Transcript Spacecraft Component Sterilization using Supercritical

Spacecraft Component Sterilization using
Supercritical Carbon Dioxide
Ronald Crawford
Andrzej Paszczynski
Chien Wai
Edison Shieh
Sterilization of Bacillus pumilus Spores using
Supercritical Fluid Carbon Dioxide
Containing Various Modifier Solutions
The Need for “Planetary Protection” during
Exploration of the Solar System
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.
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
Strong Release of Methane on Mars in Northern
Summer 2003. 2009. Michael J. Mumma, et al.
• Polar Ice
Science 323. (5917), pp. 1041 – 1045.
• Permafrost
• “Deep” Subsurface (source of methane?)
• Areas of Nitrogen Salt Accumulation
• Areas Maintaining Intense Localized Magnetic Fields
Current Technology and Concerns
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.
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)
Molecular
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
Solvent
Phase Diagram for Carbon Dioxide
Carbon Dioxide becomes a Supercritical fluid
At 31° C and 73 atm.
The SF-CO2 Sterilization System
Supercritical Carbon Dioxide Treatment System
C
D
B
A
B
E
E
F
$
H
G
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 extractor
D: Modifier reservoir
E: Programmable SF-CO2 extractor and venting of extractor
F: Extraction chambers
G: 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
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 50C
• At a static, moderate pressure of 100 atm
• With a short treatment exposure time
Process
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• 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)
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
Bacillus spores
• 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
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Optimized Conditions
(100% Spore Killing)
• 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 Grampositive 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
RESULTS
- 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.
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