EXPERIMENT RECORD N° 9248
BASE-B/-C - Bacterial Adaptation to Space Environment, parts B & C
  1. 2008 • ISS Increment 18
Life Sciences:
  • Cell and Molecular Biology
Kubik
Jason Hatton
jason.hatton@esa.int
N. Leys (1), M. Mergeay (1), R. Wattiez (2), J. Mahillon (3), P. Cornelis (4)
(1)  
Belgian Nuclear Research Center (SCK/CEN)
Laboratory of Microbiology
Boeretang 200
2400 MOL
BELGIUM
Tel:  
+32(0)14332726
Fax:  
+32(0)14314793
e-mail:  
nleys@sckcen.be
(2)  
UMH - Université de Mons-Hainaut
Department of Biological Chemistry
6 Avenue du Champs de Mars
7000 Mons
BELGIUM
e-mail:  
ruddy.wattiez@umh.ac.be
(3)  
UCL - Université catholique de Louvain
Laboratory of Food and Environmental Microbiology
Institute of Life Sciences - Microbiology Unit
Bt. Kellner (D +1, d.148)
2 Croix du Sud (Box 12)
1348 Louvain-la-Neuve-Belgium
BELGIUM
Tel:  
+32(0)10473370
Fax:  
+32(0)10473440
e-mail:  
jacques.mahillon@uclouvain.be
(4)  
VUB - Vrije Universiteit Brussel
Department of Microbial Interactions
Faculty of Sciences
Pleinlaan 2
1050 Brussel
BELGIUM
Tel:  
+32(0)26291906
Fax:  
+32(0)26291902
e-mail:  
Pierre.Cornelis@imol.vub.ac.be

The BASE-B/-C experiment continued the research undertaken from the BASE-A experiment. The experiment carried the name "BASE" which stands for "Bacterial Adaptation to Space Environments".

AIM
Determine how different bacterial species adapt to the space flight environment by studying the behaviour of different model bacteria in spaceflight conditions.

SPECIFIC GOALS
1.
Clearly distinguish the effect of microgravity on bacterial adaptation from other space flight factors by use of an on-board centrifuge,
2. Assess the effects of spaceflight factors on:

(a) bacterial cell growth kinetics,
(b) cell physiology,
(c) gene transfer between bacterial species,
(d) gene modifications,
(e) gene activation or repression and
(f) post-transcriptional regulation

The BASE experiment had as main objective to study in detail the effects of space flight conditions on the behaviour of bacteria. The BASE experiment studied how bacteria cope with and adapt to the different space flight environmental parameters (e.g. microgravity, cosmic radiation, space electromagnetism, space vibrations). Based on these results, an assessment was carried out to look into the following aspects: how such adaptations might influence the bacteria´s potential to contaminate and biodeteriorate the space habitat (biodegradation & biocorrosion of structural materials), what is their potential to endanger crew health, or what is their function in waste recycling or food production systems.

In the BASE project, we will study the physiology, gene expression, gene rearrangement and gene transfer of cultures of several model bacteria grown under microgravity and other space flight conditions. We will re-investigate, refine and build on data obtained in previous microbial space flight experiments (MESSAGE1, November 2002; MESSAGE 2, October 2003; Mobilisatsia/Plasmida, April 2004). In this experiment, we will test different bacteria using the same experiment set-up and analysis protocols, to identify universal or bacterium specific responses. In addition, we will investigate more specifically the effects of reduced gravity in space flight and other space flight parameters separately. We will compare a flight-1g control experiment (on centrifuge space grown cultures - 1g ) with a flight-microgravity experiment (static space grown cultures - μg) and a ground control experiment (ground grown cultures - 1g). Bacteria are of great concern for long-term human space flight missions, as they can be of danger for crew health and can cause equipment biodeterioration. However, bacteria will also be essential in waste treatment and food-production systems to support prolonged stays of humans in space. Microbial space flight experiments may enable us to better understand the behaviour of bacteria in space habitats and maybe in the future to better predict, monitor and control microbial growth and activity of microbes during short and long term flights.

