BIOLOGICAL PRETREATMENT AND ETHANOL PRODUCTION FROM OLIVE CAKE E. JURADO, H.N. GAVALA, G.N. BAROI and I.V. SKIADAS,* Copenhagen Institute of Technology (Aalborg University Copenhagen), Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Lautrupvang 5, DK 275 Ballerup, Denmark. *Corresponding author: ivs@bio.aau.dk, telephone +45 9942588, fax +45 9942594. Keywords: enzymatic hydrolysis, ethanol, olive cake, white rot fungi, Phaneroachaete chrysosporium, Ceriporiopsis subvermispora, Ceriolopsis polyzona, Thermoanaerobacter ethanolicus. Abstract Olive oil is one of the major Mediterranean products, whose nutritional and economic importance is well-known. However the extraction of olive oil yields a highly contaminating residue that causes serious environmental concerns in the olive oil producing countries. The olive cake (OC) coming out of the three-phase olive oil production process could be used as low price feedstock for lignocellulosic ethanol production due to its high concentration in carbohydrates. However, the binding of the carbohydrates with lignin may significantly hinder the necessary enzymatic hydrolysis of the polymeric sugars before ethanol fermentation. Treatment with three white rot fungi, Phaneroachaete chrysosporium, Ceriporiopsis subvermispora and Ceriolopsis polyzona has been applied on olive cake in order to investigate the potential for performing delignification and thus enhancing the efficiency of the subsequent enzymatic hydrolysis and ethanol fermentation process steps. It has been concluded that the conditions tested were not adequate for reaching satisfactory delignification and thus studying different conditions (humidity, ph and nitrogen levels) is necessary. Another possibility for lowering the cost of bioethanol is the use of microbial strains, which possess the ability for hydrolysis of complex carbohydrates. Thus, the addition of enzymes could be eliminated to a minimum extent for reaching a satisfactory degree of fermentable sugars release from a biomass. In that concept, the hydrolytic, ethanol-producing Thermoanaerobacter ethanolicus has been applied on olive cake supplemented with glucose in order to evaluate any inhibitory effect that olive cake might have on the microbial growth and metabolism. It was shown that the strain was able to grow and produce ethanol up to a TS content of g per L. Further investigation of the ph role on the ethanol yield will take place.
- INTRODUCTION Olive oil is one of the major Mediterranean products, whose nutritional and economic importance is well-known. However the extraction of olive oil yields a highly contaminating residue that causes serious environmental concerns in the olive oil producing countries and unfortunately, this is a problem which has not been solved yet []. Two different methods are mainly used for the extraction of olive oil: the pressing and the centrifugation of the precrashed olives. The centrifugation and especially the three-phase process is the most common in the olive oil industry. In the three-phase process, centrifugation is directly applied to the crashed olives mass, resulting in three phases: olive oil, wastewater (mixture of vegetation and process water) and olive cake (solid residue from the olives with about 5% water content). The olive cake (OC) could be used as low price feedstock for lignocellulosic ethanol production due to its high concentration in carbohydrates. However, the binding of the carbohydrates with lignin may significantly hinder the necessary enzymatic hydrolysis of the polymeric sugars before ethanol fermentation. Enzymes comprise an estimated 2 4% of cellulosic ethanol production costs [2] and thus they are one of the main factors affecting the financial viability of bioethanol. To decrease the use of enzymes and increase their efficiency, several physico-chemical processes have been tested as a previous step to enzymatic hydrolysis, but these processes present their own limitations as high costs, or production of inhibitory compounds. On the other hand, white rot fungi (WRF) are the most efficient lignin degraders in nature [3], which posses the unique ability to efficiently degrade lignin. Some WRF are the only organisms, which are able to perform selective delignification without consuming the valuable carbohydrates under specific conditions. Two main characteristics of the OC may make it suitable for fungal pretreatment. The