Hi all! I’d like to share some exciting developments of the project!
First off, we found a new collaborator, Dr. Jeff Bowman at the Scripps Institute of Oceanography, to assist us in bioinformatics analyses! You can find his website here:
http://www.polarmicrobes.org.
Secondly, we’re ready to pilot portions of this project in undergraduate classrooms! Starting this June, we will have undergraduate classes learn science by actually participating in it via sample collection, culturing, and describing diversity by counting colony types. More to come soon!
Last Thursday Wendy and I went out to collect our day 40 samples! So far so good- we have collected at day 12, 24, and 40, with one more to go. Here we are, returning with the treasures. What we do is to place the small cages with the plastic in large ziplock bags filled with seawater until we get to shore. That orange bag in the kayak contains the samples.
But the real cool part is that we added scanning electron microscopy (SEM) to the study. Any paper looking at microbes attaching to microplastics will show some SEM pictures, and we decided it was a good investment. Interestingly, both Wendy and myself have worked with Malcolm Wood, the EM guru at Scripps Research Institute (Wendy much more than myself), and he agreed to run our samples. So we plopped plastic samples (and controls) in glutaraldehyde and Wendy raced down to San Diego with the tubes on ice, while I raced to the lab to freeze the other samples. And to incubate the plates, of course.
Here is the image of HDPE, showing a large diatom to the right, and a number of rods to the left.
We’ll be presenting our results at the ASM conference in New Orleans, in less than 2 weeks! Our poster session will be Sunday June 4, 12.15 pm, poster board # 2594.
Thanks for all your support! We could not have done it without you.References
1. Reisser, J., Shaw, J., Hallegraeff, G., Proietti, M., Barnes, D. K. A., Thums, M., … Pattiaratchi, C. (2014). Millimeter-Sized Marine Plastics: A New Pelagic Habitat for Microorganisms and Invertebrates. PLoS ONE, 9(6), e100289. doi:10.1371/journal.pone.0100289
2. Carson, H. S., Nerheim, M. S., Carroll, K. A., & Eriksen, M. (2013). The plastic-associated microorganisms of the North Pacific Gyre. Marine Pollution Bulletin, 75(1-2), 126–132. doi:10.1016/j.marpolbul.2013.07.054
3. Zettler, E. R., Mincer, T. J., & Amaral-Zettler, L. A. (2013). Life in the “Plastisphere”: Microbial Communities on Plastic Marine Debris. Environmental Science & Technology, 130619162220002. doi:10.1021/es401288x
We have been quiet but we have been busy. After the FTIR analysis (showing signs of degradation in the plastic) and PCR of the colonies grown from the plastic on ChromAgar Vibrio (showing interesting but not pathogenic Vibrios), we decided we needed a longer deployment with more analyses, including SEM and (maybe) RNA. It took some time to figure out the logistics…sample deployment has always been the bottleneck of this project. Many players controlling coastal waters, many regulations of different areas, plus the reality that we do not have a power boat. Which is required to haul the anchor and chain for deployment and removal. Luckily, the Orange County Sheriff people decided to help us out again! So this morning we headed out to Dana Point Harbor with the cages prepped and the system ready to go. Thanks to deputies David and Terry, who took us on the boat, and my student James who did the heavy lifting.
So again we had the 3 kinds of floating plastic (#2, #4, and #5), sterilized with ethanol washes and UV, set into stainless steel cages (autoclaved). Samples were collected for controls before deployment, as well as water at the same time. To make the growth experiments a bit more standardized, a small area of the plastic is taken using a hole puncher, and placed in 1 ml of sterile sea water, vortexed, and then a set amount is plated. Next time will be in 10-12 days…we are hoping for a 2-month total sampling time. I have heard horror stories from many colleagues who take environmental samples about how their sampling systems disappear and get destroyed. So while it is exciting that we got started now, it will be 8 weeks of concern now, hoping that nobody will take the buoy and remove the system.
Fingers crossed! The resulting data will hopefully round out the material to start writing up the data. Thanks again for all your support. WE could not have done this without you.
Needless to say, we are very excited. Just received some preliminary FT-IR data (this is a chemical analysis technique that can expose changes in the structure of materials) for one of the plastics, LDPE (that would be the thin produce bag kind of plastic). We are for sure seeing some changes in peaks and valleys. The rest of the results should come next week.
