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Moderate pretreatment strategies for improvement of reducing sugar production from oil palm empty fruit bunches

Pretreatment at mild condition is the strategy to minimize energy consumption, to reduce toxic compounds influencing to further process, and to obtain the high reducing sugar yield as fermentation substrate from cellulose and hemicellulose of oil palm empty fruit bunches (OPEFB). The yield still could be increased by optimizing pretreatment condition and varying several process alternatives. In this study, the effect of acetic acid addition and its corresponding concentration (5–15%), pretreatment temperature (115–125 °C), time (60–90 minutes), and two-stage pretreatment (0.01–1M NaOH pretreatment followed by autohydrolysis and autohydrolysis followed by 0.1–1% acetic acid pretreatment) were evaluated. The residual solid after pretreatment was then enzymatically hydrolyzed by Cellic CTec2. The result showed that the addition of acetic acid was able to increase the yield of reducing sugar. In addition, the reducing sugar yield could be still increased when the temperature was escalated, but the extended pretreatment time gave the decline of reducing sugar yield. This yield, however, was still lower than this from NaOH pretreatment followed by autohydrolysis. The maximum yield of this pretreatment could be attained by 1 M NaOH concentration at 125 °C for 60 minutes with solid loading 10% (0.71 g/g-OPEFB).


Introduction
In the palm oil mill, Oil Palm Empty Fruit Bunch (OPEFB) which constitutes 90% of biomass are discarded [1,2]. OPEFB is usually burned to generate heat and power or is converted to lower the additional value product such as compost and mulch. Even though OPEFB utilization has been conducted, this solid waste is still available in abundance. Thus, the other alternatives in the utilization of the OPEFB are required to minimize the environmental issue. The OPEFB consists of valuable constituents such as Cellulose (19-43%) and Hemicellulose (14-37%) [3][4][5][6][7]. Hydrolysis of both components can generate C6 sugar (glucose) from cellulose and C 5 (xylose and arabinose) as well as C6 sugars (glucose, mannose, and galactose) from hemicellulose that these monomeric sugars are the carbon sources for fermentation to produce various fermented products like biofuel [8], biochemical [9], and biomaterials [10]. To produce such products from  [30] with the results of 48.55% cellulose, 28.06% hemicellulose, and 23.39% lignin.

Pretreatment methods
The OPEFB was pretreated with three strategies and was compared with autohydrolysis: acetic acid pretreatment, acetic acid post-treatment, and NaOH two-stage pretreatment. 10 g of solid loadings were set on 10% (g/ml) in an Erlenmeyer 300 ml. Autoclave temperature was approximately set at 115 and 125 o C and being kept at 60 and 90 min, it was counted when it reached the desired temperature. The drying temperature was set at 53-55 o C in incubator for 24 hours. Pretreated OPEFB liquor was stored in two pieces of 50 ml centrifuge tube (Biologix) and 1 ml was used for DNS method to analyze sugar reduction. This experiment was conducted in duplicate. Performance of the pretreatment method was evaluated by the yield of xylose from OPEFB and hemicellulose conversion which were calculated from the obtained concentration of xylose in the hydrolysate solution, following equation [31]: Autohydrolysis pretreatment used water as a solvent. In acetic acid pretreatment, OPEFB was solubilized in acetic acid (Merck, p. a.) at 5 and 15% (w/w) solution. Erlenmeyer cap was made from cotton and aluminum foil, then it was heated in autoclave. Pretreated solid was dried for enzymatic hydrolysis, while liquor was analyzed with DNS method. In acetic acid post treatment, OPEFB would go through two steps. The first step was autohydrolysis. Pretreated solid was dried for enzymatic hydrolysis, while liquor was separately (1 ml) analyzed with DNS method and the other would go through the post-treatment. 30 ml of liquor was placed in Erlenmeyer and was added with 10% acetic acid to get 0.1, 0.5, and 1% (w/w) solution. DNS method was done in the end. The third strategy we used was NaOH two stage pretreatment. The first stage was a delignification of OPEFB with NaOH aqueous solution at 0.01, 0.1, and 1M. Pretreated solid was dried for second stage, while liquor was dumped. Pretreated solid then went through the autohydrolysis pretreatment. Solid fraction was dried for hydrolysis while liquor was analyzed with DNS method [29].

Hydrolysis
Enzymatic hydrolysis was conducted using Cellic® CTec2. The hydrolysis was performed in 100 mL Erlenmeyer flasks in a rotary shaker incubator that were set at 150 rpm for 72 h in room temperature. The solution was adjusted to 10% (w/v) solid to liquid ratio and pH 5 prior to hydrolysis, and 1 mL of enzyme was used in every 30 mL hydrolysis solution. Unless it was specifically indicated, the hydrolysis was conducted to the whole pretreated solution containing the pretreated OPEFB solid and the spent liquor [3].

Analytical analysis
The reducing sugar in the solution was analyzed using the UV Spectrophotometer. Aquoeous dilute samples were solubilized in a solution of 25% DNS-Acid. Sample tubes were tightly capped and kept in water bath at 100 °C for 5 min. After cooling the samples, it was measured at 550 nm to determine the absorbance based on standard curve equation.

