Cellulose Isolation and Characterization of Green Seaweed C. Lentillifera from Halmahera, Indonesia

Abstract


INTRODUCTION
Seaweed is a benthic macroalgae that is plentiful in Indonesia and a source of essential nutrients.It is recorded that Indonesia is the second biggest production in seaweed in the world with the widespread cultivated land.Besides that, seaweed has fast growth, can be harvested all year round, and the cultivation process does not require large areas of land, fertilizers or pesticides (Salem & Ismail, 2021).There are approximately 911 species of seaweed in Indonesia, some with high economic value but only several species are being mass cultivated.Of all the species, one type of seaweed cultivated in Indonesia maritime waters is Caulerpa lentillifera.
C. lentillifera, or known as sea grapes is a type of green marine seaweed (Chlorophyceae) that is widespread mostly in the tropical regions in Asia, including Indonesia (Nguyen et al., 2011;Nofiani et al., 2018) This seaweed spreads to eastern Indonesia, Maluku waters, Bunguran Natuna Islands, and Nusa Tenggara (Razai et al., 2019).C. lentillifera grows in shallow seas and has general characteristics in the form of a thallus resembling a stolon, having rhizoids and assimilator with round ramuli resembling grapes.This commodity is mainly utilized in terms of food, medicine, or beauty industry because of the superior nutrition contains in it (Tapotubun et al., 2020).The chemical compositions of seaweed might be varied because of climate and environmental conditions where it was grown.So far, chemical composition of C. lentillifera from Halmahera, North Maluku, Indonesia has not been reported yet.
Furthermore, C. lentillifera contains polysaccharides as major components on the cell wall (Honwichit et al., 2022).Cellulose as one of polysaccharides in seaweed is potential for various applications, from conventional materials, such as the paper industry, to advanced materials such as bioplastic, composites, biomedical, and drug delivery (Fatriasari et al., 2019).Previous research showed that the cellulose content in Caulerpa sp. was 8.7% (Nurjanah et al., 2018).Cellulose is a polysaccharide which is consist of β-D-glucose units linked via (1→4) glycosidic bonds.Cellulose is mainly obtained from terrestrial plants, wood and non-wood sources (Joseph et al., 2023).In plants, cellulose is building the cell walls with hemicellulose and lignin (Fernández-Rodríguez et al., 2018).However, the presence of lignin makes the process of isolating cellulose in plants requires harsh chemical treatment causing slight cellulose degradation (Ververis et al., 2004).Recently, cellulose from seaweed has increased attention due to the absence of lignin in seaweed leading to purer cellulose obtained and also the extraction process under milder condition so that less degraded cellulose (Wahlström et al., 2020).To our knowledge, information about C. lentillifera and cellulose extracted from C. lentillifera have not been widely discussed in the available literatures.Therefore, this research intends to isolation of cellulose from C. lentillifera and characterization of C. lentillifera and its cellulose.

Sample Preparation
The collected seaweed was rinsed several times using tap water to remove any dirt in samples.Next, the samples were dried under the sun for 2 days followed by oven at 60ᵒc for 1 day to reduce water content.Dried C. lentillifera were grinded with blender to become coarse powder.The sample used for the extraction of cellulose was C. lentillifera powder that passes through a 16 mesh sieve.

Cellulose Extraction
The extraction of cellulose from C. lentillifera was done using method suggested by Bar-Shai (Bar-Shai et al., 2021) with some modification.C. lentillifera powder was soxhlet extracted for 6 hours at temperature of 80ᵒC using ethanol-benzene (1:2 v/v) solvent to remove extractives.Next, the samples were boiled in water with a ratio of sample and water was 1:20 (v/v) at 80ᵒC for 45 minutes to dissolve remaining impurities and increase the amount of insoluble fiber such as cellulose extracted from seaweed.The boiled water was discarded and the samples then purified by 4% H2O2 solution with ratio 1:80 (v/v) at 80ᵒC for 5 hours to eliminate any remaining green pigments and other contaminants including hemicellulose.Next, the supernatant was discarded and pulp cellulose was washed by water until neutral.Subsequently, cellulose was freezedried to become powder.Procedure of extraction cellulose was shown in Figure 1.Cellulose yield after freeze-drying procedure, was is calculated by Eq. (1).

