Bioresource Technology 152 (2014) 24–30
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Potential of bioethanol production from olive mill solid wastes Hiba Abu Tayeh a, Naim Najami a,b, Carlos Dosoretz c, Ahmed Tafesh a, Hassan Azaizeh a,d,⇑ a
Institute of Applied Research, Affiliated with University of Haifa, The Galilee Society, P.O. Box 437, Shefa-Amr 20200, Israel The Academic Arab College of Education, Haifa, Israel c Department of Environments, Water & Agriculture Engineering, Technion Institute, Haifa 32000, Israel d Tel Hai College, Upper Galilee 12208, Israel b
h i g h l i g h t s Two yeasts were isolated from OMSW, I. orientalis, and P. galeiformis/manshurica. I. orientalis showed better kinetics to xylose compared to the other strains. I. orientalis on hydrolysate supplemented with glucose showed best ethanol production. Using SSF process following pretreatment, the ethanol yield was 3 g/100 g dry OMSW.
a r t i c l e
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Article history: Received 15 September 2013 Received in revised form 24 October 2013 Accepted 28 October 2013 Available online 6 November 2013 Keywords: Olive mill solid wastes Lignocellulose degradation Fermentation process Bioethanol production
a b s t r a c t The main objective of this study was to screen endogenous microorganisms grown on olive mill solid wastes (OMSW) with the potential to ferment pentoses and produce ethanol. Two yeasts were isolated and identified as Issatchenkia orientalis, and Pichia galeiformis/manshurica. The adaptation of the strains displayed a positive impact on the fermentation process. In terms of xylose utilization and ethanol production, all strains were able to utilize xylose and produce xylitol but no ethanol was detected. Separate hydrolysis and fermentation process on hydrolysate undergo detoxification, strain I. orientalis showed the best efficiency in producing of ethanol when supplemented with glucose. Using simultaneous saccharification and fermentation process following pretreatment of OMSW, the average ethanol yield was 3 g/100 g dry OMSW. Bioethanol production from OMSW is not economic despite the raw material is cheap. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In recent decades renewable energy sources have considerable interest worldwide. Biomass energy is one of the oldest and most promising sources and includes organic waste, sewage, and energy crops, agricultural and industrial residues that can be utilized to produce bio-fuel. Biomass can be converted biologically to liquid or gaseous fuels, such as ethanol, methanol, methane and hydrogen by fermentation processes (Haagensen et al., 2009). Ethanol has attracted interest as alternative liquid fuel, especially for transportation. It has immense importance for countries which depends heavily on import of crude oil, spending a huge amount of its annual budget. Currently, bioethanol is mainly produced from biomass containing sugar or starch as sugar cane and ⇑ Corresponding author at: Institute of Applied Research, Affiliated with University of Haifa, The Galilee Society, P.O. Box 437, Shefa-Amr 20200, Israel. Tel.: +972 4 9504523/4; fax: +972 4 95044535. E-mail addresses: [email protected] (H. Abu Tayeh), najamina@gmail. com (N. Najami), [email protected] (C. Dosoretz), [email protected] (A. Tafesh), [email protected], [email protected] (H. Azaizeh). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.102
corn as raw material (first generation) (Patni et al., 2013). Because these products are used as food and feed for humans and animals, there is competition of using biomass as a food or fuel. Due to this competition, bioethanol production from lignicellulose (second generation) enthusiastically have been investigated worldwide (Lin et al., 2012). Lignocellulosic biomass, such as trees and agricultural residues is an attractive raw material for bioethanol production because of the large amount of potential sugar for fermentation and bioenergy production (Bellido et al., 2013; Choi et al., 2010; Cuevas et al., 2010; Kumar et al., 2009; Olsson and Hahn-Hägerdal, 1996). Production of olive oil symbolizes one of the most important economic agro-food sectors in the Mediterranean basin. The traditional oil industry generates two byproducts at the end of the process, olive cake (residue) and olive mill wastewater, which can cause serious environmental pollution problems (Ballesteros et al., 2001). Crude olive cake, the leftover solids following the pressing of olives, is a mixture of skin, pulp, and seeds. It comprises approximately 35% of the beginning olive weight, is rich in carbohydrates, and is available in appreciable quantities in the
H. Abu Tayeh et al. / Bioresource Technology 152 (2014) 24–30
Mediterranean area (Banat et al., 2013; El Asli and Qatibi, 2009). Global annual production of olive cake has been estimated to approach 4 108 kg of dry matter (El Asli and Qatibi, 2009). The main structural components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. Of these, only cellulose and hemicellulose can be used as raw materials to produce ethanol by fermentation of carbohydrates obtained through chemical or enzymatic hydrolysis (saccharification). The biological process for converting lignocellulose to fuel ethanol requires: (1) delignification to liberate cellulose and hemicellulose; (2) depolymerization of carbohydrate polymers to produce free sugars; and (3) fermentation of mixed hexose and pentose sugars to produce ethanol (Cuevas et al., 2010; Kumar et al., 2009; Lin et al., 2012; Matsushika et al., 2009). Hydrolysis of these polysaccharides (cellulose and hemicellulose) is usually accomplished by acid and/or enzymatic treatment. The utilization of both cellulose and hemicellulosic sugars like hexose, pentose and others present in a typical biomass hydrolysate is essential for the economical production of ethanol (Kumar et al., 2009; Olsson and Hahn-Hägerdal, 1996). The production of ethanol from pretreated material may be accomplished either by sequential hydrolysis and fermentation or by a simultaneous saccharification and fermentation (SSF) process (Ahmed et al., 2013; Cuevas et al., 2010; Oberoi et al., 2012). During the SSF process, end product inhibition can be overcome as glucose is simultaneously fermented as it is formed; another advantage is that a single reactor is used for both saccharification and fermentation processes. However, the enzymatic reaction in SSF process is operated at a temperature lower than its optimum level owing to the mismatch in optimum temperatures for hydrolysis (approx. 50 °C) and fermentation (approx. 30 °C) (Cuevas et al., 2010; Ruiz et al., 2006). The main component of lignocellulosic hydrolysates is glucose, a hexose sugar derived from cellulose and hemicellulose. Although the proportion of mono-saccharides in hemicellulose hydrolysates varies depending on the raw material and the hydrolysis procedure, they all contain both pentose sugars, such as D-xylose and L-arabinose and hexose sugars as well. D-Xylose is the second most abundant carbohydrate and its content is particularly high in grass and hardwood (Hendriks and Zeeman, 2009; Matsushika et al., 2009). One of the most effective ethanol-producing microorganisms for hexose sugars including glucose, mannose and galactose is Saccharomyces cerevisiae yeast with high ethanol productivity, high tolerance to ethanol, and tolerance to inhibitory compounds present in the hydrolysate of lignocellulosic biomass (Matsushika et al., 2009). However, this strain is unable to utilize xylose for growth or fermentation in order to produce ethanol. Instead, this strain metabolizes D-xylulose, an isomerization product of D-xylose. Some yeast strains have been reported to ferment xylose into ethanol, but the rate and yield of ethanol production from xylose in these xylose-utilizing yeast strains are considerably low compared to their glucose fermentation process (Ferreira et al., 2011; Lin et al., 2012). Therefore, genetic engineering and/or adaptation may be promising methods to develop sufficient xylose fermentation in S. cerevisiae. To date, numerous studies regarding the metabolic engineering of S. cerevisiae for xylose utilization have been reported, and many reviews have already addressed the current advancement in metabolic engineering of xylose-fermenting strains and factors which affect xylose metabolism in yeasts (Matsushika et al., 2009). The objectives of the current study were to isolate yeast strains from olive mill solid wastes (OMSW) with the potential to utilize xylose and produce ethanol to be compared with other commercial known yeast strains. In addition to adapt the different yeast isolates to produce bioethanol from the OMSW hydrolysate.
