Applied Microbiology and Biotechnology

, Volume 103, Issue 1, pp 201–209 | Cite as

Current strategies and future prospects for enhancing microbial production of citric acid

  • Wei Hu
  • Wen-jian Li
  • Hai-quan YangEmail author
  • Ji-hong ChenEmail author


Aspergillus niger and Yarrowia lipolytica are highly important in citric acid (CA) production. To further minimize the cost of CA bio-production using A. niger and Y. lipolytica, some strategies (e.g., metabolic engineering, efficient mutagenesis, and optimal fermentation strategies) were developed to enhance CA production and low-cost carbon sources were also utilized to decrease CA bio-production cost. In this review, we summarize the recent significant progresses in CA bio-production, including metabolic engineering, efficient mutagenesis and screening methods, optimal fermentation strategies, and use of low-cost carbon sources, and future prospects in this field are also discussed, which could help in the development of CA production industry.


Aspergillus Niger Yarrowia lipolytica Citric acid Production Strategies 


Funding information

This work was supported financially by the National Natural Science Foundation of China (No. 11605259), the western talents program of the Chinese Academy of Sciences (Y706030XB0), and the Open Project Program of the Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, China (KLCCB-KF201802).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not involve any studies with human participants performed by any of the authors.


  1. Angumeenal AR, Venkappayya D (2013) An overview of citric acid production. LWT Food Sci Technol 50(2):367–370Google Scholar
  2. Arslan NP, Aydogan MN, Taskin M (2016) Citric acid production from partly deproteinized whey under non-sterile culture conditions using immobilized cells of lactose positive and cold-adapted Yarrowia lipolytica B9. J Biotechnol 231:32–39PubMedGoogle Scholar
  3. Auta HS, Abidoye KT, Tahir H, Ibrahim AD, Aransiola SA (2014) Citric acid production by Aspergillus niger cultivated on parkia biglobosa fruit pulp. Int Scholarly Res Not 2014:762021PubMedCentralGoogle Scholar
  4. Barrington S, Kim JW (2008) Response surface optimization of medium components for citric acid production by Aspergillus niger NRRL 567 grown in peat moss. Bioresour Technol 99(2):368–377PubMedGoogle Scholar
  5. Barrington S, Kim JS, Wang L, Kim JW (2009) Optimization of citric acid production by Aspergillus niger NRRL 567 grown in a column bioreactor. Korean J Chem Eng 26(2):422–427Google Scholar
  6. Becker J, Lange A, Fabarius J, Wittmann C (2015) Top value platform chemicals: bio-based production of organic acids. Curr Opin Biotechnol 36:168–175PubMedGoogle Scholar
  7. Berovic M, Legisa M (2007) Citric acid production. Biotechnol Annu Rev 13:303–343PubMedGoogle Scholar
  8. Burk MJ, Van Dien S (2016) Biotechnology for chemical production: challenges and opportunities. Trends Biotechnol 34(3):187–190PubMedGoogle Scholar
  9. Cao S, Zhou X, Jin WB, Wang F, Tu RJ, Han SF, Chen HY, Chen C, Xie GJ, Ma F (2017) Improving of lipid productivity of the oleaginous microalgae Chlorella pyrenoidosa via atmospheric and room temperature plasma (ARTP). Bioresour Technol 244:1400–1406PubMedGoogle Scholar
  10. Cavallo E, Charreau H, Cerrutti P, Foresti ML (2017) Yarrowia lipolytica: a model yeast for citric acid production. FEMS Yeast Res 17(8):fox084Google Scholar
