# Biosynthesis, Characterization of Some Combined Nanoparticles, and Its Biocide Potency against a Broad Spectrum of Pathogens

Authors: Marwa Eltarahony; Sahar Zaki; Marwa ElKady; Desouky Abd-El-Haleem
Journal: Journal of Nanomaterials
 (2018)

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## Abstract

The development of environmentally benign procedures for the synthesis of metallic nanoparticles (NPs) is a vital aspect in bionanotechnology applications for health care and the environment. This study describes the biosynthesis of Ag, Co, Ni, and Zn NPs by employing nanobiofactoryProteus mirabilis strain 10B. The physicochemical characterization UV-visible spectroscopy, scanning electron microscopy-energy-dispersive X-ray microanalysis (EDX), X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS) technique including ζ potential, and polydispersity index (PDI) confirmed the formation of pure, stable monodisperse quasi-spherical oxide NPs of corresponding metals. The antimicrobial activity of biofabricated NPs was assessed against Gram-negative and Gram-positive bacteria, biofilm, yeast, mold, and algae via a well diffusion method. The results displayed significant antagonistic activity in comparison to their bulk and commercial antibiotics. Interestingly, the combined NPs exhibited promising synergistic biocide efficiency against examined pathogens which encourages their applications in adjuvant therapy and water/wastewater purification for controlling multiple drug-resistant microorganisms. To the best of our knowledge, no previous study reported the synthesis of semiconductor NPs by Proteus mirabilis and the biocide potency of combined NPs against a broad spectrum of pathogens not reported previously.


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## Body

## 1. Introduction

Microbial pollutants are the most dreadful cause for a wide range of infectious diseases which lead to an increase in the rate of hospitalization, morbidity, and mortality. The disease-causing agents (bacteria, fungi, viruses, algae, and protozoa) could be transmitted through water purification systems, contaminated medical devices such as catheters and dental materials, and food manufacturing machines, which create obvious threat for human health and the ambient ecosystem [1]. In developing countries, approximately 12 million people die annually due to consumption of water contaminated with various microbes as pointed out by Oves et al. [2]. Notably, the microbial population is naturally capable of developing resistance against commercial antibiotic drugs, besides their ability to organize biofilm structures which formed 15% aggregates of microbial cells embedded in 85% of the extracellular matrix which comprises glycolipids, polysaccharides, proteins, and DNA which undoubtedly leads to ineffectiveness of drugs [2, 3].Currently, nanotechnology which is described as “the sixth revolutionary technology” after the industrial revolution, nuclear energy revolution, green revolution, information technology revolution, and biotechnology revolution [4] opens the door for multidrug-resistant microorganism (MDR) defeat by virtue of metal nanomaterials’ leading-edge nature. Recently, membrane and polymers incorporated nanoparticles (NPs) which were developed for the water purification system [2], and NP-coated fabrics [5], bandages, walls, bed linen, surfaces, and medical equipment were examined as magic cure against microbial contamination [6, 7]. Among others, transition metal oxide NPs were deemed particularly attractive for the application of a new class of antimicrobial agents. Interestingly, several studies speculated that metals and metal oxide NPs utilize multiple mechanisms simultaneously in the microbial combating battle, placing MDR microorganisms in a critical position to develop resistance. However, antibiotics, especially bacterial drugs, induce cell death by cell wall inhibition (β-lactams), RNA synthesis (rifamycins), DNA replication (quinolones), or protein synthesis (macrolides) [8]. In this context, The US FDA has already approved some metal oxides such as ZnO as safe antimicrobial agents against bacteria, fungi, and virus [9]. Additionally, conjugation of antibiotic and metal NPs in “combination therapy” against pathogens exhibited a promising solution to stop MDR crisis [2, 10].In the framework of this research topic, particularly, for biocompatibility and biosafety in biological systems, the most important criterion is the NP synthesis approach. Classically, metallic NPs have been fabricated by well-established physical, chemical, and hybrid methods. Although NP yield of these methods was high with controlled size, morphology, and dispersion, the employment of hazard flammable chemicals and high temperature limit medical and environmental application due to contamination from precursor residuals. In recent decades, the legislation on waste electrical/electronic equipment (WEEE) and restriction of hazardous substances (RoHS) has been issued by the European Union. Thus, the timeline for exploiting the nature’s secret is coming for the employment of a benign, green, cost-effective, and medically/environmentally biocompatible approach. The green synthesis of NPs dedicates to the use of biological hosts including, but not limited to, bacteria, fungi, actinomycetes, algae, yeast, and plants [11]. The biological method for metal NP fabrication by microorganisms can be either intracellular or extracellular. Generally, in both cases, the proteins that are involved in cell metabolism are considered the responsible instrument for the reduction and subsequent conversion of metal ions into metal NPs [12]. Bacteria attract immense interest in NP synthesis by short generation times and easy manipulation [13]. Numerous bacterial genera as Pseudomonas sp., Bacillus mojavensis, Achromobacter sp. Rhodobacter, Klebsiella, and Lactobacillus have been used to synthesize compound NPs [14–17].In the light of the aforementioned, this study is aimed at the synthesis of Ag, Co, Ni, and Zn NPs by utilizingProteus mirabilis strain 10B as a bacterial nanofactory in an ecofriendly approach. The biosynthesized NPs were characterized using optical observation, UV-Vis spectrophotometry, XRD, EDX, TEM, zeta potential, and PDI. The antagonistic efficiency of the biosynthesized NPs was examined against pathogenic bacteria (Gram-positive and Gram-negative), biofilms (Gram-positive and Gram-negative), and eukaryotes (mold, yeast, and algae). No study to the best of the authors’ acquaintance has so far been reported regarding the biosynthesis of Ag, Co, Ni, and Zn NPs by using Proteus mirabilis. Additionally, no previous report recorded the effect of combined as-prepared NPs on broad-spectrum pathogens.


## 2. Materials and Methods

### 2.1. Bacterial Strain, Growth Conditions, and Synthesis of Ag/Co/Ni/Zn NPs

The bacterial strainProteus mirabilis 10B was procured from existing indoor strain collection that is concerned with denitrification study, which has been submitted to GenBank under the accession number KY964505 [18].The bacterium lawn (0.5McFarland≈108CFU/ml) was allowed to grow in nutrient broth (NB) (1.5% peptone, 0.3% yeast extract, 0.05% NaCl, and 0.01% glucose, final pH 7.0) followed by addition of equivalent 3 mM NP precursors Ni(NO3)2·6H2O, Co(NO3)2·6H2O, and Zn(NO3)2·6H2O and 1.5 mM of AgNO3 (Sigma-Aldrich). The cultures were incubated at 30°C under shaking conditions (Stuart orbital shaker). The cells at the stationary phase that include NPs were collected by centrifugation at 10,000 ×g for 20 min. The NPs were extracted from cells after disruption by using TSE buffer [17] through mild osmotic shock in a procedure reported by Samadi et al. [19]. The extracted NPs were dried at 100°C for 2 hr. The dried NPs were purified by washing 3 successive times by ethanol 70% and double-distilled water as described by Metz et al. [20]. The purified NPs were subjected for subsequent characterization and applications.


### 2.2. Characterization of Biosynthesized NPs

The biosynthesis of NPs was preliminarily observed optically by the visual color change throughout the incubation period. In parallel, control experiments (bacterial growth medium containing metal precursor and without bacteria) were incubated typically as in the test experiments. A UV-visible diffused reflection spectrum of the bacterially synthesized NPs was recorded using a Labomed model UV-Vis double-beam spectrophotometer in a wavelength range of 200–800 nm at room temperature. Scanning electron microscopy-energy-dispersive X-ray microanalysis (EDX) was performed for chemical composition analysis using JEOL JSM-6360LA, from Japan (Faculty of Science, Alexandria University). X-ray diffraction (XRD) analysis of nanoparticles was carried out on an X-ray diffractometer (Shimadzu 7000, USA) that operates with Cu Kα radiation (λ=0.15406nm) generated at 30 kV and 30 mA with a scan rate of 2°/min for 2θ values over a wide range of Bragg angles 10°≤2θ≤80 for identifying and evaluating crystallinity of NPs. Transmission electron microscopy (TEM) was employed to determine the morphology and particle size of as-synthesized NPs using JEOL JEM-1230, from Japan (Faculty of Science, Alexandria University). Dynamic light scattering (DLS) technique using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK; Faculty of Pharmacy, Alexandria University) was used for the determination of hydrodynamic diameter and polydispersity index (PDI) of diluted samples. The measurements were performed at 25°C, at a fixed scatter angle of 173°. Additionally, the zeta potential was measured for estimating colloidal stability using Zetasizer Nano ZS (Malvern Instruments, Worcestershire), and data were analyzed by Zetasizer software 6 [21].