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2. Methods 2.1. Raw material Olive mill solid waste (OMSW), the solid residue from the traditional olive oil production process, was obtained from Dabburia, Galilee region of Israel after the harvest months of the year 2009/2010. OMSW was dried at 45 °C, milled to particle size between 0.25–1 mm, and stored at room temperature until being used. 2.2. Content test Content test was performed to quantify the potential of a feedstock (raw material) and thus test the effectiveness of pretreatment to the total content of cellulose and hemicellulose. Wall sugar content was tested after water extraction, testing the solid percentage and the ash content was performed according to standard analytical procedures as described in protocols of the NREL. The composition of the OMSW used throughout the experiments included: extractive, lignin, acid insoluble, cellulose, hemicellulose, xylose and galactose, arabinose, acetyl, ash, with dry matter percentage of 18.3 ± 1.7, 40.2 ± 5.1, 39.5 ± 5.1, 18.4 ± 0.2, 15.9 ± 0.1, 14.1 ± 0.1, 1.8 ± 0.0, 2.7 ± 0.1, 4.5 ± 1.2, respectively. 2.3. Pretreatment of OMSW The biomass at a solid loading of 7.5% (w/v) was mixed with diluted sulfuric acid (final concentration of 2% (v/v)) at 100 °C, with the residence time of 2 h. After treatment, the reactor was removed from heating jacket and cooling to room temperature. The pretreated material was separated by filtration into two fractions, i.e., the solid insoluble residue and the liquid fraction (hydrolysate). The composition of the hydrolysate regarding main sugars (glucose and xylose) and inhibitors compounds (furfural, acetic acid, and formatic acid), is shown in Table 1. Other sugars, like galactose, arabinose and mannose, as well as hydroxymethylfurfural (HMF), were also present in hydrolysates at lower concentration (Cara et al., 2008). 2.4. Enzymatic hydrolysis After dilute acid pretreatment, enzymatic hydrolysis was performed using cellulases (Cellulase Complex-Ecostone L900 and b-glucosidase (Novozyme 188S from Trichoderma reesei were purchased from Sigma) at 50 °C and 100 rpm for 24 h in a water bath shaker. In order to maintain the pH at 4.8 in the mixture, 0.05 M sodium citrate buffer (pH 4.5) was added. 2.5. Detoxification of diluted sulfuric acid hydrolysate with activated carbon Detoxification of diluted sulfuric hydrolysate was carried out using 5% (w/v) activated carbon in an incubated water bath shaker at 50 °C for 30 min, according to the methodology described by Díaz et al. (2009). Then the mixture was cooled to room temperature, sodium hydroxide was added until the desired pH (4.5) was achieved. Afterward was centrifuged to remove the solid material and kept below 4 °C for further analysis. 2.6. Yeast strains Three strains of yeast were used in this study; one was purchased from ATCC, S. cervisiae (24860 ATCC, designated as S.C.),
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Table 1 Composition of the initial hydrolysate (mg/l) obtained after sulfuric acid (2%) pretreatment of OMSW followed by activated charcoal detoxification. Hydrolysate composition (mg/l) Without detoxification Activated charcoal detoxification a
Glucose
Xylose
Acetic acid
Formic acid
Furfural
a
8809.7 9687.0
2131.2 1886.0
530.5 298.1
56.0 1.4
1670.5 1907.0
Average of duplicated samples.
two strains were isolated from OMSW and were identification at Hy labs (Israel) and CBS (Holland): – Pichia galeiformis A. Endo & Goto – (designated as G). – P. Kudriavzevii Boidin, Pignal & Besson – (designated X). 2.7. Inoculums’ preparation The yeasts were grown at 35 °C on YPG and YPX agar (Huang et al., 2009) and supplemented with 20 g/l xylose, 10 g/l yeast extract, 20 g/l peptone, and 20 g/l agar. A single colony was chosen after 2 days and cultured in YPX medium overnight to prepare a culture with the desired cell concentration. 2.8. Adaptation process Adaptation was performed by sequential transfer of cultures to diluted hydrolysate medium with increasing concentration of hydrolysate, which means increasing concentration of inhibitors. Hydrolysate is the liquid which was obtained after filtration of the 2% sulfuric acid pretreatment OMSW. The initial hydrolysate concentration was 3.5% (w/v) and reached to 6.5% (w/v), with inoculums’ concentration of 1 g/l, and supplemented with 3.5 g/l KH2PO4, 5 g/l K2HPO4, 3.5 g/l (NH4)2HPO4, 0.25 g/l MgSO4:7H2O, 0.015 g/l CaCl2:2H2O, 0.0005 g/l Thiamine, 1 ml trace metals (Causey et al., 2003). Xylose concentration was fixed at 1 g/l. The adaptation process was performed in a 50 ml flask with 25 ml of medium. Each culture was grown at pH 5.0 for 48–72 h at 35 °C in a rotary shaker. Culture aliquot of 0.25 ml from one shake flask was transferred to the following shake flask. Cells were grown twice at the same hydrolysate dilution before transferring the cells to more concentrated hydrolysate. 2.9. Analytical methods During the experiments, samples were periodically taken for sugars, ethanol and cell growth determinations. The cellular growth was determined by measuring the fermentation broth using UV Spectrophotometer at 600 nm (Bellido et al., 2011), and was correlated to a calibration curve. Each fermentation sample was filtered through a 0.45 lm filter. Glucose, xylose, xylitol, and ethanol concentrations were determined by high-performance liquid chromatography (HPLC) (Thermo Scientific finnigan surveyor, equipped with a Shimadzu refractive index detector and Rezex-ROA column (150 7.8 mm) at 65 °C. Sulphuric acid (0.005 M) was used for elusion at a flow rate of 0.6 ml/min (Bellido et al., 2011; Cara et al., 2008; Wang et al., 2013). For furfural and HMF determinations, the HPLC system was equipped with a UV detector (Diode array) at 280 nm, at the same operation conditions which was previously described. 3. Results and discussion Yeast strains isolation was conducted by adding pure glucose and xylose as a carbon source to the OMSW by enrichment technique according to Limtong et al. (2007). Thus we were able to
isolate two strains of yeast, Issatchenkia orientalis – which was isolated on the carbon source xylose, so it was marked X and Pichia galeiformis/manshurica – which was isolated on glucose, so it was, marked G. The two strains were identified at two different places the first in Hy labs (Israel) and the other was at Centraalbureau voor Schimme lcultures (CBS), (The Netherlands). The identification processes shows that they belong to the Pichia family, that able to utilize xylose. To characterize the ability of X and G strains to produce ethanol from defined sources (glucose and xylose) and treated OMSW, they were compared to the well known isolate S. cervisiae (ATCC 24860), which efficiently known to produce ethanol from hexoses. During the first 24 h, the yeast X seems to utilized the glucose and produced in average 6 g/l of ethanol which is equivalent to 78% of the theoretical yield, however the glucose utilizing for the others yeast strains was obtained after 120 h and with low ethanol production compared to the yeast X (Fig. 1). Glycerol was produced as byproduct as well. Ethanol and glycerol concentration began to decrease after the fermentation process was over, and the OD, which marks the cell density of the yeast, continues to rise which indicated that the glycerol and ethanol are used as a carbon source for cell growth, where this is compatible with the results shown by Kötter and Ciriacy (1993), Slininger et al. (1987) and Zhao et al. (2008), but in their studies they used the yeast Pachysolen tannophilus 1771. It might be that during taking the samples the oxygen entered into the flasks when the glucose was consumed, which facilitates that the yeast utilize the ethanol as a carbon source. When using xylose as a sole carbon source, the yeast X showed a better ability compared to the other yeasts although utilization was very slow during the first 48 h in the formation of xylitol which is intermediate material of xylose pathway (Fig. 1) and no ethanol production was measured. The lag phase of the yeast strains G and S.C. is longer than 48 h, and only after that the log phase began as indicated by the sugar consumption and increase cell biomass. According to Moniruzzaman et al. (1997) and Karhumaa et al. (2007) there is an accumulation of xylitol and this is due to an imbalance of cofactor (NAD+ and NADPH) which is required for the enzyme (XR) compared to enzyme Xylitol Dehedrogenase (XDH) (Figs. 1 and 2). They also assessed that the insertion of oxygen shocks (pulses) which inhibits the enzyme XR and thus prevents the accumulation of xylitol. 3.1. Fermentation of mixed sugars, glucose and xylose The results show that glucose utilization rate by yeast X is faster as compared with yeast G and S.C. (Figs. 1 and 2). When the carbon source was glucose and xylose, ethanol was produced only by the yeast X, but the byproduct glycerol produced by the three yeast strains despite the differences of kinetics of each strain (Fig. 2). It is obvious that the xylose was consumed significantly only after ethanol and glycerol were previously consumed. Xylose utilization by the yeast X was at the rate of 82% after 240 h, compared to 21% by the yeast G. The kinetics of the yeast X in this case could be compared with the case of which the xylose is a sole carbon, so expect slower kinetics in the case of mixed sugars and this similar to
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0
1 0
0 0
24
Glucose
3 2 1 0
10 5 0
Xylose
120
time(h) Xylitol
xylose, xylitol (g/l)
15
OD
xylose, xylitol(g/l)
6 5 4
20
48
120
4 3
10
2
5
1
240
0 0
OD
Ethanol
6
20
5 4
15
3
10
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5
1
0
0 0
24
Xylose
OD
48
48
OD
120
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time(h) Ethanol
Glycerol
Yeast G(Xylose)
25
240
24
Glucose
Glycerol
Yeast X(Xylose)
25
24
48
5
15
time(h)
Glycerol
Yeast S.C.(Xylose)
0
24
6
0
120
25
6
20
5 4
15
3
10
2
5
1
0
0 0
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time (h) Xylitol
xylose, xylitol (g/l)
Glucose
48 120 240 time(h) OD Ethanol
5
OD
0
3 2
10
7
20
ethanol, glycerol (g/l)
5
15
25 glucose(g/l), OD
10
7 6 5 4
20
ethanol, glycerol(g/l)
15
Yeast G(Glucose)
25
glucose(g/l), OD
20
ethanol, glycerol(g/l)
7 6 5 4 3 2 1 0
25 glucose(g/l), OD
Yeast X(Glucose)
OD
Yeast S.C.(Glucose)
OD
24
48 144 time(h) Xylose Xylitol
240 OD
Fig. 1. Fermentation results using the yeast strains X, G, or S.C. in defined medium with a sole carbon source, glucose or xylose at 35 °C. Results are mean values of two replicates where the deviations from the average values were less than 10%.
