Detoxification of Rice Straw and Olive Tree Pruning Hemicellulosic

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Detoxification of rice straw and olive tree pruning hemicellulosic hydrolysates employing Saccharomyces cerevisiae and its effect on the ethanol production by Pichia stipitis Bruno Guedes Fonseca, Juan Gabriel Puentes, Soledad Mateo, Sebastian Sánchez, Alberto J. Moya, and Ines C. Roberto J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 30 Aug 2013 Downloaded from http://pubs.acs.org on August 30, 2013

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Journal of Agricultural and Food Chemistry

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Detoxification of rice straw and olive tree pruning

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hemicellulosic hydrolysates employing Saccharomyces

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cerevisiae and its effect on the ethanol production by Pichia

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stipitis

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Bruno Guedes Fonseca,† Juan Gabriel Puentes,‡ Soledad Mateo,‡ Sebastian Sánchez,‡ Alberto J.

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Moya,‡ Inês Conceição Roberto*†

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†

Department of Biotechnology, Engineering College of Lorena, University of São Paulo, Lorena,

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Brazil

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‡

Department of Chemical, Environmental and Materials Engineering, University of Jaén, Jaén, Spain

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*Corresponding author:

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Tel.: +55 12 3159 5026; fax: +55 12 3153 3165

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Email address: [email protected]

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ABSTRACT

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The aim of this work was to study the ability of Saccharomyces cerevisiae (baker’s yeast) to

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metabolize a variety of aromatic compounds found in rice straw (RSHH) and olive tree pruning

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(OTHH) hemicellulosic hydrolysates, obtained by acid hydrolysis at different sugar and toxic

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compound concentrations. Initially, the hydrolysates were inoculated with S. cerevisiae (10 g L-1)

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and incubated at 30 °C under agitation at 200 rpm for 6 hours. The results showed that this yeast

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was able to utilize phenolic and furan compounds in both hemicellulose hydrolysates. Next, the

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treated hydrolysates were inoculated with Pichia stipitis NRRL Y-7124 to evaluate the effect of

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biotransformation of aromatic compounds on ethanol production, and better fermentation results

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were obtained in this case compared to untreated ones. The untreated hemicellulose hydrolysates

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were not able to be fermented when they were incubated with Pichia stipitis. However, in RSHH

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treated, ethanol (YP/S) and biomass (YX/S) yields and volumetric ethanol productivity (QP) were 0.17

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g g-1, 0.15 g g-1 and 0.09 g L-1 h-1, respectively. The OTHH-treated showed less favorable results

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compared to RSHH, but fermentation process was favored with regard to untreated hydrolysate.

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These results showed that the fermentation by P. stipitis in untreated hydrolysates was strongly

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inhibited by toxic compounds present in the media, and that treatment with S. cerevisiae promoted a

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significant reduction in their toxicities.

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KEYWORDS Biological detoxification, biotransformation of the aromatic compounds, rice straw,

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olive tree pruning, hemicellulosic hydrolysates, Saccharomyces cerevisiae

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INTRODUCTION Lignocellulosic biomass such as rice straw and olive tree pruning are inexpensive sources of

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polysaccharides that can be degraded in monomeric sugars.1,2 One of the most abundant

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lignocellulosic feedstocks in developing countries, including Brazil, is the straw of various cereals,

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and the rice straw is being reported as one of the greatest byproducts of agricultural activities.3 The

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world production of rice crop for 2012 has reached a record of 488 million tons of grain, generating

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680 million tons of straw distributed around the world, with a potential production of energy of 205

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billion liters per year. 4 Debris from olive tree pruning represents a great volume of biomass in

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Spain, especially in Andalucia, where annual production reaches 3.9 million tons, varying between

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1,700 and 3,000 kg/(ha year), depending on culture conditions.2,5 Due to the large volumes of this

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residue and the environmental damage associated with this biomass, it has been suggested the

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possibility of valuing these byproducts from the agricultural industry.1,2

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The major organic constituents of lignocellulosic materials are cellulose (30-50%),

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hemicellulose (20-40%) and lignin (15-30%).6 The hemicellulosic fraction of the residues can be

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recovered by dilute acid hydrolysis, generating mainly D-xylose monomers in the rice straw

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hemicellulosic hydrolysate,1 and combination of D-xylose and D-glucose monomers in olive tree

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pruning hydrolysate, as well.2 Besides these monosaccharides, several inhibitory compounds are

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also formed during hydrolysis processes, including some products of sugar degradation (furfural and

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5-hydroxymethylfurfural – 5-HMF), phenolic compounds derived from lignin degradation, and

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acetic acid, derived from hemicellulosic structure.1,2,7 The main phenolic compounds present in rice

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straw and olive tree pruning hemicellulosic hydrolysates are vanillin, vanillyl alcohol, vanillic acid,

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ferulic acid and coumaric acid.2,8,9 In addition, the olive tree hemicellulosic hydrolysate may also

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contain others phenolic compounds such as oleuropein, tyrosol, hydroxytyrosol and elenolic acid,

