Available online at ScienceDirect. Energy Procedia 68 (2015 )

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1 Available online at ScienceDirect Energy Procedia 68 (2015 ) nd International Conference on Sustainable Energy Engineering and Application, ICSEEA 2014 The effect of substrate loading on simultaneous saccharification and fermentation process for bioethanol production from oil palm empty fruit bunches Eka Triwahyuni a, *, Muryanto a, Yanni Sudiyani a, Haznan Abimanyu a a Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan Puspiptek Serpong, Tangerang Selatan 15314, Indonesia Abstract Increasing energy demand and concern about increased greenhouse gas emissions make lignocellulosic biomass increasingly to be recognized as having great potential for biofuel and biomaterial production based on the biorefinery concept. Oil Palm Empty Fruit Bunches (EFBs) is one of the major solid wastes in the palm oil industries as a source of lignocellulosic biomass. Cellulose is the highest component of EFBs that can be converted to ethanol. The aim of this research was to investigate the different strategies for high substrate loading on SSF process of bioethanol production from EFBs. Increasing substrate loading is one of the most important challenges to make bioethanol production more economical. This research used two methods to increase the substrate concentration loading on Simultaneous Saccharification and Fermentation (SSF) i.e: direct variation of substrate concentration loading and substrate loading gradually to obtain a high-concentration substrate. A range of substrate loading was from 15% to 25% (g.ml -1 ). The SSF process was carried out at 32 o C, ph 4.8, and 150 rpm for 72 hours. The result shows that the highest concentration of ethanol can be produced by a high concentration of substrate loading gradually. The highest ethanol concentration was g.l -1 (80.21% ethanol yield) by using 25% (g.ml -1 )substrate loading gradually, 18 FPU/g substrate enzyme Cellic Ctec2 and 20% Cellic Htec2 (based on volume of Cellic Ctec2), and 1% (g.ml -1 ) yeast Saccharomyces cereviceae in SSF process. Whereas, 20% (g.ml -1 ) concentration substrate loading by directly or gradually produce almost same ethanol concentration The The Authors. Authors. Published Published by Elsevier by Elsevier Ltd. This B.V. is an open access article under the CC BY-NC-ND license ( Peer-review under responsibility of Scientific Committee of ICSEEA Peer-review under responsibility of Scientific Committee of ICSEEA 2014 Keywords:simultaneous saccharification and fermentation (SSF); substrate loading; high-concentration ethanol; high-concentration substrate * Corresponding author. Tel.: ; fax: address: ekatriwahyuni@gmail.com The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( Peer-review under responsibility of Scientific Committee of ICSEEA 2014 doi: /j.egypro

2 Eka Triwahyuni et al. / Energy Procedia 68 ( 2015 ) Introduction Energy supply not only affects nation s energy security, but also affects sustainable development. The global rise in energy demand and concern about increased greenhouse gas emissions make lignocellulosic biomass increasingly to be recognized as having great potential for biofuel and biomaterial production based on the biorefinery concept [1]. The advantages of lignocellulosic biomass as potential energy sources are their neutral carbon balance, renewable character, large availability, independence of geographic location and improvement of local economy derived from cultivation [2]. The chemical components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. Cellulose and hemicellulose can be converted to sugars (C-6 and C-5) through biological or chemical conversion, and these sugars can be fermented to ethanol or other valuable chemicals [3]. However, lignin is a polymer of phenolic nature became an inhibitor of enzymatic reactions. Lignin not only prevents cellulase from forming cellulose but also