Colonization characteristics of bacterial communities on plastic debris

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Colonization characteristics of bacterial communities on plastic debris, influenced by environmental factors and polymer types in the Haihe Estuary of Bohai Bay, China Wenjie Li, Ying Zhang, Nan Wu, Ze Zhao, Weian Xu, yongzheng ma, and Zhiguang Niu Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Environmental Science & Technology

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Colonization characteristics of bacterial communities on plastic

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debris, influenced by environmental factors and polymer types

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in the Haihe Estuary of Bohai Bay, China

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Wenjie Li1, Ying Zhang2, Nan Wu1, Ze Zhao1, Wei’an Xu1, Yongzheng Ma1*,

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Zhiguang Niu 1*

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1

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China

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2

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Key Laboratory of Environmental Technology for Complex Trans-Media Pollution,

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College of Environmental Science and Engineering, Nankai University, Tianjin

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300350, China

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*Corresponding

School of Marine Science and technology, Tianjin University, Tianjin 300072,

MOE Key Laboratory of Pollution Processes and Environmental Criteria / Tianjin

authors: [email protected] (Y. Ma); [email protected] (Z. Niu)

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TOC

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Abstract: The colonization characteristics of bacterial communities on microplastics

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or plastic debris (PD) have generated great concern in recent years. However, the

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influence of environmental factors and polymer types on the formation of bacterial

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communities on PD in estuarine areas is less studied. To gain additional insights, five

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types of PD (polyvinyl chloride, polypropylene, polyethylene, polystyrene and

ACS Paragon Plus Environment


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polyurethane) were exposed for three-time periods (two weeks, four weeks and six

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weeks) in the Haihe Estuary. 16S rRNA gene sequencing was used to identify the

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bacterial communities on PD, in seawater and sediment samples. The results indicate

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that the average growth rate of biofilm is affected by nutrients (total nitrogen and total

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phosphorus) and salinity. Furthermore, salinity is the primary factor affecting

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bacterial diversity of the colonies on PD. In addition, the genera of bacteria show

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selectivity towards the PD polymer type and tend to colonize their preferred substrate.

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Compared with seawater and sediment, PD could be carriers for enrichment of Vibrio

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in the estuarine environment with salinity ≥ 26 (± 2‰), which might increase the

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ecological risk of PD in marine environments.

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Key words: plastic debris, environmental factors, polymer types, Haihe Estuary

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1 Introduction

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Global plastic production reached 335 million tons in 2016, and this value is

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exhibiting a continuing upward trend.1 This production inevitably leads to large

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amounts of plastic waste discharged into the environment, which has been a pervasive

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feature, ranging from coastal estuaries to oceanic gyres and down to the abyssal

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seafloor.2-4 Jambeck et al. predicted that unless waste management infrastructure is

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improved, the cumulative quantity of plastic waste available to enter the ocean from

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land will increase by an order of magnitude in 2025.5 Over time, a culmination of

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physical, biological and chemical processes could reduce the structural integrity of

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plastic

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microplastics (with the diameter ≤5 mm) and nanoplastics (≤ 100 nm). With the

products,6-10

resulting

in

plastic

debris,


including

macroplastics,11

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properties of chemical stability and low bioavailability, plastic debris (PD) could exist

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for a long time in the ocean, thus affecting the marine environment.12

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As a unique substrate, PD can adsorb microorganisms in the ocean and form the

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“Plastisphere”.13 Microbial communities on PD exist in the form of biofilms,14 which

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were investigated by sampling or in-situ experiments (Figure S1). Many studies have

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shown that the composition of microbial communities on PD are significantly

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different from those of the surrounding environments (water and sediment) or other

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natural media, both in marine or freshwater ecosystems,15-17 and are obviously

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affected by position, time and plastic type.16-22 In addition, biofilms attached to PD

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had unique metabolic functions that were different from that of the surrounding

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environments. Bryant et al. pointed out that the over-expression of the Che gene, a

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secretory system gene, and the nifH gene in the biofilm on PD reflected the obvious

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enrichment chemotaxis, frequent intercellular interactions and nitrogen fixation

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compared with the free bacterial communities in seawater.23 Dussud et al. found that

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transportation across the cellular membranes, cell motility and biodegradation of

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metabolites in the PD biofilms from the Mediterranean were significantly higher than

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those of planktonic bacterial communities in seawater.19 Furthermore, PD could also

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act as a carrier to transport of microorganisms, including pathogenic bacteria,

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plastic-degrading bacteria and harmful algae species.24,25 In 2003, Maso et al. first

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proposed that marine microplastics could be used as migration tool for Ostreopsis sp.

