# Exploration of space to achieve scientific breakthroughs

Binod Prasad, Peter Richter, Nithya Vadakedath, Rocco Mancinelli, Marcus

M. Krüger, Sebastian M. Strauch, Daniela Grimm, Philippe Darriet,

Jean-Paul Chapel, Jacob A. Cohen, et al.

► To cite this version:

Binod Prasad, Peter Richter, Nithya Vadakedath, Rocco Mancinelli, Marcus M. Krüger, et al. Exploration of space to achieve scientific breakthroughs. Biotechnology Advances, 2020, 43, pp.1-33. 10.1016/j.biotechadv.2020.107572. hal-02881231

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https://hal.science/hal-02881231

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Exploration of Space to Achieve Scientific Breakthroughs

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Binod Prasadª, Peter Richterª, Nithya Vadakedathᵇ, Rocco Mancinelliᶜ, Marcus Krügerᵈ, Sebastian M.

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Strauchᶜ, Daniela Grimmf, Philippe Darrietg, Jean-Paul Chapelh, Jacob Coheni, Michael Leberta,j,#

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ªDepartment of Biology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany

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ᵇCSIR - Institute of Microbial Technology, Chandigarh, India

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ᶜBay Area Environmental Research Institute, Ames Research Center, Mountain View, California, USA

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ᵈClinic for Plastic, Aesthetic and Hand Surgery, Otto von Guericke University, Magdeburg, Germany

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ᵉUniversity of the Region of Joinville - Univille, Joinville, Brazil

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fDepartment of Biomedicine, Aarhus University, Aarhus, Denmark

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gInstitut des Sciences de la Vigne et du Vin, Villenave d'Ornon, France

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hPaul Pascal Research Institute (CRPP), Bordeaux University, Pessac, France

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iNational Aeronautics and Space Administration, Ames Research Center, Mountain View, California, USA

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jSpace Biology Unlimited SAS, Bordeaux, France

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Running title: Space for Revolutionizing Biological Research

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#Address correspondence to M. Lebert, michael.lebert@fau.de
<table><tr><td>37</td><td>Contents</td><td></td></tr><tr><td>38</td><td></td><td></td></tr><tr><td>39</td><td>1. Introduction</td><td>4</td></tr><tr><td>40</td><td>1.1. Scope of the review</td><td>4</td></tr><tr><td>41</td><td>1.2. Effects of human activities on the global ecosystem</td><td>5</td></tr><tr><td>42</td><td>1.3. Approaches to tackle problems of the fast-changing world</td><td>6</td></tr><tr><td>43</td><td>2. Evolution and mechanisms of evolution</td><td>7</td></tr><tr><td>44</td><td>2.1. What happens to an organism if a constant important stimulus is no longer impinging?</td><td>8</td></tr><tr><td>45</td><td>2.2. How does evolution work?</td><td>8</td></tr><tr><td>46</td><td>2.3. Processes modulating evolution of species</td><td>9</td></tr><tr><td>47</td><td>2.3.1. Mutation — the major force of the evolution of species</td><td>10</td></tr><tr><td>48</td><td>2.3.2. Role of HGT in the evolution of species</td><td>11</td></tr><tr><td>49</td><td>2.3.3. Epigenetic gene regulation in the evolution of species</td><td>13</td></tr><tr><td>50</td><td>2.4. Stress accelerates evolution</td><td>14</td></tr><tr><td>51</td><td>3. Space environment as a toolbox for application-oriented biological research</td><td>15</td></tr><tr><td>52</td><td>3.1. Gravity and microgravity</td><td>16</td></tr><tr><td>53</td><td>3.1.1. Sedimentation</td><td>16</td></tr><tr><td>54</td><td>3.1.2. Diffusion and convection</td><td>16</td></tr><tr><td>55</td><td>3.1.3. Shear forces</td><td>17</td></tr><tr><td>56</td><td>3.2. Space radiation</td><td>17</td></tr><tr><td>57</td><td>4. Simulated and real microgravity platforms</td><td>18</td></tr><tr><td>58</td><td>5. Microbiological studies in space environment or simulated microgravity conditions</td><td>19</td></tr><tr><td>59</td><td>5.1. Conclusions from observed effects in microbes</td><td>22</td></tr><tr><td>60</td><td>6. Microgravity and cancer research</td><td>23</td></tr><tr><td>61</td><td>7. Tissue engineering in space</td><td>24</td></tr><tr><td>62</td><td>7.1. Cartilage tissue engineering</td><td>24</td></tr><tr><td>63</td><td>7.2. Tissue engineering of bone</td><td>25</td></tr><tr><td>64</td><td>7.3. Growing blood vessels in space</td><td>25</td></tr><tr><td>65</td><td>8. Plants and algae in real or simulated microgravity</td><td>26</td></tr><tr><td>66</td><td>8.1. Plant-based studies</td><td>26</td></tr><tr><td>67</td><td>8.2. Algae-based studies</td><td>30</td></tr><tr><td>68</td><td>8.3. Conclusions from observed effects on plants and algae</td><td>31</td></tr><tr><td>69</td><td>9. Colloids in microgravity</td><td>31</td></tr><tr><td>70</td><td>10. Future perspectives of space research</td><td>33</td></tr><tr><td>71</td><td>Acknowledgements</td><td>34</td></tr><tr><td>72</td><td>References</td><td>34</td></tr><tr><td>73</td><td></td><td></td></tr><tr><td>74</td><td></td><td></td></tr></table>
75 ABSTRACT

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77 Living organisms adapt to changing environments using their amazing flexibility to remodel themselves
78 by a process called evolution. Environmental stress causes selective pressure and is associated with
79 genetic and phenotypic shifts for better modifications, maintenance, and functioning of organismal
80 systems. The natural evolution process can be used in complement to rational strain engineering for the
81 development of desired traits or phenotypes as well as for the production of novel biomaterials through
82 the imposition of one or more selective pressures. Space provides a unique environment of stressors
83 (e.g., weightlessness and high radiation) that organisms have never experienced on Earth. Cells in the
84 outer space reorganize and develop or activate a range of molecular responses that lead to changes in
85 cellular properties. Exposure of cells to the outer space will lead to the development of novel variants
86 more efficiently than on Earth. For instance, natural crop varieties can be generated with higher nutrition
87 value, yield, and improved features, such as resistance against high and low temperatures, salt stress,
88 and microbial and pest attacks. The review summarizes the literature on the parameters of outer space
89 that affect the growth and behavior of cells and organisms as well as complex colloidal systems. We
90 illustrate an understanding of gravity-related basic biological mechanisms and enlighten the possibility
91 to explore the outer space environment for application-oriented aspects. This will stimulate biological
92 research in the pursuit of innovative approaches for the future of agriculture and health on Earth.
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95 **Keywords:** Space, Evolution, Microgravity, Stress response, Mutation, Horizontal gene transfer,
96 Epigenetics, Tissue engineering, Cancer research, Colloids
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# 1. Introduction

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The scientific approach lends itself to the creation of definitions or rules that box life into specific lanes. However, upon further investigations, these lanes require repeated adjustments due to life being an ever-evolving continuous spectrum of possibilities that are based on a few absolute natural laws. The result is an ambiguous division between what is living and what is not. Life can be further thought of as a magnification of physical and chemical laws driven by local energy and order states overlaid by selection history and constraints. In recent years, and with the advent of molecular biology, the classical definition of species and life has come to be questioned. While Darwin's inheritance, variation, and selection still play a key role in the process of life's evolution, the source of the inheritance, variation, and process of selection has expanded. This expansion can result in a selection of inherited variability that is not associated with perceived optimal fitness at the time of selection. Key processes that can lead to such results include genetic hitchhiking, genetic drift via bottleneck or founder event, horizontal gene transfer (HGT) and, to a lesser extent, gene flow and epigenetics. Besides, some changes in the DNA do not seem to have any effect on amino acid translation or fitness and maybe deemed silent or neutral mutations, respectively. When gauging fitness and speciation we must consider the spatial and time aspect that is the distribution over time. Speciation occurs in either a gradual consistent pace (gradualism) or abrupt pulses (punctuated equilibria) with the main difference being the time scale of the speciation events. Punctuated events can be seen in places that have undergone rapid environmental changes (e.g., the Hawaiian Islands). The space environment with its gravitational and radiation changes affords such a novel abrupt environmental change for terrestrial life. This environment can be used to decipher underlying pathways that are not apparent on Earth, test countermeasures for observed changes, and develop new organisms with novel attributes.

Since the late 1950s space technology has developed spacecraft for transporting terrestrial life into low Earth orbit (LEO) to study, in situ, its responses to selected conditions of space (Brinckmann, 2007; Clément and Slenzka, 2006; Horneck et al., 2010; Nickerson et al., 2004). The space shuttle (Baglioni et al., 2007; Horneck et al., 1984b; Horneck et al., 1984a) and the space stations, e.g., the Russian MIR (Rettberg et al., 2002) and the International Space Station (ISS) (Baglioni et al., 2007) can truly remove the influence of gravity in an experimental setting in the LEO. The orbital microgravity environment provided by these spaceships has allowed us to perform unique experiments to study the influence of microgravity on living systems that could not be carried out on Earth. Changed environmental factors onboard a spacecraft include weightlessness (technically called “microgravity”), ionizing and non-ionizing solar or cosmic radiations, temperature extremes, and high vacuum. Among these, microgravity and cosmic radiations are of particular interest for scientific research. Regardless of the various primary experimental objectives and subsequent, sometimes-disparate conclusions drawn from the data, the majority of investigations to date have indicated that microgravity has a significant influence on cell growth and behavior. With technological advancement in molecular science and analytics, the focus of attention in modern space and gravitational biology is switching to explore the influence of microgravity on living systems at the cellular and molecular levels. Emphasis is on the investigation of genetic stability, growth, development, reproduction, aging, life span, behavior, and orientation of plants and animals in space environmental conditions. In recent years, studies under simulated or real microgravity resulted in significant outcomes in cancer research and cell and tissue engineering among others.

## 1.1. *Scope of the review*

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This review covers why we need to think out of the box and explore space environmental conditions to achieve breakthroughs for a sustainable future. The review explains the possibility of exploration of the space environment to accelerate the development of new varieties of crops or other species and novel substances using the natural evolution process. We discuss the molecular machinery and mechanisms that may lead to the evolution of organisms in space's unique environment. We consider the effects of the environment, constrained selection process, unrestricted and extensive gene expression, mutation, gene exchange, and epigenetics on the biology, adaptation, and evolution of cells or organisms in space. Technically, natural evolutionary mechanisms can be employed to obtain strains with desired traits (self-guided evolution) by setting controlled environmental and selection factors. A review of literature relevant to the effects of microgravity on growth, behavior, or survival of microbes,
plants, and cell and tissue cultures is provided. We also cover the need to study colloids in real
microgravity conditions to unveil some key physicochemical aspects, like the natural movement of
colloids that experiments performed on Earth, in the presence of gravity may mask, alter, or simply
hinder. Taken altogether, this review encompasses the different aspects of space biology that have been
studied to date. By providing a comprehensive review of this currently unfolding space research
discipline, we intend to illustrate organisms' responses to the space environment, enlighten a new
understanding of biology, and recognize the potential of the space environment for boosting biological
research in the pursuit of new solutions for the future of agriculture and health on Earth. The review not
only explains gravity-related basic biological mechanisms at the cellular level but also gives a picture
to explore space for application-oriented aspects, such as to produce and develop an array of products
for food, pharmaceutics, cosmetics, home-care with longer shelf lives and new functionalities.

