a continuous-flow membrane bioreactor. Bioresource Technology, 200: 1055–1059

[12] D'Alessio M , Nordeste R , Doxey A C , Charles T C . (2017). Transcriptome analysis of polyhydroxybutyrate cycle mutants reveals discrete loci connecting nitrogen utilization and carbon storage in Sinorhizobium meliloti. mSystems, 2(5): e00035–17

[13] Dircks K , Henze M , van Loosdrecht M C M , Mosbaek H , Aspegren H . (2001). Storage and degradation of poly-β-hydroxybutyrate in activated sludge under aerobic conditions. Water Research, 35(9): 2277–2285

[14] Graf D R H , Jones C M , Hallin S . (2014). Intergenomic comparisons highlight modularity of the denitrification pathway and underpin the importance of community structure for N₂O emissions. PLoS One, 9(12): e114118

[15] Hegarty T W . (1973). Temperature coefficient (Q10), seed germination and other biological processes. Nature, 243: 305–306

[16] Henze M, Gujer W, Mino T, Van Loosedrecht M (2000). Activated Sludge Models ASM1, ASM2, ASM2d and ASM3. Scientific & Technical Report IWA Publishing, 9. Diepoldsau, Switzerland: IWA Publishing ISBN: 9781900222242

[17] Heo S , Liu Y Q . (2021). Dependence of poly-β-hydroxybutyrate accumulation in sludge on biomass concentration in SBRs. Science of the Total Environment, 797: 149138

[18] Hu Q , Fan L R , Gao D W . (2017). Pilot-scale investigation on the treatment of cellulosic ethanol biorefinery wastewater. Chemical Engineering Journal, 309: 409–416

[19] Huang W , Zhou J , He X , He L , Lin Z , Shi S , Zhou J . (2021). Simultaneous nitrogen and phosphorus removal from simulated digested piggery wastewater in a single-stage biofilm process coupling anammox and intracellular carbon metabolism. Bioresource Technology, 333: 125152

[20] Hyatt D , Chen G L , Locascio P F , Land M L , Larimer F W , Hauser L J . (2010). Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics, 11(1): 119

[21] IPCC . (2019). 2019 Refinement to the 2006 IPCC Guidelines for National Greenhouse Gas Inventories, Chapter 6: Wastewater Treatment and Discharge. Bangkok: IPCC, 72

[22] Jia Y , Zhou M , Chen Y , Hu Y , Luo J . (2020). Insight into short-cut of simultaneous nitrification and denitrification process in moving bed biofilm reactor: effects of carbon to nitrogen ratio. Chemical Engineering Journal, 400: 125905

[23] Jiang X , Mira D , Cluff D L . (2018). The combustion mitigation of methane as a non-CO₂ greenhouse gas. Progress in Energy and Combustion Science, 66: 176–199

[24] Jones C M , Graf D R , Bru D , Philippot L , Hallin S . (2013). The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. ISME Journal, 7(2): 417–426

[25] Jones M , Stephenson T . (1996). The effects of temperature on enhanced biological phosphate removal. Environmental Technology, 17(9): 965–976

[26] Kang D D , Froula J , Egan R , Wang Z . (2015). MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities. PeerJ, 3(8): e1165

[27] Kent T R , Bott C B , Wang Z W . (2018). State of the art of aerobic granulation in continuous flow bioreactors. Biotechnology Advances, 36(4): 1139–1166

[28] Langmead B , Salzberg S L . (2012). Fast gapped-read alignment with Bowtie 2. Nature Methods, 9(4): 357–359

[29] Larriba O , Rovira-Cal E , Juznic-Zonta Z , Guisasola A , Baeza J A . (2020). Evaluation of the integration of P recovery, polyhydroxyalkanoate production and short cut nitrogen removal in a mainstream wastewater treatment process. Water Research, 172: 115474

[30] Li D , Liu C M , Luo R , Sadakane K , Lam T W . (2015). MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics (Oxford, England), 31(10): 1674–1676

[31] Li W , Cai Z Y , Duo Z J , Lu Y F , Gao K X , Abbas G , Zhang M , Zheng P . (2017). Heterotrophic ammonia and nitrate bio-removal over nitrite (Hanbon): performance and microflora. Chemosphere, 182: 532–538

[32] Li W , Li H , Liu Y D , Zheng P , Shapleigh J P . (2018a). Salinity-aided selection of progressive onset denitrifiers as a means of providing nitrite for anammox. Environmental Science & Technology, 52(18): 10665–10672

