National Aeronautics and Space Administration

A Researcher’s Guide to:

Microbial Research
2

This International Space Station (ISS) Researcher’s Guide is published by the NASA ISS Research

Integration Office.

Authors:

Audrie A. Colorado, Ph.D.

Sarah L. Castro-Wallace, Ph.D. 

C. Mark Ott, Ph.D.

Previous Contributing Authors:

David J. Smith, Ph.D.

Executive Editor: Bryan Dansberry 

Technical Editor: Carrie Gilder 

Designer: Cory Duke

Published: June 2013

Revision: July 2021

Cover and back cover: 

a. Astronaut Dr. Peggy Whitson evaluates microbial growth on a surface sample kit (SSK) contact

slide within the microgravity science glovebox (MSG) as part of the Genes in Space-3 investigation.

The SSK contact slides are used as part of the Crew Health Care System’s routine microbial

monitoring of the ISS. In a spaceflight first, Dr. Whitson collected cells from the colonies and placed

them into miniPCR for DNA extraction and gene amplification. Following this, she used the MinION

(not shown) to sequence the DNA amplified from these organisms. The identifications obtained

onboard the ISS matched those determined on the ground following nominal processing of the

returned SSK slide. This marked the first time unknown organisms were collected, cultured, and

identified off Earth.

b. Back Cover 1: Computer-generated bacteria image.

c. Back Cover 2: Computer-generated virus image.
3

An astronaut holding a Microbial Air Sampler (MAS) Petri Dish on the ISS. The MAS is used for the collection of cabin 

air atmosphere for evaluation of the microbial load.

Orbiting the Earth at almost 5 miles per second, a structure exists that is 

nearly the size of a football field and weighs almost a million pounds. The 

International Space Station (ISS) is a testament to international cooperation 

and significant achievements in engineering that has supported the 

advancement of scientific knowledge and technology development for over 

19 years. The ISS is a truly unique research platform providing groundbreaking research opportunities for commercial, government and academic 

users. The possibilities of what can be discovered by conducting research on 

the ISS are endless and have the potential to contribute to the greater good 

of life on Earth and future long-duration deep space exploration missions.

As we increase utilization of ISS as a National Laboratory, now is the 

time for investigators to propose new research and to make discoveries 

unveiling novel responses that could not be defined using traditional 

approaches on Earth.

The Lab is Open
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1. Microgravity, or weightlessness, alters many observable phenomena 

within the physical and life sciences. Systems and processes affected by 

microgravity include surface wetting and interfacial tension, multiphase 

flow and heat transfer, multiphase system dynamics, solidification, and 

fire phenomena and combustion. Microgravity induces a vast array of 

changes in organisms ranging from bacteria to humans, including global 

alterations in gene expression and 3-D aggregation of cells into tissue-like 

architecture.

2. Extreme conditions in the ISS environment include exposure to extreme 

heat and cold cycling, ultra-vacuum, atomic oxygen, and high energy 

radiation. Testing and qualification of materials exposed to these extreme 

conditions have provided data to enable the manufacturing of longlife reliable components used on Earth as well as in the world’s most 

sophisticated satellite and spacecraft components.

3. Low Earth orbit affords ISS a unique vantage point with an altitude 

of approximately 240 miles (400 kilometers) and an orbital path over 

90 percent of the Earth’s population. This can provide improved 

spatial resolution and variable lighting conditions compared to the sunsynchronous orbits of typical Earth remote-sensing satellites.

Unique Features of the ISS 

Research Environment
6

Microbiology Research Priorities on the ISS 7

The ISS as a Microbial Research Platform

Benefits of an ISS Microbial Research Platform

Unexpected Microbial Responses to Spaceflight Culture 12

Microbiology of the Built Environment 16

Microbial diversity, concentration and antibiotic resistance 

Biofilms and biofouling in water processing systems

Astronaut Microbiomes 21

Initiating Ground-Based Research – Spaceflight Analogs 23

An Example of a Spaceflight Analog – The Rotating Wall-Vessel Bioreactor

Insights Gained form Microbial Culture within the Rotating

Wall-Vessel Bioreactor

What Principal Investigators Should Know About 

Conducting Research on the ISS 28

Funding, Developing, and Launching Research to ISS

ISS U.S. National Laboratory

Other Government Agencies

International Funding Sources

Citations 32

Acronyms 43

Table of Contents
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We are on the cusp of the next giant leap in space exploration and related scientific 

research. The private sector has reinvigorated the space race, and several countries 

have affirmed their intentions of developing a robust human spaceflight program. 

The United States has targeted full utilization of the International Space Station 

(ISS) and set its exploration sights beyond low-Earth orbit. As we determine 

our destinations for the next generation of spaceflight, several questions remain 

unanswered as to the effects of the spaceflight environment on human physiology 

and the microorganisms that will inevitably accompany them. Previous spaceflight 

studies have demonstrated that microgravity can enable better understanding 

of fundamental biology and accelerate advancements in healthcare and medical 

technologies. These benefits are critical, not only for human deep-space exploration, 

but also for improving quality of life on Earth.

A human is both an individual organism and an entire ecosystem, including 

microorganisms in, on, and around the body, in which the microbial cells 

are roughly equal in number to the human cells. For the most part, these 

microorganisms are beneficial to their human host or are otherwise innocuous. 

Given the right set of conditions, many otherwise benign microorganisms can 

become pathogenic. Therefore, potential pathogens have been present on all 

NASA missions (Rogers 1986, Castro 2004). Protective measures such as stringent 

microbial monitoring, prudent vehicle design, and preflight crew quarantine are 

used to decrease the risk of infectious disease during missions (Johnston 1969, 

Rogers 1986). 

Over the past 50 years, a combination of operational experience, spaceflight and 

ground-based research have provided tremendous insight into infectious disease 

risk as well as necessary preventative measures (Johnston 1969, Taylor 1972, Taylor 

1976, Facius 1978, Fang 1997, Nickerson 2004, Ott 2004). Significant strides 

to define and mitigate the source of microbial contamination aboard spacecraft 

and to document the responses of numerous microorganisms to the spaceflight 

environment have allowed the identification of critical gaps in our understanding 

of how this environment impacts microbial ecology, the microbial genotypic and 

phenotypic characteristics, and their interactions with plant and animal hosts. 