Yeast G(GLU+XYLOSE)
glucose, xylose (g/l)
2 1.5 1 0.5 0 24
48
120
240
Time(h) Xylose Glycerol
Glucose Xylitol
Ethanol OD
Yeast X(GLU+XYLOSE)
2 1.5 1 0.5 0
Glucose Xylitol
24
48
120
Time(h) Xylose Glycerol
240
ethanol, glucose, xylose (g/l)
2.5
16 14 12 10 8 6 4 2 0
xylitol, glycerol (g/l), OD
ethanol, glucose, xylose (g/l)
Yeast S.C. (GLU+XYLOSE) 16 14 12 10 8 6 4 2 0
2.5 2 1.5 1 0.5 0 0
Ethanol OD
xylitol, glycerol (g/l), OD
0
0
xylitol, glycerol (g/l), OD
2.5
16 14 12 10 8 6 4 2 0
Glucose Xylitol
24
48 120 Time(h) Xylose Glycerol
240 Ethanol OD
Fig. 2. Fermentation results using the yeast strains X, G, or S.C. in defined medium with a mixture of glucose and xylose as carbon source at 35 °C. Results are mean values of two replicates where the deviations from the average values were less than 10%.
what was shown by Zhao et al. (2008). In the case of the strains G and S.C., it seems that xylose is inhibiting the ethanol production process. 3.2. Fermentation of chemically hydrolysed OMSW The hydrolysate was treated with activated carbon and subsequently was neutralized by sodium hydroxide. After neutralization and before the fermentation process, 1.2% glucose was added into
the hydrolysate medium. The results shows different behaviors of the strain X as compared with strains G and S.C. (Fig. 3). The yeast X utilized almost all the glucose within the first 24 h and produced glycerol, xylitol and ethanol. Glucose utilization in yeast G and S.C. was very slowly and ethanol production as well and therefore was no accumulation of xylitol or glycerol as byproducts (Fig. 3). The maximum ethanol production was obtained during the hydrolysate fermentation was calculated as 0.49, 0.44, 0.43 g ethanol/g sugar utilized using the different yeast strains X, G, S.C.,
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16 14 12 10 8 6 4 2 0
1.2 1 0.8 0.6 0.4 0.2
xylitol, glycerol (g/l)
glucose, xylose, ethanol (g/l)
Yeast X(HYDRO)
0 0
24
48
120
240
Time(h) Glucose
Xylose
Ethanol
Xylitol
Glycerol
Yeast S.C.(HYDRO)
0.8 0.6 0.4 0.2 0 0
24
48
120
240
1.2
16 14 12 10 8 6 4 2 0
1 0.8 0.6 0.4 0.2 0 0
24
Time(h) Glucose
Xylose
Ethanol
xylitol, glycerol (g/l)
1
glucose, xylose, ethanol (g/l)
1.2
16 14 12 10 8 6 4 2 0
xylitol, glycerol (g/l)
glucose, xylose, ethanol (g/l)
Yeast G(HYDRO)
48
120
240
Time(h)
Xylitol
Glycerol
Glucose
Xylose
Ethanol
Xylitol
Glycerol
Fig. 3. Fermentation of the OMSW hydrolysate (liquid phase following pretreatment with sulfuric acid) by the yeasts X, G, S.C. Two replicates were used and the measurements were done in duplicates and the deviation from the average values were less than 10%.
respectively. Other researchers have shown that the recombinant Escherichia coli FBR15 utilized xylose and produced 0.47 g ethanol/g sugar (El Asli and Qatibi, 2009).
3.3. Impact of detoxified hydrolysate on the fermentation process Following OMSW hydrolysate detoxification process (Table 1), a slight decrease in acetic acid and formic acid concentrations has been obtained (about 11% and 44%, respectively) which is in agreement with other reports showing fermentative performance improvements in detoxified hydrolysates (over-limed) (Amartey and Jeffries, 1996; Díaz et al., 2009). In contrast, furfural was reduced by more than 97% as a consequence of the detoxification treatment by activated charcoal. Other inhibitory compounds may be present in the hydrolysates, like phenolic compounds derived from lignin or other extractives, and may also be removed by detoxification methods (Parajó et al., 1998). In addition, the HMF that was present in lower concentrations, its level was reduced after activated charcoal (data not shown). No sugar losses were detected after detoxification processes, even a slight increase in sugar concentration was observed during these experiments. The results showed positive effects on the fermentation process and ethanol yield produced by X and G strains following OMSW hydrolysate detoxification process (Fig. 4). Without detoxification, only the X yeast compared with G yeast utilized small amount of glucose and xylose within the first 24 h and produced ethanol (Fig. 4, A and B). Following detoxification it has been observed that glucose and xylose were utilized accompanied with ethanol production by both strains X and G within the first 24 h (Fig. 4, C and D). These results indicated that OMSW hydrolysate detoxification before fermentation is necessary in order to produce ethanol.
3.4. Fermentation of enzymatic hydrolysed OMSW During the fermentation process 1.5% glucose and 0.45% xylose were supplemented into the enzymatic OMSW hydrolysate medium. The results showed that ethanol production using the X, G, S.C. yeast strains was of 3.08, 0.54, 0.58 mg ethanol/g utilized glucose/h), respectively. This indicated that the isolated strain X was superior compared with the other two strains. 3.5. Xylose utilization As have been shown, the isolated yeasts utilize xylose but they do not produce ethanol. Therefore, the xylose utilization of the different strains during the fermentation process was compared using different media. The highest xylose utilization was observed by using the yeast X grown in defined medium with xylose as a sole carbon source where the utilization was 93% after 240 h, and in the medium supplemented with glucose and xylose the utilization decreased to 81%, and in the medium of the enzymatic hydrolysis and hydrolysate the utilization was 25% and 2% respectively. This indicates that there are some inhibitors in these growth media. 3.6. Ethanol production from OMSW using SSF process The X, G and S.C. strains were grown using the SSF process in reactors to which the enzymes that consisted of the cellulose complex were added as well. The SSF process was conducted at anaerobic conditions with 6.7% solid concentration and incubated at 35 °C for 240 h. The released glucose by the SSF process was utilized simultaneously by the yeasts which produce ethanol during the process (Fig. 5). The final ethanol concentration produced by the three yeasts were similar and it was about 30 mg/g dried OMSW. However, the yeast X behaves differently than the strains
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B
yeast X
16
glucose, xylose, ethanol (g/l)
glucose, xylose, ethanol (g/l)
A 14 12 10 8 6 4 2 0 0
23
94
yeast G
16 14 12 10 8 6 4 2 0
165
0
Time (h) Glucose
D
yeast X 16 14 12 10 8 6 4 2 0 0
24
48 Time (h)
Glucose
Xylose
120
94
Time (h)
Glucose
Ethanol
glucose, xylose, ethanol(g/l)
glucose, xylose, ethanol(g/l)
C
Xylose
23
Xylose
165
Ethanol
yeast G 16 14 12 10 8 6 4 2 0 0
240
24
48
120
240
Time (h) Glucose
Ethanol
Xylose
Ethanol
Fig. 4. Fermentation of OMSW hydrolysate using X and G strains. Upper panel (A, B), OMSW hydrolysate without detoxification before the fermentation process. Lower panel (C and D), OMSW hydrolysate with detoxification before fermentation process. Bars represent average ± standard deviation of 3 replicates.
