Preparation of Immobilized Sulfate-Reducing Bacteria-Microalgae Beads for Effective Bioremediation of Copper-Containing Wastewater

  • Yongchao Li
  • Xiaoyan Yang
  • Bing Geng


A strain of Desulfovibrio sp. sulfate-reducing bacteria (SRB) was isolated from a sludge sample. Novel immobilized SRB beads with microalgae (Chlorella vulgaris, Scenedesmus obliquus, Selenastrum capricornutum, and Anabaena spiroides) as the carbon source were prepared and then used to treat wastewater containing 60 mg/L Cu(II) and 600 mg/L sulfate in batch experiments. The microalgae were first degraded by co-existing fermentative bacteria into fatty acids, which then served as a carbon source for SRB. The solution chemical oxygen demand was significantly lower with microalgae substrates than with ethanol as a substrate. Different immobilization methods were evaluated with an orthogonal design, which indicated that the compositional parameters for preparing immobilized beads with an optimal sulfate reduction rate were polyvinyl alcohol (2%), sodium alginate (1%), calcium chloride (6%), silica sand (1%), and a 50-mL volume of SRB suspension. SRB activity in the immobilized beads was distinctly enhanced compared with that of suspended SRB. At an initial pH of 5.5, 72.4–74.4% of sulfate and over 91.7% of Cu(II) were removed, indicating that immobilized SRB beads with plentiful low-cost microalgae as a nutrient source may be an efficient method for acid mine drainage treatment.


Cu(II) Microalgae Sulfate-reducing bacteria Immobilization Bioremediation 


Funding information

This work is financially supported by the National Water Pollution Control and Treatment Science and Technology Major Project in China (2014ZX07510-001 and 2015ZX07103-007) and the National Natural Science Foundation of China (No. 41471399, 41101474 and 51504094).