As we look toward human interplanetary exploration, the importance of this 

knowledge is imperative. With the increases in both the occupancy and duration of 

humans aboard the ISS, these knowledge gaps are becoming better defined.

Microbiology Research 

Priorities on the ISS
8

 There is much to gain by employing the microgravity environment of spaceflight 

as a basic research platform. Life on Earth evolved in the presence of gravity. 

Therefore, performing research in the reduced gravity of spaceflight holds the 

potential to determine how this physical force shaped terrestrial life. Previous 

spaceflight and ground-based spaceflight analog research has established that 

even microorganisms, the smallest Earth-based life forms, are intrinsically able to 

respond to changes in this force (Dickson 1991, Mishra 1992, Nickerson 2000, 

Nickerson 2004). 

While over 50 years of microbial research has been performed in spaceflight, a 

thorough understanding of microbial responses to spaceflight culture and how 

the spaceflight environment stimulates these responses is only beginning to be 

understood. Microgravity as a research tool, coupled with current molecular 

technology, provides researchers the opportunity to establish how variations in this 

physical force affect microbial life at the cellular, molecular and evolutionary levels. 

This potential is not surprising since innovative answers to complicated medical, 

environmental, and agricultural questions have arisen from assessing the properties 

of microorganisms in many extreme environments on Earth (Nickerson 2004). 

Similarly, the study of microbes in the spaceflight environment holds considerable 

potential for future basic research and industrial applications. Investigations into 

microbial ecology, genotypic and phenotypic properties, and the infectious diseasecausing potential of microorganisms in the spaceflight environment may unveil 

novel mechanisms that can not be elucidated using traditional approaches on Earth, 

where gravity may be restricting our discovery of unique cellular responses.

The ISS as a Microbial Research Platform 

The ISS provides a unique research platform that enhances our knowledge of the 

effects of gravity on microorganisms. This unique environment allows a better 

understanding of microbial mechanisms and interdependencies that would 

normally be masked by gravity, enabling insight into fields including microbial 

physiology, microbial interactions, molecular microbiology and microbial ecology. 

The ISS is also an ideal setting to study microorganisms in a complex contained, 

isolated, “island-like” ecosystem. Many scientific studies have focused on either 

complex ecosystems that are not well controlled in a classical experimental sense or 

very simple ecosystems that are well controlled but severely limited in dimension 

and/or diversity. To date, complex controlled ecosystems have not persisted for 
9

long periods, so studying microbial dynamics within them has been necessarily a 

short-term endeavor. Since its initial launch in the commencement of construction 

in orbit, ISS has been a relatively closed system with microbial exchange occurring 

during a small number of cargo resupply missions and crew changes. Factors 

influencing microbial growth and response are well monitored and recorded, 

including environmental conditions such as temperature and humidity as well as 

crew diet and activity. New ISS modules and transported cargo are also evaluated 

for microbial diversity and concentration. 

Benefits of an ISS Microbial Research Platform

The ISS research platform provides an opportunity to broaden our understanding 

of the unique microbial responses of microorganisms cultured during spaceflight. 

This aspect of the ISS research platform distinguishes it from every other available 

facility since no other platform can provide this microgravity environment. As the 

microorganisms are adapting their responses to this novel environment, information 

can be gathered that provides unique insight into microbial regulation and function 

that cannot be discerned using traditional methods on Earth.

The use of the ISS as a microbial research platform drives experiments that 

could decrease infectious disease risk during the human exploration of space, 

advance the application of beneficial purposes for microorganisms (e.g., waste 

remediation, probiotics), and provide unique insight into basic microbial functions 

and interactions that could be translated to studies for scientists and commercial 

entities on Earth. Translation of spaceflight findings has already begun to take 

place as scientists and corporations investigate the use of ISS microbial findings to 

better understand virulence profiles, antibiotic and disinfectant resistance, biofilm 

formation, and biodegradation properties.

As NASA travels beyond low-Earth orbit to planets such as Mars, insight from ISS 

microbial research will influence our approach to exploration. NASA proposes to 

support advanced research focused on discovering and characterizing fundamental 

mechanisms used by microorganisms and microbial communities to adapt to 

the diverse challenges of the spaceflight environment highlighted in Table 1. 

Understanding how spaceflight and gravity alter microbial responses, their exchange 

of genetic material, and their expected concentrations and distribution is vital in the 

search for extraterrestrial while also protecting other planets from microbial transfer.
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Table 1. Opportunities for Microbial Research on the ISS

The ISS also offers an unprecedented opportunity to advance indoor microbial 

ecology research. It provides an experimental platform for controlling two of the 

largest contributors to indoor microbial diversity: ventilation source and occupancy 

load. Research has shown that ventilation source significantly impacts microbial 

diversity indoors, with mechanically ventilated rooms harboring more potential 

airborne pathogens than naturally ventilated rooms (Kembel 2012). It has also been 

demonstrated that human occupancy load impacts the abundance and diversity of 

airborne microbes (Qian 2012). 

Together, these findings suggest that tremendous knowledge would be gained by 

conducting experiments in a highly controlled environment like the ISS where the 

ventilation source and occupancy load can be systematically analyzed. By sampling 

the built environment microbiome and human microbiome in the ISS jointly, 

it would be possible to tease apart how microbes are exchanged among humans, 

indoor air, and indoor surfaces. 

Subject Area Investigations of spaceflight environment alterations

Microbial 

Physiology

Microbial growth profiles

Response to stressors

Motility

Microbial metabolism

Microbial Ecology Relative roles of microbial ecology and evolution

The human and plant microbiome as a subset of the ISS microbiome

Microbial interactions with the environment over time

Microbial population dynamics and dynamics of succession

Stabilities of closed-model communities (not ambient)

Mechanisms of community change/biogeography

Selection pressure and generational aspects within selected microbes versus communities

Microbial populations occurring naturally in the environment (air, surfaces, water)

Spread of identified strains as a result of the spaceflight environment

Molecular 

Microbiology

Microbial genomic diversity and evolution

Microbial sensing

Microbial transcriptome, proteome, or metabolome 

Microbial 

Interactions

Microbe-microbe interactions

Host-microbe interactions

Plant-microbe interactions

Biofilm formation or function (single species and mixed populations) 
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These findings are important for future spaceflight missions, as well as terrestrial 

homes and offices. Indoor microbial communities might be intimately connected to 

human health (Burge 1995, Mitchel 2007, Srikanth 2008), including the spread of 

acute respiratory disease (Cohen 2000, Smith 2000, WHO 2007, Glassroth 2008) 

and the increase in the occurrence of asthma symptoms (Ross 2000, Eggleston 

2009, Schwartz 2009). 

