Examination the Hydrolysis Feasibility of OPEFB Biomass Using Aspergillus niger as Cellulase Enzyme-producing Fungus

produce use biofuels alternative energy more environmentally friendly and sustainable 12 ） Biofuel be defined as fuel derived from biomass and produces relatively little CO 2 gas so it is more environmentally friendly 13 − 15 ） . One such biofuel is bioethanol derived lignocellulosic biomass waste a very attractive raw material for researchers to continue to explore and optimize the production process an alternative energy Abstract: The objective of this study was to obtain optimization results from the biological hydrolysis of Oil Palm Empty Fruit Bunches (OPEFB) using Aspergillus niger ( A. niger ) BIOTROP 2173 isolated from grain. Optimized hydrolysis parameters include temperature, pH and time. The hydrolysis process was carried out by growing A. niger on OPEFB powder (± 30 mesh) through two schemes, namely hydrolysis on OPEFB pretreatment with 10% NaOH and hydrolysis on OPEFB non-pretreatment. The optimization results show that the best hydrolysis process of A. niger BIOTROP 2173 occurs in OPEFB pretreatment. The optimum conditions for temperature, pH and time obtained are 40℃, 6 and 24 hours, respectively. Although the amount of reducing sugar produced was lower than the OPEFB non-pretreatment, the performance of the cellulase enzyme during the hydrolysis process of OPEFB pretreatment was very good, with a fast hydrolysis rate. These results indicate that the performance of A. niger BIOTROP 2173 in the hydrolysis process is influenced by the pretreatment stage. The optimum conditions obtained then became a reference in the production of reducing sugar based on A. niger BIOTROP 2173. The amount of reducing sugar produced from OPEFB pretreatment was 0.94 mg.mL –1 , while OPEFB non-pretreatment was 15.83 mg.mL


Introduction
Nowadays, the biggest challenge of many countries is the problem of energy crisis which is a priority for people s survival 1,2 . The projected demand for energy in 2050 almost triples due to an increase in accordance with human development. It seems that the energy problem will still be the main topic that must be solved together 3,4 . Efforts to obtain alternative energy have long been explored to reduce dependence on fossil energy resources 5 7 . In 2018, energy demand in Indonesia is very dependent on fossil fuels, this is evidenced by the total final energy consumption without traditional biomass of 114 million tons of oil equivalent MTOE derived from 40 of transportation, 36 of the industry, 16 of households, 6 of the com-can be renewable and sustainable development 16 18 . Cellulose which has been separated from lignocellulosic biomass can be converted to glucose through the hydrolysis process and then converted to bioethanol through the saccharification process. OPEFB is palm oil waste with abundant lignocellulosic content 19,20 . In our previous study, we have implemented the OPEFB biomass pretreatment process to produce bioethanol even though quantitative bioethanol production is still low 16, 21 25 . We are pleased to develop OPEFB biomass waste because it is abundant biomass waste and every 1 ton of CPO production produces OPEFB lignocellulose waste which reaches 1.1 tons 26,27 . The application of renewable biofuel energy sources needs to be increased given the limited sources of fossil fuels and expensive fuels. OPEFB lignocellulose waste is abundant biomass material that can be applied to obtain biofuels that are cheaper, environmentally friendly, and as a renewable and sustainable material 28 30 . In the production of bioethanol, we need to go through several steps such as pretreatment, hydrolysis, fermentation and product separation 31,32 . So in this study, we focus on the optimization of hydrolysis under biological science related to utilizing β-glucosidase enzyme derived from A. niger before implementing in the saccharification process fermentation .
Several hydrolysis processes have been widely studied by researchers using enzymatic, acid and base conditions 33,34 . They have advantages like short time duration, low-cost, and simple preparation. In fact, the loss is to damage environmental pollution, where acid and base solutions can damage the aquatic environment and threaten the life of aquatic biota. Meanwhile, major impediments to exploiting the commercial potential of cellulose materials through enzymatic degradation pathway are slow reaction rates, lack of an ideal reactor system and complexity of interfacial heterogonous hydrolysis influenced by various factors e.g. structure and composition of cellulosic materials, cellulase adsorption and desorption, enzyme inhibition by cellobiose and glucose 35,36 . However, enzymatic hydrolysis makes for a safer environment because it is prone to denaturation. Thus, the hydrolysis process is very important to study because lignin content in lignocellulose may hamper the hydrolysis process. Hydrophobic of lignin can disturb the hydrolysis by hindering the enzyme to reach the cellulose and by binding to the enzyme and acting as inhibitors. In a previous study by 37 reported that the hydrolysis process was carried out by growing A. niger on OPEFB which functions to produce cellulase and increase the level of sugar consumption. This is reasonable considering that sugar is the main source of microorganisms 38,39 . In addition, cellulase from A. niger has high β-glucosidase activity. This is in agreement with 40 where cellulase enzyme from A. niger has the β -glucosidase activity of 168 U/mL which was ten-fold as compared to that in celluclast 1.5 litres. 36 have also reported that using A. niger EFB1 can reduce sugar and polyose production in proportion to the reduction in OPEFB and hemicellulose cellulose content by 89.32 and 48.17 after enzymatic degradation under optimal conditions. In this work, optimization of the OPEFB hydrolysis process by using A. niger as a producer of cellulase enzymes was studied. In general, we went through several steps, namely the pretreatment process under alkaline conditions NaOH and then hydrolysis process using A. niger Fig. 1 . Study of temperature changes, pH, time, and cellulase enzyme activity were evaluated to review the effectiveness of the hydrolysis process using OPEFB to produce glucose components. The method we use in the production of cellulase enzymes is Solid-State Fermentation. Although this method is the same as reported by 41 44 , however we use different sources. The source of the cellulase enzyme that we use is grain residue. This residue is waste that can pollute the environment. So this condition makes our study more economical. Nevertheless, the results obtained from this study are able to provide the Fig. 1 Schematic diagram of the hydrolysis process by comparing non-pretreatment and pretreatment of OPEFB using A. niger, 1 physical pretreatment, 2 pretreatment process, 3 10 NaOH solution prepared, 4 separation of black liquor and residue, 5 purification of residue, 6 hydrolysis process using A. niger, 7 glucose analysis using UV-Vis spectrophotometer.
best picture of the biological process of OPEFB hydrolysis.
In this study, we also specifically studied the effect of OPEFB pretreatment on A. niger performance in producing reducing sugars.