  11. Çelik G, Uçar FB, Akpinar O, Çorbaci C (2014) Production of citric and isocitric acid by Yarrowia lipolytica strains grown on different carbon sources. Turk J Biochem 39(3):285–290Google Scholar
  12. Darvishi F, Nahvi I, Zarkesh-Esfahani H, Momenbeik F (2009) Effect of plant oils upon lipase and citric acid production in Yarrowia lipolytica yeast. J Biomed Biotechnol 2009:562943PubMedPubMedCentralGoogle Scholar
  13. Dhillon GS, Brar SK, Verma M, Tyagi RD (2011) Recent advances in citric acid bio-production and recovery. Food Bioprocess Technol 4(4):505–529Google Scholar
  14. Drake JW, Charlesworth B, Charlesworth D, Crow JF (1998) Rates of spontaneous mutation. Genetics 148(4):1667–1686PubMedPubMedCentralGoogle Scholar
  15. Ferreira P, Mota M, Belo I (2015) Citric acid production by Yarrowia lipolytica from crude glycerol: influence of oxygen mass transfer rate (OTR). J Biotechnol 208:S48–S48Google Scholar
  16. Ferreira P, Lopes M, Mota M, Belo I (2016a) Oxygen mass transfer impact on citric acid production by Yarrowia lipolytica from crude glycerol. Biochem Eng J 110:35–42Google Scholar
  17. Ferreira P, Lopes M, Mota M, Belo I (2016b) Oxygen transfer rate and pH as major operating parameters of citric acid production from glycerol by Yarrowia lipolytica W29 and CBS 2073. Chem Pap 70(7):869–876Google Scholar
  18. Förster A, Jacobs K, Juretzek T, Mauersberger S, Barth G (2007) Overexpression of the ICL1 gene changes the product ratio of citric acid production by Yarrowia lipolytica. Appl Microbiol Biotechnol 77(4):861–869PubMedGoogle Scholar
  19. Fu GY, Lu Y, Chi Z, Liu GL, Zhao SF, Jiang H, Chi ZM (2016) Cloning and characterization of a pyruvate carboxylase gene from Penicillium rubens and overexpression of the gene in the yeast Yarrowia lipolytica for enhanced citric acid production. Mar Biotechnol 18(1):1–14PubMedGoogle Scholar
  20. Guilherme AA, Pinto GAS, Rodrigues S (2008) Optimization of trace metals concentration on citric acid production by Aspergillus niger NRRL 2001. Food Bioprocess Technol 1(3):246–253Google Scholar
  21. Hamdy HS (2013) Citric acid production by Aspergillus niger grown on orange peel medium fortified with cane molasses. Ann Microbiol 63(1):267–278Google Scholar
  22. Hasunuma T, Okazaki F, Okai N, Hara KY, Ishii J, Kondo A (2013) A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology. Bioresour Technol 135:513–522PubMedGoogle Scholar
  23. Hattori T, Kino K, Kirimura K (2009) Regulation of alternative oxidase at the transcription stage in Aspergillus niger under the conditions of citric acid production. Curr Microbiol 58(4):321–325PubMedGoogle Scholar
  24. Hou WL, Bao J (2018) Simultaneous saccharification and aerobic fermentation of high titer cellulosic citric acid by filamentous fungus Aspergillus niger. Bioresour Technol 253:72–78PubMedGoogle Scholar
  25. Hou L, Liu L, Zhang H, Zhang L, Zhang L, Zhang J, Gao Q, Wang D (2018) Functional analysis of the mitochondrial alternative oxidase gene (aox1) from Aspergillus niger CGMCC 10142 and its effects on citric acid production. Appl Microbiol Biotechnol 102(18):7981–7995Google Scholar
  26. Hu W, Liu J, Chen J, Wang S, Lu D, Wu Q, Li W (2014) A mutation of Aspergillus niger for hyper-production of citric acid from corn meal hydrolysate in a bioreactor. J Zhejiang Univ Sci B 15(11):1006–1010PubMedPubMedCentralGoogle Scholar
