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Bioconversion of rapeseed straw: enzymatic hydrolysis of whole slurry and co-fermentation by an ethanologenic Escherichia coli JUAN CARLOS LÓPEZ-LINARES, Inmaculada Romero, Cristobal Cara, and Eulogio Castro Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b02308 • Publication Date (Web): 19 Oct 2016 Downloaded from http://pubs.acs.org on October 24, 2016
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Bioconversion of rapeseed straw: enzymatic hydrolysis of whole slurry and co-fermentation by an ethanologenic Escherichia coli Juan Carlos López-Linares, Inmaculada Romero*, Cristóbal Cara, Eulogio Castro Department of Chemical, Environmental and Materials Engineering, University of Jaén, Campus Las Lagunillas, 23071 Jaén, Spain *Corresponding author: E-mail address:
[email protected]. Phone: +34 953213644
ABSTRACT Rapeseed straw, containing about 50% of carbohydrates was used as a low-cost raw material for bioethanol production.Since xylose represents about 30% of total sugars in this feedstock,it is crucial the use of microorganisms capable of metabolizing both pentoses and hexoses. In this work, rapeseed straw was pretreated with sulfuric acid at previously determined optimal conditions. Then, the whole slurry obtained after pretreatment was enzymatically hydrolysed and the resulting sugar solution was co-fermented by Scheffersomyces stipitisCBS654 and Escherichia coliMM160. Prior to fermentation, different detoxification methods were tested in order to increase the fermentability of the hydrolysate. Only the detoxification by ion-exchange resins achieved a fermentable hydrolysate due to the almost complete removal of phenolics and acetic acid as well as high reduction in levels of furans and formic acid. The best results of ethanol production corresponded to the fermentation of rapeseed straw hydrolysate withE. coliafter detoxification by ion-exchange resins. In this case, an ethanol concentration of 40 g/Lwas reached that corresponds to 85% of the theoretical ethanol yield. The use of this new process configuration for rapeseed straw by enzymatic saccharification of whole
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slurry and subsequent separation allowed the fermentation of all sugars in a single stage avoiding the problems associated to the presence of solids in the fermentation. KEYWORDS:agricultural residue,slurry, co-fermentation, xylose, ethanol
1. INTRODUCTION Rapeseed straw is an agricultural residue rich in cellulosic and hemicellulosic sugars - above 55%,1,2whichis currently considered as an interesting feedstock for secondgeneration ethanol productionwithout other interesting applications. Its production has increased in recent years because of the growing interest on rapeseed oil for biodiesel production.2 In 2014, 35.8 and 6.7 million hectares of rapeseed were cultivated worldwide and in the European Union, respectively.3Consequently, about 42 million tons of rapeseed straw were generated in the European Union in accordance with estimationsby Arvaniti.4 The main steps involved in the biological conversion of lignocellulosic biomass into bioethanol are pretreatment, enzymatic hydrolysis and fermentation. Pretreatment with dilute acid at high temperatures and pressures, hydrolyzes hemicellulose into monomers and open the structure for subsequent enzymatic hydrolysis step by cellulase enzymes. Acid pretreatment is a low cost pretreatment with high efficiency.5 Nevertheless, the main drawback of this pretreatment can be the generation of inhibitory compounds, such as furan aldehydes, furfural and 5-hydroxymethylfurfural (HMF), carboxylic acids (acetic and formic acid) and lignin degradation products (phenolic compounds).6The production of these compounds varies according to the lignocellulosic feedstock and pretreatment conditions and they affect to cell growth in the fermentation process.7 In order to reduce inhibition issues in hydrolysates and slurries, some detoxification method may be necessary. Different techniques such as vacuum evaporation,
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overliming, ion-exchange resins, activated charcoal, organic solvent extraction, addition of reducing agents, and treatment with laccases or microorganisms, among others, have been widely used.8, 9 Using slurries obtained from biomass pretreatment have some advantages, such as the utilization of a single vessel, eliminating process steps for solid–liquid separation and sugar cleanup after pretreatment, subsequent separate fermentation steps, and exotic metallurgy.10Moreover, after pretreatment and enzymatic hydrolysis of the whole slurry a mixture of pentoses (xylose, arabinose) and hexoses (glucose, galactose) is generated. To achieve greater ethanol production and to make the process more viable from an economic point of view11 it is crucialto make full use of sugars in the lignocellulosic biomass. The conversion by fermentation of the hemicellulosic sugars released by hydrolysis of the hemicellulose fraction is typically more difficult than the conversion of the glucose released by hydrolysis of cellulose.5 Among the natural pentose-fermenting yeasts Scheffersomyces stipitis is able to provide high ethanol yields from pentose sugars. It has been reported as one of the most promising yeast for pentose fermentation due to its low nutrient requirements, high resistance to contaminants and potential cleanup of some toxins.12S. stipitis has been used as fermenting microorganism for lignocellulosic hydrolysates from olive tree pruning biomass,13,14 sunflower seed hull,15 red oak wood16 or sugarcane bagasse.17 Recombinant microorganisms such as Escherichia coli have been developed to generate strains verytolerant to toxic compounds,mainly acetic acid, andable to achieve high ethanol productions.10In this context, slurries from different lignocellulosic residues like sugarcane bagasse,18,19 Eucalyptus,20 sweet sorghum bagasse19 or corn stover21 have been successfully fermented with genetically engineered E. coli strains. In
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addition, E. coli has been tested previously with hemicellulosic liquors from rapeseed straw22 and olive tree pruning biomass.23 The conversion process of rapeseed straw including pretreatment, enzymatic hydrolysis of the whole slurry and fermentation of both C5 and C6-sugars was carried out in thiswork. The purpose of this study was to evaluate the fermentability to ethanol of cellulosic and hemicellulosic hydrolysates from rapeseed straw highly concentrated by S. stipitis (CBS 6054) and ethanologenic E. coli (MM160) after a detoxification step.
