Selecting for Chlamydomonas reinhardtii fitness in a liquid algal growth system compatible with the International Space Station Veggie plant growth chamber

A biological life support system for spaceflight would capture carbon dioxide waste produced by living and working in space to generate useful organic compounds. Photosynthesis is the primary mechanism to fix carbon into organic molecules. Microalgae are highly efficient at converting light, water, and carbon dioxide into biomass, particularly under limiting, artificial light conditions that are a necessity in space photosynthetic production. Although there is great promise in developing algae for chemical or food production in space, most spaceflight algae growth studies have been conducted on solid agar-media to avoid handling liquids in microgravity. Here we report that breathable plastic tissue culture bags can support robust growth of Chlamydomonas reinhardtii in the Veggie plant growth chamber, which is used on the International Space Station to grow terrestrial plants. Live cultures can be stored for at least one month in the bags at room temperature. The gene set required for growth in these photobioreactors was tested through a short-wave ultraviolet light (UVC) mutagenesis and selection experiment with wild-type (CC-5082) and cw15 mutant (CC-1883) strains. Genome sequencing identified UVC-induced mutations, which were enriched for transversions and nonsynonymous mutations relative to natural variants among laboratory strains. Genes with mutations indicating positive selection were enriched for information processing genes related to DNA repair, RNA processing, translation, cytoskeletal motors, kinases, and ABC transporters. These data suggest modification of signal transduction and metabolite transport may be needed to improve growth rates in this spaceflight production system.

A biological life support system for spaceflight would capture carbon dioxide waste produced by 14 living and working in space to generate useful organic compounds. Photosynthesis is the primary 15 mechanism to fix carbon into organic molecules. Microalgae are highly efficient at converting 16 light, water, and carbon dioxide into biomass, particularly under limiting, artificial light 17 conditions that are a necessity in space photosynthetic production. Although there is great 18 promise in developing algae for chemical or food production in space, most spaceflight algae 19 growth studies have been conducted on solid agar-media to avoid handling liquids in 20 microgravity. Here we report that breathable plastic tissue culture bags can support robust 21 growth of Chlamydomonas reinhardtii in the Veggie plant growth chamber, which is used on the 22 International Space Station to grow terrestrial plants. Live cultures can be stored for at least one 23 month in the bags at room temperature. The gene set required for growth in these 24 photobioreactors was tested through a short-wave ultraviolet light (UVC) mutagenesis and 25 selection experiment with wild-type (CC-5082) and cw15 mutant (CC-1883) strains. Genome 26 sequencing identified UVC-induced mutations, which were enriched for transversions and 27 nonsynonymous mutations relative to natural variants among laboratory strains. Genes with 28 mutations indicating positive selection were enriched for information processing genes related to 29 DNA repair, RNA processing, translation, cytoskeletal motors, kinases, and ABC transporters. 30 These data suggest modification of signal transduction and metabolite transport may be needed 31 to improve growth rates in this spaceflight production system. 32

Introduction 33
Microalgae grow by converting light, water, and CO 2 into biomass. Algae have long been 34 proposed for space life support systems to recycle CO 2 and provide food either directly or 35 indirectly to astronauts (Brechignac and Schiller, 1992;Ai et al. 2008;Niederwieser et al., 2018;36 Matula and Nabity, 2019). Many species of microalgae are photosynthetically efficient under the 37 limiting light and low volume conditions necessary in space production (Kliphuis et al., 2012). 38 As single cell organisms, microalgae are easy to cultivate with minimal requirements, and have 39 great potential to yield value-added products. 40 Both eukaryotic and prokaryotic species, such as Chlorella vulgaris and Arthrospira platenis, 41 respectively are generally regarded as safe (GRAS) for human consumption (Caporgno and 42 Mathys, 2018). Algae have nutritional benefits with high levels of antioxidants and protein with 43 essential amino acids (Buono et al., 2014). Algae are also rich in ω-3 fatty acids, such as 44 eicosapentaenoic acid and docosahexaenoic acid (Salem and Eggersdorfer, 2015 Island, FL, USA) with a ratio of red: green: blue of 8:1:1. All lighting was 80-100 μ mol/m 2 /s. 102 The UVC mutagenesis dose that caused ~10% cell survival was determined by transferring 7 mL 103 of early-log phase culture at OD 600 = 0.45 to 15 cm sterile petri plates in a sterile laminar flow 104 hood. The petri plates were opened in a GS Gene Linker UV Chamber (Bio-Rad, Hercules, CA, 105 USA) and exposed to increasing doses of UVC light from germicidal bulbs. Petri plates were 106 closed, wrapped in aluminum foil, and agitated overnight in the dark at 50 rpm. Mutagenized 107 cultures were plated on TAP agar plates with non-treated samples diluted 1:5 prior to plating. 108 Colonies were grown under continuous light, counted, and normalized relative to non-109 mutagenized cultures. 110