FTIR Measurements
Characterization of powder and cellulose of C. lentillifera was conducted using the FT-IR ATR (Fourier Transform Infrared Attenuated Total Reflectance) method by Spectrum Two Perkin Elmer FTIR in the range of 4,000 cm −1 to 400 cm −1 to determine their functional groups.

X-Ray Diffraction (XRD) Measurements
XRD test of C. lentillifera powder and C. lentillifera cellulose was carried out using Shimadzu Scientific Instruments XRD-MaximaX 7000, at a range of 2 theta angle from 10° to 60°.The crystallinity index (Xc) was obtained using the Eq. (2).
where Ac is area of all the crystalline peaks, and Aa+Ac represents the total of all the crystalline and amorphous peaks.

Morphological Studies
Field emission-scanning electron microscopy (FE-SEM) was used to see the surface morphology of C. lentillifera powder.The test was carried out by using FE-SEM Thermo Scientific Quattro S, which was operating at a voltage of 1 kV at magnifications of 1000x.

Chemical Composition Analysis
In brief, C. lentillifera powder heated in the oven at 105ᵒC for 24 hours and 550ᵒC for 6 hours for moisture and ash contents analysis, respectively.
Extractives content analysis was carried out using soxhlet method with ethanol-benzene (1:2 v/v) solvent at temperature of 80°C for 6 hours.Holocellulose content analysis was performed by adding 25% NaCIO2 and glacial acetic acid into extractive-free sample, then heated at 80˚C.Afterward, the sample was washed with cold water and acetone, then dried to obtain hollocellulose.Then, into the holocellulose sample was added by 17% NaOH and aquades, and reacted for 45 min, then rinsed with 8.3% NaOH and aquades.Next, the sample was added to 10% acetic acid and neutralized to get α-cellulose.The hemicellulose content was obtained from the subtraction of holocellulose content by α-cellulose content.

Extraction of Cellulose
The extraction procedure of cellulose from C. lentillifera started with grinding to enhance the surface area for better chemical reaction (Sundari & Ramesh, 2012).The next step was soxhlet extraction using etanol-benzene solution to remove extractive, followed by boiling process to dissolve remining impurities.The boiling process resulted in an increase in insoluble fiber levels such as cellulose (Yuanita, 2010).The sample then purified by using hydrogen peroxide to eliminate any remaining green pigments and other contaminants including hemicellulose, which is more preferred in a biorefinery process from a green chemistry point of view.The resulting cellulose extracted from C. lentillifera was clear white with the yield of 31.13%, which is higher than cellulose obtained from other Caulerpa sp. up to 22.61% (Nurjanah et al., 2018).The extracted cellulose from C. lentillifera was presented in Figure 2.

Chemical Composition of C. Lentillifera
The chemical composition of C. lentillifera collected from Halmahera, Indonesia is presented in Table 1.The extractives, cellulose, and hemicellulose contents of C. lentillifera from Halmahera are higher than of C. lentillifera from Japan, except of ash content.The chemical component level differences of C. lentillifera might be influenced by their habitat, maturity level, and environmental conditions (Ito & Hori, 1989).The moisture content of C. lentillifera from Halmahera after drying process was 11.94±0.4%,whereas the ash content of C. lentillifera was 31.62±0,4%.The higher ash content was associated with the higher of mineral elements contained in C. lentillifera (Ratana-arporn & Chirapart, 2006).Extractives mostly consist of low molecular compounds soluble in liquids of low polarity such as fats, phenolics, resin acids, and waxes (Pecha & Garcia-Perez, 2020;Rabemanolontsoa & Saka, 2013).In this study, C. lentillifera contains 11.53±1,44% of extractives content.Total of α-cellulose and hemicellulose in C. lentillifera from Halmahera were 7.95±1,64% and 35.57±0.37%respectively.In seaweed, hemicellulose is the predominant carbohydrate.The majority of the hemicellulosic saccharides in seaweed may be caused by the polysaccharides from C. lentillifera were heteropolysaccharides, such as mannose, glucose, galactose, and xylose, which were the main constituents of hemicellulose (polyose) (Honwichit et al., 2022;Konishi et al., 2012;Long et al., 2020).

