Enzymatic saccharification and liquid state fermentation of hydrothermal pretreated Tunisian Luffa cylindrica (L.) fibers for cellulosic bioethanol production

Accepted Manuscript Enzymatic saccharification and liquid state fermentation of hydrothermal pretreated Tunisian Luffa cylindrica (L.) fibers for cellulosic bioethanol production Kaouther Zaafouri, Manel Ziadi, Aida ben Hassen-Trabelsi, Sabrine Mekni, Balkiss Aïssi, Marwen Alaya, Moktar Hamdi PII:

S0960-1481(17)30736-X

DOI:

10.1016/j.renene.2017.07.108

Reference:

RENE 9083

To appear in:

Renewable Energy

Received Date: 24 February 2017 Revised Date:

29 June 2017

Accepted Date: 25 July 2017

Please cite this article as: Zaafouri K, Ziadi M, ben Hassen-Trabelsi A, Mekni S, Aïssi B, Alaya M, Hamdi M, Enzymatic saccharification and liquid state fermentation of hydrothermal pretreated Tunisian Luffa cylindrica (L.) fibers for cellulosic bioethanol production, Renewable Energy (2017), doi: 10.1016/ j.renene.2017.07.108. 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.

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Graphical Abstract

Optimization of hydrothermal pretreatment of LC fibers:

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Enzymati pretreated LC AP

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Temperature = 96°C / Time = 54 minutes.

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Crude (a,b) and milled (c) Luffa cylindrica (LC) fibers.

α-cellulose, Hemicelluloses and Lignins

Liquid fraction characterization: total and reducing sugars dete

Reducing sugars = 33.55 g/kg.

Highest reducing s Sumizyme A

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45.80%: 20.76%: 13.15%.

pH= 4.0±0.2; Temp

3 Liquid state fermentation LSF of LC hydrolysates and distillation.

The conversion yield of reducing sugars to ethanol = 88.66%. The ethanol conversion efficiency = 1.58% and its volumetric yield = 70 %.

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Enzymatic saccharification and liquid state fermentation of

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hydrothermal pretreated Tunisian Luffa cylindrica (L.) fibers for

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cellulosic bioethanol production

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Kaouther Zaafouria, Manel Ziadia, Aida ben Hassen-Trabelsib, Sabrine Meknia,c, Balkiss

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Aïssia,c, Marwen Alayaa,b, Moktar Hamdia.

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a

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INSAT. Carthage University, 2 Boulevard de la terre, B.P. 676, 1080 Tunis, Tunisia.

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b

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CRTEn, Borj-Cedria Technopark, B.P. N°95 2050 - Hammam Lif , Tunisia.

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Laboratory of Microbial Ecology and Technology. LETMi-INSAT. The National Institute of Applied Sciences and Technology

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Laboratory of Wind Energy Control and Waste Energy Recovery, LMEEVED, Research and Technology Center of Energy,

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c

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Borj Cedria, Borj-Cedria Technopark, B.P. N° 1003 – Hammam-lif- 2050 – Hammam-Lif – Tunisia.

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Abstract

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The main drivers to develop biorefineries are the energetic and environmental crisis.

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Consequently, bioenergies have become a scientific and industrial trend. In North

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Africa, especially in Tunisia, Luffa cylindrica (LC) is a promising energy crop

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providing lignocellulosic biomass for biofuels production. Three principal fractions

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compose LC biomass, viz.: α-cellulose (45.80±1.3), hemicellulose (20.76±0.3) and

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lignins (13.15±0.6). The hydrothermal pretreatment of LC fibers was carried out at

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96°C for 54 minutes. After pretreatment, the reducing sugars amount reached 33.55

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g/kg. The subsequent enzymatic saccharification was performed during 1 hour at a

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temperature of 60°C, by means of two commercial enzymes AP2 and SPC. The enzyme

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AP2 seemed to be more suitable for the enzymatic hydrolysis of pretreated LC fibers by

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allowing the release of 59.4 g/kg of reducing sugars, which correspond to a reducing

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sugars recovery about 93.29%. After that, the liquid state fermentation (LSF) was

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achieved with Saccharomyces cerevisiae strain during 24 hours, in sterile and non-

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sterile conditions at 30°C, pH 4.8±0.2 and stirring 250 rpm, in order to conclude about

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Department of Biotechnology and environment sciences, High Institute of Environmental Science and Technology (HIEST) of

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ACCEPTED MANUSCRIPT the influence of contamination microflora on fermentation efficiency. After the LSF

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step, 88.66% of reducing sugars were transformed into alcohol with a conversion rate of

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1.58% and a volumetric yield about 70 % in sterile conditions. Thus, this work confirms

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that the potential conversion yield of cellulosic bioethanol is 1 Ton (dry matter) of LC

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fibers to 13.8545 kg (= 3.6599 Gallon) of biofuel.

