Optimization of Saccharification and Fermentation Process in Bioethanol Production from Oil Palm Fronds Academic research paper on "Agriculture, forestry, and fisheries"

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Procedia Chemistry 16 (2015) 141 - 148

Optimization of Saccharification and Fermentation Process in Bioethanol Production from Oil Palm Fronds

Eka Triwahyunia*, Sri Hariyantib, Deliana Dahnuma, Muhammad Nurdinb, Haznan

Abimanyua

aResearch Centerfor Chemistry, Indonesian Institute of Sciences, Kawasan Puspiptek Serpong, Tangerang Selatan 15314, Indonesia bFaculty of Mathematic and Science, University of Halu Oleo, Kendari, Sulawesi Tenggara 93232, Indonesia

Abstract

Oil Palm Frond (OPF) is one of lignocellulosic biomass, which can be utilized as raw material for bioethanol production. Bioethanol is produced as alternative energy to substitute gasoline. There are four steps in bioethanol production from OPF, i.e pretreatement, saccharification, fermentation and purification process. In this study, optimization of saccharification and fermentation process for OPF was investigated. Two methods and the variations of enzyme concentration were carried out in the saccharification and fermentation process. Separate hydrolysis and fermentation process (SHF) and simultaneous saccharification and fermentation process (SSF) were conducted to produce ethanol optimally. Variations of enzyme concentration used in this process were 10, 20, 30 and 40 FPU/g substrate. The result shows that the highest ethanol concentration can be obtained in SSF process with 30 FPU/g substrate of enzyme concentration. The process produced 59.20 g/L ethanol (95.95% yield ethanol) at 96 h of SSF process.

©2015 The Authors.Publishedby Elsevier B.V. Thisis an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of Research Center for Chemistry, Indonesian Institute of Sciences

Keywords: Bioethanol, Oil Palm Frond, Separate Hydrolysis and Fermentation, Simultaneous Saccharification and Fermentation

1. Introduction

Indonesia is one of the largest producers of palm oil which has a production growth of Crude Palm Oil (CPO) during 2008-2012 of 7.65% per year1. In 2013/2014, the production of palm oil Indonesia was 30.5xl06 MT and estimated that will increase to 33xl06MT in 2014/20152. The rapid growth of palm oil production increased the amount of biomass residues. The biomass residues in palm oil plantation, among others, oil palm trunks (OPT), oil palm empty fruit bunches (OPEFB) and oil palm fronds (OPF). OPT is obtained when replanting, OPEFB is a waste of CPO production while OPF is obtained during pruning for harvesting fresh fruit bunch (FFB), therefore it is available daily. So that the most abundant biomass from oil plant plantation is not OPEFB or OPT but OPF. Kelly-Yong et al.3 reported that each hectare of oil palm plantation produces 10.88 tons of OPF on the average. Indonesia

* Corresponding author. Tel.: +6221-7560929; fax: +6221-7560549 E-mail address: ekatriwahyuni@gmail.com

1876-6196 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of Research Center for Chemistry, Indonesian Institute of Sciences doi: 10.1016/j .proche.2015.12.002

produce approximately 115.76 million tons of OPF for 2014/2015, which area planted estimates 10.64 million hectares2. Commonly, OPFs are left rotting between the rows of palm trees, mainly for soil conservation, erosion control and ultimately the long-term benefit of nutrient recycling4. OPF as a lignocellulosic materials that can also be utilized to produce various beneficial products such as biofuels, animal feeds, and healthy food products. Generally, lignocellulosic biomass consists of three main groups of polymer that are cellulose, hemicelluloses and lignin in which cellulose and hemicelluloses can be converted into biofuels5. The OPF has been reported consisting of 38.8% cellulose, 36.4% hemicellulose and 19.3% lignin6.