Another unique benefit of the ISS as an experimental, complex, closed ecosystem 

is that over time the microbial communities present on the space station are likely 

to become increasingly dominated by human-associated microbes. Within the 

confines of the ISS, the environmental control and life support system maintains a 

homeostatic environment suitable for sustaining the human crew. This environment 

also acts to sustain and select a human-associated microbiome that persists on the 

ISS across all missions. 

As has been observed in other environmentally controlled and human-engineered 

constructs such as office buildings and airplanes, the microbiome will change over 

time in diversity (i.e., number of different types or species of microorganisms 

present) and structure (i.e., the relative composition of different types or species). 

The relative abundance of human-associated bacteria, including those that could 

potentially cause disease, is higher indoors than outdoors. Since the ISS is relatively 

closed, the microbial diversity is stable throughout the interior of the station such 

that the dispersion of new microorganisms can be tracked and the impact of their 

addition to the station microbial community can be evaluated. This premise may 

also make possible investigations into changes in the astronaut microbiome. 
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The short generation time of microorganisms makes them uniquely suited for 

studies assessing responses to altered environmental conditions. Microbial cells 

were among the first Earth-based life forms to be sent into the microgravity 

environment of space. These early investigations established that bacteria and fungi 

remained viable and capable of reproducing while also setting a precedent for 

conducting research in the spaceflight microgravity environment. Although more 

than 100 spaceflight experiments involving microorganisms have been conducted 

over the past 50 years, significant gaps remain in our knowledge about how this 

environment impacts microbial ecology, microbial genotypic and phenotypic 

characteristics, and host-microbe interactions.

In 1960, prior to the flight of Yuri Gagarin, scientists from the Union of Soviet 

Socialist Republics (USSR) launched E. coli, Aerobacter aerogenes, and Staphylococcus

species into orbit aboard an unmanned satellite (Zhukov-Verezhnikov 1962, 

Zhukov- Verezhnikov 1963). It was this experiment that lead to the conclusion 

that the microgravity environment of space did not affect the viability of the 

microorganisms (Zhukov-Verezhnikov 1962, Zhukov-Verezhnikov 1963). In an 

important subsequent experiment, the USSR launched E. coli aboard Vostok 2 in 

1961, which resulted in the identification of a variant colony type that was reported 

to be a result of spaceflight factors (Klemparskaya 1964). 

In 1967, NASA launched the unmanned Biosatellite 2, which exposed 

various biological specimens, including E. coli and Salmonella enterica serovar 

Typhimurium, to the microgravity environment of space for 45 hours (Mattoni 

1968, Mattoni 1971). For both microorganisms, an increase in population density 

was noted for the in-flight samples (Mattoni 1968, Mattoni 1971). Bacillus 

subtilis was cultured aboard Apollo 16 and 17 and resulted in the finding that 

microgravity did not affect the developmental process of spore formation (Bucker 

1975). However, when assessed after culture aboard the Apollo-Soyuz Test Project, 

the colony forming ability of B. subtilis spores was found to be reduced among 

spaceflight samples (Facius 1978). With evidence mounting that bacteria were able 

to sense and respond to the microgravity environment of spaceflight, the concern 

of both the U.S. and USSR space programs shifted to how these variations could 

impact crew health.

Unexpected Microbial 

Responses to Spaceflight 

Culture 
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Over the course of numerous spaceflights, researchers from various countries 

have analyzed changes in antibiotic resistance in E. coli and Staphylococcus aureus

(Tixador 1983, Tixador 1985, Tixador 1985, Lapchine 1987). The minimal 

inhibitory concentration (MIC) of oxacillin, chloramphenicol, and erythromycin 

for S. aureus and colistin and kanamycin for E. coli were evaluated among inflight cultures as compared to controls (Tixador 1985, Tixador 1985). These 

investigations documented increased bacterial resistance to all antibiotics tested for 

both S. aureus and E. coli. The researchers observed a thickening of the cell wall 

that accompanied the increase in resistance of S. aureus once returned from flight 

(Tixador 1985, Tixador 1985). Various other microbial properties were recorded 

during this time, including increased conjugation in E. coli (Ciferi 1988) and 

increased growth kinetics in B. subtilis (Mennigmann and Lange 1986) in response 

to microgravity.

The space shuttle era brought an enhanced capability to perform biological 

research within the microgravity environment of space and to delve further into 

the implications for human health. In 2006, taking advantage of this opportunity, 

investigators launched several microorganisms including S. Typhimurium aboard 

Space Shuttle Atlantis (STS-115) in an attempt to define the impact of spaceflight 

culture on the disease-causing potential of the microorganisms (Wilson 2007, 

Crabbé 2011). The results of the study reported that mice infected with bacteria 

grown in flight displayed a significantly decreased time to death, increased percent 

mortality, and decrease in the lethal dose (Figure 1 D) (Wilson 2007). Analysis 

of the fixed returned samples revealed differential expression of a large number of 

genes and identified a regulatory protein that was mechanistically associated with 

the spaceflight response of the organism (Wilson 2007). This was the first report 

elucidating both the molecular response connected with a regulatory mechanism 

and alterations in bacterial virulence because of growth in the spaceflight 

microgravity environment.

