From Rags to Riches: the Fermentation Potential of Agave Leaf Residues in the Brazilian Semi-arid

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P. David, Guilherme P. Nogueira, Jade R. dos Santos, Beatriz O. Vargas, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5369383/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract CAM plants are promising biomasses to assure energy security and biofuel supply in the current changing climate scenario. Their high sugar content and strengthened tolerance to high temperatures and droughts makes them attractive alternatives to classic fuel sources. In Brazil, sisal ( Agave sisalana ), is cultivated in semiarid regions for fiber production. However, fibers represent only 4% of the plant’s leaves, with the remaining majority being discarded. This work, then, aims to explore this residue’s potential for bioethanol production. For this, low-input fermentations of a fibrous Brazilian agave accession leaves were explored. A maximum ethanol yield of 54.47% (11.64 g.L − 1 ) was obtained with Kluyveromyces marxianus . Isolating endogenous microbiota activity and fermentation inhibitors (i.e. saponins) revealed major operational challenges. Nevertheless, the results demonstrate that bioethanol production from agave residues is not only attainable but also promising. The unexplored bioethanol potential from this residue in the Brazilian semiarid could yield 489 L.ha − 1 .yr − 1 , totalizing 639 million liters of fuel, in the last decade. Agave Saccharomyces cerevisiae bioethanol waste semi-arid Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Brazil, the world’s second-largest bioethanol producer, and a leading country in integrating renewables into its energy matrix [ 1 , 2 ], has relied on sugarcane for bioethanol production since the 1970s [ 3 ]. Despite the increasing production of second-generation and maize-based ethanol (Conab, n.d.; Silva and Castañeda-Ayarza, 2021), sugarcane remains the dominant feedstock in the bioethanol sector, a position that is both crucial and vulnerable. Recent years have seen sugarcane confronted with significant climatic challenges, including periods of severe drought and unexpected fires or frosts [ 5 – 8 ], underscoring the need for feedstock diversification. Agave is a promising new feedstock in this context. Thanks to its specialized Crassulacean Acid Metabolism (CAM), which minimizes water loss, agave exhibits exceptional tolerance to high temperatures and intense droughts [ 9 – 11 ]. This enables it to thrive on hostile lands where few other plants, particularly food crops, can survive [ 10 , 11 ]. Data from the tequila industry, in Mexico, indicate that ethanol can be produced from agave at high yields (5,000–6,000 L ethanol ha⁻¹ yr⁻¹), with a productivity comparable to that of sugarcane in Brazil, while requiring significantly less water (300–800 mm yr⁻¹, nearly 25% of the water needed for sugarcane) [ 12 , 13 ]. Although the main agave varieties cultivated in Brazil (such as Agave sisalana ) differs from that in Mexico ( A. tequilana ) and is used for fiber rather than ethanol production, the fibrous accessions still contain significant sugar content [ 14 , 15 ]. Agave already plays an important role in the Brazilian economy, particularly in Northeast region, where it is cultivated in a non-centralized manner by small-scale farmers [ 11 , 13 , 16 ]. A. sisalana fibers represent only 4% of its leaves and are the only part of the plant that is harvested for sisal confection, while the remaining 96% of the leaves are discarded [ 17 , 18 ]. These residues are rich in fructan-type carbohydrates called inulin or agavin, that could be fermented after hydrolysis into biofuels and other biobased products [ 19 , 20 ]. In this work we aimed to explore the ethanol production potential from the residual leaf juice of a Brazilian agave accession, Agave sp. IAC4, a “sisalana-like” agave, through fermentation with natural yeast strains. The valorization of such waste may not only include bioethanol production from the liquid part, but also biogas or biochar from the solid fraction (Fig. 1 ). 2. Materials and Methods 2.1 Raw material collection and characterization Agave leaves were obtained from Agave sp. IAC4, a fibrous Brazilian accession possibly remnant from the hybridization experiments conducted in 1958 [ 21 ] at the Agronomic Institute of Campinas (IAC, Campinas - SP, Brazil) [ 22 ]. The biomass was collected at IAC (22° 53' 40.92" S, 47° 3' 46.44" W) on June 1st, 2023. The collected leaves had comparable sizes between them and belonged to the outermost part of the plant. This material was mechanically crushed on a sugarcane juicer (Engenhos para cana B120 alto elétrico, Botini) and sifted to remove great solids from the liquid phase. This juice was immediately frozen at -20 ºC. Juice pH and BRIX were assessed immediately after juice collection. Total reducing sugars (TRS) content was evaluated after acid hydrolysis with 1% v/v sulfuric acid at 120°C for 20 minutes [ 23 ]. Sugar quantification was obtained through high-performing liquid chromatography (HPLC) and is detailed in section 2.4. pH was analyzed using a pH meter (Hanna 21, Hanna Instruments) and º Brix was measured using a refractometer (RHB32, AKSO Produtos Eletrônicos Ltda.). 2.2 Preparation of culture media Juice was thawed at ambient temperature and transferred to 50 mL falcons. Samples were centrifuged at 6000 rpm for 5 minutes. Supernatant was transferred to Schott flasks and ampicillin was added at a 1:1000 ratio to prevent bacterial contamination. Autoclaved agave leaf juice was obtained by autoclaving raw substrate at 120 ºC for 20 minutes. Liquid YPD 2% (10 g.L − 1 yeast extract, 20 g.L − 1 peptone and 20 g.L − 1 glucose) was used for yeast pre-inoculum before fermentation. 