Biodegradation of polyethylene mulching films by a co-culture of Acinetobacter sp. strain NyZ450 and Bacillus sp. strain NyZ451 isolated from Tenebrio molitor larvae

https://doi.org/10.1016/j.ibiod.2020.105089Get rights and content

Highlights

  • A consortium of two bacteria from the Tenebrio molitor larvae gut degrades PE.

  • The co-culture reduced 18% mass of PE mulching films in 30 days.

  • The two isolates form a tight biofilm on the PE mulching film surface.

  • PE undergoes oxidation after being treated with two strains.

  • Acinetobacter sp. strain NyZ450 is predominant in the consortium.

Abstract

Microbial degradation of polyethylene (PE) mulching films has attracted much attention, but to date its relatively rapid biodegradation was limited. Here, a consortium of two bacterial isolates, Acinetobacter sp. strain NyZ450 and Bacillus sp. strain NyZ451 from the gut of PE-feeding Tenebrio molitor larvae (mealworms), was suggested to utilize PE, although each strain had no such ability alone. This consortium removed approximately 18% of PE film after 30 days at 23 °C. High-temperature gel permeation chromatography analysis indicated that the number-, weight-, and size-averaged molecular weights of PE were decreased by 14%, 24% and 21% from their respective initial values of 27,494 Da, 179,374 Da and 697,487 Da after a 20-day incubation with this consortium. Under scanning electron microscope, a biofilm formed by these strains was also observed on the film's surface. Fourier transform infrared spectroscopy analysis indicated that the films incubated with these two strains or either of them were all oxidized compared with the control without inoculation. Plate colony counting and electron microscopic observation showed that strain NyZ450 was predominant in the co-culture. This study enriched microbial resources for PE degradation and provided an example for the interaction of two bacterial strains for enhanced PE degradation.

Introduction

Hundreds of millions of tons plastics were produced every year (PlasticsEurope, 2018). Because of the nature of high molecular weight, highly stable covalent bond and highly hydrophobic, petroleum-based plastics especially “C–C” inert structural backbone plastics are recalcitrant to be broken down in the environment (Peixoto et al., 2017; Wei and Zimmermann, 2017). More than eight billion tons of plastics exist on the Earth and more than half of them were discarded in the environment (Geyer et al., 2017). Polyethylene (PE, (CH2–CH2) n) is a typical representative of recalcitrant “C–C” backbone plastics. Based on the differences in the density, degree of branching and availability of functional groups on the surface, the most common PE types are low-density polyethylene (LDPE), high-density polyethylene (HDPE), linear low-density polyethylene (LLDPE), low molecular weight polyethylene (LMWPE) and cross-linked polyethylene (XLPE). It is well known that PE is one of the most widely used petroleum-based plastics all over the world. PE-based mulching films which were composed of LDPE and LLDPE have been heavily used in agriculture (Hochmuth et al., 2015; Lamont, 2017). But dumped mulching films were very difficult to be recycled, resulting in severe crop failures and environmental problems (Kasirajan and Ngouajio, 2012). One of the promising ways to make the PE degradable and recyclable may be through microbial versatile activities, which have been proved to be one of productive methods to get rid of xenobiotics in the past (Raddadi and Fava, 2019).

A previous report indicated that only 0.2%–0.5% PE film was removed after 10 years in landfills by 14C labeling and tracking the release of 14CO2 (Albertsson and Karlsson, 1988). The degradability of PE basically depends on its molecular weight, crystallinity and pretreatments. The molecular weight of PE polymers is measured using the number-, weight-, and size-averaged molecular weights (Mn, Mw and Mz). Generally, the higher the molecular weight and crystallinity of PE, the harder it is to be biodegraded. Different from HDPE, LDPE and LLDPE exhibit a lower crystallinity because of having more branch structures, making them less recalcitrant to biodegradation (Fontanella et al., 2010). On the other hand, the pretreatment is also an effective way to accelerate PE degradation. For example, the addition of photosensitizers in PE accelerated its disintegration in the environment (Albertsson and Karlsson, 1988). The degradation of PE can also be accelerated to some extent by the pretreatments with UV light, heat and mechanical shear (Albertsson and Karlsson, 1990).