[33] Li W , Liu S , Zhang M , Zhao H P , Zheng P . (2018b). Oxidation of organic electron donor by denitratation: performance, pathway and key microorganism. Chemical Engineering Journal, 343: 554–560

[34] Li W , Zhuang J L , Zhou Y Y , Meng F G , Kang D , Zheng P , Shapleigh J P , Liu Y D . (2020). Metagenomics reveals microbial community differences lead to differential nitrate production in anammox reactors with differing nitrogen loading rates. Water Research, 169: 115279

[35] Liu B , Mao Y , Bergaust L , Bakken L R , Frostegård A . (2013). Strains in the genus Thauera exhibit remarkably different denitrification regulatory phenotypes. Environmental Microbiology, 15(10): 2816–2828

[36] Lopez M L D , Lin Y Y , Sato M , Hsieh C H , Shiah F K , Machida R J . (2022). Using metatranscriptomics to estimate the diversity and composition of zooplankton communities. Molecular Ecology Resources, 22(2): 638–652

[37] Lycus P , Lovise Bøthun K , Bergaust L , Peele Shapleigh J , Reier Bakken L , Frostegård Å . (2017). Phenotypic and genotypic richness of denitrifiers revealed by a novel isolation strategy. ISME Journal, 11(10): 2219–2232

[38] Ma C , Jensen M M , Smets B F , Thamdrup B . (2017). Pathways and controls of N₂O production in nitritation-anammox biomass. Environmental Science & Technology, 51(16): 8981–8991

[39] Nurk S , Meleshko D , Korobeynikov A , Pevzner P A . (2017). metaSPAdes: a new versatile metagenomic assembler. Genome Research, 27(5): 824–834

[40] Obruca S, Sedlacek P, Mravec F, Krzyzanek V, Nebesarova J, Samek O, Kucera D, Benesova P, Hrubanova K, Milerova M, Marova I (2017). The presence of PHB granules in cytoplasm protects non-halophilic bacterial cells against the harmful impact of hypertonic environments. New Biotechnology, 39(Pt A): 68–80

[41] Olm M R , Brown C T , Brooks B , Banfield J F . (2017). dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication. ISME Journal, 11(12): 2864–2868

[42] Olm M R , Crits-Christoph A , Bouma-Gregson K , Firek B A , Morowitz M J , Banfield J F . (2021). inStrain profiles population microdiversity from metagenomic data and sensitively detects shared microbial strains. Nature Biotechnology, 39(6): 727–736

[43] Pagáčová P , Galbová K , Drtil M , Jonatová I . (2010). Denitrification in USB reactor with granulated biomass. Bioresource Technology, 101(1): 150–156

[44] Pertea M , Pertea G M , Antonescu C M , Chang T C , Mendell J T , Salzberg S L . (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology, 33(3): 290–295

[45] Philippot L , Andert J , Jones C M , Bru D , Hallin S . (2011). Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N₂O emissions from soil. Global Change Biology, 17(3): 1497–1504

[46] Qiu S J , Liu J J , Zhang L , Zhang Q , Peng Y Z . (2021). Sludge fermentation liquid addition attained advanced nitrogen removal in low C/N ratio municipal wastewater through short-cut nitrification-denitrification and partial anammox. Frontiers of Environmental Science & Engineering, 15(2): 26

[47] Quagliano J C , Miyazaki S S . (1997). Effect of aeration and carbon/nitrogen ratio on the molecular mass of the biodegradable polymer poly-β-hydroxybutyrate obtained from Azotobacter chroococcum 6B. Applied Microbiology and Biotechnology, 48(5): 662–664

[48] Ren T , Chi Y , Wang Y , Shi X , Jin X , Jin P . (2021). Diversified metabolism makes novel Thauera strain highly competitive in low carbon wastewater treatment. Water Research, 206: 117742

[49] Roco C A , Bergaust L L , Bakken L R , Yavitt J B , Shapleigh J P . (2017). Modularity of nitrogen-oxide reducing soil bacteria: linking phenotype to genotype. Environmental Microbiology, 19(6): 2507–2519

[50] Rombouts J L , Mos G , Weissbrodt D G , Kleerebezem R , Van Loosdrecht M C M . (2019). Diversity and metabolism of xylose and glucose fermenting microbial communities in sequencing batch or continuous culturing. FEMS Microbiology Ecology, 95(2): fiy233

