1102 *B. thuringiensis* strains between 1 g and the three different simulated microgravity conditions established using the RWV, the RPM, and a superconducting magnet. The spread and increase of antibiotic resistance and bacterial virulence in spaceflight could be linked to efficient gene transfer events (Senatore et al., 2018; Timmery et al., 2011; Tirumalai et al., 2019). Horneck et al., (2010) suggested the need to investigate microbial adaptation and evolution via transferable genetic elements, like phages, extrachromosomal DNA, and transposons in the space environment. On the other hand, it is shown that space environmental conditions can influence the frequency of mutation in microbial cells. Short-term spaceflights experiments have shown no effect on the mutation rate or repair activity in *E. coli* (Harada et al., 1997) and S. cerevisiae (Pross and Kiefer, 1999) but a long-term experiment has shown a pronounced effect on mutation frequency (Fukuda et al., 2000). After 40 days aboard the Russian space station MIR, the mutation rate for a cloned bacterial gene was found to be 2- to 3-fold higher than the ground control (Fukuda et al., 2000). *Alternation* in frequency and spectrum of spontaneous mutation has also been reported in *Staphylococcus epidermidis* and *B. subtilis* cells flown aboard the ISS. Compared to ground controls the mutation rate of the *rpoB* gene leading to rifampicin resistance was noted to be significantly higher in the spaceflight samples (Fajardo-Cavazos et al., 2018; Fajardo-Cavazos and Nicholson, 2016). These findings suggest that space environment induces stresses on bacterial cells causing changes in mutagenic potential and gene transfer events.

1119 The exploration of the unique environment of outer space (microgravity and radiation) to improve microbial strains for the production of biotechnology-relevant secondary metabolites gained interest because space environment favored microbial growth (shortened lag phase, faster growth rate, prolonged exponential phase, higher cell density, and proliferation in presence of antibiotic concentrations), promoted efficient DNA exchange, and elicited a wide spectrum of metabolic responses. Due to the high potential of the space environment to mold organisms with desired traits, Horneck et al., (2010) proposed the commercial application of space for the development of new pharmaceuticals, which would enhance our understanding of how bioprocesses occur under the absence of the ever-present gravity to design schemes and approaches to develop novel compounds. Improvement of the production of secondary metabolites, such as actinomycin D (Lam et al., 2002), monorden (Lam et al., 1998), and kanglemycin C (an immunosuppressant) (Zhou et al., 2006) by space cultured *Streptomyces plicatus*, *Humicola fuscoatra* WC5157, and *Nocardia mediterranei* var. *kanglensis*, respectively, have been reported. Recently, Huang et al., (2015) investigated the effects of microgravity on antibiotic production by *Streptomyces coelicolor* A3 and found that microgravity conditions altered secondary metabolism and resulted in the production of more bioactive substances highly active against *B. subtilis*. Several groups have also studied the effect of simulated microgravity using LSMMG. However, ground-based facilities do not produce results as observed in spaceflight experiments. Experiments to test the production of antibiotics using the RWV, which partially simulate the weightless environment, resulted in inhibition of antibiotics, such as the microcin B17 by *E. coli* (Fang et al., 1997a), β-lactam by *Streptomyces clavuligerus* (Fang et al., 1997c), and rapamycin by *S. hygroscopicus* (Fang et al., 2000). In addition, no effect of simulated gravity was noticed in the case of gramicidin-S production by *B. brevis* (Fang et al., 1997b). The response of these organisms to actual spaceflight experiments to produce antibiotics are not performed to draw conclusive results on actual microgravity effects. In addition, pilot experiments have been conducted to develop new vaccines against diarrhea-causing strains of *Salmonella* aboard the ISS (Horneck et al., 2010). Taken together, these studies suggest that space microgravity environment can alter secondary metabolism in microbes, suggesting potential production, development, and commercial application of compounds, such as antibiotics.

1147

## 5.1. Conclusions from observed effects in microbes

1149

1150 Majority of the studies conducted till date indicate that microgravity has a strong influence on microbial growth and behavior and could lead to the evolution of properties, which does not occur on Earth, such as a column-and-canopy structure of bacterial biofilms and attainment of a growth stage that facilitates circumvention of the immune system. The mechanisms responsible for the observed biological responses, however, are not yet fully understood. We are, however, just beginning to scratch the surface in unraveling the molecular and genetic mechanisms by which gravity influences microbial growth and behavior. The major influence of a lack of gravity is a direct effect of the environment surrounding the cell simply because there is no convection only diffusion. This suggests that in
microgravity the observed changes in microbial growth and behavior are primarily indirect. Future studies focused on studying microbial behavior (e.g., biofilm production, antibiotic resistance, etc.) in conjunction with genetic studies on the regulation of the genes known to be involved with a particular behavior will lead to a clear understanding of the role gravity plays in microbial growth, development, and behavior. Studies on the interactions of both harmful and beneficial microbes with humans under altered gravity conditions need to be emphasized. Experiments with recent molecular tools are likely going to advance our knowledge and understanding and provide an intriguing foundation for the exploration of the space environment to develop novel products.

6. Microgravity and cancer research

Spaceflight affects human physiology in both a positive and a negative direction. In addition to the variety of the observed negative biological effects on the human body, caused by a long-term spaceflight (reduction in bone density, loss of muscle mass, diminished cardiac function, distorted balance, impaired vision and others (White and Averner, 2001), several studies indicate that microgravity may have an anticancer effect (Krüger et al., 2019c). The low-gravity environment significantly affects key cellular processes, including gene transcription and intercellular contacts (Hughes-Fulford et al., 2015; Lewis et al., 1998; Martinez et al., 2015) and thus influences cell growth, cell cycle, self-renewal (Arun et al., 2019) and differentiation of cancer cells without the need for drugs (Chen et al., 2019; Jessup et al., 2000; Vidyasekar et al., 2015). Microgravity slows proliferation and elevates apoptosis of different cancer cell types derived from lymphoma (Kim et al., 2017), glioma (Zhao et al., 2017), osteosarcoma (Wei et al., 2013), and different carcinomas (Chang et al., 2013; Grimm et al., 2002; Kopp et al., 2018; Kossmehl et al., 2002; Kossmehl et al., 2003; Masiello et al., 2014; Qian et al., 2008). Cytoskeletal alterations occur very early in cancer cells (Corydon et al., 2016a; Nassef et al., 2019) and are often suggested to be an effect of gravitational unloading (Vorselen et al., 2014). The impaired cytoskeleton of cancer cells in microgravity is assumed to be attributed to cell death (Arun et al., 2017; Vidyasekar et al., 2015). Qian et al., (2008) reported that simulated-microgravity affects cell migration and adhesion of breast cancer cells. Furthermore, microgravity has been reported to cause stress signaling due to mechanical forces of tensegrity (Ingber, 1999; Ingber, 2014), cellular oxidative and endoplasmic reticulum stresses (Morabito et al., 2017). Hodgkin's lymphoma cells exposed to microgravity showed dysfunctional mitochondria leading to reduced ATP levels and autophagy (Jeong et al., 2018), which was found elevated in various cell types after annulling gravity (Sambandam et al., 2014). Autophagy is involved in stemness regulation of human stem cells and contributes to the pluripotency of cancer stem cells (Kenific et al., 2016; Sharif et al., 2017). Experiments using simulated microgravity produced by an RPM (Kopp et al., 2015) as well as space missions, such as SimBox/Shenzhou-8 (Ma et al., 2013) or CellBox-2 (Melnik et al., 2018), indicated that thyroid carcinoma cells are considerably less aggressive in microgravity than on Earth. Prostate cancer cells were also found to be less aggressive and showed changes in lipid second messengers and signal transduction after exposure to simulated microgravity using the NASA-developed High Aspect Ratio Vessel (HARV) (Clejan et al., 1996; Clejan et al., 2001). This finding will now be evaluated in real microgravity. In addition, breast, prostate, and thyroid carcinoma cells form unique morphological features when exposed to simulated microgravity (Fig. 7A) - a further sign of differentiation (Corydon et al., 2016b; Kopp et al., 2015; Lelkes et al., 1998).

