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Francinaldo Leite da Silva, Alan de Oliveira Campos, Davi Alves dos Santos, Sérgio
Dantas de Oliveira Júnior, Carlos Eduardo de Araújo Padilha, Francisco Caninde de
Sousa Junior, Gorete Ribeiro de Macedo, Everaldo Silvino dos Santos
PII: S0960-1481(17)30927-8
DOI: 10.1016/j.renene.2017.09.064
Reference: RENE 9262
Please cite this article as: da Silva FL, de Oliveira Campos A, dos Santos DA, de Oliveira Júnior
SéDantas, de Araújo Padilha CE, de Sousa Junior FC, de Macedo GR, dos Santos ES, Pretreatments
of Carnauba (Copernicia prunifera) straw residue for production of cellulolytic enzymes by
Trichorderma reesei CCT-2768 by solid state fermentation, Renewable Energy (2017), doi: 10.1016/
j.renene.2017.09.064.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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1 Pretreatments of Carnauba (Copernicia prunifera) straw residue for production of
3 Francinaldo Leite da Silvaa,b, Alan de Oliveira Camposa, Davi Alves dos Santosa, Sérgio
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7 University of Rio Grande do Norte (UFRN), Natal-RN, Brazil.
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8 b. Instituto Federal de Educação Ciência e Tecnologia da Paraíba Campus Picuí (IFPB)
9 Picuí/PB, Brazil.
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10 *Corresponding author: everaldo@eq.ufrn.br. Address: UFRN - Centro de Tecnologia -
11 CT - Avenida Senador Salgado Filho, 3000 - Lagoa Nova, Natal - RN, zipcode: 59064-
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12 741
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13 Abstract
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14 In this study the effect of pretreatments, such as hydrothermal (HT) with hydrogen
16 Carnauba (Copernicia prunifera) straw residue—a plant native to Brazil that is used to
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17 produce wax —as well as the use of the pretreated biomass for the production of
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19 using solid state fermentation (SSF) were evaluated. The untreated and pretreated
21 ray diffraction, and scanning electron microscopy assays. A kinetic study was carried out
22 to estimate the best time for producing cellulases (FPase and CMCase) and xylanases.
23 HP-A pretreatment was the only one that simultaneously reduced hemicellulose (removal
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24 of 60.72%), lignin (removal of 50.71%) and pretreatment yield of 59.28%. FPase (0.9
25 U/g) and CMCase (13 U/g) production in the case of this pretreatment was optimum,
26 while AL pretreatment was ideal for xylanase (99.5 U/g). The use of Carnauba
27 (Copernicia prunifera) straw residue coupled with HP-A pretreatment and SSF shows
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29 Keywords:
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30 Carnauba straw, solid state fermentation, pretreament, cellulase, xylanase, lignocellulosic
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31 residue
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Hydrogen Peroxide-Alkaline; Acid-Alkaline pretreatment (AA) and Alkaline
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34 pretreatment (AL)
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35
1. Introduction
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36
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37 In recent years, the growing risk of fossil fuel shortage and high degree of global
38 pollution has given impetus to the searching for sustainable energy resources across the
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39 world. Several studies have focused on the use of agricultural waste products as an
40 energy source [1–3]. These products constitute important carbon sources that can be
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41 recycled to generate food, chemicals and biofuels such as cellulosic ethanol. However,
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42 the production of cellulosic ethanol requires fermentation of sugars derived from the
45 lignocellulosic biomass; nevertheless, the production cost of these enzymes has been an
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47 identifying microorganisms that secrete these enzymes with higher efficiency and
50 enzymatic attack due to physical and chemical barriers, mainly formed by lignin and
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51 hemicellulose, that inhibit enzymatic hydrolysis. The highly crystalline and recalcitrant
52 nature of lignocellulose and the presence of lignin are the main factors that hamper
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53 hydrolysis and microbial fermentation [9]. To overcome these problems, numerous
54 studies have explored different pretreatments using chemical, physical, and biological
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55 agents or combinations of these. Amongst these, alkaline pretreatment has been used very
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56 commonly as it can effectively solubilize lignin. On the other hand, acid pretreatment has
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57 been shown to be more efficient in the removal of hemicellulose. Hemicellulose is also
59 hydrogen peroxide, has been shown to be quite efficient because it allows high sugar