OPEFB pretreatment
Firstly, physical treatment has been carried out by destructing OPEFB into small fibrous, dried, and sifted to 30 mesh filter. This condition we call as non-pretreatment unpretreated OPEFB because it is not yet included in the pretreatment process Fig. 1 . Subsequently, the pretreatment of OPEFB has applied by weighing as much 500 g non-pretreatment OPEFB and adding a 10 NaOH solution by using a chemical explosive reactor CHEMEX with a temperature of 150 for 3 hours. After that, it is filtered to separate the OPEFB residue and black liquor by using separation pump then the residual OPEFB pretreatment is washed by distilled water until pH 7 and pressed with a hydraulic press to reduce the water content 10 . It was analyzed to observe the chemical constituents such as lignin, cellulose, and hemicellulose compounds by using high-performance liquid chromatography HPLC refers to the National Renewable Energy Laboratory NREL procedure 45 .
Where the OPEFB residue 1 mm diameter and water content 10 was weighed as much as 0.3 g and added into glass tube. Then, it added with 72 H 2 SO 4 and hydrolyzed for 2 hours at a temperature of 20 . Every 15 min., it is homogeneous using a vortex tool and transferred into a 100 mL Schott bottle which containing 42 mL distilled water and re-hydrolyzed in an autoclave for 60 min. at 121 . Afterwards, the sample was cooled to ambient temperature and filtered with a Buchner filter and 0.45 µm filter paper. The filtrate is neutralized with CaCO 3 until the pH neutral and then filtered using a 0.2 µm pore filter using a syringe into the HPLC vial autosampler. The filtrate was analyzed for cellulose and hemicellulose using HPLC Aminex HPX 87H column 300 7.8 mm at 65 with a mobile phase of 5 mM H 2 SO 4 and a flow rate of 0.6 mL.min 2 and a refractory index detector refractory index detector waters 2414 T: 40 10 .

OPEFB hydrolysis using Aspergillus niger
In this study, we use of A. niger isolated from grain which is obtained from Phytopathology Laboratory, Southeast Asian Regional Center for Tropical Biology SEAMEO BIOTROP , Bogor. In this step, OPEFB residue from the pretreatment process was weighed 250 g then inserted into 8 Erlenmeyers and added 0.05 M citrate buffer. It is inserted into an autoclave for 1 hour with a temperature of 120 . Subsequently, it is put into a laminar airflow system to apply the hydrolysis process using A. niger. It is entered into the incubator shaker at a temperature 30 , 35 , 40 , 45 and the time variation of 24, 48, and 72 hours. These steps also apply for non-pretreatment OPEFB Fig. 1 6 . After that, the decantation process was applied to separate the water content from OPEFB to the glass tube and covered with aluminium foil which aims to maintain the safety of liquid from spilling when the centrifuge process. Finally, we determine the cellulase enzyme activity of the OPEFB sample using a UV-Vis spectrophotometer, in which the sample was added the 3.0 mL DNS solution and heated on warm water at a 30 for 30 minutes to increase the rate reaction. Absorbance is determined based on linearity curves for various concentrations of glucose standards Fig. 3 .

