Antimicrobial Efficacy of Biogenic Cobalt and Copper Nanoparticles against Pathogenic Isolates

antibacterial reported the antibacterial activity of biogenic NPs using various metals salts as Co, nickel oxide, ZnO NPs, and titani-um dioxide NPs against Escherichia coli studies Abstract: Biogenic synthesis of cobalt (Co) and copper (Cu) nanoparticles (NPs) was performed using the bacterial strains Escherichia coli and Bacillus subtilis . Prepared NPs were confirmed by a color change to maroon for CoNPs and green for CuNPs. The NPs characterization using FTIR showed the presence of functional groups, i.e., phenols, acids, protein, and aromatics present in the Co and CuNPs. UV-vis spectroscopy of E. coli and B. subtilis CuNPs showed peaks at 550 and 625 nm, respectively. For E. coli and B. subtilis CoNPs, peaks were observed at 300 nm and 350 nm, respectively. Antibacterial and antifungal activity of B. subtilis and E. coli Co and CuNPs was determined at 100 mg/mL concentration against two bacterial strains at 5, 2.5, and 1.5 mg/mL against fungal two strains F. oxysporum and T. viridi , respectively. B. subtilis CuNPs showed significantly higher inhibition zones (ZOI=25.7-29.7 mm) against E. coli and B. subtilis compared to other biogenic NPs. Likewise, B. Subtilis CuNPs showed lower MIC (4.3 ± 6.3) and MBC (5.3 mg/mL) values against both tested isolates. Antifungal activity of B. subtilis and E. coli CuNPs and CoNPs showed a concentration-dependent decrease in ZOI. Among all biogenic NPs, B. subtilis CoNPs showed the highest ZOI (25-30 mm) against F. oxysporum followed by E. coli CuNPs with maximum ZOI (20-27 mm) against T. viridi . Again, B. subtilis CoNPs and E. coli CuNPs showed lowest MIC and MFC values against both fungal isolates. In conclusion, the current study showed that biogenically synthesized B. subtilis Cu or CoNPs can be used as effective antimicrobial agents due to their potential antibacterial and antifungal potential.

reported increased anti-bacterial potential of NPs against bacterial strains with an increase in NPs concentration 9 .
Likewise, biogenic synthesis of copper Cu NPs is more under consideration than the other methods, using bacteria because of the requirement of mild conditions such as pH, temperature and inherent ability of bacteria to convert toxic metals to non toxic ones. Biogenically synthesized CuNPs exhibit antimicrobial activity against many Grampositive and Gram-negative bacteria types. The bacterial growth of E. coli and B. subtilis was also observed to be retarded by CuNPs. Recent studies have also revealed that CuNPs can act as an anti-cancerous agent and have the potential to be used for waste water treatment 10 .
Bacteria have been reported to possess greater potential for nanoparticles production. Bacterial interaction with hazardous metals causes them to convert harmful metal ions to non-toxic metal oxides for their survival. Reportedly, different bacteria generate various essential thiol-containing chemicals that function as a capping agent for NPs, making them table by preventing oxidation, agglomeration, and aggregation. To date, there is no detailed understanding of mechanism behind the nanoscale change. Experimental parameters such as temperature, simple downstream processing, pH, and a short creation period all play important role in bacteria driven NPs synthesis and stability 11 .
Owing to increased microbial resistance against antibiotics, promising antibacterial potential of bacterial driven NPs synthesis and little information of about E. coli as well as B. subtilis basis NPs potential, the current study has been designed to synthesize biogenically Cu and Co NPs using aforementioned bacterial isolates. Following their characterization using FTIR and UV-Vis spectroscopy, the in vitro antimicrobial antibacterial and antifungal potential of synthesized Cu, and CoNPs, was tested against bacterial and fungal isolates.

Microbial strain, metal salts, and culture media
Bacterial isolates Bacillus subtilis and Escherichia coli and fungal strains Fusarium oxisporum and Trichoderma viridi were obtained from Microbiology Lab, Department of Zoology, Government College University, Lahore, Pakistan. NPs were synthesized using copper sulfate CuSO 4 and cobalt sulphate CoSO 4 , obtained from the Department of Zoology and Chemistry, Government College University, Lahore, Pakistan. Nutrient agar and nutrient broth were prepared and sterilized by autoclaving.

