Biodegradation of polyethylene by the marine fungus Parengyodontium album - ScienceDirect

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Highlights

Parengyodontium album was isolated from plastic debris in the North Pacific Subtropical Gyre.

P. album is capable of mineralizating UV-treated polyethylene (PE) into CO2.

Over a time interval of 9 days, mineralization of the UV-treated PE occurs at a rate of 0.044 % /day-1.

Despite the high mineralization rate, incorporation of the PE-derived carbon into fungal biomass is only minor.

Abstract

Plastic pollution in the marine realm is a severe environmental problem. Nevertheless, plastic may also serve as a potential carbon and energy source for microbes, yet the contribution of marine microbes, especially marine fungi to plastic degradation is not well constrained. We isolated the fungus Parengyodontium album from floating plastic debris in the North Pacific Subtropical Gyre and measured fungal-mediated mineralization rates (conversion to CO2) of polyethylene (PE) by applying stable isotope probing assays with 13C-PE over 9 days of incubation. When the PE was pretreated with UV light, the biodegradation rate of the initially added PE was 0.044 %/day. Furthermore, we traced the incorporation of PE-derived 13C carbon into P. album biomass using nanoSIMS and fatty acid analysis. Despite the high mineralization rate of the UV-treated 13C-PE, incorporation of PE-derived 13C into fungal cells was minor, and 13C incorporation was not detectable for the non-treated PE. Together, our results reveal the potential of P. album to degrade PE in the marine environment and to mineralize it to CO2. However, the initial photodegradation of PE is crucial for P. album to metabolize the PE-derived carbon.

Graphical abstract

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Introduction

Conventional plastics are man-made, often petroleum-derived polymers that are highly durable, chemically inert, and designed to resist degradation. Plastic production globally exceeds 400 million tons per year (PlasticsEurope, 2019), and a substantial fraction of the plastic enters the marine environment (Wayman and Niemann, 2021). Both, plastic production and littering are expected to increase in the future, possibly tripling by 2060 (Lebreton and Andrady, 2019). No marine environment has been left untouched; plastic litter has been detected in surface waters (Lebreton et al., 2018; Vaksmaa et al., 2021a), the midwater column (Egger et al., 2020; Vaksmaa et al., 2022; Pabortsava and Lampitt, 2020), sediments (Kane et al., 2020) and beaches (Piperagkas et al., 2019) from arctic latitudes (Peeken et al., 2018; Obbard et al., 2014) to the tropics (Santos et al., 2009; Ivar do Sul et al., 2014). Floating plastic apparently accumulates in semi-enclosed water bodies (Cozar et al., 2015) and subtropical gyres (Cozar et al., 2014; Eriksen et al., 2014). Particularly high plastic concentrations are found in the North Pacific Subtropical Gyre (also referred to as the North Pacific Garbage Patch), which is estimated to contain more than80,000 tons of floating plastic debris (Lebreton et al., 2018).

Polyethylene (PE) is the most abundantly produced plastic type (Geyer et al., 2017) and has been identified as the dominant floating polymer type in the ocean, too (Enders et al., 2015; Erni-Cassola et al., 2019; Adamopoulou et al., 2021). Other important plastic types afloat in the sea are polypropylene (PP) and to a lesser degree polystyrene (PS) and polyethylene terephthalate (PET). It has been estimated that 1.5 to 4.1 % of the globally produced plastic enters the ocean; hence, 57–160 million metric tons (Mt) of plastic should be afloat at the ocean surface (Wayman and Niemann, 2021). However, most measured and modeled amounts of floating marine plastic debris only account for a small fraction of the expected amounts (Eriksen et al., 2014; van Sebille et al., 2015; Kaandorp et al., 2023). This phenomenon has been coined as the ‘missing plastic paradox’ (Thompson et al., 2004).