OPF for biofuels production become one of the interesting research topics to be developed by considering the abundant availability in nature. Biofuels are one of renewable energy for fossil fuel substitution in which the lack of fossil fuels has become an important issue in the world lately. OPF can be used as raw material for bioethanol that is a fuel substitute for gasoline or can be blended with gasoline. The process of bioethanol production can be through biochemical or thermochemical processes. Biochemical conversion uses biocatalysts such as enzymes and microbial cell to convert biomass first to an intermediate sugar stream and then to ethanol and co-product. Whereas thermochemical conversion reduces biomass to a fundamental chemical building block, syngas (CO, H2), that can be converted into ethanol7. Several researches about utilization of OPF for biofuel have been reported. Alkaline pretreatment conditions of OPF for the reducing sugar production has been reported by Sabrina et al.8, production of sugars from dried OPF fiber has been recently reported by Fazilah et al.9 and Goh et al.10, involving the conversion of cellulose and hemicellulose into glucose and xylose through hydrothermal treatment followed by enzymatic hydrolysis.

Bioethanol production from OPF using biochemical process consists of pretreatment, saccharification and fermentation process. The pretreatment process aims to break down the lignin structure and disrupt the crystalline structure of cellulose, so that the acids or enzymes can easily access and hydrolyze the cellulose11. Saccharification process purposed to convert cellulose into glucose using enzymatic hydrolysis and then glucose will be converted into ethanol using micro-organism on fermentation process7. Two method for saccharification and fermentation process will be investigated in this study, i.e separate hydrolysis and fermentation process (SHF) and simultaneous saccharification and fermentation process (SSF). SHF involves two sequential step, enzymatic saccharification and fermentation that allow working at optimal operating conditions for enzyme (50°C) and microorganisms (32°C). Whereas in SSF, glucose produced from hydrolysis is simultaneously metabolized by microorganism to produce ethanol, thereby alleviating problems caused by product inhibition12. This study will investigate the optimization of saccharification and fermentation process from OPF that includes the variation of process (SHF and SSF) and the variation of enzymes loading to determine the optimal amount of enzymes to obtain the highest ethanol yield.

2. Materials and Methods

2.1. Material

In this research, oil palm frond (OPF) was collected from PTPN VIII palm oil plantation, Malimping, Pandeglang, Banten. The enzymes were provided by Novozymes Korea Ltd that consist of Cellic® Ctec2 and Cellic® Htec2. The cellulase activity of Cellic® Ctec2 was measured by the Filter Paper Assay13 and was expressed as Filter Paper Unit (FPU). The enzyme activity was 144 FPU/mL for Cellic® CTec2. Commercial instant dry yeast Saccharomyces cerevisiae were applied to the fermentation process. All reagents used in this study were of analytical grade.

2.2. Pretreatment Process

OPF was chopped and milled into particle size ±3 mm for the length of fiber, then dried up to a moisture content about 10%. After that, OPF was pretreated using 10% NaOH solutions. The ratio of NaOH solutions and OPF was 5:1. Pretreatment process was carried out at temperature 150oC and 4-7 kg/cm2 of pressure for 30 minutes. OPF-treated was washed by water until neutral pH.

2.3. Saccharification and FermentationProcess

Optimization of saccharification and fermentation process on bioethanol production from OPF will be investigated using two process methods, i.e (a) Separate Hydrolysis and fermentation (SHF), and (b) Simultaneous Saccharification and Fermentation (SSF). The enzymes variation was also applied in the SHF and SSF process.

2.3.1. Separate Hydrolysis and Fermentation Process

SHF involves two sequential stages, enzymatic saccharification and fermentation. The saccharification process was carried out in a 250 mL Erlenmeyer flask in a shaking incubator (150 rpm, 50°C) for 72 h. The volume of slurry was 100 mL with concentration of substrate loading was 15% (g/mL OPF dry based). The OPF-treated and 0.05 M buffer citrate (pH 4.8) were loaded in Erlenmeyer flask. The variation of enzyme loading were 10, 20, 30 and 40 FPU/g substrate of Cellic® Ctec2 and 20% Cellic® Htec2 (this value was based on the amount of Cellic® Ctec2 loaded). Sample 1 mL was withdrawn from saccharification medium every 24 h for analyzing reducing sugars, glucose and xylose.