2.3 Fermentation Single colonies of Saccharomyces cerevisiae or Kluyveromyces marxianus were inoculated in liquid YPD 2%, where they grew overnight at 30°C and 200 rpm. S. cerevisiae strains used were BG-1, CAT-1, PE-2 and SA-1, all native from the Brazilian bioethanol industry [ 3 ]. The K. marxianus strain was Km3344 (NCYC 744) [ 24 ]. Fermentation was carried out in 250 mL sterilized Erlenmeyer flask containing 100 mL of agave leaf juice. All flasks were sealed with rubber stoppers for semi-anaerobic growth of strains. Strains were inoculated at an initial optical density (OD 600nm) of 1.0 in the fermentation media where they grew at 30°C and 150 rpm for 100 hours. 1 mL samples were collected from flasks at chosen time points for HPLC analysis. All assays were carried out in triplicates. 2.4 Analytical methods Fermentation samples underwent HPLC analysis for the quantification of sugars (sucrose, glucose and fructose), and ethanol. Samples were diluted at a 1:20 ratio with MilliQ water, filtered to HPLC vials through a 0.22 µm PVDF membrane and subjected to liquid chromatography assays in a cation exchange column (Aminex HPX-87H, Bio-Rad Laboratories) in HPLC (e2795 Separations Module, Waters) with a mobile phase of 5 mM sulphuric acid (H 2 SO 4 ). 3. Results and Discussion 3.1 Agave sp . IAC4 leaf juice characterization The leaves from which the juice was extracted were collected from many individuals of a sisalana-like agave, Agave sp. IAC4 accession. Biomass characterization was performed to infer total reducing sugars (TRS) content, pH and °Brix (Table 1 ). TRS concentrations (sucrose, glucose and fructose) in agave leaf juice were assessed before and after acid hydrolysis of the medium. Considering the TRS in hydrolyzed medium, theoretical maximum ethanol yield obtained for fermentations is 21.37 g.L − 1 . Table 1 Characterization of Agave sp. IAC4. Analysis Concentration (g.L -1 ), pH or °Brix in natura hydrolyzed Total Reducing Sugars (TRS) Sucrose 6.17 2.84 Glucose 6.06 16.03 Fructose 5.41 22.87 pH 4.3 Brix 7.0 3.2 The fermentation potential of Agave sp. IAC4 leaves To assess the best media treatment to assure highest ethanol yield, autoclaved, raw and hydrolyzed leaf juice were tested using the S. cerevisiae strain PE-2, a well-established high-performance yeast from the sugarcane bioethanol industry [ 25 – 27 ]. Also, S. cerevisiae is the yeast of choice in the tequila industry for agave fermentation after cooking or hydrolysis of agave piñas . The results are presented in Fig. 2 . Ethanol production in autoclaved agave leaf juice revealed challenging and a small ethanol amount was produced (0.73 ± 0.51 g. L − 1 ) (Fig. 2 A), while in untreated medium it displayed modest titers (5.22 ± 0.10 g. L − 1 ) (Fig. 2 B). Fermentation of hydrolyzed agave leaf juice was also carried out, but no ethanol was observed after 100 hours (data not shown). These results reveal that heat or acid treatment of this substrate renders inhibitory molecules that hinder fermentation. Also, the low fermentation efficiency (29.57%) in raw media, even though it contained a considerable amount of reducing sugars (which, in contrast to fructans, can be readily assimilated by S. cerevisiae ), suggests the presence of native molecules in the substrate that can be toxic to this yeast. Agave biomass is recognized for the presence of steroidal saponins [ 28 , 29 ], which strongly impacts yeast viability [ 30 ]. The presence of foam in the Agave sp. IAC4 leaf juice corroborates with this observation. A. sisalana saponins have already been described [ 17 , 31 ], and given this plant’s possible relationship with the accession IAC4 – since it was created in a breeding program aimed at enhancing fiber content in agave hybrids in Brazil [ 21 , 22 ]– the steroidal content could be extrapolated for the last. As it was previously observed, the presence of saponins in sisal biomass hydrolysis prevented ethanol production [ 32 ], corroborating with the results here presented. To assess whether other yeasts are capable of yielding higher ethanol titers in untreated media, fermentation of this substrate was carried out using other S. cerevisiae strains (BG-1, SA-1 and CAT-1 [ 3 ]) and the K. marxianus yeast Km3344 [ 24 ]. K. marxianus is a microorganism with endogenous fructan-hydrolysis enzymes and one of the most commonly found yeasts in agave [ 33 , 34 ]. In this assay, ethanol production was evaluated after 96 hours of fermentation (Fig. 3 ). The results pointed to the outstanding performance of K. marxianus for the fermentation of the agave leaf juice, producing 11.64 ± 0.49 g. L − 1 of ethanol (54.47% yield), whereas all S. cerevisiae strains were notably less efficient (ranging from 0.93 ± 0.33 g. L − 1 to 3.44 ± 0.27 g. L − 1 ethanol, as it was observed in Fig. 3 ). K. marxianus ’ preeminent performance in the evaluated media implies that, like other endogenous microorganisms found in agave juice, this yeast is well adapted to the media’s phytochemicals and complex ecology. It is also noteworthy that fermentation without cell inoculum (blank) produced similar ethanol titers in comparison to cultures with inoculated S. cerevisiae , suggesting ethanol productivity for this inoculum is related to endogenous microbiota only. Our results are in accordance with previous findings of fermentation of A. tequilana leaf residues and both shed light on the great potential of a biomass currently deemed as residue. Corbin et al. (2015) have evaluated the fermentation potential of untreated A. tequilana liquid leaf residue using S. cerevisiae , rendering a maximum of 13.8 g. L 1 ethanol (66% efficiency), which represents a potential over 691 L. ha − 1 . yr − 1 in ethanol productivity [ 35 ]. Also, a study carried out by the same group indicated that no-input fermentations, that is, spontaneous fermentations of untreated Agave tequilana leaf juice yielded 9.0 g. L 1 of ethanol (32% efficiency). In the same study, the exploration of low-input fermentations with S. cerevisiae strains from the beverage industry or less studied yeast strains such as K. marxianus , Opuntia stricta and Candida akabanensis revealed a potential ethanol productivity of 2687 L. ha − 1 . yr − 1 , that could reach up to 3053 L. ha − 1 . yr − 1 [ 36 ]. 