In previous studies, some insects such as yellow mealworms (Tenebrio molitor larvae) (Brandon et al., 2018; Yang et al., 2015a, b; Yang et al., 2020), superworms (Zophobas atratus larvae) (Peng et al., 2020), Indian meal moths (Plodia interpunctella) (Yang et al., 2014) and greater wax moths (Galleria mellonella) (Bombelli et al., 2017), and soil animal such as land snails (Achatina fulica) (Song et al., 2020) were reported for their special nature of eating plastics including polystyrene and polyethylene, and their gut microbes were demonstrated to be essential for plastics digestion. So far, surprisingly large numbers of bacterial genera from the soil (Peixoto et al., 2017) or the gut of insect (Yang et al., 2014) have been reported as possible PE degraders. Nevertheless, the PE catabolic abilities from reported PE-degrading bacterial isolates were all extremely weak. Discarded PE mulch films are a major source of plastics pollution in soil, but reports on the relatively rapid biodegradation of PE mulching films are still limited. For instance, Bacillus aryabhattai 5–3 only removed 3.85% of PE mulching films after a 30-day incubation (Hou et al., 2019). Pseudomonas sp. AKS2, a soil bacterial isolate, was able to remove 5% 20-μm thick LDPE carrier bags from the market after a 45-day incubation (Tribedi and Sil, 2013). As to the gut bacteria, Enterobacter asburiae YT1 and Bacillus sp. YP1 could individually remove approximately 6.1% and 10.7% of LDPE films (Mw = 88,200 Da) after 60 days (Yang et al., 2014), Rhodococcus ruber C208 removed 4% films of branched LDPE (Mw = 191,000 Da) containing UV photosensitizer pretreated with UV irradiation after 30 days (Orr et al., 2004), and Pseudomonas sp. E4 removed 4.9–28.6% LMWPE films (Mw = 1700–23,700 Da) after 80 days (Yoon et al., 2012). However, there are limited reports on the relatively rapid biodegradation of agricultural PE-based mulching film by microbes. The reports on biodegradation mechanism of C–C backbone plastics such as PE, especially those on the related genes and enzymes, are extremely scarce. It was reported that some lignin degrading enzymes such as laccase were active on PE plastics with low activities (Santo et al., 2013). Another example is that an alkane hydroxylase from Pseudomonas sp. E4 expressed in E. coli BL21 showed activity for low molecular weight polyethylene in compost (Yoon et al., 2012).

In addition, the occurrence of microbial interaction cannot be neglected in the natural environment or the gut of insect (Sakazawa et al., 1981; Scherlach and Hertweck, 2018). Considering that the plastic nature of high molecular weight, high hydrophobicity and inert covalent bond, the limited catabolic ability of reported single degraders has restricted their PE degrading efficiency. The utilization by microbial consortia has shown an improved performance in the biodegradation for PE plastics (Esmaeili et al., 2013; Mukherjee et al., 2016; Roy et al., 2008). Therefore, characterization of the interaction among different bacterial strains is an important process during the plastic degradation by bacterial consortia. This study reports the biodegradation of PE mulching films by the combination of two bacterial isolates from mealworm guts and demonstrates their relationship between the two strains for PE biodegradation. This will fill a gap in our understanding of the PE mulching film degradation by a bacterial consortium and also enrich bacterial resources for plastic degradation.

Section snippets

LDPE particles and PE mulching film, bacterial strains and medium

LDPE particles (the number-, weight- and size-averaged molecular weights (Mn, Mw and Mz) were 9,285 Da, 68,220 Da and 348,240 Da, respectively) with no additives were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). PE mulching film was sourced from commercially available products. The property of the obtained PE mulching film was characterized by attenuated total reflection fourier transformed infrared (ATR-FTIR, Nicolet 6700, Thermo Scientific, USA) and high-temperature gel permeation

Isolation and classification of PE-degrading bacterial strains

The gut suspensions of PE-feeding Tenebrio molitor larvae were used for the enrichment of PE-degrading bacterial strains. After repeated enrichments, two bacterial strains with different morphological characteristics on LB agar plates were chosen for further studies. They were designated Acinetobacter sp. strain NyZ450 (GenBank accession number MT459299 for its 16S rRNA gene) and Bacillus sp. strain NyZ451 (MT459300), respectively, according to the phylogenetic analysis of their 16S rRNA genes.

Growth of a consortium of strains NyZ450 and NyZ451 with PE particles

Discussion

Despite the discarded PE mulching films being a major source of plastics pollution in soil, reports on the relatively rapid biodegradation of PE mulching films were still limited. Furthermore, although many bacterial isolates from the soil (Peixoto et al., 2017) or the gut of insects (Yang et al., 2014) were reported to be PE degraders, the PE catabolic abilities of the pure cultures were all extremely weak and need to be further improved.

In this study, the degradation of PE mulching films was

Conclusions

In this study, a couple of PE-degrading bacteria Acinetobacter sp. strain NyZ450 and Bacillus sp. strain NyZ451 were isolated from the gut of PE-feeding Tenebrio molitor larvae. The co-culture of strains NyZ450 and NyZ451 was capable of growing with PE as the sole carbon source and removing about 18% of PE mass after a 30-day incubation. The consortium of two strains caused an obvious decrease in molecular weights of PE mulching films (14%, 24% and 21% reduction in Mn, Mw and Mz, respectively),

Ethical approval

This article does not include any studies of human participants or animals by the authors of this investigation.

Declaration of competing interest

The 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.

Acknowledgments

This study was supported by the Science and Technology Commission of Shanghai Municipality (grant 17JC1403300), the National Natural Science Foundation of China (grants 91951106 and 31670107) and the National Key R&D Program of China (grant 2018YFA0901200). We also thank the Instrumental Analysis Center of Shanghai Jiao Tong University for its technical assistance.

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