However, most mechanisms by which microgravity induces these cellular responses are still poorly understood and will be a subject of future research in space medicine. In this regard, microgravity-based research provides a very special culture environment to detect changes in proteins representing new targets for drug development in cancer. Many of these proteins and available drugs were found by analyzing multiple pilot studies with the help of semantic analyses (Bauer et al., 2017; Bauer et al., 2018). Using these results, scientists will be able to target or silence individual genes in cancer cells with pharmacological substances to inhibit selected signaling pathways. This way, they may be able to elucidate cellular changes in cancer development. The first success was achieved with MCF-7 breast cancer cells: metastasis-like spheroid formation was prevented by c-Src inhibition but promoted by blocking E-cadherin activity (Sahana et al., 2018). After annulling gravity, cells can arrange themselves scaffold-free into large three-dimensional (3D) aggregates without any signs of necrosis (Fig. 7B) (Ingram et al., 1997; Kopp et al., 2015).
1213 The extracellular matrix of these multicellular tumor spheroids comprises a complex 3D
1214 network of cell-cell and cell-matrix interactions that differs in amount and composition from
1215 corresponding monolayer cultures (Ulbrich et al., 2011). It affects the distribution and function of
1216 hormones and growth factors and thus fundamentally determines the regulation of cell growth,
1217 differentiation, and apoptosis (Kunz-Schughart, 1999). The model system 'Multicellular Spheroid'
1218 (MCS) mimics metastasis of solid tumors and delivers valuable information about cancer progression
1219 *in vivo* and is often used for pharmacological drug testing (Braddock, 2019; Zanoni et al., 2019). The
1220 special 3D architecture of tumor spheroids might facilitate the penetration and action of drugs more
1221 closely mimicking the situation of a tumor in the human body. In addition, the tumor spheroids and the
1222 more complex cancer organoids that were grown from breast tumor (Kaur et al., 2011; Kunz-Schughart
1223 et al., 2001) or prostate tumor cells (Zhau et al., 1997) together with fibroblasts have become important
1224 *ex vivo* models to study cancer key processes like neoangiogenesis, tumor cell invasion, and to
1225 investigate tumor-microenvironment and cell-cell-interactions (Hamilton, 1998; Kunz-Schughart, 1999;
1226 Kunz-Schughart et al., 2004).

1227 Microgravity, in space or produced by special devices on Earth, provides a unique environment
1228 for cancer research that has allowed insights in cancer biology that would not have been possible under
1229 normal cell culture conditions. The results already influence conventional cancer research and contribute
1230 to the understanding of cancer progression and the development of new drugs and cancer treatment
1231 strategies.

1232 Upcoming projects will examine the roles of both microgravity and cosmic radiation in tumor
1233 growth and development onboard the ISS and the Chinese Space Station (CSS). The studies of cosmic
1234 radiation, the main concern for space exploration by humans, will also help with understanding the
1235 cancer risk for astronauts on long-duration stays on space stations, or longer journeys, such as to Mars.
1236

1237 7. Tissue engineering in space
1238

1239 Microgravity-based scaffold-free tissue engineering is a novel technique in translational
1240 regenerative medicine. For this purpose, the cells are exposed to microgravity in space or to devices
1241 simulating microgravity conditions on Earth, so-called ground-based facilities (GBF), acknowledged
1242 among others by ESA (European Space Agency) and NASA. A large number of studies performed in
1243 real and simulated microgravity revealed that one part of the cultured cells of various cell types detaches
1244 from the bottom of the culture dish and starts forming 3D aggregates or MCS (Grimm et al., 2018),
1245 while another part remains growing as a 2D monolayer on the bottom of the culture flask. These MCS
1246 resemble the organs or original tissues from which the cells originally derived. In this section, we review
1247 the current knowledge about tissue engineering of cartilage, bone, and tube formation by endothelial
1248 cells in space and on ESA-GBF.
1249

1250 7.1. Cartilage tissue engineering
1251

1251 Diseases of the cartilage comprise osteoarthritis, a frequent cause of chronic pain, and a disorder
1252 with increased prevalence worldwide due to the aging population. Unfortunately, no causal treatment is
1253 available, and the major objective is to treat the pain and to attenuate the disease progression. Therefore,
1254 new research focusing on the chondrocytes is mandatory and new ideas are welcome. Microgravity-
1255 based approaches of cartilage tissue engineering have been performed in space on the MIR space station.
1256 Bovine chondrocytes were grown in 3D polyglycolic acid matrices at 1 g and cultured in rotating
1257 bioreactors for three months. One of these reactors was subsequently sent to space (MIR) where the cell
1258 culture continued to grow for another four months. In parallel, the remaining samples were kept as
1259 ground controls. Both culture conditions resulted in functional cartilage tissues, but the cartilage sample
1260 cultured in space revealed a lower size and mechanical strength (Freed et al., 1997; Freed et al., 1999;
1261 Freed and Vunjak-Novakovic, 1997).

1263 Other researchers performed a scaffold-free experiment with porcine chondrocytes, which were
1264 cultured in special cylindrical culture chambers. The cells were exposed to real microgravity on the ISS,
1265 to simulated microgravity on an RPM and 1 g on Earth. The ISS samples revealed a reduced amount of
1266 extracellular matrix proteins together with a reduced cell density compared to the 1 g controls
1267 (Stamenković et al., 2010). Various experiments with chondrocytes have been performed to engineer
1268 cartilage constructs using ESA GBF (Aleshcheva et al., 2016). Ohyabu et al., (2006) demonstrated that
1269 rabbit bone marrow mesenchymal stem cells cultured in an RWV bioreactor formed large cylindrical
1270 cartilaginous pieces without scaffolds. Another interesting approach was the production of hyaline
1271 cartilage using a RWV (Sakai et al., 2009). Human mesenchymal stem cells (MSC) exposed to simulated
1272 microgravity revealed an increased proliferation, and among others, a higher expression of collagen II
1273 compared to 1 g-samples (Yuge et al., 2006). Only the simulated-microgravity samples were able to
1274 form hyaline cartilage after transplantation in cartilage defective mice (Yuge et al., 2006).
1275

1276 Taken together, cartilage tissue engineering with and without scaffolds is an elegant technique
1277 to produce viable, functional cartilage constructs (Fig. 7C) (Grimm et al., 2018). Tissue engineering of
1278 comparable constructs is not possible with conventional static 1g-culture conditions. Furthermore, future
1279 research should increase scaffold-free approaches in space accompanied by GBF studies, avoiding
1280 potential pitfalls associated with non-autologous biomaterials. A more personalized approach should be
1281 introduced by studying patient's chondrocytes after, for example, an injury to engineer autologous
1282 cartilage.
1283

## 7.2. Tissue engineering of bone
1284

1285 Microgravity-induced osteopenia and bone loss is a serious health problem for long-term space
1286 missions (Grimm et al., 2016). Astronauts lose between 1 and 2% of weight-bearing bone mass during
1287 long-term missions (Cappellesso et al., 2015). These changes in bone structure and metabolism are still
1288 an important health concern of humans in space today (Chatani et al., 2015; Grimm et al., 2016; Vico et
1289 al., 2000). Despite this knowledge, it is of interest that simulated microgravity also induces bone
1290 formation. Bone tissue engineering using microgravity-based methods is currently a hot topic in
1291 regenerative medicine. The incidence of bone diseases is increasing because of aging populations,
1292 obesity, and reduced physical activity (Amini et al., 2012). The destruction of bone tissue is often a
1293 result of trauma or disease. The use of stem cells, specialized cells, scaffolds, and growth factors to
1294 improve bone formation are discussed as possible therapeutic strategies. Tissue-engineered bone
1295 fragments from human osteoblasts might serve as a potential alternative to the conventional use of bone
1296 grafts.
1297

