Fermentable sugars recovery from grape stalks for bioethanol production

DOI: https://doi.org/10.1016/j.renene.2013.06.006 © 2013. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/(opens in new tab/window) 1 FERMENTABLE SUGARS RECOVERY FROM GRAPE STALKS FOR BIOETHANOL PRODUCTION I. Egüés a, L. Serrano a,*, D. Amendola b, D.M. De Faveri b, G. Spigno b, J. Labidi a a Chemical and Environmental Engineering Department, University of the Basque Country, Plaza. Europa 1, 20018, Donostia-San Sebastián, Spain b Institute of Oenology and Agro-Food Engineering, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84-29122 Piacenza, Italy * Corresponding author. Tel.: +34-943017125; fax: +34-943017140. E-mail adress: lui[email protected] (L. Serrano) Abstract Three different processes were investigated for the recovery of fermentable sugars from grape stalks: autohydrolysis at 121 °C before and after a pre-washing step and acid hydrolysis (2% H2SO4 w/w) after a pre-washing step. Moreover, optimal conditions of a charcoal-based purification process were determined by experimental design. All hydrolysates, with their corresponding synthetic liquors were used as fermentation substrates for the production of metabolites by the yeast: Debaryomyces nepalensis NCYC 1026. The main fermentation product was ethanol, where a maximum production of 20.84 g/L, a conversion yield of 0.35 g ethanol/g monomeric sugars and a productivity of 0.453 g/Lh were obtained from non-purified autohydrolysate liquor. In all cases, ethanol production and cell growth were better in non-purified liquors than in synthetic liquors. These results could be influenced by the presence of other sugars in the hydrolysates, with higher concentration in non-purified ones. Keywords: Grape stalks, hydrolysis, sugars, purification, fermentation, ethanol 2 1. Introduction Grape is one of the most important fruit crops and viticulture is one of the most important agricultural activities. The principal solid residues produced in grape juice and wine making processes are stalks and grape marc. Grape stalks are the skeleton of the grape bunch and consist in lignified tissues [1]. Its composition is tannins, lignin, cellulose and hemicelluloses principally. The upgrading of this by-product through the use of its components could entail economical and environmental improvements for the industry. In concrete, hemicelluloses, which link the lignin and cellulose, are not being isolated for industrial use, but they have a high potential and they could be used in many applications. The hemicelluloses are made up of pentoses (β-D-xylose, α-L-arabinose), hexoses (β-D-mannose, β-D-glucose, α-D-galactose) and/or uronic acids (α-Dglucoronic, α-D-4-O-methylgalacturonic and α-D-galacturonic acids) where the hydroxyl groups of sugars can be partially substituted with acetyl groups [2]. Many studies have been focused in the hemicelluloses hydrolysates use to obtain biofuels, especially bioethanol, [3-5] and chemicals like xylitol [6], furfural [7] or lactic acid [8]. The composition and the concentration of hydrolysates rich in hemicelluloses, depend on the used raw material and the applied extraction process. Environmental-friendly technologies such as autohydrolysis process, which uses water as the only reagent, have gained interest for hemicelluloses sugars recovery. For that reason, in this study, hydrolysis process was studied using water as principal reagent for grape stalks fermentable sugars extraction. Preliminary experiments were performed to determine autohydrolysis optimum conditions in order to recover the maximum fermentable sugars from grape stalks. Furthermore, previous studies [9] showed the presence of non-structural carbohydrates, 3 which are sugars derived from impregnate grape juice. For this reason, a raw material washing step before autohydrolysis process was also studied in order to know the contribution of these sugars. Before fermentation study, toxic compounds which are typically generated during the hydrolysis process should be removed to improve the bioconversion of hydrolysates. Adsorption on