4. Perspective for agave leaves’ residues valorization The Brazilian Northeast concentrates all the country’s sisal fiber production, totalizing 91.9 t of fiber in 2022 produced from different fibrous agave accessions, scattered across 98.4 kha [ 37 ]. By compilating the national fiber production for the last documented decade (2013–2022) (Fig. 4 a), it is estimated that 28.3 Mt of residues (96% in mass, per leaf) were neglected, contributing to methane emissions [ 38 ] and negatively affecting working conditions for manual labor [ 39 ]. On Fig. 4 b, it is depicted the missed ethanol potential for each year in the last documented decade, considering the residue’s liquid fraction (80% of the leaves) and this work’s fermentation conditions, considering the theoretical and feasible volumetric yields of 2.70% and 1.47%, respectively. Over the decade, a range of 348–639 ML could have been produced, and despite sisal production being decreased by half after 2017, an average of 28.5 ML could still be obtained yearly. corresponding to between 267 to 489 L.ha − 1 of ethanol. Compared to sugarcane (7500 L.ha − 1 ) [ 40 ] and second-crop maize (2300 L.ha − 1 ) [ 41 ], this accounts for 3.6%-6.5% and 12%-21%, respectively. The Northeastern state of Bahia houses 96% of the national sisal production [ 37 ], and this estimated agave ethanol potential could represent between 8.4–15.5% of the state demand for this biofuel [ 42 ], currently supplied by sugarcane and second-crop maize ethanol, majoritarily from the Brazilian Southeast and Center-West regions. Additionally, this ethanol volume could replace from 0.06–0.10% of Bahia’s demand for gasoline. In terms of carbon intensity for agave bioethanol, an initial estimate of 16.4–21.8 gCO 2e .MJ − 1 could be drawn. This range considers the life cycle emissions from: i) agricultural and transportation operations, allocated (mass) between fiber and fermentable sugars in the juice [ 13 ]; ii) industrial processing into ethanol [ 43 ]; and iii) sulfuric acid use for agave juice pretreatment, calculated using ecoinvent 3.8 datasets, considering the global market average, on SimaPro 9.4. In substitution to gasoline, the avoided emissions could have represented from 490 to 973 ktCO 2e in Bahia in the period of 2013–2022. Yearly, this corresponds to up to a quarter of Salvador (Bahia’s capitol city) total emissions and, at least, 70% of its energy-related emissions [ 44 ]. Also, this could mitigate the totality of Salvador’s Carnaval emissions, a week-long festivity that attracts 1.65 million tourists and employs 32 thousand workers every year [ 45 ]. In financial terms, this ethanol volume corresponds to a missed revenue of 0.7–1.3 billion Brazilian Reais (BRL) (2,04 BRL.L − 1 , average for the period (CEPEA, 2024), in addition to 50–99 million BRL from decarbonization credits (average price 102.18 BRL (Datagro, 2024) in the RenovaBio program [ 48 ]. Revenue that could correspond to half of sisal fiber’s[ 37 ]. 5. Conclusions Our work sheds light on the relevance of agave residue valorization in the Brazilian bioeconomy. We show that despite the challenges posed by the chemical and biological composition of agave juice, agave can promptly be used to its full potential in the Brazilian semi-arid with high impact in the circular economy with lowered carbon footprint. Strategies for valorization of this biomass relies on the centralization of its production, in contrast to what is currently observed, and full exploration of its residues in a biorefinery context not only to produce ethanol, but also biogas or biochar from the solid fractions. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by the National Agency of Petroleum, Natural Gas and Biofuels (ANP), Brazil. The PD&I Clauses; the Shell Brasil Ltda. The Fundação de Amparo à Pesquisa no Estado de São Paulo (FAPESP) (ACPD: 2022/09349-5), and National Council for the Improvement of Higher Education (CAPES) (JRS: 142340/2020-0). Author’s contributions ACDP and BOV collected and processed the biomass. ACDP and JRS performed the fermentation assays. GPN estimated the carbon intensities for agave bioethanol production and calculations for biofuel missed potential. ACDP, GPN and FSBM wrote the manuscript. FSBM, MFC, and GAGP supervised this work. FSBM was responsible for the conceptualization of this manuscript. Funding for this work was obtained by GAGP. All authors reviewed the manuscript. Acknowledgements The authors thank all the members of our laboratory for their support and scientific advice. We also thank Shell Brasil and the ANP (National Agency of Petroleum, Natural Gas and Biofuels) for their strategic support through regulatory incentives for Research, Development & Innovation. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 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David","email":"","orcid":"","institution":"Universidade Estadual de Campinas - Campus Cidade Universitaria Zeferino Vaz: Universidade Estadual de Campinas","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"C. P.","lastName":"David","suffix":""},{"id":373087702,"identity":"251275c0-f4ec-4901-b445-cc7891f19482","order_by":1,"name":"Guilherme P. Nogueira","email":"","orcid":"","institution":"Universidade Estadual de Campinas - Campus Cidade Universitaria Zeferino Vaz: Universidade Estadual de Campinas","correspondingAuthor":false,"prefix":"","firstName":"Guilherme","middleName":"P.","lastName":"Nogueira","suffix":""},{"id":373087703,"identity":"5ff90c1c-14bc-47f9-b1cf-80630a6e4506","order_by":2,"name":"Jade R. dos Santos","email":"","orcid":"","institution":"Universidade Estadual de Campinas - Campus Cidade Universitaria Zeferino Vaz: Universidade Estadual de Campinas","correspondingAuthor":false,"prefix":"","firstName":"Jade","middleName":"R. dos","lastName":"Santos","suffix":""},{"id":373087704,"identity":"c85c879e-4b22-4cdc-b3aa-2d8808ad0573","order_by":3,"name":"Beatriz O. Vargas","email":"","orcid":"","institution":"Universidade Estadual de Campinas - Campus Cidade Universitaria Zeferino Vaz: Universidade Estadual de Campinas","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"O.","lastName":"Vargas","suffix":""},{"id":373087705,"identity":"22df4c45-5abb-4194-9506-165e07d8d4cb","order_by":4,"name":"Marcelo F. Carazzolle","email":"","orcid":"","institution":"Universidade Estadual de Campinas - Campus Cidade Universitaria Zeferino Vaz: Universidade Estadual de Campinas","correspondingAuthor":false,"prefix":"","firstName":"Marcelo","middleName":"F.","lastName":"Carazzolle","suffix":""},{"id":373087706,"identity":"b96bc6d6-a468-4419-b4b6-80d2f0223fcb","order_by":5,"name":"Goncalo Pereira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYNCCAgY5BgbGBjDbgDgtBgzGpGtJbECwCQB5996DH34Y2KRvuN3c+OFnzh0Gc+kD+LUYnjmXLNljkJa74c7BZsnebc8YLPsSCGiZkWPGwGNwOHfDjcQ2Bt5thxkMzhBwGEgL4x+Dw+kGQC2Mf4nRIi+RY8YMtCUBpIWZKFsMeM4YS8sYpBnOvJHYLC277RmPZQ8hW9p7DD++qbCR57uR/vDj22135Mx5CNlyAJV/gJAGoC0NaFoI6hgFo2AUjIKRBwAblETg3P44qwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4140-3482","institution":"University of Campinas - UNICAMP","correspondingAuthor":true,"prefix":"","firstName":"Goncalo","middleName":"","lastName":"Pereira","suffix":""},{"id":373087707,"identity":"6a271e0d-abeb-46f6-bf72-c8b7087bb44c","order_by":6,"name":"Fellipe S. B. de Mello","email":"","orcid":"","institution":"Universidade Estadual de Campinas - Campus Cidade Universitaria Zeferino Vaz: Universidade Estadual de Campinas","correspondingAuthor":false,"prefix":"","firstName":"Fellipe","middleName":"S. B.","lastName":"de Mello","suffix":""}],"badges":[],"createdAt":"2024-10-31 22:27:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5369383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5369383/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68916733,"identity":"51d88b2b-6bf0-4680-9ac2-b1b3b130a9ed","added_by":"auto","created_at":"2024-11-13 13:00:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":335563,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the current and potential use of agave leaves residue in Brazil. In red, the current use of agave leaves in Brazil for fiber production, where its residues are discarded. In green, the proposed use of this residue. In this new scenario, leaves are entirely used: its fibers are still employed for sisal confection, while the remaining 96% is separated in liquid phase, for bioethanol production, and solid, for biogas or biochar production\u003c/p\u003e","description":"","filename":"BR01AgaveProductionBrazilScheme.png","url":"https://assets-eu.researchsquare.com/files/rs-5369383/v1/8b53edbf8aadb52dc495229f.png"},{"id":68916730,"identity":"ac6e43d6-8342-4158-b3ad-9d32dcfa5e6c","added_by":"auto","created_at":"2024-11-13 13:00:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66426,"visible":true,"origin":"","legend":"\u003cp\u003eFermentation of \u003cem\u003eAgave \u003c/em\u003esp. IAC4 leaf residues with different pre-treatment using \u003cem\u003eS. cerevisiae\u003c/em\u003e strain PE-2. Dark circles represent ethanol concentration, dark triangles, sucrose; light circle, glucose; light triangle, fructose. Data are represented as means and standard deviation of three replicates. \u003cstrong\u003ea. \u003c/strong\u003eFermentation of autoclaved agave leaf juice. \u003cstrong\u003eb.\u003c/strong\u003e Fermentation of raw untreated agave leaf juice\u003c/p\u003e","description":"","filename":"BR02MediaTreatment.png","url":"https://assets-eu.researchsquare.com/files/rs-5369383/v1/9b7281632231c4ea0da468ef.png"},{"id":68916734,"identity":"a343a796-c3ec-420e-937e-1691ea6a5a85","added_by":"auto","created_at":"2024-11-13 13:00:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20482,"visible":true,"origin":"","legend":"\u003cp\u003eEthanol concentration after 96 hours fermentation of raw untreated \u003cem\u003eAgave \u003c/em\u003esp. IAC4 leaf residues using different yeast strains. BG-1, CAT-1, PE-2 and SA-1 are industrial bioethanol \u003cem\u003eS. cerevisiae\u003c/em\u003e, and Km3344 is a \u003cem\u003eK. marxianus \u003c/em\u003estrain. Data for each replicate are represented as circles in each bar, with standard deviation for three replicates\u003c/p\u003e","description":"","filename":"BR03Strains.png","url":"https://assets-eu.researchsquare.com/files/rs-5369383/v1/cfdcfed8b12cc6e3b5d1c2dc.png"},{"id":68916731,"identity":"e196dc32-1703-4a25-9862-fa5167734d02","added_by":"auto","created_at":"2024-11-13 13:00:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57024,"visible":true,"origin":"","legend":"\u003cp\u003eEthanol production potential from agave leaf residues in the Brazilian Northeast. \u003cstrong\u003ea\u003c/strong\u003e. Sisal (fiber) production in the last documented decade (IBGE, 2022). \u003cstrong\u003eb\u003c/strong\u003e. Ethanol yield from the residual liquid fraction (80% in mass of agave leaves) considering theoretical and maximum yield observed in this study for Agave sp. IAC4\u003c/p\u003e","description":"","filename":"BR04FiberEthanol.png","url":"https://assets-eu.researchsquare.com/files/rs-5369383/v1/37b749ee401046484ff03c1d.png"},{"id":77644313,"identity":"f1b5b130-ec0a-4c56-aa32-2cfed42065fa","added_by":"auto","created_at":"2025-03-03 22:45:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1030734,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5369383/v1/364b1985-9197-4068-9ee0-b2bb4ec20e92.