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1.2. *Effects of human activities on the global ecosystem*

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Earth has immense resources and opportunities for the development of different technologies
and products to meet the current needs of the global population. However, human activities, in particular,
the overuse of fossil fuels, uncontrolled deforestation, intensive land use for agriculture, and enormous
animal production, are dramatically affecting the global climate and causing considerable stress to land
resources, including water, soil, nutrients, plants, and animals (Dupuy et al., 1976; Grossi et al., 2019;
Masson-Delmotte et al., 2018). Global temperature rise (~0.85°C in the last 130 years) continues to
result in shrinking of ice sheets, glacial retreat, sea-level rise, extreme weather events, ocean
acidification, soil salination, desertification, and reduced availability of drinking water (Masson-
Delmotte et al., 2018). These changes are affecting agricultural productivity and food availability, safety,
and quality at global, regional, and local levels (Brown et al., 2015). While the human population is
continually growing, there is no unlimited availability of natural resources such as arable land and
freshwater, which is further amplifying the threats of climate change on food security. According to the
World Economic Forum, our food supplies will be under far greater pressure with the global population
expected to reach 9.8 billion by 2050 (Godfray et al., 2010). The demand for food will be 60% higher
than it is today. With the current state-of-the-art agricultural practices and productivity, it will be
impossible to meet the demands. Furthermore, in the last several decades, there is a significant decrease
in the number of cultivated crop species as well as in the diversity within major crops (Cardinale et al.,
2012; Nordin and Nordin, 2017). Plant domestication and subsequent selection in agriculture have
limited the genetic diversity of modern crops and increased their susceptibility to multiple diseases and
extreme environmental stresses (weather and soil conditions) (Huang et al., 2016). Furthermore, the
stress due to the changing environment is weakening plants' defense mechanisms, making them more
vulnerable to several pathogens, such as viruses, bacteria, fungi, oomycetes ("egg fungi"), nematodes,
and parasitic plants (Dean et al., 2012; Savary et al., 2019; Vurro et al., 2010). These pathogens' range
and distribution are increasing with climate change (Anderson et al., 2004; Backlund et al., 2008),
causing extensive crop damage. For instance, a study on climate change-related spreading of downy
mildew (Plasmopara viticola) concluded that the production costs in vineyards are increasing in a range
of 20-50% due to higher costs in disease management (Francesca et al., 2006). Increasing the genetic
diversity within cropping systems by the use of variety or species mixtures offer several potential
advantages, such as limiting disease development, increasing yield stability, and improving resilience
to abiotic factors (Newton, 2016; Savary et al., 2019; Schöb et al., 2015). Furthermore, it delivers other
ecosystem services including greater biodiversity. Unpredictable weather events, poor crop yields,
augmented pathogenesis, serious consequences of uncontrolled agrochemical utilization, and increasing
food insecurity have set off alarms that we cannot afford to ignore. It is important to focus on the
development of stress-resistant crops that are well adapted to thrive in a changing environment and have
stable yields under stress conditions to ensure future food security. The need is to deploy the rich
biodiversity by generating a suite of new varieties of crops, not just one.

Climate change is also intensifying health problems that already pose a major burden to
susceptible populations and will bring new and emerging health issues soon. According to an assessment
by World Health Organization (WHO), between 2030 and 2050, climate change is expected to cause
approximately 250,000 additional deaths per year from malnutrition, malaria, diarrhea, and heat stress.
The socioeconomic costs of health problems are considerable and will increase over time. Besides,
misuse and overuse of conventional antibiotics in humans, animals, and plants are accelerating the

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209 development and spread of antimicrobial resistance. Drug-resistant diseases already cause at least 210 700,000 deaths globally per year, a figure that could increase to 10 million per year by 2050 if no action 211 is taken (Interagency Coordination Group on Antimicrobial Resistance, 2019). Antimicrobial resistance 212 could cause the death of 2.4 million people in high-income countries between 2015 and 2050 if no 213 sustainable efforts are made to control it. The discovery and development of novel antibiotic compounds 214 have been slow and our arsenal of effective antibiotics is dwindling. The WHO has recommended 215 prioritizing research on the discovery and development of new antibiotics to deal with the serious 216 problem of drug-resistant infections (World Health Organization, 2017). There is an urgent need for the 217 development of new methods and novel classes of potent drugs to combat antimicrobial-resistant 218 pathogens. 219

220 **1.3. Approaches to tackle problems of the fast-changing world**
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222 Researchers from all disciplines have constantly attempted developing novel strains or bio-
223 products by both conventional approaches and emerging technologies, but the results are not as
224 envisioned. Conventional screening programs have increasingly resulted in the rediscovery of already
225 known species, strains, and metabolites (Wohlleben et al., 2016). It is assumed that only a few new
226 metabolites are left to be isolated from nature's repository but needs massive screening programs (Baltz,
227 2007; Monciardini et al., 2014). Furthermore, the discovery and process development of natural
228 chemicals for pharmaceutical, agricultural, and related applications are labor- and resource-intensive as
229 they are produced in low yields in native organisms, and sometimes even in multiple forms, thus
230 complicating their identification, isolation, and structural elucidation (Pickens et al., 2011). Besides,
231 many of the natural chemicals are structurally complex, which makes their chemical synthesis difficult
232 and therefore restricting their commercial utilization. These limitations within the natural product
233 discovery led to the development and expansion of innovative methods, such as genetic or metabolic
234 engineering, for producing novel biomaterials. Traditional breeding methods rely on hybridization and
235 recombination to stack favorable alleles together. This process is usually time-consuming and labor-
236 intensive, especially when the crop is not fully or newly domesticated.

237 The modern techniques, like genetic or metabolic engineering that include the manipulation of
238 genetic material or targeted modification of metabolic pathways of an organism using recombinant DNA
239 technology (Courchesne et al., 2009; King et al., 2016; Pickens et al., 2011; Yang et al., 2007) have
240 created a foothold in scientific discovery and development. In the recent years, several genetic toolbox
241 and methods have been developed and implemented for strain improvements such as cloning and
242 expression, shuttle vectors, transformation, reporter genes, selection markers, RNA interference, and
243 DNA microarrays (Amiri and Karimi, 2019; Doron et al., 2016; Prasad et al., 2014; Yadav et al., 2018;
244 Yao et al., 2015; Zhang et al., 2018). Genetic engineering strategy has enabled the improvement of
245 species and natural product titers, production of a new characteristic in an organism, and generation of
246 a wide variety of novel compounds for pharmaceutical and agricultural sectors, but has several
247 limitations. There is still the need for optimization to achieve titers, yields, and productivities that are
248 sufficient for industrial applications. Successful transformation is the main hurdle that prevents the
249 genetic modification of many species. Unfortunately, standard transformation methods successfully
250 used in model organisms often fail when applied to other organisms. Many of the strains developed by
251 genetic or metabolic engineering are unstable and lose engineered properties over time, thus hampering
252 the commercial application (Prasad et al., 2014; Prasad et al., 2019a; Prasad et al., 2019b). It is a general
253 observation that a successful transgene integration does not always result in the efficient expression of
254 foreign genes. This is generally attributed to inadequate recognition of the promoter that drives gene
255 expression, transgene silencing, post-transcriptional gene silencing, and/or bias in codon usage (Prasad
256 et al., 2019a). Furthermore, for genetic or metabolic engineering, it is important to consider every
257 biochemical network in the cell as well as the cellular responses to genetic changes and environmental
258 stimuli (Yang et al., 2007). For this, we need to have complete knowledge of the cell and its complex
259 metabolic and regulatory networks, which is impossible in the near future. Thus, our partial and flawed
260 knowledge of organism metabolism, physiology, and regulatory networks limits the development of
261 competent strains with a high titer, yield, and/or productivity through genetic or metabolic engineering.
262 Rapid progress in genetic engineering shows the development of clustered regularly interspaced short
263 palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system, which allows robust and
264 flexible genome editing and gene regulation in a multitude of model organisms and cell types, but has
some technical limitations, like the efficiency of Cas9 activity, target site selection, and off-target effects (Vasiliou et al., 2016). Although many of the advances in the genome-engineering territory are viewed as huge scientific achievements, their use is vigorously debated and restricted in most countries, which is a major obstacle for their widespread adoption.

Evolutionary engineering is a complementary approach to conventional techniques and imitates the natural evolution process to increase the tolerance of organisms to stress (biotic or abiotic), improve cell performance, improve utilization of a target substrate, improve the production of compounds, or produce new natural chemicals (Jin et al., 2016; Lee et al., 2015; Zhang et al., 2016). In this approach, the natural evolutionary process is rapid, directed, rational, and efficient for the improvement of strains or development of desired traits through the imposition of one or more selective pressures and is associated with genetic, physiological, and morphological changes (Sharma et al., 2019). This approach takes a broad view of the complex cellular network and is the most effective tool for developing multiple or complex traits that are a result of the interaction of several genes or for which genes are unknown. In this review, we term this process as ‘self-guided evolution’. In fact, evolutionary engineering is the most ancient tool for the development of novel phenotypes. It has long been used in agriculture, animal farming, and in recent times for the production of antibiotics and other industrially important biomaterials. For example, penicillin from *Penicillium chrysogenum* and the yeast *Saccharomyces cerevisiae* for brewing, winemaking, and ethanol fermentation at an industrial scale. Space can be explored as a platform to induce the self-guided evolution of organisms (Fig. 1). Exposure of an organism to the unique space environment (discussed in Section 3) will increase evolutionary rate and processes and result in genetically diverse phenotypes, which would not form under a load of gravity. This is because organisms never experienced the absence of gravity during their whole evolution. Subjecting organisms in the space environment could produce new valuable strains of plants, yeasts, bacteria, and others and products of pharmaceutical and agricultural relevance (Horneck et al., 2010; Huang et al., 2015). Also, the observed differentiation effects on cancer cells or the induction of 3D-organoids from cell cultures under microgravity conditions are very promising applications for commercial use of space-born technology with high return values. Before we discuss the potential of space and relevant experiments, we provide a brief overview of how evolution works and the different processes involved in bringing evolutionary changes to organisms.

## 2. Evolution and mechanisms of evolution

The term ‘evolution’ stands for patterns of change in living beings and the process of natural selection responsible for these patterns. Living things reproduce and pass on their characteristics to their offspring. In a population, individuals that are poorly adapted are less likely to reproduce. However, over time, the characteristics that help the survival of an individual become more common, and a species gradually changes. Given enough time, these small changes can add up to the extent that a new species altogether can evolve. Thus, organisms adapt to their habitats by evolving or developing special features that help them to survive. Taken as a whole, this is evolution and it occurs only when there is a change in gene frequency within a population over time. These genetic differences are heritable and passed on to the next generations. According to Gingerich, (2001), three-time scales are of interest in evolution: (i) the generational time scale, the change from one generation to the next, (ii) the micro-evolutionary time scale regarding changes within 2 – 100 generations, and (iii) the macro-evolutionary time scale dealing with changes of patterns in a scale of hundred and more generations.

Evolution is not always a slow process, but also may happen more quickly. The evolution of a species occurs due to gradualism, punctuated equilibrium, or a combination of both (Rhodes, 1983; Saylo et al., 2011; Scoville, 2019). Gradualism is a more gradual selection and variation, which is hard to notice over a short period. In punctuated equilibrium, changes are sudden and huge. In the latter case, there is a period of little change, and then one or a few huge changes occur, often through mutations in the genes of a few individuals. Mutations that result in punctuated equilibrium are helpful to the individuals in their environments because these mutations allow the survival of those that have them. Evolution by punctuated equilibrium occurs because of huge or sudden changes in the environmental conditions, which causes strong selective pressure. This results in more rapid changes, leading to genetic and phenotypic shifts in the organisms in a matter of generations.
## 2.1. What happens to an organism if a constant important stimulus is no longer impinging?

The dramatic effect of an absence of a constant environmental parameter on the evolution of organisms can be seen in creatures that move from light-exposed habitats into dark environments, such as caves, deep sea, or temporally shift their activity into the night. Rétaux and Casane, (2013) analyzed all available publications about eye loss in cave inhabiting animals and concluded that genetic drift, as well as the selection of eyeless phenotypes, is the reason for eye degeneration. Interesting is the adaptation of many deep-sea fishes to their reduced light habitat: many of them did not reduce their eyes but increased the capabilities of their visual systems. Some deep-sea fish developed the ability to view color. In *Diretmus argenteus*, 38 genes for synthesis of rhodopsin were found making this fish the vertebrate bearing the highest number of genes for visual pigment synthesis (Musilova et al., 2019). In addition, many deep-sea organisms have developed strategies to replace the absence of light by producing own light utilizing bioluminescence (Haddock et al., 2010), which plays a role in communication, camouflage, or attraction of prey. Loss or reduction of light as an environmental stimulus has also led to the evolution of completely new features in animals, such as echolocation in bats and whales, or sideline organs in fish and some amphibians. An impression of the potential of evolution in the case of the absence of gravity can be seen in aquatic organisms. Although water as habitat cannot be compared to microgravity, it shares the reduced mechanical load acting onto a submerged body. Living permanently in water under reduced mechanical load, living organisms often strongly reduced stabilizing elements in their body, for e.g., in whales, which originated from dog-like land animals about 50 million years ago (Thewissen et al., 2009). However, various other factors, such as respiratory organs, increased thermal conduction, a necessity to develop new organs for locomotion (e.g., fins) affect the shape of aquatic organisms. These examples indicate the mighty potential that the space environment may have on the evolution of an exposed organism.

## 2.2. How does evolution work?

In living cells, genes encode proteins, and proteins guide cell function and structure. Phenotypic characters are formed during the development process by the complicated interaction of a large number of different genes, which is coordinated by molecular mechanisms. Signal molecules activate signaling pathways and complex gene interactions occur as a form of gene regulatory networks to develop phenotypic characters (Carroll et al., 2009; Davidson, 2006). Usually, many signaling pathways are involved in the development of a phenotypic character. As the complexity of the phenotypic character increases, the proportion of genes involved in signaling pathways or genetic networks increases (Nei and Rooney, 2005; Vogel and Chothia, 2006). The development of phenotypic characters depends on the function of various physiological processes or characters. Similarly, the function of physiological characters is based on the development of morphological characters. Both characters cannot be separated easily.

