Rhodococcus bacteria as a promising source of oils from olive mill wastes

  • O. Marisa Herrero
  • María S. Villalba
  • Mariana P. Lanfranconi
  • Héctor M. AlvarezEmail author
Original Paper


The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2–2.7 g/l) and lipids (77–83% of CDW, 1.8–2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1–1.6 g/l of cell biomass and 7.1–14.0% of CDW, 0.1–0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.


Olive mill wastes Bioconversion Lipid production Engineered Rhodococcus 



The technical assistance of Enrique Rost for GC analyses and Pedro Torrecillas for chemical oxygen demand analyses is gratefully acknowledged.


This work was funded by Projects PIP2015-16 Nro 0529, PICT2012 Nro 2031, and Oil m&s SA company. Alvarez H.M and Lanfranconi M.P. are career researchers of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11274_2018_2499_MOESM1_ESM.docx (1.5 mb)
Supplementary material 1 (DOCX 1565 KB)


  1. Aguilera M, Quesada MT, Aguila VG, Morillo JA, Rivadeneyra MA, Romos-Cormenzana A, Monteoliva-Sanchez M (2008) Characterization of Paenibacillus jamilae strains that produce exopolysaccharide during growth on and detoxification of olive mill wastewaters. Bioresour Technol 99:5640–5644CrossRefPubMedGoogle Scholar
  2. Alvarez HM (2010) Biotechnological production and significance of triacylglycerols and wax esters. In: Kenneth N, Timmis (eds) Microbiology of hydrocarbons, oils, lipids, and derived compounds, vol 3, Chap. 44. Springer Verlag, Heidelberg, pp 2995–3002Google Scholar
  3. Alvarez HM, Steinbüchel A (2010) Physiology biochemistry and molecular biology of triacylglycerol accumulation by Rhodococcus. In: Alvarez HM (ed) Biology of Rhodococcus, Microbiology Monographs Series. Springer, Berlin, pp 263–290CrossRefGoogle Scholar
  4. Alvarez HM, Kalscheuer R, Steinbüchel A (2000) Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126. Appl Microbiol Biotechnol 54:218–223CrossRefPubMedGoogle Scholar
  5. APHA (2005) Standard methods for the examination of water and wastewater. American Public Health Association, WashingtonGoogle Scholar
  6. Azbar N, Bayram A, Filibeli A, Muezzinoglu A, Sengul F, Ozer A (2004) A review of waste management options in olive oil production. Crit Rev Environ Sci Tec 34:209–247CrossRefGoogle Scholar
  7. Barbera AC, Maucieria C, Cavallarob V, Ioppoloa A, Spagnaa G (2013) Effects of spreading olive mill wastewater on soil properties and crops, a review. Agric Water Manag 119:43–53CrossRefGoogle Scholar
  8. Bellou S, Makria A, Sarris D, Michosa K, Rentoumia P, Celikc A, Papanikolaou S, Aggelis G (2014) The olive mill wastewater as substrate for single cell oil production by Zygomycetes. J Biotechnol 170:50–59CrossRefPubMedGoogle Scholar
  9. Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly (β-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977–1982PubMedPubMedCentralGoogle Scholar
  10. Cheirsilp B, Louhasakul Y (2013) Industrial wastes as a promising renewable source for production of microbial lipid and direct transesterification of the lipid into biodiesel. Bioresour Technol 142:329–337CrossRefPubMedGoogle Scholar
  11. Chiavola V, Farabegoli G, Rolee E (2010) Combined biological and chemical-physical process for olive mill wastewater treatment. Desalination Water Treat 23:135–140CrossRefGoogle Scholar
  12. Darvishi F (2012) Microbial biotechnology in olive oil industry. In: Dimitrios B (ed) Olive oil—constituents, quality, health properties and bioconversions. InTech Publisher, Croatia, pp 309–330Google Scholar
  13. Dinamarca AM, Aguirre J, Espinoza G, Canale C, Ojeda J (2014) Biodesulfurization of dibenzothiophene and gas oil using a bioreactor containing a catalytic bed with Rhodococcus rhodochrous immobilized on silica. Biotechnol Lett 36:1649–1652CrossRefGoogle Scholar
