Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Biogeographic pattern of the nirS gene-targeted anammox bacterial community and composition in the northern South China Sea and a coastal Mai Po mangrove wetland

  • 64 Accesses


Functional genes, namely hzo/hao, nirS, hzs, and ccs gene, are efficient with high specificity for detecting anammox bacteria. Sc-nirS and An-nirS primer sets were proposed for targeting Scalindua/non-Scalindua anammox bacterial groups; previously, they have not been assessed for biogeographic study on marine-terrestrial transitional systems, specifically marine and terrestrial ecosystems. Here, we report phylogenetic distribution pattern of anammox bacteria in both northern South China Sea (nSCS) and Mai Po wetland (a coastal mangrove) using nirS gene-based primers. A well-delineated biogeographic distribution pattern from ocean to continental shelf was evident by combining both gene-based analyses as previously depicted using 16S rRNA as the biomarker. Furthermore, factors affecting the abundance and composition of An-nirS genes in Mai Po wetland were identified as substrate (NO3/NO2 concentration) and anoxic/oxic condition in association to depth. An-nirS gene abundance was from 2.6 × 103 to 1.2–1.4 × 104 copies/g dry sediment in nSCS; and it was around 5 × 103 and 1–2 × 104 copies/g dry sediment in surface and subsurface sediments of Mai Po wetland, respectively. In addition, nirS gene abundance and distribution pattern of denitrifiers and anammox bacteria in the wetland indicates a competition relationship between them. Mangrove vegetation affected the community composition of An-nirS gene considerably, and a more homogeneous distribution pattern was observed in the mangrove forest than intertidal mudflats. Sc/An-nirS gene-based biogeographic insights on anammox bacteria have shed lights on the compositional and potential functional dynamics and emphasized the importance of molecular tools on refining the current microbial ecological patterns.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Braak C, Šmilauer P (2002) CANOCO reference manual and CanoDraw for Windows user’s guide: software for canonical community ordination (version 4.5). Microcomputer Power, Ithaca

  2. Byrne N, Strous M, Crépeau V, Kartal B, Birrien J-L, Schmid M, Lesongeur F, Schouten S, Jaeschke A, Jetten M (2008) Presence and activity of anaerobic ammonium-oxidizing bacteria at deep-sea hydrothermal vents. ISME J 3(1):117–123

  3. Cao H, Hong Y, Li M, Gu J-D (2011a) Diversity and abundance of ammonia-oxidizing prokaryotes in sediments from the coastal Pearl River estuary to the South China Sea. Antonie Van Leeuwenhoek 100(4):545–556

  4. Cao H, Hong Y, Li M, Gu J-D (2011b) Phylogenetic diversity and ecological pattern of ammonia-oxidizing archaea in the surface sediments of the Western Pacific. Microb Ecol 62(4):813–823

  5. Cao H, Hong Y, Li M, Gu J-D (2012a) Community shift of ammonia-oxidizing bacteria along an anthropogenic pollution gradient from the Pearl River Delta to the South China Sea. Appl Microbiol Biotechnol 94(1):247–259. https://doi.org/10.1007/s00253-011-3636-1

  6. Cao H, Hong Y, Li M, Gu J-D (2012b) Lower abundance of ammonia-oxidizing archaea than ammonia-oxidizing bacteria detected in the subsurface sediments of the Northern South China Sea. Geomicrobiol J 29(4):332–339

  7. Cao H, Auguet J-C, Gu J-D (2013) Global ecological pattern of ammonia-oxidizing archaea. PLoS One 8:e52853. https://doi.org/10.1371/journal.pone.0052853

  8. Cha M, Young L, Wong K (1997) The fate of traditional extensive (gei wai) shrimp farming at the Mai Po Marshes Nature Reserve, Hong Kong. Hydrobiologia 352(1–3):295–303

  9. Cheung K, Poon B, Lan C, Wong M (2003) Assessment of metal and nutrient concentrations in river water and sediment collected from the cities in the Pearl River Delta, South China. Chemosphere 52(9):1431–1440. https://doi.org/10.1016/S0045-6535(03)00479-X

