Valorization of an agroextractive residue—Carnauba straw—for the production of bioethanol by simultaneous saccharification and fermentation (SSF)

Accepted Manuscript Valorization of an agroextractive residue—Carnauba straw—for the production of bioethanol by simultaneous saccharification and fermentation (SSF) Francinaldo Leite da Silva, Alan de Oliveira Campos, Davi Alves dos Santos, Emilianny Rafaely Batista Magalhães, Gorete Ribeiro de Macedo, Everaldo Silvino dos Santos PII:

S0960-1481(18)30546-9

DOI:

10.1016/j.renene.2018.05.025

Reference:

RENE 10077

To appear in:

Renewable Energy

Received Date: 20 October 2017 Revised Date:

16 March 2018

Accepted Date: 5 May 2018

Please cite this article as: da Silva FL, de Oliveira Campos A, dos Santos DA, Batista Magalhães ER, de Macedo GR, dos Santos ES, Valorization of an agroextractive residue—Carnauba straw—for the production of bioethanol by simultaneous saccharification and fermentation (SSF), Renewable Energy (2018), doi: 10.1016/j.renene.2018.05.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Valorization of an agroextractive residue—carnauba straw—for the production of bioethanol by simultaneous saccharification and fermentation (SSF) Francinaldo Leite da Silvaa,b, - [email protected]

Davi Alves dos Santosa, - [email protected]

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Alan de Oliveira Camposa, - [email protected]

Gorete Ribeiro de Macedoa, - [email protected]

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Everaldo Silvino dos Santosa,* - [email protected]

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Emilianny Rafaely Batista Magalhãesa, - [email protected]

. Laboratory of Biochemical Engineering, Chemical Engineering Department, Federal University of Rio

Grande do Norte (UFRN), Natal-RN, Brazil.

. Instituto Federal de Educação Ciência e Tecnologia da Paraíba Campus Picuí (IFPB) Picuí/PB, Brazil.

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*Corresponding author: [email protected]. Address: UFRN - Centro de Tecnologia - CT - Avenida

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Senador Salgado Filho, 3000 - Lagoa Nova, Natal - RN, zipcode: 59064-741

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ACCEPTED MANUSCRIPT

Hydrotermal (HT) Acid Alkaline (AA) Alkaline (AL)

Enzymatic Hydrolysys

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Carnauba

Chemical composition

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Pretreatments:

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Resíduo untreated (UN-T)

Simultaneous Saccharification and Fermentation (SSF)

ACCEPTED MANUSCRIPT Valorization of an agroextractive residue—carnauba straw—for the production of

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bioethanol by simultaneous saccharification and fermentation (SSF)

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Francinaldo Leite da Silvaa,b, Alan de Oliveira Camposa, Davi Alves dos Santosa,

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Emilianny Rafaely Batista Magalhãesa, Gorete Ribeiro de Macedoa, Everaldo Silvino

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dos Santosa,*

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a

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Grande do Norte (UFRN), Natal-RN, Brazil.

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b

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*Corresponding author: [email protected]. Address: UFRN - Centro de Tecnologia - CT - Avenida

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. Laboratory of Biochemical Engineering, Chemical Engineering Department, Federal University of Rio

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. Instituto Federal de Educação Ciência e Tecnologia da Paraíba Campus Picuí (IFPB) Picuí/PB, Brazil.

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Senador Salgado Filho, 3000 - Lagoa Nova, Natal - RN, zipcode: 59064-741

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Abstract

In this study, the use of carnauba straw residue to produce cellulosic ethanol by

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simultaneous saccharification and fermentation (SSF) with three industrial yeasts was

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investigated. Hydrothermal (HT), alkaline (AL), and acid–alkaline (AA) pretreatments

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were carried out on the residues, and the efficiency of the enzymatic hydrolysis of these

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residues was evaluated. The SSF was performed using Saccharomyces cerevisiae UFLA

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CA11, Saccharomyces cerevisiae CAT-1, and Kluyveromyces marxianus ATCC-36907

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at 35, 40, and 45 °C. The AL pretreatment resulted in the best removal of lignin and

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hemicellulose. In addition, enzymatic hydrolysis of the dry residue treated with AL

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converted 64.43% of the lignocellulosic biomass to sugars. SSF of the AL-pretreated

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residue using Kluyveromyces marxianus ATCC-36907 cultivated at 45 °C produced

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7.53 g/L ethanol.