  14. DuBois M, Gilles K, Hamilton J, Rebers P, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  15. Felsenstein J (1985) Phylogenies and the comparative method. Am Nat 125:1–15CrossRefGoogle Scholar
  16. Folch J, Lees M, Sloane-Stanley G (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 199:833–841Google Scholar
  17. Gouda MK, Omar SH, Aouad LM (2008) Single cell oil production by Gordonia sp. DG using agroindus-trial wastes. World J Microbiol Biotechnol 24:1703–1711CrossRefGoogle Scholar
  18. Hall T (1999) BioEdit: a user friendly biological sequence alignment editor and analysis program for Windows 95/98 NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  19. Hernández MA, Arabolaza A, Rodríguez E, Gramajo H, Alvarez HM (2013) The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630. Appl Microbiol Biotechnol 97:2119–2130CrossRefPubMedGoogle Scholar
  20. Hernández MA, Comba S, Arabolaza A, Gramajo H, Alvarez HM (2015) Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains. Appl Microbiol Biotechnol 99:2191–2207CrossRefPubMedGoogle Scholar
  21. Herrero OM, Alvarez HM (2016) Whey as a renewable source for lipid production by Rhodococcus strains: physiology and genomics of lactose and galactose utilization. Eur J Lipid Sci Technol 188:262–272CrossRefGoogle Scholar
  22. Herrero OM, Moncalian G, Alvarez HM (2016) Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production. Microbiology 162:384–397CrossRefPubMedGoogle Scholar
  23. Hughes E, Benemam J (1997) Biological fossil CO2 mitigation. Energy Convers Manag 38:467–473CrossRefGoogle Scholar
  24. Ivshina IB, Vikhareva EV, Richkova MI, Mukhutdinova AN, Karpenko JN (2012) Biodegradation of drotaverine hydrochloride by free and immobilized cells of Rhodococcus rhodochrous IEGM 608. World J Microbiol Biotechnol 28:2997–3006CrossRefPubMedGoogle Scholar
  25. Kalscheuer R, Arenskötter M, Steinbüchel A (1999) Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalcanoic acids). Appl Microbiol Biotechnol 52:508–515CrossRefPubMedGoogle Scholar
  26. Kumar P, Sussela MR, Toppo K (2011) Physico-chemical characterization of algal oil: a potential biofuel. Asian J Exp Biol Sci 2:493–497Google Scholar
  27. Kurosawa K, Wewetzer SJ, Sinskey AJ (2014) Triacylglycerol production from corn stover using a xylose-fermenting Rhodococcus opacus strain for lignocellulosic biofuels. J Microb Biochem Technol 6:254–259CrossRefGoogle Scholar
  28. Kurosawa K, Radek A, Plassmeier JK, Sinskey AJ (2015) Improved glycerol utilization by a triacyglycerol-producing Rhodococcus opacus strain for renewable fuels. Biotecnol Biofuels 8:3–11CrossRefGoogle Scholar
  29. Lanfranconi MP, Alvarez HM (2017) Rewiring neutral lipids production for the de novo synthesis of wax esters in Rhodococcus opacus PD630. J Biotechnol 260:67–73CrossRefPubMedGoogle Scholar
  30. MacEachran DP, Sinskey AJ (2013) The Rhodococcus opacus TadD protein mediates triacylglycerol metabolism by regulating intracellular NAD(P)H pools. Microb Cell Factor 12:104CrossRefGoogle Scholar
  31. Mantzavinos D, Kalogerakis N (2005) Treatment of olive mill effluents: part I. Organic matter degradation by chemical and biological processes: an overview. Environ Int 31:289–295CrossRefPubMedGoogle Scholar
  32. McNamara CJ, Anastasiou CC, O’Flaherty V, Mitchell R (2008) Bioremediation of olive mill wastewater. Int Biodeterior Biodegrad 61:127–134CrossRefGoogle Scholar
  33. Morillo JA, Antizar-Ladislao B, Monteoliva-Sánchez M, Ramos-Cormenzana A, Russell NJ (2009) Bioremediation and biovalorisation of olive mill wastes. Appl Microbiol Biotechnol 82:25–39CrossRefPubMedGoogle Scholar
  34. Mustacchi R, Knowles CJ, Li H, Dalrymple I, Sunderland G, Skibar W, Jackman SA (2005) The effect of whole cell immobilisation on the biotransformation of benzonitrile and the use of direct electric current for enhanced product removal. Biotechnol Bioeng 91:436–440CrossRefPubMedGoogle Scholar