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known to present antioxidant properties, and antimicrobial properties by denaturing proteins and



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inactivating enzymes.9-12 The amount and type of toxic compounds generated depends on both

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severity pretreatment and biomass source, and their effects on Pichia stipitis fermentation have been

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studied.8,13,14 For example, Díaz et al.13 reported a decrease of 35% in ethanol volumetric production

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when P. stipitis CECT 1922 was cultivated in synthetic medium containing D-glucose (20.0 g L-1)

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and D-xylose (15.0 g L-1), and 2.0 g L-1 of furfural. Evaluating the ethanol production by P. stipitis

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CBS 5773 in sugarcane bagasse hemicellulosic hydrolysate, Roberto et al.14 observed that furfural

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concentrations above 2.0 g L-1 inhibited cell growth. However, at concentrations below 0.5 g L-1, the

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aldehyde acted as carbon source, with positive effect on the cell growth.

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It is important to identify the potential inhibitors present in the hemicellulose hydrolysates

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before proposing a method or a sequence of methods for detoxification. This knowledge not only

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helps to choose an efficient and low-cost treatment but also let to establish conditions for hydrolysis

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that minimize its formation.7,8,15-17 Such strategies include physical, chemical and/or biological

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methods. Regarding to physical methods, the adsorption of inhibitors using activated carbon is

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effective in removing acetic acid and phenolic compounds, allowing better fermentation efficiency.

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However, these methods can remove substantial quantities of sugars in the medium by

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degradation.7,15 Chemical detoxification methods, which are based on pH change, have also been

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extensively studied. In these methods, the reduction of the toxicity of the hydrolysates is related to

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precipitation of metal ions and/or decomposition of furans which are instable under certain pH

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conditions. These methods alone do not provide satisfactory results and are often employed in

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combination with physical methods.7,15,16

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The use of microorganisms and/or enzymes for detoxification of hydrolysates has been

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explored as a method able to overcome the toxicity of the hemicellulosic hydrolysates. In fact,

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studies using laccase to detoxify different hydrolysates, by oxidative polymerization of phenolic

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compounds, showed increase in fermentation parameters compared with the untreated media.15,17

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Jonsson et al.,17 evaluating fermentation of wood cellulosic hydrolysate by S. cerevisiae, and ACS Paragon Plus Environment Page 4 of 31

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Chandel et al.,15 fermenting hemicellulosic hydrolysate from sugarcane bagasse with Candida

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shehatae NCIM 3501, observed an increase on ethanol volumetric productivity (0.8 to 2.7 g L-1 h-1)

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and xylitol (0.14 to 0.27 g L-1 h-1), respectively. Using Issatchenkia occidentalis CCTCC M 206097

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as detoxifying-agent and Candida tropicalis CCTCC M 205067 as fermentation-agent of

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hydrolysate from sugarcane bagasse hemicellulosic, Hou-Rui et al.18 verified an increase of 50%

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xylitol volumetric productivity (1.03 for 2.01 g L-1 h-1). However, I. occidentalis specie consumed

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D-xylose, the main sugar present in the hemicellulosic hydrolysate.18,19

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In the present study, we propose the use of baker's yeast Saccharomyces cerevisiae to

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detoxify hemicellulosic hydrolysates from rice straw and olive tree pruning, since this

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microorganism is capable of converting furans and phenolic aldehydes into their corresponding

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alcohols, which are considered less toxic to the metabolism of various fermentative microorganisms,

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include P. stipitis.20,21 In addition, S. cerevisiae does not consume D-xylose, the main sugar present

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in most plant biomass hemicellulose hydrolysates, which makes the advantageous process. The aim

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of this study is to contribute to the development of a biological detoxification procedure decreasing

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the toxicity of the hemicellulosic hydrolysates and to establish suitable conditions for the

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bioconversion of this hydrolysate fraction in ethanol.

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Materials and methods

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Raw material

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Rice straw was collected in Lorena, São Paulo state, Brazil. The material was sun-dried until

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approximately 10% moisture, milled to attain particles of about 1.0 cm in length and 1.0 mm in

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thickness. The olive tree pruning biomass consisted of leaves and branches (1:2 ratio) from trunks

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olive trees and was collected in an olive grove situated in Jaén, Spain. The material was air-dried

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until 5% moisture, milled, screened to select the fraction of particles with diameter from 0.425-0.60



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mm and homogenized in a single lot. The average chemical composition of rice straw and olive tree

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pruning, determined according to methodology previously described by Browning 22, are shows in

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Table 1.

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Hydrolysis Procedure Rice straw hydrolysis experiments were carried out in a 350-L stainless steel reactor, at

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120 °C, 50 rpm, during 30 min, and hydrolytic reaction of the olive tree pruning residue was carried

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out in autoclave, at 120 °C for 90 min. Both biomasses were impregnated with sulfuric acid solution

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at 1.0 and 2.0% w v-1 at solid/acid solution ration of 1:10, according to Roberto et al.1 and Mateo et

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al.,23 respectively. The slurries obtained after hydrolysis were separated by centrifugation (2000xg,

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20 min) and the liquid phases (hemicellulosic hydrolysates) were concentrated under vacuum at

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70 °C in order to obtain approximate concentrations of furans and phenolics compounds (6 and 3

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fold increase of the rice straw and olive tree pruning hemicellulosic hydrolysates, respectively). The

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volume of the hemicellulosic hydrolysates obtained after hydrolysis-reaction was divided into 3

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equal parts and concentrated under vacuum separately.