activated charcoal could be an effective and low cost technique [10] depending on the optimization in the treatment variables. Therefore, in this study, an experimental design was applied to optimise the purification process with activated charcoal, investigating the influence of temperature, time, stirring rate and pH on the removal rates of lignin degradation products (LDP), colour (C) and sugars. Finally, the hydrolysates were tested as fermentation substrates. For industrial applications, it is very important that the microorganism has the capacity to metabolize most of the sugars present in the hydrolysate, to obtain an efficient bioconversion of all the sugars into industrial interest metabolites. However, many wild-type yeast strains cannot utilize determinate sugars for their metabolism and are easily inhibited by toxic compounds generated during the hemicelluloses hydrolysis. For this reason, in this work the yeast Debaryomyces nepalensis NCYC 1026 was selected. In fact, this yeast, originally isolated from rotten apple, is known for its ability to metabolize both hexoses and pentoses sugars simultaneously and to grow also in non highly purified media [11]. 2. Materials and Methods 2.1 Raw material and autohydrolysis conditions The raw material used in this work was a mixture of grape stalks from two different Italian red cultivars, Bonarda and Barbera, kindly supplied by a wine-making factory in Piacenza (Italy) in the 2010 vintage. The samples were collected immediately 4 after the operation of pressing/destemming, oven dried at 60 ºC for 24 h, finally ground in a mill and sieved to obtain the 1– 4 cm size fraction. Chemical composition of the raw material, given on an oven dry weight basis, was determined according to TAPPI standards [12] and bibliographic procedures. Ashes (T211 om-93), hot water (T264 cm-97) and 1% NaOH solubility (T212 om-98), ethanol– toluene extractives (T204 cm-97), lignin (T222 om-98), holocellulose [13], cellulose and hemicelluloses [14] contents were determined. The hydrolysis treatments were carried out using unwashed grape stalks in a vertical autoclave, which supports a maximum temperature of 121 ºC and a maximum pressure of 198.67 kPa. The autohydrolysis process time was varied from 60 to 90, 105, 120 and 180 min, keeping constant the autohydrolysis temperature at 121 ºC and a solid/liquid ratio of 1:8 (w/v) in all experiments to find out the best conditions to get the maximum concentration of fermentable sugars. For the raw material washing step, the grape stalks were suspended in distilled water (solid/liquid ratio of 1:10/ w/v) in an erlenmeyer flask and maintained under agitation at room temperature for 2 h. The solid phase was recovered by filtration, oven dried at 50 ºC and used then for hydrolysis process at determined optimum conditions. Finally, the effect of sulphuric acid as autohydrolysis process catalyser (2% w/v) was studied to observe its effect on hemicellulosic stalks sugars extraction at autohydrolysis optimum conditions. All these experiments (schematised in Fig.1) allowed obtaining different sugar concentration liquors, providing information about the effect of different hydrolysis condition had on different fermentable sugars fractions recovery. 2.2 Hydrolysis liquor purification A 24 full-factorial design with three coded levels leading 19 experiments was made to study the effect of four different activated charcoal treatment variables (with a 5 fixed charcoal:hydrolysate ratio 1:40): pH, stirring rate, contact time, and temperature. The range and levels (low -1, medium 0, high +1) of the variables investigated in this study were: pH 2-5-8, stirring rate 150-200-250 rpm; contact time 10-35-60 min; temperature 25-35-50 ºC. The conditions were chosen in according to previous studies and the work of Mussatto and Roberto [15]. Three assays in the centre point were carried out to estimate the random error of the experimental design. The removal rates of lignin degradation products (LDP) and colour (C) were taken as the responses of the experimental design. All purification treatments were carried out in 250 ml Erlenmeyer flaks that contained 50 ml of unwashed grape stalk hydrolysate and were agitated on an orbital shaker during the purification treatment. After each treatment, the precipitate was removed by centrifugation at 5000 x g for 15 min and filtered with glass fibre filter before the HPLC analysis. After hydrolysate purification, the pH of all liquors was adjusted at pH ~ 5.4 with NaOH 5 N and then sterilized in autoclave at 121 ºC for 15 min for their application in fermentable processes. 