2. EXPERIMENTAL SECTION 2.1. Raw material. Rapeseed straw (6% moisture content) was collected from fields of the province of Seville, Spain, after seed harvest. As soon as it was collected, the feedstock was air dried, milled to particle sizes smaller than 0.4 cm using a laboratory hammer mill (Retsch, SM-100, Haan, Germany), homogenized and stored in a dry place until use. On average, the raw material showed the following composition (% dry weight basis): cellulose 31.5 ± 0.27; hemicellulose 17.4 ± 0.13 (xylan, 13.2 ± 0.12; galactan, 1.9 ± 0.11; arabinan, 1.2 ± 0.02; mannan, 1.2 ± 0.04); acid insoluble lignin, 16.2 ± 0.49; acid soluble lignin, 1.6 ± 0.05; acetyl groups, 3.4 ± 0.07; ash, 6.7 ± 0.26 and extractives 15.4 ± 1.32.24
2.2. Sulfuric acid pretreatment. Sulfuric acid pretreatment of rapeseed straw was carried out in a 1 L reactor (Parr Instr. Co., IL, USA). Rapeseed straw and an aqueous sulfuric acid solution 0.5% (w/v) were mixed at a solid loading of 6% (w/v) at 180ºC for 20 min. These conditions were previously optimized by Castro et al.25as the most adequate in terms of hemicellulosic sugars recovery in the liquor and enzymatic hydrolysis yield, simultaneously. The working temperature was reached in 35 min while
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the cooling time until room temperature was 7 min. Slurry with 2.8% (dry weight) insoluble solids content resulted after acid pretreatment of the rapeseed straw.
2.3. Enzymatic hydrolysis. The whole slurry of rapeseed straw obtained after pretreatment was hydrolysed with a cellulolytic complex (Cellic CTec3) kindly provided by Novozymes A/S (Denmark).The enzyme loading used of this cellulolytic complex was 15 FPU/g substrate. To alleviate end-product inhibition by cellobiose, fungal β-glucosidase (Novozym 188, Novozymes A/S) was added at an enzyme loading of 15 IU/g substrate. The pH was adjusted to 5 with solid KOH in the presence of 0.05 M sodium citrate buffer. The enzymatic hydrolysis was carried out in 1 L Erlenmeyer flasks, being the total working volume of 600 mL with 2.8% of insoluble solid loading. Twelve reaction flasks were incubated at 50ºC in an orbital shaker (Certomat-R, BBraun, Germany) at 150 rpm for 72 h to obtain enough volume of hydrolysate. Two milliliter samples were withdrawn at 0, 24, 48 and 72 h, and were centrifuged at 10,500 rpm (Sigma 1–14 Centrifuge) for 10 min. Glucose concentrations in the sample supernatant were determined by HPLC and compared to commercial cellulose (Sigmacell) controls with the samesolid loading. Additionally, blanks of the enzyme were prepared and analyzed by HPLC in order to subtract the sugar content since the commercial enzymes contain glucose in monomeric and oligomeric form. After enzymatic hydrolysis, liquid and solid phases were separated by vacuum filtration. The insoluble solids, mainly lignin, were washed with distilled water, dried at 40ºC and analyzed for sugar and lignin content. The liquid fraction of slurry (hydrolysate) was analyzed for glucose determination and toxic compounds such as acetic acid, furfural, HMF, formic acidand phenolic compounds.
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Additionally, a test of enzymatic hydrolysis (by triplicate) was performed for comparative purposes using the acid-pretreated solidas substrate. These enzymatic testswere performed at the same conditions described above at 5% solid loading and a total working volume of 25 mL.