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Hemicellulosic hydrolysates biodetoxification

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The assays of biodetoxification were realized in triplicate. The pH of hemicellulosic

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hydrolysates was adjusted to 5.5 with NaOH (pellets), followed by centrifugation at 1000xg for 15

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minutes to remove the insoluble materials. First, the liquors were treated with 10 g L-1 of lyophilized

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commercial Saccharomyces cerevisiae (Dr. Oetker®). The yeast was previously hydrated in distilled

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water for 10 min at room temperature, with cellular viability greater than 85%. The

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biodetoxification experiments were performed in shaker at 30 °C, 200 rpm for 6 hours, using 250

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mL Erlenmeyer flasks filled with 50 mL of hemicellulosic hydrolysates.

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Pichia stipitis inoculum preparation

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Cultures of the Pichia stipitis NRRLY-7124 were maintained on malt extract agar slants at

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4 °C. The inoculum was prepared by transfer of yeast cells in the maintenance medium to 250 mL

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Erlenmeyer flasks containing 50 mL of the medium composed by (g L-1): xylose (30.0),

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MgSO4.7H2O (1.0), (NH4)2HPO4 (3.0), KH2PO4 (19.0) and yeast extract (3.0). The inoculated flasks

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were incubated in rotary shaker at 30 °C under stirring at 200 rpm for 24 hours. Next, the cells were

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recovered by centrifugation (1100xg for 20 minutes) and resuspended in sterile distilled water in

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order to obtain the cell concentrated suspension which was used as inoculum.

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Medium and fermentation conditions

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Fermentations assays were realized in triplicate carried out in untreated and treated

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hemicellulosic hydrolysates. First, the pH of hemicellulosic hydrolysates was adjusted to 5.5 with

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NaOH (pellets). Next, the hydrolysates were centrifuged to remove solid residue. The hemicellulosic

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hydrolysate from rice straw was supplemented with yeast extract (3.0 g L-1),24 and the olive tree

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hydrolysate with (g L-1): MgSO4.7H2O (1.0), (NH4)2SO4 (3.0), KH2PO4 (2.0), peptone (3.6), and

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yeast extract (4.0).23 The experiments were carried out in 125 mL Erlenmeyer flasks containing 50

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mL of hemicellulosic hydrolysates and inoculated with 1 g L-1 of cells. The flasks were incubated in

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a rotator shaker at 30 °C, 200 rpm for 96 hours.

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Analytical methods Sugars, acetic acid, and ethanol concentrations were determined by high performance liquid

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chromatography (HPLC) in Waters (Milford, MA, USA) equipped with a refractive index detector

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and a Bio-Rad Aminex HPX-87H column (300×7.8 mm) (Hercules, CA, USA). Operation

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conditions included: temperature of 45 °C, 0.005 M sulfuric acid as eluent in a flow of 0.6 mL min-1

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and sample volume of 20 µL. Dry-weight (g L-1) calibration was determined by the absorbance of ACS Paragon Plus Environment Page 7 of 31


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the cell suspension at a wavelength of 600 nm. Total furans were determined by spectrophotometric

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method described by Martinez et al.25 The total content of phenolic compounds in hemicellulosic

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hydrolysates was estimated by the Folin-Ciocalteu modified method26 using gallic or caffeic acids as

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standard. The ultraviolet-spectrums (UV) of the hydrolysates were carried out in the range of 450-

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200 nm, with a pitch of 5 nm using quartz cuvettes. The spectrum determination was made with

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hydrolysate diluted with alkaline water (pH around 12), and distilled water used as blank (pH 12).

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RESULTS AND DISCUSSION

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Raw Materials composition

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The chemical composition and the percentage contents of rice straw and pruning olive

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employed in this study are presented in Table 1. It is found that among the structural components,

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cellulose corresponded to a greater portion of the material, and hemicellulose present in rice straw

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was 41% higher than that found for the olive tree pruning. It also observes in Table 1 that the ash

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rice straw content is 40% higher than the olive tree pruning. This behavior was expected, since rice

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straw when compared with other lignocellulosic materials, straw ash has above. For example, the

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ash content present in malt bagasse and olive pruning, reported by Roberto et al.1 and Mateo et al.27,

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were 11.4 and 3.8%, respectively. This peculiarity of rice straw can be explained by the fact that the

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material contains a high content of silicon dioxide (SiO2), which confers high-level resistance

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against pathogenic microorganisms, however, complicates the process of degradation.3,5

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When comparing the results of rice straw characterization in the present study with other

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works reported in the literature, it appears that the cellulose, hemicellulose and lignin contents are