Glucose
glucose, xylose, ethanol(g/l)
X(SSF)
Xylose Ethanol
2.5 2 1.5 1 0.5 0 0
24
48
120
240
TIME (h) Glucose Ethanol
2.5 2 1.5 1 0.5 0 0
24
48
120
240
TIME (h)
Glucose
G(SSF)
Xylose
glucose, xylose, ethanol(g/l)
glucose, xylose, ethanol(g/l)
S.C.(SSF)
Xylose Ethanol
2.5 2 1.5 1 0.5 0 0
24
48
120
240
TIME (h)
Fig. 5. SSF process using the three yeast strains and b-glucosidase and Cellulase Complex (Ecostone L900). Two replicates were used and the measurements were done in duplicates and the deviation from the average values were less than 10%.
G and S.C., and utilized most of the glucose after 24 h and the maximum glucose utilization was obtained after 120 h. However, the strains G and S.C. utilized most of the glucose after 120 h and the maximum glucose utilization was obtained after 240 h. Using SSF process, it is possible to produce in average 3 g/l ethanol from 100 g of OMSW (calculated data). Ruiz et al. (2006) showed that they achieved 7.5 g ethanol per 100 g olive tree wood,
and sunflowers resulted in 8 g per 100 g. In addition to the fact that raw material is different (different cellulose and hemicellulose percent) also pretreatment conditions were different in both cases, they used the steam explosion method at 230 °C, also the yeast strain was different as well. Therefore the comparison of the results with others from the literature is not always possible. Even the OMSW is different and depends on the source of olives grown
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in each country. Ruiz et al. (2006) showed that they can get 95.1 l ethanol/ton olive tree (pruning), and in the current study 38 l ethanol/ton OMSW was obtained which still very low and not economic for using OMSW for bioethanol production. The yield could be improved by utilizing the pentoses (mainly xylose) for ethanol production which still needs more investigation. Therefore, the obtained results so far suggest that OMSW is not a suitable feedstock for ethanol production as demonstrated by our data and by others (Ruiz et al., 2006). It might be a better idea to evaluate the use of OMSW in an anaerobic digestion to produce biogas followed by a composting process to produce fertilizer to enable the sustainable production of future olive crops. 4. Conclusions Two yeast strains were isolated from OMSW, I. orientalis, and P. galeiformis/manshurica. All strains were able to utilize xylose and produce xylitol but not ethanol. I. orientalis showed better kinetics and less sensitivity to xylose repression for ethanol production from glucose compared to P. galeiformis/manshurica and S. cervisiae. Separate hydrolysis and fermentation process on hydrolysate undergo detoxification, strain I. orientalis showed best efficiency in producing of ethanol when supplemented with glucose. Using SSF process following pretreatment of OMSW, the average ethanol yield was low as 3 g/100 g dry OMSW. Acknowledgements We thank the Ministry of Science of Israel for partial funding of this study in the framework of Regional Research and Development programs, project code 59. Special thanks go to the Technion Institute of Israel for partial scholarship supporting of Ms. Hiba Abu Tayeh for accomplishing her MSc studies. References Ahmed, I.N., Nguyen, P.L.T., Huynh, L.H., Suryadi, I., Ju, Y.H., 2013. Bioethanol production from pretreated Melaleuca leucadendrion shedding bark – simultaneous saccharification and fermentation at high solid loading. Bioresour. Technol. 136, 213–221. Amartey, S., Jeffries, T., 1996. An improvement in Pichia stipitis fermentation of acidhydrolysed hemicellulose achieved by overliming (calcium hydroxide treatment) and strain adaptation. World J. Microbiol. Biotechnol. 12, 281–283. Ballesteros, I., Oliva, J.M., Saez, F., Ballesteros, M., 2001. Ethanol production from lignocellulose byproducts of olive oil extraction. Appl. Biochem. Biotechnol. 91– 93, 237–252. Banat, F., Pal, P., Jwaied, N., Al-Rabadi, A., 2013. Extraction of olive oil from olive cake using soxhlet apparatus. Am. J. Oil Chem. Technol. 1, 2326–6570. Bellido, C., Bolado, S., Coca, M., Lucas, S., González-Benito, G., García-Cubero, M.T., 2011. Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis. Bioresour. Technol. 102, 10868–10874. Bellido, C., González-Benito, G., Coca, M., Lucas, S., García-Cubero, M.T., 2013. Influence of aeration on bioethanol production from ozonized wheat straw hydrolysates using Pichia stipitis. Bioresour. Technol. 133, 51–58. Cara, C., Ruiz, E., Oliva, J.M., Sáez, F., Castro, E., 2008. Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzyme saccharification. Bioresour. Technol. 99, 1869–1876. Causey, T.B., Zhou, S., Shanmugam, K.T., Ingram, L.O., 2003. Engineering the metabolism of Escherichia Coli W3110 for the conversion of sugar to redox-
neutral and oxidized products: homoacetate production. Appl. Biol. Sci. 100 (3), 825–832. Choi, G.-W., Kang, H.-W., Moon, S.-K., 2010. Continuous ethanol production from cassava through simultaneous saccharification and fermentation by selfflocculating yeast Saccharomyces Cervisiae CHFY0321. Appl. Biochem. Biotechnol. 160 (5), 1517–1527. Cuevas, M., Sánchez, S., Bravo, V., García, J.F., Baeza, J., Parra, C., Freer, J., 2010. Determination of optimal pre-treatment conditions for ethanol production from olive-pruning debris by simultaneous saccharification and fermentation. Fuel 89 (10), 2891–2896. Díaz, M.J., Ruiz, E., Romero, I., Cara, C., Moya, M., Castro, E., 2009. Inhibition of Pichia stipitis fermentation of hydrolysates from olive tree cuttings. World J. Microbiol. Biotechnol. 25, 891–899. El Asli, A., Qatibi, A., 2009. Ethanol production from olive cake biomass substrate. Biotechnol. Bioprocess Eng. 14, 118–122. Ferreira, A.D., Mussatto, S.I., Cadete, R.M., Rosa, C.A., Silvio, S.S., 2011. Ethanol production by a new pentose-fermenting yeast strain, scheffersomyces stipitis UFMG-IMH 43.2, isolated from the Brazilian forest. Yeast 28, 547–554. Haagensen, F., Skiadas, I.V., Gavala, H.N., Ahring, B.K., 2009. Pre-treatment and ethanol fermentation potential of olive pulp at different dry matter concentrations. Biomass Bioeng. 33, 1643–1651. Hendriks, A.T.W.M., Zeeman, G., 2009. Review-pretreatment to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10–18. Huang, C.F., Lin, T.H., Guo, G.L., Huang, W.S., 2009. Enhanced ethanol production by fermentation of rice straw hydrolysate without detoxification using a newly adapted strain of Pichia stipitis. Bioresour. Technol. 100 (17), 3914–3920. Karhumaa, K., Fromanger, R., Hahn-Hägerdal, B., Gorwa-Grauslund, M.F., 2007. High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 73, 1039–1046. Kötter, P., Ciriacy, M., 1993. Xylose fermentation by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 38, 776–783. Kumar, S., Singh, S.P., Mishra, I.M., Adikari, D.K., 2009. Review – recent advances in production of bioethanol from lignocellulosic biomass. Chem. Eng. Technol. 32 (4), 517–526. Limtong, S., Sringiew, C., Yongmanitchai, W., 2007. Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus. Bioresour. Technol. 98, 3367–3374. Lin, T.H., Huang, C.F., Guo, G.L., Hwang, W.S., Huang, S.L., 2012. Pilot-scale ethanol production from rice straw hydrolysates using xylose-fermenting Pichia stipitis. Bioresour. Technol. 116, 314–319. Matsushika, A., Inoue, H., Kodaki, T., Sawayama, S., 2009. Mini review – ethanol production from saccharomyces cerevisiae strains: current state and perspectives. Appl. Microbiol. Biotechnol. 84, 37–53. Moniruzzaman, M., Dien, B.S., Skory, C.D., Chen, Z.D., Hespell, R.B., Ho, N.W.Y., Dale, B.E., Bothast, R.J., 1997. Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain. World J. Microbiol. Biotechnol. 13, 341–346. Oberoi, H.S., Babbar, N., Sandhu, S.K., Dhaliwal, S.S., Kaur, U., Chadha, B.S., Bhargav, V.K., 2012. Ethanol production from alkali-treated rice straw via simultaneous saccharification and fermentation using newly isolated thermotolerant Pichia kudriavzevii HOP-1. J. Ind. Microbiol. Biotechnol. 39, 557–566. Olsson, L., Hahn-Hägerdal, B., 1996. Fermentation of lignocellulosic hydrolysates for ethanol production. Enzym. Microb. Technol. 18, 312–331. Parajó, J.C., Domínguez, H., Domínguez, J.M., 1998. Biotechnological production of xylitol. Part 3: operation in culture media made from lignocellulose hydrolysates. Bioresour. Technol. 66, 25–40. Patni, N., Pillai, S.G., Dwivedi, A.H., 2013. Wheat as a promising substitute of corn for bioethanol production. Proc. Eng. 51, 355–362. Ruiz, E., Cara, C., Ballesteros, M., Manzanares, P., Ballesteros, I., Castro, E., 2006. Ethanol production from pretreatment olive tree wood and sunflower stalks by an SSF process. Appl. Microbiol. Biotechnol. 129–132, 631–643. Slininger, P.J., Bolen, P.L., Kurtzman, C.P., 1987. Pachysolen tannophilus: properties and process considerations for ethanol production from D-xylose. Enzym. Microb. Technol. 9, 5–15. Wang, K., Yang, H., Wang, W., Sun, R.C., 2013. Structural evaluation and bioethanol production by simultaneous saccharification and fermentation with biodegraded triploid poplar. Biotechnol. Biof. 6, 42. Zhao, L., Zhang, X., Tan, T., 2008. Influence of various glucose/xylose mixtures on ethanol productionby pachysolen tannophilus. Biomass Bioeng. 32, 1156–1161.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Potential of bioethanol production from olive mill solid wastes Hiba Abu Tayeh a, Naim Najami a,b, Carlos Dosoretz c, Ahmed Tafesh a, Hassan Azaizeh a,d,⇑ a