In addition the information will help to gather knowledge about the bacteria used in the MELiSSA (Micro-ecological life-support system) project in order to gain understanding of closed life-support systems, and consequently a knowledge base for European development of regenerative life-support systems which could prove valuable when planning future long duration expeditions to the Moon and Mars.

BASE-B/C is part of the BIO-4 mission (together with Xenopus and ROALD) which was launched into space onboard of Soyuz TMA-13 on 12 October 2008. The BIO missions provide flight opportunities for biology experiments requiring a short duration (less than 10 days) and limited on-orbit resources. They make use of the Russian Soyuz spacecraft for both launch and recovery of the experiment samples. BASE-B/C landed with Soyuz TMA-12 on 24 October 2008.

RELATED RESEARCH
BASE-A - Bacteria Adaptation to Space Environment - part 1

ISS "Astrolab" Long Duration Mission - 2006

MESSAGE 2 - Microbiological Experiment on Space Station About Gene Expression
ISS 7S (Soyuz TMA-3) Spanish "Cervantes" Mission - 2003

MESSAGE 1 - Microbiological Experiment on Space Station About Gene Expression

ISS 5S (Soyuz TMA-1) Belgian "Odissea" Mission - 2002


Fig. 1: Experiment Concept Diagram.

Each experiment container should have two replica chambers permitting addition of bacterial innoculum to the culture medium. Also there will be two more chambers without fixation to permit live cultures to be retrieved post flight.

Fig. 2: Mission Concept.

BIOLOGICAL SAMPLES
BASE-B (Anaerobic culture)
I. Ralstonia metallidurans CH34 (ATCC43123)
II. Bacillus turingiensis sv. kurstaki HD73 (ATCC35866)
III. Pseudomonas aeruginosia PA01 (ATCC 15692)

BASE-C (Aerobic culture)
IV. Cupriavidus metallidurans CH34 (ATCC43123)
V. Rodhospirillum rubrum S1H (ATCC25903)

Experiment protocol (see Fig. 5: Functional Objectives (FO) for time/temperature margins):
   - Biological material prepared at SCK/CEN & shipped at room temperature to Baikonur
   - ´Late Access´ - Samples prepared at Baikonour: preparations started at Launch-3d
   - Filling of ECs for space & parallel ground control -> in-flight activation & fixation
   - 5 experimental steps after launch;
   - Pre-activation storage : +4°C to +28°C

Activation: mixing cells with growth medium, 28°C for all cultures
Cultivation: Growth & multiplication of cells, 28°C for all cultures, up to 6 days
Fixation: Mixing cells with fixatives
Post-fixation storage: refrigeration of ECs at +6deg
Ambient Download of experiment samples in Soyuz, maximum duration = 24h
´Early Retrieval´ - Sample return: from landing to SCK maximum Landing +36h
active refrigeration & insulation of all ECs & Petri dishes during return to laboratory (to prevent post-flight growth)

GENERAL EXPERIMENT PROCEDURE
Parameters measured
- Proteome (RNAlater II fixed samples, post flight analysis);
- Transcriptome s (RNAlater II fixed samples, post flight analysis);
- Physiology (non-fixed samples post flight analysis);
- Gene transfer (non-fixed samples, post flight analysis);
- Ionizing radiation dose (passive Low LET & High LET track detectors);
- General vibration/acceleration environment around experiment samples;
- Optional: O2 and CO2 concentration during active phase of experiment and culture turbidity (note this capability is not currently foreseen in the current hardware development).

Ground reference experiment(s)
- Control #1: Performed simultaneous with flight experiment, using the same source bacterial cultures.
- Control #2: Performed post flight using temperature/time profile from flight experiment.

Fig. 3: Detailed Experiment Timeline and associated Functional Objectives (FO).
Fig. 4: Flow Diagram with Functional Objectives (FO) indicated.
Fig. 5: List of Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline.

Science deliverables
- RNAlater fixed bacterial culture samples for transcriptome and proteome analysis.
- Live bacterial cell cultures, refrigerated, for physiology and gene transfer analysis.
- Passive ionizing radiation dosimeters, exposed to the space radiation environment close to the experiment samples.
- Recorded data on vibration/acceleration environment around experiment samples.
- Recorded data on temperature environment around experiment samples.
- Recorded data on temperature & centrifuge settings in KUBIK.