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within the range of variation 35 to 43%, 23 to 26% and 13 to 25%, respectively. As for the ash

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content, the material of the present study showed a value much lower than other values reported in

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the literature (approximately 12%).1,3,5,6,8 For the olive tree pruning, it is found that the levels of


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cellulose (25 to 31%), hemicellulose (13 to 23%) and lignin (18 to 24% ) are also within the

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variation range reported in the literature.2,1323,27 The differences in the composition observed for this

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type of residue may be associated with the ratio of wood: sheet generated after pruning process.23

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Hemicellulosic hydrolysates composition The composition of the rice straw (RSHH) and olive tree pruning (OTHH) hemicellulosic

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hydrolysates obtained by acid catalysis is showed in Table 2. As can be seen, the sugars and

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inhibitor compounds concentrations in hydrolysates varied with raw materials employed as well as

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the conditions that have been employed in the hydrolysis process.

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Total sugar concentration presented in RSHH and OTHH after dilute acid treatment were of

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18.9 and 23.7g L-1, respectively. It is verified that D-xylose (14.0 g L-1) as the highest concentration

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monomer in the RSHH. On the other hand, in OTHH both D-xylose (9.3 g L-1) and D-glucose (10.6

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g L-1) were the major products obtained. In this hydrolysate, the D-glucose:D-xylose ratio may

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depend on the leaves and branches proportion present in the olive tree pruning. In fact, Mateo et al.23

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showed that the leaves and branches mixture of olive tree pruning contain a 1.3/1.0

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hemicellulose/cellulose ration, while this fraction goes to 1.0/1.0 when olive tree pruning leaves free

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is used. The authors also found that in leaves of the olive tree pruning hemicellulosic fraction is

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twice than cellulose. In other work, Mateo et al.27 show that higher D-glucose and D-xylose yields in

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olive tree pruning hemicellulosic acid hydrolysate, with leaves/branches proportion near to one,

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were 10.0 and 9.4% respectively, obtained hydrolysis conditions were 190 °C, 0 min and 1% of

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H2SO4. These results are very similar to those obtained in present work.

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Table 2 also shows that D-glucose concentration obtained in OTHH is 4.2 times greater than

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in RSHH. While D-xylose was the main monosaccharide in RSHH, and the pentoses proportion in

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this hydrolysate was 1.3 times higher than in OTHH. This can be justified because of the different

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nature of these biomasses. Besides sugars concentration, Table 2 shows that the hydrolysates contain ACS Paragon Plus Environment Page 9 of 31


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compounds potentially harmful to the fermentation process, such as acetic acid, furans and phenolic

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compounds. It is noticed that OTHH has higher concentrations of these toxic/inhibitory compounds.

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In both hemicellulosic hydrolysates the total phenolic compounds represents the main problem as its

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concentration is highest.

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The concentration under vacuum process promoted proportional variations to the

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concentration factor only in the D-glucose and D-xylose levels, in both hemicellulosic hydrolysates

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(6.0 and 3.0 times for RSHH and OTHH, respectively), according to Table 2. The total fermentable

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sugars (D-glucose + D-xylose) concentrations were 101.6 and 62.6 g L-1 for RSHH and OTHH,

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respectively, while L-arabinose concentration in both hydrolysates was approximately 15.8 g L-1.

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Regarding inhibitor compounds, it was observed that the concentration of acetic acid increased too

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much in OTHH, corresponding 81%. The increase in total furans was 76 and 11%, with about 0.85 g

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L-1 for both hydrolysates, and phenolic compounds levels, after the process of concentration,

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reached near 10.0 g L-1 in both concentrated hydrolysates. The non-proportional increase of these

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inhibitor compounds after concentration process can be explained by the different boiling-points

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under vacuum of them. So during concentration step, acetic acid and furfural get more easily

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volatilized.28

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The presence of these toxic compounds in hemicellulosic hydrolysates pose problems for the

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bioconversion of sugars to ethanol by Pichia stipitis.2,7,8 For this reason, the RSHH and OTHH were

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submitted to biodetoxification procedures employing S. cerevisiae, aiming a reduction of these

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compounds concentration, taking in advantage the very similar concentration of total furans and

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phenolics in both concentrated hemicellulosic hydrolysates.

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Biodetoxification of the hemicellulosic hydrolysates The reduction percentages in sugars and inhibitors concentration present in hemicellulosic hydrolysates from rice straw and olive tree treated with S. cerevisiae are showed in Figure 1. As can ACS Paragon Plus Environment Page 10 of 31

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be seen, that S. cerevisiae consumed 88 and 20% of the D-glucose, with ethanol production of 5.5

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and 1.5 g L-1 in RSHH and OTHH, respectively. As expected, the pentoses were non-assimilated, in

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both hemicellulosic hydrolysates, due to a lack of the enzyme responsible for D-xylose and L-

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arabinose assimilation.29

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It is verified also in Figure 1 that the acetic acid concentration increased slightly after

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detoxification process. This increase can be attributed to the reduction of the furans and phenols