  1. Akbari, M., Hallajisani, A., Keshtkar, A. R., Shahbeig, H., & Ghorbanian, S. A. (2015). Equilibrium and kinetic study and modeling of Cu(II) and Co(II) synergistic biosorption from Cu(II)-Co(II) single and binary mixtures on brown algae C. indica. Journal of Environmental Chemical Engineering, 3, 140–149.CrossRefGoogle Scholar
  2. APHA-AWWA-WEF. (1998). Standard methods for examination of water and wastewater (20th ed.). Washington DC: American Public Health Association.Google Scholar
  3. Bayramoğlu, G., & Arica, A. M. (2009). Construction a hybrid biosorbent using Scenedesmus quadricauda and Ca-alginate for biosorption of Cu(II), Zn(II) and Ni(II): kinetics and equilibrium studies. Bioresource Technology, 100, 186–193.Google Scholar
  4. Benson, D. A., Boguski, M. S., Lipman, D. J., Ostell, J., Ouellette, B. F., Rapp, B. A., & Wheeler, D. L. (1999). GenBank. Nucleic Acids Research, 27, 12–17.CrossRefGoogle Scholar
  5. Bilal, M., Shah, J. A., Ashfaq, T., Gardazi, S. M. H., Tahir, A. A., Pervez, A., Haroon, H., & Mahmood, Q. (2013). Waste biomass adsorbents for copper removal from industrial wastewater—a review. Journal of Hazardous Materials, 263, 322–333.CrossRefGoogle Scholar
  6. Boshoff, G., Duncan, J., & Rose, P. D. (2004). The use of micro-algal biomass as a carbon source for biological sulphate reducing systems. Water Research, 38, 2659–2666.CrossRefGoogle Scholar
  7. Cai, L. M., Xu, Z. C., Qi, J. Y., Feng, Z. Z., & Xiang, T. S. (2015). Assessment of exposure to heavy metals and health risks among residents near Tonglushan mine in Hubei, China. Chemosphere, 127, 127–135.CrossRefGoogle Scholar
  8. Cao, J. Y., Zhang, G. J., Mao, Z. S., Li, Y. Y., Fang, Z. H., & Yang, C. (2012). Influence of electron donors on the growth and activity of sulfate-reducing bacteria. International Journal of Mineral Processing, 106-109, 58–64.CrossRefGoogle Scholar
  9. Choi, E., & Rim, J. M. (1991). Competition and inhibition of sulfate reducers and methaneproducers in anaerobic treatment. Water Science and Technology, 23, 1259–1264.Google Scholar
  10. Costa, M. C., Santos, E. S., Barros, R. J., Pires, C., & Martins, M. (2009). Wine wastes as carbon source for biological treatment of acid mine drainage. Chemosphere, 75, 831–836.CrossRefGoogle Scholar
  11. Costa, J. M., Rodriguez, R. P., & Sancinetti, G. P. (2017). Removal sulfate and metals Fe+2, Cu+2, and Zn+2 from acid mine drainage in an anaerobic sequential batch reactor. Journal of Environmental Chemical Engineering, 5, 1985–1989.CrossRefGoogle Scholar
  12. Das, B. K., Roy, S., Dev, S., Das, D., & Bhattacharya, J. (2015). Improvement of the degradation of sulfate rich wastewater using sweetmeat waste (SMW) as nutrient supplement. Journal of Hazardous Materials, 300, 796–807.CrossRefGoogle Scholar
  13. Flores-Chaparro, C. E., Ruiz, L. F. C., Torre, M. C. A. D. L., Huerta-Diaz, M. A., & Rangel-Mendez, J. R. (2017). Biosorption removal of benzene and toluene by three dried macroalgae at different ionic strength and temperatures: algae biochemical composition and kinetics. Journal of Environmental Management, 193, 126–135.CrossRefGoogle Scholar
  14. Fuge, R., Pearce, F. M., Pearce, N. G., & Perkins, W. T. (1993). Geochemistry of Cd in the secondary environment near abandoned metalliferous mines, Wales. Applied Geochemistry, 8, 29–35.CrossRefGoogle Scholar
  15. Gonçalves, M. M., da Costa, A. C., Leite, S. G., & Sant'Anna Jr., G. L. (2007). Heavy metal removal from synthetic wastewaters in an anaerobic bioreactor using stillage from ethanol distilleries as a carbon source. Chemosphere, 69, 1815–1820.CrossRefGoogle Scholar
  16. Hao, T., Xiang, P., Mackey, H. R., Chi, K., Lu, H., Chui, H., van Loosdrecht, M. C. M., & Chen, G. H. (2014). A review of biological sulfate conversions in wastewater treatment. Water Research, 65, 1–21.CrossRefGoogle Scholar
  17. Henriques, B., Rocha, L. S., Lopes, C. B., Figueira, P., Duarte, A. C., Vale, C., Pardal, M. A., & Pereira, E. (2017). A macroalgae-based biotechnology for water remediation: simultaneous removal of Cd, Pb and Hg by living Ulva lactuca. Journal of Environmental Management, 191, 275–289.CrossRefGoogle Scholar
  18. Hlabel, P., Maree, J., & Bruinsma, D. (2007). Barium carbonate process for sulphate and metal removal from mine water. Mine Water and the Environment, 26, 14–22.CrossRefGoogle Scholar
