Natural environment is a wealthy source of bionanofactories that invested in green approaches as the fabrication of biomimetic nanomaterials. The current study points out the importance of microbial activity in metal bioremediation, green synthesis of NPs, and global biogeochemical cycles of bioactive metals as well. It describes for the first time the synchronous biosynthesis of zero- (intracellular) and one-dimensional (extracellular) copper oxide nanoparticles (CuO-NPs) via
Transition metal oxide NPs are a significant group of semiconductors that fall in the circle of scientists’ interest because of their wide and diverse applications. Copper oxide NPs (CuO-NPs) (e.g., CuO, Cu2O, or Cu3O4) are among semiconducting compounds, particularly p-type with a monoclinic and cubic structures that attract more of a concern [
Additionally, several investigations reported the antimicrobial activity of CuO-NPs against hospital-acquired infections and plant pathogens which encourage their investment in antibacterial paints/coatings, hospital equipment, skin products, food packaging, and plant disease management [
Nowadays, CuO-NPs substituted other noble metals such as Au, Ag, and Pt in the aforementioned applications that could be attributed to their availability at least 10-fold in a cheaper price than those of precious metals, relative physicochemical stability, and easy mixing with other polymers [
In view of the brilliant application prospects of CuO-NPs and these unusual properties, a lot of investigations were achieved to prepare them. Generally, the synthesis approach often controls the size and shape of nanomaterials to be convenient for a particular application. The synthesis of CuO-NPs was performed either by top-down or bottom-up routes, including physical, chemical, and hybrid methods [
Based on the synthesis location, the microbially synthesized NPs could be classified into intracellular or extracellular. The intracellular mechanism involves the diffusion of metal ions into the cell followed by their reduction by cell wall enzymes [
This study had the objective to focus the scope of microbial-metal interaction through biofabrication of CuO-NPs via
The bacterial strain
The optical property of as-synthesized CuO-NPs was studied by UV-Vis spectroscopy with Labomed model UV-Vis double beam spectrophotometer in a wavelength range of 200–800 nm at room temperature, as a preliminary step. The structural characteristic properties of both types of CuO-NPs were determined using the following techniques: (1) scanning electron microscope-energy-dispersive X-ray microanalysis (EDX) for chemical composition analysis, (2) X-ray diffraction analysis (XRD) for identifying and evaluating crystallinity of NPs using X-ray diffractometer (Shimadzu 7000, USA) that operates with scan rate of 2°/min for 2
The well diffusion assay was applied to evaluate the antibacterial and antifungal activity of as-synthesized CuO-NPs on bacterial and fungal species listed in Table
