Environmental Science and Pollution Research

, Volume 25, Issue 36, pp 35960–35970 | Cite as

Fumaric acid production using renewable resources from biodiesel and cane sugar production processes

  • Aikaterini Papadaki
  • Harris Papapostolou
  • Maria Alexandri
  • Nikolaos Kopsahelis
  • Seraphim Papanikolaou
  • Aline Machado de Castro
  • Denise M. G. Freire
  • Apostolis A. KoutinasEmail author
Sustainable Waste Management


The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.


Bioprocess Fumaric acid Cane sugar Molasses Rhizopus arrhizus Soybean cake 


Funding information

This work was funded by Petrobras (Brazil) (project 2012/00320-2) and the National Council for Scientific and Technological Development of the Ministry of Science, Technology, and Innovation (CNPq/MCTI) through the Special Visiting Researcher fellowship (process number: 313772/2013-4).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Alexandri M, Papapostolou H, Vlysidis A, Gardeli C, Komaitis M, Papanikolaou S, Koutinas AA (2016) Extraction of phenolic compounds and succinic acid production from spent sulphite liquor. J Chem Technol Biotechnol 91:2751–2760. CrossRefGoogle Scholar
  2. Ashraf S, Sikander A, Haq I (2015) Acidic pre-treatment of sugarcane molasses for molasses for citric acid production by Aspergillus niger NG-4. Int J Curr Microbiol Appl Sci 4:584–595Google Scholar
  3. ASTM D1976-12 (2012) Standard test method for elements in water by inductively-coupled argon plasma atomic emission spectroscopy. ASTM International, West Conshohocken, PA. CrossRefGoogle Scholar
  4. Carta FS, Soccol CR, Ramos LP, Fontana JD (1999) Production of fumaric acid by fermentation of enzymatic hydrolysates derived from cassava bagasse. Bioresour Technol 68:23–28. CrossRefGoogle Scholar
  5. Cazetta ML, Celligoi MAPC, Buzato JB, Scarmino IS (2007) Fermentation of molasses by Zymomonas mobilis: effects of temperature and sugar concentration on ethanol production. Bioresour Technol 98:2824–2828. CrossRefGoogle Scholar
  6. Chen M, Zhao Y, Yu S (2015) Optimisation of ultrasonic-assisted extraction of phenolic compounds, antioxidants, and anthocyanins from sugar beet molasses. Food Chem 172:543–550. CrossRefGoogle Scholar
  7. de Souza PM, Bittencourt ML de A, Caprara CC, de Freitas M, de Almeida RPC, Silveira D, Fonseca YM, Ferreira EX, Pessoa A, Magalhães PO (2015) A biotechnology perspective of fungal proteases. Braz J Microbiol 46:337–346. CrossRefGoogle Scholar
  8. Das RK, Brar SK, Verma M (2015) Effects of different metallic nanoparticles on germination and morphology of the fungus Rhizopus oryzae 1526 and changes in the production of Fumaric acid. BioNanoSci 5:217–226. CrossRefGoogle Scholar
  9. Das RK, Brar SK, Verma M (2016) Recent advances in the biomedical applications of fumaric acid and its ester derivatives: the multifaceted alternative therapeutics. Pharmacol Rep 68:404–414. CrossRefGoogle Scholar
  10. Delgenes JP, Moletta R, Navarro JM (1996) Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae. Enzym Microb Technol 19:220–225. CrossRefGoogle Scholar
  11. Dimou C, Kopsahelis N, Papadaki A, Papanikolaou S, Kookos IK, Mandala I, Koutinas AA (2015) Wine lees valorization: Biorefinery development including production of a generic fermentation feedstock employed for poly(3-hydroxybutyrate) synthesis. Food Res Int 73:81–87. CrossRefGoogle Scholar
  12. Eurostat (2016) Renewable energy statistics. Accessed 15 October 2017
  13. FAOSTAT (2014) Accessed 16 October 2017
  14. Faustino H, Gil N, Baptista C, Duarte AP (2010) Antioxidant activity of lignin phenolic compounds extracted from kraft and sulphite black liquors. Molecules 15:9308–9322CrossRefGoogle Scholar
  15. Fu Y-Q, Li S, Chen Y, Xu Q, Huang H, Sheng X-Y (2010) Enhancement of fumaric acid production by Rhizopus oryzae using a two-stage dissolved oxygen control strategy. Appl Biochem Biotechnol 162:1031–1038. CrossRefGoogle Scholar