1297 Various research teams used the NASA-developed RWV in bone tissue engineering studies.
1298 These RWV-derived bone constructs from osteoblasts and stem cells were comparable to natural bone
1299 and had demonstrated an improved *in vivo* effectiveness in bone repair in animal studies (Cerwinka et
1300 al., 2012; Cui et al., 2018; Han and Dai, 2016; Jin et al., 2010; Sikavitsas et al., 2002; Ulbrich et al.,
1301 2014). Clarke et al., (2013) had developed a new method for tissue engineering of bone. The authors
1302 used successfully a low shear stress rotating bioreactor, such as a HARV culture system. Mann et al.,
1303 (2019) exposed human fetal osteoblast (hFOB 1.19) cells to an RPM for 7 and 14 days. This new study
1304 was the first that demonstrated a scaffold-free approach to engineer 3D bone constructs on the RPM
1305 (Fig. 7D). The next step will be to investigate these constructs in an *in vivo* animal model.
1306

1306 Taken together, new discoveries have been published in the field of microgravity-based bone
tissue engineering. Due to the huge impact of musculoskeletal medical conditions on Earth and for future
1307 space exploration, further studies on the influence of microgravity on osteoblasts and stem cells are
1308 important. This novel technology first can be used to study the mechanisms behind spaceflight-related
1309 bone loss and second, the engineered bone constructs might be suitable for the repair of bone diseases
1310 such as osteonecrosis or bone injuries.
1311

## 7.3. Growing blood vessels in space
1312

1313 Tissue engineering of scaffold-free small biocompatible blood vessels, which are applicable for
1314 vascular reconstruction purposes in transplantation, plastic, aesthetic, and hand surgery (Fig. 7E), is an
1315 important medical research topic. A key benefit of this technique is the scaffold-free engineering of 3D
1316 tubes without any foreign materials. Under normal static gravity (1g) conditions in Earth-based
1317 laboratories, a 2D monolayer of endothelial cells (ECs) is comparable to the inner surface of blood
1318 vessels (intima) but does not completely show the vascular characteristics found in vivo.
1319

1320 Sanford et al., (2002) cultured bovine aortic ECs in an RWV for up to 30 days. They found large
1321 tissue-like aggregates (containing 20 or more beads) after 30 days. This was the first report of a unique
1322 growth pattern for ECs, resulting in enhanced NO production and barrier properties. Another group used
1323 a perfusion system and developed a cell culture model for a small-diameter vessel engineered from
1324
adipose tissue stromal cells together with human umbilical vein endothelial cells using a tube-like gelatin scaffold (Frerich et al., 2006; Frerich et al., 2008). Human umbilical cord blood stem cells (CBSC) were exposed to an RWV for 14 days (Chiu et al., 2005). CD34+ mononuclear cells were cultured in the bioreactor with or without microcarrier beads (MCB) and vascular endothelial growth factor. RWV cultures showed increased proliferation and 3D tissue-like aggregates. At day 4, CD34+ cells without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. The CD34+ CBSC is capable to differentiate in a vascular endothelial cell phenotype and to form 3D tissue structures when exposed to the RWV (Chiu et al., 2005). Another study focused on MSCs exposed to simulated microgravity for 72 h. The authors demonstrated that MSC was able to differentiate into endothelial-like cells, express endothelial-specific molecules (Flk-1 and vWF), and form a capillary network (Zhang et al., 2013). Using a clinostat, (Shi et al., 2016) showed that clinorotation induced cell migration and tube formation in ECs in vitro depending on caveolin-1. A large number of studies of our team investigating ECs exposed to simulated microgravity conditions had demonstrated that ECs can form 3D tube-like structures (intima constructs) (Dittrich et al., 2018; Grimm et al., 2009; Grimm et al., 2010; Infanger et al., 2006; Ma et al., 2013; Ma et al., 2014; Ulbrich et al., 2008). In microgravity, the *de novo* construction of scaffold-free, functional, organo-typic tissues has a great advantage, because ECs grow as spheroids and as 3D tubular structures without requiring any artificial matrix or scaffolds (Grimm et al., 2018). These tubes can be cultured for more than 35 days. Interestingly, they do not develop necrosis or stop growing (Dittrich et al., 2018). Human endothelial cells of the EAhy926 cell line had been sent to the ISS (SpaceX CRS-8 mission) in 2016 to determine the impact of real microgravity on 3D growth (Pietsch et al., 2017). The ESA-SPHEROIDS project confirmed the formation of 3D vessel constructs (Krüger et al., 2019b; Pietsch et al., 2017). Cell culture in space was possible with a specifically designed automatic hardware, which allowed programmed cell feeding and fixation (Pietsch et al., 2017). A recent study reported the impact of clinorotation on endothelial progenitor cells (EPC) (Hagiwara et al., 2018). The gene expression analysis revealed an increase in angiogenic factors, including vascular endothelial growth factor. Moreover, the initial cultivation on a 3D clinostat followed by cultivation under 1g remarkably improved the EPC expansion rates and the angiogenic potential (Hagiwara et al., 2018). This interesting approach should be investigated in real microgravity in future space experiments.

In summary, microgravity-based research contributes to new developments in regenerative medicine using stem cells or ECs. Co-culture experiments of ECs with vascular smooth muscle cells and fibroblasts on an RPM will be a further step to engineer a complete, functional vessel. These constructed vessels can be co-cultured with cancer MCS and might be suitable for the use in pharmacological drug testing to reduce the number of animal experiments, or for radiation experiments.

# 8. Plants and algae in real or simulated microgravity

## 8.1. Plant-based studies

Same as in animals, genetic programs and networks control plant growth and development. Plants use a variety of stimuli, such as light, temperature, gravity, and water to orient themselves in their natural habitat. Mechano-perception is crucial for the regulation of plant growth and development as well as for its life. Mechanical touch perception enables adaptation to environmental conditions, such as wind. In trees, the weight of a branch induces the increased formation of wood at its onset to overcome the notch stress. Responses to stimuli such as gravity and light are important for the orientation of root and stem growth in plants to anchor and absorb water and minerals as well as to orient their leaves towards sunlight (Kordyum, 2014; Kordyum and Chapman, 2017). These factors are the most stable and reliable signal utilized by plants for their survival throughout evolution. Physiological orientations towards light and gravity are termed as phototropism and gravitropism, respectively. Microgravity experiments allow us to elucidate the mechanisms of gravitropism in plants. A notable number of spaceflight experiments with plants (monocots and eudicots) in vivo and in vitro (organ, tissue, cell, and protoplast cultures) have been performed on board satellites, space shuttles, and orbital stations. These studies show that gravity and other spaceflight factors influence plants at the organismal, cellular, subcellular, and molecular levels, leading to changes in orientation, response to stress, development, metabolism, and growth. These experiments have contributed to the understanding of gravity perception, signal transduction, intrinsic cell-division processes, adaptation to microgravity, and gravity-induced distribution of organelles in plants and accompanying morphometric traits (Correll et al., 2013; Ferl et
1381 al., 2002; Kiss, 2000; Kordyum and Chapman, 2017; Paul et al., 2012a; Paul et al., 2012b; Stutte et al., 2006).

