Interaction of Chlorella vulgaris: Cell attachment on nanocellulose-based hydrogel for sustainable microalgae cultivation

Wan Jusoh, W. N. L., Sajab, M. S., Mohd Yasin, N. H., Abdul, P. M., and Takriff, M. S. (2025). "Interaction of Chlorella vulgaris: Cell attachment on nanocellulose-based hydrogel for sustainable microalgae cultivation," BioResources 20(1), 201–218.

Abstract

This study explores the potential of nanocellulose-based hydrogel for microalgae cell attachment. The hydrogel, composed of cellulose nanofibrils (CNF) and carboxymethyl cellulose (CMC), indicates feasibility in biomedical and cell culturing applications. It qualifies as an excellent substrate for 3D printing bioink utilizing the liquid deposition modelling technique. The growth of Chlorella vulgaris cells in Bold Basal Media (BBM) solution exhibits similar trends for both the standard culture and the culture media incorporating the CNF/CMC hydrogel. It was observed that the ionic-bond crosslinkers of H₂SO₄ and CaCl₂ in hydrogel formation were more conducive to cell adhesion compared to using covalent-bond crosslinkers of epichlorohydrin. Microalgae cell attachment exhibited the increase of green hue on the hydrogel surfaces, which shows the potential of cell to grow in the presence of CNF/CMC hydrogel.

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Interaction of Chlorella vulgaris: Cell Attachment on Nanocellulose-Based Hydrogel for Sustainable Microalgae Cultivation

Wan Nazihah Liyana Wan Jusoh ,^a,b Mohd Shaiful Sajab ,^a,b,* Nazlina Haiza Mohd Yasin ,^c Peer Mohamed Abdul ,^a,b and Mohd Sobri Takriff ^d,a,b

DOI: 10.15376/biores.20.1.201-218

Keywords: Adhesion; Cellulose; Crosslinker; Chlorella vulgaris; Hydrogel

Contact information: a: Research Center for Sustainable Process Technology (CESPRO), Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia; b: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia; c: Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM, Bangi, Selangor, Malaysia; d: Department of Mechanical and Nuclear Engineering, College of Engineering, University of Sharjah, United Arab Emirates;

*Corresponding author: mohdshaiful@ukm.edu.my

GRAPHICAL ABSTRACT

INTRODUCTION

Biomaterials have emerged as a prominent field of study in recent years because of their unique properties and wide-ranging applications. These materials can exist in both active and passive states, and they can be either naturally derived or biologically produced (Troy et al. 2021; Liu et al. 2023). Their remarkable compatibility with living organisms and biological systems has led to their extensive use in the biomedical field, particularly in tissue engineering and drug delivery (Langer 2000; Jusoh et al. 2022; Liu et al. 2022). Alongside the growing interest in biomaterials, hydrogels, a soft material with high water content that mimics the extracellular matrix, have also gained significant attention (González-Díaz and Varghese 2016; Norioka et al. 2021). Hydrogels find applications in diverse areas such as biomedical, agricultural, sensor technology, and ecological preservation (Pyarasani et al. 2018; Correa et al. 2021).

Cellulose-based hydrogel, a groundbreaking addition to the hydrogel family, is derived from cellulose and its derivatives. It is produced from local resources rich in cellulose such as kenaf, cotton, oil palm, microalgae, and bacteria (Hua et al. 2020; Naomi et al. 2020; Chopra and Manikanika 2022; Yap et al. 2023). Despite solubility and hydrophilicity challenges (Ciolacu et al. 2020; Zou et al. 2022), these hydrogels are gaining attention due to their excellent properties such as high mechanical strength, biocompatibility, quick degradation rate, and the fact that they are safe (Kabir et al. 2018; Cui et al. 2019; Zainal et al. 2021; Ban et al. 2022; Chen et al. 2022). The application of hydrogel is intrinsically linked to biological entities and living cells, which necessitate a high-water content, biocompatible material with minimal toxicity (Mantha et al. 2019; Dattilo et al. 2023). Some studies have been conducted to understand the interaction of cellulose with living cells. Even though the attachment of living cells on the cellulose plant cell wall may not be optimal, it facilitates cell-to-cell interaction, supports bacterial attachment, and aids bacterial tolerance (Jusoh et al. 2022).