2.3 Fermentation conditions D. nepalensis NCYC 1026 used in this study was supplied as freeze-dried strain by NCYC, National Collection of Yeast Cultures, UK. According to the given instructions, the yeast was activated in 10 ml malt extract (Oxoid), incubating for 5 days in an orbital shaker (HT Infors AG CH-4103, Switzerland) at 100 rpm and at 25 ºC. The activated yeast was then divided into 3 aliquots and 50 mL of fresh malt extract was added to each one. For three days, each day 20 ml of new fresh malt extract were added until a concentration of 1.07x108 cell/ml was obtained. After that, the strain was centrifuged, washed and grown in two different synthetic media supplemented by similar sugars 6 composition of the hydrolysis liquors obtained in this study: 30 g/L of glucose + 30 g/L of fructose (with 6.5 g/L yeast nitrogen base, YNB from DifcoTM) for L1, its purified L’1 liquor, L2 and its purified L’2 liquor, and 12 g/L glucose + 2.50 g/L fructose (with 6.5 g/L YNB) for L3 and its purified L’3 liquor. After three days of growth a known volume of the first semi-synthetic culture medium was transferred into 50 ml of L1, L’1, L2, and L’2 experimental liquors, corresponding to 1x106 cell/ml initial concentration, whereas for the second culture medium a known volume was transferred into 50 ml of L3 and L’3 liquors always to obtain a 1x106 cell/ml initial concentration. Additional synthetic liquors (B1, B2 and B3), with the same sugars concentration of L1, L2 and L3 hydrolysates, were also prepared with standard sugars (Carlo Erba, Italy) to compare the fermentation yield and the growth in synthetic liquors with original liquors. All the inoculated liquors (in 250 ml Erlenmeyer flasks) were incubated in the orbital shaker at 140 rpm and 25 ºC during the experiments. Each trial was carried out in duplicate. Aliquots of 1 mL were periodically collected from different incubated liquors to observe the cell growth, sugars consumption and metabolite productions. For this objective, the aliquots were centrifuged at 8200 x g for 10 second to precipitate the yeast and to obtain the supernatant. 2.4 Analytical procedures To determine the lignin degradation products, the samples were diluted (1:1000) in distilled water and analyzed at 280 nm by a UV-1601 Shimadzu spectrophotometer. For the colour determination, the samples were diluted (1:50) and analyzed at 440 nm. The cell growth was evaluated measuring the optical density of culture at 600 nm (OD600), whereas the relation between absorbance and cell concentration (evaluated by cell counting in Burker camera under optic microscope) was previously determined to obtain a calibration curve. 7 The sugars consumption was evaluated from supernatant using Megazyme kit assays for D-Glucose, D-Fructose and D-Xylose determination, whereas the by-products and other sugars components were analyzed by High Performance Liquid Chromatography (HPLC) Jasco LC Net II/ADC equipped with a refractive index detector and a photodiode array detector. A Phenomenex Rezex ROA HPLC column (300 mm x 7.8 mm) was used for the experiment, and 0.005 N H2SO4 prepared with 100 % deionised and degassed water was used as mobile phase (0.35 mL/min flow, 40 ºC and injection volume 40 µL). High purity standards of arabinose, galacturonic acid, lactic acid, xylitol and ethanol (supplied by Fluka, Sigma Aldrich and Panreac), were used for the calibration curves. 