Tectona grandis Capped Silver-Nanoparticle Material Effects on Microbial Strains Inducing Microbiologically Influenced Corrosion

1Mechanical Engineering Department, Covenant University, Ota, Nigeria 2Chemical and Metallurgical Engineering Department, Tshwane University of Technology, Pretoria, South Africa 3Chemical Engineering Department, Covenant University, Ota, Nigeria 4Petroleum Engineering Department, Covenant University, Ota, Nigeria 5Department of Civil Engineering and Building, Vaal University of Technology, Vanderbijlpark, South Africa


Introduction
Microbiologically influenced corrosion (MIC) can refer to changes in electrochemical reactions at the surface of a metallic material maintaining interface with system of microorganisms, conglomerated into biofilm, which induce corrosion process of the metallic material [1,2].Many metallic materials, including stainless steel, and many environments, for example, domestic water, wastewater, marine, food processing, and oil and gas and industrial chemical, are susceptible to the material degradation of MIC [1][2][3][4].This has made MIC related crises a cost-gulping phenomenon, in billions of dollars of direct cost, in many countries, while in the natural gas industries alone, MIC has accounted for about a third of corrosion failures [3,5].Attachment of microbial strains unto metallic surfaces, leading to the formation of biofilm colony of microbes on the metal, has been identified as one of the major causative mechanism of microbiologically influenced corrosion (MIC) [1,2,4].That the microbial attachment, the biofilm formation, and subsequent MIC induced corrosion damage of metallic material all occur on the material surface has been drawing research attention towards microbial and material surfaces interactions among the other conditions necessary for MIC attacks of metallic materials [1,6,7].Microbes can refer to the entire evolutionary genus of microorganisms including bacteria, Archaea (methanogens), and Eukaryota (fungi), all of which are causative agents of MIC attacks on metallic materials [4].However, motility of bacteria strains, especially the conveniently flagellated strains, enhances them with the special advantage of pioneering initial attachment to the metallic surface before attracting other secondary colonizers of microorganism, for forming MIC inducing biofilm [2,7].Once the mutual interactions of the colonies are established in the biofilm, resistance to MIC control approaches could be enhanced with consequence propagation of the corrosion degradation process.It is for this reason that attempts at making the initiation of MIC due to methods that could preclude or delay the initial attachment to the metallic surface are being opined as a corrosion-protection/control approach that will go a long way in MIC mitigation.
For instance, coating is a corrosion control method that can be applied to different types of metallic materials with suitable corrosion-protection additives [8] for which the use of nanotechnology based methods via nanoparticle usage is being proffered as a novel approach [9].Notable research advances in this direction include the identification of the antimicrobial property of silver for its usage in the form of silver-nanoparticle dispersed in polymer or included in coating applications for metallic corrosion-protection and antibacterial resistance improvement [10,11].Of pertinent relevance to the current discourse is the use, in [12], of silvernanoparticle material that had been synthesized from plants as reducing and capping agent for inhibition of both microbial growth and metallic corrosion in acidic chloride environment.Such synthesis of metallic nanoparticle via natural bioresource approach is known to offer benefits of environmental friendliness and ecocompatibility, especially due to the nonusage of toxic chemical for the synthesizing process.
Tectona grandis is a natural plant from which leaf-extract had been used successfully also for inhibiting mild steel and stainless steel corrosion in reported works [13,14].In addition to these, Tectona grandis leaf-extract has been employed recently for investigating the growth inhibition on MIC inducing microbes [15].In spite of these, however, there is dearth of study in which the antimicrobial property from silver had been annexed with the green benefits that could be derivative from Tectona grandis usage as a precursor and capping agent for silver-nanoparticle synthesis.In contrast, extracts from many natural plants have been employed in studies for biologically mediated synthesis of silver-nanoparticle material [16][17][18][19].Among these are leaves of Prunus persica by [16], Aloe vera by [17], and Azadirachta indica by [19].In addition to these, the cited work in [18] detailed a review on the use of extracts from 40 plants, among which 27 are leaf-extracts, as a precursor for reducing silver from its compound to its nanoparticle.All these reports from the literature detailed antimicrobial effects of the biosynthesized silver nanoparticle, but none include use of Tectona grandis for the silver nanoparticle synthesis.Notable details of antimicrobial activity, from some of the cited works that also tested antimicrobial effect of the plant extract, for example, the report on Azadirachta indica in [19], include the indication that the plant extract usage alone resulted in "no zone" at inhibiting growth of the studied microbial strains.In sharp comparison, extracts from the fruit [20] and from the leaf [21] of Tectona grandis inhibited growth of different strains of microbes.Apart from these, Tectona grandis leafextract was also shown to be capable of improvement of antimicrobial potency when it was used in conjunction with tetracycline in [22].These garner supports for the choice of Tectona grandis leaf-extract for use as a precursor for plant extract capped silver nanoparticle in this study.
More especially, motivation for the present study was drawn from the reported work in [22] wherein Tectona grandis leaf-extract was actually used for silver-nanoparticle synthesis but with antibacterial study conducted on only one Gram-positive (Staphylococcus aureus) and only one Gramnegative (Escherichia coli) strains of microbes.The positive results from that study indicate that more works need to be carried out for assessing the effectiveness of this bionanoparticle material on more types of microbes, especially those known to induce MIC.Such more types of microbes need to necessarily include Pseudomona aeruginosa (a flagellated, motile Gram-negative microbial strain), for instance, for this strain is known to usually pioneer attachment to metals for biofilm formation and eventual MIC attack on metallic materials [2,7,23,24].Therefore the objective of this paper was to investigate effect of Tectona grandis capped silvernanoparticle on the inhibition of the growth of microbial strains inducing microbiologically influenced corrosion of metallic materials.