The viability and growth potential of cells can be assessed through their attachment to the hydrogel surfaces, which also provides insights into the toxicity of the hydrogel. Cell growth is environment-dependent and cannot thrive in harsh and highly toxic conditions. Several studies have explored cell attachment potential on cellulose hydrogels from various sources such as birch kraft pulp, diethylaminoethyl cellulose, seaweed, nata de coco, and Acetobacter culture (Tan et al. 2016; Johns et al. 2018; Loh et al. 2018; Pajorova et al. 2020; Bar-Shai et al. 2021; Vel et al. 2022). Bacterial cellulose hydrogel demonstrates effective cell attachment, viability, and cell transfer for keratinocytes and fibroblasts, making them suitable for wound healing applications. The hydrophilic nature of hydrogel enhances cell attachment (Loh et al. 2018). Meanwhile, for the growth and attachment of marine macroalgae, the prepared scaffolds were found to exhibit high topographical cues affecting cell attachment and interaction, thus demonstrating non-toxic properties with increased cell viability using fibroblasts (Bar-Shai et al. 2021).

Despite numerous studies focusing on the interaction of hydrogel with cells, there are still gaps in understanding the prospects involving cellulose-based hydrogel especially the exploration of cell attachment using cellulose, those derived from oil palm biomass. The conditions for growth and the survival ability of cells with the cellulose-based hydrogel are yet to be identified. Therefore, this work aims to understand the potential of cellulose-based hydrogel in providing a conducive environment for cell growth and promoting the attachment of microalgae cells, specifically Chlorella vulgaris. The oil palm biomass was chosen as the raw material for hydrogel fabrication due to its strength, low cost, and high availability. The C. vulgaris is cultured and grows in conjunction with the cellulose-based hydrogel for eight days, and the optical density (OD) of the media and color effect of the hydrogel is recorded. The impact of hydrogel crosslinkers of ionic-bond, (H₂SO₄ and CaCl₂) and covalent-bond (epichlorohydrin (ECH)) on microalgae attachment is also examined. It was discovered that the growth of microalgal cells within encapsulated hydrogel promoted the growth duration due to easy access and high circulation of nutrients.

EXPERIMENTAL

Materials

Cellulose was extracted from the raw material, oil palm empty fruit bunch (OPEFB) fiber, which ranges in size from 106 to 500 µm. The preparation of cellulose and cellulose ink involved the use of 90% formic acid, 30% hydrogen peroxide (H₂O₂), iron (0) sourced from iron (II) sulfate heptahydrate (Fe₂SO₄·7H₂O), lithium hydroxide, urea, sodium carboxymethyl cellulose, sulfuric acid (H₂SO₄), and calcium chloride (CaCl₂), all obtained from Merck, Darmstadt, Germany. The Bold Basal Medium are the combination of macronutrient, alkaline ethylenediaminetetraacetic acid (EDTA) solution, acid iron solution, boron solution, and trace metal solution by following the previous study (Anderson 2005).

Preparation of Nanocellulose-based Hydrogel

The fractionation of cellulose and lignin from oil palm empty fruit bunch (OPEFB) fibers was conducted using an organosolv extraction process, as established in previous studies (Sajab et al. 2019; Mohan et al. 2021). Initially, lignin was extracted from OPEFB using 90% formic acid in a three-neck flask equipped with a condenser. The extraction was performed at a 30:1 ratio for 2 h at 90 °C. Excess organosolv lignin was filtered through a vacuum filter, and the pretreated pulp was washed with deionized water. To purify the cellulose from the pretreated pulp, undissolved lignin and hemicellulose were removed by catalytic oxidation using 5% w/v of 30% hydrogen peroxide and 10 mg/L of Fe(II) at 90 °C for 24 h. The purified cellulose pulp was then separated and thoroughly washed with deionized water until it appeared white.