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and Coolia sp.24 Virksek et al. identified a fish pathogen (Aeromonas salmonicida) on

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plastic debris for the first time in the Italian North Adriatic Sea.25 Moreover, the



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conditional pathogen Vibrio was found on PD in the North Atlantic,13 Scottish

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beaches,26 the North Sea and the Baltic Sea.27 Previous studies have also shown that

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there were some potential plastic-degrading bacteria including Pseudomonas sp.,

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Arthrobacter sp.,28 Kocuria palustris,29 Bacillus cereus,30 Bacillus gottheilii31 and

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Rhodococcus sp.,32that settled on PD (Table S1).

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Although the colonization characteristics of bacterial communities on plastic

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debris have been investigated in the sea, few studies have illustrated the key factors in

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determining the growth rate of biofilm and bacterial composition in estuarine areas. In

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addition, whether PD could provide a suitable living environment and improve the

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survival strategy of bacterial communities from the river to the sea remains unknown.

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Haihe Estuary, located in Bohai Bay, has many functional zones (including

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Tianjin Port, the estuary of the Dagu River, a water park, and an industrial park),

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resulting in a natural experimental field for investigations of the environmental effects

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on the bacterial communities of PD. Moreover, Bohai Bay is a semi-enclosed bay, and

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the pollution of microplastics in Bohai Bay is more severe than in other bays of

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China.33 Previous studies have utilized two strategies for studying bacterial

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communities on PD: sampling and in-situ experiments. In sampling experiments, PD

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was randomly sampled in the field, and the source of PD was unclear because of the

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migration of PD in the environment.17,20,34 Consequently, to avoid the influence of

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random migration of PD on bacterial communities, we established an in-situ exposure

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experiment in Haihe Estuary for three time periods (two weeks, four weeks and six

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weeks) to elucidate the colonization characteristics of bacterial communities on PD


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that are affected by local environmental factors and polymer types.

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There are four objectives of this study. 1) Investigate the spatial and temporal

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influences affecting the growth rate of biofilm and the composition of bacterial

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communities on PD in Haihe Estuary. 2) Determine the main environmental factors

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influencing biofilm formation and composition of PD bacterial communities. 3)

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Explore the influence of polymer types on the composition and metabolic function of

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bacterial communities from the Haihe River to the Bohai Bay. 4) Compare the

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abundance of the pathogenic bacteria colonized on PD to seawater (SW) and sediment

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(SD) samples in estuarine areas.

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2 Materials and methods

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2.1 Selection of in-situ sites

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Due to the strong metabolic activity of microorganisms during the summer, the

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experiment was conducted from July 2018 to September 2018. A total of 9 in-situ

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sites were selected for this experiment (Figure 1). S1, S2, S3, S4, and S5, defined as

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the interaction zones between the freshwater and seawater (F-S zones), were selected

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to explore the effects of environmental factors on bacterial communities attached to

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PD from the Haihe River to the Bohai Bay. The statistical results showed that the

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annual average precipitation in Haihe River Basin was 500~600 mm. The annual

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average surface runoff is 60~70 mm, and the runoff coefficient is 0.10~0.15.35,36 S1 is

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in front of the Haihe tidal gate, and S2 is behind Haihe tidal gate, which is often open

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in summer. Therefore, the two sites are the most obvious areas of freshwater and

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seawater mixing. S3, S4, and S5 are in-situ sites downstream of the Haihe Estuary,



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and S5 is almost seawater and less influenced by anthropogenic activities. The

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bacterial communities on PD from S6, S7, S8 and S9 are also investigated since these

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in-situ sites are special zones (Sp zones) deeply influenced by anthropogenic

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activities. S6 is located at the Tianjin Port Coal Terminal, and S7 is at the Tianjin Port

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Passenger Station. S8 is located at the Dagu draining river estuary, where all the

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treated wastewater from the southern part of Tianjin city enters into the Bohai Bay. S9

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is located at the Lingang Petroleum Industrial Zone. The location coordinates and

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environmental parameters (temperature, dissolved oxygen, salinity, pH, total nitrogen

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(TN) and total phosphorus (TP)) of each in-situ site were collected and are

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summarized in Table S2.

