Microbial Monitoring in the International Space Station and Its Application on Earth

Abstract

The space habitat is a confined environment with a simple ecosystem that consists mainly of microorganisms and humans. Changes in the pathogenicity and virulence of bacteria, as well as in astronauts’ immune systems, during spaceflight may pose potential hazards to crew health. To ensure microbiological safety in the space habitat, a comprehensive analysis of environmental microbiota is needed to understand the overall microbial world in this habitat. The resulting data contribute to evidence-based microbial monitoring, and continuous microbial monitoring will provide information regarding changes in bioburden and microbial ecosystem; this information is indispensable for microbiological management. Importantly, the majority of microbes in the environment are difficult to culture under conventional culture conditions. To improve understanding of the microbial community in the space habitat, culture-independent approaches are required. Furthermore, there is a need to assess the bioburden and physiological activity of microbes during future long-term space habitation, so that the “alert” and/or “action” level can be assessed based on real-time changes in the microbial ecosystem. Here, we review the microbial monitoring in the International Space Station–Kibo, and discuss how these results will be adapted to the microbial control in space habitation and pharmaceutical and food processing industries.

1. INTRODUCTION

The space habitat is a confined environment with a simple ecosystem that consists mainly of microorganisms and humans. Recent studies revealed that microbes in the space habitat were mainly derived from humans and that some microorganisms brought in from the Earth have adapted to the environment.^1,2) It has been reported that, under microgravity, the biofilm development process and biofilm architectures differ from those observed on Earth.³⁾ The pathogenicity and virulence of bacteria change during spaceflight,⁴⁾ and astronauts’ immune systems may be weakened by stress.⁵⁾ These changes in the host–parasite relationship in the space habitat may pose potential hazards to crew health. To ensure microbiological safety in the space habitat, a comprehensive analysis of environmental microbiota is needed to understand the overall microbial world in this habitat. The resulting data contribute to evidence-based microbial monitoring, and continuous microbial monitoring will provide information regarding changes in bioburden and microbial ecosystem; this information is indispensable for microbiological management.

2. BACTERIAL MONITORING IN THE INTERNATIONAL SPACE STATION (ISS)

To ensure microbiological safety in long-term space habitation, it is necessary to understand the microbiota, bioburden, and microbial dynamics in the environment. Evidence-based recognition of the current microbiological situation in the space environment is indispensable for the assessment of potential microbiological risks. The National Aeronautics and Space Administration (NASA) and the Russian Federal Space Agency have been continuously monitoring microbes in the ISS.^1,6,7) The majority of microbes in the environments are hardly cultured under conventional culture conditions.^8,9) To understand the real microbial world, culture-independent approaches are required. In the past decade, the use of high-throughput sequencers has expanded in the field of environmental microbiology, facilitating these culture-independent analyses of bacterial abundance and community structures.

In Japan, since 2009, our team has collaborated with Japan Aerospace Exploration Agency (JAXA) to perform continuous monitoring of environmental microbes in the Japanese Experiment Module “Kibo” on the ISS (Experiment name, “Microbe”).^2,10) Astronauts sampled microbes from the surface in the Kibo using optimized sampling methods (Fig. 1). Samples were stored in the MELFI (Minus Eighty degree Celsius Laboratory Freezer for ISS) on the ISS, transported to Earth, and analyzed in our laboratory. In addition to conventional culture methods, the bacterial abundance and community structure of the microorganisms were analyzed using quantitative PCR and 16S ribosomal RNA (rRNA) gene targeted amplicon sequencing methods, respectively (Fig. 2).

Fig. 1. Photographs of Sampling Points in Kibo²⁾

(Color figure can be accessed in the online version.)

Fig. 2. Procedure for the Microbiological Analysis of Kibo Samples

The results showed that most bacteria present in the Kibo were constituents of human microbiota, and that these bacteria became established through long-term operations in the Kibo (Fig. 3). These bacteria might have originated from astronauts. However, the bacterial abundance was close to the limit of quantification (10²–10³ cells/cm²), approximately one-tenth of bacterial abundance in the ground laboratory, which indicated that microbiological control in the Kibo was appropriate (Table 1). Because bacteria in the ISS are generally considered to be human-derived bacteria, it is difficult to eliminate microbes while people are present, even in a well-controlled environment. Therefore, it is important to properly manage and coexist with microorganisms, based on data collected in the space habitat.

Fig. 3. Bacterial Community Structure on the Interior Surfaces in Kibo²⁾

(a) At the phylum level. (b) Expanding beta- and gamma-proteobacteria and Firmicutes to the family level. (Color figure can be accessed in the online version.)