  19. Hsu, H. F., Jhuo, Y. S., Kumar, M., Ma, Y. S., & Lin, J. G. (2010). Simultaneous sulfate reduction and copper removal by a PVA-immobilized sulfate reducing bacterial culture. Bioresource Technology, 101, 4354–4361.CrossRefGoogle Scholar
  20. Hungate, R. E., & Macy, J. (1973). The roll-tube method for cultivation of strict anaerobes. Bulletins from the Ecological Research Committee, 3, 123–126.Google Scholar
  21. Kieu, H. T. Q., Müller, E., & Horn, H. (2011). Heavy metal removal in anaerobic semi-continuous stirred tank reactors by a consortium of sulfate-reducing bacteria. Water Research, 45, 3863–3870.CrossRefGoogle Scholar
  22. Lane, D. J. (1991). 16S/23S rRNA sequencing. In E. Stackebrandt & M. Goodfellow (Eds.), Nucleic acid techniques in bacterial systematics (pp. 115–175). New York: Wiley.Google Scholar
  23. Lens, P. N. L., Visser, A., Janssen, A. J. H., Pol Hulshoff, L. W., & Lettinga, G. (1998). Biotechnological treatment of sulfate-rich wastewaters. Critical Reviews in Environmental Science and Technology, 28, 41–88.CrossRefGoogle Scholar
  24. Li, Y. C., Hu, X. X., & Ren, B. Z. (2016). Treatment of antimony mine drainage: challenges and opportunities with special emphasis on mineral adsorption and sulfate reducing bacteria. Water Science and Technology, 73, 2039–2051.CrossRefGoogle Scholar
  25. Li, X., Dai, L. H., Zhang, C., Zeng, G. M., Liu, Y. G., Zhou, C., Xu, W. H., Wu, Y., Tang, X. Q., Liu, W., & Lan, S. M. (2017). Enhanced biological stabilization of heavy metals in sediment using immobilized sulfate reducing bacteria beads with inner cohesive nutrient. Journal of Hazardous Materials, 324, 340–347.CrossRefGoogle Scholar
  26. Liamleam, W., & Annachhatre, A. P. (2007). Electron donors for biological sulphate reduction. Biotechnology Advances, 25, 452–463.CrossRefGoogle Scholar
  27. Liu, G. M., Ren, N. Q., Wang, A. J., Wang, X., Du, D. Z., & Chen, M. (2004). The fermentation type of acidogenic bacteria and their cooperation with SRB in an acidogenic sulfate-reducing reactor. Acta Scientiae Circumstantiae, 24, 782–788.Google Scholar
  28. Ma, S. C., Zhang, H. B., Ma, S. T., Wang, R., Wang, G. X., Shao, Y., & Li, C. X. (2015). Effects of mine wastewater irrigation on activities of soil enzymes and physiological properties, heavy metal uptake and grain yield in winter wheat. Ecotoxicology and Environmental Safety, 113, 483–490.CrossRefGoogle Scholar
  29. Macías, F., Caraballo, M. A., Nieto, J. M., Rötting, T. S., & Ayora, C. (2012). Natural pretreatment and passive remediation of highly polluted acid mine drainage. Journal of Environmental Management, 104, 93–100.CrossRefGoogle Scholar
  30. Madzivire, G., Petrik, L. F., Gitari, W. M., Ojumu, T. V., & Balfour, G. (2010). Application of coal fly ash to circumneutral mine waters for the removal of sulphates as gypsum and ettringite. Minerals Engineering, 23, 252–257.CrossRefGoogle Scholar
  31. Martins, M., Faleiro, M. L., Silva, G., Chaves, S., Tenreiro, R., & Costa, M. C. (2011). Dynamics of bacterial community in up-flow anaerobic packed bed system for acid mine drainage treatment using wine wastes as carbon source. International Biodeterioration & Biodegradation, 65, 78–84.CrossRefGoogle Scholar
  32. McCauley, C. A., O’Sullivan, A. D., Milke, M. W., Weber, P. A., & Trumm, D. A. (2009). Sulfate and metal removal in bioreactors treating acid mine drainage dominated with iron and aluminum. Water Research, 43, 961–970.CrossRefGoogle Scholar
  33. Mothe, G. K., Pakshirajan, K., & Das, G. (2017). Heavy metal removal from multicomponent system by sulfate reducing bacteria: mechanism and cell surface characterization. Journal of Hazardous Materials, 324, 62–70.CrossRefGoogle Scholar
  34. Mwesigye, R. A., & Tumwebaze, B. S. (2017). Water contamination with heavy metals and trace elements from Kilembe copper mine and tailing sites in western Uganda; implications for domestic water quality. Chemosphere, 169, 281–287.CrossRefGoogle Scholar
  35. Pagnanelli, F., Cruz Viggi, C., Cibati, A., Uccelletti, D., & Palleschi, C. (2012). Biotreatment of Cr(VI) contaminated waters by sulphate reducing bacteria fed with ethanol. Journal of Hazardous Materials, 199-200, 186–192.CrossRefGoogle Scholar
  36. Postgate, J. R. (1984). The sulfate-reducing bacteria (2nd ed.). Cambridge: Cambridge Univ. Press.Google Scholar
