Seasonal bacterial community dynamics in a crude oil refinery wastewater treatment plant

  • Pedro Soares-Castro
  • Trilok C. Yadav
  • Signe Viggor
  • Maia Kivisaar
  • Atya Kapley
  • Pedro M. SantosEmail author
Environmental biotechnology


The biological treatment of oil refinery effluents in wastewater treatment plants (WWTPs) relies on specialized bacteria contributing to remove organic load, nitrogen, sulfur, and phosphorus compounds. Knowledge about bacterial dynamics in WWTPs and how they affect the performance of the wastewater treatment is limited, particularly in tropical countries. The bacterial communities from three compartments of an oil refinery WWTP in Uran, India, were assessed using 16S-metabarcoding, in winter and monsoon seasons, upstream (from the surge pond) and downstream the biotower (clarifier and guard pond), to understand the effects of seasonal variations in WWTP’s efficiency. The organic load and ammonia levels of the treated wastewater increased by 3- and 9-fold in the monsoon time-point. A decreased abundance and diversity of 47 genera (325 OTUs) comprising ammonia and nitrite oxidizing bacteria (AOB, NOB, denitrifiers) was observed in the monsoon season downstream the biotower, whereas 23 OTUs of Sulfurospirillum, Desulfovibrio, and Bacillus, putatively performing dissimilatory nitrate reduction to ammonia (DNRA), were 3-fold more abundant in the same compartments (DNRA/denitrifiers winter ratio < 0.5 vs. monsoon ratio around 3). The total abundance of reported sulfate- and sulfite-reducing bacteria also increased 250- and 500-fold downstream the biotower, in the monsoon time-point. Bacteria performing DNRA may thus outcompete denitrification in this WWTP, limiting the biodegradation process. The alterations detected in bacterial populations involved in the removal of nitrogen and sulfur species evidenced a reduced quality of the released wastewater and may be good candidates for the following monitoring strategies and optimization of the wastewater treatment.


Refinery wastewater treatment Metabarcoding Denitrification Dissimilatory nitrate reduction to ammonia (DNRA) Bacterial community 16S rRNA gene amplicon sequencing 



The authors would like to acknowledge the management of the Uran WWTP and Dr. JS Sharma (general manager at ONGC) for the coordination of the sample collection, as well as Jörg Becker and João Sobral for the sequencing services provided at the Instituto Gulbenkian de Ciência.

Author’s contributions

SV, MK, AK, and PMS designed the study. TCY and AK were involved in WWTP sample collection, chemical analysis, and DNA extraction. PS-C and PMS prepared samples for high-throughput sequencing. PS-C and PMS analyzed the sequencing data. PS-C, SV, MK, AK, and PMS drafted the manuscript. All the authors read, revised, and approved the manuscript.

Funding information

This work was supported by the ERA-NET project WRANA (Inn-INDIGO/0004/2014), which included the postdoctoral grant of P.S.-C (grant BPD1/wrana/2017), and by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.

Supplementary material

253_2019_10130_MOESM1_ESM.xlsx (121 kb)
ESM 1 (XLSX 120 kb)
253_2019_10130_MOESM2_ESM.pdf (258 kb)
ESM 2 (PDF 257 kb)