2.4. Conditioning of acid hydrolysate. Different detoxification methods, such as activated charcoal, ion-exchange resins and overliming were studied to evaluate their effect on the reduction of the concentration of toxic compounds in rapeseed straw hydrolysate obtained after enzymatic hydrolysis of whole slurry. After that, detoxified hydrolysates were concentrated by evaporation to obtain a highly concentrated sugar solution. 2.4.1. Activated charcoaladsorption. The pH of the hydrolysate was adjusted to 0.8 by addition of concentratedH2SO4. Then, the hydrolysate was mixed with powder activated charcoal (100 mesh particle size - Sigma-Aldrich) in a 3.5% (w/v) ratio. The suspension was incubated in a rotary shaker (Certomat-R, B-Braun, Germany) at 45ºC, 200 rpm for 1 h. The liquid fraction was recovered by vacuum filtration and its content in sugars and inhibitors was measured. 2.4.2. Ion-exchange resintreatment. Prior to the treatment, the resins were washed with distilled water and dried at 35ºC.The hydrolysate was treated with ion-exchange resins (Microionex MB200) in a plastic column (7 cm wide x 13 cm length) at room temperature. Previously, the pH of the hydrolysate was adjusted to 6 by addition of solid KOH. The ratio resins:hydrolysate was 0.2 g resin per mL liquor. After detoxification, the composition of hydrolysate was determined. 2.4.3. Overlimingprocess. Overliming of acid hydrolysate was carried out with Ca(OH)2 at 50ºC. The base was added to the hydrolysate until pH 10, maintaining it on a shaking
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incubator at 200 rpm for 30 min.26Afterward, concentratedH2SO4 was added until pH 2.5 and the precipitate was separated by centrifugation at 3,500 rpm for 10 min (Rotina 420, Hettich Zentrifugen, Germany). The filtrate was analysed to determine its composition in sugars and inhibitory compounds. 2.4.4. Concentration. Detoxified and non-detoxified hydrolysates were fourfold concentrated by evaporation at 60ºC under vacuum conditions. The aim of this stage was to obtain a solution with high sugar concentration, reducing at the same time its content in volatile compounds such as acetic acid and furfural that results toxic for the fermentative microorganisms.The initial pH of these hydrolysates was adjusted to 2.5 by addition of 96% H2SO4 (w/v) or solid KOH, depending on the case. Temperature and pH used in this stage were selected according to previous experiences (data not shown). The liquors obtained after each detoxification treatment and after concentration by evaporation were characterized in order to determine their levels of sugars and toxic compounds. Detoxifiedand fourfold concentratedhydrolysates were used as fermentation medium.Likewise, the composition of raw hydrolysate after concentration was determined and its fermentability was evaluated.
2.5. Microorganisms and inocula. Escherichia colistrain MM160, a kind gift by Dr. Ingram from University of Florida (USA), and Scheffersomyces stipitis strain CBS 6054 were the microorganisms employed in the fermentation experiments. The use of two different ethanologenic microorganisms allowed to compare theperformance of fermentation of cellulosic and hemicellulosic hydrolysate obtained after enzymatic hydrolysis of the whole slurry. E. coli MM160 was kept in 40% glycerol stocks at -80ºC. Cells wereincubated in 250 mL Erlenmeyer flasks using 75 mL AM1 culture medium27 and sterilized by
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filtration(Millipore GP 0.22 µm, Millipore, Ireland). The inoculum was grown at 37ºC in an orbital incubator at 180 rpm for 24 h, centrifuged (3500 rpm, 10 min), washed and added to the culture medium. S. stipitisCBS 6054 was preserved in agar slants. The inoculum was prepared in 250 mL Erlenmeyer flasks using75 mL culture medium composed of 5 g/L yeast extract, 1 g/L NH4Cl, 1 g/L KH2PO4, 0,5 g/L MgSO4·7H2O and 40 g/L xylose. This medium was sterilized in autoclave at 121ºC for 20 min.The cell was grown at 30ºC in ashaking incubator at 180 rpm for 24 h, and processed as forE.coli.
2.6. Fermentation of rapeseed straw hydrolysates. Fermentation of hydrolysates was performed by E. coli and S. stipitis (see Section 2.5 for details) and results were compared.In the fermentation assays with E. coli MM160,hydrolysates were supplemented with salts as described for the growth medium (except xylose) and sterilized at 112ºC for 15 min. Fermentation assays were carried out at 37ºC, 400 rpm (magnetic stirring) and pH 7 for 96 h. Initial cell concentration was around 0.6 g/L, based on the absorbance at 620 nm. 300 mL glass flasks with pH probe were used, containing 150 mL of fermentation liquor. A water bath was utilized to keep the temperaturewhile pH was controlled by 2M KOH addition. Each flask was provided with a robber cap including two holes for venting and sampling. Fermentation assaysby S. stipitis CBS 6054 were performed in 250 mL Erlenmeyer flasks containing 100 mLof fermentation medium. It was previously supplemented with the medium described above for S. stipitis strain except xylose and then, it was sterilized as for E. coli. Initial biomass concentration was approximately 1 g/L. Fermentation tests were performed in a rotary shaker at 30ºC, 150 rpm and pH 6.5 for 96 h.
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Samples were taken at different times and centrifuged at 11,500 rpm for 10 min to determine cell growth, sugars uptake and ethanol production. Duplicate experiments were performed andaverage values and standard deviations are shown. To evaluate the performance of the fermentation stage, ethanol yields were expressed as grams ethanol per gram of consumed sugars and referred to the theoretical ethanol yield (0.51 g ethanol/g glucose).
2.7. Analytical procedures. The solid fractions obtained after acid pretreatment and after enzymatic hydrolysis of the whole slurry were characterized according to NRELmethodology as described in López-Linares et al.28 Sugar content in the hydrolysates was determined by high performance liquid chromatography (HPLC) in a Waters 2695 liquid chromatograph (Mildford, MA, EEUU) equipped with a refractive index detector (Waters 2414). A Transgenomic CHO-782 carbohydrate analysis column operating at 70ºC with ultrapure water as a mobile-phase (0.6 mL/min) was used. The concentration of acetic acid, formic acid, furfural and HMF in the hydrolysates were also determined using the HPLC system with refractive index detector mentioned above but using a Bio-Rad HPX-87H column at 65ºC, and 5 mM H2SO4 as mobile phase at a flow rate of 0.6 mL/min. Total phenolic compounds concentration wasdetermined by the Folin-Ciocalteu procedure.29 Fermentation samples were analysed for sugar and ethanol concentrations using the same HPLC equipment described for inhibitors determination. Cell content was estimated by filtration of fermentation samples, using 0.2 µm cellulose nitrate filters (Sartorius stedim Biotech, Göttingen, Germany). The biomass concentration was determined as the ratio between the mass of dried biomass and the volume of filtered
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inoculum. All analytical determinations were carried out in triplicate. Average results and the relative standard deviations (below 1%) are shown.
