Document Type: Research Paper


1 Laboratory of Kinetic and Molecular Dynamic, Institute of Chemistry, Federal University of Bahia, Campus de Ondina, Salvador, Bahia, 40170-290, Brazil

2 Laboratory of Microbiology, Wageningen University, Dreijenplein 10, Wageningen, 6703 HB, The Netherlands

3 Laboratory of Biotechnology and Ecology of Microorganisms, Department of Biointeraction, Federal University of Bahia, Bahia, Brazil.

4 Laboratory of Kinetic and Molecular Dynamic, Department of Chemistry, Federal University of Bahia, Bahia, Brazil.


Crude glycerol (CG) is an abundantly available and cheap by-product from biodiesel production. Value-added applications for CG are highly wanted by industry and several processes such as the use of CG for enhanced oil recovery have been proposed. The aim of this study was to evaluate the sulfide production of sulfate-reducing bacteria (SRB) indigenous to oil reservoirs using CG as substrate. The samples of CG were obtained from a biodiesel production plant, processing castor beans, soybeans, cotton, and waste oils and fats. Growth tests were performed in Postgate medium, with different types and concentrations of CG, and a mixed inoculum of SRB isolated from the produced water of a mature oil well of Bahia (Brazil). The experiment was monitored by measuring the concentration of sulfide using a colorimetric method. The results showed that SRB grew and produced more than 250 ppm sulfide at CG concentrations of 2%. However, at CG concentrations of 3% or higher, the biogenic production of sulfide was reduced. The study demonstrates that CG will likely stimulate SRB in oil fields, whenever CG is present at lower concentrations. Maintaining CG concentrations inhibitive to SRB will not certainly be achievable throughout oil reservoirs. Dosing CG to oil fields may lead to problems associated with souring in longer terms. The utilization of CG by SRB could in turn be interesting for other biotechnological processes, e.g. metal recovery processes based on precipitation with biologically formed sulfide.


      [1]     Hansen C. F., Hernandez A., Mullan B. P., Moore K., et al., “A Chemical Analysis of Samples of Crude Glycerol from the Production of Biodiesel in Australia, and the Effects of Feeding Crude Glycerol to Growing-finishing Pigs on Performance, Plasma Metabolites and Meat Quality at Slaughter,” Anim. Prod. Sci., 2009, 49, 154-161.##

      [2]     Hazimah A. H., Ooi T. L., and Salmiah A., “Recovery of Glycerol and Diglycerol from Glycerol Pitch,” J. Oil Palm Res., 2003, 15, 1-5.##

      [3]     Sandun F., Sushil A., Kiran K., and Ranjitha B., “Glycerol Based Automotive Fuels from Future Biorefineries,” Fuel, 2007, 86, 2806-2809.##

      [4]     Yazdani S. S. and Gonzalez R., “Anaerobic Fermentation of Glycerol: a Path to Economic Viability for the Biofuels Industry,” Curr. Opin. Biotechnol., 2007, 18, 213-219.##

      [5]     Johnson D. T. and Taconi K. A., “The Glycerin Glut: Options for the Value-added Conversion of Crude Glycerol Resulting from Biodiesel Production,” Environ. Progress, 2007, 2, 338-348.##

      [6]     Silva G. P., Mack M., and Contiero J., “Glycerol: A Promising and Abundant Carbon Source for Industrial Microbiology,” Biotechnol. Adv., 2009, 27, 30-39.##

      [7]     Quintella C. M., Borges S. M. S., Almeida P. M. M., Musse A. P. S., et al., “Secondary Recovery of Crude Oil with Glycerin, Co-product of the Biodiesel Production,” Anais Rio Oil & Gas Expo. and Conference, Rio de Janeiro, 2006.##

      [8]     Muyzer G. and Stams A. J. M., “Ecology and Biotechnology of Sulphate-reducing Bacteria,” Nature Rev. Microbiol., 2008, 6, 441-454.##

      [9]     Postgate J. R., The Sulphate-reducing Bacteria, Cambridge: Cambridge University Press, 1984.##

    [10]    Hubert C., Nemati M., Jenneman G., and Voordouw G., “Corrosion Risk Associated with Microbial Souring Control Using Nitrate or Nitrite,” Appl. Microbiol. Biotechnol., 2005, 68, 272-282.##