Defibrillation of cellulose was achieved through mechanical shearing using a high-speed homogenizer (IKA T25 Digital, Staufen, IKA Germany). A 0.7 wt% cellulose suspension was fibrillated at 25,000 rpm in controlled cycles for a total of 30 min, ensuring the system temperature remained below 70 °C to prevent thermal hydrolysis of cellulose. For the preparation of CMC ink solution, 4.5 wt% of CMC was mixed with deionized water at 40 °C and stirred until fully dissolved. The prepared solutions of CNF and CMC were then vigorously stirred using a high-speed homogenizer (IKA T25 Digital, Staufen, IKA Germany) at 5,000 rpm for 10 min until a complete solution and thorough mixing were achieved.

Nanocellulose-based Hydrogel Formation

A liquid deposition modeling (LDM) was selected for 3D printing of cellulose-based hydrogel at various concentrations, given its aptitude for extruding ink from the liquid phase. The paste extruder (Structur3d Printing in Kitchener, Canada) was integrated with commercial 3D printer (Ultimaker 2+, Ultimaker, Geldermalsen, Netherlands). The design was created using computer-aided design (CAD) and sliced with Ultimaker Cura 4.5 (Ultimaker, Geldermalsen, Netherlands) following to the Discovery configuration. All printings were conducted using a nozzle with a diameter of 0.84 mm.

In the formation of hydrogel, two types of crosslinkers were utilized: 3% H₂SO₄ and CaCl₂, and epichlorohydrin (ECH). For the regeneration process using 3% H₂SO₄ and CaCl₂, the 3D-printed material was immersed in H₂SO₄ for several hours, followed by CaCl₂. Concurrently, the ECH was combined with the CNF/CMC solution prior to the printing process at the volume of 5% from the total solution. After the printing was completed, the 3D-printed material was left to dry at room temperature until it was completely dry. All the 3D materials were rinsed with distilled water before being used in subsequent experiments.

Preparation of C. vulgaris Inoculum

Bold’s Basal Medium, the nutrient source for C. vulgaris cells, was prepared as per a previous study (Anderson 2005). All materials and tools were sterilized using an autoclave at 121 °C for 15 min. Subsequently, 10% of the C. vulgaris stock culture was added to the 1.0 L sterilized BBM. The cells in this inoculum were allowed to grow under white light and aeration using air pump. Cell growth using the OD value is fitted into a kinetic model equation, which applies the cell mitosis approach and cell agglomeration method (Beruto et al. 2014; Paladino and Neviani 2020).

In the microalgal cell attachment study, the 3D-printed nanocellulose-based hydrogel was immersed in the microalgal culture, following the details outlined in Table 1 for different crosslinkers and culture conditions. The experiment was carried out over eight days at room temperature. For microalgal culture conditions in the BBM medium, it was conducted under two different media conditions: new culture and media with an OD of 0.8. However, for encapsulation conditions, the experiment was performed only in the new culture. This condition was applied for both types of crosslinkers. The OD of the culture and the image of the 3D-printed nanocellulose-based hydrogel were recorded daily.

Table 1. Formulation of 3D-printed Nanocellulose-based Hydrogel and Microalgae Culture Condition

Characterization

The OD of the culture media was analysed using a spectrophotometer (DR1900, HACH, Loveland, CO, USA) at the wavelength of 750 nm and the color changes by a digital camera (Griffiths et al. 2011; Scarponi et al. 2021). The structures of microalgae and CNF/CMC hydrogel were examined using optical microscope (B-380 series, OPTIKA microscope, Bergamo, Italy) and a field emission scanning electron microscope (FESEM) (Merlin Compact, Zeiss Pvt Ltd, Oberkochen, Germany). Prior to analysis, the samples were placed on an aluminium mount and coated with iridium using a sputter coater to avoid electrical charging. A computer aided imaging analysis is conducted by using open source, Fiji software (https://imagej.net/software/fiji/), with the incorporation of processing package, ImageJ (Version 2.15.1) for quantitative analysis of the cell growth. ImageJ is the image processing software commonly used for visualizing, validating, and quantifying the image data into more understandable form according to the experimental needs. The image and color of the CNF/CMC hydrogel and microalgal cells were identified and classified based on their color and tones (Carbone et al. 2017). The color channel is set depends on the desired color analysis and is presented in the black and white image.