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. CrossRefGoogle Scholar
  2. Andersen KSS, Kirkegaard RH, Karst SM, Albertsen M (2018) ampvis2: an R package to analyse and visualise 16S rRNA amplicon data. bioRxiv:299537.
  3. Arndt D, Xia J, Liu Y, Zhou Y, Guo AC, Cruz JA, Sinelnikov I, Budwill K, Nesbø CL, Wishart DS (2012) METAGENassist: a comprehensive web server for comparative metagenomics. Nucleic Acids Res 40:W88–W95. CrossRefGoogle Scholar
  4. Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto J-M (2011) Enterotypes of the human gut microbiome. Nature 473:174–180. CrossRefGoogle Scholar
  5. Aslan S, Dahab M (2008) Nitritation and denitritation of ammonium-rich wastewater using fluidized-bed biofilm reactors. J Hazard Mater 156:56–63. CrossRefGoogle Scholar
  6. Association APH, Association AWW, Federation WPC, Federation WE (1965) Standard methods for the examination of water and wastewater, 18th edn. American Public Health Association, Washington, DCGoogle Scholar
  7. Ballinger SJ, Head IM, Curtis TP, Godley AR (1998) Molecular microbial ecology of nitrification in an activated sludge process treating refinery wastewater. Water Sci Technol 37:105–108. CrossRefGoogle Scholar
  8. Bougard D, Bernet N, Chèneby D, Delgenès J-P (2006) Nitrification of a high-strength wastewater in an inverse turbulent bed reactor: effect of temperature on nitrite accumulation. Process Biochem 41:106–113. CrossRefGoogle Scholar
  9. Bramucci M, Nagarajan V (2006) Bacterial communities in industrial wastewater bioreactors. Curr Opin Microbiol 9:275–278. CrossRefGoogle Scholar
  10. Chang Y-I, Cheng H-P, Lai S-H, Ning H (2014) Biodegradation of naphthalene in the oil refinery wastewater by enriched activated sludge. Int Biodeterior Biodegradation 86:272–277. CrossRefGoogle Scholar
  11. Chen Y, Lan S, Wang L, Dong S, Zhou H, Tan Z, Li X (2017) A review: driving factors and regulation strategies of microbial community structure and dynamics in wastewater treatment systems. Chemosphere 174:173–182. CrossRefGoogle Scholar
  12. Daims H, Lebedeva EV, Pjevac P, Han P, Herbold C, Albertsen M, Jehmlich N, Palatinszky M, Vierheilig J, Bulaev A, Kirkegaard RH, von Bergen M, Rattei T, Bendinger B, Nielsen PH, Wagner M (2015) Complete nitrification by Nitrospira bacteria. Nature 528:504–509. CrossRefGoogle Scholar
  13. Dalsgaard T, Bak F (1994) Nitrate reduction in a sulfate-reducing bacterium, Desulfovibrio desulfuricans isolated from rice paddy soil: sulfide inhibition, kinetics, and regulation. Appl Environ Microbiol 60:291 LP–291297Google Scholar
  14. Deng S, Li D, Yang X, Xing W, Li J, Zhang Q (2016) Biological denitrification process based on the Fe(0)–carbon micro-electrolysis for simultaneous ammonia and nitrate removal from low organic carbon water under a microaerobic condition. Bioresour Technol 219:677–686. CrossRefGoogle Scholar
  15. Dixon P (2003) VEGAN, a package of R functions for community ecology. J Veg Sci 14:927–930. CrossRefGoogle Scholar
  16. Dong LF, Sobey MN, Smith CJ, Rusmana I, Phillips W, Stott A, Osborn AM, Nedwell DB (2010) Dissimilatory reduction of nitrate to ammonium, not denitrification or anammox, dominates benthic nitrate reduction in tropical estuaries. Limnol Oceanogr 56:279–291. CrossRefGoogle Scholar
  17. Eisenmann E, Beuerle J, Sulger K, Kroneck PMH, Schumacher W (1995) Lithotrophic growth of Sulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia. Arch Microbiol 164:180–185CrossRefGoogle Scholar
  18. Fernández I, Dosta J, Fajardo C, Campos JL, Mosquera-Corral A, Méndez R (2012) Short- and long-term effects of ammonium and nitrite on the Anammox process. J Environ Manag 95:S170–S174. CrossRefGoogle Scholar
  19. Foley JA, DeFries R, Asner GP, Barford C, Bonan G, Carpenter SR, Chapin FS, Coe MT, Daily GC, Gibbs HK, Helkowski JH, Holloway T, Howard EA, Kucharik CJ, Monfreda C, Patz JA, Prentice IC, Ramankutty N, Snyder PK (2005) Global consequences of land use. Science 309:570 LP–570574. CrossRefGoogle Scholar
