Introduction

We wanted to tackle microplastic pollution, a global issue that affects both rich and poor countries, including those where having access to large research facilities and resources is not trivial. Our solution is using a bacterial biofilm to capture microplastics. To demonstrate that this is a promising approach, we needed to develop measurements to quantify the biofilm produced and the microplastic captured. We tried to come up with measurements that could be reproduced by several iGEM teams without need for high expertise, or expensive machinery and reagents. We came up with a strategy to measure biofilm biomass by vacuum filtering a bacterial culture and then weighting the filter content. Similarly, in order to quantify microplastic capturing, we avoided the most common (yet very expensive and skill-based) techniques such as quantum cascade laser (QCL) and Raman spectroscopy. Instead, we adjusted our quick and easy protocol to get the mass of the plastics instead of the bacterial biomass. We describe all our protocols.
→ Read more: Experiments

Our measurements are potentially useful to all projects related to biofilm growth and quantification (importantly, also with systems different from Curli fibers and hosts different from E. coli), and to microplastic capture via biofilm entrapment. With minor adaptations they could be useful for other kinds of plastic trapping and degradation projects, and potentially applicable to other pollutant microparticles.
In this page we showcase how we employed these measurements in our project, to characterize the successful production of Curli fibers by an E. coli strain that constitutively over expresses the csg genes from a plasmid, but has the csg genes deleted from its genome. As reference value, we used the same strain not carrying the engineered plasmid. The differences in biofilm production is reflected in the amount of microplastics that can be captured by the two strains. We qualitatively observed the microplastics captured under the microscope, but we also established a measurement to precisely quantify the amount of plastic captured.

Measurements

Our project aims to capture microplastics through the properties of biofilm. Biofilm is created by bacteria that grow both on solid surfaces and in liquid medium. These bacteria are wrapped in an extracellular matrix of polymeric substances, which allows them to adapt to various environments.

In order to characterize biofilm production, we have first relied on existing protocols. One of the most common techniques is Congo red staining, based on the attachment of the dye to the Curli fibers. The staining of E. coli colonies provides a rapid assessment of the biofilm production with the naked eye, with positive samples being strongly red and negative samples being just faintly coloured. The qualitative observation can be confirmed by image analysis, and can be complemented by quantitative measurement of the Congo red fluorescence via a plate reader.

We were concerned that this method would not be easily adoptable by all iGEM teams, notably those with limited access to reagents and specialized equipment. Furthermore, while staining and accurate quantification of small samples can be performed in the lab, the implementation of biofilm at large scale would require more scalable techniques to quantify biomass production in the order of magnitude of kilograms or more. Ideally, this measurement should be performed easily even by people with little or no experience in molecular biology.

Therefore, we came up with a method that only requires tubes, a vacuum pump, a filter, an oven and a weighing scale (Figure 1).

Figure 1: Schematic representation of the biomass quantification and the microplastic capture quantification using a vacuum pump system. A: after inoculation of the E. coli strain of interest in a 50 ml Falcon tube with LB +/- antibiotic and microplastics, incubate the tubes at 37°C for 24 h. B: prepare the vacuum pump system, after weighing the filter to be used, to start the two tests: for the first test (“Biomass quantification of biofilm”) pour the content of the tubes in the vacuum system; for the second test (“Quantification of microplastics captured by a biofilm”) centrifuge the tubes, lyse the cells and pour the contents into the vacuum flask. C: for each sample, remove the filter from the vacuum pump system and place it in an empty Falcon tube. D: Incubate the Falcon tube containing the filters at 40-50°C for at least 3 h. E: weight each filter. F: data analysis.

The idea is to use the vacuum system with a filter characterized by a pore size that retains the biofilm attached to microplastics, but not individual cells. The cells that are not embedded in the biofilm will pass through the filter. Comparing the filter weight before and after use enabled us to quantify the biomass of the biofilm that had been entrapped.
→ Read more: Experiments: protocol for biomass quantification.

We performed the appropriate pilot tests and control experiments, such as filtering water or culture medium without cells, to ensure that the weighing process is accurate and robust. Then, we compared the biofilm production in our two strains: M037, which does not produce the Curli fiber, due to the deletion of csg operon in the genome; and M037 pC3, which produces Curli fibers, thanks to the plasmid pC3 constitutively expressing the csg genes (Figure 2).

Figure 2: Quantification of biomass produced by M037 Δcsg and M037 pC3 strain, which contains the plasmid pC3 constitutively expressing the csg operons (Welch two samples t-test: p-value=0.026). This significant result shows us the overexpression of Curli fibers produced by the pC3 plasmid.