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Figure 1. In-situ experimental sites in Haihe Estuary. S1, S2, S3, S4 and S5 are the interaction

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zones between the freshwater and seawater (F-S zones). S6, S7, S8, and S9 are the special zones

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(Sp zones) deeply influenced by anthropogenic activities: S6 is located at the Tianjin Port Coal

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Terminal; S7 is located at the Tianjin Port Passenger Station; S8 is located at the estuary of Dagu

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draining river; S9 is located at the Lingang Petroleum Industrial Zone.


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2.2 In-situ experimental process

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Previous studies have shown that polyethylene, polypropylene and polystyrene

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are the main types of plastic debris in water samples and sediments of Bohai

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Bay.33,37,38 Therefore, the samples used in this experiment included polyethylene (PE),

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polypropylene (PP) and polystyrene (PS), as well as polyvinyl chloride (PVC) and

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polyurethane (PU), which were purchased from Aladdin Biochemical Technology

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Co., Ltd. (Shanghai, China). PVC, PE, PP and PU are transparent sheets while PS is

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white foam. Additionally, the sizes of PD are all 50.0 mm × 50.0 mm × 1.0 mm. For

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each type of PD, ten pieces of PD were packed in 40 mesh nylon filter bags (200 mm

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× 200 mm), which also contained several green nylon cords to distinguish plastic

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types. Five nylon filter bags containing five different types of PD (PE, PP, PS, PVC

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and PU) were bundled into one group and were bound with weights (3 kg) to ensure

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that the samples could be located on the interface between seawater and sediment. At

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each in-situ site, there were three groups, one of which was harvested at two weeks,

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four weeks and six weeks. Each obtained PD was packed in labeled sterile vacuum

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bags. In addition, three replicate seawater samples were collected at each in-situ site.

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Per replicate, 2 L seawater samples were collected with a water sampler at the depth

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of 30 cm beneath the surface.17,19 Potential microplastics and other impurities in the

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water samples were removed using a quantitative membrane (80~120 μm). Then, the

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filtered water samples were passed through a 0.22 μm Millipore polycarbonate

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membrane (Merck Millipore, Billerica, USA). At each in-situ site, three replicate

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sediment samples from the upper sediment layer (0-5 cm) were collected using a Van



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Veen grab sampler.17 Approximately 100 g of grab-collected sediment was sampled in

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a sterile 100 mL falcon tube. The collected seawater, sediment and PD samples were

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transported to the laboratory in ice bags within 2 h. Three types of travel blank

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samples (sterilized DI-water, blank quartz sand, and virgin plastic debris (PVC, PP,

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PE, PS and PU)) were used as controls during the sample collection. The filter paper,

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sediments and PD were stored at -20 °C in the laboratory without any treatment.

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2.3 Determination of biofilm content

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The principle of biofilm content determination is that optical density is

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proportional to the quantity of biofilms on the surface of PD.16,39 Following a previous

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study,16 each PD was rinsed three times with sterilized DI-water, and then three

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triplicate samples were placed in sterilized culture dishes. Next, each PD was

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air-dried at room temperature at least for 45 minutes and stained with crystal violet

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(1% w/v) for 45 minutes.16 The stained PD was further washed three times with

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DI-water and air-dried at room temperature for another 45 minutes. Each PD was cut

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into four pieces of similar size (25.0 ± 1.0 mm × 25.0 ± 1.0 mm × 1.0 mm) and placed

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into a 50 mL sterilized centrifuge tube (diameter = 30.0 mm), to which 10 mL ethanol

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(95% v/v) was added. Finally, the ethanol solution was transferred to a quartz

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colorimetric dish (1 cm) to measure the optical density at 595 nm using a UV-Vis

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spectrophotometer (DR6000 HACH Company USA).

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The average growth rate of biofilm was calculated using the following equation.

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OD represents optical density; OD (2) represents the biomass on each type of PD at

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each in-situ site after two-week exposure; OD (4) represents the biomass on each type


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of PD at each in-situ site after four-week exposure; OD (6) represents the biomass on

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each type of PD at each in-situ site after six-week exposure; ν represents the average

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growth rate of biofilm with the unit of “1/w”(w represents week); t represents

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exposure time.