    [11]    Coetser S. E. and Cloete T. E., “Biofouling and Biocorrosion in Industrial Water Systems,” Crit. Rev. Microbiol., 2005, 31, 213-232.##

    [12]    Estublier A., Dino R., Schinelli M. C., Barroux C., et al., “CO2 Injection inBuracica-long-term Performance Assessment,” Energy Procedia., 2011, 4, 4028-4035.##

    [13]    American Society of Testing and Material, Standard Test Method for Sulphate-reducing Bacteria in Water and Water-formed Deposits, West Conshohocken: American Society of Testing and Material Annual Book of Standards (ASTM, D4412), 2002.##

    [14]    American Public Health Association, Standard Methods for the Examination of Water and Wastewater (13th ed.), Washington: American Public Health Association, 1973.##

    [15]    Clesceri S., Greenberg A. E., and Eaton A. D., Standard Methods for the Examination of Water and Wastewater (20th ed.), Washington: American Public Health Association, American Water Works Association, Water Environmental Federation, 1998.##

    [16]    Daims H., Stöcker K., and Wagner M., Fluorescence In Situ Hybridization for the Detection of Prokaryotes, in Advanced Methods in Molecular Microbial Ecology, Osborn A. M., Smith C. J., ed. Abingdon: BIOS Scientific Publishers, 2005, 213-239.##

    [17]    Amann R. I., Devereux R., Key R., and Stahl D. A., “Molecular and Microscopic Identification of Sulfate-reducing Bacteria in Multispecies Biofilms,” Appl. Environ. Microbiol., 1992, 58, 614-623.##

    [18]    Manz W., Eisenbrecher M., Neu T. R., and Szewyk U., “Abundance and Spatial Organization of Gram-negative Sulfate Reducing Bacteria in Activated Sludge Investigated by In Situ Probing with Specific 16S rRNA Targeted Oligonucleotides,” FEMS Microbiol. Ecol., 1998, 25, 43-61.##

    [19]    Devereux R., Kane M. D., Winfrey J., and Stahl D. A., “Genus and Group-specific Hybridization Probes for Determinative and Environmental Studies of Sulfate Reducing Bacteria,” Syst. Appl. Microbiol., 1992, 15, 601-609.##

    [20]    Santos A. F. J., Batista L. L. F., Lima J. B. T., Almeida R. C. C., et al., “Evaluation of the Fluorescence in Situ Hybridization Technique for Detection of Eubacteria and Sulfate-reducing Bacteria from Samples of Water in Oil Fields,” Chem. Eng. Trans., 2010, 20, 139-144.##

    [21]    Gherna R. L., “Preservation,” in Manual of Methods for General Microbiology, Gerhard P., ed. Wshington: American Society for Microbiology, 1981, 208-217.##

    [22]    Francisco D. E., Mah R. A., and Rabin A. C., “Acridine Orange Epifluorescence Technique for Counting Bacteria in Natural Waters,” Trans. Am. Microsc. Soc., 1973, 92, 416-421.##

    [23]    Trüper H. G. and Schlegel H. G., “Sulfur Metabolism in Thiorhodeaceae. I. Quantitative Measurements on Growing Cells of Chromatium Okenii,” Antonie van Leeuwenhoek, 1964, 30, 225-238.##

    [24]    Bastos L. C. S. and Pereira P. A. P., “Influence of Heating Time and Metal Ions on the Amount of Free Fatty Acids and Formation Rates of Selected Carbonyl Compounds During the Thermal Oxidation of Canola Oil,” J. Agric. Food Chem., 2010, 58, 12777-12783.##

         [25]         Journal of Plastic,” Internet:, Nov. 9, 2010 [Nov. 9, 2010].##

    [26]    Feron V. J., Til H. P., de Vrijes F., Wouterson R. A., et al., “Aldehydes: Occurrence, Carcinogenicity Potential, Mechanism of Action and Risk Assessment,” Mutat. Res., 1991, 259, 363-385.##

    [27]    Ma T. H. and Harris M. M., “Review of the Genotoxicity of Formaldehyde,” Mutat. Res., 1988, 196, 37-59.##

    [28]    Fink T.R. and Worner P., “Pollution Prevention Opportunities in Oil and Gas Production, Drilling, and Exploration,” Internet: 03/02975.pdf, 1993 [Dec. 23, 2014].##

