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3141
3142 **Figure Legends**

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

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

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

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

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

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

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

3192
3193 **Fig. 8. Simplified scheme of the physiological mechanism involved in the gravitaxis of Euglena gracilis.** If a vertically upward swimming cell (A) deviates from its vertical movement path (B and C), mechano-sensitive membrane patches are activated due to forces generated by the weight of the cell body (C). This triggers a physiological signal cascade (orange insert) in which calcium channels, a particular calmodulin (CAM 2, activated by increasing concentrations of calcium ions), a not yet
3198 identified adenylate cyclase are involved. Increased levels of cAMP, synthesized from activated
3199 adenylate cyclase activates a protein kinase A, which most likely phosphorylates flagellar proteins. This
3200 results in reorientation strokes of the flagella, aligning the cell back to the vertical position (D). In
3201 addition, a recently identified flagellar protein EgPCDUF4201 is involved in gravitaxis, but its role in
3202 the signal transduction chain is yet to be determined. The Figure is adopted and modified according to
3203 (Nasir et al., 2018). Parts of the figure were drawn by using pictures from Servier Medical Art
3204 (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License
3205 (https://creativecommons.org/licenses/by/3.0/).
3206
<figure><img src="image_3.png" /></figure>

Exploration of Space to Achieve Scientific Breakthroughs

1

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3

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

4

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

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6

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

7

ᵇCSIR - Institute of Microbial Technology, Chandigarh, India

8

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

9

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

10

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

11

fDepartment of Biomedicine, Aarhus University, Aarhus, Denmark

12

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

13

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

14

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

15

16

jSpace Biology Unlimited SAS, Bordeaux, France

17

18

Running title: Space for Revolutionizing Biological Research

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

#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**

76
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.
93
94

95 **Keywords**: Space, Evolution, Microgravity, Stress response, Mutation, Horizontal gene transfer,
96 Epigenetics, Tissue engineering, Cancer research, Colloids
# 1. Introduction

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*

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.

164

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

166

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
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**
221

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

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.
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## 2.1. What happens to an organism if a constant important stimulus is no longer impinging?

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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.

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## 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 role.
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*
431
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, the majority of genotypic mutations that affect phenotype are harmful (Eyre-Walker and Keightley, 2007). Therefore, an important question arises: what keeps a check on the mutation rate? An array of data shows that natural selection and genetic drift plays a significant role in determining genomic mutation rates across a wide range of taxa (Baer et al., 2007; Eyre-Walker and Keightley, 2007; Nei, 2013; Vos et al., 2015). Selection functions mostly in large populations, while drift prevails during periods of small population size (Ohta, 1992). For instance, because of its large population size, natural selection is more effective in reducing genomic mutation rates in the unicellular eukaryote *C. reinhardtii* than in the multicellular eukaryote *D. melanogaster* (Sniegowski and Raynes, 2013). Mutations and selection are dependent on each other. When the effective population size is large, selection tends to remove deleterious mutations that have negative fitness effects on populations and brings the mutation rate down to zero. However, the reduction is counterbalanced by an increase in mutation rates to facilitate ongoing adaptation. Thus, natural selection adjusts mutation rates to an optimum level by maintaining a balance between deleterious mutations and mutations that act as a source of genetic variation required for adaptation to a changing environment (Lynch, 2010a; Sung et al., 2012; Vos et al., 2015). This suggests that strongly selected advantageous mutations are exponentially distributed. There exists a variation in natural selection among individuals or species, which is associated to the differential regulation of DNA polymerases that have varying fidelities in replication, differences in mutagen exposure, and abilities of species to neutralize or block mutagens, recognize and repair DNA damage, and modulate or buffer mutational effects (Baer et al., 2007).

508 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).

515 **2.3.2. Role of HGT in the evolution of species**

517 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).

524 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).