adsorbs the enzyme, thereby inactivating it for cellulose hydrolysis [4]. One of the largest lignocellulosic sources in Indonesia is wastes from oil palm industry, such as oil palm empty fruit bunches (EFBs) or frond. Oil palm production in Indonesia increased from million tons in 2008 to million tons in 2012 [5]. The dry weight of EFBs is about 8% of the dry weight of FFB (Fresh Fruit Bunches) [6], or 39% of the weight of CPO produced [7]. The chemical component of EFBs is 44.21% cellulose, 16.68% hemicelluloses and 35.51% lignin [8]. Cellulose is the highest component of EFBs that can be converted to ethanol. The production of second generation bioethanol from lignocellulosic can be carried out by three major step, i.e: pretreatment of raw material, enzymatic hydrolysis of cellulose to sugars, and biological conversion of sugars to ethanol [9]. Pretreatment process is crucial for achieving effective hydrolysis of substrates. Mechanical pretreatments or fractionation of lignocellulosic in aqueous alkali and acid to improve the enzymatic accessibility [10]. Hydrolysis and fermentation process can be achieved by several process strategies. They include separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and prehydrolysis and simultaneous saccharification and fermentation (PSSF) [11]. SHF involves two sequential step, enzymatic saccharification and fermentation that allows working at optimal operating conditions for enzyme and microorganisms. However, the products formed during the hydrolysis step in SHF, such as cellobiose and glucose, can inhibit the cellulase enzyme as well as the fermenting microorganisms. Whereas in SSF, glucose produced from hydrolysis is simultaneously metabolized by microorganism, thereby alleviating problems caused by product inhibition[12]. Moreover, the SSF process has other advantages such as reduced operation all costs, lower enzyme requirement and increased productivity [13]. The economical processes are required in ethanol production from lignocellulosic. When the concentration of ethanol after fermentation is lower than 7 % (v/v), extensive energy must be consumed in subsequent distillation processes. However, it is difficult to produce ethanol with a concentration higher than 7 % (v/v) from lignocellulosic biomass through ordinary fermentation processes [14]. Increasing substrate loading in hydrolysis and fermentation step is one of the most important challenges produces ethanol more economical [15]. However, high substrate concentration has also causes a larger levels of inhibiting compounds [16], diffusional enzymes problems [17], endproduct inhibition [18],stirring and mixing limitations by viscosity increase[19] or possible mass transfer limitations appearing above 20% insoluble solids concentration [20]. Thus, in this study it will be investigated the different strategies for high substrate loading in SSF process of bioethanol production from EFBs. A range of substrate loading from 15% to 25% (g.ml -1 ) was investigated to determine its effect on ethanol concentration in SSF process. 2. Materials and methods 2.1. Materials In this study, oil palm empty fruit bunches (EFBs) fiber was collected from a Palm Oil Mill, Musi Banyuasin, South Sumatra, Indonesia. Enzymes (Cellic Ctec2 and Cellic Htec2) were provided by Novozymes Korea Ltd. The Cellic Ctec2 was used for saccharification of cellulose into glucose. The β-glucosidase enzyme supplementation was not necessary due to the highactivity of β-glucosidase present in Cellic Ctec2. The Cellic Htec2 (hemicellulase enzyme) was added to hydrolyse the xylooligosaccharides and improve the cellulose saccharification. The cellulase activity of Cellic Ctec2 was measured by the Filter Paper assay [21]and was