EVT mutagenesis 111
Colonies from TAP agar plates were scraped and suspended in 600 μL liquid TAP media and 112 adjusted to an optical density of 1 at 600 nm using a visible light spectrophotometer (SmartSpec 113 3000, Bio-Rad, Hercules, CA, USA). The Chlamydomonas suspension was used to inoculate 114 TAP liquid media at a 1:250 dilution, i.e. 0.2 mL of OD 600 =1 suspension was added to 50 mL 115 TAP media. WT was inoculated on day 1 of the experiment and cw15 was inoculated on day 2. 116 The cultures were grown in flasks until reaching an OD 600 of 0.4-0.5 on day 4. Non-mutagenized 117 cells from the culture were sampled for whole genome sequencing by centrifuging 2 mL of the 118 culture and freezing the cell pellet at -80°C until DNA was extracted. For each mutagenesis, 7 119 mL of early-log phase culture was exposed to 8 mJ of UV light. The mutagenized cells were then 120 used to inoculate 100 mL of TAP media in a PermaLife tissue culture bag in a sterile laminar 121 flow hood. Inoculated tissue culture bags were held at room temperature in the dark for 7 d and 122 then transferred to the Veggie growth chamber. 123

EVT culture conditions and selection 124
The Veggie chamber was set with the reservoir at maximum distance from the lighting. The red 125 light was set to 'medium'; the blue light was set to 'low'; and the green light was set to 'on'. The 126 bellows were closed during growth cycles, and the fan was set to 'low'. The initial mutagenized 127 cultures were grown for 7 d in Veggie. The cultures were then passaged by transferring 1 mL of 128 culture to a bag containing fresh TAP media using a sterile syringe. The second culture was 129 grown for 6 d and then passaged to a third tissue culture bag for 6 d of growth. During each 130 passage, 2 mL of culture was sampled, centrifuged, and the cell pellet was frozen at -80°C until 131 DNA was extracted. The remaining cultures were stored in a soft stowage, Cargo Transport Bag 132 (CTB) until 36 d after the initial inoculation. For each dark-stored culture, 2 mL was sampled for 133 DNA extraction. 134

Biomass measurement 135
Dry biomass was determined by transferring all of the remaining culture volume into two 50 mL 136 centrifuge tubes. The cells were centrifuged with the supernatant media remove and the cell 137 pellets were lyophilized overnight. Dry weights were measured on an analytical balance. 138 Biomass for each culture bag is the average of the two technical replicates. 139

