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Sobri, Z., Mohamed Asa’ari, A. Z., Yacob, N., H'ng, P. S., Abdullah, L. C., and Zainudin, E. S. (2021). "In situ formation of zinc oxide on bamboo bleached pulp in preparation of antibacterial paper: Effect of precursors addition," BioResources 16(3), 6121-6134.


An approach of green in situ synthesis single-step method was applied to produce antibacterial paper. The objective was to investigate the effect of precursor addition on the formation of zinc oxide particles using an in situ single-step method. Zinc chloride concentrations of 0.1, 0.3, 0.5, and 0.7 M were prepared and added into a solution of algae extract and bamboo pulp. The prepared pulps were tested and made into handsheets using a papermaking machine based on TAPPI T205 (2006). Morphological observation of treated papers was conducted using a field emission scanning electron microscope (FESEM). An average of 400 to 570 nm zinc oxide spherical-shaped particle was observed on the fibers of paper. The percentage of element composition of the treated paper were 15.08% to 34.08% of zinc and 17.45% to 32.59% of oxygen captured via scanning electron microscopy with energy dispersive X-ray (SEM-EDX) analysis. The crystallinity test was performed using X-ray dispersion (XRD). A higher percentage of precursors exhibited a more amorphous structure. A measurement of more than 30% increment of inhibition zone was obtained from 10.00 to 25.00 mm against S. aureus, S. choleraesuis, and E. coli. Precursors addition of more than 0.3 M would have the most potential to enhance the growth of zinc oxide via in situ preparation, hence providing better antibacterial properties of the prepared papers.

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In situ Formation of Zinc Oxide on Bamboo Bleached Pulp in Preparation of Antibacterial Paper: Effect of Precursors Addition

Zakiah Sobri,a Ainun Zuriyati Mohamed Asa’ari,a,* Norzita Yacob,b Paik San H’ng,a Luqman Chuah Abdullah,a and Edi Syams Zainudin c,*

An approach of green in situ synthesis single-step method was applied to produce antibacterial paper. The objective was to investigate the effect of precursor addition on the formation of zinc oxide particles using an in situ single-step method. Zinc chloride concentrations of 0.1, 0.3, 0.5, and 0.7 M were prepared and added into a solution of algae extract and bamboo pulp. The prepared pulps were tested and made into handsheets using a papermaking machine based on TAPPI T205 (2006). Morphological observation of treated papers was conducted using a field emission scanning electron microscope (FESEM). An average of 400 to 570 nm zinc oxide spherical-shaped particle was observed on the fibers of paper. The percentage of element composition of the treated paper were 15.08% to 34.08% of zinc and 17.45% to 32.59% of oxygen captured via scanning electron microscopy with energy dispersive X-ray (SEM-EDX) analysis. The crystallinity test was performed using X-ray dispersion (XRD). A higher percentage of precursors exhibited a more amorphous structure. A measurement of more than 30% increment of inhibition zone was obtained from 10.00 to 25.00 mm against S. aureus, S. choleraesuis, and E. coli. Precursors addition of more than 0.3 M would have the most potential to enhance the growth of zinc oxide via in situ preparation, hence providing better antibacterial properties of the prepared papers.

Keywords: Algae; Zinc oxide; Bamboo; Bleached pulp; In situ preparation; Papermaking

Contact information: a: Pulp and Paper & Pollution Control Program, Laboratory of Biopolymer and Derivatives, Institute of Tropical Forestry and Forest Products (INTROP), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; b: Malaysian Nuclear Agency, Bangi 43000 Kajang, Selangor, Malaysia; c: Laboratory of Biocomposites, Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;

* Corresponding authors:;