RESULTS AND DISCUSSION

CNF/CMC Hydrogel

The extracted CNF and hydrogel CNF/CMC were characterized by morphology to understand the biomaterial structure. Figure 1(a) shows the CNF micrograph in which the structure was more dispersed and formed fibrils like a spider web (Sajab et al. 2019). The observed size of the CNF fibrils was smaller compared to cellulose, showing that the size reduction occurred during the mechanical disintegration process. The production of CNF was also evidenced by the TEM analysis with the observed size in between 15 and 50 nm, which is also supported by previous study (Kargarzadeh et al. 2018). The SEM image of CNF/CMC hydrogel is shown in Fig. 1(c) after the regeneration process. High porosity structure of hydrogel can be seen clearly in Fig. 1(d) from TEM analysis. The increase of pores in the hydrogel will help for a better absorption and attachment based on higher surface area available (Annabi et al. 2010).

Fig. 1. Morphology analysis after cellulose extraction (a) CNF (SEM); (b) CNF/CMC (SEM); (c) CNF (TEM); (d) CNF/CMC (TEM).

In this study, all formulated hydrogels were 3D printed with a simple structure. A square shape with a central hole, measuring 15 × 15 mm², was selected for the cell adhesion experiment. Figure 2 depicts the 3D-printed nanocellulose-based hydrogel prior to the crosslinking process. The 3D-printed nanocellulose-based hydrogel post-crosslinking, using two types of crosslinkers: ionic-bonded (H₂SO₄ and CaCl₂) and covalent-bonded (ECH) crosslinkers. The H₂SO₄ and CaCl₂ crosslinkers were applied after the 3D-printing process by immersing the 3D-printed nanocellulose-based hydrogel into the crosslinking solutions. In contrast, ECH was incorporated into the CNF/CMC hydrogel ink formulation prior to the printing process.

This procedure resulted in a minor alteration in the hydrogel’s ink for 3D printing. Consequently, the formulation ratio of CNF to CMC had to be adjusted from 4.5% CNF/4.5% CMC to 6% CNF/5% CMC to ensure better printing and shape fidelity. The 3D-printed nanocellulose-based hydrogel was left to dry at room temperature until a constant weight was observed. For both crosslinkers, a washing process was necessary to eliminate any undesired chemicals before proceeding with the next experiment.

Fig. 2. 3D-printed CNF/CMC hydrogels in square shape for cell adhesion and different types of crosslinking reactions

Microalgae Growth Cells

The microalgal species, C. vulgaris, was selected for this study due to its potential health benefits as a dietary supplement, broad range of applications, and adaptable cultivation (Allaguvatova et al. 2019; Bito et al. 2020). The growth of C. vulgaris in BBM was monitored over 20 days by recording the OD and color of the culture media. The growth curve, plotted in Fig. 2(a), was derived from normal cultivation using 10% (v/v) of the initial C. vulgaris stock culture. The lag phase was bypassed, and the culture directly entered the log phase due to the suitability of microalgae to adapt in the BBM. The curve has a similar pattern with the sigmoid shape based on the microbial growth, which was predicted based on the Eq. 1 shown below (Beruto et al. 2014; Paladino and Neviani 2020),

(1)

where c₀ is the initial inoculum concentration (g/L), k₁, is the specific rate constant, and k_ɑ, is a specific rate constant representing the total average probability that cells have to form clusters. The exponential phase lasted approximately 11 days before transitioning to the stationary phase. This indicates that the microalgae actively grew for 11 days, with a remarkable increase in OD reading from 0.2 to 1.2. The stationary phase saw only gradual changes in OD, from 1.2 to 1.5, over the course of nine days since it already achieved zero growth during this period.