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aldehydes to their respective alcohols by yeast S.cerevisiae These reactions are catalyzed by non-

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specific reductase-enzymes, such as alcohol dehydrogenase, which leads to an increase in

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intracellular acetaldehyde levels and subsequent oxidation of the aldehyde to acetic acid. 13,20,21

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Baker’s yeast was able to reduce the total furans in RSSH and OTHH by 50 and 35%, with

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residual concentrations of 0.44 and 0.54 g L-1, respectively. These results were expected, since yeast

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has metabolic capacity to reduce furan aldehydes to their corresponding alcohols.30-35 However, it

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was observed no reduction in total phenolic compounds concentration in the RSHH (Figure 1a), and

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only 5% in OTHH (Figure 1b). This fact may be associated with the reaction of Folin-reagent and

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phenolic compounds. The reaction occurs between the hydroxyl groups present in these compounds

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and S. cerevisiae shows capability of reducing phenolic aldehydes and acids groups to their

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respective alcohols, which can compromise the analysis.36 So in order to confirm the reduction in

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the furans and phenolic compounds concentration, ultraviolet spectra were performed before and

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after the hydrolysates biotransformation. This technique can provide information regarding the

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presence of phenolic compounds derived from lignin and furans resulting from the degradation of

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carbohydrates, and permits to evaluate changes in concentration or even changes in the chemical

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structure of these molecules.8,25,37,38

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Figure 2 shows ultraviolet spectrum of the hemicellulosic hydrolysates before and after

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biological detoxification by S. cerevisiae, and it was verified a decrease of peaks in the 250-400 nm

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regions in both hydrolysates employed. In the present study, it was observed an absorptive reduction ACS Paragon Plus Environment Page 11 of 31


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of 36 and 17% at 280 nm for RSHH and OTHH, respectively. According to Martinez et al.,25 most

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of the reduction in 280 nm is attributable to furfural and/or 5-HMF, since this region is verified as

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maximum absorption area of these inhibitors, and only a small part of the soluble lignin is

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quantified. It is also known that the reduction-products of furans compounds have greater

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ultraviolet-absorptive in the 190-210 nm regions.39 Some studies have reported a reduction of 5-

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HMF to alcohol-HMF,30-32 as well as the furfural reduction to furfuryl-alcohol under anaerobic and

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aerobic when S. cerevisiae is used.30,33,34 Liu et al.31 suggest that the aldehyde functional group

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present on the furan ring is the cause of toxicity of these compounds, however their reduced forms

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are less inhibitory to fermentative yeasts, such as P. stipitis.

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Regarding the soluble products from acid-degradation of lignin, the spectra range among

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320–400 nm is attributed to aromatic compounds having aromatic ring mediated conjugation

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between hydroxyl and carbonyl groups. The peak with a maximum at approximately 300 nm is

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characteristic of non-conjugated hydroxyl groups.8 In the present study the UV-profiles show for

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RSHH and OTHH reductions of 36 and 13% respectively, for both regions (300-350 nm). These

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results suggest a reduction in the low molecular weight phenolic compounds concentrations present

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in the hemicellulosic hydrolysates, which was not observed for the Folin-reagent methodology. This

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reduction in UV-absorptivity was expected, since S. cerevisiae has the metabolic capacity to convert

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aldehyde and phenolic acid in their corresponding alcohols. Mathew et al.40 indicated that the ferulic

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acid decarboxylation to produce 4-vinyl-guaiacol leads to an increase in the maximum UV-

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absorptivity in the 210 nm region. The authors suggested that this change is due to phenolic alcohols

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formation. Karmakar et al.41 analyzed the degradation product of the ferulic acid by employing the

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bacterium Bacillus coagulans, and detected 4-vinyl-guaiacol, as well as a decrease in UV-

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absorptivity at the 290-310 nm regions.

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On the other hand, the reduction percentages of phenol and furan compounds were higher at RSHH than OTHH. This may be explained attending to the different acetic acid concentrations in ACS Paragon Plus Environment Page 12 of 31

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theses hydrolysates. It is significative that a double acetic acid concentration in OTHH cause a half

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reduction of aromatic compounds compared with RSHH.

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Although reductions in furan and phenolic compounds in the present study have not been

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completed, one advantage to use S. cerevisiae as the detoxifying agent is that this yeast does not

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consume the main sugar present in hemicellulose hydrolysates (D-xylose). Some studies of

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biological detoxification have reported total reduction of these toxic compounds, but also with

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consumption of pentoses. For example, Zhang et al.42 reported that Amorphotheca resinae ZN1

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demonstrated the ability to degrade completely furans contained in corn stover hemicellulosic

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hydrolysate; however, the strain consumed 57% of D-xylose, making the method disadvantageous

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because the loss of this sugar compromises the subsequent fermentation step.

