Journal of Soils and Sediments

, Volume 19, Issue 1, pp 439–450 | Cite as

Biodegradation of biodiesel and toluene under nitrate-reducing conditions and the impact on bacterial community structure

  • Hugo RibeiroEmail author
  • Joana Gomes da Silva
  • João Jesus
  • Catarina Magalhães
  • Joana M. Dias
  • Anthony S. Danko
Sediments, Sec 2 • Physical and Biogeochemical Processes • Research Article



Biodiesel is a renewable fuel that can be mixed with toluene and be accidentally released into anoxic ecosystems and impact soil microbial communities. Therefore, the aim of the present work was to examine, under nitrate-reduction conditions, the biodegradation of toluene in the presence of two different types of biodiesel (sunflower and rapeseed), and their impact on the bacterial community structure.

Materials and methods

Sediment samples were spiked individually with toluene, biodiesel, and their blends in laboratory-designed microcosms. Sunflower oil biodiesel was produced in the laboratory, while rapeseed oil biodiesel was a commercial product. Degradation of biodiesels and blends was monitored by directly measuring the substrate or indirectly by determining nitrate removal during the course of the experiment. Denitrification rates were estimated with the acetylene inhibition technique. Denaturing gradient gel electrophoresis was used to assess impacts on the bacterial community structure exposed to biodiesels, blends, and toluene.

Results and discussion

The results of this study showed that toluene and biodiesel were completely degraded within 10 days. Biodiesel significantly affected the bacterial community structure at a similar magnitude, independently of its origin. Additionally, toluene impacted the bacterial community and denitrification process to a lower extent than biodiesel and a clear decrease in the relative bacterial richness and diversity was shown in samples with biodiesel and blends. To the best of our knowledge, this is one of the first reports describing degradation of biodiesel alone and blends under nitrate-reducing conditions, and also the effects of these compounds on the denitrification process. In addition, due to the recently discovered “oxygenic denitrification” process, the acetylene inhibition technique and nitrous oxide quantification may not be the most adequate tool to estimate denitrification rates. Further detailed studies are advised to understand whether the identified bacterial community shift impacts ecosystem functions.


Our results help to understand the biodegradation of toluene, biodiesel, and their blends in sediments under nitrate-reducing conditions and might be important in implementing bioremediation strategies in anoxic environments.


Acetylene inhibition technique Biodiesel Bioremediation Microbial community structure Soil Toluene 



The authors acknowledge the anonymous reviewers and editors for their valuable comments and suggestions, which were helpful in improving the manuscript.

Funding information

The authors acknowledge the Portuguese Science and Technology Foundation (FCT) for the following financial support: the research grant SFRH/BPD/112485/2015 and other funds within the project PTDC/AAG-TEC/4403/2012, financed by national funds through FCT/MEC (PIDDAC) and co-financed by FEDER through COMPETE (POFC) FCOMP-01-0124-FEDER-027941 and the Project UID/EQU/00511/2013-LEPABE (Laboratory for Process Engineering, Environment, Biotechnology and Energy – EQU/00511) by FEDER funds through Programa Operacional Competitividade e Internacionalização – COMPETE2020. This research was also partially supported by the Structured Program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Line ECOSERVICES).

Supplementary material

11368_2018_2079_MOESM1_ESM.jpg (255 kb)
ESM 1 (JPG 254 kb)
11368_2018_2079_MOESM2_ESM.xls (104 kb)
ESM 2 (XLS 103 kb)


