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Microwave-assisted pretreatment using alkali metal salt in combination with orthophosphoric acid for generation of enhanced sugar and bioethanol

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The microwave-assisted alkali salt (NaCl, MgCl2, and KCl) in combination with H3PO4 pretreatment of rice straw followed by enzymatic hydrolysis with in-house and commercial enzyme cocktail (cellulase and xylanase) was investigated. The effect of pretreatment on the structure and composition of RS was investigated using XRD and FTIR analysis, and the result suggested strategic structural changes in the RS structure. The maximum CrI of 51.3% was obtained with KCl–H3PO4 pretreatment system which is in line with maximum saccharification per biomass of 79% using commercial xylanase and cellulase enzyme cocktail (ComCK–XylCell). Comparable saccharification per biomass of 74% was obtained with in-house xylanase and cellulase enzyme cocktail (CrCK–XylCell). The simultaneous saccharification and fermentation by KCl–H3PO4 pretreated system of RS resulted the maximum sugar yield of 30.6 and 26.9% and ethanol yield of 12.2 and 11.9 g/L using ComCK–XylCell and CrCK–XylCell cocktail, respectively, along with Saccharomyces cerevisiae MTCC 173. The hydrolysis of pretreated RS pulp using in-house enzyme cocktail resulted in high sugar and ethanol yield suggesting that the studied alkali metal in combination with weak acid such as H3PO4 can serve as a cost-effective and environment friendly pretreatment method for high production of sugar and ethanol.

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  1. 1.

    Van Dyk JS, Pletschke BI (2012) A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—factors affecting enzymes, conversion and synergy. Biotechnol Adv 30:1458–1480

  2. 2.

    Himmel ME, Ding S-Y, Johnson DK et al (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science (80-) 315:804–807

  3. 3.

    Sewsynker-Sukai Y, Suinyuy TN, Kana EBG (2018) Development of a sequential alkalic salt and dilute acid pretreatment for enhanced sugar recovery from corn cobs. Energy Convers Manag 160:22–30

  4. 4.

    Shen Z, Zhang K, Si M, Liu M, Zhuo S, Liu D, Ren L, Yan X, Shi Y (2018) Synergy of lignocelluloses pretreatment by sodium carbonate and bacterium to enhance enzymatic hydrolysis of rice straw. Bioresour Technol 249:154–160

  5. 5.

    McKendry P (2002) Energy production from biomass (part 1): overview of biomass. Bioresour Technol 83:37–46

  6. 6.

    Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83:1–11

  7. 7.

    Taherzadeh MJ, Karimi K (2008) Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int J Mol Sci 9:1621–1651

  8. 8.

    Chaturvedi V, Verma P (2013) An overview of key pretreatment processes employed for bioconversion of lignocellulosic biomass into biofuels and value added products. 3 biotech 3:415–431. https://doi.org/10.1007/s13205-013-0167-8

  9. 9.

    Kumar B, Bhardwaj N, Agrawal K et al (2020) Current perspective on pretreatment technologies using lignocellulosic biomass: an emerging biorefinery concept. Fuel Process Technol 199:106244

  10. 10.

    Zhu L, O’Dwyer JP, Chang VS et al (2010) Multiple linear regression model for predicting biomass digestibility from structural features. Bioresour Technol 101:4971–4979

  11. 11.

    Binod P, Satyanagalakshmi K, Sindhu R et al (2012) Short duration microwave assisted pretreatment enhances the enzymatic saccharification and fermentable sugar yield from sugarcane bagasse. Renew Energy 37:109–116

  12. 12.

    Hendriks A, Zeeman G (2009) Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 100:10–18

  13. 13.

    Ravindran R, Jaiswal AK (2016) A comprehensive review on pre-treatment strategy for lignocellulosic food industry waste: challenges and opportunities. Bioresour Technol 199:92–102

  14. 14.

    Ethaib S, Omar R, Kamal SMM, Biak DRA (2015) Microwave-assisted pretreatment of lignocellulosic biomass: a review. J Eng Sci Technol 2:97–109

  15. 15.

    Tsubaki S, Azuma J (2011) Application of microwave technology for utilization of recalcitrant biomass. In: in advances in induction and microwave heating of mineral and organic materials S. Grundas (Ed.). InTech, p 697e722

  16. 16.

    Gong G, Liu D, Huang Y (2010) Microwave-assisted organic acid pretreatment for enzymatic hydrolysis of rice straw. Biosyst Eng 107:67–73

  17. 17.