Planned analyses

  • Physiology analysis
    The size, shape and membrane and intracellular composition of the cells grown under space flight conditions will be investigated using flow cytometry and several biochemical profiling techniques (substrate use profiling, antimicrobial resistance profiling, etc.)
  • Metabolome analysis (substrates used, metabolites produced)
    - The remaining concentrations of the substrates in the growth medium (carbon source, nitrogen source, phosphorus source, etc.) is analysed to investigate the metabolic activity of the cells grown in space flight conditions.
    - In addition the production of intracellular and extracellular metabolites (organic acids), energy and carbon storage compounds (PHB), or signalling molecules (quorum sensing molecules) will be investigated to assess the metabolic and communication activity under space flight conditions.
    - Mainly commercially available colorimetric spectrometry analysis kits and chromatographic techniques will be used for substrate and metabolite quantification and identification.
  • Proteome analysis (proteins)
    The total protein pool present in the cells grown under space flight conditions will be analysed to investigate which genes were actively translated in structural or functional proteins, and thus which physiological and metabolic processes were (in)activated. Various techniques protein profiling and identification tools will be used including 2D-gel electrophoresis, MALDITOF, HPLC-MS-MS.
  • Transcriptome analysis (RNA)
    The total RNA pool present in the cells grown under space flight conditions will be analysed to investigate which genes were actively transcribed, and thus which physiological and metabolic processes were (in)activated. Full bacteria genome DNA microarrays (in house produced or commercially available from Affimetrix) and RT-qPCR tools will be used.
  • Genome and gene transfer analysis
    Several of the test bacteria contain multiple mobile genetic elements (plasmids, insertion sequences, transposons, genomic islands, etc.). The stability and rearrangements of these mobile genetic elements in the space grown cells will be investigated. Several genome typing techniques and gene transfer techniques will be used such as PCR with specific primers and sequencing or conjugation, transformation, transduction experiments.

Expected results
Via these experiments the scientist team hopes to detect the changes in microbial physiology, metabolism or genetic information due to the space flight conditions. If changes are observed, an assessment of the possible impact of these changes for biosafety (crew health, spacecraft environment and life support system functioning) will be made.


The preliminary results indicate that none of the four test bacteria were able to proliferate in space during the incubation in the KUBIK. The reason remains unclear, as all cells were viable, present in the culture medium and temperature data indicated 28ºC. In addition, all parallel ground control cultures did grow. Only one of the bacteria that were packed in Biokit 2 for return showed growth in the non-fixed cultures. We suspect that growth occurred in these samples after incubation in the KUBIK during the 16.5 h additional storage at ambient temperature in the Soyuz before return. As four different bacteria and different culture media were involved, a ´biological´ cause of failure can be excluded. The four different bacteria and different culture media prepared by different research groups used in the flight experiment units were checked post-flight and have prove to be correct. In addition samples were prepared by different science teams, minimizing also the potential of human error as cause of experiment failure. Moreover, all tested bacteria have shown growth on ground in the same experiment conditions during the parallel ground control experiments and in space in previous flight experiments. Also the activation (adding cells to culture medium) and fixation (adding fixative to culture) seem to have taken place, and temperature readings indicated correct incubation temperature. Although the experiment failure root cause could not be identified, it is suspected that perhaps a possible error in the timeline, which may have caused a too short incubation time between automatic activation and fixation or removal from the KUBIK, may have limited the cell proliferation. Unfortunately, as no bacteria proliferated during flight, insufficient biomass was obtained to continue cell and molecular analysis. Thus the samples from the BASE-B and BASE-C flight experiment cannot be exploited any further and no trustable scientific data could be obtained.
click on items to display

Fig. 1: Experiment Concept Diagram.

Fig. 2: Mission Concept.

Fig. 3: Detailed Experiment Timeline and associated Functional Objectives (FO).

Fig. 4: Flow Diagram with Functional Objectives (FO) indicated.

Fig. 5: List of Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline.

Fig. 6: Kubik Experiment Hardware.
 
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