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ACCEPTED MANUSCRIPT Keywords

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Carnauba straw, bioethanol, pretreatment, lignocellulosic residue, SSF

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Abbreviations

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UT-R, Untreated Carnauba waste; HT, hydrothermal pretreatment; AA, acid–alkaline

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pretreatment; AL, alkaline pretreatment; SSF, simultaneous saccharification and

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fermentation

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1. Introduction

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Carnauba is a plant native to the Brazilian northeast (Copernicia prunifera

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(Miller) H. E. Moore). Its main characteristic is its resistance to the climatic conditions

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of this semi-arid region; therefore, carnauba provides an important economic resource

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for extractive farmers during dry period. The main economic value of carnauba is the

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extraction of the wax that covers its leaves, especially young leaves, and is known

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internationally as “carnauba wax” [1,2]. According to data from the Brazilian Institute

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of Geography and Statistics (IBGE), in 2016 alone 17300 tons of wax powder were

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produced [3]. In addition, a mass balance of wax production demonstrated that 1000

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straws are required to produce 7.8 kg of wax powder, generating approximately 352210

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tons of lignocellulosic waste annually [4,5]. The straw waste after wax extraction

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consists of a material rich in lignocellulosic residue, with potential for biotechnological

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application in biorefining [6]. Thus, this biomass could be used to produce ethanol in

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situ coupled to the carnauba wax industry.

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Lignocellulosic wastes have been the subject of many studies in recent years, as

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these wastes can undergo biodegradation to generate value-added chemicals such as

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butanol, xylitol, and cellulosic ethanol [7–9]. Cellulosic ethanol is important as a clean

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ACCEPTED MANUSCRIPT alternative fuel in light of the growing demand for fossil fuels, which are nonrenewable

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and emit large concentrations of CO2 into the atmosphere, worsening the greenhouse

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effect and consequently increasing of global temperature. In addition to being a

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renewable energy source, the production of cellulosic ethanol does not necessarily

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require an increase of the amount of land devoted to monoculture cultivation.

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Additionally, cellulosic ethanol production would not affect the food supply once there

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is a wide variety of residual biomass available worldwide for bioconversion [10–12].

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However, lignocellulosic material requires pretreatment to achieve the efficient

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conversion of polysaccharides into fermentable sugars [13].

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Several pretreatments have been used to improve the hydrolysis of biomass. These

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pretreatments are necessary because the lignocellulosic biomass is recalcitrant, which

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reduces the accessibility and digestibility of the lignocellulolytic enzymes [14,15].

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Physical, chemical, or combined pretreatments have been described in the scientific

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literature, including steam explosion, microwave, ultrasonic, hydrothermal, dilute acid,

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alkaline, and oxidative pretreatments [16–18]. In general, they these methods modify

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the physical structure and chemical composition of the lignocellulosic biomass, mainly

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by altering the cellulose chains, removing lignin or hemicellulose, or inducing both of

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these changes simultaneously. However, the production of cellulosic ethanol is also

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complicated by a combination of other factors: the reduction of the amount of inhibitors

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formed as a result of pretreatment of the biomass; the need for enzymatic complexes

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with a high degree of synergism and low inhibition by feedback; the selection of

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fermenting microorganisms that are tolerant to variation in pH and temperature and are

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capable of metabolizing both hexoses and pentoses; and the development of a suitable

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fermentation strategy that allows the maximum yield of ethanol [19–22].

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ACCEPTED MANUSCRIPT Different strategies have been employed for the fermentation of sugars from

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lignocellulosics into ethanol, including separate hydrolysis and fermentation (SHF),

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simultaneous saccharification and fermentation (SSF), and semi-simultaneous

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saccharification and fermentation (SSSF) [23]. SHF has a high hydrolysis rate, but

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involves a higher amount of inhibitors, which increases the number of processing steps

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required, and thus, the final cost of the ethanol. In contrast, in SSF less inhibitors

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formation occurs, and the process is carried out in a single step. SSSF has been

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presented as an alternative method to reduce the negative effects of SHF and SSF

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[24,25]. However, the efficiency of these processes depends on the microorganism

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strains used during fermentation.

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In this context, this study aims to investigate the enzymatic hydrolysis and

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cellulosic ethanol production from carnauba straw residues subjected to hydrothermal

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(HT), alkaline (AL), and combined acid and alkaline (AA) pretreatments. Fermentation

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was carried out by an SSF process using three different yeast strains: Saccharomyces

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cerevisiae UFLA CA11, Saccharomyces cerevisiae CAT-1, and Kluyveromyces

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marxianus ATCC-36907.