3 140 Eka Triwahyuni et al. / Energy Procedia 68 ( 2015 ) expressed as Filter Paper Unit (FPU). The enzyme activity was 144 FPU/mLfor Cellic CTec2. Commercial instant dry yeast Saccharomyces cereviceae were applied to the SSF process Pretreatment Process EFBs fiber was dried and milled to a particle size ±3 mm, then treatedby 10% NaOH solutions. The ratio of NaOH solutions and EFBs was 5:1. Pretreatment process was carried out at temperatures 150 o C and 4-7 kg/cm 2 of pressure for 30 minutes. EFBs-treated was washed with water until neutral ph and dried to a moisture content below 10% SSF process of EFB-treated for high-concentration bioethanol production Ethanol was produced through SSF method. One method to produce high-concentration ethanol was by increasing the substrate concentration loading into SSF process. This research used two methods to increase the substrate concentration loading, i.e.: Variation of substrate concentration loading directly SSF was carried out in a250 ml Erlenmeyer flask in a shaking incubator (150 rpm, 32 o C) for 72 h. The total volume of working slurry was 100 ml. The variations of substrate concentration loading were 15, 20, and 25% (g.ml -1,EFBs-treated dry based). EFBs-treated and 0.05 M buffer citrate (ph 4.8) were loaded in Erlenmeyer flask. The enzyme loading amount was 30 FPU/g substrate (Cellic Ctec2) and (this value was based on the amount of Cellic Ctec2 loaded). The yeast loading amount was 1% (g.ml -1 ). Sample (1 ml) was withdrawn from SSF medium every 24 h, centrifuged and analyzed for glucose, xylose and ethanol Substrate loading gradually to obtain a high-concentration substrate The initial concentration of substrate loading was 15% (g.ml -1 ). EFBs-treated and 0.05 M buffer citrate (ph 4.8) were loaded in Erlenmeyer flask. The enzyme loading amount was 30 FPU g/substrate (Cellic Ctec2) and 20% Cellic Htec2 (this value was based on the amount of Cellic Ctec2 loaded). The yeast loading amount was 1% (g.ml -1 ). After 24 h of SSF process, substrate was added up to 20% and 25% g.ml -1 without the addition of enzyme and yeast. SSF was carried out in a250 ml Erlenmeyer flask using a shaking incubator (150 rpm, 32 o C) for 72 h. Sample (1 ml) was withdrawn from SSF medium every 24 h, centrifuged and analyzed for glucose, xylose and ethanol. Flow chart of experimental set up can be seen in Fig Analytical methods Cellulose, hemicelluloses and lignin contents of EFBs-untreated and EFBs-treated were determined by the National Renewable Energy Laboratory (NREL) using standard biomass analytical procedures [22]. The composition of the product from SSF was determined by measuring glucose, xylose, and ethanol concentrations by high-performance liquid chromatography (HPLC). The HPLC (Waters, USA) system was equipped with a Bio-Rad Aminex HPX-87C column, a guard column, an automated sampler, a gradient pump, and a refractive index detector. The mobile phase was deionized water at a flow rate of 0.6 ml/min and oven temperature was maintained at 80 C.

4 Eka Triwahyuni et al. / Energy Procedia 68 ( 2015 ) Fig. 1. Flow chart of the experimental process 2.5. Ethanol yield calculations (1) Where [EtOH] f is Ethanol concentration at the end of SSF (g/l), [EtOH] i is Ethanol concentration at the beginning of SSF (g/l) which is zero, [Biomass] is Dry biomass concentration at the beginning of SSF (g/l), f is cellulose fraction of dry biomass (g/g), 0.51 isconversion factor for glucose to ethanol based on stoichiometric biochemistry of yeast, is converts cellulose to equivalent glucose. 3. Results and discussion 3.1. Characteristic of EFBs In bioethanol production based on lignocellulosic materials, which has sugars as intermediates, it needs to break down the feedstock s structure and obtain sugars from cellulose and hemicellulose. Hence pretreatment is necessary to prepare the feedstock in order to improve conversion of sugars [23]. Removal of non-cellulose components by pretreatment is beneficial to increasing in cellulose content. In this study, EFBs was treated by alkali solutions (10% NaOH solutions).the chemical component of EFBs-untreated and EFB-treated used in this study was listed in Table 1.

