Demonstrate

PET Microfibre Filtering

In the prototyping and testing phase we were successfully able to capture plastic microfibres within our filter attached to the outlet of the washing machine. This is shown below in the video of the microsieve capturing microfibres as well as several photos of the captured microfibres collected within our filter prototype during a wash.

In testing the wash, , set for one hour and twenty four minutes, contained two large polyester fleeces which were known to release significant amounts of microfibres. These were captured and contained within several filter models with pore sizes of 40, 70 and 100 microns. The mass of microfibres captured by each filter is shown below along with the flow rates for the wash.

These findings demonstrated that the filter serves its purpose as it filters out the majority of microplastics, therefore reducing the amount that makes its way into the oceans.

Figure 1: Graph showing the flow rate flowing through the filter against the mesh size used.

This success was subsequently built upon through further testing. The filter prototype was used to capture the microfibres released from 5 consecutive washes without cleaning out the filter. This was carried out without incident and importantly with little to no effect on the flow rate, as shown in the table below, meaning the filter was not clogging or backing up the system. Showing theuse of the filter in consecutive washes without the need for cleaning which is a big step towards demonstrating the viability of the product for use in commercial washing machines in which it would not be feasible to clean out the filter after every wash.

	1st Wash	2nd Wash	3rd Wash	4th Wash	5th Wash
Flow Rate 1 (L/min)	11.9	12.4	12.1	12.2	12.1
Flow Rate 2 (L/min)	11.8	12.2	11.9	12.1	11.9
Flow Rate 3 (L/min)	11.5	11.2	11.7	11.8	11.5
Mean Flow Rate (L/min)	11.7	11.9	11.9	12.0	11.8

Finally, an added success comes in the fact that, as shown above, models utilising different mesh sizes all succeeded in capturing microplastics with no drop in flow rates. The significance being that we are not restricted in our design options whilst still being able to achieve success.

This shows that the filter that we designed and manufactured serves its purpose and acts as an ideal platform to utilise the PETase, BHETase and MHETase that we engineered.

Please consult the Hardware page for more details on the testing procedure and results.

Plastic Degradation

We have been able to demonstrate that our PETase enzymes are able to break down PET fibres that were obtained using the filter system designed and demonstrated above. From the filter system 35 µg of fibres was incubated in 500 µl of enzyme at a range of concentrations (50 to 2000 µM) in 50 mM NaPhosphate buffer pH7.5. The fibres were incubated with the enzyme solution for 76 hours before the reaction was terminated by heating at 80 °C for 15 mins and the reaction analysed by HPLC.

For the HPLC assay samples (10 µl) was analysed using a high-performance liquid chromatography system (HPLC, Agilent 1200) using an Eclipse Plus C18 column (Agilent, UK). The mobile phase was 99.9 % Water with 0.1 % Formic Acid at a flow rate of 0.8 ml min-1 and the effluent monitored at 240 nm. The typical elution condition was 10 min with 20% - 80% acetonitrile. The amounts of products (BHET, MHET and TPA) were calculated by comparison to a standard curve. All samples were analysed in triplicate and the data averaged and standard errors calculated.

Three of the rationally designed enzymes were able to degrade microfibres

PETase S121E_D186H_R280A
PETase T88A_S121E_D186H_R280A
PETase T88A_S93M_S121E_W159F_D186H_R280A

An example HPLC trace showing PET Fibres obtained from the washing machine filter without enzyme to confirm no auto-hydrolysis of PET fibres.

An example HPLC trace showing PET Fibres obtained from the washing machine filter with enzyme to confirm hydrolysis of PET fibres.

PETase S121E_D186H_R280A

Figure 2: The breakdown of PET fibres harvested from our filter into its constitutive parts with a change in enzyme concentration over a 76 hour period.

T88A_S121E_D186H_R280A (SP1)

Figure 3: The breakdown of PET fibres harvested from our filter into its constitutive parts with a change in enzyme concentration over a 76 hour period.

PETase T88A_S93M_S121E_W159F_D186H_R280A (SP2)

Figure 4: The breakdown of PET fibres harvested from our filter into its constitutive parts with a change in enzyme concentration over a 76 hour period.

The PETase S121E_D186H_R280A was able to degrade the highest amount of PET fibres into BHET, MHET and TPA over a range of enzyme concentrations over a 76 hour period and has been demonstrated to be effective for use in our filter system.

Electrical Design

The following is the code which allows us to wirelessly control the delivery of the enzymes as well as the water flow into our filter unit. The code is specific to the Arduino microcontroller, an open source electronic device which is useful for controlling small scale electronic devices. The code on the Arduino controls a relay and 3 valves turning them on and off which both restrict water input from the washing machine outlet and opens the enzyme vat allowing our enzyme solution to fully submerge the trapped microplastic and degrade them. A prototype setup of the filter system can be seen below, as well as some of the code used by the Arduino for valve control during basic testing.