After 72 h of saccharification process, the hydrolyzate was added 1% (g/mL) dry yeast for fermentation process. The fermentation process was conducted at temperature 32°C, 150 rpm for 72 h in a shaking incubator. Sample 1 mL was withdrawn from fermentation medium every 24 h for analyzing product of ethanol, glucose and xylose remaining.

2.3.2. Simultaneous Saccharification and Fermentation Process

SSF involves the simultaneous process of saccharification and fermentation process. The enzymes and yeast simultaneous added at the beginning process. The process was carried out in a 250 mL Erlenmeyer flask in a shaking incubator (150 rpm, 32°C) for 96 h. The concentration of substrate was 15% (g/mL) at 100 mL total volume of slurry. The variation of enzyme loading were 10, 20, 30 and 40 FPU/g substate of Cellic® Ctec2 and 20% Cellic® Htec2 (this value was based on the amount of Cellic® Ctec2 loaded). 0.05 M citrate buffer was added in Erlenmeyer until the total volume of 100 ml (substrate, enzymes and buffer citrate) to keep the process conditions at pH 4.8. 1% (g/mL) dry yeast was added in SSF process. Sample 1 mL was withdrawn from SSF medium every 24 h for analyzing product of ethanol, reducing sugars, glucose and xylose remaining.

2.4. Analysis

Chemical component (cellulose, hemicelluloses and lignin) of OPF-untreated and OPF-treated were determined using standard biomass analytical procedures from the National Renewable Energy Laboratory (NREL)14.

The reducing sugar during saccharification process was analyzed using DNS assay method15. Reagent A was a mixture of 2.5 g DNS (3,5-dinitrosalicylic acid) and 50 mL NaOH 2 M, while Reagent B was a mixture of 75 g Potassium Sodium Tartrate (Rochelle Salt) and 125 mL of destiled water and was shaken under heating until total dissolution. Subsequently, reagent A was added into reagent B and homogenized under heating until dissolved completely, and after cooling, distilled water was added until the volume of the solution 250 mL. For analysis, an aliquot 500 ^g.mL"1 of the sample was added 1 mL of DNS, and shaken in vortex and boiled for 5 minutes. After cooling, the solution was added 3.75 mL of destiled water, and shaken again. Then, the solution was analised in spectrophotometer at 540 nm. The amount of reducing sugar released per ml was estimated from a standard curve prepared with known glucose concentration.

The product composition 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 80oC.

2.5. Ethanol yield calculation

The ethanol yield was calculated as:

% ceiluiose conversion =

[EtOH], - [EtOH],:

■*100%

0.51(/tb:<mMssH.Ill]'

Where:

[EtOH]f: Ethanol concentration at the end of SSF (g/L)

[EtOH];: Ethanol concentration at the beginning of SSF (g/L) which is zero

[Biomass]: Dry biomass concentration at the beginning of SSF (g/L), f: cellulose fraction of dry biomass (g/g) 0.51: convertion factor for glucose to ethanol based on stoichiometric biochemistry of yeast 1.111: converts cellulose to equivalent glucose

3. Result and Discussion

3.1. OPF Characterization

Generally, the whole branch of OPF consists of petiole and leaflets16. However, in this study, we used the OPF only from the petiole part, since the leaflet should remain in the plantation for soil conservation, erosion control and nutrient recycling. The composition of monomeric sugar and other major component in raw OPF and OPF after pretreatment is shown in Table 1. The pretreatment method used in this study was alkali pretreatment that has been identified as one of the best chemical pretreatment methods for delignification of lignocellulosic biomass17. The mechanism of alkaline pretreatment is believed to be saponification of intermolecular ester bonds crosslinking xylan hemicellulose and other components, for example, lignin and other hemicelluloses. The porosity of the lignocellulosic materials increases with the removal of these crosslinks18. The critical parameters for alkaline pretreatment are reaction temperature, time and alkali loading19. In this study, pretreatment process was conducted at temperature 150oC and 4-7 kg/cm2 of pressure for 30 minutes using 10% NaOH solutions.

