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

Prokaryotic community profiling of local algae wastewaters using advanced 16S rRNA gene sequencing


Algae biomass-fed wastewaters are a promising source of lipid and bioenergy manufacture, revealing substantial end-product investment returns. However, wastewaters would contain lytic pathogens carrying drug resistance detrimental to algae yield and environmental safety. This study was conducted to simultaneously decipher through high-throughput advanced Illumina 16S ribosomal RNA (rRNA) gene sequencing, the cultivable and uncultivable bacterial community profile found in a single sample that was directly recovered from the local wastewater systems. Samples were collected from two previously documented sources including anaerobically digested (AD) municipal wastewater and swine wastewater with algae namely Chlorella spp. in addition to control samples, swine wastewater, and municipal wastewater without algae. Results indicated the presence of a significant level of Bacteria in all samples with an average of approximately 95.49% followed by Archaea 2.34%, in local wastewaters designed for algae cultivation. Taxonomic genus identification indicated the presence of Calothrix, Pseudomonas, and Clostridium as the most prevalent strains in both local municipal and swine wastewater samples containing algae with an average of 17.37, 12.19, and 7.84%, respectively. Interestingly, swine wastewater without algae displayed the lowest level of Pseudomonas strains < 0.1%. The abundance of some Pseudomonas species in wastewaters containing algae indicates potential coexistence between these strains and algae microenvironment, suggesting further investigations. This finding was particularly relevant for the earlier documented adverse effects of some nosocomial Pseudomonas strains on algae growth and their multidrug resistance potential, requiring the development of targeted bioremediation with regard to the beneficial flora.

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

Fig. 1
Fig. 2
Fig. 3


  1. Arashiro LT, Rada-Ariza AM, Wang M, van der Steen P, Ergas SJ (2017) Modelling shortcut nitrogen removal from wastewater using an algal-bacterial consortium. Technology, Water Science & in press

  2. Aristidou A, Penttilä M (2000) Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechnol 11:187–198

  3. Christi Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–396

  4. Congressional Research Service (CRS). Agricultural-based biofuels: overview and emerging issues. (2013) https://www.fas.org/sgp/crs/misc/R41282.pdf

  5. Davis R, Fishman D, Frank ED, Wigmosta MS, Aden A, Coleman AM, Pienkos PT, Skaggs RJ, Venteris ER, Wang MQ (2012) Renewable diesel from algal lipids: an integrated baseline for cost, emissions, and resource potential from a harmonized model. ANL

  6. Davis RE, Fishman DB, Frank ED, Johnson MC, Jones SB, Kinchin CM, Skaggs RL, Venteris ER, Wigmosta MS (2014) Integrated evaluation of cost, emissions, and resource potential for algal biofuels at the national scale. Environ Sci Technol 48(10):6035–6042

  7. Day J, Thomas M, Achilles-Day UE, Leakey RJ (2012) Early detection of protozoan grazers in algal biofuel cultures. Bioresour Technol 114:715–719

  8. Ferrel J, Sarisky-Reed V (2010) National algal biofuels technology roadmap. U.S. Department of Energy (DOE). Office of Energy and Renewable Energy. OBP

  9. Goldemberg J (2007) Ethanol for a sustainable energy future. Science 315(5813):808–810

  10. Issa A (1999) Antibiotic production by the cyanobacteria Oscillatoria angustissima and Calothrix parietina. Environ Toxicol Pharmacol 8(1):33–37

  11. Kim JD, Kim B, Lee CG (2007) Alga-lytic activity of Pseudomonas fluorescens against the red tide causing marine alga Heterisugma akashiwo. Biol Control 41(3):296–303

  12. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41(1):e1

  13. Lee E, Jalalizadeh M, Zhang Q (2015) Growth kinetic models for microalgae cultivation: a review. Algal Res 12:497–512

  14. Limayem A, Gonzalez F, Micciche A, Haller E, Nayak B, Mohapatra SS (2016) Molecular identification and nanoremediation of microbial contaminants in algal systems using untreated wastewater. J Environ Sci Health Part B 51(12):868–872

  15. Magalhães MJTL, Pontes G, Serra PT, Balieiro A, Castro D, Pieri FA, Crainey JL, Nogueira PA, Orlandi PP (2016) Multidrug resistant Pseudomonas aeruginosa survey in a stream receiving effluents from ineffective wastewater hospital plants. BMC Microbiol 16(1):193–200

  16. Mandal S, Mallick N (2011) Waste utilization and biodiesel production by the green microalga Scenedesmus obliquus. Appl Environ Microbiol 77(1):374–377

  17. Mata TM, Martins AA, Caetano NS (2010) Microalgae for biodiesel production and other applications: a review. Renew Sustainable Energy Rev 14(1):217–232

  18. Mulbry WW, Wilkie AC (2001) Growth of benthic freshwater algae on dairy manures. J Appl Phycol 13(4):301–306

  19. Mulbry W, Kondrad S, Buyer J (2008) Treatment of dairy and swine manure effluents using freshwater algae: fatty acid content and composition of algal biomass at different manure loading rates. J Appl Phycol 20(6):1079–1085

  20. Pandey PK, Soupir ML (2011) Escherichia coli inactivation kinetics in anaerobic digestion of dairy manure under moderate, mesophilic and thermophilic temperatures. AMB Express 1:18

  21. Pandey PK, Biswas S, Vaddella VK, Soupir ML (2015) Escherichia coli persistence kinetics in dairy manure at moderate, mesophilic, and thermophilic temperatures under aerobic and anaerobic environments. Bioprocess Biosyst Eng 38(3):457–467