2.5
Whole genome sequencing 140 DNA was extracted from the flash-frozen and dark-stored cell pellets as described with some 141 minor modifications (Newman et al., 1990 We tested commercial FEP tissue culture bags for the ability to support microalgae growth 200 without agitation or active mixing of gases with liquids. Under LED lighting, the bags are able to 201 support robust growth ( Figure 1A-B). Time courses of growth show a 2 d delay in the bags with 202 log phase between 4-6 d and stationary phase at 8 d for both WT and cw15 strains ( Figure 1C-203  D). These results indicate that FEP tissue culture bags provide sufficient gas exchange to support 204 microalgae, but that Chlamydomonas laboratory strains are better adapted to grow in flasks. 205 To select for mutations that would improve growth in tissue culture bags, we determined the 206 dose-response for cell lethality in a UVC light chamber. Figure 1E shows that 6-10 mJ of UVC 207 exposure is sufficient to kill ~90% of WT cells. Similar results were obtained for cw15, and we 208 concluded that 8 mJ of UVC would give sufficient DNA damage to induce mutations in both 209 strains without risking excessive cell death and culture failure during spaceflight. 210 An EVT was completed at the Kennedy Space Center (Figure 2 Veggie reservoir ( Figure 2B). The bags were left without any agitation except during passages 217 ( Figure 2C). To complete a passage, culture bags were removed from the reservoir, agitated 218 manually, and 1 mL of culture was transferred to fresh media for a new growth cycle ( Figure 2D-219 F). An additional 2 mL of culture was sampled and cell pellets were frozen to preserve a DNA 220 sample without dark storage. 221 The remaining culture was stored in a closed CTB to simulate ambient storage on the ISS and 222 return of live cultures to Earth ( Figure 3A). At 36 d after the initial inoculation, all culture bags 223 were sampled for DNA extraction and biomass assessment. The WT strain showed higher 224 biomass compared to the cw15 cell wall mutant, and there was a trend for increased biomass 225 with additional culture passages. Increased biomass with the latter passages may reflect loss of 226 biomass due to dark storage or selection for faster growth during the experiment. 227 Whole genome sequencing of the pre-mutagenized cultures, frozen cell pellets, and dark stored 228 cultures was completed with an average read depth of 16x. Variants consisting of single 229 nucleotide polymorphisms (SNP) and short insertion-deletion (InDel) polymorphisms were 230 called with CRISP using pooled sample parameters (Figure 4). After removing missing data (≥ 231 30%) and monomorphic polymorphisms (MAF=0), we detected 73,573 WT variants and 79,455 232 cw15 variants. Filtering to remove low-depth and low-quality reads reduced WT and cw15 233 polymorphic variants to 48,380 and 53,939, respectively. 234 Plotting the variant density across the genome identified known hotspots of natural variation 235 among laboratory strains ( Figure 5, Gallaher et al., 2015). The WT strain is a sequence-verified 236 clone of CC-1690, and the polymorphic variants identified overlapping peaks on chromosomes 237 2, 6, and 9. In addition, there were peaks that overlapped with other natural variants on 238 chromosomes 3 and 12. The cw15 strain derives from a cross with CC-1690 and a similar pattern 239 of natural variant peaks was observed. These natural variants may represent spontaneous 240 mutations that are easily tolerated in Chlamydomonas or regions of the genome that are difficult 241 to align with high confidence. In either case, known natural variants are likely to have little 242 signal of selection due to induced UVC mutagenesis. We removed all exact matches to natural 243 variants to obtain 5,286 WT and 5,873 cw15 novel variants, which are more evenly distributed 244 across the genome ( Figure 5). 245 The novel variants show a different spectrum of base changes than natural variants ( Figure 6A). 246 There is a relative decrease in transitions and an increase in transversions with the 247 complementary mutations of A>C and T>G as well as C>G and G>C predominating. These data 248 suggest the novel variants represent mutations caused by induced mutagenesis instead of the 249 endogenous spectrum of Chlamydomonas variants. In addition, the novel mutations are found at 250 a higher allele frequency indicating the novel mutations have more sequence read support across 251 samples than natural variants ( Figure 6B). We conclude that novel variants better represent the 252 UVC mutagenized sites. 253 Centrifugation and freezing cell pellets for DNA sampling requires more extensive astronaut 254 time and limiting resources on the ISS. We compared the mutations recovered from samples that 255 had been frozen at the time of passage and those from live cultures that had been stored in the 256 dark. There were no significant differences in sequencing depth based on the storage conditions 257 ( Figure 5C-D). However, dark storage decreased the number of mutations recovered in passage 258 1, which were cultures stored for 22 d prior to sampling. Student's t-tests showed a significant 259 reduction of mutations detected for the WT strain (p = 0.003), but the reduction was non-260 significant for cw15 (p = 0.07). For frozen stored libraries, the number of novel mutations 261 detected in each passage was nearly constant indicating sequencing depth was limiting for 262 mutant detection. These results suggest that changes in allele frequency over culture passages is 263 not a reliable indicator of selection for this experiment. 264 To assess the effects of novel mutation on protein coding sequences, SnpEff was used to identify 265 protein coding changes. Natural variants were enriched for synonymous mutations, while the 266 novel variants were enriched for protein coding changes ( Figure 7A). These results are consistent 267 with an increased frequency of deleterious mutations after UV mutagenesis. Individual genes 268 were tested for selection based on nucleotide diversity (π) using SNPGenie. Novel variants were 269 enriched for genes showing evidence of positive selection with about 46% of genes tested having 270 π N > π S , compared to 29% of all variants ( Figure 7B). These results are consistent with the 271 enrichment for nonsynonymous mutations resulting from UV mutagenesis. 272 Based on GO term enrichment analyses, the positively selected genes in WT and cw15 both 273 show significant enrichments for terms associated with purine nucleotide binding and hydrolase 274 activity ( Figure 7C). The individual genes with these GO terms represent information processing 275 functions in DNA damage repair, RNA processing, translation, cytoskeletal motors, and signal 276 transduction (Supplementary Table 1). The WT libraries also had enrichment for terms 277 associated with small molecule transporters with individual genes predominantly being ABC 278 transporters. 279 The WT strain also had significant enrichments for genes under purifying selection (Figure 8). 280 More than half of these genes are predicted to function in regulating the levels of cyclic 281 nucleotide second messengers suggesting second messenger signal transduction may be under 282 selective pressure in in the culture bag growth system. The cw15 strain had no significantly-283 enriched GO terms for genes under purifying selection. 284