### 2.3. Nanoparticles’ Inhibitory Effect against Planktonic Pathogens

The well diffusion assay was applied to assess antibacterial and antifungal activity of as-synthesized NPs on bacterial and fungal species. A single colony was grown overnight in nutrient broth for bacterial inoculum preparation, and turbidity was adjusted to 0.5 McFarland standards. The fungal inoculum was cultivated in Sabouraud dextrose broth for 72 h. Mueller-Hinton agar (MHA) (Sigma-Aldrich) plates were swabbed with 0.1 ml of each culture suspension, and bacterially synthesized NPs (100 and 200μg/ml) were impregnated to a center well with a diameter of 8 mm. The plates were incubated at 37°C for 24 h (bacteria) (Labnet 311D incubator) and 25°C for 72 h (fungi) (Labnet 311D incubator). The zone of inhibition (ZOI) was measured by subtracting the well diameter from the total inhibition zone diameter and expressed in millimeters. The antimicrobial activity of antibiotics (rifamycin, streptomycin, and tetracycline for prokaryotes and nystatin for eukaryotes) in addition to NP precursors (100 and 200 μg/ml) was also examined comparatively as conventional control for the antimicrobial assay [7, 15, 22].


### 2.4. Inhibitory Effect against Biofilm Formation

Spectrophotometric tissue culture plate assay was performed to investigate the biofilm inhibition of bothP. aeruginosa and S. aureus. Sterile 96-well polystyrene microtiter plate wells were inoculated with 100 μl of bacterial cell suspension. The respective concentrations (150 and 300 μg/ml) of NPs, antibiotics, and NP precursors were added into the wells. Two controls were examined in parallel (positive control wells: medium containing bacterial suspension and negative control wells: sterile medium only). Microtiter plates were covered and incubated under stationary conditions at 37°C for 24 hours. After the incubation time, the well content was discarded, washed, processed by crystal violet, and solubilized with ethanol as in Namasivayam et al. [10]. The absorbance of the ethanol-solubilized mixture at 595 nm using a plate reader (Tecan Infinite M200, Switzerland) was determined, and biofilm inhibition percentage was calculated by the following equation:
(1)Biofilminhibition%=A−A0A×100,where A represents the absorbance of the positive control wells and A0 reveals the absorbance of the treated wells with an antimicrobial agent [23].


### 2.5. Inhibitory Effect against Algae (Chlorella vulgaris)

The algicidal effect of NPs was studied by adding 150 and 300μg/ml compared to exact concentrations of antibiotic on C. vulgaris growth. Algae were cultivated and incubated as presented by Ilavarasi et al. [24]. The cell density of the culture was determined by counting with a hemocytometer under a light microscope (Olympus BH2, Japan). The inhibition percentage was calculated as in (1).


### 2.6. Application of Combined NPs in Water and Wastewater

The bacterial suppression potential of combined NPs against bacterial load present in various samples from water bodies and wastewater manufactories was studied. The samples (Table1) were collected in March 2017 and subjected to treatment with combined NPs (150 μg/ml) for different contact times (30, 60, and 120 min) [17]. The bacterial count was determined using the pour plate method and expressed as CFU/ml. The bacterial load within each sample without any treatment was determined as positive control plates. The suppression percentage at each time interval was calculated according to
(2)Suppressionpercentage=numberofcoloniesincontrol−numberofcoloniesintreatment/numberofcoloniesincontrol∗100.Table 1
Application of combined NPs in real water and wastewater samples from different sources.
Sample type
Sample name
Bacterial suppression (%)
30 min
60 min
120 min
Agricultural wastewater
Bahig Canal
48.5
54.5
72.1
Industrial wastewater
Tolombat, Almax
33.6
49.4
75.0
Municipal wastewater
Borg El Arab sewage plant
73.8
97.1
98.7
Sewage 21 k-region
75.2
83.7
94.7
Freshwater
Almahmoudia Canal
67.6
80.7
94.6
Lakewater
Mariout Lake
36.7
53.1
74.0
Seawater
Al Max Sea
39.3
56.1
74.8
Salt mine water
Elmahahat
19.9
32.1
42.4


## 2.1. Bacterial Strain, Growth Conditions, and Synthesis of Ag/Co/Ni/Zn NPs

The bacterial strainProteus mirabilis 10B was procured from existing indoor strain collection that is concerned with denitrification study, which has been submitted to GenBank under the accession number KY964505 [18].The bacterium lawn (0.5McFarland≈108CFU/ml) was allowed to grow in nutrient broth (NB) (1.5% peptone, 0.3% yeast extract, 0.05% NaCl, and 0.01% glucose, final pH 7.0) followed by addition of equivalent 3 mM NP precursors Ni(NO3)2·6H2O, Co(NO3)2·6H2O, and Zn(NO3)2·6H2O and 1.5 mM of AgNO3 (Sigma-Aldrich). The cultures were incubated at 30°C under shaking conditions (Stuart orbital shaker). The cells at the stationary phase that include NPs were collected by centrifugation at 10,000 ×g for 20 min. The NPs were extracted from cells after disruption by using TSE buffer [17] through mild osmotic shock in a procedure reported by Samadi et al. [19]. The extracted NPs were dried at 100°C for 2 hr. The dried NPs were purified by washing 3 successive times by ethanol 70% and double-distilled water as described by Metz et al. [20]. The purified NPs were subjected for subsequent characterization and applications.


## 2.2. Characterization of Biosynthesized NPs

The biosynthesis of NPs was preliminarily observed optically by the visual color change throughout the incubation period. In parallel, control experiments (bacterial growth medium containing metal precursor and without bacteria) were incubated typically as in the test experiments. A UV-visible diffused reflection spectrum of the bacterially synthesized NPs was recorded using a Labomed model UV-Vis double-beam spectrophotometer in a wavelength range of 200–800 nm at room temperature. Scanning electron microscopy-energy-dispersive X-ray microanalysis (EDX) was performed for chemical composition analysis using JEOL JSM-6360LA, from Japan (Faculty of Science, Alexandria University). X-ray diffraction (XRD) analysis of nanoparticles was carried out on an X-ray diffractometer (Shimadzu 7000, USA) that operates with Cu Kα radiation (λ=0.15406nm) generated at 30 kV and 30 mA with a scan rate of 2°/min for 2θ values over a wide range of Bragg angles 10°≤2θ≤80 for identifying and evaluating crystallinity of NPs. Transmission electron microscopy (TEM) was employed to determine the morphology and particle size of as-synthesized NPs using JEOL JEM-1230, from Japan (Faculty of Science, Alexandria University). Dynamic light scattering (DLS) technique using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK; Faculty of Pharmacy, Alexandria University) was used for the determination of hydrodynamic diameter and polydispersity index (PDI) of diluted samples. The measurements were performed at 25°C, at a fixed scatter angle of 173°. Additionally, the zeta potential was measured for estimating colloidal stability using Zetasizer Nano ZS (Malvern Instruments, Worcestershire), and data were analyzed by Zetasizer software 6 [21].


## 2.3. Nanoparticles’ Inhibitory Effect against Planktonic Pathogens

The well diffusion assay was applied to assess antibacterial and antifungal activity of as-synthesized NPs on bacterial and fungal species. A single colony was grown overnight in nutrient broth for bacterial inoculum preparation, and turbidity was adjusted to 0.5 McFarland standards. The fungal inoculum was cultivated in Sabouraud dextrose broth for 72 h. Mueller-Hinton agar (MHA) (Sigma-Aldrich) plates were swabbed with 0.1 ml of each culture suspension, and bacterially synthesized NPs (100 and 200μg/ml) were impregnated to a center well with a diameter of 8 mm. The plates were incubated at 37°C for 24 h (bacteria) (Labnet 311D incubator) and 25°C for 72 h (fungi) (Labnet 311D incubator). The zone of inhibition (ZOI) was measured by subtracting the well diameter from the total inhibition zone diameter and expressed in millimeters. The antimicrobial activity of antibiotics (rifamycin, streptomycin, and tetracycline for prokaryotes and nystatin for eukaryotes) in addition to NP precursors (100 and 200 μg/ml) was also examined comparatively as conventional control for the antimicrobial assay [7, 15, 22].


## 2.4. Inhibitory Effect against Biofilm Formation

Spectrophotometric tissue culture plate assay was performed to investigate the biofilm inhibition of bothP. aeruginosa and S. aureus. Sterile 96-well polystyrene microtiter plate wells were inoculated with 100 μl of bacterial cell suspension. The respective concentrations (150 and 300 μg/ml) of NPs, antibiotics, and NP precursors were added into the wells. Two controls were examined in parallel (positive control wells: medium containing bacterial suspension and negative control wells: sterile medium only). Microtiter plates were covered and incubated under stationary conditions at 37°C for 24 hours. After the incubation time, the well content was discarded, washed, processed by crystal violet, and solubilized with ethanol as in Namasivayam et al. [10]. The absorbance of the ethanol-solubilized mixture at 595 nm using a plate reader (Tecan Infinite M200, Switzerland) was determined, and biofilm inhibition percentage was calculated by the following equation:
(1)Biofilminhibition%=A−A0A×100,where A represents the absorbance of the positive control wells and A0 reveals the absorbance of the treated wells with an antimicrobial agent [23].


