In this section, several developments concerning nanocellulose based membrane filters capable of removing microbes will be reviewed. An important aspect of the modification of nanocellulose materials is to increase the binding affinity of the materials towards microbes. There are a number of studies that focused on filtration removal of viruses and bacteria; however, very limited studies have been done concerning other types of microbes such as fungi, algae and protozoa.
Ensnarement of viruses is one of the most crucial steps in biopharmaceutical and clinical processes and applications (Lebrun 2017). Of the various types of microbes, virus is among the smallest and most difficult to deal with as compared to other microbes.
Exploration of nanocellulose as a filtration material against several types of viruses has received much research attention. As mentioned earlier, several viruses including COVID-19 are airborne viruses that can be dispersed and spread through human nasal or saliva secretions from an infected person. Therefore, in order to minimize infection risks from viruses, an efficient, robust and affordable air-borne virus removal filter is an urgent requirement. Multiple research articles were published recently with regards to this type of air filter.
Several factors such as filters pore size, thickness, number of layers, size of the virus, the charge on the filter surface, its ionic strength and surface chemistry are usually influence the efficiency of air filtration process (Metreveli et al. 2014). Generally, the use of size-exclusion type filtration has several benefits such as flexibility and ease of use since it provides virus removal predictability through its physical properties, allows for the filtration of viral markers enabling easy validation of the filtration process and does not use toxic or mutagenic chemicals for viral inactivation (Burnouf and Radosevich 2003; Metreveli et al. 2014; Asper et al. 2015b).
Gustafsson et al. (2018) evaluated filter papers made from nanocellulose in a mille-feuille arrangement of varying thicknesses using a simulated wastewater matrix to explore its ability to remove viruses for drinking water purification applications. The filtrations of various samples of simulated wastewater with its total suspended solid content being 30 nm latex particles as surrogate waste material and 28 nm ΦX174 bacteriophages as the viral contamination. The authors examined the performance of these filter papers at a pressure of 1 and 3 bar with varying thicknesses of 9 and 29 µm. The data they obtained found that filter paper made from 100% nanocellulose has the capacity to efficiently remove even the smallest of viruses, with up to 99.9980–99.9995% efficiency.
Manukyan et al. (2019) fabricated nanocellulose-based mille-feuille type filter paper for use in upstream applications for serum-free growth media filtration and it was designed to remove ΦΧ174 bacteriophages. The filter performance was evaluated based on its ability to filter small-medium sized virus using varying thicknesses of the fabricated filter paper (i.e. 11 and 33 µm), as well as by varying the operating pressures, (i.e. 1 and 3 bar). Based on their results, the 33 µm thick filter showed more stability and had better virus removal as compared to the 11 µm thick filter although their flux was nominally lower. The findings of this study suggest that nanocellulose filter paper would be a viable alternative for the filtration of large volumes of cell culture media in upstream bioprocessing.
Asper et al. (2015) in their study used filter paper composed of 100% CNF to remove xenotropic murine leukaemia virus (xMuLV). The results of this filtration of xMuLV suggested that the nanocellulose filter paper was useful for removal of endogenous rodent retroviruses and retrovirus-like particles during the production of recombinant proteins.
Metreveli et al. (2014) reported that the nanocellulose filter was able to remove Swine Influenza A Virus (SIV) which had a particle size of 80–120 nm and it is shown in Fig. 7. The latex beads and SIV particle are observed as stacked structures on the surface of the porous filter paper membrane.
Previous researches on surface modification of nanocellulose have led to the improvement of filtration efficiency against viruses. Electrostatic interaction between nanocellulose and viruses is improved dramatically with the incorporation of quaternary compounds as discussed in Sect. 4 above. For instance, viruses like coronavirus have negatively charged surface and would interact with the cationic or anionic charge of the nanocellulose-quaternary compounds (Leung and Sun 2020). Figure 8 shows a schematic diagram of coronavirus structure with proteins embedded in its bilayer membrane and negatively-charged lipid head groups protruding to the outer side of membrane.