3. RESULTS AND DISCUSSION 3.1. Sulfuric acid pretreatment. The composition of both solid and liquid fractions from the acid-pretreatment of rapeseed straw was determined in order to evaluate the cellulose and hemicellulose content in the solid fraction and the level of sugars and inhibitory compounds in the prehydrolysates (Table 1).As expected, the most part of glucose remained in the cellulose fraction of pretreated straw. Thus, acid-pretreatment involved a significant increase of cellulose content about 46%, yielding pretreated straw with 58.3% of cellulose. As shown, glucose was released at a concentration below of 2 g/L, and probably it should be non-structural glucose whilst the main sugar in liquid fraction was xylose, representing almost 50% of total sugars.The level of toxic compounds was not significant, being the main one acetic acid (2.5 g/L) from the solubilisation of acetyl groups contained in the hemicellulose structure. The presence of furfural from dehydration of pentose sugars is also common in lignocellulosic hydrolysates.The concentration of this toxic compound in the acid prehydrolysate of rapeseed straw was 1.8 g/L. Nevertheless, the concentrations of sugars and toxic compounds in lignocellulosic prehydrolysates depend on the feedstock as well as on the pretreatment conditions.30
3.2. Enzyme hydrolysis of pretreated materials. The whole slurry resulting after sulfuric acid pretreatment was hydrolysedwith cellulases and β-glucosidase at standard conditions. The presence of by-products from the pretreatment in the liquid fraction of the slurry may result in poor performance of hydrolysis due to enzyme inhibition.Some
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authors have reported a strong negative effect of furans, phenols and low molecular weight acids, mainly formic acid on the cellulolytic activity.31 In this work, acid pretreated slurry did not require dilution since the presence of inhibitory compounds was not important(Table 1). Nevertheless, previous assays of enzymatic hydrolysis using acid-pretreated straw showed better performance (Figure 1), which could be related to the presence of these toxic compounds in the slurry that affectedslightly to the enzymatic activity. Thus, lignin-derived phenolic compounds have been reported as inhibitors of cellulose during enzymatic hydrolysis.32 However, as can be seen in Figure 1, glucose was released from cellulose fraction in acid-pretreated slurryyielding aglucose concentration of 14 g/Lafter 72 h. This concentration corresponds to a yield of 67.6 g glucose/100 g glucose in raw straw whilsta yieldof 75.4 g/100 gwas attained using acid-pretreated solid as substrate. Therefore, only a drop in yield of 10% was observed when working with whole slurry instead of pretreated solid. This indicates that the presence of compounds from the degradation of sugars and lignin in the slurry formed during the pretreatment (Table 1) were not very toxic for the enzymes. It is worth mentioning that initial glucose concentration in the hydrolysate derived from the pretreatment (about 2 g/L) was subtracted from glucose concentration measured in the hydrolysate to take account of the glucose concentration only from cellulose. According to the time course of slurry hydrolysis, the increase in glucose production was not relevant by increasing the process time from 48 to 72 h. It suggests the enzymatic hydrolysis time could be reduced to 48 h. After the hydrolysis of whole slurry, a sugar solution (referred to as hydrolysate) composed by hemicellulosic sugars released on the pretreatment plus the glucose released by enzymatic action was obtained. Obviously, this hydrolysate also
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contentsinhibitory products from the sugar degradation and acetic acid from the hydrolysis of the acetyl groups of hemicelluloses.