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Key words: Luffa cylindrica, Hydrothermal pretreatment, Enzymatic saccharification,

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Liquid state fermentation LSF, Cellulosic bioethanol.

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1. Introduction

Nowadays, biorefineries start to replace petrochemistry to solve the environmental and

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fossil energy issues [1]. With a global production reaching 200 billion metric tons a

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year, Biomass and its byproducts represent great potential feedstocks for energy

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conversion technologies [2]. Lignocellulosic biomass is renewable, more abundant and

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cheapest resource worldwide.

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In the tropical, subtropical and countries with a moderate climate like in Tunisia, Luffa

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cylindrica (LC) is a promising energy crop providing lignocellulosic biomass for

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biofuels production. LC is an annual herbaceous fibrous and climbing plant from the

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cucurbitaceous family with a plant growth yield reaching 62000 LC fruits/ha (20 to 25

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fruits/plant). Although, this yield depends highly on the climate [3].

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To produce 2G-bioethanol from lignocellulosic biomass, three main steps are required,

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viz.: pretreatment, enzymatic saccharification, fermentation and distillation [4].

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Pretreatment step plays three important roles, i.e.: lignins destruction; hydrolysis of

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hemicelluloses and modification of cellulose, which will improve the subsequent

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enzymatic hydrolysis [5,6].

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The enzymatic saccharification is catalyzed by the synergistic action of four cellulases

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cellobiohydrolases, exo-1,4-β-glucanases - that will hydrolyze celluloses into

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cellobiose-and β-glucosidases that hydrolyze cellobiose into glucose. Bioconversion of

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pretreated lignocellulosic biomass needs multiple enzyme activities. The monomeric

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sugars liberated from enzymatic saccharification are converted into ethanol thanks to

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some microorganisms. Saccharomyces cerevisiae is the most employed yeast for

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ethanol producing from hexoses, since it is resistant to low pH, high temperatures, and

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various inhibitors [7]. Other yeasts could produce ethanol through hexoses recovery,

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mainly from xylose e.g.: Pichia stipitis, Candida shehatae, Kluveromyces marxianus

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and Pachysolen tannophilus. Some bacteria could also fermented monomeric sugars to

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produce alcohols, such as: Zymomonas mobilis and Escherichia coli [4,8]. Several types

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of fermentation processes have been studied, e.g.: batch, continuous, continuous with

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cell recycling, fed-batch and repeated-batch culture designs [5]. In order to obtain a fuel

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grade or anhydrous ethanol, many distillation and dehydration processes are employed

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[4].

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In this context, the main goal of this work is to study the 2G-bioethanol process

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feasibility from LC fibers by means of maximizing the enzymatic saccharification of the

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pretreated substrate and testing the alcoholic fermentation of biomass hydrolysates.

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2. Materials and methods

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2.1. Luffa cylindrica (LC) raw fibers: sampling and pretreatment

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Luffa cylindrica raw fibers were collected from a private farm with an intensive crop in

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the region of Monastir located in the Tunisian Sahel (center-east of Tunisia) in January

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2014, by sampling manually the fresh crude fruits. Then, they were milled with a

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kitchen grinder (Figure 1). The hydrothermal pretreatment of LC fibers was performed

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and published by the authors [9]. The pretreated fibers were stocked in glass bottles at

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4°C for the subsequent enzymatic saccharification and fermentation.

