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EXPERIMENT RECORD N° 9693
Biorock - Extraterrestrial Geomicrobiological Package Test Bed for the ISS
 
|   Life Sciences:
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  |   Rene Demets rene.demets@esa.int |
K.W. Finster (1), C. Cockell (2), N. Nicholson (2), C.M. Loudon (2), D. Cullen (3), R. Möller (4), E. Rabbow (4), P. Rettberg (4), N. Leys (5), R. van Houdt (5), B. Byloos (5)
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BACKGROUND
Microbes can interact with rock. By lowering the pH, particular microbes are able to extract cations (Fe++, Mg++, Ca++ etc.) from the rock surface. The released ions are absorbed across the cell membrane and then incorporated, as minor but essential components, into the microbe’s proteins and other macromolecules. For many microbes the energy for ion extraction is provided by organic nutrients; they need food to accomplish this task.
Microbes interacting with rock can be considered as miners at microscopic scale. An ion-providing rock can eventually be covered by an extensive multilayer of microbial miners, called a biofilm. The biofilm accelerates the weathering of the rock. Biomining is nowadays well recognized and is used for instance in the copper industry.
The BIOROCK experiment investigates how gravity affects the interaction between microbes and rock in a liquid medium. The results will help to develop future equipment to support biomining on the Moon (0.17 g), Mars (0.38 g) and on asteroids (micro-g). The latter have recently been identified as the prime target for extra-terrestrial biomining.
Microgravity can be considered as an end-member condition to understand how mixing regimes such as convection currents change the three-component problem of the interaction of liquid, rocks and micro-organisms. BIOROCK is expected to gain basic physical insights into this problem, with many future applications other than mining (for example in life support systems involving microbial components).
Justification for the need of this space experiment
The minimum duration of low gravity and microgravity required for the BIOROCK experiment is in the order of weeks. There is no technique available on ground that can offer reduced gravity conditions for such a long time.
DETAILED OBJECTIVES
The interaction between microbes and rocks in a liquid phase can be affected by reduced gravity in more than one way. The reduction of thermal convection in low-gravity, and its absence in microgravity, will minimize the natural stirring in liquids and gases. This may restrict the supply of food and oxygen to the bacteria and hence lead to a suppression of growth, proliferation and mining performance. The first goal of BIOROCK is to verify and quantify this assumption. The second goal is to find out if morphological and genetic changes occur in the biofilms.
SPECIFIC GOALS
The goal of the BIOROCK experiment is to investigate the effects of altered gravity on the rock/microbe/liquid system as a whole. The objective is not to investigate whether bacteria can detect changes in gravity (as was demonstrated for some protists*). The questions and hypotheses that BIOROCK seeks to address are in particular:
1. Do Martian gravity and microgravity affect microbially-induced rock alteration?Hypothesis: Martian gravity and microgravity have both an impact on mixing regimes and therefore on microbe-mineral interactions.
2. Do Martian gravity and microgravity induce alterations in biofilms formed by microbes associated with rocks?Hypothesis: Space conditions change the structure and morphology of microbial biofilms formed on solid rocks substrates from which they are gathering nutrients.
3. Do Martian gravity and microgravity induce alterations in gene expression and mutation rates of microbes associated with rocks?Hypothesis: The space environment changes the microbe/mineral environment and hence the gene expression of rock-dwelling microorganisms.
4. To what extent is the (change of) mining performance in Martian gravity and microgravity dependent on the biological species?
Previous flight experiments
The interaction between microbes and rock was for the first time explored under microgravity conditions in 2014 on Foton-M4 in a pilot experiment from SCK-CEN.
Indirect precursors are the BIOFILTER and OCLAST experiments, performed in 2005 and 2007 on Foton-M2 (16 days in orbit) and Foton-M3 (12 days in orbit).
In both cases, cell cultures interacted with and partly consumed solid substrates.
In the BIOFILTER experiment (MAP AO-99-LS-019) microbes were grown on stainless steel, Kapton foil, polypropylene and aluminum tape. Especially the Kapton foil was heavily damaged by the bacteria. In the OCLAST experiment (NSS-95-5-I) osteoclasts were cultured in vitro on chips of bone. In space, the resorption of bone by the osteoclast turned out to be stronger than on ground (Tamma et al. 2009).