Changes in nucleotide sequences and coordination of genes expression at different times and dimensions in the development process play important roles in evolution. To understand the evolution of physiological or morphological characters, it is important to know the mechanism of gene regulation in the developmental process. In general, two essential cellular processes - transcription (DNA to RNA) and translation (RNA to protein) are involved in the production of proteins. The expression of thousands of genes and their coordinated regulation determine what a cell can do. The control of gene expression is important to all organisms. Activation of specific genes and the amount of protein or RNA product production are regulated by the complex interactions between genes, RNA molecules, regulatory proteins, and other components of the gene expression system. A gene is composed of protein-coding sequences and the regulatory region. During gene expression, the regulatory region that consists of the cis-regulatory elements and the transcription promoter (TATA box) initiates the transcription of the coding region of messenger RNAs (mRNAs). The basic components of gene expression regulation are the transcription factors and co-regulators, and the cis-regulatory elements, which serve as a docking site for transcription factors (Yilmaz and Grotewold, 2010). The transcription factors generated by different pathways interact with one another in the development process. Regulatory proteins that affect RNA polymerase activity bind to DNA sequences in the control regions of the genes that they regulate through specific protein-protein interactions, transfer signals to the transcriptional machinery, and results in specific rates of gene expression. In eukaryotes, chromatin structure also plays a significant
376 role in modulating gene expression. Recent studies have shown non-coding RNAs (ncRNAs) (e.g., 377 microRNAs (miRNAs), short interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs)) as key 378 components in the regulation of gene expression as well (Gomes et al., 2013; Nei, 2013; Patil et al., 379 2014; Yilmaz and Grotewold, 2010).

380 Evolution of genes and ultimately characters occur through changes (nucleotide changes or gene 381 duplication) in the protein-coding or regulatory regions of DNAs or both. Mutation and selection play 382 an important role in the evolution of characters. However, new functional genes may also evolve by 383 employing or co-opting other genes for a new function (gene co-option) or by adopting foreign genes 384 that come from outside the individual (i.e., HGT). Changes in the protein-coding region result in the 385 alternation of the amino acid sequence of the encoded protein. This alternation sometimes changes 386 protein function, and this change may affect physiological or phenotypic characters. Amino acid 387 substitutions vary extremely among different proteins. A strong relationship exists between the 388 functional constraints of gene products and the rates of amino acid or nucleotide substitutions (Nei, 389 2005). For instance, the amino acid substitution rate is much lower in significant proteins, such as 390 hemoglobin and cytochrome c than in less important protein, like fibrinopeptides. In addition, the 391 evolution rate is much lower in the active site of the protein than other regions to maintain the same 392 function. A study on the interspecific comparison of the evolution of protein molecules (e.g., cytochrome 393 c, hemoglobin, and insulin) involved in various physiological functions showed that the amino acid 394 substitutions occurring in the active sites primarily lead to the functional change of these proteins (King 395 and Jukes, 1969; Nei, 2005) and most amino acid substitutions in structural proteins are relatively 396 neutral. This study supports the basic principle of the evolution of proteins controlling physiological 397 characters (Nei, 1987; Nei, 2005). Pseudogenes, for example, globin gene families, which do not have 398 any function, evolve at the fastest rate because of the non-existence of selective constraints. 399

400 Many developmental biologists emphasized that morphological evolution is a result of 401 mutations of regulatory genes and not the structural genes (Carroll et al., 2009; Davidson, 2006; King 402 and Wilson, 1975). In contrast, Nei, (1987) proposed the major gene effect hypothesis, which states that 403 morphological evolution occurs by small mutations of major effects in the structural or regulatory 404 regions of the DNA. Studies on the evolutionary changes of the protein-coding regions of genes involved 405 in morphological evolution show that the pattern of amino acid substitution is similar to the genes 406 involved in physiological characters (Hoekstra et al., 2006; Liao et al., 2010; Nei, 2013). However, most 407 amino acid substitutions do not affect physiological characters noticeably, while in the case of 408 morphological characters, a few amino acid substitutions can cause dramatic alternations. Nucleotide 409 deletion/insertion, exon deletion, exon shuffling, and transposon insertion may also cause functional or 410 structural changes in the case of morphogenes (Hoekstra and Coyne, 2007; Nei, 2013; Reid and Ross, 411 2011; Xu et al., 2012). Hoekstra et al., (2006) reported that a single amino acid substitution in the *MC1R* 412 gene contributes to the adaptive beach color pattern in the beach mouse, *Peromyscus polionatus*. 413 Mutation in the gene *SBE1* that is responsible for causing wrinkled seeds in pea (Bhattacharyya et al., 414 1993) is due to the insertion of a transposon. Albinism in cavefish is caused by the loss of function 415 mutations (exon deletion) in the *OCA2* gene, which operates during the first step of the melanin 416 synthesis pathway (Bilandžija et al., 2013). These examples suggest that new mutations are important 417 for adaptation of organisms to the challenging or new environment and the innovative changes are 418 incorporated into the genome or spread through the population by natural selection (elimination of 419 previous genotypes) or by genetic and genomic drift. It appears that natural selection plays a more 420 important role in phenotypic evolution than in physiological or molecular evolution, but is secondary to 421 mutation.

421 Evolution is associated with both direct and indirect selection. Strong selection pressure for 422 resistance imposed by the use of antibiotics led to the propagation of antibiotic resistance in bacteria. 423 This points to direct selection as an underlying evolutionary mechanism (Barlow, 2009). Indirect 424 selection can be seen for example in animals living in caves. The selection pressure induced by the dark 425 environment in caves reduced eye size and pigmentation but promoted the development of other sensory 426 and neuromodulatory systems in the cavefish *Astyanax* for the orientation in a dark environment (Rétaux 427 and Casane, 2013). One may also speculate that the useless neuronal capacity for the procession of visual 428 signals is reduced in favor of other sensory elements, such as olfactory.

429
430 2.3. *Processes modulating evolution of species*
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### 2.3.1. *Mutation — the major force of the evolution of species*

Genetic variation is vital to evolutionary change. Without it, no subsequent change can be accomplished (Hershberg, 2015). Genetic variation in an organism is primarily generated by mutation (Nei, 2013; Wayne and Miyamoto, 2006), which, in the simplest form, is a change of DNA sequence that could not be repaired by the cellular repair system. A mutation can result from a single base-pair substitution (point mutation), an insertion of a transposable element in a regulatory sequence, a gene deletion, or a duplication of a particular gene, chromosome, or the entire genome. Point mutations, whereby a single nucleotide base is changed, inserted, or deleted from a DNA sequence, perhaps change a single protein and thus seem unimportant (Nei, 2013). However, many tiny point mutations over time affect the history of species. On the other hand, mutations that alter specific segments of genes or whole chromosomes are commonly associated with species differences or genetic diseases. In most cases, mutations have little or neutral effects, while harmful mutations reduce the fitness of an organism. Sometimes, the change in the DNA sequences is useful (advantageous mutations) and has a positive effect (Eyre-Walker, 2006). Although rare, this can lead to significant evolutionary change. The genotype generated by this mutation has higher fitness than the pre-existing ones and may form an innovative phenotype, allowing organisms to adapt better to their environment. The new genetic variant (mutant) which feeds, grows, or reproduces more effectively becomes more abundant within the population over time. Thus, mutations lead to changes in an organism, making it biologically different and unique from the original population.

Numerous studies acknowledge that organisms evolve systems to respond to environmental cues by controlling mutation rates. Mutation rate data is vital for understanding patterns of molecular diversity within species and divergence among species (Lynch, 2010a). High-throughput genome sequencing and mutation accumulation experiments, coupled with comparative approaches and constant theoretical progress are rapidly expanding our knowledge on natural mutation variation both within and among species. There is substantial evidence that mutation rates vary widely among different forms of life (Baer et al., 2007; Sung et al., 2012). In a recent study, 36 whole-genome single nucleotide polymorphism datasets from 16 species of bacteria sampled over periods extending over 2000 years were compared to estimate and compare their evolutionary rates (Duchêne et al., 2016). The evolutionary rates in the most rapidly evolving bacteria, such as *Enterobacterium faecium*, *Staphylococcus aureus*, and *Acinetobacter baumannii* were noted to be between $10^{-5}$ and $10^{-6}$ substitutions per site year$^{-1}$. These rates were found to be within the range estimated for other rapidly evolving bacterial species (Holt et al., 2012; Kennemann et al., 2011; Pérez-Losada et al., 2007; Ward et al., 2014) and similar to the rates estimated for slowly evolving DNA viruses (Duchêne et al., 2014; Firth et al., 2010). The pathogenic bacteria *Mycobacterium leprae* and *Yersinia pestis* show very low change of base pairs with estimates of ~$10^{-8}$ substitutions per site year$^{-1}$ and are considered as slow evolving bacteria (Duchêne et al., 2016; Kay et al., 2015). However, the unicellular eukaryote *Chlamydomonas reinhardtii* (large genome) exhibits an extremely low per-base mutation rate of $6.76 \times 10^{-11}$ per cell division (Ness et al., 2012). The analysis shows somatic mutation rate is higher than germline mutation rate in humans (Lynch, 2010a, 2010b), in a wide range of tissue types in mouse and rat, in the medaka fish (Winn et al., 2000), and in *Drosophila melanogaster* (Garcia et al., 2007). The average mutation rate in human somatic cells ($1.02 \times 10^{-9}$ per nucleotide site per cell division) is higher than *S. cerevisiae* and *Escherichia coli* (Lynch, 2006; Lynch et al., 2008). The mutation rates in the unicellular microbes *S. cerevisiae* and *E. coli* are not much different from the germline mutation rates of multicellular species, such as *D. melanogaster* and *Arabidopsis thaliana* (Lynch, 2010a). In addition, the mutation rate differs across the nuclear genome within a species and mainly depends on the base composition, DNA repair domains, recombination rate, and gene expression and density.

The proportion of advantageous or beneficial, neutral, and deleterious mutations vary among species (Baer et al., 2007; Eyre-Walker and Keightley, 2007). Analysis suggests that about 30, 16, and 2.8% of the amino-acid-changing (non-synonymous) mutations in hominids, *Drosophila* spp., and enteric bacteria, respectively, are effectively neutral (Charlesworth and Eyre-Walker, 2006; Eyre-Walker et al., 2002), and the remaining amino-acid mutations are deleterious (Eyre-Walker and Keightley, 2007). The rate of effectively neutral mutations occurring outside protein-coding sequences (in non-coding DNA) is estimated to be at least 95% in humans and around 50% in *D. melanogaster* (Eyre-Walker and Keightley, 2007). The percentage of advantageous mutations are rare and estimated to be 0% in *E. coli* (Elena et al., 1998) and in the RNA bacteriophage φ6 (Burch et al., 2007), 4% in vesicular stomatitis virus (Sanjuán et al., 2004), and 6% in *S. cerevisiae* (Thatcher et al., 1998). Studies
488 indicate that deleterious mutations are far more common than advantageous ones (Drake, 2006). Also,
489 the majority of genotypic mutations that affect phenotype are harmful (Eyre-Walker and Keightley,
490 2007). Therefore, an important question arises: what keeps a check on the mutation rate? An array of
491 data shows that natural selection and genetic drift plays a significant role in determining genomic
492 mutation rates across a wide range of taxa (Baer et al., 2007; Eyre-Walker and Keightley, 2007; Nei,
493 2013; Vos et al., 2015). Selection functions mostly in large populations, while drift prevails during
494 periods of small population size (Ohta, 1992). For instance, because of its large population size, natural
495 selection is more effective in reducing genomic mutation rates in the unicellular eukaryote C. reinhardtii
496 than in the multicellular eukaryote D. melanogaster (Sniegowski and Raynes, 2013). Mutations and
497 selection are dependent on each other. When the effective population size is large, selection tends to
498 remove deleterious mutations that have negative fitness effects on populations and brings the mutation
499 rate down to zero. However, the reduction is counterbalanced by an increase in mutation rates to
500 facilitate ongoing adaptation. Thus, natural selection adjusts mutation rates to an optimum level by
501 maintaining a balance between deleterious mutations and mutations that act as a source of genetic
502 variation required for adaptation to a changing environment (Lynch, 2010a; Sung et al., 2012; Vos et
503 al., 2015). This suggests that strongly selected advantageous mutations are exponentially distributed.
504 There exists a variation in natural selection among individuals or species, which is associated to the
505 differential regulation of DNA polymerases that have varying fidelities in replication, differences in
506 mutagen exposure, and abilities of species to neutralize or block mutagens, recognize and repair DNA
507 damage, and modulate or buffer mutational effects (Baer et al., 2007).
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509 The speed and mode of evolution are dependent on the rate at which genomes accumulate
510 random mutations. Sometimes evolution works much more quickly. Treatments of cells by high-energy
511 sources such as radiation (X-rays, and nuclear radiation), chemicals, etc., induce deleterious genetic
512 changes, but also may cause advantageous ones. Exposure of bacteria to mutagens or stress (mutation-
513 inducing chemicals, UV-radiation, or antibiotics) increases bacterial mutation rates to evolve and adapt
514 to the challenging environmental conditions (Cox and Battista, 2005; MacLean et al., 2013; MacLean
515 and Buckling, 2009).
516
517 2.3.2. Role of HGT in the evolution of species
518 HGT, also known as lateral gene transfer, is the transfer of genetic material between organisms
519 independent of sexual reproduction. HGT events can result in uptake of whole genes, ‘genomic islands’,
520 operons, or plasmids and an instant acquisition of new functions that could be beneficial for adaptation
521 to novel niches. Thus, HGT enables organisms to increase their genetic diversity and evolutionary fitness
522 probably due to the gain of new genes, the gain of paralogs of existing genes, and the gain of
523 phylogenetically distant orthologous gene (Koonin et al., 2001; Kuo and Ochman, 2009; Rocha, 2008;
524 Sieber et al., 2017).
525 Horizontal gene exchange is more common in closely related organisms but also occurs between
526 distantly related organisms, contributing to evolution (Fig. 2) (Andersson, 2005; Boto, 2010;
527 Zhaxybayeva et al., 2006; Zhaxybayeva et al., 2007). Gene exchange is reported between bacteria and
528 archaea (Gophna et al., 2004; Koonin et al., 1997; Nelson et al., 1999; Rest and Mindell, 2003), between
529 bacteria and eukaryote (Anderson and Seifert, 2011; Brown et al., 1998; Dröge et al., 1998; Guljamow
530 et al., 2007; Watkins and Gray, 2006), from archaea to eukaryote (Andersson et al., 2003), and within
531 bacteria (Felmingham and Washington, 1999), archaea, and eukaryote (Nedelcu et al., 2008; Richards
532 et al., 2006). For instance, the development of antibiotic-resistant and pathogenic strains due to HGT
533 between bacterial species (Davies and Davies, 2010; Lerminiaux and Cameron, 2019). Another example
534 is the formation of crown galls (woody protrusions) on the trunks of fruit trees because of the transfer
535 of bacterial DNA (tumor-inducing plasmid) of the *Agrobacterium* genus to plant somatic cells and the
536 expression of this DNA (Gelvin, 2003; Kondo et al., 2002; Pitzschke and Hirt, 2010).
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538 It is often difficult to conclude the exact mechanisms of gene transfer between organisms as
539 they are rare evolutionary events and identified by their consequences and not at the time of happening.
540 The molecular basis of inter-domain HGT has been largely understood by studies of genes in model
541 species or cell lines. The fundamental steps involved in a successful gene transfer between two lineages
542 include the transfer of the foreign genetic material into the host cell as naked DNA, incorporation of the
543 transferred gene in the recipient nucleus or existence as an extrachromosomal DNA, and expression into
544 a protein that must provide a function for selection in a population. In prokaryotes, HGT is generally
545 mediated by three potential mechanisms – transformation, conjugation, and transduction (Jain et al.,