To confirm these findings and further our understanding of factors influencing 

spaceflight culture-mediated changes in virulence, a follow-up investigation was 

performed on Space Shuttle Endeavor (STS-123) to again assess the response 

of S. Typhimurium to the spaceflight environment. This set of experiments 

included culturing S. Typhimurium in various different types of growth media 

in the spaceflight environment (Figure 1 E). The findings from this spaceflight 

experiment confirmed the previous reports of increased virulence of the bacteria 
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Figure 1. Astronauts A) Heidi Stefanyshyn-Piper activates S. Typhimurium to grow in flight during the MICROBE 

experiment on STS-115 and B) Don Gories activates S. Typhimurium to grow in-flight as part of the MDRV experiment 

on STS-123. After growth in the spaceflight environment, the bacterial samples were returned to Earth and immediately 

used in a mouse model of salmonellosis. The results from both experiments revealed that S. Typhimurium becomes 

more virulent as a result of growing in the microgravity conditions of space. The experiments also described the 

composition of the growth medium as an important factor controlling the change in virulence (Wilson 2007,

Wilson 2008).
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(Wilson 2008). Furthermore, the data from this assessment revealed that media ion 

concentration dramatically influences the spaceflight-related virulence response of 

S. Typhimurium (Wilson 2008).

In addition to microgravity, the spaceflight environment has a unique radiation 

background. Several spaceflight experiments have investigated the impact of this 

radiation on microbial organisms (de Serres 1969, Berry and Volz 1979, Bouloc and 

D’Ari 1991, Horneck 2010); however, topics such as alterations in mutational rates 

and how these mutations could alter the phenotype of the organisms is generally 

understudied.
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While the ISS is generally closed to external influences, microorganisms 

are continuously introduced on the ISS, providing a novel platform for the 

investigation of human and environmental microbiomes. Since the ISS is not a 

completely closed system, the low frequency of exchange of people and materials 

with the outside and the potential for characterization of the microbiology of 

materials brought to station makes control of microbial inputs to this unique 

system more achievable. The ISS offers opportunities to study the dynamics 

of microbial populations and communities in the absence of uncontrolled 

introduction of microorganisms from unknown sources. While other ecosystems, 

such as homes or submarines, have some of the characteristics of ISS, none can 

match this platform’s unique isolation from contaminating contacts. This isolation 

provides the opportunity to gain insight into the interactions between humans and 

environmental organisms and changes in microbial communities through mutation 

or genetic exchange with minimal external interference. 

Microbial diversity, concentration, and antibiotic resistance 

Most of our understanding of the microbial diversity aboard spacecraft has relied on 

culturing microorganisms using a relatively few types of growth media. Generally, 

the environmental data indicate that the potable water, air, and surfaces to which 

the crew is exposed are free of obligate pathogens; however, opportunistic pathogens 

such as Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and S. aureus are not 

uncommon (Pierson 1996, Castro 2004, Pierson 2012). 

Spaceflight food is another potential source of microorganisms aboard spacecraft. 

While the incidence of contamination is low, preflight analyses of food samples 

have indicated the presence of organisms such as Salmonella enterica serovar 

Typhimurium (S. Typhimurium), S. aureus, Enterobacter cloacae and Cronobacter 

sakazakii (unpublished data). Figure 2 details the relative abundance of bacterial and 

fungal strains isolated from the air and surfaces of the ISS before and during flight. 

Since these findings are based on cultured organisms, only a part of the picture of the 

microbial diversity of spacecraft has been captured. By coupling current molecular 

methods with the ISS platform, a higher resolution of this picture has the potential 

to be viewed.

While multiple preflight measures are in place to limit microbial contamination 

aboard spacecraft, the limitations of these conventional monitoring methods 

were demonstrated by a comprehensive media-based and microscopic analysis of 

microorganisms isolated from free-floating water collected behind panels aboard 

Microbiology of the 

Built Environment
17

the Mir Space Station (Ott 2004). Several medically significant organisms that were 

not commonly isolated during standard operational monitoring were identified, 

including Legionella species, Serratia marcescens, and E. coli.

Figure 2. The abundance of A) bacterial and B) fungal strains isolated from air and surfaces from the International Space 

Station environment before and during flight. Isolates are categorized by genera and relative abundance (Pierson 2012).

A

B
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Further microscopic examination of these samples revealed the presence of amoeba

resembling Acanthamoeba or Hartmanella species and ciliated protozoa resembling

Stylonychia species (Ott 2004). This finding reinforced the need for a more thorough

investigation of the microbial diversity of spaceflight habitats, especially at time

points later in their service life.

Recent studies have taken modern molecular approaches to characterize the

microbial ecology of the built environment aboard the ISS. The ISS environment

is predominantly composed of human-associated microorganisms (Mora 2019,

Blaustein 2019). Findings also concluded that the microbial community composition

on the ISS is not much different than terrestrial homes and persists across spaceflight

expeditions even with the change of crew and payloads (Lang 2017, Singh 2018,

Blaustein 2019).

Modern molecular approaches have also been used onboard the ISS with the goal

of near real-time microbial profiling of the environment. The emergence of portable

molecular biology tools such as miniPCR bio’s miniPCRTM and Oxford Nanopore

Technologies’ MinIONTM has made in situ analysis a reality with both systems being

tested onboard the ISS in 2016 (Boguraev 2017, Castro-Wallace 2017).

With the validation of the first off-Earth polymerase chain reaction (PCR) and DNA

sequencing, methods were developed to apply this technology to the identification of

microbes onboard the ISS. In 2017, an astronaut collected cells from culture slides

used for routine microbial monitoring, extracted and amplified DNA, generated

sequencing libraries, and sequenced the resulting DNA with the MinION (Burton

2020). From the three colonies selected by the crewmember, two identifications

were obtained. Upon return of the culture slide to the ground and nominal

processing, the same identities were confirmed, as two of the colonies were the same

organism (Burton 2020). This outcome resulted in the first collection, culture, and 

identification of unknown microorganisms off Earth.

To enable a more rapid assessment of the environment and to extend understanding

beyond the limitations of traditional culture processes, a direct swab-to-sequencer,

culture-independent method was developed and tested onboard the ISS beginning

in 2018 (Stahl-Rommel 2021). As with previous investigations where samples were

returned to Earth for molecular analysis, the data displayed a high similarity to that

of the human microbiome. While unsurprisingly higher in diversity and difficult to

culture organisms, the data also paralleled that of historic culture-based sampling

(Figure 3.) (Stahl-Rommel 2021).
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Preventive measures during spaceflight missions continue to provide protection to 

the crew. Implementation of a preflight quarantine period, which began during the 

Apollo program, has significantly limited the number of obligate pathogens that are 

carried into the spacecraft by the crew. Other efforts to reduce the infectious disease 

risk to the crew include but are not limited to: routine screening of spaceflight food 

for the presence of harmful microorganisms, HEPA filters installed on the ISS, 

and treatment of water system with biocides. However, the risk of obligate and 

opportunistic pathogens on spacecraft has not been eliminated.