62nd ESA Parabolic Flight Campaign - 2015
RELATED RESEARCH
ISS Increment 64 - 2020
Academic Lecture
BioRock: Wee Miners in Space
Talk by Dr Rosa Santomartino, Postdoctoral Research Associate in Microbial Astrobiology at The University of Edinburgh - Streamed live on 3 July 2020
Microorganisms such as bacteria are everywhere on Earth’s biosphere, including the human body, and will necessarily follow humans on their journey during space exploration. As they play many important roles in biological processes on Earth, they will be crucial in space. For instance, microorganisms will be essential components of extraterrestrial life support systems. Possible applications include the extraction of useful elements from extraterrestrial rocks, an industrial process called biomining, and soil formation. During this online presentation, Dr Rosa Santomartino talked about how microorganisms could help us in the establishment of extraterrestrial human settlements, with a particular focus on biomining. She showed the results from the first space biomining experiment, BioRock, that was performed on the International Space Station in summer 2019, and gave an outlook on future microbiological applications in space.
The experiment hardware was launched on 25 July 2019 (22:01 GMT) on board the SpaceX-18 cargo craft.
The bacteria arrived at the Space Station in a dehydrated, dormant state.
The organisms are given ‘food’ to restore cell growth and left to grow on basalt at 20°C.
After three weeks, the samples will be preserved and stored at 4°C while they await their return to Earth.
Following unberthing from the International Space Station (ISS) and release from the Space Station Remote Manipulator System (SSRMS) at 14:59 UTC on 27 August 2019, the SpaceX’s CRS-18 Dragon spacecraft splashed down in the Pacific Ocean around 20:20 UTC.
Dragon CRS-18 brought back to Earth the following European experiment samples:
- deep-frozen samples of the Amyloid Aggregation experiment;- deep-frozen microbes of the BioRock experiment;- algae of German Aerospace Center DLR’s PhotoBioreactor;- Matiss-2 experiment.
Back on Earth, researchers will map out how altered states of gravity affect the rock and microbes as a whole, as well as which microbe is the best candidate for mining in space. It is hoped these results will shine light on extra-terrestrial biomining technologies and life-support systems involving microbes for longer duration spaceflight.
Protocol Overview
Pre-flight preparations in investigator’s lab
• Manufacturing of basalt slides• Preparation of cultures on basalt slides• Preparation of culture medium• Loading and assembly of the BMRs
Mission Operations
• Transport of BMRs to the launch site• Upload• On-board storage• Supply of culture medium: start of mining• Photography part 1• Separation of BMRs over three g-levels: μg, 1g, 0.38g• Provision of stable culturing temperature (incubator)• Supply of fixative: end of mining• Photography part 2• On-board storage• Download• Post-fixation on ground of all test samples• Delivery of BMRs to the investigator’s lab
Post-flight analysis in investigator’s lab
• Analysis of biofilms• Analysis of culture medium• Analysis of basalt slides
Four sets of Biomining Reactors (BMRs): 3 in space, 1 on ground
The BIOROCK experiment is proposed to be physically configured as a collection of small culturing devices, in this document referred to as Biomining Reactors (BMRs). Inside each BMR microbial cultures are grown, attached as a biofilm onto the surface of a flat piece of rock. The biofilm-covered rock is surrounded by culture medium that provides organic food, oxygen and some minerals (the missing minerals are provided by the rock).
Three microbial species have been selected to do the mining work. Each individual BMR will be occupied by a single species. For statistical reasons, three BMRs are required for each species (3 x 3 = 9 BMRs).
Considering that the culture medium itself may contribute to the weathering of the rock (see f.i. Wu et al. 2008) the core set of nine will be supplemented by three more BMRs for reference, without organisms but with rock substrate and with culture medium. That makes (3 x 3) + 3 = 12 BMRs per set.
Four sets of 12 BMRs (in total: 48) will be required in total as illustrated in Figure 1.
1. One set will be exposed to microgravity;2. A second set will be exposed to Martian gravity on an on-board centrifuge;3. A third set will be exposed to 1g on an on-board centrifuge;4. A fourth set shall be kept on ground in parallel to the flight experiment.