The results were further corroborated by the color changes observed in the culture media, as depicted in Fig. 2(b). The initial pale-yellow color on day 0 transitioned to dark green by day 8, a hue that persisted until day 11. From day 12 onwards, the culture media began to display a brownish color, eventually turning completely brown by day 18. This color shift indicates a decline in microalgae cell growth as the green pigment in the cell was disrupted and turned brown. This brown colour also indicates cells that are non-viable due to nutrients deficiency and environmental changes (Ganuza et al. 2016; Valdez et al. 2018). As shown in Fig. 2(a), during the stationary phase, the C. vulgaris cells exhibited a smaller gap in OD readings over several days due to interrupted cell growth. In the subsequent cell adhesion experiment, an OD of 0.7 to 1.2 was maintained, indicating that the C. vulgaris was in the exponential phase and exhibited a vibrant green color. Moreover, the capability of microalgae to grow also was explored by the addition of crosslinkers into culture media, as shown in Figs. 2(a,b). The presence of CaCl₂ reduced the growth of microalgal cells, as indicated by lowering the OD value in which it only reached 0.6 on day 10 and gradually decreased afterwards even though the culture media was able to turn green. Meanwhile, the presence of H₂SO₄ and ECH causing the death of microalgae cells on the same day was shown by the decrease in plotted OD value and colour changes in Fig. 3(a,b). The findings from the toxicological study showed that some materials used in the fabrication of hydrogel will interfere and are not suitable for cell growth. The prepared hydrogel needs to undergo a thorough cleaning process before being used in the cell applications.

Fig. 3. The observation of (a) optical density and (b) color intensity of the growth of C. vulgaris in BBM, CaCl₂, H₂SO₄, and ECH for 5 to 20 days

Under the optical microscope, the cellular structure of C. vulgaris is spherical, exhibiting a green coloration attributed to the presence of chlorophyll, as depicted in Fig. 3(a). Further analysis using FESEM provided high-resolution micrograph images, offering an intricate view of the C. vulgaris cells’ surface, as shown in Fig. 3(b). The FESEM images confirmed the preservation of the microalgae cells’ morphology, with their characteristic cell shape and size remaining consistent.

Fig. 4. The (a) optical microscope and (b) micrograph images of C. vulgaris

Effect of CNF/CMC Hydrogels in Microalgae Cultivation

Cell attachment refers to the ability of a single cell to adhere to other cells or materials. In this context, the study focuses on the ability of microalgae cells to adhere to cellulose hydrogel. This study helps in understanding the interactions and behavior of cells with biomaterials. The surface of the cellulose hydrogel plays an important role in providing suitable conditions for cell attachment. The 3D-printed cellulose hydrogel, with the presence of two different crosslinker, dual crosslinker (H₂SO₄ and CaCl₂) and ECH, was immersed in microalgae culture to study the cell attachment capability.

The growth of C. vulgaris at different culture conditions alongside the 3D-printed nanocellulose-based hydrogel was monitored based on OD readings and the color of the hydrogel over an eight-day period. In the study of the impact of CNF/CMC hydrogels, the cultivation was divided into three scopes, each designed to explore different aspects of microalgae growth in relation to CNF/CMC nanocellulose-based hydrogel. The CNF/CMC nanocellulose-based hydrogel was tested for its impact on the growth of microalgae in a fresh culture of C. vulgaris (10% of the original culture). Subsequently, the attachment of cells on the hydrogel surface was observed using initial media at exponential phase. C. vulgaris is cultivated until it reaches an exponential phase before being transferred and combined with the 3D printed hydrogel across different categories. The encapsulation of cells within the hydrogel matrix was analyzed by injection of 1.0 mL of C. vulgaris within the hydrogel to study the cell-hydrogel interactions.

Figures 5(a) and 5(b) display the OD readings of the effect of microalgae growth in the presence of CNF/CMC with different formulations and crosslinkers. By using crosslinkers, H₂SO₄ and CaCl₂, (Fig. 5(a)), the readings began to reach 1.0 to 1.2 on day 4 until day 6, which is earlier compared to the normal growth. However, for the ECH crosslinker, the 10% stock culture directly increased at the early stage of cultivation with the reading of 0.1 – 1.0 (Fig. 5(b)). The growth rate was observed to increase more rapidly compared to H₂SO₄ and CaCl₂ especially for CNF-2. However the OD readings of CNF/CMC-2 and CNF/CMC/BBM-2 hydrogels only showed an increase up to 0.8, possibly due to incomplete washing process of hydrogel. The hydrogel acts as a scaffold and facilitates the initial cell attachment during the culture process, which resulted in a significant increase in growth rate (Salehi et al. 2024). Increases in the cell growth rate cause growth limiting factors to occur earlier especially in terms of nutrient sources. This also contributes to the faster transition from exponential phase to the stationary phase.

