Biogenic Nanoparticles Silver and Copper and Their Composites Derived from Marine Alga Ulva lactuca: Insight into the Characterizations, Antibacterial Activity, and Anti-Biofilm Formation

Bacterial pathogens cause pain and death, add significantly to the expense of healthcare globally, and pose a serious concern in many aspects of daily life. Additionally, they raise significant issues in other industries, including pharmaceuticals, clothing, and food packaging. Due to their unique properties, a great deal of attention has been given to biogenic metal nanoparticles, nanocomposites, and their applications against pathogenic bacteria. This study is focused on biogenic silver and copper nanoparticles and their composites (UL/Ag2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs) produced by the marine green alga Ulva lactuca. The characterization of biogenic nanoparticles UL/Ag2 O-NPS and Ul/CuO-NPs and their composites Ul/Ag/Cu-NCMs has been accomplished by FT-IR, SEM, TEM, EDS, XRD, and the zeta potential. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments were conducted to prove antibacterial activity against both Gram-positive and Gram-negative bacteria and anti-biofilm. The FTIR spectroscopy results indicate the exiting band at 1633 cm−1, which represents N–H stretching in nanocomposites, with a small shift in both copper and silver nanoparticles, which is responsible for the bio-reduction of nanoparticles. The TEM image reveals that the Ul/Ag/Cu-NCMs were hexagonal, and the size distribution ranged from 10 to 35 nm. Meanwhile, Ul/CuO-NPs are rod-shaped, whereas UL/Ag2 O-NPS are spherical. The EDX analysis shows that Cu metal was present in a high weight percentage over Ag in the case of bio-Ag/Cu-NCMs. The X-ray diffraction denotes that Ul/Ag/Cu-NCMs, UL/CuO-NPs, and UL/Ag2 O-NPS were crystalline. The results predicted by the zeta potential demonstrate that Ul/Ag/Cu-NCMs were more stable than Ul/CuO-NPs. The antibacterial activity of UL/Ag2 O-NPS, Ul/Ag/Cu-NCMs, and UL/CuO-NPs was studied against eleven Gram-negative and Gram-positive multidrug-resistant bacterial species. The maximum inhibition zones were obtained with UL/Ag2 O-NPS, followed by Ul/Ag/Cu-NCMs and Ul/CuO-NPs in all the tested bacteria. The maximum anti-biofilm percentage formed by E. coli KY856933 was obtained with UL/Ag2 O-NPS. These findings suggest that the synthesized nanoparticles might be a great alternative for use as an antibacterial agent against different multidrug-resistant bacterial strains.


