Techniques for cleaning and sterilizing

Many techniques are available to the spacecraft engineer to ensure that a spacecraft has its bioload reduced and its biorelevant contamination minimized to acceptable levels (Ulrich, 1966). It is rare to find a situation that merits the application of only one method, and in general a suite of methods is chosen with particular processes being applied to specific subsystems according to their compatibility; see Debus et al. (2002), for a flight mission example.

14.3.1 Filtration and intrinsically clean assembly

Clean assembly techniques require that the bioload of components is monitored and tracked throughout the build process, with items being stored in sterile containers along with witness plates. Rigorous traceability of processes, such as soldering and fastener attachment, are needed with bioload reduction and monitoring being applied to tools and build environments where necessary, to ensure that the bioload of the finished spacecraft is understood with confidence during its assembly.

14.3.2 Thermal stress

Most known microbes cannot endure temperatures much in excess of 110 °C when alive, and few can survive in habitats with wide temperature ranges. However, in the dormant spore phase, both fungi and bacteria can endure wider temperature extremes, with extreme cold being far less of a threat than extreme heat. Heating both desiccates the already water-depleted spore, and damages the

Table 14.2. An abbreviated description of current COSPAR planetary protection regulations

Category Mission target

Comments

The Sun, The Moon, Venus, certain classes of asteroids Bodies with no direct relevance to the study of life or chemical evolution

The gas giant planets, comets, TNOs, carbonaceous asteroids. Mission targets relevant to the origin of life or chemical evolution. If contamination is taken to such bodies, future missions should not have their science compromised

Mars, Europa. Mission targets are significant to the study of life's origin and chemical evolution

Landers to Mars and Europa. Spacecraft to these destinations could jeopardize the scientific return of future missions

Mars landers without payload to study extant life

Essentially no steps have to be taken to ensure compliance Terrestrial biota are killed by the destination's environments, and no in-situ biota are expected

No specific changes are needed for missions hardware or design Documents should be prepared that detail the post-mission and failure contingencies for the mission

Detailed spacecraft construction documentation needed, may include an inventory of organic matter onboard. Cleanroom assembly, and implementation of bioburden reduction procedures beyond clean working. Orbit biasing to lower collision risks or whole craft bioload to be <5 X 105 spores

Requires more detailed documentation than Category III, assays of bioburden, a probability of contamination analysis, and an organic matter inventory. Extra requirements may include trajectory biasing, assembly in cleanrooms, bioload reduction, and partial sterilization of landed hardware. The requirements are akin to those of the Viking landers, with the exception of whole spacecraft sterilization

Bioload to be no greater than Viking lander pre-sterilization levels, compliance to Category IV in general. Bioburden on exposed surfaces to be an average of <300 spores m~2, and the total vehicle surface burden <3 X 105 spores

Category Mission target

Comments

Mars landers with payload for the study of extant life

Mars landers with payload for the study of extant life which visit regions of special scientific interest; such regions are places where terrestrial microbes may thrive or where native life may prosper

Either the whole landed spacecraft is to be as sterile as the Viking landers, or to limits dictated by the payload's detection limit. Or, the sub-systems in the sample acquisition chain should be sterilized and prevented from being contaminated by other hardware. Bioload on exposed surfaces to be similar to Viking lander levels (by inference, a total of 30 spores)

If the craft lands in a special region1 then the entire landed craft must be sterile to Viking lander levels (>112 °C for 30 hours). If the craft lands outside this area, parts that can contact the region (wheels, arms, sensor covers, etc.) must be sterilized to Viking lander levels. The whole landed system may need to be sterilized if non-nominal arrival could cause contamination

V All sample return missions to the

Earth or the Moon. A subcategory 'unrestricted Earth return' applies for material from bodies thought to have no native biota

For restricted Earth return, any sample should be contained using a verifiable and fail-safe method after sample acquisition. No uncontained material from the mission's target shall be returned to Earth - the so-called 'breaking of the chain' of contact. Example missions would be those that deliver material from Mars or Europa to Earth

1 Such as an ice-rich region of Mars, or an area showing extant hydrothermal activity.

DNA of the microbe. Thus heating is frequently used as a robust method of irrevocably disabling a microbe since the volume, rather than just the surface, of an object can be sterilized. Figure 14.3 shows the response of two bacilli to prolonged heating, the spores of Bacillus subtilis var. niger are often used as candidates for establishing thermal-kill procedures for hardware because of their high thermal resistance.