508
509 The speed and mode of evolution are dependent on the rate at which genomes accumulate
510 random mutations. Sometimes evolution works much more quickly. Treatments of cells by high-energy
511 sources such as radiation (X-rays, and nuclear radiation), chemicals, etc., induce deleterious genetic
512 changes, but also may cause advantageous ones. Exposure of bacteria to mutagens or stress (mutation-
513 inducing chemicals, UV-radiation, or antibiotics) increases bacterial mutation rates to evolve and adapt
514 to the challenging environmental conditions (Cox and Battista, 2005; MacLean et al., 2013; MacLean
515 and Buckling, 2009).
516
517
518 2.3.2. Role of HGT in the evolution of species
519 HGT, also known as lateral gene transfer, is the transfer of genetic material between organisms
520 independent of sexual reproduction. HGT events can result in uptake of whole genes, ‘genomic islands’,
521 operons, or plasmids and an instant acquisition of new functions that could be beneficial for adaptation
522 to novel niches. Thus, HGT enables organisms to increase their genetic diversity and evolutionary fitness
523 probably due to the gain of new genes, the gain of paralogs of existing genes, and the gain of
524 phylogenetically distant orthologous gene (Koonin et al., 2001; Kuo and Ochman, 2009; Rocha, 2008;
525 Sieber et al., 2017).
526 Horizontal gene exchange is more common in closely related organisms but also occurs between
527 distantly related organisms, contributing to evolution (Fig. 2) (Andersson, 2005; Boto, 2010;
528 Zhaxybayeva et al., 2006; Zhaxybayeva et al., 2007). Gene exchange is reported between bacteria and
529 archaea (Gophna et al., 2004; Koonin et al., 1997; Nelson et al., 1999; Rest and Mindell, 2003), between
530 bacteria and eukaryote (Anderson and Seifert, 2011; Brown et al., 1998; Dröge et al., 1998; Guljamow
531 et al., 2007; Watkins and Gray, 2006), from archaea to eukaryote (Andersson et al., 2003), and within
532 bacteria (Felmingham and Washington, 1999), archaea, and eukaryote (Nedelcu et al., 2008; Richards
533 et al., 2006). For instance, the development of antibiotic-resistant and pathogenic strains due to HGT
534 between bacterial species (Davies and Davies, 2010; Lerminiaux and Cameron, 2019). Another example
535 is the formation of crown galls (woody protrusions) on the trunks of fruit trees because of the transfer
536 of bacterial DNA (tumor-inducing plasmid) of the *Agrobacterium* genus to plant somatic cells and the
537 expression of this DNA (Gelvin, 2003; Kondo et al., 2002; Pitzschke and Hirt, 2010).
538
539
540 It is often difficult to conclude the exact mechanisms of gene transfer between organisms as
541 they are rare evolutionary events and identified by their consequences and not at the time of happening.
542 The molecular basis of inter-domain HGT has been largely understood by studies of genes in model
543 species or cell lines. The fundamental steps involved in a successful gene transfer between two lineages
544 include the transfer of the foreign genetic material into the host cell as naked DNA, incorporation of the
545 transferred gene in the recipient nucleus or existence as an extrachromosomal DNA, and expression into
546 a protein that must provide a function for selection in a population. In prokaryotes, HGT is generally
547 mediated by three potential mechanisms – transformation, conjugation, and transduction (Jain et al.,

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The speed and mode of evolution are dependent on the rate at which genomes accumulate random mutations. Sometimes evolution works much more quickly. Treatments of cells by high-energy sources such as radiation (X-rays, and nuclear radiation), chemicals, etc., induce deleterious genetic changes, but also may cause advantageous ones. Exposure of bacteria to mutagens or stress (mutation-inducing chemicals, UV-radiation, or antibiotics) increases bacterial mutation rates to evolve and adapt to the challenging environmental conditions (Cox and Battista, 2005; MacLean et al., 2013; MacLean and Buckling, 2009).

### 2.3.2. Role of HGT in the evolution of species

HGT, also known as lateral gene transfer, is the transfer of genetic material between organisms independent of sexual reproduction. HGT events can result in uptake of whole genes, 'genomic islands', operons, or plasmids and an instant acquisition of new functions that could be beneficial for adaptation to novel niches. Thus, HGT enables organisms to increase their genetic diversity and evolutionary fitness probably due to the gain of new genes, the gain of paralogs of existing genes, and the gain of phylogenetically distant orthologous gene (Koonin et al., 2001; Kuo and Ochman, 2009; Rocha, 2008; Sieber et al., 2017).

Horizontal gene exchange is more common in closely related organisms but also occurs between distantly related organisms, contributing to evolution (Fig. 2) (Andersson, 2005; Boto, 2010; Zhaxybayeva et al., 2006; Zhaxybayeva et al., 2007). Gene exchange is reported between bacteria and archaea (Gophna et al., 2004; Koonin et al., 1997; Nelson et al., 1999; Rest and Mindell, 2003), between bacteria and eukaryote (Anderson and Seifert, 2011; Brown et al., 1998; Dröge et al., 1998; Guljamow et al., 2007; Watkins and Gray, 2006), from archaea to eukaryote (Andersson et al., 2003), and within bacteria (Felmingham and Washington, 1999), archaea, and eukaryote (Nedelcu et al., 2008; Richards et al., 2006). For instance, the development of antibiotic-resistant and pathogenic strains due to HGT between bacterial species (Davies and Davies, 2010; Lerminiaux and Cameron, 2019). Another example is the formation of crown galls (woody protrusions) on the trunks of fruit trees because of the transfer of bacterial DNA (tumor-inducing plasmid) of the *Agrobacterium* genus to plant somatic cells and the expression of this DNA (Gelvin, 2003; Kondo et al., 2002; Pitzschke and Hirt, 2010).

It is often difficult to conclude the exact mechanisms of gene transfer between organisms as they are rare evolutionary events and identified by their consequences and not at the time of happening. The molecular basis of inter-domain HGT has been largely understood by studies of genes in model species or cell lines. The fundamental steps involved in a successful gene transfer between two lineages include the transfer of the foreign genetic material into the host cell as naked DNA, incorporation of the transferred gene in the recipient nucleus or existence as an extrachromosomal DNA, and expression into a protein that must provide a function for selection in a population. In prokaryotes, HGT is generally mediated by three potential mechanisms – transformation, conjugation, and transduction (Jain et al.,
544 2002). Transformation is the ability of cells to obtain foreign DNA from the extracellular environment and incorporate it into the genome of a cell. Transduction is the process of movement of genes from one species to another via viruses. Conjugation involves cell-to-cell contact for the transfer of genetic material to a recipient cell, which is facilitated by a tube-like structure known as the pilus. The process of conjugation was thought to exist in closely related bacteria because of the compatibility, similar codon usage, and higher success rate for homologous recombination (Beiko et al., 2005; Ochman et al., 2000).
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549 However, there is considerable evidence that distantly related bacteria can potentially conjugate with one another and exchange DNA (Cordero and Hogeweg, 2009; Lerat et al., 2005). These processes are less dominant in eukaryotes. Physical contact of symbiotic or host-parasite relationships, as well as transfection of a virus, have been implicated for the DNA transfer of unrelated species into eukaryotic cells (Gogarten, 2003).
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555 HGT includes the successful transfer of genetic material as well as the existence of the transferred genetic material throughout generations (Boto, 2010). After genes are transferred, natural selection determines which of them spread throughout populations (Gogarten et al., 2002; Pál et al., 2005). Genes that have useful functions are preserved, whereas useless ones are generally removed (positive selection) (Hao and Golding, 2008; Nakamura et al., 2004). Thus, the impact of HGT in the evolution of an organism is dependent on the number of transferred genes, their successful maintenance in the recipient genomes over generations, and the extent of the phenomena concerning the evolutionary time framework and phylogenetic distance between organisms (Boto, 2010). The long-term persistence of horizontally transferred genes confirms that they confer a selective advantage to recipient organisms.
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564 There is substantial evidence that the transfer of even a single or few genes gives the recipient new functions, allowing them to exploit and adapt to the new environment (Fournier and Gogarten, 2008).
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569 For example, several anaerobes have acquired many metabolic genes from bacteria for their adaptation to anaerobic environments (Keeling and Palmer, 2008). The acquisition of genes involved in the catabolism of complex carbohydrates by rumen ciliates (Ricard et al., 2006) and the rumen fungus *Orpinomyces joyonii* (Garcia-Vallvé et al., 2000) from bacteria have facilitated their colonization and adaptation to the anaerobic environment of the ruminant gut. The pathogenesis of certain intracellular parasitic bacteria, such as *Chlamydia* and *Rickettsia* is anticipated to be due to the HGT of the chloroplast-type ATP/ADP translocases from plants, which allows them to scavenge ATP from the host (Koonin et al., 2001).
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574 Comparative analyses of data from whole-genome sequencing projects suggest that HGT has significantly influenced the evolutionary mechanism throughout the history of life and emergence of the three domains of life: Archaea, Bacteria, and Eukarya (Andersson et al., 2003; Boto, 2010; Boucher et al., 2003; Brown, 2003; Daubin et al., 2003; Richards et al., 2006; Soucy et al., 2015). With advances in sequencing technologies, the information on the importance of HGT in microbial evolution continues to expand (Choi and Kim, 2007; Dagan et al., 2008; Koonin and Wolf, 2008; Pál et al., 2005; Treangen and Rocha, 2011). In many prokaryotes, gene gain due to HGT is considered to be a more significant process than gene duplication (Dagan et al., 2008; Treangen and Rocha, 2011; Vos et al., 2015), while the relevance of HGT in gene loss is not adequately understood (Kurland et al., 2003; Polz et al., 2013).
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583 The last few years of attained results show that HGT plays a far superior role in the evolution of archaea and bacteria than formerly thought (Kloesges et al., 2011; Sieber et al., 2017; Smillie et al., 2011; Vos et al., 2015; Wiedenbeck and Cohan, 2011). The genes and species that are associated with HGT between the archaea and the bacteria are highly diverse (Brown, 2003). Koonin et al., (2001) reported that between 1.6 and 32.6% of the genes of each microbial genome are acquired by HGT indicating that a high level of gene transfer exists for most bacterial and archaeal genomes. Almost 24% of the genes in the bacterium *Thermotoga maritima* that grow well at 80°C are more similar to hyperthermophilic archaeal genes than other bacterial genes (Nelson et al., 1999). *T. maritima* acquired genes from archaea through extensive HGT. Both cultures share reverse gyrase gene, which perhaps provides them adaptation to live at very high temperatures (Forterre et al., 2000). Significant differences in gene content (over 30%) among strains of *E. coli* demonstrate that HGT has a considerable effect on genome evolution even at the species level (Hayashi et al., 2001). Dagan et al., (2008) analyzed evolutionary history among 539,723 genes distributed across 181 sequenced prokaryotic genomes and found that at least 81 ± 15% of genes in each genome were involved in HGT. They indicated that HGT has very low barriers and can substantially affect all gene families among prokaryotes over long evolutionary time scales.
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The impact of HGT in eukaryotic evolution remains controversial, in particular when multicellular eukaryotes are concerned (Huang, 2013; Ku et al., 2015; Salzberg et al., 2001; Stanhope et al., 2001). It is anticipated that HGT plays a less prominent role in the evolution of eukaryotes than in prokaryotes. A group of researchers supports that gene inheritance in eukaryotes is vertical (Ku et al., 2015) and that eukaryotic genomes show no evidence for either continuous or recent gene acquisitions from prokaryotes (Ku and Martin, 2016). However, the last decade has seen an increasing number of reports on the HGT of genes into eukaryotic genomes from both endosymbionts and free-living organisms (Alsmark et al., 2013; Andersson, 2005; Dunning Hotopp et al., 2007; Hall and Dietrich, 2007; Kishore et al., 2013; Mackiewicz et al., 2013; Marin et al., 2007; Soanes and Richards, 2014). These studies indicate that HGT had a strong impact in modulating both unicellular and multicellular eukaryote evolution. Significant numbers of HGT events contributing to genome evolution have been reported in rumen ciliates (Ricard et al., 2006), the parasitic protozoan *Entamoeba histolytica* (Loftus et al., 2005) and *Trichomonas vaginalis* (Carlton et al., 2007), the anaerobic parasite *Blastocystis* (Eme et al., 2017), the diatom *Phaeodactylum tricornutum* (Bowler et al., 2008), and the multicellular *Hydra magnipapillata* (Chapman et al., 2010). The phylogenomic analysis revealed that the metabolic repertoire of *E. histolytica* and *T. vaginalis* is shaped by the HGT of bacterial genes into their genome (Carlton et al., 2007; Loftus et al., 2005). Extensive gene transfer events have provided the diatom *P. tricornutum* with novel possibilities for metabolite management and the perception of environmental signals to thrive in oceans (Bowler et al., 2008). In a recent study, Eme et al., (2017) identified that *Blastocystis* has horizontally transferred genes both from prokaryote and eukaryote donors and the genes are involved in diverse metabolic pathways facilitating adaptation of the parasite to the gut environment, infection, and circumvention of host defenses. The gain of genes from bacteria and other eukaryotes has also resulted in the evolution of bdelloid rotifers (Gladyshev et al., 2008), fungi (Marcet-Houben and Gabaldón, 2010; Richards et al., 2006), and even plants (Huang and Gogarten, 2008). Analysis of prokaryotic-derived HGTs in 60 fully sequenced genomes showed that inter-domain transferred genes in fungi mostly included bacterial arsenite reductase, catalase, different racemases, and peptidoglycan metabolism enzymes (Marcet-Houben and Gabaldón, 2010). The coffee berry borer beetle, *Hypothenemus hampei*, which is a devastating pest of coffee, gained the bacterial *HhMAN1* gene through HGT to hydrolyze coffee berry galactomannana and colonize the coffee berries (Acuña et al., 2012). Genes that are horizontally transferred from eukaryotes to bacteria or archaea are functionally diverse and do not belong to just one or a few categories (Koonin et al., 2001; Koonin and Wolf, 2008). An example of gene transfer from eukaryote to bacteria, mainly proteobacteria, is the glutaminyl-tRNA synthetase gene, which is not present in other bacteria, the archaea (archaebacteria), and organelles (Brown and Doolittle, 1999).