Introduction
Nanotechnology, in combination with biology, provides a promising field of nanobiotechnology.This includes living entities, such as cyanobacteria, bacteria, viruses, algae, acti-nomycetes, yeasts, fungi, and plants [1][2][3].The green biogenesis of nanoparticles (NPs) utilizing either algae or plant extracts has appeared to be an economical and environmentally beneficial technique [4][5][6].Specifically, macro-algae cover various bioactive contents with multiple constructions and promising biological applications [7,8].Due to their potential utility in the bio-manufacture of nanomaterials, algae have been referred to as "nanofactories".The main benefit of this environmentally friendly fabrication is the lack of hazardous solvents and reagents, which prevents the production of waste and byproducts during the synthesis of metal nanoparticles.In addition, the use of algae fosters the goal of using natural, renewable resources [9,10].The process of metal stabilization and reduction is supported by a variety of substances found in the crude extracts of macroalgae, such as amines, amides, alkaloids, terpenoids, pigments, proteins, etc. [11,12].Ulva lactuca is a type of green seaweed comprised of highly active substances with antitumor, antioxidant, hypocholesterolemic, and antimicrobial capabilities [13].Ag-NPs can be fabricated using U. lactuca [14].Ag-NPs fabricated using U. lactuca have antibacterial activities against Escherichia coli and Pseudomonas aeruginosa [15].The biosynthesis of nanoparticles by U. lactuca is a reliable, economical, and eco-friendly process for the synthesis of metallic nanoparticles [16].
A nanocomposite is a composite material in which at least one of the materials is one dimension in size, around 10-9 nm [17].They have high-performance materials that exhibit rare characteristics.Their perspective is so notable that they are valuable in many fields [18], from packaging to biomedical applications [19].The antifungal effect resulting from the copper-silver-chitosan nanocomposite was more significant than that of other nanoparticles tested, which has had an essential influence on the growth of Candida albicans in laboratory conditions compared to other nanoparticles [20].Different studies showed that the as-synthesized Ag/Cu-NMCs could be an efficient substitute for an antimicrobial agent against drug-resistant microbes and plant-pathogenic bacteria [21,22].The Ag-Cu-Co oxide caused a maximum zone of inhibition against E. coli that was found to be 25 mm [23].The fabricated nanostructured surfaces (Ag-CuxO nanostructures) exhibit excellent bactericidal efficiency against E. coli (Gram-negative) and Staphylococcus aureus (Gram-positive) [24].Good antibacterial activity was noted against both bacteria in milk (Escherichia coli and Staphylococcus aureus) with a sensitivity of 40-90% at CuO/Ag concentrations ranging from 6 to 200 mg/100 mL [25].CuO/Ag 2 O NCPs synthesized by the leaf extract of Eichhornia crassipes show good antibacterial activities against four human pathogenic bacteria, such as K. pneumonia, E. coli, S. epidermides, and S. aureus [26].
Due to their unique characteristics, such as superior electrical conductivity, low electrochemical migration behavior, a high melting point, and, most importantly, low cost, copper nanoparticles have applications in many areas, such as industries, medicine, electronics, etc. [27,28].For more than 200 years, copper has been known for its ability to inhibit the growth of microorganisms and lower microbial concentrations by 99.9% [29,30].Numerous studies have shown that copper nanoparticles show excellent antimicrobial activity against many bacteria [28,31].Moreover, nano-copper oxide (Cu-O) works as an essential antimicrobial agent against Staphylococcus aureus, Escherichia coli, Vibrio cholera, Pseudomonas aeruginosa, Bacillus subtilis, and Syphillis typhus [32][33][34][35].The reputation of copper has expanded lately with the COVID-19 pandemic, when it was noticed that SARS-CoV-2 on copper surfaces decomposes more rapidly (in four hours) than other materials, such as plastic and stainless steel [36].
This study aims to investigate the efficacy of the marine green alga Ulva lactuca, collected from the Jeddah seashore, as reducing and stabilizing agents for the biofabrication of UL/Ag 2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NMCs.We have compared the characteristics of silver and copper nanoparticles and their composites.We have also determined the antibacterial activities of biofabricated nanoparticles and their composites against some multi-drug-resistance bacterial strains.

Results and Discussion
2.1.Characterisation of Ul/Ag 2 O-NPS, Ul/Ag/Cu-NCMs, and Ul/CuO-NPs 2.1.1.Fourier Transform Infrared (FTIR) of UL/Ag 2 O-NPS, Ul/Ag/Cu-NCMs and Ul/CuO-NPs Fourier transform infrared spectroscopy was conducted to reveal the functional groups in the molecules characterized during the production of the newly synthesized nanoparticles, which are responsible for the stabilization and coating of the nanoparticles in all three nanoparticles.The FTIR spectra bands of the silver nanoparticles are shown in Figure 1 and Table 1 .The band at 1633 cm −1 , which is present in all of the synthesized nanoparticles (UL/Ag 2 O-NPS, Ul/Ag/Cu-NCMs, and Ul/CuO-NPs) with a small shift, represents the N-H stretching band, which denotes the active group that acts as reducing and stabilizing agents in biofabricated nanoparticles.Phytochemical compounds found in biomass, such as alcohols, aldehydes, alkanes, and epoxy groups or ether groups, can be responsible for the nucleation process to reduce Ag+ to Ag • [37].The biosynthesis of nanoparticles may be triggered by several compounds, such as carbonyl groups, phenolics, flavonones, terpenoids, amides, amines, proteins, pigments, alkaloids, and other reducing agents present in the biological extracts [11].
Molecules 2023, 28, x FOR PEER REVIEW 3 of 22 determined the antibacterial activities of biofabricated nanoparticles and their composites against some multi-drug-resistance bacterial strains.Fourier transform infrared spectroscopy was conducted to reveal the functional groups in the molecules characterized during the production of the newly synthesized nanoparticles, which are responsible for the stabilization and coating of the nanoparticles in all three nanoparticles.The FTIR spectra bands of the silver nanoparticles are shown in Figure 1 and Table 1 with a small shift, represents the N-H stretching band, which denotes the active group that acts as reducing and stabilizing agents in biofabricated nanoparticles.Phytochemical compounds found in biomass, such as alcohols, aldehydes, alkanes, and epoxy groups or ether groups, can be responsible for the nucleation process to reduce Ag+ to Ag° [37].The biosynthesis of nanoparticles may be triggered by several compounds, such as carbonyl groups, phenolics, flavonones, terpenoids, amides, amines, proteins, pigments, alkaloids, and other reducing agents present in the biological extracts [11].The SEM image has been used to illustrate the size and shape of the produced nanoparticles.As shown in the sample images in Figure 2A-C, the surface is rough due to the presence of Cu, Ag, and Ag combined with Cu.The modification in morphology and coarseness of the surface was due to the foundation of nanoparticles [57].

