BioEnergy Research

, Volume 12, Issue 2, pp 419–432 | Cite as

Sequential Enzymatic and Mild-Acid Hydrolysis of By-Product of Carrageenan Process from Kappaphycus alvarezii

  • Fernando Roberto Paz-Cedeno
  • Eddyn Gabriel Solórzano-Chávez
  • Levi Ezequiel de Oliveira
  • Valéria Cress Gelli
  • Rubens Monti
  • Samuel Conceição de Oliveira
  • Fernando MasarinEmail author


Kappaphycus alvarezii is a red macroalgae widely used to produce carrageenan. The carrageenan processing produces a by-product rich in glucan which has been reported as easily hydrolyzed with enzymes, but the hydrolysate forms a gel at usual fermentation temperatures. The purpose of this study was to evaluate the enzymatic hydrolysis integrated with a mild-acid treatment of the by-product to obtain a hydrolysate rich in monomeric sugars. Using an enzyme load of 10 FPU g−1 of by-product, close to 100 and 14.7% of glucan and galactan conversion were reached, respectively. Increasing the enzyme load to 100 FPU g−1 raised the galactan conversion to 30%. The mild-acid treatment after enzymatic hydrolysis was satisfactory, increasing the glucose and galactose concentrations, without producing significant amounts of fermentation inhibitors and avoiding the formation of a gel structure. The statistical analysis showed that the main effects on the response were negative for the three independent variables, meaning that the selectivity (S) becomes lower when experimental conditions at the higher levels are used (longer time, higher temperature, and acid concentration). Therefore, the integrated enzymatic and acid hydrolysis of the by-product becomes a promising technological route to produce monomeric sugars for bioethanol or fine chemical production.


Kappaphycus alvarezii By-product Carrageenan Chemical composition Enzymatic and acid hydrolysis 





Refractive Index Detector


National Renewable Energy Laboratory


3,5-Dinitrosalicylic acid


Filter paper units


International Units


High-performance liquid chromatography system


Reverse-phase column HYPERSIL C18


Variable of selectivity



FAPESP, CNPq, PROPe-UNESP, and Programa de Apoio ao Desenvolvimento Científco da Faculdade de Ciências Farmacêuticas da UNESP-PADC supported this work. We appreciate, also, the support of German Enrique Pesantez (Bachelor of Science in Electrical Engineering) by providing resources for the English revision of the paper.

Availability of Supporting Data

Supporting data used in the publication of this paper can be provided upon request.

Authors’ Contributions

EGSC and FRPC performed the chemical and enzymatic hydrolysis analyses of the samples, data interpretation, and review of the manuscript. VCG provided the experimental macroalgae strains and performed the field trials, data interpretation, and review of the manuscript. LEO, RM, SCO, and FM participated in the design of the study, data interpretation, mathematical modeling, and review of the manuscript. All authors read and approved the final manuscript.


This work was supported by FAPESP (contract number 2014/05969-2), CNPq (contract number 440385/2014-8), PROPe—UNESP (contract number 506), and Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP-PADC (contract number 2013/19-1).

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no competing interests.

Ethical Approval and Consent to Participate

Not applicable.

Consent for Publication

All authors read and approved the final manuscript.

Supplementary material

12155_2019_9968_MOESM1_ESM.docx (2.4 mb)
ESM 1 (DOCX 2.42 mb)


