Impeding Effect of Polystyrene Microplastic Pollutants on Hg2+ Uptake Potential of Aspergillus flavus

Imania Ghaffar1, Ali Hussain2*, Arshad Javid1 and Shahid Mehmood1

1Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Outfall Road, Lahore, Pakistan

2Institute of Zoology, University of the Punjab, Quaid-i-Azam Campus, Lahore, Pakistan

3Department of Poultry Production, University of Veterinary and Animal Sciences, Outfall Road, Lahore, Pakistan

ABSTRACT

Microplastic pollution has aroused up to intimidating level around the globe and has become a focal point for researchers. However, data regarding the effects of microplastics on structure and function of filamentous fungal species is very scarce. Fungi have made a prominent mark in the area of heavy metals’ bioremediation. This study attempts to check the influence of microplastic pollutants on Hg2+ uptake potential of metal-resistant Aspergillus flavus at laboratory scale under pre-optimized conditions. A. flavus showed a remarkable potential of remediating simulated wastewater, i.e., 100% Hg2+ reduction was achieved at 25 mg/L of the added metal in 15 days of incubation. On higher concentrations like 75 and 100 mg/L, A. flavus showed almost negligible reduction of Hg2+ but this strain was able to tolerate Hg2+ up to 200 mg/L. Polystyrene microbeads at a concentration of 100 mg/L reduced the metal uptake potential of A. flavus up to 21%. Polystyrene microparticles might have formed aggregates on fungal mycelia blocking the attachment sites for heavy metals. Our findings will be helpful in designing an efficient bioremedial system mediated by the pollution-resistant microflora. More research is required to check the possible effects of microplastic pollutants on the fungal mycelia to exploit their maximum potential.

Article Information

Received 30 January 2023

Revised 18 March 2023

Accepted 10 April 2023

Available online 13 May 2023

(early access)

Published 08 June 2024

Authors’ Contribution

IG performed experiments and prepared the first draft of the article. AH supervised the work and finalized the article. AJ helped in data analysis.

SM assisted in data compilation.

Key words

Bioremediation system, Biosorption, Fungal remediation, Heavy metals, Microplastic pollutants

DOI: https://dx.doi.org/10.17582/journal.pjz/20230130070108

* Corresponding author: [email protected]

0030-9923/2024/0004-1725 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Rapid industrialization and development result in several environmental concerns. A variety of pollutants are damaging environment but heavy metal pollution is a serious distress due to its persistence and non-biodegradability (Ayele et al., 2021). Major industries that contribute in heavy metal pollution include metal-manufacturing plants, smelting and mining sites, tanneries and coating and painting industries. Other than anthropogenic activities some natural sources like volcanic eruptions, geysers, deep-sea vents, and forest fires also result in metal contamination of the environment (Tchounwou et al., 2012; Ghaffar et al., 2023). In addition to environmental pollution heavy metals are also a threat to human beings and all other living organisms. Some metals have a potential of chronic toxicity even at lower concentrations (Ayele et al., 2021; Ghaffar et al., 2023). To deal with heavy metal pollution bioremediation has highly been recommended by the researchers because of its sustainable and cost-effective nature (Ghaffar et al., 2023). Currently, biosorption nature of fungus has been explored to some extent (Ayele et al., 2021). Reports have suggested that fungi have successfully been exploited for the remediation of several heavy metals including Ni, Cr, Pb, Hg, Zn, and Cd (Chaurasia et al., 2023; Sharma et al., 2023).

Three groups (mushrooms, molds and yeast) of this eukaryotic microorganism are considered important in various applications (Mohmand et al., 2011; Carris et al., 2012). Fungal cell wall is of prime importance in bioremediation it has been revealed that its structure has high metal-binding properties than other biosorption agents. Fungal biomass shows extensive tolerance towards high metal concentrations and low pH (Ghaed et al., 2013). Dead and alive both types of fungal biomass exhibit sorption qualities (Ayele et al., 2021). Biosorption by live fungal biomass is an active process, in this process external and internal metabolism like volatilization, detoxification, bioaccumulation and chelation occur while inactivated (dead) biomass displays passive adsorption i.e., only cell surface binding occurs (Javanbakht et al., 2014; Cai et al., 2016). For the removal of metals from a liquid medium filamentous fungal species are more effective. The commonly used fungal species for the treatment of heavy metals include yeast (Saccharomyces, Penicillium), mushrooms and molds (Rhizopus, Aspergillus) (Pansuphaphol et al., 2016; Alothman et al., 2020; Gajewska et al., 2022).

