Comparative assessment of acid and enzyme pretreatment of Spirodella polyrhiza for bioethanol production

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Research Paper 01/06/2021
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Comparative assessment of acid and enzyme pretreatment of Spirodella polyrhiza for bioethanol production

Asma Khattak, Zulfiqar Ali Malik, Abdul Haq, Qurrat ul ain Rana, Haji Khan, Fazl ur Rehman, Samiullah Khan, Aamer Ali Shah, Malik Badshah
Int. J. Biosci.18( 6), 103-119, June 2021.
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Duckweeds are considered more sustainable compared to conventional crops due to no feed versus fuel competition. However, the feedstocks used for bioethanol production require an efficient pretreatment to increase the sugar yield resulting in reduced cost of the process. To combat low sugar yield challenges, five isolates named AG 1, AG 2, AG 3, AG 4 and AG 5 were isolated from soil. The bacterial isolate AG 3 (Bacillus AS1 MK321577) showed the highest amylase activity with the DNS method. The Bacillus AS1 MK321577 strain was evaluated for saccharification of Spirodella polyrhiza followed by bioethanol production via Saccharomyces cerevisiae. The specific activity of amylase was found to be 0.396 U/mg/min after 24 h incubation. Maximum amylase activity was observed in boiled potato peels as carbon sources in the production media at pH 6 and 45 ºC. Placket-Burman’s design for optimization of physical and nutritional parameters was used. The parameters selected for optimization were KH2PO4, pH, inocula, CaCl2, MgCl2, temperature, starch, MgSO4.7H2O, reaction time, NaCl and NaNO3. The current study reports that Bacillus AS1 MK321577 strain has the highest amylase activity. Acid pretreatment of S. polyrhiza was also carried out and compared with the amylase pretreatment. Overall, the highest reducing sugars yield (0.432 mg/ml) and highest bioethanol production (99%) were observed in the amylase pretreated sample. In comparison to acidic pretreatment, enzymatic pretreatment is a more environmentally friendly process and resulted in a 15% enhanced yield of reducing sugars.


Abd-Elaziz AM, Karam EA, Ghanem MM, Moharam ME, Kansoh AL. 2020. Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization. International journal of biological macromolecules 148, 292-301.

Abootalebi SN, Saeed A, Gholami A, Mohkam M, Kazemi A, Nezafat N, Mousavi SM, Hashemi SA, Shorafa E. 2020. Screening, characterization and production of thermostable alpha-amylase produced by a novel thermophilic Bacillus megaterium isolated from pediatric intensive care unit. Journal of Environmental Treatment Techniques 8(3), 952-960.

Bhatt B, Prajapati V, Patel K, Trivedi U. 2020. Kitchen waste for economical amylase production using Bacillus amyloliquefaciens KCP2. Biocatalysis and Agricultural Biotechnology 26, p.101654.

Chen G, Fang Y, Huang J, Zhao Y, Li Q, Lai F, Xu Y, Tian X, He K, Jin Y, Tan L. 2018. Duckweed systems for eutrophic water purification through converting wastewater nutrients to high-starch biomass: comparative evaluation of three different genera (Spirodela polyrhiza, Lemna minor and Landoltia punctata) in monoculture or polyculture. RSC Advances 8(32), 17927-17937.

Djekrif-Dakhmouche S, Gheribi-Aoulmi Z, Meraihi Z, Bennamoun L. 2006. Application of a statistical design to the optimization of culture medium for α-amylase production by Aspergillus niger ATCC 16404 grown on orange waste powder.  Journal of Food Engineering 73(2), 190-197.

ElSharayidi MS, Dewidar A, Shafik H. 2021. Molecular characterization of thermostable hydrolytic Enzymes producing bacteria isolated from hot spring of Ras Sedr, South Sinai, Egypt. Alfarama Journal of Basic & Applied Sciences 2(1), 60-69.

Fromm HJ. 2013. Initial rate enzyme kinetics. Springer Science & Business Media 22.

Fasiku SA, Ogunsola OF, Fakunle A, Olanbiwoninu AA. 2020. Isolation of Bacteria with Potential of Producing Extracellular Enzymes (Amylase, Cellulase and Protease) from Soil Samples. Journal of Advances in Microbiology 21-26.

Giordano PC, Beccaria AJ, Goicoechea HC. 2011. Significant factors selection in the chemical and enzymatic hydrolysis of lignocellulosic residues by a genetic algorithm analysis and comparison with the standard Plackett-Burman methodology. Bioresource Technology 102(22), 10602–10610.

Haq A, Adeel S, Khan A, ul ain Rana Q, Khan MA, Rafiq M, Ishfaq M, Khan S, Shah AA, Hasan F, Ahmed S. 2020. Screening of Lipase-Producing Bacteria and Optimization of Lipase-Mediated Biodiesel Production from Jatropha curcas Seed Oil Using Whole Cell Approach. BioEnergy Research 13(4), 1280-96.

Harun R, Liu B, Danquah MK. 2011. Analysis of process configurations for bioethanol production from microalgal biomass. Progress in Biomass and Bioenergy Pro‐duction. InTech 27, p 395-409.

Hutcheon GW, Vasisht N, Bolhuis A. 2005.  Characterisation of a highly stable α-amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles 9(6), 487-495.

Izmirlioglu G, Demirci A. 2016. Improved simultaneous saccharification and fermentation of bioethanol from industrial potato waste with co-cultures of Aspergillus niger and Saccharomyces cerevisiae by medium optimization. Fuel 185, 684-691.

Johnvesly B, Naik GR. 2001. Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process biochemistry. 37(2), 139-144.