Characterisation of
The TEM image shows that UL/Ag 2 O-NPS are spherical in shape and range from 10 to 45 nm (Figure 2D and Table 2).The image proves that UL/Ag 2 O-NPS are polydispersed and range in size from 15 to 20 nm, as shown in the frequency histogram (Figure 2G).The Ag-NPs synthesized by Ulva fasciata were spherical and 50 nm in size [58]; by Enteromorpha flexuosa were 2-32 nm/circular [59]; by Padina tetrastromatica were 14 nm/spherical [60]; and by Gracilaria corticata were 8-46 nm/spherical [61].
The TEM image of the Ul/CuO-NPs shows they are rod-shaped, one-dimensional nanoparticles; the length of the rod ranged from 46 to 65 nm, and the width ranged from 10 to 40 nm (Figure 2E).The Ul/CuO-NPs are polydispersed and range in size, according to width, from 20 to 25 nm (Figure 2E and Table 2).The size of the synthesized copper nanoparticles using Zingiber officinale and Curcuma longa rhizome extract was in the nanorange of approximately 20 to 100 nm [62], and the average size of the copper nanoparticles synthesized by Eucalyptus leaf extract ranged from 10 to 130 nm.In contrast, the size of the mint leaf extracts of the copper nanoparticles ranged from 23 to 39 nm [63].The results estimated that there was some aggregation of Ul/CuO-NPs; some nanoparticles were well distributed, while most were found in the agglomerated form [63].The results agree with Ramzan et al. [64] when using Cedrus deodara aqueous extract to synthesize the copper nanoparticles; only a few spherical-shaped and well-separated particles were found, and most of them were agglomerated.Amaliyah et al. [65] used Piper retrofractum Vahl extract to synthesize spherical Cu-NPs with the propensity to form a random aggregate.Moreover, Mahmoud et al. [66] utilized orange peels to synthesize CuO-NPs, which appear to aggregate.Meanwhile, when Ag and Cu were loaded to form Ul/Ag/Cu-NCMs, the shape and size changed to a hexagonal shape, and they were well distributed in the solution with a size range of 10-35 nm (Figure 2G and Table 2).The image denotes polydispersed Ul/Ag/Cu-NCMs with a frequency in the range of 15 to 20 nm with 39%.The image demonstrates there is a core shell around the UL/Ag 2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs; the shell appears faint and dark around the nanoparticles and is denoted by arrows in the images.The shell may be related to organic compounds derived from U. lactuca.The core-shell nanomaterials can be extended to the class of nanomaterials having distinct boundary materials covering (either fully or partially) the inner component.Coreshell nanoparticles (core-shell NPs) are structures that frequently combine the characteristics of the two (or more) materials used, with the shell controlling the surface properties of the particles while encasing the core entirely [67].
The results indicate that U. lactuca is an excellent reducing and stabilizing agent.López-Ubaldo et al. [68] reported the size of Ag/Cu-NCMs biosynthesized by Ricinus communis was in the range of 10-25 nm and spheroid in shape.The size of Ag/Cu-NCMs green synthesized by Opuntia ficusindica, as indicated by Rocha-Rocha et al. [69], ranged from 10 nm to 20 nm.Mohamad et al. [70] stated that leaf palm extract efficiently reduced and stabilized agents for the biosynthesis of Cu/Ag-Nano composites.The supernatant of Rhodopseudomonas capsulate showed high efficiency when it was used to synthesize Cu/Ag-NCMs.3b.The EDX analysis of the Ul/CuO-NPs revealed that there was 37.94% copper, 45.61% oxygen, and 16.46% carbon.The four peaks are C, O, Cu, and Ag, with weight percentages of 28.91, 42.66, 27.47 and 0.96, respectively in Ul/Ag/Cu-NCMs.The extensive presence of Cu over Ag may be related to the methods of synthesis that use 1:9 Ag:Cu in the synthesis of nanocomposites.The appearance of different elements, such as oxygen 42.66% and carbon 17.08%, may be due to the organic compounds found in the alga extract [71].
Molecules 2023, 28, x FOR PEER REVIEW 7 of 22 appearance of different elements, such as oxygen 42.66% and carbon 17.08%, may be due to the organic compounds found in the alga extract [71].3a).These results confirm the formation of UL/Ag2 O-NPS as nanocrystals, and the crystal size ranged from 12.2 to 36.7.3a).These results confirm the formation of UL/Ag 2 O-NPS as nanocrystals, and the crystal size ranged from 12.2 to 36.7.The major crystalline peak was obtained at 2 theta 29.841 with an intensity of 100% and a size of 12.2 nm.The X-ray diffraction of the Ag-NPs biofabricated by Turbinaria urbinate showed two intense peaks at 27.94 and 32.27, which correspond to Miller indices of 110 and 111 [72].(c)   The XRD spectra of Ul/CuO-NMPs derived from U. lactuca reveal the crystal lattice structure of the biosynthesized Ul/CuO-NPs (Figure 4b and Table 3b).The XRD spectrum analysis for Ul/CuO-NPs shows diffraction peaks at 2Ө The zeta potential is an analysis method that measures the number of electric charges on the nanoparticle's surface.The zeta potential is an analysis method that measures the number of electric charges on the nanoparticle's surface.The antibacterial activity of UL/Ag 2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs derived from U. lactuca in different volumes (10, 30, and 100 µL at the same concentration of 1 mg/mL) was tested against 11 bacterial strains of Gram-negative and Gram-positive multidrug-resistant bacteria: Streptococcus mutans ATCC 25175 and Lactobacillus acidophilus CH-2 [79].The results indicate that the different volumes and types of nanoparticles had significant effects on the antibacterial activity.The higher volumes (100 µL/well) possessed higher antibacterial activities than 30 and 10 µL/well (Figure 6 and Table 4).The maximum zone of inhibition for the volume (100 µL) of Ul/Ag 2 O-NPs was recorded against Streptococcus mutans ATCC 25175 (17 mm), followed by Ul/Ag/Cu-NCMs (14 mm).No significant effect of Ul/CuO-NPs (100 µL) and Ul/Ag-NPs with volume of 10 and 30 µL were noted.The lowest inhibition zone was recorded with Ul/CuO-NPs (10 and 30 µL).The UL/Ag 2 O-NPS showed the highest antibacterial activity (18.3 mm at 100 µL) against Lactobacillus acidophilus CH-2, followed by Ul/Ag/Cu-NCMs (16 mm at 100 µL).The Ul/CuO-NPs showed the lowest antibacterial activity against Staphylococcus aureus ATCC6538 and Staphylococcus aureus in the inhibition zone (7 mm at 10, and 30 µL).The Ul/Ag-NPs and Ul/Cu-NPs possessed better antibacterial activity against Staphylococcus aureus than the Ul/Ag/Cu-NCMs with a volume of 100 µL.The Ul/Ag 2 O-NPs denoted the highest antibacterial activity against Klebsiella pneumoniae KY856924, Acinetobacter KY856930, E. coli KY856933, and Enterobacter KY856934 at 100 µL/well, but the results showed that the Ul/CuO-NPs had the highest antibacterial activity against E. coli KY856932, and Ul/Ag/Cu-NCMs had higher activity against Enterobacter aerogenes at 100 µL/well.This study demonstrates that the UL/Ag 2 O-NPS had more antibacterial activity in most tested bacteria, except E. coli KY856932 and Enterobacter aerogenes.The higher antibacterial activity of UL/Ag 2 O-NPS over Ul/CuO-NPs and Ul/Ag/Cu-NCMs may be due to Ul/Ag 2 O-NPs having a positive charge.The positively charged Ag-NPs displayed the largest inhibitory zones against all the strains tested and in all the concentrations employed.In any concentration examined, neutral and negatively charged AgNPs did not exhibit enough antibacterial action against P. vulgaris [80].The positively charged BPEI-AgNPs were the most toxic NPs, whereas the more negatively charged citrate-AgNPs were the least hazardous [81].Bacteria with a negative surface charge come into contact with nanoparticles with a positive zeta potential, and the electrostatic forces that result encourage a stronger attraction and interaction between the two organisms and may even allow for bacterial membrane penetration [82].Also possible are the low-release Ul/CuO-NPs and Ul/Ag/Cu-NCMs.The Cu-NPs may possess antibacterial activity by denaturing the proteins and enzymes of bacteria [83].An investigation was conducted by Chandraker et al. [84] and an excellent result was seen with a 12.43 mm zone of inhibition for the antibacterial activity of green Cu-NPs produced utilizing an aqueous leaf extract of Ageratum houstonianum against the Gram-negative bacterium Escherichia coli.A strong antibacterial effect of Cu-NPs on Gram-positive Bacillus subtilis and Gram-negative Escherichia coli was also mediated by an aqueous extract of Curcuma longa.When compared to Gram-positive bacteria, the Gram-negative bacteria had a greater zone of inhibitory growth, which suggests that the Gram-positive bacteria are more sensitive to Cu-NPs [85].A previous study performed by Jayandran et al. [86] compared the antibacterial activities of Cu-NPs synthesized using curcumin against two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria to the antibacterial activity of pure curcumin.Fascinatingly, the zone of inhibition recognized for Cu-NPs against S. aureus and B. subtilis showed higher antibacterial activity than the normal drug, chloramphenicol.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assays
The MIC value can be used to assess the effectiveness of nanomaterials.The MIC is described as the lowest antimicrobial compound concentration that inhibits a microorganism's growth [87].The results in Table 5 demonstrate that, after 24 h of incubation at 37 °C, the MIC value of UL/Ag2 O-NPS was 0.062 mg/L with Streptococcus