3. Results and Discussion The composition of the grape stalks (weight percentage) used in this work was: 22.61±2.14% extractives, 32.35±0.31% lignin, 12.19±0.52% cellulose, 26.43±0.42% hemicelluloses (13.35% glucose and 13.08% fructose) and 6.11± 0.43% ashes. This high fructose concentration in the raw material derived from grape juice which impregnated stalks during destemming operations. 3.1 Autohydrolysis Unwashed grape stalks were used to find out the optimum conditions for maximum fermentable sugars extraction. Table 1 shows the results of sugars concentrations in the liquors of the trials aimed to study the best conditions of autohydrolysis in order to maximize the glucose and fructose content. All the liquors presented an acid pH value due to the organic acid formed in the autohydrolysis process. The optimal conditions chosen to use the autohydrolysis liquor as a fermentative medium were 121 ºC, 90 min, 1/8 because this experiment presented the highest fermentable sugar concentration. 8 3.2 Autohydrolysis liquor purification experimental design Autohydrolysis liquor contained, in addition to fructose and glucose as the major sugars, several compounds that are toxic to yeasts, namely, acetic acid, furfural, hydromethylfurfural (HMF), and lignin degradation products. The results of the experimental design (Table 2) showed that the removal of colour and lignin degradation products was dependent on the conditions employed in the treatment of the unwashed grape stalk hydrolysate with activated charcoal. The hydrolysate colour is directly related to the presence of lignin degradation products (phenolic compounds), so a loss of colour in the treatment with activated charcoal is obtained by removing these compounds from the hydrolysate. Lignin degradation products and colour removal were strongly influenced by the temperature and pH. pH 2 produced better results than pH 5 and 8, probably as a result of the low formation of phenolate ions at low pH and the fact that these ions are poorly adsorbed on activated charcoal [16]. High temperatures promoted an increase in the density of the packing of phenolic molecules in the activated charcoal pores [17] producing a high removal of phenolic compounds, so the colour intensity of the hydrolysate also decreased. The conditions presented in the experiment 11 (pH=2, 250 rpm, 10 min, 50 ºC) were selected as the best conditions for removal the colour and lignin degradation products with the objective of the application of hydrolysate in fermentation processes after the purification process. The selected purification conditions were applied to the liquors from hydrolysis of grape stalks, hydrolysis of washed grape stalks and acid hydrolysis of washed grape stalks. The results are presented in Table 3. 15 List of tables Table 1. Autohydrolysis conditions in autoclave (all experiments at 121 ºC and 1/8 ratio solid/liquid) and sugar concentration of obtained initial liquors from unwashed grape stalks. Table 2. Experimental design for the colour removal (CR) and lignin degradation products removal (LDPR) in the treatment of unwashed grape stalk hydrolysates with activated charcoal. Table 3. Characterization of different concentrated hydrolysate liquors before and after purification process at optimal conditions: liquors obtained from grape stalks autohydrolysis (L1 and purified L’1), liquors obtained from washed grape stalks autohydrolysis (L2 and purified L’2) and liquors obtained from washed grape stalks using 2% H2SO4 (w/w) acid hydrolysis process (L3 y L’3). Table 4. Ethanol maximum production yields and volumetric productivities by D. nepalensis in studied concentrated liquors. 16 Table 1 Experiments Glucose (g/L) Fructose (g/L) pH 60 min 5.50±0.50 6.80±0.60 3.92±0.10 90 min 8.30±0.80 9.60±0.50 3.98±0.10 105 min 7.40±1.10 8.70±1.00 3.89±0.10 120 min 7.05±1.25 8.40±1.15 3.86±0.10 180 min 8.20±1.50 8.90±1.10 3.87±0.10 17 Table 2 Experiment aX 1 bX 2 cX 3 dX 4 CR (%) LDPR (%) 1 -1 -1 -1 -1 65.5 91 2 +1 -1 -1 -1 43.7 87.4 3 -1 +1 -1 -1 51.5 89.9 4 +1 +1 -1 -1 53.7 92.4 5 -1 -1 +1 -1 44.8 87.3 6 +1 -1 +1 -1 50.8 87.9 7 -1 +1 +1 -1 54.9 87.4 8 +1 +1 +1 -1 44.1 80.5 9 -1 -1 -1 +1 75.1 74.2 10 +1 -1 -1 +1 60.3 80 11 -1 +1 -1 +1 83.4 100 12 +1 +1 -1 +1 43.6 73.2 13 -1 -1 +1 +1 80.6 100 14 +1 -1 +1 +1 71.3 95.8 