  10. Dalsgaard T, Thamdrup B, Canfield DE (2005) Anaerobic ammonium oxidation (anammox) in the marine environment. Res Microbiol 156(4):457–464. https://doi.org/10.1016/j.resmic.2005.01.011

  11. Dang H, Zhou H, Zhang Z, Yu Z, Hua E, Liu X, Jiao N (2013) Molecular detection of Candidatus Scalindua pacifica and environmental responses of sediment anammox bacterial community in the Bohai Sea, China. PLoS One 8(4):e61330

  12. Dittmar T, Lara RJ (2001) Do mangroves rather than rivers provide nutrients to coastal environments south of the Amazon River? Evidence from long-term flux measurements. Mar Ecol Prog Ser 213:67–77

  13. Dong LF, Naqasima Sobey M, Smith CJ, Rusmana I, Phillips W, Stott A, Mark Osborn A, Nedwell DB (2011) Dissimilatory reduction of nitrate to ammonium, not denitrification or anammox, dominates benthic nitrate reduction in tropical estuaries. Limnol Oceanogr 56(1):279

  14. Fernandes SO, Bonin PC, Michotey VD, Garcia N, LokaBharathi PA (2012a) Nitrogen-limited mangrove ecosystems conserve N through dissimilatory nitrate reduction to ammonium. Sci Rep:2. https://doi.org/10.1038/srep00419

  15. Fernandes SO, Michotey VD, Guasco S, Bonin PC, Bharathi PA (2012b) Denitrification prevails over anammox in tropical mangrove sediments (Goa, India). Mar Environ Res 74:9–19. https://doi.org/10.1016/j.marenvres.2011.11.008

  16. Fierer N, Jackson RB (2006) The diversity and biogeography of soil bacterial communities. Proc Natl Acad Sci U S A 103(3):626–631. https://doi.org/10.1073/pnas.0507535103

  17. Fisher J, Acreman M (1999) Wetland nutrient removal: a review of the evidence. Hydrol Earth Syst Sci 8(4):673–685

  18. Flores-Mireles AL, Winans SC, Holguin G (2007) Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots. Appl Environ Microbiol 73(22):7308–7321

  19. Gori F, Tringe SG, Kartal B, Marchiori E, Jetten MSM (2011) The metagenomic basis of anammox metabolism in CandidatusBrocadia fulgida’. Biochem Soc Trans 39(6):1799–1804. https://doi.org/10.1042/Bst20110707

  20. Green JL, Bohannan BJ, Whitaker RJ (2008) Microbial biogeography: from taxonomy to traits. Science 320(5879):1039–1043. https://doi.org/10.1126/science.1153475

  21. Han P, Gu J-D (2013) More refined diversity of anammox bacteria recovered and distribution in different ecosystems. Appl Microbiol Biotechnol 97(8):3653–3663

  22. Han P, Gu J-D (2015) Further analysis of anammox bacterial community structures along an anthropogenic nitrogen-input gradient from the riparian sediments of the Pearl River Delta to the deep-ocean sediments of the South China Sea. Geomicrobiol J. https://doi.org/10.1080/01490451.2014.1001502

  23. Harhangi HR, Le Roy M, van Alen T, Hu B-l, Groen J, Kartal B, Tringe SG, Quan Z-X, Jetten MS, den Camp HJO (2012) Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria. Appl Environ Microbiol 78(3):752–758

  24. Hill AR, Cardaci M (2004) Denitrification and organic carbon availability in riparian wetland soils and subsurface sediments. Soil Sci Soc Am J 68(1):320–325

  25. Hong Y-G, Li M, Cao H, Gu J-D (2011) Residence of habitat-specific anammox bacteria in the deep-sea subsurface sediments of the South China Sea: analyses of marker gene abundance with physical chemical parameters. Microb Ecol 62(1):36–47

  26. Hu B, Shen L, Xu X, Zheng P (2011) Anaerobic ammonium oxidation (anammox) in different natural ecosystems. Biochem Soc Trans 39(6):1811