Figure 5(c) presents the physical appearance of the 3D-printed nanocellulose-based hydrogel after cultivation. This was indicated by the greenish color on the biomaterial surface, which signifies the presence of microalgal cells. Cell attachment on the CNF/CMC hydrogel was more pronounced with the use of dual crosslinking, H₂SO₄ and CaCl₂, compared to ECH. The presence of green colour on the hydrogel surface could be clearly observed, especially for CNF/CMC-1 and CNF/CMC/BBM-1. In the case of the different hydrogel crosslinkers, although ECH crosslinker produced a more rigid 3D-printed nanocellulose-based hydrogel, it also prevented the microalgae from attaching to the surface, as shown by the lack of color changes. Only a small green spot of microalgae is visible at the fracture point of the hydrogel. This suggests that the microalgal cell adhesion on the CNF/CMC hydrogel was more pronounced in the presence of H₂SO₄ and CaCl₂ than ECH and could be related to the surface condition of the hydrogel. From the literature it is stated that materials with hydrophobic properties and high surface roughness tend to attract cells for attachment (Chang and Wang 2011; Ferrari et al. 2019; Majhy et al. 2021). This may explain why H₂SO₄ or CaCl₂ as crosslinker produced the hydrogel with higher surface roughness and hydrophobicity than ECH. However, the surface characteristic has not yet been analyzed. From the collected images, the results clearly show that the new culture medium aids in cell attachment. A higher intensity of green color can be clearly observed, especially in the CNF/CMC hydrogel with H₂SO₄ and CaCl₂, dual crosslinking.

Fig. 5. The optical density of the new culture media for 8 days with different hydrogels crosslinker of (a) H₂SO₄ and CaCl₂, (b) ECH crosslinking and (c) physical appearance of 3D printed nanocellulose-based hydrogel

Microalgal Cell Adhesion on CNF/CMC Hydrogels

In this context, the study focuses on the capability of microalgal cells to attach to a nanocellulose-based hydrogel. This experiment aids in understanding the interaction and behavior of the cells with the biomaterial. Figures 6(a) and 6(b) present the cell attachment impacts on the hydrogels in the exponential phase culture medium. The optical density was maintained between 0.7 to 1.2 as the C. vulgaris is in the active phase and showed a deep green colour. As shown in Fig. 6(a,b), the OD reading declines in the early 3 days before it started to increase, possibly because the microalgae cells adaptation with the new environment in the presence of hydrogel. However, cells reactivated after the adaptation period in which early 3 days. This phenomenon may indicate an attachment interaction between the microalgae and the substrate. The surface of the nanocellulose-based hydrogel played a crucial role in providing suitable conditions for cell binding.

Fig. 6. The optical density of the exponential phase culture media for 8 days with different hydrogels crosslinker of (a) H₂SO₄ and CaCl₂, (b) ECH crosslinking and (c) physical appearance of 3D printed nanocellulose-based hydrogel

Moreover, adhesion of C. vulgaris was observed in this study based on the color of the hydrogel after being immersed in culture media (see Fig. 6(c)). However, culture media in exponential phase exhibited minimal to no color alteration from day 1 to day 6. Notably, the new culture conditions of CNF/CMC and CNF/CMC/BBM (Fig. 4) exhibited a higher color intensity. The adherence of C. vulgaris escalated with the cell growth in the culture medium. As the cells proliferated, they sought out available surfaces to latch onto, facilitating their growth. However, when cell growth was consistent and the OD remained in the exponential phase, cell adhesion became less preferred, as optimal growth had been attained and cell division began to wane. As depicted in Fig. 6(c), C. vulgaris cells demonstrated a preference for the CNF/CMC hydrogel over CNF alone. These experimental findings were supported by some previous study on the high potential of microalgal cell attachment using different biomaterial, as summarized in Table 2.