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2. Materials and methods

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2.1. Microorganisms

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Three microorganisms were used in the fermentation process for the production

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of ethanol from carnauba straw residues: Saccharomyces cerevisiae UFLA CA11, a

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yeast that was previously isolated from the spontaneous fermentation of sugarcane juice

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in different stills located in cities in the state of Minas Gerais, Brazil [26];

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Saccharomyces cerevisiae CAT-1, which is an industrial yeast used in distilleries in

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Brazil, and was supplied by LNF Latino Americana Co., Brazil [27]; and finally,

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Kluyveromyces marxianus ATCC-36907. The microorganisms were kept in glycerol at

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the Biochemical Engineering Laboratory (LEB) in the Federal University of Rio Grande

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do Norte, Brazil.

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2.2. Lignocellulosic biomass

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Carnauba straw was supplied by “Carnauba Viva,” a non-governmental

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organization (Assu, Rio Grande do Norte – Brazil). The residues were processed to

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extract the carnauba wax by the extractive farmers of the NGO. Upon arrival to the lab,

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the residues were washed thoroughly using water to remove particulate materials and

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sugar residues. Subsequently, the residues were dried at 70 °C for 48 h, ground in a mill

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(Willye, TE - 680, Tecnal), sifted to 20 mesh and stored at room temperature (25 °C).

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3.2 Biomass pretreatment

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The carnauba straw residue obtained after the extraction of carnauba wax by the

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extractive farmers was pretreated using one of three different pretreatments:

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hydrothermal (HT), alkaline (AL), or alkaline–acid (AA) pretreatment. For all

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pretreatments, a solids load of 20% (w/v) was used. For the HT pretreatment, a mixture

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consisting of water and carnauba residue was prepared. The mixture was heated to 121

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°C for 30 min in an autoclave, and then washed until the pH reached a value of

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approximately 7.0; three washes were typically required. The AL pretreatment was

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performed in a 4% NaOH solution (w/v). This mixture was also heated to 121 °C for 30

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min [14]. Subsequently, the residue was washed with water until a pH value of

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approximately 7.0 was achieved; in this case, twelve washes were usually required.

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Finally, the AA pretreatment consisted of two steps, combining the AL and AA

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pretreatments. In the first step, the pretreatment process was the same as in the AL

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ACCEPTED MANUSCRIPT method. After the washing process in the first step, the residue was pretreated again by

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submersion in a 2% (v/v) H2SO4 solution. The mixture was then heated to 121 °C for 30

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min, and subsequently, the residue was washed with water until a pH of approximately

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7.0 was achieved; thus, nine washes were required. Next, part of pretreated residue was

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used while still wet, and another part was dried at 50 °C for 24 h for a preliminary

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hydrolysis test.

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3.3 Analysis of the substrate composition

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The compositional analysis of substrate (natural and pretreated carnauba fiber)

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after pretreatment followed a standard NREL protocol [28–31]. A 0.3 g sample of dried

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and extractive-free substrate was placed in a pressure tube to which 3.0 mL of a 72% by

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weight H2SO4 solution was added. The mixture was left at 30 °C for 1 h and stirred with

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a glass rod every 10 min. Subsequently, 84.0 g of deionized water was added to achieve

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a concentration of 4% H2SO4 by weight. The samples were then heated at 120 °C for 1 h

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in an autoclave. The resulting solution was filtered and used for HPLC sugar analysis.

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The precipitate was washed with water to a neutral pH, then dried at 105 °C and

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weighed to determine the amount of Klason lignin.

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3.4 Enzymatic hydrolysis of the biomass after different pretreatments

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3.4.1 Enzymes and enzymatic activity

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In the study of the hydrolysis of untreated and pretreated carnauba residues, the

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cellulase enzymes from Trichoderma reesei ATCC 26921, supplied from Sigma-

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Aldrich (USA), and β-glucosidase NS-22118 and xylanase NS-22036, both acquired

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from Novozymes (Denmark), were used. The total cellulase enzymatic activity was

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expressed as filter paper activity (FPU), and the activities of the β-glucosidase enzymes

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ACCEPTED MANUSCRIPT were estimated according to the method of Ghose [32]. The total cellulases and β-

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glucosidase enzymatic activities (FPU and CBU, respectively) were defined as the

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amount of enzyme amount necessary to produce 1.0 µmol of glucose per minute under

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the assay conditions. In addition, xylanase activity was also estimated according to the

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method of Ghose [32]. In this case, the xylanase activity (FXU) was defined as the

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amount of enzyme required to produce 1.0 µmol of xylose per minute under the test

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conditions.

