Production of Biosynthesized Silver Nanoparticles using Metarhizium anisopliae fungus for the Treatment of Petroleum Pollutants in Water

In this study, a silver nanoparticle (AgNPs) was created using a biological technique from an extract of the fungus Metarhizium anisopliae . The characteristics of the prepared AgNPs were identified by utilizing the optical, ultraviolet, and infrared absorbance spectroscopy. The shape, size, and charge distribution on the particles were determined by using scanning electron microscopy and zeta voltage analysis .The analysis of biological activity of the silver nanoparticles showed its effectiveness in treating pollutants, as confirmed by the reduction of higher than 93% weight of crude oil in contaminated water samples. The crude oil mass was effectively transformed into the gelatinous mass that lacks consistency and emulsification. The chemical analysis of NP-treated and untreated crude oil-contaminated water samples was performed using gas chromatography mass spectrometry (GC MASS). The results displayed the emergence of 55 graphic peaks, each of them indicating a chemical compound, in the control sample, while in the study sample, about 51 of these peaks disappeared and the area of the remaining 4 peaks was reduced. The silver nanoparticles' capability to maintain their effectiveness under cryogenic storage conditions for six months was tested and compared to that of the fungal isolation before the production of the silver nanoparticles. The results showed no significant changes in the shape, size, and efficiency of the silver nanoparticles in the treatment of oil pollutants in water. The results indicated the higher efficiency of the silver nanoparticles, as compared to chemicals, in treating petroleum pollutants as well as enhancing the solubility, emulsification, and degradation of hydrocarbons . In addition, the AgNPs are characterized by the availability of inexpensive, easy, fast to produce, and environmentally friendly production materials, as compared to the usage of chemical products that are highly toxic to aquatic organisms, expensive to produce, and highly accumulative in the ecosystem, i.e. environmentally unsafe.

https://dx.doi.org/10.21123/bsj.2023.8577P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal spills and various oil residues to water surfaces.Oil is one of the most dangerous types of pollutants, because of the high quantities of its derivatives as well as its toxic substances that exert remarkably harmful impacts on humans and animals.The toxicity of oil is due to its composition of many hydrocarbons, such as benzene, which is a carcinogenic hydrocarbon.In addition, oil contains ether and gasoline, which are dangerous compounds when available in high concentrations.Moreover, phenolic compounds contained in the oil are, upon inhalation, among the causative agents of allergy 1 .
Collectively, these impacts can lead to the death of aquatic organisms.Furthermore, oil, especially crude oil, contains hydrogen sulfide and other toxic gases that are emitted when hydrocarbon particles evaporate or dissolve 2 .Biodegradation is considered one of the most important and safe mechanisms to eliminate oil pollutants from water 3 .This process is achieved by microorganisms, e.g.bacteria, fungi, and algae, through their consumption of hydrocarbons present in the oil.The degree of consumption depends on the nature of the microorganism 4 .The process of biological treatment is accomplished through the decomposition of oil hydrocarbon compounds by microorganisms and their conversion into simple and environmentally friendly compounds, such as carbon, carbon dioxide, nitrogen, phosphorous, and others 5 .Fungal species, e.g.Aspergillus, have recently been utilized in the field of biological treatment and disposal of environmental pollutants resulting from oil waste.It was found that these species tend to consume hydrocarbons present in petroleum products 6 .
Nanotechnology technique deals with particles ranging from 1 to 100 nm 7 .Nanomaterials differ from their larger counterparts in terms of their physical, chemical, and structural characteristics, the nanomaterials' extraordinary magnetic, electrical, and optical characteristics and surface activity are due to their nanoscale shape and size 8 .
There are many studies in field of biosynthesis of nanoparticles using different biological materials, like study of Shah et al, silver nanoparticles (AgNPs) were synthesized by using Silybum marianum plant extract 9 and Abd-Elhady et al, used biological approach to synthesis Extracellular Silver Nanoparticles from Streptomyces aizuneusis 10 .
At the present time, new innovative methods have been introduced to control and reduce environmental pollution, including nanotechnology which improves the properties of fungi at low costs 11 .The present study aims at synthesizing and diagnosing silver nanoparticles produced from the extract of the fungus Metarhizium anisopliae.The effectiveness of these NPs in treating oil pollutants in water, as well as their storage potential, were determined.

