Biotechnology and Bioprocess Engineering 2009, 14: 118-122 DOI/10.1007/s12257-008-0071-y
Ethanol Production from Olive Cake Biomass Substrate Abdelghani El Asli1* and Abdel-Illah Qatibi2 2
1 School of Science and Engineering, Al Akhawayn University, PO Box 1846, 53000 Ifrane, Morocco Anaerobic Microbiology Team (E02B26), Sciences and Techniques Faculty, Cadi Ayyad University, PO Box 549, 40000 Marrakech, Morocco
Abstract The inexpensive production of sugars from lignocellulose is an essential step for the use of biomass to produce fuel ethanol. Olive cake is an abundant by-product of the olive oil industry and represents a potentially significant lignocellulosic source for bioethanol production in the Mediterranean basin. Furthermore, converting olive cake to ethanol could add further value to olive production. In the present study, olive cake was evaluated as a feedstock for ethanol production. To this end, the lignocellulosic component of the olive cake was dilute-acid pretreated at a 13.5% olive-cake loading with 1.75% (w/v) sulfuric acid and heating at 160°C for 10 min. This was followed by chemical elimination of fermentation inhibitors. Soluble sugars resulting from the pretreatment process were fermented using E. coli FBR5, a strain engineered to selectively produce ethanol. 8.1 g of ethanol/L was obtained from hydrolysates containing 18.1 g of soluble sugars. Increasing the pretreatment temperature to 180°C resulted in failed fermentations, presumably due to inhibitory by-products released during pretreatment. © KSBB Keywords: ethanol, lignocellulose, fermentation, olive cake
INTRODUCTION According to the Kyoto agreements, total CO2 emissions from industrialized nations are to be reduced by 5% by 2010, relative to the 1990 level. In the longer term, a reduction of more than 50% will be required to stabilize the CO2 level in the atmosphere. One of the major strategies to achieve these objectives is the large-scale substitution of petrochemical fuels and products with CO2-neutral alternatives derived from biomass. Fuel ethanol contributes little net carbon dioxide to the atmosphere, and therefore its use helps to fulfill the commitments of the Kyoto protocol. Efficient ethanol production processes and inexpensive substrates are the keys for successful production of bioethanol. The common ethanol production process that uses crops such as sugar cane and corn are well established; however, utilization of alternate lignocellulosic substrates such as wood, agricultural residues (e.g., straw, corn stover, cane bagasse) or forestry wastes (e.g., sawdust, pulp mill residues) as well as some solid industrial and municipal wastes (e.g., newsprint, paper, cardboard packing) [1,2] could make bioethanol more competi tive with fossil fuels. Use of alternate lignocellulosic substrates *Corresponding author Tel: +212-61-069160 Fax: +212-35-862030 e-mail:
[email protected]
would also avoid the use of food crops for fuel production. However, the pretreatment of this lignocellulosic biomass releases fermentation inhibitors that must be eliminated, either chemically or biologically, before sugar fermentation [3,4]. Production of olive oil symbolizes one of the most important economic agro-food sectors in the Mediterranean basin. The traditional oil industry generates two downstream byproducts, olive cake (residue) and olive mill wastewater, which can cause serious environmental pollution problems. 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 Mediterranean area [5,6]. Global annual production of olive cake has been estimated to approach 4 × 108 kg. Olive residues have been characterized as a possible source of animal feed or other valuable products [7-11]. Ballesteros et al. [2] used waste from a two-step centrifugation process, adopted in Spain for extraction of olive oil, for ethanol fermentation. In the present study, olive cake from a traditional olive oil extraction unit was evaluated as a carbon source for the production of ethanol. This article describes the acid and heat pretreatment of olive cake and the fermentation of soluble sugars obtained during this pretreatment. The raw material was pretreated with dilute acid and treated chemically, by overliming, to
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remove fermentation inhibitors. The sugars released during pretreatment were fermented to produce ethanol using a recombinant ethanol-producing bacterium. Hydrolysate supernatants were also tested for the ability to support growth of a fungal strain with intrinsic resistance to fermentation inhibitors.
Table 1. Composition of olive cake used in this study
MATERIALS AND METHODS
overnight culture grown in liquid LB medium containing 2% xylose. Cells were washed with phosphate buffer (25 mM each KH2PO4 and Na2HPO4, pH 6.8) to remove residual xylose and then suspended in hydrolysate to achieve a 10% (v/v) inoculum in each flask. Fermentations also contained LB medium and MOPS (pH 7) at final concentrations of 1X and 50 mM, respectively. Fermentations (5 mL) were carried out in duplicate in 10 mL Erlenmeyer flasks that were capped with a rubber stopper and vented with a needle. Cultures were incubated at 30°C with gentle mixing and sampled periodically for measurement of sugar and ethanol content.
