Plastic pollution is something that we hear about often these days. Increased global plastic production coupled with the fact that plastics can take centuries to biodegrade produces a major problem. You might visualize the plastic pollution problem in terms of sea turtles with plastic bags over their heads, but these macroplastics—large plastic items such as bags and bottles—represent only one facet of the larger problem. A second, often overlooked portion of the problem is pollution by microplastics. Microplastics, like those in Figure 1, are defined as plastics less than 5 mm in diameter and can come either from degradation of larger plastic debris or products that directly use these small plastics.¹ Microplastics are incredibly prevalent in our oceans, having been found to be four hundred times more prevalent than macroplastics, by weight.²

But, don’t take our word for it; listen as Brandis Friedman of WTTW Chicago reports on "Tiny plastic microbeads pile up into a problem for the Great Lakes".

We agree with David St. Pierre of the Metropolitan Water Treatment Plant of Greater Chicago “If we deal with [the issue of microbeads] on front end, we take care of it before it’s a problem by eliminating it as a pollutant source…[this would be a] very inexpensive way to deal with the problem.”⁴ However, facial scrubs and toothpastes are not the only products releasing microplastics into the environment. They can also come from polystyrene packaging, discarded fishing line, and importantly, washing machine effluent. Synthetic clothing is becoming increasingly common throughout the world, and these nylon, polyester, and acrylic fabrics release small fibers into the water during washing which are inadequately collected by the machine or water treatment plants, thus releasing them to the environment. Sampling has shown that a single synthetic garment can release as many as 1900 fibers in a single wash, which amounts to a massive amount of pollution when one considers the millions of synthetic clothes that are washed on a daily basis. ⁵

But what is the real danger of microplastics? Not enough research has been done to truly get a grasp on their widespread effects, but many concerning revelations have been made. Many plastics are manufactured along with certain additives like dyes and plasticizers. Degradation of larger plastics into microplastics can expose these often harmful chemicals to the environment. Besides those pollutants the plastic is made with, microplastics have been seen to have an increased affinity for harmful persistent organic pollutants (POPs) that they encounter in treatment plants and the marine environment. One study has shown that a polychlorinated biphenyl (a POP) levels were 10⁶ times higher on polypropylene pellets than in the surrounding seawater.⁶ To compound the problem, the plastics are likely to release the POPs again in the presence of high levels of organic material, as found in the digestive tract of animals, for instance, a phenomenon that could be more intense for less hydrophobic polymers such as those from synthetic clothing.⁷ This means that microplastics can concentrate the POPs in the ocean, and when the microplastics are ingested by plankton or larger animals and those animals are eaten by others, the microplastics release the concentrated pollutants to the animals’ tissues. This intensified bioaccumulation of harmful chemicals affects not only marine life, but everything that eats marine life, including humans.

As was mentioned by David St. Pierre in the video above, there is currently no cost effective way to remove microplastics from the effluent at wastewater treatment plants. Our project works to solve this problem through the creation of a biofilter to remove microplastics. It has two unique parts which each tackle a different aspect of the filter. On the one hand, we are attempting to create a strain of E. coli which will form a biofilm along the surfaces of a filter to increase the concentration of bacteria within the filter and minimize the amount of bacteria lost in the flow-through. The second aspect of our project is to have those E. coli secrete a nylon degrading enzyme into the water which will degrade the microplastics passing through into their constituent monomers and prevent the polymer from leaving the treatment plant where it can wreak havoc.

Biofilm Formation: The Basis for Our Biofilter

A biofilm is a physiological state of bacterial communities where the cells are encased in an extracellular polymeric matrix. This makes the colony incredibly stable and resistant to mechanical dislodging as well as immunological attack and other harmful conditions. E. coli, as well as other eubacteria, use the polysaccharide poly-1,6-N-acetyl-D-glucosamine (PGA) as a primary adhesin between cells of a biofilm. In E. coli, the creation and export of this polymer requires the four genes of the pgaABCD operon.9 pgaA and pgaB have been shown to be necessary for export of the PGA polysaccharide, while pgaC and pgaD are used in the biosynthesis of the molecule.10 All four genes are necessary for biofilm formation, and overproduction of PGA has been shown to increase biofilm formation in E. coli.11

SEM image of the exopolysaccharide connections that hold a biofim together8

The biofilm formation portion of our project focuses on the overproduction of the pgaABCD operon gene products in order to increase biofilm formation. This is done through two pathways—the insertion of a plasmid containing the entire operon into the cell, or the insertion of a plasmid containing the transcriptional activator ''NhaR''. NhaR is a DNA binding protein within the LysR family of transcriptional regulators which has been shown to be necessary for transcription of the pgaABCD operon.¹² NhaR is transcribed in unaltered E. coli in the presence of monovalent cations and alkaline conditions, leading to biofilm production under these stressful conditions.¹³ Expression of NhaR under a constitutive promoter on a plasmid enables us to create a biofilm under all conditions within our filter. The adhesive nature of this biofilm will then prevent our cells from being swept away in the flow of the water through the filter, enabling long term functionality within a water treatment plant.

Overview:

A BioBrick of the nhaR gene in E.coli bacteria is supposed to increase their biofilm formation. When expressed, the nhaR gene acts as a transcriptional regulator of the pga operon. The pga operon increases cell-cell adhesion when expressed. This increased cell-cell adhesion will increase the biofilm formation in the bacteria. Therefore, the aim of the crystal violet assay is to quantify the amount of biofilm produced by these bacteria.

Mechanism of the assay: Crystal violet binds to polysaccharides of the biofilm matrix. By measuring the binding to adhering biofilm, the amount of biofilm produced can be quantified. A stock solution of crystal violet is incubated with the culture, the supernatant is removed, bound crystal violet is mobilised with an acetic acid solution and the absorption is determined. The absorption values determine the amount of biofilm present in the culture at the time of staining.

Methodology

Crystal violet trials were conducted with E.coli BL 21 (DE3) cultures grown with both LB and soybroth media on 96 well plates. The 96 well plates’ columns were divided with different concentrations of IPTG on the control (circular pSB1C3, and on the nhaR BioBrick with the IPTG inducible promoter. No IPTG was added to the nhaR BioBrick with the constitutive promoter. Chloramphenicol was added to all samples. Experimental constants: Strain of E. coli used: BL-21. Amount of culture: 100 µL. Amount of Chloramphenicol 20: .01 µL per well. Experimental variables: nhaR with IPTG inducible promoter vs. nhaR with constitutive promoter. The IPTG inducible promoter used was BBa_K215000 while the constitutive promoter used was BBa_J23100. Media used: LB vs Soybroth. To learn more about the specific protocol we used, click here

Figure 1.C0, C1, C2, C3: Control with 0, 100, 1,000, 10,000 µM IPTG, N0, N1, N2, N3: nhaR BioBrick with IPTG inducible promoter with 0,100, 1,000, 10,000 µM IPTG, NC: nhaR BioBrick with constitutive promoter, with 0 µM IPTG.