ARID ZONE JOURNAL OF ENGINEERING, TECHNOLOGY & ENVIRONMENT AZOJETE September 2023. Vol. 19(3):561-572 Published by the Faculty of Engineering, University of Maiduguri, Maiduguri, Nigeria. Print ISSN: 1596-2490, Electronic ISSN: 2545-5818 www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 561 ORIGINAL RESEARCH ARTICLE DELIGNIFICATION TRENDS IN PRETREATMENT OF Musa paradisiaca PSEUDO-STEM AND THE PRODUCTION OF BIOETHANOL F. K. Owofadeju, T. E. Kolajo* and O. A. Onibudo Department of Wood Products Engineering, University of Ibadan, Ibadan, Nigeria *Corresponding author’s email address: tolukolajo@yahoo.com 1.0 Introduction Biomass utilization in the production of bioenergy provides an alternative to the use of fossil fuels and, as a result, reduces greenhouse gas emissions and restoring ecosystem balance (Ben Iwo et al., 2016). Biomass for bioenergy also alleviates concerns about the depletion of nonrenewable petroleum resources used in the production of fuels, chemicals, and other bio-products. A rational approach to the use of agricultural and forestry residues has also been necessary. First generation biofuels were created by fermenting the sugar found in grain starches like corn, sorghum, cassava, and barley as well as the sugar in sugar cane and beets (Alalwan et al., 2019; Dahman et al., 2019) as evidenced by the world's top two producers of ethanol, United States and Brazil, which use corn and sugarcane respectively to produce about three-quarters of the world's ethanol (Yesmin et al., 2020; Fu et al., 2022). This has led to competition between the economies of food and energy. Second generation biofuels investigate the hydrolysis and fermentation of lignocellulose carbohydrates (Baruah et al., 2018; Garba 2021) using feedstocks ARTICLE INFORMATION ABSTRACT This study was conducted to assess the potential of pre-treated Musa paradisiaca pseudo- stem as an alternative source of raw material for ethanol production. The pseudo-stem was pre-treated with NaOH and NaOH-AQ at varying time and liquor concentrations using a digester. Cellulose yield (CY) was determined gravimetrically, Residual Klason Lignin (RKL) was evaluated in accordance with TAPPI T236 standards and the effect of the pre-treatment variables were examined. Acid hydrolysis using dilute solutions of HCL and H2SO4 followed pre-treatment, while fermentation was achieved by the addition of an active yeast strain (Saccharomyces cerevisae). The ethanol produced was distilled, qualitative analysis was conducted using the Fourier Transform Infrared Spectroscopy (FTIR) and other standard methods. There was a general decrease in CY and RKL with increasing pulping time and alkali charge, using both NaOH and NaOH-AQ. CY ranged from 2.4 – 4.2g and 3.2 - 5.0g while RKL ranged from 2.46%-3.9% and 1.94-3.00% for both NaOH and NaOH-AQ pre-treatments respectively. However, there was significant difference between the effects of liquor concentration and time on CY at p=0.05. The addition of 0.1% anthraquinone to soda pulping liquor significantly increased CY and decreased RKL. The difference in glucose yields between the two dilute acid hydrolyses was not significant. Various qualitative tests on bio-ethanol showed traits very similar to the laboratory grade ethanol while FTIR confirmed the presence of ethanol. It is therefore proven that NaOH and NaOH-AQ are suitable pretreatment methods in the production of bioethanol from Musa paradisiaca. © 2023 Faculty of Engineering, University of Maiduguri, Nigeria. All rights reserved. Submitted 26 May, 2023 Revised 12 July, 2023 Accepted 20 July, 2023 Keywords: Dilute acid hydrolysis anthraquinone pre- treatment bioethanol delignification http://www.azojete.com.ng/ file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com tolukolajo@yahoo.com Arid Zone Journal of Engineering, Technology and Environment, Sept, 2023; Vol. 