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3.4.2 Enzymatic hydrolysis

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The enzymatic hydrolysis of the pretreated and untreated carnauba straw residue

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was performed according to the method of Yang [33] and Araujo [34]. It should be

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highlighted that the AA-pretreated residue was not hydrolyzed, since a low yield was

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observed after this pretreatment. The solids loading used for the enzymatics hydrolysis

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was 4% (w/v). The solids were loaded into 250 mL Erlenmeyer flasks, and sodium

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citrate buffer (50 mM, pH 4.8) and a 0.01% (w/v) sodium azide solution were added to

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prevent microbial growth during hydrolysis [35]. The enzymatic loading was 20.0 FPU

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per gram of residue, 20.0 CBU per gram of residue, and 10.0 FXU per gram of residue

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[34]. The experiments were carried out in a rotary incubator agitated at 150 rpm at 50

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°C for 96 h. The collected samples were placed in a boiling water bath for 5 min to

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inactivate the enzymes and, therefore, to end the reaction. The samples were then

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centrifuged at 10000 rpm (5804R-Eppendorf) for 10 min. The liquid fraction was

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filtered using 20.0 µm membranes (Millipore) and stored at −20 °C for use in

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determination of the sugars. The assays were performed in triplicate. After the initial

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hydrolysis with dry bagasse, another hydrolysis was performed to compare the dry

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bagasse and wet bagasse after pretreatment. The hydrolysis conditions were the same as

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described above. However, the moisture of the wet bagasse was measured, and the mass

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was adjusted to the dry weight. Cellulose conversion was estimated using Eq. (1)

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according to the report of Zhou [36].

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  (%) = 

 (/)×.

 ! (⁄)

# × 100 Eq. (1)

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3.5 Fermentative process

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3.5.1. Inoculum preparation

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S. cerevisiae UFLA CA11, S. cerevisiae CAT-1, and K. marxianus ATCC-

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36907 were maintained in Petri dishes containing PDA (potato dextrose agar) culture

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medium at 30 °C for 24 h. The inoculating strains were subsequently grown in 250 mL

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Erlenmeyer flasks using 100 mL of a sterile culture medium containing 50 g/L glucose,

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1 g/L (NH4)2SO4, 0.5 g/L KH2PO4, 0.25 g/L MgSO4, 10 g/L yeast extract, and 10 g/L

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peptone at 30 °C on an orbital shaker operating at 150 rpm for 12 h, and then

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centrifuged. The inoculated cell concentration was 0.2 g/L. Subsequently, the cells were

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inoculated into 48 mL of the culture medium to start the SSF process.

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3.5.2 Saccharification and simultaneous fermentation (SSF)

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SSF experiments were carried out using the dried AL-pretreated carnauba

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residue. SSF was performed with 4% (w/v) of the pretreated solids in 48 mL of a 50

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mM sodium citrate buffer (pH 5.0), using the enzymes mentioned above. The enzyme

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loading was 20 FPU, 20 CBU, and 10 IU per gram of pretreated residue supplemented

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with 1 g/L (NH4)2SO4, 0.5 g/L KH2PO4, 0.25 g/L MgSO4, 2 g/L yeast extract, and 1 g/L

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peptone [34]. The SSF process was initiated by adding the enzymes and microorganism

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strains (S. cerevisiae UFLA CA11, S. CAT-1 and K. marxianus ATCC-36907) at each

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reaction, followed by incubation at 35 °C, 40 °C and 45 °C on an orbital shaker at 150

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rpm. Samples were withdrawn at 0, 6, 12, 24, 36, and 48 h. The concentrations of

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ethanol and sugars were determined by HPLC. All determinations were performed in

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triplicate. The ethanol yield was estimated using Eq. (2), assuming that all the potential

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glucose in the pretreated solids was available for fermentation [37].

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&'ℎ)* (%) =

.;<<(=×>×<.<<)

× 100

Eq. (2)

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where:

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3.6 Analytical methods

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components were carried out using HPLC (Acela, Thermo Scientific) using a Shim-

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Pack SCR-101H column (Shimadzu Co., Japan) operating at 65 °C. Sulfuric acid (5.0

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mM) was used as the mobile phase with a flow rate of 0.6 mL/min, and 20.0 µL of the

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sample was injected into the column. The samples were prefiltered using 20.0 µm

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membranes (Millipore). During the hydrolysis, the total reducing sugars formation was

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also assayed using a spectrophotometer (Thermo Spectronic) by the 3,5-dinitrosalicylic

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acid method [38]

w = weight of the total dry biomass in SSF;

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f = proportion of the cellulose fraction of the dry biomass (g/g);

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[Ethanol]produced (g/L) = (ethanol)t – (ethanol)0;

3.7 Statistical analysis

Statistical analysis was performed in triplicate. The influence of different

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pretreatments and the performance of the dried and wet biomass hydrolysis was assayed

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by Tukey tests using Statistica 7.0 (StaSoft Inc., USA, 2005).

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3. Results and discussion

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3.1 Composition of the untreated and pretreated carnauba straw In this study, the potential of the agroextractive residue produced by the

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extraction of carnauba straw wax in the production of cellulosic ethanol was evaluated.