3.3. Conditioning of cellulosic and hemicellulosic hydrolysate before fermentation. After the enzymatic hydrolysis of the whole slurry, total sugars in liquid fraction accounted for 24.9 g/L, 9.9 g/L hemicellulosic sugars released in the pretreatment (Table 1) and 14 g/L glucose from enzymatic hydrolysis. This liquid fraction, or raw hydrolysate, was concentrated about fourfold by evaporation at 60ºC reaching a sugar solution with more than 88 g/L. Table 2 shows the composition (sugars and inhibitory compounds) of the raw hydrolysate before and after concentration. The objective of this stage was to reach a concentrated sugar solution since a diluted ethanol solution implies high cost of distillation process.33 However, as can be appreciated in Table 2, the concentration of the raw hydrolysate also implied a significant increase in the concentrations of these compounds (2.5 g/L formic acid, 5.5 g/L acetic acid, 2.1 g/L HMF and 4.9 g/L phenolics) except furfural that was completely removed because of its high volatility. After the concentration of raw hydrolysate, it could not be fermented by either microorganism used in this work. For this reason, prior to fermentation, different detoxification methods such as ion-exchange resin, activated charcoal and overliming were used to increase the fermentability of the hydrolysate by reducing the negative effects of the toxic compounds on the fermentation performance. Detoxification can be expensive since it involves an additional stage in the process. An alternative strategy to reduce inhibition problems might be the use of a large inoculum although it is not considered a good solution on an industrial scale. Another
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possibility would be the use of microorganisms more resistant to toxic compounds that did not require this stage.30 Similarly to raw hydrolysate, the three detoxified hydrolysates were also fourfold concentrated prior to fermentation to obtainhighly concentrated sugar solutions.Detoxification stage was previous to concentrationstage to avoid significant increasesin the concentration of toxic compounds.Table 2 summarizes the composition of the detoxified hydrolysates before and after concentration. The effectiveness of the three methods was evaluated by comparing toxic compound concentrations before and after detoxification and by checking the fermentability of detoxified hydrolysates after concentration. As can be seen in Table 2, detoxification by activated charcoal resulted very efficient to remove phenolic compounds (87%) and furans (furfural, 91.5% and HMF, 90%) without significant sugar loss.However, this method resulted less efficient for organic acids since it removed 40% offormic acid and only 17% of acetic acid.Other authors also reported high removal of furans and phenolic compounds to detoxify corncob prehydrolysates by activated charcoal.34 As regards overliming, it is considered a cheap detoxification method that removes toxic compounds by precipitation. When sulfuric acid is used for pretreatment, calcium sulphateis formed.30Detoxification of rapeseed straw hydrolysate by overliming achieved to remove 70% of furans while the rest of toxic compounds were practically unaltered after the treatment. Moreover, this methodresulted in sugar loss of about 30%(Table 2). Overliming has been proved as an efficient method to remove furans of hydrolysates from sugarcane bagasse26 and brewery’s spent grain.35 Concerning the use of ion-exchange resins, it has been reported as an efficient method to remove acetic acid.36As can be seen in Table 2, this method eliminatedalmost
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completely acetic acid and phenolic compounds in the rapeseed straw hydrolysate. Furthermore, detoxification by resins also yielded an important reduction in levels of other inhibitory compounds, such as furfural (84%), HMF (68%) and formic acid (71%) without significant sugar loss (Table 2). Thereby, it appears that the most efficient detoxification method tested for rapeseed straw hydrolysate was the ion-exchange resins.
3.4. Fermentation by Scheffersomycesstipitis. Glucose and xylose are the main sugars in the composition of rapeseed straw (Table 1). For this reason, the co-fermentation of both sugars is essential to increase the effectiveness of the bioethanol production. The process configuration used in this work allowed the bioconversion of both C5 and C6 sugars of rapeseed straw in a single fermentation stage. Moreover,since solid fraction was removed from the slurry by filtration after enzymatic hydrolysis, the problems associated with the presence of solids in the fermentation medium were avoided. The fermentability of the concentrated raw hydrolysate was evaluated without detoxification with the xylose-fermenting yeast Scheffersomyces stipitisbut no sugars consumption was observed. This indicates that inhibitory compounds such as furfural, HMF and the organic acids, formic acid and acetic acid, were present at concentrations that lead to complete inhibition of the yeast. Likewise, the fermentability of the three detoxified hydrolysates was tested with S.stipitis. Rapeseed straw hydrolysate detoxified by activated charcoal and then concentrated was scarcelyfermented by S. stipitis.Glucose consumption startedonly after 96 h fermentation time and only 37% of this monosaccharide had beenconsumed after 300 h, while the rest of sugars remained in the medium. This poor performance of fermentation yielded an ethanol production as low as 2 g/L aftersuch a long time(Figure 2a).This