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assays
The MIC value can be used to assess the effectiveness of nanomaterials.The MIC is described as the lowest antimicrobial compound concentration that inhibits a microorganism's growth [87].The results in Table 5  The MBC was 0.5 mg/mL of Ul/CuO-NPs and Ul/Ag/Cu-NCMs with Streptococcus mutans ATCC 25175, as well as of Ul/Ag/Cu-NCMs with Staphylococcus aureus ATCC 25923 and of UL/Ag 2 O-NPS with Klebsiella pneumonia KY856924 and E. coli KY856932.The MBC values were more than 1 mg/mL of Ul/CuO-NPs with Lactobacillus acidophilus CH-2, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis, Klebsiella pneumoniae KY856924, Acinetobacter KY856930, E. coli KY856932, and Enterobacter KY856934; the MBC values were also more than 1 mg/mL of Ul/Ag/Cu-NCMs with Staphylococcus aureus ATCC6538, Staphylococcus epidermidis ATCC 12228, Klebsiella pneumoniae KY856924, Acinetobacter KY856930, E. coli KY856932, Enterobacter KY856934, and Enterobacter aerogenes.It was revealed that the Ag-NPs had a more vital antibacterial capacity than the Cu-NPs, suggesting that Ag ions are more effective antimicrobial agents than Cu ions [89,90].The Ag-NPs also exhibited greater antibacterial potency against different strains of E. coli and S. aureus, as well as against fungi [91], which may be a result of their more potent interaction with the polysaccharides and proteins found on cell walls [92].Because Cu-NPs have a lower antibacterial capacity than Ag-NPs, the possibility of an oxide layer on them has been suggested [93,94].