1382 In earlier investigations of gravity and microgravity, solely the measurement of physiological
1383 or anatomical parameters was possible. Currently, an arsenal of molecular biological methods is
1384 available to investigate transcription levels, protein expression and modification, or metabolic profiles.
1385 Kimbrough et al., (2004) carried out whole-genome microarray analysis of *Arabidopsis* root apices after
1386 gravity stimulation (reorientation) and mechanical stimulation to investigate the effect of gravity and
1387 mechanical forces on plants. Out of 22,744 monitored genes, 1730 (7.6%) and 1691 (7.5%) genes were
1388 found to be altered in transcript abundance in response to gravity or mechanical stimulation,
1389 respectively. An increase in transcription factors, genes encoding cell wall modifying enzymes
1390 (xyloglucan endotransglycosylases, and pectinesterases), transporters (ions, sugar, purines), and genes
1391 involved in the response to other environmental stresses (dehydration, cold, and pathogen response)
1392 were observed after gravity stimulation. The severe effect of just reversing the position of a polar plant
1393 organ towards the gravity vector was demonstrated with the gynophore (a stalk supporting the female
1394 part of a flower) of peanut (Li et al., 2013). Some of the gynophores were positioned upside-down,
1395 while the others were kept in their normal position and changes in transcriptome and proteins were
1396 detected. After 6 and 12 h, more than 170 and 420 genes were found to display altered expression levels,
1397 respectively. The proteomic analysis performed via 2D-gel electrophoresis and subsequent MALDI-
1398 TOF analysis revealed that especially proteins involved in stress or plant defense as well as proteins of
1399 the carbohydrate metabolism were influenced due to reversing the position of gynophores. Several
1400 studies have reported the effects of gravity on the transcriptome and proteome of *Arabidopsis*
1401 (Hausmann et al., 2014; Martzivanou et al., 2006; Martzivanou and Hampp, 2003). Hyper-gravity of 7
1402 x g for 1 h resulted in up- or down-regulation of around 200 genes among 4100 analyzed genes of A.
1403 *thaliana* (Martzivanou and Hampp, 2003). Mainly genes of major pathways, genes of proteins involved
1404 in cellular organization and cell wall formation, phosphorylation events, and signaling were altered. The
1405 fraction of stress response as well as genes involved in gravity-sensing was considerably lower.
1406 Significant changes in the expression of various genes in *A. thaliana* due to the effect of subsequent
1407 short-term microgravity phases (each about 20 s) achieved during parabolic flight maneuvers have also
1408 been reported (Paul et al., 2011). Transcriptome profiling revealed that parabolic flight caused changes
1409 in gene expression by prominence of genes associated with signal transduction, stress, and biotic and
1410 abiotic stimuli.
1411

1412 Various experiments with *A. thaliana* callus cultures as a test system have been performed under
1413 different accelerations and microgravity conditions as well (Babbick et al., 2007; Barjaktarovic et al.,
1414 2009; Barjaktarović et al., 2009; Hausmann et al., 2014; Martzivanou and Hampp, 2003). It seems likely
1415 that also in plant cell cultures, acceleration or microgravity induces a variety of unspecific effects
1416 ranging from changes in transcription level to protein translation and modification as well as changes in
1417 the metabolome. Proteome analysis (2D-gel electrophoresis and subsequent ESI-MS/MS) of
1418 *Arabidopsis* callus cells following simulated microgravity (RPM) as well as hypergravity revealed that
1419 a variety of proteins changed. Largely, proteins involved in radical scavenging and detoxification of
1420 reactive oxygen species (ROS), primary metabolism, and signaling were found to be different from
1421 controls (Barjaktarović et al., 2009). ROS generation is likely due to the activity of calcium-dependent
1422 NADPH oxidases (Hausmann et al., 2014) or linked to auxin signaling (Tan et al., 2011). Transcription
1423 levels of ROS-related enzymes, like ascorbate- and glutathione peroxidases, which are involved in ROS
1424 homeostasis of cells altered during microgravity (Barjaktarovic et al., 2009; Barjaktarović et al., 2009).
1425 Significant changes in protein phosphorylation, mainly in stress proteins are also reported (Barjaktarovic
1426 et al., 2009). Calcium-dependent genes also changed in short and long-term microgravity exposure
1427 (Hausmann et al., 2014; Paul et al., 2012b; Salmi et al., 2011; Salmi and Roux, 2008).

1428 Wang, (2006) investigated the effect of vertical clinorotation on the proteome of *Arabidopsis*
1429 calli (horizontally rotated calli served as a control). Among others, stress response proteins, enzymes of
1430 the carbohydrate metabolism (e.g., glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate-
1431 isomerase), signaling proteins, and transcription factors were found to be altered. Determination of
1432 clinostat effects on the proteome of intact plants (wild type and a *pin2* mutant, which exhibits a gravity-
1433 insensitive root growth phenotype) came to a similar result (Tan et al., 2011). Altered gravity conditions
1434 significantly elevated the expression of certain classes of proteins, like those involved in detoxification
1435 (GST 6, NAD⁺-ADH, GS-FDH), in chaperone functions (HSP70) and in mediating resistance to stress
1436 factors correlated with energy metabolism (mitochondrial ATP synthase, putative inosine-5'-
1437 monophosphate dehydrogenase, and isocitrate dehydrogenase) in both wild type and *pin2* mutant (Tan et al., 2011).

1438 During a parabolic flight different molecular and physiological parameters were investigated (Neef et al., 2015). During microgravity, ROS molecules and cytosolic calcium increased in *A. thaliana* cell cultures (Ca²⁺-sensor YC3.6-cells). Phosphorylation analysis revealed that in hyper-g, superoxide dismutase, ascorbate peroxidase, glycerol kinase, phosphoglyceromutase, annexin, a microtubule-associated protein and ATP-dependent NAD(P)H hydrate hydratase, serine/threonine phosphatase (protein regulation), UDP glucose dehydrogenase, V-Type proton ATPase, Hsp70, and malate-dehydrogenase genes were influenced. Under microgravity, phosphorylation of triose-P isomerase, inorganic pyrophosphatase, pyruvate dehydrogenase, citrate synthase, Hsp70, CML14 (Ca²⁺-binding protein), phosphoenolpyruvate carboxykinase, fructokinase, and dehydrogenase ferritin-2 were found to be altered compared to the control. While only a few proteins changed by varying gravitational conditions, the effect on gene expression (measured with Affymetrix GeneChip expression analysis) was much more pronounced. Gene expression in hyper-g and microgravity showed a strong overlap in 1378 up- and 1769 down-regulated genes. In addition, many genes were exclusively altered in microgravity (396 up and 485 down) or hyper-g (413 up and 637 down directly at the onset of 1.8g). Genes involved included calcium-binding, signaling (calcium Ins(1,4,5,.)P3), stress response, and detoxification (e.g., catalase). Microarray analysis of *A. thaliana* calli cultures subjected to magnetic levitation, which “simulates” different gravity levels (microgravity, 0.1 g, 1 g, and hyper-g) has revealed changes in stress and metabolic genes among others in high gradient magnetic fields (Manzano et al., 2012). Also, Paul et al., (2013) found significant stress responses (effects on heat shock proteins and chaperons) of *Arabidopsis* tissue cultures in space.

1459 Correll et al., (2013) investigated the transcriptome of *A. thaliana* seedlings in space (TROPI-2 experiment on the ISS) and found that the transcription of 982 genes differed from ground controls. 1460 Among these genes, 135 and 143 were noted to be up- and down-regulated at least two-fold, 1461 respectively. Next to many undefined genes, a large number of genes involved in salt stress, oxidative 1462 stress, detoxification, biotic-induced stress, water stress, and defense response (and other stress 1463 responses) were among the differentially expressed ones. In addition, changes in the actin cytoskeleton 1464 organization that among other functions is involved in the transport of cell wall components to the cell 1465 periphery on exposure to microgravity were noted. In a spaceflight experiment (BRIC-16 on STS-131, 1466 2010), Paul et al., (2012b) analyzed and compared the transcriptome of *A. thaliana* seedlings and cell 1467 cultures. While the seedlings reacted moderately over a variety of functions, the cell cultures reacted 1468 mainly with the transcription of stress response and heat shock proteins. For instance, the expression of 1469 the heat shock transcription factor HSFA2 or the heat shock protein Hsp70 was noted to be 20-fold 1470 higher in cell cultures. This shows that even undifferentiated plant cells perceive the absence of gravity 1471 as stress but do so in a more general sense than plant seedlings, which have specialized organs to sense 1472 their environment and react. In seedlings, among others, genes involved in pathogen response and 1473 wounding, stress response (cold and drought stress), auxin-mediated root development, and calcium 1474 signaling were also found to be different compared to the ground control. During a long-term experiment 1475 onboard the ISS, Paul et al., (2013) investigated the seedlings of *A. thaliana* and found 1480 genes to 1476 be differentially expressed from 1g controls (centrifuge). Also, gene expression was found to be organ- 1477 specific. About 181 genes were found to be exclusively altered in roots, only 114 in the hypocotyl, and 1478 only 113 in the leaves. Leaves, hypocotyl, and roots shared only a few genes, which were altered in all 1479 organs (26 of 408). Differentially expressed genes codes for proteins of touch-wounding response, cell 1480 wall remodeling, growth hormone, and signal transduction. Perhaps altered hormone-signaling affect 1481 the cell wall remodeling genes, influencing the growth and cell expansion (Schüler et al., 2015). 1482