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818; www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 562 such as corn stalks, rice stalks, plantain pseudo-stems, banana pseudo-stems, poplar and willow trees, grasses such as hemp and switchgrass, capable of producing two rounds of harvests per year continuously without the need for replanting, and biomass found in municipal wastes (USEIA, 2022). The plantain pseudo-stem has been identified as a biomass that can be used for a variety of purposes, including bioethanol production (Ahmad and Danish, 2018). It is the leftover plantain bunch material that has been harvested and allowed to rot. This feedstock is cheap, plentiful, and renewable. The presence of lignin between and within the cell-wall of cellulose microfibrils, which results in recalcitrance during hydrolysis, makes it, like other biomass feedstocks, difficult to process. To hydrolyze and ferment lignocellulose sugars into ethanol, lignin must be eliminated (Islam et al., 2019). The optimization of ethanol production processes from lignocellulosic biomass is regarded as important research from both an industrial and a scientific standpoint (Ramaraj and Unpaprom, 2019). One of the primary reason bioethanol has struggled to compete favorably with fossil fuels is the lack of a commercially viable manufacturing cost. This begins with the selection of appropriate pretreatment methods to overcome lignin's recalcitrance. The presence of lignin in lignocelluloses has hampered bioethanol production (Zhao and Zhao, 2018). For the purpose of removing lignin from the cellulose matrix, a number of pretreatment procedures have been investigated, including diluted acid pretreatment (Li et al., 2016; Thomas et al, 2017; Liu et al., 2018), ultrasound pretreatment (Ivetic et al., 2017; Luzzi et al., 2017), ionic liquids (Liu et al., 2016; Raj et al., 2018), Since biomass differs in morphology, chemical composition, and structure, a suitable, appropriate, and commercially viable pretreatment method for each specific biomass is required. In this study, plantain pseudo-stems were pre-treated with soda (NaOH) and soda/anthraquinone (NaOH-AQ) before being hydrolyzed and fermented to produce bioethanol. The pretreatment variables' effects on cellulose yield and residual lignin were investigated and a t-test was conducted to ascertain the significant levels of the differences. 2.0 Materials and Methods 2.1 Collection and Preparation Pseudo stems from a mini-plantation was harvested at the University of Ibadan in Nigeria, located at latitude 7.440N and longitude 3.900E. The pseudo-stems were collected, and the leaf was identified in the University of Ibadan's Herbarium as belonging to the Plantae kingdom, Musaceae family, Musa genus and paradisiaca species. For easier pulping, the stems were cut into 3-5 cm chips. 2.2 Pretreatment Dissolution and serial dilutions of sodium hydroxide pellets with de-ionized water were used to prepare the NaOH pulping liquor while 0.1% anthraquinone were combined with sodium hydroxide to create NaOH-AQ liquor. The samples were pretreated in a 6-liter chemical reactor file:///C:/user/Downloads/azojete143/www.azojete.com.ng file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com Owofadeju et al: Delignification Trends in Pretreatment of Musa Paradisiaca Pseudo-Stem and the Production of Bioethanol. AZOJETE, 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818, www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 563 built for low temperature biomass pretreatments (Kolajo and Onilude, 2016). The biomass to liquor ratio was 1 to 15 w/v while a temperature of 120°C, was maintained throughout the pulping experiment as adopted by Kolajo (2021). Pulping duration was varied (30, 45 and 60 minutes) at 10-, 12-, and 14% alkali concentrations. The resulting cellulose substrate was washed with distilled water before being refined in a lab refiner for a minute and then put through a 0.1mm sieve. The pulp yield was measured gravimetrically following electronic oven drying to a constant weight. 2.3 Dilute Acid Hydrolyses Separately, 0.5g of cellulose substrate was mixed with 50 ml of 0.2M HCL and H2SO4 in accordance with Camargo et al. (2019). The reaction was run at 25°C for two hours. The reaction was stopped by neutralizing the acid while additionally changing the pH by adding 25ml of concentrated potassium hydroxide solution. The resulting glucose mixture was filtered to remove impurities and then fermented. 