Fungal Isolate
A local fungal isolate, identified as Metarhizium anisopliae, was grown in Potato Dextrose Agar-PDA culture medium sterilized with an autoclave for 15 minutes.Two grams of tetracycline were added per liter of medium to prevent bacterial growth.The culture dishes were incubated in an incubator (Memmert) at 26±2 ˚C and a relative humidity of 75±5 % for a week, until a complete fungal growth was achieved, according to the method described in 12 .

Crude Oil Sample
Oil samples were obtained from Kirkuk oil fields according to the following specifications: The American Petroleum Institute (API) gravity at 60 Fº

Preparation of Fungal Suspension:
The fungal suspension was prepared by pouring 5 ml of sterile distilled water to a petri dish containing the fungal colonies upon their complete growth on the culture medium.The spores were separated by an Lshaped harvester.The fungal suspension was filtered through a Whatman grade 1 filter paper after adding another 5 ml of distilled water to the sides of the filter paper to eliminate the mycelium and obtain the fungal cells (Fungal stock suspension), based on the method of 13 .

Cell Number Counting in the Fungal Suspension:
Following the method of 14 , the counting chamber of a Neubauer hemocytometer was utilized to count the number of cells.One drop (0.1 ml) of the stock fungal suspension was placed on the slide, which was covered with a cover slip prior to counting the number of cells in the four squares of the chamber.
An optical microscope was used to examine the slide at 40 x power.The calculations were made according to the following equation:

Preparation of Metarhizium anisopliae extract:
To obtain its biomass, the fungus isolate was grown in a petri dish containing sterile potato agar and dextrose medium.The culture was incubated at 26 ± 2 ˚C and relative humidity of 75 ± 5 % for 7 days.Four discs of the grown colonies were collected, with a diameter of 5 mm, and inoculated within a sterile liquid dextrose potato broth (PDB) medium.Two grams of tetracycline per liter of medium were added, followed by the incubation of the flasks in a shaker incubator (100 cycles per minute) at 26 ± 2 ˚C for 21 days under wet conditions.Fig. 1 shows the preparation of the biomass of Metarhizium anisopliae.The fungal biomass was harvested after 21 days of growing using a sterile filter unit (Buchner funnel and sterile Whatman No.1 filter paper) under vacuum pressure and aseptic filtration conditions.The harvest was washed with distilled water three times, followed by a washing step with deionized distilled water.Cell breakage technique with ultrasound for 5 minutes and 30 seconds intervals in one run was applied to break down the fungal cells.The harvest was filtered using 0.45 µm Millipore filters.The https://dx.doi.org/10.21123/bsj.2023.8577P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal filtrate was kept at 26 ± 2 ˚C and relative humidity of 75 ± 5 % until use, according to the method described in 15 .

Preparation of the Silver Nanoparticles
The aqueous solution of silver nitrate at a concentration of 50 mmol was prepared as follows: The solution was heated to 60 ° C for 10 minutes with stirring, using a water bath and ultrasound waves.
The silver nanoparticle was prepared by distilling 50 ml of fungal extract into 950 ml of silver nitrate solution in a vibrating incubator at 60 ° C for 120 hours.The color changes were monitored, as evidenced by the formation of silver nanoparticles via the biological reduction process, according to the method of 16 .

UV-VIS Spectrophotometry
The optical characteristics of the nanoparticle, silver nitrate, and fungal biomass extract, were determined separately by 10 times dilution of 2 ml of the prepared solutions with deionized water to reduce the false readings.The samples were transferred for examination by spectrophotometry and ultraviolet waves, with the appropriate wavelength being fixed within a range of 190 to 850 nm 17 .

