Enzymatic hydrolysis of dilute alkaline pretreated Chlorella sp. biomass for biosugar production and fed-batch bioethanol fermentation

Main Article Content

Mohd Asyraf Kassim Kean Meng Tan Noor Aziah Serri


This study investigated the factor that affect enzymatic hydrolysis of dilute alkaline pretreated Chlorella sp. biomass and its potential as a bioethanol feedstock. The enzymatic hydrolysis optimization of dilute alkaline pretreated Chlorella sp. biomass was carried out prior to fermentation process. The enzymatic hydrolysis was performed using cellulase cocktail from Trichoderma longibrachiatum. The enzymatic hydrolysis parameters such as pH, temperature, enzyme and biomass concentrations that affect hydrolysis performance were also investigated. The optimization of enzymatic hydrolysis was carried out using the central composite design (CCD) approach. The study indicated that the highest reducing sugar 413.42 ± 7.62 mg gbiomass-1, which corresponding to 84.33% hydrolysis yield was achieved when the hydrolysis was performed using 12.5 g L-1 biomass at 45 °C and initial pH 5.0 for 96 h of incubation. The enzymatic hydrolysis kinetic parameters (Vmax and Km) of the pretreated biomass were also been determined. The analysis indicated that Vmax and Km values for enzymatic hydrolysis of the biomass were 0.18 mgmL-1min-1 and 26.34 mg mL-1 respectively. The hydrolysate generated from the enzymatic hydrolysis was further fermented for bioethanol production using Saccharomyces cerevisiae in batch and fed-batch fermentation modes. The fed-batch fermentation resulted slightly higher bioethanol concentration of 1008.48 mg L-1, corresponding to bioethanol yield of 0.43 ggsugar-1 and 0.081 ggbiomass-1 compared to batch fermentation. The results revealed that optimization of enzymatic hydrolysis process could enhance reducing sugar production from dilute alkaline pretreated Chlorella sp. biomass. The results obtained also demonstrated that fed-batch fermentation of alkaline pretreated Chlorella sp. hydrolysate from enzymatic hydrolysis process improve the efficiency of ethanolic fermentation in term of ethanol concentration production and yield.


Article Details

Research Articles


[1] Behera S, Singh R, Arora R, Sharma NM, Shukla M, Kumar S. Scope of algae as third generation biofuels. Frontiers in Bioengineering and Biotechnology. Front Bioeng Biotechnol. 2014;2:90.

[2] Mongkolchaiarunya S, Vaithanomsat P, Chuntranuluk S. Effect of nitrogen source on ethanol production from weeds by simultaneous saccharification and fermentation process. Asia Pac J Sci Technol. 2016;21:210-213.

[3] Alam F, Mobin S, Chowdhury H. Third generation biofuel from algae. Procedia Eng. 2015;105:763-768.

[4] van den Broek LAM, Wagemakers MJM, Vershoor AM, Frissen AE, Haveren J, Blaauw R, Mooibroek H. Microalgae as renewable raw material for bioproducts: Identification and biochemical composition of microalgae from a raceway pond in The Netherlands. In: Popa V, Volf I, editors. Biomass as Renewable Raw Material to Obtain Bioproducts of High-Tech Value. US: Elsevier; 2018.p.39-68.

[5] Chen CY, Zhao X, Yen H, Ho S, Cheng C, Lee D, Bai F, Chang J. Microalgae-based carbohydrates for biofuel production. Biochem Eng J. 2013;78:1-10.

[6] Suthkamol S, Srinorakutara T, Butivate E, Orasoon K. Comparison of SHF and SSF process for ethanol production from alkali-acid pretreated sugarcane trash. Asia Pac J Sci Technol. 2016;21:229-235.

[7] Eldalatony MM, Kabra AN, Hwang JH, Govindwar SP, Kim KH, Kim H, Jeon GH. Pretreatment of microalgal biomass for enhanced recovery/extraction of reducing sugars and proteins. Bioprocess Biosyst. Eng. 2016;39:95-103.

[8] Miranda JR, Passarinho PC, Gouveia L. Pre-treatment optimization of Scenedesmus obliquus microalga for bioethanol production. Bioresour Technol. 2012;104:342-348.

