Biosecurity and Martian Legal Review

Introduction

The use of genetically modified organisms (GMO) in industry is a controversial topic terrestrially, with concerns over their interactions with wild organisms and unknown long-term impacts on health. It is therefore logical that there would be even graver concerns with using GMOs in space, an environment where terrestrial life is largely untested and the bacterial reaction to microgravity is poorly understood. There is recent evidence that bacterial response to microgravity could even be considered dangerous due to its persister like reaction making it highly antibiotic resistant.

However, the use of GMOs is now considered a possible solution to many of the issues currently impeding further space travel. NASA are exploring both the use of genetically modified algae in integrated life support systems and the use of GMOs to synthesise useful materials for constructing and maintaining habitats is widely documented.

To facilitate the expansion of synthetic biology into the extra-terrestrial arena, there would need to be a paradigm shift in the legal framework surrounding GMO use and a reconsideration of the function of planetary protection laws. Comprehensive risk-benefit analysis would need to be undertaken in order to rework any legislation, compensating the risks of cross contamination with highly efficient life support systems and habitat creation. To do this, it must first be understood what current measures are in place and how these would need to be reworked to facilitate change.

Laws governing GMOs in Space with specific reference to Mars

Mars poses unique risk when considering contamination; life has yet to be found on Mars and as such we have no idea how it would interact with terrestrial organisms or even if it uses DNA as genetic material. As such it could pose a unique danger to human life, as is often popularised in the media. However, the reverse is also true. Our highly sophisticated immune systems and the array of microorganisms we bring with us could feasibly outcompete any life that there is on Mars. This would deprive us of the opportunity to study a completely new tree of life and answer questions on how life originally formed on our own planet.

Many of the laws governing space itself take reference from those governing Antarctica, meaning no country can claim sovereignty over the territory and it becomes an area purely for research purposes. Whilst these laws are currently in place and the international nature of current space operations such as aboard the ISS reign, it is unlikely that claiming territory will become an issue. However, this may change in the future as exploration expands and colonisation becomes a distinct possibility.

Planetary Protection Protocols

The design of the planetary protection protocols (PPP) for Mars are based on a two-way system, firstly that no terrestrial organic material will come into contact with the Martian Biosphere and secondly that no Martian organic material will come into contact with the Earths biosphere. NASA categorises each Mission depending on both the type of planetary body and the mission type. These categorisations are determined by the Space Studies Board and international policy guidelines. Missions are categorised I-V and the cleanliness of the spacecraft will depend upon the category the mission is given. For example, a category V mission will undergo more sterilisation procedures than a category I mission. Planetary protection guidelines then feed into mission design through the work of the committee on space research (COSPAR), who shape the design, development, and operation of spacecraft and missions headed for Mars.

COSPAR has designated that Mars itself has its own subdivisions of categorisations for Missions;

IVa – Lander/ probe – ‘Lander systems not carrying instruments for the investigations of extant Mars life

IVb – Lander/probe – ‘Lander systems designed to investigate extant Martian life’

IVc- Lander/probe – ‘Missions investigating Martian Special Regions, even if they do not include life detection experiments. Martian Special Regions include those within which terrestrial organisms are likely to replicate and those potentially harbouring extant Martian Life.’

All Mars missions are categorised as IV minimum. There is currently no categorisation for a manned mission to Mars, however NASA is currently planning safety procedures for such an eventuality.

Whilst all of Mars is considered significant in terms of planetary protection, as referenced in category IVc missions, there is a system of regions of special interest on Mars called ‘Martian Special Regions’. COSPAR defines a special region as ‘areas on Mars where terrestrial life may have the potential to proliferate if introduced’. In 2013 the special regions science analysis group (SR-SAG2) was established to update the areas of Mars defined as ‘special regions’ based upon the latest discoveries of both the Martian surface and extremophile organisms. The report was reviewed by both NASA and the ESA before being published in 2014. The main finding of the report was to add methane sources to the physical parameters indicating a special region. They also determined that Maps should not be relied upon to indicate whether a landing site was in a special region and instead that each landing site should be considered for its own suitability to form a special region. They also recommended that sites considered ‘special regions’ should be reconsidered every two years based upon the recent advances in science.

