A new approach to wood protection: Preliminary study of biologically synthesized copper oxide nanoparticle formulation as an environmental friendly wood protectant against decay fungi and termites

In last decade, nanoparticles have been reported to resolve many impeding technological challenges in various fields of science like engineering, medicine, water purification, catalysis etc. to name among a few (Klefenz 2004, Goodsell 2004, Chan and Nie 1998, Colvin et al. 1994, Tan et al. 2006, Lee et al. 2008, Pissuwan et al. 2006, Cai et al. 2008, Cao 2004). One such area in which nanoparticles, mainly metal nanoparticles have proved to be instrumental is wood protection. The metal nanoparticles used in wood protection are generally copper, zinc and boron. Lot of literature is available on the use of nanometals for wood protection (Clausen 2007, Clausen et al. 2010, Kartal et al. 2009, Akhtari and Arefkhani 2010, Nair et al. 2017). Terzi et al. (2016) reported the role of nanoparticles of ZnO, B₂O_3, CuO, TiO₂, CeO₂ and SnO₂ in prevention of fungal decay, mold growth and termite attack on wood along with their effect on weathering properties and water repellency. Mishra et al. (2018) reported the impact of nanoparticles on the wood coating and preservation treatments based on survey of the registered patents. The report gave an overview of the scientifically most followed trends on nanoparticle utilization in wood science and wood protection depicted by recently filed patents. Even though nanoparticles have addressed various issues of wood science in a more efficient way than the traditional methods of employing metal salts, commercially available nanoparticles are synthesized by physical and chemical methods. The disadvantage of physical synthesis method is that they are energy intensive and are expensive. Chemical synthesis methods involve the use of many toxic chemicals which limits the applications of chemically synthesized nanoparticles, especially in medical field and are not environmental friendly (Vijayaraghavan and Ashokkumar 2017). Search for a biocompatible, non-toxic and ecofriendly method of nanoparticle synthesis lead to the development of biological synthesis route for production of nanoparticles. Biological synthesis of nanoparticles involves the use of biomaterials such as bacteria (Hulkoti and Taranathm 2014), fungi (Dhillon et al. 2012), yeast (Moghaddam et al. 2015), virus (Dujardin et al. 2003), microalgae (Schrofel et al. 2011), macroalgae (Singaravelu et al. 2007) and plant extracts (Mittal et al. 2013). Of all these biomaterials, plant extracts are considered more advantageous for the synthesis due to their availability, renewable nature, simplicity of process, efficiency, rate and stability of synthesized nanoparticles and cost effectiveness (Vijayaraghavan and Ashokkumar 2017, Iravani 2011).

Wood science researchers so far have not explored the possibility of utilizing biologically synthesized nanomaterials for wood protection. Plant extracts contain numerous organic molecules like carbohydrates, proteins, vitamins, alkaloids, enzymes etc. These molecules can act as either potential reducing or stabilizing agents for the synthesis of nanoparticles. In addition to the ecofriendliness and cost effectiveness, plant extract based synthesis combines the intrinsic properties of plants with the biocidal properties of nanoparticles via the synergistic effect. The plant extracts which have both wood preservative properties and are capable of synthesizing metal nanoparticles are to be selected. The synergistic effect of the plant extract with the biocidal nature of metal nanoparticles also contributes to the efficacy of a wood protectant. There are several factors that affect synthesis of nanoparticles using plant extracts, its characterization and application namely origin of plant material, method of extraction employed and the solvents used. The temperature employed, pH-value and the reaction time also plays an important role in the size and shape of nanoparticles produced (Vijayaraghavan and Ashokkumar 2017).