Materials and Microbial Growth Conditions
Olive cake, the solid residue from the traditional olive oil production process, was obtained in Fès, Morocco. Escherichia coli FBR5 is a recombinant strain that carries the Zymomonas mobilis pyruvate decarboxylase and alcohol dehyrogenase genes for the selective production of ethanol [12]. E. coli FBR5 was grown at 35°C in liquid LB medium (5 g yeast extract, 10 g tryptone, and 5 g NaCl per L) containing 0.4% xylose. Coniochaeta ligniaria NRRL30616 is a fungal strain that can grow on furfural and 5-hydroxymethylfurfural (HMF) as its sole sources of carbon [13]. C. ligniaria NRRL30616 was grown in olive cake hydrolysate supernatant supplemented with 0.1% (NH4)2SO4 as a nitrogen source. A 10% (v/v) inoculum of C. ligniaria was added following harvesting and washing the cells from of an overnight YP-glucose culture. Cultures were incubated at 30°C with shaking and sampled periodically for analysis of furfural and HMF content. Sugar stock solutions were prepared in deionized water and filter sterilized. Furfural and HMF stock solutions were prepared in methanol. Pretreatment
Ground olive cake was suspended in 0~4% sulfuric acid at 10~20% (w/v) and loaded into a 2-in. Schedule 80316 stainless steel pipe reactor with threaded end caps. The mixture was heated to 160~180°C, incubated for 10 min in a fluidized heating bath, and then quickly cooled to room temperature in a water bath. Solids were removed by centrifugation for 20 min at 15,000 g and then washed with one-tenth volume of sterile water. Wash liquid was combined with the original supernatant and the pH was adjusted with Ca(OH)2 to 6.5. Hydrolysate supernatants were filter sterilized. Chemical Elimination of Inhibitory Compounds
The dilute-acid hydrolysate supernatant was adjusted to pH 10 with Ca(OH)2, then NaSO2 (1 mg/mL) was added. The mixture was held at 90°C for 30 min in a water bath, then cooled to room temperature. The pH was then reduced to 7 with HCl, and precipitates were removed by passing the supernatant through a 0.45 µm filter. Fermentations
E. coli FBR5 was collected by centrifugation from an
Klason lignin Cellulose Xylan Arabinan
Olive cake (% db) 28.1 17.9 20.9 1.9
Analytical Methods
Moisture was measured by drying samples at 105°C until they reached a stable weight. Xylose and arabinose were determined by hydrolyzing the biomass with trifluoroacetic acid and analyzing for production of free sugars by highperformance liquid chromatography (HPLC) as described previously [14]. Cellulose content was determined using American Society for Testing and Materials (ASTM) method E1758-95. Sugars and ethanol concentrations were determined using a HPLC system equipped with refractive index detection. Furfural and HMF were quantified using reverse phase HPLC with ultraviolet detection at 277 nm as previously described [13].
RESULTS AND DISCUSSION Feedstock Composition
Table 1 shows the composition of the solid residue obtained from a traditional olive oil extraction unit. The residue is comprised of pressed olives, including stones. Olive cake is 95% (w/w) dry and essentially consists of strongly lignified cell walls (28.1%), is rich in cellulose (15.9%) and xylan (20.9%) on a dry basis, and contains a small amount of arabinan (1.9%). Dilute-acid Pretreatment
To solubilize the olive cake material during pretreatment, a range of H2SO4 concentrations and temperatures were tested. The resulting concentration of sugars and fermentation inhibitors (furfural and HMF) were measured (Fig. 1) to determine the optimal pretreatment conditions that liberated the most sugar while minimizing formation of inhibitors. The amount of acid added was increased [up to 4% (w/v)]
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A
A
B B
C
C
D
Fig. 1. Effect of acid loading on (A) final pH, (B) furans, and (C) sugar yield. All pretreatments shown were performed at 180°C and a 20% (w/v) loading of solids for 10 min. Standard deviations are shown for furans and sugars. Measurements were done in triplicate and the deviation from the average values were less than 10%.
beyond that typically used for dilute-acid pretreatment of corn stover, because at lower acid loadings the pH of the olive cake hydrolysate was higher than desirable to achieve
Fig. 2. Fermentation of soluble sugars in olive cake hydrolysate by E. coli FBR5. 13.5% (w/v) olive cake was pretreated with 1.25 (●), 1.5 (●), or 1.w5 (●) (w/v) H2SO4 for 10 min at 160°C. (A) glucose, (B) xylose, (C) arabinose, and (D) ethanol concentrations during fermentation. Measurements were done in triplicate and the deviation from the average values were less than 10%.