  20. Goddard AD, Moir JWB, Richardson DJ, Ferguson SJ (2008) Interdependence of two NarK domains in a fused nitrate/nitrite transporter. Mol Microbiol 70:667–681. CrossRefGoogle Scholar
  21. Guo F, Ju F, Cai L, Zhang T (2013) Taxonomic precision of different hypervariable regions of 16S rRNA gene and annotation methods for functional bacterial groups in biological wastewater treatment. PLoS One 8:e76185. CrossRefGoogle Scholar
  22. Hai R, Wang Y, Wang X, Li Y, Du Z (2014) Bacterial community dynamics and taxa-time relationships within two activated sludge bioreactors. PLoS One 9:e90175. CrossRefGoogle Scholar
  23. Hazen TC, Stahl DA (2006) Using the stress response to monitor process control: pathways to more effective bioremediation. Curr Opin Biotechnol 17:285–290. CrossRefGoogle Scholar
  24. Hu M, Wang X, Wen X, Xia Y (2012) Microbial community structures in different wastewater treatment plants as revealed by 454-pyrosequencing analysis. Bioresour Technol 117:72–79. CrossRefGoogle Scholar
  25. Jin R-C, Yang G-F, Zhang Q-Q, Ma C, Yu J-J, Xing B-S (2013) The effect of sulfide inhibition on the ANAMMOX process. Water Res 47:1459–1469. CrossRefGoogle Scholar
  26. Jones ZL, Jasper JT, Sedlak DL, Sharp JO (2017) Sulfide-induced dissimilatory nitrate reduction to ammonium supports anaerobic ammonium oxidation (anammox) in an open-water unit process wetland. Appl Environ Microbiol 83:e00782–e00717. Google Scholar
  27. Joshi NA, Fass JN (2011) Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (version 1.33). Available at
  28. Ju F, Zhang T (2015) Bacterial assembly and temporal dynamics in activated sludge of a full-scale municipal wastewater treatment plant. ISME J 9:683–695. CrossRefGoogle Scholar
  29. Kim D-J, Lee D-I, Keller J (2006) Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH. Bioresour Technol 97:459–468. CrossRefGoogle Scholar
  30. Kim YM, Park D, Lee DS, Park JM (2007) Instability of biological nitrogen removal in a cokes wastewater treatment facility during summer. J Hazard Mater 141:27–32. CrossRefGoogle Scholar
  31. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD (2013) Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol AEM 79:01043-13. Google Scholar
  32. Kuenen JG (2008) Anammox bacteria: from discovery to application. Nat Rev Microbiol 6:320–326. CrossRefGoogle Scholar
  33. Kumar M, Lin J-G (2010) Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal - strategies and issues. J Hazard Mater 178:1–9. CrossRefGoogle Scholar
  34. Lackner S, Terada A, Smets BF (2008) Heterotrophic activity compromises autotrophic nitrogen removal in membrane-aerated biofilms: results of a modeling study. Water Res 42:1102–1112. CrossRefGoogle Scholar
  35. Liu T, Liu S, Zheng M, Chen Q, Ni J (2016) Performance assessment of full-scale wastewater treatment plants based on seasonal variability of microbial communities via high-throughput sequencing. PLoS One 11:e0152998. CrossRefGoogle Scholar
  36. Louca S, Parfrey LW, Doebeli M (2016) Decoupling function and taxonomy in the global ocean microbiome. Science 353:1272–1277. CrossRefGoogle Scholar
  37. Lu H, Chandran K, Stensel D (2014) Microbial ecology of denitrification in biological wastewater treatment. Water Res 64:237–254. CrossRefGoogle Scholar
  38. Lydmark P, Almstrand R, Samuelsson K, Mattsson A, Sörensson F, Lindgren P-E, Hermansson M (2007) Effects of environmental conditions on the nitrifying population dynamics in a pilot wastewater treatment plant. Environ Microbiol 9:2220–2233. CrossRefGoogle Scholar
  39. Mania D, Heylen K, Spanning RJM, Frostegård Å (2014) The nitrate-ammonifying and nosZ-carrying bacterium Bacillus vireti is a potent source and sink for nitric and nitrous oxide under high nitrate conditions. Environ Microbiol 16:3196–3210. CrossRefGoogle Scholar
  40. McMurdie PJ, Holmes S (2013) phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8:e61217. CrossRefGoogle Scholar
  41. Meerbergen K, Van Geel M, Waud M, Willems KA, Dewil R, Van Impe J, Appels L, Lievens B (2017) Assessing the composition of microbial communities in textile wastewater treatment plants in comparison with municipal wastewater treatment plants. Microbiologyopen 6:e00413. CrossRefGoogle Scholar