//Add dependencies
// IRremote - Version: Latest 
#include 
#include 
#include 
#include 
#include         

//Predefine Variables
int X;
int Y;
int Z;    //Define variables X, Y and z
int in1 = 8;  
int in2 = 9;
int in3 = 10;  
int in4 = 11;  //Define Valve pinout
float TIME = 0;
float FREQUENCY = 0;
float WATER = 0;
float TOTAL = 0;
float LS = 0;   //Add floats for dependent variables
const int input = A0;  //Sets constant input value of "A0"
int Win = 13;   //Defines WiFi input pin
LiquidCrystal lcd(7, 6, 5, 4, 3, 2);  //Define LCD pinout
IRrecv irrecv(Win); //create new instance of receiver on WiFi input pin
decode_results results;  //Returns results from decoder 

//Begin setup
void setup()
{
  Serial.begin(9600);
  lcd.begin(16, 2);
  lcd.clear();
  lcd.setCursor(0, 0);
  lcd.print("Water Flow Meter");
  lcd.setCursor(0, 1);
  lcd.print("**************");
  delay(2000);    //Displays "Water Flow Meter" for 2 seconds before enabling loop
  irrecv.enableIRIn();  //Enables WIn
  pinMode(input, INPUT);  
  pinMode(Win, INPUT);   //Sets pin modes for "input" and "WIn" to "input"
  pinMode(in1, OUTPUT);
  pinMode(in2, OUTPUT);
  pinMode(in3, OUTPUT);
  pinMode(in4, OUTPUT);  //Sets pin modes for valve pins to "output"
  digitalWrite(in1, HIGH);
  digitalWrite(in2, HIGH);
  digitalWrite(in3, HIGH);
  digitalWrite(in4, HIGH);  //Sets all valves to high signal by default
}

//Begin Loop
void loop()
{

  X = pulseIn(input, HIGH);  //X reads for HIGH pulse on the input pin
  Y = pulseIn(input, LOW);   //Y reads for LOW pulse on the input pin
  TIME = X + Y;   //Time is recorded for length of HIGH or LOW pulse on input pin
  FREQUENCY = 1000000 / TIME;
  WATER = FREQUENCY / 7.5;
  LS = WATER / 60;   //Calculating values for LCD from X and Y
  if (FREQUENCY >= 0)
  {
    if (isinf(FREQUENCY))
    {
      lcd.clear();
      lcd.setCursor(0, 0);
      lcd.print("VOL. :0.00");
      lcd.setCursor(0, 1);
      lcd.print("TOTAL:");
      lcd.print( TOTAL);
      lcd.print(" L");  //Displays values of 0 for zero flow, volume (would div/0 otherwise)
    }
    else
    {
      TOTAL = TOTAL + LS;
      Serial.println(FREQUENCY);
      lcd.clear();
      lcd.setCursor(0, 0);
      lcd.print("VOL.: ");
      lcd.print(WATER);
      lcd.print(" L/M");
      lcd.setCursor(0, 1);
      lcd.print("TOTAL:");
      lcd.print( TOTAL);
      lcd.print(" L");  //Displays output variables on LCD
    }
  }
  if (irrecv.decode(&results)){
    irrecv.resume();
  } 
 
  //Test Loop - 5s toggle
  if (digitalRead(Win) == HIGH) {  

    digitalWrite(LED_BUILTIN, HIGH);
    digitalWrite(in1, LOW);
    digitalWrite(in2, LOW);
    digitalWrite(in3, LOW);
    digitalWrite(in4, LOW);
    delay(5000);
  }
   if (digitalRead(Win) == LOW) {  

    digitalWrite(LED_BUILTIN, LOW);
    digitalWrite(in1, HIGH);
    digitalWrite(in2, HIGH);
    digitalWrite(in3, HIGH);
    digitalWrite(in4, HIGH);
    delay(5000);
  }
  delay(500);  //Toggles all valves every 5 seconds with a 500ms delay in between each toggle
}
}

The WiFi module integrated into the final filtration system would monitor how much enzyme solution has been passed into the filter, based on the number of washes. It will then communicate this information to the user via an app interface, that we have published, giving them the peace of mind about refilling the system.

The prototype app will show the user how many fibres have been degraded and the number of fish that have benefited from using the PETexe filter, it gives direct feedback about the positive impacts they are having on the environment, showing them that they are making a real difference!

To fully appreciate the app please download it or come see it in action at our demonstration table.