TITLE: Microplastics in marine environments: protocol for isolating natural biofilms from seawater-incubated particles
AUTHORS: Meril Massot, elsa gadoin, Andreja Rajkovic, stephanie.bedhomme

[DESCRIPTION]
Microplastics, upon which microbial communities arise rapidly, have emerged as a significant source of pollution in the ocean1. Studying these communities is crucial to understand their potential impacts on marine ecosystems, as well as their role as vector for potential pathogens2, 3 , and reservoir for antimicrobial resistance genes4, 5.

The proposed workflow delineates an approach for incubating microplastics particles in seawater, ensuring reproducibility while controlling the origin, incubation duration, and weathering conditions of microplastics. The workflow details the preparation of the incubation device, sample collection, and subsequent laboratory processes to obtain the natural marine biofilms associated with microplastics. Our processing method helps minimize contamination by naturally occurring microparticles in marine environments and allows for an accurate estimate of microplastics concentration by direct counting.

The aim of this publication is to provide a standardized protocol to facilitate comparisons between natural communities associated with microplastic pollution.

References
1.  Bowley, J., Baker-Austin, C., Porter, A., Hartnell, R. & Lewis, C. Oceanic Hitchhikers – Assessing Pathogen Risks from Marine Microplastic. Trends Microbiol. 29, 107–116 (2021).

2. Kirstein IV., Kirmizi S., Wichels A., Garin-Fernandez A., Erler R., Löder M., Gerdts G., – Dangerous hitchhikers? Evidence for
potentially pathogenic Vibrio spp. on microplastic particles. Mar Environ Res .120, 1–8 (2016).

3. Pedrotti ML., De Figueiredo Lacerda AL., Petit S., Ghiglione JF., Gorsky G., Vibrio spp and other potential pathogenic bacteria associated to microfibers in the North-Western Mediterranean Sea. PLOS ONE 17:e0275284. (2022).

4. Wu X., Pan J., Li M., Li Y., Bartlam M., Wang Y., Selective enrichment of bacterial pathogens by microplastic biofilm. Water Res 165, 114979. (2019).

5. Liang H., de Haan WP., Cerdà-Domènech M., Méndez J., Lucena F., García-Aljaro C., Sanchez-Vidal A., Ballesté E., Detection of faecal bacteria and antibiotic resistance genes in biofilms attached to plastics from human-impacted coastal areas. Environ Pollut. 319:120983. (2023).

[BEFORE_START]
This protocol was developed to investigate natural communities associated with microplastics within the size range of 30 to 50 µm. It involves adapting the mesh size of the bags and the PluriStrainer to match the fraction size of the microplastics under study. When considering including a control material such as glass in the experiment, similar procedure can be used if control particles size range of 30 to 50 µm. 
Reconstituted Instant Ocean is used as sterile seawater to process the samples back from the field. 



PRE-IMPLEMENTATION

Obtain information on the number of microparticles per gram of powder or per milliliter of suspension to control the input.
Prepare 900 mL of sterile seawater per sample

[GUIDELINES]
When working in the lab, always wear a lab coat and disposable gloves
Clean each working surface with ethanol 70% before starting and between sample processing 
Handle the samples using sterile tweezers, ensuring sterilization between samples using ethanol 70%

[STEPS]
SECTION: SAMPLE PREPARATION
1. MICROPLASTIC PARTICLES CONDITIONING
Microplastics are enclosed in nylon mesh bags with a mesh size inferior to microparticule size to ensure their entrapment. Each bag will contain one type of plastic polymer. Three replicates per polymer and per site should be conducted.
SECTION: ON-FIELD SETUP
3. INCUBATION
Securely fasten the oyster cage to an exposed surface, ensuring that the bottom of the bags reaches the appropriate depth
Allow the microplastics to incubate for the appropriate amount of time


 

SECTION: SAMPLE PREPARATION
1.1. MICROPLASTICS CONTAINER PREPARATION 1/2
Fold a 25 µm mesh-nylon bag in the opposite direction of the stitching to achieve a pyramidal shape
Pyramidal shape avoids any collapsing of the bag in order to provide sufficient space for microorganisms to colonize the surface of microplastics

 
Glue a horizontal line, 1 cm in height, onto 75% of the total bag width. Position the sealed line at a height of 4 cm from the bottom
Use waterproof superglue designed for plastic materials. Exercise caution to avoid complete sealing until the addition of microplastics.
  
Apply glue to the stitches beneath the horizontal line to prevent any unintended dispersal of microplastics


 

SECTION: SAMPLE PREPARATION
1.3. MICROPLASTICS CONTAINER PREPARATION 2/2
Finish to glue the 1 cm-height line in order to seal the fourth side of the bag 
Verify that the fourth side sealed with glue is hermetically closed by applying pressure with a pipet tip
Fold the bag above the glued section and staple it
Punch two holes above the glued section in order to insert ty-rap later
Immerse the bags in sterile milliQ water for 5 min to allow particles with a diameter under 25 µm to pass through the mesh, preventing any site contamination.
Expose the bags to UV light treatment for 30 min to achieve sterilization

 
SECTION: MICROSCOPICAL EXAMINATION OF MICROPLASTICS AND THEIR ASSOCIATED BIOFILMS
9. ESTIMATION OF MICROPLASTIC CONCENTRATION & VISUAL CHECKING FOR COLONIZATION

Objective: Quantify a potential leakage of microplastics and obtain an accurate estimate of microplastics concentration for subsequent experiments. Ensure homogenous distribution of microplastics on and around the Malassez grid before commencing the counting process. 