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behaviour of the yeast can be attributed to the presence in the medium of high level of acetic acid, 4.8 g/L (Table 2), since S. stipitis is very sensible to the toxic effect of this compound.16,37Even worse behaviour of the yeast was observed in the fermentation of hydrolysate detoxified by overliming, without sugars consumptionand ethanol production (data not shown). Likewise, as can be seen in Table 2,high concentrations of acetic acid in this hydrolysate (6.8 g/L) and phenolic compounds (4.6 g/L) might be the reason for this complete inhibition of the yeast.Previous reports on fermentation by S. stipitis showed strong inhibition in the cell growth fermenting synthetic medium with 5 g/L of acetic acid16along withsevere toxic effect of phenolics even at low levels.17,38Moreover, high concentrations of formic acid (3.6 g/L) remained in the rapeseed straw hydrolysate after overliming and this could contribute to a synergistic effect on cell growth inhibition. Probably, the removal of acetic acid and the reduction in levels of phenolics and formic acid by ion-exchange resins allowed the fermentation of rapeseed straw hydrolysate with S. stipitis (Figure 2b). As can be appreciated, during the fermentation glucose was the preferred monosaccharide, being depleted from the culture medium in 48h. Xylose was metabolized at a slower rate compared to glucose and only 33% of XGM (sum of xylose, galactose and mannose) had been consumed at that time. This preferential consumption of glucose instead of xylose has been reported by several authors.12,16,39 S. stipitis showed maximum ethanol yield, 0.40 g/g (79.1% of theoretical yield) at that time with the highest productivity 0.652 g/Lh(0.563 g/Lh for glucose and 0.089 g/L for XGM) and 4.6 g/L cell concentration. Although the consumption of total sugar increased up to 95% and residual XGM only accounted for 4.8 g/L in the mediumafter 96 h fermentation time, the ethanol productionincrease was not significant (33.3 g/L at
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96 h vs31.3 g/L at 48 h). It is worth mentioning that S. stipitis could not metabolize arabinose and consumption of this sugar was not observed through the time course of fermentation. The same behaviour was reported by other authors who performed the fermentation of prehydrolysates fromsugarcane bagasse17 or olive tree pruning biomass37with the same yeast. These authors also determinedthat the initial concentrations of arabinose in the hydrolysateswere detected in the medium at the end of fermentation. As can be observed in Figure2b, ethanol was produced mainly from glucose, and one it was exhausted no ethanol production from pentose catabolism was observed.This might be related to the relative amounts of both sugars glucose and xylosein the medium. Thus, the amount of glucose in the rapeseed straw hydrolysate used as medium was about 3 times higher than the amount of xylose. Some authors have reported that low concentrations of glucose are necessary to an efficient utilization of xyloseby xylose-fermenting microorganisms such asP.stipitis,12E. coli40 and recombinant S. cerevisiae.21It is worth mentioning that complete ethanol assimilation was observed after 96 h whilst residual xylose and arabinose were in the medium, similarly to that observed by other authors.17,41Terán-Hilares et al.39 attained similar results to those presented in this work using sugarcane bagasse hemicellulosic hydrolysates by S. stipitis although with an initial glucose-to-xylose ratio of 2. Gupta et al.34obtained an ethanol solution of 14 g/L and 0.31 g/g (61% of the theoretical ethanol yield) after fermenting hemicellulosic hydrolysates of corncob detoxified by activated charcoal with P. stipitis. The same yeast used in this work has been tested with hemicellulosic hydrolysates of olive tree pruning after resin treatment with maximum ethanol production of 6.5 g/L and 61% of theoretical ethanol yield after 28 h of fermentation.37
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3.5. Fermentation by Escherichia coli. Likewise S. stipitis, undetoxified rapeseed straw hydrolysate could not be fermented after concentration with ethanologenic Escherichia coli MM160, which can be attributed tothehigher concentrationof toxic compounds due to evaporation (Table 2). The fermentability of the three detoxified rapeseed straw hydrolysatesafter concentration was evaluated with this microorganism.Thus, hydrolysatesdetoxified by overliming or activated charcoal were toxic forE. coliand were not fermented. Probably, this behaviour of the microorganism could be related to the presence of toxic compounds in these hydrolysates after detoxification and concentration. Thus, these detoxification methods although very efficient in reducing the level of furans,were not capable of reducing the level of other toxic compounds as organic acids (acetic and formic acid) or phenolic compounds in the case of overliming (Table 2). Therefore, by concentrating these hydrolysates by evaporation the levels of these compounds were considerably increased. Although E. coli is highly resistant to the presence of acetic acid, formic acid concentrations higher than 2 g/L in both hydrolysates could be the cause of the microorganism inhibition.Furthermore, overliming did not reduce the level of phenolic compounds remaining in the hydrolysate at 4.6 g/L of these compounds and this could increase the inhibitory effect on the microorganism. Phenolic compounds have been considered the most toxic compounds in lignocellulosic hydrolysates even at low concentrations.38The additive or synergistic effect of these compounds can increase significantly microbial inhibition for E. coli.42 By the contrary, detoxification of hydrolysate by ion-exchange resins and then concentrationresulted in a sugar solution fermentable by E. coli. The fermentation profile for the rapeseed straw hydrolysate treated by resins showed that the glucose was