The maximum inhibition zone of different concentrations of biogenic CuO-NPs, metal precursor, and antibiotics against planktonic pathogens.
Microorganism | Concentration | Zone of inhibition (ZOI) (mm) | |||||||
---|---|---|---|---|---|---|---|---|---|
CuO-NP type | Metal precursor | Antibiotics | |||||||
Class | Strain | Intracellular | Extracellular | Rifamycin | Tetracycline | Streptomycin | Nystatin | ||
Fungi | 100 | 5.3 ± 03 | 0.3 ± 0.03 | 1.2 ± 0.3 | ND | ND | ND | 5.7 ± 0.8 | |
200 | 8.5 ± 0.5 | 0.4 ± 0.02 | 2 ± 0.6 | ND | ND | ND | 9.1 ± 1.2 | ||
100 | 6.9 ± 0.5 | 0.3 ± 0.05 | 1.5 ± 0.6 | ND | ND | ND | 6.8 ± 0.6 | ||
200 | 10 ± 0.7 | 0.5 ± 0.1 | 2.1 ± 0.4 | ND | ND | ND | 9.9 ± 0.5 | ||
Gram-negative bacteria | 100 | 3.8 ± 0.8 | 0 ± 0 | 0.8 ± 0.2 | 5.1 ± 0.5 | 6.1 ± 0.5 | 5.2 ± .8 | ND | |
200 | 5.3 ± 0.9 | 0.2 ± 0.09 | 1.1 ± 0.6 | 8.9 ± 0.3 | 10.3 ± 0.9 | 9.6 ± 1.6 | ND | ||
100 | 3.2 ± 0.6 | 0 ± 0 | 0.8 ± 0.3 | 5.5 ± 0.6 | 6.3 ± 0.8 | 5.5 ± 0.7 | ND | ||
200 | 4.7 ± 0.6 | 0.2 ± 0.07 | 1.2 ± 0.5 | 8.1 ± 1.0 | 10.1 ± 1.3 | 9.7 ± 1.1 | ND | ||
100 | 3.1 ± 0.2 | 0.1 ± 0.03 | 1 ± 0.4 | 5.7 ± 0.7 | 6.3 ± 0.8 | 5.5 ± 0.7 | ND | ||
200 | 5.4 ± 0.3 | 0.3 ± 0.03 | 1.8 ± 0.6 | 8.3 ± 0.8 | 9.2 ± 1.3 | 9.3 ± 1.3 | ND | ||
Gram-positive bacteria | 100 | 5.8 ± 0.8 | 0.5 ± 0.05 | 1.7 ± 0.7 | 7.4 ± 1.2 | 8.7 ± 0.8 | 7.5 ± 1.0 | ND | |
200 | 7 ± 1.1 | 0.6 ± 0.1 | 2.6 ± 0.8 | 13.5 ± 1.5 | 14.2 ± 1.3 | 12.2 ± 1.3 | ND | ||
100 | 8.3 ± 1.2 | 0.4 ± 0.08 | 0.8 ± 0.07 | 7.8 ± 0.6 | 8.2 ± 0.8 | 8.4 ± 0.6 | ND | ||
200 | 10.6 ± 1.5 | 0.8 ± 0.08 | 1.3 ± 0.3 | 14.2 ± 0.9 | 15.5 ± 1.5 | 13.8 ± 1.1 | ND | ||
100 | 7.5 ± 0.7 | 0.2 ± 0.03 | 1.2 ± 0.5 | 6.7 ± 0.3 | 8.3 ± 0.5 | 7.6 ± 0.8 | ND | ||
200 | 9.7 ± 0.3 | 0.3 ± 0.05 | 1.8 ± 0.9 | 12.3 ± 1.0 | 14.7 ± 1.3 | 13.2 ± 1.5 | ND | ||
100 | 4.8 ± 0.6 | 0.3 ± 0.07 | 1.4 ± 0.5 | 6.3 ± 0.7 | 7.9 ± 0.7 | 6.7 ± 0.8 | ND | ||
200 | 6.3 ± 0.8 | 0.5 ± 0.03 | 2.5 ± 0.7 | 9.5 ± 1.1 | 12.2 ± 0.6 | 10.5 ± 0.5 | ND |
ND: not detected.
A colorimetric tissue culture plate assay was performed for studying the ability of as-synthesized CuO-NPs to inhibit biofilm activity of both
The algicidal effect of both types of CuO-NPs, NP precursors, and antibiotic (150 and 300
This study sought the biogenic synthesis of CuO-NPs by
Optical properties of CuO-NPs (a) and UV-Vis absorption spectra of intracellular (b) and extracellular (c) CuO-NPs synthesized by strain 10B; NPs: intra/extracellular CuO-NPs, Ctrl-1: media with copper precursor Cu(NO3)2, and Ctrl-2: media without NP precursor Cu(NO3)2.
Herein, spectral analysis revealed that the SPR absorption maximum peaks of the intracellular and extracellular CuO-NPs occurred at 275 and 430 nm, respectively (Figures
A commonly used characterization technique for crystallographic identity and the phase purity of the examined material is XRD. Figures
XRD crystallographic pattern of biosynthesized CuO-NPs; (a) intracellular and (b) extracellular and (c) JCPDS card number 73-1917.