Biofilms and biofouling in water processing systems

The advancement of the current spacecraft water disinfection system is a major 

concern to take into consideration for future spaceflight missions. Biofilm 

formation aboard the ISS was first documented on STS-95 (Mclean 2001). Bacteria 

that form biofilms can often withstand environmental stressors such as disinfectants 

and antibiotics. The protective niche biofilms offer bacteria can also harbor 

infectious organisms that can potentially threaten crew health (Donlan 2002). 

0

25

50

75

100

JEM Air Grid Node1 S4 PMM Curtain

Location

Relative Abundance

Haemophilus

Streptococcus

Staphylococcus

Cutibacterium

Granulicatella

Other

Gemella

Bacillus

Neisseria

Anaerococcus

Veillonella

Peptoniphilus

Lactobacillus

Aggregatibacter

Dolosigranulum

Selenomonas

Finegoldia

Campylobacter

Facklamia

Abiotrophia

Aerococcus

Fastidiosipila

Fenollaria

ID

0

25

50

75

100

JEM Air Grid Node1 S4 PMM Curtain

Location

Relative Abundance

16S ID

Haemophilus parainfluenzae

Other

Staphylococcus saccharolyticus

Streptococcus mitis

Staphylococcus caprae

Streptococcus australis

Streptococcus salivarius

Cutibacterium acnes

Streptococcus parasanguinis

Staphylococcus epidermidis

Staphylococcus hominis

Streptococcus pneumoniae

Streptococcus infantis

Streptococcus dentisani

Granulicatella adiacens

Neisseria perflava

Finegoldia magna

Aggregatibacter segnis

Lactobacillus crispatus

Campylobacter ureolyticus

Peptoniphilus harei

Streptococcus sanguinis

Anaerococcus nagyae

Facklamia hominis

Gemella haemolysans

Abiotrophia defectiva

Aggregatibacter aphrophilus

Staphylococcus capitis

Figure 3. Microbial profiles generated from three locations onboard the ISS through use of the culture-independent, 

swab-to-sequencer method of the top 15 A) genus- and B) suggested species-level identifications. Data were generated 

onboard following sample collection, processing, and sequencing. Raw sequence data were downlinked to Earth for 

analysis. (Stahl-Rommel 2021)
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Microorganisms play a pivotal role in the functioning of key spacecraft systems such 

as the ISS water system (Pierson 2012). Microbial contamination due to biofilms 

within the water system could be catastrophic since the system has multiples uses 

such as providing potable drinking water, irrigating plants grown for consumption, 

and aiding in crew hygiene. Elevated bacterial concentrations have been detected in 

both the Russian and U.S. potable water systems (Bruce 2005, Pierson 2012). There 

are also reports of biofilm growth and associated biofouling of filters, membranes, 

and the stainless steel water line associated with spacecraft water systems (Carter 

2017, Diaz 2019). These events further support the necessity to prevent or control 

microbial growth, inhibit or prevent biofilm formation, and prevent microbiallyinduced biofouling in design of future spacecraft systems. 
21

Astronaut Microbiomes

With the exception of sending microorganisms into orbit for research purposes, 

significant care is taken to reduce the levels of microbes in the spaceflight 

environment. Stringent preflight microbiological monitoring and remediation of 

NASA spacecraft has been performed throughout the human spaceflight program 

(Johnston 1969, Rogers 1986, Castro 2004). However, the combination of the crew 

members’ microbiota and the inability to ensure complete sterility of the craft and 

cargo results in the coexistence of humans and microorganisms in the spaceflight 

environment. 

In-flight data acquired during Apollo, Skylab, and the Mir Space Station missions 

increased our knowledge of the impact of spacecraft habitation on the crew and 

vehicle microbiota. Findings from these early spaceflight programs were critical to 

the design of later spacecraft and in establishing microbiological acceptability limits 

for the in-flight environment. While this information and the insight gained from 

the space shuttle and ISS programs has proven critical in our approach to mitigating 

microbial risk to crewmembers and their vehicle, several questions still remain.

Microbiological evaluations of the crew members were in place since for the first 

manned Apollo flight with the goal of characterizing the microbial load of astronauts 

preparing for lunar surface exploration (Taylor 1972). During early Apollo missions, 

a thorough microbial baseline was established for each astronaut to facilitate the 

identification of any possible terrestrial contaminants in returned lunar samples. 

Studies conducted during the later Apollo missions were designed to identify and 

prepare for possible microbial-associated issues arising as a result of the lengthier 

Skylab program (Taylor 1972). The findings of these early investigations included 

identifying trends such as increases in the number of sites on a crew member’s body 

that organisms were isolated from, the quantity of those organisms, and increased 

levels of microbes in the environment (Johnston 1969, Taylor 1972). These early 

studies also documented the incidence of microbial transfer between crew members 

and the spacecraft environment (Taylor 1972). The knowledge gained resulted 

in operational and engineering activities to control the environment of the crew 

concerning crew contacts (quarantine), food, water, and air.

More recent studies have collected data over longer time periods and sampling more 

body sites with the implementation of longer duration spaceflight missions. One 

study analyzed 16S and metagenomics sequencing to characterize astronauts’ gut, 

skin, nasal and oral microbiomes over 6+ months before, during and after flight 
22

(Voorhies 2017). Results from inflight samples revealed that the overall composition 

of bacterial communities from the gut, skin, and nose significantly change in space. 

Moreover, for some crew members, intestinal microbial diversity increased under 

microgravity conditions, most likely promoted by the new spaceflight diet and/or 

the crowded living conditions of the ISS that might favor a more fluid interchange 

of microbial flora among crew members (Voorhies 2017). The NASA Twins study 

also found more changes in microbial community composition and function were 

found during spaceflight (Garrett-Bakelman 2019). These findings warrant further 

investigation into longer duration spaceflight impacts on crew health.
23

Microgravity cannot be created on Earth; however, aspects of the microgravity 

environment can be mimicked by use of ground-based simulators. Numerous 

ground-based methods of simulating the microgravity environment of spaceflight 

have been developed and implemented to overcome the constraints that 

accompany biological gravitational research. Ground-based simulators have proven 

indispensable as tools for preparing spaceflight experiments and have generated 

independent investigations. For example, parabolic flights and drop towers are 

means of providing “free fall” for a limited amount of time. 