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Effect of biodetoxification on ethanol production The treatment efficiency of hydrolysates with S. cerevisiae was evaluated by Pichia stipitis

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regarding the sugar consumption, cell growth and ethanol conversion (Figures 3 and 4). Figure 3

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shows the sugars consumption by P. stipitis in RSHH and OTHH, respectively. D-xylose

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consumption by P. stipitis was not observed in both untreated hemicellulose hydrolysates, and that

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only 25% of the D-glucose was consumed in the OTHH, with no hexose consumption in the RSHH.

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Once both hydrolysates were biodetoxified, D-glucose was completely consumed by P.

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stipitis. This consume requires just less than 24 h for RSHH and 96 h for OTHH. The D-xylose

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consumption includes a lag phase of 24 h for RSHH and 48 h for OTHH. After this time, D-xylose

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consumption increased to approximately 55% after 96 hours fermentation assays, with a

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consumption rate of approximately 0.49 and 0.16 g L-1 h-1, for RSHH and OTHH respectively. In

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OTHH, it was observed that the D-glucose and D-xylose consumption occurred simultaneously,

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beginning after 48 hours of the assay. This behavior was not observed by Agbogbo et al.43 when

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growing P. stipitis CBS 6054 in media containing different proportions of these sugars (60.0 g L-1 of ACS Paragon Plus Environment Page 13 of 31


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total sugars). The authors found that first the yeast consumed all the D-hexose present in the

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medium to finally start D-xylose consumption, and the D-glucose consumption rate was always

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higher than that of D-xylose, as observed in the present study. This delay in sugar consumption can

314

be associated with yeast adaptation phase in medium containing high acetic acid concentration. In

315

the present study, the acetic acid initial concentration in OTHH was 4.1 g L-1, which represents acid

316

concentration of approximately 50% higher than in RSHH. Diaz et al.13 observed that the presence

317

of acetic acid in a fermentation medium with D-glucose (20.0 g L-1) and D-xylose (15.0 g L-1)

318

affects P. stipitis metabolism, even at the lowest concentration tested (3.0 g L-1). Another study

319

demonstrates that acetic acid concentrations of 3.5 g L-1 completely inhibit the growth of P. stipitis

320

and ethanol production.44 According to Bellido et al.,44 the D-xylose consumption was more affected

321

by the acid presence than D-glucose, being more pronounced as the concentration of acetic acid

322

increased. Another fact that would justify the low D-xylose consumption by P. stipitis in OTHH is

323

the presence of D-glucose at higher concentrations which could lead to enzymes catabolic repression

324

that utilize D-xylose.29

325

Figure 4 shows that the biotransformation favored P. stipitis growth and ethanol production

326

in both hydrolysates. These results can be explained by reduction in the total furan and phenolic

327

concentration. It can be seen that the biotransformation improved P. stipitis growth by 4.5 and 3.6

328

times in RSHH and OTHH, respectively, compared with untreated results. The growth rate in

329

treated-RSHH was approximately 0.08 g L-1 h-1, i.e. 25% higher than observed for treated-OTHH,

330

where we again observed an adaptation phase of near 48 hours.

331

The ethanol production by Pichia stipitis during fermentation of the hemicellulosic

332

hydrolysates also can be observed in Figure 4. It was verified an ethanol production (final minus

333

initial concentration) of 8.3 and 5.9 g L-1 in RSHH and OTHH, respectively. In the OTHH

334

fermentation (Figure 4b) the ethanol concentration started with 1.2 g L-1, and after 48 h decrease to

335

0.72 g L-1, reaching 7.1 g L-1 at the fermentation end time (96 h). According to Figure 4a, this ACS Paragon Plus Environment Page 14 of 31

Page 15 of 31


336

profile was not observed for RSHH, P. stipitis produced 13.8 g L-1 in the fermentative process. The

337

ethanol effect in the D-glucose and D-xylose fermentability by P. stipitis was studied by Meyrial et

338

al.45 The authors demonstrated that the alcoholic fermentation of P. stipitis NRRL Y-7124 in a

339

medium containing D-glucose or D-xylose (50.0 g L-1), can be affected depending of the ethanol

340

initial concentration. It was also observed that ethanol concentrations down to 10 g L-1, do not affect

341

yeast growth and ethanol volumetric production.

342

The fermentation parameters such as ethanol (YP/S) and biomass (YX/S) yield factors, ethanol

343

volumetric productivity (QP) and ethanol efficiency ( ) are given in Table 3. Ethanol yield factor

344

(grams per gram) was defined as the ratio between ethanol concentration (grams per liter) and total

345

substrate consumed (grams per liter). Ethanol volumetric productivity (grams per liter per hour) was

346

calculated as the ratio between the ethanol concentration (grams per liter) and the fermentation time

347

(hour). Cell yield factor (grams per gram) was defined as the ratio between cell concentration and

348

total substrate consumed (grams per liter). The efficiency of sugar conversion to ethanol ( , %) was

349

determined as the ratio between YP/S (g g-1) and the theoretical value (0.51 g g-1) of this parameter.

