The Effects of Ozonolysis on Oil Palm Fruit Mesocarp Delignification

Oil palm fruit mesocarp (OPFM) is a solid by-product containing cellulose, potentially serving as a raw material for biofuel. The cellulose content of this solid by-product can be extracted through delignification. Therefore, this study aimed to investigate the application of ozone for OPFM delignification to break down lignin bonds in the material. During the analysis, ozonolysis delignification was influenced by particle size, oxygen flow rate, and reaction time. Ozone flowrate analyzed using the Iodometric method. Cellulose, hemicellulose, and lignin content of raw material and treated samples were analyzed using the α – cellulose, γ – cellulose, and the Kappa method. The results showed that by using a particle size of 100 mesh, and a flow rate of 2 Lmin -1 for 15 min, ozone de - graded 42.03% lignin, 15.89% hemicellulose, and concentrated 62.85% cellulose. SEM and FTIR results showed the removal of hemicellulose and lignin from OPFM with ozonolysis delignification. Furthermore, XRD analysis showed the crystallinity degree of the high cellulose yield.


INTRODUCTION
Oil palm fruit mesocarp (OPFM) is a solid waste from oil palm production, posing a significant environmental challenge.The palm oil industry produces a lot of OPFM as waste.Generally, these industries utilize OPFM only as fuel for boilers and are not fully utilized.Combusted OPFM causes environmental impacts without any more economical and sustainable utilization.Currently, Indonesia is the world's largest oil palm producer, exporting 55.6% of global palm oil demand by 2023 (United State Department of Agriculture, 2023).OPFM contains 60% cellulose, a potential source of biofuel, 17.9% hemicellulose, and 11% lignin (Simão et al., 2019;Sreekala et al., 1997).Cellulose is a renewable energy source that can be converted into glucose and fermented into bioethanol.However, the presence of lignin content requires delignification as the upper layer of lignocellulose covers the cellulose bonds, inhibiting the conversion ability of the catalyst.This shows that delignification accelerates cellulose hydrolysis, where the rate of lignin removal corresponds with the process efficiency.In this study, the delignification process uses the ozonolysis method by including ozone (O 3 ) as a catalyst to oxidize, dissolve, and destroy the lignocellulose cell wall (Keris-Sen and Gurol, 2017).Initially, ozone gas is flowed into ozonolysis reactor filled with OPFM, playing an effective role without producing toxic waste or damaging the structure of cellulose and hemicellulose during delignification process (Mardawati et al., 2018).Additionally, ozone is a strong oxidant and highly reactive in degrading lignin (Kamel et al., 2015), which is considered a clean and environmentally friendly technology (Sulfahri et al., 2020) capable of breaking cellulose bonds in biomass without including harmful chemicals (Wan and Saidina, 2021).
Based on these advantages, ozonolysis method is considered suitable for degrading lignin.
Ozone that is widely used in the delignification process is generated from the conversion of oxygen using an ozone generator.Furthermore, it flows into a fixed bed reactor containing OPFM mixed with distilled water to optimize the reaction between raw material particles and minimize waste (Bhattarai et al., 2015).this study, ozone was produced using a corona discharge ozone generator with model AZ-1000MG-G, 220 V/50 Hz AC voltage source.It is capable of converting oxygen into ozone with oxygen sourced from pure oxygen as input.The input flow is connected to an oxygen supply tube that has been equipped with a flowmeter.Therefore, the flow rate of the input oxygen can be adjusted or varied to achieve the optimum flow rate.Ozone production was analyzed using the Iodometric method.During the delignification process, ozone comes into contact with OPFM in an ozonolysis reactor, with particle size significantly influencing the contact area of ozone to the sample.The particle size of tiny samples can simplify ozone to degrade lignin, showing the need for physical treatment before the delignification process.This study aimed to analyze the effects of ozonolysis delignification on lignin degradation to enhance concentrate cellulose.Therefore, the contents of cellulose, hemicellulose, and lignin were analyzed before and after delignification in this study.Specifically, cellulose, hemicellulose, and lignin content were analyzed using the α-cellulose, γ-cellulose, and the Kappa method, respectively.SEM, FTIR, and XRD analyses were also conducted to analyze the effect of ozone delignification on OPFM.

Materials
OPFM as raw material was collected from Bangka, Indonesia.The samples collected were cleaned and dried to reduce the moisture content, followed by cutting into small pieces to facilitate the pulverization process and blending to reach the size of 80 mesh and 100 mesh.The pro-analyst chemicals used included NaOH, distilled water, potassium dichromate, H 2 SO 4 , ferroin indicator, ferrous ammonium sulfate, KMNO 4 , KI, sodium thiosulfate, and amylum.