  35. Niaounakis M, Halvadakis CP (2006) Olive processing waste management literature review and patent survey. Waste Management Series, Vol 5. Elsevier, Amsterdam, pp 514Google Scholar
  36. Öner ET (2013) Microbial production of extracellular polysaccharides from biomass. In: Fang Z (ed) Pretreatment techniques for biofuels and biorefineries. Springer, Berlin, pp 35–56CrossRefGoogle Scholar
  37. Papanikolaou S, Chevalot I, Komaitis M, Marc I, Aggelis G (2002) Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures. Appl Microbiol Biotechnol 58:308–312CrossRefPubMedGoogle Scholar
  38. Paraskeva P, Diamadopoulos E (2006) Technologies for olive mill wastewater (OMW) treatment: a review. J Chem Technol Biotechnol 81:1475–1485CrossRefGoogle Scholar
  39. Ramos MJ, Fernández CM, Casas A, Rodríguez L, Pérez A (2009) Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour Technol 100:261–268CrossRefPubMedGoogle Scholar
  40. Roig A, Cayuela ML, Sánchez-Monedero MA (2006) An overview on olive mil wastes and their valorization methods. Waste Manag 26:960–969CrossRefPubMedGoogle Scholar
  41. SanGiovanni JP, Chew EY (2005) The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina. Prog Retin Eye Res 24:87–138CrossRefPubMedGoogle Scholar
  42. Sarris D, Giannakis M, Philippoussis A, Komaitis M, Koutinas AA, Papanikolaou S (2013) Conversions of olive mill wastewater-based media by Saccharomyces cerevisiae through sterile and non-sterile bioprocesses. J Chem Technol Biotechnol 88:958–969CrossRefGoogle Scholar
  43. Schlegel HG, Kaltwasser H, Gottschalk G (1961) A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies. Arch Mikrobiol 38:209–222CrossRefPubMedGoogle Scholar
  44. Seto M, Kimbara K, Shimura M, Hatta T, Fukuda M, Yano K (1995) A novel transformation of polychlorinated biphenyls by Rhodococcus sp. strain RHA1. Appl Environ Microbiol 61:3353–3358PubMedPubMedCentralGoogle Scholar
  45. Spurr AR (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43CrossRefPubMedGoogle Scholar
  46. Subramaniam R, Dufreche S, Zappi M, Bajpai R (2010) Microbial lipids from renewable resources: production and characterization. J Ind Microbiol Biotechnol 37:1271–1287CrossRefPubMedGoogle Scholar
  47. Suttinun O, Müller R, Luepromchai E (2010) Cometabolic degradation of trichloroethene by Rhodococcus sp. strain L4 immobilized on plant materials rich in essential oils. Appl Environ Microbiol 76:4684–4690CrossRefPubMedPubMedCentralGoogle Scholar
  48. Villalba MS, Alvarez HM (2014) Identification of a novel ATP-binding cassette transporter involved in long-chain fatty acid import and its role in triacylglycerol accumulation in Rhodococcus jostii RHA1. Microbiology 160:1523–1532CrossRefPubMedGoogle Scholar
  49. Vogt B, Berker R, Mayer F (1995) Improved contrast by a simplified post-staining procedure for ultrathin sections of resin-embedded bacterial cells: application of ruthenium red. J Basic Microbiol 35:349–355CrossRefGoogle Scholar
  50. Wältermann M, Luftmann H, Baumeister D, Kalscheuer R, Steinbüchel A (2000) Rhodococcus opacus PD630 as a source of high-value single cell oil? Isolation and characterization of triacylgycerols and other storage lipids. Microbiology 146:1143–1149CrossRefPubMedGoogle Scholar
  51. Youdim KA, Martin A, Joseph JA (2000) Essential fatty acids and the brain: possible health implications. Int J Dev Neurosci 18:383–399CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • O. Marisa Herrero
    • 1
  • María S. Villalba
    • 1
  • Mariana P. Lanfranconi
    • 1
  • Héctor M. Alvarez
    • 1
    Email author
  1. 1.Instituto de Biociencias de la Patagonia (INBIOP)Universidad Nacional de la Patagonia San Juan Bosco y CONICETComodoro RivadaviaArgentina

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