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Table 2. Ethanol fermentation of soluble sugars in olive cake dilute-acid hydrolysatesa H2SO4 (% w/v)
Temperature (°C)
1.25 1.50 1.75 1.25 1.50
160 160 160 180 180
Initial
Final
Initial
Final
Initial
Final
Ethanol (% w/v)
Metabolic c efficiency (%)
Process d efficiency (%)
1.8 3.2 6.0 30.2 34.8
0.01 0.06 0.1 14.3 18.1
0.2 0.3 0.4 1.5 1.7
0 0 0 0.82 0.95
1.14 1.44 1.81 1.39 1.71
0.03 0.02 0.08 1.66 1.73
0.48 0.67 0.81 0.02 0.01
84.8 92.5 91.8 − −
82.6 91.2 87.7 − −
Furfural (mM)
HMF (mM)
Soluble sugars (% w/v) b
a
Olive cake at 13.5% (w/v) was pretreated at the indicated acid concentration and temperature for 10 min. 51 h. c Metabolic efficiency is the percent of theoretical ethanol yield, based on sugars consumed. d Process efficiency is the percent of theoretical ethanol yield, based on total sugars available. b
optimal pretreatment effects. Apparently, the organic content of olive cake biomass has considerable buffering capacity beyond that present in other biomass feedstocks such as corn stover. Increasing the acid concentration resulted in a lower final pH and an increased release of pentoses from the olivecake; however, some sugar degradation occurred, as evidenced by the increased concentration of HMF and, particularly, furfural in the hydrolysates generated with higher acid loadings. Results for the pretreatments performed at 180°C and 20% (w/v) loading of solids are shown in Fig. 1; similar trends were observed at 160 and 170°C. In the pretreatment of olive cake, approximately 45% of the material was recovered as solids (results not shown). Pretreatment of olive cake at 10% (w/v) loading resulted in the release of more sugar than at a 20% loading (not shown), indicating limitations of these pretreatment conditions for solubilizing hemicellulose at higher loadings of solids. This is consistent with the results reported by Ballesteros et al. [15], who worked with residues from a two-step centrifugation process for extraction of olive oil. In order to obtain an indication of the fermentability of hydrolysates, a fungal strain selected for growth on furans and corn stover dilute-acid hydrolysate [13] was tested for growth in olive cake hydrolysates. The strain, C. ligniaria NRRL30616, grew and removed furfural and HMF from neutralized hydrolysates that had been pretreated with acid concentrations up to 1.75% (not shown). At 2.0% and higher acid concentrations, the NRRL30616 strain failed to grow over a 24-h period. Presumably, the combination of furans and other inhibitory compounds (e.g., lignin derivatives) under these conditions prevented growth of this inhibitor-tolerant strain. Simultaneous Saccharification and Fermentation
Hydrolysate from pretreated olive cake was fermented using the ethanol-producing E. coli strain FBR5. Pretreatment conditions of 160 or 180°C for 10 min with 1.25, 1.5, or 1.75% (w/v) H2SO4 were evaluated. A loading of solids at 13.5% (w/v) was used. Fermentation of these supernatants is summarized in Fig. 2 and Table 2. Pretreatment at 160°C, followed by overliming, generated a hydrolysate supernatant that was efficiently fermented by E. coli FBR5. All of the glucose was consumed during the fermentation of the 160°C hydrolysates within 20 h, while conversion of
the pentose sugars, xylose and arabinose, required an extra day. The highest acid concentration, 1.75% (w/v), generated the highest levels of free sugars, and correspondingly, the most ethanol was also produced from this hydrolysate. The ethanol yield was 0.45 g of ethanol produced per 1.0 g of sugar present at the start of fermentation and 0.47 g of ethanol per 1.0 g of sugar consumed. The theoretical yield was 0.51 g ethanol/g glucose or xylose. Pretreatment at 180°C resulted in a hydrolysate that could not be fermented by E. coli FBR5 (Table 2), even though an attempt was made to remove inhibitors by overliming. The concentration of furfural and HMF was much higher in the 180°C hydrolysates when compared to the 160°C hydrolysates (Table 2). Presumably, some of the furans and other inhibitory compounds derived from lignocellulose remained in the hydrolysate and prevented successful fermentation under these conditions. In any case, the concentration of soluble sugars was lower in the 180°C hydrolysates, and this was likely due to degradation of sugars under the more severe conditions of higher temperature and increased acid loading. The composition of the olive cake, which is a mixture of skin, pulp, and seeds from olives, indicated that cellulose and xylan are its predominant components. The xylose content of the olive cake is about 92% of the hemicellulosic sugars and 50% of the total olive cake sugars. Taking into consideration the sugar composition of the olive cake and the sugar yield in the supernatants, it can be concluded that lignocellulosic olive cake may be a suitable feedstock for ethanol production. Fermentation of the solids, using enzymatic hydrolysis of pretreated cellulose and simultaneous saccharification and fermentation, would be expected to further increase the fermentation yields. Acknowledgements We wish to thank Dr. Nancy Nichols and Dr. Bruce Dien from the USDA. This work was supported by the Al Akhawayn University seed money project, the Fulbright commission, and the USDA National Center for Agricultural Utilization Research, Peoria, Illinois, USA.
Received April 22, 2008; accepted July 3, 2008
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