That said, there is still lot to do. Lots of data to analyze, and hopefully a new deployment next month. Stay tuned!
Few days ago we used the MOBIO Biostic kit to isolate DNA I extracted from microbial colonies grown on ChromAgar Vibrio with the goal of identifying them using PCR. Colonies had been collected and frozen at -20 oC. Super excited to see what comes up! =)
Thanks to your contributions, we were able to purchase the Geneious software package to validate our analyses. I’m still in the midst of pre-processing for our sequences–since we have millions of reads and low quality data can result in inaccurate species identification, it’s important to use only the highest quality data to identify species. So I’m busily combing through our data to eliminate poor quality reads and sequences that are too small to be useful. Happy Friday and we hope to update you again soon!
View of Dana Point Harbor, just North of our sampling site.
The team has been quiet since the ISME conference, but that does not mean we have been idle…on the contrary! We have been teaching and dealing with a number of other things that popped up as fall started. However the afterglow of the ISME conference, which validated our research and inspired new ideas has stayed with us. We finally met last Tuesday for a strategic meeting, and made a few decisions as to how to move forward.
We’ll do a few other experiments from the material of the last ocean run, specifically some chemical analysis of the plastic and also DNA extraction from the colonies grown on the ChromAgar Vibrio. We will also rerun the bioinformatics analysis again, and hope to start using Geneious soon, thanks to your backing! Other plans involve blueprinting of the cages with some improvements, so we can offer the design to whoever wants to follow our approach. One major goal is to assemble enough data for publication.
With the long warm summer that we have had in SoCal, seems like we should be fine for another sampling tun in the near future. We decided on a longer time frame, probably up to 2 months and with more time in-between to see if some of the changes we saw towards day 28 were outliers or beginning tendencies.
In summary, we are excited and looking forward the next stage. We’ll keep you posted!
ISME16 started yesterday afternoon with the keynote address and today it was in full swing. It is my first time and so far I have been impressed with the scheduling (there seems to be plenty of time to attend the presentations, and look at the posters, and talk to other people). I attended a number of interesting lectures, but learned the most by talking to people, especially today during the poster session. Found a researcher from Belgium, Caroline De Tender, who had done a similar study on immersed polyethylene. So we chatted and compared notes of our methodologies and ideas. Interestingly enough, there seemed to be nobody else doing the same kind of research at the meeting, although I did get some ideas from people doing biofilm and biofouling research. American Gut’s Rob Knight came by, which was quite flattering, and I was very happy to see the SDSU crew from the metagenomics workshop, including Liz Dinsdale herself, Matt Haggerty, Mike Doano, and others. Unfortunately I have to return early as I am teaching a class, so if you arrived to this page from scanning the barcode in my poster, please check it out and contact us! This other barcode is the one leading to the bigger pdf image of the poster.
This week Ana and Rachel attended a workshop on metagenomics hosted by Liz Dinsdale at San Diego State University.
From DNA extraction all the way through sequencing and analysis, we were able to produce metagenomes from shotgun sequencing from some of our samples and hope that they will add some interesting comparisons to our 16S data set.
We also learned how to use a lot of really cool analysis tools to visualize these complex data sets, so when our next sequencing run is completed we’ll be able to explore our data in multiple ways and Rachel will be able to create some really eye-catching figures and charts.
We hit our funding goal yesterday, thanks to all of you who donated to this project…thank you! We are so touched by the number of people who felt moved by the project and supported us.
So what is next? We are currently in the process of crunching the data of our second run, and prepare the poster for the ISME conference in 2 weeks (!). Next week Ana and Rachel will be at SDSU for a metagenomics workshop, which we hope will give us more insight and local networking. Once back, probably it would be a good time for a second deployment before the winter storms roll around.
One of our backers wrote a message interested in bringing the system to high school/college kids. That would be in line with the original idea of this project, which was to have project that could be implemented as a citizen science project, engaging students and the community in something they care about.
We’ll keep you posted with what we are doing and planning. The project will have an extended goal as there are always more things we could do…we are thinking data loggers to track climate conditions, electron microscopy, more locations. And obviously, we are open to collaborations! Thanks again!!!!