  1. Aktas DF, Lee JS, Little J, Ray RI, Davidova IA, Lyles CN, Suflita JM (2010) Anaerobic metabolism of biodiesel and its impact on metal corrosion. Energy Fuel 24:2924–2928CrossRefGoogle Scholar
  2. Allison SD, Martiny JB (2008) Resistance, resilience, and redundancy in microbial communities. Proc Natl Acad Sci U S A 105:11512–11519CrossRefGoogle Scholar
  3. Atashgahi S, Hornung B, Waals MJ, Rocha UN, Hugenholtz F, Nijsse B, Molenaar D, van Spanning R, Stams AJM, Gerritse J, Smidt H (2018) A benzene-degrading nitrate-reducing microbial consortium displays aerobic and anaerobic benzene degradation pathways. Sci Rep 8:4490CrossRefGoogle Scholar
  4. Borges JM, Dias JM, Danko AS (2014) Influence of the anaerobic biodegradation of different types of biodiesel on the natural attenuation of benzene. Water Air Soil Pollut 225:1–10CrossRefGoogle Scholar
  5. Carvalho MF, Jorge RF, Pacheco CC, De Marco P, Castro PML (2005) Isolation and properties of a pure bacterial strain capable of fluorobenzene degradation as sole carbon and energy source. Environ Microbiol 7:294–298CrossRefGoogle Scholar
  6. Chen CS, Shu YY, Wu SH, Tien CJ (2015) Assessing soil and groundwater contamination from biofuel spills. Environ Sci Process Impact 17:533–542CrossRefGoogle Scholar
  7. Chikere CB, Okpokwasiki GC, Chikere BO (2011) Monitoring of microbial hydrocarbon remediation in the soil. 3 Biotech 1:117–138CrossRefGoogle Scholar
  8. Clarke KR, Gorley RN (2006) Primer v6: user manual/tutorial, PRIMER-E, PlymouthGoogle Scholar
  9. Clarke KR, Somerfield PJ, Gorley RN (2008) Testing null hypotheses in exploratory community analyses: similarity profiles and biota-environmental linkage. J Exp Mar Biol Ecol 366:56–69CrossRefGoogle Scholar
  10. Colla TS, Andreazza R, Bücker F, de Souza MM, Tramontini L, Prado GR, Frazzon APG, Camargo FAO, Bento FM (2014) Bioremediation assessment of diesel–biodiesel-contaminated soil using an alternative bioaugmentation strategy. Environ Sci Pollut Res 21:2592–2602CrossRefGoogle Scholar
  11. Corseuil HX, Hunt CS, Santos RCF, Alvarez PJJ (1998) The influence of the gasoline oxygenate ethanol on aerobic and anaerobic BTX biodegradation. Water Res 32:2065–2072CrossRefGoogle Scholar
  12. Corseuil HX, Monier AL, Gomes APN, Chiaranda HS, do Rosario M, Alvarez PJJ (2011) Biodegradation of soybean and castor oil biodiesel: implications on the natural attenuation of monoaromatic hydrocarbons in groundwater. Ground Water Monit R 31:111–118CrossRefGoogle Scholar
  13. Cyplik P, Schmidt M, Szulc A, Marecik R, Lisiecki P, Heipieper HJ, Owsianiak M, Vainshtein M, Chrzanowski Ł (2011) Relative quantitative PCR to assess bacterial community dynamics during biodegradation of diesel and biodiesel fuels under various aeration conditions. Bioresour Technol 102:4347–4352CrossRefGoogle Scholar
  14. DeMello JA, Carmichael CA, Peacock EE, Nelson RK, Arey JS, Reddy CM (2007) Biodegradation and environmental behavior of biodiesel mixtures in the sea: an initial study. Mar Pollut Bull 54:894–904CrossRefGoogle Scholar
  15. Demirbaş A (2008) Biodegradability of biodiesel and petrodiesel fuels. Energ Source Part A 31:169–174CrossRefGoogle Scholar
  16. Dias JM, Alvim-Ferraz MCM, Almeida MF (2008) Comparison of the performance of different homogeneous alkali catalysts during transesterification of waste and virgin oils and evaluation of biodiesel quality. Fuel 87:3572–3578CrossRefGoogle Scholar
  17. Ettwig KF, Butler MK, Le Paslier D, Pelletier E, Mangenot S, Kuypers MMM et al (2010) Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464:543–548CrossRefGoogle Scholar
  18. Evans PJ, Mang DT, Young LY (1991) Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures. Appl Environ Microbiol 57:450–454Google Scholar
  19. Green SJ, Leigh MB, Neufeld JD (2015) Denaturing gradient gel electrophoresis (DGGE) for microbial community analysis. In: McGenity T, Timmis K, Nogales B (eds) Hydrocarbon and lipid microbiology protocols. Springer, Heidelberg, pp 77–99CrossRefGoogle Scholar