1483 Changes in transcription in plants after transfer to microgravity are not surprising considering 1484 the report of Millar and Kiss, (2013), where, transcription and metabolomics during tropistic responses 1485 were investigated. In this study, seedlings were irradiated unidirectional with red or blue light to measure 1486 phototropic responses and tilted towards the gravity vector to induce gravitropic responses. In all 1487 tropisms, genes for carbohydrate and secondary metabolism were found to be downregulated, while 1488 different genes involved in amino acid metabolism were found to be upregulated. Changes in gene 1489 expression were highest in gravitropism compared to different phototropisms. Especially genes coding 1490 for proteins of subcellular localization, proteins with binding function, defense genes, and metabolic 1491 genes were more profoundly altered during gravitropism. It was found that various metabolites, which 1492 are typical for biotic and abiotic stress, such as asparagine, phenylalanine (interaction between ethylene
1493 and auxin), threonine, sucrose (correlation to osmosis), etc. were increased upon all tropistic reactions.
1494 In a very recent study, Vandenbrink et al., (2019) reported down-regulation of light-associated pathways
1495 (e.g., photosynthesis-antenna proteins, photosynthesis, porphyrin, and chlorophyll metabolism) in A.
1496 *thaliana* grown in a microgravity environment and irradiated with a blue light onboard the ISS compared
1497 to 1 g control. In contrast, pathways involved in ribosome biosynthesis, which has been associated with
1498 elements controlling cell growth and proliferation (Kamal et al., 2018), and oxidative phosphorylation
1499 were upregulated. The authors suggest a connection between gravity and phototropism in *Arabidopsis*.
1500

In other investigated plants, acceleration changes led to changes in protein patterns or
1501 transcription profiles as well. The transcription profiles of spores of the fern *Ceratopteris richardii*
1502 germinating in microgravity and 1 g analyzed during the NASA space shuttle mission STS-93 revealed
1503 changes in the mRNA expression of roughly 5% of genes analyzed (Salmi and Roux, 2008). Various
1504 genes were noted to be altered, e.g., transferases, hydrolases, and heat shock proteins. Bushart et al.,
1505 (2013) also identified potential modulators (calcium pumps and apyrases) for gravity response in
1506 *Ceratopteris*. Jin et al., (2015) analyzed rice calli grown on the Chinese satellite Shenzhou-8. In
1507 comparison to 1 g ground and 1 g inflight controls, they found 627 microgravity responsive transcripts,
1508 mostly involved in the cell wall structure, primary metabolism, transcription, calcium regulation, and
1509 others. Stutte et al., (2006) investigated the effect of microgravity on dwarf wheat (leaf ultrastructure,
1510 biochemical, physiological, and transcription levels) grown in real microgravity (located in a Biomass
1511 Production System (BPS)-device) during a space shuttle flight and subsequent installation on the ISS.
1512 Interestingly only marginal changes in leaf ultrastructure (e.g., thinner cross section and higher cell
1513 density) and chloroplast (more ovoid shape, slightly different thylakoid structure-no effect of
1514 photosynthetic performance) compared to a 1 g-ground control were detected (Stutte et al., 2006).
1515 Transcription was not significantly impaired. Xu and Guo (Xu and Guo, 2013) investigated RPM-effects
1516 on six conserved miRNAs in *Solanum lycopersicum*. After 30 days of RPM, the expression level in six
1517 investigated miRNAs was found to be increased after transient reduced expression before. Putative
1518 targeted proteins of this miRNA are involved in physiological and metabolic processes (protein kinases,
1519 oxidoreductase), transcription factors, and others. Real microgravity resulted in the detection of 19
1520 differentially expressed proteins in *Oryza sativa* investigated using 2D-gel electrophoresis and mass
1521 spectrometry (Ma et al., 2007). Four of these proteins could be identified: hydrogen peroxide oxidase,
1522 ascorbate peroxidase, a manganese-stabilizing protein, and a ribosomal protein.
1523

Recent studies have indicated that epigenetic mechanisms (DNA methylation and histone
1524 modification) control gene expression in plants in gravitational alterations indicating that gravitational
1525 changes are sensed as stress by plants (Kamal et al., 2018; Ou et al., 2010; Xu et al., 2018; Zhou et al.,
1526 2019). The regulation of genes expression via epigenetic modification affects plant responses to
1527 environmental stresses as discussed before. Kamal et al., (2018) performed experiments with cell
1528 cultures of *Arabidopsis* under simulated micro- and Martian-gravity (RPM-treatment) as well as under
1529 hypergravity (centrifugation) and observed that samples exposed to artificial microgravity and a lower
1530 extend cells exposed to Martian-gravity showed an increased DNA methylation level compared to 1 g
1531 and hypergravity conditions. Also, this study revealed effects of simulated microgravity or simulated
1532 Martian gravity on the cell cycle (lower fraction of cells in G1-phase), nucleolus structure (decrease of
1533 cells showing compact nucleolus structure and increase of fibrillary structure) as well as changes in
1534 levels of proteins involved in cell cycle regulation. Recently, Xu et al., (2018) investigated the
1535 methylation pattern changes in *Arabidopsis* seedlings exposed to real microgravity conditions onboard
1536 the Chinese recoverable scientific satellite SJ-10. They found widespread methylation of genes involved
1537 in metabolic pathways, biosynthesis of secondary metabolites, ribosome pathway, starch and sucrose
1538 metabolism, phenylalanine biosynthesis, purine, glutathione, amino sugars, and nucleotide sugar
1539 metabolism), proteasomes, nucleotide excision repair, and ribosomes among others. Ou et al., (2010)
1540 found significant changes in genetics, as well as DNA methylation frequencies (hyper- and hypo-
1541 methylation), in rice plants (*O. sativa* L.) germinated from seeds kept in space for 18 days. Among the
1542 assessed 460 genomic loci, the genetic change frequencies ranged from 0.7% to 6.7%, while
1543 hypermethylation frequencies were about 1.95% for CG and 1.44% for CNG. The hypomethylation
1544 frequencies were noted to be 0.76% (CG) and 0.80% (CNG). In a very recent whole-genome survey of
1545 DNA methylation, Zhou et al., (2019) found differentially methylated genes in microgravity-exposed
1546 *Arabidopsis* (ISS).
1547

Chinese space researchers combined space, biological, and plant-breeding technologies for
1548 screening and identifying diverse new varieties of plants through a space mutation breeding program
carried out from 1987 to 2005 (Chengzhi, 2011; He et al., 2006; Xianfang et al., 2004). They pointed out that the space environment may result in genetic mutagenesis of seeds or seedlings and thus open the route to develop new varieties of plants. He et al., (2006) summarized the progress of this space mutation breeding program and reported that Chinese scientists and agricultural biologists sent more than 1000 varieties of different plant seeds (crops, floricultural plants, vegetables, fruits, medicinal plants, and forest trees) into space. The space-exposed seeds grown on Earth showed positive physiological and genetic variations. Through this 'space breeding' program, Chinese research institutes (70 involved) identified more than 200 promising new varieties of plants exhibiting high quality, high yield, and disease resistance properties. Many of the space-breed plant varieties are cultivated in large scales in some provinces in China (extended up to 270000 hectares) and are bringing social-economic benefits (Xianfang et al., 2004).