Table 1 . Chemical Component of OPF

No. Component %OPF-untreated %OPF-treated

1 Cellulose 33.47 72.59

2 Hemicellulose 13.95 8.04

3 Lignin 30.16 14.09

4 Ash 0.23 0.06

5 Extractive 22.20 5.21

Based on component analysis in Table 1., cellulose was the main component in OPF followed by lignin, extractive and hemicellulose. After pretreatment process, the composition of OPF changed, i.e. decreased lignin and hemicelluloses content, while increased cellulose content. Delignification reached 53.28%, while cellulose content increased to 72.5 9%. Then, cellulose from pretreatment result will be converted into ethanol using SHF and SSF process.

3.2. Separate Hydrolysis and Fermentation Process (SHF)

SHF is one method of saccharification and fermentation process that involves two sequential steps: enzymatic saccharification and fermentation process. Enzymatic saccharification of OPF used cellulase and xylanase enzymes that consist of Cellic® Ctec2 and Cellic® Htec2. Cellic® CTec2 is complex enzymes that consist of cellulase, p-glucosidase, and hemicellulase whereas Cellic® HTec2 consists of endoxylanase with high specificity toward soluble hemicellulose and cellulose20. So, beside glucose as main product in sacchrification process, hemicellulose also can be converted to xylose. The process was conducted at a temperature 50oC for 72 h. After substrates are hydrolyzed to glucose and subsequently fermented to ethanol. The fermentation process used Saccharomyces cerevisiae at a temperature 32°C for 72 h. The result of SHF process can be seen in Fig. 1 and 2.

Fig 1. The Enzymatic Saccharification of OPF with variation of enzyme concentration; (a) 10 FPU/g substrate; (b) 20 FPU/g substrate; (c) 30

FPU/g substrate; (d) 40 FPU/g substrate

Fig 1. shows the result of saccharification process. Glucose, xylose and reducing sugar concentration was increasing during saccharification process. Reducing sugars (RS) are the end products of many biological processes and enzymatic reactions. Many polysaccharide-degrading enzymes are commonly assayed by quantifying the amount of RS released during the analysis21'22. Glucose, xylose and others derivative of cellulose and hemicelluloses include polysaccharides have been degraded by enzyme. So, in Fig 1. it can be seen that the concentration of reducing sugar was higher than glucose and xylose concentration. RS can be investigated by the DNS method employing glucose as the standard. DNS reacts with free carbonyl group of the RS under alkaline condition, forming 3-amino-5-nitrosalicylic acid, an aromatic compound with maximum absorption at 540 nm, allowing a quantitative spectrophotometer measurement of the amount of RS present23.

The highest concentration of glucose, xylose and reducing sugar was obtained at the 72 hour process. In 10 FPU/g substrate of enzyme concentration, the highest concentration of glucose, xylose and reducing sugar were 77.45, 10.7, and 120.63 g/L respectively. The highest concentration of glucose, xylose and reducing sugar were 88.79, 11.66 and 132.09 g/L for 20 FPU/g substrate of enzyme concentration. In 30 FPU/g substrate of enzyme concentration, the highest concentration of glucose, xylose and reducing sugar were 101.32, 13.83, and 154.59 g/L. Then, the highest concentration of glucose, xylose and reducing sugar were 100.45, 14.58, and 145.70 g/L. From these data, it can be seen that the products concentration almost same for 30 and 40 FPU/g substrate of enzyme concentration. Moreover, using 30 FPU/g substrate of enzyme can produce higher glucose and reducing sugar concentration than using 40 FPU/g substrate. So that the optimum of enzyme concentration used in the saccharification process was 30 FPU/g substrate.

Fig 2. shows the ethanol production and glucose reduction during fermentation process. The highest of ethanol concentration were 39.36 g/L (at 72 h process, 10 FPU/g substrate of enzyme concentration), 46.63 g/L (at 48 h process, 20 FPU/g substrate of enzyme concentration), 52.76 g/L (at 48 h process, 30 FPU/g substrate of enzyme concentration), and 48.97 g/L (at 24 h process, 40 FPU/g substrate of enzyme concentration). So that the highest of ethanol concentration can be produced in the 30 FPU/g substrate of enzyme concentration.

