Proof of Concept

Expression and Integration of Protein into Bacterial Cellulose Using a Co-culture

One of the key objectives of our project is to be able to integrate our antimicrobial protein and our biodegradable plastic Polyhydroxybutyrate (PHB) into our bacterial cellulose (BC). To streamline the integration process our team went forward with a co-culture. The co-culture contains recombinant E. coli that produces nisin and PHB and the other is the BC-producing bacteria, K. xylinus. Our team used green fluorescence protein (GFP) to prove how proteins and molecules can be integrated as the BC is produced.

The preliminary tests with GFP producing E. coli (GFP E. coli) included the addition of GFP E. coli in a test tube with K. xylinus producing BC. The BC produced in the test tube were green as outlined in Figure 1, indicating that GFP was present within the BC biofilm.

Figure 1. Three preliminary co-culture in a test tube with K. xylinus producing bacterial cellulose and GFP E. coli. Samples are shown under a UV light.

The next iteration of this test was to set up our co-culture systems on a petri dish, which provided a larger surface area to better observe the distribution of GFP throughout the palette. The distribution of GFP is evident in Figure 2, where there are different levels of brightness of the GFP, indicating that the protein has been produced as the layers of BC were secreted. Folding the BC to create a thick layer allowed for a more vibrant green from the GFP E. coli, making the presence of GFP more prominent.

Figure 2. Co-culture in a petri dish. Petri dish on the left is a control with bacterial cellulose containing no GFP E. coli. Petri dish on the right contains GFP E. coli integrated within the bacterial cellulose fibers. Samples are shown under a UV light.

Figure 3. Folded bacterial cellulose produced in a co-culture. Samples are shown under a UV light.

Therefore, these tests have demonstrated that E. coli producing a protein in a co-culture will be integrated within BC fibers and remain there.

However, BC serves as a fruit packaging material for the team. For this reason all bacteria needs to be removed from the packaging. We used an autoclave machine which is used to sterilize lab equipment using high temperature steam. The efficacy of the autoclave BC was tested by autoclaving our BC samples and then using aseptic technique placing a piece of that sterilized BC onto fresh agar plate to observe for bacterial growth. This technique of sterilization was deemed effective as no bacteria grew on our agar plate post autoclaving BC.

This technique of sterilization was then tested on our co-culture BC as high temperatures have the ability to sterilize samples but also the ability to denature proteins.When the autoclaved co-culture BC was observed under UV light the green from GFP E. coli was still present. Hence, we knew that GFP protein was retained amongst the BC fibers, even after sterilization and purification, and a similar process will be used for integrating nisin into BC.

Figure 4. A control bacterial cellulose which has not been autoclaved and does not have any GFP E. coli present. On the right is an autoclaved bacterial cellulose sample consisting of lysed E. coli cells and GFP remaining on the BC. Samples are shown under a UV light.

Immobilization of Nisin into Co-cultured Bacterial Cellulose and Characterization of Antimicrobial Properties

To test Cellucoat’s preservative properties, we combined several elements of our project to demonstrate our project’s potential for a preservative packaging. Elements included using our lab-grown BC produced in a co-culture which was then sterilized using our autoclave and NaOH treatments (click here), as well as immobilizing nisin into BC, and characterizing its antimicrobial properties in a petri dish and on the surface of fruit (click here).

Using our co-cultured BC, we immobilized nisin into the material and conducted a series of Kirby-Bauer disc diffusion tests against Bacillus subtilis. This experiment demonstrated Cellucoat’s ability to combine its production and preservation aspects, and its ability as a packaging to kill gram-positive bacteria.

Figure 5. Kirby-Bauer disc diffusion test comparing nisin (N), water (W), thymol (T), and phenol (P), against B. subtilis. Results were used for analysis using KB-Perry

We also used our co-cultured BC with immobilized nisin to test our products application as a fruit packaging. We created a timelapse video that spanned over the course of 12 days and observed whether our packaging would prevent rotting and spoilage on grapes. After 12 days, we removed our BC samples to reveal that they had noticeably prevented deterioration compared to the uncovered surface of the grape.