The entrapment of the virus onto nanocellulose matrix is due to the presence of electrostatic force attraction between the negatively charged virus particle and the positively charged nanocellulose membrane. Several studies have shown successful results in filtering negatively charged viruses using cationic nanocellulose. For example, Mi et al. (2020) developed filtration setup using modified CNC with a positively charged guanidine group to adsorb porcine parvovirus and Sindbis virus and completely filter out those viruses from water. It is interesting to point out that this filtration system has exceeded the Environement Protection Agency (EPA) virus removal standard requirement for portable water. In addition to the electrostatic interaction between the virus and guanidine group, Meingast and Heldt (2020) outlined that the complete virus removal from water was also due to the protonated guanidine groups on the cationic CNC forming ionic and hydrogen bonds with the proteins and lipids on the virus surface.
Other than that, Rosilo et al. (2014) in their study observed a very high affinity binding between the cationic CNC (known as CNC-g-P(QDMAEMA)s) mixture and cowpea chlorotic mottle virus (CCMV) and norovirus-like particles in water dispersions. Of note, this cationic CNC mixture was prepared by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylamino ethyl methacrylate) and its subsequent quaternization of the polymer pendant amino groups.
In addition, the anionic CCMVs could also be removed using functionalized lignin with a quaternary amine. In their study, they found that the CCMVs would form agglomerated complexes with cationic lignin (Rivière et al. 2020). Therefore, suggesting its potential use as material in the development of membrane filter for the removal of CCMVs.
Besides that, Sun et al. (2020) reported that covalent modification on CNF (i.e. functionalization of nanocellulose) using polyglutamic acid (PGS) and mesoporous silica nanoparticles (MSNs) resulted in successful filtration of EV71 virus and Sindbis virus. This is particularly due to the interaction between two exposed positively charged amino acids (His10 and Lys14) and the negatively charged MSNs on the modified CNF (Sun et al. 2020).
In other related study, nanocellulose was functionalized using citric acid in the fabrication of nanofiltration based filter paper production for virus removal applications (Quellmalz and Mihranyan 2015). The addition of environmental-friendly and non-toxic citric acid in the fabrication is to improve the wet strength properties of paper filter as it acts as a cross-linking agent for the nanocellulose. The citric acid cross-linking of nanocellulose was proved to be beneficial in developing paper-based sterile (virus removal) industrial filters since it managed to efficiently remove 20 nm Au nanoparticles from a feed solution as reported in the study (Quellmalz and Mihranyan 2015).
Table 4 summarizes the development of nanocellulose-based membrane filtration material for virus removal that have been discovered/explored so far. In addition to the guanidine groups, lignin, nanoparticles, and citric acid, nanocellulose could also be functionalized with several other compounds such as small organic molecules, porphyrin dendrimers and others polymers in order to make it positively or negatively charged (Sunasee and Hemraz 2018). However, it is important to note that not all of these examples have been tested as a filter to remove viruses.
Table 4
Nanocellulose developed filtration material for virus removal
Microbes | Type of nanocellulose | Functionalization | Findings | Reference |
A/swine/Sweden/9706/2010 (H1N2) - Swine influenza | BNC | Not applicable | • The newly developed non-woven, µm-thick filter paper consisting of crystalline BNC able to remove virus particles solely based on the size-exclusion principle, with a log 10 reduction value ≥ 6.3, thereby matching the performance of industrial synthetic polymer virus removal filters currently in use. | (Metreveli et al. 2014) |
Xenotropic murine | BNC | Not applicable | • The developed BNC filter paper could remove the endogenous rodent retroviruses and retrovirus-like particles. | (Asper et al. 2015a) |
MS2 viruses | BNC | Not applicable | • This study highlights the efficiency of the nanocellulose-based filter paper in removing/filtering out the ΦX174 bacteriophage with value of5 − 6 log virus clearance (28 nm; pI 6.6). | (Wu et al. 2019) |
ColiphagesΦX174 | BNC | Not applicable | • The nanocellulose-based filter paper exhibited 5 − 6 log virus clearance of MS2 viruses (27 nm; pI 3.9). This study also showed the possibility of producing cost-efficient viral removal filters (i.e. manufacturing process). | (Wu et al. 2019) |
Parvoviruses | BNC | Not applicable | • The developed filter was the first non-woven, wet-laid filter paper composed of 100% native cellulose. This study showed that the non-enveloped parvoviruses could be eliminated using this filter. | (Gustafsson et al. 2016) |
EV71 | CNF | Polyglutamic acid and mesoporous silica nanoparticles | • This study showed that the modified microfibers could strongly adsorb the epitope of the EV71 capsid which is useful for virus removal | (Sun et al. 2020) |
Sindbis virus | CNC | Guanidine | • Functionalization of guanidine on CNC resulted in over 4 log removal value against the Sindbis virus. | (Mi et al. 2020) |
Porcine parvo virus | CNC | Guanidine | • Authors also revealed that functionalization of guanidine on CNC managed to remove the Porcine parvo virus with over 4 log removal value. | (Mi et al. 2020) |
5.2 Bacteria
The development of nanocellulose as filtration material against bacteria also been widely discovered. Generally the diameter of most waterborne bacteria is larger than 0.2 µm (Ma et al. 2011). Thereby, it would be easy for nanocellulose-based membrane filters to entrap most bacteria species using the size-exclusion mechanism. Moreover, as discussed in Sect. 4 earlier, modification of nanocellulose by surface functionalization can also be done to increase the removal efficiency of bacteria. In this review, we highlight several findings concerning bacterial removal using nanocellulose based membrane filters.