  27. Hu Z, Speth DR, Francoijs K-J, Quan Z-X, Jetten M (2012) Metagenome analysis of a complex community reveals the metabolic blueprint of anammox bacterium “Candidatus Jettenia asiatica”. Front Microbio 3. https://doi.org/10.3389/fmicb.2012.00366

  28. Huang X, Huang L, Yue W (2003) The characteristics of nutrients and eutrophication in the Pearl River estuary, South China. Mar Pollut Bull 47(1):30–36

  29. Humbert S, Tarnawski S, Fromin N, Mallet M-P, Aragno M, Zopfi J (2010) Molecular detection of anammox bacteria in terrestrial ecosystems: distribution and diversity. ISME J 4(3):450–454. https://doi.org/10.1038/ismej.2009.125

  30. Humbert S, Zopfi J, Tarnawski SE (2012) Abundance of anammox bacteria in different wetland soils. Environ Microbiol Rep 4(5):484–490. https://doi.org/10.1111/j.1758-2229.2012.00347.x

  31. Jetten M, Cirpus I, Kartal B, van Niftrik L, Van De Pas-Schoonen K, Sliekers O, Haaijer S, Van Der Star W, Schmid M, Van De Vossenberg J (2005) 1994–2004: 10 years of research on the anaerobic oxidation of ammonium. Biochem Soc Trans 33(Pt 1):119–123

  32. Jetten MS, Lv N, Strous M, Kartal B, Keltjens JT, Op den Camp HJ (2009) Biochemistry and molecular biology of anammox bacteria. Crit Rev Biochem Mol Biol 44(2–3):65–84

  33. Kartal B, Geerts W, Jetten MS (2011) Cultivation, detection, and ecophysiology of anaerobic ammonium-oxidizing bacteria. Methods Enzymol 486(Part A):89–108. https://doi.org/10.1016/B978-0-12-381294-0.00004-3

  34. Kraft B, Strous M, Tegetmeyer HE (2011) Microbial nitrate respiration–genes, enzymes and environmental distribution. J Biotechnol 155(1):104–117

  35. Lam P, Lavik G, Jensen MM, van de Vossenberg J, Schmid M, Woebken D, Gutiérrez D, Amann R, Jetten MS, Kuypers MM (2009) Revising the nitrogen cycle in the Peruvian oxygen minimum zone. Proc Natl Acad Sci U S A 106(12):4752–4757. https://doi.org/10.1073/pnas.0812444106

  36. Li M, Gu J-D (2011) Advances in methods for detection of anaerobic ammonium oxidizing (anammox) bacteria. Appl Microbiol Biotechnol 90(4):1241–1252

  37. Li M, Cao H, Hong Y-G, Gu J-D (2011a) Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes. Microbes Environ 26(1):15–22. https://doi.org/10.1264/jsme2.ME10131

  38. Li M, Ford T, Li X, Gu J-D (2011b) Cytochrome cd1-containing nitrite reductase encoding gene nirS as a new functional biomarker for detection of anaerobic ammonium oxidizing (Anammox) bacteria. Environ Sci Technol 45(8):3547–3553

  39. Li M, Hong Y-G, Cao H-L, Gu J-D (2011c) Mangrove trees affect the community structure and distribution of anammox bacteria at an anthropogenic-polluted mangrove in the Pearl River Delta reflected by 16S rRNA and hydrazine oxidoreductase (HZO) encoding gene analyses. Ecotoxicology 20(8):1780–1790

  40. Li M, Cao HL, Hong YG, Gu JD (2013a) Using the variation of anammox bacteria community structures as a bio-indicator for anthropogenic/terrestrial nitrogen inputs in the Pearl River Delta (PRD). Appl Microbiol Biotechnol 97(22):9875–9883. https://doi.org/10.1007/s00253-013-4990-y

  41. Li M, Hong Y, Cao H, Gu J-D (2013b) Community structures and distribution of anaerobic ammonium oxidizing and nirS-encoding nitrite-reducing bacteria in surface sediments of the South China Sea. Microb Ecol 66(2):281–296. https://doi.org/10.1007/s00248-012-0175-y