2.4 Fermentation and Distillation Fermentation was performed at 37oC. The experiment was conducted in a DNP-9052-1A Thermostat Incubator. A 500ml glucose solution made from HCL and H2SO4 hydrolyzed cellulose substrates was mixed with 1.0g of an active yeast strain (Saccharomyces cerevisiae). The yeast was mixed evenly, the mixture was labeled, and it was placed in the incubator for 48 hours (Kolajo, 2021). The resulting ethanol was distilled at 78oC using a simple batch distillation method according to ASTM D86. 2.5 Qualitative Tests For the qualitative analysis, the FTIR device (Buck Scientific Infrared Spectrometer M350) was used (Gallignani et al., 1994; Conklin et al., 2014). A Buck Scientific Infrared Spectrometer with the model number M530 was used to gather the infrared (IR) spectra of the solutions over a 500–4000 cm–1 range. The absorbed radiation was transformed into rotational and/or vibrational energy by the sample molecules. The resulting signal, which was displayed as a spectrum at the detector typically ranges from 400 to 4000 cm-1, representing the molecular fingerprint of the sample. The samples were compared to the pure ethanol's infrared spectra, which were obtained. The ASTM D7795-12 standard was also used for chemical tests (acidity and alkalinity) as well as physical tests (density, boiling point, odor, and color). 3.0 Results and Discussion 3.1 Cellulose Yield of Pretreated Plantain Pseudo-stem The highest yield obtained for the plantain pseudo-stem using only NaOH and NaOH-AQ pretreatments was 4.2g at 10% concentration and 30 minutes, and 5.1g at 10% concentration and 30 minutes. Tables 1 and 2 show that the yield for both NaOH and NaOH-AQ pulping was in the range of 2.4 - 4.2g and 3.2 - 5.0g, respectively. The cellulose yield difference between NaOH and NaOH-AQ pulping is significant at p-value of 0.05, indicating that the addition of AQ to NaOH pretreatment significantly yielded a higher cellulose volume for hydrolysis, as also reported by Khristova et al. (2006) and Lois-Correa (2012). http://www.azojete.com.ng/ file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com Arid Zone Journal of Engineering, Technology and Environment, Sept, 2023; Vol. 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818; www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 564 3.2 Residual Klason Lignin (RKL) of Pretreated Plantain Pseudo-stem For the NaOH and NaOH-AQ pulping of the samples, the RKL of the pre-treated biomass ranged from 2.46 to 3.9% and 1.94 to 3%, respectively (Tables 1 and 2). For the NaOH and NaOH-AQ pulping of the samples, the pretreatment with the lowest RKL was 2.46 and 1.94%; this indicates an average of over 60% lignin removal in both schedules. However, according to Xu et al. (2011), the amount of RKL is within the range that permits cellulose hydrolysis. The results of the analysis of variance reveal a significant difference between the RKL values of pulping with NaOH and NaOH-AQ. Table 1: Pretreatment Variables and Pulp Yields using NaOH Conc. (%) Time (mins) Yield (g) Kappa No. RKL 10 30 4.27 ± 0.06 30.17 3.900 10 45 4.03 ± 0.15 29.00 3.770 10 60 3.17 ± 0.15 21.10 2.743 12 30 3.80 ± 0.10 26.10 3.393 12 45 3.67 ± 0.21 21.40 2.782 12 60 3.07 ± 0.06 18.90 2.457 14 30 3.47 ± 0.15 23.40 3.042 14 45 2.70 ± 0.10 20.10 2.623 14 60 2.47 ± 0.06 18.90 2.457 Table 2: Pretreatment variables and Pulp Yields using NaOH-AQ Conc. (%) Time (mins) Yield (g) Kappa No. RKL 10 30 5.13 ± 0.15 23.10 3.003 10 45 4.93 ± 0.12 22.20 2.886 10 60 4.37 ± 0.06 19.20 2.496 12 30 4.53 ± 0.06 22.20 2.886 12 45 4.37 ± 0.15 18.90 2.457 12 60 4.00 ± 0.10 15.90 2.067 14 30 3.83 ± 0.15 19.20 2.496 14 45 3.57 ± 0.06 16.50 2.145 14 60 3.234 ± 0.15 14.90 1.937 3.3 Effect of Pretreatment on Cellulose Yield and RKL 3.3.1 Effect of Pretreatment Time on Cellulose Yield The effects of pretreatment time on the yield of pre-treated plantain pseudo-stem for NaOH and NaOH-AQ are depicted in Figures 1 and 2. As the cooking time and alkali concentration increased, the cellulose yield decreased. Kolajo and Onilude (2019) speculate that this drop-in yield may be caused by the biomass's extractives and other non-fibrous components dissolving. As a pseudo-stem, there are a number of other cell types present besides cellulose fibres (Patel file:///C:/user/Downloads/azojete143/www.azojete.com.ng file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com Owofadeju et al: Delignification Trends in Pretreatment of Musa Paradisiaca Pseudo-Stem and the Production of Bioethanol. AZOJETE, 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818, www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 565 and Patel, 2022; Kolajo et al., 2023) which are likely to be dissolved by the cooking liquor. The yield from the pretreatment may be impacted as the carbohydrate content of the biomass dissolves over time. 10% 12% 14% 0 1 2 3 4 5 Liquor concentration Y ie ld ( g ) 30 mins 45 mins 60 mins Figure. 