To enable analysis of microbial response to aspects of the microgravity environment 

for greater amounts of time, other analogs have been developed using a variety of 

technology such as clinostats, rotating-wall vessels, random positioning machines, 

and magnetic levitation (Klaus 2001, Nickerson 2004, Herranz 2013). While these 

simulators do not eliminate the force of gravity, they reproduce many characteristics 

of the environment produced in true microgravity.

An Example of a Spaceflight Analog – The Rotating Wall-Vessel 

Bioreactor 

Each spaceflight analog system has unique advantages and disadvantages (Klaus 

2001, Buels 2009, Dijkstra 2011). Of the simpler systems, the rotating-wall vessel 

Initiating Ground-Based 

Research – Spaceflight

Analogs

Figure 4. The Rotating-Wall Vessel 

Bioreactor (Synthecon, Houston, Texas). 

(A) Image of the NASA-designed RWV 

apparatus. (B) RWV culture system in the 

incubator with the respective base units 

and power supply systems. (C) The altered 

positioning of the RWV that results in the 

two culture orientations, depicting the axis 

of rotation. The LSMMG environment is 

achieved by rotation of the RWV on an 

axis parallel to the ground, whereas the 

axis of rotation in the control orientation is 

perpendicular to the ground. (D) Depiction 

of the orbital path of a cell when cultured in 

the LSMMG orientation. The combination 

of the clock-wise solid body rotation of 

the media and the sedimentation effect, 

whereby gravity and lack of motility causes 

a cell to settle to the bottom of the vessel, 

results in the continuous suspension of the 

cell in an orbit.
24

(RWV) bioreactor has been increasingly used to enhance our understanding of 

microbial responses that may be occurring during spaceflight (Fang 1997, Nickerson 

2000, Lynch 2004, Nauman 2007, Crabbé 2008, Castro 2011). The RWV 

(Figure 4 A) is an optimized form of suspension culture in which cells are grown 

in physiologically relevant, low-fluid-shear conditions. These low-shear effects of 

the fluid on the cells has resulted in the adoption of the term “Low-Shear Modeled 

Microgravity” (LSMMG) for use in accurately describing the environment produced 

by the RWV bioreactor (Wilson 2002). A cell in liquid media in microgravity 

experiences two unique aspects important in modeling this environment: 1) 

remaining in a constant state of suspension and 2) experiencing a quiescent 

surrounding, devoid of shearing, turbulent forces (Klaus 1997). The RWV bioreactor 

effectively models these aspects of the microgravity culture environment.

Insights Gained from Microbial Culture Within the Rotating 

Wall-Vessel Bioreactor

The RWV bioreactors were initially intended as a spaceflight analog for eukaryotic 

cells (Wolf 1991, Hammond 2001) but have since been used to examine bacteria 

(Fang 1997, Nickerson 2000, Crabbé 2008, Castro 2011), fungi (Johanson 2002), 

and archaea (Dornmayr- Pfaffenhuemer 2011) in response to this environment. 

In the mid-1990s, Fang and colleagues were the first to put a bacterium inside the 

RWV. The team was primarily focused on the effects of LSMMG on secondary 

metabolite production (Fang 1997, Fang 1997). Over the course of their studies, 

they noted that the modeled microgravity environment of the RWV did not 

alter gramicidin production from Bacillus brevis (Fang 1997), decreased betalactam production by Streptomyces clavuligerus (Fang 1997), inhibited Streptomyces 

hygroscopicus’ production of rapamycin (Fang 2000), and prevented microcin B17 

production from E. coli (Fang 2000). A summary of certain bacterial, fungal, 

and archaeal responses to the simulated microgravity conditions within the RWV 

bioreactors since the work of Fang and colleagues can be found in Table 2.

Pioneering work by Nickerson and colleagues expanded this area of research by 

connecting the LSMMG response of an enteric pathogen, S. Typhimurium, to 

a human host and the spaceflight environment (Nickerson 2000, Wilson 2002, 

Wilson 2002). The conditions within the RWV were found to have profound 

effects on the behavior of S. Typhimurium, including an increase in its virulence 

potential (Nickerson 2000). Mice challenged with LSMMG-cultured S. 

Typhimurium suffered an increased percent mortality, increased time to death, and 

required a lower LD50 as compared to control cultures (Nickerson 2000). The 
25

Microorganism Response to Modeled Microgravity 

within the RWV Bioreactor

Reference

S. Typhimurium 3339 - Increased: virulence in a mouse model; resistance to 

acid, thermal, and osmotic stress; macrophage survival

- Decreased: LPS production; resistance to oxidative 

stress; Hfq expression

- Differential gene expression

Nickerson, 2000

Wilson, 2002 

Wilson, 2002b 

Wilson, 2007

S. Typhimurium 14028 - Increased: virulence in a mouse model and cellular 

invasion

- Differential gene expression

Chopra, 2006

Pacello, 2012

S. Typhimurium D23580 - Altered virulence profile and pathogenesis-related 

stress responses

Yang, 2016

E. coli AMS6 - Increased biofilm formation and resistance to osmotic, 

ethanol and antibiotic stress

Lynch, 2006

E. coli E2348/69 - Increased intimin production Carvalho, 2005

E. coli MG1655 - Decreased growth

- Differential gene expression

Tucker, 2007

E. coli K12 - Differential gene expression Vukanti, 2008

Tirumalai, 2017

E. coli 083:H1 - Increased resistance to thermal and oxidative stress 

and adhesion to epithelial cells

Allen, 2008

E. coli O157:H7 - Increased cell size; membrane fluidity

- Decreased resistance to thermal stress

Kim, 2014

Kim, 2016

E. coli ATCC25922 - Increased expression of efflux pump genes; decreased 

antibiotic susceptibility

Xu, 2015

Klebsiella pneumoniae - Thicker biofilm formation; increased productin of 

cellulose

- Differential gene expression

Wang, 2016

Mycobacterium marinum - Differential gene expression; Role for SigH identified

- Increased sensitivity to oxidative stress

Abshire, 2016

P. aeruginosa PA01 - Increased: biofilm formation; elastase production, and 

rhamnolipid production; alginate production; resistance 

to oxidative and thermal stress; Hfq expression

- Differential gene expression

Crabbé, 2008

Crabbé, 2010

Streptococcus pneumoniae 

TIGR4

- Differential gene expression Allen, 2006

Allen, 2007

Streptococcus mutans - Differential gene expression

- Increased sensitivity to oxidative stress

Orsini, 2017

Table 2. Examples of Microbial Responses to Modeled Microgravity
26

success of the flight analog studies using the RWV resulted in the aforementioned 

two spaceflight experiments involving S. Typhimurium. One outcome of these 

investigations was the documented increased virulence of the bacterium in response 

to spaceflight, paralleling the bacterium’s response to LSMMG as produced by the 

RWV (Nickerson 2000, Wilson 2007) and validating its use as a spaceflight analog.

