Effect of Microplastics on Gut Bacterial Community of the

Earthworm Pheretima guillelmi

Qiongqiong Shang

, Mingxia Tan

and Jie Chi

School of Environmental Science and Engineering, Tianjin University, Tianjin, 300350, PR, China

Keywords: Earthworm, Microplastics, Intestinal.

Abstract: Earthworm is an important part of the soil ecosystem. Study on the responses of earthworm gut bacteria to

microplastics (MPs) is still lacking. In this work, the effects on non-biodegradable polyethylene (PE) and

biodegradable poly (butylene adipate-co-terephthalate) (PBAT) MPs on bacterial community of earthworm

gut were investigated. The results showed that the number of operational Taxonomic Units (OTUs) and α

diversity indexes in treatments with MPs were higher than those in treatment without MPs. The number of

OTUs was in the order of treatments with aged-PE MPs > treatments with unaged-PE MPs > treatments with

PBAT MPs. The number of OTUs in treatments with lower MPs concentration was higher than that in

treatments with higher MPs concentration. Addition of MPs increased the relative abundances of genera

Ensifer, Bacteroides and Bacillus, but decreased the relative abundances of genera Salmonella, Escherichia-

Shigella and Paracoccus. Therefore, MPs significantly impact the microbial community of earthworm gut,

which is related to types of MPs.

1 INTRODUCTION

Soil animals are widely distributed all over the world

and play a key role in soil health and biodiversity

(BARDGETT 2014). Among them, earthworms are

the largest invertebrates in the soil, which can

enhance soil structure and fertility (BERTRAND

2015). Most of the ecological functions of

earthworms are connected to their internal microbial

communities, which are very sensitive to external

environmental interference (ZHANG 2022).

However, our research on the intestinal microflora of

soil animals has just begun, and the understanding of

the composition diversity and ecological functions of

the earthworms gut bacteria is still lacking, and

further research is required.

Microplastics (MPs) are widely present in the soil

environment, and absorb organic pollutants and metal

pollutants in the soil, even have a certain impact on

animals and microorganisms in soils. At the same

time, exposure to MPs disturbs the growth of

earthworms (HUERTA 2016), thereby affecting their

intestinal microbial community. However, the

concentration and properties of MPs are different, and

the effects on earthworms are also distinct.

https://orcid.org/0000-0002-1939-4009

Therefore, in this research, two types of MPs,

non-biodegradable polyethylene (PE) and

biodegradable poly (butylene adipate-co-

terephthalate) (PBAT), were selected and earthworms

(Pheretima guillelmi) were the target species to study

the effects of different types and different amounts of

MPs particles on the structure of the earthworm gut

bacterial community.

2 MATERIALS AND METHODS

2.1 Preparation and Characterization

of Materials

Polyethylene (PE) and poly (butylene adipate-co-

terephthalate) (PBAT)with particle size ranges of

104-178 μm are purchased from Dongguan Plastic

Technology Co. Ltd. The MPs were washed with

methanol for 48 hours (changing methanol every 24

hours), and then dried in a fume hood at room

temperature. Aged PE (APE) was prepared by 18%

(v/w) H

and UV exposure (HUFFER 2018). The

functional groups of MPs were determined by FTIR

260

Shang, Q., Tan, M. and Chi, J.

Effect of Microplastics on Gut Bacterial Community of the Earthworm Pheretima guillelmi.

DOI: 10.5220/0011199300003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 260-264

ISBN: 978-989-758-595-1

spectrometer (Varian Excalibur HE 3100). The

carbonyl index (CI) is often used as an indicator of

the presence of carbonyl groups (SONG 2017). The

calculation of CI of three types of MPs was

determined based on the absorbance at 1720, 2850

-1

: CI=Abs (1720 cm

-1

) / Abs (2850 cm

-1

). The

crystallinity of MPs was determined by X-ray

diffraction (XRD) of Bruker company.

The soil was collected from the Beiyang Park

Campus, Tianjin University, China. After air-dried,

the soil samples were sieved through a 2-mm mesh.

The basic physicochemical properties of the soil

samples were as follows: pH 8.08, total organic

content 1.50%, and composition of 6.09% sand,

68.33% silt and 25.58% clay.

The earthworm Pheretima guillelmi was selected

for this work. The earthworms were first incubated in

soil for 7~14 days to adapt to the laboratory

conditions, maintaining soil moisture content at

70%~85% during culture. Before use, healthy

earthworms were selected and placed on hydrated

filter paper at 20 ±3 °C for 24 h to clear their gut

contents.

2.2 Experiments Methods

There seven treatments were set up, including soils

without MPs (CK), soil +0.2% (w/w) MPs

(i.e.PE,APE and PBAT) and soil +2% (w/w) MPs

(i.e.PE,APE and PBAT). Portions of 200 g soil with

or without MPs were added into a series of 500 mL

glass beakers (distilled water was added into the

beakers to keep soil moisture content of 85%). Each

treatment was replicated 3 times. Five healthy

earthworms (have obvious reproductive ring, sexual

maturity) were washed with distilled water, weighed

and added into the beakers, which covered gauze to

prevent the earthworm escaping. These beakers were

placed in an intelligent illumination incubator (3200

Lux) with 12 h light/12 h dark at 20 ±3 °C for 28 days.

The earthworms were removed from soil on day 28,

washed with distilled water and placed in alcohol.

After being inactivated, the earthworm was dissected

and the gut was removed to measure the microbial

community structure (MA 2017).