Institute of Applied Research, Affiliated with University of Haifa, The Galilee Society, P.O. Box 437, Shefa-Amr 20200, Israel The Academic Arab College of Education, Haifa, Israel c Department of Environments, Water & Agriculture Engineering, Technion Institute, Haifa 32000, Israel d Tel Hai College, Upper Galilee 12208, Israel b
h i g h l i g h t s Two yeasts were isolated from OMSW, I. orientalis, and P. galeiformis/manshurica. I. orientalis showed better kinetics to xylose compared to the other strains. I. orientalis on hydrolysate supplemented with glucose showed best ethanol production. Using SSF process following pretreatment, the ethanol yield was 3 g/100 g dry OMSW.
a r t i c l e
i n f o
Article history: Received 15 September 2013 Received in revised form 24 October 2013 Accepted 28 October 2013 Available online 6 November 2013 Keywords: Olive mill solid wastes Lignocellulose degradation Fermentation process Bioethanol production
a b s t r a c t The main objective of this study was to screen endogenous microorganisms grown on olive mill solid wastes (OMSW) with the potential to ferment pentoses and produce ethanol. Two yeasts were isolated and identified as Issatchenkia orientalis, and Pichia galeiformis/manshurica. The adaptation of the strains displayed a positive impact on the fermentation process. In terms of xylose utilization and ethanol production, all strains were able to utilize xylose and produce xylitol but no ethanol was detected. Separate hydrolysis and fermentation process on hydrolysate undergo detoxification, strain I. orientalis showed the best efficiency in producing of ethanol when supplemented with glucose. Using simultaneous saccharification and fermentation process following pretreatment of OMSW, the average ethanol yield was 3 g/100 g dry OMSW. Bioethanol production from OMSW is not economic despite the raw material is cheap. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In recent decades renewable energy sources have considerable interest worldwide. Biomass energy is one of the oldest and most promising sources and includes organic waste, sewage, and energy crops, agricultural and industrial residues that can be utilized to produce bio-fuel. Biomass can be converted biologically to liquid or gaseous fuels, such as ethanol, methanol, methane and hydrogen by fermentation processes (Haagensen et al., 2009). Ethanol has attracted interest as alternative liquid fuel, especially for transportation. It has immense importance for countries which depends heavily on import of crude oil, spending a huge amount of its annual budget. Currently, bioethanol is mainly produced from biomass containing sugar or starch as sugar cane and ⇑ Corresponding author at: Institute of Applied Research, Affiliated with University of Haifa, The Galilee Society, P.O. Box 437, Shefa-Amr 20200, Israel. Tel.: +972 4 9504523/4; fax: +972 4 95044535. E-mail addresses: [email protected] (H. Abu Tayeh), najamina@gmail. com (N. Najami), [email protected] (C. Dosoretz), [email protected] (A. Tafesh), [email protected], [email protected] (H. Azaizeh). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.102
corn as raw material (first generation) (Patni et al., 2013). Because these products are used as food and feed for humans and animals, there is competition of using biomass as a food or fuel. Due to this competition, bioethanol production from lignicellulose (second generation) enthusiastically have been investigated worldwide (Lin et al., 2012). Lignocellulosic biomass, such as trees and agricultural residues is an attractive raw material for bioethanol production because of the large amount of potential sugar for fermentation and bioenergy production (Bellido et al., 2013; Choi et al., 2010; Cuevas et al., 2010; Kumar et al., 2009; Olsson and Hahn-Hägerdal, 1996). Production of olive oil symbolizes one of the most important economic agro-food sectors in the Mediterranean basin. The traditional oil industry generates two byproducts at the end of the process, olive cake (residue) and olive mill wastewater, which can cause serious environmental pollution problems (Ballesteros et al., 2001). Crude olive cake, the leftover solids following the pressing of olives, is a mixture of skin, pulp, and seeds. It comprises approximately 35% of the beginning olive weight, is rich in carbohydrates, and is available in appreciable quantities in the
H. Abu Tayeh et al. / Bioresource Technology 152 (2014) 24–30
Mediterranean area (Banat et al., 2013; El Asli and Qatibi, 2009). Global annual production of olive cake has been estimated to approach 4 108 kg of dry matter (El Asli and Qatibi, 2009). The main structural components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. Of these, only cellulose and hemicellulose can be used as raw materials to produce ethanol by fermentation of carbohydrates obtained through chemical or enzymatic hydrolysis (saccharification). The biological process for converting lignocellulose to fuel ethanol requires: (1) delignification to liberate cellulose and hemicellulose; (2) depolymerization of carbohydrate polymers to produce free sugars; and (3) fermentation of mixed hexose and pentose sugars to produce ethanol (Cuevas et al., 2010; Kumar et al., 2009; Lin et al., 2012; Matsushika et al., 2009). Hydrolysis of these polysaccharides (cellulose and hemicellulose) is usually accomplished by acid and/or enzymatic treatment. The utilization of both cellulose and hemicellulosic sugars like hexose, pentose and others present in a typical biomass hydrolysate is essential for the economical production of ethanol (Kumar et al., 2009; Olsson and Hahn-Hägerdal, 1996). The production of ethanol from pretreated material may be accomplished either by sequential hydrolysis and fermentation or by a simultaneous saccharification and fermentation (SSF) process (Ahmed et al., 2013; Cuevas et al., 2010; Oberoi et al., 2012). During the SSF process, end product inhibition can be overcome as glucose is simultaneously fermented as it is formed; another advantage is that a single reactor is used for both saccharification and fermentation processes. However, the enzymatic reaction in SSF process is operated at a temperature lower than its optimum level owing to the mismatch in optimum temperatures for hydrolysis (approx. 50 °C) and fermentation (approx. 30 °C) (Cuevas et al., 2010; Ruiz et al., 2006). The main component of lignocellulosic hydrolysates is glucose, a hexose sugar derived from cellulose and hemicellulose. Although the proportion of mono-saccharides in hemicellulose hydrolysates varies depending on the raw material and the hydrolysis procedure, they all contain both pentose sugars, such as D-xylose and L-arabinose and hexose sugars as well. D-Xylose is the second most abundant carbohydrate and its content is particularly high in grass and hardwood (Hendriks and Zeeman, 2009; Matsushika et al., 2009). One of the most effective ethanol-producing microorganisms for hexose sugars including glucose, mannose and galactose is Saccharomyces cerevisiae yeast with high ethanol productivity, high tolerance to ethanol, and tolerance to inhibitory compounds present in the hydrolysate of lignocellulosic biomass (Matsushika et al., 2009). However, this strain is unable to utilize xylose for growth or fermentation in order to produce ethanol. Instead, this strain metabolizes D-xylulose, an isomerization product of D-xylose. Some yeast strains have been reported to ferment xylose into ethanol, but the rate and yield of ethanol production from xylose in these xylose-utilizing yeast strains are considerably low compared to their glucose fermentation process (Ferreira et al., 2011; Lin et al., 2012). Therefore, genetic engineering and/or adaptation may be promising methods to develop sufficient xylose fermentation in S. cerevisiae. To date, numerous studies regarding the metabolic engineering of S. cerevisiae for xylose utilization have been reported, and many reviews have already addressed the current advancement in metabolic engineering of xylose-fermenting strains and factors which affect xylose metabolism in yeasts (Matsushika et al., 2009). The objectives of the current study were to isolate yeast strains from olive mill solid wastes (OMSW) with the potential to utilize xylose and produce ethanol to be compared with other commercial known yeast strains. In addition to adapt the different yeast isolates to produce bioethanol from the OMSW hydrolysate.