## 2.5. Inhibitory Effect against Algae (Chlorella vulgaris)

The algicidal effect of NPs was studied by adding 150 and 300μg/ml compared to exact concentrations of antibiotic on C. vulgaris growth. Algae were cultivated and incubated as presented by Ilavarasi et al. [24]. The cell density of the culture was determined by counting with a hemocytometer under a light microscope (Olympus BH2, Japan). The inhibition percentage was calculated as in (1).


## 2.6. Application of Combined NPs in Water and Wastewater

The bacterial suppression potential of combined NPs against bacterial load present in various samples from water bodies and wastewater manufactories was studied. The samples (Table1) were collected in March 2017 and subjected to treatment with combined NPs (150 μg/ml) for different contact times (30, 60, and 120 min) [17]. The bacterial count was determined using the pour plate method and expressed as CFU/ml. The bacterial load within each sample without any treatment was determined as positive control plates. The suppression percentage at each time interval was calculated according to
(2)Suppressionpercentage=numberofcoloniesincontrol−numberofcoloniesintreatment/numberofcoloniesincontrol∗100.Table 1
Application of combined NPs in real water and wastewater samples from different sources.
Sample type
Sample name
Bacterial suppression (%)
30 min
60 min
120 min
Agricultural wastewater
Bahig Canal
48.5
54.5
72.1
Industrial wastewater
Tolombat, Almax
33.6
49.4
75.0
Municipal wastewater
Borg El Arab sewage plant
73.8
97.1
98.7
Sewage 21 k-region
75.2
83.7
94.7
Freshwater
Almahmoudia Canal
67.6
80.7
94.6
Lakewater
Mariout Lake
36.7
53.1
74.0
Seawater
Al Max Sea
39.3
56.1
74.8
Salt mine water
Elmahahat
19.9
32.1
42.4


## 3. Results and Discussion

### 3.1. Biosynthesis and Characterization of NPs

This study explores the biogenic synthesis of numerous metallic nanoclusters byP. mirabilis strain 10B. The reaction mixture of ionic solutions (metal salts) was analyzed primarily along with their respective controls by visual observation; the solution color changed from pale yellow to black or dark brown in the cells and surrounding media of Ag, Co, and Ni (Figure 1). Alternatively, in the case of Zn diverse starches like haziness and white clusters were noticed. In parallel control experiments, no particularly notable changes were observed, suggesting that the biotransformation of metal ions occurs only in the presence of the reducing agent (bacteria) to relevant NPs. The results obtained are in accordance with Thanh et al. [25] and Manokari and Shekhawat [26].Figure 1
Visual inspection of NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs. Ctrl: control (media containing metal ions without bacteria).The bacterially synthesized NPs have optical and physical properties that are related to shape, size, concentration, and agglomeration state, which were studied by applying valuable analysis techniques such as UV-Vis spectroscopy, EDX, XRD, TEM, and zeta potential for identifying and characterizing as-synthesized NPs.


### 3.2. UV Spectroscopic Analysis

The signature of colloidal particles particularly noble metals was monitored through UV-Vis spectroscopy which deliberates being a preliminary stage for nanocrystal characterization. As illustrated in Figure2(a), a strong and narrow surface plasmon absorption peak (SPR) was observed at wavelengths 400–430 nm. According to Mie’s theory, small spherical nanocrystals should exhibit a single surface plasmon band; however, anisotropic particles should exhibit two or three bands, depending on their size, morphology, configuration, and dielectric environment of the prepared nanoparticles [16]. The SPR phenomenon arises because the metallic NPs physically absorbed light, and as a result of this absorption, conduction electrons of metal undertake coherent oscillation.Figure 2
UV-Vis absorption spectra of NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)On the other hand, two absorption bands were observed in wavelength ranges of 250–350 and 400–580 nm of a cobalt-containing sample (Figure2(b)). As in Farhadi et al. [27], the first band can be assigned to the O2− → Co2+ charge transfer process and the second one to the O2− → Co3+ charge transfer, which suggested formation of cobalt oxide. However, the UV-Vis optical absorption spectra of Ni and Zn reaction mixture appeared at 370 and 380 nm, respectively, indicating an almost uniform size of the NPs as highlighted by Sathyavathi et al. [21] and Selvarajan and Mohanasrinivasan [28].


### 3.3. Energy-Dispersive X-Ray Analysis (EDX)

EDX is a compositional analysis approach which gives a qualitative as well as quantitative status of elements that may be involved in formation of NPs [29]. As can be seen in Figure 3, the elemental profile of as-fabricated NPs exhibited typical characteristic elemental peaks at approximately 3 keV, 7 keV, 6–7 keV, and 8–10 keV which was attributed to Ag, Co, Ni, and Zn, with atomic percentages 50, 21, 9, and 39%, respectively, and confirms the formation of their corresponding nanomaterial. The existence of other elements accompanied with biosynthesized NPs could also be noticed in high percentage specially sulfur and phosphorus, suggesting conjugation of bacterial biomolecules that contain polar phosphorus backbones as DNA, RNA, ATP, and phospholipids. However, sulfur is an important structural and functional component of amino acids as methionine and cysteine [17]. Furthermore, some signals of Na, K, Ca, and Cu were detected, proposing that they are constituents of an amino acid functional group that still adhered to the nanoparticles. Additionally, a peak for Al was also revealed due to the Al stub used to place the sample in the instrument [30].Figure 3
EDX profile of biosynthesized NPs by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)


### 3.4. X-Ray Diffraction (XRD)

The crystallographic identity and crystalline nature quality in addition to the phase purity of the examined material were determined by X-ray diffraction (XRD). A comparison of the XRD pattern of surveyed samples with the standard Joint Committee of Powder Diffraction Standards (JCPDS) file confirmed that the particles formed in our experiments were Ag2O, Co3O4, NiO, and ZnO nanocrystals as elucidated in Table 2. The X-ray difractograms of biosynthesized NPs are illustrated in Figure 4. Generally, the diffraction peaks of all as-synthesized NPs appeared sharp, clearly distinguishable, and broad, which indicates the ultrafine nature and small crystallite size. The XRD spectrum containing no other phase indicates the purity of the sample [31].Table 2
XRD diffraction peaks and corresponding crystallographic planes matched with JCPDS.
NP type
Bragg reflections (2θ degree) and corresponding Miller indices
Crystalline phase
Standard JCPDS card number
Reference
Ag2O
27.94° (100), 32.27° (110), 46.34° (200), 54.9° (211), 57.6° (211), and 67.48° (220)
Face-centered cubic (FCC)
76–1393
[30]
Co3O4
19.2° (111), 31.1° (220), 44.9° (400), and 55.9° (422)
Face-centered cubic (FCC)
073–1701
[32, 33]
NiO
37.1° (111), 43.5° (200), and 62.4° (220)
Face-centered cubic (FCC)
47–1049
[34, 35]
ZnO
31.6° (100), 34.35° (002), 36.2° (101), 47.5° (102), 56.4° (110), 62.8° (103), 67.8° (200), 68.9° (112), and 76.9° (004)
Hexagonal (wurtzite structure)
89–1397
[15, 28]Figure 4
XRD crystallographic pattern of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)


### 3.5. Dynamic Light Scattering (DLS) andξ Potential

DLS technique was employed to evaluate the time-dependent oscillation of scattered light in dispersed nanoparticles owing to Brownian motion, which is mainly based on their hydrodynamic diameters (size distribution). Moreover, it also describes the degree of uniformity, either homogeneity or heterogeneity of particle size distribution through the polydispersity index (PDI). PDI is a dimensionless parameter which can also estimate NP aggregation. Its values range from 0 (highly uniform, monodisperse, and finer particle size distribution) to 1 (highly polydisperse with a very broad particle size distribution). In general, values of 0.3 and below are considered to be acceptable and reveal a homogenous distribution and values higher than 0.7 refer to heterogeneous dispersion of the samples and are usually not suitable to be measured by DLS [36]. In this study, the particle size distribution curves of biosynthesized AgNPs, CoNPs, NiNPs, and ZnNPs are illustrated in Figure 5. It shows the Z-average of 5, 57, 93, and 48 nm for AgNPs, CoNPs, NiNPs, and ZnNPs, respectively. It is notable that Z-average is the mean hydrodynamic size of the collection of particles measured by DLS [37].Figure 5
Particle size distribution curve of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)Notably, the size measurement by DLS seems to be larger than TEM measurement (as seen later), which could be attributed to that DLS assesses the size of overall aqueous medium accompanying with NPs as referred by Venditti et al. [38]. On the other hand, PDI values recorded 0.329, 0.381, 411, and 0.337 for AgNPs, CoNPs, NiNPs, and ZnNPs, respectively, which implies homogenous dispersity of NPs regarding to the small size of monodisperse NPs.The zeta potential considers being a pivotal criterion for the determination of colloidal stability and gives an insinuation about the degree of repulsion between adjacent similarly charged particles in dispersion. Theξ potential magnitude is indicative of long-term stability, since the stability range lies between +30 and −30 mV which means that particles with ξ potentials more positive than +30 mV or more negative than −30 mV are considered stable [39]. In this study, the ξ potential value of dispersed biosynthesized NPs appeared clearly to be advantageous by recording −54, −52.5, −43.1, and −53.4 mV for AgNPs, CoNPs, NiNPs, and ZnNPs, respectively (Figure 6). Based on the ranking table of colloid stability behavior in relation to the zeta potential referred by Vishwakarma [40], our as-prepared NPs exhibited good stability by the considerable repulsive force that is present between ultrafine particles which results in Brownian motion that retains particles away from disposition to come into aggregates or flocculates.Figure 6
Zeta potential analysis of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)Accordingly, the negative singe of the zeta potential indicates that the particles are warped with anionic biomolecules as nucleic acid residues (DNA-RNA) and also negatively charged amino acids as glutamate and aspartate [39], which provide constancy by acting as capping, stabilizing, and functionalizing agents.