This research reports the synthesis of copper oxide nanoparticles using plant extracts and testing its efficacy as a wood preservative. Leaf extracts of Neem (Azadirachta indica), Pongamia (Pongamia pinnata), Lantana (Lantana camara) and extract of orange peel (Citrus reticulata) were used to synthesize copper nanoparticles. All these plants have reported wood preservative properties (Macias et al. 2005, Sotannde et al. 2011, Machado et al. 2013, Venmalar 2017, Gupta et al. 2017) and are reported in the synthesis of metal nanoparticles (Ansilin et al. 2016, Sundrarajan et al. 2015, Majumder 2012).

The synthesized CuO nanoparticles (CuO nps) using plant leaf extracts were characterized by SEM coupled with EDAX. SEM image confirmed the presence of CuO nanoparticles that are spherical in nature. The particles were well separated without agglomeration and the particle size remained within a range of 136 nm to 380 nm in the case of CuO nps synthesized using A. indica leaf extract. In the case of CuO nps synthesized using P. pinnata leaf extract and C. reticulata peel extracts the particle sizes were in the range of 466nm to 2 µm and 259 nm to 519 nm respectively. The particle size of CuO nps synthesized using L. camara leaf extract was in the range of 33 nm to 46 nm. EDAX profile also confirmed the presence of copper and oxygen in major fraction (Figure 1).

Figure 1:
Biologically synthesized CuO nanoparticles using plant extracts were characterized by SEM coupled with ED. a) Particle size of the synthesized copper oxide nanoparticles using Azadirachta indica leaf extract was between 136 mm and 380 nm. b) Particle size of the synthesized copper oxide nanoparticles using Pongamia pinnata leaf extract was between 466 mm and 2um. c) Particle size of the synthesized copper oxide nanoparticles using Lantana camara leaf extract was between 33 nm and 46 nm. d) Particle size of the synthesized copper oxide nanoparticles using Citrus reticulata leaf extract was between 259 nm and 519 nm.

Initiation of growth in control plates inoculated with Trametes hirsuta (white rot) and Oligoporus placenta (brown rot) was observed on 4^th day of the experiment. On the 9^th day the experiment was terminated as the control plates showed complete growth (Figure 2). The percentage of inhibition for T. hirsuta and O. placenta were calculated for all the plates (Table 1).

Figure 2:
Plates showing inhibition of growth on Trametes hirsuta Wulf. ex Fr. and Oligoporus placenta (Fr.) Gilb. & Ryvarden at the end of the experiment (9^th day) by CuO-L. camara np formulation

There was no inhibition of fungal growth at concentrations 0,25; 0,5; 1; 5; 7 and 10% in all the plates till the end of the experiment in the case of plates having A. indica, P. pinnata, and orange peel (C. reticulata) extracts. Plates with CuO nanoparticles synthesized using A. indica, P. pinnata and orange peel (C. reticulata) extracts also showed no inhibition at concentrations ranging from 0,25; 0,5; 1; 5; 7 and 10%. Plates containing precursor solution of CuSO₄^.5H₂O showed inhibition of up to 17% at a concentration of 10% which is not significant. L. camara leaf extract exhibited an inhibition of 9,66 % at a concentration of 10% against the white rot fungus T. hirsuta.

Table 1:
Effect of biologically synthesized CuO nanoparticle against the wood decay fungi Trametes hirsuta) and Oligoporus placenta*.

In plates with CuO nanoparticles synthesized using L. camara leaf extract there was 48,88 % inhibition on the growth of the white rot fungus T. hirsuta and 22,96 % inhibition on the growth of brown rot fungus O. placenta at the end of experiment at a concentration of 7%. At 10% concentration there was complete inhibition of growth (100%) against T. hirsuta and an inhibition of 34,44 % was observed against O. placenta. It revealed that even though CuO nanoparticle synthesized by L. camara leaf extract completely inhibited the growth of T. hirsuta , it is not capable of inhibiting the growth of O. placenta completely at a concentration of 10%. For further studies 10% of the sample is taken as minimal inhibitory concentration (MIC).