1: The effect of pretreatment time on Cellulose yield using NaOH 10% 12% 14% 0 2 4 6 Liquor concentration Y ie ld ( g ) 30 mins 45 mins 60 mins Figure 2: The effect of pretreatment time on Cellulose yield using NaOH-AQ 3.3.2 Effect of Liquor Concentration on Cellulose Yield An increase in alkali charge resulted in a decrease in the cellulose yield obtained from pretreatment at constant temperature and pretreatment duration. This pattern can be seen in the pulping of the plantain pseudo-stem with NaOH and NaOH-AQ (Figures 1 and 2). The increase in alkali charge indicated by the graphs may be the cause of this. Because the alkali affects both the lignin and carbohydrate components of the pseudo stem, an increase in alkali charge led to a corresponding decrease in yields. These observations correlate with other findings in http://www.azojete.com.ng/ file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com Arid Zone Journal of Engineering, Technology and Environment, Sept, 2023; Vol. 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818; www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 566 literature (Jie et al., 2022, Kolajo 2021) who reported that total pulp yield is decreased as active alkali charge is increased. 3.3.3 Effect of Time and Liquor Concentration on RKL Figures 3 and 4 show how RKL is impacted by both time and alcohol concentration. As the time was extended, the lignin content further decreased, resulting in a lower Kappa number and RKL. A rapid decrease in RKL was observed between 45 to 60 minutes of pretreatment at 10% concentration in contrast to 12 and 14% alkaline charges. This trend was similar in both NaOH and NaOH-AQ pretreatments. This may be attributed to the dissolution of the non-fibrous components of the biomass first, before delignification. This then implies that pulping at 10% alkali charge will require a longer duration for delignification to occur. However, RKL across the liquor concentrations also decreased as the time was increased, indicating lignin dissolution. Figure 3: Effect of Time and NaOH Concentration on RKL of Plantain Pseudo-stem 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 R K L TIME (MINS) Conc 10 Conc 12 Conc 14 file:///C:/user/Downloads/azojete143/www.azojete.com.ng file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com Owofadeju et al: Delignification Trends in Pretreatment of Musa Paradisiaca Pseudo-Stem and the Production of Bioethanol. AZOJETE, 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818, www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 567 Figure 4: Effect of Time and NaOH-AQ Concentration on RKL of Plantain Pseudo-stem 3.4 Qualitative Tests The bioethanol produced is a clear, colorless liquid that boiled between 79 and 82oC. The tested density varied from 0.83g/mL to 0.85g/mL. After distillation at between 80 and 82oC, there was complete miscibility with water and no residue was left behind. The pH ranged from 7.5 to 8.0. The test results supported the presence of ethanol as reported by Suleiman et al. (2017) and Kolajo (2021) when compared to laboratory grade ethanol (Analar® chemicals). 