By comparing sets 3. and 4., contributions by non-gravity related space effects like launch vibrations and cosmic irradiation can be identified.
Organisms
Three microbial species have been selected for the BIOROCK experiment. They are:
• Bacillus subtilis var. subtilis (DSM 10)
Motility: sedentary (= immotile).Niche: commonly found in soil.Resting state: upon desiccation B. subtilis produces endospores.Note: B. subtilis can optionally swim using a flagellum, but not under the conditions defined for BIOROCK.B. subtilis will be prepared by DLR, Cologne.
• Sphingomonas desiccabilis (DSMZ 16792)
Motility: sedentary (= immotile).Niche: desert soil crust.Resting state: upon desiccation no spores are formed.S. desiccabilis will be prepared by the University of Edinburgh.
• Cupriavidus metallidurans CH34 (DSMZ 2839)
Motility: sedentary (= immotile).Niche: heavy metal plants in Belgium.Resting state: upon desiccation no spores are formed.C. metallidurans will be prepared by SCK-CEN, Mol.
The selection of the three organisms was based on several criteria:
1) they are robust to desiccation, enabling an upload under dry, dormant conditions,2) they are tolerant of thermal excursions,3) they have previously been implicated in microbe-rock interactions and thus rock weathering,4) they can be easily grown and manipulated in the laboratory, thus lending themselves to experimental use,5) all are risk group 1 (non-pathogen lowest category).
All three species are heterotrophic, meaning they need organic food. Heterotrophs are relatively easy to grow compared to chemolithotrophs. The latter do not need food because they are capable of tapping energy from inorganic salts. Chemolithotrophs such as Acidithiobacillus sp. have been used in biological mining operations but they grow specifically at a very low pH, which causes problems with experimental equipment.
Rock substrate
The bacteria will be grown on basalt. Basalt has been selected because the physical and chemical properties are very well known. Basalt is biocompatible and has successfully been tested in biomining experiments on ground.
Culture medium
The cultures will be uploaded in a desiccated, dormant state. The experiment shall be started in space by providing R2A liquid culture medium. R2A, a standard medium for growing microorganisms, has the following composition:
Component Concentration (g/L)
yeast extract: 0.5
peptone: 0.5
casamino acids: 0.5
glucose: 0.5
soluble starch: 0.5
Na-pyruvate: 0.3
K2HPO4: 0.3
MgSO4.7H2O: 0.05
all at pH 7.2
The organic molecules provided by the R2A medium are consumed by the bacteria. R2A contains in total 2.8 g of usable organic food per liter (= all components in above list except K2HPO4 and MgSO4.7H2O).
Note: In the actual experiment a water-diluted version of R2A might be used, with all its constituents at a final concentration that is two times lower.
Oxygen
All three organisms require oxygen.Microbes can sometimes be cultured under anaerobic conditions but growth, proliferation and mining capabilities are slowed down under such circumstances.
Fixative
The experiment shall be terminated in space by providing a chemical fixing solution to the cultures. The fixative that has been selected for BIOROCK is NOTOXhisto.
Culturing at μg, 1g, 0.38g
Temperature: 18°C - 22°C
Duration: Minimum - 15 days / Preferred - 21 days / Maximum - 25 days
On-board storage and download
Temperature: 2.0°C - 8.0°C / no freezing permitted / preferred: 4.0°C
Duration: Max. 4 weeks
GROUND REFERENCE EXPERIMENT
A reference experiment will be conducted on ground in parallel with the flight experiment, following to the as-planned timeline and parameters. Another ground experiment will be conducted when the flight experiment is over and all on-board recordings have been made available to the science team. In this second reference experiment, the timeline and parameters as actually monitored on board of the ISS will be reproduced. The same BMRs can be used for the first and the second ground reference experiment.
GROUND-BASED TEST RUN in spring 2019
In preparation for the experiment, researchers performed a "dry run" on Earth ahead of BioRock’s launch to the Space Station aboard a Space-X cargo resupply mission in July.
Cells of one of three organisms that will be used for BioRock were inoculated and dried on a sample of basalt, then given ‘food’ to restore cell growth. The biofilm was left to grow for three weeks at 20°C, then preserved and stored at 4-6°C for one month. Researchers finally observed the sample under a fluorescent microscope to assess its performance.