This quantitative method for assessing biofilm production is simple, fast, cheap and skill-independent. The absence of requirements such as expensive equipment, materials and chemicals limits the inter-lab variability, similarly the small amount of technical skills needed reduces the inter-user variability.
Importantly, since it does not rely on the specificity of the Curli system, it could be useful for characterizing all parts involved in biofilm production, including molecular systems different from Curli, and organisms different from E. coli.
The second challenge of our project was to quantify the microplastic content in solution, before and after treatment with our biofilm. Several powerful techniques for microplastic identification exist, but they are typically extremely expensive and time consuming, requiring complex equipment and strong expertise. Some of the experts we interviewed advised us to consider quantum cascade laser (QCL) and Raman spectroscopy.

Instead, we tried to adopt some simpler and yet informative techniques. First, we established a protocol, to qualitatively observe via fluorescence microscopy the microplastics entrapped in the biofilm. Staining of Curli fibers with Congo red and of microplastics with Nile Blue or Nile Red easily allowed us to identify the two components, (Figure 3) using a fluorescence microscope (objective: 100x).
→ Read more: Experiments, Microplastics protocols

Figure 3: Microplastics stained with Nile Blue and Curli fibers stained with Congo red. Photos A) and B) are taken with a fluorescence microscope (Exposure set to 100 ms, Objective: 100x), while C) is the composite image of A) and B). A) Photo taken at an excitation wavelength of 550 nm and emission wavelength of 605 nm. We can see Congo red fluorescence staining Curli fibers especially at the top of the image. Due to an overlap of the wavelengths, we can also see Nile Blue fluorescence staining the microplastic in the bottom right corner. B) Photo taken at an excitation wavelength of 605 nm and emission wavelength of 670 nm. We can see Nile Blue fluorescence (microplastic in the bottom right corner). C) When the two images are merged we can see the biofilm (in red) that has captured a piece of microplastic (in purple).

Direct observation is very important to investigate the interaction of biofilm with the microplastics, but it provides little information about the capturing capacity of the system. Counting microplastic particles would be a time-consuming and error-prone option. Furthermore, we are aware that not all iGEM teams might have easy access to chemicals such as the dyes and instruments such as a fluorescence microscope.
Therefore, we modified our biofilm quantification assay to a quantitative method to measure the mass of plastics entrapped in the biofilm. In this case, after incubating the biofilm with microplastics, we centrifuge it, in order to separate entrapped and free plastic particles. Captured plastics settle at the bottom, with the biofilm pellet, while the rest remain in the supernatant. The pellet is recovered and resuspended, then the cells are lysed, so the biofilm gets destroyed. Everything is passed through the filter, but in this case all biomass should flow through, while only microplastics that were associated with the biofilm should be retained.
→ Read more: Experiments, Biofilm protocols

We performed once again the appropriate controls, such as testing the retention capacity of the filter (i.e. the amount of microplastic lost during the filtering process) and the efficiency of the lysis process (i.e. the absence of biomass content in the filter upon lysis). We then moved on to quantify the microplastics captured by biofilm produced by our 2 strains: M037 Δcsg and M037 containing pC3. We used 2 types of microplastics for the quantification: PE (polyethylene) and PP (polypropylene) with 3 biological replicates per sample. We measured a significant difference between the two strains for both plastic types. This indicates that the measurement and the plastic capturing work robustly.

Figure 4: The first two barplots show the ability of the strain overexpressing Curli fibers (M037 pC3) to capture PP microplastics compared to the strain with the csg operon deletion (M037), not producing curli fiber (Welch two samples t-test: p-value=0.003). The next two barplots show the ability of the M037 pC3 strain to catch PE microplastic compared to the M037 strain (Welch two samples t-test: p-value=0.02). We detected a significant difference for the two types of microplastics. That shows us that the overexpression of Curli fibers allows us to capture more microplastics.

We believe that the true strength of this protocol is its adaptability: being system-independent, it can be applied not only to projects investigating capture of microplastics via a biofilm. Any entrapment strategy could leverage this method, as well as groups trying to degrade microplastics. A simple variant is to measure by subtraction, i.e. by quantifying the mass of plastic particles that are left in the sample following a given treatment.
In summary, this protocol could be extremely valuable to characterize all parts and systems linked to the microplastic issue. Furthermore, it could easily be adopted to measure capture and/or degradation of other pollutant particles, simply by adjusting the filter size.
Although we did not address many key questions linked to our system during the limited timeframe of our project (e.g., reproducibility in different media, robustness under different environmental conditions, efficiency compared to other strategies), we could answer them thanks to our protocols.

Table of contents

Introduction

Measurements