  27. Hu W, Li W, Chen J (2017) Recent advances of microbial breeding via heavy-ion mutagenesis at IMP. Lett Appl Microbiol 65(4):274–280PubMedGoogle Scholar
  28. Ikram-ul-Haq, Khurshid S, Ali S, Ashraf H, Qadeer MA, Rajoka MI (2001) Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses. World J Microbiol Biotechnol 17(1):35–37Google Scholar
  29. Jiang Y, Liu KM, Zhang HS, Wang YL, Yuan QQ, Su N, Bao J, Fang X (2017) Gluconic acid production from potato waste by Gluconobacter oxidans using sequential hydrolysis and fermentation. ACS Sustain Chem Eng 5(7):6116–6123Google Scholar
  30. Juturu V, Wu JC (2016) Microbial production of lactic acid: the latest development. Crit Rev Biotechnol 36(6):967–977PubMedGoogle Scholar
  31. Kamzolova SV, Finogenova TV, Morgunov IG (2008) Microbiological production of citric and isocitric acids from sunflower oil. Food Technol Biotechnol 46(1):51–59Google Scholar
  32. Karasu-Yalcin S, Bozdemir MT, Ozbas ZY (2010) Effects of different fermentation conditions on growth and citric acid production kinetics of two Yarrowia lipolytica strains. Chem Biochem Eng Q 24(3):347–360Google Scholar
  33. Kumar D, Verma R, Bhalla TC (2010) Citric acid production by Aspergillus niger van. Tieghem MTCC 281 using waste apple pomace as a substrate. J Food Sci Tech Mys 47(4):458–460Google Scholar
  34. Kumari KS, Babu IS, Rao GH (2008) Process optimization for citric acid production from raw glycerol using response surface methodology. Indian J Biotechnol 7(4):496–501Google Scholar
  35. Kutyla-Olesiuk A, Wawrzyniak UE, Ciosek P, Wróblewski W (2014) Electrochemical monitoring of citric acid production by Aspergillus niger. Anal Chim Acta 823:25–31PubMedGoogle Scholar
  36. Li CL, Yang HL, Xia XL, Li YJ, Chen LP, Zhang M, Zhang L, Wang W (2013) High efficient treatment of citric acid effluent by Chlorella vulgaris and potential biomass utilization. Bioresour Technol 127:248–255PubMedGoogle Scholar
  37. Li Q, Huang B, He Q, Lu J, Li X, Li Z, Wu H, Ye Q (2018) Production of succinate from simply purified crude glycerol by engineered Escherichia coli using two-stage fermentation. Bioresour Bioprocess 5(1):41Google Scholar
  38. Liu XY, Chi Z, Liu GL, Madzak C, Chi ZM (2013) Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin. Mar Biotechnol 15(1):26–36PubMedGoogle Scholar
  39. Liu XY, Lv JS, Zhang T, Deng YF (2015a) Citric acid production from hydrolysate of pretreated straw cellulose by Yarrowia lipolytica SWJ-1b using batch and fed-batch cultivation. Prep Biochem Biotechnol 45(8):825–835PubMedGoogle Scholar
  40. Liu XY, Wang XF, Xu JX, Xia J, Lv JS, Zhang T, Wu Z, Deng YF, He JL (2015b) Citric acid production by Yarrowia lipolytica SWJ-1b using corn steep liquor as a source of organic nitrogen and vitamins. Ind Crop Prod 78:154–160Google Scholar
  41. Liu YN, Li QG, Zheng P, Zhang ZD, Liu YF, Sun CM, Cao GQ, Zhou WJ, Wang XW, Zhang DW, Zhang TC, Sun JB, Ma YH (2015c) Developing a high-throughput screening method for threonine overproduction based on an artificial promoter. Microb Cell Factories 14:121Google Scholar
  42. Liu XY, Xu JX, Xia J, Lv JS, Wu Z, Deng YF (2016) Improved production of citric acid by Yarrowia lipolytica using oleic acid as the oxygen-vector and co-substrate. Eng Life Sci 16(5):424–431Google Scholar