The ‘missing plastic paradox’ is partially caused by the fact that plastic input scenarios into the ocean are modeled (Eriksen et al., 2014; Kaandorp et al., 2023; Lebreton et al., 2019) which is associated with uncertainties. Furthermore, plastic redeposition at the shore (Lebreton et al., 2019; Onink et al., 2021) and export of plastic from the ocean surface to the midwater column and deep-sea, and its eventual burial into sediments (Egger et al., 2020; Pabortsava and Lampitt, 2020; Kane et al., 2020; Peng et al., 2020), remove considerable amounts of plastic from the ocean surface and water column. Additionally, plastic fragmentation leads to the formation of small, micro and nanoscale particles, which are not routinely measured and thus not accounted for in global budgets (Koelmans et al., 2015; Ter Halle et al., 2017; Andrady, 2011; Materić et al., 2022). Both physicochemical and biological processes have been shown to break down plastics. UV-induced photooxidation is relevant in breaking down plastics and affecting the biodegradation of plastics by microbes (Delre et al., 2023; Gewert et al., 2015; Goudriaan et al., 2023). Although the role of microbes in the degradation of plastics has been demonstrated in a number of recent studies (Zeghal et al., 2021; Vaksmaa et al., 2021b; Oliveira et al., 2020), biodegradation rates and the key microbes involved are still not well constrained, particularly for the marine environment.

Marine microbes potentially involved in plastic degradation are often studied by investigating the microbial colonization and biofilm community composition. The intent is to reveal potential plastic degraders by comparing microbial communities on different types of polymers and investigating taxa with high relative abundance or potential to degrade complex hydrocarbons (Vaksmaa et al., 2021a; Oberbeckmann et al., 2018; Latva et al., 2022; Dudek et al., 2020a; Miao et al., 2019). A second approach focuses on isolation of plastic degraders to measure their plastic degradation abilities. However, this approach often focuses on bacteria but not on eukaryotes (Joshi et al., 2022; Sekiguchi et al., 2011; Nag et al., 2021). Plastic degradation kinetics are often determined based on weight loss of the polymer. Additionally, chemical changes in the polymer structure and changes in surface morphology are measured by Fourier-transform infrared spectroscopy and by scanning electron- or atomic force microscopy, respectively. However, these approaches are rather unsensitive, semi-quantitative and they do not distinguish between physicochemical and biological degradation. Respiratory measurements applied to measure CO2 formation and O2 consumption are quantitative and allow determining biological activity; however they cannot be solely linked to plastic degradation (Zeghal et al., 2021; Vaksmaa et al., 2021b). Using assays with isotopically labeled polymers allows sensitive tracing isotopically labeled matter from the polymer into degradation products and microbial biomass (Goudriaan et al., 2023; Vaksmaa et al., 2023a).

Several studies demonstrated that biofilms on plastic marine debris (PMD) do not only contain bacteria but also eukaryotes, including fungi (Zeghal et al., 2021; Zettler et al., 2013; Muthukrishnan et al., 2019; Marsay et al., 2022). Fungi on different plastic surfaces were described from a variety of marine habitats, for example on PE incubated on the seafloor in the North Sea (De Tender et al., 2017). Further findings were reported for PE and PS in the Baltic Sea (Kettner et al., 2017) as well as for PE, PP, PS and polyurethane (PUR) in surface waters of the Western South Atlantic and the Antarctic Peninsula (Lacerda et al., 2020).

Marine fungi, in contrast to terrestrial fungi, are less investigated; their role and metabolic potential in the ocean are thus not well resolved (Zeghal et al., 2021; Richards et al., 2012; Vaksmaa et al., 2023b). However, fungi in general have the ability to degrade vast amounts and variety of compounds including complex hydrocarbons such as lignin and cellulose (Goodell et al., 2020; Bourbonnais et al., 1995; Coughlan et al., 1990), pollutants such as Dichloro-Diphenyl-Trichloroethane (DDT), polycyclic aromatic hydrocarbons, or even Trinitrotoluene (TNT), among other environmentally hazardous compounds (Harms et al., 2011; Anasonye et al., 2015; Haritash and Kaushik, 2009; Andersson and Henrysson, 1996; Mitra et al., 2001). Fungi can be considered as the ‘masters of degradation’ because they utilize a plethora of digestive enzymes, making them potential candidates for plastic degradation, too (Zeghal et al., 2021; Vaksmaa et al., 2021b; Vaksmaa et al., 2023b). Indeed, several terrestrial fungi belonging to the genera of Penicillium, Trichoderma, Aspergillus and Paecilomyces have been shown to degrade plastic polymers (Sowmya et al., 2015; Sowmya et al., 2014; Yamada-Onodera et al., 2001; Zhang et al., 2020; Sheik et al., 2015). In contrast, in the marine environment, only a few fungal species have been identified to degrade PE, including Zalerion maritimum (Paço et al., 2017), Alternaria alternata FB1 (Gao et al., 2022) and Rhodotorula mucilaginosa (Vaksmaa et al., 2023a), while Cladosporium halotolerans 6UPA1 was shown to degrade PUR (Zhang et al., 2022).