    [29]    Voordouw G., Armstrong S. M., Reimer M. F., Fouts B., et al., “Characterization of 16S rRNA Genes from Oil Field Microbial Communities Indicates the Presence of a Variety of Sulfate-reducing, Fermentative, and Sulfide-oxidizing Bacteria,” Appl. Environ. Microbiol., 1996, 62, 1623-1629.##

    [30]    Magot M., Basso O., Tardy-Jacquenod C., and Caumette P., “Desulfovibrio Bastinni sp. nov. and Desulfovibrio Gracilis sp. nov., Moderatety Halophilic, Sulfate-reducing Bacteria Isolated from Deep Subsurface Oilfield Water,” Int. J. Syst. Evol. Microbiol., 2004, 54, 1693-1697.##

    [31]    Miranda-Tello E., Fardeau M. L., Fernandéz L., Ramiréz F., et al., “Desulfovibrio Capillatus sp. nov., a Novel Sulfate-reducing Bacterium Isolated from an Oil Field Separator located in the Gulf of Mexico,” Anaerobe, 2003, 8, 97-103.##

    [32]    Stams A. J. M., Hansen T. A., and Skyring G. W., “Utilization of Amino Acids as Energy Substrates by Two Marine Desulfovibrio Strains,” FEMS Microbiol. Ecol., 1985, 31, 11-15.##

    [33]    Kremer D. R. and Hansen T. A., “Glycerol and Dihydroxyacetone Dissimilation in Desulfovibrio Strains,” Arch. Microbiol., 1987, 147, 249-256.

    [34]    Esnault G., Caumette P., and Garcia J. L., “Characterization of Desulfovibrio Giganteus sp. nov., a Sulfate-reducing Bacterium Isolated from a Brackish Coastal Lagoon,” Syst. Appl. Microbiol., 1988, 10, 147-151.##

    [35]    Finster K., Coates J. D., Liesack W., and Pfennig N., “Desulfuromonas Thiophila sp. nov., a New Obligately Sulfur-reducing Bacterium from Anoxic Freshwater Sediment,” Int. J. Syst. Bacteriol., 1997, 47, 754-758.##

    [36]    Alazard D., Joseph M., Battaglia-Brunet F., Cayol J. L., et al., “Desulfosporosinus Acidiphilus sp. nov.: a Moderately Acidophilic Sulfate-reducing Bacterium Isolated from Acid Mining Drainage Sediments,” Extremophiles, 2010, 14, 305-312.##

    [37]    Sánchez-Andrea I., Stams A. J., Amils R., and Sanz J. L., “Enrichment and Isolation of Acidophilic Sulfate-reducing Bacteria from Tinto River Sediments,” Environ. Microbiol. Reports., 2013, 5, 672-678.##

    [38]    Parshall G. W. and Ittel S. D., “Homogeneous Catalysis: the Application and Chemistry of Catalysis by Soluble Transition Metal Catalysts,” New York: John Willey and Sons, 1992.##

    [39]    Allison J. N., Goddard III W. A., “Oxidative Dehydrogenation of Methanol to Formaldehyde,” J. Cat., 1985, 92, 127-135.##

    [40]    Feijoo G., Soto M., Mendez R. E., and Lema J. R., “Sodium Inhibition in the Anaerobic Digestion Process: Antagonism and Adaptation Phenomena,” Enz. Microbial. Technol., 1995, 17, 180-188.##

    [41]    Bashir B. H. and Matin A., “Combined Effect of Calcium and Sodium on Potassium Toxicity in Anaerobic Treatment Processes,” Electron. J. Environ. Agric. Food Chem., 2004, 4, 670-676.##

    [42]    Chaves E. S., Ramos J. C., Fontana K. B., Modolon S., et al., “Simple and Fast Method for the Determination of Na and K in Raw Glycerin from Biodiesel Production by Flame Atomic Emission Spectrometry,” Br. J. Anal. Chem., 2010, 1, 24-59.##

    [43]    Jensen T., Kvist T., Mikkelsen M. J., Christensen P. V., et al., “Fermentation of Crude Glycerol from Biodiesel Production by Clostridium Pasteurianum,” J. Ind. Microbiol. Biotechnol., 2012, 39, 709-717.##

    [44]    Dabrock B., Bahl H., and Gottschalk G., “Parameters Affecting Solvent Production by Clostridium Pasteurianum,” Appl. Environ. Microbiol., 1992, 58, 1233-1239.##