Overall, there is abundant evidence to demonstrate that extensive genetic exchange has occurred in both prokaryotic and eukaryotic organisms and HGT has played a significant role in shaping our complex and remarkable biological world. HGT could potentially change genomes more rapidly than point mutation and has important implications in the evolution of organisms, such as adaptation to novel niches. Genes acquired by HGT have clear functional or environmental significance for the recipient. The acquisition of foreign genes via HGT allows organisms to thrive in new and competitive environments. As a globally important evolutionary mechanism, HGT could be used to generate novel strains that would allow them to potentially exploit and adapt to new and challenging environments.

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## 2.3.3. *Epigenetic gene regulation in the evolution of species*

Epigenetics represents the changes in gene expression and function that occur without alteration in the DNA sequence (Casadesús and Low, 2006; Crisp et al., 2016; Eichten et al., 2014; Ito, 2014). Phenotype variations due to epigenetic processes are stable and inherited to the offspring. Epigenetic processes involve DNA-methylation, histone modification, and non-coding RNA (ncRNA) activity. DNA methylation includes the addition of a methyl group to specific nucleotides catalyzed by DNA methyltransferases (Casadesús and Low, 2006; Law and Jacobsen, 2009). Histone modification is a post-translational modification of the histone proteins via acetylation, methylation, phosphorylation, and ubiquitylation (Gendrel and Colot, 2005; Ito, 2014), which can influence gene expression by modifying chromatin structure or employing histone modifiers (Bannister and Kouzarides, 2011). Histone modifications act in processes such as chromosome packaging, transcriptional activity, and DNA damage or repair. The ncRNAs, such as short ncRNAs (<30 bp; miRNAs, siRNAs, and piRNAs) and the long ncRNAs (>200 bp) are also shown to regulate gene expression at the transcriptional and post-
transcriptional level by playing a role in histone modification, DNA methylation targeting, heterochromatin formation, and gene silencing (Crisp et al., 2016; Ito, 2014; Palazzo and Lee, 2015). DNA methylation change, histone modification, and chromatin and small interfering RNA (siRNA) abundance are all perturbed (induced or blocked) by both biotic and abiotic environmental conditions, resulting in the alternation of gene expression (Boyko et al., 2010; Ding et al., 2012; Khraiwesh et al., 2012; Sani et al., 2013; Zong et al., 2013).

Epigenetic effects are not only common but they can also underlie and influence many aspects of evolution (Lind and Spagopoulou, 2018) in several organisms including plants, animals, and microbes. This is true both for epigenetic effects that are visible only in a single generation as well as for transgenerational epigenetic inheritance (Fig. 3). Epigenetic regulation enables a rapid increase in variation within a species in times of environmental stresses or cues, providing an opportunity for adaptation to the arising selection pressures. The passing on of these variations to the offspring establishes epigenetic inheritance (Holliday, 2006), which is important for adaptation, above all in organisms with limited genetic variation. Plants have developed innovative approaches to survive harsh environmental stresses (Khraiwesh et al., 2012; Zhu et al., 2008; Zong et al., 2013). Among others, the efficient strategy is the epigenetic regulation of gene activity throughout their lifespan. In plants, DNA methylation and histone modification affect various biological processes in the current as well as in subsequent generations, such as growth and development, flowering time, fertility, and morphology. They regulate the silencing of transgenes and transposons as well as control genomic imprinting (Bai and Settles, 2014; Crisp et al., 2016; Ito, 2014). Stress-induced epigenetic changes can be inherited through cell division (meiotic and mitosis) (Calarco et al., 2012; Eichten et al., 2014; Feng et al., 2010). Earlier investigation showed that the DNA methylation pattern is not passed to the next generation (Monk, 1995) in mammalian cells but later studies indicated that epigenetic modifications of some genes can be transmitted through the germline (Anway et al., 2005; Crolley et al., 2006; Rakyan et al., 2003). Like in many eukaryotes, epigenetic regulation by DNA methylation (adenine instead of cytosine) also influences various cellular functions in bacteria (Casadesús and Low, 2006). It governs virulence pattern in a wide range of pathogens, controls cell cycle, provides signal for DNA replication, repair of DNA, chromosome segregation, exchange of plasmids, and bacteriophage genome packaging, and regulates activities of transposase and certain promoters as well as expression of genes (Bhagwat and Lieb, 2002; Casadesús and Low, 2006; Løbner-Olesen et al., 2005; Roberts, 1985; Wion and Casadesús, 2006).

Collectively, epigenetic modifications enable (i) survival of organism in changing environments without the need for genetic alterations (Burggren, 2016), (ii) adaptation to the new environment by the development of rapid variation due to higher rate of methylation or demethylation (i.e., the epimutation rate) of DNA and histones compared to genetic mutations (van der Graaf et al., 2015), and (iii) adaptation of small populations with a limited genetic variation or of asexual organisms to new environments. Thus, epigenetics provides a means for the development of variations within a species that can adapt at times of stress or deprivation.

## 2.4. *Stress accelerates evolution*

The interaction between an organism and the environment is central in evolution (Fig. 4). Stress is the major force driving the evolution of living organisms (Nevo, 2011; Parsons, 2005). Parsons defined stress as “an environmental factor causing potential changes to biological systems with the potential for impacts on evolutionary processes” (Parsons, 2005). Stress plays a significant role in facilitating adaptation by allowing better modifications, maintenance, and functioning of organismal systems to the ever-changing environment (Badyaev, 2005; Rutherford, 2003). Organisms become extinct when they fail to change and evolve adaptive mechanisms to diminish the detrimental effects of environmental changes.

Living cells are constantly subject to a myriad of external and internal stresses that affect their performance and well-being. External stresses are naturally occurring or man-made changes in abiotic factors, such as temperature, light, other climatic factors, and chemical components, and biotic aspects including competition, predation, and parasitism (Bijlsma and Loeschcke 2005). Internal stresses consist of genetic architecture changes in organisms brought naturally or artificially by numerous mutagenic agents, such as oxygen-free radical metabolic by-products (Baer et al., 2007; Bijlsma and Loeschcke, 1997). Both external and internal stresses result in a wide array of biological responses ranging from genotypic to physiological to phenotypic. Based on the intensity, frequency, and uniformity of stress, these responses are either associated with the development of stress tolerance, stress resistance, and
novel traits, or the reduction, migration, and extinction of a population. Evolutionary change is not always associated with phenotypic changes. Under a normal range of environments, stress result only in the expression and accumulation of novel genetic variation and physiological adaptations and no changes in morphology (Badyaev, 2005). Moderate stress is vital for normal growth and differentiation of cells and organisms (Badyaev, 2005; Clark and Fucito, 1998; Müller, 2003). For example, gravity influences the development of cells and organisms profoundly and subtly in terms of shape, function, and behavior (Bizzarri et al., 2015). Extreme environmental changes disrupt normal development, function, and may cause mass extinction events but are also associated with evolutionary changes and the origin of new species adapted to new environments (Nevo, 2011; West-Eberhard, 2003). Harsh environments occasionally lead to novel phenotypic alternations and simultaneously initiate strong phenotypic selection (directional selection) for survival and succession in changing and new environments (Badyaev, 2005; Rutherford, 2003; Wright, 2004). Examples of such shifts in populations include responses to selection arising from human activities, such as the evolution of antibiotic resistance in bacteria, pesticide resistance in insects, and heavy metals resistance in plants and invertebrates (Davies and Davies, 2010; Hoffmann and Hercus, 2000; Lerminiaux and Cameron, 2019; McKenzie and Batterham, 1994). Nonetheless, the mechanisms behind the relationship between extreme environments and phenotypic evolution are rarely understood.

Organisms' ability to recognize, assess, and react to stressors, in particular to those that are extreme, occasional, and shorter than the generation time of an organism and associated with phenotypic evolution is quite remarkable. It is intriguing to see how an organism survives stress, accommodate stress-induced modifications, and reproduce without reducing its functionality. With the increase in evolutionary studies of stress, it is widely recognized that adaptation of an organism to ever-changing environments is generally associated with an increase of recombination and mutation rates (genetic variance) and amplification of phenotypic variation (Badyaev, 2005; Hoffmann and Merilä, 1999; Horne et al., 2014; MacLean et al., 2013; Wright, 2004). Stress increases mutations rates in prokaryotes as well as in eukaryotes, which is probably the reason for discontinuous (punctuated) evolution (Velkov, 2002). The mutation rate in bacteria under stress is up to 100 to 200-fold higher compared to non-stressed cells (Foster, 2000). This increase is due to (i) activation of an SOS-regulon, which lead to the formation of a DNA polymerase IV that produces many errors during DNA-replication, (ii) activation of the homologous recombination system that is associated with single- and double-strand breaks in DNA by recombination enzymes, (iii) activation of double-strand break repair system, which restores replication of a stalled or damaged replication fork, and (iv) transient switch-off of the mismatch repair system (Bruijn, 2016; Kuzminov, 1999; McKenzie et al., 2000; Motamedi et al., 1999). Furthermore, stress increases the efficiency of interspecific gene transfer. Plasmids are considered as evolutionary tools as they can accelerate evolution by increasing mutations of their own and chromosomal genes as well (Velkov, 2002). The adaptive mutation rate is higher in plasmid than chromosomal genes (Rosenberg, 2001). In stress, bacterial cells also suppress their own restriction-modification system transiently to improve their chances of gaining new foreign genes, thereby facilitating evolutionary changes (Kelleher and Raleigh, 1995; Velkov, 2002). In eukaryotes, the heat shock protein Hsp90 enables the native folding of proteins (e.g., signal transduction and morphogenetic proteins) under normal conditions. However, under stress, this protein is involved in other important use that leads to increased accumulation of multiple mutations in vital genes (Rutherford, 2000).

