3.1 Isolation and identification of fungi
In the present study, the results of the isolation and identification of fungi indicated that 23 genera were isolated from Tigris River water samples during the two seasons. All the isolated fungal species were purified using the agar plate pour method and identified and characterized primarily according to the external appearance, backdrop, and color of the colonies on PDA media. Light microscopy was then used to investigate the features and characteristics of the hyphae, conidia, conidiophores and spore shapes of the isolated fungi.
During this study, 524 fungal colonies were isolated from all water samples collected in the dry and wet seasons. Their occurrence was quantified, and they were subsequently identified, resulting in the isolation of 23 fungal genera. The results of the present study indicated that the culture features of the fungal isolates, including term surface characteristics; reverse, color, edge and diameter forward representation of the surface; and reverse features of the isolated fungus, were identified (Fig. 3) via primary screening. After that, each fungal genus was isolated in special petri dishes (Fig. 4).
Figure 3
Figure 4
Microscopic observations of the fungal isolates are shown in Fig. 5, which shows the conidia, conidiophores and spores.
Figure 5
Among the isolated genera, Aspergillus spp. was consistently found to be the most common among the fungi isolated during the study period, with over 56 isolates (10.69%), which had hyaline conidiophores, septate hyphae and a redial conidial head bearing spores, followed by Penicillium spp. Fifty of the isolates (9.54%) were isolated via microscopy, with conidiophores, septate hyphae and secondary branches. Fusarium spp. had septate hyphae shaped of multiseptate canoe attached to the conidiophores, and Alternaria spp. was the fourth most common genus (34, 6.49%), as shown in Table 1.
3.2. Investigation of the Fungal Growth Ability on CMC Agar Medium and Cellulose
The results of the primary assay of enzymatic activity showed that 57 fungal isolates out of the total obtained isolates had the ability to degrade the substrate CMC agar medium to smaller oligosaccharides or monosaccharides through the production of an extracellular enzyme called hemicellulase. The fungal isolates that grew on CMC media exhibited a halo zone around the colonies, whereas the fungal isolates that grew without any halo zone formed around the fungal colonies (Fig. 6). The current results imply that a large amount of hemicellulase is produced by Aspergillus spp. and Penicillium spp. This enzymatic activity is important for the development of biotechnological applications in industry, according to Gomashe et al. (2013).
Figure (6):
The sizes of the halos around the fungal colonies varied according to the type of fungal genus and species. The size of the halo zone ranged from 75 mm to 60 mm in the fungal genera Aspergillus, Penicillium, Fusarium, and Alternaria, and the size of the fungal zone ranged from 30 mm to 20 mm in the following genera: Cladosporium, Trichoderma, Rhizopus, Mucor, Botrytis, Aureobasidium and Chalaropsis.
A total of 41 fungal isolates that were able to grow on cellulose were obtained (Fig. 7). The examination was based on the growth of the fungus, and its growth was compared with that of fungi grown on modified Czapek dox agar.
Figure (7):
There were previous studies on the enzymatic hydrolytic ability of Aspergillus fungi that were isolated from the Paranaense rainforest (Argentina), and the ability of these fungi to undergo cell degradation was evaluated using Congo staining and fluorescence panel tests for carboxymethylcellulase, beta-glucosidase, and cellobiohydrolase; all the results were positive. This study demonstrated the ability of Aspergillus fungi to process cellulosic biomass (Díaz et al. 2021).
In a study by El Bergadi et al. (2014), 31 fungi were isolated from an old library in the city of Fez in Morocco, and nine isolates were obtained with a positive result in terms of CMC degradation. The most common species were Mucor racemosus, Aspergillus niger, Aspergillus oryzae, Mucor racemosus and Penicillium chrysogenum, as were other less common species, such as Aspergillus melleus. Hypocrea lixii and Schizophyllum commune.