Figure 6. Results for 12 day timelapse of nisin and BC paper spot test to protect grapes from spoilage. Spot 1 shows nisin immobilized onto BC paper, spot 2 shows BC only, and spot 3 shows nisin only.

Integrating PHB into Bacterical Cellulose and Characterizing its Properties

After conducting HP interviews and testing the mechanical properties and strength of the BC our team was planning on using as the material for Cellucoat, it was found that BC is very weak. For comparison, when less than 2 mm thick, is 500x weaker than plastic of the same thickness, and has a strength akin to that of paper.

To remedy the poor mechanical strength of BC, our team decided to integrate PHB into the BC in a manner similar to a plasticizing agent that creates a more durable final material. The manner in which the team planned on doing this was through a co-culture with an E. coli strain recombinantly producing and secreting PHB directly into the BC fibers. However, the PHB-producing E. coli was not prepared yet, and our team needed a way to prove outside of literature that PHB can improve the mechanical properties of our material.

Hence, our team decided to use pure solid PHB that came as granules and dissolve it into a 0.25M solution with 100% Acetic acid. 1.5 mL of the 0.25 g PHB per 1 mL of acetic acid solution (0.25 g/mL) was poured into BC palettes that have been culturing for three weeks at 30 degrees and being fed using a 24 hour schedule with 4 mL of HS media. After both samples have grown, they were autoclaved, purified using NaHCO3 solution and air dried.

Figure 7. Image of BC (right) and BC and PHB composite (left) after being purified for four days with a 0.5M NaHCO3 treatment. The samples were left out to air dry for three days and then were cut up and sent to uniaxial testing.

After drying, the samples were cut to size for uniaxial tests. The uniaxial testing looked at the tensile strength, stiffness, and stretchability of the BC and BC and PHB composites. Based on these results, BC showed an ultimate tensile strength 3.780MPa higher than PHB-BC, which means that BC only could withstand a larger force before tearing. The Young’s Modulus of pure BC was also 758.2MPa higher than composite PHB-BC. This value indicates that pure BC is the stiffer of the two polymers, and is less flexible. Finally, the maximum elongation of composite PHB-BC was 0.02868 higher than pure BC, meaning the PHB and BC composite was more flexible and able to be stretched.

The reduction in strength of the PHB BC composite can be attributed to the uneven distribution of PHB within the BC due to the ex situ method used to incorporate PHB. Furthermore, 100% glacial acetic acid has an approximate pH of 2.3 which was observed to cause a phenomenon called acid hydrolysis of the BC. Acid hydrolysis cellulose is a classic way to break down cellulose into glucose and can be done using a concentrated acid. This may have also contributed to the less than optimal strength performance of the BC and PHB composite, as the BC itself has been broken down and weakened from the incorporation of PHB using a strong acid as a solvent.

Taken together, these uniaxial tests provide sufficient proof that the concept of integrating PHB provides a benefit to the properties of the packaging material, as it increases the flexibility and stretchability. These findings further motivate the use of a co-culture approach to incorporate the PHB in situ as the BC fibers form.

Production of a BC Box With BioSculpting

While producing bacterial cellulose remains relatively simple, turning the material into something beyond a biomaterial sheet was a challenge. However, in order to demonstrate that BC could serve as an effective packaging material, creating a prototype was of the utmost importance. From our HP interviews, we decided to create a clamshell alternative to target a widespread, near-ubiquitous form of packaging.

To create this, we utilized molding and corrugation techniques to create BC cardboard, a layered form of BC that provides additional strength to supplement our PHB production. Using this material, we were able to produce numerous box prototypes in a variety of sizes to demonstrate the feasibility of our production method.

In order to further develop BC as a viable material for packaging, we optimized parts of the production and purification pipeline, producing BC that was stronger, more transparent, and less brittle than raw BC. We did this through purification using NaHCO3 solution and through air drying, developing a material much more comparable to the plastics used in clamshell packaging. By taking this optimized BC and combining it with the BC cardboard workflow, we were able to produce a strong, durable box, serving as an effective proof of concept for the BioSculpting protocols.