The Citrate Metabolism in Homo- and Heterofermentative LAB: A Selective Means of Becoming Dominant over Other Microorganisms in Complex Ecosystems


The citrate metabolism has been extensively studied in lactic acid bacteria (LAB) for its aroma compound production. Among the 4-carbon (C4) by-products obtained from citrate fermentation, diacetyl is one of the better known products for its contribution to the buttery aroma of dairy products. A lot of documents deal with ways to improve diacetyl concentration in food matrices. Apart from these organoleptic advantages, in a microbial ecosystem, the citrate metabolism gives selective advantages to citrate positive microorganisms. Citrate metabolism allows the LAB to use another carbon source for their growth, withstand acidic conditions and generate a “proton motive force” (PMF). Moreover, the citrate/glucid co-metabolism leads to the fast release of organic compounds known for having bacteriostatic effects. Under specific conditions, the Cpathway liberates diacetyl which is bacteriostatic. In this review we first describe the citrate metabolism and the enzymes involved in the two homo- and heterofermentative LABLc diacetylactisandLeuconostocspp. Moreover, the way to shift the metabolic pathway toward the production of aromatic compounds is discussed for both of these fermentative types of bacteria. Finally, the selective advantages of citrate metabolism for LAB in complex microbial ecosystems are delineated.

Share and Cite:

Laëtitia, G. , Pascal, D. and Yann, D. (2014) The Citrate Metabolism in Homo- and Heterofermentative LAB: A Selective Means of Becoming Dominant over Other Microorganisms in Complex Ecosystems. Food and Nutrition Sciences, 5, 953-969. doi: 10.4236/fns.2014.510106.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] García-Quintáns, N.G., Repizo, G., Magni, C., López, P., Mayo, B. and Pérez-Martínez, G. (2008) Citrate Metabolism and Aroma Compound Production in Lactic acid Bacteria. In: Mayo B., López P., Pérez-Martínez G., Eds., Molecular Aspects of Lactic acid Bacteria for Traditional and New Applications, Research Signpost, Kerala, 65-88.
[2] Bott, M. (1997) Anaerobic Citrate Metabolism and Its Regulation in Enterobacteria. Archives of Microbiology, 167, 78-88.
[3] Hugenholtz, J. (1993) Citrate Metabolism in Lactic Acid Bacteria. FEMS Microbiology Reviews, 12, 165-178.
[4] Beuvier, E. and Buchin, S. (2004) Raw Milk Cheeses. In: Fox, P. McSweeney, P. Cogan, T. and Guinee T., Eds., Cheese: Chemistry, Physics and Microbiology, Academic Press, London, 319-345.
[5] Bartowsky, E.J. and Henschke, P.A. (2004) The “Buttery” Attribute of Wine—Diacetyl—Desirability, Spoilage and Beyond. International Journal of Food Microbiology, 96, 235-252.
[6] Cabral, M.E., Mukdsi, M.C.A., Medina De Figueroa, R.B. and González, S.N. (2007) Citrate Metabolism by Enterococcus faecium and Enterococcus durans Isolated from Goat’s and Ewe’s Milk: Influence of Glucose and Lactose. Canadian Journal of Microbiology, 53, 607-615.
[7] De Vos, W.M. and Hugenholtz, J. (2004) Engineering Metabolic Highways in Lactococci and Other Lactic Acid Bacteria. Trends in Biotechnology, 22, 72-79.
[8] Hemme, D. and Foucaud-Scheunemann, C. (2004) Leuconostoc, Characteristics, Use in Dairy Technology and Prospects in Functional Foods. International Dairy Journal, 14, 467-494.
[9] Minervini, F., et al. (2010) Robustness of Lactobacillus plantarum Starters during Daily Propagation of Wheat Flour Sourdough Type I. Food Microbiology, 27, 897-908.
[10] Hugenholtz, J. and Kleerebezem, M. (1999) Metabolic Engineering of Lactic Acid Bacteria: Overview of the Approaches and Results of Pathway Rerouting Involved in Food Fermentations. Current Opinion in Biotechnology, 10, 492-497.
[11] Kleerebezem, M., Hols, P. and Hugenholtz, J. (2000) Lactic Acid Bacteria as a Cell Factory: Rerouting of Carbon Metabolism in Lactococcus lactis by Metabolic Engineering. Enzyme and Microbial Technology, 26, 840-848.
[12] Bourel, G., Henini, S., Krantar, K., Oraby, M., Diviès, C. and Garmyn, D (2001) Sugar Citrate Cometabolism in Leuconostoc mesenteroides. Le Lait, 81, 75-82.