Microplastics are omnipresent, i.e., they are literally found in every ecosystem (i.e., water, air, soil) and all living forms. Reports have suggested that synthetic microfibers are also present in air other than water and beaches (Sutton et al., 2016; Horn et al., 2019). Taking in to account the shape of microplastics they can be found in the form of fibers, pellets, foams, fragments, granules or films. Fibers originate from synthetic clothes; pellets are small spheres manufactured to prepare bigger plastic items and foam is coming in the environment from single-use styrofoam containers. Periodic break down of plastic made bottles and other objects made from plastic result in the formation of plastic fragments in the environment. Granules intrude in environment from the products used in personal care products e.g., toothpaste whereas plastic bags or food packaging materials are the main sources of plastic films. Pellets and granules are similar in shape (i.e., round), but pellets are much larger than granules (Lujan-Vega et al., 2021; Ghaffar et al., 2022a). Microplastics can handicap biosorption potential and growth of microbes by inducing structural and functional changes (Ghaffar et al., 2022a, b). Keeping in view the omnipresence and hazardous nature of microplastics, it is obvious that they can handicap the in-situ bioremediation. As fungi are one of the most potent microbes used for the removal of heavy metals so the present study is conducted to assess the impeding effect of microplastics on Hg2+ uptake potential of a metal-resistant fungal strain.

MATERIALS AND METHODS

Sample collection

To isolate a metal-resistant fungal strain sample was collected from a highly polluted drain known as Hudiara drain located in Lahore, Pakistan. This drain receives several types of anthropogenic and industrial discharge, providing an optimum environment to the pollution resistant microbes. Sampling of wastewater was performed using precise protocols of sterility and hygiene. While sampling some parameters of wastewater such as pH, humidity and temperature were measured as 7.4, 70% and 32 °C, respectively. The sample obtained from the wastewater drain was then carefully taken to PG (post-graduate) Laboratory, Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Lahore, Pakistan to perform analysis and further examination.

Isolation of pure culture of metal-resistant fungal strain

The collected sample was spread over MEA (malt extract agar) plates amended with various concentrations (up to 200 mg/L) of metal (Hg) and incubated in a dark incubator (OMEGA 1-52) for 3-5 days at 30 °C. Some fungal species appeared on plates after 5 days of incubation out of which the most resistant one was picked and pure cultured. Pure culturing was performed by inoculating MEA containing sterile petri plates with fungal spores in the center and incubating for 5 days in a dark incubator (OMEGA 1-52), the procedure was repeated if required. The strain was further proceeded for identification and experimentation.

Morphological identification of the fungal isolate

For morphological identification, color and texture of the fungal colony was checked as macroscopic features. For microscopic features fungal mycelia were stained with Lactophenol cotton blue and studied under microscope. Diba et al. (2007) was followed to identify the morphology of the isolated fungal strain.

Molecular level identification of the fungal isolate

Fungal strain was identified at molecular level by 18S rRNA gene sequencing. Dneasy® plant mini kit (Qiagen, Hilden, Germany) was used to isolate DNA of the freshly cultured fungal mycelia. Amplification of 18S rRNA was performed by using primer pairs for amplifying regions ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) (Zhang et al., 2010). In a in a thermal cycler (Hamburg 22331, Germany) the initial denaturation was conducted at 95°C for 5 min followed by 35 cycles of denaturation (94°C for 30 s), annealing (50°C for 30 s), extension of primer (72°C for 2 min) and the final extension (72°C for 7 min). Electrophoresis was then performed by using stained (C21H20BrN3) agarose gel to separate the PCR product. The PCR product was then purified with the help of Gene Purification Kit (Hunan Runmei Gene, CE, ISO 13485, China) and the amplicon obtained was then got sequenced and compared with the most alike sequences by using NCBI blast tool.