  42. Moreno-Vivián C, Cabello P, Martínez-Luque M, Blasco R, Castillo F (1999) Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol 181:6573 LP–6576584Google Scholar
  43. Muszyński A, Tabernacka A, Miłobędzka A (2015) Long-term dynamics of the microbial community in a full-scale wastewater treatment plant. Int Biodeterior Biodegradation 100:44–51. CrossRefGoogle Scholar
  44. Nakano MM, Zuber P (1998) Anaerobic growth of a “strict aerobe” (Bacillus subtilis). Annu Rev Microbiol 52:165–190. CrossRefGoogle Scholar
  45. Palacin-Lizarbe C, Camarero L, Hallin S, Jones CM, Cáliz J, Casamayor EO, Catalan J (2019) The DNRA-denitrification dichotomy differentiates nitrogen transformation pathways in mountain lake benthic habitats. Front Microbiol 10:1229. CrossRefGoogle Scholar
  46. Potter LC, Millington P, Griffiths L, Thomas GH, Cole JA (1999) Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth? Biochem J 344(Pt 1):77–84Google Scholar
  47. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO (2012) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596. CrossRefGoogle Scholar
  48. Rehr B, Klemme J-H (1989) Competition for nitrate between denitrifying Pseudomonas stutzeri and nitrate ammonifying enterobacteria. FEMS Microbiol Lett 62:51–57. CrossRefGoogle Scholar
  49. Rognes T, Flouri T, Nichols B, Quince C, Mahé F (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. CrossRefGoogle Scholar
  50. Smith VH (2003) Eutrophication of freshwater and coastal marine ecosystems a global problem. Environ Sci Pollut Res 10:126–139CrossRefGoogle Scholar
  51. Strohm TO, Griffin B, Zumft WG, Schink B (2007) Growth yields in bacterial denitrification and nitrate ammonification. Appl Environ Microbiol 73:1420 LP–1421424. CrossRefGoogle Scholar
  52. Sun Y, De Vos P, Heylen K (2016) Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis. BMC Genomics 17:68. CrossRefGoogle Scholar
  53. Turk O, Mavinic DS (1989) Stability of nitrite build-up in an activated sludge system. JWPCF 61:1440–1448. Google Scholar
  54. van den Berg EM, van Dongen U, Abbas B, van Loosdrecht MCM (2015) Enrichment of DNRA bacteria in a continuous culture. ISME J 9:2153–2161. CrossRefGoogle Scholar
  55. Warnes GR, Bolker B, Bonebakker L, Gentleman R, Huber W, Liaw A, Lumley T, Maechler M, Magnusson A, Moeller S (2009) gplots: various R programming tools for plotting data. version 3.0.1. Available at
  56. Wu J, Zhang J, Jia W, Xie H, Gu RR, Li C, Gao B (2009) Impact of COD/N ratio on nitrous oxide emission from microcosm wetlands and their performance in removing nitrogen from wastewater. Bioresour Technol 100:2910–2917. CrossRefGoogle Scholar
  57. Xing W, Li D, Li J, Hu Q, Deng S (2016) Nitrate removal and microbial analysis by combined micro-electrolysis and autotrophic denitrification. Bioresour Technol 211:240–247. CrossRefGoogle Scholar
  58. Xue J, Schmitz BW, Caton K, Zhang B, Zabaleta J, Garai J, Taylor CM, Romanchishina T, Gerba CP, Pepper IL (2019) Assessing the spatial and temporal variability of bacterial communities in two Bardenpho wastewater treatment systems via Illumina MiSeq sequencing. Sci Total Environ 657:1543–1552. CrossRefGoogle Scholar
  59. Yadav TC, Pal RR, Shastri S, Jadeja NB, Kapley A (2015) Comparative metagenomics demonstrating different degradative capacity of activated biomass treating hydrocarbon contaminated wastewater. Bioresour Technol 188:24–32. CrossRefGoogle Scholar
  60. Yang Q, Wang J, Han X, Xu Y, Liu D, Hao H, Li X, Guo Y, Niu T, Qi S (2014) Analysis of the bacterial community in a full-scale printing and dyeing wastewater treatment system based on T-RFLP and 454 pyrosequencing. Biotechnol Bioprocess Eng 19:191–200. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.CBMA – Centre of Molecular and Environmental BiologyUniversity of MinhoBragaPortugal
  2. 2.CSIR-National Environmental Engineering Research Institute (CSIR-NEERI)NagpurIndia
  3. 3.Institute of Molecular and Cell BiologyUniversity of TartuTartuEstonia

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