3.5 Fourier Transform Infrared Spectroscopy Analysis (FTIR) The results of the FTIR analysis of the bioethanol made from plantain stem biomass are shown in Table 3. The vibrational frequencies for (O-H), (C-H), and (C-O) for sample A, as depicted in Figure 5, were noted at 3467 cm-1, 2780 cm-1, and 1209 cm-1, respectively. Figure 6 illustrates the vibrational frequencies for (O-H), (C-H), and (C-O) for sample B at 3189 cm-1, 2775 cm-1, and 1072 cm-1. Table 3. The Infrared Spectra of Bioethanol Obtained from Plantain Pseudo Stem (𝒄𝒎−𝟏) Sample code Acids O-H C-H C-O A HCl 3467 2780 1209 B H2SO4 3189 2775 1072 All produced bioethanol spectra from the hydrolysis of both hydrochloric and sulphuric acids contained bands in the 3189–3467 cm-1 range for the hydroxyl functional group (OH) (Forough et al., 2013). While the stretching vibrations of (C-O) were responsible for the absorbance peaks around 1072–1209 cm-1, the stretching vibrations of (C-H) were responsible for the absorbance peaks at 2775–2780 cm-1. The samples' similar bands served as proof that they were all bioethanol samples. Numerous functional groups, including hydroxyl (OH stretch), methyl (CH3 stretch), 0 0.5 1 1.5 2 2.5 3 3.5 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 R K L TIME (MINS) Conc 10 Conc 12 Conc 14 http://www.azojete.com.ng/ file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com Arid Zone Journal of Engineering, Technology and Environment, Sept, 2023; Vol. 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818; www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 568 and alkane (CH2 stretch), are present in bioethanol. The infrared spectra of bioethanol show transmittance and a peak corresponding to a functional group. These functional groups can also be seen in the spectra of pure ethanol (Ogidi, 2020). In the (O-H) stretching region between 3000 cm-1 and 3600 cm-1 are reported to have distinct broad bands in the infrared spectra of pure ethanol and blends, according to Corsetti et al. (2015). Additional ethanol-specific traits can be found in the fingerprint region. Figure 5: FTIR for Hydrolyzed Substrate using HCl Figure 6: FTIR for Hydrolyzed Substrate using H2SO4 1000200030004000 Wavenumbers 0 50 100 P er ce n t T ra n sm it ta n ce 3 8 5 9 .2 0 3 4 6 7 .2 0 3 1 9 4 .4 0 2 7 7 6 .8 0 2 3 6 4 .8 0 2 0 2 3 .2 0 1 6 7 6 .8 0 1 2 0 9 .6 0 1 0 2 9 .6 0 7 4 1 .6 0 1000200030004000 Wavenumbers 3 8 5 0 .4 0 3 1 8 8 .8 0 2 7 7 5 .2 0 2 3 5 3 .6 0 1 8 3 9 .2 0 1 0 7 2 .0 0 7 0 8 .0 0 file:///C:/user/Downloads/azojete143/www.azojete.com.ng file:///C:/Users/Engr.%20Samuel/Documents/Engr%20Oyeniyi/azojete/AZOJETE%20ARCHIVE/UPLOAD/VOL%2019%20NO%203/tolukolajo@yahoo.com Owofadeju et al: Delignification Trends in Pretreatment of Musa Paradisiaca Pseudo-Stem and the Production of Bioethanol. AZOJETE, 19(3):561-572. ISSN 1596-2490; e-ISSN 2545-5818, www.azojete.com.ng Corresponding author’s e-mail address: tolukolajo@yahoo.com 569 3.6 Conclusion There is a general decrease in cellulose yield and residual klason lignin as pretreatment time and concentration increased in both soda and soda anthraquinone pulping processes. This trend also confirmed that lignin condensation did not occur. Cellulose yield is increased significantly with the addition of 0.1% anthraquinone to soda pulping liquor with a higher decrease in Residual Klason Lignin. There was sufficient delignification as confirmed by the subsequent hydrolysis and fermentation into ethanol. The properties of bioethanol produced compared well with laboratory grade ethanol. This has established that plantain pseudo-stem wastes can be utilized for bioethanol production using low temperature and cost-effective pretreatments that can be explored for biomass having similar morphology. Funding: No financial support was received for this research. Competing Interests: The authors have no competing interests to declare. Credit Authorship Contribution: Owofadeju F.K.: Experimentation, Supervision, Funding; Kolajo T.E.: Conceptualisation, Experimentation, Supervision, Funding, Writing – Review and Editing, Onibudo O.A.: Experimentation, Funding, Writing – Original draft. REFERENCES Ahmad, T. and Danish, M. 2018. Prospects of plantain waste utilization in wastewater treatment: A review. Journal of Environmental Management, 206: 330-348 Alalwan, HA., Alminshid, AH. and Aljaafari HAS. 2019. Promising evolution of biofuel generations, Subject review, Renewable Energy Focus, 28: 127-139. doi.org/10.1016/j.ref.2018.12.006. Baruah, J., Nath, BK., Sharma, R., Kumar, S., Deka, RC., Baruah, DC. and Kalita, E. 2018. Recent Trends in the Pretreatment of Lignocellulosic Biomass for Value-Added Products. Frontiers in Energy Research, 6(141): 1-19. doi:10.3389/fenrg.2018.00141 Ben-Iwo, J., Manovic, V. and Longhurst, P. 2016. 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