Optimization of growth parameters

Fungal strain was allowed to grow in different conditions to determine the optimum level of different parameters. For this purpose, malt extract agar (MEA) was used and following parameters were optimized as:

Temperature

Fungal mycelia were incubated at 20 °C, 30 °C and 40 °C on petri plates in a dark incubator (OMEGA 1-52) and growth was observed. Maximum growth was observed at 30 °C. Temperature was further narrowed down at 28 °C, 29 °C, 31 °C, and 32 °C to achieve the accuracy. At every temperature colonies were counted on regular basis up to 5 days.

pH

pH for the fungal mycelia was set at 5, 7 and 9 and petri plated inoculated with fungal spores were incubated for 5 days in a dark incubator (OMEGA 1-52) and it was detected that number of colonies was the highest at pH 5. Then the growth was further checked at pH 4.8, 4.9, 5.1 and 5.2 by counting the number of colonies for 5 days.

Incubation period

Sterile petri plates containing growth medium of fungi along with fungal spores were incubated at 30 °C in a dark incubator (OMEGA 1-52) and the number of colonies was observed after every day for consecutive 5 days.

Experimental design

The fungal-microplastic interaction was checked on the basis of the concentration of Hg adsorbed by Aspergillus flavus. Experimentation was carried out in three groups to check the impact of polystyrene microbeads on the efficiency of A. flavus to adsorb Hg2+. Microplastics (0.5µm) used for this purpose were purchased from Poly Sciences Europe GmbH (Eppelheim, Germany). The 1st group deals with the interaction of fungi with microplastics (i.e., experimental flasks with inclusion of microbeads 100 mg/L). While the 2nd group only contained microplastics (100 mg/L) and heavy metal to check the microplastic-metal interaction and the 3rd group was kept as control i.e., without inclusion of microbeads. All experimentation was conducted in triplicates. In all three sets Hg concentrations of 25, 50, 75 and 100 mg/L were added. Tolerance of the fungal isolate was pre-assessed and it was revealed that this strain was able to withstand Hg up to 200 mg/L.

The fungal spore suspension was prepared by adding a loop full of freshly cultured fungal mycelia and sporangia into a 250mL Erlenmeyer flask containing sterile Basal medium. The flask was placed in dark incubator for 3-5 days in a dark incubator (OMEGA 1-52). 10% (v/v) of fungal spore suspension was used as inoculum for group 1 and group 3 harboring about 109 mg/L spores. While group 2 remained un-inoculated. To prepare concentrations of Hg, a compound of HgSO4 was used. Basal medium ((gL-1) NaCl, 0.01; K2HPO4, 1.5; MgCl2.7H2O, 0.05) was used to prepare all the mentioned concentrations of the mercury (II) and microplastics and pH was maintained at 4.8.

The fungal mycelia were permitted to uptake Hg2+ from artificially prepared wastewaters with the inclusion and/or exclusion of polystyrene microbeads in a 15 days trial. All experimental and control flasks were placed in dark incubator at 29 °C. Our results only showed the final concentration of Hg2+ biosorbed by A. flavus with the inclusion and/or exclusion of polystyrene microparticles. The final concentration of Hg2+ was achieved by subtracting the Hg2+ concentration absorbed in group 2 from group 1.

Data analysis

10 mL of sample was removed periodically (i.e., after every 5 days) from all the experimental and control flasks, and filtered with a Whatman cellulose filter paper. Filtrates were analyzed by using Atomic Absorption Spectrophotometer (CE-2041, UK) to check the varying concentration of Hg.

Statistics of the analyzed data

Statistical analysis of Hg concentrations adsorbed by fungal hyphae in all groups were carried out by using R software. Means were considered significant at p-value<0.05. Differences between Hg concentration of both control and experimental groups were compared by using T-test. Results are graphically expressed with the help of origin software 6.0.