### 3.6. Transmission Electron Microscopy (TEM)

For decades, TEM has been a powerful tool in microbiological researches for high-resolution ultrastructural studies of microorganisms and their components [41]. The size, shape, and morphologies of the as-prepared NPs and its producing biofactory were characterized by TEM. Particles with higher electron density will appear darker in the TEM-negative film. As highlighted in Figure 7, strain 10B behaved differently with metal precursors. Remarkably, the AgNP micrograph (Figure 7(a)) exhibited numerous, tiny, roughly globular, uniform NPs with particle size 8.86 nm scattered as seeds like in a monodisperse pattern at the periplasmic space of bacterial cells. Nonetheless, large aggregates of quasi-spherical CoNPs, NiNPs, and ZnNPs appeared to be engulfed in the cytoplasmic compartment and held by cytoplasmic proteins in a spider-net thread-like shape recording 22.1, 35.9, and 19.1 nm, respectively, in diameter.Figure 7
TEM micrograph of strain 10B-synthesized NPs: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)Interestingly, strain 10B was easily handled and manipulated with different heavy metals disparately and without much difficulty according to their toxicity or benefiting. This selective interaction with metals resulted in production of NPs in different cellular localizations [42]. Generally, the acquisition of heavy metals takes place in bacteria through harnessing of uptake systems (unspecific or specific) which proceeded by the chemiosmosis gradient across the cytoplasmic membrane of bacteria. The expression of these uptake systems is either constitutive or inducible. ATP-binding cassette transporters (ABC system) coordinate a metal traffic process of nearly every biologically required transition metal ion from the extracellular milieu to the cell cytosol interiorly [43]. Consequently, the accumulation of heavy metal ions within the microbial cell occurs by metabolic-dependent biosorption processes, which includes intracellular compartmentalization [44].Once metal ions enter the cell, it begins transformation reactions to encounter their toxicity. Oxidation reduction reaction is the most pivotal enzymatic detoxification mechanism for bacterial transformation of metals to their nanoscale [42]. The respiratory enzymes (oxidoreductase) which were membrane-bounded proteins such as NADH-dependent nitrate reductase or NAD-linked dehydrogenases were proposed to initiate the reduction process.In both enzymes with different scenarios, the final result was analogous, where NADH and NADPH are coenzymes which act as electron carriers mediating several and reversible electron transfers (accepting or losing) in the electron transfer chain throughout the metabolic process. During the biological oxidation/reduction reaction, the enzymes may shuttle electrons to the metal ions that are capable of undergoing redox reaction via multiple changes in their oxidation state, which finally leads to metal NP formation [45]. This mechanism seems to be similar to the formation of magnetic Fe3O4 particles by magnotatic bacteria [46]. In our study, the oxide form of NPs was produced due to the oxygen present in the medium and incubation condition or other oxidizing agents produced by bacterial cells as reported by Dhoondia and Chakraborty [47].The silver ions were described as “oligodynamic” owing to their higher bactericidal competency at minute concentrations [48]. Accordingly, strain 10B during the stationary growth phase accumulated the biosynthesized AgNPs at the periplasm for external extrusion by the efflux system to maintain homeostasis [46]. As recorded by Ma et al. [43], the periplasm of Gram-negative bacteria has dual function according to bacterial sensitivity and ambient condition; under favorable circumstances, the periplasm behaves as storage compartment for biologically essential metal ions detached from the cytosol. However, under acute toxicity, it eliminates metals away from the cell through the Czc-ABC efflux pumping process.Remarkably, formation of metal-protein complexes sequestered in the cytoplasm considers being another basic mechanism of heavy metal resistance according to Cerasi et al. [49] which proposed to be exploited by strain 10B in cytoplasmic synthesis of CoNPs, NiNPs, and ZnNPs. Metallothioneins (MTs) and glutathione (GSH) are important metal-binding proteins that conduct efficiently in metal scavenging (detoxification), storage, and metal homeostasis maintenance [50, 51]. The nanofactory 10B seems to store CoNPs, NiNPs, and ZnNPs as essential micronutrients which play vital catalytic, regulatory, and structural roles in proteins [52]. It is noteworthy to mention the significance of nickel ion (Ni2+) in nickel-metallo enzyme synthesis such as urease, Ni superoxide dismutase, hydrogenase, methyl coenzyme reductase, and carbon monoxide dehydrogenase which resides in the cytoplasm [53].Besides, more than 300 known bacterial enzymes require zinc for their catalytic functions as protein structure stabilization/folding, management of gene expression, DNA replication/repair, response to oxidative stress, biosynthesis of amino acids, extracellular peptidoglycan synthesis, cofactor of virulence-related proteins, and maintenance of the intracellular redox buffering of the cell [49, 54]. On the other hand, cobalt is known as a constituent of cobalamin cofactor (B12) that is crucial for fatty acid catabolism and methyl transfer reactions [43].The conclusive outcome is ascertained that the bacterial mechanism for metal resistance/detoxification is somehow involved in the NP biosynthesis process. The silver detoxification machinery could typically bind and reduce other divalent ion systems and thus resulted in NP formation [55]. Also, such metal-resistant nanobiofactory participates mainly in biogeochemical cycling of those metal ions [52] and could be utilized in bioremediation of metal polluted areas as well [56].