  20. He JZ, Ge Y, Xu Z, Chen C (2009) Linking soil bacterial diversity to ecosystem multifunctionality using backward-elimination boosted trees analysis. J Soils Sediments 9:547–554CrossRefGoogle Scholar
  21. He Z, Feng Y, Zhang S, Wang X, Wu S, Pan X (2018) Oxygenic denitrification for nitrogen removal with less greenhouse gas emissions: microbiology and potential applications. Sci Total Environ 621:453–464CrossRefGoogle Scholar
  22. Heuer H, Krsek M, Baker P, Smalla K, Wellington EM (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl Environ Microbiol 63:3233–3241Google Scholar
  23. Ivone VM, Conceição E, Olga CN, Célia MM (2013) Bacterial diversity from the source to the tap: a comparative study based on 16S rRNA gene-DGGE and culture-dependent methods. FEMS Microbiol Ecol 83:361–374CrossRefGoogle Scholar
  24. Jakeria MR, Fazal MA, Haseeb ASMA (2014) Influence of different factors on the stability of biodiesel: a review. Renew Sust Energ Rev 30:154–163CrossRefGoogle Scholar
  25. Jayamani I, Cupples AM (2012) Effect of isobutanol on toluene biodegradation in nitrate amended, sulfate amended and methanogenic enrichment microcosms. Biodegradation 24:657–663CrossRefGoogle Scholar
  26. Jesus J, Castro F, Niemelä A, Borges MT, Danko AS (2015) Evaluation of the impact of different soil salinization processes on organic and mineral soils. Water Air Soil Pollut 226:1–12CrossRefGoogle Scholar
  27. Ji X, Long X (2016) A review of the ecological and socioeconomic effects of biofuel and energy policy recommendations. Renew Sust Energ Rev 61:41–52CrossRefGoogle Scholar
  28. Jørgensen C, Flyvbjerg J, Arvin E, Jensen BK (1995) Stoichiometry and kinetics of microbial toluene biodegradation under denitrifying conditions. Biodegradation 6:147–156CrossRefGoogle Scholar
  29. Kim DJ, Park MR, Lim DS, Choi JW (2013) Impact of nitrate dose on toluene degradation under denitrifying condition. Appl Biochem Biotechnol 170:248–256CrossRefGoogle Scholar
  30. Laboratory U S (1954) Diagnosis and improvement of saline and alkali soils Washington, D.C., US Dept. of AgricultureGoogle Scholar
  31. Langenhoff AAM, Zehnder AJB, Schraa G (1996) Behaviour of toluene, benzene, and naphthalene under anaerobic conditions in sediment columns. Biodegradation 7:267–274CrossRefGoogle Scholar
  32. Lee JS, Ray RI, Little BJ (2010) An assessment of alternative diesel fuels: microbiological contamination and corrosion under storage conditions. Biofouling 26:623–635CrossRefGoogle Scholar
  33. Lisiecki P, Chrzanowski Ł, Szulc A, Ławniczak Ł, Białas W, Dziadas M, Owsianiak M, Jacek S, Cyplik P, Marecik R, Jeleń H, Heipieper HJ (2014) Biodegradation of diesel/biodiesel blends in saturated sand microcosms. Fuel 116:321–327CrossRefGoogle Scholar
  34. Liu CM, Wu SY (2016) From biomass waste to biofuels and biomaterial building blocks. Renew Energy 96:1056–1062CrossRefGoogle Scholar
  35. Magalhaes C, Joye SB, Moreira RM, Wiebe WJ, Bordalo AA (2005) Effect of salinity and inorganic nitrogen concentrations on nitrification and denitrification rates in intertidal sediments and rocky biofilms of the Douro River estuary, Portugal. Water Res 39:1783–1794CrossRefGoogle Scholar
  36. Meneghetti LR, Thomé A, Schnaid F, Prietto PD, Cavelhão G (2012) Natural attenuation and biostimulation of biodiesel contaminated soils from southern Brazil with different particle sizes. J Environ Sci Eng 2B:155–162Google Scholar
  37. Meyer DD, Beker SA, Bücker F, Peralba MCR, Frazzon APG, Osti JF, Andreazza R, Camargo FAO, Bento FM (2014) Bioremediation strategies for diesel and biodiesel in oxisol from southern Brazil. Int Biodeterior Biodegrad 95:356–363CrossRefGoogle Scholar