5 142 Eka Triwahyuni et al. / Energy Procedia 68 ( 2015 ) Table 1. Chemical Component of EFBs No. Component %EFB-untreated %EFB-treated 1 Cellulose(a) Hemicellulose(a) Lignin Ash Extractive Pretreatment using alkali solutions such as NaOH, Ca(OH) 2 or ammonia can remove lignin and a part of hemicelluloses, and efficiently increase the accessibility of enzyme to cellulose [25]. The mechanism of alkaline pretreatment is believed to be saponification of intermolecular ester bonds crosslinking xylan hemicelluloses and other components, for example, lignin and other hemicellulose. The porosity of the lignocellulosic materials increases with the removal of the crosslinks [26]. Based on HPLC sugar analysis that can be seen in Table 1, cellulose increased to 75.05%, and hemicelluloses decreased to 10.19% after pretreatment process. Delignification was reached 69.43%. Cellulose will be converted to ethanol using cellulolitic enzymeand yeast Saccharomycess cereviceae on SSF process Effect of variation of substrate concentration loading directly on SSF process In an attempt to increase ethanol concentration at high substrate loading, EFBs-treated was subjected to SSF process by S. cerevisiae at a temperature of 32 o C. SSF was conducted at 15%, 20% and 25% (g.ml -1 ) substrate loading to evaluate ethanol production. The enzymes and yeast simultaneously inserted at the beginning of SSF process. The sugar released from enzymatic hydrolysis are immediately consumed by yeast for ethanol production on SSF process. Enzymatic hydrolysis rates can be maximized by reducing the product inhibition. SSF gives higher ethanol yields from cellulose than SHF and requires lower amounts of enzymes [27]. In this study, the product of SSF were analyzed using HPLC to determine the amount of ethanol formed and glucose and xylose remaining, shown in Fig. 2.The result indicates that 15% (A) and 20% (B) substrate concentration loading almost the same tendency, whereas 25% (C) showed a slow fermentation rate and low concentration of ethanol production. A and B could be stirred and mixed very well, so sample (1 ml) could be withdrawn from SSF medium from 24 h to 72 h of SSF process. Meanwhile, C could not be stirred and mixed perfectly because of the high viscosity, so sample could only be withdrawn at 72 h of SSF process. At 15% (A) and 20% (B) substrate loading, glucose could be optimum converted into ethanol in SSF process. The highest ethanol concentration at 72 h SSF were g/l (A) and g/l (B).According to literature, the threshold of economic profitability corresponds to bioethanol concentrations in the fermentation broth in the range 4 5 volume percent. Achieving this threshold entails the utilization of media containing 15 20% solids (on dry basis) [28]. Therefore, increasing substrate loading required to produce high-concentration ethanol so that the process is more profitable. In this study, substrate loading was increased to 25% (C) for producing higherconcentration ethanol. However, at 25% (C) substrate loading ethanol could not be optimally produced, which has remaining glucose g/l. So it required an additional time of SSF process to convert the remaining glucose into ethanol. It is because high solid loadings may result in limited cellulose conversions in enzymatic hydrolysis [17] or in SSF stages, owing to mass transfer limitation. The efficiency of enzymatic digestibility and fermentation is significantly reduced at high concentrations of pretreated biomass, as mixing becomes difficult with increased viscosity [29]. Therefore, the next step substrate will be loaded gradually in order to obtain high-concentration substrate with a stable viscosity.

