Spacecraft Component Sterilization usingSupercritical Carbon Dioxide

Sterilization of Bacillus pumilus Spores using Supercritical Fluid Carbon Dioxide

Containing Various Modifier Solutions

Ronald CrawfordAndrzej Paszczynski

Chien WaiEdison Shieh

In order to embark on a planetary exploration of the Martian surface for microbial life, great care must be taken to prevent any bio-contamination introduced by the visiting spacecraft.

The Need for “Planetary Protection” during Exploration of the Solar System

It is essential to not contaminate its environment with terrestrial biomolecules and/or life forms.

Anonymous. 2002. COSPAR Planetary Protection Policy. 255 http://www.cospar.org/scistr/PPPPolicy.htm

Where Might Extant Life be Found on Mars?

• In or on Rocks (e.g., varnishes)• Caves• Polar Ice• Permafrost• “Deep” Subsurface (source of methane?)• Areas of Nitrogen Salt Accumulation• Areas Maintaining Intense Localized Magnetic

Fields

Strong Release of Methane on Mars in Northern Summer 2003. 2009. Michael J. Mumma, et al. Science 323. (5917), pp. 1041 – 1045.

Many spacecraft are assembled in clean room facilities and subjected to sterilization treatments to eliminate bacterial spores and vegetative cells.

• Alcohol Wipes• Heating (Viking Missions)• Hydrogen Peroxide• Ultraviolet Light

Great Concern: Bacillus species (spore-formers)

• Highly resistant to sterilization treatments: heat, UV radiation, H2O2 treatment, chemical disinfection, and starvation.

• B. pumilus strain SAFR 032: Survives standard decontamination protocols of the Jet Propulsion Laboratory spacecraft assembly facility.

• SAFR 032 exhibits extreme resistance to simulated Mars UV irradiation and liquid H2O2 treatment when compared with other Bacillus species.

Current Technology and Concerns

Requirements for Sterilization of Spacecraft or Spacecraft Components that May Contact the

Martian Surface

• Kill resistant microbial forms such as Bacillus spores• Cause no damage to sensitive electronics and other

spacecraft components / materials• Cost effective and scalable to large size

• Not dangerous to assembly facility personnel (e.g., ethylene oxide)

Malachite Green Spore Stain of a Bacillus species

Supercritical Carbon Dioxide

Definition: A supercritical fluid is any substance at a temperature and pressure above its thermodynamic critical point.

• A supercritical fluid can diffuse through solids like a gas, and dissolve materials like a liquid.

• Close to the critical point, small changes in pressure or temperature result in large changes in density.

• Supercritical fluids can be good substitutes for organic solvents

• Most commonly used and studied are carbon dioxide (decaffeination; biodiesel production by transesterificaiton; dry cleaning) and water (power generation).

• Properties can be modified by addition of “modifiers.

Critical Properties of Various Solvents

(Reid et al, 1987)

SolventMolecular weight

Critical temperature

Critical pressure

Critical density

g/mol K MPa (atm) g/cm3

Carbon dioxide (CO2)

44.01 304.1 7.38 (72.8) 0.469

Water (H2O) 18.02 647.3 22.12 (218.3) 0.348

Phase Diagram for Carbon Dioxide

Carbon Dioxide becomes a Supercritical fluidAt 31° C and 73 atm.

The SF-CO2 Sterilization System

Supercritical Carbon Dioxide Treatment System

A: Compressed liquid CO2 tank with siphon

B: ISCO programmable syringe pumps and controllers electronic for pumps and extractor C: Programmable controllers electronic for pumps and extractorD: Modifier reservoir E: Programmable SF-CO2 extractor and venting of extractor

F: Extraction chambersG: Venting with controlled flow rate through restrictor used during dynamic extraction stages,for possible collection of SF-CO2 extract

H: Four-liter stainless steel SF-CO2 sterilization chamber

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B B

E

E

G

H

A

D

F

C

Research Objective

Define optimal sterilization conditions to eliminate spores of Bacillus pumilus strain SAFR 032 from

metal and electronics.

• Achievable with various low concentrations of SF- CO2 modifiers

• At a constant, moderate temperature of 50C

• At a static, moderate pressure of 100 atm

• With a short treatment exposure time

Process

• Spores of two Bacillus pumilus strains were used• ATCC 7061 strain (control)• SAFR 032 strain (resistance target)

• Sterilization of spores performed on• A metal surface (US dimes)• A plastic surface (casing of electronic flash drives)

[SanDisk, Cruzer Multipack, USB 2.0 Flash Drive, 2GB]• Representative NASA spaceflight-qualified metal

(2cm * 1cm coupon sheets; NASA JPL)

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Bacillus spores

Process• Pure Bacillus pumilus spores was inoculated on the various surfaces through a liquid suspension. The liquid was then evaporated in a desiccation chamber to leave the spore deposits

• Two Bacillus pumilus strains were used on the US dime and electronic flash drives

• ATCC 7061 (control)• SAFR 032 (resistant target)

• Only the resistant strain SAFR 032 was used for the experiments involving the NASA metal coupon sheets.