Tectona grandis Leaf-Extract Biosynthesis of Silver-
Nanoparticle Material.Tectona grandis (T.grandis), Verbenaceae, was subjected to standard procedure detailed in [15] for obtaining pasty form of leaf-extract.This procedure includes drying in a well aerated room maintained at 20 ∘ C (by a 5-ton air-conditioning unit) and grinding it into small bits before wrapping in Whatman5 filter paper for placing into a condenser-equipped soxhlet extractor utilizing CH 3 OH (methanol) for solvent medium.Out of the T. grandis leafextract paste obtained from the procedure detailed, 25 mg was dissolved and made up to a volume of 1000 ml using 0.1 M AgNO 3 , that is, silver nitrate (Sigma Aldrich5).As detailed in [5,15], after 48 hrs, sample was obtained from the dissolution, which was then centrifuged at 3,500 rpm for 15 minutes in the Laboratory Centrifuge, Model SM-80-2 obtained from Surgifield5 (England).The resultant supernatant was then poured out before the biosynthesized nanoparticle residue got transferred to a watch glass for air drying and afterwards collection in Eppendorf tubes.These were stored at room temperature for further use as the biosynthesized T. grandis capped silver nanoparticle in the study.

Characterisation of the Biosynthesized Silver-Nanoparticle
Material.Sample from the biosynthesized T. grandis capped silver-nanoparticle was prepared and then characterised using a Pro X PHENOM6, Model 800-07334 (Phenom-world5, Netherlands) scanning electron microscopy and energy dispersive spectroscopy (SEM + EDS) instrument.For this characterisation, the sample was placed on a sample stub for loading onto the stage of the Quorum Sputter Coater, Model Q150R ES, obtained from Quorum Technologies Lim-ited5 (England), and for coating the sample with gold [5,15].This was followed by the removal of the sample stub, from the coating instrument, for placing on standard sample holder mounted on the Pro X PHENOM, Model 800-07334 obtained from Phenomworld (Netherlands).On this instrument, the biosynthesized T. grandis capped silver-nanoparticle was subjected to the scanning electron microscopy and energy dispersive spectroscopy (SEM + EDS) analyses.

Inhibition Study of MIC Inducing Microbial Strains.
Staphylococcus aureus (S. aureus) and Escherichia coli (E.coli) were used, respectively, as Gram-positive and Gramnegative microbial strains in this study.Reasons for choosing these microbial strains include the motivation to facilitate comparison of test-results from this study with what obtained from the previously cited work by other researchers [22].In addition, however, Bacillus spp.and Micrococcus varians (M.varians) were included among the Gram-positive while Pseudomonas aeruginosa (P.aeruginosa) and Serratia spp.were among the Gram-negative microbial strain in the present study.These isolates were obtained from the culture collection centre in the Biotechnology Unit of Department of Applied Biological Sciences, Covenant University, Ota, Ogun State, Nigeria, and were maintained on nutrient broth and incubated overnight at 37 ∘ C between 18 and 20 hrs [25,26].From these cultures of microbial strains, 2 ml, of the microbes, was collected into sterile tube for making up with sterile distilled H 2 O until matching turbidity standard of 0.5% Mcfarland [27].
Each ensuing mixture of test-organisms was then used for seeding sterile nutrient agar plates, via the agar well diffusion method.Wells were bored into the seeded nutrient agar using 9 mm sterile cork borer.A gram by mass of the T. grandis capped silver nanoparticle was then dissolved and thoroughly mixed in 10 ml of C 2 H 6 OS (Dimethyl sulfoxide; DMSO).From this dissolution, 0.2 ml was obtained using sterile pipette, for dispersing into the well that had been bored onto the agar plate.This was followed by incubating the plates at 37 ∘ C for 24 hrs, before measuring, in mm unit, the zones of inhibition that resulted from this procedure [28].
For a positive control of antimicrobial effects, 10 g Gentamicin, from Abtek Biologicals Limited6 (Liverpool, UK), was utilized.The antibiotic chemical was used also, that is, just as the synthesized bionanoparticle material, in the seeded agar plates for the microbial growth inhibition study.This approach was to facilitate the comparison of the inhibition results obtained from the biosynthesized nanoparticle with that obtained from the antibiotic chemical usage.