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2.2. Analytical methods

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2.2.1. Lignocellulosic composition of LC fibers

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The lignocellulosic composition of LC fibers, have been determined according to the

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gravimetric method using specific chemicals previously detailed in the literature [10]

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with some modifications related to the initial sample weight for simplicity and

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repeatability. Firstly, 10 grams of LC milled fibers are defatted with a mixture of

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toluene and ethanol (2v/v) for 6 hours at room temperature. Secondly, the defatted LC

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fibers were treated with 200 mL of water at 80°C for 2 hours to extract the water-

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soluble polysaccharides. After that, the lignins content determination required a

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simultaneous treatment of the collected solid fraction from the previous step with the

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sodium hypochlorite and the acetic acid at pH 4 for 2 hours at 75°C. Then, for the

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extraction and the purification of the α-cellulose, the holocellulose fraction obtained

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from the previous acid treatment, was purified with 600 mL of sodium hydroxide (10%

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weight/volume) for 10 hours at 20°C under stirring conditions. The previous reaction

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mix was filtrated, the permeate was neutralized with HCl chlorhydric acid (6M), until

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reaching a pH about 5.5 and then precipitated with 450 mL of ethanol (95° alcoholic

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degree). The obtained pellets were washed with ethanol (70° alcoholic degree), then

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dried in a ventilated oven at 50°C, in order to obtain the hemicellulosic fraction. All the

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experiments of the lignocellulosic characterization of LC fibers were carried out in

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triplicate.

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2.2.2. Total sugars determination

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ACCEPTED MANUSCRIPT The total sugars amount of the studied samples was carried out referring to Dubois

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method [11] by using a phenol solution (5% w/v) and concentrated sulfuric acid H2SO4

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(96%-98% v/v). Then, the sample incubation was performed in a boiling water-bath at

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100°C during 5 minutes. The absorbance of each sample was measured at a wavelength

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λ=480 nm using a spectrophotometer UV-visible type Jenway®. The total sugars

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concentration of each sample was calculated according to the standard curve previously

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established with the same protocol detailed above, using glucose as a reference sugar

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(Figure 2).

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2.2.3. Reducing sugars determination

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The reducing sugars content was measured according to the method of Miller (1959)

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[12] by mixing the studied sample with the 3-5,dinitrosalicylic acid DNS reagent

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prepared with the Potassium Sodium tartrate (KNaC4H4O6·4H2O) and the Sodium

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Hydroxide (NaOH). The reaction was carried out in a boiling water-bath at 100°C

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during 15 minutes. After the reaction cooling, the absorbance of each sample was

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determined at a wavelength about 540 nm using a spectrophotometer UV-visible type

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Jenway®. The reducing sugars concentration of each sample was measured according to

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the standard curve previously elaborated with Miller protocol described above, using

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glucose as a reference sugar (Figure 3).

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2.2.4. Ethanol determination

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The ethanol concentration of distilled samples resulting from fermentation step was

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determined by the high-performance liquid chromatography using Agilent® equipment

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with inverse C18 column (PRONTOSIL 120-5-C18-AQ 5.0Xm (250 mm x 4.0mm) and

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sulfuric acid 1 mM as mobile phase; at 25°C with a flow of 0.3 mL/min (analysis time:

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30 min and injection volume: 20µL) according to the protocol previously detailed in the

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2.3. Experimental methodology

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2.3.1. Enzymatic saccharification of pretreated LC fibers

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The enzymatic saccharification was carried out in 250 mL Erlenmeyer flasks containing

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100 mL of total reaction mix volume of LC pretreated fibers, using separately two

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commercial enzymes for a comparative purpose: Sumizyme AP2 (Pectinase; Cellulases

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and Hemicellulase activities: 54000 unit/g) and Sumizyme SPC (Pectinase and

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Cellulase activities: 6,000 u/g -1.000 u/g) provided as a powder by Shin Nihon

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Chemical Co., Ltd. (Japan). The “Enzyme:Substrate” ratios were 0.2% (w/w) and

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0.005% (w/w) for AP2 and SPC respectively with taking into consideration the

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lignocellulosic biomass dry matter content. Enzymes were previously dissolved in 1 mL

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of sodium acetate buffer solution (0.1 M) (pH 4.0±0.2). The enzymatic saccharification

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was conducted for 1 hour at 60°C and the reaction was stopped by increasing the

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temperature to 85°C for 15 minutes. The monitoring of enzymatic hydrolysis was

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achieved through reducing sugars measurement.