Chemical constituents of OPEFB pretreatment process
In the initial stage, we determined the chemical content of OPEFB biomass before committing of the pretreatment process. The purpose of this stage is to represent the chemical constituents contained in the OPEFB biomass waste. Table 1 shows the chemical constituents of OPEFB waste before going through the pretreatment process. The high cellulose content of 36.59 explains that the largest chemical constituents in OPEFB are cellulose, lignin, and hemicellulose. The same result was shown by 37 that before pretreatment, chemical contents such as cellulose, hemicellulose, and lignin were also examined with values of 35.2 , 17.9 , and 24.1 , respectively. According to 25 also reported that in OPEFB biomass waste contains high lignin and cellulose compounds, this of course provides the advantage to be converted into renewable bioenergy materials. Unfortunately, the lignin content in lignocellulose can also inhibit the hydrolysis process. Therefore, chemical pretreatment must be carried out to increase the cellulose content and increase swelling, increase the internal surface of the cellulose and decrease the degree of crystallinity and further increase lignin disruption. Lignin has hydrophobic properties so that it interferes with the hydrolysis process by inhibiting the enzyme to react cellulose and by binding to the enzyme and acting as an inhibitor. In addition, it can also reduce cellulose hydrolysis by passively binding to cellulolytic enzymes. Analysis of ash content as complementary data representing undetectable chemical constituents in OPEFB biomass. The mechanism of enzymatic hydrolysis of OPEFB into glucose reducing sugar is a complicated process because the cellulase enzyme consists of at least endoglycanase EG , cellobiohydrolase CBH , and β-glucosidase βG . However, this complex process can be simplified into two stages as shown in Fig. 2. The first stage is a heterogeneous reaction between the insoluble substrate cellulose and the dissolved enzyme. This stage produces oligosaccharides which are dissolved through the synergistic action of CBH and EG which is considered as the control step for the whole hydrolysis. The second stage is a homogeneous reaction to the breakdown of oligosaccharides into glucose which is catalyzed by βG.

Optimization of the hydrolysis process 3.2.1 Standard curve to analyze of reducing sugar
Determination of the standard curve to obtain reducing sugar content in each variation is to vary the concentration of 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mg.mL 1 from a standard solution of glucose 100 mg.L 1 . The purpose of this step is to determine the level of reducing sugar in each variation by plotting linear regression to the absorbance obtained from the UV-Vis spectrophotometer analysis. Based on Fig.  3, it can be seen that linear regression is obtained with the equation y 0.1629x-0.1665 and a correlation coefficient of 0.998. This study applies the Somogyi-Nelson method, which is a method for determining reducing sugar levels. In principle, reducing sugars will be reduced by Cu 2 to Cu ions, then it will reduce arsenomolybdate compounds to form greenish-blue complexes. The Somogyi-Nelson method is more specific if it is used to determine reducing sugar levels in samples that have a mixture of sugar compounds in it, compared to the anthrone-sulfate method 46    B , optimization at both hydrolysis temperature shows that the highest reducing sugar content is at 40 . It is due to A. niger included in the mesophyll microbe group with optimum growth at 35-37 . This is evidenced by an increase in temperature at 45 which reduces the production of reducing sugar content, this is also certainly correlated with cellulase enzyme production. In the same studies by 40 and 47 , the cellulase enzyme showed high stability at 37 , however, the activity of β -glucosidase enzyme decreased by approximately 28 , 20 , and 45 after 48, 72 and 96 h incubation at 50 , respectively. So it can be concluded that temperature optimization is carried out well at 40 which plays a role in maintaining the stability of enzymes such as endoglycanase and β-glucosidase as variables that are responsible for the hydrolysis process.
Although at this stage we studied the hydrolysis process at various temperatures as shown in Fig. 4 , our focus was on a temperature of 40 to observe the OPEFB hydrolysis process by A. niger BIOTROP 2173 with a duration of 24, 48 and 72 hours. This is a form of data comparison in optimizing the duration of time required for the hydrolysis process using A. niger. Based on Fig. 4 A , it can be seen that reducing sugar content in non-pretreatment tends to decrease with increasing time. This condition can be related to the optimum performance time and the amount of lignin in the OPEFB non-pretreatment. The optimum time occurs from hydrolysis for 24 hours with the amount of reducing sugar produced is 5.33 mg.mL 1 . The decrease in the amount of reducing sugar can be caused by the need for A. niger for the reducing sugar itself. A. niger requires reducing sugars for growth and the production of other metabolic products. The decrease in reducing sugar concentration is further caused by the growth of A. niger in the aerobic phase before the anaerobic stage which will then convert the sugar into ethanol. As in Figs. 4 A and 4 B also shows the optimum time produced from hydrolysis for 24 hours with the amount of reducing sugar produced is 2.42 mg.mL 1 . Although from the graph it can be seen that the hydrolysis for 72 hours produces higher reducing sugars, this time is not the optimum hydrolysis time. Apart from what we mentioned earlier that A. niger requires reducing sugars for growth and other metabolism, it is necessary to think about that A. niger under certain conditions will produce reducing sugars. This condition occurs at 72 hours of hydrolysis. As for the decrease in the amount of reducing sugars at 48 hours of hydrolysis, this can be related to A. niger need for reducing sugars.