XRD Analysis of C. Lentillifera and Extracted Cellulose
From XRD graphs in Figure 3, there are some peaks with high intensities correspond to various salt crystals presented on the surface of C. lentillifera (Bulota & Budtova, 2015;Long et al., 2020).Peaks at 2Ɵ of 27.3ᵒ, 31.7ᵒ, 45.5ᵒ, and 56.6ᵒ were confirmed as diffraction peak of halite sodium chlorite (NaCl) crystal (Bao et al., 2017).The XRD patterns of potassium chloride (KCl) contain two main peaks at 28.5ᵒ and 40.9ᵒ (Ismail et al., 2022).
After going through the removal extractives, boiling, and bleaching process, the C. lentillifera cellulose XRD graphs, still showing some peaks of amorphous-like cellulose.The extracted cellulose from C. lentillifera displays three peaks at 2Ɵ of 13.5ᵒ reflection assigned to the (1 0 1) crystallographic plane, broad peak at 2Ɵ of 18.8ᵒ -19.9ᵒ reflection assigned to amorphous region of cellulose, and at 2Ɵ of 22.8ᵒ reflection assigned to the (0 0 2) or (2 0 0) crystallographic plane of cellulose I allomorph (Popescu et al., 2011).Cellulose extraction of other green seaweed, Ulva lactuca also resulted amorphous-like cellulose.It is concluded that the cellulose microfibrils as cell wall constituents, interwoven with xylan or mixed xylan-glucan polymers (Lahaye et al., 1994).The crystallinity index of cellulose from C. lentillifera was 32%.The low crystallinity of extracted cellulose probably because it is a mixtures of cellulose with a xylose-glucose (xyloglucan) polysaccharide, which  affected formation of cellulose crystals leading to a low crystallinity (Wahlström et al., 2020).Additionally, some seaweeds contain cellulose as a major component of their cell walls, which tends to make them highly crystalline, while others do not have cellulose as a main composition of their cell walls, resulting in low cellulose content and crystallinity in their cell walls (Mihranyan et al., 2004).

FTIR Analysis of C. Lentillifera and Extracted Cellulose
The infrared spectrum of C. lentillifera and extracted cellulose shown broad absorbance around 3400 -3300 cm -1 , which were associated to the hydroxyl groups stretching (Becerra et al., 2023).Moreover, there was peak at around 2919 cm −1 mainly represent to the symmetrical or antisymmetrical stretching of C-H on methyl and methylene from the aliphatic groups on cellulose or hemicellulose (Chaudhari, 2016).The absorbance at 1647-1645 cm -1 demonstrated stretching of the O-H as a result of the cellulose structure absorbing water (Arnata et al., 2020).The peak at 1520 cm -1 in IR spectra of C. lentillifera powder represent the absorption of amide band II (N-H bending coupled with C-N stretching) from proteins (Long et al., 2020).After treatment to obtain cellulose, The peak at 1520 cm -1 disappeared, which means that protein was disappear successfully from the cellulose fraction.
Tabel 2. IR spectrum of functional groups of the sample based on the absorption band (cm −1 ).C-0 (cm −1 ) C-5 (cm The increase in peak sharpness after bleaching at around 1419, 1324, 1165, and 908 cm −1 owing to increase of cellulose purity levels (Arnata et al., 2020).The peak at 908 cm -1 is associated with the cellulosic β-glycosidic linkages (Joseph et al., 2023).FT-IR spectra on C. lentillifera powder and extracted cellulose are presented in Figure 4.The typical functional groups with conforming bands are displayed in Table 2.

SEM Analysis of C. Lentillifera
Figure 5 shows SEM micrograph of green macroalgae C. lentillifera.C. lentillifera depicts large number of elliptically shaped diatoms, as if forming a split right in the middle like a coffee bean.Previous study showed similar result of C. lentillifera in rich colonization of elliptically shaped diatoms (Khan et al., 2022).

Figure 3 .
Figure 3. XRD analysis of raw material C. lentillifera and extracted cellulose.

Figure 4 .
Figure 4. FTIR analysis of raw material C. lentillifera and extracted cellulose.