    Merino-Pérez O, Martínez-Palou R, Labidi J, Luque R (2015) Microwave-assisted pretreatment of lignocellulosic biomass to produce biofuels and value-added products. In: Production of Biofuels and Chemicals with Microwave. Springer, pp 197–224

  18. 18.

    Intanakul P, Krairiksh M, Kitchaiya P (2003) Enhancement of enzymatic hydrolysis of lignocellulosic wastes by microwave pretreatment under atmospheric pressure. J Wood Chem Technol 23:217–225

  19. 19.

    Qing Q, Zhou L, Huang M, Guo Q, He Y, Wang L, Zhang Y (2016) Improving enzymatic saccharification of bamboo shoot shell by alkalic salt pretreatment with H2O2. Bioresour Technol 201:230–236

  20. 20.

    Qing Q, Zhou L, Guo Q, Huang M, He Y, Wang L, Zhang Y (2016) A combined sodium phosphate and sodium sulfide pretreatment for enhanced enzymatic digestibility and delignification of corn Stover. Bioresour Technol 218:209–216

  21. 21.

    Sewsynker-Sukai Y, Kana EBG (2017) Optimization of a novel sequential alkalic and metal salt pretreatment for enhanced delignification and enzymatic saccharification of corn cobs. Bioresour Technol 243:785–792

  22. 22.

    Kumar B, Bhardwaj N, Verma P (2019) Pretreatment of rice straw using microwave assisted FeCl3-H3PO4 system for ethanol and oligosaccharides generation. Bioresour Technol Rep 7:100295

  23. 23.

    Bhardwaj N, Chanda K, Kumar B et al (2017) Statistical optimization of nutritional and physical parameters for xylanase production from newly isolated Aspergillus oryzae LC1 and its application in the hydrolysis of lignocellulosic agro-residues. BioResources 12:8519–8538

  24. 24.

    Kumar B, Bhardwaj N, Alam A et al (2018) Production, purification and characterization of an acid/alkali and thermo tolerant cellulase from Schizophyllum commune NAIMCC-F-03379 and its application in hydrolysis of lignocellulosic wastes. AMB Express 8:173

  25. 25.

    Bhardwaj N, Kumar B, Agarwal K, Chaturvedi V, Verma P (2019) Purification and characterization of a thermo-acid/alkali stable xylanases from Aspergillus oryzae LC1 and its application in Xylo-oligosaccharides production from lignocellulosic agricultural wastes. Int J Biol Macromol 122:1191–1202

  26. 26.

    Trindade WG, Hoareau W, Megiatto JD, Razera IA, Castellan A, Frollini E (2005) Thermoset phenolic matrices reinforced with unmodified and surface-grafted furfuryl alcohol sugar cane bagasse and curaua fibers: properties of fibers and composites. Biomacromolecules 6:2485–2496

  27. 27.

    Guimarães JL, Frollini E, Da Silva CG et al (2009) Characterization of banana, sugarcane bagasse and sponge gourd fibers of Brazil. Ind Crop Prod 30:407–415

  28. 28.

    Segal L, Creely JJ, Martin AE Jr, Conrad CM (1959) An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text Res J 29:786–794

  29. 29.

    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428

  30. 30.

    Verma P, Watanabe T, Honda Y, Watanabe T (2011) Microwave-assisted pretreatment of woody biomass with ammonium molybdate activated by H2O2. Bioresour Technol 102:3941–3945. https://doi.org/10.1016/j.biortech.2010.11.058

  31. 31.

    Ai Y, Yu Z, Chen Y et al (2016) Rapid determination of the monosaccharide composition and contents in tea polysaccharides from Yingshuang green tea by pre-column derivatization HPLC. J Chemother 2016:5

  32. 32.

    Nisha M, Shankar M, Krishnan N et al (2016) Direct estimation of ethanol as a negative peak from alcoholic beverages and fermentation broths by reversed phase-HPLC. Anal Methods 8:4762–4770

  33. 33.

    Cheng Y-S, Zheng Y, Yu CW, Dooley TM, Jenkins BM, Vander Gheynst J (2010) Evaluation of high solids alkaline pretreatment of rice straw. Appl Biochem Biotechnol 162:1768–1784

  34. 34.

    Zheng Y, Lee C, Yu C et al (2013) Dilute acid pretreatment and fermentation of sugar beet pulp to ethanol. Appl Energy 105:1–7

  35. 35.

    Laureano-Perez L, Teymouri F, Alizadeh H, Dale BE (2005) Understanding factors that limit enzymatic hydrolysis of biomass. Appl Biochem Biotechnol 124:1081–1099

  36. 36.