### 3.7. Inhibitory Effect against Planktonic Pathogens

The antimicrobial activities of examined NPs in different concentrations (100–200μg/ml) which were assessed on the basis of clearance zone in comparison with standard antibiotics and metal precursors are presented in Table 3. The diameter of inhibition zone ranged from 4.0 to 9.5 mm, 1.8 to 10.3 mm, and 3.2 to 13.4 mm for fungi, Gram-negative bacteria, and Gram-positive bacteria, respectively. Notably, the antimicrobial action of all examined biogenic NPs was rated “good” since the zone of inhibition was >1 mm as reported by Prasad et al. [11]. It is evident from Table 3 that Gram-positive bacteria intrinsically were more susceptible to all examined antimicrobial agents than Gram-negative ones were. This could be attributed to the structural and compositional differences of the outer bacterial wall; Gram-negative bacteria have an additional lipopolysaccharide layer comparable to Gram-positive ones. The uniqueness of the penetration mechanism of this extra layer could dramatically alter the suppression caused by antimicrobial agents. It is plausible to speculate that the presence of powerful resistance mechanisms like multiple efflux pump in Gram-negative bacteria such as the CzcCBA system [11] contributes in cobalt/zinc/cadmium resistance as mentioned by Lee et al. [57]. Also, plenty of negative charges in the lipopolysaccharide layer repel the negatively charged NPs and hence block the availability of cell wall-binding sites for NPs. Moreover, the outer thick peptidoglycan layer of the Gram-positive bacteria cell wall has better permeability than the Gram-negative one which makes the bacteria more susceptible to harmful substances such as toxins [58]. Consequently, the bacterial resistance/susceptibility rate is governed by both cell wall structure and resistance of bacteria to the reactive oxygen species produced by the action of an antimicrobial agent [59]. Therefore, Gram-negative bacteria require a significantly higher concentration of antimicrobial agent to be eradicated.Table 3
Antimicrobial activity through the maximum inhibition zone of different concentrations of biogenic NPs, metal precursors, and antibiotics in parallel to a collective activity effect against planktonic pathogens.
Microorganism
Concentration (μg/ml)
Zone of inhibition (ZOI, mm)
Class
Strain
NP type
Metal precursor
Collective activity effect (120μg/ml)
Antibiotics
Ag
Co
Ni
Zn
Ag
CO
Ni
Zn
Rif.
Tet.
Strp.
Nyst.
Fungi
Aspergillus bracelleuse (ATCC 16404)
100
4.8
3.9
4.0
4.5
2.2
0.9
0.8
0.8
9.3
ND
ND
ND
5.3
200
9.0
7.3
6.9
8.5
5.6
1.5
1.3
1.5
ND
ND
ND
9.5
Candida albicans (ATCC 10231)
100
5.3
4.3
4.1
4.9
4.5
1.0
0.9
1.2
9.8
ND
ND
ND
6.1
200
9.5
7.5
7.6
9.1
6.5
1.8
1.8
2.1
ND
ND
ND
10.2
Gram-negative bacteria
Pseudomonas aeruginosa (ATCC 27853)
100
4.8
2.4
2.5
2.1
3.2
0.9
1.0
0.9
7.8
5.3
6.6
5.8
ND
200
10.3
5.1
4.9
4.8
5.5
1.4
1.4
1.7
9.1
10.7
9.3
ND
Salmonella typhi (ATTC 700931)
100
4.2
2.5
2.4
2.2
3.5
1.0
0.8
1.0
7.9
5.2
6.9
5.9
ND
200
8.7
4.1
4.5
4.2
5.8
1.8
1.8
1.4
8.8
10.5
9.2
ND
E. coli (ATCC 25922)
100
4.6
2.4
2.3
1.8
3.0
1.2
0.8
0.8
7.4
5.3
6.8
5.7
ND
200
9.7
4.8
4.7
4.3
6.2
2.1
1.8
1.7
8.4
9.8
9.2
ND
Gram-positive bacteria
Clostridium perfringens (ATCC 13124)
100
5.5
3.8
4.1
3.9
4.7
1.5
1.8
1.7
10.3
7.0
8.5
7.5
ND
200
12.0
7.2
6.9
7.0
7.6
3.4
3.2
3.2
13.3
14.5
12.4
ND
Enterococcus faecalis (ATCC 29212)
100
4.5
2.4
2.3
2.5
3.7
0.8
0.8
0.8
6.3
7.4
6.1
ND
200
9.3
5.3
5.0
5.1
7.4
2.1
2.3
2.3
8.6
9.8
12.3
10.3
ND
Bacillus cereus (ATCC 7464)
100
6.3
4.0
4.3
4.2
4.7
1.8
1.4
1.5
12.8
7.5
8.7
8.1
ND
200
13.4
8.8
8.1
8.5
8.0
4.1
4.0
4.2
14.8
15.8
13.7
ND
Staphylococcus aureus (ATCC 25923)
100
5.8
3.2
3.5
3.3
4.8
1.8
1.3
1.5
11.5
6.7
8.0
7.3
ND
200
11.7
7.0
7.1
6.9
8.0
3.9
4.0
4.3
12.6
14.7
13.6
NDIt is noteworthy that an efficient antimicrobial activity was more pronounced by all types of as-synthesized NPs as compared to their precursors. Besides, a potent antimicrobial pattern of AgNPs was observed against all examined pathogenic prokaryotes and eukaryotes comparable to the others. Absolutely, the larger surface area (surface/volume ratio) associated with ultrafine AgNPs permits more closely interactions with microbial cells; hence, it enhances the cytotoxicity to the microorganisms than the large-sized nanoclusters do [1, 59]. Undoubtedly, the extent of NPs’ lethal effect relies on the concentration of applied NPs and the initial microbial concentration. In contrast to our results, a negligible inhibitory effect of 100 μg/ml AgNPs with a particle size of 12–40 nm was recorded on E. coli batch cultures (105–108 CFU/ml) [60]. However, AgNPs (120 μg/ml) with a particle size range of 2.26–10.34 nm were sufficient enough to inhibit the growth of E. coli MTCC-1302 at initial concentration (103–104 CFU/ml) [61].Further, CoNPs, NiNPs, and ZnNPs showed moderate, reproducible, and almost equal biocide activity suggesting that they have dominant inhibitory targets focused on diverse microbial metabolic pathways. Briefly, the NPs’ inhibitory mechanisms include disorganization of the building composition of the cell wall/cell membrane, increasing the membrane permeability, causing dysfunction of essential proteins by reacting with thiol groups, impairing DNA replication, and producing elevated levels of reactive oxygen species (ROS) such as hydrogen peroxide which in turn induce oxidative stress [2, 7, 22]. Interestingly, our results suggested that ZnNPs preferentially exhibited antifungal rather than antibacterial properties which come into agreement with previous studies by Manokari et al. [26] and Roberson et al. [62].Moreover, the biocide activity of NPs increases linearly with increase in NP concentration. Thus, it is obvious from the data that the antimicrobial activities are dose-dependent. Our result is coincident with Pandian et al. [22] who reported the dose-dependent manner of Ni-NPs that exerted antibacterial and antifungal activities against a wide range of pathogens. Lastly, the collective activity of four biosynthesized NP types with equivalent participation (30 μg/ml for each) resulted in enhancement of antagonistic activity against all studied pathogens. From our perspective, the synergetic effect of combined NPs imitates and approximates the antibiotic influence which boosts its application in adjuvant therapy to defeat several multiple antibiotic-resistant microorganisms. As indicated by Ashajyothi et al. [63], the ability of metals to target multiple sites in an organism makes them superior to conventional antibiotics.


### 3.8. Inhibitory Effect against Biofilm Formation andC. vulgaris

Currently, the biofilm is considered to be among the most serious issues which medical devices and water disinfection applications are facing in particular, since it exerts several mechanisms simultaneously to resist different stress factors up to 1000 times in comparison to planktonic cells [64]. Thus, NP strategies were developed to replace or enhance antibiotic treatment in solving this medical and environmental problem. In this study, nearly all features that were observed on planktonic pathogen upon exposure to various treatments (individual NPs, combined NPs, metal precursors, and antibiotics) are considered to be predominant with biofilm pathogens and even algae as evident from Table 4. Generally, sufficient antibiofilm and algicidal activities were exhibited by all types of as-prepared NPs especially AgNPs. The inhibition percentage ranged from moderate to potent and increased with gradual increase in NP concentration.Table 4
Biocide activity of NPs, metal precursors, and antibiotics in addition to collective activity influence on biofilm and algal growth.
Biofilm type
P. aeruginosa
S. aureus
C. vulgaris
Treatment (μg/ml)
Inhibition (%)
NP type
Ag
150
60.25
70.62
56.37
300
89.16
94.57
94.56
Co
150
44.77
48.09
24.40
300
76.78
80.65
49.28
Ni
150
47.82
49.98
39.71
300
74.24
83.98
59.81
Zn
150
49.95
55.13
44.50
300
73.68
83.56
73.21
Combined
Ag, Co, Ni, and Zn
150
78.52
85.73
75.18
Metal precursor
Ag
150
44.68
50.32
38.79
300
63.68
78.64
65.87
Co
150
25.97
34.71
7.08
300
57.01
61.14
28.23
Ni
150
27.69
38.54
−16.75
300
51.98
65.14
4.31
Zn
150
21.46
30.11
−46.41
300
50.76
59.04
−11.00
Antibiotics
Rifamycin
150
52.74
59.68
ND
300
81.18
93.23
Tetracycline
150
55.61
63.74
300
87.61
97.36
Streptomycin
150
50.18
57.69
300
79.47
90.85
Nystatin
150
ND
53.46
300
97.48Notwithstanding that the sessile microbial cells of biofilm protect themselves by embedding in a self-produced extracellular polymeric matrix (DNA, proteins, and polysaccharides), NPs inhibit biofilm efficiently through the damage of a planktonic phase, a chemical communication mechanism that held sessile state in aggregated form (quorum sensing), and increase in hydrophobicity which blocks the initial adhesion of free living cells in the biofilm as referred by Lee et al. [57] and Franci et al. [65]. In consistency with our results, Vincent et al. [66] and Sangani et al. [67] reported the potent antibiofilm effects of Ni-NPs and ZnO-NPs. Otherwise, the enhancement of V. cholerae biofilm by addition of ZnO-NPs was mentioned by Salem et al. [68].What is more, in contrast to our study, the stimulatory influence of ZnO-NPs (200 mg/l) was noticed with 35% cell viability ofC. vulgaris growth after 72 h incubation as referred by Miazek et al. [69]. The drastic alteration in the cellular division processes of C. vulgaris accompanied with application of NPs was recorded by Gong et al. [70]. This moves in accordance to our result, where 75% reduction in algal growth was observed by employing combined NPs. Consequently, the exploitation of NPs in controlling algal blooms will restrict their consecutive adverse environmental problems such as odoriferous, unsightly scums, toxicity of water bodies, and eutrophication [71].On the other hand, considerable stimulation in algal growth was observed with some metal precursors (Ni and Zn) and weak inhibition caused by cobalt salt even at high concentrations. These micronutrients are indispensable elements for algal metabolism and physiological function such as photosynthesis and respiration [72].In consideration of the foregoing, the robust synergetic effect of combined NPs against a wide range of pathogenic microbial forms opens promising avenues for their exploitation as antimicrobial agents in unlimited biomedicine, pharmaceutical product, and environmental applications.


### 3.9. Application of Combined NPs in Water and Wastewater

As observed in Table1, a noticeable bacterial suppression was exhibited by combined NPs predominantly with increasing contact time. Virtually, the biocide activity of combined NPs in municipal wastewater, freshwater, and agricultural wastewater was higher than in salt water, particularly Elmalahat. This could be attributed to the presence of suspended solids which adversely influence on binding strength between NPs and bacterial surface. Obviously, the existence of Cl− in the samples may interact with different metal ions (Ni2+, Co2+, Ag+, and Zn2+) that are released from combined NPs which resulted in production of NiCL2, CoCL2, AgCl, and ZnCL2. In addition, the presence of cations such as K+, Na+, Ca2+, and Mg2+ could be adsorbed on negatively charged NPs which neutralize their surface forming large flocculates and thus losing monodispersity. Consequently, the effective dose that was designed for bacterial eradication required to be adjusted according to the type of the samples and their content [73].