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exhausted within 72 h whilst 14 g/L of XGM remained in the medium at that time (Figure 3). The glucose uptake rate was estimated from Figure3 to be 0.98 g/Lh whilst for XGM the uptake rate was only 0.28 g/Lh at that time. Concerning arabinose, only about 40% of this sugar was consumed after 72 h fermentation time although its initial concentration in the medium was not significant, below 4% of total sugars. Similarly to S. stipitis, a clear preference of E. coli to consume glucose in presence of other sugars was observed.Preferential utilization of glucose first and then xylose and arabinose was also reported in the fermentation of corn stover hydrolysates with another recombinant strain of E. coli.21According to these authors, this behaviour of E. colican be due to the catabolic repression of glucose and arabinose on the utilization of xylose.Pedraza et al.43 also detected that the consumption rate for xylose was lower than the one for glucose when fermenting corncob hydrolysates with E. coli MS04 although xylose was exhausted only after 36 h. The complete use of sugars can be due to an initial glucoseto-xylose ratio lower than 1. This microorganism was able to metabolize 85% of total sugars in the fermentation medium with final ethanol concentration of 40 g/L and cell concentration of2.3 g/L at 72 h fermentation time. In spite of the incomplete fermentation, an ethanol yield of 0.43 g ethanol/g glucose (85% of theoretical yield) was attained at that time. These results compare favourably with those reported by Arvaniti et al.44 after fermenting the whole slurry of rapeseed straw pretreated by wet oxidation yielding a maximum ethanol production of 67%.The results achieved in this work were similar to those reportedwith corncob using similar process configuration, e.g., enzymatic hydrolysis of the whole slurry and fermentation by E. coli MS04.43 As can be appreciated in Figure3, although almost 90% of total sugars in the medium were consumed after 90 h fermentation time, an increase of ethanol productionwas not
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observed. This fact can be related to the tolerance of E. coli to high ethanol concentrations. Some authors have reported product inhibition for ethanol concentrations of above 35 g/L with E. coli MS0445 and 45 g ethanol/L with E. coli LY01.46 Figure 4 shows the overall process material balance for ethanol production from rapeseed strawwith E. coliaccording to the process configuration used in this work.Rapeseed straw wasacid-pretreated at previously determined optimal conditions (180ºC, 0.5%w/v H2SO4, 20 min) obtaining a slurry with 2.8% of insoluble solids content, mainly cellulose and lignin. Sugar solution resulting after enzymatic hydrolysis of this whole slurry was fermented with E. coli after detoxification by resins and concentration stages. According to the sugars content in raw rapeseed straw, 54.3 g/100 g and considering 0.51g ethanol/g sugar as the theoretical ethanol yield, a maximum production of 27.7 g ethanol/100 g straw could be obtained. However, as can be appreciated in the Figure 4, some sugars loss occurred through the bioconversion process of rapeseed straw. Thus, about 5 g glucose remained as cellulose in the solid after enzymatic hydrolysis as well as 0.8 g of xylose. Moreover, the presence of residual sugars in the fermentation broth, about 7 g, must be also taken into account. Therefore, all in all, a maximum process yield of 21.3 g ethanol/g straw could be obtained. However, only a process yield of 16.6 g ethanol/g straw was attained although it is worth mentioning that the final concentration of alcoholic solution was as high as 39.5 g/L making the process more feasible economically.The fact of obtaining lower overall yield can be related to product inhibition effect.The co-fermentation of the slurry from steam pretreated bagasse with the same E. coli strain resulted in an ethanol yield of 0.21 g ethanol/g bagasse with an alcoholic concentration of 30 g/L.10Saha reported an
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ethanol yield of 0.29 g/g corn from acid pretreated corn stover with E. coli FBR5 although the ethanol concentration was as low as 28.9 g/L.47
4. CONCLUSION The results of this study confirm that the detoxification by ion-exchange resins was more effective than activated charcoal and overliming for reducing fermentation inhibition. The process configuration used in this work allowed the bioconversion of all sugars present in straw in a unique stage by the co-fermentation of pentoses and hexoses using ethanologenic microorganisms capable of metabolizing both kinds of sugars. Both microorganisms S. stipitis and E. coli were capable of fermenting cellulosic and hemicellulosic hydrolysates of acid pretreated rapeseed straw after resins treatment to produce ethanol with yields higher than 0.40 g/g. However, the best results for ethanol production were achieved by E. coli since it reached the minimum ethanol concentration required for distillation purposes (4% by volume) with 85% of the theoretical ethanol yield. Further research will be focused on improvement of xylose utilization by E. colisince the complete utilization of sugars is necessary to drive process economics in the bioethanol production from rapeseed straw.
ACKNOWLEDGMENTS The authors thank support from University of Jaén (Plan de Apoyo).
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Figure captions Figure 1. Time course of glucose concentration attained by enzymatic hydrolysis of acid-pretreated straw (5% solids) and whole slurry (2.8% solids). Figure 2a. Fermentation of rapeseed straw hydrolysate by Scheffersomyces stipitis after activated charcoal detoxification. Figure2b. Fermentation of rapeseed straw hydrolysate by Scheffersomyces stipitis after ion-exchange resins treatment to remove inhibitors fermentation. Figure3. Fermentation of rapeseed straw hydrolysate by Escherichia coli after ionexchange resins treatment to remove inhibitors fermentation. Figure4. Material balance flow diagram of the overall process for ethanol production from rapeseed strawby enzymatic hydrolysis of the whole slurry and co-fermentation of the resulting hydrolysate.