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The evaluation was based on the use of agricultural waste for ethanol production

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through micro-distillery and biorefinery methods. Thus, three different pretreatments

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were used to optimize the enzymatic hydrolysis of the carnauba biomass: hydrothermal

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(HT), alkaline (AL), and alkaline acid (AA) pretreatment. Fig. 1 shows the comparison

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of the chemical composition and biomass residue after the pretreatments per 100 grams

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of dry residue. The HT pretreatment showed a higher residue yield (59.07 g per 100 g of

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untreated residue) and a greater removal of hemicellulose (75.60%), but it was not

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effective in the removal of lignin. On the other hand, the AA pretreatment showed low

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reduction (23.24 g per 100 g of untreated residue) and high loss of cellulose (57.19%).

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The AL pretreatment achieved significant removal of both lignin and hemicellulose

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(75.60% and 64.02%, respectively), demonstrating that this method was the best of the

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three assayed pretreatments for carnauba biomass.

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The UN-T showed a high cellulose content (Fig. 1). This was important, as it

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indicated that carnauba straw could be a promising substrate for ethanol production in

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micro-distilleries. The AA pretreatment was not efficient in this study, mainly due to the

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low yield of the pretreated biomass and the loss of cellulose, even after the removal of

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removing lignin and hemicellulose. The HT pretreatment removed hemicellulose, even

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at a temperature below those commonly used in other studies [18,40]. However, the low

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lignin removal in the HT pretreatment (21.04%) may compromise the biomass

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hydrolysis efficiency due to lignin remodeling in the lignocellulosic fiber after the

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ACCEPTED MANUSCRIPT biomass returns to room temperature [41,42]. In the AL pretreatment, an interesting

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relationship between the removal of lignin and hemicellulose was observed. AL has

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been one of the most commonly used pretreatments for lignocellulosics. Nevertheless,

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there seems to be a consensus among researchers that no single pretreatment is capable

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of achieving the same efficiency in the hydrolysis of different biomasses [16].

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The greater amount of pseudo-extractables generated by the pretreatments

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should also be noted. These components have been reported to be compounds and

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fragments of steroids, fatty acids, pectin, and polysaccharides, as well as fragments of

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these compounds, that have reprecipitate in the fibers and are not extracted by weak

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organic solvents such as ethanol [43]. Although not determined in this study, some of

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these pseudo-extractables may be remnants of carnauba straw wax, myricyl cerotate,

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which can remain in the fiber in different amounts depending on the pretreatment used

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due its apolar nature [44]. In addition, a considerable amount of ash was observed in in

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the untreated material (NT). This finding is not unexpected, as agricultural residues such

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as grasses, sugar cane, and straw have been reported to have high ash content in the

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literature [43].

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3.1 Enzymatic hydrolysis of untreated and pretreated residues

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The chemical composition of the untreated and pretreated residues was used to

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determine the best pretreatment for the enzymatic hydrolysis study of the pretreated

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carnauba straw biomass. Therefore, the AA pretreatment was discarded at this stage,

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and NT was studied only as a control. The enzymatic hydrolysis was standardized using

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the conditions quoted above in section 3.4. The enzymatic saccharification of the

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untreated and HT- and AL-pretreated residues are shown in Fig. 2.

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ACCEPTED MANUSCRIPT No difference was observed between the glucose production of the NT- and HT-

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treated residues Fig 2A. Fig. 2B shows that the glucose yields of the HT-treated residue

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and NT residue were 12.20% and 18.37% , respectively, even though the HT treatment

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removed 75.60% of the hemicellulose, as shown in Fig. 1. However, the AL

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pretreatment demonstrated 6-fold higher glucose production than NT (16.57 g/L) and a

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glucose yield of 64.43%, 3.6 times higher than that of NT, and therefore, represented

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the best pretreatment for the biomass of carnauba straw in this study. Interestingly, the

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HT pretreatment did not show any benefit for the hydrolysis of the carnauba straw

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biomass. This was probably due to its high removal of hemicellulose, which is located

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more internally in the fiber, and lower removal of lignin, which is located more

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externally in the fiber, which, together with modifications in the structure of the

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cellulose caused by the pretreatment, had a negative effect on the hydrolysis. Two

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hypotheses can be considered to explain this result. Firstly, the hydrothermal

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pretreatment was conducted at a temperature lower than 150 °C, which may not have

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allowed for high removal of lignin. Secondly, remodeling of lignin and possibly of

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myricyl cerotate (carnauba leaf wax) on the cellulosic fiber may have increased its

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amorphous character, thus reducing its crystallinity. Hydrothermal pretreatments are

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generally conducted at high pressure and temperatures (150 and 300 °C, initial pressure

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of 0–60 bar, 2–40 min) [45,46]. Studies have shown that hydrothermal pretreatment

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mainly affects the remodeling of lignin on the cellulosic fiber and the degradation of its

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components. However, the effectiveness of the pretreatments depends on the processing

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conditions and the complexity and heterogeneity of the biomass [40,46].