Commercially available CuO nanoparticles were also tested against wood decay fungi for concentrations ranging from 0.1 to 5%. An inhibition of 71,6 % was observed against T. hirsuta and 51,6% against O. placenta at a concentration of 5% copper oxide nanopowder in distilled water. There was no complete inhibition on growth of both indicating the instability of the CuO nanoparticles in distilled water, as the particles that dispersed in distilled water without adding any stabilizer tend to aggregates and settle down. It corroborates the fact that the stability of the nanoparticles in the dispersing medium is one of the main factor that describes the properties of nanoparticles (Yu and Xie 2012, Nair et al. 2017, Nair et al. 2018).

Since there was no significant level of inhibition in the plates containing L. camara leaf extract and precursor solution of CuSO₄.5 H₂O (0.025M) alone, it can be concluded that the complete inhibition on growth of T. hirsuta observed in plates containing CuO nps synthesized by L. camara leaf extract is due to the presence of CuO nanoparticles synthesized or due to the synergistic effect of the plant extract and the synthesized CuO nanoparticles. The smaller size of CuO nanoparticles synthesized by L. camara leaf extract when compared to the size of CuO nanoparticles synthesized by other plant extracts might have also contributed to the inhibition on the growth of the decay fungi. The copper oxide nanoparticles synthesized in plant extracts were found to be stabilized by the components of the plant extract maintaining the properties of nanoparticles. This is in accordance with earlier reports that CuO nanoparticles synthesized with the help of plant extracts acts as both reducing and stabilizing agents (Lee et al. 2011, Khan et al. 2017). Many plant extracts have reported to have antifungal property and have wood preservative effects, also (Tascioglu et al. 2013). Gupta et al. (2017) reported that L. camara have wood protection properties as evidenced by its ability to improve the dimensional stability of wood. Since both CuO nanoparticles and L. camara extracts had wood preservative properties, the synergistic effect of both would be resulting in an effective ecofriendly, and less expensive wood preservative.

Table 2:
Antitermite activity of biologically synthesized CuO nps plant extract formulation as per IS 4833 1993 (Graveyard test-accelerated).

There was no damage to the rubber wood samples treated with the formulation of copper nanoparticles synthesized using all the plant extract after an exposure period of six months in the field. (Table 2). Evaluation is progressing to get a better idea regarding the antitermite activity of the plant extract CuO nps formulation under field conditions.

Formulation of copper oxide nanoparticle and Lantana camara leaf extract was found to be effective as a wood protectant against two decay fungi as evidenced by growth test on agar with added test substances. This may be due to the smaller particle size of the CuO nps synthesized using L. camara extract. All the plant extract CuO np formulations are found to give protection against termites in the field for a period of six months and further evaluations are in progress for a better conclusion. Development of a sustainable plant extract based synthesis route for metal nanoparticles enables the possibility of combining the intrinsic property of plant extract and metal nanoparticles for potential application in wood protection. The resultant formulation may protect wood from biodeterioration in a more effective way without any harmful effects to the environment. However, decay tests with impregnated wood samples must follow to prove protecting activity also with wood. In view of a feasible use for wood protection it must be also investigated if the CuO nps fix to the woody cell wall because otherwise only an indoor use will be possible.

Furthermore, more fungi than only each one white rot and one brown rot species should be investigated because there are hundreds of different species involved in wood decay. In view of practice it is also remarkable that the CuO nps were more effective against a white-rot fungus because in many regions (e,g. Europe) softwoods are the main construction woods and these are mostly attacked by brown-rot fungi. Last, the costs of such wood treatment compared to other wood protection measures must be considered.

The authors are thankful to University Grants Commission, India for the financial assistance provided in the form of Post Doctoral Fellowship No.F./PDFSS-2015-17-KAR-11613 and Director, Institute of Wood Science and Technology (IWST) for providing the necessary infrastructure. The assistance of CeNSE, IISc, Bangalore, India for the SEM-EDAX analysis is gratefully acknowledged.