3 RESULTS AND DISCUSSIONS

3.1 Characterization of Microplastics

The FTIR fingerprints appear to be quite different for

MPs derived (Fig. 1). Compared to the spectra of PE

and APE, the APE has an obvious peak in the

carbonyl region (1870~1540cm

-1

). The CI increased

from 0.04 (PE) to 0.27 (APE). It is indicating that the

polarity of the PE increased after aging. In addition,

the peak of PBAT spectra in the carbonyl region is

more obvious, and the CI of PBAT is 0.92, and the

polarity is the strongest.

According to the degree of crystallinity, MPs can

be divided into crystalline state, semi-crystalline state

and amorphous state (amorphous). Amorphous MPs

also include glassy state and rubber state (GUO

2012). The X-ray diffractograms of PE/APE/PBAT

composites are shown in Fig.2. The crystallinity of

MPs varies. As semi-crystalline plastics, PE has

obvious crystallization peaks, APE also shows

obvious crystallization peaks. Through jade data

processing, PE crystallinity is 56.85%, while APE is

61.67%. Compared with PE, the crystallinity of APE

increased slightly. PBAT has no obvious

crystallization peak and is a rubber polymer.

Figure1: FTIR spectra of microplastics.

Effect of Microplastics on Gut Bacterial Community of the Earthworm Pheretima guillelmi

261

Figure 2: XRD of microplastics.

3.2 Effects of Microplastics on

Earthwormsgut Bacteria

At the end of the experiment, 16SrDNA sequences

ofthe samples of earthworms guts have been tested,

and the results of OTUs analysis were as shown in

Table 1. The results show that, compared with the

CK, the number of OTUs in treatments with MPs

significantly increased. The number of OTUs of the

low concentration treatments were higher than that of

the high at the same type of MPs. The variation trend

of α diversity index (Shannon and Simpson) of the

earthworm gut bacterial community was essentially

consistent with the number of OTUs.

Figure 3 shows the top 10 phyla in the relative

abundances of the earthworm gut bacteria. It can be

seen that the relative abundances of

Verrucomicrobiota and Firmicutes in the earthworm

gut were the highest for all cultivation. Compared

with CK, the relative abundances of Firmicutes,

Crenarchaeota, Bacteroidota, Actinobacteriota and

Planctomycetes increased significantly in those

treatments with MPs with the highest increase of

Firmicutes by 3.28~23.78%, while the relative

abundances of Proteobacteria significantly reduced

by 53.70%~69.10%. And the relative abundances of

Bacteroidota increased when the concentration of

MPs increased.

Table 1: The number of OTUs and α diversity index of earthworm gut bacteria.

Group OTUs Shannon Simpson

CK 357 2.135 0.588

PE-0.2 1774 7.297 0.979

PE-2 1163 6.768 0.982

APE-0.2 945 4.045 0.829

APE-2 778 5.952 0.958

PBAT-0.2 764 5.534 0.939

PBAT-2 752 7.034 0.973

Figure 3: Thebacterial community composition of earthworm gut is shown as relative abundance (%) at the phylum levels.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

262

Figure 4: The bacterial community composition of earthworm gut is shown as relative abundance (%) at the genera levels.

Figure 4 lists the top 20 bacterial genera in the

relative abundance of the earthworm gut bacteria,

belonging to 6 phyla, namely Verrucomicrobia,

Proteobacteria, Actinobacteria, Planctomycetes,

Bacteroidetes and Firmicutes. Additionally,

compared with CK, the relative abundance of Ensifer,

Bacteroides, and Bacillus in the earthworm gut

increased significantly after added MPs, while the

relative abundances of Salmonella, Escherichia-

Shigella and Paracoccus decreased significantly.

Many studies have reported that earthworms

produce more intestinal secretions when they ingest

materials without rich fresh organic matter.

Therefore, MPs addedto the soil may stimulate

earthworms to produce more intestinal mucus.

Simultaneously, the bacteria in the gut of earthworms

is stimulated, which has a significant effect on the

consumption of pollutants (HUERTA 2016). In this

study, the number of OTUs and α diversity indexes of

the earthworm gut bacteria increased significantly

after adding MPs to the soil where earthworms live.

Addition of MPs increased the relative abundances of

genera Ensifer, but decreased the relative abundances

of genera Salmonella. Earthworms change the

growing environment of their own gut microbial

communities by swallowing soil containing MPs.

4 CONCLUSIONS

In this work, the changes of earthworms gut bacteria

were observed for a 28-day experiment in the MPs

added soil. The results showed that the microbial

community structure of earthworms gut was

significantly affected by non-biodegradable

polyethylene (PE) and biodegradable poly (butylene

adipate-co-terephthalate) (PBAT) MPs. The number

of operational Taxonomic Units (OTUs) and α

diversity indexes in treatments with MPs were higher

than those in treatment without MPs. The number of

OTUs was in the order of treatments with aged-PE

MPs > treatments with unaged-PE MPs > treatments

with PBAT MPs. The number of OTUs in treatments

with lower MPs concentration was higher than that in

treatments with higher MPs concentration. Addition

of MPs increased the relative abundances of genera

Ensifer, Bacteroides and Bacillus, but decreased the

relative abundances of genera Salmonella,

Escherichia-Shigella and Paracoccus. Therefore, MPs

significantly impact the microbial community of

earthworm gut, which is related to types of MPs.

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