Biofilm Formation Inhibition Assay
A static biofilm assay was performed to evaluate the effect of the synthesized UL/Ag 2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs derived from U. lactuca on E. coli KY856933 biofilm formation, and showed positive biofilm formation before treatment with the nanoparticles.Figure 7 shows the biofilm inhibition percentage at different concentrations of nanoparticles (1, 0.5, 0.25, 0.12, and 0.06 mg/mL).The results obtained in the microtiter plates show that the highest inhibition percentage was 87.02 with 0.5 mg/mL UL/Ag 2 O-NPS, followed by 1.0 mg/mL UL/Ag 2 O-NPS (86.31%).The same inhibition percentage was obtained with 0.25 mg/mL UL/Ag 2 O-NPS, and with 1.0 mg/mL Ul/Ag/Cu-NCMs, the inhibition was 85.5%.The lowest percentage of biofilm inhibition was 44.38 with 0.06 mg/mL Ul/CuO-NPs.The order of anti-biofilm strength formed by Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 43300 with biosynthesis nanocomposite was Ag-TiO 2 > TiO 2 -Ag > Cu-Ag > Ag-Cu [95].The bio-Ag-NPs inhibited biofilm formation by both Escherichia coli and S. aureus at concentrations of 4 µg/mL [96].The Ag-NPs demonstrated notable anti-biofilm activity against S. aureus biofilms and powerful bactericidal effects against P. aeruginosa and S. aureus, respectively [97].The minimal inhibitory concentration of the produced Ag-NPs against biofilm-forming Staphylococcus aureus verified their concentration-dependent inhibition (MIC) [98].