3.3. Promoting Fungal Growth and Biomass Accumulation on HDPE and PS
The results of the present study revealed that among 41 fungal isolates grown on MPs as the sole carbon source under optimum conditions, visual observation of HDPE and PS samples treated with fungal isolates revealed limited fungal colonization, while visible growth was observed primarily in areas inoculated with A. carbonarius, Eurotium sp., A. westerndijkiae, and A. glaucus. These fungal genera exhibited heavy growth and greater biomass accumulation on HDPE substrates than on PS substrates. The results indicate the selective nature of fungal decomposition toward these plastics, suggesting potential differences in plastic degradability between different fungal species. The results of this study contribute to our understanding of fungal biodegradation capabilities and provide insight into developing sustainable plastic waste management strategies.
The macroscopic and microscopic characteristics of the fungal isolates, including their probable identities, strongly differed among the four fungal isolates (Table 2).
Pigments and coloration, including green and whitish spores with septate and nonseptate hyphae, were observed in the fungal isolates (Fig. 8). The four fungal isolates were investigated for protease, pectinase, lipase, laccase and amylase production, as shown in Table 2. These fungal enzymes play an important role in utilizing and breaking down the chemical bonds of plastic polymers and using them as a carbon source.
Figure (8):
During a laboratory experiment conducted by the researchers Pramila and Ramesh (2011), where fungal isolates from the sea were exposed to growth in a medium containing low-density polyethylene (LDPE) as the sole source of carbon, an increase in the weight of the fungi was observed. This is evidence of fungal consumption of MPs as a carbon source, and the fungus was identified as an Aspergillus spp.
Additionally, during a laboratory experiment conducted by the researchers Ameen et al. 2015, fungal isolates were taken from tidal water and sediment collected from mangrove trees on the Red Sea coast of Saudi Arabia; six fungal isolates demonstrated the ability to grow on pieces of LDPE, and the growth of fungi on the surface of the pieces was detected by SEM.
3.4. Detection of Fungal Enzymes
The production enzymes that could induce the biodegradation of HDPE and PS, A. carbonarius and Eurotium sp., showed promising results (Table 3 and Fig. 9). The ability of these microbes to degrade organic and inorganic materials such as cellulose, hemielluloses, lignin and starch could enable the aforementioned fungi to quickly degrade polymers (Kumar et al. 2013); therefore, the growth and MP biodegradation of the 2 fungal isolates were tested in the present study.
A previous study showed the ability of fungi to decompose MPs via enzymes secreted by fungi, such as laccases and peroxidases, which are used for decomposing lignin, and the ability of fungi to decompose polyvinyl chloride (PVC) and polyethylene (PE). Polyurethane (PUR) and polyethylene terephthalate (PET) are degraded by esterase enzymes such as lipases and cutinases (Temporiti et al. 2022).
Varshney et al. (2023) reported the ability of several fungal species, such as Colletotrichum fructicola, Trichoderma viride, Cephalosporium sp., Stagonosporopsis citrulli, Diaporthe Italiana and Aspergillus nomius, to decompose plastic polymers of various types, as fungal strains consume plastic polymers as the sole source of carbon and convert them into environmentally friendly carbon compounds. The above studies agree with the current study.
Figure 9
3.5 Examination of HDPE and PS Disc Using Light Microscopy
The ability of the obtained fungal isolates to grow and utilize HDPE and PS as carbon and nitrogen sources was tested. The growth of A. carbonarius and Euroitum sp. in HDPE- and PS-containing media indicated that the isolates could utilize HDPE and PS as the sole carbon and nitrogen sources for growth. Based on these results, it was speculated that when HDPE and PS were used together, the metabolic activity of the fungi increased since they could use HDPE and PS films (Fig. 10A), which clearly indicates the growth of fungal mycelia on HDPE and PS molecules, further indicating and suggesting that exopolymer substances on the surface of fungi might be involved in this process. Since HDPE and PS are not soluble, the hydrophobicity of exopolymer substances on mycelia is essential in the absorption process. Seneviratne et al. (2006) reported that fungi are considered to have a greater ability to degrade MPs because they secrete hydrophobic proteins, bind to polymer surfaces, grow faster and can penetrate various substances (Kim and Rhee 2003) (Fig. 10B).
