Potential of industrial by-products and wastes from the Iberian Peninsula as carbon sources for sulphate-reducing bacteria

  • J. D. Carlier
  • L. M. Alexandre
  • A. T. Luís
  • M. C. CostaEmail author
Original Paper


Industrial by-products and wastes from Portugal and Spain were tested for the first time as carbon sources/electron donors for sulphate-reducing bacteria. Cultures in mineral medium supplemented with the tested substrates were monitored, and sulphate reduction efficiency is discussed in light of substrates compositions, dosages and corresponding chemical oxygen demand/[SO42−] ratios. The results reveal the ability of those substrates to feed SRB and confirm that testing doses targeting ratios of 1.5 and values close to this was a good strategy to optimize sulphate reduction activity. As expected, this activity was faster for substrates that have in their composition simple compounds (such as low-chain alcohols and organic acids) and/or compounds that can be rapidly degraded (such as sugars), though it also occurred in a longer-term perspective with substrates composed mainly of slowly degradable compounds (such as cellulose and lignin). Thus, this work demonstrates the potential of new substrates and respective required doses to feed SRB bioreactors in long-term passive bioremediation processes or faster more active processes. That is, it opens the way for the use of such substrates in the treatment of sulphate-rich waters, as the acid mine drainage generated in some mines on the Iberian Pyrite Belt region, and it encourages further experiments to evaluate the use of SRB-based processes to treat the industrial wastewaters successfully tested in this work themselves, specially the olive mill wastewater which is still a problem for many small olive oil producers.


Chemical oxygen demand/[SO42−] ratio Electron donors Organic substrates Sulphate reduction 



The authors also wish to acknowledge the company Águas do Algarve, namely Eng. Joaquim Freire and Eng. Patrício Fontinha, for supplying the WWTP sludge used to enrich cultures with SRB and companies that supplied the substrates tested as sources of carbon sources/electron donors (and the persons involved in this supply): Orange juice plant LARA—Laranja do Algarve S.A. (Eng. Pedro Pacheco), Factory of candies Dulciora (anonymous collaboration), Sugar processing plant Azucarera (Eng. Maria Hernandez Garcia), Olive mill Lagar Santa Catarina (Eng. Renato Rocha), Águas Públicas do Alentejo, WWTP Mina de São Domingos (Eng. Olga Martins), Paper and paper pulp factories Navigator Company (Eng. Luis Medeiros Machado and Eng. Patrícia Castellano Rodrigues).


The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) managed by REA-Research Executive Agency ( under Grant Agreement No. 619101; and the Portuguese Foundation for Science and Technology (FCT) through the Project UID/Multi/04326/2019.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13762_2018_2197_MOESM1_ESM.pdf (275 kb)
Supplementary material 1 (PDF 275 kb)