  38. Milano J, Ong HC, Masjuki HH, Chong WT, Lam MK, Loh PK, Vellayan V (2016) Microalgae biofuels as an alternative to fossil fuel for power generation. Renew Sust Energ Rev 58:180–197CrossRefGoogle Scholar
  39. Mukumbuta I, Uchida Y, Hatano R (2018) Evaluating the effect of liming on N2O fluxes from denitrification in an Andosol using the acetylene inhibition and 15N isotope tracer methods. Biol Fertil Soils 54:71–81CrossRefGoogle Scholar
  40. Müller JB, Ramos DT, Larose C, Fernandes M, Lazzarin HS, Vogel TM, Corseuil HX (2017) Combined iron and sulfate reduction biostimulation as a novel approach to enhance BTEX and PAH source-zone biodegradation in biodiesel blend-contaminated groundwater. J Hazard Mater 326:229–236CrossRefGoogle Scholar
  41. Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73:127–141CrossRefGoogle Scholar
  42. Nakatsu CH (2007) Soil microbial community analysis using denaturing gradient gel electrophoresis. Soil Sci Soc Am J 71:562–571CrossRefGoogle Scholar
  43. Nalgundwar A, Paul B, Sharma SK (2016) Comparison of performance and emissions characteristics of DI CI engine fueled with dual biodiesel blends of palm and jatropha. Fuel 173:172–179CrossRefGoogle Scholar
  44. Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amamm RI, Ludwig W, Backhaus H (1996) Sequence heterogeneities of genes encoding 16 rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643CrossRefGoogle Scholar
  45. Pasqualino JC, Montane D, Salvado J (2006) Synergic effects of biodiesel in the biodegradability of fossil-derived fuels. Biomass Bioenergy 30:874–879CrossRefGoogle Scholar
  46. Pires ACC, Cleary DFR, Almeida A, Cunha Â, Dealtry S, Mendonça-Hagler LCS, Smalla K, Gomes NCM (2012) Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples. Appl Environ Microbiol 78:5520–5528CrossRefGoogle Scholar
  47. Portugal F, Dias JM, Ribeiro H, Magalhães C, Mucha AP, Danko AS (2017) Anaerobic biodegradation of ethylic and methylic biodiesel and their impact on benzene degradation. Clean - Soil Air Water 45:1600264CrossRefGoogle Scholar
  48. Ramos DT, Silva MLB, Chiaranda HS, Alvarez PJJ, Corseuil HX (2012) Biostimulation of anaerobic BTEX biodegradation under fermentative methanogenic conditions at source-zone groundwater contaminated with a biodiesel blend (B20). Biodegradation 24:333–341CrossRefGoogle Scholar
  49. Ramos DT, da Silva MLB, Nossa CW, Alvarez PJ, Corseuil HX (2014) Assessment of microbial communities associated with fermentative–methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20). Biodegradation 25:681–691CrossRefGoogle Scholar
  50. Restrepo-Flórez JM, Bassi A, Rehmann L, Thompson MR (2013) Effect of biodiesel addition on microbial community structure in a simulated fuel storage system. Bioresour Technol 147:456–463CrossRefGoogle Scholar
  51. Ribeiro H, Almeida CMR, Magalhães C, Bordalo AA, Mucha AP (2015) Salt marsh sediment characteristics as key regulators on the efficiency of hydrocarbons bioremediation by Juncus maritimus rhizospheric bacterial community. Environ Sci Pollut Res 22:450–462CrossRefGoogle Scholar
  52. Ribeiro H, Mucha AP, Azevedo I, Salgado P, Teixeira C, Almeida CMR, Joye SB, Magalhães C (2016) Differential effects of crude oil on denitrification and anammox, and the impact on N2O production. Environ Pollut 216:391–399CrossRefGoogle Scholar
  53. Ribeiro H, de Sousa T, Santos J, Sousa AGG, Teixeira C, Salgado P, Monteiro MM, Mucha AP, Almeida CMR, Torgo L, Magalhães C (2018) Potential of dissimilatory nitrate reduction pathways in polycyclic aromatic hydrocarbon degradation. Chemosphere 199:54–67CrossRefGoogle Scholar
  54. Sajjadi B, Raman AAA, Arandiyan H (2016) A comprehensive review on properties of edible and non-edible vegetable oil-based biodiesel: composition, specifications and prediction models. Renew Sust Energ Rev 63:62–92CrossRefGoogle Scholar
  55. Schaefer CE, Yang X, Pelz O, Tsao DT, Streger SH, Steffan RJ (2010) Anaerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials. Chemosphere 81:1111–1117CrossRefGoogle Scholar