first characteristic is the activation of the WRF s enzymatic system by the presence of polyphenolic compounds in olive-mill effluents [4] in combination with the existing pectinase activity [5] and its high dry matter content (up to 5%). Several studies have shown a better performance of the WRF in presence of elevated oxygen levels [6, 7]. Also, the substrate must possess enough moisture to support growth and metabolism of microorganism [8]. Another possibility for lowering the cost of bioethanol is the use of microbial strains, which possess the ability for hydrolysis of complex carbohydrates. Thus, the addition of enzymes could be eliminated to a minimum extent for reaching a satisfactory degree of fermentable sugars release from a biomass. The aim of the present work was to study the capability of selected fungi either to perform delignification or to alter the lignin structure of the olive cake and thus enhancing the efficiency of the subsequent enzymatic hydrolysis and ethanol fermentation process steps. Three WRF strains were selected based on previous studies [9,,, 5] and screened for their ability to grow on OC and to facilitate sugars release during enzymatic hydrolysis: Phaneroachaete chrysosporium, Ceriporiopsis subvermispora and Ceriolopsis polyzona. Also, the hydrolytic, ethanol-producing Thermoanaerobacter ethanolicus has been applied on olive cake supplemented with glucose in order to evaluate any inhibitory effect that olive cake might have on the microbial growth and metabolism. The research performed so far is part of a more general research frame, which is shown in figure. 2- MATERIALS AND METHODS 2.-Analytical Methods Dry matter content was determined as total solids (TS) concentration and was carried out according to Standard Methods [2]. Detection and quantification of sugar monomers,
glucose and xylose as well as end-fermentation products, i.e. ethanol, acetic and lactic acids was made by HPLC-RI equipped with an Aminex HPX-87H column (BioRad) at 6 C. A solution of 4mmol L - H 2 SO 4 was used as eluent at a flow rate of.6ml min -. Prior to HPLC analysis, ml of sample was acidified with μl of 2% H 2 SO 4. Centrifugation at, rpm for min followed by filtration through a.45μm membrane filter was then applied. Three groups of carbohydrates were analyzed in the olive pulp: the st group was the total carbohydrates, including those bound in the lignocellulosic biomass, the 2nd group was the soluble carbohydrates and the last one was the simple sugars. An overview and details of the methodology used for carbohydrates and lignin determination can be found in [3]. olive cake pretreatment, biological (fungal) or physicochemical (alkali treatment) A. SHF with Celluclast.5L & Novozyme-88 and T. ethanolicus enzymatic hydrol. ethanol prod. B. SSF with Celluclast.5L & Novozyme-88 and T. ethanolicus enzymatic hydrolysis and ethanol production (SSF) C. DMC with Celluclast.5L & Novozyme-88 and T. ethanolicus direct microbial conversion (DMC) (no enzymes addition) Figure : Research outline for bioethanol production from olive cake (SHF: separate hydrolysis and fermentation, SSF: simultaneous saccharification and fermentation). 2.2-Enzymatic hydrolysis The possibility of applying enzymatic hydrolysis for release of the sugars contained in the olive cake was assessed using two different combinations of enzymes. The assessment was done as batch tests with 5 g TS olive cake and different enzyme loadings. The first enzymes combination was a mixture of Celluclast.5L (an enzyme mixture containing cellulase and hemicellulase activities, CAS number: 92-54-8) and Novozyme 88 (an enzyme containing beta-glucosidase activity, MDL number: MFCD3628) and the second, which was named Novozymes Biomass Kit, consisted of five different enzymes (NS53, NS5, NS52, NS53, NS222) containing cellulase, beta-glucosidase, arabinase, hemicelluloses, pectinase and xylanase activities. All enzymes were kindly provided by Novozymes A/S (Bagsværd, Denmark). Enzyme activity was measured by Filter Paper assay [4, 5]. Novozyme 88 was supplemented with Celluclast at a ratio of :3 (volume based) while the NS53:NS5:NS52:NS53:NS222 ratio was 5:.5::.25:5. The olive cake was distributed to infusion flasks before being heated to 2 C C for 2 min. After cooling, enzyme loadings of.,.34 and.58 g of each of the two enzymes combinations per g TS were added to the batches. The enzymatic hydrolysis was tested at 5 C with agitation at 2 rpm for a period of four. Glucose and xylose concentrations were determined after the completion of the experiment. All batch tests were run in triplicates. For enzymatic hydrolysis of the fungal pretreated olive cake (described in the following) a mixture of the Novozymes Biomass kit was used at a loading of 5 FPU per g TS olive cake. The enzymatic hydrolysis was performed at the conditions described above. 