  1. Almendros AI, Martín-Lara MA, Ronda A, Pérez A, Blázquez G, Calero M (2015) Physico-chemical characterization of pine cone shell and its use as biosorbent and fuel. Bioresour Technol 196:406–412. CrossRefGoogle Scholar
  2. Amaral C, Lucas MS, Coutinho J, Crespí AL, do Rosário Anjos M, Pais C (2008) Microbiological and physicochemical characterization of olive mil wastewaters from a continuous olive mill in Northeastern Portugal. Bioresour Technol 99:7215–7223. CrossRefGoogle Scholar
  3. Annachhatre AP, Suktrakoolvait S (2001) Biological sulphate reducing using molasses as a carbon source. Water Environ Res 73:118–126. CrossRefGoogle Scholar
  4. Azbar N, Bayram A, Filibeli A, Muezzinoglu A, Sengul F, Ozer A (2004) A review of wastes management options in olive oil production. Crit Rev Environ Sci Technol 34:209–247. CrossRefGoogle Scholar
  5. Baena S, Fardeau ML, Labat M, Ollivier B, Garcia JL, Patel BKC (1998) Desulfovibrio aminophilus sp. nov., a novel amino acid degrading and sulfate reducing bacterium from an anaerobic dairy wastewater lagoon. Syst Appl Microbiol 21(4):498–504. CrossRefGoogle Scholar
  6. Bechard G, Yamazaki H, Gould WD, Bedard P (1994) Use of cellulosic substrates for the microbial treatment of acid mine drainage. J Environ Qual 23:111–116. CrossRefGoogle Scholar
  7. Buzzini AP, Pires EC (2007) Evaluation of a upflow anaerobic sludge blanket reactor with partial recirculation of effluent used to treat wastewaters from pulp and paper plants. Bioresour Technol 98:1838–1848. CrossRefGoogle Scholar
  8. Cao J, Zhang G, Mao Z, Li Y, Fang F, Chao Y (2012) Influence of electron donors on the growth and activity of sulphate-reducing bacteria. Int J Miner Process 106–109:58–64. CrossRefGoogle Scholar
  9. Castro JM, Moore JN (2000) Pit lakes: their characteristics and the potential for their remediation. Environ Geol 39:1254–1260. CrossRefGoogle Scholar
  10. Chamkh F, Spröer C, Lemos PC, Besson S, El Asli AG, Bennisse R, Labat M, Reis M, Qatibi AI (2009) Desulfovibrio marrakechensis sp. nov., a 1,4-tyrosol-oxidizing, sulphate-reducing bacterium isolated from olive mill wastewater. Int J Syst Evol Microbiol 59:936–942. CrossRefGoogle Scholar
  11. Chen W, Horan NJ (1998) The treatment of a high strength pulp and paper mill effluent for wastewater re-use. Environ Technol 19:163–171. Google Scholar
  12. Choudhary RP, Sheoran AS (2011) Comparative study of cellulose waste versus organic waste as substrate in a sulphate reducing bioreactor. Bioresour Technol 102:4319–4324. CrossRefGoogle Scholar
  13. Clements RL (1964) Organic acids in citrus fruits. I. Varietal differences. J Food Sci 29:276–280. CrossRefGoogle Scholar
  14. Costa MC, Duarte JC (2005) Bioremediation of acid mine drainage using acidic soil and organic wastes for promoting sulphate-reducing bacteria activity on a column reactor. Water Air Soil Pollut 165:325–345. CrossRefGoogle Scholar
  15. Costa JM, Rodriguez RP, Sancinetti GP (2017) Removal sulfate and metals Fe+2, Cu+2, and Zn+2 from acid mine drainage in an anaerobic sequential batch reactor. J Environ Chem Eng 5(2):1985–1989. CrossRefGoogle Scholar
  16. Das BK, Gauri SS, Bhattacharya J (2013) Sweetmeat waste fractions as suitable organic carbon source for biological sulfate reduction. Int Biodeterior Biodegrad 82:215–223. CrossRefGoogle Scholar
  17. Dev S, Roy S, Bhattacharya J (2017) Optimization of the operation of packed bed bioreactor to improve the sulfate and metal removal from acid mine drainage. J Environ Manag 200:135–144. CrossRefGoogle Scholar
  18. Elliott P, Ragusa S, Catcheside D (1998) Growth of sulphate-reducing bacteria under acidic conditions in an upflow anaerobic bioreactor as a treatment system for acid mine drainage. Water Res 32:3724–3730. CrossRefGoogle Scholar
  19. Hao T, Xiang P, Mackey HR, Chi K, Lu H, Chui H, van Loosdrecht MCM, Chen GH (2014) A review of biological sulphate conversions in wastewater treatment. Water Res 65:1–21. CrossRefGoogle Scholar
  20. Hokkanen S, Bhatnagar A, Sillanpää M (2016) A review on modification methods to cellulose-based adsorbents to improve adsorption capacity. Water Res 91:156–173. CrossRefGoogle Scholar
  21. Hussain A, Qazi JI (2012) Biological sulphate reduction using watermelon rind as a carbon source. Biologia (Pakistan) 58:85–92Google Scholar
  22. Hussain A, Qazi J (2016) Application of sugarcane bagasse for passive anaerobic biotreatment of sulphate rich wastewaters. Appl Water Sci 6(2):205–211. CrossRefGoogle Scholar
  23. Jiang Y, Qin Y, Yu F, Li H, Liu K (2018) Is COD/SO4 2− ratio responsible for metabolic phase-separation shift in anaerobic baffled reactor treating sulfate-laden wastewater? Int Biodeterior Biodegrad 126:37–44. CrossRefGoogle Scholar