350

The fermentations of biotransformation hydrolysates are characterized by quick kinetics with

351

larger yield and productivity. The biomass yield increased approximately 46% in OTHH when

352

compared with untreated hydrolysate. It was also verified that biomass and ethanol yields, and

353

ethanol production efficiency in RSHH were approximately 15% superior to in OTHH. On the other

354

hand, the ethanol volumetric productivity was 33% greater at RSHH than at OTHH, 0.09 and 0.06 g

355

L-1 h-1, respectively. However, comparing with the untreated hydrolysates, both had improved their

356

fermentative parameters, since no ethanol production was observed for the untreated hydrolysates.

357

As previously discussed, one explanation for the low ethanol productivity values obtained may be

358

associated with the presence of acetic acid. According to Diaz et al.,13 in D-glucose (20.0 g L-1) and

359

D-xylose (15.0 g L-1) medium, the acid acts more negatively in ethanol volumetric productivity than

360

in P. stipitis growth. The authors observed that in the presence of 3.0 g L-1 acetic acid the ethanol ACS Paragon Plus Environment Page 15 of 31


Page 16 of 31

361

volumetric productivity was 22% lower than in the control without acetic acid (0.38 and 0.49 g L-1

362

h-1, respectively). It was also observed that 6.0 g L-1 acetic acid completely inhibit the ethanol

363

production, in 24 hours fermentation process.

364

The results of this study showed that biological detoxification of RSHH and OTHH could be

365

successfully performed by S. cerevisiae thus removing and/or reducing the inhibitory compounds

366

and allowing the ethanol production by P. stipitis.

367

In conclusion, sugars and toxic compounds concentrations present in hemicellulose

368

hydrolysates depend on the type of raw material as well as the conditions that have been employed

369

in the hydrolysis. The method and the conditions of biotransformation process also should be

370

specific for each hydrolysate. The biological detoxification of RSHH and OTHH by S. cerevisiae is

371

a very interesting method because it reduces the inhibitory compounds without affecting the D-

372

xylose levels in the medium. S. cerevisiae has a versatile and apparently inducible enzyme

373

mechanism for biotransformation of inhibitory to less inhibitory compounds; so this yeast provides a

374

new alternative for biological treatment method. It is remarkable that to P. stipitis fermentation it is

375

not necessary the total removal of inhibitors compounds since this yeast allows little concentrations

376

of those compounds and a right biotransformation must preserve a good quality of sugars

377

hydrolysates. However, although this process improved the ethanol fermentation of RSHH and

378

OTHH by P. stipitis, the optimization of detoxification conditions is necessary, so that it can reduce

379

the concentrations of aromatic compounds and acetic acid.

380 381

ABBREVIATIONS USED

382

RSHH, rice straw hemicellulosic hydrolysate; OTHH, olive tree hemicellulosic hydrolysate; YP/S,

383

ethanol yield; YX/S, biomass yield; QP, volumetric ethanol productivity; 5-HMF, 5-

384

hydroxymethylfurfural; UV, ultraviolet;

, efficiency.

385


Page 17 of 31


386

ACKNOWLEDGEMENTS

387

The authors acknowledge the financial support of “Ciência Sem Fronteiras” of the “Conselho

388

Nacional de Desenvolvimento Científico e Tecnológico” (CNPq), “Coordenação de

389

Aperfeiçoamento de Pessoal de Nível Superior” (CAPES) and “Fundação de Amparo à Pesquisa do

390

Estado de São Paulo” (FAPESP) (Brazil) and the University of Jaén (Bioprocesses Research

391

Group).

392 393

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395

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acid hydrolyzate to ethanol by encapsulated S. cerevisiae. J. Biotechnol. 2006, 125(3), 377-384.

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generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae

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ZN1, and the consequent ethanol fermentation. Biotechnol. Biofuels. 2010, 3(26), 1-15.

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glucose/xylose mixtures using Pichia stipitis. Process Biochem. 2006, 41, 2333–2336.

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Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia

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stipitis. Bioresource Technol. 2011, 102(23), 10868-10874.

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Meyrial, V.; Delgenes, J. P.; Romieu, C.; Moletta, R.; Gounot, A. M. Ethanol tolerance and

508

activity of plasma membrane ATPase in Pichia stipitis grown on D-xylose or on D-glucose. Enzyme

509

Microb. Tech. 1995, 17, 535-540.

510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530


Page 23 of 31

531


FIGURE LEGENDS

532 533

Figure 1. Sugars and inhibitor compounds concentrations in (a) RSSH and (b) OTHH, before (dark)

534

and after (grey) biotransformation treatment by Saccharomyces cerevisiae.

535 536

Figure 2. Ultraviolet-spectrum profile before and after the biotransformation treatment by

537

Saccharomyces cerevisiae in (a) RSHH and (b) OTHH. (blue) Untreated and (orange) treated.

538 539

Figure 3. Sugars consumption by Pichia stipitis in (a) RSHH (b) OTHH treated (solid) by

540

Saccharomyces cerevisiae and untreated (hollow). D-glucose (square) and D-xylose (circle).

541 542

Figure 4. Biomass and ethanol production by Pichia stipitis in (a) RSHH (b) OTHH treated (solid)

543

by Saccharomyces cerevisiae and no-treated (hollow). Biomass (triangle) and ethanol (star).