The RWV bioreactor technology has also been used to develop 3-D intestinal cell 

culture models as predictive human surrogates to study host-enteric pathogen 

interactions to unveil novel infectious disease mechanisms and in vivo-like 

outcomes not observed using traditional cell cultures (Nickerson 2001, Barrila 

2010, Drummond 2016, Barrila 2017). The RWV provides physiologically relevant 

low fluid shear conditions that enable 3-D architecture critical to recapitulate 

key aspects of the differentiated form and function of parental tissues in vivo 

(Barrila 2010). The first reported use of RWV-derived 3-D cell culture models 

of human intestinal epithelium was to study the early stages of S. Typhimuriuminduced enteric salmonellosis (Nickerson 2001). These 3-D models were further 

advanced by incorporation of phagocytic macrophages, a critical immune cell in the 

Microorganism Response to Modeled Microgravity 

within the RWV Bioreactor

Reference

S. aureus N315 - Increased: biofilm formation; susceptibility to whole blood

- Decreased: growth; carotenoid production; resistance 

to oxidative stress; Hfq expression

Castro, 2011

S. aureus RF1, RF6, RF11 - Decreased: carotenoid production; hemolytic activity

- Differential gene expression

Rosado, 2010

S. aureus 25923 - Increased: growth and membrane integrity Vukanti, 2012

Yersina pestis KIMD27 - Decreased: Hela cell rounding Lawal, 2010

Haloferax mediterranei - Increased antibiotic resistance

- Differential pigment production and protein expression

Dornmayr-Pfaffenhuemer, 

2011

Halococcus dombrowskii - Decreased cell aggraegation

- Differential pigment production and protein expression

Dornmayr-Pfaffenhuemer, 

2011

Vibrio fischeri - Differential gene expression

- Altered host-symbiote relationships

Foster 2013

Casaburi 2017

Saccharomyces cerevisiae - Increased aberrant budding

- Differential gene expression

Johanson, 2002

Purevdorj-Gage, 2006

Sheehan, 2007

Candida albicans - Increased: filamentous growth; biofilm formation; 

antimicrobial resistance; aggregation; random budding

- Differential gene expression

Altenburg, 2008

Searles, 2011

Crabbé, 2013
27

Salmonella infection process, to better reproduce the multicellular complexity of the 

parental tissue encountered during infection (Barrila 2017). 

In addition to the similarities between S. Typhimurium cultured in-flight and 

within the RWV bioreactor, other commonalities have been demonstrated. 

For example, scanning electron microscopy images revealed an unidentified 

extracellular matrix around S. Typhimurium cells following spaceflight culture 

(Wilson 2007) in response to the modeled microgravity conditions within the 

RWV bioreactor (Figure 5 A). P. aeruginosa, S. aureus, E. coli, and C. albicans have 

all demonstrated increased biofilm formation (Lynch 2006, Crabbé 2008, Castro 

2011, Searles 2011). 

With multiple reports of changes in phenotype following exposure to both true 

microgravity and simulated microgravity, differences in gene expression in response 

to culture in these environments is not unexpected. The most surprising find was 

the identification of the involvement of Hfq, an RNA chaperone protein that exerts 

post-transcriptional regulation by binding messenger RNA with small non- coding 

RNA (Valentin-Hansen 2004), with the mechanism governing the spaceflight 

response of S. Typhimurium (Wilson 2007). The role for Hfq was validated with 

the RWV bioreactor and has since been shown to be involved in the modeled 

microgravity response of both P. aeruginosa and S. aureus (Crabbé 2010, Castro 

2011). While first identified in spaceflight, the use of the RWV bioreactor on Earth 

produced evidence that suggests that the ability to sense and respond to mechanical 

stimuli such as microgravity and simulated microgravity may be evolutionarily 

conserved among structurally diverse prokaryotes.

Figure 4. Increased extracellular matrix production as a result of A) spaceflight culture in S. Typhimurium and modeled 

microgravity in B) P. aeruginosa and in C) S. aureus (Wilson et al. 2007, Crabbe et al. 2008, Castro et al. 2011).
28

What Principal Investigators

Should Know About

Conducting Research on

the ISS

Supporting research in science and technology is an important part of NASA’s 

overall mission. NASA solicits research through the release of NASA Research 

Announcements (NRA), which cover a wide range of scientific disciplines. All NRA 

solicitations are facilitated through the web-based NASA Solicitation and Proposal 

Integrated Review and Evaluation System (NSPIRES) http://nspires.nasaprs.com/ 

external/. Registering with NSPIRES allows investigators to stay informed of newly 

released NRAs and enables submission of proposals. NSPIRES supports the entire 

lifecycle of NASA research solicitations and awards, from the release of new research 

calls through the peer review and selection process.

In planning the scope of their proposal, investigators should be aware of available 

resources and the general direction guiding NASA research selection. NASA 

places high priority on recommendations from the latest National Research 

Council’s NRC Decadal Survey. In addition, principal investigators (PIs) should 

be aware that spaceflight experiments may be limited by a combination of power, 

crew time, or volume constraints. Launch and/or landing scrubs, causing delays 

or rescheduling, are not uncommon, so alternative implementation scenarios 

should be considered in order to reduce any risk of pre-launch payload impacts or 

post-landing samples vulnerability from these scrubs. Preliminary investigations 

using ground-based simulators may be necessary to optimize procedures before 

spaceflight. In addition, many experiments require unique hardware to meet the 

needs of the spaceflight experiment. 

