ENVIRONMENTAL MONITORING

04
Microbial Survival in Compressed
Gases Under Fast Decompression
to Normal Atmospheric Conditions
a review by: Frank Panofen, Particle Measuring Product Line Manager - Sterility Assurance , Microbiology

Background Microbes are not humans Findings from high-pressure studies:

Microbiological gas testing systems are
commercially available. Serratia and
Bacterial cultures
Monitoring compressed process gases and air in
in food products Carnobacterium spp
direct contact with pharmaceutical drugs during
survive conditions
manufacturing: require 2500- 3000
• is vital to the quality and safety of the product, bar* to inactivate3
and (low O2, <0°C, 7 mbar)5
• a key requirement under GMP regulations for Microbial cells survive the volatile
external pressures endured in Lessened microbial
aseptic manufacturing. Cells treated at
multiple harsh environments,
The sampling methodology used for compressed pressures of <1000 growth & viability on
including extreme deep-sea
gases involves the sudden decompression of environments.7 bar* showed no metabolic processes in
compressed gas right before taking a sample for vivo are seen
Microbes conserve the properties of
microbial counting. with pressures higher
necessary for life to originate under viability3
than 1000 bar*5
Issue harsh conditions.6
*1 Bar = 0.1 Mpa = 14.5 PSI
• It has been proposed that asserts that the
decompression step could significantly
deteriorate microbial cells and affect their ability Microbes Survive Under Compression and Decompression
to generate colony forming units1.
300 bar 300 bar | Escherichia coli and Corynebacterium xerosis survive

This speculation is proven false through a review A comparison of bacterial recovery from compressed gases
of regulations, applications and the numerous using the MAS100 CG and a standard air filter method found1: 250 bar

scientific literature related to microbial survival • 92% recovery using the MAS100 CG
200 bar

under a broad range of environmental pressure • Bacterial cells maintained suitable growth, even up to 10 bar during 150 bar

conditions. the incubation. 100 bar

• Compression cycle did not influence the viability of the tested

50 bar
microorganisms.
10 bar | 10 to 1 bar found in a CG sample
0
• Hypothesis from study: sampling prior to decompression avoided
potential microbial cell damage and lowered viability. This
hypothesis is refuted by multiple studies. Decompression-sensitive bacteria study
Facts Escherichia coli and Corynebacterium xerosis survive
Gram-negative, gas vacuolate bacteria (M. aquaticus,
P. pneumaticum, and M. glaucopis) are known to be very
In pharmaceutical environments, microorganisms rapid decompression from 300 bar susceptible to decompression.4
are exposed to pressure reductions: • Compare to decompression from 10 to 1 bar found in a CG sampler
• Populations of these cells were saturated with Ar, N2 or He up to 100
• Typically from 2.5 to 1.1 bar bar. Gas phases of the vesicles remained intact.
Cells without gas vesicles were not harmed by • Upon rapid decompression to atmospheric pressure, the vesicles
• A maximum of 10 bar decreased to 1.1 bar in a decompression from up to 300 bar.4 expanded and ruptured the cells.
worst-case scenario • Viable counts indicated minimum pressures were between 25 - 50 bar.
• Majority of the cell envelopes were ruptured at pressures between
Microorganisms are resilient under variable 50 - 100 bar.
compression and decompression environments.

Gas Applications Used in

Pharmaceuticals and Biotechnology Instrumentation Selection Instrumentation Financials
Cryopreservation & Lyophilization Blanketing
and Return of Investment (ROI)
Liquid Nitrogen, Carbon Dioxide, Nitrogen Rare measurement vs. investment
Cryogenic Storage Solutions
ph Control Frequency of monitoring compressed gases
Aseptic Packaging Carbon Dioxide
RCS High Flow, MerckSigm
• Classification: Performed monthly (1-6 months) or quarterly
Nitrogen
(6-12 months)
Pressure Transfer
Pressure & Leak Testing Nitrogen • Routine testing:  EU-2x year, US-1x year
Carbon Dioxide, Helium, Nitrogen Testing is not frequent and investing into dedicated equipment with all
Filtration, Separation & Purification associated costs like validation, maintenance and repair, is difficult to
Process Chilling Variety of CG
Liquid Carbon Dioxide, Liquid Nitrogen financially justify.
Analysis & Instrumentation Alternatives to dedicated instrument investment
Purging CG Mixtures
MiniCapt® Mobile, PMS Particle
Measuring Systems
MAS-100 CG, MBV AG
Carbon Dioxide, Nitrogen Option 1 - Service Provider
Sample scheduling and control of data and measurements can become
Wastewater Treatment difficult without quality communication and agreements between
Oxygen Decompression of gases has no influence on the viability
parties.
of microorganisms. Instrument selection is based on the
following parameters: Option 2 - Multipurpose Instrument
Instruments are readily available and their validation maintained due to
Summary • Size and weight - Lifting heavy instruments to a sampling point or
scheduled maintenance and, if necessary, repair. Additional costs only
from the accessory.
Sampling after decompression parallels a typical pushing a huge enclosure below a filling line should be minimized.
1 Sandle, T. (2015). Microbiological Assessment of Compressed Gases in Pharmaceutical Facilities.
scenario of using compressed gases during different • Preferred usage of mobile instruments/handhelds Journal of Validation Technology. Institute of Validation Technology (IVT Network.com).
stages of the manufacturing process. • Ease of use 2 Zingre, H. and Meier, R. (2013). Detection of Microorganisms in Compressed Gases. MBV AG
Application Note. Stäfa, Switzerland.
Microorganisms under hyperbaric or hydrostatic • Software/data management - GMP compliance 3 FDA. (2014). Kinetics of Microbial Inactivation for Alternative Food Processing Technologies - High
Pressure Processing (HPP). A report of the Institute of Food Technologists for the Food and Drug
pressures ranging from 1 to 10 bar resulted in no • Cleaning/disinfection - No contribution to the particle load Administration of the U.S. Department of Health and Human Services. Updated December 2014.
significant reduction of viability. 4 Hemmingsen, B. B. & Hemmingsen, E. A. (1980). Rupture of the Cell Envelope by Induced
Intracellular Gas Phase Expansion in Gas Vacuolate Bacteria. Journal of Bacteriology. 143 no.
Decompression from 10 bar to normal atmospheric 2:841-846.
pressure does not harm microorganisms. “...sampling device shall be selected according to the area 5 Nicholson, W. (2013). Growth of Carnobacterium spp. from permafrost under low pressure,
temperature, and anoxic atmosphere has implications for Earth microbes on Mars. pnas
Decompression occurring before or after agar plate being monitored. The selection for a particular application 110(2):666- 671.

impaction does not decrease viability and recovery of shall take into consideration the following factors… effect
CFUs. of the sampling device on the process or environment to
be monitored …” ISO 146987
6 Picard A., & Daniel I. (2013). Pressure as an environmental parameter for microbial life–a review.
Biophysical Chemistry. 183, 30-41 PMID: 23891571.
7 ALVIN. (2016). Project ALVIN Life around deep sea vents.
http://www.amnh.org/explore/curriculum-collections/deep-sea-vents/pressure-in-the-deep-
seas/ American Museum of Natural History.