62 Brazil and it is used in the Brazilian semiarid region for the extraction of carnauba wax.
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64 lignocellulosic residues with great potential as a carbon source for the production of
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66 residues is being used extensively to maximize the efficiency of biomass hydrolysis, its
67 use to enhance the production of cellulase from agricultural waste is still rare. In this
68 study, we evaluated the effect of four pretreatment methods on the carnauba straw and the
70 fermentation (SSF) fermentation using the fungus Trichoderma reesei CCT2768. To the
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71 best of our knowledge, this is the first study in which Carnauba is being used to produce
74 2.1. Microorganism
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75 The microorganism used in the fermentation process for the production of crude
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76 enzyme extract was the filamentous fungus Trichoderma reesei CCT2768, obtained from
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78 Brazil.
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79 2.2. Lignocellulosic material
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80 The residue of carnauba straw, main biomass of this study was supplied by the
82 (RN), Brazil. The other complementary wastes, namely residues from sugarcane, green
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83 coconut, and cashew were procured from Usina Estivas (Arês, Brazil), Natal (Arês,
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84 Brazil), and Cione Co. (Ceara State, Brazil), respectively. The residues were washed
85 thoroughly with water to remove particulate materials and sugar residues. Thereafter, the
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86 residues were dried at 70 °C for 48 hours, ground in a Wiley mill (mill type Willye, TE -
89 HT pretreatment was carried out using 20% (w/v) solid immersed in water. The
90 mixture was heated at 121 °C for 30 minutes and subsequently washed with water until
91 the pH value reached around 7.0[13,14]. The AL pretreatment was performed using 20%
92 (w/v) residue in a solution of 4% NaOH (w/v). This mixture was also heated at 121 °C
93 for 30 minutes [15]. Subsequently the residue was washed with water until the pH value
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94 reached around 7.0. The AA pretreatment was performed in two steps. Firstly, 20% (w/v)
95 of the residue was submerged in 2% (v/v) H2SO4 solution. Then the mixture was heated at
96 121 °C for 30 minutes [16]. Thereafter the residue was washed ten times with water until
97 pH value reached around 7.0. In the second stage of pretreatment it was the same as the
98 AL one. The HP-A pretreatment was performed using 4% (w/v) of biomass in a 7.35%
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99 (v/v) hydrogen peroxide solution. The pH of hydrogen peroxide solution was adjusted to
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100 11.5 using NaOH. The mixture was stirred at 150 rpm for 1 hour at room temperature
101 [11,17]. The residue was then washed with water until the pH value reached around 7.0.
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102 Post different pretreatments, all the samples were dried at 50 °C for 24 hours.
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103 2.4. Characterization of untreated and pretreated Carnauba biomass
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104 The cellulose, hemicellulose, lignin, ash, moisture contents, and extractives were
105 evaluated from both the untreated fiber and pretreated biomass according to NREL
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107 2.5. Analysis of functional groups by Fourier transform infrared spectroscopy (FTIR)
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108 FTIR analysis was used to identify the main constituent organic groups of lignin
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109 and hemicellulose. The FTIR absorption spectra was obtained in the range of 400-4000
110 cm-1 using the Fourier transform infrared spectrophotometer (FT-65 IR-spectrum, Perkin
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113 Pretreated waste and untreated carnauba straw, previously dried in an oven
114 (moisture below 10%) were visualized under a scanning electron microscope (Phillips
115 XL-30ESEM, USA) using a 15 kV electron beam. The electron micrographs were
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117 2.7. X-ray analysis to determine crystallinity
119 (XRD) (XRD-6000, Shimadzu, Japan) analysis, with the following X-ray source
120 parameters: Cu-Kα radiation, 30.0 kV voltage, and 15 mA of electric current. One scan
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121 with continuous 2θ interval of 4.0 - 70.0 ° was applied with a rate of 2.0 degree per
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123 The crystallinity index (%CI) of the fibers was calculated according Segal [19], as shown
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124 in Eq. (1).