[51] Sanford R A , Wagner D D , Wu Q , Chee-Sanford J C , Thomas S H , Cruz-García C , Rodríguez G , Massol-Deyá A , Krishnani K K , Ritalahti K M , Nissen S , Konstantinidis K T , Löffler F E . (2012). Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils. Proceedings of the National Academy of Sciences of the United States of America, 109(48): 19709–19714

[52] Sarmiento-Pavía P D, Sosa-Torres M E (2021). Bioinorganic insights of the PQQ-dependent alcohol dehydrogenases. European Journal of Biochemistry, 26(2–3): 177–203

[53] Schalk-Otte S , Seviour R J , Kuenen J G , Jetten M S M . (2000). Nitrous oxide (N₂O) production by Alcaligenes faecalis during feast and famine regimes. Water Research, 34(7): 2080–2088

[54] Scherson Y D , Wells G F , Woo S G , Lee J , Park J , Cantwell B J , Criddle C S . (2013). Nitrogen removal with energy recovery through N₂O decomposition. Energy & Environmental Science, 6(1): 241–248

[55] Scherson Y D , Woo S G , Criddle C S . (2014). Production of nitrous oxide from anaerobic digester centrate and its use as a co-oxidant of biogas to enhance energy recovery. Environmental Science & Technology, 48(10): 5612–5619

[56] Shen X Y , Zhuge Y Y , Liu Y D , Shapleigh J P , Li W . (2021). Long-term effects of acetylene on denitrifying N₂O production: Biomass performance and microbial community. Journal of Water Process Engineering, 42: 102137

[57] Sun Y , Angelotti B , Brooks M , Wang Z W . (2021). Feast/famine ratio determined continuous flow aerobic granulation. Science of the Total Environment, 750: 141467

[58] Tian H , Xu R , Canadell J G , Thompson R L , Winiwarter W , Suntharalingam P , Davidson E A , Ciais P , Jackson R B , Janssens-Maenhout G . . (2020). A comprehensive quantification of global nitrous oxide sources and sinks. Nature, 586(7828): 248–256

[59] Trainer M A , Capstick D , Zachertowska A , Lam K N , Clark S R D , Charles T C . (2010). Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti. BMC Microbiology, 10(1): 92

[60] Wang J P , Liu Y D , Meng F G , Li W . (2020). The short- and long-term effects of formic acid on rapid nitritation start-up. Environment International, 135: 105350

[61] Wang Q , He J . (2020). Complete nitrogen removal via simultaneous nitrification and denitrification by a novel phosphate accumulating Thauera sp. strain SND5. Water Research, 185: 116300

[62] Xu Z , Dai X , Chai X . (2018). Effect of different carbon sources on denitrification performance, microbial community structure and denitrification genes. Science of the Total Environment, 634: 195–204

[63] Yang Q , Cui B , Zhou Y , Li J , Liu Z , Liu X . (2020). Impact of gas-water ratios on N₂O emissions in biological aerated filters and analysis of N₂O emissions pathways. Science of the Total Environment, 723: 137984

[64] Zhang J , Zhang Y , Quan X . (2012). Electricity assisted anaerobic treatment of salinity wastewater and its effects on microbial communities. Water Research, 46(11): 3535–3543

[65] Zhou F , Xiao W Z , Zhou K Y , Zhuang J L , Zhang X , Liu Y D , Ni B J , Shapleigh J P , Zhou M , Luo X Z , Li W . (2022). Performance characteristics and community analysis of a single-stage partial nitritation, anammox and denitratation (SPANADA) integrated process for treating low C/N ratio wastewater. Chemical Engineering Journal, 433: 134452

[66] Zhuang J L , Sun X , Zhao W Q , Zhang X , Zhou J J , Ni B J , Liu Y D , Shapleigh J P , Li W . (2022). The anammox coupled partial-denitrification process in an integrated granular sludge and fixed-biofilm reactor developed for mainstream wastewater treatment: Performance and community structure. Water Research, 210: 117964

[67] Zhuang J L , Zhou Y Y , Liu Y D , Li W . (2020). Flocs are the main source of nitrous oxide in a high-rate anammox granular sludge reactor: insights from metagenomics and fed-batch experiments. Water Research, 186: 116321

[68] Zhuge Y Y , Shen X Y , Liu Y D , Shapleigh J , Li W . (2020). Application of acidic conditions and inert-gas sparging to achieve high-efficiency nitrous oxide recovery during nitrite denitrification. Water Research, 182: 116001

RIGHTS & PERMISSIONS

Higher Education Press