  24. Kim YM, Kim S, Han TU, Park YK, Watanabe C (2014) Pyrolysis reaction characteristics of Korean pine (Pinus Koraiensis) nut shell. J Anal Appl Pyrol 110:435–441. CrossRefGoogle Scholar
  25. Kiran MG, Pakshirajan K, Das G (2017) An overview of sulfidogenic biological reactors for the simultaneous treatment of sulfate and heavy metal rich wastewater. Chem Eng Sci 158:606–620. CrossRefGoogle Scholar
  26. Kiyuna LS, Fuess LT, Zaiat M (2017) Unraveling the influence of the COD/sulfate ratio on organic matter removal and methane production from the biodigestion of sugarcane vinasse. Bioresour Technol 232:103–112. CrossRefGoogle Scholar
  27. Koschorreck M, Wendt-Potthoff K, Bozau E, Herzsprung P, Geller W, Schultze M (2007) In: Cidu R, Frau F (eds) IMWA symposium 2007: Water in mining environments. OAI, Cagliari, Italy, 27–31st May 2007Google Scholar
  28. Kumar RN, McCullough CD, Lund MA (2011) How does storage affect the quality and quantity of organic carbon in sewage for use in the bioremediation of acidic mine waters? Ecol Eng 37:1205–1213. CrossRefGoogle Scholar
  29. Lefticariu L, Walters ER, Pugh CW, Bender KS (2015) Sulfate reducing bioreactor dependence on organic substrates for remediation of coal-generated acid mine drainage: field experiments. Appl Geochem 63:70–82. CrossRefGoogle Scholar
  30. Li YL, Wang J, Yue ZB, Tao W, Yang HB, Zhou YF, Chen TH (2017) Simultaneous chemical oxygen demand removal, methane production and heavy metal precipitation in the biological treatment of landfill leachate using acid mine drainage as sulfate resource. J Biosci Bioeng 124(1):71–75. CrossRefGoogle Scholar
  31. Liamleam W, Annachatre AP (2007) Electron donors for biological sulfate reduction. Biotechnol Adv 25:452–463. CrossRefGoogle Scholar
  32. Logan MV, Reardon KF, Figueroa LA, McLain JET, Ahmann DM (2005) Microbial community activities during establishment, performance, and decline of bench-scale passive treatment systems for mine drainage. Water Res 39:4537–4551. CrossRefGoogle Scholar
  33. Lowson RT (1982) Aqueous oxidation of pyrite by molecular oxygen. Chem Rev 82:461–497. CrossRefGoogle Scholar
  34. Lu X, Zhen G, Ni J, Kubota K, Li YY (2017) Sulfidogenesis process to strengthen re-granulation for biodegradation of methanolic wastewater and microorganisms evolution in an UASB reactor. Water Res 108:137–150. CrossRefGoogle Scholar
  35. MacPherson R, Miller JDA (1963) Nutritional studies on Desulfovibrio desulfuricuns using chemically defined media. Microbiology 31:365–373. Google Scholar
  36. Maree JP, Gerber A, Hill E (1987) An integrated process for biological treatment of sulphate-containing industrial effluents. J Water Pollut Control Fed 59:1069–1074Google Scholar
  37. Martins M, Faleiro ML, Barros RJ, Veríssimo AR, Costa MC (2009) Biological sulphate reduction using food industry wastes as carbon sources. Biodegradation 20:559–567. CrossRefGoogle Scholar
  38. Michailides MK, Tekerlekopoulou AG, Akratos CS, Coles S, Pavlou S, Vayenas DV (2015) Molasses as an efficient low-cost carbon source for biological Cr(VI) removal. J Hazard Mater 8:95–105. CrossRefGoogle Scholar
  39. Miran W, Jang J, Nawaz M, Shahzad A, Jeong SE, Jeon CO, Lee DS (2017) Mixed sulfate-reducing bacteria-enriched microbial fuel cells for the treatment of wastewater containing copper. Chemosphere 189:134–142. CrossRefGoogle Scholar
  40. Muhammad SN, Kusin FM, Madzin Z (2017) Coupled physicochemical and bacterial reduction mechanisms for passive remediation of sulfate- and metal-rich acid mine drainage. Int J Environ Sci Technol. Google Scholar
  41. Mulopo J (2016) Pilot scale assessment of the continuous biological sulphate removal from coal acid mine effluent using grass cutting as carbon and energy sources. J Water Process Eng 11:104–109. CrossRefGoogle Scholar
  42. Muyzer G, Stams AJ (2008) The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol 6(6):441–454. CrossRefGoogle Scholar
  43. Neculita CM, Zagury GJ (2008) Biological treatment of highly contaminated acid mine drainage in batch reators: long-term treatment and reactive mixture characterization. J Hazard Mater 157:359–366. CrossRefGoogle Scholar
  44. Neculita CM, Zagury GJ, Bussiere B (2007) Passive treatment of acid mine drainage in bioreactors using sulphate-reducing bacteria: critical review and research needs. J Environ Qual 36:1–16. CrossRefGoogle Scholar
  45. O’Flaherty V, Mahony T, O’Kennedy R, Colleran E (1998) Effect of pH on growth kinetics and sulphide toxicity thresholds of a range of methanogenic, syntrophic and sulphate-reducing bacteria. Process Biochem 33(5):555–569. CrossRefGoogle Scholar