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eFrom Rags to Riches: the Fermentation Potential of Agave Leaf Residues in the Brazilian Semi-arid\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBrazil, the world\u0026rsquo;s second-largest bioethanol producer, and a leading country in integrating renewables into its energy matrix [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], has relied on sugarcane for bioethanol production since the 1970s [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite the increasing production of second-generation and maize-based ethanol (Conab, n.d.; Silva and Casta\u0026ntilde;eda-Ayarza, 2021), sugarcane remains the dominant feedstock in the bioethanol sector, a position that is both crucial and vulnerable. Recent years have seen sugarcane confronted with significant climatic challenges, including periods of severe drought and unexpected fires or frosts [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], underscoring the need for feedstock diversification.\u003c/p\u003e \u003cp\u003eAgave is a promising new feedstock in this context. Thanks to its specialized Crassulacean Acid Metabolism (CAM), which minimizes water loss, agave exhibits exceptional tolerance to high temperatures and intense droughts [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This enables it to thrive on hostile lands where few other plants, particularly food crops, can survive [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Data from the tequila industry, in Mexico, indicate that ethanol can be produced from agave at high yields (5,000\u0026ndash;6,000 L ethanol ha⁻\u0026sup1; yr⁻\u0026sup1;), with a productivity comparable to that of sugarcane in Brazil, while requiring significantly less water (300\u0026ndash;800 mm yr⁻\u0026sup1;, nearly 25% of the water needed for sugarcane) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although the main agave varieties cultivated in Brazil (such as \u003cem\u003eAgave sisalana\u003c/em\u003e) differs from that in Mexico (\u003cem\u003eA. tequilana\u003c/em\u003e) and is used for fiber rather than ethanol production, the fibrous accessions still contain significant sugar content [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAgave already plays an important role in the Brazilian economy, particularly in Northeast region, where it is cultivated in a non-centralized manner by small-scale farmers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eA. sisalana\u003c/em\u003e fibers represent only 4% of its leaves and are the only part of the plant that is harvested for sisal confection, while the remaining 96% of the leaves are discarded [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These residues are rich in fructan-type carbohydrates called inulin or agavin, that could be fermented after hydrolysis into biofuels and other biobased products [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this work we aimed to explore the ethanol production potential from the residual leaf juice of a Brazilian agave accession, \u003cem\u003eAgave sp.\u003c/em\u003e IAC4, a \u0026ldquo;sisalana-like\u0026rdquo; agave, through fermentation with natural yeast strains. The valorization of such waste may not only include bioethanol production from the liquid part, but also biogas or biochar from the solid fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Raw material collection and characterization\u003c/h2\u003e \u003cp\u003eAgave leaves were obtained from \u003cem\u003eAgave\u003c/em\u003e sp. IAC4, a fibrous Brazilian accession possibly remnant from the hybridization experiments conducted in 1958 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] at the Agronomic Institute of Campinas (IAC, Campinas - SP, Brazil) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The biomass was collected at IAC (22\u0026deg; 53' 40.92\" S, 47\u0026deg; 3' 46.44\" W) on June 1st, 2023. The collected leaves had comparable sizes between them and belonged to the outermost part of the plant. This material was mechanically crushed on a sugarcane juicer (Engenhos para cana B120 alto el\u0026eacute;trico, Botini) and sifted to remove great solids from the liquid phase. This juice was immediately frozen at -20 \u0026ordm;C. Juice pH and BRIX were assessed immediately after juice collection. Total reducing sugars (TRS) content was evaluated after acid hydrolysis with 1% v/v sulfuric acid at 120\u0026deg;C for 20 minutes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Sugar quantification was obtained through high-performing liquid chromatography (HPLC) and is detailed in section 2.4. pH was analyzed using a pH meter (Hanna 21, Hanna Instruments) and \u0026ordm; Brix was measured using a refractometer (RHB32, AKSO Produtos Eletr\u0026ocirc;nicos Ltda.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of culture media\u003c/h2\u003e \u003cp\u003eJuice was thawed at ambient temperature and transferred to 50 mL falcons. Samples were centrifuged at 6000 rpm for 5 minutes. Supernatant was transferred to Schott flasks and ampicillin was added at a 1:1000 ratio to prevent bacterial contamination. Autoclaved agave leaf juice was obtained by autoclaving raw substrate at 120 \u0026ordm;C for 20 minutes. Liquid YPD 2% (10 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yeast extract, 20 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e peptone and 20 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose) was used for yeast pre-inoculum before fermentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fermentation\u003c/h2\u003e \u003cp\u003eSingle colonies of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e or \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e were inoculated in liquid YPD 2%, where they grew overnight at 30\u0026deg;C and 200 rpm. \u003cem\u003eS. cerevisiae\u003c/em\u003e strains used were BG-1, CAT-1, PE-2 and SA-1, all native from the Brazilian bioethanol industry [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The \u003cem\u003eK. marxianus\u003c/em\u003e strain was Km3344 (NCYC 744) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Fermentation was carried out in 250 mL sterilized Erlenmeyer flask containing 100 mL of agave leaf juice. All flasks were sealed with rubber stoppers for semi-anaerobic growth of strains. Strains were inoculated at an initial optical density (OD 600nm) of 1.0 in the fermentation media where they grew at 30\u0026deg;C and 150 rpm for 100 hours. 