And it performed beautifully - see Figure 4. A patch of biofilm is visible to the right of the central cavity, which is the basalt’s natural porosity.
The results of the dry run show that the experimental conditions for BioRock, from the choice of the organism to the storage temperature and timing, are appropriate. This experiment also gave researchers the first clues as to what would be most interesting to focus on when samples return from space.
see ESA web story: http://www.esa.int/spaceinimages/Images/2019/03/BioRock
PLANNED POST-FLIGHT ANALYSES
All three components of the rock/microbe/liquid system will be analysed after the flight: the rock substrate, the biofilm and the culturing fluid. In detail:
- macroscopic analyses (rock material, biofilm formation),- pH measurements of the culture medium,- microscopic imaging (SEM, TEM, EM, light microscopy - staining),- biochemical investigations (determination of cation release rates by ion chromatography and inductively coupled plasma emission spectroscopy, medium changes by HPLC-MS),- microbiological analyses (e.g. quantification; cultivation-independent approaches),- genome and proteomic analyses,- biofilm studies (formation, stability, quantification of biomass and structure).
The following division of analysis is foreseen:
Biofilm Structure
Analysis of structure, morphology and 3D internal structure of biofilms: Kai Finster (Aarhus), Charles Cockell (Edinburgh) and Ralf Möller (Cologne).
Techniques: SEM, TEM, AFM, Confocal microscopy, Raman, staining.
Post-flight characterization of the microbial physiology
Cultivation studies, phenotypical characterization, microbial resistance, microgravity induced mutagenesis: Ralf Möller (Cologne).
Techniques: Growth and cultivation approaches, mutation screening, resistance analyses,
gene expression by transcriptional profiling.
Elemental release rates
Analysis of release rates of all major cations and trace elements into solution: Rob Van Houdt (Mol), Charles Cockell (Edinburgh). Solutions to be examined by ICP-MS.
Organics analysis
Analysis of organics production to identify potential agents of rock weathering: Charles Cockell (Edinburgh).
Analysis of metabolites in solutions: Kai Finster (Aarhus).
Proteomes of cells
Analysis of protein changes in cells in response to microgravity and presence of micro-bemineral interactions: Rob Van Houdt (Mol), Charles Cockell (Edinburgh).
We carried out a mining experiment on the International Space Station to test hypotheses on the bioleaching of REEs from basaltic rock in microgravity and simulated Mars and Earth gravities using three microorganisms and a purposely designed biomining reactor. Sphingomonas desiccabilis enhanced mean leached concentrations of REEs compared to non-biological controls in all gravity conditions. No significant difference in final yields was observed between gravity conditions, showing the efficacy of the process under different gravity regimens. Bacillus subtilis exhibited a reduction in bioleaching efficacy and Cupriavidus metallidurans showed no difference compared to non-biological controls, showing the microbial specificity of the process, as on Earth. These data demonstrate the potential for space biomining and the principles of a reactor to advance human industry and mining beyond Earth.
For more details, please, consult Reference Document no 22 (also see PDF in Attachment section):
C.S. Cockell, R. Santomartino, K. Finster, A.C. Waajen, L.J. Eades, R. Moeller, P. Rettberg, F.M. Fuchs, R. Van Houdt, N. Leys, I. Coninx, J. Hatton, L. Parmitano, J. Krause, A. Koehler, N. Caplin, L. Zuijderduijn, A. Mariani, S.S. Pellari, F. Carubia, G. Luciani, M. Balsamo, V. Zolesi, N. Nicholson, C.M. Loudon, J. Doswald-Winkler, M. Herová, B. Rattenbacher, J. Wadsworth, R.C. Everroad, R. Demets, (2020), "Space station biomining experiment demonstrates rare earth element extraction in microgravity and Mars gravity", Nature Communications, 11, DOI: 10.1038/s41467-020-19276-w, pp. 5523.
Expected results
The results are expected to provide a qualitative and quantitative comparison of bacterial/rock interactions taking place at terrestrial gravity, Martian gravity and microgravity.