## 3.1. Biosynthesis and Characterization of NPs

This study explores the biogenic synthesis of numerous metallic nanoclusters byP. mirabilis strain 10B. The reaction mixture of ionic solutions (metal salts) was analyzed primarily along with their respective controls by visual observation; the solution color changed from pale yellow to black or dark brown in the cells and surrounding media of Ag, Co, and Ni (Figure 1). Alternatively, in the case of Zn diverse starches like haziness and white clusters were noticed. In parallel control experiments, no particularly notable changes were observed, suggesting that the biotransformation of metal ions occurs only in the presence of the reducing agent (bacteria) to relevant NPs. The results obtained are in accordance with Thanh et al. [25] and Manokari and Shekhawat [26].Figure 1
Visual inspection of NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs. Ctrl: control (media containing metal ions without bacteria).The bacterially synthesized NPs have optical and physical properties that are related to shape, size, concentration, and agglomeration state, which were studied by applying valuable analysis techniques such as UV-Vis spectroscopy, EDX, XRD, TEM, and zeta potential for identifying and characterizing as-synthesized NPs.


## 3.2. UV Spectroscopic Analysis

The signature of colloidal particles particularly noble metals was monitored through UV-Vis spectroscopy which deliberates being a preliminary stage for nanocrystal characterization. As illustrated in Figure2(a), a strong and narrow surface plasmon absorption peak (SPR) was observed at wavelengths 400–430 nm. According to Mie’s theory, small spherical nanocrystals should exhibit a single surface plasmon band; however, anisotropic particles should exhibit two or three bands, depending on their size, morphology, configuration, and dielectric environment of the prepared nanoparticles [16]. The SPR phenomenon arises because the metallic NPs physically absorbed light, and as a result of this absorption, conduction electrons of metal undertake coherent oscillation.Figure 2
UV-Vis absorption spectra of NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)On the other hand, two absorption bands were observed in wavelength ranges of 250–350 and 400–580 nm of a cobalt-containing sample (Figure2(b)). As in Farhadi et al. [27], the first band can be assigned to the O2− → Co2+ charge transfer process and the second one to the O2− → Co3+ charge transfer, which suggested formation of cobalt oxide. However, the UV-Vis optical absorption spectra of Ni and Zn reaction mixture appeared at 370 and 380 nm, respectively, indicating an almost uniform size of the NPs as highlighted by Sathyavathi et al. [21] and Selvarajan and Mohanasrinivasan [28].


## 3.3. Energy-Dispersive X-Ray Analysis (EDX)

EDX is a compositional analysis approach which gives a qualitative as well as quantitative status of elements that may be involved in formation of NPs [29]. As can be seen in Figure 3, the elemental profile of as-fabricated NPs exhibited typical characteristic elemental peaks at approximately 3 keV, 7 keV, 6–7 keV, and 8–10 keV which was attributed to Ag, Co, Ni, and Zn, with atomic percentages 50, 21, 9, and 39%, respectively, and confirms the formation of their corresponding nanomaterial. The existence of other elements accompanied with biosynthesized NPs could also be noticed in high percentage specially sulfur and phosphorus, suggesting conjugation of bacterial biomolecules that contain polar phosphorus backbones as DNA, RNA, ATP, and phospholipids. However, sulfur is an important structural and functional component of amino acids as methionine and cysteine [17]. Furthermore, some signals of Na, K, Ca, and Cu were detected, proposing that they are constituents of an amino acid functional group that still adhered to the nanoparticles. Additionally, a peak for Al was also revealed due to the Al stub used to place the sample in the instrument [30].Figure 3
EDX profile of biosynthesized NPs by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)


## 3.4. X-Ray Diffraction (XRD)

The crystallographic identity and crystalline nature quality in addition to the phase purity of the examined material were determined by X-ray diffraction (XRD). A comparison of the XRD pattern of surveyed samples with the standard Joint Committee of Powder Diffraction Standards (JCPDS) file confirmed that the particles formed in our experiments were Ag2O, Co3O4, NiO, and ZnO nanocrystals as elucidated in Table 2. The X-ray difractograms of biosynthesized NPs are illustrated in Figure 4. Generally, the diffraction peaks of all as-synthesized NPs appeared sharp, clearly distinguishable, and broad, which indicates the ultrafine nature and small crystallite size. The XRD spectrum containing no other phase indicates the purity of the sample [31].Table 2
XRD diffraction peaks and corresponding crystallographic planes matched with JCPDS.
NP type
Bragg reflections (2θ degree) and corresponding Miller indices
Crystalline phase
Standard JCPDS card number
Reference
Ag2O
27.94° (100), 32.27° (110), 46.34° (200), 54.9° (211), 57.6° (211), and 67.48° (220)
Face-centered cubic (FCC)
76–1393
[30]
Co3O4
19.2° (111), 31.1° (220), 44.9° (400), and 55.9° (422)
Face-centered cubic (FCC)
073–1701
[32, 33]
NiO
37.1° (111), 43.5° (200), and 62.4° (220)
Face-centered cubic (FCC)
47–1049
[34, 35]
ZnO
31.6° (100), 34.35° (002), 36.2° (101), 47.5° (102), 56.4° (110), 62.8° (103), 67.8° (200), 68.9° (112), and 76.9° (004)
Hexagonal (wurtzite structure)
89–1397
[15, 28]Figure 4
XRD crystallographic pattern of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)


## 3.5. Dynamic Light Scattering (DLS) andξ Potential

DLS technique was employed to evaluate the time-dependent oscillation of scattered light in dispersed nanoparticles owing to Brownian motion, which is mainly based on their hydrodynamic diameters (size distribution). Moreover, it also describes the degree of uniformity, either homogeneity or heterogeneity of particle size distribution through the polydispersity index (PDI). PDI is a dimensionless parameter which can also estimate NP aggregation. Its values range from 0 (highly uniform, monodisperse, and finer particle size distribution) to 1 (highly polydisperse with a very broad particle size distribution). In general, values of 0.3 and below are considered to be acceptable and reveal a homogenous distribution and values higher than 0.7 refer to heterogeneous dispersion of the samples and are usually not suitable to be measured by DLS [36]. In this study, the particle size distribution curves of biosynthesized AgNPs, CoNPs, NiNPs, and ZnNPs are illustrated in Figure 5. It shows the Z-average of 5, 57, 93, and 48 nm for AgNPs, CoNPs, NiNPs, and ZnNPs, respectively. It is notable that Z-average is the mean hydrodynamic size of the collection of particles measured by DLS [37].Figure 5
Particle size distribution curve of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)Notably, the size measurement by DLS seems to be larger than TEM measurement (as seen later), which could be attributed to that DLS assesses the size of overall aqueous medium accompanying with NPs as referred by Venditti et al. [38]. On the other hand, PDI values recorded 0.329, 0.381, 411, and 0.337 for AgNPs, CoNPs, NiNPs, and ZnNPs, respectively, which implies homogenous dispersity of NPs regarding to the small size of monodisperse NPs.The zeta potential considers being a pivotal criterion for the determination of colloidal stability and gives an insinuation about the degree of repulsion between adjacent similarly charged particles in dispersion. Theξ potential magnitude is indicative of long-term stability, since the stability range lies between +30 and −30 mV which means that particles with ξ potentials more positive than +30 mV or more negative than −30 mV are considered stable [39]. In this study, the ξ potential value of dispersed biosynthesized NPs appeared clearly to be advantageous by recording −54, −52.5, −43.1, and −53.4 mV for AgNPs, CoNPs, NiNPs, and ZnNPs, respectively (Figure 6). Based on the ranking table of colloid stability behavior in relation to the zeta potential referred by Vishwakarma [40], our as-prepared NPs exhibited good stability by the considerable repulsive force that is present between ultrafine particles which results in Brownian motion that retains particles away from disposition to come into aggregates or flocculates.Figure 6
Zeta potential analysis of biosynthesized NPs synthesized by strain 10B: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)Accordingly, the negative singe of the zeta potential indicates that the particles are warped with anionic biomolecules as nucleic acid residues (DNA-RNA) and also negatively charged amino acids as glutamate and aspartate [39], which provide constancy by acting as capping, stabilizing, and functionalizing agents.