(<0 (d) Fig 2. The fermentation process of OPF with variation of enzyme concentration; (a) 10 FPU/g substrate; (b) 20 FPU/g substrate; (c) 30 FPU/g

substrate; (d) 40 FPU/g substrate

3.3. Simultaneous Saccharification and Fermentation Process (SSF)

Simultaneous saccharification and fermentation (SSF) process was conducted in this study. SSF believed have several advantages, i.e increase of hydrolysis rate by reducing end product inhibition of cellulose, lower enzyme requirement, higher ethanol yield, lower requirement for sterile conditions, shorter process time and cost reductions by eliminating expensive reaction and separation equipment24. In this process, The enzymes and yeast simultaneous added at the beginning process. Glucose produced from hydrolysis is simultaneously metabolized by microorganism, thereby alleviating problems caused by product inhibition in SSF process25. In Fig. 3 it can be seen ethanol production during SSF process. Fig 3. shows that the highest ethanol concentration was produced in SSF process using 30 FPU/g substrate of enzyme concentration. In this process, glucose released by the hydrolyzing enzymes is consumed immediately by the fermenting microorganism (Saccharomyces cerevisiae) present in the culture, and a low concentration of sugars is maintained in the media, thus reducing the problem of end product inhibition of cellulose24.

(<0 (d) Fig 3. The SSF process of OPF with variation of enzyme concentration; (a) 10 FPU/g substrate; (b) 20 FPU/g substrate; (c) 30 FPU/g substrate;

(d) 40 FPU/g substrate

The comparison of ethanol production from two methods of saccharification and fermentation process (SHF and SSF) and variation of enzyme concentration can be seen in Table 2.

Table 2. Ethanol production in variation of saccharification and fomentation process and enzyme concentration

No. Process Methods Variation of enzyme concentration Time of process (h) and Theoretical Final ethanol Ethanol

(FPU of Cellic® Ctec2/g substrate) temperature (°C) ethanol conc. conc. (g/L) yield (%)

1. Separate Hydrolysis and Fermentation (SHF) 10 72 h of saccharification (50 °C) 72 h of fermentation (32 °C) 61.70 39.36 63.79

20 44.63 72.34

30 51.74 83.86

40 47.97 77.75

2 Simultaneous Saccharification and Fermentation (SSF) 10 96 h of SSF (32 °C) 61.70 47.83 77.52

20 45.59 73.89

30 59.20 95.95

40 53.76 87.13

From Table 2. it can be seen that ethanol yield in SHF process lower than in SSF process. It may be caused by the accumulation of the hydrolysis products in the enzymatic process which causes feedback inhibiton of the cellulolytic enzyme system. Another major problem in SHF is microbial contaminations due to the longer incubation time in hydrolysis23. The highest ethanol concentration was 59.20 g/L in SSF process with 30 FPU/ g substrate of enzyme concentration during 96 h process., which it was different approximately 2.5 g/L with theoretical ethanol concentration (61.70 g/L). The ethanol yield obtained was 95.95%. From those data SSF process requires a shorter time and lower temperatures than SHF to obtain a higher ethanol yield. So that SSF process is an important challenges to produces ethanol more economically.

Conclusion

This study showed that simultaneous saccharification and fermentation process can produce ethanol optimally. The highest ethanol concentration can be obtained in SSF process with 30 FPU/g substrate of enzyme concentration. The process produced 59.20 g/L ethanol (95.95% yield ethanol) at 96 h of SSF process. Using appropriate method and dose of enzyme could increase ethanol concentration in saccharification and fermentation process.

Acknowledgement

The authors thank to Hendris Hendarsyah, Novita Ariam and Imi Fitria for invaluable assistances in this work. This research was funded by National Priority Project of of Indonesian Institute of Sciences (LIPI) of fiscal year 2014.

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