Proposed implementation

Introduction

Due to limited resources in space, and the fact that long-term space missions will keep needing medical therapeutic biomaterials for their crew, it is prudent to have a biomaterial that is versatile and can also be produced on board or in the exoplanet. A versatile material for use in medicine today is cellulose, which can be produced both by bacteria and plants. While it is convenient to use plants to obtain cellulose on earth, where space and resources are readily available, space requires different solutions. Because of this, we focused on using the bacterium, Komagataeibacter xylinus, to produce cellulose. While cellulose is a versatile and useful material, it is less than optimal as a biomedical material due to its high crystallinity and relatively low biocompatibility [7]. Because of this, we focused on solving this by producing a copolymer of chitin and cellulose, which we call CellulALT, in situ from simple sugars.

K. xylinus using glucose and n-acetylglucosamine as monomers to create the polymer CellulALT.

Device design

Our material will be produced as a hydrogel, which is the normal way K. xylinus produce cellulose. Our modification includes the expression of four genes from Saccharomyces cerevisiae by the transformation of K. xylinus. With these four genes, the concentration of UDP-acetylglucosamine (UDP-GlcNAc) will increase and allow for it to enter the cellulose synthesis complex, incorporating UDP-GlcNAc in addition to normal cellulose monomers, UDP-glucose (UDP-Glc).

The hydrogel is harvestable using normal separation techniques such as centrifugation and purified in a simple acid bath followed by CO₂ purification. The product can then be used directly as a hydrogel or further processed into pills, scaffolding materials, etc.

Device production

The material can be produced in any bioreactor that contains a supply of oxygen and a source of glucose. In addition, the bacteria should have a source of essential trace metals and available sulfur and nitrogen. Current and potential constraints and possible solutions will be discussed later in this section.

Human safety

Neither cellulose nor chitin are very toxic on their own and would therefore not be considered to have hazards when used by or in humans for biomedical practices. The largest potential hazard with this product, when used in medical devices, is the long-term chronic effects due to immune reactions [6]

Product disposal

Both cellulose and chitin are biodegradable when in proximity to cellulases and chitinases. Due to this, it is unlikely that the released product could stay in the environment for longer than a short time before it is completely degraded by microbes if available.

Implementation plan

Upon successful engineering of our bacteria which can produce CellulALT, it could be an indispensable part of space exploration and an integral part of biomaterial production here on Earth. Here, we will discuss the following:

A) Therapeutic implementation on Earth and in Space and proposed end users
B) Challenges for production in space, and our proposed solutions for them.
C) Challenges for production on Earth, and our proposed solutions for them.
D) Insight from our iGEM partner, team UniCAMP
E) Need on Earth vs need in space

A) Therapeutic implementation on Earth and in Space

Our co-polymer, CellulALT would be used mainly by medical experts for the treatment of various ailments. Our co-polymer already has multiple potential uses today on Earth, and even more possible uses in space in the future. A summary of the variety of possible uses can be explained as follows:

The various possible applications of our co-polymer “CellulALT”.

Sustained drug delivery material:

human practices

Lysozyme acting on n-acetylglucosamine to break down the co-polymer.

Our co-polymer in hydrogel form could be infused with drugs. The drugs will be contained within the 3-dimensional hydrogel matrix. CellulALT as a vector for drugs.

Once inside the body, various enzymes such as lysozymes and chitinases could break down the CellulALT matrix by targeting the n-acetylglucosamine monomers. Over time, the biodegradation of the copolymer matrix would then result in the release of the drug from the matrix. CellulALT for disease treatment.

A sustained drug delivery method such as this could result in the following:

Lessen the need for repeated intake of a drug
Ensure more precise delivery of drug to target tissues
Possibly make the need to repeatedly wash cancer wounds with anti-cancer drugs redundant

Probable end users can be:

Cancer patients recovering from surgery who would have CellulALT in their wounds to help keep dosing the wound tissue to ensure the death of any any leftover cancer tissue or cell.
Space surgeons: Even for non-cancer surgical treatments, a non-fluidic drug dosing tool like CellulALT would be indispensable to maintain repeated on-site dosing in the microgravity environment. CellulALT as a solid hydrogel has less chance of displacement.