  37. Quan, L. M., Khanh, D. P., Hira, D., Fujii, T., & Furukawa, K. (2011). Reject water treatment by improvement of whole cell anammox entrapment using polyvinyl alcohol/alginate gel. Biodegradation, 22, 1155–1167.CrossRefGoogle Scholar
  38. Russell, R. A., Holden, P. J., Wilde, K. L., & Neilan, B. A. (2003). Demonstration of the use of Scenedesmus and Carteria biomass to drive bacterial sulfate reduction by Desulfovibrio alcoholovorans isolated from an artificial wetland. Hydrometallurgy, 71, 227–234.CrossRefGoogle Scholar
  39. Sahinkaya, E., & Yucesoy, Z. (2010). Biotreatment of acidic zinc-and copper-containing wastewater using ethanol-fed sulfidogenic anaerobic baffled reactor. Bioprocess and Biosystems Engineering, 33, 989–997.CrossRefGoogle Scholar
  40. Sahinkaya, E., Gunes, F. M., Ucar, D., & Kaksonen, A. H. (2011). Sulfidogenic fluidized bed treatment of real acid mine drainage water. Bioresource Technology, 102, 683–689.CrossRefGoogle Scholar
  41. Sánchez-Andrea, I., Sanz, J. L., Bijmans, M. F., & Stams, A. J. (2014). Sulfate reduction at low pH to remediate acid mine drainage. Journal of Hazardous Materials, 269, 98–109.CrossRefGoogle Scholar
  42. Seiler, H. G., Sigel, H., Sigel, A., & Townshend, A. (1988). Handbook on toxicity of inorganic compounds. New York: Marcel Dekker.Google Scholar
  43. Sheoran, A. S., Sheoran, V., & Choudhary, R. P. (2010). Bioremediation of acid-rock drainage by sulphate-reducing prokaryotes: a review. Minerals Engineering, 23, 1073–1100.CrossRefGoogle Scholar
  44. 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. Molecular Biology and Evolution, 28, 2731–2739.CrossRefGoogle Scholar
  45. Tang, J. C., Gong, G. C., Su, H., Wu, F. H., & Herman, C. L. (2016). Performance evaluation of a novel method of frost prevention and retardation for air source heat pumps using the orthogonal experiment design method. Applied Energy, 169, 696–708.CrossRefGoogle Scholar
  46. Wakeman, K. D., Erving, L., Riekkola-Vanhanen, M. L., & Puhakka, J. A. (2010). Silage supports sulfate reduction in the treatment of metals-and sulfate-containing waste waters. Water Research, 44, 4932–4939.CrossRefGoogle Scholar
  47. Wang, W., Kang, Y., & Wang, A. (2013). One-step fabrication in aqueous solution of a granular alginate based hydrogel for fast and efficient removal of heavy metal ions. Journal of Polymer Research, 20, 101–111.CrossRefGoogle Scholar
  48. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173, 697–703.CrossRefGoogle Scholar
  49. Xiao, R., & Zheng, Y. (2016). Overview of microalgal extracellular polymeric substances (EPS) and their applications. Biotechnology Advances, 34, 1225–1244.CrossRefGoogle Scholar
  50. Yan, H., & Pan, G. (2002). Toxicity and bioaccumulation of copper in three green microalgal species. Chemosphere, 49, 471–476.CrossRefGoogle Scholar
  51. Yang, W. C., Tang, Q. Z., Wei, J. M., Ran, Y. J., Chai, L. Y., & Wang, H. Y. (2016). Enhanced removal of Cd(II) and Pb(II) by composites of mesoporouscarbon stabilized alumina. Applied Surface Science, 369, 215–223.CrossRefGoogle Scholar
  52. Yuvaraja, G., Subbaiah, M. V., & Krishnaiah, A. (2012). Caesalpinia bonducella leaf powder as biosorbent for Cu(II) removal from aqueous environment: Kinetic and isotherms. Industrial & Engineering Chemistry Research, 51, 11218–11225.CrossRefGoogle Scholar
  53. Zhang, M. L., & Wang, H. X. (2014). Organic wastes as carbon sources to promote sulfate reducing bacterial activity for biological remediation of acid mine drainage. Minerals Engineering, 69, 81–90.CrossRefGoogle Scholar
  54. Zhang, L., Cai, Z. J., Yang, J. M., Yuan, Z. W., & Chen, Y. (2015). The future of copper in China—a perspective based on analysis of copper flows and stocks. Science of the Total Environment, 536, 142–149.CrossRefGoogle Scholar
  55. Zhang, M. L., Wang, H. X., & Han, X. M. (2016). Preparation of metal-resistant immobilized sulfate reducing bacteria beads for acid mine drainage treatment. Chemosphere, 154, 215–223.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Civil EngineeringHunan University of Science and TechnologyXiangtanChina
  2. 2.Agricultural Clear Watershed Group, Institute of Environment and Sustainable Development in AgricultureChinese Academy of Agricultural Sciences (CAAS)BeijingChina

Personalised recommendations