Ozonolysis delignification
OPFM powder was weighed for 5 g each and wetted with 10% H 2 O by weight of the material pH of 3 (Hermansyah et al., 2021).The wetted sample was put into the ozone reactor and the flow rate was adjusted with variations of 1 Lmin -1 , 2 Lmin -1 , and 3 Lmin -1 .The reaction time variation in the ozone reactor was varied for 5 min, 10 min, and 15 min, followed by connecting the power cord to the ozone generator.Initially, the voltage regulator was set to the lowest point (zero), to maintain low voltage (220 V).The oxygen gas flow rate was adjusted as required and kept stable.This was followed by a gradual increase of mains voltage until an intended value was reached.Before ozonizing the biomass, ozone content was measured by the iodometric method and a 2% KI solution was put into the first analysis vessel.During the bubbling stage, ozone-containing gas was poured into the analysis tube and the flow of ozone-containing gas was stopped.Subsequently, a portion of the solution present in the analysis tube was titrated with Na 2 S 2 O 3 solution using a starch indicator.Ozone that did not react in the reactor flowed into the second analysis vessel filled with 2% KI solution.The ozone content of ozonation reaction was analyzed as in the first analysis vessel using the Iodometric method.The delignified sample was soaked with 100 mL of 5% NaOH at room temperature for 60 min, filtered, and spelled with hot aquadest until the pH was neutral.Furthermore, each sample was dried using ovens for 24 hours at 105 ºC to achieve 0% moisture content.

Ozone production analysis
The 200 mL KI solution that passed through ozone was added with 10 mL H 2 SO 4 to form a darker-colored solution, followed by the immediate addition of H 2 SO 4 and titrated with 0.2 N Na 2 S 2 O 3 .Furthermore, 0.5 ml of 2% amylum was added after the color turned pale yellow, causing the solution to turn dark blue.The titration was continued until the color changed colorless and the volume of Na 2 S 2 O 3 was measured.The formula used to compute the ozone concentration of the sample solution is expressed as follows: (1)

SEM, FTIR, and XRD analysis
The raw material and product were examined using a Tescan Vega 4 LMH scanning electron microscope to obtain the surface morphology.Furthermore, the samples were examined using a Prestige 21 FTIR instrument in the range of 500-4000 cm -1 to show the absorption band of cellulose, hemicellulose, and lignin.X-ray diffraction was used to show the high amount of cellulose structure, and determine crystallinity degree as well as particle size.

RESULTS AND DISCUSSION
The effect of oxygen flow rate and reaction time on ozone production Oxygen as feedstock was fed into the ozone generator, producing ozone, which flowed to the analyzer column.This column was filled with KI solution and ozone was dissolved in KI.To achieve optimum production, variations were made to the flow rate and reaction time.The oxygen flow rate used varied at the rates of 1, 2, and 3 Lmin -1 with variations in reaction time of 5, 10, and 15 min.As shown in Figure 1, the highest ozone concentration produced was 9.56 mg/L at a reaction time of 15 min with an oxygen flow rate of 1 Lmin -1 .The longer ozonolysis time caused more ozone to form, which was abundant at a smaller oxygen (O 2 ) flow rate of 1 Lmin -1 .This phenomenon was attributed to the equilibrium of the ozone formation process, where the reaction proceeded rapidly because the limiting reagent was the amount of ozone, instead of the oxygen.Increasing the oxygen flow rate can increase the ozone capacity, as more oxygen molecules are available for ozone formation.However, excessive flow rates may result in a lower ozone capacity due to the formation of less reactive ozone species or the generation of unwanted byproducts (Yulianto et al., 2019).Furthermore, the reaction time can significantly impact the ozone concentration and lead to higher energy efficiencies.Longer reaction times generally lead to higher ozone concentrations, as more oxygen molecules have the opportunity to react and form ozone (Shrestha et al., 2015).Regardless of the amount of oxygen in the generator, the quantity of ozone formed depended on