  46. Ollivier B, Cord-Ruwisch R, Hatchikian EC, Garcia JL (1988) Characterization of Desulfovibrio fructosovorans sp. nov. Arch Microbiol 149:447–450. CrossRefGoogle Scholar
  47. Parshina SN, Sipma J, Henstra AM, Stams AJ (2010) Carbon monoxide as an electron donor for the biological reduction of sulphate. Int J Microbiol 2010:319527. CrossRefGoogle Scholar
  48. Pereira R, Ribeiro R, Gonçalves F (2004) Plan for an integrated human and environmental risk assessment in the S. Domingos mine area (Portugal). Hum Ecol Risk Assess 10:543–578. CrossRefGoogle Scholar
  49. Postgate JR (1984) The sulphate-reducing bacteria, 2nd edn. Cambridge University Press, CambridgeGoogle Scholar
  50. Prasad D, Wai M, Bérubé P, Henry JG (1999) Evaluating substrates in the biological treatment of acid mine drainage. Environ Technol 20:449–458. CrossRefGoogle Scholar
  51. Prasetyo EN, Rodríguez RD, Lukesch B, Weiss S, Murkovic M, Katsoyannos E, Sygmund C, Ludwig R, Nyanhongo GS, Guebitz GM (2015) Laccase–cellobiose dehydrogenase-catalyzed detoxification of phenolic-rich olive processing residues. Int J Environ Sci Technol 12:1343–1352. CrossRefGoogle Scholar
  52. Rambabu N, Panthapulakkal S, Sain M, Dalai AK (2016) Production of nanocellulose fibers from pinecone biomass: evaluation and optimization of chemical and mechanical treatment conditions on mechanical properties of nanocellulose films. Ind Crop Prod 83:746–754. CrossRefGoogle Scholar
  53. Raunkjaer K, Jacobsen TH, Nielsen PH (1994) Measurement of pools of protein, carbohydrate and lipid in domestic wastewater. Water Res 28:251–262. CrossRefGoogle Scholar
  54. Relvas JMRS, Barriga FJAS, Longstaffe FJ (2006) Hydrothermal alteration and mineralization in the Neves-Corvo volcanic-hosted massive sulfide deposit, Portugal. II. Oxygen, hydrogen, and carbon isotopes. Econ Geol 101(4):791–804. CrossRefGoogle Scholar
  55. Rivas B, Torrado A, Torre P, Converti A, Dominguez JM (2008) Submerged citric acid fermentation on orange peel autohydrolysate. J Agric Food Chem 56:2380–2387. CrossRefGoogle Scholar
  56. Shofinita D, Feng S, Langrish TAG (2015) Comparing yields from the extraction of different citrus peels and spray drying of the extracts. Adv Powder Technol 26:1633–1638. CrossRefGoogle Scholar
  57. van Haandel AC, van der Lubbe JGM (2007) Handbook biological waste water treatment—design and optimization of activated sludge systems. Quist Publishing, LeidschendamGoogle Scholar
  58. Vasquez Y, Escobar MC, Neculita CM, Arbeli Z, Roldan F (2016) Selection of reactive mixture for biochemical passive treatment of acid mine drainage. Environ Earth Sci 75:576. CrossRefGoogle Scholar
  59. Vela FJ, Zaiat M, Foresti E (2002) Influence of the COD to sulphate ratio on the anaerobic organic matter degradation kinetics. Water SA 28:213–216. CrossRefGoogle Scholar
  60. Wang A, Ren N, Wang X, Lee D (2008) Enhanced sulphate reduction with acidogenic sulphate-reducing bacteria. J Hazard Mater 154:1060–1065. CrossRefGoogle Scholar
  61. Willow MA, Cohen RRH (2003) pH, dissolved oxygen, and adsorption effects on metal removal in anaerobic bioreactors. J Environ Qual 32:1212–1221. CrossRefGoogle Scholar
  62. Wolicka D, Borkowski A (2009) Phosphogypsum biotransformation in cultures of sulphate reducing bacteria in whey. Int Biodeterior Biodegrad 63:322–327. CrossRefGoogle Scholar
  63. Zerrouki S, Rihani R, Bentahar F, Belkacemi K (2015) Anaerobic digestion of wastewater from the fruit juice industry: experiments and modeling. Water Sci Technol 72:123–134. CrossRefGoogle Scholar
  64. Zhang M, Wang H (2014) Organic wastes as carbon sources to promote sulphate reducing bacterial activity for biological remediation of acid mine drainage. Miner Eng 69:81–90. CrossRefGoogle Scholar
  65. Zhang QH, Jin PK, Ngo HH, Shi X, Guo WS, Yang SJ, Wang XC, Wang X, Dzakpasu M, Yang WN, Yang L (2016) Transformation and utilization of slowly biodegradable organic matters in biological sewage treatment of anaerobic anoxic oxic systems. Bioresour Technol 218:53–61. CrossRefGoogle Scholar

Copyright information

© Islamic Azad University (IAU) 2019

Authors and Affiliations

  • J. D. Carlier
    • 1
  • L. M. Alexandre
    • 1
  • A. T. Luís
    • 1
    • 3
  • M. C. Costa
    • 1
    • 2
    Email author
  1. 1.CCMARUniversity of AlgarveFaroPortugal
  2. 2.Faculty of Sciences and TechnologiesUniversity of AlgarveFaroPortugal
  3. 3.GeoBioTec Research Unit and Geosciences DepartmentUniversity of AveiroAveiroPortugal

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