Scanning-Electron-Microscopic (SEM) Analysis SEM.
The image of the T. grandis capped silver-nanoparticle by the SEM instrument is shown in Figure 1(a) and analyses employing ParticleMetric5 rendering facility of the SEM instrument in Figure 1(b).Results from the ParticleMetric analysis indicated that for 157 particles the SEM facility could pick for the rendering in Figure 1(b), circle equivalent diameters, in m unit, range from (median = 48.7:average = 57.5) to (median = 8.26: average = 9.95).Figure 1(b) however depicted regions of uncoloured clusters that represent regions of particles beyond the identifiable range of the SEM system and indicate that nanoscale range of particles was obtained.

Energy-Dispersive-Spectroscopic (EDS) Characterisation.
Results from the EDS characterisation of elemental constituents from the T. grandis capped silver nanoparticle are presented in Figure 2.
From the EDS characterisation in Figure 2, up to three spikes of silver (Ag), among the prominent spikes in the figure, could be identified.By this, it was established that silvernanoparticle material was obtained.The other spikes among which carbon, oxygen, and nitrogen are also prominent or prevalent corroborate elemental constituents of biocompatible compounds from the T. grandis leaf-extract as could be inferred from biochemical characterisation studies of other plant leaf-extracts in [29][30][31][32][33].It could be noted that Figure 3 also includes linear plots of standard susceptibility criteria, as either sensitive or resistant, by the Gram-positive or Gram-negative microbes to the Gentamicin chemical, used as the antibiotic control for this study.These linear plots are for aiding direct interpretation of the zone of microbial growth inhibition obtained from the biosynthesized nanoparticle and the antibiotic control as per the susceptibility criteria from [34], according to microbial susceptibility interpretation for the standardized single-disc method [35].From this, it could be deduced that all the three Gram-positive microbial strains for the study exhibited were sensitive to the T. grandis capped silver-nanoparticle material, by the zone of inhibition that was greater than 15 mm obtained for these microbes.Compared to this, only two of the Gram-positive microbial strains studied were sensitive to the Gentamicin (control antibiotic chemical), among which the Bacillus spp.just reached the 15 mm bar of zone of inhibition.The third Gram-positive strain of microbe for the study, S. aureus, was resistant to the Gentamicin control.In an almost similar manner, all the three Gramnegative strains of microbes for the study were sensitive to the T. grandis capped silver-nanoparticle material compared to only two, E. coli and Serratia spp., that were sensitive to the Gentamicin control antibiotic.The P. aeruginosa was resistant to the Gentamicin chemical, as per the set susceptibility criteria interpretation for the single-disc method in [34,35].

Conclusions
By the results in the study, it is established that the T. grandis capped silver nanoparticle material, to which all the microbial strains studied were sensitive, outperformed the Gentamicin chemical on the inhibition of the growth of MIC inducing microbial strains studied.By this, it is opined that the T. grandis capped silver nanoparticle material could be suitable for use in MIC control applications for the corrosion-protection of metallic materials designed to operate in the MIC environment.However, further studies are recommended for investigating specific applications to which the biosynthesized nanoparticle material would be applied for situating antimicrobial effects for that condition of application.

Figure 1 :
Figure 1: SEM + EDS analyses of the T. grandis capped silver-nanoparticle material.(a) Image from scanned electron microscope.(b) Clusters of particles colouring by ParticleMetric rendering of the scanned microscopic image.

Figure 2 :Figure 3 :
Figure 2: EDS characterisation of the bionanoparticle from the SEM facility.
Studied.Results of the growth inhibition effects by the T. grandis capped silver-nanoparticle on the studied MIC inducing microbial strains are presented in Figure 3, wherein Figure 3(a) depicts results from the Gram-positive and Figure 3(b) results from the Gramnegative microbial strains.