544 545 546 547 548 549 550 551 552 553



Page 24 of 31

554

TABLES

555

Table 1 – Chemical characterization of rice straw and olive tree pruning used in the present study

556

Content (% on dry matter) a

Component

557

Rice straw

Olive tree pruning

Cellulose

35.1 ± 0.6

24.9 ± 0.5

Hemicellulose

25.9 ± 0.5

15.3 ± 0.1

Acid-insoluble lignin

13.3 ± 1.9

17.6 ± 0.1

Acid-soluble lignin

5.1 ± 0.1

1.7 ± 0.1

Acetyl Groups

1.5 ± 0.1

2.0 ± 0.1

Ash

5.5 ± 0.1

3.4 ± 0.1

a

Average of at least three determinations.

558

559

560

561

562

563

564


Page 25 of 31


565

Table 2 Concentration of sugars and toxic compounds of the rice straw and olive tree pruning

566

hemicellulosic hydrolysates in their original and concentrated forms

567

RSHH

Concentration (g L-1) a

Original

Concentrated

Original

Concentrated

D-glucose

2.5 ± 0.1

14.4 ± 0.5

10.6 ± 0.2

33.8 ± 0.2

D-xylose

14.0 ± 0.6

87.2 ± 1.4

9.3 ± 0.2

28.8 ± 0.7

L-arabinose

2.4 ± 0.1

15.9 ± 0.8

3.8 ± 0.1

15.7 ± 0.2

Acetic acid

1.4 ± 0.1

1.8 ± 0.3

2.2 ± 0.1

4.0 ± 0.1

0.21 ± 0.01

0.87 ± 0.03

0.74 ± 0.04

0.83 ± 0.03

2.8 ± 0.1

10.5 ± 0.3

3.6 ± 0.2

9.3 ± 0.3

Furans b Phenolics c 568

a

OTHH

Average of at least three determinations, b Martinez et al.25, c Singleton et al.26.

569 570 571 572 573 574 575 576



Page 26 of 31

577

Table 3 Effect of biodetoxification method on fermentation parameters of the rice straw (RSHH)

578

and olive tree (OTHH) hemicellulosic hydrolysates by P. stipitis

579

RSHH

Fermentative Parameters a

Untreated

Treated

Untreated

Treated

YX/S (g g-1)

0

0.15

0.07

0.13

YP/S (g g-1)

0

0.17

0

0.14

QP (g L-1 h-1)

0

0.09

0

0.06

0

33.3

0

27.0

(%) 580 581 582

OTHH

a

Average of at least three determinations , Ethanol yield (YP/S), Biomass yield (YX/S), Ethanol volumetric productivity (QP), Efficiency ( )

583 584 585 586 587 588 589 590 591 592 593



-1 Phenolics Compounds (g L )

Sugars and

90

2.5

a

75

Initial Final 2.0

60 1.5 45 1.0 30 0.5

15 0

-1 Acetic acid and Furans (g L )

Page 27 of 31

0.0

s e e e d ans cos Xylos binos enolic ic aci r u u l F t ra DPh D-G Ace L-A

594

-1 Phenolics Compounds (g L )

Sugars and

35

b

30

3.5 25

3.0

20

2.5

15

2.0 1.5

10 1.0 5

0.5

0

596

4.5 Initial Final 4.0

-1 Acetic acid and Furans (g L )

595

0.0

s e e e d ans cos Xylos binos enolic ic aci r u u l F t ra DPh D-G Ace L-A

597 598 599

Figure 1

600



1.0

a

Page 28 of 31

Untreated Treated

Absorbance

0.8

0.6

0.4

0.2

0.0 200

250

300

350

400

450

Wavelength (nm)

601

1.0

b

Untreated Treated

Absorbance

0.8

0.6

0.4

0.2

0.0 200

250

300

350

400

450

Wavelength (nm) 602 603 604 605

Figure 2

606


Page 29 of 31


607

-1

D-glucose and D-xylose (g L )

90

a

75 60 45 30 15 0 0

24

48

72

96

72

96

Time (h) 608 609

-1

D-glucose and D-xylose (g L )

35

b

30 25 20 15 10 5 0 0

24

48

Time (h) 610 611 612

Figure 3

613



Page 30 of 31

614

a

-1

Biomass and Ethanol (g L )

15

12

9

6

3

0 0

24

48

72

96

72

96

Time (h) 615 616

b

7

-1

Biomass and Ethanol (g L )

8

6 5 4 3 2 1 0 0

24

48

Time (h) 617

618

Figure 4

619


Page 31 of 31


TOC Graphic

620

621 622 623 624

S. cerevisiae lyophilized

0.8

non-biodetoxified hydrolysate biodetoxified hydrolysate

Absorbance

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 200

250

300

350

400

450

500

Wavelength (nm)

Hemicellulosic Hydrolysate


Detoxification of Rice Straw and Olive Tree Pruning Hemicellulosic

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