Figure (10):
3.6 Determining the Optimal Conditions for Fungal Growth on Microplastics (pH, temperature and incubation period)
The degradation was determined by calculating the weight loss in the HDPA and PS discs before and after fungal treatment:
After 30, 40 and 50 days of incubation at 28, 30 and 32°C at pH 5, 5.5 and 6, two fungi were isolated from HDPE and PS degraded by A. carbonarius (Table 4) and Eurotium sp. (Table 5) under continuous shaking.
Table (1): List of identified fungal genera in Tigris River during the study period.
Fungal genera
|
No of fungal isolates
|
occurrence %
|
Aspergillus
|
56
|
10.69
|
Penicillium
|
50
|
9.54
|
Fusarium
|
41
|
7.82
|
Alternaria
|
34
|
6.49
|
Cladosporium
|
25
|
4.77
|
Trichoderma
|
27
|
5.15
|
Candida
|
21
|
4.01
|
Rhizopus
|
27
|
5.15
|
Mucor
|
17
|
3.24
|
Botrytis
|
16
|
3.05
|
Aureobasidium
|
21
|
4.01
|
Chalaropsis
|
19
|
3.63
|
Keratinophilic
|
16
|
3.05
|
Exophiala
|
15
|
2.86
|
Cryptococcus
|
15
|
2.86
|
Acremonium
|
17
|
3.24
|
Eurotium
|
23
|
4.39
|
Rhodotorula
|
15
|
2.86
|
Paecilomyces
|
14
|
2.67
|
Phoma
|
14
|
2.67
|
Scedosporium
|
13
|
2.48
|
Sporothrix
|
13
|
2.48
|
Verticillium
|
15
|
2.86
|
Table (2): Characteristics of fungal isolates used for HDPA and PS biodegradation
No
|
Fungi identified
|
Macroscopic and microscopic characteristics
|
1
|
A. carbonarius
|
Un sexual state, basal mycelium white, conidial heads globos to radiate, walled smooth to rough. Vesicles globos to subglobs, aspergilla biseriate
|
2
|
A. glaucus
|
Un sexual state, filamentous fungi, thin walled, conidial heads which radiate to somewhat columnar, mycelium are septate
|
3
|
- westerdijkiae
|
Un sexual state, filamentous fungi, smooth and hyaline.
|
4
|
Eurotium sp.
|
Sexual state, cleistothecia bright yellow fruit body, ascomata globos to subglobs, asci globs to subglobs, ascospore one celled, conidiophores smooth, conidia rough.
|
Table (3): degradation enzymatic for MPs disk of fungal isolates (Aspergillus carbonarius, Emericella, Aspergillus westerdijkiae, Eurotium)
Pectinase
|
Protease
|
Lipase
|
CMCase
|
Laccase
|
Amylase
|
Cellulase
|
Fungi isolate
|
+
|
+
|
+
|
-
|
+
|
+
|
-
|
A. carbonarius
|
-
|
-
|
-
|
+
|
-
|
+
|
-
|
Emericella
|
-
|
-
|
+
|
+
|
-
|
-
|
-
|
A. westerdijkiae
|
-
|
-
|
+
|
+
|
+
|
+
|
+
|
Eurotium
|
Table(4): Comparative Assessment of Microplastic Degradation by Aspergillus carbonarius via Weight Loss Analysis
Aspergillus carbonarius is considered to be the major producer of ochratoxin A. Aspergillus niger occurs in a range of foods
|
MPs
|
Temp.