536 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
545 and incorporate it into the genome of a cell. Transduction is the process of movement of genes from one
546 species to another via viruses. Conjugation involves cell-to-cell contact for the transfer of genetic
547 material to a recipient cell, which is facilitated by a tube-like structure known as the pilus. The process
548 of conjugation was thought to exist in closely related bacteria because of the compatibility, similar codon
549 usage, and higher success rate for homologous recombination (Beiko et al., 2005; Ochman et al., 2000).
550 However, there is considerable evidence that distantly related bacteria can potentially conjugate with
551 one another and exchange DNA (Cordero and Hogeweg, 2009; Lerat et al., 2005). These processes are
552 less dominant in eukaryotes. Physical contact of symbiotic or host-parasite relationships, as well as
553 transfection of a virus, have been implicated for the DNA transfer of unrelated species into eukaryotic
554 cells (Gogarten, 2003).

555 HGT includes the successful transfer of genetic material as well as the existence of the
556 transferred genetic material throughout generations (Boto, 2010). After genes are transferred, natural
557 selection determines which of them spread throughout populations (Gogarten et al., 2002; Pál et al.,
558 2005). Genes that have useful functions are preserved, whereas useless ones are generally removed
559 (positive selection) (Hao and Golding, 2008; Nakamura et al., 2004). Thus, the impact of HGT in the
560 evolution of an organism is dependent on the number of transferred genes, their successful maintenance
561 in the recipient genomes over generations, and the extent of the phenomena concerning the evolutionary
562 time framework and phylogenetic distance between organisms (Boto, 2010). The long-term persistence
563 of horizontally transferred genes confirms that they confer a selective advantage to recipient organisms.
564 There is substantial evidence that the transfer of even a single or few genes gives the recipient new
565 functions, allowing them to exploit and adapt to the new environment (Fournier and Gogarten, 2008).
566 For example, several anaerobes have acquired many metabolic genes from bacteria for their adaptation
567 to anaerobic environments (Keeling and Palmer, 2008). The acquisition of genes involved in the
568 catabolism of complex carbohydrates by rumen ciliates (Ricard et al., 2006) and the rumen fungus
569 Orpinomyces joyonii (Garcia-Vallvé et al., 2000) from bacteria have facilitated their colonization and
570 adaptation to the anaerobic environment of the ruminant gut. The pathogenesis of certain intracellular
571 parasitic bacteria, such as Chlamydia and Rickettsia is anticipated to be due to the HGT of the
572 chloroplast-type ATP/ADP translocases from plants, which allows them to scavenge ATP from the host
573 (Koonin et al., 2001).

574 Comparative analyses of data from whole-genome sequencing projects suggest that HGT has
575 significantly influenced the evolutionary mechanism throughout the history of life and emergence of the
576 three domains of life: Archaea, Bacteria, and Eukarya (Andersson et al., 2003; Boto, 2010; Boucher et
577 al., 2003; Brown, 2003; Daubin et al., 2003; Richards et al., 2006; Soucy et al., 2015). With advances
578 in sequencing technologies, the information on the importance of HGT in microbial evolution continues
579 to expand (Choi and Kim, 2007; Dagan et al., 2008; Koonin and Wolf, 2008; Pál et al., 2005; Treangen
580 and Rocha, 2011). In many prokaryotes, gene gain due to HGT is considered to be a more significant
581 process than gene duplication (Dagan et al., 2008; Treangen and Rocha, 2011; Vos et al., 2015), while
582 the relevance of HGT in gene loss is not adequately understood (Kurland et al., 2003; Polz et al., 2013).
583 The last few years of attained results show that HGT plays a far superior role in the evolution of archaea
584 and bacteria than formerly thought (Kloesges et al., 2011; Sieber et al., 2017; Smillie et al., 2011; Vos
585 et al., 2015; Wiedenbeck and Cohan, 2011). The genes and species that are associated with HGT
586 between the archaea and the bacteria are highly diverse (Brown, 2003). Koonin et al., (2001) reported
587 that between 1.6 and 32.6% of the genes of each microbial genome are acquired by HGT indicating that
588 a high level of gene transfer exists for most bacterial and archaeal genomes. Almost 24% of the genes
589 in the bacterium Thermotoga maritima that grow well at 80°C are more similar to hyperthermophilic
590 archaeal genes than other bacterial genes (Nelson et al., 1999). T. maritima acquired genes from archaea
591 through extensive HGT. Both cultures share reverse gyrase gene, which perhaps provides them
592 adaptation to live at very high temperatures (Forterre et al., 2000). Significant differences in gene content
593 (over 30%) among strains of E. coli demonstrate that HGT has a considerable effect on genome
594 evolution even at the species level (Hayashi et al., 2001). Dagan et al., (2008) analyzed evolutionary
595 history among 539,723 genes distributed across 181 sequenced prokaryotic genomes and found that at
596 least 81 ± 15% of genes in each genome were involved in HGT. They indicated that HGT has very low
597 barriers and can substantially affect all gene families among prokaryotes over long evolutionary time
598 scales.
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.