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126 The I002 is the intensity of the diffraction peak at 22.5° and Iam is the intensity of the
129 The biomass residues were sterilized by autoclaving at 121 °C for 15 minutes, and
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130 inoculated from a sporulated culture containing the conidia spores of T. reesei CCT2768
132 The spore concentration was 1x107 spores/g solid medium for both pretreated and
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133 untreatread residues [20]. The SSF was performed in a bacteriological BOD incubator at
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134 a temperature of 30 °C for 168 hours. Samples were collected every 24 hours for the
136 terms of the moisture content established at 60% and pH of the nutrient saline [21]. The
137 enzymes were extracted from the flasks after a predetermined time period with the
138 addition of 25 mL of acetate buffer (200 mM, pH 5.0) to the fermented solid substrate
139 and mixing with a glass rod. The fermentate was subjected to stirring at 160 rpm for 30
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140 minutes. Subsequently, the extract was clarified by filtration and centrifugation at 11750
141 g for 10 minutes. The supernatant containing the enzyme extract was used for analysis of
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144 The endoglucanase activity (CMCase) was determined according to Ghose [22],
145 using a solution of carboxymethylcellulose (4%) in sodium citrate buffer (pH 4.8). The
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146 total cellulase activity (FPase) was determined using filter paper (Whatman No. 1)
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147 according to Ghose [22]. Xylanase activity was performed in the same manner as
148 CMCase activity, and the substrate consisted of 1.0% xylan solution (Sigma-Aldrich,
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149 USA) using a 5-minute incubation period. Reducing sugars were quantified using the 3,5-
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150 dinitrosalicylic acid method (DNS) according to Miller [23]. For all enzymes, one unit of
151 enzyme activity was defined as the amount of enzyme required to release the equivalent
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152 to 1 µmol of glucose per minute under the assay conditions. The enzyme productivity
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153 values were expressed as U/g dry substrate employed in the fermentation medium.
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155 Thermal stability of cellulase enzymes secreted by the fungus T. reesei was
156 analyzed by determining enzymatic activity after incubation in a water bath at different
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157 temperatures between 30-70 °C at 5 °C intervals for 1 hour. The residual activity was
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158 determined under optimal pH and temperature conditions. The initial activity was
159 assumed to be 100% and used to calculate the enzyme activity as a percentage of the
160 initial activity. The pH stability of the enzymes was estimated at pH values between 2.0 -
161 11.0 using different buffers (50 mM) in assays with a 1-hour incubation. The following
162 buffers were used: glycine-HCl buffer (pH 2.0 and 3.0); sodium citrate buffer (pH 4.0 and
163 6.0); sodium phosphate buffer (pH 6.0 to 8.0) and glycine-NaOH buffer (pH 9.0 and
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164 11.0) [20]. The enzyme activity obtained at pH 4.8 was used to calculate the relative
167 Statistical analysis were performed by using Statistica 7.0 [24] using the Turkey’s
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168 test for three independent samples at 5% level of significance (p≤0.05).
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169 3. Results and Discussion
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170 3.1. Chemical characterization of untreated and pretreated Carnauba waste
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untreated (UT-R) and treated waste. Table 1 illustrates the chemical composition and
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173 Table 2 shows the pretreatment yield, removed lignin and hemicelulose and cellulose
174 loss. HT pretreatment was efficient in the removal of hemicellulose, wherein the HT
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175 pretreatment removed 75.08% hemicellulose compared to untreated control (Table 2). In
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176 comparison, efficient removal of lignin was observed with AA pretreatment, which
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177 reduced lignin content in waste by up to 84.55% when compared to the untreated waste.
178 However, AA pretreatment shows low yield and high loss rate of cellulose during pretreatment.
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179 This effect is quite undesirable, because it makes the process impracticable. AL pretreatment
180 removed 75.60% of lignin and 65.54% of hemicellulose, and yield greater than AA, being
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181 43.30%. Expectedly, HP-A reduced lignin and hemicellulose by 50.71% and 60.72%,
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182 respectively, as compared to untreated waste. This result was expected since several
183 studies have shown the ability of HP-A to reduce lignin and hemicellulose contents
184 [11,17,25]. We highlight that “others” in the Table 1 was considered components not
185 determined in this study such as: remained sterol, fatty acids, pectin, fragments of lignin
186 and polyscchacarides re-precipitated on fibers, in the case of the pretreatments. In some
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188 3.2. FTIR analysis
189 Pretreatments have the ability to change the chemical structure and functional
190 groups present in biomass, thus modifying its properties. FTIR analysis was used to
191 determine changes in Carnauba lignocellulosic waste before and after pretreatments.