Zinc oxide is a multifunctional mineral due to its physical and chemical properties. It exists as a mineral named zincite and has been commercially synthesized for commercial purpose (Mirzaei and Darroudi 2017). Its non-toxicity and compatibility to human skin has placed zinc oxide in a wide range of applications such as in pharmaceuticals (Pronin et al. 2014), paints (Osmond 2019), textiles (Rajendra et al. 2010; Belay et al. 2020), and the electronic industries (Balogun et al. 2020). Among all material particles, zinc oxide is well known for having the most varied particle structures (Kołodziejczak-Radzimska and Jesionowski 2014), especially one-dimensional structures such as needles, ribbons, tubes, wires (Nikoobakht et al. 2013), rods (Jaisai et al. 2012), prisms (Wirunmongkol et al. 2013), sheets (Lu et al. 2017), triangles (Nagarajan and Kuppusamy 2013), squares (Mishra and Sharma 2015), and combs (Xu et al. 2012). Besides that, zinc oxide appears in two-dimensional and three-dimensional structures. The two-dimensional structures including nanoplate and nanopellets while flower, dandelion, snowflakes, coniferous urchin-like and polygonal (Zhou et al. 2013; Namvar and Rahman 2013) are the zinc oxide shapes that are grouped in three-dimensional structures. The shape, size, and spatial structure can be tailored by adjusting methods during the preparation of zinc oxide. In general, three methods are applied to prepare zinc oxide: (1) chemical, (2) physical, and (3) biological. Controlled precipitation, the sol-gel method, solvothermal and hydrothermal methods, and the method using emulsion and microemulsion environments are categorized as chemical methods (Kolodziejczak-Radzimska and Jesionowski 2014). The biological method, which is regarded as a green method, uses substrates including plant extracts, microorganisms, enzymes, and bacteria.

The biological or green method in processing zinc oxide has attracted many researchers in biosensors, pharmaceuticals, cancer treatment, and magnetic resonance imaging (MRI) areas (Kulkarni and Muddapur 2014). This is due to its eco-friendly, non-toxic, and safe reagent that is preferred in the biologically mediated preparatory method compared to the chemical and physical methods (Bhuyan et al. 2015). In addition, efficiency in cost and synthesizing particles without pressure has led to truly green chemistry (Nagarajan and Kuppusamy 2013; Sutradhar and Saha 2015). Equation 1 shows the reaction in zinc oxide formation (Sutradhar and Saha 2015). A study conducted by Jamdagni et al. (2016) succeeded in producing 12 to 32 nm of zinc oxide particles in aggregates from Nyctanthes arbortristis (flower) extract. This occurred when a bioactive compound contained in biomaterial reduced the metal element to form metal oxide. In examples, this can successfully be achieved by gentle warming and incubation that could activate quinones in Cyprus sp. (mesophytes) to reduce its zinc oxide particle size (Kirthi et al. 2013). Algae, also known as bionanofactories, can be synthesized nanoparticles with high stability and important sources in producing metallic-nanoparticles (Ramaswamy et al. 2016). Macroalgae such as seaweeds that have become part of habitual diet in Asian countries can be classified into three subcategories: Phaeophyta (brown seaweed), Rhodophyta (red seaweed), and Chlorophyta (green seaweed) (Brownlee et al. 2012).

Zn2+ + 2e Zn0 + Air (O2) ZnO (1)

In manufacturing antibacterial paper, a coating technique is usually selected by incorporating antibacterial agent in coating film that is later coated on the paper surface. Another coating technique was applied by Akrami et al. (2015). The essential oils from cumin and a thyme-like plant that have antibacterial properties were extracted and applied on paper surfaces and presented good antibacterial activity against food-borne bacteria (Akrami et al. 2015). Prasad et al. (2010) also applied a coating technique and observed that no bacterial growth was observed for zinc oxide-coated paper. Other than the coating technique, an in situ bio-mediated precipitation method was also practiced. A study conducted by El-Samahy et al. (2017) produced antibacterial paper by incorporating chitosan and nanocellulose via in situ approaches that resulted in strong antibacterial activity of paper. In terms of applying inorganic material as antibacterial agent, Maślana et al. (2021) boosted antibacterial performance of their cellulose-based paper sheet by using titanium dioxide particles. In addition, Li et al. (2021) also focused on in situ growth of nano-ZnO/GQDs on their cellulose paper to repel against water and bacteria.