[13] Hoefnagel, M.H.N., et al. (2002) Metabolic Engineering of Lactic Acid Bacteria, the Combined Approach: Kinetic Modelling, Metabolic Control and Experimental Analysis. Microbiology, 148, 1003-1013.
[14] Hugenholtz, J., et al. (2000) Lactococcus lactis as a Cell Factory for High-Level Diacetyl Production. Applied and Environmental Microbiology, 66, 4112-4114.
[15] Lopez de Felipe, F., Kleerebezem, M., de Vos, W.M. and Hugenholtz, J. (1998) Cofactor Engineering: A Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase. Journal of Bacteriology, 180, 3804-3808.
[16] Magni, C., De Mendoza, D., Konings, W.N. and Lolkema, J.S. (1999) Mechanism of Citrate Metabolism in Lactococcus lactis: Resistance against Lactate Toxicity at Low pH. Journal of Bacteriology, 181, 1451-1457.
[17] Hache, C., Cachon, R., Waché, Y., Belguendouz, T., Riondet, C., Deraedt, A. and Diviès, C. (1999) Influence of LactoseCitrate Co-Metabolism on the Differences of Growth and Energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides and Leuconostoc mesenteroides ssp. cremoris. Systematic and Applied Microbiology, 22, 507-513.
[18] Sánchez, C., Neves, A.R., Cavalheiro, J., Dos Santos, M.M., García-Quintáns, N., López, P. and Santos, H. (2008) Contribution of Citrate Metabolism to the Growth of Lactococcus lactis CRL264 at Low pH. Applied and Environmental Microbiology, 74, 1136-1144.
[19] Konings, W.N. (2002) The Cell Membrane and the Struggle for Life of Lactic Acid Bacteria. Antonie van Leeuwenhoek, 82, 3-27.
[20] Marty-Teysset, C., Lolkema, J.S., Schmitt, P., Diviès, C. and Konings, W.N. (1996) The Citrate Metabolic Pathway in Leuconostoc mesenteroides: Expression, Amino Acid Synthesis, and α-Ketocarboxylate Transport. Journal of Bacteriology, 178, 6209-6215.
[21] Martín, M., Magni, C., López, P. and de Mendoza, D. (2000) Transcriptional Control of the Citrate-Inducible citMCDEFGRP Operon, Encoding Genes Involved in Citrate Fermentation in Leuconostoc paramesenteroides. Journal of Bacteriology, 182, 3904-3912.
[22] García-Quintáns, N.G., Magni, C., De Mendoza, D. and López, P. (1998) The Citrate Transport System of Lactococcus lactis subsp. lactis biovar. diacetylactis Is Induced by Acid Stress. Applied and Environmental Microbiology, 64, 850857.
[23] Martín, M.G., Sender, P.D., Peiru, S., De Mendoza, D. and Magni, C. (2004) Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar. diacetylactis CRL264. Journal of Bacteriology, 186, 5649-5660.
[24] Cogan, T.M. (1981) Constitutive Nature of the Enzymes of Citrate Metabolism in Streptococcus lactis subsp. diacetylactis. Journal of Dairy Research, 48, 489-495.
[25] García-Quintáns, N.G., Repizo, G., Martin, M., Magni, C. and Lopez, P. (2008) Activation of the Diacetyl/Acetoin Pathway in Lactococcus lactis subsp. lactis biovar. diacetylactis CRL264 by Acidic Growth. Applied and Environmental Microbiology, 74, 1988-1996.
[26] Drici, H., Gilbert, C., Kihal, M. and Atlan, D. (2010) Atypical Citrate-Fermenting Lactococcus lactis Strains Isolated from Dromedary’s Milk. Journal of Applied Microbiology, 108, 647-657.
[27] Kaneko, T., Suzuki, H. and Takahashi, T. (1987) The Effects of Metal Ions on Diacetyl Production by Streptococcus lactis subsp. diacetylactis 3022. Agricultural and Biological Chemistry, 51, 2315-2320.
[28] Mellerick, D. and Cogan, T.M. (1981) Induction of Some Enzymes of Citrate Metabolism in Leuconostoc lactis and Other Heterofermentative Lactic Acid Bacteria. Journal of Dairy Research, 48, 497-502.
[29] Ramos, A., Jordan, K.N., Cogan, T.M. and Santos, H. (1994) 13C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis. Applied and Environmental Microbiology, 60, 1739-1748.