## 3.6. Transmission Electron Microscopy (TEM)

For decades, TEM has been a powerful tool in microbiological researches for high-resolution ultrastructural studies of microorganisms and their components [41]. The size, shape, and morphologies of the as-prepared NPs and its producing biofactory were characterized by TEM. Particles with higher electron density will appear darker in the TEM-negative film. As highlighted in Figure 7, strain 10B behaved differently with metal precursors. Remarkably, the AgNP micrograph (Figure 7(a)) exhibited numerous, tiny, roughly globular, uniform NPs with particle size 8.86 nm scattered as seeds like in a monodisperse pattern at the periplasmic space of bacterial cells. Nonetheless, large aggregates of quasi-spherical CoNPs, NiNPs, and ZnNPs appeared to be engulfed in the cytoplasmic compartment and held by cytoplasmic proteins in a spider-net thread-like shape recording 22.1, 35.9, and 19.1 nm, respectively, in diameter.Figure 7
TEM micrograph of strain 10B-synthesized NPs: (a) AgNPs, (b) CoNPs, (c) NiNPs, and (d) ZnNPs.
(a)
(b)
(c)
(d)Interestingly, strain 10B was easily handled and manipulated with different heavy metals disparately and without much difficulty according to their toxicity or benefiting. This selective interaction with metals resulted in production of NPs in different cellular localizations [42]. Generally, the acquisition of heavy metals takes place in bacteria through harnessing of uptake systems (unspecific or specific) which proceeded by the chemiosmosis gradient across the cytoplasmic membrane of bacteria. The expression of these uptake systems is either constitutive or inducible. ATP-binding cassette transporters (ABC system) coordinate a metal traffic process of nearly every biologically required transition metal ion from the extracellular milieu to the cell cytosol interiorly [43]. Consequently, the accumulation of heavy metal ions within the microbial cell occurs by metabolic-dependent biosorption processes, which includes intracellular compartmentalization [44].Once metal ions enter the cell, it begins transformation reactions to encounter their toxicity. Oxidation reduction reaction is the most pivotal enzymatic detoxification mechanism for bacterial transformation of metals to their nanoscale [42]. The respiratory enzymes (oxidoreductase) which were membrane-bounded proteins such as NADH-dependent nitrate reductase or NAD-linked dehydrogenases were proposed to initiate the reduction process.In both enzymes with different scenarios, the final result was analogous, where NADH and NADPH are coenzymes which act as electron carriers mediating several and reversible electron transfers (accepting or losing) in the electron transfer chain throughout the metabolic process. During the biological oxidation/reduction reaction, the enzymes may shuttle electrons to the metal ions that are capable of undergoing redox reaction via multiple changes in their oxidation state, which finally leads to metal NP formation [45]. This mechanism seems to be similar to the formation of magnetic Fe3O4 particles by magnotatic bacteria [46]. In our study, the oxide form of NPs was produced due to the oxygen present in the medium and incubation condition or other oxidizing agents produced by bacterial cells as reported by Dhoondia and Chakraborty [47].The silver ions were described as “oligodynamic” owing to their higher bactericidal competency at minute concentrations [48]. Accordingly, strain 10B during the stationary growth phase accumulated the biosynthesized AgNPs at the periplasm for external extrusion by the efflux system to maintain homeostasis [46]. As recorded by Ma et al. [43], the periplasm of Gram-negative bacteria has dual function according to bacterial sensitivity and ambient condition; under favorable circumstances, the periplasm behaves as storage compartment for biologically essential metal ions detached from the cytosol. However, under acute toxicity, it eliminates metals away from the cell through the Czc-ABC efflux pumping process.Remarkably, formation of metal-protein complexes sequestered in the cytoplasm considers being another basic mechanism of heavy metal resistance according to Cerasi et al. [49] which proposed to be exploited by strain 10B in cytoplasmic synthesis of CoNPs, NiNPs, and ZnNPs. Metallothioneins (MTs) and glutathione (GSH) are important metal-binding proteins that conduct efficiently in metal scavenging (detoxification), storage, and metal homeostasis maintenance [50, 51]. The nanofactory 10B seems to store CoNPs, NiNPs, and ZnNPs as essential micronutrients which play vital catalytic, regulatory, and structural roles in proteins [52]. It is noteworthy to mention the significance of nickel ion (Ni2+) in nickel-metallo enzyme synthesis such as urease, Ni superoxide dismutase, hydrogenase, methyl coenzyme reductase, and carbon monoxide dehydrogenase which resides in the cytoplasm [53].Besides, more than 300 known bacterial enzymes require zinc for their catalytic functions as protein structure stabilization/folding, management of gene expression, DNA replication/repair, response to oxidative stress, biosynthesis of amino acids, extracellular peptidoglycan synthesis, cofactor of virulence-related proteins, and maintenance of the intracellular redox buffering of the cell [49, 54]. On the other hand, cobalt is known as a constituent of cobalamin cofactor (B12) that is crucial for fatty acid catabolism and methyl transfer reactions [43].The conclusive outcome is ascertained that the bacterial mechanism for metal resistance/detoxification is somehow involved in the NP biosynthesis process. The silver detoxification machinery could typically bind and reduce other divalent ion systems and thus resulted in NP formation [55]. Also, such metal-resistant nanobiofactory participates mainly in biogeochemical cycling of those metal ions [52] and could be utilized in bioremediation of metal polluted areas as well [56].


## 3.7. Inhibitory Effect against Planktonic Pathogens

The antimicrobial activities of examined NPs in different concentrations (100–200μg/ml) which were assessed on the basis of clearance zone in comparison with standard antibiotics and metal precursors are presented in Table 3. The diameter of inhibition zone ranged from 4.0 to 9.5 mm, 1.8 to 10.3 mm, and 3.2 to 13.4 mm for fungi, Gram-negative bacteria, and Gram-positive bacteria, respectively. Notably, the antimicrobial action of all examined biogenic NPs was rated “good” since the zone of inhibition was >1 mm as reported by Prasad et al. [11]. It is evident from Table 3 that Gram-positive bacteria intrinsically were more susceptible to all examined antimicrobial agents than Gram-negative ones were. This could be attributed to the structural and compositional differences of the outer bacterial wall; Gram-negative bacteria have an additional lipopolysaccharide layer comparable to Gram-positive ones. The uniqueness of the penetration mechanism of this extra layer could dramatically alter the suppression caused by antimicrobial agents. It is plausible to speculate that the presence of powerful resistance mechanisms like multiple efflux pump in Gram-negative bacteria such as the CzcCBA system [11] contributes in cobalt/zinc/cadmium resistance as mentioned by Lee et al. [57]. Also, plenty of negative charges in the lipopolysaccharide layer repel the negatively charged NPs and hence block the availability of cell wall-binding sites for NPs. Moreover, the outer thick peptidoglycan layer of the Gram-positive bacteria cell wall has better permeability than the Gram-negative one which makes the bacteria more susceptible to harmful substances such as toxins [58]. Consequently, the bacterial resistance/susceptibility rate is governed by both cell wall structure and resistance of bacteria to the reactive oxygen species produced by the action of an antimicrobial agent [59]. Therefore, Gram-negative bacteria require a significantly higher concentration of antimicrobial agent to be eradicated.Table 3
Antimicrobial activity through the maximum inhibition zone of different concentrations of biogenic NPs, metal precursors, and antibiotics in parallel to a collective activity effect against planktonic pathogens.
Microorganism
Concentration (μg/ml)
Zone of inhibition (ZOI, mm)
Class
Strain
NP type
Metal precursor
Collective activity effect (120μg/ml)
Antibiotics
Ag
Co
Ni
Zn
Ag
CO
Ni
Zn
Rif.
Tet.
Strp.
Nyst.
Fungi
Aspergillus bracelleuse (ATCC 16404)
100
4.8
3.9
4.0
4.5
2.2
0.9
0.8
0.8
9.3
ND
ND
ND
5.3
200
9.0
7.3
6.9
8.5
5.6
1.5
1.3
1.5
ND
ND
ND
9.5
Candida albicans (ATCC 10231)
100
5.3
4.3
4.1
4.9
4.5
1.0
0.9
1.2
9.8
ND
ND
ND
6.1
200
9.5
7.5
7.6
9.1
6.5
1.8
1.8
2.1
ND
ND
ND
10.2
Gram-negative bacteria
Pseudomonas aeruginosa (ATCC 27853)
100
4.8
2.4
2.5
2.1
3.2
0.9
1.0
0.9
7.8
5.3
6.6
5.8
ND
200
10.3
5.1
4.9
4.8
5.5
1.4
1.4
1.7
9.1
10.7
9.3
ND
Salmonella typhi (ATTC 700931)
100
4.2
2.5
2.4
2.2
3.5
1.0
0.8
1.0
7.9
5.2
6.9
5.9
ND
200
8.7
4.1
4.5
4.2
5.8
1.8
1.8
1.4
8.8
10.5
9.2
ND
E. coli (ATCC 25922)
100
4.6
2.4
2.3
1.8
3.0
1.2
0.8
0.8
7.4
5.3
6.8
5.7
ND
200
9.7
4.8
4.7
4.3
6.2
2.1
1.8
1.7
8.4
9.8
9.2
ND
Gram-positive bacteria
Clostridium perfringens (ATCC 13124)
100
5.5
3.8
4.1
3.9
4.7
1.5
1.8
1.7
10.3
7.0
8.5
7.5
ND
200
12.0
7.2
6.9
7.0
7.6
3.4
3.2
3.2
13.3
14.5
12.4
ND
Enterococcus faecalis (ATCC 29212)
100
4.5
2.4
2.3
2.5
3.7
0.8
0.8
0.8
6.3
7.4
6.1
ND
200
9.3
5.3
5.0
5.1
7.4
2.1
2.3
2.3
8.6
9.8
12.3
10.3
ND
Bacillus cereus (ATCC 7464)
100
6.3
4.0
4.3
4.2
4.7
1.8
1.4
1.5
12.8
7.5
8.7
8.1
ND
200
13.4
8.8
8.1
8.5
8.0
4.1
4.0
4.2
14.8
15.8
13.7
ND
Staphylococcus aureus (ATCC 25923)
100
5.8
3.2
3.5
3.3
4.8
1.8
1.3
1.5
11.5
6.7
8.0
7.3
ND
200
11.7
7.0
7.1
6.9
8.0
3.9
4.0
4.3
12.6
14.7
13.6
NDIt is noteworthy that an efficient antimicrobial activity was more pronounced by all types of as-synthesized NPs as compared to their precursors. Besides, a potent antimicrobial pattern of AgNPs was observed against all examined pathogenic prokaryotes and eukaryotes comparable to the others. Absolutely, the larger surface area (surface/volume ratio) associated with ultrafine AgNPs permits more closely interactions with microbial cells; hence, it enhances the cytotoxicity to the microorganisms than the large-sized nanoclusters do [1, 59]. Undoubtedly, the extent of NPs’ lethal effect relies on the concentration of applied NPs and the initial microbial concentration. In contrast to our results, a negligible inhibitory effect of 100 μg/ml AgNPs with a particle size of 12–40 nm was recorded on E. coli batch cultures (105–108 CFU/ml) [60]. However, AgNPs (120 μg/ml) with a particle size range of 2.26–10.34 nm were sufficient enough to inhibit the growth of E. coli MTCC-1302 at initial concentration (103–104 CFU/ml) [61].Further, CoNPs, NiNPs, and ZnNPs showed moderate, reproducible, and almost equal biocide activity suggesting that they have dominant inhibitory targets focused on diverse microbial metabolic pathways. Briefly, the NPs’ inhibitory mechanisms include disorganization of the building composition of the cell wall/cell membrane, increasing the membrane permeability, causing dysfunction of essential proteins by reacting with thiol groups, impairing DNA replication, and producing elevated levels of reactive oxygen species (ROS) such as hydrogen peroxide which in turn induce oxidative stress [2, 7, 22]. Interestingly, our results suggested that ZnNPs preferentially exhibited antifungal rather than antibacterial properties which come into agreement with previous studies by Manokari et al. [26] and Roberson et al. [62].Moreover, the biocide activity of NPs increases linearly with increase in NP concentration. Thus, it is obvious from the data that the antimicrobial activities are dose-dependent. Our result is coincident with Pandian et al. [22] who reported the dose-dependent manner of Ni-NPs that exerted antibacterial and antifungal activities against a wide range of pathogens. Lastly, the collective activity of four biosynthesized NP types with equivalent participation (30 μg/ml for each) resulted in enhancement of antagonistic activity against all studied pathogens. From our perspective, the synergetic effect of combined NPs imitates and approximates the antibiotic influence which boosts its application in adjuvant therapy to defeat several multiple antibiotic-resistant microorganisms. As indicated by Ashajyothi et al. [63], the ability of metals to target multiple sites in an organism makes them superior to conventional antibiotics.