In our previous study (Vaksmaa et al., 2023a) on the marine yeast Rhodotorula mucilaginosa, we applied a stable isotope probing approach by using 13C-labeled PE and tracing the plastic-derived 13C into the mineralization end product CO2 and into biomass. A similar approach for measuring plastic degradation was previously used for tracing 13C-label from substrate PE into CO2 and biomass of bacteria (Goudriaan et al., 2023), into the lacustrine food chain (Taipale et al., 2019) or from 13C-labeled bioplastic into soil microbes and CO2 (Zumstein et al., 2018).

We isolated the fungus Parengyodontium album from biofilms covering floating plastic fragments, collected during a scientific expedition to the North Pacific Subtropical Gyre. Our main aim for this study was to investigate the capability of this fungus to degrade PE. For this, we incubated P. album with non-treated and UV-treated 13C-labeled PE and traced the PE-derived carbon from the polymer source to the terminal oxidation product CO2, which allowed us to calculate fungal-mediated PE mineralization rates. Furthermore, we used nanometer-scale secondary ion mass spectrometry (nanoSIMS) and analyzed the fatty acids stable carbon isotopic composition to evaluate the assimilation of the PE-derived 13C into P. album biomass.

Section snippets

Sampling and isolation of P. album

PMD was sampled from the North Pacific Garbage Patch, located in the North Pacific Subtropical Gyre, with a Manta trawl operated from M/S Maersk Transporter during The Ocean Cleanup's North Pacific Mission 3 in December 2019 (Egger et al., 2021). PMD items retrieved from the Manta trawls were placed in seawater and kept at room temperature until further treatment in our onshore laboratories. Using sterile tweezers, single PMD items were then stamped onto solid Murashige and Skoog (MS) media

Identification of the fungal isolate and microscopical investigations

The identification of the fungal species by sanger sequencing of the ITS region and blasting against NCBI database revealed that the fungus was P. album (see supplementary material). This fungus belongs to the Ascomycota phylum, Cordycipitaceae family, and was renamed from Tritirachium album (Limber, 1940) to Engyodontium album (deHoog, 1978) and more recently to Parengyodontium album (Tsang et al., 2016), described in detail (Leplat et al., 2020).

Epifluorescence microscopy was carried out on

Discussion

Some plastic degrading microbes have been discovered over the last years (Goudriaan et al., 2023; Tanasupawat et al., 2016; Verma and Gupta, 2019), so that it is very likely that microbial plastic degradation can remove at least some plastic from the marine realm (Wayman and Niemann, 2021; Jacquin et al., 2019). However, it is poorly constrained to which extent microbial plastic degradation occurs in the marine environment and how important that process is. The role of fungi, which have been

Outlook: marine fungi and plastic degradation

Fungi represent a ubiquitous group of organisms in terrestrial habitats, lacustrine systems, and marine environments (Tian et al., 2018; Pietryczuk et al., 2018; Bahram et al., 2018; Rojas-Jimenez et al., 2020), although research on marine fungi is still in its infancy (Zeghal et al., 2021; Vaksmaa et al., 2021b). Fungi exhibit the remarkable ability to adapt to diverse and challenging environmental conditions and have been found to occupy a diverse array of ecological niches within the ocean,

Funding

This work has been supported by the European Research Council (ERC-CoG grant no. 772923, project VORTEX) and the Dutch Research Council (grants no. OCENW.XS23.1.071, VI.Veni.212.029, OCENW.XS21.4.079). The NanoSIMS facility at Utrecht University was financed through a large infrastructure grant by the Dutch Research Council (grant no. 175.010.2009.011).

CRediT authorship contribution statement

A. Vaksmaa: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Funding acquisition, Conceptualization. H. Vielfaure: Writing – review & editing, Visualization, Methodology, Investigation. L. Polerecky: Writing – review & editing, Visualization, Methodology, Investigation. M.V.M. Kienhuis: Writing – review & editing, Visualization, Methodology, Investigation. M.T.J. van der Meer: Writing – review & editing, Visualization, Methodology, Investigation.

Declaration of competing interest

ME is employed by The Ocean Cleanup, a non-profit organization aimed at advancing scientific understanding and developing solutions to rid the oceans of plastic. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.