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192 Fig.1 shows the FTIR spectra for both untreated and pre-treated wastes and Table 3
193 summarizes the chemical functional groups that were found according to peaks in FTIR
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194 spectra, as well as the measurement of relative changes due to pretreatment compared to
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196 The band at 2920 cm-1 (-CH stretching) was found in all pretreated groups and
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197 was attributed to cellulose, and hemicellulose. This observation was as per expectation.
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198 The pretreatment with hydrogen peroxide caused minimal chemical changes. The
199 hemicellulose can be detected best in the 1735 cm-1 (C = O) band that has been attributed
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200 to acetyl groups since they are related to their conjugates, xylan, for example [27]. It was
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201 observed a reduction of this peak in the AA, AL and the HP-A pretreatments, indicating
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202 that in these pretreatments there was reduction of hemicellulose. However, the HT
203 pretreatment did not change this band. Bands among 1662, 1604, and 1400 cm-1 (C = C)
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204 were assigned to the vibration of the aromatic skeleton of lignin [28–30]. The AL
205 pretreatment showed the greatest changes for the three bands attributed to lignin. As
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206 expected, the HT pretreatment did little to change the concentration of lignin in
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207 accordance with the analyzed spectrum. The band 1056 cm-1 (C-O) located between the
208 bands 1200 and 1035 cm-1 has been related to stretching of cellulose and hemicellulose
209 [27]. Finally, the band 898 cm-1 is characteristic of the glycosidic β-(1-4) bonds that
210 occur in cellulose and attributed to the amorphous cellulose region. Pretreatment AL
211 presented the highest intensity for this band, Table 3 [29].
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212 FTIR results of this study corroborate the observation that AL pretreatment (Table
213 1 and Table 2), followed by AA pretreatment, were the most efficient in removing lignin
214 and hemicellulose, consequently considerably modifying the crystalline structure of the
215 cellulose. The HP-A pretreatment showed a moderate change in the crystalline structure
216 of cellulose, but the changes were more intense than that observed using the HT
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217 pretreatment.
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218 3.3. Crystallinity index as determined by XRD
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219 In many studies the CI of lignocellulosic waste has been used as in indicator of the
220 changes in the structure and crystallinity of cellulose biomasses [31]. The crystallinity of
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221 organic waste is an important factor for saccharification by enzymes as well as microbial
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222 attack. Crystalline cellulose is more recalcitrant than amorphous cellulose, the former
224 Fig. 2 shows the X-ray diffractogram for the pretreated and untreated Carnauba
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225 straw. Through the analysis of the spectra and calculation of CI, it was observed that all
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227 34.6%, which increased to 49.4%, 47.34% and 40.1% after the AL, AA, and HP-A
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229 The effect of the alkaline pretreatment has been widely associated with the
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230 removal of lignin and hemicellulose (to a smaller extent), whereas the use of acid
232 monosaccharides. Furthermore, the HP-A pretreatment removes lignin and some of the
233 hemicellulose. Cellulose is mainly crystalline despite some part being amorphous;
234 however, both lignin and hemicellulose are amorphous [2,33]. Expectedly, the reduction
235 of hemicellulose and lignin can increase the crystallinity of pretreated waste. Thus, the
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236 results of XRD analysis reinforce the results of the chemical characterization (Table 1).
237 In contrast, the HT pretreatment solubilizes hemicellulose and a small portion of lignin
238 [33,34]. In this study, the HT pretreatment was effective in removing hemicelluloses of
239 approximately 75% but to lignin it reduced in a lower extension about 21% (Table 2).
240 This could explain the reduction in crystallinity to 26.77%, which may be related to the
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241 rearrangement of lignin after solubilization of hemicelluloses [34].
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242 3.4. Morphological changes before and after the pretreatment
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243 Scanning electron microscopy (SEM) was performed to analyze the
244 morphological changes of Carnauba straw waste caused by various pretreatments. For
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245 each type of pretreatment, three images at different magnifications are presented in Fig.