Based on several years of studies on plant systems under different acceleration and microgravity conditions it is evident that gravity affects several mechanisms in plants. Plant systems develop or activate a range of molecular responses to raise their defenses upon exposure to gravity-related conditions. These conditions change directly or indirectly the information in the genome, which influence the transcription process and eventually result in the production of changed proteins. Gene activity is affected by non-genetic mechanisms, and the level of stress-induced transcripts alters. In addition, certain repressive RNA transcripts activate and signaling molecules vital for cell survival and adaptation accumulate. The combination of all these responses changes the global systemic organization of plants for the potential recovery, development, and growth under gravity-related conditions. For instance, *A. thaliana* germinated in space but plants grew smaller and roots showed different growth patterns (Millar et al., 2011; Paul et al., 2012a; Paul et al., 2012b). Altered acceleration leads to changes in gene expression which is associated with specific responses in whole plants, seedlings, and plant cell cultures, and differs between different plant organs (Paul et al., 2012a; Paul et al., 2012b; Paul et al., 2013) but share some common gene changes. For example, genes involved in root hair development and generation, cell wall remodeling, cell wall modifying enzymes, actin cytoskeleton association, metabolism and response to ROS, calcium signaling, and general stress responses (Correll et al., 2013; Fengler et al., 2015; Fengler et al., 2016; Hausmann et al., 2014; Mazars et al., 2014a; Mazars et al., 2014b; Paul et al., 2005; Paul et al., 2011; Paul et al., 2013; Schüler et al., 2015; Schüler et al., 2016; Zhang et al., 2015; Zhang and Zheng, 2015). The involvement of the second messengers ROS, Ca²⁺, and heat shock proteins, as well as distinct changes in the transcriptome, indicates that microgravity is a significant stressor for plants. In summary, whole plants seem to be less affected by microgravity-induced physical phenomena than cell cultures because (i) the cells possess a high internal pressure (turgor) caused by the osmotic pressure of the huge internal vacuole which pushes the cytoplasm against a rigid cell wall and (ii) because of intracellular cytoplasma-streaming, which mixes the cytoplasm also in the absence of microgravity.

## 8.2. Algae-based studies

Intensive gravitational biology work over many years has made green macroalgae *Chara* a well-established model system for studies on polarized growth and gravity-sensing in plants (Braun et al., 1999; Braun et al., 2004; Braun and Limbach, 2006; Braun and Richter, 1999; Braun and Sievers, 1994). Microgravity experiments during a parabolic flight campaign revealed that the physical contact to a hypothetical receptor protein is responsible for signal transduction and not the force applied by the statoliths on the lower membrane (Limbach et al., 2005). Many studies have been conducted with unicellular photosynthetic organisms. The single cellular green algae *C. reinhardtii* shows precise gravitactic behavior. Although non-gravitactic mutants exist (Yoshimura et al., 2003), it is still controversially discussed, whether this phenomenon is based on active physiological mechanisms or solely on physical effects such as buoyancy or bottom-heaviness (Häder et al., 2006b; Häder et al., 2006a). Mathematic models and calculations support physical mechanisms (Hagen et al., 2014). In recent years, the unicellular flagellate *Euglena gracilis* has become a model organism for gravitational research. Its behaviors (phototaxis and gravitaxis, i.e., response to the direction of light and gravity as stimuli) have been extensively investigated during various space experiments (Nasir et al., 2014; Strauch et al., 2010) because *E. gracilis* use physiological mechanisms to sense gravity and light for orientation in its habitat. Several experiments in microgravity helped to develop a working hypothesis of the mechanisms involved in gravitaxis. According to it, mechano-sensitive channels are periodically opened
1605 when the cells deviate from a vertical path upwards (Lebert and Häder, 1996, 1997a, 1997b). This allows
1606 for the influx of calcium (Richter et al., 2001; Richter et al., 2002), which in turn activates a calmodulin
1607 (Daiker et al., 2010; Häder et al., 2009b). Subsequently, an adenylate cyclase increases the intracellular
1608 cAMP levels, thereby activating a specific protein kinase A (PKA) (Daiker et al., 2011; Streb et al.,
1609 2002; Tahedl et al., 1998). Curiously, the phototactical signal transduction chain also leads to that PKA
1610 (Häder et al., 2005; Häder et al., 2017; Ntefidou et al., 2003). A molecule interacting with PKA was
1611 found in the flagellum (Nasir et al., 2018) (Fig. 8).

1612 After a spaceflight aboard the Chinese Satellite Shenzhou-8, *Euglena* cells were analyzed for
1613 possible adaptations to microgravity (Nasir et al., 2014). Genes related to oxidative stress or heat shock
1614 proteins were upregulated, as well as genes suspected to play a role in gravitaxis, like calmodulin,
1615 calcium-binding and other signal proteins, and proteins involved in cell cycle control. Of the tested 32
1616 genes, 18 were up-regulated and one down-regulated, while the rest remained unchanged. Recent
1617 advances in the sequencing of the nuclear and mitochondrial genome of *Euglena* (Ebenezer et al., 2019)
1618 allowed microarray construction and investigate the transcription pattern in the course of a parabolic
1619 flight campaign (Krüger et al., 2019a). The study supported that significant changes in gene expression
1620 can be achieved within a very short time. Different signal transduction elements, such as mitogen-
1621 activated kinase kinase kinase NPK1, hybrid sensor histidine kinase/response regulator, adenylate
1622 guanylate cyclase domain-containing protein, etc., were found to be regulated as well as transport
1623 proteins, phosphorylases, primary metabolism, and DNA and protein modification genes. Microgravity
1624 might also affect the photosynthesis and perception of light: genes involved in these processes were
differently expressed, even though the experiment was performed in darkness (Daiker et al., 2011; Nasir
1625 et al., 2018). Some of the altered transcripts showed similarities to those observed in *A. thaliana*
1626 (Hausmann et al., 2014; Martzivanou et al., 2006). Taken together, the study contributed to the
1627 understanding of the short-term effects of microgravity and different accelerations on gene expression,
1628 which can be used as a reference data set for further on-ground and spaceflight-related studies.
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8.3. *Conclusions from observed effects on plants and algae*

In plants and algae, like in animal cells, the current knowledge about gravity perception **and** the connected signal transduction chains is very limited. However, photosynthetic organisms (plants, plant cell cultures, and algae) and human or animal systems share similar patterns of “disturbed-gravity-induced” changes in certain classes of proteins or gene expression. For instance, signal transduction chains involving calcium are affected in plants and animals alike (Hashemi et al., 1999; Kordyum, 2003; Paul et al., 2012b). Six orthologue genes between *Drosophila melanogaster* and *Caenorhabditis elegans* were found to be differentially expressed (Leandro et al., 2007). Therefore, there appears to be something like a common response to **spaceflight**. Unfortunately, differences in the experimental setups (duration, hardware, limited numbers of experiments and replicates, quality of microgravity, etc.) make it very difficult to compare results of space experiments and draw a definite interpretation and conclusion. Future space experiments **need** to be performed under standardized conditions to attain reproducible results and **conclusions** (Ruyters and Braun, 2014; Wolff et al., 2014). A general database should be developed comprising of genomics, proteomics, metabolomics, and transcriptomics datasets obtained from space experiments and corresponding ground studies of different organisms.