6 Eka Triwahyuni et al. / Energy Procedia 68 ( 2015 ) glucose & xylose concentration (g/l) ethanol concentration (g/l) time (h) A-glucose B-glucose C-glucose A-xylose B-xylose C-xylose A-ethanol B-ethanol C-ethanol Fig. 2. Ethanol, glucose and xylose concentration of EFBs-treated at (A) 15 % (g.ml-1) substrate loading, (B) 20% (g.ml-1) substrate loading, (C) 25% (g.ml-1) substrate loading, with 30 FPU/g substrate of Cellic CTec2 and Cellic HTec2 (20% of Cellic CTec2 loaded), 1% (g.ml-1) dry yeast, using the SSF process 3.3. Effect of Substrate loading gradually to obtain a high-concentration substrate The production of bioethanol with substrate loading gradually to obtain a high-concentration substrate becomes possible to produce high-concentration ethanol, where the viscosity decreases with the formation of glucose and ethanol, facilitate mixing through enzymatic digestion and the progress of fermentation [30]. In this study, the initial substrate loading was 15% with enzyme and yeast loading. Substrate was added until 20% (D) and 25% (E) after 24 h of SSF process without the addition of enzyme and yeast. The result can be seen in Fig. 3, which shows that A, D, and E substrate concentration loading almost the same tendency. Glucose can be converted into high-concentration ethanol. The addition of substrate gradually can make the enzymes work more optimally. During 24 h of SSF, the enzymes work to convert 15%substrate into glucose that simultaneously converted to ethanol by yeast. After 24 h of SSF, substrate was added until 20%(D) and 25% (E) so that enzymes and yeast will work to convert the fresh substrate. Without the addition of enzymes and yeast, D and E could be stirred and mixed perfectly so sample (1 ml) could be withdrawn from SSF medium every 24 h. The data of ethanol production with the variation of substrate concentration and enzymes loading can be seen in Table 2. Table 2 shows that substrate loading gradually produces higher ethanol yield than substrate loading directly. At 20% (g.ml -1 ) substrate loading, B and D produce almost the same concentration of ethanol on different enzymes loading. D requires fewer enzymes than B, that is 22.5 FPU/g substrate of Cellic Ctec2. Whereas at 25% (g.ml -1 ) substrate loading, C and E produce a different ethanol concentration. E (25% substrate loading gradually) almost produces ethanol yield two times from C (25% substrate loading directly). E produce high-concentration ethanol (80.21% ethanol yield) with enzymes loaded lower than C. In the substrate loading directly (C), cellulose could not be converted optimally into glucose because of enzyme diffusion problems and limitations of the stirring and mixing by viscosity increase. Substrate loading directly gives a higher viscosity than substrate loading gradually so that enzyme and yeast could not work optimally. Based on the data in Table 2, substrate and enzymes loading which are believed to play important roles in selected variables (ethanol concentration, ethanol yield). Based on Collins [31] in

7 144 Eka Triwahyuni et al. / Energy Procedia 68 ( 2015 ) Coyle [32] estimation that the cost of enzymes for cellulosic ethanol production was 14.5% of the overall production costs. Minimizing enzymes (cellulase) dosage is important for cost reduction of lignocellulosic ethanol production. glucose & xylose concentration (g/l) time (h) A-glucose D-glucose E-glucose A-xylose D-xylose E-xylose A-ethanol D-ethanol E-ethanol Ethanol concentration (g/l) Fig.3.Ethanol, glucose and xylose concentration of EFBs-treated at (A) 15 % (g.ml-1) substrate loading, (D) 20% (g.ml-1) substrate loading ( 15% at 0 h & 5% at 24 h), (E) 25% (g.ml-1) substrate loading (15% at 0 h & 10% at 24 h) in SSF process Table 2. Variation of substrate concentration and enzymes loading for Ethanol Production type Substrate loading (g) Substrate loading ratio (% g.ml-1) Enzyme loading (FPU/g substrate) Enzyme loading (ml) SSF time (h) A 15 (at 0 h) (Cellic Ctec2) (8.43) B 20 (at 0 h) (Cellic Ctec2) (9.92) C 25 (at 0 h) (Cellic Ctec2) (5.77) D 15 (at 0 h) (Cellic Ctec2) (at 24 h) (9.69) E 15 (at 0 h) (Cellic Ctec2) (at 24 h) (10.57) final Ethanol conc. (g/l) or (% v/v) Ethanol yield (%)