The effect of ozonolysis delignification on OPFM
OPFM was delignified using ozone with flow rate variations of 1 Lmin -1 , 2 Lmin -1 , and 3 Lmin -1 for 5, 10 and 15 min.After this process, cellulose, hemicellulose, and lignin content were analyzed, as shown in Table 1.The results showed that the particle size of the sample affected the reduction of lignin content in OPFM, leading to a high amount of cellulose.Furthermore, ozonolysis delignification process was effective in degrading lignin and hemicellulose bonds by 60.21% in the 100-mesh sample for 10 minutes using 2 Lmin -1 oxygen.The amount of lignin removed for each sample is shown in Figures 2 to 4, where the maximum degraded content was 45.98% in the 100-mesh sample with an ozone flow rate of 1 Lmin -1 for 15 min of reaction.A previous study stated that the amount of biomass surface area when reacting with ozone significantly affected lignin degradation (Rodolfo Travaini et al., 2016).
The results showed that the largest lignin removal was obtained in the sample with a 100-mesh size.The particle size of OPFM has a very significant effect on achieving the optimum ozonolysis process.The smaller particles interact with ozone, making the degradation of lignin becomes more preferable, improving the ability of ozone to break lignin bonds in OPFM, and resulting in reduced surface area.Therefore, ozone reacted more easily with OPFM to break lignin bonds, as observed in a previous study on  Furthermore, lignin degradation increases as the amount of ozone reacted increases due to the powerful oxidizing properties of ozone.This is because a higher flowrate can provide a more consistent and intense exposure of ozone to the lignin, leading to a higher rate of radical formation and subsequent lignin degradation (Baig et al., 2015;Anggoro et al., 2022).When lignin comes into contact with ozone, it can be degraded rapidly, leading to the break of aromatic rings and the generation of carboxyl groups (Wan et al., 2021).This process is facilitated by the formation of radicals, which are very reactive species and react close to where lignin was formed.The radicals can degrade cellulose at acidic pH but primarily react with lignin leading to its destruction.Therefore, increasing ozone consumption can generate the lignocellulose structure by breaking lignin bonds (García-Cubero et al., 2009; Rodolfo Travaini et al., 2016) and degrading the carbohydrate bonds in the biomass (Binder et al., 1980).Ozone also interacts with lignin breakdown products, producing aliphatic acids and low  (Neely, 1984).Ozone mostly dissolved lignin and mildly solubilized the hemicellulose portion, resulting in minimal cellulose loss (Travaini et al., 2015).Lee et al conducted a study on coastal Bermuda grass and observed that a 31.3%increase in the amount of lignin degraded raised the ozone reaction by 26.4% (Lee et al., 2010).The quantity of ozone that reacts determines the influence of reaction time on the ozonolysis process.Therefore, the efficiency of the delignification process will continue to increase until optimum time is reached (Schultz-Jensen et al., 2011).
In this study, the optimum reaction time for ozonolysis delignification was 15 min with 42.03% of lignin removal.The reaction time influences the extent of lignin degradation achieved during the treatment process, with longer reaction times generally leading to more extensive lignin removal (Baig, 2022;Hermansyah et al., 2021).However, this increased the expense of delignification.This study took less reaction time with high lignin removal compared to prior studies.The 50% delignification was accomplished using 2.7% (w/w) wheat straw over a 2 h reaction (García-Cubero et al., 2012).The ozonolysis delignification also treated bagasse sugarcane and yielded 92.5% cellulose after 120 min (Rodolfo Travaini et al., 2013).The ozone delignification reaction involves the insertion of an oxygen atom into carbon-hydrogen bonds, thus disintegrating the lignin structure (Vitasari, 2008).Ozone is highly reactive toward compounds with double bonds and functional groups, making it highly effective in breaking down the complex structure of lignin (M'Arimi et al., 2020).The interaction of ozone with the OPFM surface has a significant impact on the adsorption process due to the chemical and structural changes that occur during the ozonation process.The ozone treatment breaks down the lignin, which is the most outermost layer of the biomass, allowing for better access to the cellulose and hemicellulose components.This increased accessibility enhances the adsorption process by providing a more porous and reactive surface for the adsorbate to bind to (Suhada et al., 2021;Valdés et al., 2002).