[9] Khan MI, Lee MG, Shin JH, Kim JD. Pretreatment optimization of the biomass of Microcystis aeruginosa for efficient bioethanol production. AMB Express 2017;7:1-19.

[10] Hernández D, Rano B, Coca M, Garcia-Gonzalez MC. Saccharification of carbohydrates in microalgal biomass by physical, chemical and enzymatic pre-treatments as a previous step for bioethanol production. Chem Eng J. 2015;262:939-945.

[11] Harun R, Danquah MK. Influence of acid pre-treatment on microalgal biomass for bioethanol production. Process Biochem. 2011;46:304-309.

[12] Kim JS, Lee YY, Kim TH. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour Technol. 2016;199:42-48.

[13] Mahdy A, Mendez L, Ballesteros M, Gonzalez-Fernadez C. Autohydrolysis and alkaline pretreatment effect on Chlorella vulgaris and Scenedesmus sp. methane production. Energy. 2014;78:48-52.

[14] Chen Y, Steven MA, Zhu Y, Holmes J, Xu H. Understanding of alkaline pretreatment parameters for corn stover enzymatic saccharification. Biotechnol Biofuels. 2013;6:1-10.

[15] Harun R, Danquah MK. Enzymatic hydrolysis of microalgal biomass for bioethanol production. Chem Eng J. 2011;168:1079-1084.

[16] Choi SP, Nguyen MT, Sim SJ. Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production. Bioresour Technol. 2010;101:5330-5336.

[17] Kassim MA, Bhattacharya S. Dilute alkaline pretreatment for reducing sugar production from Tetraselmis suecica and Chlorella sp. biomass. Process Biochem. 2016;51:1757-1766.

[18] Singh A, Bishnoi NR. Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production. Appl Microbiol Biotechnol. 2012;93:1785-1793.

[19] Lee OK, Kim AL, Seong DH, Lee CG, Jung YT, Lee JW, Lee EY. Chemo-enzymatic saccharification and bioethanol fermentation of lipid-extracted residual biomass of the microalga, Dunaliella tertiolecta. Bioresour Technol. 2013;132:197-201.

[20] Trivedi N, Gupta V, Reddy CR, Jha B. Enzymatic hydrolysis and production of bioethanol from common macrophytic green alga Ulva fasciata Delile. Bioresour Technol. 2013;150:106-112.

[21] Ho SH, Danquah MK, Zhang S, Zhang X, Wu M, Chen XD, Ng I, Jing K, Lu Y. Characterization and optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E. Bioresour Technol. 2013;135:157-165.

[22] Zeng X, Danquah MK, Zhang S, Zhang X, Wu M, Chen XD, Ng IS, Jing K, Lu Y. Autotrophic cultivation of Spirulina platensis for CO2 fixation and phycocyanin production. Chem Eng J. 2012;183:192-197.

[23] Kurpan Nogueira DP, Silva AF, Aroujo OQF, Chaloub RM. Impact of temperature and light intensity on triacylglycerol accumulation in marine microalgae. Biomass Bioenergy. 2015;72:280-287.

[24] Nielsen SS. Phenol-sulfuric acid method for total carbohydrates. In: S.S. Nielsen, editor. Food analysis laboratory manual. US: Springer; 2009. p. 47-53.

[25] López CV, Garcia MC, Fernandez FG, Bustos CS, Chisti Y, Sevilla JM. Protein measurements of microalgal and cyanobacterial biomass. Bioresour Technol. 2010;101:7587-7591.

[26] Pradeep GC, Choi YH, Choi YS, Seong CN, Cho SS, Lee HJ, Yoo JC. A novel thermostable cellulase free xylanase stable in broad range of pH from Streptomyces sp. CS428. Process Biochemistry. 2013;48:1188-1196.

[27] Chen CY, Lee PJ, Tan CH, Lo YC, Huang CC, Show PL, Lin CH, Chang JS. Improving protein production of indigenous microalga Chlorella vulgaris FSP-E by photobioreactor design and cultivation strategies. Biotechnol J. 2015;10:905-914.

[28] Chia MA, Lombardi AT, MelãO MDGG. Growth and biochemical composition of Chlorella vulgaris in different growth media. An. Acad Bras Cienc. 2013;85:1427-1438.

[29] Ohse S, Dernerm RB, Ozorio RA, Correa RG, Furlong EB, Cunha PCR. Lipid content and fatty acid profiles in ten species of microalgae. Idesia 2015;33:93-101.