    Lü J, Zhou P (2011) Optimization of microwave-assisted FeCl3 pretreatment conditions of rice straw and utilization of Trichoderma viride and Bacillus pumilus for production of reducing sugars. Bioresour Technol 102:6966–6971. https://doi.org/10.1016/j.biortech.2011.04.044

  37. 37.

    Wang L, Han G, Zhang Y (2007) Comparative study of composition, structure and properties of Apocynum venetum fibers under different pretreatments. Carbohydr Polym 69:391–397

  38. 38.

    Kumar R, Mago G, Balan V, Wyman CE (2009) Physical and chemical characterizations of corn Stover and poplar solids resulting from leading pretreatment technologies. Bioresour Technol 100:3948–3962

  39. 39.

    Guo G-L, Chen W-H, Chen W-H et al (2008) Characterization of dilute acid pretreatment of silvergrass for ethanol production. Bioresour Technol 99:6046–6053

  40. 40.

    Moodley P, Kana EBG (2017) Microwave-assisted inorganic salt pretreatment of sugarcane leaf waste: effect on physiochemical structure and enzymatic saccharification. Bioresour Technol 235:35–42

  41. 41.

    Jin S, Zhang G, Zhang P, Li F, Wang S, Fan S, Zhou S (2016) Microwave assisted alkaline pretreatment to enhance enzymatic saccharification of catalpa sawdust. Bioresour Technol 221:26–30

  42. 42.

    Lai LW, Idris A (2016) Comparison of steam-alkali-chemical and microwave-alkali pretreatment for enhancing the enzymatic saccharification of oil palm trunk. Renew Energy 99:738–746

  43. 43.

    Singh R, Tiwari S, Srivastava M, Shukla A (2014) Microwave assisted alkali pretreatment of rice straw for enhancing enzymatic digestibility. J Energy 2014:7

  44. 44.

    Banerjee D, Mukherjee S, Pal S, Khowala S (2016) Enhanced saccharification efficiency of lignocellulosic biomass of mustard stalk and straw by salt pretreatment. Ind Crop Prod 80:42–49

  45. 45.

    Amini N, Haritos VS, Tanksale A (2018) Microwave assisted pretreatment of eucalyptus sawdust enhances enzymatic saccharification and maximizes fermentable sugar yield. Renew Energy 127:653–660

  46. 46.

    Tsegaye B, Balomajumder C, Roy P (2019) Optimization of microwave and NaOH pretreatments of wheat straw for enhancing biofuel yield. Energy Convers Manag 186:82–92

  47. 47.

    Hu Z, Wen Z (2008) Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment. Biochem Eng J 38:369–378

  48. 48.

    Ramadoss G, Muthukumar K (2015) Influence of dual salt on the pretreatment of sugarcane bagasse with hydrogen peroxide for bioethanol production. Chem Eng J 260:178–187

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The authors acknowledge the support for instrumentation facility FTIR from Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Bombay, XRD from Department of Physics, Central University of Rajasthan (CURAJ), HPLC from School of Chemical Sciences, Central University of Rajasthan (CURAJ). Authors are also thankful to DST-FIST (SR/FST/LSI-676/2016) for necessary infrastructure facility generated at Department of Microbiology, CURAJ. NB acknowledges University Grants Commission for providing Rajiv Gandhi National Fellowship for doctoral studies. BK acknowledges Jawaharlal Nehru Memorial Fund, New Delhi, CSIR-SRF for providing funding for Doctoral Studies.


This work was financially supported by the Department of Biotechnology, Government of India through Project BT/304/NE/TBP/2012 and BT/PR7333/PBD/26/373/2012.

Author information

Pradeep Verma (PV) has played a vital role in conceptualization of research idea. Nisha Bhardwaj (NB) has conducted the laboratory work and prepared the rough draft of MS. Bikash Kumar (BK) has performed the formal analysis of the results and writing of the MS. The experimental, writing, and formal analysis was supervised by PV. In addition, PV’s role has key in acquisition of the financial supports for the project leading to this publication. All authors have given approval to the final version of the manuscript.

Correspondence to Pradeep Verma.

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Bhardwaj, N., Kumar, B. & Verma, P. Microwave-assisted pretreatment using alkali metal salt in combination with orthophosphoric acid for generation of enhanced sugar and bioethanol. Biomass Conv. Bioref. (2020). https://doi.org/10.1007/s13399-020-00640-1

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  • Microwave-assisted pretreatment
  • Alkali metals
  • Orthophosphoric acid
  • Ethanol