  43. Lotfy WA, Ghanem KM, El-Helow ER (2007) Citric acid production by a novel Aspergillus niger isolate: I. Mutagenesis and cost reduction studies. Bioresour Technol 98(18):3464–3469PubMedGoogle Scholar
  44. Maitan-Alfenas GP, Visser EM, Guimarães VM (2015) Enzymatic hydrolysis of lignocellulosic biomass: converting food waste in valuable products. Curr Opin Food Sci 1:44–49Google Scholar
  45. Max B, Salgado JM, Rodríguez N, Cortés S, Converti A, Domínguez JM (2010) Biotechnological production of citric acid. Braz J Microbiol 41(4):862–875PubMedPubMedCentralGoogle Scholar
  46. Mirbagheri M, Nahvi I, Emtiazi G, Darvishi F (2011) Enhanced production of citric acid in Yarrowia lipolytica by triton X-100. Appl Biochem Biotechnol 165(3–4):1068–1074PubMedGoogle Scholar
  47. Moeller L, Zehnsdorf A, Aurich A, Barth G, Bley T, Strehlitz B (2013) Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation. Eng Life Sci 13(2):163–171Google Scholar
  48. Mostafa YS, Alamri SA (2012) Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger. Saudi J Biol Sci 19(2):241–246PubMedPubMedCentralGoogle Scholar
  49. Mourya S, Jauhri KS (2000) Production of citric acid from starch-hydrolysate by Aspergillus niger. Microbiol Res 155(1):37–44PubMedGoogle Scholar
  50. Niu J, Arentshorst M, Nair PDS, Dai ZY, Baker SE, Frisvad JC, Nielsen KF, Punt PJ, Ram AFJ (2016) Identification of a classical mutant in the industrial host Aspergillus niger by systems genetics: LaeA is required for citric acid production and regulates the formation of some secondary metabolites. G3 (Bethesda) 6(1):193–204Google Scholar
  51. Papamkolaou S, Galiotou-Panayotou M, Fakas S, Komaitis M, Aggelis G (2008) Citric acid production by Yarrowia lipolytica cultivated on olive-mill wastewater-based media. Bioresour Technol 99(7):2419–2428Google Scholar
  52. Rakicka M, Lazar Z, Rywinska A, Rymowicz W (2016) Efficient utilization of inulin and glycerol as fermentation substrates in erythritol and citric acid production using Yarrowia lipolytica expressing inulinase. Chem Pap 70(11):1452–1459Google Scholar
  53. Rodrigues C, Vandenberghe LPD, Teodoro J, Pandey A, Soccol CR (2009) Improvement on citric acid production in solid-state fermentation by Aspergillus niger LPB BC mutant using citric pulp. Appl Biochem Biotechnol 158(1):72–87PubMedGoogle Scholar
  54. Rymowicz W, Fatykhova AR, Kamzolova SV, Rywinska A, Morgunov IG (2010) Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes. Appl Microbiol Biotechnol 87(3):971–979PubMedGoogle Scholar
  55. Rywinska A, Rymowicz W (2010) High-yield production of citric acid by Yarrowia lipolytica on glycerol in repeated-batch bioreactors. J Ind Microbiol Biotechnol 37(5):431–435PubMedGoogle Scholar
  56. Rywinska A, Rymowicz W (2011) Continuous production of citric acid from raw glycerol by Yarrowia lipolytica in cell recycle cultivation. Chem Pap 65(2):119–123Google Scholar
  57. Rywinska A, Rymowicz W, Marcinkiewicz M (2010) Valorization of raw glycerol for citric acid production by Yarrowia lipolytica yeast. Electron J Biotechnol 13(4):1–9Google Scholar
  58. Santos R, Prata A (2009) Influence of agitation, aeration and time of spore germination on the citric acid production from hemicellulosic hydrolysate in submerged fermentation. New Biotechnol 25:S225–S226Google Scholar