## 3.8. Inhibitory Effect against Biofilm Formation andC. vulgaris

Currently, the biofilm is considered to be among the most serious issues which medical devices and water disinfection applications are facing in particular, since it exerts several mechanisms simultaneously to resist different stress factors up to 1000 times in comparison to planktonic cells [64]. Thus, NP strategies were developed to replace or enhance antibiotic treatment in solving this medical and environmental problem. In this study, nearly all features that were observed on planktonic pathogen upon exposure to various treatments (individual NPs, combined NPs, metal precursors, and antibiotics) are considered to be predominant with biofilm pathogens and even algae as evident from Table 4. Generally, sufficient antibiofilm and algicidal activities were exhibited by all types of as-prepared NPs especially AgNPs. The inhibition percentage ranged from moderate to potent and increased with gradual increase in NP concentration.Table 4
Biocide activity of NPs, metal precursors, and antibiotics in addition to collective activity influence on biofilm and algal growth.
Biofilm type
P. aeruginosa
S. aureus
C. vulgaris
Treatment (μg/ml)
Inhibition (%)
NP type
Ag
150
60.25
70.62
56.37
300
89.16
94.57
94.56
Co
150
44.77
48.09
24.40
300
76.78
80.65
49.28
Ni
150
47.82
49.98
39.71
300
74.24
83.98
59.81
Zn
150
49.95
55.13
44.50
300
73.68
83.56
73.21
Combined
Ag, Co, Ni, and Zn
150
78.52
85.73
75.18
Metal precursor
Ag
150
44.68
50.32
38.79
300
63.68
78.64
65.87
Co
150
25.97
34.71
7.08
300
57.01
61.14
28.23
Ni
150
27.69
38.54
−16.75
300
51.98
65.14
4.31
Zn
150
21.46
30.11
−46.41
300
50.76
59.04
−11.00
Antibiotics
Rifamycin
150
52.74
59.68
ND
300
81.18
93.23
Tetracycline
150
55.61
63.74
300
87.61
97.36
Streptomycin
150
50.18
57.69
300
79.47
90.85
Nystatin
150
ND
53.46
300
97.48Notwithstanding that the sessile microbial cells of biofilm protect themselves by embedding in a self-produced extracellular polymeric matrix (DNA, proteins, and polysaccharides), NPs inhibit biofilm efficiently through the damage of a planktonic phase, a chemical communication mechanism that held sessile state in aggregated form (quorum sensing), and increase in hydrophobicity which blocks the initial adhesion of free living cells in the biofilm as referred by Lee et al. [57] and Franci et al. [65]. In consistency with our results, Vincent et al. [66] and Sangani et al. [67] reported the potent antibiofilm effects of Ni-NPs and ZnO-NPs. Otherwise, the enhancement of V. cholerae biofilm by addition of ZnO-NPs was mentioned by Salem et al. [68].What is more, in contrast to our study, the stimulatory influence of ZnO-NPs (200 mg/l) was noticed with 35% cell viability ofC. vulgaris growth after 72 h incubation as referred by Miazek et al. [69]. The drastic alteration in the cellular division processes of C. vulgaris accompanied with application of NPs was recorded by Gong et al. [70]. This moves in accordance to our result, where 75% reduction in algal growth was observed by employing combined NPs. Consequently, the exploitation of NPs in controlling algal blooms will restrict their consecutive adverse environmental problems such as odoriferous, unsightly scums, toxicity of water bodies, and eutrophication [71].On the other hand, considerable stimulation in algal growth was observed with some metal precursors (Ni and Zn) and weak inhibition caused by cobalt salt even at high concentrations. These micronutrients are indispensable elements for algal metabolism and physiological function such as photosynthesis and respiration [72].In consideration of the foregoing, the robust synergetic effect of combined NPs against a wide range of pathogenic microbial forms opens promising avenues for their exploitation as antimicrobial agents in unlimited biomedicine, pharmaceutical product, and environmental applications.


## 3.9. Application of Combined NPs in Water and Wastewater

As observed in Table1, a noticeable bacterial suppression was exhibited by combined NPs predominantly with increasing contact time. Virtually, the biocide activity of combined NPs in municipal wastewater, freshwater, and agricultural wastewater was higher than in salt water, particularly Elmalahat. This could be attributed to the presence of suspended solids which adversely influence on binding strength between NPs and bacterial surface. Obviously, the existence of Cl− in the samples may interact with different metal ions (Ni2+, Co2+, Ag+, and Zn2+) that are released from combined NPs which resulted in production of NiCL2, CoCL2, AgCl, and ZnCL2. In addition, the presence of cations such as K+, Na+, Ca2+, and Mg2+ could be adsorbed on negatively charged NPs which neutralize their surface forming large flocculates and thus losing monodispersity. Consequently, the effective dose that was designed for bacterial eradication required to be adjusted according to the type of the samples and their content [73].


## 4. Conclusions

To summarize, this study for the first time demonstrates the employment ofProteus mirabilis strain 10B as a biotemplate in fabrication of multiple compound NPs (Ag, Co, Ni, and Zn). The biosynthesized NPs were physicochemically characterized using UV-Vis spectroscopy, XRD, EDX, TEM, ζ potential, and PDI. The biosynthesized NPs displayed an antagonistic activity against a wide range of microbial pathogens (Gram-positive, Gram-negative, and anaerobic bacteria, mold, yeast, biofilm, and algae). The present study evaluates the promising potency of combined NPs against various pathogens which sparks an immense interest in multiple applications. Also, up to our knowledge, the synergistic biocidal activity of combined NPs on various microbial populations has not been studied before.



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*Source: 5263814-2018-08-28.xml*