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ν=

∆𝑂𝐷 ∆𝑡

=

𝑂𝐷(6) ― 𝑂𝐷(2) 4

2.4 DNA extraction and 16S rRNA sequencing

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Each PD was rinsed three times with sterilized DI-water. For each type of PD,

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three replicates were cut with scissors into small pieces less than 7.5 mm, and then

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placed in 10 ml sterilized centrifugal tubes (diameter = 15.0 mm). The total DNA of

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bacterial communities on PD and sediment samples was extracted using the Power

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Soil DNA Isolation Kit (Mobio Laboratories, Carlsbad, CA). For the seawater

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samples, the total DNA of bacterial communities on filter paper was extracted by the

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Power Water DNA Isolation Kit (Mobio Laboratories, Carlsbad, CA) according to the

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instruction. The extracted DNA of all samples was stored at -20 °C prior to

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sequencing.

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2.5 Sequence processing and data analysis

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Bacterial communities on PD were detected by next-generation amplicon

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sequencing of 16S rRNA genes. PCR was applied to amplify the V4 hypervariable

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region of bacterial and archaeal 16S rRNA genes by using the primer 515F (5’-

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GTGCCAGCMGCCGCGGTAA-3’)

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GGACTACHVGGGTWTCTAAT-3’). The 50 μL reaction system contained 25 μL

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PCR Master Mix with HF Buffer, 3 μL of each primer, 10 μL gDNA and 6 μL

and

the

reverse


primer

806R

(5’-


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DI-H2O. The PCR amplification conditions were as follows: initial denaturation of 30

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s at 98 °C, 25 cycles of (98 °C, 15 s; 58 °C, 15 s; and 72 °C, 15 s), and a final

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extension step at 72 °C for 1 minute. PCR products were purified using an Agarose

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Gel DNA Purification Kit (Qiagen, Chatsworth, CA) and then were subjected to

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Illumina MiSeq4000 platform at GUHE Info Technology Co., Ltd. (Hangzhou,

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China).

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2.6 Data analysis and statistics

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The data for each sample were split from the original data according to the

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barcode and primer sequences based on the Standard Operation Procedure. Vsearch

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v2.4.4 was used to stitch the reads of each sample to obtain the original Tags data

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(Raw Tags).40 The sequences that were shorter than 150 bp and of low quality (quality

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score < 20) were removed from the raw sequence data. Operational taxonomic units

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(OTUs) were defined as clusters sharing 97% sequence identity. The OTU statistics

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and graphical output were conducted by Vsearch v2.4.4. The representative OTU

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sequence was annotated with species based on the SILVA128 database.41 Alpha

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diversity was estimated by Chao 1, Simpson and Shannon indexes using QIIME

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software, which was used to compare OTUs abundance and evenness among samples,

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and then ranked abundance curve and dilution curve were generated. In addition, the

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relative abundance data for bacterial communities in seawater (SW), plastic debris

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(PD) and sediment (SD) were standardized by Z-score standardization. The difference

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between bacterial communities from all in-situ sites and three substrates (SW, PD,

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SD) were compared by principal component analysis (PCA) and principal


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co-ordinates analysis (PCoA) based on Bray-Curtis similarities or weighed UniFrac

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distances.42-44 PCA and PCoA were all carried out in R with the vegan package.

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Correlations between bacterial composition (average growth rate of biofilm and alpha

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diversity) and environmental factors were explored by redundancy analysis (RDA).

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RDA was carried out in R with the vegan 2.3-5 package. Meanwhile, based on R

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Package Vegan, PERMANOVA (Permutational multivariate analysis of variance)

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was used to distinguish markers for the composition of microbial community among

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samples.45 Furthermore, LEfSe analysis was used to screen marker species with

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different classification levels. This method combined linear discriminant analysis with

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the nonparametric Kruskal-Wallis test (package 'stats' version 3.2.0) and the

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Wilcoxon rank sum test. In addition, bacterial community function was predicted by

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FAPROTAX and Bugbase software.46,47 Var test, one-way ANOVA and two-way

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ANOVA were used to evaluate the significant differences among samples. The post

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hoc analysis was followed by Tukey's post hoc tests. p < 0.05 indicated that the

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factors were significantly different.

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3 Results and discussion

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3.1 Effect of environmental factors on biofilm attached to PD

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The PCR product concentration of the travel blank samples (sterilized DI-water,

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blank quartz sand, and virgin plastic debris (PVC, PP, PE, PS and PU)) was low, and

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all DNA was used for sequencing. For each travel blank sample, the number of reads

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was

Colonization characteristics of bacterial communities on plastic debris

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