pH variations test
Variation in pH has been carried out by correlating the optimization temperature with reducing glucose content as tested using a UV-Vis spectrophotometer. In addition, native enzyme or protein monomer undergoes a conformational change commonly called denaturation caused by pH changes, organic solvents, heat, protein concentration, shaking, or the presence of other proteins or chemical compounds. In some cases, lead to the exposure of sticky hydrophobic areas. These areas increase the propensity of the monomer to aggregate or stick to each other, causing it to become active The active monomers or unfolded proteins begin to aggregate forming oligomers that ultimately lead to insoluble fibrils or amorphous aggregates 40 . Figure 5 shows how the pH is related to the amount of reducing sugar until it reaches the optimum condition. In general, both OPEFB hydrolysis non-pretreatment Fig. 5 A and OPEFB hydrolysis with pretreatment Fig. 5 B showed the same pattern, namely hydrolysis activity was slow at a more acidic pH pH 5-6.5 for OPEFB . non pretreatment and pH 5-5.5 for pretreatment OPEFB . Even though the hydrolysis pattern is the same, the amount of reducing sugar and the optimum pH produced is different. The highest amount of reducing sugar was produced from Fig. 4 Temperature variation in the hydrolysis process by using A. niger, A non-pretreatment process, B pretreatment process.
hydrolysis of OPEFB non-pretreatment which was 15.82 mg.mL 1 . As for the hydrolysis of OPEFB by pretreatment, the optimum reducing sugar was 0.94 mg.mL 1 . The optimum pH of these two treatments, respectively, is pH 7 and pH 6. The interesting thing from Fig. 5 is how the relationship between hydrolysis time and pH and reducing sugar. The hydrolysis time can be related to the hydrolysis rate. So that, even though Fig. 5A shows the highest sugar hydrolysis results, but the hydrolysis rate is slow. Unlike the case with Fig. 5B which shows a fast hydrolysis rate until optimum conditions are obtained. This situation is due to removing some of the lignin due to the pretreatment process using 10 NaOH. The drastic decrease in the amount of reducing sugar after the optimum pH illustrates that the loss of the active side of the cellulase enzyme from A. niger is due to denaturation. This denaturation occurs slowly. 3.2.4 Effect of temperature on cellulase enzyme activity Furthermore, cellulase enzyme activity was analyzed in each treatment by adding 1.0 mL of sample into a glass tube followed by the insertion of 6.0 mm Whatman paper and the addition of 1 mL of 0.05 M citrate buffer. It was put on a water bath to be heated to 30 for 30 min and added 3.0 mL DNS solution then analyzed using a UV-Vis spectrophotometer. Figure 6 shows the relationship between hydrolysis temperature and cellulase enzyme activity. As it is understood that temperature will affect the work of enzymes in the production of reducing sugars. Many factors cause enzyme activity to be disrupted, one of which is the high lignin content of OPEFB. This is shown in Fig. 6 A , where at 24 hours of hydrolysis the enzyme activity shows an irregular pattern. This is influenced by the EFB used, namely the non-pretreatment EFB. When looking at the data shown in Table 1, you will find that non-pretreatment OPEFB contains 26.53 lignin. A very different result is shown in Fig. 6 B , where the OPEFB used was the 10 NaOH pretreatment OPEFB. The use of OPEFB pretreatment causes a regular pattern of enzyme activity.  This can be observed at the hydrolysis temperature of 40 , which is the optimum temperature of this work.

Conclusions
Based on these results, we can conclude the optimization of hydrolysis to the next step for the saccharification process to produce bioethanol can be using A. niger to produce high glucose levels at 40 with a pH value of 6-7. We also report that high reducing sugar levels are found in non-delignification of OPEFB. Meanwhile, the addition of A. niger can cause a decrease in glucose levels due to the lignin content in lignocellulose can inhibit the hydrolysis process. Lignin has hydrophobic properties so that it interferes with the hydrolysis process to react with cellulose and by binding to enzymes and acting as inhibitors.