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2.3.2. Liquid state fermentation of LC hydrolysates and distillation

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The best enzymatic saccharification reaching the maximum reducing sugars amount was

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selected for the fermentation step. A volume of 100 mL of LC hydrolysates were

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inoculated with 10% (v/v) of 12 hours-old pre-culture of commercial yeast

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Saccharomyces cerevisiae supplied by Rayen® food company (Béja-Tunisia), grown on

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Sabouraud broth. The fermentation was conducted at 30°C and pH 4.8±0.2, during 24

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hours at 250 rpm of agitation in sterile and non-sterile conditions; in order to conclude

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about the effect of the microbial contamination flora on the final bioethanol yield. To

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increase the final ethanol concentration, the fermentation broth distillation was

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3. Results and discussion

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3.1. Lignocellulosic characterization of LC fibers

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The lignocellulosic characterization of LC fibers is summarized in Table 1. The studied

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LC fibers are rich in α-cellulose (45.80±1.3)% and in hemicelluloses (20.76±0.3) %

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with a small amount of polysaccharides 7.86%±0.1. Besides, their lignins content is

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about 13.15%±0.6. As it can be seen, the lignocellulosic composition of the Tunisian

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LC fibers is almost similar to those of Brazilian (63-65.5% of α-cellulose) and Algerian

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LC fibers (45% of cellulose) [14,15,16]; and also to those of some other lignocellulosic

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biomasses having an α-cellulose content ranging from 39.23% to 55.9% [17, 18], such

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as: Tunisian and Algerian Alfa (Stipa tenacissima) stems; Posidonia oceanica fibers;

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non-wood substrate (Prosopis alba, etc…); some other wood (Pinus pinaster); rush;

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palm leaflets and stip. Certainly, LC composition depends on various factors, such as:

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species, variety, soil type, weather conditions, plant age, etc [15]. These findings

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confirm that LC fibers are suitable lignocellulosic energy crops for 2G-bioethanol

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production.

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3.2. Enzymatic saccharification of pretreated LC fibers

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The variation of reducing sugars amounts during the enzymatic saccharification with

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both SPC and AP2 enzymes is illustrated by Figure 4. Referring to the dry matter

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content of LC fibers (5.5%±0.33), the highest reducing sugars concentration is about

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59.4 g/kg for the saccharification carried out by Enzyme AP2. Although, the enzymatic

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hydrolysis of LC pretreated fibers performed with Enzyme SPC liberates around 37

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g/kg of reducing sugars, which correspond to a reducing sugars recovery about 93.29%.

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The current finding demonstrates that the reducing sugars amount reached is higher than

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ACCEPTED MANUSCRIPT those of steam pretreated agricultural residues (viz.: Triticale, Canadian prairie spring

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wheat (SW), durum wheat, feed barley, malt barley, oat and flax straws) ranging from

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30% to 70% [19] and also than those of pretreated cassava stems ranging from 55.40%

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to 86.6% [20].

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3.3. Liquid state fermentation LSF of LC hydrolysates

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The liquid state fermentation LSF of LC hydrolysates was monitored by the reducing

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sugars measurements as shown in Figure 5. Based in above results, it is possible to

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conclude that the reducing sugars conversion yield to ethanol is around 88.66% in

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sterile conditions. Thus, the ethanol conversion efficiency is 1.58% and its volumetric

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yield 70 % which is higher than several cellulosic ethanol yields, previously listed in the

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literature for various lignocellulosic feedstocks [21, 22, 23, 24, 25].

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4. Conclusion

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This study highlighted that for the enzymatic saccharification of hydrothermal

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pretreated Tunisian LC fibers carried out with AP2 enzyme, the reducing sugars

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concentration reached the highest value of 59.4 g/kg. Afterthat, the reducing sugars

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conversion rate to bioethanol is about 88.66%. Consequently, the potential conversion

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yield of 2G-bioethanol is 1 Ton (dry matter) of LC fibers to 13.8545 kg (= 3.6599

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Gallon) of biofuel. Thus, the current work confirms that Luffa cylindrica could be

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considered as a promising energy crop for 2G-bioethanol production in Tunisia.