641

## 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.

692

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).

753

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

755

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.

767
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769. **Gravity and microgravity**
770

771 Gravity is one of the four basic forces in physics and has two effects on an object as a function
772 of its mass: displacement (motion) and/or deformation (weight). It is the reason for the mutual mass-
773 dependent attraction of objects. The magnitude of the attractive force between two bodies of mass is
774 determined from Newton's law of gravitation: F₁₂ = (G m₁m₂) r⁻², where G is the gravitational constant
775 (6.672 × 10⁻¹¹ Nm²kg⁻²), F₁₂ is the force resulting from the attraction by a particle of mass (m₁) on another
776 mass particle (m₂) a distance (r) away. Force accelerate bodies. The acceleration effect of the small body
777 (e.g., human) on a huge mass (e.g., Earth) is so negligible that the high-mass object attracts the body.
778 The gravity field of Earth accelerates masses at the Earth's radius with about 9.81 m/s² (differs
779 concerning geographic position). At Earth's surface, the path of an object to the Earth center is inhibited
780 and gravity becomes noticeable as weight force (F= m a, where, m is mass and a is gravitational
781 acceleration). The outcome of this relationship is so ubiquitous in our daily lives that weight is usually
782 not considered as a variable to be experimentally manipulated. The fact that the Earth's gravity gives
783 weight to attract matter leads to various phenomena, such as the formation of water bodies and
784 atmosphere, hydrostatic pressure, sedimentation, convection, and increased shear forces. In addition, the
785 physical solubility of gases such as oxygen in water depends on acceleration. Since the origin of life, all
786 organisms are constantly exposed to the vector of gravity. Organisms, above all terrestrial ones,
787 developed structures to counteract the impact of gravitational forces.

788 To annul gravity on Earth is not easy. Only conditions of free fall enable real microgravity
789 conditions. Microgravity means acceleration conditions close to weightlessness (Horneck et al., 2010;
790 Klaus, 2004). Weightlessness leads to pronounced physical changes in the sample environment, such as
791 no sedimentation, no hydrostatic pressure difference within the samples or the surrounding medium in
792 open containers, no buoyancy-driven convection: transport of metabolites reduced to the range of
793 diffusion, and low shear forces. Acceleration directly influences the properties of membranes, as has
794 been determined in experiments with defined vesicles and living cells (Kohn et al., 2017; Sieber et al.,
795 2016). In cells as well as in vesicles, membrane fluidity increases significantly in microgravity, which,
796 for example, influences the uptake and effects of drugs.
797

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3.1.1. Sedimentation

799
800 Under microgravity, no sedimentation takes place. Various organisms use intracellular heavy
801 particles (e.g., amyloplasts or otoliths) or the whole cell body as statoliths to sense the vector of gravity.
802 This phenomenon is based on the higher density of those statoliths which sediment within the less dense
803 cytoplasm. Without gravity or external acceleration this movement stops.