9. Colloids in microgravity

Grape juice and its fermented form, wine, are complex **multi-component** fluids containing
1650 sugars, different alcohols, hundreds of different tannins, or polyphenols prone to various attractive and
1651 repulsive interactions (hydrophobic, van der Waals, electrostatic, hydrogen bonding). The tannins are
1652 likely to aggregate (haze formation) with time (ageing), **thus** making the red wine a meta-stable colloidal
dispersion: a system of fine particles suspended in a fluid (Zanchi et al., 2008). **Other common examples**
1653 of colloidal dispersion than wine are paints, inks, mayonnaise, ice cream, milk, and biological fluids
1654 such as blood. Although these systems are routinely produced, used, and studied, we still have much to
1655 learn about the underlying properties of colloidal systems. **Hence,** understanding and controlling them
1656 is a central issue in many technological and scientific fields as it will foster academic and industrial
1657 research to **fine-tune** their physico-chemical structures to put forward new functionalities and products.
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The force of gravity may in certain cases impact the colloidal phase behavior as well as dynamical processes. It can, for example, destabilize a dispersion through either the rising toward the liquid/fluid interface of some of the components lighter than the dispersing fluid as in oil-in-water emulsions (creaming) or the sedimentation of the denser ones as silica particles in water. Indeed, at the colloidal level, the so-called gravitational length $l_g = \frac{k_B T}{G}$, where $k_B T$ is the thermal energy source of the Brownian motion and $G = \frac{\pi D^3 \Delta \rho g}{6}$ the buoyant weight (with D the colloidal diameter, $\Delta \rho$ the density mismatch between the colloid and the solvent, and g the gravitational acceleration. It characterizes at what height gravity will start to play an important role in the equilibrium phase behavior (Royall et al., 2005). In a gravitational field, the balance between diffusive forces, pushing the particles up, and gravity, pulling them down, results in hydrostatic equilibrium, the sedimentation profile, where the volume fraction of non-interacting colloids (dilute regime) $\phi = \frac{\pi \rho}{6 D^3}$ decays exponentially with height as $\phi(z) = \phi_0 \exp(-z/l_g)$, with a decay length, $l_g$, historically determined by Perrin in his 1916 seminal work on the measurement of the Boltzman's constant $k_B$ (Perrin, 1916). It follows that if the size of a colloid in the dispersion is somehow below 1µm (with small $\Delta \rho$), its motion will be mainly diffusive with a negligible impact of gravity-driven convection (sedimentation or creaming). A useful (dimensionless) quantity to weigh the influence of gravity in colloidal dynamics is the Péclet number (Pe) that measures the ratio between convective and diffusive transport in a gravitational field (El Masri et al., 2012; Okubo et al., 2000):

$$P_e = \frac{D}{2l_g} = \frac{\pi D^4 \Delta \rho g}{12k_B T} = \frac{\tau_B}{\tau_S}$$

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Where, $\tau_B$ and $\tau_S$ are the time scales for Brownian diffusion and sedimentation (creaming). When $P_e << 1$, gravity may be neglected, when $P_e >> 1$ the colloidal dispersion will be strongly affected by gravity. Indeed, for 1µm silica particles in water (~$\Delta \rho = 1$ g cm$^{-3}$) the sedimentation rate due to gravity is 100 times larger than Brownian diffusion. By selecting the right particle size and matching the particle-solvent density one can significantly reduce the influence of gravity in laboratory experiments. It is however very difficult to reduce $\Delta \rho$ significantly below $10^{-3}$ g.cm$^{-3}$ without extreme temperature control (better than 0.01°C) required to reach the condition of microgravity. Furthermore, for optical studies, the fluid and the disperse component should be index-matched which practically excludes that they are density-matched at the same time, something impossible to achieve for all the different colloidal systems used and studied every day all over the world. Colloidal stability is a key issue in various domains ranging from soft-matter, physical chemistry, colloidal and macromolecular science to material science and biotechnology through micro and synthetic biology as it relates to end-user products, drugs.

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These systems are often structured by attractive forces between building blocks such as micelles, macromolecules, proteins, globules, nanoparticles in the form of weak, flowable gels. These structures in turn age through coaserning where they diffuse to encounter and then compact or merge. When they can no longer support the gravitational stresses, they collapse, sediment, or sink, often abruptly without warning (delayed collapse) with a dynamic much faster than Brownian motion. Consequently, all the motions are biased by hydrodynamics as in the separation of emulsions into layers of different density for example. This pinpoints the difficulties to fully capture and understand the fundamentals of coaserning but also crystallization (Okubo et al., 2000; Zhu et al., 1997), aggregation (Veen et al., 2012), gelation (Manley et al., 2004), crystal nucleation and growth, late stage of spinodal decomposition (Bailey et al., 2007), the collapse of tenuous structures, the spatial distribution of aerosols, interfaces, bubbles, and droplets shapes.

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Another interesting aspect is the possibility to construct synthetic cells with life-like properties to tackle key societal challenges, such as environmental cleanup, energy production, and drug delivery but also to bring new insights concerning the possible pathways responsible for the origin of life. This bottom-approach aim at producing artificial compartments drawing on a combination of natural biomolecules and synthons to mimic cell structure and functions by incorporating i) cellular extracts containing proteins and nucleic acids required for DNA replication and ii) enzymes to develop biochemical reactions and reactors. To date, two types of compartments have been used to assemble synthetic cell models, namely membrane-bound vesicles and membrane-free micron-sized droplets formed by all-in-water emulsions (ATPS, complex coacervates) (Douliez et al., 2018; Dzieciol and Mann, 2012). The former ensure long-term solute encapsulation and confinement of biomolecules but
1713 the lipid bilayer limits molecular exchange, while the latter allows spontaneous solute uptake but the
1714 lack of membrane leads to low confinement times and then support facile solute exchange. Bioreactors
1715 capable of synthesizing proteins have been developed using vesicles (Ciancaglini et al., 2012; Noireaux
1716 and Libchaber, 2004), while all-in-water emulsions have been suggested as a step toward the production
1717 of protocell models (Dzieciol and Mann, 2012). The design of synthetic cells holds great promise but
1718 their studies and development are facing the same problems as encountered with a more classical
dispersion. The coarsening of the compartments leading to fast sedimentation or macroscopic phase
1719 separation if not correctly stabilized, large scale convection taking over the diffusion of small
1720 biomolecules, nutriment, and actives.
1721

1722 Real microgravity conditions are then needed to unveil some key physicochemical aspects like
1723 the natural movement of colloids that experiments performed on Earth, in the presence of gravity may
1724 mask, alter, or simply hinder. Studies in microgravity will open the routes for the pharmaceutical, food,
1725 agricultural, and chemical companies to develop a wide range new products (DiFrancesco and Olson,
1726 2015). Since more than 30 years, numerous scientific experiments on colloids in microgravity have been
1727 carried out on aircraft (and Space Shuttles) (Lee and Israelsson, 2001; Okubo et al., 1999; Okubo et al.,
1728 2000; Sprenger et al., 1993; Tomita et al., 2018; Zhu et al., 1997). If the characteristic time scale of the
1729 given experiment is longer (an aging process for example that needs to unfold slowly and evenly) then
1730 the unique capabilities of the ISS is an ideal location to study more deeply the various physical, physico-
1731 chemical aspects and mechanism(s) at play (vide supra) in colloid science in microgravity. In particular,
1732 ISS has developed the so-called Light Microscopy Module (LMM), a modified commercial and highly
1733 flexible, state-of-the-art light imaging microscope facility that provides researchers with powerful
diagnostic hardware and software with the capability of remotely acquiring and downloading digital
1734 images and videos across many levels of magnification. This provides a unique environment to observe
1735 colloidal self-assembly in 3-D and microgravity for a variety of shapes, sizes, and particle interactions
1736 to build understanding and drive colloidal engineering (Bailey et al., 2007; Manley et al., 2004;
1737 McPherson and DeLucas, 1999; Safari et al., 2017; Veen et al., 2012).
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10. Future perspectives of space research