Synthesis of biogenic cobalt and copper nanopar-
ticles CoNPs and CuNPs Biogenic synthesis of CoNPs and CuNPs was performed extracellularly using a pre-established protocol 12 . In brief, purified bacterial isolates were inoculated in nutrient broth and incubated for 24 hours at 37 . Both bacterial cultures were centrifuged at 5000 rpm for 10 minutes under sterilized conditions. Pellet was discarded and the supernatant was collected in separate flasks. 3.5 g w/v of CoSO 4 was added into the flask, mixed thoroughly, and kept at 37 in an incubator for 48 hours until a magenta deposition was seen 13 , the same procedure was repeated by using bacterial culture and CuSO 4 until a bluish-green deposition was seen, indicating formation of CuNPs. Control flasks with nutrient broth only were run in parallel and prepared NPs solution was stored at 4 .
Cu and CoNPs were oven-dried to get powder form following the method 13 . Cu and Co NP solution was poured into sterilized falcon tubes aseptically and centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded and pellet was stored. Pellet was placed in sterilized Petri dishes and placed in an incubator at 37 for three days until the Cu and CoNPs were dried completely. The dried Cu and CoNPs were collected in Eppendorfs and stored in refrigerator at 4 for further use. Prepared Cu and CoNPs were green and pink in color, respectively.

Characterization of Co and CuNPs
The characterization of Cu and CoNPs was performed using ultraviolet-visible spectroscopy UV-vis; UV-1700, Shimadzu and Fourier-transform infrared spectroscopy FTIR; Bruker Alpha Platinum ATR . For UV-vis spectroscopy, Co and CuNPs solutions were used by absorbing light emitted by substances, thus showing the UV-visible absorption spectrum of Co and CuNPs 14 . FTIR study was performed using the dry powder form of prepared NPs and showed different functional groups present in the Co and CuNPs 12 .

Antibacterial assay of Co and CuNPs
Agar well diffusion method was used to study antibacterial study of Cu and CoNPs 12 . Muller Hinton Agar MHA was prepared and autoclaved. media and Petri plates were incubated for 24 hours at 37 . Dried CoNPs of both, B. subtilis and E. coli were prepared in solution form by adding 100 mg of CoNPs and CuNPs, each into 1 mL sterilized distilled water. Rifampicin 100 µg/mL was used as positive control.
6 mm wells were made in each petri dish containing MHA with the help of a sterilized cork borer. An antibacterial assay was performed against two bacterial isolates, B. subtilis and E. coli. Using sterilized cotton bud, fresh suspensions of E. coli and B. subtilis were spread on plates. 100 µL of E. coli CoNPs and B. subtilis CoNPs were added to the respective wells. 100 µL autoclaved distilled water and rifampicin were added as negative and positive controls, respectively. Same procedure was repeated for CuNPs. Plates were left for 10 minutes before transferring them into the incubator at 37 . After 24 hours, the plates were observed for zones of inhibition ZOI , which were noted in milli meter mm . MBC is defined as the concentration that kills 99.99% of the original inoculum. For this purpose, nutrient agar was prepared, poured in petri plates and allowed to solidify. 10 µL from MIC test tubes with no visible turbidity was spread evenly on plates in replicates. The plates were incubated for 24 hours at 37 for 24 hours results were recorded 12 .

Antifungal activity of Co and CuNPs
Antifungal activity of the Co and CuNPs was performed against two fungal strains, i.e., F. oxisporum and T. viridi. Potato dextrose broth PDB was prepared, autoclaved and transferred into 2 separate falcon tubes. Already maintained fungal cultures of F. oxisporum and T. viridi were sub-cultured in PDB using an inoculating loop in a laminar airflow. The cultures were placed in incubator at 37 for 24 hours.
Potato dextrose agar PDA was prepared, autoclaved, and poured into four sterilized petri dishes. 6 mm wells were made and fresh cultures of F. oxisporum and T. viridi were spread uniformly on respective petri dishes in replicates. Afterwards, 100 µL of E. coli and B. subtilis CoNPs were added into wells of each plate. Wells containing 100 µL autoclaved distilled water and voriconazole solution were marked as negative and positive controls, respectively. Same procedure was performed for the CuNPs. The plates were left for 10 minutes before transferring them into incubator at 37 for 24 hours and the results were noted.

MIC and Minimum fungicidal concentration MFC for
fungi The MIC of both E. coli CoNPs and B. subtilis CoNPs against F. oxisporum and T. viridi was performed by preparing NPs solution. In brief, 12 test tubes were taken for each of two E. coli and B. subtilis CoNPs and 1 mL sterilized PDB was added. The optical density O.D of each culture was adjusted to 0.5 McFarland turbidity standard. For E. coli CoNPs, 10 µL of F. oxisporum was poured in 6 test tubes and 5, 10, 15, 20, and 25 µL of the E. coli CuNPs solution was added. Likewise, 10 µL of T. viridi was added into the second set of 6 test tubes and 5, 10, 15, 20, and 25 µL of the E. coli CoNPs solution was added into five test tubes. One test tube in each set containing fungal inoculum in PDB but without respective NPs was used as control. Exact process was repeated for B. subtilis CoNPs. These test tubes were left in the incubator at 37 for 24 hours under shaking conditions and results were noted. PDB was prepared, autoclaved, and poured into plates for MFC determination until solidification occurred 10 µL from the MIC test tubes with no visible turbidity was spread evenly on prepared plates. The plates were incubated for 24 hours at 37 and results were noted 13 .