|
Weight of MPs disk before treatment
(Control)
|
Lose weight after 30 day
|
Lose weight after 40 day
|
Lose weight after
50 day
|
pH
|
PS
|
28°C
|
0.5 gm
|
0.0009
|
0.0014
|
0.0025
|
pH=5
|
|
30°C
|
0.5 gm
|
0.0012
|
0.0018
|
0.0034
| |
|
32°C
|
0.5 gm
|
0.0010
|
0.0014
|
0.0022
| |
|
28°C
|
0.5 gm
|
0.0018
|
0.0024
|
0.0036
|
pH=5.5
|
|
30°C
|
0.5 gm
|
0.0023
|
0.0032
|
0.0041
| |
|
32°C
|
0.5 gm
|
0.0021
|
0.0033
|
0.0038
| |
|
28°C
|
0.5 gm
|
0.0011
|
0.0014
|
0.0019
|
pH=6
|
|
30°C
|
0.5 gm
|
0.0013
|
0.0017
|
0.0022
| |
|
32°C
|
0.5 gm
|
0.0012
|
0.0015
|
0.0019
| |
HDPE
|
28°C
|
0.5 gm
|
0.0156
|
0.0163
|
0.0177
|
pH=5
|
|
30°C
|
0.5 gm
|
0.0166
|
0.0172
|
0.0189
| |
|
32°C
|
0.5 gm
|
0.0157
|
0.0166
|
0.0169
| |
|
28°C
|
0.5 gm
|
0.0182
|
0.0189
|
0.0209
|
pH=5.5
|
|
30°C
|
0.5 gm
|
0.0198
|
0.0232
|
0.0236
| |
|
32°C
|
0.5 gm
|
0.0179
|
0.0199
|
0.0201
| |
|
28°C
|
0.5 gm
|
0.0177
|
0.0183
|
0.0198
|
pH=6
|
|
30°C
|
0.5 gm
|
0.0181
|
0.0193
|
0.0232
| |
|
32°C
|
0.5 gm
|
0.0168
|
0.0176
|
0.0188
| |
Table 5 Comparative Assessment of Microplastic Degradation by Eurotium Fungi via Weight Loss Analysis
Eurotium fungi is a genus of fungi belonging to the family Trichocomaceae
|
MPs
|
Temp.
|
Weight of MPs disk before treatment
(Control)
|
Lose weight after 30 day
|
Lose weight after 40 day
|
Lose weight after
50 day
|
|
PS
|
28°C
|
0.5 gm
|
0.0014
|
0.0023
|
0.0031
|
pH=5
|
|
30°C
|
0.5 gm
|
0.0012
|
0.0015
|
0.0021
|
|
|
32°C
|
0.5 gm
|
0.0011
|
0.0013
|
0.0017
|
|
|
28°C
|
0.5 gm
|
0.0013
|
0.0021
|
0.0027
|
pH=5.5
|
|
30°C
|
0.5 gm
|
0.0011
|
0.0014
|
0.0020
|
|
|
32°C
|
0.5 gm
|
0.0009
|
0.0012
|
0.0016
|
|
|
28°C
|
0.5 gm
|
0.0013
|
0.0019
|
0.0023
|
pH=6
|
|
30°C
|
0.5 gm
|
0.0010
|
0.0014
|
0.0019
|
|
|
32°C
|
0.5 gm
|
0.0008
|
0.0010
|
0.0014
|
|
HDPE
|
28°C
|
0.5 gm
|
0.0144
|
0.0185
|
0.0251
|
pH=5
|
|
30°C
|
0.5 gm
|
0.0138
|
0.0177
|
0.0231
|
|
|
32°C
|
0.5 gm
|
0.0131
|
0.0168
|
0.0216
|
|
|
28°C
|
0.5 gm
|
0.0139
|
0.0175
|
0.0232
|
pH=5.5
|
|
30°C
|
0.5 gm
|
0.0127
|
0.0163
|
0.0220
|
|
|
32°C
|
0.5 gm
|
0.0110
|
0.0146
|
0.0203
|
|
|
28°C
|
0.5 gm
|
0.0128
|
0.0134
|
0.0178
|
pH=6
|
|
30°C
|
0.5 gm
|
0.0111
|
0.0123
|
0.0165
|
|
|
32°C
|
0.5 gm
|
0.0103
|
0.0117
|
0.0154
|
|
3.7 Analyzing LDPE and PS discs Using Scanning Electron
Microscopy
Scanning electron microscopy was used to examine the physical surface topography of the two plastic types (HDPE and PS) at each sampling unit. The plastic samples were subjected to different magnifications (Figs. 11 and 12) to observe the surface morphology and biodegradation. Examination of the control HDPE and PS films revealed smooth and featureless surfaces (Figs. 11 and 12).