Bamboo is a member of a large woody grass family and is a fast-growing plant that can be found naturally in subtropics and temperate zones such as Asia. It is reported that bamboo can grow three times faster than eucalyptus with 1,300 to 1,400 species around the world (Liese and Kohl 2015; Yeasmin et al. 2015; Tripathi et al. 2018) with 120 species in Asia that include 59 species in Malaysia. The most common bamboo species such as Semantan (Gigantochloa scortechicinii) and Gading (Bambusa vulgaris) exhibit excellent strength capacity for building material compared to wood and concrete except concrete has better stiffness (Janssen 1981; van der Lugt et al. 2006; Dhanush et al. 2021). Other than being a good building material, a strong utensil, and current hygiene instrument as a toothbrush, bamboo is also an ideal material in papermaking (Chen et al. 2019; Hammett et al. 2001). Bamboo can be applied in producing advanced material such as antibacterial fabric. Zhang et al. (2013) improved antibacterial activity of bamboo fabric at 99% performance degree against S. aureus and E. coli by synthesizing zinc oxide. Another study conducted by Yin et al. (2017) improved the porosity properties of bamboo tissue paper’s (napkins) surface by incorporating chitosan. In addition, Zhang et al. (2017) improvised bamboo antibacterial performance using zinc oxide and graphene oxide. The antibacterial bamboo is also expected to be a potential material in the textile industry.

Looking at the small number of studies that concentrated on the in situ bio-mediated preparation of zinc oxide, a study was conducted to be simpler in a single step and easier than the usual method. This study focused on the effect of precursor concentration, which is believed to be a great influencer to zinc oxide in situ performance.


Raw Materials and Chemicals

The 2 m of Semantan bamboo culms (Gigantochloa scortechicinii) were purchased from Sungai Siput, Perak, Malaysia and ground into chips until 2 to 3 cm width and length. Air-dried algae of Rhodophyta were acquired from Semporna, Sabah, Malaysia. The chemicals involved in the study were zinc chloride (ZnCl2), sodium hydroxide (NaOH), sodium chlorite (NaClO2), acetic acid (CH3COOH), and hydrogen peroxide (H2O2). All chemicals were purchased from RandM Chemicals (Selangor, Malaysia).

Preparation of Bleached Pulps of Bamboo

These bamboo chips were cooked using a soda pulping technique with 17% sodium hydroxide in a twin digester (GIST, Daejeon, South Korea) at 170 °C for 90 min. After completing the pulping process, the pulp was washed and screened using a Somerville Shive Content Analyser (Frank-PTI, Birkenau, Germany). The next step was the bleaching process, in which delignification (D) and extraction (E) processes as D1EPD2 sequences were applied in a 70 °C water bath. The consistency of unbleached pulp was 10% which was placed in a transparent plastic bag containing 2.00% of NaClO2 and 3.00% of CH3COOH throughout the D1 stage for 180 min. The bleaching process continued with the extraction step, in which a mixture of NaOH at 1.50% and H2O2 at 1.00% was applied to the pulps for 90 min. Finally, the D2 stage involved the immersion of pulps for another 90 min by adding NaClO2 and CH3COOH at 1.25% and 3.00% concentrations accordingly. The pulps were washed between bleaching sequences. Prior to further experiment, the bleached pulp was beaten by using a PFI-mill Beater according to the TAPPI T248 (2000) standard method reaching 350 to 400 mL.

Pulp and Paper Characterization

Approximately 500 g of dried algae was washed and oven-dried at 60 °C overnight. The dried alga was ground using a blender to obtain algae powder, which was then sieved with 40-mesh sifter to collect a uniform particle size. Later, 2 g of dried seaweed was heated with 200 mL of distilled water until it boiled before filtering it, of which the effluent was taken into the next experiment. The preparation of green zinc oxide nanoparticles via in situ approaches was conducted by incorporating 2.4 g of bleached pulps in the 100 mL of extract solution. Prior to that, the extract solution was prepared by adding different concentrations of pre-cursor, namely zinc chloride, and heated for 4 h at 50 °C in a water bath. Finally, the samples were rinsed with distilled water and proceeded to the papermaking by using handsheet machine. The papermaking process was referred to the TAPPI T205 (2006), producing a 60 g/m2 piece of paper. The list of samples prepared in this study is shown as in Table 1.