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[14] | L.L. Wu, A.D. Jacobson, H.C. Chen, M. Hausner, (2008), "Characterization of elemental release during microbe-basalt interactions at T = 28°C", Geochimica et Cosmochimica Acta, 71, 9, DOI: https://doi.org/10.1016/j.gca.2007.02.017, pp. 2224-2239. |
[15] | A.L. Gronstal, V. Pearson, A. Kappler, C. Dooris, M. Anand, F. Poitrasson, T.P. Kee, C.S. Cockell, (2009), "Laboratory experiments on the weathering of iron meteorites and carbonaceous chondrites by iron-oxidising bacteria", Meteoritics & Planetary Science, 44, DOI: 10.1111/j.1945-5100.2009.tb00731.x, pp. 233-248. |
[16] | R. Tamma, G. Colaianni, C. Camerino, A. Di Benedetto, G. Greco, M. Strippoli, R. Vergari, A. Grano, L. Mancini, G. Mori, S. Colucci, M. Grano, A. Zallone, (2009), "Microgravity during spaceflight directly affects in vitro osteoclastogenesis and bone resorption", The FASEB Journal, 23, 8, DOI: 10.1096/fj.08-127951, pp. 2549-2554. |
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[20] | B. Byloos, I. Coninx, O. Van Hoey, C.S. Cockell, N. Nicholson, V. Ilyin, R. Van Houdt, N. Boon, N. Leys, (2017), "The impact of space flight on survival and interaction of Cupriavidus metallidurans CH34 with basalt, a volcanic moon analog rock", Frontiers in Microbiology, 8, DOI: 10.3389/fmicb.2017.00671, pp. 671. |
[21] | R. Santomartino, A.C. Waajen, W. de Wit, N. Nicholson, L. Parmitano, C.M. Loudon, R. Moeller, P. Rettberg, F.M. Fuchs, R. Van Houdt, K. Finster, I. Coninx, J. Krause, A. Koehler, N. Caplin, L. Zuijderduijn, V. Zolesi, M. Balsamo, A. Mariani, S.S. Pellari, F. Carubia, G. Luciani, N. Leys, J. Doswald-Winkler, M. Herová, J. Wadsworth, C.R. Everroad, B. Rattenbacher, R. Demets, C.S. Cockell, (2020), "No Effect of Microgravity and Simulated Mars Gravity on Final Bacterial Cell Concentrations on the International Space Station: Applications to Space Bioproduction", Frontiers in Microbiology, 11, DOI=10.3389/fmicb.2020.579156, pp. 2414. |
[22] | C.S. Cockell, R. Santomartino, K. Finster, A.C. Waajen, L.J. Eades, R. Moeller, P. Rettberg, F.M. Fuchs, R. Van Houdt, N. Leys, I. Coninx, J. Hatton, L. Parmitano, J. Krause, A. Koehler, N. Caplin, L. Zuijderduijn, A. Mariani, S.S. Pellari, F. Carubia, G. Luciani, M. Balsamo, V. Zolesi, N. Nicholson, C.M. Loudon, J. Doswald-Winkler, M. Herová, B. Rattenbacher, J. Wadsworth, R.C. Everroad, R. Demets, (2020), "Space station biomining experiment demonstrates rare earth element extraction in microgravity and Mars gravity", Nature Communications, 11, DOI: 10.1038/s41467-020-19276-w, pp. 5523. |
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 Figure 1: Experiment concept. The BIOROCK experiment is proposed to be conducted in four sets of 12 BMRs (= Biomining Reactors). Except for their biological contents, all BMRs are identical. | Figure 2: Experiment Flow Diagram from storage to storage. | Figure 3: Experiment flow with two centrifuges available (top) or just one (bottom). In the latter case terrestrial g and Martian g can only be provided sequentially, with the experiment spread out over two successive sessions. As illustrated, exposure to o-g can also be divided over in two successive runs. |
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Figure 4: In this image, taken with a fluorescent microscope, Sphingomonas desiccabilis is growing on basalt. Sphingomonas desiccabilis is one of three microbes chosen for the BIOROCK experiment. Microbes are able to weather down a rock from which they can extract ions. This natural process enables biomining, in which useful metals are extracted from rock ores. credit: UK Centre for Astrobiology/University of Edinburgh-R. Santomartino | Figure 5: ESA astronaut Luca Parmitano sets up the Biorock experiment by installing experiment containers in the small temperature-controlled Kubik incubators onboard the ISS. This experiment will continue to run in Kubik, unleashing a microbe on a basalt rock and assessing the biofilm that forms over the rock as the organism grows. Observing the rock-microbe system in space will help researchers understand the potential for biomining on other planetary bodies like asteroids, where new resources could be unearthed. | Figure 6: ESA astronaut Luca