  59. Schneider M, Zimmer GF, Cremonese EB, Schneider RDD, Corbellini VA (2014) By-products from the biodiesel chain as a substrate to citric acid production by solid-state fermentation. Waste Manag Res 32(7):653–660PubMedGoogle Scholar
  60. Show PL, Oladele KO, Siew QY, Zakry FAA, Lan JCW, Ling TC (2015) Overview of citric acid production from Aspergillus niger. Front Life Sci 8(3):271–283Google Scholar
  61. Sindhu R, Binod P, Pandey A (2016) Biological pretreatment of lignocellulosic biomass. Bioresour Technol 199:76–82PubMedGoogle Scholar
  62. Sun XW, Wu HF, Zhao GH, Li ZM, Wu XH, Liu H, Zheng ZM (2018) Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing. Bioprocess Biosyst Eng 41(7):1029–1038PubMedGoogle Scholar
  63. Tan MJ, Chen X, Wang YK, Liu GL, Chi ZM (2016) Enhanced citric acid production by a yeast Yarrowia lipolytica over-expressing a pyruvate carboxylase gene. Bioprocess Biosyst Eng 39(8):1289–1296PubMedGoogle Scholar
  64. Themelis DG, Tzanavaras PD (2001) Reagent-injection spectrophotometric determination of citric acid in beverages and pharmaceutical formulations based on its inhibitory effect on the iron (III) catalytic oxidation of 2,4-diaminophenol by hydrogen peroxide. Anal Chim Acta 428(1):23–30Google Scholar
  65. Torrado AM, Cortés S, Salgado JM, Max B, Rodríguez N, Bibbins BP, Converti A, Domínguez JM (2011) Citric acid production from orange peel wastes by solid-state fermentation. Braz J Microbiol 42(1):394–409PubMedPubMedCentralGoogle Scholar
  66. Urak S, Yeniay O, Karasu-Yalcin S (2015) Optimization of citric acid production from a carrot juice-based medium by Yarrowia lipolytica using response surface methodology. Ann Microbiol 65(2):639–649Google Scholar
  67. Wakai S, Arazoe T, Ogino C, Kondo A (2017) Future insights in fungal metabolic engineering. Bioresour Technol 245:1314–1326PubMedGoogle Scholar
  68. Wang LF, Wang ZP, Liu XY, Chi ZM (2013) Citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 and purification of citric acid. Bioprocess Biosyst Eng 36(11):1759–1766PubMedGoogle Scholar
  69. Wang L, Cao ZL, Hou L, Yin LH, Wang DW, Gao Q, Wu ZQ, Wang DP (2016) The opposite roles of agdA and glaA on citric acid production in Aspergillus niger. Appl Microbiol Biotechnol 100(13):5791–5803PubMedGoogle Scholar
  70. Wang BS, Li H, Zhu LH, Tan FL, Li YR, Zhang L, Ding ZY, Shi GY (2017) High-efficient production of citric acid by Aspergillus niger from high concentration of substrate based on the staged-addition glucoamylase strategy. Bioprocess Biosyst Eng 40(6):891–899PubMedGoogle Scholar
  71. Wang SZ, Sun XX, Yuan QP (2018) Strategies for enhancing microbial tolerance to inhibitors for biofuel production. Bioresour Technol 258:302–309PubMedGoogle Scholar
  72. Woinaroschy A, Nica A, Ofiteru ID, Lavric V (2010a) Kinetic models for citric acid production. Rev Chim-Bucharest 61(10):979–982Google Scholar
  73. Woinaroschy A, Ofiteru ID, Nica A (2010b) Optimal control of fed-batch bioreactors for citric acid production. Rev Chim-Bucharest 61(8):788–792Google Scholar
  74. Xie G, West TP (2009) Citric acid production by Aspergillus niger ATCC 9142 from a treated ethanol fermentation co-product using solid-state fermentation. Lett Appl Microbiol 48(5):639–644PubMedGoogle Scholar