Extraction of Algae
At the end of January, in the winter, Ulva lactuca (Figure 8) was gathered from the seashore (21°.637086N and 39°.101631E) in Jeddah, Saudi Arabia (2020).One gram of airdried U. lactuca was mixed with 100 mL of deionized (D.D.) water to create the Ag-NPS and Ag-Cu NCMS.This mixture was then boiled for an hour and filtered, and the remaining 100 mL of D.D. water was added.For the CuO-NPs, 100 mL of D.D. water was

Extraction of Algae
At the end of January, in the winter, Ulva lactuca (Figure 8) was gathered from the seashore (21

Synthesis of Silver/Copper Nanocomposites (Ul/Ag/Cu-NCMs) by U. lactuca Extract
Briefly, 60 mL of distilled and deionized water were combined with 9 mM of copper II sulfate and 1 mM of silver nitrate (D.D. water).Drop by drop, 40 mL of aqueous algal extract was added to the Ag/Cu solution, followed by 0.2 M NaOH, which produced a grayish precipitate that was left in a magnetic stirrer for 2 h [70].To remove the contaminants, the mixture was centrifuged, the supernatant was eliminated, and the grayish precipitate was washed twice with 50 mL of D.D. water and once more with 50 mL of ethanol by repeated centrifugation.After one day of air drying, silver/copper nanoparticle powder was produced.

Biosynthesis of Silver Nanoparticles Using Alga Aqueous Extract
Alga extract (30 mL) was injected drop by drop with a syringe on 70 mL of 1 mM  Briefly, 60 mL of distilled and deionized water were combined with 9 mM of copper II sulfate and 1 mM of silver nitrate (D.D. water).Drop by drop, 40 mL of aqueous algal extract was added to the Ag/Cu solution, followed by 0.2 M NaOH, which produced a grayish precipitate that was left in a magnetic stirrer for 2 h [70].To remove the contaminants, the mixture was centrifuged, the supernatant was eliminated, and the grayish precipitate was washed twice with 50 mL of D.D. water and once more with 50 mL of ethanol by repeated centrifugation.After one day of air drying, silver/copper nanoparticle powder was produced.

Biosynthesis of Silver Nanoparticles Using Alga Aqueous Extract
Alga extract (30 mL) was injected drop by drop with a syringe on 70 mL of 1 mM AgNO 3 on a magnetic stirrer until the color changed from clear to brown to darker brown, after ten minutes.

Biosynthesis
Copper Oxide Nanoparticles (Ul/CuO-NPs) by U. lactuca Extract A faint blue powder of copper nanoparticles was obtained after air drying for one day.
Copper II sulfate, CuSO 4 (9 mM), was added to 70 mL distal and deionized water (D.D. water) under constant stirring.Thirty ml of aqueous algal extract was added drop by drop to the copper solution.After 10 min of stirring, 0.2 M NaOH was added, changing the color from blue to greenish; this was then placed in a magnetic stirrer for 2 h [99].The greenish precipitate was filtered and washed twice with 50 mL of D.D. water and then with 50 mL of ethanol to eliminate the impurities.