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References

191

[1] S.H. Mood, A.H. Golfeshan, M. Tabatabei, G.S. Jouzani, G.H. Najafi, M. Gholami,

192

D.M. Ardjman, Lignocellulosic biomass to bioethanol, a comprehensive review with a

193

focus on pretreatment, Renew. Sust. Energ. Rev.27 (2013) 77–93.

194

[2] P. Phitsuwan, K. Sakka, K. Ratanakhanokchai, Improvement of lignocellulosic

AC C

EP

TE D

M AN U

SC

RI PT

171

8

ACCEPTED MANUSCRIPT biomass in planta: a review of feedstocks, biomass recalcitrance, and strategic

196

manipulation of ideal plants designed for ethanol production and processability,

197

Biomass Bioenerg.58 (2013) 390–405.

198

[3] UNFAO Food and Agriculture Organization of the United Nations, Minor oil crops,

199

Part I - Minor edible oil crops, prepared by B.L. Axtell from research by R.M. Fairman.

200

Intermediate Technology Development Group Rugby, UK. M-17. ISBN 92-5-103128-2,

201

1992.

202

[4] A. Limayem, S. C. Ricke, Lignocellulosic biomass for bioethanol production:

203

current

204

Prog.Energy.Combust.Sci.38(4) (2012) 449–467.

205

[5] O.J. Sanchez, C.A. Cardona, Trends in biotechnological production of fuel ethanol

206

from different feedstocks, Bioresour. Technol.99 (2008) 5270–5295.

207

[6] M. Balat, Production of bioethanol from lignocellulosic materials via the

208

biochemical pathway, Energy Convers. Manage. 52, (2011) 858-875.

209

[7] R. Cripwell, L. Favaro, S.H. Rose, M. Basaglia, L. Cagnin, S. Casella, W.V. Zyl,

210

Utilisation of wheat bran as a substrate for bioethanol production using recombinant

211

cellulases and amylolytic yeast, Appl. Energ.160 (2015) 610–617.

212

[8] F.A. Gonçalves, H.A. Ruiz, E.S. dos Santos, J.A. Teixeira, G.R. de Macedo,

213

Bioethanol production by Saccharomyces cerevisiae, Pichia stipitis and Zymomonas

214

mobilis from delignified coconut fibre mature and lignin extraction according to

215

biorefinery concept, Renew. Energ.94 (2016) 353–365.

216

[9] K. Zaafouri, M. Ziadi, A. Ben Hassen-Trabelsi, S.Mekni, Balkiss Aïssi, M. Alaya,

217

L. Bergaoui, M. Hamdi, Optimization of Hydrothermal and Diluted Acid Pretreatments

218

of Tunisian Luffa cylindrica (L.) Fibers for 2G Bioethanol Production through the Cubic

potential

issues

and

future

prospects,

AC C

EP

TE D

M AN U

perspectives,

SC

RI PT

195

9

ACCEPTED MANUSCRIPT Central Composite Experimental Design CCD: Response Surface Methodology,

220

Biomed. Res. Int. (2017), Article ID 9524521, https://doi.org/10.1155/2017/9524521.

221

[10] X.F. Sun, R.C. Sun, J. Tomkinson, M.S. Baird, Preparation of sugarcane bagasse

222

hemicellulosic succinates using NBS as a catalyst. Carbohydr. Polym.53 (2003) 483–

223

495.

224

[11] B. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colormetric method

225

for determination of sugars and relative substances, Anal. Chem.28 (1956) 350–356.

226

[12] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing

227

sugar, Anal. Chem.3 (1959) 426–428.

228

[13] V.B. Hugo, LI. Felipe, The unified metabolism of Gluconacetobacter entanii in

229

continuous and batch processes, Process. Biochem.42 (2007) 1180–1190.

230

[14] G. Siqueira, J. Bras, A. Dufresne. Luffa cylindrica as a lignocellulosic source of

231

fiber, microfibrillated cellulose and cellulose nanocrystals: Luffa as a cellulose source,

232

BioResources.5 (2) (2010) 727–740.

233

[15] V.O.A. Tanobe, T.H.D. Sydenstricker, M. Munaro, S.C. Amico, A comprehensive

234

characterization of chemically treated Brazilian sponge-gourds (Luffa cylindrica),

235

Polymer. Test.24 (4) (2005) 474–482.