[30] Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, S.D. and Sorokin, A. (2001) The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp. lactis IL1403. Genome Research, 11, 731-753.
[31] Dartois, V., Phalip, V., Schmitt, P. and Diviès, C. (1995) Purification, Properties and DNA Sequence of the D-Lactate Dehydrogenase from Leuconostoc mesenteroides subsp. cremoris. Research in Microbiology, 146, 291-302.
[32] Cocaign-Bousquet, M., Garrigues, C., Loubiere, P. and Lindley, N.D. (1996) Physiology of Pyruvate Metabolism in Lactococcus lactis. Antonie van Leeuwenhoek, 70, 253-267.
[33] Fitzgerald, R.J., Doonan, S., McKay, L.L. and Cogan, T.M. (1992) Intracellular pH and the Role of D-Lactate Dehydrogenase in the Production of Metabolic End Products by Leuconostoc lactis. Journal of Dairy Research, 59, 359-367.
[34] Garrigues, C., Loubiere, P., Lindley, N.D. and Cocaign-Bousquet, M. (1997) Control of the Shift from Homolactic Acid to Mixed-Acid Fermentation in Lactococcus lactis: Predominant Role of the NADH/NAD+ Ratio. Journal of Bacteriology, 179, 5282-5287.
[35] Snoep, J.L., Teixeira de Mattos, M.J., Starrenburg, M.J.C. and Hugenholtz, H. (1992) Isolation, Characterization, and Physiological Role of the Pyruvate Dehydrogenase Complex and Alpha-Acetolactate Synthase of Lactococcus lactis subsp. lactis biovar. diacetylactis. Journal of Bacteriology, 174, 4838-4841.
[36] Henriksen, C.M. and Nilsson, D. (2001) Redirection of Pyruvate Catabolism in Lactococcus lactis by Selection of Mutants with Additional Growth Requirements. Applied Microbiology and Biotechnology, 56, 767-775.
[37] Melchiorsen, C.R., Jokumsen, K.V., Villadsen, J., Johnsen, M.G., Israelsen, H. and Arnau, J. (2000) Synthesis and Posttranslational Regulation of Pyruvate Formate-Lyase in Lactococcus lactis. Journal of Bacteriology, 182, 47834788.
[38] Condon, S. (1987) Responses of Lactic Acid Bacteria to Oxygen. FEMS Microbiology Letters, 46, 269-280.
[39] Thomas, T.D., Turner, K.W. and Crow, V.L. (1980) Galactose Fermentation by Streptococcus lactis and Streptococcus cremoris: Pathways, Products, and Regulation. Journal of Bacteriology, 144, 672-682.
[40] Hugenholtz, J. and Starrenburg, M.J.C. (1992) Diacetyl Production by Different Strains of Lactococcus lactis subsp. lactis var. diacetylactis and Leuconostoc spp. Applied Microbiology and Biotechnology, 38, 17-22.
[41] Pudlik, A.M. and Lolkema, J.S. (2011) Citrate Uptake in Exchange with Intermediates in the Citrate Metabolic Pathway in Lactococcus lactis IL1403. Journal of Bacteriology, 193, 706-714.
[42] Phalip, V., Schmitt, P. and Divies, C. (1995) Purification and Characterization of the Catabolic α-Acetolactate Synthase from Leuconostoc mesenteroides subsp. cremoris. Current Microbiology, 31, 316-321.
[43] Boumerdassi, H., Desmazeaud, M., Monnet, C., Boquien, C.Y. and Corrieu, G. (1996) Improvement of Diacetyl Production by Lactococcus lactis ssp. lactis CNRZ 483 through Oxygen Control. Journal of Dairy Science, 79, 775-781.
[44] Cogan, J.F., Walsh, D. and Condon, S. (1989) Impact of Aeration on the Metabolic End-Products Formed from Glucose and Galactose by Streptococcus lactis. Journal of Applied Microbiology, 66, 77-84.
[45] Monnet, C., Phalip, V., Schmitt, P. and Divies, C. (1994) Comparison of α-Acetolactate Synthase and α-Acetolactate Decarboxylase in Lactococcus spp. and Leuconostoc spp. Biotechnology Letters, 16, 257-262.
[46] Cogan, T.M., Fitzgerald, R.J. and Doonan, S. (1984) Acetolactate Synthase of Leuconostoc lactis and Its Regulation of Acetoin Production. Journal of Dairy Research, 51, 597-604.