  1. 1.
    Renewable Fuel Association (2018) World fuel ethanol production. Available at: Accessed 8 Feb 2019
  2. 2.
    Masarin F, Gurpilhares DB, Baffa DC et al (2011) Chemical composition and enzymatic digestibility of sugarcane clones selected for varied lignin content. Biotechnol Biofuels 4:55. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Mendes FM, Siqueira G, Carvalho W, Ferraz A, Milagres AMF (2011) Enzymatic hydrolysis of chemithermomechanically pretreated sugarcane bagasse and samples with reduced initial lignin content. Biotechnol Prog 27:395–401. CrossRefPubMedGoogle Scholar
  4. 4.
    Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96:673–686. CrossRefPubMedGoogle Scholar
  5. 5.
    Hendriks ATWM, Zeeman G (2009) Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 100:10–18. CrossRefPubMedGoogle Scholar
  6. 6.
    Wi SG, Kim HJ, Mahadevan SA, Yang DJ, Bae HJ (2009) The potential value of the seaweed Ceylon moss (Gelidium amansii) as an alternative bioenergy resource. Bioresour Technol 100:6658–6660. CrossRefPubMedGoogle Scholar
  7. 7.
    Martone PT, Estevez JM, Lu F, Ruel K, Denny MW, Somerville C, Ralph J (2009) Discovery of lignin in seaweed reveals convergent evolution of cell-wall architecture. Curr Biol 19:169–175. CrossRefPubMedGoogle Scholar
  8. 8.
    Ge L, Wang P, Mou H (2011) Study on saccharification techniques of seaweed wastes for the transformation of ethanol. Renew Energy 36:84–89. CrossRefGoogle Scholar
  9. 9.
    John RP, Anisha GS, Nampoothiri KM, Pandey A (2011) Micro and macroalgal biomass: a renewable source for bioethanol. Bioresour Technol 102:186–193. CrossRefPubMedGoogle Scholar
  10. 10.
    Wei N, Quarterman J, Jin YS (2013) Marine macroalgae: an untapped resource for producing fuels and chemicals. Trends Biotechnol 31:70–77. CrossRefPubMedGoogle Scholar
  11. 11.
    Masarin F, Cedeno FRP, Chavez EGS, de Oliveira LE, Gelli VC, Monti R (2016) Chemical analysis and biorefinery of red algae Kappaphycus alvarezii for efficient production of glucose from residue of carrageenan extraction process. Biotechnol Biofuels 9(122):122. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Roldán IUM, Mitsuhara AT, Munhoz Desajacomo JP, de Oliveira LE, Gelli VC, Monti R, Silva do Sacramento LV, Masarin F (2017) Chemical, structural, and ultrastructural analysis of waste from the carrageenan and sugar-bioethanol processes for future bioenergy generation. Biomass Bioenergy 107:233–243. CrossRefGoogle Scholar
  13. 13.
    Paz Cedeno FR, Solorzano Chavez EG, de Oliveira LE et al (2018) Perspectives for the production of bioethanol from macroalgae biomass. In: Brienzo M (ed) Bioethanol and beyond. Nova Science Publishers, New York, pp 203–230Google Scholar
  14. 14.
    Estevez JM, Ciancia M, Cerezo AS (2004) The system of galactans of the red seaweed, Kappaphycus alvarezii, with emphasis on its minor constituents. Carbohydr Res 339:2575–2592. CrossRefPubMedGoogle Scholar
  15. 15.
    Campbell R, Hotchkiss S (2017) Carrageenan industry market overview. In: Hurtado AQ, Critchley AT, Neish IC (eds) Tropical seaweed farming trends, problems and opportunities: focus on Kappaphycus and Eucheuma of commerce. Springer International Publishing, Cham, pp 193–205CrossRefGoogle Scholar
  16. 16.
    FAO (2018) FAO yearbook. Fishery and aquaculture statistics 2016. Table B-92: red seaweeds. Available at: Accessed 8 Feb 2019
  17. 17.
    Meinita MDN, Kang JY, Jeong GT, Koo HM, Park SM, Hong YK (2012) Bioethanol production from the acid hydrolysate of the carrageenophyte Kappaphycus alvarezii (cottonii). J Appl Phycol 24:857–862. CrossRefGoogle Scholar
  18. 18.
    McHugh DJ (2003) A guide to the seaweed industry. Available at: Accessed 8 Feb 2019
  19. 19.
    Brienzo M, Arantes V, Milagres AMF (2008) Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus. Fungal Biol Rev 22:120–130. CrossRefGoogle Scholar
  20. 20.
    Henning Jørgensen JBK, CF (2007) Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels Bioprod Biorefining 1:119–134. CrossRefGoogle Scholar
  21. 21.
    Park JH, Hong JY, Jang HC, Oh SG, Kim SH, Yoon JJ, Kim YJ (2012) Use of Gelidium amansii as a promising resource for bioethanol: a practical approach for continuous dilute-acid hydrolysis and fermentation. Bioresour Technol 108:83–88. CrossRefPubMedGoogle Scholar
  22. 22.