Comparison Pretreatment Types: Autohydrolysis and Acetic Acid Pretreatment
Reducing sugar extraction by autohydrolysis was principally catalysed by acetic acid obtained from degradation of hemicellulose structure. Thus, further analysis to investigate the effect of acetic acid with the main purpose to increase reducing sugar yield was required. The result showed that the addition of acetic acid declined the amount of reducing sugar in spent liquor (figure 1). This might be caused by the further decomposition of reducing sugar during pretreatment. The impact was the decrease of reducing sugar recovery and inhibitor formation such as furfural and HMF. When the temperature and time were elevated by 10 °C and 30 minutes, respectively, the reducing sugar yield in spent liquor after autohydrolysis tended to go down. In the contrary, this from acetic acid pretreatment increased by 0.02 g/g-OPEFB. The using of this strategy was higher than Harahap [29] who obtained maximum xylose yield on 0.04 g xylose/g OPEFB. This proved that more severe condition of acetic acid could increase reducing sugar yield. The graph shows optimum condition while temperature set on 125 o C and time on 60 min. This result may obtain because of furfural production when the pretreatment set at 125 o C and 90 min, so yield are decreased [32]. The increasing temperature of 5% acetic pretreatment from 115 °C to 125 °C led to the decline of reducing sugar yield. Conversely, significant increase of the reducing sugar yield occurred in the higher concentration of acetic acid (15%) at 125 °C. The combination of concentration and temperature increase positively affected to the yield. When the time was extended from 60 to 90 minutes, however, the yield decreased for all pretreatment. In conclusion, 15% acetic acid pretreatment gave the highest reducing sugar yield (0.40 g/g-OPEFB) as compared to two other pretreatment.

Two stage pretreatment: autohydrolysis followed by acetic acid pretreatment
The yield of reducing sugar from autohydrolysis still could be increased by dilute acetic acid pretreatment accompanied with heating to the spent liquor. Figure 2 showed that the yield increased after autohydrolysed liquor was further treated by acetic acid pretreatment. The higher acetic acid concentration was used, the more reducing sugar yield was obtained at 115 °C for 60 minutes. This result showed that the spent liquor from autohydrolysis still contained oligomer of reducing sugar. Harahap [29] used xylanase for post-treatment of autohydrolysis, and the result showed the same tendency where the reducing sugar yield increased particularly for xylose. Hence, post-treatment of the spent liquor was needed. When autohydrolysis temperature was increased by 10 °C, the maximum reducing sugar from the post-treatment was achieved at 0.5% acetic acid (0.05 g/g-OPEFB). After autohydrolysis was performed at the longer duration (up to 90 minutes), however, no significant increase occurred. From this strategy, the use of acetic acid hydrolysis of spent oligomeric compounds in liquor can increase sugar yield, but it is not significant. It may cause by intrinsic property difference of cellulose and hemicellulose [32]. Vallejos [33] got bit higher productivity of xylose from sugarcane bagasse post-treatment strategy with 0.46 g sugar/g OPEFB. He uses 3%(w/w) of H2SO4. Lower productivity can caused of OPEFB has lower cellulose and hemicellulose compounds and lignin is higher [34].

Two stage pretreatment: NaOH pretreatment followed by autohydrolysis
Delignification is aimed to reduce lignin contents in OPEFB. Uses of NaOH must be sufficient, or the condensation reaction will be occurs [35]. When the lignin is reduced, access to cellulose are easier and sugar yield will increase. But, uses of lignin may cause hemicellulose removal [36]. The results of two-stage pretreatment are shown in figure 3. First stage pretreatment effect on yield not significantly increase, but the bigger impact is obtained from hydrolysis. It may cause by ease access for enzyme to cellulose [37]. The highest reducing sugar yield from this strategy obtained 0.73 g sugar/g OPEFB from 1M NaOH + Autohydrolysis at 125 o C and 60 min. Addition of NaOH concentration was significantly increase (p = 0.0031) on sugar yield, but on time and temperature are not significant (p = 0.244 and 0.375). Optimum lignin release and hemicellulose holding when OPEFB a first stage pretreated [38]. Our results higher than Zulkiple [39] has obtained fermentable sugar at 0.0439 g sugar/g OPEFB with using NaOH treatment in 10% solid loadings, 120 o C and 120 min.

Conclusions
Compared to OPEFB pretreatment using either one stage pretreatment and acetic acid two stage pretreatment, NaOH two stage pretreatment was more appropriate to be applied in particular for producing reducing sugar via enzymatic hydrolysis. The best pretreatment condition were obtained from NaOH two stage pretreatment at 125 °C for 60 min, giving the maximum reducing sugar yield of 0.73 g/g OPEFB. The difference temperature in this study showed an increased reducing sugar yield towards increased of temperature. However, difference time residence was declined the highest reducing sugar yield of the previous investigation.