25
2. Methods 2.1. Raw material Olive mill solid waste (OMSW), the solid residue from the traditional olive oil production process, was obtained from Dabburia, Galilee region of Israel after the harvest months of the year 2009/2010. OMSW was dried at 45 °C, milled to particle size between 0.25–1 mm, and stored at room temperature until being used. 2.2. Content test Content test was performed to quantify the potential of a feedstock (raw material) and thus test the effectiveness of pretreatment to the total content of cellulose and hemicellulose. Wall sugar content was tested after water extraction, testing the solid percentage and the ash content was performed according to standard analytical procedures as described in protocols of the NREL. The composition of the OMSW used throughout the experiments included: extractive, lignin, acid insoluble, cellulose, hemicellulose, xylose and galactose, arabinose, acetyl, ash, with dry matter percentage of 18.3 ± 1.7, 40.2 ± 5.1, 39.5 ± 5.1, 18.4 ± 0.2, 15.9 ± 0.1, 14.1 ± 0.1, 1.8 ± 0.0, 2.7 ± 0.1, 4.5 ± 1.2, respectively. 2.3. Pretreatment of OMSW The biomass at a solid loading of 7.5% (w/v) was mixed with diluted sulfuric acid (final concentration of 2% (v/v)) at 100 °C, with the residence time of 2 h. After treatment, the reactor was removed from heating jacket and cooling to room temperature. The pretreated material was separated by filtration into two fractions, i.e., the solid insoluble residue and the liquid fraction (hydrolysate). The composition of the hydrolysate regarding main sugars (glucose and xylose) and inhibitors compounds (furfural, acetic acid, and formatic acid), is shown in Table 1. Other sugars, like galactose, arabinose and mannose, as well as hydroxymethylfurfural (HMF), were also present in hydrolysates at lower concentration (Cara et al., 2008). 2.4. Enzymatic hydrolysis After dilute acid pretreatment, enzymatic hydrolysis was performed using cellulases (Cellulase Complex-Ecostone L900 and b-glucosidase (Novozyme 188S from Trichoderma reesei were purchased from Sigma) at 50 °C and 100 rpm for 24 h in a water bath shaker. In order to maintain the pH at 4.8 in the mixture, 0.05 M sodium citrate buffer (pH 4.5) was added. 2.5. Detoxification of diluted sulfuric acid hydrolysate with activated carbon Detoxification of diluted sulfuric hydrolysate was carried out using 5% (w/v) activated carbon in an incubated water bath shaker at 50 °C for 30 min, according to the methodology described by Díaz et al. (2009). Then the mixture was cooled to room temperature, sodium hydroxide was added until the desired pH (4.5) was achieved. Afterward was centrifuged to remove the solid material and kept below 4 °C for further analysis. 2.6. Yeast strains Three strains of yeast were used in this study; one was purchased from ATCC, S. cervisiae (24860 ATCC, designated as S.C.),
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Table 1 Composition of the initial hydrolysate (mg/l) obtained after sulfuric acid (2%) pretreatment of OMSW followed by activated charcoal detoxification. Hydrolysate composition (mg/l) Without detoxification Activated charcoal detoxification a
Glucose
Xylose
Acetic acid
Formic acid
Furfural
a
8809.7 9687.0
2131.2 1886.0
530.5 298.1
56.0 1.4
1670.5 1907.0
Average of duplicated samples.
two strains were isolated from OMSW and were identification at Hy labs (Israel) and CBS (Holland): – Pichia galeiformis A. Endo & Goto – (designated as G). – P. Kudriavzevii Boidin, Pignal & Besson – (designated X). 2.7. Inoculums’ preparation The yeasts were grown at 35 °C on YPG and YPX agar (Huang et al., 2009) and supplemented with 20 g/l xylose, 10 g/l yeast extract, 20 g/l peptone, and 20 g/l agar. A single colony was chosen after 2 days and cultured in YPX medium overnight to prepare a culture with the desired cell concentration. 2.8. Adaptation process Adaptation was performed by sequential transfer of cultures to diluted hydrolysate medium with increasing concentration of hydrolysate, which means increasing concentration of inhibitors. Hydrolysate is the liquid which was obtained after filtration of the 2% sulfuric acid pretreatment OMSW. The initial hydrolysate concentration was 3.5% (w/v) and reached to 6.5% (w/v), with inoculums’ concentration of 1 g/l, and supplemented with 3.5 g/l KH2PO4, 5 g/l K2HPO4, 3.5 g/l (NH4)2HPO4, 0.25 g/l MgSO4:7H2O, 0.015 g/l CaCl2:2H2O, 0.0005 g/l Thiamine, 1 ml trace metals (Causey et al., 2003). Xylose concentration was fixed at 1 g/l. The adaptation process was performed in a 50 ml flask with 25 ml of medium. Each culture was grown at pH 5.0 for 48–72 h at 35 °C in a rotary shaker. Culture aliquot of 0.25 ml from one shake flask was transferred to the following shake flask. Cells were grown twice at the same hydrolysate dilution before transferring the cells to more concentrated hydrolysate. 2.9. Analytical methods During the experiments, samples were periodically taken for sugars, ethanol and cell growth determinations. The cellular growth was determined by measuring the fermentation broth using UV Spectrophotometer at 600 nm (Bellido et al., 2011), and was correlated to a calibration curve. Each fermentation sample was filtered through a 0.45 lm filter. Glucose, xylose, xylitol, and ethanol concentrations were determined by high-performance liquid chromatography (HPLC) (Thermo Scientific finnigan surveyor, equipped with a Shimadzu refractive index detector and Rezex-ROA column (150 7.8 mm) at 65 °C. Sulphuric acid (0.005 M) was used for elusion at a flow rate of 0.6 ml/min (Bellido et al., 2011; Cara et al., 2008; Wang et al., 2013). For furfural and HMF determinations, the HPLC system was equipped with a UV detector (Diode array) at 280 nm, at the same operation conditions which was previously described. 3. Results and discussion Yeast strains isolation was conducted by adding pure glucose and xylose as a carbon source to the OMSW by enrichment technique according to Limtong et al. (2007). Thus we were able to