Fourier Transform Infrared (FTIR) Spectroscopy
This method was utilized to determine the active groups of both the fungal biomass extract alone and its mixture with silver nitrate solution (silver nanoparticles).These active groups function as encapsulating and reducing agents for silver nanoparticles.The samples were prepared by depositing a drop of each sample on a glass slide and drying it at 60 ° C in an oven for 30 minutes.The product was made into a paste using a high-viscosity liquid substance (Nujol paraffin oil).A small amount of the paste was placed between two discs of potassium bromide to form a very thin layer, which was examined with a Fourier transform infrared device 18 .

Scanning Electron Microscopy (SEM)
The structural characteristics of the samples were determined in terms of size and shape.The samples were prepared by mounting drops of the silver nanoparticles solution separately on glass slides, followed by drying at 60 ° C for 30 minutes and staining with crystal violet for one minute.The specimens were washed with distilled water and fixed with Giemsa stain for one minute.They were then coated with gold in an atmosphere saturated with argon gas under pressure of 50 Pa and a current of 50 mA for 50 seconds.The specimens were examined with an SEM at 30000 X magnification 19 .

Zeta Potential
The stability of silver nanoparticles was evaluated by using a zeta potential device with a measurement range of -160 to +160 mV 20 .

Storability of the Nanoparticle
A sample of the silver nanoparticles was placed in clean and sterilized containers and stored at 4 ° C. Six months after production, the effectiveness of the stored nanoparticle was tested in terms of causing phenotypic changes, emulsification, and disintegration of crude oil.This was performed by incubating a mixture of crude oil-contaminated water samples with the nanoparticle for one week and comparing the results with those of the control sample (crude oil-contaminated water without nanoparticle).

Non-specialized Chemical Tests
The bio-nanoparticle was chemically tested for its efficiency in breaking down crude oil.The silver nanoparticles were added to 50 ml of sterile distilled water, and then 1 ml of the crude oil was added in 250 ml conical flasks.The mixture was placed in a https://dx.doi.org/10.21123/bsj.2023.8577P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal shaking incubator at 30 ° C at a 50 rpm for 14 days, with the process being repeated twice.The mixture was placed in special tubes and centrifuged at 10,000 rpm for 20 minutes to remove impurities.The supernatant was combined with hexane extract and a few drops of hydrochloric acid were added until the pH of the medium reached 2. The hydrocarbons were extracted by adding the solvent chloromethane, after placing the sample in a separation funnel, to separate the water layer from the hydrocarbon layer which is dissolved in the solvent.The last layer was filtered through a filter paper containing anhydrous sodium sulfate (Na2SO4) as a desiccant agent and utilizing a clean beaker to collect the filtrate.The solvent was evaporated by using a rotary evaporator at room temperature, and the sample was weighed to calculate the weight of the remaining hydrocarbons.
The method was repeated using the control sample.The rate of degradation was determined according to the method of 21 .through the equation below: Crude oil degradation rate = Weight of remaining crude oil / Weight of control crude oil *100.