Antibacterial Assay
The antibacterial activity of UL/Ag 2 O-NPS, Ul/Ag/Cu-NCMs, and Ul/CuO-NPs was investigated against eleven pathogenic bacterial strains.Six Gram-negative bacteria (Klebsiella pneumoniae KY856924, Acinetobacter KY856930, E. coli KY856932, E. coli KY856933, Enterobacter KY856934, and Enterobacter aerogenes), which were isolated from different medical samples and identified as multidrug-resistant in a previous study [78,100], in addition to five pathogenic Gram-positive control strains (Klebsiella pneumoniae KY856924, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, and Streptococcus mutans ATCC 25175) were utilized.All the bacterial strains were cultured in nutrient broth at 37 • C for 24 h.The bacterial strains were spread on Mueller-Hinton agar (MHA) using a sterile cotton swab.Wells were made in the agar plates and filled with 10, 30, and 100 µL of 20 mg/mL UL/Ag 2 O-NPS, Ul/Ag/Cu-NCMs, and Ul/CuO-NPs, respectively.The plates were incubated at 37 • C for 24 h, and the zone of inhibition was observed after 24 h of incubation.

Minimum Inhibitory Concentration (MIC) Assays
The minimum inhibitory concentrations (MIC) of green synthesized nanoparticles were determined in a 24-well culture plate using standard broth micro-dilution methods to verify the antibacterial activity.Two-fold dilutions of UL/Ag 2 O-NPS, Ul/Ag/Cu-NCMs, and Ul/CuO-NPs in concentrations ranging from 1 mg/mL to 0.062 mg/mL were prepared to determine the MIC in the nutrient broth media.The results were observed and compared to the negative control after 24 h at 37 • C of incubation.

Minimum Bactericidal Concentration (MBC) Assays
After the MIC determination of the nanoparticles, the MBC was conducted by transferring 10 µL from each overnight MIC culture plate well to sterile Mueller-Hinton agar (MHA) fresh plates.Viable growth was determined after 24 h at 37 • C. The lowest concentration with no visible growth on the MHA plate was recorded as the MBC value.

Biofilm Formation Inhibition Assay
The biofilm inhibition assay was performed as described previously by Heikens et al. [101] and Hamed et al. [102] with the following modifications: one colony of overnight plate-grown E. coli KY856933 bacterial culture (which showed positive biofilm formation on Congo red agar) was resuspended in 5 mL tryptic soy broth (TSB) supplemented with 0.25% glucose on a shaking incubator at 37 • C to an OD of 660.
The bacterial cultures were diluted with fresh media (1:20) and supplemented with different concentrations (1, 0.5, 0.25, 0.12, and 0.06 mg/mL) of UL/Ag 2 O-NPS, Ul/CuO-NPs, and UL/Ag/Cu-NMCs separately, in addition to a culture without nanoparticles as a positive control.Then, 200 µL of each concentration was aliquoted into the wells of a 24-well flat-bottom polystyrene microtiter plate and incubated for 24 h at 37 • C without agitation.After incubation, the bacteria were removed, the wells were washed with 200 µL phosphate buffer saline (PBS), and the plates were dried for 1 h at room temperature.
A total of 100 mL of 0.2% Gram's crystal violet was added to each well for 15 min after the first hour.After the stain was eliminated, 200 µL of PBS were used to wash the plates three times.At room temperature, the plates were dried for 15 min.Each well received one milliliter of ethanol (95%) before being incubated for 15 min.At 570 nm, the reaction mixture was spectrophotometrically measured.

% of inhibition =
OD in control − OD in treatment OD in control * 100

Statistical Analysis
The experiments were performed in triplicate and the results were considered as the means ± standard error of the mean.An analysis of variance (ANOVA) was performed on all of the data.Duncan's multiple range tests were used to determine the significance of the variable mean differences (p ≤ 0.05), SPSS version 16.