Scaffolds for tissue regeneration:
Bacterial cellulose has also been tested for its potential as a scaffold to aid in proper tissue regeneration with promising results. So, CellulALT could also be used for the same purpose with the added benefit that it has the potential for easier biodegradation.

Probable end users:
Patients requiring scaffolds: CellulALT may have the potential to act as a biodegradable scaffold for various tissues requiring physical support during recovery. The potential slow-release capability has the added benefit that compounds that can aid in regeneration could be added to the CellulALT scaffold. The compounds would be released as the CellulALT degrades. The end result could be better healing with no need for removal of scaffold.

CellulALT as a scaffold in tissue regeneration.

Biodegradable bandages:
Wounds produce biodegrading enzymes such as lysozymes that could easily break down CellulALT over time. Hence, in using it as a bandage or wound dressing, it can be left on and the body itself would take care of its removal as the tissue heals.

Probable end use can be:
To treat chronic wounds: People with chronic wounds would benefit from a potentially biodegradable bandage made from CellulALT as it would not require frequent removal like traditional bandages.
Skin grafts: Skin grafts could be supported by CellulALT while they regenerate together with the tissue they are applied on.

Dietary fiber:
Both bacterial cellulose (the polymer of glucose) and chitin (the polymeer of n-acetylglucosamine) have been researched for their potential use as dietary fiber substitutes to plants. Hence, the co-polymer CellulALT, which is made from monomers of these two polymers, could serve the same purpose.

B) Challenges for production in space, and our proposed solutions for them:

1) Oxygen demand: A challenge for the use of our engineered bacterium would be the need for oxygen as it is an aerobic bacterium [1, 2]. In space, oxygen a limited resource necessary for humans as well as other life for maintaining food sources. This was one of the concerns that was raised by experts as can be seen on our Integrated Human Practices section. Input from expert that led us in considering this problem: Ingrid Bakke (See Integrated Human Practices) Our solutions-
- a) Photosynthetic aid: Oxygen demands from non-human sources could be offset by photosynthetic organisms on board. If the colony is within the Goldilocks Zone of a star in terms of light, solar energy could be harnessed by photosynthetic plants and bacterium.
- b) Culturing strains capable of growing in O₂ deficient conditions: In cases where photosynthetic organisms cannot be relied upon for oxygen production due to distance from a star, it may be necessary to create strains of K. xylinus that have reduced oxygen consumption whilst still being functional [3]. In addition, bacterium is easier to store when its need as a producer is not needed, as it can easily be cryopreserved and expanded when necessary.
2) Space in Space: Building separate housing units where the bacteria would be grown away from the other functioning units would create problems. Separating compartments in outer space and on exoplanets would result in more compartments that are at risk of malfunctioning, require more resources to build, protect and contain them. Our solution: Integrating the compartments where the bacteria is grown with compartments where humans live would solve these problems. Instead of creating a unit where the bacteria, or even other things are grown, and another where the humans live, the two can be joined together. The problem and solutions with energy demands can even be explained as a topic of its own in the next point.
3) Energy Cost: The optimal pH for the growth of the bacteria and its polymer production is around 30°C. Assigning separate energy aliquots for the maintenance of this optimal temperature for the growth compartments could be costly for long duration space missions.
Our solutions:
- a) Growth compartments integrated with living spaces: The bacterial growth compartments could be integrated with living compartments for humans that have temperatures close to the optimal temperature for the bacteria. The connected bacteria and human compartments could equilibrate in temperature. This will also ensure that heat energy used to maintain human life will not be lost without use before dissipating into the surrounding environment.
- b) Renewable energy sources: Renewable energy sources like sunlight are still available outside of earth and so the energy obtained from this will be of use to maintain temperature. Even outside of the reach of sufficient solar light, other renewable energy sources like geothermal energy from the exoplanet where the mission is stationed would be an option. It is not likely that space exploring humans will choose to be locations that are completely devoid of materials that they would need.
4) Nutrients for growing the bacteria: In terms of nutrient requirements, the optimal carbon source is simple sugars like monosaccharides and disaccharides. While these can be produced abundantly on earth from photosynthetic plants, those options become limited in space. The bacteria also need a nitrogen source. The availability of these resources needs to be considered as space explorers who are going on decades to possibly centuries long missions cannot bring everything that they will ever need for that mission. So, besides resources like food that they would already need, carrying extra food for the bacteria may not be an option.
a) Standard media: In the case that the long exploration mission is not just a space colonization mission, but also a scientific mission, considerations could be made for also carrying the reagents for standard HS media that the bacteria can use, that other scientific experiments would also use.
b) Organic waste on board: Komagataeibacter xylinus and even other species from the genus can use organic waste to obtain essential elements for their growth and bacterial cellulose formation. Although K. xylinus may prefer simpler compounds for bacterial cellulose formation, the lack of these in heterotrophic organic waste could be circumvented by integrating anerobic waste digestion systems that have been researched for space. They could break down waste to make available the simple compounds that our bacteria could use more efficiently to make the co-polymer. Additionally, the integration of an anaerobic waste management system would mean that there is not an extra oxygen demand. Furthermore, the use of organic waste to create a polymer that is again biodegradable would result in a sustainable process for creation of therapeutic materials.