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246 3. Fig. 3A, B and C show the morphology of the untreated residue. The morphology of
247 the straw waste after AA pretreatment is shown in Fig. 3D, E and F. In Fig. 3F, it can be
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248 clearly seen that there has been exposure of pulp fibers due to the removal of
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249 hemicellulose and lignin by the combined pretreatment of an acid (2% H2SO4) and a base
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250 (4% NaOH). Images of the alkali pretreatment are shown in Fig. 3G, H, and I. The alkali
251 pretreatment apparently severed and removed lignin of the cellulosic structure. A
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252 comparison between HT pretreated residue (Fig. 3J, K, and L) and untreated control
253 reveals changes in morphology due to HT pretreatment. These changes were attributed to
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254 the removal of hemicellulose as shown in Table 2. Finally, Fig. 3M, N and O, show the
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255 changes caused by HP-A pretreatment. In Fig. 3O exposure of pulp fibers after the
258 The SEM analysis revealed the ability of pretreatments to modify the
259 morphological structure of lignocellulosic residues. The changes observed with the
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260 alkaline pretreatment (Fig.3F) showed that the removal of lignin increased the exposure
261 and separation of cellulose fibers. Similar results have been reported previously for
262 sugarcane residues pretreated with different concentrations of NaOH [35]. Although SEM
263 images showed differences in the fibers after hydrothermal pretreatment, hemicellulose
264 reduction along with an increase in the concentration of lignin in comparison to the
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265 untreated waste was observed. This proportional increase in lignin can be attributed to the
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266 highest removal of hemicelluloses. Additionally, the HT pretreatment removed cellulose
267 which certainly contributed to decreased crystallinity as mentioned above in the analysis.
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268 3.5. Pretreated Carnauba waste-induced production of lignocellulolytic enzymes
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269 The pretreated Carnauba biomass was used as a carbon source for inducing the
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270 production of lignocellulolytic enzymes by the filamentous fungus T. reesei CCT-2768.
271 Production was performed using a SSF process in an Erlenmeyer flask for 168 hours. The
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272 best FPase activity was observed when alkaline hydrogen peroxide-pretreated waste was
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273 used, with FPase activity reaching 0.9 U/g after 72 hours of fermentation as shown in Fig.
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274 4A. Interestingly, the second best inducer was the residue from the hydrothermal
275 pretreatment, showing activity of 0.5 U/g after 48 hours of fermentation. Enzymatic
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276 extracts produced in presence of the fiber samples subjected to the AL and AA
277 pretreatments showed low FPase activity, which was even lesser than the activity of the
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278 enzyme extract of untreated waste. FPase activity in the HP-A pretreated residue extract
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279 showed higher CMCase activity (13 U/g), with this peak production occurring after 96
280 hours of fermentation, as is shown in 4B. The results for xylanase activity showed that
281 the extract using fiber subjected to AA pretreatment led to a peak of 99.5 U/g, after 48
282 hours of fermentation, followed by 94.2 U/g for the enzymatic extract for the HP-A
283 pretreatment as shown in Fig.5C. Secretion of total protein by T. reesei CCT2768 was
284 also more efficient using the HP-A pretreatment, showing the largest peak of protein
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285 secretion, 1.6 mg/g after 96 hours of fermentation (Fig. 4D). Overall, our results show
286 that the HP-A pretreatment was the best for producing cellulolytic enzymes, although it
287 was not the most aggressive or the mildest. Additionally, it effectively removed lignin
288 and hemicellulose at the same time. Studies have shown that pretreatments are able to
289 reduce the recalcitrance of lignocellulosic material, improving the digestibility and the
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290 access of microorganisms to cellulose [9,10,14] Accordingly, removal of both lignin and
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291 hemicelluloses may have favored the access of fungus to cellulosic structure, thus
292 favoring a greater secretion. Furthermore, pretreatment using alkaline hydrogen peroxide
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293 produces less recalcitrant inhibiting substances which may explain the results obtained in
294 this extract [11]. In addition to increased secreted amount, another important factor to
295
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note is the time spent in the production of enzymes. The extract with hydrogen peroxide
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296 was able to show an FPase activity peak at 72 hours of fermentation. Other kinetic studies
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297 using different strains of T. reesei induced by different residues show the best times in the
298 range of four to eight days (or 96 to 192 hours) [4]. Decreasing enzyme production time
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300 operating costs. The enzymatic extract induced by pretreated alkaline straw showed high
301 xylanase production in 48 hours. The alkaline pretreatment works mainly by removing
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302 the lignin and does not considerably alter the concentration of hemicellulose in the fibers
303
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304 hemicellulose. It is possible that in AL pretreatment, the efficient removal of lignin favors
305 the attack of the residual hemicellulose by xylanases, which would explain why the