1 mL samples were collected from flasks at chosen time points for HPLC analysis. All assays were carried out in triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analytical methods\u003c/h2\u003e \u003cp\u003eFermentation samples underwent HPLC analysis for the quantification of sugars (sucrose, glucose and fructose), and ethanol. Samples were diluted at a 1:20 ratio with MilliQ water, filtered to HPLC vials through a 0.22 \u0026micro;m PVDF membrane and subjected to liquid chromatography assays in a cation exchange column (Aminex HPX-87H, Bio-Rad Laboratories) in HPLC (e2795 Separations Module, Waters) with a mobile phase of 5 mM sulphuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 \u003cem\u003eAgave sp\u003c/em\u003e. IAC4 leaf juice characterization\u003c/h2\u003e \u003cp\u003eThe leaves from which the juice was extracted were collected from many individuals of a sisalana-like agave, \u003cem\u003eAgave\u003c/em\u003e sp. IAC4 accession. Biomass characterization was performed to infer total reducing sugars (TRS) content, pH and \u0026deg;Brix (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). TRS concentrations (sucrose, glucose and fructose) in agave leaf juice were assessed before and after acid hydrolysis of the medium. Considering the TRS in hydrolyzed medium, theoretical maximum ethanol yield obtained for fermentations is 21.37 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of \u003cem\u003eAgave sp.\u003c/em\u003e IAC4.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eAnalysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eConcentration (g.L\u003csup\u003e-1\u003c/sup\u003e), pH or \u0026deg;Brix\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ein natura\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehydrolyzed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTotal Reducing Sugars (TRS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFructose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eBrix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The fermentation potential of \u003cem\u003eAgave\u003c/em\u003e sp. IAC4 leaves\u003c/h2\u003e \u003cp\u003eTo assess the best media treatment to assure highest ethanol yield, autoclaved, raw and hydrolyzed leaf juice were tested using the \u003cem\u003eS. cerevisiae\u003c/em\u003e strain PE-2, a well-established high-performance yeast from the sugarcane bioethanol industry [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Also, \u003cem\u003eS. cerevisiae\u003c/em\u003e is the yeast of choice in the tequila industry for agave fermentation after cooking or hydrolysis of agave \u003cem\u003epi\u0026ntilde;as\u003c/em\u003e. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Ethanol production in autoclaved agave leaf juice revealed challenging and a small ethanol amount was produced (0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 g. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), while in untreated medium it displayed modest titers (5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 g. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Fermentation of hydrolyzed agave leaf juice was also carried out, but no ethanol was observed after 100 hours (data not shown). These results reveal that heat or acid treatment of this substrate renders inhibitory molecules that hinder fermentation. Also, the low fermentation efficiency (29.57%) in raw media, even though it contained a considerable amount of reducing sugars (which, in contrast to fructans, can be readily assimilated by \u003cem\u003eS. cerevisiae\u003c/em\u003e), suggests the presence of native molecules in the substrate that can be toxic to this yeast.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAgave biomass is recognized for the presence of steroidal saponins [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], which strongly impacts yeast viability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The presence of foam in the \u003cem\u003eAgave\u003c/em\u003e sp. IAC4 leaf juice corroborates with this observation. \u003cem\u003eA. sisalana\u003c/em\u003e saponins have already been described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and given this plant\u0026rsquo;s possible relationship with the accession IAC4 \u0026ndash; since it was created in a breeding program aimed at enhancing fiber content in agave hybrids in Brazil [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u0026ndash; the steroidal content could be extrapolated for the last. As it was previously observed, the presence of saponins in sisal biomass hydrolysis prevented ethanol production [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], corroborating with the results here presented.\u003c/p\u003e \u003cp\u003eTo assess whether other yeasts are capable of yielding higher ethanol titers in untreated media, fermentation of this substrate was carried out using other \u003cem\u003eS. cerevisiae\u003c/em\u003e strains (BG-1, SA-1 and CAT-1 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]) and the \u003cem\u003eK. marxianus\u003c/em\u003e yeast Km3344 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eK. marxianus\u003c/em\u003e is a microorganism with endogenous fructan-hydrolysis enzymes and one of the most commonly found yeasts in agave [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this assay, ethanol production was evaluated after 96 hours of fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results pointed to the outstanding performance of \u003cem\u003eK. marxianus\u003c/em\u003e for the fermentation of the agave leaf juice, producing 11.