  75. Xu J, Chen YQ, Zhang HJ, Tang L, Wang K, Zhang JH, Chen XS, Mao ZG (2014) Production of citric acid using its extraction wastewater treated by anaerobic digestion and ion exchange in an integrated citric acid-methane fermentation process. Bioprocess Biosyst Eng 37(8):1659–1668PubMedGoogle Scholar
  76. Yadegary M, Hamidi A, Alavi SA, Khodaverdi E, Yahaghi H, Sattari S, Bagherpour G, Yahaghi E (2013) Citric acid production from bagasse through solid state fermentation method using Aspergillus niger mold and optimization of citric acid production by taguchi method. Jundishapur J Microb 6(9):e7625Google Scholar
  77. Yalcin SK, Bozdemir MT, Ozbas ZY (2009) A comparative study on citric acid production kinetics of two Yarrowia lipolytica strains in two different media. Indian J Biotechnol 8(4):408–417Google Scholar
  78. Yalcin SK, Bozdemir MT, Ozbas ZY (2012) Effects of initial medium pH and temperature on growth and citric acid production kinetics of a novel domestic Yarrowia lipolytica strain. New Biotechnol 29:S62–S62Google Scholar
  79. Yin X, Li JH, Shin HD, Du GC, Liu L, Chen J (2015) Metabolic engineering in the biotechnological production of organic acids in the tricarboxylic acid cycle of microorganisms: advances and prospects. Biotechnol Adv 33(6):830–841PubMedGoogle Scholar
  80. Yu D, Shi Y, Wang Q, Zhang X, Zhao Y (2017) Application of methanol and sweet potato vine hydrolysate as enhancers of citric acid production by Aspergillus niger. Bioresour Bioprocess 4(1):35PubMedPubMedCentralGoogle Scholar
  81. Yu B, Zhang X, Sun WJ, Xi X, Zhao N, Huang ZC, Ying ZJ, Liu L, Liu D, Niu HQ, Wu JL, Zhuang W, Zhu CJ, Chen Y, Ying HJ (2018) Continuous citric acid production in repeated-fed batch fermentation by Aspergillus niger immobilized on a new porous foam. J Biotechnol 276:1–9PubMedGoogle Scholar
  82. Zhang HJ, Zhang JH, Xu J, Tang L, Mao ZG (2014a) A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production. Biochem Eng J 90:206–213Google Scholar
  83. Zhang X, Zhang XF, Li HP, Wang LY, Zhang C, Xing XH, Bao CY (2014b) Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl Microbiol Biotechnol 98(12):5387–5396PubMedGoogle Scholar
  84. Zhang HJ, Xu J, Su XF, Bao JW, Wang K, Mao ZG (2017) Citric acid production by recycling its wastewater treated with anaerobic digestion and nanofiltration. Process Biochem 58:245–251Google Scholar
  85. Zhang N, Jiang JC, Yang J, Wei M, Zhao J, Xu H, Xie JC, Tong YJ, Yu L (2018) Citric acid production from acorn starch by tannin tolerance mutant Aspergillus niger AA120. Appl Biochem Biotechnol.
  86. Zheng X, Zheng P, Zhang K, Cairns TC, Meyer V, Sun J, Ma Y (2018) 5S rRNA promoter for guide RNA expression enabled highly efficient CRISPR/Cas9 genome editing in Aspergillus niger. ACS Synth Biol.
  87. Zhi XH, Yang HJ, Berthold S, Doetsch C, Shen JQ (2010) Potential improvement to a citric wastewater treatment plant using bio-hydrogen and a hybrid energy system. J Power Sources 195(19):6945–6953Google Scholar
  88. Zhou PP, Meng J, Bao J (2017) Fermentative production of high titer citric acid from corn stover feedstock after dry dilute acid pretreatment and biodetoxification. Bioresour Technol 224:563–572PubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Institute of Modern PhysicsChinese Academy of SciencesLanzhouChina
  2. 2.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

Personalised recommendations