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The carnauba straw residue pretreated with 4% NaOH showed a better response

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to enzymatic hydrolysis. The effect of the alkaline pretreatment is mainly related to

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delignification [16]. However, significant removal of lignin and hemicellulose were 12

ACCEPTED MANUSCRIPT observed in the AL pretreatment, as can be seen in Fig. 1. The removal of both

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components was essential for increasing the hydrolysis yield. Studies have shown that

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AL pretreatment modifies the uronic ester bonds of 4-O-methyl-D-glucuronic acid,

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promoting changes in the bonding to the xylan skeleton, producing charged carboxyl

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groups and cleaving the bonds with the lignin and hemicelluloses. Increased porosity of

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the fiber and the contact surface has been observed as a result, which promotes greater

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cellulose accessibility and in turn maximizes enzymatic hydrolysis [17,47,48]

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3.2 Comparison of the enzymatic saccharification of untreated and pretreated HT and

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AL, dry (D) and wet (W)

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After pretreatment, a portion of the wet bagasse (W) was used in the hydrolysis,

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and another part was dried (D) at 50 °C for 24 h. The total moisture of both residues

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was measured so that an amount corresponding to 4% (m/v) biomass on a dry basis

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could be used in the enzymatic hydrolysis; the results are shown in Table 1. The NT

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residue was used as a control. However, there was no distinction between wet and dry

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untreated residues. For the AL pretreatment, the glucose production (14.87 g/L) and

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glucose yield (73.20%) were higher when dry residue was used.

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Despite having some advantages, such as the avoiding the drying step, the use of

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the wet residue hinders its storage. In addition, it can result in greater contamination by

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microorganisms due to the high moisture content. On the other hand, it is important to

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note that certain drying conditions may cause changes in the fiber structure after

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pretreatment. However, due to the complexity and heterogeneity of lignocellulosic

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biomass, it is possible that for different biomasses, different effects may be obtained

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during hydrolysis [17]

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3.3 SSF using Saccharomyces cerevisiae UFLA CA11

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evaluated for the production of cellulosic ethanol using the AL-pretreated carnauba

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straw residue. Fig. 3A and Fig. 3B show that the fermentation performance was

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improved when temperatures of 35 °C (4.77 g /L ethanol) and 40 °C (4.58 g/L ethanol)

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were used. An analysis of the residual accumulation of glucose and xylose reducing

324

sugars at the end of the fermentation kinetics indicated that lower concentrations of

325

glucose (0.85 g/L) and xylose (1.39 g/L) were observed at 35 °C (Fig. 3A). However,

326

the highest concentrations of residual reducing sugars, 5.4 g/L and 3.7 g/L of glucose

327

and xylose, respectively, were observed at 45 °C (Fig. 3C). These results suggest that

328

for this strain, the optimum SSF temperature should be between 35 °C and 40 °C under

329

the conditions used in this study.

330

3.4 SSF using Saccharomyces cerevisiae CAT-1

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Saccharomyces cerevisiae CAT-1 is a strain widely used in ethanol production

332

industries in Brazil. This is the main yeast used because of its high resistance to pH

333

shocks, during breaks in the fermentation process and the recycling process [27,49].

334

The performance of CAT-1 in the SSF process using carnauba straw residue pretreated

335

with 4% NaOH was evaluated. For this strain, higher ethanol production was observed

336

at 35 ° C, yielding 6.48 g/L with glucose and xylose accumulations of 0.14 g/L and 1.14

337

g/L, respectively (Fig. 4A). Lower performance was observed at elevated temperatures,

338

as shown in Fig. 4C, with the ethanol production decreasing and the residual glucose

339

increasing at 45 °C.

340

3.5 SSF using Kluyveromyces marxianus ATCC-36907

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The challenge in the production of cellulosic ethanol through SSF has been to

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balance the efficiency of saccharification and the conversion of sugars into cellulosic 14

ACCEPTED MANUSCRIPT ethanol. Therefore, some studies in the literature have utilized Kluyveromyces

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marxianus. This strain differs from others in its thermotolerance and ability to

345

metabolize xylose in addition to glucose. Kluyveromyces marxianus ATCC-36907 gave

346

different results than the other strains assayed in the SSF over 48 h. Although the

347

ethanol production peak occurred 12 h after the start of the fermentation, similarly to the

348

other strains studied, the ethanol production was higher at temperatures of 40 °C and 45

349

°C (7.29 g/L and 7.52 g/L, respectively), as can be seen in Fig. 5.