    Ra CH, Jeong GT, Shin MK, Kim SK (2013) Biotransformation of 5-hydroxymethylfurfural (HMF) by Scheffersomyces stipitis during ethanol fermentation of hydrolysate of the seaweed Gelidium amansii. Bioresour Technol 140:421–425. CrossRefPubMedGoogle Scholar
  23. 23.
    Modig T, Lidén G, Taherzadeh MJ (2002) Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem J 363:769–776. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Horiuchi JI, Shimizu T, Tada K, Kanno T, Kobayashi M (2002) Selective production of organic acids in anaerobic acid reactor by pH control. Bioresour Technol 82:209–213. CrossRefPubMedGoogle Scholar
  25. 25.
    Papanikolaou S, Chatzifragkou A, Fakas S, Galiotou-Panayotou M, Komaitis M, Nicaud JM, Aggelis G (2009) Biosynthesis of lipids and organic acids by Yarrowia lipolytica strains cultivated on glucose. Eur J Lipid Sci Technol 111:1221–1232. CrossRefGoogle Scholar
  26. 26.
    Meynial-Salles I, Dorotyn S, Soucaille P (2008) A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity. Biotechnol Bioeng 99:129–135. CrossRefPubMedGoogle Scholar
  27. 27.
    Song H, Lee SY (2006) Production of succinic acid by bacterial fermentation. Enzym Microb Technol 39:352–361. CrossRefGoogle Scholar
  28. 28.
    Willke T, Vorlop KD (2001) Biotechnological production of itaconic acid. Appl Microbiol Biotechnol 56:289–295. CrossRefPubMedGoogle Scholar
  29. 29.
    Ha SJ, Wei Q, Kim SR, Galazka JM, Cate J, Jin YS (2011) Cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain. Appl Environ Microbiol 77:5822–5825. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Aguirre-von-Wobeser E, Figueroa FL (2001) Photosynthesis and growth of red and green morphotypes of Kappaphycus alvarezii (Rhodophyta) from the Philippines. Mar Biol 138:679–686. CrossRefGoogle Scholar
  31. 31.
    Dawes CJ, Lluisma AO, Trono GC (1994) Laboratory and field growth studies of commercial strains of Eucheuma denticulatum and Kappaphycus alvarezii in the Philippines. J Appl Phycol 6:21–24. CrossRefGoogle Scholar
  32. 32.
    Muñoz J, Freile-Pelegrín Y, Robledo D (2004) Mariculture of Kappaphycus alvarezii (Rhodophyta, Solieriaceae) color strains in tropical waters of Yucatan, Mexico. Aquaculture 239:161–177. CrossRefGoogle Scholar
  33. 33.
    Hayashi L, de Paula EJ, Chow F (2007) Growth rate and carrageenan analyses in four strains of Kappaphycus alvarezii (Rhodophyta, Gigartinales) farmed in the subtropical waters of São Paulo state, Brazil. J Appl Phycol 19:393–399. CrossRefGoogle Scholar
  34. 34.
    Hayashi L, Oliveira EC, Bleicher-Lhonneur G, Boulenguer P, Pereira RTL, von Seckendorff R, Shimoda VT, Leflamand A, Vallée P, Critchley AT (2007) The effects of selected cultivation conditions on the carrageenan characteristics of Kappaphycus alvarezii (Rhodophyta, Solieriaceae) in Ubatuba Bay, São Paulo, Brazil. J Appl Phycol 19:505–511. CrossRefGoogle Scholar
  35. 35.
    Hames B, Scarlata C, Sluiter A (2008) Determination of protein content in biomass: laboratory analytical procedure (LAP). Report no. NREL/TP-510-42625. Available at: Accessed 8 Feb 2019
  36. 36.
    Lourenço SO, Barbarino E, De-paula JC et al (2002) Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds. Phycol Res 50:233–241. CrossRefGoogle Scholar
  37. 37.
    Sluiter A, Hames B, Ruiz R, et al (2005) Determination of ash in biomass: laboratory analytical procedure (LAP). Report No. NREL/Tp-510-42622. Available at: Accessed 8 Feb 2019
  38. 38.
    Craigie JS, Leigh C (1978) Carrageenans and agars. In: Hellebust JA, Craigie JS (eds) Handbook of phycological methods. Cambrige University Press, New York, pp 109–131. CrossRefGoogle Scholar
  39. 39.
    Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268. CrossRefGoogle Scholar
  40. 40.
    Araujo A, Ward OP (1990) Extracellular mannanases and galactanases from selected fungi. J Ind Microbiol 6:171–178. CrossRefGoogle Scholar
  41. 41.
    Hartree EF (1972) Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem 48:422–427. CrossRefPubMedGoogle Scholar
  42. 42.
    Oliveira SC, Paz-Cedeno FR, Masarin F (2017) Kinetic modeling of monomeric sugars formation during the enzymatic hydrolysis of the residue generated in the carrageenan production from algal biomass. XXI Simpósio Nacional de Bioprocessos SINAFERM. Aracaju, Sergipe. 3–6 September 2017. Accessed 8 Feb 2019
  43. 43.