In Table 14.3 the survival rates are shown for Bacillus subtilis var. spores in different settings. It is notable that embedded spores tend to survive heating better than exposed organisms.

Table 14.3. The resistance of spores to heating at 120 °C in different settings (after Bruch, 1964)

Spore and environment D10 (hours)

Bacillus subtilis var. niger in asbestos patching cement 2.1

Bacillus subtilis var. niger in solid rocket propellant 2.5

Bacillus subtilis var. niger on paper: vacuum/air at 1 bar 0.3/0.91

Sg o hJ

Bacillus subtilis var. niger Bacillus stearothermophilus

Bacillus subtilis var. niger Bacillus stearothermophilus

120 130

180 190

Figure 14.3. The resistance of spores from two common bacterial species to dry heat.

120 130

160 170

180 190

140 150

Temperature ( C)

Figure 14.3. The resistance of spores from two common bacterial species to dry heat.

Heating brings with it the possibility of damage to components from mechanical tolerances being exceeded by expansion, and by degradation of material properties. For landers that are subject to whole-craft heating there are obvious benefits in establishing a low bioload prior to the final heat-treatment; a lower temperature/ duration process can then be used to meet the specific COSPAR regulation.

14.3.3 Radiation exposure

Both corpuscular and electromagnetic radiation can harm living and dormant cells. Damage to cellular molecules can occur either through the reaction of radiation-formed ions and radicals, or by direct absorption of the radiation. In each case the depth to which the radiation penetrates depends on the energy of the radiation and its ability to lose energy to the surroundings by scattering or by ionformation. Of the many forms of radiation, hadronic (protons, neutrons, atomic nuclei) particles are able to generate ion-tracks most readily, with charged particles

Table 14.4. The features of the types of radiation pertinent to this chapter

Radiation

Effect on biological matter

Comment

Germicidal ultra-violet (UV) light with wavelengths between 100 nm and 300 nm

Beta particles

Protons and alpha particles

UV is absorbed by the DNA bases cytosine and thymine which can then link to each other rather than to their complementary adenines on the opposite side of the DNA

Ion-pairs are generated

Intense ion-trails produced, leading to lethal chemistries upon recombination

Produced by various discharge lamps (mercury, xenon, deuterium, hydrogen). Can damage elastomers and plastics

Limited penetration depths

Modest penetration depths for plausible energy particles

Gamma rays

DNA cleavage

High doses can alter glasses and damage semiconductor junctions being the most efficient at ion-production. Such radiation has a high linear energy transfer (LET). To a lesser extent, electromagnetic radiation can also generate tracks of ions or radicals, but their weaker interaction with matter results in deeper penetration distances for the same particle energy, and so the induced ion-pairs are more sparsely scattered. The DNA repair mechanisms in cells are best able to mend single breaks in the molecule, and so radiations that generate dense localized ion-tracks are more likely to kill a cell or render a dormant spore unrevivable. Table 14.4 shows some features of the types of radiation pertinent to this chapter.

For biological material the most important species formed by radiation are the OH and O radicals, and an organism is harmed at the molecular level either by direct damage to its DNA or through the effect of radicals formed from water during the ionization process.

There are two main drawbacks to the use of radiation as a sterilizing agent; ionizing radiation can alter the electronic properties of semiconductors in an irrevocable manner, leading to memory cells that are unwriteable (frozen bits) or otherwise damaged junctions. In practice, semiconductors used for planetary spacecraft are often encapsulated in 'radiation-hard' packages so as to operate at higher background radiation levels. However, the trend in using more complex and modern integrated circuits leads to smaller junctions which in turn are more susceptible to radiation damage. A modern EEPROM wafer would have at least one junction irrevocably destroyed after exposure to 200 grays of b radiation (Shaneyfelt et al. 1994). This same dose would kill only nine-tenths of an E. coli colony, and a smaller reduction in more resistant species or spores (Figure 14.4).

