Week 5: BUSY BUSY BUSY

This week for s-stem I have been doing my maintenance duty. The first day was pretty hard, only because I had to get rid of all the old bacteria tubes.  The smell was about ten times worse then the ugly smell of the incubators (barf!). I had to separate the glass tubes from the plastic caps,  the caps go to the autoclave and the glass tube is disposed of in the proper glass bin.  The dead bacteria is simply poured down the drain. My second task was to put all the clean glassware back on the shelf, a much more pleasant task.  The past couple weeks have been very very busy with WAESO as my team preps for the upcoming conference.  So so much to do.

WAESO Conference Abstract

It was recently discovered that drinking water all around the world contains microplastics that have made their way from wastewater into the environment and back to drinking water treatment plants. Microplastics are any piece of plastic smaller than 5 mm. They are small enough that they do not get filtered out during the water treatment process so they end up in our water supply and can continue back on a cycle of contamination. Microplastics are found in 94% of the tap water in the United States. The human and ecosystem health implications of this are not fully known however this is an emerging issue that will most likely require innovation and the design of strategies to reduce the amount of  microplastics contamination in our recreational and drinking water supply. One proposed solution is the use of microbes to aid in the biodegradation of microplastics. Several studies have shown that Pseudomonas putida and Pseudomonas fluorescens have been found to grow on polyvinyl chloride (PVC), while Pseudomonas stutzeri has been found to grow on hydroxybutyrate. Studies have shown that, in addition to colonizing microplastics, Pseudomonas species may play a role in the decomposition of plastics such as polyvinyl alcohol (PVA), polypropylene and polythene. This raised the question of, what types of microplastics can different Pseudomonas species colonize and biodegrade? The purpose of this project was to determine if Pseudomonas stutzeri along with other Pseudomonas species (Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens) are capable of colonizing plastic types PVC (plastic type 3), Low-density Polyethylene (LPDE) (plastic type 4), Polypropylene (PP) (plastic type 5), and Polystyrene (plastic type 6). The initial goal of this project was to establish protocols for the colonization, isolation, and genetic identification of the Pseudomonas species from each of the four microplastics tested.

To achieve this goal,  Pseudomonas stutzeri was grown on Pseudomonas Isolation Agar (PIA) and Pseudomonas fluorescens (PF) agar to determine the characteristic colony morphology. ribosomal-16S primers were also used to identify the genetic relationships between P. stutzeri, P. aeruginosa, P. putida, and P. fluorescens. SEM was used to image the microplastics that were co-incubated with the different Pseudomonas species at 20x, 620x, 1150x, 4400x, and 7500x. Future studies will be used to determine which Pseudomonas species can aid in the biodegradation of microplastics PVC 3, LPDE 4, PP 5, and PS 6 .

Pseudomonas Stutzeri and Micro Plastics

            This research project was already in progress a semester before I joined in. When Dr. Cotter first explained the research to me I was very excited to get the chance to aid in such an amazing and necessary project. The significance of this topic is huge globally. Micro plastics are found in almost every environment on the planet. What are micro plastics? Plastic debris can come in all shapes and sizes, but those that are less than five millimeters in length (or about the size of a sesame seed) are called “microplastics.” (NOAA) It is critical that we know about micro plastics and how they affect the ecosystem. It should be obvious that microplastics are not a natural part of our diets, so it should be alarming when we see statistics like this one from a Nature.com article titled River plastic emissions to the world’s oceans stating, “We estimate that between 1.15 and 2.41 million tonnes of plastic waste currently enters the ocean every year from rivers…” (Lebreton, L.C.M. et al) How do we begin to solve this global issue?

            Enter the Pseudomonas. “Pseudomonas stutzeri is a gram-negative bacterium… The presence of P. stutzeri in virtually all environments has led to it being called "almost universal." Soil and marine waters are two environments where P. stutzeri can be found.” (Baughman, W. et al) Pseudomonas stutzeri’s abundant presence in the environment makes it the perfect candidate to possibly colonize and degrade microplastics. Several studies have already investigated microbial degradation of plastics with success.

            My research question is: Can Pseudomonas stutzeri colonize micro plastics 3, 4, 5, and 6? Research will be done through experiments and observation.

Name	I/D/C	Symbol	Units	Description
Plastics 3,4,5, & 6	Independent	-	-	PVC, LDPE, PP, & PS-E
The colonization of microplastics	Dependent	-	-	Positive=growth Negative=no growth
Inoculating TSA with microplastics for evidence of Pseudomonas growth	Control	-	-	Growth either on microplastics or TSA.

            My hypothesis is that P. stutzeri will colonize micro plastics 3, 4, 5, & 6. At the end of my experiments I may find that P. stutzeri will not colonize micro plastics 3, 4, 5, & 6 and that may bring on further experimentation with other microplastics in the future.

Works cited

 

Baughman, W., Balaze, K., and Bruce, A., students of Prof. Jay Lennon at Michigan State University. (24 April 2012) Pseudomonas stutzeri. 8 February 2018. https://microbewiki.kenyon.edu/index.php/Pseudomonas_stutzeri

NOAA. (10 October 2017).  What are Microplastics? 8 February 2018. https://oceanservice.noaa.gov/facts/microplastics.html

Lebreton, L.C.M., Van der Zwet, J., Damsteeg, JW., Slat, B., Andrady, A., Reisser, J. (07 June 2017). River plastic emissions to the world’s oceans. 8 February 2018. http://www.nature.com.ezproxy.pc.maricopa.edu/articles/ncomms15611

Week 2: Unknown Bacteria

This was my first official week in the lab and I absolutely loved it! I'm learning so much and can not wait to learn more. I was given a bacteria sample only known to me as B8675-C and I was tasked with preforming as series of tests to determine exactly what the bacteria was. On the fourth day of testing, with much excitement, I was able to determine that my bacteria was Bacillus subtilis. I plan on doing further testing in the lab tomorrow with the Bacillus subtilis. The series of testing was as follows: 1) Isolation Test  2)Gram staining  3)Endospore  4)Fermentation