Statistical analysis
Data was analyzed using mean and standard error of mean SEM . One-way analysis of variance ANOVA followed by the Post Hoc Tukey test was used to determine the statistical significance at p ≤ 0.05.

Visual observation Color change
Visual characterization showed a visible change in the color from red to magenta. Later these NPs were dried, and their color changed from magenta to light burgundy, proving the formation of CoNPs Fig. 1a . Cell free supernatant of B. subtilis and E. coli and CuSO 4 salt were incubated for 48 hrs. Color change from blue to green the confirming the formation of CuNPs Fig. 1a .

Characterization of Co and CuNPs
Following the visual observation, Co and CuNPs were characterized by ultraviolet-visible spectroscopy UV-Vis and Fourier transform infrared techniques FTIR .

UV-Vis Spectroscopy
Uv-vis spectroscopy was employed to determine the absorption of Co and CuNPs at room temperature   coli CuNPs and B. subtilis CuNPs showed distinct absorption peaks at 500 nm and 625 nm, respectively Fig. 1b .

Fourier transform infrared techniques FTIR
The physical and chemical composition of the CoNPs was studied by Fourier transform infrared FTIR spectroscopy. The FTIR spectra were recorded between 500 and 4000 cm 1 . For E. coli CoNPs Fig. 2a , the notable peaks were observed at 827, 1072, 1398, 1558, 2358, and 3261 cm 1 . Peaks at 827, 1072, and 1398 cm 1 showed the -CH bond, C-N, and C-O bond stretching in amino acid. The peak at 1558 cm 1 showed the -OH bond stretch. A peak at 3261 cm 1 showed the presence of the OH group. Notable peaks observed in the FTIR graph for B. subtilis CoNPs were at 1031, 1155, 1369, 1558, 2328, and 3292 cm 1 , as shown in Fig. 2b. The peaks at 1031 and 1155 cm 1 showed the presence of aromatic groups. The peak at 1369 cm 1 showed the stretching due to the vibration of the nitrogen group. The peak at 1558 cm 1 showed the formation of the alkene group, the peak at 2320 cm 1 Fig. 2c . A peak at 3447 cm 1 was due to the presence of N-H and O-H groups. The peak at 1633 cm 1 indicated the N-H group. Peak at 1558 cm 1 showed the presence of carboxyl group. Peak observed at 1398 cm 1 confirmed presence of C-N group. C-H bond due to presence of glucose residues was confirmed by peak at 1072 cm 1 Fig. 2c Fig. 2d . Peak at 1558 cm 1 indicated presence of carboxyl group. Broad peak at 3564 cm 1 showed the presence of hydrogen-bonded groups of alcohol -OH , phenols and amines -N-H of amide. 1643 1 cm peak showed presence of amide carbonyl groups. The peak at 668 cm 1 gave information about presence of aromatic groups Fig. 2d .

Antibacterial assay of Co and CuNPs
High surface-to-volume ratios and nano scale sizes are distinguishing characteristics of biogenic NPs, which improve their reaction with pathogenic microbes.

MIC and MBC of Co and CuNPs
MIC and MBC of biogenically synthesized Co and CuNPs were tested against both Gram-positive and Gram-negative strains at various concentrations 5-25 mg/mL . In general, B. subtilis CoNPs were more susceptible to E. coli and  3.5 Antifungal activity of biogenically synthesized Co and CuNPs Antifungal activity of E. coli and B. subtilis Co and CuNPs was studied at three concentrations 5.0, 2.5, and 1.5 mg/mL against F. oxysporum and T. viridi. Antifungal activity of the CoNPs against fungal strains F. oxysporum and T. viridi was shown in Table 3 16 . Overall, a concentration-dependent decrease in antifungal activity of NPs was observed against fungal strains. B. subtilis CoNPs showed strong inhibition of F. oxysporum with significantly p ≤