Process• All sterilization treatments were performed in triplicate

• All experiments analyzed against two separate controls • A control containing no deposited spores that does not undergo

sterilization • A control containing deposited spores that undergoes sterilization

• Physical conditions of sterilization system • Constant pressure of 100 atm • Constant temperature of 50°C • Treatment time of 45 minutes

• 10 minutes dynamic cycle • 30 minutes static cycle • 5 minutes dynamic cycle

• The samples containing deposited spores from ATCC 7061 strain was placed in Chamber 1• The samples containing deposited spores from SAFR 032 strain was placed in Chamber 2

Process

• After each sterilization treatment the US dimes, metal coupons, and electronic flash drives were placed in TSA Petri dishes

• Circular strokes were used to transfer any bacterial spores onto the TSA medium.

• After the strokes, the materials were placed at the center of the plates, and then placed in a 30°C incubator

• The presence of viable spores (outgrowth as colonies) were monitored for the next five days

Results

Control Sterilization with effective modifier conditions

Optimized Conditions(100% Spore Killing)

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• Modifier Conditions • Four different modifier chemicals were used

• Hydrogen peroxide (H2O2) • tert-butyl hydroperoxide (CH3)3COOH

• formic acid (HCOOH) • Triton X-100

• They were added to either water or methanol, which served as the solvent

• During the sterilization process the modifiers were continuously added to SF-CO2 at a controlled and pre-determined concentration

• The lowest effective concentrations were established for each modifier

Optimized Conditions(100% Spore Killing)

• 3.3% water containing 3% H2O2

• 3.3% water containing 3% tert-butyl hydroperoxide

• 3.3% 50/50 water/methanol containing 3% H2O2

*Final concentration of both peroxides was 0.099% v/v in SF-CO2 - water/methanol mixture

Optimized Conditions(100% Spore Killing)

• 3.3 % water mixed with 10% methanol containing 0.5% formic acid

*0.016% v/v HCOOH in SF-CO2/water/methanol mixture

•3.3% water containing 10% methanol, 1% formic acid and 2% H2O2

*0.033% v/v HCOOH and 0.066% v/v H2O2 in SF-CO2/water mixture

Optimized Conditions(100% Spore Killing)

• 10% methanol containing 12 % H2O2

• 10% methanol containing 12% tert-butyl hydroperoxide

*Final concentration of peroxides was 1.2% v/v in SF-CO2/methanol mixture

• 10% methanol containing 6% H2O2 and 6% tert-butyl hydroperoxide

*Final concentration was 0.6% v/v H2O2 and 0.6% v/v tert-butyl hydroperoxide in SF-CO2 methanol mixture

Optimized Conditions(100% Spore Killing)

• The Triton X-100 modifier experiments failed to achieve complete killing of spores in either strain at the highest percentage input tested (1% w/v in water)

• Triton X-100 does not dissolve well in water and at 1% weight/volume

• It also produced a strong foaming effect at this concentration during dynamic phase of sterilization

BacLight Fluorescent Assay of the B. pumilus spores-Mixture of two different stains that allows fluorescent representation of live vs. dead spores

-The two strains are SYTO 9 (detection of all spores, live and dead), and Propidium Iodide (detection of only dead spores)

SYTO 9-Nucleic acid stain that permeates the cell membrane, giving off a large fluorescence in the green wavelength region. Can be used to stain RNA and DNA in both Gram-negative and in our Gram-positive B. pumilus bacterial spores

Propidium Iodide -Nucleic acid stain that is membrane impremeant and works as an intercalating agent, thus indicating that our sterilization system does somehow compromise the cell membrane

RESULTS

-Under the fluorescent microscope, the viable spores only display a green fluorescent outline of the spores structure, while in dead spores, the stain penetrates through and the entire structure fluoresces green as well as red

-In unsterilized samples, only a few spores may be displaying the Propidium Iodide red wavelength, but in sterilized samples, an almost majority percentage fluoresces red as well

-The small percentage that aren’t supposedly have still intact membranes although they fail to display any germination when incubated on a nutrient agar plate, indicating successful sterilization

- A high degree of killing, yet mild and gentle on the spore structure

- A key advantage as SF-CO2 containing modifiers can be used as an effective sterilization system for sensitive electronics and other components.

RESULTS

Testing of sterilization of sensitive electronic components without

contributing to destruction

Wikipedia Photo

• Tested effect of sterilization process on various electronic devices • Tested effects on performance and structure of the electronic devices (compatibility tests)•Sterilization procedures were performed on

•Computer memory flash drives (SanDisk, Cruzer Multipack, USB 2.0 Flash Drive, 2GB)

•MIT Lincoln chips 2D and 3D (H. Hess, University of Idaho Department of Electrical and Computer Engineering)

-Processed multiple B. pumilus SAFR-032 spore samples from JPL NASA

-Metal circular disks each with a 1 x 105 deposited spores

-Treated them through our SF-CO2 sterilization system and verified complete sterilization of deposited spores

-Sterilized samples were sent back to JPL who took pictures of them under their scanning electron microscope

Dipicolinic acid (DPA) ??

- Metal chelating chemical compound which composes 5% to 15% of the dry weight of bacterial spores; it is unique to spores

- Believed to be released from the spore core upon the spore coat being perforated

- Previous research have implicated it as responsible for heat and chemical resistance of endospores

Future Research

-Genome map and proteomic comparison of B. pumilus SAFR 032 vs. other B. pumilus strains

-What gene or set up genes gives B. pumilus SAFR 032 its incredible resistance?

-Statistical understanding of relationship between temperature, pressure, SF-CO2, treatment time, and modifiers in its cooperative ability at log reduction of spores

-Analysis of other possible chemical or enzymatic release from spores during sterilization

- Modifiers that create a very basic pH condition

Thank You