236

[16] Y. Laidani, S. Hanini, G. Mortha, G. Heninia, Study of a Fibrous Annual Plant

237

Luffa cylindrica for paper Application Part I: Characterization of the Vegetal, Iran. J.

238

Chem. Chem. Eng.31 (4) (2012) 119–129.

239

[17] Z. Marrakchi, R. Khiari, H. Oueslati, E. Mauret, F. Mhenni, Pulping and

240

papermaking properties of Tunisian Alfa stems (Stipa tenacissima)-Effects of refining

241

process, Ind. Crop. Prod.34 (2011) 1572–1582.

242

[18] S. Hamza, H. Saad, B. Charrier, N. Ayed, F. Charrier-El Bouhtoury, Physico-

AC C

EP

TE D

M AN U

SC

RI PT

219

10

ACCEPTED MANUSCRIPT chemical characterization of Tunisian plant fibers and its utilization as reinforcement for

244

plaster based composites, Ind. Crop. Prod.49 (2013) 357–365.

245

[19] R.P. Chandra, V. Arantes, J. Saddler, Steam pretreatment of agricultural residues

246

facilitates hemicelluloses recovery while enhancing enzyme accessibility to cellulose,

247

Bioresour. Technol.185 (2015) 302–307.

248

[20] P.A. Kouteu Nanssou, Y.J. Nono, C. Kapseu, Pretreatment of cassava stems and

249

peelings by thermohydrolysis to enhance hydrolysis yield of cellulose in bioethanol

250

production process, Renew. Energ.97 (2016) 252–265.

251

[21] Y. Liu, Y. Zhang, J. Xu, Y. Sun, Z. Yuan, J. Xie, Consolidated bioprocess for

252

bioethanol production with alkali-pretreated sugarcane bagasse, Appl. Energ.157 (2015)

253

517–522.

254

[22] D. Mazaheri, S.A. Shojaosadati, S.M. Mousavi, P. Hejazi, S. Saharkhiz, Bioethanol

255

production from carob pods by solid-state fermentation with Zymomonas mobilis, Appl.

256

Energ.99 (2012) 372–378.

257

[23] S.T. Thomsen, M. Jensen, J.E. Schmidt, Production of 2nd generation bioethanol

258

from lucerne: Optimization of hydrothermal pretreatment, BioResources. 7(2) (2012)

259

1582–1593.

260

[24] S.K. Thangavelu, A.S. Ahmed, F.N. Ani, Bioethanol production from sago pith

261

waste using microwave hydrothermal hydrolysis accelerated by carbon dioxide, Appl.

262

Energ.128 (2014) 277–283.

263

[25] G. Baskar, R. Naveen Kumar, X. Heronimus Melvin, R. Aiswarya, S. Soumya,

264

Sesbania aculeate biomass hydrolysis using magnetic nanobiocomposite of cellulase for

265

bioethanol production, Renew. Energ.98 (2016) 23–28.

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Acronym table

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LC: Luffa cylindrica.

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LSF: Liquid State Fermentation.

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AP2: Commercial Cellulolytic Enzyme.

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SPC: Commercial Cellulolytic Enzyme.

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Highlights

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1. The maximum reducing sugars amount is 59.4 g/kg for the hydrolysis made with

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AP2.

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2. Ethanol conversion efficiency is 1.58% and its volumetric yield 70 %.

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3. The potential conversion yield of 1 Ton of LC fibers is 3.6599 Gallon of biofuel.

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ACCEPTED MANUSCRIPT Table 1. Lignocellulosic characterization of Tunisian LC fibers

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Value (%)

Component

7.86±0.1

Lignins

13.15±0.6

α-cellulose

45.80±1.3

20.76±0.3

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Hemicelluloses

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Water-soluble polysaccharides

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ACCEPTED MANUSCRIPT Figures caption

Fig.1. Luffa cylindrica (LC) crude sponges (a) and milled fibers (b). Fig.2. The standard curve for the total sugars determination.

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Fig.3. The standard curve for the reducing sugars determination. Fig.4. Variation of reducing sugars amounts during the enzymatic saccharification with both SPC ( ) and AP2 (

) enzymes.

).

AC C

EP

TE D

M AN U

conditions (

) and non-sterile

SC

Fig.5. Variation of reducing sugars amounts during the LSF in sterile (

6