[47] McCourt, J.A. and Duggleby, R.G. (2006) Acetohydroxyacid Synthase and Its Role in the Biosynthetic Pathway for Branched-Chain Amino Acids. Amino Acids, 31, 173-210.
[48] Cocaign-Bousquet, M., Garrigues, C., Novak, L., Lindley, N.D. and Loublere, P. (1995) Rational Development of a Simple Synthetic Medium for the Sustained Growth of Lactococcus lactis. Journal of Applied Microbiology, 79, 108116.
[49] Godon, J.J., Delorme, C., Bardowski, J., Chopin, M.C., Ehrlich, S.D. and Renault, P. (1993) Gene Inactivation in Lactococcus lactis: Branched-Chain Amino Acid Biosynthesis. Journal of Bacteriology, 175, 4383-4390.
[50] Goupil-Feuillerat, N., Cocaign-Bousquet, M., Godon, J.J., Ehrlich, S.D. and Renault, P. (1997) Dual Role of AlphaAcetolactate Decarboxylase in Lactococcus lactis subsp. lactis. Journal of Bacteriology, 179, 6285-6293.
[51] Aymes, F., Monnet, C. and Corrieu, G. (1999) Effect of α-Acetolactate Decarboxylase Inactivation on α-Acetolactate and Diacetyl Production by Lactococcus lactis subsp. lactis biovar. diacetylactis. Journal of Bioscience and Bioengineering, 87, 87-92.
[52] Park, S.H., Xing, R. and Whitman, W.B. (1995) Nonenzymatic Acetolactate Oxidation to Diacetyl by Flavin, Nicotinamide and Quinone Coenzymes. Biochimica et Biophysica Acta, 1245, 366-370.
[53] Rondags, E., Stien, G., Germain, P. and Marc, I. (1996) Kinetic Study of the Chemical Reactivity of α-Acetolactate as a Function of pH in Water, and in Fresh and Fermented Culture Media Used for Lactococcus lactis spp. lactis biovar. diacetylactis Cultivation. Biotechnology Letters, 18, 747-752.
[54] Crow, V.L. (1990) Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation. Applied and Environmental Microbiology, 56, 1656-1665.
[55] Rattray, F.P., Walfridsson, M. and Nilsson, D. (2000) Purification and Characterization of a Diacetyl Reductase from Leuconostoc pseudomesenteroides. International Dairy Journal, 10, 781-789.
[56] Bassit, N., Boquien, C.Y., Picque, D. and Corrieu, G. (1993) Effect of Initial Oxygen Concentration on Diacetyl and Acetoin Production by Lactococcus lactis subsp. lactis biovar. diacetylactis. Applied and Environmental Microbiology, 59, 1893-1897.
[57] Gilliland, S.E., Anna, E.D. and Speck, M.L. (1970) Concentrated Cultures of Leuconostoc citrovorum. Applied Microbiology, 19, 890-893.
[58] Pack, M.Y., Vedamuthu, E.R., Sandine, W.E., Elliker, P.R. and Leesment, H. (1968) Effect of Temperature on Growth and Diacetyl Production by Aroma Bacteria in Singleand Mixed-Strain Lactic Cultures. Journal of Dairy Science, 51, 339-344.
[59] Petit, C., Vilchez, F. and Marczak, R. (1989) Influence of Citrate on the Diacetyl and Acetoin Production by Fully Grown Cells of Streptococcus lactis subsp. diacetylactis. Current Microbiology, 19, 319-323.
[60] Rattray, F.P., Myling-Petersen, D., Larsen, D. and Nilsson, D. (2003) Plasmid-Encoded Diacetyl (Acetoin) Reductase in Leuconostoc pseudomesenteroides. Applied and Environmental Microbiology, 69, 304-311.
[61] Pedersen, M.B., Gaudu, P., Lechardeur, D., Petit, M.A. and Gruss, A. (2012) Aerobic Respiration Metabolism in Lactic Acid Bacteria and Uses in Biotechnology. Annual Review of Food Science and Technology, 3, 37-58.
[62] Marty-Teysset, C., Lolkema, J.S., Schmitt, P., Divies, C. and Konings, W.N. (1995) Membrane Potential-Generating Transport of Citrate and Malate Catalyzed by CitP of Leuconostoc mesenteroides. Journal of Biological Chemistry, 270, 25370-25376.
[63] Poolman, B., Bosman, B., Kiers, J. and Konings, W. (1987) Control of Glycolysis by Glyceraldehydes-3-Phosphate Dehydrogenase in Streptococcus cremoris and Streptococcus lactis. Journal of Bacteriology, 169, 5887-5890.