2.3-Biological (fungal) pre-treatment P. chrysosporium strain DSM 699, C. subvermispora strain CBS 347.63 and C. polyzona strain MUCL 38443 were obtained from the Deutsche Sammlung von Microorganismen und Zellkulturen (DSMZ), CBS-KNAW fungal biodiversity centre and Belgian Co-ordinated
Collections of Micro-organisms (BCCM), respectively. The medium used for growing the cultures was the DSM 9 for P. chrysosporium and MA2 (malt agar 2%) for C. subvermispora and C. polyzona. Agar-grown cultures were maintained at -8 C in % glycerol in water. P. chrysosporium, C. subvermispora and C. polyzona cultures were first regrown in agar plates (5 g of agar per L medium for P. chrysosporium and 2 g per L for C. subvermispora and C. polyzona). Subsequently they were transferred in autoclaved (2 C) infusion bottles containing 3 ml of their respective growth medium and placed in a rotary shaker at 5 rpm at 37, 32 and 2 C respectively. The rubber stopper of the infusion bottles was equipped with a glass tube filled with cotton in order to ensure sterile oxygen transfer. The cultures were harvested, rinsed with water and homogenized prior to olive cake inoculation (% v/w). Delignification experiments with P. chrysosporium and C. polyzona: A thin layer of g olive cake was added in 2 infusion bottles and autoclaved at 2 C for 2 min. Subsequently, the bottles were inoculated with the fungi and incubated at 37 C in a rotary shaker at 5 rpm. Triplicates were analyzed for lignin and carbohydrates content at, 4,, 5, 9 and 25 after inoculation. One triplicate control with olive cake without inoculation was running in parallel. Sugar release experiments with P. chrysosporium, C. subvermispora and C. polyzona pretreatment followed with enzymatic treatment: Sugars release after 7 and 4 fungal pretreatment of the olive cake followed by 4 enzymatic hydrolysis was investigated as shown in figure 2. Also, experiments with P. Chrysosporium grown in medium without peptone (elimination of the nitrogen content) and forced aeration using an air compressor have been carried out as well. After 4 of enzymatic treatment all triplicates were analyzed for free and soluble sugars content. Autoclaved olive cake control vials, no fungi addition, x6 vials inoculated with fungi, x2 after 7 and 4 after 7 and 4 enzyme addition, 5 FPU per g TS autoclavation at 2 C for 2 min free and soluble sugars analysis Figure 2. Experimental set-up for fungal pre-treatment followed by enzymatic hydrolysis. 2.4-Microbial batch fermentation tests T. ethanolicus, strain DSM 2246 was obtained from the Deutsche Sammlung von Microorganismen und Zellkulturen (DSMZ) culture collection and was maintained in DSM 6 medium. Stock cultures were stored at -8 C in % glycerol and inoculation cultures were transferred once before use. All cultures were grown in a N 2 atmosphere, at 7 C. Experiments with T. ethanolicus on glucose medium supplemented with different olive cake contents were carried in triplicates in serum vials sealed with rubber stoppers. Glucose and products concentrations as well as ph were followed throughout the experiment. 3- RESULTS AND DISCUSSION 3.-Olive cake characterization and enzymatic hydrolysis Olive cake was characterized by a 5,7 ±,4 % TS content and a ph value of 5,5. The total carbohydrates in olive pulp were composed by glucose and xylose (3,3 ±,2 and 6,4 ±,3 g per g TS, respectively) with the soluble and simple sugars comprising a small fraction