### 3. Space environment as a toolbox for application-oriented biological research

Space is a unique environment in many aspects (Horneck et al., 2010). It provides a combination of stressors that organisms have never experienced on Earth (Fig. 5). Certain changes are noticeable only in space. Studies conducted so far show that space environment leads to alternations in genetics and variations in morphology and physiology, producing negative or positive effects on the organisms. For example, space conditions increases virulence, reduces the susceptibility of pathogenic microorganisms against antimicrobial agents, and enhances bacterial growth (reduced lag phase and enhanced exponential phase) (Horneck et al., 2010; Klaus et al., 1997; Mermel, 2012). Space offers the extraordinary environmental conditions of high radiation of galactic and solar origin, high vacuum, extreme temperatures, and microgravity (Horneck et al., 2010). The radiation in LEO is strong enough to increase the mutation rate (about 100-fold compared with that on Earth), and microgravity forces
766

biological systems to reorganize, adapt, and evolve. Microgravity is the absence of gravity and leads to pronounced physical changes in a sample or system environment.

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## 3.1. Gravity and microgravity

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Gravity is one of the four basic forces in physics and has two effects on an object as a function of its mass: displacement (motion) and/or deformation (weight). It is the reason for the mutual mass-dependent attraction of objects. The magnitude of the attractive force between two bodies of mass is determined from Newton's law of gravitation: $F_{12} = (G m_1 m_2) r^{-2}$, where G is the gravitational constant $(6.672 \times 10^{-11} \text{ Nm}^2\text{kg}^{-2})$, $F_{12}$ is the force resulting from the attraction by a particle of mass ($m_1$) on another mass particle ($m_2$) a distance ($r$) away. Force accelerate bodies. The acceleration effect of the small body (e.g., human) on a huge mass (e.g., Earth) is so negligible that the high-mass object attracts the body. The gravity field of Earth accelerates masses at the Earth's radius with about $9.81 \text{ m/s}^2$ (differs concerning geographic position). At Earth's surface, the path of an object to the Earth center is inhibited and gravity becomes noticeable as weight force ($F=ma$, where, $m$ is mass and $a$ is gravitational acceleration). The outcome of this relationship is so ubiquitous in our daily lives that weight is usually not considered as a variable to be experimentally manipulated. The fact that the Earth's gravity gives weight to attract matter leads to various phenomena, such as the formation of water bodies and atmosphere, hydrostatic pressure, sedimentation, convection, and increased shear forces. In addition, the physical solubility of gases such as oxygen in water depends on acceleration. Since the origin of life, all organisms are constantly exposed to the vector of gravity. Organisms, above all terrestrial ones, developed structures to counteract the impact of gravitational forces.

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To annul gravity on Earth is not easy. Only conditions of free fall enable real microgravity conditions. Microgravity means acceleration conditions close to weightlessness (Horneck et al., 2010; Klaus, 2004). Weightlessness leads to pronounced physical changes in the sample environment, such as no sedimentation, no hydrostatic pressure difference within the samples or the surrounding medium in open containers, no buoyancy-driven convection: transport of metabolites reduced to the range of diffusion, and low shear forces. Acceleration directly influences the properties of membranes, as has been determined in experiments with defined vesicles and living cells (Kohn et al., 2017; Sieber et al., 2016). In cells as well as in vesicles, membrane fluidity increases significantly in microgravity, which, for example, influences the uptake and effects of drugs.

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### 3.1.1. Sedimentation

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Under microgravity, no sedimentation takes place. Various organisms use intracellular heavy particles (e.g., amyloplasts or otoliths) or the whole cell body as statoliths to sense the vector of gravity. This phenomenon is based on the higher density of those statoliths which sediment within the less dense cytoplasm. Without gravity or external acceleration this movement stops.

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### 3.1.2. Diffusion and convection

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Diffusion is essentially based on the Brownian motion and results finally in an even mixing of all types of molecules in a given limited volume. Diffusion is fast only over small distances (scale of a cell). In contrast, convection depends on specific density differences in a given volume. It allows a very effective mass transport over large distances. Diffusion is not gravity-dependent, while convection rate is. The diffusion rate of a particle depends on its diffusion coefficient $D$ [$D = (k T)/(4 \pi \eta a)$], where $k$ is the Boltzmann constant $(1.3806488 \times 10^{-23} \text{ J/K})$ divided by NA (Avogadro number: $6.022 \times 10^{23}$), $T$ is the temperature in Kelvin (K), $\eta$ is the viscosity of the medium, and $a$ is the radius of the particle. Typical values for 'D' are about $5 \times 10^{-6} \text{ cm}^2\text{s}^{-1}$ for small molecules, such as sucrose, $6 \times 10^{-7} \text{ cm}^2\text{s}^{-1}$ for large molecules and values below $10^{-10} \text{ cm}^2\text{s}^{-1}$ for organelles. The square distance of the migration of a particle can be calculated by $d^2 = 2 D t$. The time ($t$) for a particle to be translocated depends on the square of the distance ($d$). This means that diffusion processes are rather slow. For example, the time of a protein needed to travel 3 mm from an effector to a receptor cell would last about 2 h.

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Convection is a gravity-dependent buoyancy-driven effect. The specific weight of air or fluids (except water at its point of an anomaly) becomes lower with increasing temperature. With uneven temperature distribution (e.g., presence of a heating source), heated water or air begins to move upward, while it is replaced by colder air and water. Convection results in a circulation, which also mixes the air or water and leads to a better distribution of oxygen or metabolites. No convection occurs when gravity
822 is absent. This may lead to severe effects on the macroscopic and eventually on the microscopic scale.
823 The availability of oxygen is impaired when particle translocation is solely based on diffusion. Stout et
824 al., (2001) clearly showed that hypoxia occurs in the root zone of *Brassica rapa* under microgravity.
825 Among others, the activity of ADH (alcohol dehydrogenase) was found to be increased several-fold in
826 microgravity indicating anaerobiosis compared to 1g. Not only oxygen supply but also nutrient uptake
827 as well as dissipation of metabolic waste products are most likely hampered in microgravity. As
828 mentioned before, many studies under real and simulated microgravity reported that metabolic enzymes
829 were affected. The lack of convection is an indirect effect of microgravity. To overcome these problems
830 convection needs to be replaced by air and fluid streams (which is, of course, difficult when roots are
831 embedded in agar and also introduces a force in a force-free environment). Most likely non-motile
832 organisms or cells are more sensitive against depleting-effects in their direct environment. Motile
833 organisms can constantly change their position from depleted to non-depleted sites. In this respect, it
834 would be interesting to compare e.g., the development of motile and non-motile bacteria in not-stirred
835 containers under microgravity.

836 The high viscosity of the cytoplasm reduces the diffusion speed so that the Peclet number (ratio
837 between convection and diffusion) is >1 (Todd, 1989). This means that convection contributes
838 considerably to movement within the cytoplasm (assuming the cells do not show plasma streaming).
839 Convection and diffusion are not only important for mass transport but also for heat transport. In cells
840 with a high-energy conversion ratio, it would be theoretically possible that focal heat-producing cellular
841 components, such as mitochondria, the nucleus during DNA-replication, or centrosomes may become
842 overheated under microgravity, because of a misbalance between convectional and diffusional heat
843 transport. Overheating of mitochondria may account for intrinsic-induced apoptosis due to cytochrome
844 c release from damaged mitochondria [space-related apoptosis and other effects are reviewed among
845 others in Majima et al., (2009)]. Indeed, in a variety of publications effects on abnormal distribution or
846 ultrastructure of mitochondria were reported (Bell et al., 1992; Klymchuk, 2007; Mao et al., 2013;
847 Nikawa et al., 2004; Philpott et al., 1990; Popova, 2003). To prove this, we propose a simple experiment
848 in which temperature inside a cell will be measured spatially in microgravity and under 1 g. Recently
849 different methods for temperature detection have been developed, based on fluorescence lifetime-
850 imaging of thermosensitive fluorophores (Okabe et al., 2012) or sub-micrometer thermometry using
851 nanodiamonds (Kucsko et al., 2013).
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### 3.1.3. Shear forces

854 Shear forces are strongly reduced under microgravity. The random positioning machine (RPM)
855 leads to increased shear forces and stress on biological systems because the samples are constantly
856 moved in the field of gravity. Clinorotation, in contrast, provides an environment with very low shear
857 forces (Hauslage et al., 2017). The luminescent dinoflagellate *Pyrocystis noctiluca*, which produces
858 bioluminescence when experiencing shear forces, was subjected to clinorotation. The authors
859 constructed hardware, which can be fixed on different "simulated-gravity"-devices and recorded shear
860 force-induced light signals from the dinoflagellates. Random-positioning was found to induce strong
861 luminescence indicating the significant presence of shear forces. In contrast, no luminescence was
862 induced upon clinorotation. This indicates that clinorotation is probably the closest match to real
863 microgravity which can be achieved on Earth in terms of shear forces.
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### 3.2. Space radiation

866 The main fraction of space radiation reaching LEO consists of protons from solar particle events
867 and high-energy galactic cosmic rays (mainly hydrogen ions and helium ions and some high-atomic-
868 number nuclei and high-energy nuclei), as well as other high-energy solar particle events (Häder et al.,
869 2009a; Horneck et al., 2010; Suparta and Zulkeple, 2014). The ionizing radiation (γ-ray, x-ray, β-ray,
870 α-radiation, and neutron radiation) in LEO is considerably increased compared to Earth, with the
871 perceived radiation strongly dependent on the orbit of the spacecraft. We calculated the yearly dose in
872 millisieverts during different LEO space missions according to the data provided in a corresponding
873 NASA report (space shuttle, Skylab, and ISS) (Moreno-Villanueva et al., 2017). The perceived level of
874 ionizing radiation is between 85 times (space shuttle mission 41-C: about 0.69 mSv/d) and 250 times
875 (Skylab 4: about 2.04 mSv/d) that at ground level (about 0.0082 mSv/d). Polar orbits or frequent
876 passages through the so-called South Atlantic Anomaly, where the Earth inner Van Allen belt is thin
877 (only about 190-200 km), will exceed the values of the above-mentioned missions multiple times. The
878 investigation on the influence of space ionizing radiation on different organisms is an important field of
879 Ionizing radiation interacts with DNA and may lead to stable and inheritable genetic alternations,
880 changes in gene function, metabolism, physiology, growth, shape, and structure. The effects of radiation
881 on organisms depend on the quality and quantity of radiation, and cell age and characteristics (Horneck
882 et al. 2010; Matallana-Surget and Wattiez 2013). Space radiation cannot be simulated on Earth to its full
883 complexity.

884

885 **4. Simulated and real microgravity platforms**
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886

887 Based on free fall, different measures are available to achieve real microgravity. These include
888 drop towers (microgravity time about 5 s), aircraft parabolic flights (microgravity time about 20 – 30 s),
889 sounding rockets (microgravity time 5 – 13 min), and orbital platforms such as satellites or space stations
890 (microgravity time weeks to years) (Fig. 6). The “microgravity carriers” differ widely in terms of
891 microgravity-time and microgravity-quality, as well as cost and accessibility (Pletser, 2004; Ruyters and
892 Friedrich, 2006). Drop tower experiments and aircraft parabolic flight experiments are relatively
893 reasonably priced and accessible in a foreseeable time scale (weeks or months). Residual accelerations
894 are very low during drops, while they are higher in parabolic flights. Additionally, the typical
895 acceleration profile of a parabola includes two phases of 1.8 g before and after the µg-phase. Sounding
896 rockets or orbital carriers demand a long time of experiment preparation and are quite cost-intensive.
897 Due to the high costs of those technical microgravity opportunities, extensive ground-based data sets
898 are required for the in-depth analysis of the microgravity experiments.

899 To inhibit the orientation of organisms to the vector of gravity, different devices have been
900 developed by which the gravity vector is randomized. The principle of randomizing the acceleration
901 vector is the rotation of the samples around one or two axes. These methods are often referred to as
902 “simulated microgravity”. In any case, it is important to emphasize that “simulated” or “functional”
903 microgravity is different from real microgravity because (omnidirectional) acceleration forces are still
904 acting on the samples. In contrast to real microgravity, no gravitational unloading is achieved. Next to
905 averaging the vector of gravity, these devices inhibit the sedimentation of the samples. Most common
906 devices for averaging the vector of gravity are:

907 • 2D-clinostat and 3D-clinostat, where the samples are rotated around one and two axes, respectively
908 (Hemmersbach et al., 2006). A clinostat reproduces the quiescent, unstirred fluid conditions that
909 are achievable on orbit.