Detection of Crude Oil Decomposition Using Gas-Mass Chromatography
A volume of 1 ml of crude oil was added to 50 ml of sterile distilled water in a 200 ml conical flask.After stabilization, the nanoparticle was added to the mixture in two replicates.In addition, the negative control (water with crude oil without nanoparticles) and positive control (water with nanoparticles without crude oil) samples were prepared.The flasks were placed in a shaking incubator at 30 ° C at a rotation speed of 100 rpm for 14 days.They were then placed in special tubes in a centrifuge for half an hour at a rotation speed of 12000 rpm to precipitate the impurities.
The filtrate was dissolved by adding hexane solvent in the ratio of 1: 1 volume in the separation flasks, that were shaken for a while and left in a vertical position until the separation process was completed when the hexane layer was collected.The samples were concentrated with a rotary evaporator at a temperature of 40 ° C. The samples, which now represent the remainder of the hydrocarbons, were tested for the level of dissociation and decomposition of crude oil components by the nanoparticle.For this analysis, a Gas-Chromatography device (Shimadzu Model 2014) was used, with a 30 m long capillary column of the CPSIL5-CB type, into which 0.4 mL of each sample was injected.The conditions used to operate the device involved nitrogen as carrier gas at a flow rate of 25 milliliters / minute, while the FID detector temperature was set at 270 ° C.
The separation column was prepared with an initial temperature of 80 ° C and programmed to raise the temperature at a rate of 5 ° C / minute to a temperature of 260 ° C, until it reaches the end of the total time of 60 minutes, the peaks of the used compounds were compared with the peaks of standard materials that appear at the same retention time.The area was calculated to estimate the amount of hydrocarbons remaining after silver nanoparticles decomposition according to the following equations 22 : Percentage of residual hydrocarbons = sample area / total area * 100

UV-VIS Spectroscopy
The optical and spectroscopic characteristics of the prepared silver nanoparticles were studied.The presence of color changes was observed when the biomass extract of M. anisopliae was added to silver nitrate solution and compared to the solution containing the fungus biomass extract without silver nitrate AgNO3, which did not demonstrate any color changes.These colors ranged from light brown to dark brown, as displayed in Fig. 2. It was also observed that the color change process was stabilized after 120 hours at a temperature of 26 ± 2 ° C. The occurrence of these changes is evidence of the formation of silver nanoparticles while showing of this particular color is due to the excitation of the surface plasmon of the particles formed, i.e. surface plasmon resonance (SPR) 23,24 .This property appears on the surfaces of some metals as a result of the collective movement of free electrons in the nanoparticle when light falls on them.The direction of electron movement changes over time with the same oscillation of the incident optical electromagnetic wave.This characteristic is clearly found in silver, gold, and copper, provided that the nanomaterial is colloidal, in the visible light region.It is responsible for changing the colors of these elements when they reach the nanoscale and has many properties and applications.This characteristic is based on the particle size, shape, and medium in which the particles are present 25 .
The above-mentioned results agree with those reported by 26 , during his study of the effects of silver nanoparticles prepared by biological methods.He recorded the presence of color changes, upon adding the biomass extract of B. Bassiana to a silver nitrate solution, within 120 hours at a temperature of 25 ° C.These color changes also appeared when preparing silver nanoparticles using the fungi species of Aspergillus terreus HA1N and Penicillium expansum HA2N, and Fusarium oxysporum 27,28 .The color changes occur within 5 days of the reaction and remain constant for 60 days.
Ultraviolet spectroscopy was used to measure the surface plasmon excitation of the silver nanoparticles in solution, by recording the highest absorbance at the wavelength of 293.68 nm for the silver nanoparticles prepared by the extract of M. anisopliae as illustrated in Figs. 3, 4, and 5.The ultraviolet spectrum of silver nanoparticles produced by Penicillium citrinum at a wavelength of 265 nm was reported to indicate the presence of tyrosine and tryptophan residues, thus confirming that the proteins secreted by the fungus are important for the formation and stability of the silver nanoparticles 29 .The fungus Aspergillus ochraceus recorded the highest absorbance at the wavelength of 280 nm.This indicates that the fungus released its enzymes into the fungal filtrate 30 .The highest absorbance at the wavelength of 260 -290 nm is due to the presence of amino acids and phenylalanine, along with tyrosine and tryptophan present in the protein.This is evidence of the release of proteins and enzymes by the fungus to the aqueous medium, which has led to the suggestion of the mechanism by which silver ions are reduced, by 31 .The above-mentioned studies reflect the high activity of the biomass extract of M. anisopliae in reducing substances by the presence of fungal enzymes, including anthraquinones, naphthoquinones, and nitrate reductase, that enhance the reduction of nitrates to nitrites and ammonia 32 .Fungi are characterized by their ability to assist the formation of larger quantities of silver nanoparticles, as compared to bacteria, due to their ability to secrete a large amount of enzymes that reduce silver into metal particles and form silver nanoparticles 33 .