of them (4 ±,6 and,34 ±,5 g-soluble and free glucose per g TS and,9 ±,22 and,94 ±,4 g-soluble and free xylose per g TS, respectively). The low content of free and soluble sugar compared to the total carbohydrates implies that efficient hydrolysis should considerably increase the availability of sugars for ethanol fermentation. Lignin content is also high (45,8 g per g TS) and therefore reduction of lignin will further facilitate the efficiency of hydrolysis and ethanol fermentation steps. The enzymatic hydrolysis or raw olive cake with both enzyme mixtures (Celluclast.5L- Novozyme-88 and Novozymes Biomass Kit) significantly increased the soluble sugars concentration (Figure 3). Based on the results obtained, the use of the Novozymes Biomass Kit was more effective for olive cake hydrolysis and therefore the Novozymes Biomass kit was used for all subsequent experimental tests at a loading of 5 FPU per g TS. 3, 2,,,5, glucose xylose control () 8, 29,7 5 FPU per g TS (a) 3, 2,,,5, glucose xylose control () 6,2 2,3 35,8 FPU per g TS Figure 3: Soluble sugars released from olive cake after enzymatic hydrolysis with (a) Celluclast.5L mixed with Novozyme-88 and (b) Novozymes Biomass Kit 3.2-Biological (fungal) pre-treatment The results from the delignification experiments with P. chrysosporium and C. polyzona are shown in Figure 4. It seems that after of fungal growth some delignification started to occur in the experiments with P. chrysosporium reaching approximately 5%, while at the same time a decrease of total glucose content was observed while xylose content remained unaltered. On the other hand, the experimental data with C. polyzona showed that no lignin degradation or total carbohydrates decrease took place. It is noticeable that the standard deviation of lignin and total carbohydrates was considerably high, thus not allowing for solid conclusions to be drawn. (b) 5 48 46 lignin - P. chrysosporium lignin-c. polyzona 25 2 glu - P. chrysosporium glu-c. polyzona xyl-p. chrysosporium xyl-c. polyzona 44 5 42 4 5 38 4 5 9 25 4 5 9 25 Figure 4. Lignin, total-glucose and total xylose content in g per g TS versus time in during delignification experiments with P. chrysosporium and C. polyzona. In figures 5a, 5b and 5c, soluble sugars release after enzymatic treatment of fungal pre-treated olive cake by the three strains is shown. In all three cases studied, the sugars release was lower or comparable to the control (no fungal pre-treatment). Data from experiments with P. Chrysosporium grown in medium without peptone (elimination of the nitrogen content) were comparable with the ones shown in figure 5a. Also, forced aeration did not influence the
sugars release. Investigation of delignification potential at different conditions, i.e. different humidity, ph and nitrogen levels, is necessary. 3 control without sterilisation with sterilization (a) 3 control without sterilisation with sterilization (b) 2 2,5,5 glucose-7 glucose-4 xylose-7 xylose-4 glucose-7 glucose-4 xylose-7 xylose-4 5 4 control without sterilisation with sterilization (c) 3 2 glucose-7 glucose-4 xylose-7 xylose-4 Figure 5. Soluble sugars release in g per g TS after enzymatic treatment of fungal pretreated olive cake by (a) P. chrysosporium, (b) C. subvermispora and (c) C. polyzona. 3.3-Microbial batch fermentation tests Glucose and products concentration as well as ph values in the experiments with T. ethanolicus supplemented with glucose and different amounts of olive cake are shown in Table. Complete consumption of glucose was observed in the experiments with up to g TS olive cake added per L of medium within three, while the vials with 5 and 25 g TS per L showed limited and no consumption, respectively. It is noticeable that the higher the amounts of olive cake the lower the ethanol yield was (.9,. and.63 g per g in the control vials and vials supplements with 5 and g TS per L olive cake, respectively). This can be attributed either to the lower ph values and/or to potential presence of inhibitors in the olive cake. It is more likely that the ethanol yield was dependent on the ph values while the absence of microbial activity at 25 g TS olive cake per L was due to inhibitory effects since a ph of 5,5 allowed still for microbial growth. Future studies will include experiments with high TS concentration and controlled ph in order to clarify this issue. Glucose at time Substrate in g/l Products concentration in g/l and ph values after 3 Glucose after 3 Lactic acid Acetic acid Ethanol ph Control 5,47 ±,39,74 ±,4,22 ±,2,5 ±,6 6,5 5 g TS/L 6,4 ±,68 2,84 ±,27,25 ±,2,65 ±,5 5,7 g TS/L 7,33 ±,3 4 ±,6,23 ±,46 ±,6 4,8 5 g TS/L 8,97 ±,74 3,8 ±,54 2,9 ±,3,33 ±,2 4,8 25 g TS/L,7 ± 9 9 ±,49,2 ±, 5,5 Table. Glucose and products concentration as well as ph values in the experiments with T. ethanolicus supplemented with glucose and different amounts of olive cake.
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