Conclusions
In this experimental study, Ulva lactuca aqueous extract was successfully used to synthesize silver and copper nanoparticles and their composites.Their characterization was achieved using FTIR, SEM, TEM, EDX, XRD, and the zeta potential.The FTIR analysis reveals that the functional groups of nanoparticles and their composites differ.The TEM image demonstrates changes in the shape and size of the silver and copper nanoparticles and their composites, which were biofabricated by green alga U. lactuca.The zeta potential denotes that the UL/Ag 2 O-NPS have a positive charge, while the Ul/CuO-NPs and Ul/Ag/Cu-NPs have a negative charge.The fact that the UL/Ag 2 O-NPS were more effective against the tested pathogenic bacteria than both the Ul/CuO-NPs and Ul/Ag/Cu-NPs may be due to the positive charge of UL/Ag 2 O-NPS, as well as the small size of the nanoparticles.The inhibition of biofilm formation by E. coli KY856933 was the highest with the applied UL/Ag 2 O-NPS, followed by the Ul/CuO-NPs and Ul/Ag/Cu-NMPs.

2. 1 . 3 .
Energy Dispersive X-ray (EDX) of UL/Ag 2 O-NPS, Ul/CuO-NPs, and Ul/Ag/ Cu-NCMs Derived from U. lactuca Energy dispersive X-ray (EDX) is a vital analytical procedure used to verify the sample's elemental composition.Ag and O were found, by weight, in UL/Ag 2 O-NPS at 54.42 and 47.58%, respectively.The chemical composition of the prepared Ul/CuO-NPs was identified by EDX analysis, as shown in Figure

2 . 1 . 5 .
Figure 5 demonstrates the zeta potential of UL/Ag 2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs derived from U. lactuca.The results show that the zeta potential value of UL/Ag 2 O-NPS has a positive charge (+1.14), while the zeta potential values of Ul/CuO-NPs and Ul/Ag/Cu-NCMs have negative charges (−0.195 and −3.69, respectively).The zeta potential values differ according to the methods used and plant synthesis.The silver nanoparticles formed by the fungus Trichoderma viride had a positive charge [77].The zeta potential values of the Ag-NPs produced by photosynthesis by Oscillatoria limnetica had a highly positive charge [78].Molecules 2023, 28, x FOR PEER REVIEW 10 of 22 the crystalline UL/Ag2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs derived from U. lactuca.Zeta Potential of UL/Ag2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs Derived from U. lactuca Figure 5 demonstrates the zeta potential of UL/Ag2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs derived from U. lactuca.The results show that the zeta potential value of UL/Ag2 O-NPS has a positive charge (+1.14), while the zeta potential values of Ul/CuO-NPs and Ul/Ag/Cu-NCMs have negative charges (−0.195 and −3.69, respectively).The zeta potential values differ according to the methods used and plant synthesis.The silver nanoparticles formed by the fungus Trichoderma viride had a positive charge [77].The zeta potential values of the Ag-NPs produced by photosynthesis by Oscillatoria limnetica had a highly positive charge [78].

Figure 7 .
Figure 7. Anti-biofilm studies of green synthesized Ul/Ag2 ONPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs in the presence of biofilm-forming of E. coli KY856933.Different letters denote the significant values of mean; bars are St-error.

Figure 7 .
Figure 7. Anti-biofilm studies of green synthesized Ul/Ag 2 ONPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs in the presence of biofilm-forming of E. coli KY856933.Different letters denote the significant values of mean; bars are St-error.
• .637086N and 39 • .101631E) in Jeddah, Saudi Arabia (2020).One gram of air-dried U. lactuca was mixed with 100 mL of deionized (D.D.) water to create the Ag-NPS and Ag-Cu NCMS.This mixture was then boiled for an hour and filtered, and the remaining 100 mL of D.D. water was added.For the CuO-NPs, 100 mL of D.D. water was combined with six grams of air-dried U. lactuca alga, boiled for one hour, filtered, and then the remaining 100 mL of D.D. water was added.Molecules 2023, 28, x FOR PEER REVIEW 16 of 22 combined with six grams of air-dried U. lactuca alga, boiled for one hour, filtered, and then the remaining 100 mL of D.D. water was added.

Table 1 .
Absorption peaks assigned to the active groups of Ul/Ag 2 O-NPS, Ul/Ag/Cu-NCMs, and Ul/CuO-NPs derived from U. lactuca.

Table 2 .
The main differences between biofabricated nanoparticles by U. lactuca.

Table 4 .
Cont.Different letters in each column are significant value (p ≤ 0.05).