Table 1. Bacterial Abundance on Interior Surfaces in Kibo Determined by Fluorescent Microscopy and Quantitative PCR²⁾

	Microbe-I		Microbe-II		Microbe-III
	TDCa)	qPCRb)	TDC	qPCR	TDC	qPCR
Outer surface of incubator	2 × 103	4 × 103	2 × 102	<1 × 102	2 × 102	<1 × 102
Air diffuser	9 × 102	2 × 103	<2 × 102	3 × 102	<2 × 102	<1 × 102
Handrail	7 × 102	5 × 102	<2 × 102	1 × 102	2 × 102	<1 × 102
Air return grill	NTc)	NT	<2 × 102	1 × 102	<2 × 102	<1 × 102
Internal surface of incubator	NT	NT	<2 × 102	1 × 102	<2 × 102	<1 × 102

Unit: cells/cm². a) TDC, total direct counting with fluorescent microscopy. b) qPCR, quantitative PCR. c) NT, not tested.

As previously mentioned, microbial monitoring at the ISS is performed by transporting samples collected in the ISS to a ground laboratory for analysis. During long-term space habitation, onboard and real-time microbial monitoring is indispensable for rapid responses to microbiological hazards. State-of-the-art newly developed rapid microbiological methods will facilitate on-site monitoring and analysis of microbes in real time. In the near future, the bioburden and physiological activity of microbes will be determined aboard the ISS, so that the “alert” and/or “action” level can be judged in real time based on changes in the microbial ecosystem.

3. CURRENT STATUS OF MICROBIAL MONITORING AT NASA AND THE EUROPEAN SPACE AGENCY

NASA conducted Microbial Tracking Experiment 1 (MT-1) from 2015 to 2017 and Microbial Tracking Experiment 2 (MT-2) from 2017. MT-1 was performed to catalog the environmental microbiome of the ISS and MT-2 was performed to catalog and characterize potential human-related disease-causing microorganisms. In MT-1, culture methods were compared to molecular methods to characterize the diversity of bacteria and fungi on the ISS.⁷⁾ NASA has a database of molecular analysis from space experiments, named “GeneLab,” which is open to the scientific research community. The molecular data sets from MT-1 and MT-2 experiments were placed in the NASA GeneLab to encourage future innovation. In Europe, the European Space Agency module “Columbus” is regularly screened for surface contamination with the same procedures used by NASA.

NASA is promoting molecular methods for space microbe studies, which are useful for analysis because they facilitate understanding of the microbial world on the ISS. DNA amplification by a mini-PCR system was launched on the ISS in 2016 and samples of zebrafish DNA were amplified using a plasmid prepared in the ground laboratory. The sample was processed by PCR and a genetic analysis was performed while in orbit using the Wetlab-2 project. A very small stick-size DNA sequencer, MinION (Oxford Technologies) was launched to the ISS, which could analyze DNA without large errors due to microgravity.

A new NASA Translational Research Institute was established to develop the “Omics in Space (OIS)” project, which involves monitoring of physiological and immunological conditions that affect humans during spaceflight missions. NASA is developing automatic instrumentation for nucleic acid extraction and sequencing methodologies in space (e.g., PCR, DNA sequencing, and sample manipulation systems). Without returning samples to the ground, microorganisms can be monitored and identified in space in the near future.

4. GROUND APPLICATIONS

Modern microbiological methods for microbial monitoring are rapid and provide more precise information than conventional methods. In particular, 16S rRNA gene targeted amplicon sequencing can reveal the detailed microbial characteristics of the target environment.¹¹⁾ These data provide important information to manage microbes both in space and on the ground. These methods are included in the Japanese Pharmacopoeia 17th edition, published in 2016.¹²⁾ In the pharmaceutical and food processing industries, these new methods will be applied for microbiological quality control and environmental monitoring. With protocol standardization and validation of newly developed microbiological methods or rapid microbiological methods, maximum and minimum thresholds can be defined for environmental quality control of air, water, and surfaces in microbiologically controlled facilities, such as pharmaceutical manufacturing fields. These culture-independent new methods will lead to a new era of microbiological control in space and on earth.

5. CONCLUSION

Human activities have expanded to space environments and space agencies are planning long stays on lunar bases, as well as exploration of Mars. NASA is working with other space agencies to develop a small spaceship, known as the Space Gateway, that will orbit the Moon. This commute from Earth to this spaceship will be approximately 5 d (250000 miles); the ship will be the base for astronauts’ expeditions on the Moon, and future human missions to Mars. The data obtained from microbiological studies in the ISS and simulated environments are indispensable for manned space exploration. The findings regarding the relationship between humans and microorganisms obtained in space, which is the challenging ultimate living environment, will also be useful on the ground.

Acknowledgments

This review was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, KAKENHI (Grant-in-Aid for Scientific Research on Innovative Areas) Grant Number JP15H05946.

Conflict of Interest

The authors declare no conflict of interest.

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