308 HP-A pretreatment induced cellulolytic enzyme synthesis to the greatest extent.
309 The stability of FPase and CMCase (extracted from the SSF of HP-A pretreated biomass)
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310 at different temperatures and pH was analyzed. For this, the activity of these enzymes at a
312 materials and methods. In Fig. 5A it can be seen that at the temperature of 55 °C, FPase
313 activity reaches a level of about 55% of optimal, while 75% CMCase activity was
314 achieved, but both these dropped to 25% when exposed to 60 °C for 1 hour. Similar
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315 results were obtained using Trichoderma C4 and Trichoderma atroviride 676 [36]. The
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316 use of enzymes showing thermostability at high temperatures is of great interest for
317 bioprocesses.
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318 In Fig. 6B, the stability of FPase and CMCase activity in the pH range from 2 to
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319 11 is shown. An interesting result can be seen for FPase activity, which has stability
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320 above 80% in the pH range of 2-6. This indicates preferential activity at acidic pH.
321 Further, FPase activity remains at around 50% between the pH values of 6 to 11. The
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322 CMCase activity showed higher relative activity at alkaline pH. In the pH range of 7 to
323 11, CMCase activity remained above 80%, moreover, in the pH range from 2 to 6 activity
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324 remains high with 75% relative activity. This result is very encouraging since enzymes
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325 are involved in a wide pH range, allowing greater biotechnological application [20].
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327 To the best of our knowledge in the current study it was the first one to examine
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328 Carnauba straw residue pretreated with alkaline hydrogen peroxide for using as an
329 inducer. The induction of enzymes by lignocellulolytic carbon sources derived from
330 agricultural residues is an important approach to minimize the cost of enzyme production.
331 These enzymes can be applied in various biotechnological processes, with special
332 attention to the production of cellulosic ethanol. We compared the use of Carnauba straw
333 untreated (UT-R) and pretreated with alkaline hydrogen peroxide (HP-A) with three other
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334 untreated and pretreated agricultural wastes: green coconut (CT), sugarcane (SC) and
335 cashew (CW). The hydrogen peroxide pretreatments were carried out under the same
336 conditions for all residues, following the same methodology for the Carnauba straw as
337 mentioned in section 2.3. Enzymes were produced for 72 hours by SSF, the best FPase
338 activity being using HP-A (Fig. 4A). Fig. 6A shows FPase activity for the different
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339 wastes used. It can be seen that the enzyme extract produced with the residue pretreated
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340 using the HP-A demonstrated twice more activity than untreated. However, when
341 comparing HP-A with the pretreated residues it was observed that HP-A has activity
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342 seven-fold higher than SC and CW. Although HP-A has FPAse activity greater than CT,
343 this difference was not significantly. Although, induced CMCase activity was significantly
344
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higher to HP-A, even comparing all untreated and pretreated residues (Fig. 6B). Fig. 6C shows
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345 the results of xylanase activity. Untreated carnauba and cashew straw residues showed
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346 low xylanase activity when compared to other residues, except when compared to
347 pretreated SC that had reduced ability to induce xylanases. The HP-A residue showed
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348 higher xylanase production (82 U/g), followed by untreated sugarcane residue (65 U/g)
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349 and pretreated coconut residue (74 U/g), respectively. Fig. 6C shows the results for
350 xylanase activity. The residue of Carnauba straw and untreated cashew showed low
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351 xylanase activity, when compared to other wastes. The Carnauba straw residue treated
with alkaline hydrogen peroxide had the highest xylanase production (72 U/g) it is
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352
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353 slightly above the residue of sugarcane (65 U/g) and coconut residue (57 U/g),
354 respectively. Finally, the secretion of total proteins by T. reesei CCT2768 was equally
355 significant in HP-A and pretreated CT, 1.75mg/g and 1.72mg/g, respectively.