Table 1. Samples Prepared for the Study

Characterization of Zinc Oxide Nanoparticles and Zinc Oxide Paper

The formation and distribution of zinc oxide on papers was observed using field emission scanning electron microscope (FESEM) (Model: JSM 7600F, JEOL, Tokyo, Japan). The energy dispersive X-ray (EDX) was applied to confirm the elements that attached on the fibers. The properties of zinc oxide-paper were characterized via X-ray dispersion (XRD) (PW 3040/60 MPD X’pert High Pro Panalytical, Phillips, Omaha, NE, USA) while antibacterial testing was conducted against three types of bacteria explicitly as Staphylococcus aureus (positive strain bacteria), Salmonella choleraesuis, and Escherichia coli (negative strain bacteria) using the agar well diffusion method (Balouiri et al. 2016).


Morphological Observation of Zinc Oxide Paper

Morphological observation of zinc oxide paper is exhibited as micrographs shown in Fig. 1 via 500×, 1,500×, and 10,000× magnifications. Flocs of elements were observed on the fiber surfaces of the papers. This was confirmed after spotting the elements using FESEM-EDX, which represented the elements of zinc and oxygen as shown in Fig. 2 and Table 2.

Table 2 exhibits scanning electron microscopy with energy dispersive X-ray (SEM-EDX) analysis of the spotted area for two elements, namely Zn and O, that appeared on paper surfaces. Figure 2 shows the percentage weight of zinc and oxygen for B1, B3, B5, and B7, which ranged from 15.08% to 34.08%, and 17.45% to 32.59%, respectively. Different weight, size, and shape of zinc oxide particles are the outcomes of interactions between bioactive compounds in algae like phenols, and metal atoms from precursor were added into the systems (Shao et al. 2004).

The zinc oxide particles were measured with a range of 400 nm to 570 nm. They appeared in spherical form (Fig. 3), as achieved by Vinay and Chandrasekhar (2021), Vijayakumar et al. (2018) and Yedurkar et al. (2016). Chronologically, zinc oxide particles were synthesized by reducing Zn2+ from zinc chloride with the bioactive compounds present in algae extract (Sadatzadeh et al. 2018). After in situ preparation stage, the pulps were poured into the handsheet machine tank before filtering the pulp slurry to leave a sheet of paper on the wire mesh. Several parts of the elements of zinc oxide were believed to be flushed away, which researchers continue to find ways to protect as much zinc oxide from heavy dislodgements. As the findings of these preliminary experiments appear very beneficial, the retention of additives will be studied and applied in future experiments.

Fig. 1. FESEM micrographs of (a) B1, (b) B3, (c) B5, and (d) B7 with magnification of 500X (left column) and 1,500X (right column); the presence of zinc oxide is shown in the circles.

Table 2. SEM-EDX Analysis of Flocs in Terms of Two Elements that Appeared on Paper Surfaces

Fig. 2. SEM-EDX analysis of flocs in terms of two elements that appeared on paper surfaces

Fig. 3. Sample B3 of zinc oxide obtained in this study

Fig. 4. The intensity peaks of crystalline structure of zinc oxide particles (shown by arrows) of samples B1, B3, B5, and B7 from XRD analysis

Chemical Composition of Zinc Oxide Pulp

The zinc oxide pulp samples were dried in sheet form and were tested for their properties in terms of the crystalline structure of zinc oxide particles by using XRD. In Fig. 4, sharp and narrow diffraction peaks of zinc oxide that attached on pulp were observed within the 2θ range from 10.00° to 30.00°, which indicated high crystallinity of zinc oxide for samples B1, B3, and B5, as achieved by Prasad et al. (2010). The crystallinity of prepared zinc oxide pulp decreased as the concentration of zinc chloride increased. These were presented by sharp peaks observed at 22° of 2θ. Meanwhile, sample B7 only displayed a small peak at 22.2°, indicating a reduction of zinc oxide due to a decrease of degree of crystallinity (Ramesan et al. 2019). In addition, a broader diffraction pattern exhibited by sample B7 indicated the amorphous structure of zinc oxide was formed (Ramesan et al. 2018). This is also parallel to findings by Vijayakumar et al. (2018) and Chikkanna et al. (2019) who obtained green synthesized zinc oxide that is recorded at range 20<2>80.