Parmitano slides samples into the Kubik experiment container on the ISS. For the next three weeks, three different species of bacteria will unleash themselves on basalt slides in the Kubik centrifuge that simulates Earth and martian gravity as well as in microgravity. The aim is to test how altered states of gravity affect biofilm formation - or the growth of microbes on rocks. Microbes are able to weather down a rock from which they can extract ions. This natural process enables biomining, where useful metals are extracted from rock ores. Already a common practice on Earth, biomining will eventually take place on the Moon, Mars and asteroids as we expand our understanding and exploration of the Solar System. |
 |  |  http://www.youtube.com/wa tch?v=J8p-5VMkwgU&app=des ktop |
Figure 7: ESA astronaut Luca Parmitano is taking pictures of the BioRock experiment on the International Space Station. The BioRock experiment was the first investigation into using organisms to mine for resources in space. For three weeks, three different species of bacteria will unleash themselves on basalt slides in the Kubik centrifuge that simulates Earth and martian gravity as well as in microgravity. The research is testing how altered states of gravity affect biofilm formation - or the growth of microbes on rocks. Microbes are able to weather down a rock from which they can extract ions. This natural process enables biomining, where useful metals are extracted from rock ores. Already a common practice on Earth, biomining will eventually take place on the Moon, Mars and asteroids as we expand our understanding and exploration of the Solar System. The bacteria arrived at the Space Station on the latest Dragon resupply mission in a dehydrated, dormant state. The organisms are given ‘food’ to restore cell growth and left to grow on basalt at 20°C. After three weeks, the samples will be preserved and stored at 4°C while they await their return to Earth. Researchers will map out how altered states of gravity affect the rock and microbes as a whole, as well as which microbe is the best candidate for mining in space. It is hoped these results will shine light on extraterrestial biomining technologies and life-support systems involving microbes for longer duration spaceflight. Copyright ESA/NASA | Figure 8: BioRock bio reactor. Credit: Kayser Italia | Academic Lecture: "BioRock: Wee Miners in Space"- Talk by Dr Rosa Santomartino, Postdoctoral Research Associate in Microbial Astrobiology at The University of Edinburgh - Streamed live on 3 July 2020 Summary: Microorganisms such as bacteria are everywhere on Earth’s biosphere, including the human body, and will necessarily follow humans on their journey during space exploration. As they play many important roles in biological processes on Earth, they will be crucial in space. For instance, microorganisms will be essential components of extraterrestrial life support systems. Possible applications include the extraction of useful elements from extraterrestrial rocks, an industrial process called biomining, and soil formation. During this online presentation, Dr Rosa Santomartino talked about how microorganisms could help us in the establishment of extraterrestrial human settlements, with a particular focus on biomining. She showed the results from the first space biomining experiment, BioRock, that was performed on the International Space Station in summer 2019, and gave an outlook on future microbiological applications in space. |
 http://eea.spaceflight.es a.int/attachments/spacest ations/ID5fac640ace71f.pd f | ||
Reference Document no 22: C.S. Cockell, R. Santomartino, K. Finster, A.C. Waajen, L.J. Eades, R. Moeller, P. Rettberg, F.M. Fuchs, R. Van Houdt, N. Leys, I. Coninx, J. Hatton, L. Parmitano, J. Krause, A. Koehler, N. Caplin, L. Zuijderduijn, A. Mariani, S.S. Pellari, F. Carubia, G. Luciani, M. Balsamo, V. Zolesi, N. Nicholson, C.M. Loudon, J. Doswald-Winkler, M. Herová, B. Rattenbacher, J. Wadsworth, R.C. Everroad, R. Demets, (2020), "Space station biomining experiment demonstrates rare earth element extraction in microgravity and Mars gravity", Nature Communications, 11, DOI: 10.1038/s41467-020-19276-w, pp. 5523. |
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