356 The comparison of the induction by Carnauba straw waste with different
357 pretreatment confirms the potential of the HP-A pretreatment of producing cellulolytic
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359 production of second generation ethanol mainly due to the lower yields as shown in the
360 present study as well as by the formation of inhibitors [9,37]. Considering a renewable
361 energy approach, pretreatment influence shows different performance depending on the
362 aim of cellulolytic enzyme or ethanol production. For instance, aiming ethanol production
363 AL and AA cause significant losses on pentoses and lignin. This fact is not interesting,
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364 thus it should be reused in the biorefinery context. A strategy used to bypass such
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365 limitation is to use engineering strains to able to ferment pentoses [38,39]. For enzymes
366 production is quite important to have residual hemicelluose, for instance, aiming the
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367 xylanase production as shown in the present study. Additionally, lignin can be burnt to
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369 4. Conclusion
370 This study showed that pretreatments changed the physical and chemical
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371 characteristics of the Carnauba straw waste. The HP-A pretreatment is more efficient in
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372 removing lignin and hemicellulose at the same time. Moreover, under the conditions
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373 studied HP-A treated straw induced the production of FPase (0.9 U/g) and CMCase (13
374 U/g) within 96 hours in T. reesei CCT2768 during SSF, better than the other
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375 pretreatments. Overall, the Carnauba residue pretreated with HP-A produced more
376 cellulolytic enzymes than Carnauba, sugarcane, cashews and coconut untreated used in
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377 this study. The enzymes produced have great thermal and pH stability. Finally, the use of
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378 Carnauba agricultural waste pretreated with HP-A is a promising process to produce
380 Acknowledgments
381 The authors thank CAPES and Brazilian National Council for Research (CNPq) for
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383
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393 anacardo (anacardium occidentale L.) mediante secador solar de radiación directa,
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395 [4] G.H. Hansen, M. Lübeck, J.C. Frisvad, P.S. Lübeck, B. Andersen, Production of
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548 Table captions
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549 Table 1: Chemical composition of the biomass before and after pretreatment.
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550 Pretreatments: UT-R (untreated waste) HT (hydrothermal), HP-A (alkaline hydrogen
551 peroxide), AL (alkaline) and AA (alkaline - acid). The same letters indicate no
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UT-R 23.96 ± 0.75d 11.84 ± 0.82b 32.79 ± 0.91b 11.62 ± 0.45a 7.69 ± 0.05a 12.1 ± 0.37e
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HT 34.46 ± 0.21b 4.36 ± 0.45d 38.3 ± 1.38a 3.22 ± 0.44b 6.32 ± 0.17b 13.34 ± 0.48d
HP-A 32.51 ± 0.54c 7.85 ± 0.04c 27.27 ± 0.57c 2.53 ± 0.08b 5.39 ± 0.05c 24.45 ± 0.13a
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AA 44.31 ± 0.93 a 17.56 ± 0.32a 21.81 ± 0.85d 0.73 ± 0.16c 0.87 ± 0.05e 14.72 ± 0.18c
AL 45.16 ± 0.72a 17.51 ± 0.43a 18.49 ± 1.07e 0.94 ± 0.29c 1.90 ± 0.05d 16.0 ± 0.66b
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559 Table 2: Biomass yield and removing main compounds after pretreatment
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574 Fig.1. FTIR analysis of the Carnauba waste before and after pretreatment. The
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575 represented spectra correspond UT-R (untreated), HT (hydrothermal pretreatment), HP-A
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576 (alkaline hydrogen peroxide pretreatment), AA (acid alkali pretreatment) and AL
578 Fig.2. Diffractogram of untreated and pretreated Carnauba waste. UT-R - waste; B -
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584 Fig.4. Production of cellulolytic enzymes using different pretreatments of carnauba leaf
585 waste as carbon source for T. reesei CCT2768. The kinetics of production cellulolytic
586 enzymes induced by untreated waste (UT-R), hydrothermal pretreatment (HT), acid
587 alkaline pretreatment (AL), hydrogen peroxide pretreatment (HP-A) and acid alkaline
588 pretreatment, were produced in SSF for 168 hours at 30 °C. Each value represents the
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590 Fig.5. Thermal and pH stability of concentrated cellulolytic enzymes produced by T.
591 reesei CCT2768 induced waste carnauba pretreated with alkaline hydrogen peroxide.
592 Each value represents the mean and standard deviation of three experiments.
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594 (CS) untreated and pretreated using the HP-A pretreatment and three other agricultural
595 wastes inducing carbon sources untreated and pretreated: CT (coconut waste), SC
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