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Microbes attaching to plastic bags in the ocean

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A recently started project exploring microbial populations attaching to plastic when inmersed in the ocean. Floating plastic (#2, #4, and #5) are deployed in coastal waters for varying times, and DNA extracted for 16S rRNA metagenomic sequencing. Validation of the sampling methodology and preliminary data are shown.

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Microbes attaching to plastic bags in the ocean

  1. 1. Exploring the Microbes attaching to Plastic Bags in Local Beaches A community Based Research Project Ana Maria Barral, Ph.D. National University 2015
  2. 2. Background • Long-term effect of plastic in the oceans has been and is studied • Not much is known what is the effect of plastic bags on local beaches shorter term • The “plastisphere” present high content of the genus Vibrio: pathogens and/or plastic degraders? • Hypothesis: floating plastic bags on beaches will present distinct microbial populations compared to water.
  3. 3. Plastic types tested Floating plastic types: • #2 HDPE (produce bags, milk jugs) • #4 LDPE (most grocery bags, juice cartons) • #5 PP (yogurt & margarine tubs) PE: polyethylene PP: polypropylene
  4. 4. Pilot study (March 2015) • Goal: Validation of the system: Time frame (4 weeks) Sterilization methodology DNA extraction Sequencing • Location: Agua Hedionda Lagoon, Carlsbad
  5. 5. Pilot location: Agua Hedionda Lagoon • 400 acre coastal wetland, home to a rich ecosystem • Mixture of salt and fresh water
  6. 6. Setup of sampling system • Ocean quality stainless steel cages (316) • Folded and secured with zip ties • Attached to a floating device, held approximately 20 inches under surface with weights Bart’s Iron Design Capo Beach
  7. 7. Sterilization procedure • Plastic squares approx. 7 cm x 7 cm • Soaked in 70% EtOH 5 minutes • UV-irradiated in Germicidal Cabinet • Stored in sterile autoclave bags • Cages were autoclaved
  8. 8. Experiment Setup • Day 0 samples: Sterilized plastic controls (directly frozen from pouches) • Cages & plastic assembled just before deployment • 2 plastic samples per time point (reproducibility) • Water samples also collected • Collected weekly for 3 weeks • Samples placed on ice and stored at -20 oC till processing
  9. 9. Setting up the cages
  10. 10. Deploying the samples
  11. 11. DNA extraction • For plastic samples, MO-BIO PowerSoil Kit was used • For #2 samples, the whole plastic sample could be processed • #4 and #5, smaller pieces • Yield: 0.09-9 ug/ml (rec. 2 ug/ml) • For water samples, MO-BIO Power Water
  12. 12. 16S rRNA metagenomic sequencing • Omega Bio-Tek, GA • Illumina MiSeq platform • Primers for the V3/V4 region (Klindworth 2013) • Price tag for 15 samples + 3 controls (free): $1350 • Turnaround time: supposedly 3 weeks, it was over 1 month • Controls: sterile plastic & no sample (DNA kit), internal blanks (Omega)
  13. 13. DNA reads
  14. 14. Samples vs Control
  15. 15. Samples vs. Controls
  16. 16. Summary system validation • Good quality DNA was collected to run metagenomic DNA sequencing • Blanks and controls had much lower read number & completely different populations from the samples => what was collected on plastic came from the water. • Replicate read numbers were very similar, except Cyanobacteria in 2 cases: unequal exposure to sunlight?
  17. 17. Metagenomic data • Diversity at different taxonomic levels (from kingdom to species) for each sample • For ex. plastic #2 at 3rd week: • Kingdom Phylum
  18. 18. Dendogram of genus level classifications Samples cluster similarly per the sampling day #2 and #5 are slightly more similar to each other than to #4
  19. 19. Main bacterial genera #2 #4 #5
  20. 20. Some interesting results • Ruegeria lacuscaerulensis is a typical bacterioplankton, originally described from a lagoon in Iceland. It was consistently expressed on all three plastic types at all sampling times. • Winogradskyella echinorum is a species originally isolated from a sea urchin. • Rickettsia marmionii, observed on plastic #2 and #4 during the first week of sampling, has been described to cause one type of human spotted fever. • Vibrios were observed at low numbers in all samples
  21. 21. Ongoing study (ocean waters) • More robust design to withstand waves & surf • Larger cages (lobster trap type) contain the small sampling cages • Attached to sturdy buoy or anchored • Sampling of plastic & H2O for DNA sequencing • Swabbing of samples on culture media
  22. 22. Sampling site: Doheny State Beach, Dana Point In a beach area frequented by surfers and beachgoers Close to the San Juan River mouth Support from the OC Sheriff’s Department!
  23. 23. Culturing of samples • To combine genetic characterization of microbial populations with morphological and physiological methods • 2 media: – Salt Water Nutrient Agar (general medium) – ChromAgar Vibrio specifically for the isolation & identification of Vibrio species (95% per CAV)
  24. 24. Very preliminary data • Day 8 samples cultured on Salt Water Agar (24h)
  25. 25. More very preliminary data • Day 8 samples cultured on ChromAgar Vibrio (24h) H2O #5 All 3 plastics showed mauve and blue colonies, indicative of V. parahemolyticus and V. cholerae/V.vulnificus, respectively.
  26. 26. What is next? • Ocean run: – Results of cultures, possible identification of Vibrio cultures (?) – Metagenomic sequencing of plastic and water – Specific primers for Vibrios or other genera of interest • Research: – Increase sampling using a crowdsourced approach – “Ready-to-deploy” cage sets – EM of plastic? – Chemical analysis of plastic?
  27. 27. Educational possibilities • Engaging topic that many care about (ocean, pollution, water safety) • Different levels for different courses: – General Bio lab (experimental design, DNA isolation, simple culturing) – Microbiology (cultures, microbial identification) – Molecular Biology (DNA isolation, PCR) – Bioinformatics (analysis)
  28. 28. Acknowledgments • Dean Carol Richardson • Dr. Emelia DeForce • Dr. Wendy Ochoa • David Slingluff • Avi from Bart’s Iron and Jeff from SD Lobsterport • Michelle Hills and Brett Naftzger • Sgt. Mike Scalise from OC Sheriff Department • Dr. James Leichter from SOI • Dr. Rachel Simmons
  29. 29. Questions?

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