[64] Konings, W.N., Lolkema, J.S. and Poolman, B. (1995) The Generation of Metabolic Energy by Solute Transport. Archives of Microbiology, 164, 235-242.
[65] Belguendouz, T., Cachon, R. and Diviès, C. (1997) pH Homeostasis and Citric Acid Utilization: Differences between Leuconostoc mesenteroides and Lactococcus lactis. Current Microbiology, 35, 233-236.
[66] Cogan, T.M. (1987) Co-Metabolism of Citrate and Glucose by Leuconostoc spp.: Effects on Growth, Substrates and Products. Journal of Applied Microbiology, 63, 551-558.
[67] Kimoto, H., Nomura, M. and Suzuki, I. (1999) Growth Energetics of Lactococcus lactis subsp. lactis biovar diacetylactis in Cometabolism of Citrate and Glucose. International Dairy Journal, 9, 857-863.
[68] Starrenburg, M. and Hugenholtz, J. (1991) Citrate Fermentation by Lactococcus and Leuconostoc spp. Applied and Environmental Microbiology, 57, 3535-3540.
[69] Schmitt, P. and Divies, C. (1992) Effect of Varying Citrate Levels on C4 Compound Formation and on Enzyme Levels of Leuconostoc mesenteroides subsp. cremoris Grown in Continuous Culture. Applied Microbiology and Biotechnology, 37, 426-430.
[70] Moon, N.J. (1983) Inhibition of the Growth of Acid Tolerant Yeasts by Acetate, Lactate and Propionate and Their Synergistic Mixtures. Journal of Applied Bacteriology, 55, 453-460.
[71] Shelef, L.A. (1994) Antimicrobial Effects of Lactates: A Review. Journal of Food Protection, 57, 445-450.
[72] Rossland, E., Langsrud, T., Granum, P.E. and Sorhaug, T. (2005) Production of Antimicrobial Metabolites by Strains of Lactobacillus or Lactococcus Co-Cultured with Bacillus cereus in Milk. International Journal of Food Microbiology, 98, 193-200.
[73] De Vuyst, L. and Vandamme, E.J. (1994) Antimicrobial Potential of Lactic Acid Bacteria. In: De Vuyst, L. and Vandamme, E.J., Eds, Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and Applications, Blackie Academic and Professional, London, 91-142.
[74] Jay, J.M. (1982) Antimicrobial Properties of Diacetyl. Applied and Environmental Microbiology, 44, 525-532.
[75] Güler, Z. and Gürsoy-Balci, A.C. (2011) Evaluation of Volatile Compounds and Free Fatty Acids in Set Types Yogurts Made of Ewes’, Goats’ Milk and Their Mixture Using Two Different Commercial Starter Cultures during Refrigerated Storage. Food Chemistry, 127, 1065-1071.
[76] Dalmasso, M., Prestoz, S., Rigobello, V. and Demarigny, Y. (2008) Behavior of Lactococcus lactis subsp. lactis biovar. diacetylactis in a Four Lactococcus Strain Starter during Successive Milk Cultures. Food Science and Technology International, 14, 469-477.
[77] Piard, J. and Desmazeaud, M. (1991) Inhibiting Factors Produced by Lactic Acid Bacteria. 1. Oxygen Metabolites and Catabolism End-Products. Lait, 71, 525-541.
[78] Veringa, R.A., Verburg, E.H. and Stadhouders, J. (1984) Determination of Diacetyl in Dairy Products Containing AlphaAcetolactic Acid. Netherlands Milk and Dairy Journal, 38, 251-263.
[79] Xanthopoulos, V., Picque, D., Bassit, N., Boquien, C.Y. and Corrieu, G. (1994) Methods for the Determination of Aroma Compounds in Dairy Products: A Comparative Study. Journal of Dairy Research, 61, 289-297.
[80] Macciola, V., Candela, G. and De Leonardis, A. (2008) Rapid Gas-Chromatographic Method for the Determination of Diacetyl in Milk, Fermented Milk and Butter. Food Control, 19, 873-878.
[81] Jimeno, J., Lazaro, M.J. and Sollberger, H. (1995) Antagonistic Interactions between Propionic Acid Bacteria and Non-Starter Lactic Acid Bacteria. Le Lait, 75, 401-413.
[82] Demarigny, Y. (2012) Fermented Food Products Made with Vegetable Materials from Tropical and Warm Countries: Microbial and Technological Considerations. International Journal of Food Science & Technology, 47, 2469-2476.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.