  42. Liang Y, Wong M (2003) Spatial and temporal organic and heavy metal pollution at Mai Po Marshes Nature Reserve, Hong Kong. Chemosphere 52(9):1647–1658

  43. Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71(12):8228–8235

  44. Martiny JBH, Bohannan BJM, Brown JH, Colwell RK, Fuhrman JA, Green JL, Horner-Devine MC, Kane M, Krumins JA, Kuske CR, Morin PJ, Naeem S, Ovreas L, Reysenbach AL, Smith VH, Staley JT (2006) Microbial biogeography: putting microorganisms on the map. Nat Rev Microbiol 4(2):102–112. https://doi.org/10.1038/nrmicro1341

  45. Mohan SB, Schmid M, Jetten M, Cole J (2004) Detection and widespread distribution of the nrfA gene encoding nitrite reduction to ammonia, a short circuit in the biological nitrogen cycle that competes with denitrification. FEMS Microbiol Ecol 49(3):433–443

  46. Mulder A, Graaf A, Robertson L, Kuenen J (1995) Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol Ecol 16(3):177–184

  47. Palmer K, Horn MA (2012) Actinobacterial nitrate reducers and proteobacterial denitrifiers are abundant in N2O-metabolizing palsa peat. Appl Environ Microbiol 78(16):5584–5596. https://doi.org/10.1128/aem.00810-12

  48. Reef R, Feller IC, Lovelock CE (2010) Nutrition of mangroves. Tree Physiol 30(9):1148–1160. https://doi.org/10.1093/treephys/tpq048

  49. Rich JJ, Dale OR, Song B, Ward BB (2008) Anaerobic ammonium oxidation (anammox) in Chesapeake Bay sediments. Microb Ecol 55(2):311–320. https://doi.org/10.1007/s00248-007-9277-3

  50. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ (2009) Introducing Mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23):7537–7541. https://doi.org/10.1128/AEM.01541-09

  51. Schmid MC, Hooper AB, Klotz MG, Woebken D, Lam P, Kuypers MM, Pommerening-Roeser A, Op Den Camp HJ, Jetten MS (2008) Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium-oxidizing bacteria. Environ Microbiol 10(11):3140–3149

  52. Seo J, Jang I, Gebauer G, Kang H (2014) Abundance of methanogens, methanotrophic bacteria, and denitrifiers in rice paddy soils. Wetlands 34(2):213–223. https://doi.org/10.1007/s13157-013-0477-y

  53. Smith CJ, Nedwell DB, Dong LF, Osborn AM (2007) Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments. Appl Environ Microbiol 73(11):3612–3622

  54. Sonthiphand P, Hall MW, Neufeld JD (2014) Biogeography of anaerobic ammonia-oxidizing (anammox) bacteria. Front Microbiol 5. https://doi.org/10.3389/fmicb.2014.00399

  55. Strous M, Kuenen JG, Jetten MSM (1999) Key physiology of anaerobic ammonium oxidation. Appl Environ Microbiol 65(7):3248–3250

  56. Takeuchi J (2006) Habitat segregation of a functional gene encoding nitrate ammonification in estuarine sediments. Geomicrobiol J 23(2):75–87

  57. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

  58. Throbäck IN, Enwall K, Jarvis Å, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49(3):401–417

  59. Van de Graaf AA, Mulder A, de Bruijn P, Jetten M, Robertson LA, Kuenen JG (1995) Anaerobic oxidation of ammonium is a biologically mediated process. Appl Environ Microbiol 61(4):1246–1251

  60. van de Vossenberg J, Woebken D, Maalcke WJ, Wessels HJ, Dutilh BE, Kartal B, Janssen-Megens EM, Roeselers G, Yan J, Speth D (2012) The metagenome of the marine anammox bacterium ‘Candidatus Scalindua profunda’ illustrates the versatility of this globally important nitrogen cycle bacterium. Environ Microbiol