Table 2. The Capability of Cell Attachment on Different Substrate Material and Microalgae Species

The image analysis of microalgal cell attachment on 3D-printed CNF/CMC hydrogel was performed by using ImageJ software as shown in Fig. 7. The attachment of microalgal cells on the 3D-printed biomaterial surfaces can be visualized by isolating and analyzing the green channels within the image data, which reveal the presence of the characteristic green pigments found in microalgae. These pigments, typically chlorophyll, are indicative of microalgal cells, and their detection through the split green channels serves as a confirmation of cell attachment on the biomaterial substrate. Previous studies have mentioned the changes of thin yellow layer to green layer as the growth of cells corresponds to the area of cells coverage generated by imaging data (Berner et al. 2015; Carbone et al. 2017). Whiter color observed on the CNF/CMC/BBM-1 formulation compared to CNF/CMC-1 which explains that the presence of BBM in the biomaterial formulation promotes the cell adherences on the hydrogel surface.

Fig. 7. The image analysis of microalgal cell attachment on 3D-printed CNF/CMC hydrogel

Encapsulation Microalgae Cell in CNF/CMC Hydrogels

Figure 8(a) illustrates the growth of encapsulated microalgae in the 3D-printed nanocellulose-based hydrogel. The CNF/CMC hydrogel encapsulated with microalgae had been cultivated in BBM media for 8 days. Initially, from day 0 to day 3, a lag phase was observed, characterized by an absence of microalgae growth within the BBM media. However, expansion of C. vulgaris from the hydrogel to the BBM media occurred on day 3, prompting the cells to grow and enter the exponential phase.

Encapsulation of C. vulgaris into the hydrogel exhibited the most noticeable color transformation from white to green, as shown in Fig. 8(b). The gradual change is likely attributable to the prolonged lag phase followed by an extended period of exponential growth experienced by the microalgae. As the C. vulgaris cells adapted to the hydrogel environment, they exhibited an increased period of attachment, which was conducive to cell division by gradually turning the hydrogel to green colour. In line with previous observations, the hydrogel’s green coloration was more pronounced when the hydrogel was crosslinked with H₂SO₄ and CaCl₂, compared to ECH. This suggests that the chemical composition of the hydrogel plays a significant role in the microalgae’s presence and growth. Decreases in cell attachment can be related to the surface characteristic of hydrogel since the OD reading of culture medium showed almost similar trend for both types of crosslinker.

Fig. 8. The optical density of the encapsulated culture media for 8 days with different hydrogels crosslinker of (a) H₂SO₄ and CaCl₂ and ECH crosslinking and (b) physical appearance of 3D printed nanocellulose-based hydrogel

CONCLUSIONS

Cellulose-based hydrogels, especially those containing CNF and CMC, show promise for various applications, including biomedical and cell culturing. This works was conducted to analyze the potential of these hydrogels for 3D printing and their interaction with living cells.

The viscosity of the ink was found to play a crucial role in achieving a well-defined printed shape. The CNF/CMC hydrogel, when crosslinked with two types of crosslinkers, achieved excellent performance for 3D printing, particularly in liquid deposition modeling. Adjusting the CNF to CMC ratio may be necessary after adding the crosslinker to achieve optimal results.
In terms of cell growth, the growth of C. vulgaris cells in BBM solution exhibited similar trends for both the standard culture and the culture media incorporating the CNF/CMC hydrogels. It was found that the use of H₂SO₄ and CaCl₂ in hydrogel formation was more conducive to cell attachment compared to using epichlorohydrin. Besides, longer lag phase would contribute to higher cell attachment, as shown by the new culture and encapsulation experiment.

ACKNOWLEDGMENTS

The authors are grateful for the support of the Universiti Kebangsaan Malaysia, Grant No. DIP-2023-002.

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Article submitted: May 16, 2024; Peer review completed: June 20, 2024; Revised version received: August 7, 2024; Accepted: October 29, 2024; Published: November 11, 2024.

DOI: 10.15376/biores.20.1.201-218