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Additionally, it was observed that after 6 h of cultivation, there was no glucose

351

accumulation, which can be an indicator of the efficiency of the strain in converting

352

glucose into ethanol. Another important factor to observe is the utilization of xylose by

353

Kluyveromyces marxianus. Xylose was consumed only at 45 °C and 24 h after the start

354

of SSF, and 18 h after the decline of glucose reserves in the medium (Fig. 5C). These

355

data suggest that xylose was preferably consumed at fermentation temperatures above

356

40 °C and after glucose depletion.

357

3.5 Comparison between the strains utilized in the SSF

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Three strains of yeast were utilized for the SSF of carnauba straw residue at

359

different temperatures in this study. As previously mentioned, for all strains and

360

conditions, the maximum ethanol production occurred 12 h after the initiation of SSF.

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Table 2 shows the comparison between these strains. In general, the CA11 and CAT-1

362

strains showed higher yields and ethanol productivities at the lowest temperature

363

studied (35 °C).

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Although these strains are widely used in the conventional ethanol production

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industry, the production of cellulosic ethanol at low fermentation temperature is not of

366

interest, as saccharification of the biomass by enzymes occurs at higher temperatures 15

ACCEPTED MANUSCRIPT [20,50,51]. However, since Saccharomyces cerevisiae CAT-1 and S. cerevisiae UFLA

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CA11 are strains that are widely used in the ethanol production industry, they have been

369

assayed in the present study for comparison with Kluyveromyces marxianus ATCC-

370

36907. Kluyveromyces marxianus ATCC-36907 was shown to be more efficient for the

371

production of cellulosic ethanol from carnauba straw residues pretreated with 4%

372

NaOH, with a yield of 132 L ethanol/t of carnauba straw. The fact this yeast tolerates

373

temperatures of 45 °C and produces a higher yield of ethanol (75.29 g/L) is very

374

interesting for SSF, since the optimum temperature for cellulolytic enzymes is around

375

50 °C [10,34]

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It should be highlighted that with the development of technologies for the

377

production of cellulosic ethanol, it is possible that the micro-distillery and biorefinery

378

concepts could be applied to residues of less globally cultivated agricultural crops, such

379

as carnauba residue. This will only be possible if solutions can be found to the problems

380

involved in the production of cellulosic ethanol, such as: (i) the costs of cellulolytic

381

enzymes; (ii) cost of pretreatments and the formation of inhibitors that reduce

382

hydrolysis efficiency; and (iii) inhibition of enzymatic hydrolysis by monomers of

383

sugars and cellobiose accumulating in the fermentation medium [16,20,52,53] .

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In this study, the pretreatment of biomass and fermentative yeast strains with

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potential for the production of cellulosic ethanol from pretreated carnauba straw was

386

carried out. The combination of AL-pretreated carnauba and an SSF process utilizing

387

Kluyveromyces marxianus provided the best performance. These results were important,

388

because SSF processes utilizing thermotolerant yeasts can reduce the number of

389

processing steps, thereby minimizing costs, reducing the possibility of contamination,

390

increasing the saccharification efficiency, and decreasing the inhibitory feedback of

391

cellulases. Additionally, this type of method should be advantageous for tropical

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ACCEPTED MANUSCRIPT countries where the temperature is higher [19,54]. This study was specific to carnauba

393

biomass, whose wax content differentiates it from most biomasses that are currently

394

being studied for the production of cellulosic ethanol. Therefore, the use of carnauba

395

straw for the production of ethanol must be closely linked to the wax extraction process

396

to be economically viable. That is, the production of cellulosic ethanol from carnauba

397

straw can be applied in situ in the context of a micro-distillery, which could be coupled

398

to the carnauba wax removal process.

4. Conclusions

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The pretreatments used on the fibers yielded different results, with AL

402

pretreatment being the most useful to increase the cellulose content of the carnauba

403

straw residue. In addition, the hydrolysis of the dried residue showed a better yield of

404

sugars than hydrolysis of the wet residue. Among the three strains studied for the

405

production of cellulosic ethanol, Kluyveromyces marxianus ATCC-3690 provided the

406

highest ethanol concentration and yield when utilized in the SSF at 45 °C. Finally,

407

carnauba straw residue can be used for the production of cellulosic ethanol by SSF

408

according to the micro-distillery and biorefinery concepts.