  61. Van Dongen U, Jetten MS, Van Loosdrecht M (2001) The SHARON®-Anammox® process for treatment of ammonium rich wastewater. Water Sci Technol 44(1):153–160

  62. Wang J, Gu J-D (2013) Dominance of Candidatus Scalindua species in anammox community revealed in soils with different duration of rice paddy cultivation in Northeast China. Appl Microbiol Biotechnol 97(4):1785–1798. https://doi.org/10.1007/s00253-012-4036-x

  63. Wang Y, Zhu G, Harhangi HR, Zhu B, Jetten MS, Yin C, Op den Camp HJ (2012) Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane-oxidizing bacteria in a paddy soil. FEMS Microbiol Lett 336(2):79–88

  64. Wang Y-F, Feng Y-Y, Ma X, Gu J-D (2013) Seasonal dynamics of ammonia/ammonium-oxidizing prokaryotes in oxic and anoxic wetland sediments of subtropical coastal mangrove. Appl Microbiol Biotechnol 97(17):7919–7934. https://doi.org/10.1007/s00253-012-4510-5

  65. Yin K, Harrison PJ (2008) Nitrogen over enrichment in subtropical Pearl River estuarine coastal waters: possible causes and consequences. Cont Shelf Res 28(12):1435–1442

  66. Yin K, Lin Z, Ke Z (2004) Temporal and spatial distribution of dissolved oxygen in the Pearl River Estuary and adjacent coastal waters. Cont Shelf Res 24(16):1935–1948

  67. Yu Y, Breitbart M, McNairnie P, Rohwer F (2006) FastGroupII: a web-based bioinformatics platform for analyses of large 16S rDNA libraries. BMC Bioinformatics 7(1):57

  68. Zhao Z, Zhuang Y-X, Gu J-D (2012) Abundance, composition and vertical distribution of polycyclic aromatic hydrocarbons in sediments of the Mai Po Inner Deep Bay of Hong Kong. Ecotoxicology 21(6):1734–1742

  69. Zhou Z, Chen J, Meng H, Dvornyk V, Gu J-D (2017) New PCR primers targeting hydrazine synthase and cytochrome c biogenesis proteins in anammox bacteria. Appl Microbiol Biotechnol 101(3):1267–1287. https://doi.org/10.1007/s00253-016-8013-7

  70. Zhou Z, Wei Q, Yang Y, Li M, Gu J-D (2018) Practical applications of PCR primers in detection of anammox bacteria effectively from different types of samples. Appl Microbiol Biotechnol 102:5859–5871. https://doi.org/10.1007/s00253-018-9078-2

Download references


Authors would like to thank Mrs. Kelly Lau for her laboratory assistant during this research. We would also like to thank Prof. Yiguo Hong (Guangzhou University), Prof. Meng Li (Shenzhen University), and Dr. Yong-Feng Wang (Guangdong Academy of Forestry Science) for the help on sample collections and physiochemical analyses.


This research was supported by a Hong Kong General Research Fund (GRF Grant No. 701913 to J-DG). Zhichao Zhou was supported by the University Postgraduate Fellowship (The University of Hong Kong). Jing Chen was supported by the Hong Kong PhD Fellowship, and is now supported by the Fundamental Research Support Funds for the Central Universities.

Author information

Correspondence to Ji-Dong Gu.

Ethics declarations

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Permission for sampling and the research to be conducted at Mai Po Nature Reserve was granted by Agriculture, Fisheries and Conservation department of the Hong Kong SAR Government.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material


(PDF 935 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhou, Z., Chen, J., Gu, W. et al. Biogeographic pattern of the nirS gene-targeted anammox bacterial community and composition in the northern South China Sea and a coastal Mai Po mangrove wetland. Appl Microbiol Biotechnol (2020). https://doi.org/10.1007/s00253-020-10415-3

Download citation


  • Anammox
  • nirS gene
  • Biogeographic distribution
  • Northern South China Sea
  • Mai Po wetland
  • Marine-terrestrial transition