1742 Life on Earth has always developed within the boundaries of gravity. In space, organisms
1743 encounter completely novel conditions, such as weightlessness and radiation. The majority of space
1744 experiments until date have revealed that microgravity has a significant influence on organisms' growth
1745 and behavior. Future space and gravitational biology research programs should focus on the exploration
1746 of the influence of microgravity on living systems at the cellular and molecular levels, as well as on
1747 genetic stability, growth, and development of animal and plant systems utilizing advanced scientific
1748 methods and tools. The space environment can be explored to develop new products for the benefit of
1749 humankind. For instance, we can generate natural crop varieties with higher nutrition and yields and
1750 improved features, such as resistance against high and low temperatures, salt stress, microbial and pest
1751 attacks, drought, and other stresses. Introduction of plants to artificial stressors in microgravity will
1752 activate the expression of dormant genes, change the global systemic organization of plants, and improve
1753 their recovery processes in subsequent stresses, like the ability to adapt rapidly to a disease-causing
1754 organism. Strains improved in space environmental conditions can be a valuable partner for
1755 conventional engineering strategies. Characterization of evolved strains and identification of the
1756 important mutations or molecular basis that enabled evolved phenotype or behavior can increase our
1757 understanding of organisms' physiology and metabolism as well as uncover the significance of
1758 molecular mechanisms contributing to strain fitness and performance. Application of emerging genome
1759 and transcriptome sequencing and analysis tools, and advanced metabolic identification and
1760 characterization methods are required to interpret the story behind the evolution-based strain
1761 improvement approach. These investigations can reveal novel strategies for improving the tolerance of
1762 strains to biotic or abiotic factors, increasing utilization of a target substrate for growth and yield, or
1763 improve the production of target compounds.
1764

1764 Several infrastructures and space operation capacities are currently present to execute various
1765 biological experiments in space. In addition, more advanced technologies are being developed. The
1766 current Dragon spacecraft by SpaceX, the Cygnus spacecraft by Orbital ATK, and the new Shepard
1767 spacecraft by Blue Origin, and the robust space platforms, such as the ISS and the Russian Earth-orbiting
1768 satellites BION and FOTON are available as well as space platforms and spacecrafts from China and
1769 **India will be accessible in the near future.** Most platforms are currently used for publicly driven space programs, serving and servicing the ISS or far more ambitious space exploration programs such as to the Moon or Mars. ISS offers the most sophisticated research facility in the world. Only highly elaborated ground-based research facilities, such as the Large Hadron Collider at CERN, can compete with the ISS in terms of complexity and operating expense. Differences in experimental setups (viz. duration, hardware, limited numbers of experiments and replicates, and microgravity quality) have limited our understanding of space research data to achieve breakthroughs in biological research. It is therefore required to carry out space experiments under defined and standardized conditions. Factors that need to be emphasized in space research are numbers, time, and on-site monitoring of relevant factors. The required changes cannot be expected to happen often, but by increasing the number of exposed subjects, and by increasing mutational pressure, one can increase the probability that the changes will occur. The longer the subjects are exposed, the higher the chance of change. However, there should be a threshold of exposure time to allow survivability of the biological system. Recent advances in online monitoring of genetic and metabolic changes will allow close tracking of occurring changes. The development of a general database consisting of datasets from different experimental analyses is highly recommended.

1785 In conclusion, space offers unique types of stressors or environmental conditions (i.e., weightlessness and high radiation) that can be used to accelerate evolutionary processes in an organism for the development of a new strain adapted to the challenging environments. Microgravity as an environmental condition never experienced before by any organism on Earth most likely increases mutations rates and influences metabolic and signal transduction pathways in a way that phenotypes may occur, which would not develop under 1g. We anticipate that the evolutionary changes in space will be considered fast. Evolution will be based on random processes with positive selection guiding the development of phenotypes with desired properties. Microgravity potentially constitutes a platform to develop valuable strains and bio-products as well as to study more deeply the various physical, physicochemical aspects, and mechanism(s) at play in colloid science. We propose to craft different varieties of organisms in space to meet Earth's demand in the age of ultimate planetary- and human- impacting threats. Something that would definitely help academia and industry to formulate a wide range of new products for food, medicine, pharmaceutics, cosmetics, home-care with longer shelf lives and new functionalities.

1799

1800 Acknowledgements

1801 The Space Biology Unlimited SAS, France, Space Cargo Unlimited, USA support this work, which is highly appreciated by the authors. The authors are also grateful to the German Aerospace Center DLR (Grant no. 50WB1923) for their support. SMS was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES).

1806

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3141
3142 **Figure Legends**

3143
3144 **Fig. 1. A simplified scheme of self-guided evolution.** Diverse genetic variants of strains can be achieved by subjecting cells in microgravity and radiation in combination with other biotic and abiotic stressors. Suitable mutants selected according to defined parameters and isolated subsequently. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

3150
3151 **Fig. 2. Scheme of horizontal gene transfer between non-related organisms.** Horizontal gene exchange is common in closely related organisms but also occurs between distantly related organisms. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

3156
3157 **Fig. 3. Stress-induced epigenetics in plants.** Epigenetic bring about new traits without altering the genetic code. Epigenetic regulation increases variation within a species in times of environmental stresses or signals (biotic and abiotic factors), thereby provide ways for adaptation to the arising selection pressures.

3161
3162 **Fig. 4. The central dogma of evolution.** In biology and ecology, environmental factors or stressors (abiotic or biotic components) affect living organisms, leading to adaptive changes over time employing evolution, which includes mutation, gene flow (horizontal gene transfer), genetic drift, epigenetic regulation, and natural selection as mechanisms of change.

3166
3167 **Fig. 5. Simplified representation of parameters cells or organisms encounters in outer space.** Lack of gravity on board of a spacecraft considerably changes the environment in which thermal convection, mechanical load as well as hydrostatic does not exist. This has significant impacts on cells in terms of gas, nutrient, and heat transfer. Space unique environment provides a combination of stressors (e.g., high radiation and weightlessness/microgravity) that organisms have never experienced on Earth. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

3175
3176 **Fig. 6. Different facilities** for the investigation of altered gravitational conditions Platforms include aircraft-based parabolic flights, 3D and 2D clinostats, random-positioning machine (RPM), sounding rocket, and drop tower. Clinostat and RPM constantly change the position of the test object to the vector of gravity. With drop tower, aircraft-based parabolic flights, and sounding rockets, microgravity conditions can be achieved ranging from few seconds to several minutes.

3181
3182 **Fig. 7. Applications of microgravity-based research on human cells.** (A) MCF-7 breast cancer cells form unique ring-like structures when grown for 14 days in s-µg produced by an RPM. (B) Schematics of cancer research using microgravity. In µg, cancer cells detach from the growth surface and form 3D MCS composed of less aggressive, more differentiated cells. Using Omics research and bioinformatics, scientists try to elucidate the mechanisms behind the alteration of the phenotype to mimic these effects with drugs. The results may be used in the development of novel cancer therapies. Furthermore, µg provides a suitable culture environment for scaffold- and necrosis-free tissue engineering of (C) cartilage, (D) bone, and (E) blood vessels. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

3192
3193 **Fig. 8. Simplified scheme of the physiological mechanism involved in the gravitaxis of Euglena gracilis.** If a vertically upward swimming cell (A) deviates from its vertical movement path (B and C), mechano-sensitive membrane patches are activated due to forces generated by the weight of the cell body (C). This triggers a physiological signal cascade (orange insert) in which calcium channels, a particular calmodulin (CAM 2, activated by increasing concentrations of calcium ions), a not yet
3198 identified adenylate cyclase are involved. Increased levels of cAMP, synthesized from activated
3199 adenylate cyclase activates a protein kinase A, which most likely phosphorylates flagellar proteins. This
3200 results in reorientation strokes of the flagella, aligning the cell back to the vertical position (D). In
3201 addition, a recently identified flagellar protein EgPCDUF4201 is involved in gravitaxis, but its role in
3202 the signal transduction chain is yet to be determined. The Figure is adopted and modified according to
3203 (Nasir et al., 2018). Parts of the figure were drawn by using pictures from Servier Medical Art
3204 (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License
3205 (https://creativecommons.org/licenses/by/3.0/).
3206