Discussion
Microbial synthesis of metal nanoparticles can take place either intracellularly or extracellularly. For intracellular synthesis of NPs, additional steps such as reactions with appropriate detergents and ultrasound treatment is required to release the synthesized NPs. However, extracellular biosynthesis is comparatively cheap and requires simpler downstream process leading to large-scale production of NPs with wider potential applications. Therefore, current study focused on extracellular synthesis of metal NPs using bacterial isolates 17 , such as B. subtilis and E. coli. Previous study has reported the greater potential of B. subtilis for synthesis of metal NPs. Likewise, El-Shanshoury 18 showed that E. coli bacteria reaction with highly reactive metal oxide NPs results in significant increase in membrane permeability, causing the bacteria incapable of regulating transport through the membrane leading to ultimate cell death. Further, metal depletion may form irregular pits in bacterial cell membrane affecting its permeability, primarily caused by progressive release of membrane proteins and LPS molecules.
Biologically synthesized CoNPs and CuNPs have been proved to be very effective and valuable compounds. They have excellent antimicrobial and antiviral activity. Biogenic synthesis of CoNPs and CuNPs is a more cost-effective and eco-friendly approach. Maximum absorption peaks of E. coli and B. subtilis CoNPs observed at 300 and 350 nm via UV-Vis spectroscopy were similar to the studies by Rahman et al. 19 . The authors showed an absorption spectrum at 319 nm as the characteristic absorption peak of CoNP. UV-Vis absorption spectrum exhibited distinct absorption peaks at 625 nm for B. subtilis and 550 nm for E. coli CuNPs. Similarly, UV-Vis spectrum observed for biogenic algal-based CuNPs from Botryococcus braunii showed peak at 258-490 nm by Rahman et al. 19 , who showed maximum absorption peak at 258 nm. Likewise, Noman et al. 20 biosynthesized Escherichia based CuNPs and showed UV-Vis absorption spectrum in the range of 300-800 nm.
The FTIR results of E. coli and B. subtilis CoNPs showed the presence of functional groups i.e., phenols, acids, protein, and polypeptides which confirmed the biogenic synthesis of CoNPs. Previously, Iqbal et al. 21 also showed the presence of -CH and OH groups in prepared CoNPs and reported that they play an essential role in their stabilization and reduction. Iqbal et al. 21 reported Peaks at 2919 and 1729 cm 1 resulted in stretching of C-H and -C O alkane and alkene groups respectively that are involved in the formation of CoNPs. FTIR spectrum of E. coli CuNPs confirmed peak at 3447 cm 1 due to the presence of N-H and O-H groups. Peaks at 1633 cm 1 , 1558 cm 1 , 1072 cm 1 and 1398 cm 1 were due to the presence of N-H group, carboxyl group, C-H bond and C-N group, respectively. In another study by Slavin et al. 22 , biogenic plantbased CuNPs from Bougainvillea plant flowers extract showed FTIR peaks at 3365, 1643, and 602 cm 1 showed groups of alcohol -OH , phenols, amines -N-H , carbonyl group and aromatic groups, respectively.
Antibacterial activity of prepared E. coli and B. subtilis CoNPs showed inhibition zones against B. subtilis and E. coli at 100 mg/mL concentration. These results are in accordance with the studies by Stanic et al. 23 15 . Authors studied the antifungal activity of CoNPs and observed 12 mm ZOI against C. albican. In a study done by Rehman et al. 24 , the antifungal assay of CoNP against C. albicans at 1 mg/mL concentration showed promising antifungal potential. Likewise, the growth inhibitory potential of Gram-positive B. subtilis and E. coli CuNPs against Gram-negative E. coli and B. subtilis bacterial strains corroborate with findings by Amer et al. 25 . Similarly, Daniel et al. 26 30 also reported MIC and MFC of biologically synthesized CuNPs against C. guilliermondii as 2 and 4 mg/mL, respectively. Additionally, Tahvilian et al. 27 published MIC and MFC of 2 and 8 mg/mL by biologically synthesized CuNPs against C. albicans.

Conclusion
Nanoparticles NPs have remarkable properties that make them useful in fields of medicine, agriculture, environment, and energy 31 . It was observed that NPs are more efficient against bacteria and fungi as compared to antibiotics and antifungal drugs. Owing to bacterial resistance to specific antibiotics, NPs have gained attention as more efficient therapeutic approach against drug-resistant bacteria and fungi 32 . Regarding antibacterial potential, among two types of biogenically synthesized NPs E. coli and B. subtilis , results showed that B. subtilis CuNPs showed significantly higher antibacterial potential with bigger ZOI and lowest MIC-MBC values. Antifungal assay showed that B. subtilis CoNPs and E. coli CuNPs were better antifungal agents with lowest MIC and MFC values. Overall, B. subtilis Co, CuNPs showed significant p 0.05 antimicrobial potential. The current study concluded that biogenically synthesized NPs are effective antimicrobial agents compared to antibiotics and could be investigated further for toxicity evaluation for various environmental and biomedical applications. Also, the CuNPs and CoNPs in this study have to clarify low cytotoxicity by cell experiments for future practical use.

Author Contributions
IL designed and supervised study. RA performed the experimental work and wrote the first draft. UH, AL, AB helped in data analysis. SS, SN, MU, MM and FR helped in revision.IL corrected the final draft. All authors approved the final version of manuscript.