8 Eka Triwahyuni et al. / Energy Procedia 68 ( 2015 ) Conclusions The experimental data showed that high-concentration ethanol could be produced by high-concentration substrate loading gradually. The highest ethanol concentration was g/l (80.21% ethanol yield) by using 25% (g.ml -1 ) substrate loading gradually,18 FPU/g substrate enzyme Cellic Ctec2 and 20% Cellic Htec2 (based on volume of Cellic Ctec2) and 1% (g.ml -1 )of yeast on SSF process. Increasing substrate loading and minimizing enzymes dosage in SSF process is an important challenges to produces ethanol more economical. Acknowledgements The authors thank to Hendris Hendarsyah and Irni Fitria, who have given valuable assistances to this work. This research was funded by National Project of Indonesian Institute of Sciences (LIPI) of fiscal year References [1] Ragauskas AJ., Williams CK., Davison BH., Britovsek G., Cairney J., Eckert CA The path forward for biofuels and biomaterials. Science 311: [2] Zhang YHP A sweet out-of-the-box solution to the hydrogen economy: is the sugar powered car science fiction. Energy Environ Sci 2009;2: [3] Badger PC Ethanol from cellulose: a general review. In: Janick J, Whipkey A (eds) Trends in new crops and new uses. ASHS Publishers, Alexandria [4] Converse A.O., Ooshima H., Burns D.S Kinetics of enzymatic hydrolysis of lignocellulosic materials based on surface area of cellulose accessible to enzyme and enzyme adsorption on lignin and cellulose. Appl Biochem Biotechnol 24 25:67 73 [5] Minister of Agriculture Agriculture Statistic Jakarta: Kementerian Pertanian. [6] Sheil D., Casson A., Meijaard E., Noordwijk MV., Gaskell J., Groves JS., Wertz K.,Kanninen The impacts and opportunities of oil palm in Southeast Asia, CIFOR, Bogor, Indonesia. [7] Yano S., Murakami K., Sawayama S., Imou K.,Yokoyama S Bioethanol production potential from oil palm empty fruit bunches in southeast asian countries considering xylose utilization, Journal of the Japan Institute of Energy, 88(10): [8] Triwahyuni E., Anindyawati T., Sarwono R., Idiyanti T., Separated Enzymatic Hydrolysis and Fermentation Palm Oil Empty Fruit Bunches for Bioethanol Production. Proceedings of International Conference on Biotechnology 2012, pp [9] Wyman CE., Dale BE., Elander RT., Holtzapple M., Ladisch MR., Lee YY Coordinated development of leading biomass pretreatment technologies. Bioresour Technol 2005;96: [10] Kang KE., Han M., Moon S-K., Kang H-W., Kim Y., Cha Y-L Optimization of alkali-extrusion pretreatment with twin-screw for bioethanol production from Miscanthus. Fuel109: [11] López-Linares JC., Romero I., Cara C., Ruiz E., Moya M., Castro E Bioethanol production from rapeseed straw at high solids loading with different process configurations. Fuel 122: [12]Alfani F., Gallifuoco A., Saporosi A., Spera A., Cantarella M Comparison of SHF and SSF processes for the bioconversion ofsteamexploded wheat straw.j. Ind. Microbiol.Biotechnol.25, [13]Chen H., Xu J., Li Z Temperature cycling to improve the ethanol production with solid state simultaneous saccharification and fermentation. Appl. Biochem. Microbiol. 43, [14] Madson PW Ethanol distillation: the fundamentals. In: Ingledew WM, Kelsall DR, Austin GD, Kluspies C (eds) The alcohol textbook. Nottingham University Publishers, Nottingham, pp [15] Modenbach AA., Nokes SE Enzymatic hydrolysis of biomass at high-solids loadings. A review. Biomass Bioenergy;56: [16] Jørgensen H., Vibe-Pedersen J., Larsen J., Felby C Liquefaction of lignocellulose at high-solids concentrations.biotechnol Bioeng;96: [17] Wang W., Kang L., Wei H., Arora R., Lee YY Study on the decreased sugar yield in enzymatic hydrolysis of cellulosic substrate at high solid loading. Appl Biochem Biotechnol;164: [18] Andric P., Meyer AS., Jensen PA., Dam-Johansen K Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes. Biotechnol Adv;28: [19] Koppram R., Tomás-Pejó E., Xiros C., Olsson L Lignocellulosic ethanol production at high-gravity: challenges and perspectives. Trends Biotechnol. [20] Hodge DB., Karim MN., Schell DJ., McMillan JD Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose. Bioresour Technol;99: [21] Adney B., Baker J Measurement of Cellulase Activities. Laboratory Analytical Procedure (LAP). Technical Report NREL/TP [22]National Renewable Energy Laboratory, Standard Biomass Analytical Procedures. procedures.html

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