Thoroughly shake the Falcon tube to homogeneize the microplastics resuspension
Pipet 10 µL of the resuspended microplastics and dispense it onto a Malassez cell. Place a coverslip onto the slide
Count the total number of microplastics in 10 squares of the Malassez cell at the appropriate objective
Multiply the count by 10,000 to obtain an estimate of the microplastic concentration per milliliter        
Calculate the mean and standard deviation over 3 independant estimations to obtain the total number of microplastics recovered per milliliter 

SECTION: ISOLATION OF PLANKTONIK BACTERIA FROM SURROUNDING SEAWATERS
10. Objective: Characterize planktonic bacterial communities composition at incubation site 

Connect a filter funnel to a vacuum source. If available, employ a multi-branch filtration system to simultaneously filter water samples
Place a 0.2 µm polycarbonate filter membrane on the filter funnel using sterile tweezers
Filter 1 liter of seawater collected at incubation site
If the membrane becomes clogged, consider filtering 2 x 500 mL of seawater using different membranes.
With sterile tweezers, transfer the membrane and place it in a 2 mL Eppendorf tube or bead-beating tube for subsequent DNA extraction
Label the tube and store it at -20°C for later DNA extraction
Discard the filtered water down the sink


SECTION: SAMPLE PREPARATION
2. INCUBATION DEVICE PREPARATION
Prepare the oyster cage following the manufacturer's instructions
Attach diving weights to the bottom of the cage using Ty-raps or ropes
The quantity of weights should be adjusted based on the strength of marine currents at each incubation site. The weights should enable the cage to minimize its movements
Attach two Ty-raps to each bag containing microplastics
Add an additional Ty-rap around all the Ty-raps within a sample group (e.g., all bags containing the same plastic type for a specific site)
It makes the samples easier to manipulate on-site
Attach groups of bags on the upper grid of the oyster cage using Ty-raps

 

SECTION: SAMPLE PREPARATION
1.2. MICROPLASTIC ADDITION
Consider color-coding the bags to facilitate polymer identification (as shown in the picture above)

ANHYDROUS FORM
weigh 50 mg  of microplastics using an analytical balance
Transfer the microplastics into the respective bags, through the unsealed part. 

SOLUTION
For microplastics in a solution, pipet 500 µL and transfer directly into the respective bag
SECTION: ON-FIELD SETUP
4. MICROPLASTICS AND SURROUNDING SEAWATER COLLECTION
Remove the oyster cage from water
Collect the bags in sterile boxes along with surrounding water 
Avoid removing bags from water to minimize stress on microorganims
Collect three separate 1-liter samples of the surrounding water using sterile glass bottles
Transport the samples to the lab in a cooler with ice packs
Store the samples at 4 °C until further processing

SECTION: RECOVERING MICROPLASTICS FROM BAGS
5. EXTERIOR BAG WASHING 

Objective: to prevent planktonic material introduction in collected samples

Scrap the external surface of the bag using a sterile blade
Rinse the bag thoroughly with 50 mL of reconstituted Instant Ocean, using a 50 mL syringe to eliminate all external debris

 
SECTION: RECOVERING MICROPLASTICS FROM BAGS
6. PLANKTONIC PARTICLE RELEASING 

Objective: Separate all particles smaller than 25 µm from the bag and reduce the number of microorganisms not associated with microplastics.

Pour 250 mL of Instant Ocean into the bottom of a sterile pipette tip box
Immerse the bag in the bath
Move the bag in the bath with 10 back-and-forth movements.
Transfer the bag to a second 250 mL bath and repeat the back-and-forth movements
Transfer the bag to a third 250 mL bath and repeat the back-and-forth movements
Use a 50 mL syringe filled with Instant Ocean to rinse the external surface of the bag, concentrating the microplastics at the bottom.
    At each step, avoid placing the bag on a contaminated surface
    
 
SECTION: RECOVERING MICROPLASTICS FROM BAGS
7. BAG CONTENT RESUSPENSION
Above a sterile 50 mL Falcon tube, make a cut in the bag below the sealed line, approximately 3 cm from the bottom
Transfer the liquid content of the bag into sterile 50 ml Falcon tube
Cut the bag vertically down the middle
Rince each cut segment above the tube using 10 mL of Instant Ocean

 
SECTION: RECOVERING MICROPLASTICS FROM BAGS
8. RESUSPENSION OF MICROPLASTICS IN STERILE SEAWATER
Discard the nylon bags
Plug a Connector Ring on a new 50 mL falcon tube and add a PluriStrainer Mini cell strainer - Mesh Size 20 µm on it
Pour the microplastic suspension onto the PluriStrainer Mini cell strainer
The microplastics and their associated biofilms are kept on the strainer while planktonic bacteria are eliminated through the mesh
Invert the PluriStrainer Mini cell strainer and attach it to a new sterile 50 mL Falcon tube. 
The microplastics are retained beneath the mesh, inside the Falcon tube
Rinse the mesh with 6 mL of Instant Ocean to resuspend the microplastics 
Collected microplastics and their associated biofilms are ready for downstream analysis