SEM analysis
SEM analysis was treated on OPFM samples before and after ozonolysis delignification process.This analysis was carried out to show the difference in morphology of lignin degradation in the sample.Based on the results, the untreated OPFM showed an absence of voids in the sample.Figure 5 shows that OPFM fiber has a hollow top layer indicating degradation of lignin bonds.This phenomenon showed the loss of lignin bonds wrapping the fiber had been partially lost due to ozonolysis delignification process, exposing the cellulose surface (Lim and Zulkifli, 2018).Additionally, the formation of voids showed the expansion of the inner surface due to missing hemicellulose bonds (Elanthikkal et al., 2010).The asymmetric and smoother appearance of OPFM fiber showed the effectiveness of ozone in degrading lignin and hemicellulose.The composition of untreated and treated OPFM show in Table 2. Based on these compositions, the amount of carbon decreased about 34.91% after ozonolysis pre-treatment, but the amount of oxygen increased about 41.94%.The reduced carbon content after pre-treatment is because a number of carbon bonds contained in lignin and hemicellulose have been degraded.carbon elements that are degraded after the ozonolysis process are dissolved in the washing stage after leaving the ozonolysis reactor.The remaining carbon was the carbon bond contained in the concentrated cellulose.Figure 5 shows the pores in the treated sample which indicates that the top cover layer has been exposed.The sample pores look larger because the hemicellulose content in the treated sample has been reduced and resulted in the formation of larger pore cavities in the treated sample.The dissolving lignin on the surface of the sample after pre-treatment was indicated by the reduction of 34.91% carbon and the oxygen increased by 41.92%, as shown in Table 2.The removed carbon was a lignin constituent component that had been degraded and dissolved in the washing process after the ozonolysis pre-treatment which aimed to neutralize the pH of the sample.

FTIR analysis
OPFM sample was analyzed using FTIR with a range of 500-4000 cm -1 , as shown in Figure 6.The results showed strong absorption band ranging from 2800-3400 cm -1 , which indicated the presence of hydrogen bond (O-H) absorption at the peak of 3335.69 cm -1 (identification of cellulose bonds).Strong intensity of absorption was also observed in the wave range of 3331.59-3335.69cm -1 in charge of absorbing O-H valence vibrational bonds on cellulose intermolecular (Schwanninger et al., 2004).This shows that ozone was able to degrade lignin bonds as the outer layer of lignocellulose well.Strong absorption at the peak of 1030.47 cm -1 showed the capacity of the wave to absorb C-H aromatic bond deformation.This showed the presence of symmetrical CH 2 bonds with hydrogen (O-H).IR wave 2849.19 cm -1 absorbed valence vibrations of CH 2 bond asymmetry, while at 2916.93 cm -1 absorbed valence vibrations of CH 2 symmetry with hydrogen bonds (O-H) (Schwanninger et al., 2004).The strong

XRD analysis
XRD analysis of OPFM presented in Figure 7 showed that the sample observed has several peaks at 2θ = 21.76,43.58, 44.61, 72.60, and 88.23.The crystallinity degree of OPFM was 95.94% with a particle size of 56.06 nm.In this study, the crystallinity degree of cellulose obtained was significantly higher compared to the results of Chieng et al.The crystallinity degree of OPFM with NaOH and continued with H 2 SO 4 delignification at 80 °C for 4 h was obtained at 77.8% (Chieng et al., 2017).Yasim-Anuar et al. obtained a crystallinity degree of 56% by delignifying OPFM using 5% (w/v) NaClO 2 at 70 °C for 90 min (Yasim-Anuar et al., 2017).The high crystallinity degree showed the elevated amount of cellulose structure to the total particles in the sample.The cellulose with higher crystallinity can be more easily processed and recycled, potentially reducing the environmental impact of the production process (Carolin et al., 2023).Furthermore, the higher levels of crystallinity in cellulose enhance mechanical properties, such as strength, flexibility, and resistance to degradation (Vanderfleet and Cranston, 2021).

CONCLUSIONS
In conclusion, this study showed that ozonolysis delignification process on OPFM was effective in degrading lignin.In this treatment, OPFM particle size and oxygen flow rate affected the interaction between ozone and lignocellulose bonds.The results showed that ozone could dissolve in moist lignocellulose and adsorb into particles at smaller particle sizes.In this study, ozone degraded lignin by 42.03% in the 100 -mesh, showing the significant effect of particle size and oxygen flow rate in lignin degradation of OPFM bonds.The degraded lignin bonds and cellulose content in OPFM were proven by SEM, FTIR, and XRD.

Acknowledgment
This study was funded under the contract between the Directorate General of Higher Education, Research, and Technology Ministry of

Figure 1 .
Figure 1.The effect of oxygen flow rate and reaction time on ozone concentration

Figure 2 .Figure 3 .
Figure 2. The effect of oxygen flow rate and reaction time on 60 mesh OPFM ozonolysis delignification

Figure 4 .
Figure 4.The effect of oxygen flow rate and reaction time on 100 mesh OPFM ozonolysis delignification

Table 1 .
Cellulose, hemicellulose, and lignin content of OPFM after the ozonolysis delignification process

Table 2 .
Compositions of untreated and after treated OPFM