Current sterilisation techniques employed by space agencies

The aim of sterilisation techniques employed by space agencies such as NASA is to minimise the biological burden placed on spacecraft. The amount this burden must be limited to depends on the mission category, with category V being the most stringent and category I the least. Category V missions must also detail their plans for safeguarding Earths biosphere from extra-terrestrial contamination, the two-way containment system. The current sterilisation techniques employed revolve around a series of clean rooms and microbial barriers, chemical cleaning and sterilisation, prevention of recontamination, preventing impacts and contamination of solar system bodies, and bioburden assay methods.

Mission categorisation also determines what sections of spacecraft need to be cleaned, for example for landers or rovers where only certain parts of the machine are exposed to the surface, only these parts must meet the required contamination protocols. This assumes that the internal sections were sterilised and assembled in a clean room of category ISO class 8 or above. This is similar to the sub- system approach employed by NASA whereby ‘only the components that contact the planetary surface are required to reach the most stringent cleanliness requirements’, this means that the lander would need to meet the requirement, but any capsule would not necessarily.

The categorisation for these missions, in particular to Mars, were developed from the biological burdens found on the Viking spacecraft. The resulting probability limits for inadvertent contamination of liquid water body, or special region of Mars, are 1x10-4. In order to minimise the risks of inadvertent contamination missions must fill out;

 Pre-launch report- describing how compliance is achieved  Post-launch report- analysing the planetary protection practices such as impact probabilities, contamination probabilities and spacecraft bioburden  End of mission report- comprehensive analysis and assessment of final decommissioning of the spacecraft  Extended mission report – should the mission require extension this will detail the timeline and purpose of the extension  Additional documentation includes implementation plans, contamination analysis plans, microbiology plans and microbial reduction plans

The use of clean rooms is significant to control the bioburden of the spacecraft and meet the stringent decontamination protocols required to build spacecraft. Spacecrafts for any mission above category II must be assembled in cleanrooms over ISO class 8 by personnel equipped with hoods, masks, surgical gloves, boots and protective suits to minimise the bioburden of any equipment. Clean rooms use laminar flow systems and advanced filtration systems to limit particle sizes in the room to maximum 0.5um, thus no microorganisms can enter the room. Clean rooms also control any particle density, temperature, humidity and pressure. These methods act as microbial barriers meaning that sterilisation occurring in clean rooms should not allow recontamination. Within the clean room all surfaces are cleaned with ethanol, and any electronic assemblies and batteries are cleaned by low-temperature hydrogen peroxide plasma. NASA approved sterilisation methods include vapor phase hydrogen peroxide (VHP) and dry heat microbial reduction (DHMR). Previous Mars Viking landers were sterilised using DHMR at sub-system and full system levels by being heated at 111.7 degrees Celsius at 1.3mg/mL humidity for 30 hours. These sterilisation methods are now standard for all category IV Mars missions. Other methods have been used at sub-system level including autoclaving inner tubing and gamma radiation sterilization of any parachute.

Whilst all these methods are used within a sterile environment, spacecraft are launched from open air launchpads thereby challenging planetary protection through recontamination. In order to prevent recontamination, spacecraft are specially packaged to prevent contamination from the open-air environment. These measures can include deployable biobarriers, filtration systems reducing particulate transport, overpressure approaches, and assembly techniques. Wrapping typically uses antistatic materials or packaging in special sterilised storage containers. One example of prevention of recontamination includes the use of Tedlar to cover the robotic arm holding the spacecraft, which is only retracted once the arm was ready for use on the Martian surface. Vents on the spacecraft can also be covered whilst in the open air to prevent contamination of the inner systems. Recontamination is the biggest hinderance to ensuring sterilisation for spacecraft whilst they are launched from Earth, and different techniques are required for different stages of launch and parts of the craft.

These prevention methods are designed to minimise contamination not only of Mars, but other icy satellites including Ganymede, Enceladus and Europa. Contamination is believed to be a high risk upon impact with planetary surfaces and to minimise this, trajectory corrections and biases are used. Several factors are considered when calculating points of impact with planets:

 Estimated bioburden at launch  Survival of contaminating organisms during cruise phase  Survival of contaminating organisms in the radiation environment adjacent to target  Probability of encountering or landing on target  Probability of surviving landing of impact on the target  Mechanisms and timescales of transport to the surface  Survival of contaminating organisms before, during and after sub-surface.