FTIR Spectroscopy
Infrared spectroscopy is a strong tool for identifying the kinds of chemical bonds in a molecule, by producing an infrared absorption spectrum that is similar to the molecular "fingerprint".A number of power unit peaks were obtained when analyzing M. anisopliae biomass extract, silver nitrate solution, as well as the prepared silver nanoparticles, as shown in Figs. 6, 7, and 8. Infrared spectroscopy demonstrated the presence of amide bonds between amide bands, through the values in the regions of 1200 to 1800 1 / cm and the presence of the Alkyl halides group (Cl), through the values in the areas 505.35, 576.72, 794.67 and 842.89 1 / cm.Carboxyl (COO-H) aggregates were recorded in regions 939.33 and 1039.63 1 / cm.The analysis also showed the presence of aromatic compounds in regions 746.45, 867.97, and 889.18 1 / cm.Also, regions of ketones (C-C) and aldehydes (C = O) appeared in regions 1139.63 and 1708.93 1 / cm.As for the presence of ethers and esters, they were indicated in the area 1192.01 1 / cm, in addition to the presence of energy regions of alkanes at 817-1689.64 1 / cm and alkanes at 1377.17, 1467.83, and 2804.45 1 / cm.Alcohols were found at the energy regions 1211.3,1236.37, and 1257.59 1 / cm.Spectroscopy also recorded the existence of a group of C = O and a group of N-H, represented by the values in the regions of 1400-17001 / cm.As for the energy regions of amines, they were located at 1639.49-1514.12 1 / cm, indicating the presence of protein on the surface of the Ag particle 34 .The area at 1014.56-1303.81indicated the presence of C-O in the active groups of esters, ethers, and alcohols, distinguished by their strong ability to bond with silver ions 31,35 .The energy units of the carboxyl and Alkyl halides groups reflect the presence of proteins, which is an indication of their important role in the biological reduction of the silver nitrate and the production of the silver nanoparticles, as well as their functions as stabilizers that envelope and prevent silver nanoparticles from aggregating.The regions of ketones, ethers, esters, and aromatics have a similar role 33 .Thus, M. anisopliae extract is characterized by being rich in active compounds that contribute to the biological reduction of the silver nitrate and the production of silver nanoparticles.Being a rich source of capping agents and stabilizing silver nanoparticles, this extract competes with chemical and physical methods that use toxic, harmful, and costly substances 36 .
Soleimani et al, 37 indicated that the aqueous extract of B. bassiana contains capping, stabilizing, and reducing agents, represented by alcoholic, protein, and phenolic groups.These groups are among the most active groups that participated in the biological reduction process, as indicated by the infrared spectroscopy values.

Examination by Scanning Electron Microscopy
The size and shape of the prepared silver nanoparticles were determined by electron microscopy.Image 1 shows that the silver nanoparticles prepared with the extract of M. anisopliae were characterized by spherical and oval shapes, while the size range was 34.7 to 141.6 nm, with an average of 76.65 nm.
The shape and size of the silver nanoparticles play a significant role in determining their physical, chemical, and biological properties. 38, found the size of the silver nanoparticles prepared by the aqueous extract of Beauveria bassiana to be in the range of 10-50 nm.The spherical shape of the silver nanoparticles produced from the aqueous extract of Penicillium citrinum appeared when examined under an electron microscope 29 .

Zeta Potential Analysis
The zeta potential is usually referred to using the Greek letter zeta (ζ).The value of zeta potential of silver nanoparticles is -75.65 mV for silver nanoparticles as shown in Fig. 9.It is an important indicator of the stability of the nanoscale solution.
The zeta potential size refers to the degree of electrostatic repulsion between adjacent, similarly charged particles in the dispersion.For sufficiently small particles, the high zeta potential value will provide stability, that is, the dispersion will resist aggregation.Therefore, colloids with high zeta potentials (positive or negative) are electrically

Detecting the Decomposition of the Crude Oil Using Non-specialized Chemical Methods
The ability to decompose crude oil was tested by calculating the quantitative loss of the crude oil, achieved following the utilization of more than 93% nanoparticle compared to that of 25% fungal isolation.This indicates a high efficiency of the silver nanoparticles in breaking down crude oil with a percentage that is double that of the fungal isolation 40 .