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3.1.2. Diffusion and convection

Diffusion is essentially based on the Brownian motion and results finally in an even mixing of
805 all types of molecules in a given limited volume. Diffusion is fast only over small distances (scale of a
806 cell). In contrast, convection depends on specific density differences in a given volume. It allows a very
807 effective mass transport over large distances. Diffusion is not gravity-dependent, while convection rate
808 is. The diffusion rate of a particle depends on its diffusion coefficient D [D = (k T)/(4 π η a)], where k
809 is the Boltzmann constant (1.3806488 x 10⁻²³ J/K) divided by NA (Avogadro number: 6.022 x 10²³), T
810 is the temperature in Kelvin (K), η is the viscosity of the medium, and a is the radius of the particle.
811 Typical values for ‘D’ are about 5 x 10⁻⁶ cm²s⁻¹ for small molecules, such as sucrose, 6 x 10⁻⁷ cm²s⁻¹ for
812 large molecules and values below 10⁻¹⁰ cm²s⁻¹ for organelles. The square distance of the migration of a
813 particle can be calculated by d² = 2 D t. The time (t) for a particle to be translocated depends on the
814 square of the distance (d). This means that diffusion processes are rather slow. For example, the time of
815 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
822 (except water at its point of an anomaly) becomes lower with increasing temperature. With uneven
823 temperature distribution (e.g., presence of a heating source), heated water or air begins to move upward,
824 while it is replaced by colder air and water. Convection results in a circulation, which also mixes the air
825 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).
852
853

### 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.
864
865

### 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
investigation on the influence of space ionizing radiation on different organisms is an important field of study. Ionizing radiation interacts with DNA and may lead to stable and inheritable genetic alternations, changes in gene function, metabolism, physiology, growth, shape, and structure. The effects of radiation on organisms depend on the quality and quantity of radiation, and cell age and characteristics (Horneck et al. 2010; Matallana-Surget and Wattiez 2013). Space radiation cannot be simulated on Earth to its full complexity.

## 4. Simulated and real microgravity platforms

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

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

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

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

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

Recently, diamagnetic levitation was thoroughly tested as a measure to produce microgravity-like conditions. In principle, all molecules in a sample should be lifted using an extremely strong magnetic field (10 - 16.5 T) in a way that the samples in the center of the solenoid are in the condition of weightlessness. The solenoid produces the magnetic field. Different magnetic fields result in different effective gravity levels. Manzano et al., (2012) constructed sample holders with different sample locations in the solenoid. With this method, simultaneously microgravity, Moon-, Martian- and hypergravity can be achieved. A “1 g-position” serves as an additional control. Magnetic levitation is presently the only facility which enables microgravity and low gravity-like effects in a stable environment (Manzano et al., 2012). Prerequisite is that all components of a sample have the same magnetic susceptibility and therefore this method is not suited for a variety of organisms. For example, since barium sulfate statoliths in *Chara* rhizoids have a far different magnetic susceptibility than the rest of the cell, investigations of gravitropism in this organism using diamagnetic levitation are not possible (Herranz et al., 2013). Also, a magnetic field alone can exert effects on living organisms. Paul et al., (2006) reported changes in gene expression patterns in *Arabidopsis* induced by strong magnetic fields. Significant effects of magnetic levitation on the orientation of *Euglena gracilis* as well as *Paramecium* cells, are also described (Hemmersbach et al., 2014). Under normal conditions, both organisms show a 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.

947

## 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.

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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

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convection required for optimal cell growth and development in reduced gravity conditions (Benoit et al., 2008; Janosi et al., 2002).

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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.

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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.

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.

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1049 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* 1081 revealed that cells subjected to short-term spaceflight differentially regulated 452 genes compared to 1082 synchronous ground controls (Crabbé et al., 2013). Among others, genes involved in cell aggregation 1083 (similar to flocculation), antifungal agent resistance, stress tolerance, ABC transportation, oxidative 1084 stress resistance, transcriptional regulation, actin cytoskeleton, and ergosterol-encoding genes were 1085 observed to be differentially expressed. Enhanced random budding was observed in spaceflight-cultured 1086 cells compared to bipolar budding for ground samples. Although microgravity induced a virulence- 1087 related phenotype, no increased virulence in a murine intraperitoneal infection model was observed. 1088 This study supported the finding that physical forces in the human body, such as low fluid-shear, play a 1089 role in the infection process, advancing our understanding of infectious diseases on Earth to develop 1090 novel treatment strategies. A study on the metabolic and phenotypic changes of the yeast *S. cerevisiae* 1091 strains under microgravity revealed the upregulation of proteins related to anaerobic conditions and 1092 alternation in budding pattern with a switch towards more random budding (van Mulders et al., 2011).

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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