To determine the effectiveness of the current techniques of sterilisation various bioburden assay methods have been developed. These aim to measure the bioburden or abundance of microorganisms on spacecraft or their components and in clean rooms. They are essential for checking that spacecraft meet the strict bioburden requirements in order to minimise contamination of solar system bodies. NASA use three primary methods;

 The NASA standard assay  Total adenosine triphosphate assay (ATP)  Limulus amebocyte assay (LAL)

The NASA standard assay ‘enumerates cultivatable heat-tolerant microorganisms where the survivors are used as a proxy for total bioburden’ and mainly finds spore-forming bacteria such as bacilli. It can be sampled using swap or wipe methods. Samples are then heat shocked following plating onto trypticase soy agar and enumerated by CFU after 72 hours. The CFU then forms a proxy for total bioburden of the spacecraft.

ATP assay is a molecular approach that measures the abundance of ATP, a biochemical common to all microorganisms. ATP is measured to be directly proportional to bioluminescence and using the bioluminescence biological contamination is estimated. However, as ATP can also be found in dead cells it is not a suitable method for giving a proxy count of the total viable bioburden of the spacecraft.

Similar to the ATP assay, the LAL assay provides a proxy for organic abundance through the measurement of lipopolysaccharides (endotoxins) which are found in high abundance in the outer- membrane of Gram-negative bacteria. This assay can also test for the presence of yeasts and mould, making it an effective measure for spacecraft bioburden but not for total viable cells.

Risk of GMOs on Mars

The extensive sterilisation protocols put into place by space agencies such as NASA means that unwanted contamination is minimised, thus the chances of interactions between GMOs and terrestrial bacteria are minimised. Because of this, there is a minimised chance of horizontal gene transfer between terrestrial microorganisms and so many of the impacts that a GMO could have in a controlled environment are predictable. This is especially true when we apply the principles of synthetic biology in tightly controlled environments such as for use in life support systems such as bioreactors. In accordance with NASA, for category IV missions only exposed components need to be sterilised and as the inside of a bioreactor is a self-contained environment which would not be exposed to the outside world, the Planetary Protection protocols would not be as strict governing GMOs in these environments. There would evidently need to be mechanisms which could kill any bacteria which may attempt to escape but within a closed system security risks would be minimal.

The main risk of contamination of Mars would come from humans themselves. Humans contain a rich microbiome containing many species of bacteria, the majority of which live in the gut but also inhabiting the mouth and the surface of the skin. These bacteria would be much harder to contain and prevent contamination of special regions on Mars. However, these bacteria are also highly evolved to live in the habitat that humans provide; warm and with high amounts of liquid. Therefore, these are not the extremophile bacteria that would be able to survive on the Martian surface or sporulate and lie dormant on the surface until suitable conditions presented themselves. So, whilst contamination of the Martian surface could be a possibility from manned missions to mars, colonisation of the surface by such bacteria is unlikely due to the conditions presented by the Martian surface itself.

Lastly, the Martian surface is believed to be inhospitable for many bacteria to thrive and as such, many species of bacteria are believed to be deep sub-surface colonies. As has been theorised by Professor Charles Cockell, this means that whilst we should aim not to contaminate the surface of Mars with terrestrial organisms, even if it should happen it is unlikely that they would interact with the Martian Biosphere and thus any Martian organisms which we found would be left intact. This fact does not negate the concept of planetary protection, which is irrefutably crucial, however it does qualify the taking of certain small risks for the advancement of our knowledge of Mars. Using synthetic Biology to help humans survive on Mars is proving to be a very feasible concept and the risks of any minute contamination happening should result in allowances being made in planetary protection protocols.

Benefits of GMOs on Mars

Genetic modification is a viable method for optimising organisms for human use. On Earth we use genetic modification for a wide range of reasons, from insulin production in diabetics to pesticide resistance in crops. It is accepted that it would be difficult for humans to survive on Mars, even for a small, well equipped research group. Genetically modifying organisms could help to alleviate many of the existing problems.

NASA is already researching how algae could be used to design closed system life support for the astronauts on Mars and, expanding upon the principle of insulin production, we could feasibly create many other chemicals which would help humans stay on Mars for longer periods of time. As previously alluded to, many of these processes need to happen in tightly regulated, enclosed environments and as such contamination risks are minimal. Furthermore, Mars has many natural resources which could make these processes highly sustainable. For example, perchlorate salts or natural minerals found in the Martian regolith which could be used by bacteria to produce products useful for humans.

In a way, the methods of surviving on Mars are similar to the methods of cultivation practices by stone age humans. We use the technology available to us to help us survive in new environments and stay for longer periods of time. Where we once used selective breeding to select for desirable traits, we now use molecular Biology to insert desirable genes into target organisms so that we may survive in a more hostile environment.