    Oliveira SC, Paz-Cedeno FR, Masarin F (2018) Mathematical modeling of glucose accumulation during enzymatic hydrolysis of carrageenan waste. In: Silva Santos A (ed) Avanços científicos e tecnológicos em bioprocessos. Atena Editora, pp 97–103.
  44. 44.
    Froment GF, Bischoff KB, De Wilde J (2010) Chemical reactor analysis and design, 3rd edn. Wiley, New YorkGoogle Scholar
  45. 45.
    Zinnai A, Venturi F, Quartacci MF, Andreotti A, Andrich G (2010) The kinetic effect of some wine components on the enzymatic hydrolysis of β-glucan. South African J Enol Vitic 31:9–13. CrossRefGoogle Scholar
  46. 46.
    Sluiter JB, Ruiz RO, Scarlata CJ, Sluiter AD, Templeton DW (2010) Compositional analysis of lignocellulosic feedstocks. 1. Review and description of methods. J Agric Food Chem 58:9043–9053. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Aguilar R, Ramírez JA, Garrote G, Vázquez M (2002) Kinetic study of the acid hydrolysis of sugar cane bagasse. J Food Eng 55:309–318. CrossRefGoogle Scholar
  48. 48.
    Yun EJ, Kim HT, Cho KM, Yu S, Kim S, Choi IG, Kim KH (2016) Pretreatment and saccharification of red macroalgae to produce fermentable sugars. Bioresour Technol 199:311–318. CrossRefPubMedGoogle Scholar
  49. 49.
    Rasmussen H, Sørensen HR, Meyer AS (2014) Formation of degradation compounds from lignocellulosic biomass in the biorefinery: sugar reaction mechanisms. Carbohydr Res 385:45–57. CrossRefPubMedGoogle Scholar
  50. 50.
    Abd-Rahim F, Wasoh H, Zakaria MR, Ariff A, Kapri R, Ramli N, Siew-Ling L (2014) Production of high yield sugars from Kappaphycus alvarezii using combined methods of chemical and enzymatic hydrolysis. Food Hydrocoll 42:309–315. CrossRefGoogle Scholar
  51. 51.
    Alftrén J, Hobley TJ (2014) Immobilization of cellulase mixtures on magnetic particles for hydrolysis of lignocellulose and ease of recycling. Biomass Bioenergy 65:72–78. CrossRefGoogle Scholar
  52. 52.
    Rodrigues AC, Haven MØ, Lindedam J, Felby C, Gama M (2015) Celluclast and Cellic® CTec2: saccharification/fermentation of wheat straw, solid–liquid partition and potential of enzyme recycling by alkaline washing. Enzym Microb Technol 79–80:70–77. CrossRefGoogle Scholar
  53. 53.
    Pellerin P, Brillouet JM (1994) Purification and properties of an exo-(1→3)-β-d-galactanase from Aspergillus niger. Carbohydr Res 264:281–291. CrossRefPubMedGoogle Scholar
  54. 54.
    Okemoto K, Uekita T, Tsumuraya Y, Hashimoto Y, Kasama T (2003) Purification and characterization of an endo-beta-(1→6)-galactanase from Trichoderma viride. Carbohydr Res 338:219–230. CrossRefPubMedGoogle Scholar
  55. 55.
    Almeida JRM, Modig T, Petersson A, Hähn-Hägerdal B, Lidén G, Gorwa-Grauslund MF (2007) Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol 82:340–349. CrossRefGoogle Scholar
  56. 56.
    Jönsson LJ, Martín C (2016) Pretreatment of lignocellulose: formation of inhibitory by-products and strategies for minimizing their effects. Bioresour Technol 199:103–112. CrossRefPubMedGoogle Scholar
  57. 57.
    Larsson S, Palmqvist E, Hahn-Hägerdal B, Tengborg C, Stenberg K, Zacchi G, Nilvebrant NO (1999) The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzym Microb Technol 24:151–159. CrossRefGoogle Scholar
  58. 58.
    Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW (2004) Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Ind Microbiol Biotechnol 31:345–352. CrossRefPubMedGoogle Scholar
  59. 59.
    Keating JD, Panganiban C, Mansfield SD (2006) Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol Bioeng 93:1196–1206. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Fernando Roberto Paz-Cedeno
    • 1
  • Eddyn Gabriel Solórzano-Chávez
    • 1
  • Levi Ezequiel de Oliveira
    • 2
  • Valéria Cress Gelli
    • 3
  • Rubens Monti
    • 4
  • Samuel Conceição de Oliveira
    • 1
  • Fernando Masarin
    • 1
    Email author
  1. 1.School of Pharmaceutical Sciences (FCF), Department of Bioprocesses and BiotechnologyUNESP—São Paulo State UniversityAraraquaraBrazil
  2. 2.Lorena School of Engineering (EEL), Department of Chemical EngineeringUSP—University of São PauloLorenaBrazil
  3. 3.North Coast Research and Development Center, Secretariat of Agriculture and Supply of the State of São PauloFisheries Institute (IP)São PauloBrazil
  4. 4.School of Pharmaceutical Sciences (FCF), Department Food and NutritionUNESP—São Paulo State UniversityAraraquaraBrazil

Personalised recommendations