Wang et al. (2013) demonstrated that a multi-layered nanofibrous microfiltration system with high flux, low-pressure drops and high retention capability against bacteria (Brevundimonas diminuta and Escherichia coli) was possible by impregnating ultrafine CNF into an electrospun polyacrylonitrile (PAN) nanofibrous scaffold supported by a poly (ethylene terephthalate) (PET) non-woven substrate. The CNF was functionalized prior to impregnation with carboxylate and aldehyde groups using TEMPO oxidation. It was observed that this CNF-based microfiltration membrane exhibited full retention capability against those bacteria.
Otoni et al. (2019) developed a cationic CNF compound using Girard’s reagent T (GRT) and shaped it into foam using several protocols such as cryo-templating to remove the ubiquitous human pathogen Escherichia coli. The porosity of this foam, which is associated directly with its surface area and pore size plays a significant role in the removal of Escherichia coli. The cryogel foams produced by this method had porosities of circa 98% and were established to be able to achieve an approximately 85% higher anti Escherichia coli activity when compared to sample foams made up of unmodified CNF. The cationic CNF using GRT demonstrated good potential for both air and liquid filtration, with excellent absorbency through functional coating. Access to safe drinking water in high- and low-income countries has become one of the biggest challenges in the world as natural resources become scarcer.
Gouda et al. (2014) invented a modified electrospun CNF containing silver nanoparticles (AgNPs) as a water disinfecting system for water purification systems. The AgNP content, its physical characterization, surface morphology and antimicrobial efficacy of the developed membrane filter was then studied. AgNP, which belongs to the group of biocidal nanoparticles, has antimicrobial properties and is commonly used due to its size quantization effect. This can cause a shift in the reactivity of metals in the nanoscale. The developed membrane filter had excellent ability to remove bacteria including Escherichia coli, Salmonella typhi, and Staphylococcus aureus with a percentage filtration of more than 91% in contaminated water samples.
Ottenhall et al. (2018) developed a CNF-based membrane filter modified with polyelectrolyte multilayers to produce multilayer cationic polyvinyl amine (PVAm) and anionic polyacrylic acid (PAA). The authors had successfully modified the CNF with cationic polyelectrolyte PVAm together with the anionic polyelectrolyte PAA in either single layers or multilayers (3 or 5 layers) using a water-based process at room temperature. Based on filtration analysis, the functionalized CNF-based membrane filters with several layers was physically able to remove more than 99.9% of Escherichia coli from water. The 3-layer membrane filter could remove more than 97% of cultivatable bacteria from natural water samples, which was a remarkable performance as compared with simple processing technique using plain nanocellulose filters.
Table 5 summarizes the effectiveness of nanocellulose-based membrane filters that have been functionalized with bioactive compounds for the removal of bacteria.