High-resolution imaging provided evidence of the physical association of mycelia (hyphae) with the surface of plastic, as demonstrated by others (Cowan et al. 2022). The removal of fungal biomass by HDPE films treated with A. Carbonarius and Eurotium sp. resulted in appreciable surface erosion, folding and pitting in the form of cracks, holes, scions and cavities (Fig. 11b). This observation is consistent with previous studies and fungal colonization (Sen and Raut 2015). After assessment of polyethylene (HDPE) and polystyrene deterioration, weight loss increased.
Figure (11):
Figure (12):
3.8 Fourier Transform Infrared Spectroscopy (FTIR) Analysis
The changes in spectral peaks due to biodegradation were determined using an FTIR spectrophotometer (SHIMADZU – Japan). The degradation of polystyrene and the high density of polyethylene were confirmed by the changes in the functional groups in the FTIR spectra. Untreated discs served as controls, and discs of HDPE and PS were treated with isolated Asp. Fumigatus and Euorotium spp.
Interpreting the FTIR spectrum of high-density polyethylene (HDPE) involves analyzing the characteristic peaks and their corresponding functional groups or molecular vibrations. The following is a general interpretation of the FTIR spectrum of HDPE (Fig. 13a):
1. CH Stretching Bands (~ 2800–3000 cm):
(2970–2990 cm− 1): This region corresponds to the stretching vibrations of C-H bonds in the methylene (CH2) groups in the polymer backbone. The presence of strong and sharp peaks in this region is characteristic of HDPE.
2. CH2 Rocking and Scissoring Bands (~ 1400–1470 cm):
(1463 cm− 1): This peak is associated with the rocking motion of CH2 groups in the polymer chain. It is typically a medium-intensity peak.
3. CH2 Deformation Bands (~ 720–730 cm):
(720 cm− 1): This region corresponds to the deformation vibrations of CH2 groups. This is another characteristic feature of HDPE.
4. Absence of Carbonyl (C = O) Peaks: HDPE is a nonpolar polymer, and therefore, no peaks should be observed in the carbonyl (C = O) stretching region, which is typically between 1700 and 1750 cm− 1. This absence distinguishes HDPE from other polymers, such as polyethylene terephthalate (PET) or polypropylene.
5. Absence of Aromatic Bands: HDPE is also devoid of any peaks in the aromatic region (approximately 1600 − 1500 cm− 1), as it does not contain aromatic rings in its structure.
6. Absence of Hydroxyl (OH) Peaks: You should not see any hydroxyl group (OH) peaks in the FTIR spectrum of HDPE, as it does not contain any hydroxyl groups in its structure.
7. Minor impurity peaks: In some cases, minor impurity peaks may be present in the spectrum, depending on the purity of the sample or any additives used in the HDPE formulation. These should be identified and attributed to their respective functional groups.
It is important to note that the specific wavenumbers and intensities of the peaks may vary slightly depending on the manufacturing process, molecular weight, and any additives in the HDPE sample. Therefore, it is essential to compare the FTIR spectrum of an HDPE sample to a reference spectrum or known standards to confirm its identity and assess any potential impurities or variations (Nishikida and Coates 2003).
HDPE after 30 days:
-
After 30 days of exposure to HDPE, we noticed that there was no noticeable change in the chemical structure of the polymer (Fig. 13b), and the evidence is the presence and persistence of all the vibrational bands with the same strength and intensity that were originally present in the pure polymer.
-
After 40 days of fungal exposure to the HDPE disc (Fig. 13c), we observed many differences in the FTIR spectrum, indicating the breaking of bonds in the old compound and the formation of new bonds, thus changing the chemical composition of the compound, as we noticed a change in the intensity of the stretching vibration present in the parent compound (decrease in concentration).