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 g. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of ethanol (54.47% yield), whereas all \u003cem\u003eS. cerevisiae\u003c/em\u003e strains were notably less efficient (ranging from 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 g. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 g. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ethanol, as it was observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eK. marxianus\u003c/em\u003e\u0026rsquo; preeminent performance in the evaluated media implies that, like other endogenous microorganisms found in agave juice, this yeast is well adapted to the media\u0026rsquo;s phytochemicals and complex ecology. It is also noteworthy that fermentation without cell inoculum (blank) produced similar ethanol titers in comparison to cultures with inoculated \u003cem\u003eS. cerevisiae\u003c/em\u003e, suggesting ethanol productivity for this inoculum is related to endogenous microbiota only.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur results are in accordance with previous findings of fermentation of \u003cem\u003eA. tequilana\u003c/em\u003e leaf residues and both shed light on the great potential of a biomass currently deemed as residue. Corbin et al. (2015) have evaluated the fermentation potential of untreated \u003cem\u003eA. tequilana\u003c/em\u003e liquid leaf residue using \u003cem\u003eS. cerevisiae\u003c/em\u003e, rendering a maximum of 13.8 g. L\u003csup\u003e1\u003c/sup\u003e ethanol (66% efficiency), which represents a potential over 691 L. ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. yr\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in ethanol productivity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Also, a study carried out by the same group indicated that no-input fermentations, that is, spontaneous fermentations of untreated \u003cem\u003eAgave tequilana\u003c/em\u003e leaf juice yielded 9.0 g. L\u003csup\u003e1\u003c/sup\u003e of ethanol (32% efficiency). In the same study, the exploration of low-input fermentations with \u003cem\u003eS. cerevisiae\u003c/em\u003e strains from the beverage industry or less studied yeast strains such as \u003cem\u003eK. marxianus\u003c/em\u003e, \u003cem\u003eOpuntia stricta\u003c/em\u003e and \u003cem\u003eCandida akabanensis\u003c/em\u003e revealed a potential ethanol productivity of 2687 L. ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. yr\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, that could reach up to 3053 L. ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. yr\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Perspective for agave leaves’ residues valorization","content":"\u003cp\u003eThe Brazilian Northeast concentrates all the country\u0026rsquo;s sisal fiber production, totalizing 91.9 t of fiber in 2022 produced from different fibrous agave accessions, scattered across 98.4 kha [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. By compilating the national fiber production for the last documented decade (2013\u0026ndash;2022) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), it is estimated that 28.3 Mt of residues (96% in mass, per leaf) were neglected, contributing to methane emissions [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and negatively affecting working conditions for manual labor [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. On Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, it is depicted the missed ethanol potential for each year in the last documented decade, considering the residue\u0026rsquo;s liquid fraction (80% of the leaves) and this work\u0026rsquo;s fermentation conditions, considering the theoretical and feasible volumetric yields of 2.70% and 1.47%, respectively. Over the decade, a range of 348\u0026ndash;639 ML could have been produced, and despite sisal production being decreased by half after 2017, an average of 28.5 ML could still be obtained yearly. corresponding to between 267 to 489 L.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of ethanol. Compared to sugarcane (7500 L.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and second-crop maize (2300 L.ha \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], this accounts for 3.6%-6.5% and 12%-21%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Northeastern state of Bahia houses 96% of the national sisal production [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and this estimated agave ethanol potential could represent between 8.4\u0026ndash;15.5% of the state demand for this biofuel [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], currently supplied by sugarcane and second-crop maize ethanol, majoritarily from the Brazilian Southeast and Center-West regions. Additionally, this ethanol volume could replace from 0.06\u0026ndash;0.10% of Bahia\u0026rsquo;s demand for gasoline. In terms of carbon intensity for agave bioethanol, an initial estimate of 16.4\u0026ndash;21.8 gCO\u003csub\u003e2e\u003c/sub\u003e.MJ\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be drawn. This range considers the life cycle emissions from: i) agricultural and transportation operations, allocated (mass) between fiber and fermentable sugars in the juice [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; ii) industrial processing into ethanol [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]; and iii) sulfuric acid use for agave juice pretreatment, calculated using ecoinvent 3.8 datasets, considering the global market average, on SimaPro 9.4.