Antibacterial Test

Referring to Table 3, all samples exhibited inhibition zones from 10.00 to 25.00 mm. All zinc oxide pulp samples showed inhibition zones with increasing diameter as the concentration of the precursors increased. The average inhibition zone for pulp samples increased from sample B1 until B7 by 35% against S. aureus, 59% against S. choleraesuis, and 58% against E. coli. Sample B1 exhibited stronger antibacterial activity against S. aureus, while sample B3 successfully inhibited both S. aureus and S. choleraesuis. Meanwhile, samples B5 and B7 exhibited stronger antibacterial activities against S. choleraesuis with more than 20 mm of inhibition zone. Referred to findings by Alswat et al. (2016), parameter of concentration, increasing amount and decreasing particle size of zinc oxide contributed to inhibition zones. Interestingly, Gudkov et al. (2021) stated that modifying the synthesis method is one of the keys to increase antimicrobial effectiveness instead of the conventional method, namely as physicochemical.

According to Sidiqi et al. (2018), the size of zinc oxide nanoparticles tested against E. coli and S. aureus in the range from 401 nm to 1.2 μm showed an increasing of antimicrobial activity as the size of particles decreased. The presence of zinc oxide particles as an antibacterial agent damaged the respiratory enzymes and cytoplasmic contents in the bacterial membrane, that resulted in the death of bacterial cells (Gunalan et al. 2012; Vijayakumar et al. 2018). The penetration properties of zinc oxide nanoparticles can be explained by reaction of the oxygen released on the zinc oxide surface with hydrogen ion to produce hydrogen peroxide that can penetrate and kill the bacteria (Fang et al. 2006; Gunalan et al. 2012). According to Ohira et al. (2008), the smaller size of zinc oxide enhanced the production of hydrogen peroxide that strongly depends on surface area of zinc oxide. Figure 5 shows the inhibition zones for B1, B3, B5, and B7 pulp samples against S. aureus, S. choleraesuis, and E. coli.

Table 3. Antibacterial Screening Test Results

Fig. 5. Inhibition zone for samples B1, B3, B5, and B7 (black circles) against (a) S. aureus, (b) S. choleraesuis and (c) E. coli


Zinc oxide-containing paper was successfully produced via in situ approach, and the study can be concluded as follows:

  1. Morphological observation showed that the size of the zinc oxide particles ranged from 400 to 570 nm with spherical shape. The percentages of element composition of the paper were 15.08% to 34.08% of zinc and 17.45% to 32.59% of oxygen.
  2. The crystallinity of zinc oxide-pulp, prepared by a bio-mediated precipitation, decreased as the concentration of zinc chloride increased. The higher percentage of precursors developed more amorphous structure, as shown by B7.
  3. Antibacterial activities against S. aureus, S. choleraesuis, and E. coli has shown acceptable inhibition regarding the increasing concentration of zinc chloride. It was found that more than a 30% increment of inhibition zone occurred with a diameter ranging from 10.00 to 25.00 mm.
  4. Overall, the addition of precursors more than 0.3 M had higher potential to enhance the growth of zinc oxide via in situ preparation, hence providing better antibacterial resistance properties.


The authors are grateful to Universiti Putra Malaysia for the financial support of this work under Putra Initiative Putra Grant Scheme vote no. 9662000 and Higher Institution Centre of Excellence (HICoE) vote no. 6369111.


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Article submitted: November 5, 2020; Peer review completed: February 6, 2021; Revised version received: June 5, 2021; Accepted: June 6, 2021; Published: July 20, 2021.

DOI: 10.15376/biores.16.3.6121-6134