-
We noticed the disappearance of the band in the area between 1400 and 1470 cm-1, which indicates the breakage of the CH2 group in the polymer chain.
-
We noticed a significant change and decrease in the intensity of the beam located in the confined area (720–730 cm− 1), which is further evidence that the fungus consumes this substance.
The appearance of new bands of strong, moderate, and weak intensity at 824, 1269, 2551, and 3454 cm− 1 indicates the formation of new effective aggregates and a change in the chemical composition of the original polymer (Rohrbach et al., 2023). This interpretation also applies to the HDPE disc after it was exposed for 50 days (Fig. 13d).
Figure (13):
Interpreting the FTIR spectrum of polystyrene involves analyzing the characteristic peaks and their corresponding functional groups or molecular vibrations. Here, a general interpretation of the FTIR spectrum of polystyrene is given (Fig. 14a).
1. Aromatic C-H Stretching Bands (~ 3080 − 3050 cm):
1. Aromatic C-H Stretching Bands (~ 3080 − 3050 cm− 1):
3050–3080 cm− 1: These peaks correspond to the stretching vibrations of aromatic C-H bonds in the phenyl rings of the polystyrene structure. They typically appear as a cluster of sharp peaks.
2. C = C Aromatic Ring Stretching Bands (~ 1590–1620 cm):
2. C = C Aromatic Ring Stretching Bands (~ 1590–1620 cm− 1):
1590–1620 cm− 1: This region is associated with the stretching vibrations of the carbon‒carbon double bonds (C = C) in the aromatic rings of the polystyrene structure.
3. Phenyl ring out-of-plane bending (~ 690–760 cm):
3. Phenyl ring out-of-plane bending (~ 690–760 cm− 1):
690–760 cm− 1: These bands correspond to the out-of-plane bending vibrations of the phenyl rings in polystyrene. They are usually medium to strong in intensity.
4. Phenyl Ring In-Plane Bending (~ 1450–1500 cm):
4. Phenyl Ring In-Plane Bending (~ 1450–1500 cm− 1):
1450–1500 cm− 1: These bands correspond to the in-plane bending vibrations of the phenyl rings in polystyrene. They are typically medium to strong in intensity.
5. Absence of Carbonyl (C = O) Peaks: Polystyrene is a nonpolar polymer and does not contain carbonyl (C = O) groups, so you should not observe any peaks in the carbonyl stretching region, which is typically between 1700–1750 cm− 1.
6. Absence of Hydroxyl (OH) Peaks: Similarly, you should not see any hydroxyl group (OH) peaks in the FTIR spectrum of polystyrene, as it does not contain hydroxyl groups in its structure.
7. Minor Impurity Peaks: Depending on the purity of the sample or any additives used in the polystyrene formulation, minor impurity peaks may be present in the spectrum. These should be identified and attributed to their respective functional groups.
It is important to note that the specific wavenumbers and intensities of the peaks may vary slightly depending on the manufacturing process, molecular weight, and any additives in the polystyrene sample. Therefore, it is essential to compare the FTIR spectrum of a polystyrene sample to a reference spectrum or known standards to confirm its identity and assess any potential impurities or variations (Nishikida and Coates 2003).
PS after 30 and 40 Days
After 30 days, after PS was exposed to the fungus and preserved in a culture medium, we noticed a great similarity between the two spectra, as it was observed that there was no change in the main bands present in PS in the regions between (690–760), (1450–1500), (1590–1620) and (3050–3080) cm− 1 where it was recorded in a disc PS that was exposed to the fungus after 30 and 40 days, which indicates that there was no change in the chemical composition of the substance; therefore, no breaking of bonds or formation of new bonds occurred (Fig. 14b, c).
PS after 50 Days
After 50 days after the fungus was exposed to PS (Fig. 14d), we noticed that the main bands present in the standard PS compound remained but at a lower intensity (lower concentration), with the appearance of some new bands in the (480 and 3444 cm− 1) region. This indicates that the fungus began to break the bonds in the styrene polymer and began to form new bonds (Rohrbach et al. 2023).
Figure (14)