15 -1 +1 +1 +1 74.9 100 16 +1 +1 +1 +1 66.1 95.7 17 0 0 0 0 59 80.4 18 0 0 0 0 53.2 81.1 19 0 0 0 0 45.3 80.6 aX1: pH normalized; bX2: stirring rate normalized; cX3: contact time normalized; dX4: temperature normalized; ranges: pH 2-5-8, stirring rate 150-200-250 rpm; contact time 10-35-60 min; temperature 25-35-50 ºC. 18 Table 3 Liquors Absorbance Concentration (g/L) Colour LDP Glucose Fructose Xylose L1 0.87 0.53 30.96±1.19 30.70±1.50 0 L’1 0.14 0.00 32.00±1.00 30.00±1.20 0 L2 0.75 0.53 6.01±0.10 5.96±0.28 0 L’2 0.07 0.09 6.05±0.13 6.15±0.20 0 L3 0.38 0.69 12.22±0.33 2.57±0.20 9.78±1.19 L’3 0.009 0.17 10.90±0.10 2.54±0.05 9.27±0.10 19 Table 4 Liquor Sugars concentration Fermentation Yields (g/L) Max ethanol production (g/L) YE/S (g/g) YE/St (g/g) Qp (g/Lh) YB/S (109cell/g) YE/B (g/109cell) B 1 60.00 13.14±1.50 0.22 - 0.07 8.03 0.03 L 1 61.66 20.84±1.25 0.34 0.14 0.45 10.57 0.03 L’ 1 62.00 13.16±1.10 0.30 0.09 0.45 14.39 0.02 B 2 12.00 1.90±0.25 0.18 - 0.09 16.17 0.01 L 2 11.97 2.66±0.30 0.22 0.01 0.12 25.56 0.01 L’ 2 12.20 2.38±0.17 0.19 0.01 0.11 28.36 0.01 B 3 24.50 3.75±0.20 0.15 (0.26)1 - 0.07 8.86 (14.96)a 0.02 L 3 24.57 5.20±0.35 0.21 (0.35)1 0.02 0.07 19.21 (31.91) a 0.01 L’ 3 22.71 1.86±0.35 0.08 (0.14)1 0.01 0.06 11.41 (19.27) a 0.01 Y E/S : ethanol yield (g ethanol per g monomeric sugar consumed) YE/St: ethanol yield (g ethanol per g of dry hydrolysed stalks) Qp: volumetric productivities (g/L of ethanol per hour) YB/S: biomass yield (x109cell biomass per g monomeric sugar consumed) YE/B: ethanol yield (g ethanol per x109cell biomass) a: Yield taking into account the production of ethanol from glucose and fructose (without xylose) 20 List of figures: Figure 1. Different hydrolysis treatments conditions used for grape stalks fermentable sugars recovery at optimal conditions: grape stalks feed (S1), insoluble autohydrolysis fraction (S’1), autohydrolysate (L1), washed grape stalks feed (S2), insoluble washed autohydrolysis fraction (S’2), washed grape stalks autohydrolysate (L2), washed grape stalks feed (S3), washed acid hydrolysis insoluble fraction (S’3), and acid hydrolysate (L3). Figure 2. Sugar consumption and by-products obtained from D. nepalensis growth in 30 g/L glucose and 30 g/L fructose of synthetic liquor B1 (2a), grape stalks autohydrolysate liquor L1 (2b), and the purified liquor L’1 (2c). Figure 3. Sugar consumption and by-products obtained from D. nepalensis growth in 6 g/L glucose and 6 g/L fructose of synthetic liquor B2 (3a), washed grape stalks autohydrolysate liquor L2 (3b), and the purified liquor L’2 (3c). Figure 4. Sugar consumption and by-products obtained from D. nepalensis growth in 12 g/L glucose, 2.5 g/L fructose and 10 g/L xylose of synthetic liquor B3 (4a), washed grape stalks acid autohydrolysate liquor L3 (4b), and the purified liquor L’3 (4c). Figure 5. Cell growth of D. nepalensis in B1, L1, L’1, B2, L2, L’2 and B3, L3, L’3 liquors represented as cell/ml with monomeric sugars consumption (Ct) respect to the initial sugar concentration (C0). 21 Fig. 1 22 Fig. 2 a) B1 0 5 10 15 20 25 30 35 0 20 40 60 80 100 120 140 160 180 200 h g/L Glucose Fructose Ethanol Lactic acid b) L 1 0 5 10 15 20 25 30 35 0 10 20 30 40 50 h g/L Glucose Fructose Ethanol Galacturonic acid Lactic acid Arabinose c) L'1 0 5 10 15 20 25 30 35 0 10 20 30 40 50 h g/L Glucose Fructose Ethanol Galacturonic acid Lactic acid Arabinose 23 Fig. 3 b) L2 0 1 2 3 4 5 6 7 0 5 10 15 20 25 h g/L Glucose Fructose Ethanol Galacturonic acid Lactic acid Arabinose c) L'2 0 1 2 3 4 5 6 7 0 5 10 15 20 25 h g/L Glucose Fructose Ethanol Galacturonic acid Lactic acid Arabinose a) B2 0 1 2 3 4 5 6 7 0 5 10 15 20 25 h g/L Glucose Fructose Ethanol Lactic acid 24 Fig. 4 c) L' 3 0 2 4 6 8 10 12 14 0 20 40 60 80 100 h g/L Glucose Fructose Ethanol Galacturonic acid Lactic acid Xylose Xylitol Arabinose b) L 3 0 2 4 6 8 10 12 14 0 20 40 60 80 100 120 140 160 180 200 h g/L Glucose Fructose Ethanol Galacturonic acid Lactic acid Xylose Xylitol Arabinose a) B 3 0 2 4 6 8 10 12 14 0 20 40 60 80 100 120 140 160 180 200 h g/L Glucose Fructose Ethanol Lactic acid Xylose Xylitol