RESULTS AND DISCUSSION

The morphological characteristics have revealed that the isolated strain showed resemblance with A. flavus. The colony color of the isolated strain was yellowish-green with powdery texture. Microscopic study has revealed that its hyphae were non-septate, shape of vesicle was globose, and phialides were biseriate loosely present all over the vesicle, and philiades were radiating from metulae. Our findings are in accordance with Afzal et al. (2013) and Okayo et al. (2020). So, the morphological characterization and BLAST search of 18S rDNA nucleotide sequence revealed that the metal-resistant fungal isolate belonged to the genus Aspergillus and species was identified as Aspergillus flavus.

Optimized parameters of the fungal isolate

Growth of A. flavus was effected by varying the parameters like temperature, pH and incubation period. To obtain the optimum pH A. flavus was incubated with different pH values and optimum growth of A. flavus was observed at pH 4.8 and optimum temperature was 29 oC among 28 oC, 29 oC, 31 oC and 32 oC. Our results are related to Kote et al. (2009) according to their report A. flavus showed optimum growth and activity at pH 5 and Samapundo et al. (2007) suggested that temperature range for A. flavus is 25 to 30 oC. Casquete et al. (2017) isolated different strains of A. flavus which showed maximum growth in range of pH from 5 to 5.5 and temperature from 25 to 30 oC. Gunasekaran (1981) reported that A. flavus showed optimum mycelial growth at pH 4.5 results of this study are almost similar to our findings while optimum temperature reported in this study was 37 oC. Gallo et al. (2016) reported that maximum expression of regulatory genes by A. flavus was at 28 oC while minimum was at 37 oC. The isolated fungal strain showed maximum number of colonies on 5th day of incubation period at optimized temperature and pH.

Effect of metal-concentrations on metal uptake potential of fungal isolate

A. flavus has a tremendous potential for the biosorption of heavy metals (Anupong et al., 2022). Present study showcases the capability of a metal-resistant strain A. flavus to remediate artificially prepared mercury polluted wastewater and results have revealed that the biosorption potential of the isolated strain reduces with an increase in the concentration of mercury (II) i.e., Hg+2 uptake potential was maximum i.e., 100% at 25 mg/L on 15th day of incubation period (Fig. 1A). At higher concentrations (75 and 100 mg/L) reduction of Hg+2 was almost negligible (Fig. 1C, D). The isolated fungal strain was able to withstand Hg (II) up to 200 mg/L but only lower concentrations were degraded efficiently. The overall metal-uptake sequence of Aspergillus flavus was as follows: 25 > 50 > 75 > 100 mg/L (Figs. 1-2). A. flavus showing 100% and 96% reduction at 25 and 50 mg/L (Fig. 1A, B), respectively of a toxic metal like mercury (II) is remarkable. Kurniati et al. (2014) reported 98.73% reduction of Hg+2 by A. flavus at 10 mg/L which is lower than our findings. Several reports have shown that A. flavus has an amazing potential to remediate a large number of heavy metals. A. flavus is able to tolerate metals like Cr and Cu up to 1000 mg/L (Dusengemungu et al., 2020; Palanivel et al., 2023). Live and dead biomass of filamentous fungi have the ability to remove mercury through various mechanisms like reduction, biosorption and bioaccumulation (Arıca et al., 2003). Villalba-Villalba et al. (2022) reported that A. flavus has high tolerance against higher concentration of Cu, Zn, and Pb and was able to withstand only lower concentrations of Hg, Cd, and Ag. Mart´ınez-Ju´arez et al. (2012) reported the removal of mercury from aqueous solution by 14 different fungal species and Mucor spp. showed the maximum reduction i.e., up to 95% at 100 mg/L and Aspergillus flavus showed comparatively lower Hg removal efficiency among all. Acosta-Rodríguez et al. (2018) reported that Aspergillus niger removed 83.2% of mercury at 100 mg/L and was able to tolerate mercury up to 2000 mg/L which shows A. niger has remarkably higher remedial potential for mercury (II) than the fungal species isolated in present study.