910 • Rotating wall vessel (RWV) bioreactors are special 2D-clinostat for liquid cultures. RWV is a
911 suspension culture system that allows cells to grow under physiologically relevant low fluid shear
912 conditions normally experienced by cells cultured in a spaceflight environment. A tube with a
913 narrow diameter is rotated around its length axis (Hammond and Hammond, 2001; Klaus, 2001).
914 The RWV bioreactor enables perfusion of nutrients to and waste from the culture.

915 • The random positioning machine (RPM), which in principle works similar to a 3D-clinostat, but
916 arbitrarily changes the movement direction (Patel et al., 2007; van Loon, 2007).

917 Recently, diamagnetic levitation was thoroughly tested as a measure to produce microgravity-
918 like conditions. In principle, all molecules in a sample should be lifted using an extremely strong
919 magnetic field (10 - 16.5 T) in a way that the samples in the center of the solenoid are in the condition
920 of weightlessness. The solenoid produces the magnetic field. Different magnetic fields result in different
921 effective gravity levels. Manzano et al., (2012) constructed sample holders with different sample
922 locations in the solenoid. With this method, simultaneously microgravity, Moon-, Martian- and hyper-
923 gravity can be achieved. A “1 g-position” serves as an additional control. Magnetic levitation is presently
924 the only facility which enables microgravity and low gravity-like effects in a stable environment
925 (Manzano et al., 2012). Prerequisite is that all components of a sample have the same magnetic
926 susceptibility and therefore this method is not suited for a variety of organisms. For example, since
927 barium sulfate statoliths in *Chara* rhizoids have a far different magnetic susceptibility than the rest of
928 the cell, investigations of gravitropism in this organism using diamagnetic levitation are not possible
929 (Herranz et al., 2013). Also, a magnetic field alone can exert effects on living organisms. Paul et al.,
930 (2006) reported changes in gene expression patterns in *Arabidopsis* induced by strong magnetic fields.
931 Significant effects of magnetic levitation on the orientation of *Euglena gracilis* as well as *Paramecium*
932 cells, are also described (Hemmersbach et al., 2014). Under normal conditions, both organisms show a
933 precise orientation behavior, termed as negative gravitaxis. However, upon exposure to a strong
magnetic field, both cell types aligned to the magnetic field lines. While *Paramecium*-cells were aligned parallel to the field lines, chloroplast-containing *E. gracilis* cells showed a perpendicular alignment towards the field lines. In addition, even upon application of very strong magnetic fields, it was not possible to levitate immobilized cells. This clearly shows that magnetic levitation is, at least for various biological systems, no substitute for real microgravity. Herranz et al., (2013) compared literature data of experiments performed under simulated microgravity with data from real microgravity. The survey revealed that the optimal simulated microgravity facility to achieve comparable results to real microgravity is specific for the investigated organism and the investigated phenomenon. However, after profound literature research, Kiss et al., (2019) strongly promote the application of ground-based gravity simulation but also recommend a very stringent and careful standardization of the experiments to allow better comparison between the data. Taken together, due to the fundamental physical differences between real and simulated microgravity, it is important to consider the applied experiments' environmental needs.

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## 5. Microbiological studies in space environment or simulated microgravity conditions

948

To study the influence of various space conditions, microbes have been sent into space for over 60 years (Clément and Slenzka, 2006; Crabbé et al., 2011; Crabbé et al., 2013; Horneck et al., 2010; Huang et al., 2015; Juergensmeyer et al., 1999; Kacena et al., 1999; Kim et al., 2013a; Kim et al., 2013b; Nickerson et al., 2004; Rosenzweig and Chopra, 2012; Taylor, 1974; Wilson et al., 2007; Wilson et al., 2008). The two main physical factors that microbes experience in space are microgravity and cosmic radiation, while other factors, like space vacuum, thermal extremes, and solar UV radiation are mitigated by spacecraft design. Besides, several studies have been performed utilizing a low-shear modeled microgravity (LSMMG) environment to understand the influence of the space environment on a range of microorganisms (Altenburg et al., 2008; Arunasri et al., 2013; Baker et al., 2004; Chopra et al., 2006; Gao et al., 2011; Huang et al., 2015; Kim et al., 2016; Lawal et al., 2013; Nickerson et al., 2000; Pacello et al., 2012; Rosenzweig et al., 2014; Searles et al., 2011; Soni et al., 2014; Tucker et al., 2007; Xu et al., 2015). These microbes include among others *Salmonella enterica* serovar Typhimurium, *Yersinia pestis*, *E. coli*, *Pseudomonas aeruginosa*, *P. fluorescens*, *Streptococcus mutans*, *Mycobacterium marinum*, *Bacillus subtilis*, *Streptomyces coelicolor*, *S. clavuligerus*, *S. hygroscopicus*, *Serratia marcescens*, *Enterobacter cloacae*, *Enterococcus faecalis*, *Rhodospirillum rubrum*, and the yeasts *Candida albicans* and *Saccharomyces cerevisiae* (Horneck et al., 2010; Senatore et al., 2018; Tirumalai et al., 2019). Microbial characters were found to be changed in real and simulated microgravity conditions including metabolism, genetic transfer, growth, morphology, stress sensitivity, and pathogenicity.

A majority of microbiological studies in space have reported similar responses in space across several bacterial species, including reduced lag phase and increased cell population (Benoit and Klaus, 2007; Horneck et al., 2010). Unexplained inconsistencies deviating from these typical findings were occasionally reported over the years. A literature survey substantially proposes that cell motility is the key variable responsible for the seemingly incongruent results (Benoit and Klaus, 2007; Horneck et al., 2010). Studies of microgravity effects on motile and non-motile cells have shown that non-motile cells exhibit a difference in growth kinetics in space, while growth kinetics of motile strains is not affected under microgravity and static conditions. This is perhaps due to the lack of convection, which limits the mass transfer between cells and the fluid environment (Klaus et al., 2004; Klaus, 2004). That means the transport of nutrients from the medium to non-motile cells as well as the convection-driven distribution of waste products from cells is reduced (the only diffusion occurs), which in turn leads to the accumulation of waste products and reduction of oxygen and nutrients around the cells. This causes specific biological responses in non-motile cells, altering their growth and behavior relative to 1 g controls. However, these do not account for motile cells as they are constantly changing their position in the medium. A strong correlation exists between motility and cell number of culture in spaceflight (Benoit and Klaus, 2007; Horneck et al., 2010). This suggests that bacteria lack gravity-dependent physiological mechanisms but are indirectly influenced by the lack of convection in microgravity conditions. Also, it is proposed that due to their small size, it is unlikely that bacteria respond to gravity using physiological mechanisms (Horneck et al., 2010; Klaus et al., 1997). Above all, the Brownian motion superimposes the gravitational force in small objects (>10 µm) (Klaus et al., 1997). Convection is present but minimized under sub-1 g accelerations and thus it is important to determine the degree of
990 convection required for optimal cell growth and development in reduced gravity conditions (Benoit et al., 2008; Janosi et al., 2002).

991 Several groups of bacteria (e.g., Proteobacteria, Firmicutes, Staphylococcaceae, and Enterobacteriaceae) are present onboard the ISS (Ichijo et al., 2016), which are mostly transferred from the bodies of the astronauts. It is important to study the relationship between humans and microbes in space habitats, as they are crucial for long-term manned space missions. Space influences microbes but microbes also influence spaceflight and may become a potential threat to the crewmembers. Microorganisms play an important role in several biological processes, e.g., in wastewater treatment, but may become problematic through the buildup of biofilms that can negatively affect life-support systems. During long-term missions, they also pose risks of increased pathogenicity and development of antibiotic resistance due to unpredictable genomic changes (Tirumalai et al., 2017), which was concluded based on a growth study of Lac+ *E. coli* cells for over 1000 generations in lysogeny broth under LSMMG. Previous and recent studies have shown that space conditions increase virulence and reduce the susceptibility of pathogenic microorganisms against antimicrobial agents (increase in minimum inhibitory concentrations) (Horneck et al., 2010; Ilyin, 2005; Kalpana et al., 2016; Lapchine et al., 1986; Mermel, 2012; Nickerson et al., 2000; Senatore et al., 2018; Tirumalai et al., 2019). *S. enterica* serovar Typhimurium exhibited increased pathogenicity in mice infected with cells exposed to simulated microgravity compared to 1 g conditions (Nickerson et al., 2000). The cells subjected to simulated microgravity were also found to be more acid-resistant, suggesting the possibility of enhanced survival in the gastrointestinal tract, as well as showed altered protein expression patterns. Low fluid shear forces as present in microgravity increase virulence, gene expression, and stress response. This was concluded based on experiments conducted in RWV bioreactor using a *Salmonella* liquid culture (Yang et al., 2016). The increase in microbial virulence and resistance against antimicrobials in the space environment could be attributed to reduced lag phase, increased population growth, and enhanced secondary metabolite production (Horneck et al., 2010). But, conflicting reports also exist showing reduced resistance and virulence in some bacteria in microgravity conditions (Hammond et al., 2013; Juergensmeyer et al., 1999). Hammond et al., (2013) assessed the effects of spaceflight, clinorotation in a 2-D clinorotation device, and static ground controls on virulence by determining the ability of pathogens *Listeria monocytogenes*, methicillin-resistant *S. aureus* (MRSA), *Enterococcus faecalis*, and *Candida albicans* to kill *Caenorhabditis elegans*. They found all four pathogens less virulent in microgravity.

1021 Moreover, the biofilm formation in a microgravity environment is different compared to normal gravity controls (Kim et al., 2013b; Mauclaire and Egli, 2010), which could be associated with increased pathogenesis and tolerance to antibiotics. Increased viable cells and thick and higher biomass biofilms with a column-and-canopy structure is observed in spaceflight compared to 1 g controls, indicating that the community-level behaviors of bacteria are highly influenced in microgravity (Kim et al., 2013b). Due to the differences in extracellular polymeric substances responsible for biofilm formation, differences in cell aggregates and clumping have been observed in *S. typhimurium* (Wilson et al., 2007) and *P. aeruginosa* (Kim et al., 2013a) cultured in space. Crabbé et al., (2011) noted differential expression of genes involved in biofilm formation and cell aggregation as well as validated microscopically the enhanced self-aggregation behavior of space flight grown *C. albicans* compared to synchronous 1 g controls. Increased biofilm formation has also been reported in *C. albicans* grown under LSMMG determined by microscopy and morphology-specific gene expression profiling (Searles et al., 2011). Enhanced biofilm formation ability has been noted in *Klebsiella pneumonia* in simulated microgravity conditions (Wang et al., 2016). A thick biofilm showing high tolerance to antibiotics, salt, and ethanol treatment has also been reported in LSMMG grown *E.coli* cells compared to controls (Lynch et al., 2006; Tucker et al., 2007). Recently, Cheng et al., (2014) reported that simulated microgravity alters the biofilm structure of *S. mutans* as well as increases its proportion within a dual-species biofilm (co-cultured with *S. sanguinis*). Taken together, the changes in microbial virulence, antibiotic susceptibility, and biofilm formation in combination with suppression of the immune system in astronauts because of various spaceflight stressors increase health-related concerns during a long-term manned space mission.

1042 Significant changes in membrane fluidity have been reported in microbial cells under reduced gravity conditions as well. Vukanti et al., (2012) reported that variations in membrane potential and integrity under modeled microgravity conditions make *E. coli* and *S. aureus* cells physiologically more active and viable compared to 1 g samples. Kim and Rhee, (2016) also showed that the simulated space
1046 environment alter physiological characteristics of a foodborne pathogen *E. coli* O157:H7. The study indicated that heat resistance of *E. coli* decreased under LSMMG conditions most likely due to the increases in membrane fluidity and downregulation of relevant heat stress genes.