To understand previous spaceflight studies, prospective PIs should 

familiarize themselves with the Space Station Research Explorer database 

(https://www.nasa.gov/stationexperiments). This database describes research 

conducted on the ISS, including that of International Partners [Canadian Space 

Agency (CSA), ESA (European Space Agency), Japan Aerospace Exploration 

Agency (JAXA), Roscosmos and others]. A detailed catalog of previous, 

current, and proposed experiments, facilities, and results, including investigator 

information, research summaries, operations, hardware information, and related 

publications is available.
29

Additionally, details pertaining to research supported by the Space Biology and 

Physical Sciences research areas within the Biological & Physical Sciences (BPS) 

Division of NASA’s Science Mission Directorate, and the Human Research Program 

in NASA’s Human Exploration and Operations (HEO) Mission Directorate can 

be located in the Task Book: Biological & Physical Sciences Division and Human 

Research Program in a searchable online database format at: https://taskbook.

nasaprs.com/Publication/welcome.cfm.

When planning microbiology experiments bound for the ISS, it is important that 

PIs understand the exposure risks to the crew members and implement the required 

levels of containment. Only microorganisms with a biosafety level of 1 or 2 are 

allowed to be flown to the ISS. Biosafety level 1 organisms usually require only one 

level of containment. Biosafety level 2 organisms are divided into two categories: 

those that are moderate risk agents associated with human diseases and those for 

which primary exposure routes include percutaneous exposure, ingestion, and 

mucous membrane exposure. Microorganisms that meet this description generally 

require two levels of containment. Biosafety level 2 organisms associated with a 

higher risk of human diseases in which a lower infectious dose, the likelihood of 

aerosolization, and/or larger amounts of agent are present may require three levels 

of containment. 

In order to fly any biological sample, an investigator must submit a biohazardous 

materials form through the NASA Biosafety Review Board (BRB). Information on 

flying biohazardous samples, including the form, can be found at: https://www.

nasa.gov/feature/hazardous-material-summary-tables-hmsts. 

Funding, Developing, and Launching Research to ISS 

Several sources of funding are available to scientists to be used for research, payload 

development, payload processing at NASA facilities, on-orbit operation, and more. 

Once a payload has been selected for development, engineering and operations, 

personnel in the ISS Program Office are available to work with payload teams 

through the design, test, certification, build, and launch phases prior to beginning 

mission operations on ISS. More detailed information on this process is available at 

the ISS Research & Technology Opportunities web page at https://www.nasa.gov/

stationopportunities. 
30

In general, NASA funding for space station use is obtained through NASA Research 

Announcements (NRAs). Funding for other government agencies, private, nonprofit, and educational use of the space station is obtained through research 

opportunities released by ISS U.S. National Laboratory. Space Station International 

Partner funding can be obtained through their respective processes. 

Potential proposers to any NASA program announcement should contact the 

relevant Program Scientist to discuss the appropriateness of their research concept 

to the specific solicitation and for contacts within the ISS Program Office to discuss 

expected development costs for their proposal budgets. 

For an overview of the types of considerations to make when planning and 

implementing a new payload, see The Quick Start Guide to Payload Design, found 

at https://www.nasa.gov/mission_pages/station/research/quick-start-guide.

ISS U.S. National Laboratory

In 2011, NASA finalized a cooperative agreement with the Center for the 

Advancement of Science in Space to manage the International Space Station U.S. 

National Laboratory (ISS National Lab). The independent, nonprofit research 

management organization ensures the station’s unique capabilities are available to 

the broadest possible cross section of U.S. scientific, technological and industrial 

communities.

The ISS National Lab develops and manages a varied research and development 

portfolio based on U.S. national needs for basic and applied research. It establishes 

a marketplace to facilitate matching research pathways with qualified funding 

sources and stimulates interest in using the national lab for research and technology 

demonstrations and as a platform for science, technology, engineering and 

mathematics education. The goal is to support, promote and accelerate innovations 

and new discoveries in science, engineering and technology that will improve life on 

Earth.

More information on ISS National Lab, including proposal announcements, is 

available at www.issnationallab.org.
31

Other Government Agencies 

Potential funding for research on the ISS is also available via governmental 

partnerships with ISS U.S. National Laboratory and includes (but is not limited to) 

such government agencies as:

• Defense Agency Research Projects Agency (DARPA)

• Department of Energy (DOE)

• Department of Defense (DOD)

• National Science Foundation (NSF)

• National Institutes of Health (NIH)

• U.S. Department of Agriculture (USDA) 

International Funding Sources

Unique and integral to the ISS are the partnerships established between the 

United States, Russia, Japan, Canada and Europe. All partners share in the greatest 

international project of all time, providing various research and experiment 

opportunities for all. These organizations – Russian space agency Roscosmos, 

Japan Aerospace Exploration Agency (JAXA), Canadian Space Agency (CSA), and 

ESA (European Space Agency), which includes Italy’s Agencia Spatiale Italiano 

(ASI), France’s Centre National d’Etudes Spatiales (CNES), and Germany’s 

Deutsches Zentrum für Luft- und Raumfahrt (DLR) or German Aerospace 

Center – provide potential funding opportunities for international scientists from 

many diverse disciplines.
32

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BRB Biosafety Review Board

BRIC Biological Research in Canisters

CASIS Center for the Advancement of Science in Space

CSA Canadian Space Agency

DNA Deoxyribonucleic Acid

ESA European Space Agency

EXPRESS EXpedite the PRocessing of Experiments for Space Station

ISS International Space Station

JAXA Japan Aerospace Exploration Agency

LSMMG Low-Shear Modeled Microgravity

MAS Microbial Air Sampler

MIC Minimal Inhibitory Concentration

NRA NASA Research Announcements

NRC National Research Council

NSF National Science Foundation

NSPIRES NASA Solicitation and Proposal Integrated Review and Evaluation System

PCR polymerase chain reaction

PI Principal Investigators

RWV Rotating-Wall Vessel

USSR Union of Soviet Socialist Republics

Acronyms
44

The Complete ISS Researcher’s 

Guide Series

1. Acceleration Environment

2. Cellular Biology

3. Combustion Science

4. Earth Observations

5. Fluid Physics

6. Fruit Fly Research

7. Fundamental Physics

8. Gene Lab

9. Human Research

10. Macromolecular Crystal Growth

11. Microbial Research

12. Microgravity Materials Research

13. Physical Sciences Informatics Systems

14. Plant Science

15. Rodent Research

16. Space Environmental Effects

17. Technology Demonstration

44
45

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