Also, the broad diffraction patterns implied the smaller crystal size of intracellularly synthesized CuO-NPs than extracellularly synthesized one. In addition, the results suggest that bacterial mediated synthesis of single-phase CuO-NPs and no diffraction peaks related to other phases were detected which indicate the phase purity [
A semiquantitative approach that identifies the elemental composition of NPs with their relative proportions (e.g., Atomic%) is EDX. The EDX pattern of intra/extracellularly synthesized CuO-NPs is represented in Figure
EDX profile of biosynthesized CuO-NPs by strain 10B (a) intracellular and (b) extracellular.
DLS is a noninvasive technique that assesses hydrodynamic diameters (size distribution) and
Particle size distribution curve for Cu-ONPs synthesized intracellularly (a) and extracellularly (b) by strain 10B.
Moreover, DLS evaluates the homogeneous/heterogeneous dispersion of NPs through polydispersity index (PDI), which measures the second moment of the size distribution of the NP population. The PDI ranges are from 0 to 1; values greater than 0.5 or close to 1 indicate that the sample has a broad size distribution, while values close to zero show a homogeneous dispersion [
Zeta potential of biosynthesized CuO-NPs; intracellular (a) and extracellular (b).
Remarkably, the negative sign of zeta potential could be attributed to the negatively charged phosphate group (PO43−) along the sugar-phosphate backbone within nucleic acid residues (DNA and RNA) and also negatively charged amino acids as aspartate and glutamate. These compounds provide stability for CuO-NPs by acting as capping, stabilizing, and functionalizing agent [
TEM micrograph of strain 10B synthesized intracellular CuO-NPs; (a) at stationary phase and (b) extracted NPs (after cell lysis).
TEM micrograph of strain 10B synthesized extracellular CuO-NPs; (a) intact cell with EPS supplied with needle and spherical shaped CuO-NPs, (b) needle- or spindle-shaped CuO-NPs (magnified view) (large view in the bottom right square), (c) sheet- or flake-shaped CuO-NPs, and (d) nanowire CuO-NPs.
In this bottom-up approach, the copper precursor salt was reduced by means of oxidation/reduction reaction into their respective NPs. This process included a cascade of steps initiated by uptake Cu ions probably via uncharacterized energy-independent channels such as OmpC porins, Zn2+ uptake systems, or some ATPases [
Apparently, the main suggestion in current work is that the bioreduction process occurred by the action of nitrate reductase, particularly, with complete exhaustion of nitrate (NO3−) and presence of nitrite (NO2−) (data not shown). During the metabolic process, the nitrate reductase and conjugated electron shuttling molecules may shuttle electrons to the metal ions, which by this way undergoing redox reaction and leading eventually to NP formation as referred by Lin et al. [
Despite the significance of copper, bacteria generated enormous protective mechanisms against high levels of it. Interestingly, cytoplasmic accumulation is an important adaptive mechanism adopted by copper-resistant bacteria for detoxification [
Another assumption that could also be proposed is that the intracellularly accumulated CuO-NPs were deposited as cytoplasmic inclusions in associated with polyphosphate granules. Evidently, large phosphorous peak with 52.6% was detected from EDX (Figure
As a whole, the metal salt reduction, nucleation, and growth were the main steps in the extracellular synthesis of CuO-NPs. In addition, the bacterial EPS was the fundamental responsible and a key location for this multistep process. As documented by Kumar et al. [
Notably, production of EPS mediates nullification of toxic compounds by sequestering and chelation of metals [
It is worth pointing out that these spherical shaped CuO-NPs conducted as the seed, embryo, or nuclei particle that subjected to growth and self-assembly within the EPS layer leading to rod (Figure
Virtually, the ingredients of 10B EPS contributed mainly in stabilization and interparticle binding of CuO-NPs to form nanowires by modifying the order of free energies for different crystallographic facets of NPs and subsequently controlling their relative growth rate kinetics through selective adsorption and chemical interactions between EPS constituents and NP faces [
Additionally, Cl− was suggested to be another abiotic parameter that could induce elongation of CuO-NP shape [
Notably, the size measurement by DLS seems to be larger than TEM measurement, which could be explained by the fact that DLS assesses the size of overall aqueous medium accompanying with NPs. Thus, overlapping in NP size occurred which resulted in the generation of the electrical double layer along with interference on charged particles [
Considering the outbreak of microbial contamination in several sectors such as food processing, water treatment systems, medical/pharmaceutical/agricultural products, and especially with daily increasing in multidrug-resistant microorganisms (MDR), the current study dealt with the antimicrobial activity of CuO-NPs against a wide spectrum of pathogens. The results of well diffusion assay revealed clear zones of inhibition for different concentrations of intra/extracellularly synthesized CuO-NPs in parallel to NP precursor and antibiotics (Table
Moreover, the inhibitory effect was more pronounced in the Gram-positive bacteria as compared to the other examined pathogens. The structural and compositional differences of the outer bacterial wall in addition to bacterial physiology and metabolism could explicate this result, where Gram-negative bacteria have additional outboard negatively charged lipopolysaccharide layer that repels the negatively charged CuO-NPs resulted in limiting cell wall binding sites available for NP binding. In addition, Gram-negative bacteria have unique multiple efflux pump system [
Meanwhile, CuO-NPs exerted a remarkable suppression against
Broadly, there were common features shared between the free-floating pathogens and their biofilms counterpart as clarified in Table
Antagonistic activities of CuO-NPs, metal precursor, and antibiotics on biofilm and algal growth.