1047 Gravity-dependent physical alterations influence metabolic pathways, gene expression, and genetics, affecting physiology and phenotype of microbes. In the last two decades, research groups are focusing on the gene expression changes (up- or down-regulation) in response to real or simulated microgravity. Microarray analysis revealed differential expression of about 100 genes mostly involved in transcriptional regulation, virulence, lipopolysaccharide synthesis, and iron-utilization in *Salmonella* under simulated microgravity (Wilson et al., 2002). Global microarray and proteomic analyses of *Salmonella* grown aboard space shuttle mission (STS-115) showed that 167 transcripts and 73 proteins changed expression with the RNA-binding protein Hfq as a global transcriptional regulator involved in the microbial response to spaceflight conditions (Wilson et al., 2007). Crabbé et al., (2011) also found Hfq as a global transcriptional regulator, while investigating the global transcriptional and proteomic responses of spaceflight-induced *P. aeruginosa*. They reported differential regulation of 167 genes and 28 proteins and found transcriptional response data similar to cells grown in microgravity analog conditions using the RWV bioreactor. Among others, genes for oxygen metabolism, virulence-related (the *lecA* and *lecB* lectin) genes, and the gene for rhamnosyltransferase (*rhlA*) involved in rhamnolipid production were induced in spaceflight. Recently, Orsini et al., (2017) investigated the effects of simulated microgravity on *S. mutans* and reported up-regulation of 153 genes and down-regulation of 94 genes (both ≥2-fold). Genes involved in carbohydrate metabolism, translation, and stress responses, as well as genes of extrachromosomal elements, were affected. When a waterborne pathogen *Mycobacterium marinum* was grown under LSMMG in high aspect ratio vessels (HARVs) using a rotary cell culture system or the control orientation (normal gravity), it transitioned from exponential phase growth earlier than the normal gravity culture. LSMMG significantly altered transcript levels and induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. In summary, these results suggest that LSMMG may simulate a nutrient-deprived environment (Abshire et al., 2016) as would be expected in a diffusion-limited environment as previously discussed. To understand how the microbial transcriptome responds to the environment of spaceflight, the model Gram-positive bacterium *B. subtilis* was flown on two separate missions to the ISS (Morrison et al., 2019). Cells were grown to late-exponential/early stationary phase, frozen, and then returned to Earth for RNA-seq analysis in parallel with matched ground control samples. The results indicated differences in oxygen availability between flight and ground control samples, likely due to diffusion limitation in microgravity, and to differences in cell sedimentation.

1080 The global transcriptional profiling of the human opportunistic fungal pathogen *C. albicans* revealed that cells subjected to short-term spaceflight differentially regulated 452 genes compared to synchronous ground controls (Crabbé et al., 2013). Among others, genes involved in cell aggregation (similar to flocculation), antifungal agent resistance, stress tolerance, ABC transportation, oxidative stress resistance, transcriptional regulation, actin cytoskeleton, and ergosterol-encoding genes were observed to be differentially expressed. Enhanced random budding was observed in spaceflight-cultured cells compared to bipolar budding for ground samples. Although microgravity induced a virulence-related phenotype, no increased virulence in a murine intraperitoneal infection model was observed. This study supported the finding that physical forces in the human body, such as low fluid-shear, play a role in the infection process, advancing our understanding of infectious diseases on Earth to develop novel treatment strategies. A study on the metabolic and phenotypic changes of the yeast *S. cerevisiae* strains under microgravity revealed the upregulation of proteins related to anaerobic conditions and alternation in budding pattern with a switch towards more random budding (van Mulders et al., 2011).

1093 Spaceflight and ground-based studies have shown that the stress environment of space (microgravity) can also affect the transfer of gene fragments or even the whole chromosome between cells. Boever et al., (2007) reported the efficient transfer of genes via plasmid-mediated conjugation in Gram-positive bacterial strain (*B. thuringiensis*) in spaceflight compared to ground control. In contrast, no differences in gene transfer were noticed between spaceflight and ground control Gram-negative species *E. coli* and *Cupriavidus metallidurans*. Another study also showed HGT of small or large plasmids in members of *Bacillus* genera (Timmery et al., 2011), suggesting that such a process could contribute to the spread of virulence potential of *Bacillus* in confined environments. However, Beuls et al., (2009) reported no significant difference in the conjugative and mobilizable transfer frequencies in
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**

1148
1149
1150 Majority of the studies conducted till date indicate that microgravity has a strong influence on
1151 microbial growth and behavior and could lead to the evolution of properties, which does not occur on
1152 Earth, such as a column-and-canopy structure of bacterial biofilms and attainment of a growth stage that
1153 facilitates circumvention of the immune system. The mechanisms responsible for the observed
1154 biological responses, however, are not yet fully understood. We are, however, just beginning to scratch
1155 the surface in unraveling the molecular and genetic mechanisms by which gravity influences microbial
1156 growth and behavior. The major influence of a lack of gravity is a direct effect of the environment
1157 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).
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The extracellular matrix of these multicellular tumor spheroids comprises a complex 3D network of cell-cell and cell-matrix interactions that differs in amount and composition from corresponding monolayer cultures (Ulbrich et al., 2011). It affects the distribution and function of hormones and growth factors and thus fundamentally determines the regulation of cell growth, differentiation, and apoptosis (Kunz-Schughart, 1999). The model system 'Multicellular Spheroid' (MCS) mimics metastasis of solid tumors and delivers valuable information about cancer progression *in vivo* and is often used for pharmacological drug testing (Braddock, 2019; Zanoni et al., 2019). The special 3D architecture of tumor spheroids might facilitate the penetration and action of drugs more closely mimicking the situation of a tumor in the human body. In addition, the tumor spheroids and the more complex cancer organoids that were grown from breast tumor (Kaur et al., 2011; Kunz-Schughart et al., 2001) or prostate tumor cells (Zhau et al., 1997) together with fibroblasts have become important *ex vivo* models to study cancer key processes like neoangiogenesis, tumor cell invasion, and to investigate tumor-microenvironment and cell-cell-interactions (Hamilton, 1998; Kunz-Schughart, 1999; Kunz-Schughart et al., 2004).

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

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

# 7. Tissue engineering in space

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

## 7.1. Cartilage tissue engineering

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

Other researchers performed a scaffold-free experiment with porcine chondrocytes, which were cultured in special cylindrical culture chambers. The cells were exposed to real microgravity on the ISS, to simulated microgravity on an RPM and 1 g on Earth. The ISS samples revealed a reduced amount of extracellular matrix proteins together with a reduced cell density compared to the 1 g controls (Stamenković et al., 2010). Various experiments with chondrocytes have been performed to engineer 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 microgravity (396 up and 485 down) or hyper-g (413 up and 637 down directly at the onset of 1.8g).

1439 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.
1440 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. Among these genes, 135 and 143 were noted to be up- and down-regulated at least two-fold, respectively. Next to many undefined genes, a large number of genes involved in salt stress, oxidative stress, detoxification, biotic-induced stress, water stress, and defense response (and other stress responses) were among the differentially expressed ones. In addition, changes in the actin cytoskeleton organization that among other functions is involved in the transport of cell wall components to the cell periphery on exposure to microgravity were noted. In a spaceflight experiment (BRIC-16 on STS-131, 2010), Paul et al., (2012b) analyzed and compared the transcriptome of *A. thaliana* seedlings and cell cultures. While the seedlings reacted moderately over a variety of functions, the cell cultures reacted mainly with the transcription of stress response and heat shock proteins. For instance, the expression of the heat shock transcription factor HSFA2 or the heat shock protein Hsp70 was noted to be 20-fold higher in cell cultures. This shows that even undifferentiated plant cells perceive the absence of gravity as stress but do so in a more general sense than plant seedlings, which have specialized organs to sense their environment and react. In seedlings, among others, genes involved in pathogen response and wounding, stress response (cold and drought stress), auxin-mediated root development, and calcium signaling were also found to be different compared to the ground control. During a long-term experiment onboard the ISS, Paul et al., (2013) investigated the seedlings of *A. thaliana* and found 1480 genes to be differentially expressed from 1g controls (centrifuge). Also, gene expression was found to be organ-specific. About 181 genes were found to be exclusively altered in roots, only 114 in the hypocotyl, and only 113 in the leaves. Leaves, hypocotyl, and roots shared only a few genes, which were altered in all organs (26 of 408). Differentially expressed genes codes for proteins of touch-wounding response, cell wall remodeling, growth hormone, and signal transduction. Perhaps altered hormone-signaling affect the cell wall remodeling genes, influencing the growth and cell expansion (Schüler et al., 2015).

1451 Changes in transcription in plants after transfer to microgravity are not surprising considering the report of Millar and Kiss, (2013), where, transcription and metabolomics during tropistic responses were investigated. In this study, seedlings were irradiated unidirectional with red or blue light to measure phototropic responses and tilted towards the gravity vector to induce gravitropic responses. In all 1452 tropisms, genes for carbohydrate and secondary metabolism were found to be downregulated, while 1453 different genes involved in amino acid metabolism were found to be upregulated. Changes in gene 1454 expression were highest in gravitropism compared to different phototropisms. Especially genes coding 1455 for proteins of subcellular localization, proteins with binding function, defense genes, and metabolic 1456 genes were more profoundly altered during gravitropism. It was found that various metabolites, which 1457 are typical for biotic and abiotic stress, such as asparagine, phenylalanine (interaction between ethylene 1458
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. thaliana* grown in a microgravity environment and irradiated with a blue light onboard the ISS compared
1496 to 1 g control. In contrast, pathways involved in ribosome biosynthesis, which has been associated with
1497 elements controlling cell growth and proliferation (Kamal et al., 2018), and oxidative phosphorylation
1498 were upregulated. The authors suggest a connection between gravity and phototropism in *Arabidopsis*.
1499

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

1524 Recent studies have indicated that epigenetic mechanisms (DNA methylation and histone
1525 modification) control gene expression in plants in gravitational alterations indicating that gravitational
1526 changes are sensed as stress by plants (Kamal et al., 2018; Ou et al., 2010; Xu et al., 2018; Zhou et al.,
1527 2019). The regulation of genes expression via epigenetic modification affects plant responses to
1528 environmental stresses as discussed before. Kamal et al., (2018) performed experiments with cell
1529 cultures of *Arabidopsis* under simulated micro- and Martian-gravity (RPM-treatment) as well as under
1530 hypergravity (centrifugation) and observed that samples exposed to artificial microgravity and a lower
1531 extend cells exposed to Martian-gravity showed an increased DNA methylation level compared to 1 g
1532 and hypergravity conditions. Also, this study revealed effects of simulated microgravity or simulated
1533 Martian gravity on the cell cycle (lower fraction of cells in G1-phase), nucleolus structure (decrease of
1534 cells showing compact nucleolus structure and increase of fibrillary structure) as well as changes in
1535 levels of proteins involved in cell cycle regulation. Recently, Xu et al., (2018) investigated the
1536 methylation pattern changes in *Arabidopsis* seedlings exposed to real microgravity conditions onboard
1537 the Chinese recoverable scientific satellite SJ-10. They found widespread methylation of genes involved
1538 in metabolic pathways, biosynthesis of secondary metabolites, ribosome pathway, starch and sucrose
1539 metabolism, phenylalanine biosynthesis, purine, glutathione, amino sugars, and nucleotide sugar
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

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. reinhardii* 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.
1629
1630

1631 8.3. Conclusions from observed effects on plants and algae
1632

1632
1633 In plants and algae, like in animal cells, the current knowledge about gravity perception and the
1634 connected signal transduction chains is very limited. However, photosynthetic organisms (plants, plant
1635 cell cultures, and algae) and human or animal systems share similar patterns of “disturbed-gravity-
1636 induced” changes in certain classes of proteins or gene expression. For instance, signal transduction
1637 chains involving calcium are affected in plants and animals alike (Hashemi et al., 1999; Kordyum, 2003;
1638 Paul et al., 2012b). Six orthologue genes between *Drosophila melanogaster* and *Caenorhabditis elegans*
1639 were found to be differentially expressed (Leandro et al., 2007). Therefore, there appears to be
1640 something like a common response to spaceflight. Unfortunately, differences in the experimental setups
1641 (duration, hardware, limited numbers of experiments and replicates, quality of microgravity, etc.) make
1642 it very difficult to compare results of space experiments and draw a definite interpretation and
1643 conclusion. Future space experiments need to be performed under standardized conditions to attain
1644 reproducible results and conclusions (Ruyters and Braun, 2014; Wolff et al., 2014). A general database
1645 should be developed comprising of genomics, proteomics, metabolomics, and transcriptomics datasets
1646 obtained from space experiments and corresponding ground studies of different organisms.
1647
1648

1649
1648 9. Colloids in microgravity

1650 Grape juice and its fermented form, wine, are complex multi-component fluids containing
1651 sugars, different alcohols, hundreds of different tannins, or polyphenols prone to various attractive and
1652 repulsive interactions (hydrophobic, van der Waals, electrostatic, hydrogen bonding). The tannins are
1653 likely to aggregate (haze formation) with time (ageing), thus making the red wine a meta-stable colloidal
1654 dispersion: a system of fine particles suspended in a fluid (Zanchi et al., 2008). Other common examples
1655 of colloidal dispersion than wine are paints, inks, mayonnaise, ice cream, milk, and biological fluids
1656 such as blood. Although these systems are routinely produced, used, and studied, we still have much to
1657 learn about the underlying properties of colloidal systems. Hence, understanding and controlling them
1658 is a central issue in many technological and scientific fields as it will foster academic and industrial
1659 research to fine-tune their physico-chemical structures to put forward new functionalities and products.
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):

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

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 ($\sim \Delta \rho = 1 \text{ g cm}^{-3}$) 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 $10^{-3} \text{ g.cm}^{-3}$ 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.

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.

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).
1738
1739
1740
1741

## 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

1765 Several infrastructures and space operation capacities are currently present to execute various
1766 biological experiments in space. In addition, more advanced technologies are being developed. The
1767 current Dragon spacecraft by SpaceX, the Cygnus spacecraft by Orbital ATK, and the new Shepard
1768 spacecraft by Blue Origin, and the robust space platforms, such as the ISS and the Russian Earth-orbiting
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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