K. xylinus using glucose and n-acetylglucosamine as monomers to create the polymer CellulALT.

5) Harvesting in space: On earth, when bacterial cellulose is formed in liquid media near the air and water interface in a compact flat mass covering the entire surface. This makes it easily harvestable right from the medium’s surface. The liquid surface can exist and be stable on Earth because of sufficient gravity. In the microgravity conditions of space, the maintenance of a stable liquid-air interface near which the polymer can form may be difficult. With no gravity to stabilize and hold down the liquid growth media, slight jostles and movements could result in uncontrolled movement of the liquid media and disturb the even formation of the polymer. This may not only make harvesting the polymer formed difficult, but also make further processing more complicated. Input from expert that led us in considering this problem: Eric Thompson (See Integrated Human Practices)

Our Solutions: a) Centrifuging compartments: The use of centrifugal force could be a possible way to create artificial gravity conditions in space. Living spaces on space exploratory vehicles would probably be centrifuged to create artificial gravity for the health and benefit of the crew. Hence, the components of that would contain the bacterial growth medium could be built/attached with the centrifuging living spaces. This will also save energy as mentioned above in the problem of energy cost. b) Exoplanet’s gravity: If the mission or colony is stationed on an exoplanet or any celestial body with sufficient gravity, that should be enough to provide much-needed stability.

C) Challenges for production on earth, and our proposed solutions for them.

As mentioned in various literature and even from an expert that we contacted (see our Integrated Human Practices), the main bottleneck in the industrial production of bacterial cellulose is the cost of producing it. Our solutions:

a) Alternative growth media: There have been several research that have investigated more cost-effective growth media for bacterial cellulose growth. For the growth of our co-polymer, these alternative “cheaper” growth media should be investigated [4, 5].
b) Organic waste: Organic waste itself could be used to grow the polymer without having to rely on standard media or even “cheaper” alternative standard media as mentioned above. Growing bacterial cellulose in mixed medium like for example, waste from the orange juice industry that our iGEM partners (UniCamp Brazil) propose to use, would of course result in a polymer that needs further purification steps before it can be used for biomedical applications. But using organic waste could be a really feasible and even sustainable method to produce our polymer which is mentioned in our Sustainable Development Impact Page.

D) Insight from our iGEM partner, iGEM team UniCAMP

With regards to the requirements of our bacteria to make the co-polymer, we used mathematical modeling that they designed for us to visualize how the co-polymer would be formed in different conditions of resource availability. The result of this can be seen in our model page.

E) Need on earth vs space

There are indeed many therapeutic applications for CellulALT, but due to its possibly relatively expensive production costs (like bacterial cellulose) as compared to other therapeutic biomaterials, its use and production is limited. On earth, we still have much cheaper alternatives from cotton to chemically formulated therapeutic materials that impede the adoption of bacterial cellulose. However, in space and beyond earth, due to lack of space and other resources, therapeutic biomaterials such as CellulALT could become an indispensable and sustainable part of space exploration.