  16. Goldberg I, Lonberg-Holm K, Bagley EA, Stieglitz B (1983) improved conversion of fumarate to succinate Escherichia coli strains amplified for fumarate reductase. Appl Environ Microbiol 1838–1847Google Scholar
  17. Harland BF, Harland J (1980) Fermentative reduction of phytate in rye, white and whole wheat breads. Cereal Chem 57:226–229Google Scholar
  18. Hashizume T, Yamagami T, Sasaki Y (1967) Constituents of cane molasses. Agri Biol Chem 31:324–329. CrossRefGoogle Scholar
  19. Hu C, Zhao X, Zhao J, Wu S, Zhao ZK (2009) Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides. Bioresour Technol 100:4843–4847. CrossRefGoogle Scholar
  20. Ito S, Barchi AC, Escaramboni B, de Oliva Neto P, Herculano RD, Azevedo Borges F, Romeiro Miranda MC, Fernández Núñez EG (2017) UV/Vis spectroscopy combined with chemometrics for monitoring solid-state fermentation with Rhizopus microsporus var. oligosporus. J Chem Technol Biotechnol 92:2563–2572. CrossRefGoogle Scholar
  21. Jang Y-S, Kim B, Shin JH, Choi YJ, Choi S, Song CW, Lee J, Park HG, Lee SY (2012) Bio-based production of C2–C6 platform chemicals. Biotechnol Bioeng 109:2437–2459. CrossRefGoogle Scholar
  22. Kachrimanidou V, Kopsahelis N, Chatzifragkou A, Papanikolaou S, Yanniotis S, Kookos I, Koutinas AA (2013) Utilisation of by-products from sunflower-based biodiesel production processes for the production of fermentation feedstock. Waste Biomass Valor 4:529–537. CrossRefGoogle Scholar
  23. Koutinas AA, Vlysidis A, Pleissner D, Kopsahelis N, Lopez Garcia I, Kookos IK, Papanikolaou S, Kwan TH, Lin CSK (2014) Valorization of industrial waste and by-product streams via fermentation for the production of chemicals and biopolymers. Chem Soc Rev 43:2587–2627. CrossRefGoogle Scholar
  24. Kumar G, Bakonyi P, Periyasamy S, Kim SH, Nemestóthy N, Bélafi-Bakó K (2015) Lignocellulose biohydrogen: practical challenges and recent progress. Renew Sust Energ Rev 44:728–737. CrossRefGoogle Scholar
  25. Kundu S, Panda T, Majumdar SK, Guha B, Bandyopadhyay KK (1984) Pretreatment of Indian cane molasses for increased production of citric acid. Biotechnol Bioeng 26:1114–1121. CrossRefGoogle Scholar
  26. Liao W, Liu Y, Frear C, Chen S (2007) A new approach of pellet formation of a filamentous fungus – Rhizopus oryzae. Bioresour Technol 98:3415–3423. CrossRefGoogle Scholar
  27. Lie S (1973) The EBC-ninhydrin method for determination of free alpha amino nitrogen. J I Brewing 79:37–41. CrossRefGoogle Scholar
  28. Liu Y-P, Zheng P, Sun Z-H, Ni Y, Dong J-J, Zhu L-L (2008) Economical succinic acid production from cane molasses by Actinobacillus succinogenes. Bioresour Technol 99:1736–1742CrossRefGoogle Scholar
  29. Martín C, Jönsson LJ (2003) Comparison of the resistance of industrial and laboratory strains of Saccharomyces and Zygosaccharomyces to lignocellulose-derived fermentation inhibitors. Enzym Microb Technol 32:386–395. CrossRefGoogle Scholar
  30. Meussen BJ, de Graaff LH, Sanders JPM, Weusthuis RA (2012) Metabolic engineering of Rhizopus oryzae for the production of platform chemicals. Appl Microbiol Biotechnol 94:875–886. CrossRefGoogle Scholar
  31. Obata Y, Senba Y, Koshiba M (1963) Detection of phenolic compounds by chromatography in beet sugar molasses. Agr Biol Chern 27:340–341CrossRefGoogle Scholar
  32. Papadaki A, Androutsopoulos N, Patsalou M, Koutinas M, Kopsahelis N, de Castro AM, Papanikolaou S, Koutinas AA (2017) Biotechnological production of Fumaric acid: the effect of morphology of Rhizopus arrhizus NRRL 2582. Fermentation 3:33. CrossRefGoogle Scholar
  33. Petruccioli M, Angiani E, Federici F (1996) Semi-continuous fumaric acid production by Rhizopus arrhizus immobilized in polyurethane sponge. Process Biochem 31:463–469. CrossRefGoogle Scholar
  34. Rauf A, Irfan M, Nadeem M, Ahmed I, Iqbal HMN (2010) Optimization of Growth Conditions for Acidic Protease Production from Rhizopus oligosporus through Solid State Fermentation of Sunflower Meal. WASET International Journal of Biotechnology and Bioengineering 4(12). dai:
  35. Rhodes RA, Moyer AJ, Smith ML, Kelley SE (1959) Production of fumaric acid by Rhizopus arrhizus. Appl Microbiol 7:74–80Google Scholar
  36. Roa Engel CA, Straathof AJJ, Zijlmans TW, Van Gulik WM, Van Der Wielen LAM (2008) Fumaric acid production by fermentation. Appl Microbiol Biotechnol 78:379–389. CrossRefGoogle Scholar
  37. Roa Engel CA, van Gulk WM, Marang L, van der Wielen LAM, Straathof AJJ (2011) Development of low pH fermentation strategy for fumaric acid production by Rhizopus oryzae. Enzym Microb Technol 48:39–47. CrossRefGoogle Scholar
  38. Scherer R, Godoy HT (2009) Antioxidant activity index (AAI) by the 2,2-diphenyl-1-picrylhydrazyl method. Food Chem 112:654–658CrossRefGoogle Scholar
  39. Sindhu R, Gnansounou E, Binod P, Pandey A (2016) Bioconversion of sugarcane crop residue for value added products - an overview. Renew Energ 98:203–215. CrossRefGoogle Scholar
  40. Teclu D, Tivchev G, Laing M, Wallis M (2009) Determination of the elemental composition of molasses and its suitability as carbon source for growth of sulphate-reducing bacteria. J Hazard Mater 161:1157–1165. CrossRefGoogle Scholar
  41. Tsouko E, Kachrimanidou V, Dos Santos AF, do Nascimento Vitorino Lima ME, Papanikolaou S, de Castro AM, Freire DM, Koutinas AA (2017) Valorization of By-Products from Palm Oil Mills for the Production of Generic Fermentation Media for Microbial Oil Synthesis. Appl Biochem Biotechnol 181:1241–1256. CrossRefGoogle Scholar
  42. Wang R, Shaarani S, Md Godoy LC, Melikoglu M, Vergara CS, Koutinas A, Webb C (2010) Bioconversion of rapeseed meal for the production of a generic microbial feedstock. Enz Microb Technol 47:77–83. CrossRefGoogle Scholar
  43. Xiao ZJ, Liu PH, Qin JY, Xu P (2007) Statistical optimisation of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate. Appl Microbiol Biotechnol 74:61–68CrossRefGoogle Scholar
  44. Xu Q, Li S, Huang H, Wen J (2012) Key technologies for the industrial production of fumaric acid by fermentation. Biotechnol Adv 30:1685–1696. CrossRefGoogle Scholar
  45. Zhang J, Geng A, Yao C, Lu Y, Li Q (2012) Effects of lignin-derived phenolic compounds on xylitol production and key enzyme activities by a xylose utilizing yeast Candida athensensis SB18. Bioresour Technol 121:369–378CrossRefGoogle Scholar
  46. Zhang K, Yu C, Yang S-T (2015) Effects of soybean meal hydrolysate as the nitrogen source on seed culture morphology and fumaric acid production by Rhizopus oryzae. Process Biochem 50:173–179. CrossRefGoogle Scholar
  47. Zhang L, Li X, Yong Q, Yang S-T, Ouyang J, Yu S (2016) Impacts of lignocellulose-derived inhibitors on L-lactic acid fermentation by Rhizopus oryzae. Bioresour Technol 203:173–180. CrossRefGoogle Scholar
  48. Zhou Y, Du J, Tsao GT (2000) Mycellial Pellet Formation by Rhizopus oryzae ATCC 20344. Appl Biochem Biotechnol 84–86:779–89Google Scholar
  49. Zhou Z, Du G, Hua Z, Zhou J, Chen J (2011) Optimisation of fumaric acid production by Rhizopus delemar based on the morphology formation. Bioresour Technol 102:9345–9349. CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Aikaterini Papadaki
    • 1
  • Harris Papapostolou
    • 1
  • Maria Alexandri
    • 1
    • 2
  • Nikolaos Kopsahelis
    • 3
  • Seraphim Papanikolaou
    • 1
  • Aline Machado de Castro
    • 4
  • Denise M. G. Freire
    • 5
  • Apostolis A. Koutinas
    • 1
    Email author
  1. 1.Department of Food Science and Human NutritionAgricultural University of AthensAthensGreece
  2. 2.Department of BioengineeringLeibniz Institute for Agricultural Engineering and Bioeconomy (ATB)PotsdamGermany
  3. 3.Department of Food TechnologyTechnological Educational Institute (TEI) of Ionian IslandsKefaloniaGreece
  4. 4.Biotechnology Division, Research and Development CentrePETROBRASRio de JaneiroBrazil
  5. 5.Biochemistry Department, Chemistry InstituteFederal University of Rio de Janeiro, Cidade UniversitáriaRio de JaneiroBrazil

Personalised recommendations