isolate two strains of yeast, Issatchenkia orientalis – which was isolated on the carbon source xylose, so it was marked X and Pichia galeiformis/manshurica – which was isolated on glucose, so it was, marked G. The two strains were identified at two different places the first in Hy labs (Israel) and the other was at Centraalbureau voor Schimme lcultures (CBS), (The Netherlands). The identification processes shows that they belong to the Pichia family, that able to utilize xylose. To characterize the ability of X and G strains to produce ethanol from defined sources (glucose and xylose) and treated OMSW, they were compared to the well known isolate S. cervisiae (ATCC 24860), which efficiently known to produce ethanol from hexoses. During the first 24 h, the yeast X seems to utilized the glucose and produced in average 6 g/l of ethanol which is equivalent to 78% of the theoretical yield, however the glucose utilizing for the others yeast strains was obtained after 120 h and with low ethanol production compared to the yeast X (Fig. 1). Glycerol was produced as byproduct as well. Ethanol and glycerol concentration began to decrease after the fermentation process was over, and the OD, which marks the cell density of the yeast, continues to rise which indicated that the glycerol and ethanol are used as a carbon source for cell growth, where this is compatible with the results shown by Kötter and Ciriacy (1993), Slininger et al. (1987) and Zhao et al. (2008), but in their studies they used the yeast Pachysolen tannophilus 1771. It might be that during taking the samples the oxygen entered into the flasks when the glucose was consumed, which facilitates that the yeast utilize the ethanol as a carbon source. When using xylose as a sole carbon source, the yeast X showed a better ability compared to the other yeasts although utilization was very slow during the first 48 h in the formation of xylitol which is intermediate material of xylose pathway (Fig. 1) and no ethanol production was measured. The lag phase of the yeast strains G and S.C. is longer than 48 h, and only after that the log phase began as indicated by the sugar consumption and increase cell biomass. According to Moniruzzaman et al. (1997) and Karhumaa et al. (2007) there is an accumulation of xylitol and this is due to an imbalance of cofactor (NAD+ and NADPH) which is required for the enzyme (XR) compared to enzyme Xylitol Dehedrogenase (XDH) (Figs. 1 and 2). They also assessed that the insertion of oxygen shocks (pulses) which inhibits the enzyme XR and thus prevents the accumulation of xylitol. 3.1. Fermentation of mixed sugars, glucose and xylose The results show that glucose utilization rate by yeast X is faster as compared with yeast G and S.C. (Figs. 1 and 2). When the carbon source was glucose and xylose, ethanol was produced only by the yeast X, but the byproduct glycerol produced by the three yeast strains despite the differences of kinetics of each strain (Fig. 2). It is obvious that the xylose was consumed significantly only after ethanol and glycerol were previously consumed. Xylose utilization by the yeast X was at the rate of 82% after 240 h, compared to 21% by the yeast G. The kinetics of the yeast X in this case could be compared with the case of which the xylose is a sole carbon, so expect slower kinetics in the case of mixed sugars and this similar to
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0
1 0
0 0
24
Glucose
3 2 1 0
10 5 0
Xylose
120
time(h) Xylitol
xylose, xylitol (g/l)
15
OD
xylose, xylitol(g/l)
6 5 4
20
48
120
4 3
10
2
5
1
240
0 0
OD
Ethanol
6
20
5 4
15
3
10
2
5
1
0
0 0
24
Xylose
OD
48
48
OD
120
240
time(h) Ethanol
Glycerol
Yeast G(Xylose)
25
240
24
Glucose
Glycerol
Yeast X(Xylose)
25
24
48
5
15
time(h)
Glycerol
Yeast S.C.(Xylose)
0
24
6
0
120
25
6
20
5 4
15
3
10
2
5
1
0
0 0
240
time (h) Xylitol
xylose, xylitol (g/l)
Glucose
48 120 240 time(h) OD Ethanol
5
OD
0
3 2
10
7
20
ethanol, glycerol (g/l)
5
15
25 glucose(g/l), OD
10
7 6 5 4
20
ethanol, glycerol(g/l)
15
Yeast G(Glucose)
25
glucose(g/l), OD
20
ethanol, glycerol(g/l)
7 6 5 4 3 2 1 0
25 glucose(g/l), OD
Yeast X(Glucose)
OD
Yeast S.C.(Glucose)
OD
24
48 144 time(h) Xylose Xylitol
240 OD
Fig. 1. Fermentation results using the yeast strains X, G, or S.C. in defined medium with a sole carbon source, glucose or xylose at 35 °C. Results are mean values of two replicates where the deviations from the average values were less than 10%.
Yeast G(GLU+XYLOSE)
glucose, xylose (g/l)
2 1.5 1 0.5 0 24
48
120
240
Time(h) Xylose Glycerol
Glucose Xylitol
Ethanol OD
Yeast X(GLU+XYLOSE)
2 1.5 1 0.5 0
Glucose Xylitol
24
48
120
Time(h) Xylose Glycerol
240
ethanol, glucose, xylose (g/l)
2.5
16 14 12 10 8 6 4 2 0
xylitol, glycerol (g/l), OD
ethanol, glucose, xylose (g/l)
Yeast S.C. (GLU+XYLOSE) 16 14 12 10 8 6 4 2 0
2.5 2 1.5 1 0.5 0 0
Ethanol OD
xylitol, glycerol (g/l), OD
0
0
xylitol, glycerol (g/l), OD
2.5
16 14 12 10 8 6 4 2 0
Glucose Xylitol
24
48 120 Time(h) Xylose Glycerol
240 Ethanol OD
Fig. 2. Fermentation results using the yeast strains X, G, or S.C. in defined medium with a mixture of glucose and xylose as carbon source at 35 °C. Results are mean values of two replicates where the deviations from the average values were less than 10%.
what was shown by Zhao et al. (2008). In the case of the strains G and S.C., it seems that xylose is inhibiting the ethanol production process. 3.2. Fermentation of chemically hydrolysed OMSW The hydrolysate was treated with activated carbon and subsequently was neutralized by sodium hydroxide. After neutralization and before the fermentation process, 1.2% glucose was added into
the hydrolysate medium. The results shows different behaviors of the strain X as compared with strains G and S.C. (Fig. 3). The yeast X utilized almost all the glucose within the first 24 h and produced glycerol, xylitol and ethanol. Glucose utilization in yeast G and S.C. was very slowly and ethanol production as well and therefore was no accumulation of xylitol or glycerol as byproducts (Fig. 3). The maximum ethanol production was obtained during the hydrolysate fermentation was calculated as 0.49, 0.44, 0.43 g ethanol/g sugar utilized using the different yeast strains X, G, S.C.,
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16 14 12 10 8 6 4 2 0
1.2 1 0.8 0.6 0.4 0.2
xylitol, glycerol (g/l)
glucose, xylose, ethanol (g/l)
Yeast X(HYDRO)
0 0
24
48
120
240
Time(h) Glucose
Xylose
Ethanol
Xylitol
Glycerol
Yeast S.C.(HYDRO)
0.8 0.6 0.4 0.2 0 0
24
48
120
240
1.2
16 14 12 10 8 6 4 2 0
1 0.8 0.6 0.4 0.2 0 0
24
Time(h) Glucose
Xylose
Ethanol
xylitol, glycerol (g/l)
1
glucose, xylose, ethanol (g/l)
1.2
16 14 12 10 8 6 4 2 0
xylitol, glycerol (g/l)
glucose, xylose, ethanol (g/l)
Yeast G(HYDRO)
48
120
240
Time(h)
Xylitol
Glycerol
Glucose
Xylose
Ethanol
Xylitol
Glycerol
Fig. 3. Fermentation of the OMSW hydrolysate (liquid phase following pretreatment with sulfuric acid) by the yeasts X, G, S.C. Two replicates were used and the measurements were done in duplicates and the deviation from the average values were less than 10%.
respectively. Other researchers have shown that the recombinant Escherichia coli FBR15 utilized xylose and produced 0.47 g ethanol/g sugar (El Asli and Qatibi, 2009).