[30] Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: A review Renewable Sustainable Energy Rev. 2010;14:217-232.

[31] Auxenfans T, Cronier D, Chabbert B, Paes G. Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment. Biotechnol Biofuels. 2017;10:1-16.

[32] Lima MA, Lavorente GB, Silva HKP, Rezende CA, Bernardinelli OD, deAzevedo ER, Gomez LD, McQueen-Mason SJ, Labate CA, Polikarpov I. Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1. Biotechnol Biofuels. 2013;6:1-17.

[33] Ramachandriya KD, Wilkins M, Atiyeh HK, Dunford NT, Hiziroglu S. Effect of high dry solids loading on enzymatic hydrolysis of acid bisulfite pretreated Eastern redcedar. Bioresour Technol. 2013;147:168-176.

[34] Andreaus J, Azevedo H, Cavaco-Paulo A. Effects of temperature on the cellulose binding ability of cellulase enzymes. J. Mol. Catal. B: Enzym. 1999;7:233-239. [35] Adebowale ARA, Sanni LO. Effects of solid content and temperature on viscosity of tapioca meal. J Food Sci Technol. 2013;50:573-578.

[36] Benoit S.M, Afizah MN, Ruttarattanamongkol K, Rizvi SSH. Effect of pH and temperature on the viscosity of texturized and commercial whey protein dispersions. Int. J. Food Prop. 2013;16:322-330.

[37] Harun R, Danquah Michael K, Forde Gareth M. Microalgal biomass as a fermentation feedstock for bioethanol production. J. Chem. Technol. Biotechnol. 2009;85:199-203.

[38] El-Dalatony MM, Kurade MB, Abou-Shanab R, Kim H, Salama ES, Jeon BH. Long-term production of bioethanol in repeated-batch fermentation of microalgal biomass using immobilized Saccharomyces cerevisiae. Bioresour Technol. 2016;219:98-105.

[39] Shokrkar H, Ebrahimi S, Zamani M. Extraction of sugars from mixed microalgae culture using enzymatic hydrolysis: Experimental study and modeling. Chem Eng Commun. 2017;204:1246-1257.

[40] Asli UA, Nwaha I, Hamid H, Zakaria ZA, Sadikin AN, Kamaruddin MJ. A kinetic study of enzymatic hydrolysis of oil palm biomass for fermentable sugar using polyethylene glycol (PEG) immobilized cellulase. Jurnal Teknologi. 2017;78:51-57.

[41] Tovar LP, Lopes ES, Macial MRW, Filho R. Determination of enzyme (cellulase from Trichoderma reesei) kinetic parameters in the enzymatic hydrolysis of H2SO2-catalyzed hydrothermally pretreated sugarcane bagasse at high-solid loading. Chem Eng Trans. 2015;43:571-576.

[42] Carvalho ML, Sousam R, Rodriguez-Zuniga UF, Suarez CAG, Rodrigues DS, Giordano RC, Giordano RLC. Kinetic study of the enzymatic hydrolysis of sugarcane bagasse. Braz. J Chem Eng. 2013;30:437-447.

[43] Phukoetphim N, Salakkam A, Laopaiboon P, Laopaiboon L. Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: Effects of sugar levels, nitrogen supplementation, and feeding regimes. Electron. J Biotechnol. 2017;26:84-92.

[44] Chang YH, Chang KS, Huang CW, Hsu CL, Jang HD. Comparison of batch and fed-batch fermentations using corncob hydrolysate for bioethanol production. Fuel. 2012;97:166-173.

[45] Laopaiboon L, Thanonkeo P, Jaisil P, Laopaiboon P. Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World J Microbiol Biotechnol. 2007;23:1497-1501.

[46] Kim SK, Nguyen CM, Ko EH, Kim IC, Kim JS, Kim JC. Bioethanol production from Hydrodictyon reticulatum by fed-batch fermentation using Saccharomyces cerevisiae KCTC7017. J Microbiol Biotechnol. 2017;27:1112-1119.

[47] Maiorella B, Blanch HW, Wilke CR. By-product inhibition effects on ethanolic fermentation by Saccharomyces cerevisiae. Biotechnol Bioeng. 1983;25:103-121.