Treatment | Concentration ( |
Inhibition% | ||
---|---|---|---|---|
CuO-NP type | ||||
Intracellular | 150 | 55.2 ± 3.4 | 82.5 ± 6.9 | 67.4 ± 8.7 |
300 | 84.1 ± 5.6 | 98.2 ± 4.8 | 92.3 ± 5.2 | |
Extracellular | 150 | 9.7 ± 2.3 | 13.6 ± 2.9 | 10.8 ± 1.2 |
300 | 17.5 ± 4.9 | 19.1 ± 7.2 | 20.6 ± 4.6 | |
Metal precursor | ||||
Cu(NO3)2 | 150 | 3.4 ± 0.9 | 10.1 ± 2.1 | −27.2 ± 6.8 |
300 | 5.18 ± 1.3 | 17.08 ± 6.1 | −5.3 ± 1.0 | |
Antibiotics | ||||
Rifamycin | 150 | 53.1 ± 6.3 | 58.3 ± 7.4 | ND |
300 | 80.8 ± 8.4 | 92.7 ± 6.7 | ||
Tetracycline | 150 | 54.9 ± 3.9 | 62.7 ± 9.8 | |
300 | 87.5 ± 10.2 | 96.1 ± 9.3 | ||
Streptomycin | 150 | 52.0 ± 6.8 | 58.0 ± 5.7 | |
300 | 78.2 ± 4.2 | 92.0 ± 10.2 | ||
Nystatin | 150 | ND | 52.5 ± 5.6 | |
300 | 96.8 ± 12.1 |
ND: not detected.
The drastic algicidal effect of intracellular CuO-NPs is illustrated in Table
Commonly, the antagonistic activity (antibacterial, antifungal, antibiofilm, and algicidal) of both types of CuO-NPs manifested a marked dose-dependent manner. It is evident that there was an increment in the inhibition strength as the CuO-NP concentration elevated. In agreement with our study, Essa and Khallaf [
Actually, the higher antagonistic activity of intracellular CuO-NPs over extracellular CuO-NPs and their precursor salt attributed to the larger surface area (surface/volume ratio) associated with ultrafine size and monodispersity with no agglomeration which subsequently leads to a faster dissolution rate (elution/releasing) of copper ions. Therefore, that allowed more tightly adherence with microbial cells and eventually cytotoxicity stimulation [
To summarize, this work for the first time demonstrates the ability of
The authors would like to confirm that all data used to support the findings of this study are included within the article.
The authors declare no conflict of interest.
The authors acknowledge the Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El Arab, Alexandria, Egypt, for supporting the activities presented in this study. Also, the authors gratefully thank Eng. Ayman Kamal for his efforts in imaging the samples by electron microscopy. This work supported completely by the Genetic Engineering and Biotechnology Institute, City of Scientific Research and Technological Applications, Borg El Arab, Alexandria, Egypt.