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TABLES Table 1. Composition of acid-pretreated rapeseed straw based on three determinations Component Solid fraction (%) Cellulose Hemicellulose (only xylose) Lignin Liquid fraction(g/L) Sugars Glucose Xylose Galactose Arabinose Mannose Inhibitors Furfural 5-HMF Formic acid Acetic acid
Concentration 58.3 ± 0.10 4.5 ± 0.11 32.0 ± 0.20
1.86 ± 0.06 4.78 ± 0.10 1.53 ± 0.13 0.84 ± 0.07 0.89 ± 0.03 1.79 ± 0.25 0.70 ± 0.12 0.87 ± 0.18 2.50 ± 0.22
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Table 2. Composition of hydrolysates* before and after detoxification and concentration. Sugars(g/L)
Inhibitors(g/L) Acetic acid 2.81 ± 0.03
HMF
Furfural
0.74 ± 0.00
Formic acid 0.88 ± 0.01
0.57 ± 0.00
1.29 ± 0.04
Total phenols 1.41 ± 0.10
1.61 ± 0.05
2.96 ± 0.02
2.48 ± 0.02
5.47 ± 0.02
2.05 ± 0.01
0.00 ± 0.00
4.85 ± 0.08
1.57 ± 0.01
0.88 ± 0.01
0.77 ± 0.02
0.53 ± 0.01
2.33 ± 0.01
0.06 ± 0.00
0.11 ± 0.00
0.18 ± 0.02
26.48 ± 0.16
6.42 ± 0.07
2.58 ± 0.05
3.38 ± 0.03
1.98 ± 0.00
4.78 ± 0.03
0.21 ± 0.01
0.00 ± 0.00
0.85 ± 0.10
18.09 ± 0.30
6.17 ± 0.08
1.41 ± 0.03
0.62 ± 0.02
0.76 ± 0.01
0.31 ± 0.01
0.26 ± 0.00
0.21 ± 0.00
0.26 ± 0.00
0.09 ± 0.00
Ion-exch. resins +Conc
71.73 ± 0.32
24.67 ± 0.32
5.53 ± 0.06
2.21 ± 0.03
2.99 ± 0.17
1.11 ± 0.02
0.93 ± 0.02
0.83 ± 0.01
0.00 ± 0.00
0.37 ± 0.01
Overliming
14.23 ± 0.04
4.98 ± 0.04
1.13 ± 0.03
0.37 ± 0.02
0.60 ± 0.01
0.88 ± 0.00
2.81 ± 0.04
0.18 ± 0.00
0.36 ± 0.00
1.32 ± 0.04
Overliming + Conc
55.51 ± 0.15
19.42 ± 0.18
4.40 ± 0.05
1.44 ± 0.06
2.34 ± 0.02
3.55 ± 0.26
6.84 ± 0.05
0.61 ± 0.01
0.00 ± 0.00
4.63 ± 0.09
Glucose
Xylose
Galactose
Arabinose
Mannose
Raw hydrolysate
16.88 ± 0.33
6.38 ± 0.02
1.50 ± 0.12
0.73 ± 0.02
Concentrated raw hydrolysate Conditioning Activated charcoal
57.83 ± 0.17
21.09 ± 0.26
4.53 ± 0.04
17.38 ± 0.04
6.51 ± 0.01
Activated charcoal+ Conc
64.54 ± 0.57
Ion-exch. resins
*Mean values and standard deviatons of three determinations
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FIGURES 30
25
20 Glucose (g/L)
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15
10 Pretreated solid 5 Slurry 0 0
24
48
72
Time (h)
Fig 1.
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60
Concentration (g/L)
50 40
Glucose XGM
30
Arabinose 20
Ethanol Cell
10 0 0
24
48
72
96
120 144 Time (h)
168
192
216
240
Fig. 2a.
70 60 Concentration (g/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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50 Glucose 40
XGM
30
Arabinose
20
Ethanol Cell
10 0 0
24
48
72 Time (h)
96
120
Fig. 2b.
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80 70 Concentration (g/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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60 50
Glucose
40
XGM Arabinose
30
Ethanol 20
Cell
10 0 0
24
48
72 Time (h)
96
120
Fig. 3.
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1
H2SO4 (96%)8.7 g
2
H2O1658.0 g
3 Rapeseed straw
ACID PRETREATMENT H2SO40.5% w/v 180 ºC 20 min 6% w/v
4 5 6 7
100 g dry weight Glucose 34.6 g Xylose 14.9 g Galactose 2.1 g Arabinose 1.4 g Mannose 1.3 g Lignin 17.9 g Extractives 15.4
Cellic CTec3 5.6 mg β-glucosidase 0.6 mg Citric acid 8.1 g Sodium citrate 13.2 g KOH (85%) 15.3 g
Slurry 1714.9g
ENZYMATIC HYDROLYSIS 50 ºC 72 h pH 5 150 rpm 2.8 % w/v
9
ION-EXCHANGE RESIN DETOXIFICATION pH 6.0 20 % w/v
11 12
KOH (85%) 5.6 g
13
Glucose 4.8 g Xylose 0.8 g Lignin 18.5 g
Liquid Glucose 28.4 g Xylose 10.7 g Galactose 2.5 g Arabinose 1.2 g Mannose 1.2 g Phenols 2.4 g Formic acid 1.5 g Acetic acid 4.7 g HMF 1.0 g Furfural 2.2 g
8
10
Solid 26.8g
Ion-exchange resin 336.7 g
14 15
Liquid
EVAPORATION 60ºC pH 2.5
Glucose 28.4 g Xylose 10.1 g Galactose 2.3 g Arabinose 1.1 g Mannose 1.3 g Phenols 0.1 g Formic acid 0.4 g Acetic acid 0.0 g HMF 0.3 g Furfural 0.4 g
XGM: Sum of xylose, galactose and mannose 16
Liquid Glucose 28.4 g Xylose 10.1 g Galactose 2.3 g Arabinose 1.0 g Mannose 1.4 g Phenols 0.1 g Formic acid 0.4 g Acetic acid 0.0 g HMF 0.3 g Furfural 0.0 g
FERMENTATION 37 ºC 72 h pH 7.0 400 rpm
(NH4)2HPO4 (98%) 1.1 g NH4H2PO4 (98%) 0.4 g MgSO4. 7H2O (99%) 0.1 g KCl (100%) 0.1 g Betaine (99%) 0.1 g Trace elements (> 98%) 0.3 g KOH (85%) 21.9 g Inoculum (E. coli) 0.3 g
Liquid Ethanol 16.6 g Glucose 0.3 g XGM 5.8 g Arabinose 0.9 g Formic acid 0.4 g Acetic acid 1.5 g HMF 0.0 g Furfural 0.0 g Cell 1.0 g
Fig. 4
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