Table 5
Nanocellulose developed filtration material for bacterial removal
Microbes | Type of nanocellulose | Functionalization | Findings | Reference |
Escherichia coli | CNC | Silver nanoparticles | • It possesses high adsorption capacity and is reusable. Beneficial in total removal of Escherichia coli. | (Suman et al. 2014) |
Bacillus subtilis and Escherichia coli | CNF | ZnO and CeO2 | • It has high anti-bacterial activity, MIC50 against Bacillus subtilis (10.6 µg ml-1) and Escherichia coli (10.3 µg ml− 1). | (Nath et al. 2016) |
Escherichia coli | BNC | Not applicable | • The significance of Brownian motion caused by microorganisms captured with BNC-based filter paper through theoretical modelling and filtration experiments was investigated by the authors. • It was found that the BNC-based filter was capable of filtering the bacteria. | (Gustafsson et al. 2018a) |
Escherichia coli, Staphylococcus aureus | CNF | Activated carbon | • The two-layer AC/OCNF/CNF membrane able to remove Escherichia coli bacteria up to ~ 96–99% and inhibits the growth of Escherichia coli and Staphylococcus aureus on the upper CNF surface | (Hassan et al. 2017) |
Escherichia coli | BNC | Silver nanoparticle | • Higher amount of silver nanoparticles loaded onto the BNC membrane surface could increase the inhibition zone hence highlighting its good antimicrobial property against Escherichia coli. | (Zelal et al. 2018) |
Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa | BNC | Silver nanoparticle | • BNC-silver nanoparticle membrane showed strong antimicrobial activity against Gram positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. | (Barud et al. 2011) |
Escherichia coli, Staphylococcus aureus | BNC | Silver nanoparticle | • The developed Ag/BNC membrane exhibited good property as antimicrobial agent against Escherichia coli and Staphylococcus aureus as the antibacterial ratio against Escherichia coli and Staphylococcus aureus reached 99.4% and 98.4%, respectively. | (Zhang et al. 2013) |
Escherichia coli | CNF | polyethersulfone (PES) membranes | • TEMPO oxidized-CNF coating is effective against Escherichia coli. The effectiveness was attributed to the pH reduction effect induced by carboxyl groups | (Aguilar-Sanchez et al. 2020) |
5.3 Other Types Of Microbes
Nanocellulose would also be able to act as a removal agent for other types of microbes which are larger in size than bacteria, such as fungi, algae and protozoa. However, it is noteworthy that there is still a lack of studies regarding this matter. To the best of our knowledge, there are no available reports on the development of a nanocellulose-based membrane filter for the removal of fungi.
Algae is also a major contributor to microbial contamination in water resources and their presence could change the taste or odour of water. For example, blue-green algae and coloured flagellates (especially the Chrysophyta and Euglenophyta genuses of algae) are the best-known algae that cause contamination in water resources. Furthermore, green algae may also be a significant contamination factor as well (Sen et al. 2013). Hence, the potential of nanocellulose should be explored by scientists to define their role as a membrane filtration material suitable for removing algae and protozoa from the contaminated water efficiently. Algae and protozoa are known to have a larger size than viruses and bacteria, thus the removal of these microbes could be effectively carried out using the size-exclusion mechanism.
However, similar to viruses and bacteria, the nanocellulose needs to be modified with other compounds such as metal nanoparticles, enzymes and proteins in order to increase its filtration efficiency (Gopakumar et al. 2018). Studies have shown that different charges between the cellular membrane of algae and protozoa do play a dominant role in the adsorption/retention of these microbes on a filtration membrane’s surface (i.e. through the electrostatic interaction principle) (Kim et al. 2017; Ottenhall et al. 2018).
Previous study carried out by Ge et al. (2016) discovered the sustainability and the most efficient approach in harvesting algae using a modified CNC. The modification was made by introducing a 1-(3-aminopropyl)-imidazole (APIm) structure as a reversible coagulant. As shown in Fig. 9, coagulation process occurs when the positively charged CNC-APIm interacts with the negatively charged Chlorella vulgaris in the presence of carbon dioxide (carbonated water). Their findings are in agreement with the works done by Qiu et al. (2019) as harvesting efficiency could reach up to 85% with only 0.2g CNC-APIm mass ratio, 5secs of CO2 sparging time, and a 50 ml/min flow rate. This signifies that the CNC-APIm complex could be an alternative to current conventional coagulants for harvesting algae in industrial applications.
Algae harvesting is important for biodiesel industry and many researches have been carried out to increase its sustainability in a global scale. For example, the capability of CNF and CNC in harvesting algae were investigated by Yu et al. (2016). In their study, they discovered that the CNF did not require any surface modification to harvest the algae as it played a role as an algae flocculant via its network geometry, something that the CNC (even cationic modified CNC) could not do. Flocculation of algae did not happen when CNC is used as the freely moving algae cannot be entrapped by nanoparticles structure formation of CNC. However, this study only focuses on the flocculation capability of CNF and CNC which could intrigue a further study on the filtration efficiency of both materials for algae harvesting. This finding indirectly could point to the development of a nanocellulose-based membrane filter for algae removal in the future.