Kannan TR, Kanagaraj C. 2019. Molecular characteristic of α-AMYLASE enzymes producing from Bacillus lichenformis (JQ946317) using solid state fermentation. Biocatalysis and Agricultural Biotechnology 20, 101240.

Kaur M, Kumar M, Sachdeva S, Puri SK. 2018. Aquatic weeds as the next generation feedstock for sustainable bioenergy production. Bioresource technology 251, 390-402.

Kokab S, Asghar M, Rehman K, Asad MJ, Adedyo O. 2003. Bio-processing of banana peel for α-amylase production by Bacillus subtilis. International Journal of Agriculture and Biology. 5(1), 36-39.

Leu SY, Zhu JY. 2013. Substrate-related factors affecting enzymatic saccharification of lignocelluloses: our recent understanding. Bioenergy Research 6(2), 405-415.

Li Y, Zhang F, Daroch M, Tang J. 2016. Positive effects of duckweed polycultures on starch and protein accumulation. Bioscience reports 36(5), e00380.

Liu Y, Chen X, Wang X, Fang Y, Huang M, Guo L, Zhang Y, Zhao H. 2018. Improving biomass and starch accumulation of bioenergy crop duckweed (Landoltia punctata) by abscisic acid application. Scientific reports 8(1), 1-9.

Luang-In V, Yotchaisarn M, Saengha W, Udomwong P, Deeseenthum S, Maneewan K. 2019. Isolation and Identification of Amylase-producing Bacteria from Soil in Nasinuan Community Forest, Maha Sarakham, Thailand. Biomedical and Pharmacology Journal 12(3), 1061-1068.

Ma YB, Zhu M, Yu CJ, Wang Y, Liu Y, Li ML, Sun YD, Zhao JS, Zhou GK. 2018. Large‐scale screening and characterisation of Lemna aequinoctialis and Spirodela polyrhiza strains for starch production. Plant Biology 20(2), 357-364.

Mushtaq Q, Irfan M, Tabssum F, Iqbal Qazi J. 2017. Potato peels: a potential food waste for amylase production. Journal of Food Process Engineering. 40(4), e12512.

Nahar K, Sunny SA. 2020. Duckweed-based clean energy production dynamics (ethanol and biogas) and phyto-remediation potential in Bangladesh. Modeling Earth Systems and Environment 6(1), 1-11.

Neha S, Mallick SA, Deepika S. 2018. Amylase producing efficiency of Bacillus species isolated from Jammu soil. Journal of Mycopathological Research. 56(2), 123-8.

Palmer T. 1985 Understanding Enzyme Ellishorwood Publisher

Pinjari AB, Kotari V. 2018. Characterization of extracellular amylase from Bacillus sp. strain RU1. Journal of Applied Biology & Biotechnology 6(3), 29-34.

Pranay K, Padmadeo SR, Jha V, Prasad B. 2019. Screening and identification of amylase producing strains of Bacillus. J Appl Biol Biotechnol. 7(04), 57-62.

Ranjan K, Lone MA, Sahay S. 2021. Detergent compatible cold-active alkaline amylases from Clavispora lusitaniae CB13. Journal of Microbiology, Biotechnology and Food Sciences 9(6), 306-310.

Raul D, Biswas T, Mukhopadhyay S, Kumar Das S, Gupta S. 2014. Production and partial purification of alpha amylase from Bacillus subtilis (MTCC 121) using solid state fermentation. Biochemistry Research International.

Rehman A. 2019. Isolation, optimization, partial purification and characterization of amylase produced by indigenously isolated Bacillus AS2 strain (Doctoral dissertation, University of Karachi, Karachi).

Rzhetsky A, Nei M. 1992. A simple method for estimating and testing minimum-evolution trees. References: Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4(4), 406– 425.

Sahnoun M, Kriaa M, Elgharbi F, Ayadi DZ,  Bejar S, Kammoun R. 2015. Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: optimization of culture conditions. International journal of biological macromolecules. 75, 73-80.

Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology Evolution. 4(4), 406–425.

Satrimafitrah P, Razak AR, Hardi J, Puspitasari DJ, Yelenggete I. 2020. Thermostable amylase activity produced by thermophilic bacteria isolated from Pulu Hotspring, Central Sulawesi. In Journal of Physics: Conference Series 1434(1), p. 012034. IOP Publishing.

Ul ain Rana Q, Khan MAN, Irfan M, Shah AA, Hasan F, Khan S, Badshah M. 2019. Starved Spirodela polyrhiza and Saccharomyces cerevisiae: a potent combination for sustainable bioethanol production. Biomass Conversion and Biorefinery, 1-10.

Ullah I, Khan MS, Khan SS, Ahmad W, Zheng L, Shah SUA, Iqbal A. 2021. Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil. Saudi journal of biological sciences 28(1), 970-979.

Wei H, Liu W, Chen X, Yang Q, Li J, Chen H. 2019. Renewable bio-jet fuel production for aviation: A review. Fuel. 254, 115599.

Wilson K. 2001. Preparation of genomic DNA from bacteria. Current protocols in molecular biology. 56(1), 2-4.

Yablokov V, Smeltsova I, Faerman V. 2013. Thermal stability of amino acids. Russian Journal of General Chemistry 83, 476-480.

Yu C, Zhao X, Qi G, Bai Z, Wang Y, Wang S, Ma Y, Liu Q, Hu R, Zhou G. 2017. Integrated analysis of transcriptome and metabolites reveals an essential role of metabolic flux in starch accumulation under nitrogen starvation in duckweed. Biotechnology for biofuels 10(1), 1-4.