  22. Parmar A, Singh NK, Pandey A, Gnansounou E, Madamwar D (2011) Cyanobacteria and microalgae: a positive prospect for biofuels. Bioresour Technol 102(22):10163–10172

  23. Praveen P, Loh KC (2015) Photosynthetic aeration in biological wastewater treatment using immobilized microalgae-bacteria symbiosis. Appl Microbiol Biotechnol 99(23):10345–10354

  24. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, ... & Glöckner FO (2012) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic acids research, gks1219

  25. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, Turner P, Parkhill J, Loman NJ, Walker AW (2014) Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 12(1):87

  26. Sheehan J, Dunahay T, Benemann J, Roessler P (1998) A look back at the U.S. Department of Energy’s aquatic species program: biodiesel from algae. NREL, Golden, Colorado. NREL/TP-580-24190

  27. Shi J, Pandey PK, Franz AK, Deng H, Jeannotte R (2016) Chlorella vulgaris production enhancement with supplementation of synthetic medium in dairy manure wastewater. AMB Express 6(1):15

  28. Slekovec C, Plantin J, Cholley P, Thouverez M, Talon D, Bertrand X, Hocquet D (2012) Tracking down antibiotic-resistant Pseudomonas aeruginosa isolates in a wastewater network. PLoS One 7(12):E49300

  29. Su Y, Mennerich A, Urban B (2011) Municipal wastewater treatment and biomass accumulation with a wastewater-born and settleable algal–bacterial culture. Water Res 45(11):3351–3358

  30. United States Department of Agriculture (2010) A USDA regional roadmap to meeting the biofuels goals of the renewable fuels standard by 2022

  31. Unnithan VV, Unc A, Smith GB (2014) Mini-review: a priori considerations for bacteria–algae interactions in algal biofuel systems receiving municipal wastewaters. Algal Res 4:35–40

  32. Von Sivers M, Zacchi G (1996) Ethanol from lignocellulosics: a review of the economy. Bioresour Technol 56(2):131–140

  33. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73(16):5261–5267

  34. Wang L, Min M, Li Y, Chen P, Chen Y, Liu Y, & Ruan R (2010a). Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Applied biochemistry and biotechnology, 162(4):1174–1186.

  35. Wang H, Liu L, Liu ZP, Qin S (2010b) Investigations of the characteristics and mode of action of an algalytic bacterium isolated from Tai Lake. J J Appl Phycol 22(4):473–478

  36. Wang B, Yang X, Lu J, Zhou Y, Su J, Tian Y, Zhang J, Wang G, Zheng T (2012) A marine bacterium producing protein with algicidal activity against Alexandrium tamarense. Harmful Algae 13:83–88

  37. Wang H, Zhang W, Chen L, Wang J, Liu T (2013) The contamination and control of biological pollutants in mass cultivation of microalgae. Bioresour Technol 128:745–750

  38. Wang M, Lee E, Zhang Q, Ergas SJ (2016) Anaerobic co-digestion of swine manure and microalgae Chlorella sp.: experimental studies and energy analysis. BioEnergy Research 9(4):1204–1215

  39. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18(1):315–322

  40. Woertz I, Feffer A, Lundquist T, Nelson Y (2009) Algae grown on dairy and municipal wastewater for simultaneous nutrient removal and lipid production for biofuel feedstock. J Environ Eng 135(11):1115–1122

  41. Yan C, Muñoz R, Zhu L, Wang Y (2016a) The effects of various LED (light emitting diode) lighting strategies on simultaneous biogas upgrading and biogas slurry nutrient reduction by using of microalgae Chlorella sp. Energy 106:554–561

  42. Yan C, Zhu L, Wang Y (2016b) Photosynthetic CO2 uptake by microalgae for biogas upgrading and simultaneously biogas slurry decontamination by using of microalgae photobioreactor under various light wavelengths, light intensities, and photoperiods. Appl Energy 178:9–18

  43. Yilmaz P, Parfrey LW, Yarza P, Gerken J, Pruesse E, Quast C, ... & Glöckner FO (2013) The SILVA and “all-species living tree project (LTP)” taxonomic frameworks. Nucleic Acids Res, gkt1209

  44. Zhou W, Qiao X, Sun J, Xing K, Tang X (2011) Ecological effect of z-qs01 strain on Chlorella vulgaris and its response to UV-B radiation stress. Procedia Environ Sci 11:741–748

Download references


We thank the Office of Research and Innovations at the University of South Florida for their financial support through their interdisciplinary grant for the Department of Cell Biology, Microbiology, and Molecular Biology (CMMB) along with the College of Pharmacy and Internal Medicine. We also thank the Department of Environmental Engineering at the University of South Florida for providing us with algae-wastewater samples.

Author information

Correspondence to Alya Limayem.

Additional information

Responsible editor: Gerald Thouand

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Limayem, A., Micciche, A., Nayak, B. et al. Prokaryotic community profiling of local algae wastewaters using advanced 16S rRNA gene sequencing. Environ Sci Pollut Res 25, 704–711 (2018). https://doi.org/10.1007/s11356-017-0078-z

Download citation


  • High-throughput Illumina16S rRNA gene amplicon sequencing
  • Algae wastewaters
  • Lytic bacteria
  • Drug resistance
  • Pseudomonas
  • Bioinformatics
  • Profiling
  • Taxonomy