3.3. Impact of detoxified hydrolysate on the fermentation process Following OMSW hydrolysate detoxification process (Table 1), a slight decrease in acetic acid and formic acid concentrations has been obtained (about 11% and 44%, respectively) which is in agreement with other reports showing fermentative performance improvements in detoxified hydrolysates (over-limed) (Amartey and Jeffries, 1996; Díaz et al., 2009). In contrast, furfural was reduced by more than 97% as a consequence of the detoxification treatment by activated charcoal. Other inhibitory compounds may be present in the hydrolysates, like phenolic compounds derived from lignin or other extractives, and may also be removed by detoxification methods (Parajó et al., 1998). In addition, the HMF that was present in lower concentrations, its level was reduced after activated charcoal (data not shown). No sugar losses were detected after detoxification processes, even a slight increase in sugar concentration was observed during these experiments. The results showed positive effects on the fermentation process and ethanol yield produced by X and G strains following OMSW hydrolysate detoxification process (Fig. 4). Without detoxification, only the X yeast compared with G yeast utilized small amount of glucose and xylose within the first 24 h and produced ethanol (Fig. 4, A and B). Following detoxification it has been observed that glucose and xylose were utilized accompanied with ethanol production by both strains X and G within the first 24 h (Fig. 4, C and D). These results indicated that OMSW hydrolysate detoxification before fermentation is necessary in order to produce ethanol.
3.4. Fermentation of enzymatic hydrolysed OMSW During the fermentation process 1.5% glucose and 0.45% xylose were supplemented into the enzymatic OMSW hydrolysate medium. The results showed that ethanol production using the X, G, S.C. yeast strains was of 3.08, 0.54, 0.58 mg ethanol/g utilized glucose/h), respectively. This indicated that the isolated strain X was superior compared with the other two strains. 3.5. Xylose utilization As have been shown, the isolated yeasts utilize xylose but they do not produce ethanol. Therefore, the xylose utilization of the different strains during the fermentation process was compared using different media. The highest xylose utilization was observed by using the yeast X grown in defined medium with xylose as a sole carbon source where the utilization was 93% after 240 h, and in the medium supplemented with glucose and xylose the utilization decreased to 81%, and in the medium of the enzymatic hydrolysis and hydrolysate the utilization was 25% and 2% respectively. This indicates that there are some inhibitors in these growth media. 3.6. Ethanol production from OMSW using SSF process The X, G and S.C. strains were grown using the SSF process in reactors to which the enzymes that consisted of the cellulose complex were added as well. The SSF process was conducted at anaerobic conditions with 6.7% solid concentration and incubated at 35 °C for 240 h. The released glucose by the SSF process was utilized simultaneously by the yeasts which produce ethanol during the process (Fig. 5). The final ethanol concentration produced by the three yeasts were similar and it was about 30 mg/g dried OMSW. However, the yeast X behaves differently than the strains
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H. Abu Tayeh et al. / Bioresource Technology 152 (2014) 24–30
B
yeast X
16
glucose, xylose, ethanol (g/l)
glucose, xylose, ethanol (g/l)
A 14 12 10 8 6 4 2 0 0
23
94
yeast G
16 14 12 10 8 6 4 2 0
165
0
Time (h) Glucose
D
yeast X 16 14 12 10 8 6 4 2 0 0
24
48 Time (h)
Glucose
Xylose
120
94
Time (h)
Glucose
Ethanol
glucose, xylose, ethanol(g/l)
glucose, xylose, ethanol(g/l)
C
Xylose
23
Xylose
165
Ethanol
yeast G 16 14 12 10 8 6 4 2 0 0
240
24
48
120
240
Time (h) Glucose
Ethanol
Xylose
Ethanol
Fig. 4. Fermentation of OMSW hydrolysate using X and G strains. Upper panel (A, B), OMSW hydrolysate without detoxification before the fermentation process. Lower panel (C and D), OMSW hydrolysate with detoxification before fermentation process. Bars represent average ± standard deviation of 3 replicates.
Glucose
glucose, xylose, ethanol(g/l)
X(SSF)
Xylose Ethanol
2.5 2 1.5 1 0.5 0 0
24
48
120
240
TIME (h) Glucose Ethanol
2.5 2 1.5 1 0.5 0 0
24
48
120
240
TIME (h)
Glucose
G(SSF)
Xylose
glucose, xylose, ethanol(g/l)
glucose, xylose, ethanol(g/l)
S.C.(SSF)
Xylose Ethanol
2.5 2 1.5 1 0.5 0 0
24
48
120
240
TIME (h)
Fig. 5. SSF process using the three yeast strains and b-glucosidase and Cellulase Complex (Ecostone L900). Two replicates were used and the measurements were done in duplicates and the deviation from the average values were less than 10%.
G and S.C., and utilized most of the glucose after 24 h and the maximum glucose utilization was obtained after 120 h. However, the strains G and S.C. utilized most of the glucose after 120 h and the maximum glucose utilization was obtained after 240 h. Using SSF process, it is possible to produce in average 3 g/l ethanol from 100 g of OMSW (calculated data). Ruiz et al. (2006) showed that they achieved 7.5 g ethanol per 100 g olive tree wood,
and sunflowers resulted in 8 g per 100 g. In addition to the fact that raw material is different (different cellulose and hemicellulose percent) also pretreatment conditions were different in both cases, they used the steam explosion method at 230 °C, also the yeast strain was different as well. Therefore the comparison of the results with others from the literature is not always possible. Even the OMSW is different and depends on the source of olives grown
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in each country. Ruiz et al. (2006) showed that they can get 95.1 l ethanol/ton olive tree (pruning), and in the current study 38 l ethanol/ton OMSW was obtained which still very low and not economic for using OMSW for bioethanol production. The yield could be improved by utilizing the pentoses (mainly xylose) for ethanol production which still needs more investigation. Therefore, the obtained results so far suggest that OMSW is not a suitable feedstock for ethanol production as demonstrated by our data and by others (Ruiz et al., 2006). It might be a better idea to evaluate the use of OMSW in an anaerobic digestion to produce biogas followed by a composting process to produce fertilizer to enable the sustainable production of future olive crops. 4. Conclusions Two yeast strains were isolated from OMSW, I. orientalis, and P. galeiformis/manshurica. All strains were able to utilize xylose and produce xylitol but not ethanol. I. orientalis showed better kinetics and less sensitivity to xylose repression for ethanol production from glucose compared to P. galeiformis/manshurica and S. cervisiae. Separate hydrolysis and fermentation process on hydrolysate undergo detoxification, strain I. orientalis showed best efficiency in producing of ethanol when supplemented with glucose. Using SSF process following pretreatment of OMSW, the average ethanol yield was low as 3 g/100 g dry OMSW. Acknowledgements We thank the Ministry of Science of Israel for partial funding of this study in the framework of Regional Research and Development programs, project code 59. Special thanks go to the Technion Institute of Israel for partial scholarship supporting of Ms. Hiba Abu Tayeh for accomplishing her MSc studies. References Ahmed, I.N., Nguyen, P.L.T., Huynh, L.H., Suryadi, I., Ju, Y.H., 2013. Bioethanol production from pretreated Melaleuca leucadendrion shedding bark – simultaneous saccharification and fermentation at high solid loading. Bioresour. Technol. 136, 213–221. Amartey, S., Jeffries, T., 1996. An improvement in Pichia stipitis fermentation of acidhydrolysed hemicellulose achieved by overliming (calcium hydroxide treatment) and strain adaptation. World J. Microbiol. Biotechnol. 12, 281–283. Ballesteros, I., Oliva, J.M., Saez, F., Ballesteros, M., 2001. Ethanol production from lignocellulose byproducts of olive oil extraction. Appl. Biochem. Biotechnol. 91– 93, 237–252. Banat, F., Pal, P., Jwaied, N., Al-Rabadi, A., 2013. Extraction of olive oil from olive cake using soxhlet apparatus. Am. J. Oil Chem. Technol. 1, 2326–6570. Bellido, C., Bolado, S., Coca, M., Lucas, S., González-Benito, G., García-Cubero, M.T., 2011. Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis. Bioresour. Technol. 102, 10868–10874. Bellido, C., González-Benito, G., Coca, M., Lucas, S., García-Cubero, M.T., 2013. Influence of aeration on bioethanol production from ozonized wheat straw hydrolysates using Pichia stipitis. Bioresour. Technol. 133, 51–58. Cara, C., Ruiz, E., Oliva, J.M., Sáez, F., Castro, E., 2008. Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzyme saccharification. Bioresour. Technol. 99, 1869–1876. Causey, T.B., Zhou, S., Shanmugam, K.T., Ingram, L.O., 2003. Engineering the metabolism of Escherichia Coli W3110 for the conversion of sugar to redox-
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