 

 

Effect of microplastic bead size on remedial potential of fungi

The smaller polystyrene microparticles (0.5µm) influenced the remedial potential of A. flavus, i.e., in experimental sets slightly lesser Hg+2 uptake was observed (Fig. 2) as compared the control group (without inclusion of microplastics) which depicts that small sized microplastics have regressive effects on fungal mycelia because they can block the attachment sites for metals and cause impairment of fungal cell wall. Studies about effects of microplastics on structure and bioremedial potential of filamentous fungi are scarce. Fan et al. (2022) reported that fungal communities are more sensitive towards microplastic pollutants than bacterial communities and microplastics declined the abundance of different fungal communities. Some studies report the effects of different size and charge of microplastics on microbes (Ghaffar et al., 2022a). Microplastics significantly reduce the growth and biosorption potential of microalgae at various concentrations (Sjollema et al., 2016; Ghaffar et al., 2022b) which is in accordance to our findings. Concentration of microplastics is directly proportional to the severity of damage (Besseling et al., 2014; Sjollema et al., 2016). In the present study, 100 mg/L of PS microbeads reduced biosorption potential of Aspergillus flavus up to 21% which is not very remarkable but statistically significant while Ghaffar et al. (2022b) reported a remarkable decrease in the metal uptake potential of microalgae at the same concentration of microbeads. Lagarde et al. (2016) showed a decreased microbial growth at 400 mg/L of microbeads. At higher concentration of metals fungal mycelia exposed with PS microbeads showed 0% removal of Hg+2 (Fig. 1D). Microplastics may also form aggregate around the fungal hyphae, ultimately repressing their biosrption potential, as aggregation of microplastics has been reported by Lagarde et al. (2016) and Ghaffar et al. (2022b).

Effect of incubation period

After 5 days of incubation Aspergillus flavus showed a remarkable removal of Hg+2 i.e., 92% and on 15th day the metal-reduction potential of Aspergillus flavus was at its fullest. While the maximum removal of heavy metals by Aspergillus sp. was reported on the 7th day of incubation by Acosta-Rodríguez et al. (2018) and Kumar and Dwivedi (2020) reported the maximum reduction of heavy metals by A. flavus on 8th day of incubation. Sharma et al. (2022) reported 70-84% Hg removal by white rot fungi in 7 days of incubation. Ozsoy (2010) reported maximum biosorption of Hg by Rhizopus oligosporus after 6 hours of incubation and Dusengemungu et al. (2020) reported that maximum biosorption of heavy metals by filamentous fungi was observed after 24 h of incubation which indicates that lag phase of our isolate is longer than the other fungal species but A. flavus was able to easily acclimatized in growth medium and showed efficient metal reduction as compared to some bacterial and microalgal species (Hussain and Qazi, 2016; Muneeb et al., 2020; Ghaffar et al., 2022b). The log phase i.e., 10-15 days showed maximum reduction and then fungal mycelia entered in stationary phase and formed spores possibly due to the scarcity of nutrients and abundance of metabolites.

Conclusions

A. flavus has an efficient remedial potential for mercury which is toxic even in minute concentrations. To achieve the maximum metal reduction determination of the optimum conditions is important. Other than optimum conditions several other pollutants can also affect the remedial efficacy of the fungal species in in-situ wastewater treatment plants. So, in this study effect of microplastic pollutants on remedial potential of A. flavus was studies at laboratory scale and it was concluded that microplastics can handicap the heavy metal removal efficiency of the fungal mycelia by blocking the attachment sites available for mercury (II). Almost no data is available regarding the effects of microplastic pollutants on filamentous fungi and their capability to remove heavy metals. Investigations are needed to determine the extent up to which microplastics cause functional stress in filamentous fungi because in present age plastics are everywhere and damaging almost all living entities.

Acknowledgement

The support of Department of Biological Sciences, University of Veterinary and Animal Sciences, Ravi Campus, Pattoki is highly acknowledged for helping in fungal identification.

Funding

External funding was not received for this study.

IRB approval

The current research work was approved by Board of Studies of Department of Wildlife and Ecology, University of Vet-erinary and Animal Sciences, Lahore.

Ethical statement

The current research work didn’t involve any animal model and thus ethical statement was not needed.

Statement of conflict of interest

The authors have declared no conflict of interest.

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