Introduction

In our ASTWS sewage treatment system, PETase-Biofilm system worked together efficiently to biodegrade the PET plastic microparticles from the water. The system combined PETase and Biofilm. PETase is used to biodegrade microplastic particles, while Biofilm is used to capture and enrich microplastic particles from the water, so as to improve the degradation efficiency of the system.

As for whether this system can achieve the desired effect, it needs to be proved in three aspects: 1) Whether PETase can effectively degrade PET plastics; 2) Whether Biofilm can capture microplastics in water; 3) Whether the degradation efficiency of PETase and Biofilm system is higher than that of the single PETase.

For the second item, we soaked the small plastic pieces in the culture medium containing E. coli with OMP gene. After 48 hours, we observed that the biofilm produced by the bacteria was successfully attached to the surface of the plastic pieces (See Fig.1). In order to obtain more direct evidence, we planned to fluorescently label the microplastics then put them into the culture medium containing E. coli with OMP gene to trace the capture process directly. However, Subject to the funding and time, this scheme has not been implemented in this project.

Fig.1 Biofilm produced by the engineered bacteria was successfully attached to the surface of the plastic pieces

For the first and third items, we need to measure the degradation rates of plastics, and there are two options: to detect the quality loss of plastics, or to detect the increase of degradation products.

The result of loss of plastic mass is the most direct proof. Theoretically speaking, as long as the plastic sample of certain mass was immersed in the culture medium with engineering bacteria, it can be weighed regularly. However, in the actual experiment, we found that the biodegradation rate of plastics was relatively slow, the mass loss of plastics was quite limited in a certain period of time, and the variation of the experiment had great impact on the results. Preliminary tests have shown that minor weight reduction of the tested plastic samples can be observed, but the reliability is disputed. In order to ensure the reliability of the data, high requirements were put forward for experimental process design, experimental operation and sensitivity of weighing equipment, so as to reduce possible measurement errors and systematic errors. Thus, samples of sufficient mass should be measured regularly with a high-sensitivity analytical balance after a long enough period of time in an environment with the highest concentration of PETase and Biofilm. Due to laboratory conditions and limited time, we abandoned this scheme.

Because of the high sensitivity of the high performance liquid chromatography (HPLC), it was more easily to detect the concentration changes of plastic degradation products in a relatively short time by HPLC. Thus we chose PET degradation product Hydroxyethyl terephthalate (MHET) [1] as the detection object and then designed the following experiments.

As the HPLC in our laboratory was broken and could not be repaired until October, we lost the last time window (Chinese National Day holiday at beginning of October) for completing the work by ourselves, so we had to send the prepared samples to the Public Service Platform For the Evolution of Innovative Drug Property (Hangzhou Leading Pharmatech.Co., Ltd)(See Fig.2), and asked them to help us complete the test.

Fig.2 Lab of Public Service Platform For the Evolution of Innovative Drug Property (Hangzhou Leading Pharmatech.Co., Ltd) for HPLC test

Experiments

1. Sample information

1.1 Standard

We use commercial Hydroxyethyl terephthalate (MHET), a monomer component after plastic degradation, as standard, CAS No. : 1137-99-1. The standard is provided by Suzhou Amante Biotechnology Co., LTD., article No. Ac-2862.

Four concentration gradients were set in the experiment, which were 0.001/0.005/0.01/ 0.05mM respectively.

1.2 Samples

There are four groups of samples to be tested

Group No. 1 – LB Medium only

Group No. 2 – LB Medium + plastics

Group No. 3 – LB Medium + E. coli containing PETase part only + plastics

Group No. 4 – LB Medium + E. coli containing both PETase and OMP part + plastics

1.3 Sample preparation

1. Each group was added with 250ml LB medium and 1g of cut-up PET plastic pieces (note: no plastic was added to sample No. 1);

2. Strains expressing PETase were inoculated in sample No. 3;

3. Strains expressing PETase and OMP were inoculated in sample No. 4;

4. Cultured for 48 hours in a shaking table at 37℃ with a rotating speed of 220rpm;

5. Took 1ml medium from each of group and centrifuged at 15000rpm for 30 min;

6. The supernatant of Samples No. 1 and No. 2 were taken as samples to be tested;

7. In order to completely remove the bacteria in samples No. 3 and No. 4 to prevent the bacteria from blocking the HPLC column, ultrafiltration tube with molecular interception of 10,000 Da was used to filter the supernatant. The filtered medium was used as the sample to be tested.

1.4 Method and experimental condition for HPLC

Instrument model: Waters-2695-2998

Column: Zorbax Extend C-18

Injection quantity: 20 ul

Column temperature box: 40 °C [2]

Flow rate: 1 mL /min [2]

Detection wavelength: 254nm [2]

Mobile phase: 70% MilliQ Water, 20% acetonitrile, and 10% formic acid [1]

Results and Discussion

1. Standards

HPLC result for standard MHET concentration gradients of 0.001/0.005/0.01/ 0.05mM were shown from Fig.3 to Fig.6 respectively. Only the peak at 3.34 min is for MHET. The peak area size was shown in Table 1. The results of MHET were used as positive control. It was very clear that the 3.34 min peak area was in direct proportion to the concentration, i.e. higher concentration of MHET caused larger peak area. (See Fig.4)

Fig.3 HPLC result of MHET Standard

Table 1. Standard concentration and corresponding HPLC peak area

Fig. 4 MHET peak area is in direct proportion to the concentration

2. Samples

The HPLC results of experimental samples group No.1 to No.4 were shown in Fig.5. Group 1 and Group 2 samples were used as blank control and negative control respectively. Table 2 listed the MHET peak aera of each sample and we could easily tell that:

1) Peak area of group 1 and group 2 were almost at same level and very low (about 1/3 area of 0.001mM MHET standard). Since group 1 is LB medium only, which should not contain any MHET, and group 2 should not contained any MHET either. Therefore, there was not any significant degrading effect from LB medium.

2) Peak area of group 3 and group 4 were much larger than group 1 and 2. Therefore, the presence of MHET in group 3 and group 4 samples indicated that the PETase produced by E. coli in group 3 and group 4 samples has successfully degraded the PET plastics.

3) Peak area of group 4 was even 1.66 times larger than that of group 3，thus, the group 4 was more efficient. During sampling, we determined that the group 3 and group 4 samples had the same OD600 value, so the difference in degradation rate caused by the number of bacteria could be ignored. At the same time, the expression level of PETase in group 4 should be slightly lower than that in the group 3, because the E. coli in group 4 has double expression of two genes. Therefore, the higher degradation efficiency of group 4 indicated that the PETase + Biofilm system that we designed was successful.

Fig. 5 HPLC result of sample group