4
Discussion 285 This EVT validated a strategy for identifying genes required by Chlamydomonas during log 286 phase growth in spaceflight. We have shown that commercial FEP tissue culture bags can be 287 used for batch culture of microalgae. Chlamydomonas is viable in these bags after prolonged 288 dark storage, which enables full genome sequencing and identification of mutant genes in the 289 culture. We have since used this strategy to grow the WT and cw15 strains during the SpaceX 290 CRS-15 mission; analysis of the spaceflight experiment is on-going. 291 Whole genome sequencing has been used to assess the mutagenic load of the bacteria, 292 Staphylococcus aureus, in a two week spaceflight exposure (Guo et al., 2015). Less than 40 293 SNPs were detected in the genome from spaceflight with similar numbers of SNPs detected on 294 ground and in spaceflight. These data suggest that exogenous mutagenesis is necessary to gain 295 adequate signal of selection in short-term competitive growth experiments. 296 A yeast selection experiment was completed in spaceflight by using a genome-wide deletion 297 collection (Nislow et al., 2015). The competitive growth experiment measured the reduction in 298 representation of bar-coded mutants over the course of ~21 mitotic generations. This type of 299 competitive growth identifies individual genes needed for growth. By contrast, UVC 300 mutagenesis has a higher genetic load and generates a more diverse array of allele types to 301 compete within the culture. Limitations to random mutagenesis are that loss-of-function 302 mutations in all genes are not represented in each biological replicate of the experiment and that 303 multiple mutations are simultaneously selected in individual cells during mitotic divisions. 304 UV light causes direct DNA damage to create cyclobutane pyrimidine dimers, and the UV 305 mutation signature is typically biased towards C>T transitions (Ikehata and Ono, 2011). 306 Chlamydomonas DNA readily forms pyrimidine dimers, and WT strains have a robust dark-307 repair pathway to repair 90-95% of the DNA damage directly within 24 h (Small, 1987 for both of these enzymes in the EVT (Pannunzio et al., 2018). Evidence for selection in double-323 strand break repair and translesion synthesis suggest that these pathways are likely relevant to the 324 UV-induced mutations observed. 325 The low sequence coverage of this experiment creates risk in using the frequency of recovery for 326 specific mutant alleles in determining whether specific genes are under positive or purifying 327 selection. The number of alleles discovered is limited by the sequencing depth, and higher 328 coverage is necessary to increase the power of the statistics to detect selection at a genome-wide 329 level. Nevertheless, we were able to classify 476 genes in the WT strain and 503 genes in the 330 cw15 strain for purifying or positive selection based on mutations within coding sequences. 331 Among these, 104 genes were enriched for molecular functions based on GO terms. In addition 332 to DNA repair pathways, these analyses revealed signal transduction and other information 333 processing functions including, chromatin reading, RNA processing, and translation to be 334 enriched within selected genes. The enriched molecular functions are predicted to be processes 335 necessary for Chlamydomonas to adapt to the UV mutagenesis, lack of media agitation, the 336 diffusion of gases across the FEP membrane, and the lighting conditions in the Veggie unit. 337 Scalable production of Chlamydomonas in spaceflight has multiple potential applications. The 338 species will accumulate lipids to about 20-25% of total biomass, which can be used as an organic 339 chemical feedstock (Becker, 2007;Xu et al., 2018). Chlamydomonas also has high protein 340 content of 40-60% of biomass making it a potential source of food. Although it is not yet 341 designated as a GRAS organism by the FDA, Chlamydomonas is non-toxic; animal feeding 342 studies show no harmful effects at 4 g algae biomass per kg body weight per day, the highest 343 consumption levels tested (Murbach et al., 2018 Baek, K., Yu, J., Jeong, J., Sim, S.J., Bae, S., and Jin, E. (2018). Photoautotrophic production of 393 macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free 394