\u003c/p\u003e \u003cp\u003eIn substitution to gasoline, the avoided emissions could have represented from 490 to 973 ktCO\u003csub\u003e2e\u003c/sub\u003e in Bahia in the period of 2013\u0026ndash;2022. Yearly, this corresponds to up to a quarter of Salvador (Bahia\u0026rsquo;s capitol city) total emissions and, at least, 70% of its energy-related emissions [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Also, this could mitigate the totality of Salvador\u0026rsquo;s Carnaval emissions, a week-long festivity that attracts 1.65\u0026nbsp;million tourists and employs 32 thousand workers every year [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In financial terms, this ethanol volume corresponds to a missed revenue of 0.7\u0026ndash;1.3\u0026nbsp;billion Brazilian Reais (BRL) (2,04 BRL.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, average for the period (CEPEA, 2024), in addition to 50\u0026ndash;99\u0026nbsp;million BRL from decarbonization credits (average price 102.18 BRL (Datagro, 2024) in the RenovaBio program [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Revenue that could correspond to half of sisal fiber\u0026rsquo;s[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur work sheds light on the relevance of agave residue valorization in the Brazilian bioeconomy. We show that despite the challenges posed by the chemical and biological composition of agave juice, agave can promptly be used to its full potential in the Brazilian semi-arid with high impact in the circular economy with lowered carbon footprint. Strategies for valorization of this biomass relies on the centralization of its production, in contrast to what is currently observed, and full exploration of its residues in a biorefinery context not only to produce ethanol, but also biogas or biochar from the solid fractions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Agency of Petroleum, Natural Gas and Biofuels (ANP), Brazil. The PD\u0026amp;I Clauses; the Shell Brasil Ltda. The Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa no Estado de S\u0026atilde;o Paulo (FAPESP) (ACPD: 2022/09349-5), and National Council for the Improvement of Higher Education (CAPES) (JRS: 142340/2020-0).\u003c/p\u003e\u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e \u003cp\u003eACDP and BOV collected and processed the biomass. ACDP and JRS performed the fermentation assays. GPN estimated the carbon intensities for agave bioethanol production and calculations for biofuel missed potential. ACDP, GPN and FSBM wrote the manuscript. FSBM, MFC, and GAGP supervised this work. FSBM was responsible for the conceptualization of this manuscript. Funding for this work was obtained by GAGP. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank all the members of our laboratory for their support and scientific advice. We also thank Shell Brasil and the ANP (National Agency of Petroleum, Natural Gas and Biofuels) for their strategic support through regulatory incentives for Research, Development \u0026amp; Innovation.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSilva ALda, Casta\u0026ntilde;eda-Ayarza JA (2021) Macro-environment analysis of the corn ethanol fuel development in Brazil. 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Accessed 2 May 2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuura MISF, Scachetti MT, Chagas MF et al (2018) RenovaCalc: M\u0026eacute;todo e ferramenta para a contabilidade da. Intensidade de Carbono de Biocombust\u0026iacute;veis no Programa RenovaBio\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Agave, Saccharomyces cerevisiae, bioethanol, waste, semi-arid","lastPublishedDoi":"10.21203/rs.3.rs-5369383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5369383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCAM plants are promising biomasses to assure energy security and biofuel supply in the current changing climate scenario. Their high sugar content and strengthened tolerance to high temperatures and droughts makes them attractive alternatives to classic fuel sources. In Brazil, sisal (\u003cem\u003eAgave sisalana\u003c/em\u003e), is cultivated in semiarid regions for fiber production. However, fibers represent only 4% of the plant\u0026rsquo;s leaves, with the remaining majority being discarded. This work, then, aims to explore this residue\u0026rsquo;s potential for bioethanol production. For this, low-input fermentations of a fibrous Brazilian agave accession leaves were explored. A maximum ethanol yield of 54.47% (11.64 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was obtained with \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e. Isolating endogenous microbiota activity and fermentation inhibitors (i.e. saponins) revealed major operational challenges. Nevertheless, the results demonstrate that bioethanol production from agave residues is not only attainable but also promising. The unexplored bioethanol potential from this residue in the Brazilian semiarid could yield 489 L.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.yr\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, totalizing 639\u0026nbsp;million liters of fuel, in the last decade.\u003c/p\u003e","manuscriptTitle":"From Rags to Riches: the Fermentation Potential of Agave Leaf Residues in the Brazilian Semi-arid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-13 13:00:31","doi":"10.21203/rs.3.rs-5369383/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c06600a3-afa3-4f01-a750-408e14e817aa","owner":[],"postedDate":"November 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T22:37:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-13 13:00:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5369383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5369383","identity":"rs-5369383","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}