Team:MichiganState/Results/Design

Results

General Anoxic HGT Biosensor Bioreactor Design

Bioreactor Design Results

Screening of Plastics

The testing initially started with Acrylonitrile Butadiene Styrene (ABS), Polylactic Acid (PLA), Standard Resin, and Dental Resin. These plastics were placed in test tubes with inoculated bacteria, Methylomicrobium alcaliphilum 20Z. Optical densities (OD) were taken every two hours to measure bacterial growth. The results of the OD’s and mass differences are in Table 1 and Table 2. Due to these results, it is concluded that ABS is toxic to the methanotroph strain resulting in low growth rates and a negative net gain. The negative mass means that the bacteria started eating at the plastic and that the plastic hindered the growth of bacteria. PLA showed high bacterial growth rates but the average next gain was close to zero. This means that on one hand, the methanotrophs did not eat at the plastic, but on the other hand, they also did not form a significant biofilm around the plastic, which is the goal. Standard Resin showed high growth rates and a positive net gain which is promising. This means that 20Z successfully lived in the test tube with the Standard Resin while additionally forming a film around the plastic, which is the goal. Dental Resin showed low growth rates but a positive net gain. The optical densities for Dental Resin were started on a Friday, so the peak growth of the bacteria may have been missed over the weekend, resulting in low measured growth rates. Dental Resin had a relatively consistent positive net gain for all three samples, leading us to believe that the 20Z strain formed a biofilm around each plastic piece. Due to these results, we will continue testing with Standard and Dental Resin to continue to evaluate which plastic reacts best with the methanotroph strain.

Table 1 Table showing Optical Densities of different plastics.


Table 2 Table showing masses of the different plastics before and after testing.

Effects of Pigmentation on Bacteria

More 20Z was cultured and inoculated with samples of Standard and Dental Resin, OD’s were again taken to measure bacterial growth. To get more accurate results, White Standard Resin, Black Standard Resin, Clear Resin, Dental Resin, and High-Temperature Resin were tested to observe the effects of pigmentation and dye on the bacteria and its growth. Four samples of each plastic were used, two were placed on shakers and two were placed in a still, sterile environment, with access oxygen. This data helped us determine which plastic would still result in a biofilm even when the bacteria is not moving, which would be the exact scenario in the landfill. The data from these tests are in Table 3 and Figure 1. Table 3 displays the mass net gain. Dental Resin and High-Temperature Resin both gained mass, leading to continued testing with them. White Resin had high optical density growth rates which also lead to further testing.

Table 3 Table showing masses of the pigmented resins before and after testing.
Figure 1a(Left) and b(Right) Chart showing Optical Densities of Black(Left) and White(Right) resin samples
Figure 1c(Left) and d(Right) Chart showing Optical Densities of Clear(Left) and Dental(Right) resin samples
Figure 1e Chart showing Optical Densities of High-Temperature resin samples

Testing Growth with Methylorubrum extorquens

Testing began next with White, Dental, and High-Temperature Resin with Methylorubrum extorquens AM1 instead of 20Z. This was done because AM1 grows much faster than 20Z, it is more visible, and our PI had a lot of experience with the strain. These factors would allow for more accurate results. All test tubes were placed on a shaker and OD’s were measured regularly. The results of these tests are displayed in Table 4 and Table 5. Table 4 displays the measured OD’s throughout the growth period, and in Table 5 mass gain represents bacterial growth. Both sets of data show that Dental and High-Temperature Resin are best with the bacterial strain.

Table 4 Table showing Optical Densities of AM1 strain


Table 5 Table showing final masses of plastics using AM1

After selecting Resin, specifically Dental and High-Temperature resin, a biofilm was grown on a cross-section of the bioreactor. Twenty agar plates were used. Ten of the plates were grown with PA1 as a test and ten were grown with AM1. Within the ten plates of each bacteria, five plates had cross-sections of the Dental Resin and five plates had cross-sections of the High-Temperature Resin. Methylorubrum extorquens PA1 was used as a test because it is very similar to AM1 but grows much faster. Within a few days, a biofilm was growing on the PA1 plates. This proved that most likely AM1 would also grow with similar results. Predictably AM1 did grow just as well as PA1 but only on the plates with Dental Resin.

The plates were left in a 38oC incubator throughout the process. They were refed with methane every day to keep the bacteria growing. Throughout the tests, AM1 was growing too slowly and in the process depleted its agar supply. The plates were remade with a larger amount of agar to keep the bacteria healthy for the entire duration of the growth.

The ten plates with PA1 grew a nice biofilm around the sides of both the Dental Resin and High-Temperature Resin. However, the results that mattered were the AM1 growth. On the Dental Resin, the AM1 flourished growing a large biofilm all around the section of the bioreactor. However, on the High-Temperature Resin, The AM1 did not grow any biofilm at all. This proved that Dental Resin would be the plastic that would make up the bioreactor since AM1 flourished.

The Dental Resin was selected as the main plastic because of the greater growth of biofilm produced in the final stages of testing. Although the OD’s of Black and High-Temperature Resin showed larger growth in Figures 1a and 1e respectively, Dental resin (Figure 1d) showed more potential growth at the end stages of the measurements. In Figure 1, the still samples (Samples 1 and 2) of Dental Resin(d) showed the highest OD’s, which simulated its environment.