Storability of the Silver Nanoparticle
The bio-nanoparticle was stored for six months in cooling, after which the phenotypic changes of the crude oil due to the effectiveness of the silver nanoparticles were studied by adding the stored nanoparticles to the crude oil sample in water.The results were compared with the crude oil sample (the silver nanoparticles-free control) in water for one week of incubation at room temperature.The appearance of crude oil emulsification was observed as a result of the effectiveness of the nanoparticle, as well as the absence of phenotypic changes in response to the silver nanoparticles stored for six months.
Singh et al, 41 found one characteristic feature of AgNPs produced by Cedecea sp.extracts which is their extreme stability.Inductively coupled plasma mass spectrometry and thermogravimetric analysis revealed that the produced AgNPs are stable for periods exceeding one year.

The Effectiveness of the Silver Nanoparticle in Decomposing Oil Pollutants
The chemical analysis of the samples under study was performed utilizing GC-MASS chromatography and compared to those of the non-biological (control) sample.The results revealed the appearance of 55 graphic peaks, each peak refers to a chemical compound in the control sample (untreated crude oil).Nevertheless, a large number of these peaks (51 peaks) disappeared and only 4 peaks appeared when examining the sample of crude oil-contaminated water treated with silver nanoparticles, but the area of these peaks was greatly reduced as shown in Figs.
10,11 and 12, Tables 1 and 2. This possibly refers to a significant reduction in the concentration of compounds, which indicates the efficiency of this method in decomposing hydrocarbon compounds 42 .
Previous studies indicated the use of this technique to detect the dissociation occurring in petroleum compounds and the identification of the kind and concentration of those decomposing compounds utilizing the GC-MASS technique 43 44 .

Conclusion
Preparation and identification of silver nanoparticles from the extract of the fungus Metarhizium anisopliae was made to increase the treatment efficiency of oil and hydrocarbon pollutants in water.The utilization of an extract of a local fungal isolate that is not harmful to the environment and available locally.
Production of a nano-scale biological from the extract of Metarhizium anisopliae was conducted for the first time, locally in Iraq and internationally, with a decomposition efficiency of oil pollutants that is much higher than that of fungal isolation alone.The product is of high economic feasibility, material availability, cost-effectiveness, and ease of preparation.
The capability of the nanoparticles in maintaining their effectiveness under cryogenic storage conditions for six months was tested and compared to that of the fungal isolate before the production of the silver nanoparticle.The results showed high efficiency and good storage capacity.
The capability of the nanoparticles in maintaining their effectiveness under cryogenic storage Number of spores = (N / 80) *10 6 * 10 where: N = the average number of cells counted in the four squares.80 = sum of the minute squares within the four counted squares.10 6 = concentration correction factor.10 = volume correction factor.

Figure 8 .
Figure 8. Infrared spectroscopy of the silver nanoparticles prepared with M. anisopliae aqueous extract

Image 1 .
A scanning electron microscope image showing the shape and size of the nanoparticle prepared with M. anisopliae extract; A = 98.2 nm, B = 65.11nm, C = 35.31nm.

Figure 9 .
Figure 9. Zeta potential analysis of silver nanoparticles prepared with M. anisopliae extract.

Figure 10 .
Figure 10.GC-MASS results of the control sample of untreated the crude oil showing the retention time and peaks of the chemical compounds.

Figure 11 .
Figure 11.GC-MASS results of a sample of the crude oil treated with silver nanoparticles, showing the decrease in the number and area of the chemical compounds' peaks.