Micrococus radiodurans

Micrococus radiodurans

Clostridium botulinum (type A) spores

Figure 14.4. The effect of p radiation on the survival of common bacteria (data from Goldblinth, 1971).

Clostridium botulinum (type A) spores

Figure 14.4. The effect of p radiation on the survival of common bacteria (data from Goldblinth, 1971).

14.3.4 Sterilizing chemicals

Certain gases, such as ethylene oxide, chlorine dioxide and paraformaldehyde are toxic to living and spore-form bacteria, either by alkylation or denaturing of vital cell architecture such as nucleic acids. The gases can be delivered to the spacecraft hardware at room temperature and pressure in some form of flow-controlled enclosure. Depending on the agent used, the enclosure around the hardware can be simple, and need only be relatively gas-tight if access to the sterilizing area is appropriately controlled. The properties of the above common sterilizing agents are listed in Table 14.5. The oxidizing nature of all of the compounds is reflected by their general incompatability with organic compounds and their toxicity.

14.3.5 Other gaseous sterilizing methods

Vigorous sterilizing agents can be generated through the electrical excitation of gases. These techniques are considered separately as they require more sophisticated equipment, such as a vacuum chamber, and some means of forming the sterilizing agent, which can be a partial plasma or a gas in an excited but neutral state. Low-pressure neutral vapours of hydrogen peroxide have already been shown to have a useful sterilizing effect (Rohatgi et al., 2001) and at low pressure can be readily ionized by an electrical discharge. The plasma's ions, such as OH~ have a high lethality for bacteria in both spore and live phase. The process occurs at total pressures of less than 10 torr and no significant heat is generated upon exposure of

Compound

Nature at room temperature

Comments

Ethylene oxide (epoxy ethane) CH2OCH2

Chlorinedioxide CIO)

Paraformaldehyde (CH20)n

Hydrogen peroxide

Colourless gas, m.p. -112°C, b.p. 10.8 °C. Density 1.5 that of air. Flash point — 20 °C, auto-ignition temperature 429 °C. Flammable in air at concentrations above 2.6% volume at room temperature

Yellowish green gas, m.p. — 59 °C, b.p. 11 °C. Vapour density 2.4 times that of air. May decompose violently at concentrations >10% by contact with sunlight, mercury, platinum-group metals, carbon monoxide, short aliphatic molecules

White crystalline powder, m.p. 120 °C. Hash point: 71 °C, auto-ignition temperature: 300 °C. Flammable in air at concentrations between 7.0-73.0% at room temperature

Clear liquid, b.p. 150°C. Vapour density 1.17 times that of air. High-concentration solutions decompose in the presence of heat and platinum-group metals.

Ether-like odour, corrosive to eyes, skin, mucosal surfaces. Acute exposure can cause headaches, nausea, convulsions. Carcinogenic in humans at high concentrations. Degrades some fluoro-elastomers, Viton®, and Buna® variants

Pungent odour, corrosive to eyes, skin, mucosal surfaces. Acute exposure can cause headaches, and nausea. A strong oxidizer; can degrade some elastomers and plastics

Formaldehyde-like odour, corrosive to eyes, skin, mucosal surfaces. Suspected to be carcinogenic in humans at high concentrations Can degrade EPDM (ethylene propylene diene, modified) polymers and Buna® valiants Modest concentration solutions used (~30%) to form vapours at low pressures. Relatively low toxicity, wide material compatibility objects to the gas, with the whole operation occurring at room temperatures. Other plasmas, of helium (Fraser et al., 1975), air (Lei et al., 2004), oxygen (Mogul et al., 2003) and nitrogen (Yoshida et al., 2003), have also shown sterilizing capability.

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