PR CONSIDERATIONS

During our visit to Dihua WWTP, the chief engineer informed us that they use the activated sludge process, which uses aerobic microbes to digest organic matter in aeration tanks. The steady influx and mixing of air provide oxygen favorable to aerobic microbes; the turbulent water can also increase the probability of PR binding to citrate-capped NPs (CC-NPs). Thus, we envision directly adding PR bacteria into existing aeration tanks. In addition, as part of the activated sludge process, WWTPs regularly cycle microbe-rich sludge back into aeration tanks to maintain the microbial populations (figure 5-1). Ideally, this would stabilize the PR bacterial population in aeration tanks, allowing this system to be low-maintenance and easily adaptable to existing infrastructure.

Applying PR bacteria in a WWTP Model

After we experimentally demonstrated that PR bacteria binds to CC-NPs, we wanted to test PR bacteria under conditions similar to a WWTP aeration tank. To learn more about the specific conditions, we visited and talked to engineers at Dihua WWTP, our local urban facility. At Dihua, wastewater is retained in aeration tanks for up to 5 hours, and a central rotor constantly churns the wastewater. To simulate these conditions, we built our own “aeration tank” using clear cylinders and a central rotor. Then, we set up three groups in separate aeration tanks: PR bacteria + distilled water, PR bacteria + CC-AgNP, or CC-AgNP solution alone (figure 5-4A). Finally, we turned on the rotor and churned the mixture for 5 hours.

In WWTPs, aeration tanks lead to secondary sedimentation tanks (figure 5-2); the addition of flocculants—polymers that aggregate suspended solids—can accelerate sedimentation. During our visit to Dihua WWTP, the engineers gave us samples of their flocculants and we added this to our simulation. After 5 hours of mixing, we stopped the rotor and added the flocculant powder used by Dihua WWTP to each tank (see timelapse video below). In the CC-AgNP cylinder, adding flocculants did not have any effect (figure 5-4B and C), suggesting that current wastewater treatment practices cannot remove NPs. In the cylinders containing PR bacteria, however, aggregated materials (including bacteria) settled to the bottom of the cylinder as expected (figure 5-4B). We centrifuged the contents of each cylinder, and observed that the pellet of the PR bacteria + CC-AgNPs mixture was orange, reflecting the presence of aggregated CC-AgNPs (figure 5-4C). In this WWTP aeration tank simulation, we show that PR bacteria pulls down CC-AgNPs.

Figure 5-4 Applying PR in a WWTP model. A) Three groups were setup and churned for 5 hours: PR + distilled water, PR + CC-AgNPs, and CC-AgNPs + distilled water. B) After 5 hours, flocculants were added and aggregated materials settled to the bottom. C) We then centrifuged the contents of each cylinder, and observed that the pellet of the PR + CC-AgNPs mixture was orange, reflecting the presence of aggregated CC-AgNPs. Experiment & Figure: Justin Y.

BIOFILM PROTOTYPE

Our goal is to design a prototype that 1) maximizes the contact area between biofilm and NPs, and 2) can be easily implemented in existing WWTP infrastructure.

Maximize NP-Biofilm Contact Area

Some aquariums already utilize biofilms grown on plastic structures called biocarriers for water purification. Commercial biocarriers use various ridges, blades, and hollow structures to maximize surface area available for biofilm attachment (figure 5-7A). With that in mind, we designed and 3D-printed plastic (polylactic acid, or PLA) prototypes with many radiating blades to maximize the area available for biofilm attachment (figure 5-7B). We used PLA because it was readily available for printing and easy to work with, allowing us to quickly transition from constructing to testing our prototype.

To test how well biofilms actually adhere and develop on our prototypes, we used BBa_K2229300 liquid cultures, since they produced the most biofilm in previous tests. After an incubation period, we observed biofilm growth and attachment to our prototypes (figure 5-7C).

Maximize Adaptability to Existing Infrastructure

We would like to implement our prototype in secondary sedimentation tanks in existing WWTPs. The water in this step is relatively calm compared to aeration tanks, which will help keep biofilm structures intact. In addition, larger particles in wastewater would already be filtered out to maximize NP removal. The director of Boswell’s WWTP told us that most sedimentation tanks use devices called surface skimmers to remove oils, which constantly rotate around a central axle; we envision attaching our prototype to the same central axle. In WWTPs that do not have a central rotor in the sedimentation tank, a motor and rod could be easily installed. The slow rotation would keep biofilm structure intact while at the same time, increase the amount of NPs that come into contact with our biofilm.

Applying Biofilm in a WWTP Model

After we experimentally demonstrated that biofilms trap NPs, we wanted to test biofilms under conditions similar to a WWTP sedimentation tank. Based on Boswell’s circular tank design, we built our own “sedimentation tanks” using clear plastic tubes, and attached biocarriers to a central spinning rotor. Three cylinders were set up: biofilm + distilled water, biofilm + AuNP, and AuNP solution alone. Here, we decided to grow biofilm directly onto biocarriers in the cylinders to minimize any disturbances. Finally, we turned on the rotor—set at a slow rotation speed—to simulate the mild movement of water in sedimentation tanks.

In this simulation, we expected to see biofilms first attach and grow on the biocarriers, and then begin trapping NPs in the tanks. After about 30 hours of mixing, the color of the AuNP solution started to change from purple to clear in the cylinder containing biofilm (figure 5-8). This suggested that enough biofilm had adhered onto the biocarrier and began removing AuNPs in the solution. In contrast, the cylinder containing only AuNP solution did not change at all (timelapse video below shows the cylinders 36 hours after the start). As the biofilm-coated biocarrier removed AuNPs from solution, we also observed more purple aggregates of AuNP sticking to the rotating biofilm biocarrier. Here, we demonstrate that our biofilm approach effectively removes NPs in a WWTP sedimentation tank model.

Figure 5-8 Biofilms effectively remove NPs in a simulated sedimentation tank. After about 30 hours of mixing, the color of the AuNP solution started to change from purple to clear (blue asterisk) in the cylinder containing biofilm. Prototype & Experiment: Yvonne W., Justin Y.

In summary, we have shown that both of our approaches, using either PR bacteria or biofilm, are successful at removing NPs in simulated WWTP conditions.

REFERENCES

Mueller, N. C., & Nowack, B. (2008). Exposure Modeling of Engineered Nanoparticles in the Environment. Environmental Science & Technology, 42(12), 4447-4453. doi:10.1021/es7029637