409

Acknowledgments

410

The authors thank CAPES and the Brazilian National Council for Research (CNPq) for

411

financial support. IFPB campus Picuí-PB is also thanked for allowing the first author

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Francinaldo Leite da Silva to move away to carry out this research.

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Compliance with Ethical Standards

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Conflict of Interest: The authors declare there is no conflict of interest.

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Ethical approval: This article does not contain any studies with human participants or

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animals performed by any of the authors.

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Genetic Dissection of Sugarcane Flowering under Equatorial Field Conditions,

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Trop. Plant Biol. 9 (2016) 252–266. doi:10.1007/s12042-016-9175-2.

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Tables and tables captions

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Table 1. Comparison of the hydrolysis of untreated (NT) and pretreated (AL) dry (D) or wet (W) carnauba straw residues.

Carnauba

Glucose

Residue

(g/L)

Xylose (g/L) c

1.06 ± 0.08

c

Arabinose

Total Sugar

Glucose Yield

(g/L)

Reducing (g/L)

(%)

3.04± 0.37

a

2.75 ± 0.03

c

19.34c

UN-T-W

1.74 ± 0.36

UN-T -D

1.74 ±0.14c

1.09 ±0.06c

3.11± 0.23a

3.10 ± 0.01c

18.04c

AL -W

12.67 ± 0.93 b

7.44 ± 0.65b

2.84± 0.41a

25.32 ± 0.03b

62.39b

AL -D

14.87 ± 0.19 a

8.31 ± 0.09a

3.27± 0.07a

33.74 ± 0.01a

73.20a

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*Values with different letters in columns represent statistically significant differences

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(p<0,05)

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Yeast strain Saccharomyces cerevisiae UFLA CA11

SSF temperature °C 35 40 45

Saccharomyces cerevisiae CAT-1

35 40 45

Kluyveromyces marxianus ATCC 36907

35 40 45

Ethanol production (g/L) 4.77 ± 0.06aA 4.58 ± 0.04aA 2.06 ± 0.13bA

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Table 2. Comparison of the SSF process among the three yeast strains studied.

47.69 ± 3.70aA 45.80 ± 0.60aA 20.63 ± 1.20bA

Ethanol Productivity (g/L.h) 0.39 ± 0.370aA 0.38 ± 0.05aA 0.17 ± 0.01bA

6.48 ± 0.38aB 5.47 ± 0.04bB 2.3 ± 0.07cB

64.85 ± 3.80aB 54.81 ± 0.6bB 23.05 ± 0.77aB

0.54 ± 0.03aB 0.45 ± 0.03bB 0.19 ± 0.01cB

3.36 ± 0.22 bC 7.29 ± 0.32aC 7.52 ± 0.11aC

33.64 ± 2.27bC 73.04 ± 3.20aC 75.29 ± 1.15aC

0.28 ± 0.01bC 0.60 ± 0.02aC 0.62 ± 0.09aC

Yield ethanol (%)

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the same temperature by different yeasts. Values with different letters in columns

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represent statistically significant differences (p<0,05).

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Figure captions

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Fig. 1. Chemical composition of untreated and pretreated Carnauba straw residue. NT:

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untreated, HT: hydrothermal pretreatment, AL: alkaline pretreatment and AA: acid

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alkaline pretreatment.

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Fig. 2. Results of enzymatic hydrolysis of carnauba straw residue. (A) Cellulose,

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xylose, arabinose and total reducing sugar (B) glucose yield. UN-T: untreated, HT:

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hydrothermal pretreatment and AL: alkaline pretreatment. Capital letters in bars

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represent statistically significant diferences (p<0.05).

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Fig. 3. Ethanol production using Saccharomyces cerevisiae UFLA CA11 in the SSF.

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The experiments were performed at three temperatures: (A) 35 °C; (B) 40 °C; (C) 45

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°C. All assays were done in triplicate.

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Fig. 4. Ethanol production using Saccharomyces cerevisiae CAT-1 in the SSF. The

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experiments were performed at three temperatures: (A) 35 ° C; (B) 40 ° C; (C) 45 ° C.

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All assays were done in triplicate.

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Fig. 5. Ethanol production using Kluyveromyces marxianus ATCC-36907 in the SSF.

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The experiments were performed at three temperatures: (A) 35 °C; (B) 40 °C; (C) 45

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°C. All assays were done in triplicate.

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ACCEPTED MANUSCRIPT HIGHLIGHTS

Use of agroextractive residue - Carnauba straw (CS) - for producing ethanol

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Alkaline pretreatment reduces hemicellulose and lignin in Carnauba residue

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Ethanol production by Simultaneous Saccharification and Fermentation (SSF)

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Kluyveromyces marxianus ATCC-36907 using SSF produces 7.53 g/L ethanol

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