Team:PuiChing Macau/Engineering
With the overall project goal to convert food waste into biodegradable and eco-friendly bioplastics, polylactic acid (PLA). Considering food waste contains different types, including Fruit & Vegetable, Meat, Carbohydrates etc. In order to achieve our goal of producing PLA in a one-step approach and promoting its efficiency, we tend to check different data of the food waste that we get. However, this requires innovative molecular design, sophisticated synthesis, and therefore long hour input and expensive resources. Therefore, we carried out the investigation to:
- To analyse the pH value of different type of food waste (Fruit & Vegetable, Meat, Carbohydrate)
- To analyse the growth of e.coli in different pH value
- To improve the production system and help increase the growth of e.coli in acidic environment
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Food Waste Analysis Protocol
Materials:
- 60g of food waste
- 60mL of deionized water
- Blender
- Filter bag/gauze (for filtration of solid food waste)
Protocol:
- Collect 60 g of food waste (e.g. starch, vegetables, fruit, or trimmings)
- Add 60mL of deionized water and 60g of food waste into a blender and blend it until most of the food waste has turned to liquid
- Pour the mixture into a filter bag/gauze to obtain the liquid filtrate
- Analyze the pH value of the liquid filtrate using a pH meter or pH test strips
Figure 1. The average pH value of the three main categories of food ( Fruit & Vegetable, Meat, Carbohydrate)
Based on the data we got in the food waste analysis, the result shows that most of the food waste performs in the range of pH4-pH7 in acidic or neutral environments, which may inhibit the survival and growth of the E.coli. In this case, we have to confirm and analyse the effect on the growth of E.coli in the acidic environment, so we built up a test (CFU (WT4 & WT7) with IPTG to mimic the growth of E. coli)
To analyse if the pH value will inhibit the growth of the E.coli using colony-forming unit (CFU) to estimate the number of e.coli grown in the Agar plate. Counting with colony-forming units requires culturing the microbes and counts only viable cells.
Materials:
- Agar Plates in pH4 & pH7 (calculate the amount of agar plates needed depends on the dilution range)
- Snap cap tubes
- Sample ( BL21(DE3)induced)
Experimental steps:
- After induction overnight, add 200ul sample into 96 well plate and measure the OD of each sample, use LB as a blank.
- calculate the amount of bacteria culture needed
e.g. sample 1 OD: 0.5 sample 2 OD: 0.6 sample 3 OD: 0.55 Use 0.5 to calculate, sample 1—>2ml，sample 2—>1.667ml，sample 3—>1.818ml
Equation: (lowest OD value among the samples)(Final volume) / (OD value)(sample volume) - Separate the samples into 2 groups: pH 7 and pH 4
e.g. sample 1: 1ml for pH7 and 1ml for pH4 - Centrifuge the samples at 5,000rpm to get the pellets
- Wash cells twice with respective pH minimal medium (Add100ul of respective pH minimal medium to the sample and centrifuge with 5000rpm)
- Resuspend the pellets in 2 ml minimal medium (pH 7 and pH 4 for each samples) in snap cap tubes
- Take 25ul from each samples and do serial dilution (For 10-1: 25ul sample + 225 ul respective pH minimal medium)
- Spread plate with the dilution 10-5, 10-6, 10-7, 10-8
Result:
Figure 2. Number of E.coli colonies (WT: BL21 control) in pH4 and pH7
From the graph, we can find out in pH4 and pH7 the total number of e.coli have a big difference, which confirms that the acidic environment inhibits the growth of E. coli. To solve the problem our team has developed an acid-tolerant system using fabB genes to try to increase the production of unsaturated fatty acids by increasing the growth of E.coli in an acidic environment.
Build & Test: CFU
CFU (WT4 & WT7) with IPTG to mimic the growth of E. coli
To analyse if the pH value will inhibit the growth of the E.coli useing colony-forming unit (CFU) to estimate the number of e.coli grown in the Agar plate, and check if the Acid-Tolerant System does help increase the growth of the E.coli in acidic environment. Counting with colony-forming units requires culturing the microbes and counts only viable cells.
Sample: BBa_K3863005 Control: BL21(DE3)
Materials:
Experimental steps:
e.g. sample 1 OD: 0.5 sample 2 OD: 0.6 sample 3 OD: 0.55 Use 0.5 to calculate, sample 1—>2ml，sample 2—>1.667ml，sample 3—>1.818ml
Equation: (lowest OD value among the samples)(Final volume) / (OD value)(sample volume)
e.g. sample 1: 1ml for pH7 and 1ml for pH4
Learn
After adding the Acid-Tolerant system that we have designed, e.coli growth have a significant increase compare to the e.coli without the system in pH4 ( the acidic environment), whereas in pH7, the Acid-Tolerant does not have negative impacts on the growth of the E.coli, the growth range is similar in both with and without the Acid-Tolerant system, which proves that the system does help increase E.coli growth in the acidic environment.
Figure 3a. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 4 (N=4, error bar: SEM)
Figure 3b. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 7 (N=4, error bar: SEM)
Improve
Based on the analysis of the first and second cycle mentioned, our data are slightly in line with the theory, we have proved that the Acid-Tolerant System we created does help to improve the growth of E.coli in pH4, for the further experiment, we aim to have more test on different pH value from pH4-pH7, more specific test to confirm the growth of the E.coli and the effect on our production system.
Furthermore, we found out that Acid-Tolerant System which comprised both FabB and FabA gene is proven to be effective for the growth of the E.coli in acidic environment, we hope that we can have further experiments on these and compare if it is more beneficial or not to add the FabA gene in our Acid-Tolerant System.