  56. Schambeck CM, Ramos DT, Chiaranda HS, Mezzari MP, Fernandes M, Corseuil HX (2015) Influência do biodiesel de soja na biodegradação anaeróbia do benzeno e tolueno. Engenharia Sanitaria e Ambiental 20:315–321CrossRefGoogle Scholar
  57. Schleicher T, Werkmeister R, Meyer-Pittroff WR (2009) Microbiological stability of biodiesel-diesel-mixtures. Bioresour Technol 100:724–730CrossRefGoogle Scholar
  58. Seitzinger SP, Nielsen LP, Caffrey J, Christensen PB (1993) Denitrification measurements in aquatic sediments: a comparison of three methods. Biogeochemistry 23:147–167CrossRefGoogle Scholar
  59. Sendzikiene E, Makareviciene V, Janulis P, Makareviciute D (2007) Biodegradability of biodiesel fuel of animal and vegetable origin. Eur J Lipid Sci Technol 109:493–497CrossRefGoogle Scholar
  60. Sgouridis F, Stott A, Ullah S (2016) Application of the 15N gas-flux method for measuring in situ N2 and N2O fluxes due to denitrification in natural and semi-natural terrestrial ecosystems and comparison with the acetylene inhibition technique. Biogeosciences 13:1821–1835CrossRefGoogle Scholar
  61. Sikkema J, de Bont JA, Poolman B (1995) Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59:201–222Google Scholar
  62. Sørensen G, Pedersen DV, Nørgaard AK, Sørensen KB, Nygaard SD (2011) Microbial growth studies in biodiesel blends. Bioresour Technol 102:5259–5264CrossRefGoogle Scholar
  63. Soriano AU, Martins LF, Ventura ESA, de Landa FHTG, Valoni ÉA, Faria FRD et al (2015) Microbiological aspects of biodiesel and biodiesel/diesel blends biodeterioration. Int Biodeterior Biodegrad 99:102–114CrossRefGoogle Scholar
  64. Stams AJM, Dijk JBV, Dijkema C, Plugge CM (1993) Growth of syntrophic propionate-oxidizing bacteria with fumarate in the absence of methanogenic bacteria. Appl Environ Microbiol 59:1114–1119Google Scholar
  65. Stolz J, Follis P, Floro RG (1995) Aerobic and Anaerobic biodegradation of the methyl esterified fatty acids of soy diesel in freshwater and soil environments. Accessed 17 Aug 2017
  66. Sydow M, Owsianiak M, Szczepaniak Z, Framski G, Smets BF, Ławniczak Ł, Lisiecki P, Szulc A, Cyplik P, Chrzanowski Ł (2016) Evaluating robustness of a diesel-degrading bacterial consortium isolated from contaminated soil. New Biotechnol 33:852–859CrossRefGoogle Scholar
  67. Thomas AO, Leahy MC, Smith JW, Spence MJ (2017) Natural attenuation of fatty acid methyl esters (FAME) in soil and groundwater. Q J Eng Geol Hydrogeol 50:301–317CrossRefGoogle Scholar
  68. Torsvik V, Øvreås L (2002) Microbial diversity and function in soil: from genes to ecosystems. Curr Opin Microbiol 5:240–245CrossRefGoogle Scholar
  69. Wu S, Yassine MH, Suidan MT, Venosa AD (2015) Anaerobic biodegradation of soybean biodiesel and diesel blends under methanogenic conditions. Water Res 87:395–402CrossRefGoogle Scholar
  70. Zedelius J, Rabus R, Grundmann O, Werner I, Brodkorb D, Schreiber F, Ehrenreich P, Behrends A, Wilkes H, Kube M, Reinhardt R, Widdel F (2011) Alkane degradation under anoxic conditions by a nitrate-reducing bacterium with possible involvement of the electron acceptor in substrate activation. Environ Microbiol Rep 3:125–135CrossRefGoogle Scholar
  71. Zhang L, Xu Z (2008) Assessing bacterial diversity in soil. J Soils Sediments 8:379–388CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Centre of Marine and Environmental Research (CIIMAR)University of PortoMatosinhosPortugal
  2. 2.Faculty of SciencesUniversity of PortoPortoPortugal
  3. 3.Centre for Natural Resources and the Environment (CERENA), Department of Mining EngineeringUniversity of Porto (FEUP)PortoPortugal
  4. 4.Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE), Department of Metallurgical and Materials EngineeringUniversity of PortoPortoPortugal

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